If skin is breached, infection remains localized and are extinguished within a few days without illness
Genetically programmed
In place even before infection
Respond in the same way to repeated exposures
Specific to structures shared by groups of microbes = cannot distinguish microbes
Recognition
- soluble proteins and cell-surface receptors bind to pathogens or to altered cells and serum
- peptides, proteins, glycoproteins, proteoglycans, peptidoglycans, nucleic acids
Recruitment
- effector mechanisms provided by effector cells to kill and eliminate pathogen
- complement serum proteins to mark pathogens

Serum proteins of complement system recognizes pathogen
Activation of serum proteins = fragment binds to pathogen and acts as marker
Fragment recruits leukocytes with surface receptors to site
Leukocyte binds to fragment and engulfs it

Cytokines

Soluble secreted proteins
Trigger and regulate innate immune response
Induces vasodilation of the endothelium
- Leakage of blood plasma
- Expansion of fluid volume = edema or swelling = pressure on nerve endings causes pain
Alters adhesive properties of endothelium
- WBCs attach to it to enter the inflamed tissue = inflammatory cells
- Increases swelling and contribute to pain
Enables cells and molecules of the immune system to be brought rapidly and in large numbers into the site of infection

Adaptive Immune System

Adds/ enhances innate immune response
- Innate immune response slows spread of infection and recruits lymphocytes
Slow response (days to weeks)
Adapts to the nuances of the infecting pathogen
Specificity and diversity
- cell-surface receptors recognize specific antigens and different portions (epitopes) of polysaccharide and macromolecules
- huge lymphocyte repertoire due to variability in structures of antigen-binding sites
  - different clones differ in their receptors and therefore specificity for antigens
Clonal selection
- Selection of lymphocytes by antigens for activation to produce specific antibody
  - specific antigen receptors exist on lymphocytes before they are presented with an antigen due to random mutations during initial maturation and proliferation
Clonal expansion
- Lymphocyte specific for an antigen undergoes proliferation after exposure
- Increase in number of cells that express identical receptors for the antigen
- Keeps pace with rapidly dividing pathogens
Immunological memory
- To elicit a stronger and faster adaptive immune response
- Exposure to antigen generates long-lived memory cells specific for the antigen
- Also called acquired or protective immunity
- Primary immune response: first exposure to pathogen
- Secondary immune response: subsequent exposures with more rapid, larger, and qualitatively different from primary response

Adaptive Immunity is better understood than innate immunity

Infections are overcome before they cause any symptoms
Inherited deficiencies and impairment of function are rare
Clinicians work together with adaptive response for a cure = more favored than innate

Hematopoiesis

Leukocyte are derived from pluripotent hematopoietic stem cell
Gives rise to precursors of lymphoid, myeloid, and erythroid lineages
Sources: yolk sac > fetal liver > spleen > bone marrow
Occurs throughput life due to short-lived nature of blood cells

Erythroid Lineage

Gives rise to erythrocytes and megakaryocytes
Megakaryocyte
- Fusion of multiple precursor cells
- Permanent residents of the bone marrow
- has small, non-nucleated fragments = platelets
  - maintain integrity of vessels
  - blood clotting

Myeloid Lineage

granulocytes
- kill pathogens and enhance inflammation
- aka polymorphonuclear leukocytes
- neutrophil
  - capture, engulfment and killing via phagocytosis
  - effector cells of innate
  - shortly-lived = pus
- eosinophil
  - against parasites and worms
- basophil
  - regulates response to parasites
Monocytes
- Circulates in blood
- Recruited by macrophages into infected tissues = matures into macrophages
  - In the presence of infection and extensive tissue damage, macrophages may be overwhelmed and die due to the number of dead pathogens and dead neutrophils
Macrophage
- Derived from embryonic stem cell
- Becomes resident tissue macrophages
- First detects infections
- Becomes activated and recruit neutrophils and other leukocytes to the site of infection for the innate response by secreting cytokines
- General scavenger cells
Dendritic cells
- Determines whether and when the innate response needs support from adaptive response
- Carry intact and degraded pathogen to lymphoid organs = initiates adaptive response
Mast Cells
- Resident in all connective tissue
- Plays a role in inflammation
- Violent spasms of smooth muscle to eject parasites from respiratory and GI tracts

Lymphoid Lineage

Natural killer cells
- Defense against viral infections
- Recruited by macrophages
- Secretes cytokines that impede viral replication
Small lymphocytes circulate in inactive forms
B cells
- Cell surface receptors: immunoglobulins
- Effector cells: plasma cells = soluble antibodies
- Can recognize antigen in epitopes
T cells
- T-cell receptors
- Cannot recognize antigen alone
- Antigen must be bound to major histocompatibility complex molecule
  - “presents” antigen to the T cell receptor

Effector Cells of Adaptive

Plasma cells
- Production of antibodies
- Circulate in blood
- Enters tissue
- Binds to antigen
Cytotoxic T cells
- Expresses CD8 receptors on its surface
- Kill cells infected with intracellular pathogen
Helper cells
- Expresses CD4 receptors
- Secrete cytokines to help activate other cells
Regulatory T cells
- Inhibit immune response by closing CD4 and CD8 responses
- Prevents unnecessary tissue damage

Humoral Immunity

Immunity due to antibodies
For extracellular microbes
Facilitate engulfment and destruction of microbes and toxins by phagocytes
Neutralization: binds to pathogen and inhibit growth, replication, or interaction with human cell receptors
Phagocytes can bind to antibody molecules
Opsonization: Antibody can coat bacteria to enhance phagocytosis

Lymphoid Tissues

Major lymphoid organs: bone marrow, thymus, spleen, adenoids, tonsils, appendix, lymph nodes, and Peyer’s patches
Primary lymphoid tissues: where lymphocytes develop and mature
Secondary lymphoid tissues: where mature lymphocytes become stimulated
Forms a network of lymphatics and collect the leaked plasma from the blood and returns it to the circulation as lymph via the thoracic duct and empties into the left subclavian vein
No pump = sluggish flow = away from peripheral tissues = driven by continual movements
- Or else edema
B cells and T cells move through both blood and lymph
- If they become activated by a pathogen, they remain in the lymph node
- Otherwise they leave in the efferent lymph and return to the blood
- Continual state of flux = lymphocyte recirculation
- To monitor the secondary lymphoid tissues for infection

Secondary Lymphoid Tissues

To establish infection, a microorganism must colonize a tissue and overwhelm the local innate immune response
Dendritic cells that are either pathogen-infected or loaded with pathogen antigens are carried in the lymph through lymphatics to the nearest lymph node (draining lymph node)
- Prevents potentially dangerous materials from entering circulation

Lymphoid follicles
- Where lymphocytes are segregated into B cell areas and T cell areas
- Pathogens and antigen-laden DC in lymph enter via afferent lymphatic vessel
- Lymph is drained via the efferent lymphatic vessel
- Dendritic cells stay in the node
  - Allows pathogens and other extraneous materials to be extracted by the resident macrophages
- Activated B cells proliferate in the node
  - Forms germinal center
- T cells are activated by antigen-bearing DC
  - Some become Helper T cells and help B cells activate into plasma cells in the node
- Lymphocyte activation, proliferation, and differentiation lead to swelling of lymph node

ADAPTIVE IMMUNE SYSTEM

Antibody Responses
- B lymphocytes or B cells
- Plasma cells
  - When B cells encounter an antigen, they become plasma cells
- Antibodies/ immunoglobulins
Cell-mediated immune responses
- T lymphocytes or t cells found in all cells except RBCs
- Major histocompatibility complex and T-Cell receptors

Spleen

Filters blood to remove damaged and senescent RBCs
Also a secondary lymphoid tissue that defends against blood-borne pathogens
- Splenic macrophages and DCs
- Stimulation of B cells and T cells from the blood
Red pulp: blood cells are monitored and elderly or damaged ones are removed
White pulp: similar to lymph node but without lymphatics

Mucosa-associated Lymphoid Tissue (MALT)

Secondary LT of mucosal tissues
Pathogens arrive by direct delivery mediated by M cells of the mucosal epithelium

Gut-associated LT (GALT)

Tonsils, adenoids, appendix, Peyer’s patches of the GI tract

Bronchial-associated LT (BALT)

Lines the respiratory epithelium

CHAPTER 2

IMMEDIATE INNATE RESPONSE

First line of defenses
- Physical barriers
- Molecular mechanisms of innate immunity
Second line of defense
- Induced mechanisms of innate
- mobilization of cells upon detection of infection
Third line of defense
- Adaptive immune response

Barriers

Skin and mucosal epithelia
Commensal microorganism = microbiota
- Deters pathogen invasion
- Invading pathogen must compete with the resident commensals for nutrients and space
Extracellular pathogens
- Accessible to soluble secreted molecules of immune system
Intracellular pathogens
- to combat, human cells are killed to interfere with the pathogen’s life cycle
- expose any pathogen released from dead cells to soluble molecules

Complement System

plasma proteins made in the liver, present in blood
induced when tissue becomes infected
opsonizes bacteria and extracellular virus particles
has proteases that circulate in the blood, lymph, and tissues
- inactive form = zymogens
Protease cleaves and activates the next protease
C3 most important proteins of the complement
- Those lacking C3 are prone to severe infections
- Has thioester bond with glycoprotein

Activation of the complement system by infection = cleavage of C3 into C3a and C3b
- C3b attaches to the pathogen’s surface = complement fixation
- Tags the pathogen for phagocyte-mediated destruction
- Organize the formation of protein complexes that damage the pathogen’s membrane
- C3a = acts as a chemoattractant to recruit phagocytes and other effector cells from the blood
- The thioester bond is exposed to the hydrophilic environment upon cleavage
  - Most will hydrolyze but a minority will react with the hydroxyl or amino group on the pathogen’s surface

Pathways

All lead to C3 activation, deposition of C3b and recruitment of effector mechanisms

Alternative Pathway

Start of infection
Binding of C3 with water = iC3
iC3 binds to factor B and is susceptible to cleavage by factor D
fragment Ba is released while fragment Bb with protease activity remains bound to iC3 = iC3Bb complex
iC3Bb cleaves C3 into C3a and C3b
high concentration of C3 in blood = activation and cleavage of C3 in large quantity = some C3b will bind to pathogen’s surface
C3 convertase = proteases that cleave and activate C3
- iC3Bb is an example

C3b fragments bound to a pathogen also binds to factor B and cleaved by factor D
- Forms C3Bb complex
- The C3 convertase of the alternative pathway
- Works right at the pathogen surface
- Binds C3 and cleaves into C3a and C3b
- Because this convertase is present on the pathogen surface, it is more efficient in fixing C3b to the pathogen surface
- Rapid formation of additional C3Bb molecules

Regulatory Proteins

Plasma protein properdin (factor P) increases complement activation
- Binds to C3Bb on pathogen surface and prevent its degradation by proteases
- Factor H plasma protein counters factor P
  - Binds C3b and facilitates further cleavage to iC3b by factor I plasma serine protease
- Combined effects of factors H and I is to decrease the amount of C3 convertase on the pathogen surface
- Those who lack factor I = immunodeficiency
  - Formation of C3Bb remains unregulated until the C3 reservoir in blood, extracellular fluid, and lymph is exhausted
  - When faced with bacterial infections, individuals with factor I deficiency fix very little C3b to bacterial surfaces, causing inadequate clearance of bacteria by phagocytes
  - More susceptible to ear infections and abscesses caused by encapsulated polysaccharide bacteria
Decay-accelerating factor (DAF)
- Binds to C3b component of the alternative C3 convertase
- Causes its dissociation and inactivation
membrane cofactor protein (MCP)
- Binding of MCP to C3b makes C3b susceptible to cleavage and inactivation by factor I
Complement control protein (CCP) modules
- DAF, MCP, factor H
- Proteins made up of CCP modules = regulators of complement activation
The combined effect of the reactions that promote and regulate C3 activation is to ensure that C3b is deposited only on pathogen surface and not on human cells
- Effective way of distinguishing human cells from microbial cells

Macrophage

First effector cell to encounter invaded tissue
Prevalent in connective tissues, linings of GI and respiratory tracts, liver (Kupffer cells), and alveoli of lungs
Macrophage cell surface receptors enhances phagocytosis

Complement receptor 1 (CR1)
- Binds to C3b fragments on pathogen surface
- Facilitates engulfment of the pathogen into endosome or phagosome
  - Fuses with lysosome which delivers toxins and enzymes to degrade the pathogen
- Opsonization
- Protects the surface of cells on which it is expressed
  - Disrupts C3 convertase by making C3b susceptible to cleavage by factor I
- Made up of CCP modules
CR3 and CR4
- Examples of integrins which contributes to adhesive interactions
- Bind iC3b fragments on microbial surfaces
- Although the iC3b fragment has no C3 convertase activity, it facilitates phagocytosis and pathogen destruction as a ligand for CR3 and CR4
- Combination of CR1, CR3 and CR4 > single complement receptor

Terminal Components

C5, C6, C7, C8, C9 proteins
Cooperate to form the membrane-attack complex (MAC)
- Large pore assembled in the pathogen membrane to disrupt its integrity

C5
- Similar to C3 but lacks a thioester bond
- Activated by the alternative C5 convertase
- Has 2 fragments of C3b and one Bb fragment = C3b2Bb
- Cleaves C5 into C5a and C5b

- C5b
  - Initiates MAC formation
  - C6 and C7 binds to C5b = forms complex to to expose hydrophobic region of C7
  - C7 inserts into the lipid bilayer
- C8 joins complex
  - C8 binds to C5b to expose its hydrophobic site and inserted into the membrane
- These events lead to the polymerization of 18 C9 molecules and MAC formation
  - Final complex: one molecule each of C5b, C6, and C7, three molecules of C8, and 18 molecules of C9

Soluble Proteins

S protein, clusterin and factor J
Prevent association of the C5b, C6 and C7 complex with the cell membranes
Homologous restriction factor (HRF) and CD59 (protectin)
- Prevents C9 recruitment to the complex by binding to C5b678 complex
- Impaired synthesis of the glycosylphosphatidylinositol tail CD59 = human disease paroxysmal nocturnal hemoglobinuria (PNH) = complement-mediated lysis of RBCs which have no DAF, HRF, or CD59 = not protected from actions of the complement
- Treatment: monoclonal antibody specific for C5 and prevent its cleavage and activation by C5 convertase

Inflammatory Peptides

C3a and C5a fragments are ligands for receptors on phagocytes, endothelial cells, and mast cells
Increases inflammation at the site of complement activation
Can induce anaphylactic shock = C3a and C5a are anaphylatoxins
- C5a more potent and stable than C3a
- Induces contraction of smooth muscle and degranulation of mast cells and basophils
  - Release of histamine and other vasoactive substances that increase capillary permeability
  - Increases exit of plasma and cells from the blood
  - C5a induces neutrophils and monocytes to adhere to vessel walls and recruit phagocytes
  - C5a increases CR1 and CR3 on surfaces

Plasma Proteins

Vessels damaged by pathogens activate the coagulation system
- Enzymes in plasma that cooperates with platelets to form blood clots
- Pathogens are immobilized in the clots and cannot enter the blood and lymph
- During clotting, platelets degranulate and release active agents to recruit immune cells, antimicrobial defenses, and tissue repair
Kinin system
- Enzymatic cascade of plasma proteins induced by damaged tissue
- Leads to the production of bradykinin
- Reduces hypertension and promotes vasodilation and smooth muscle relaxation

Protease inhibitors
- Contained in human secretions and plasma
- For pathogens that have acquired proteases (i.e., plasmin) to hide from the immune system
- Ex: alpha2-macroglobulin
- Lures microbial protease with its “bait” region to be cleaved by the protease
- Activates the thioester in a2-macroglobulin = binds and envelops the protease
- Immediately bound by specific receptors on macrophages, hepatocytes, and fibroblasts

Defensins

Antimicrobial peptide that can neutralize broad range of structurally diverse toxins
Alpha-defensins and beta-defensins
- Amphipathic (has hydrophobic and hydrophilic regions to penetrate membranes) = pore formation
When binding to microbial toxin, it promotes unfolding of the toxin = susceptible to proteases = anti-chaperones

Pentraxins

Plasma proteins that bind microorganisms and deliver them to phagocytes
Same role as antibodies

CHAPTER 3

INDUCED INNATE RESPONSES

Inflammation

Induced phase involves soluble and cellular receptors that detect the presence of a pathogen and recruit leukocytes to make an inflammatory response
Inflammation will help induce a subsequent adaptive immune response
Macrophages activated by the presence of an infection will release inflammatory cytokines

Self vs Non-Self vs Altered Self

Receptors can recognize structural features that distinguish microbial macromolecules from human macromolecules
Non-self: microbes
Altered-self: infected cells and cancerous cells
Individual cells express different combinations of receptors = functional diversity = increases likelihood that at least some of the cells will be able to mount an effective response to any given pathogen

Lectins

Carbohydrate-specific receptors found in macrophages

Surface Receptors of Resident Macrophages

Receptor-mediated endocytosis = macrophages surface receptors capture pathogens and deliver them to endosomes where the pathogen is killed and partly degraded
Endosome fuses with lysosome where extreme acidity and concentration of degradative enzymes completely degrades the pathogen
Pattern-recognition receptors (PRRs): receptors that recognize a structural feature common to many different types of pathogen
- Structural feature on microbe is called pathogen-associated molecular pattern (PAMP)
- Damage to cells or tissue that does not involve an infection: Damage-associated molecular pattern (DAMP)
Scavenger receptors: most of the PRRs of macrophages
- Trigger phagocytosis, cell adhesion and intracellular signaling to identify microbes and molecules and cause their elimination
- SR classes A-L (11 classes)
- Bind to many different ligands including surface components of microbes
- In the absence of infection, SRs remove dead and dying cells and unwanted macromolecules and cells that have died by apoptosis (damaged or infected cells)

- Some SRs are lectins
  - Mannose receptor (SR-E3) and Dectin-1 (SR-E2)
    - MR recognizes mannose and other sugars present in the glycans of bacterial surfaces and internalized by receptor-mediated endocytosis
    - Dectin recognizes beta-glucans (glucose polymers) on fungal and bacterial surfaces
  - Belongs to C-type lectin domain (CTLD)
  - Calcium ion coordinates the interaction of the carbohydrate ligand with the protein receptor

SR-A Class

Macrophage receptor with collagenous structure (MARCO)
- Recognizes bacterial lipopolysaccharide (LPS) of the outer membrane of Gram-negative bacteria
- Also binds to Gram-positive bacteria
SR-A1
- Recognizes LPS and lipoteichoic acid cell wall component of Gram-positive bacteria
- Additional ligands: hepatitis C virus, beta-amyloid, and heat shock proteins

CR3 and CR4

Binds to iC3b
Part of integrins that contribute to adhesive interactions between cells
Recognizes LPS of E.coli, Salmonella, N. gonorrhoeae, and other Gram-negative bacteria
Recognizes lipophosphoglycan of protozoan parasite

Toll-like Receptors

Toll-like receptors (TLRs) = signaling receptors present on immune cells
- Responds to a range of microbial and viral products
- Main macrophage receptor for LPS = TLR4 which recognizes the lipid A component of LPS

TLR4
- Extracellular domain binds LPS
  - Pathogen-recognition domain: has repeated leucine residues called leucine rich repeat region (LRR)
  - Variation in the number of LRRs give each type of TLR a different ligand specificity
- Cytoplasmic domain signals macrophage to transcribe genes encoding proteins needed for innate immune response
  - Signaling domain: Toll/interleukin-1 receptor (TIR) domain
- Can bind two molecules of LPS

Gram-negative Infection

Macrophages use mannose receptor to internalize and degrade bacteria and release LPS from the bacterial surface
LPS is bound by LPS-binding protein and delivers it to CD14 at the macrophage surface
TLR4 and myeloid differentiation factor 2 (MD2) forms a complex with CD14 and LPS
- MD2 only associates with extracellular domains of TLR4 but not other TLRs
The TIR domain engages with the domain of MyD88 which acts as an adaptor protein
The second domain of MyD88 engages with protein kinase IRAK4 (Interleukin-1 receptor-associated kinase 4)
- Induces IRAK4 to self-phosphorylate and dissociate from the complex and phosphorylate TRAF6 (tumor necrosis factor receptor-associated factor 6)
- Leads to the activation of inhibitor of kappa-B kinase (IKK) kinase complex
- IKK phosphorylates inhibitor of kappa-B (IkB)
- releases nuclear factor kappa-B (NFkB) to enter nucleus
  - Initiates transcriptions of genes encoding cytokines and adhesion molecules
  - When not needed, NFkB is held in the cytoplasm by IkB
- IkB degrades

NFkB Essential Modulator Deficiency (NEMO Deficiency)

Loss of y subunit (NEMO) of IKK
Impairs the activation of NFkB = impairs activation of macrophages by TLR4 signaling
X-linked = males are more susceptible

TLR Sensing

Receptors on plasma membrane
- Recognizes carbs, lipid, and protein ligands on outer surface of pathogen
Receptors within endosomal vesicle membranes
- Recognizes features of nuceic acids of pathogens and distinguish from human DNA and RNA
TLR4 and TLR1:TLR2 sense bacterial infection
TLR3 in endosomes sense viral infection

TLR4 Polymorphism

Allotype: proteins encoded by different alleles of the same gene
Causes genetic polymorphism
- More common in TLRs that recognize surface epitopes than nucleic acids due to greater diversity of carbs, lipids and proteins
Septic shock
- Bacterial infection spreads from tissue to blood and becomes systemic
- Potent activation of the innate causes vasodilation and therefore leakage of fluid throughout the body
  - Produces septic shock in which blood supply is perturbed and vital organs fail
- Caused by Gram-negative

NOD Proteins

Intracellular
Recognizes bacterial degradation products in cytoplasm
Has three different regions
- Carboxy-terminal region = pathogen-recognition domain with binding site for degraded products
- Central region: nucleotide-binding oligomerization domain (NOD domain) which enables receptors to form oligomers
- Amino-terminal region = caspase-recruitment domain (CARD)
  - contains binding site for RIPK2 (receptor-interacting serine-threonine protein kinase 2) that initiates signaling from NODs and acts as an adaptor molecule
  - 1 in NOD1 and 2 in NOD2
  - Mediates RIPK2 and NODs interaction
    - Phosphorylation of RIPK2
    - Activation of kinase TAK1
    - Phosphorylation of IKK
    - IKK activates NFkB
    - Macrophage activation
    - normally recruits caspases protease to protein complexes

Interferon Viral Response

Cytoplasmic proteins that can detect viral nucleic acids and produce Type I Interferons
- Interferes with viral replication in the infected cell
- Instructs nearby uninfected cells to fight infection
- Alerts immune system that infection is present and causes virus-infected cells to be more vulnerable to NK cells
In response to infection, one cell secretes cytokine that binds to a cytokine receptor on another cell
- Induces intracellular signals that change the behavior if second cell
- Makes contact with target cell before releasing cytokines to avoid unnecessary immune response damage to tissues
- When the cytokine and its receptor are both from same type of cell = give autocrine signals
- Cytokine and receptor are products of different cells: paracrine signals

RIG-I-like receptors (RLRs)
- Detects cytoplasmic viral RNAs
- Comprised of RIG-I and MDA-5
  - Recognizes ds vRNA
- Two CARDs
  - Interacts with mitochondrial antiviral signaling protein (MAVS)
  - Forms a dimer on mitochondrial membrane
  - MAVS engages TRAF6 which initiates signaling pathway to activation of IRF3 and IRF7
    - IRF3 initiates IFN-beta gene granscription
    - IRF7 initiates IFN-alpha gene transcription
  - secretion of Type I interferons

Secreted IFN-Beta maintains activation of infected cells (autocrine action) and binds to receptors on nearby uninfected cells (paracrine action)
Infection induces phosphorylation of IRF3 to enter nucleus
NFkB and IRF3 activates transcription of INF-beta gene followed by secretion of IFN-B
Some IFN-B are bound by IFN-B receptors (autocrine)
IRF7 is mobilized and induces production and secretion of IFN-a
IFN-B receptors of uninfected cells bind to IFN-B secreted by infected cell (paracrine)
- Induces uninfected cell to secrete IFN-B and contribute to interferon response

Plasmacytoid Dendritic Cells

Produce Type I IFNs
Present in blood and lymph tissues
Expresses TLR7
- Detects ss vRNA
Expresses TLR9
- Detects presence of unmethylated CpG motifs in ds DNA

Inflammasomes

Enable activated macrophages to release IL-1beta
Interleukin-1B
- Master regulator of inflammation
- Inactive pro-form is stored in cytoplasm by macrophages
- Activated by cleavage and secreted in response to infection
- Macrophage assembles inflammasome to cleave pro-IL-1B into active IL-1B
  - Inflammasome has NOD-like receptors (NLR) that detects infection
  - NLR protein 3 basis for NLRP3 inflammasome
    - same domains as NODs
    - recruits molecules for oligomerization of inflammasome upon sensing infection
    - procaspase 1 self cleaveage forms caspase 1 which cleaves pro-IL-1B
- Allows macrophage to respond to infection with large burst of IL-1B
- Gasdermin D
  - To allow IL-1B to escape macrophage
  - Upon sensing infection, and when pro-IL-1B is cleaved, caspase 4 cleaves gasdermin D
  - Forms pores in PM where IL-1B leaves the dying macrophafe
  - Pyroptosis = cytokine release, pore formation, death of macrophage
IL-1a
- For homeostasis
- Always expressed by healthy cells

Autoinflammatory Diseases

Chronic and recurrent bouts of systemic inflammation mediated by cells of innate immunity and do not involve adaptive immunity

Inflammation of Infected Tissue

Attracts blood-borne immune effector cells
Macrophage and neutrophils have distinct complementary properties
Macrophage
- Long-lived
- Reside in tissues
- Work from the start of infection
- Raise the alarm
Neutrophils
- Short-lived
- Dedicated killers that circulate in blood
- Awaiting call from macrophage and defend infected tissue
- First population of effector cells recruited to infected tissue

Release of IL-1B by Inflammasome

Activates macrophages
Two main effects
- Improve speed and efficiency in capturing and digesting pathogens
- Secretion of cytokines to recruit other effector cells
  - Tumor necrosis factor-a (TNF-a) = vasodilation
  - IL-6 = heat generation via metabolism of fat and muscle cells
  - CXCL8 = chemokine to attract neutrophils
  - CCL2 = chemokine to attract monocytes
  - IL-12 = recruits and activates NK cells to secrete cytokines to enhance and maintain macrophage response

Recruitment of Neutrophils

Macrophages release CXCL8 which guides neutrophils into site of infection
Adhesion molecules on leukocyte surface and tissue-cell surface drives movement from blood and tissue
In absence of infection, neutrophils move rapidly through capillaries and do not interact with vascular endothelium
Within infected tissue, vasodilation and adhesion molecules of endothelial cells allow contact with endothelium
- Rolling adhesion

TNF-a induces endothelium to express intercellular adhesion molecules 1 and 2 (ICAM-1 and ICAM-2) and neutrophils to express leukocyte function associated antigen-1 (LFA-1)
ICAMs bind CR3 and LFA-1
CXCL8 binds to neutrophil’s chemokine receptors CXCR1 or CXCR2
- Tight binding to immobilize neutrophil on endothelium
CXCL8 binding with receptor
- Allows binding with G protein
- GDP is replaced by GTP
- Activates and releases G protein from receptor
- Neutrophil leaves blood by squeezing between cells = diapedesis
- Overall process of leaving the blood = extravasation
Neutrophil migration is directed by gradient of CXCL8 bound to cell surface and ECM
- Neutrophil moves towards CXCL8 concentration at actuvated macrophage
Pyogenic bacteria = pus-forming bacteria

Neutrophil Programmed Death

They devote all their resources to storage and delivery of antimicrobial weaponry
Has receptors for recognition: CR4 and CD14

Neutrophil Granules

Order of loading of granules
Azurophilic/ primary granules
Specific/ secondary granules
Gelatinase/ tertiary granules
Secretory vesicles
Order of degranulation/ releasing of granules is reversed
- Controlled by calcium concentration
- Low levels = sufficient for secretory vesicles
- Higher level in tissue = specific and gelatinase degranulation

Bacterium is engulfed to form endosome that fuses with azurophilic, specific and gelatinase granules
Components of NADPH oxidase by specific granules facilitate a respiratory burst
- Raises pH = becomes phagosome and kills pathogen
Fuses with lysosomes
- Lowers pH and activates hydrolases to degrade pathogen
Respiratory burst
- Increase in oxygen consumption
After neutrophils have deliverd all granules, they die
- Apoptosis
- NETosis which produces neutrophil extracellular traps (NETs) that capture and kill pathogens
  - Nucleus swell and burst

Chronic Granulomatous Disease (CGD)

Genetic syndrome caused by defective forms of genes encoding NADPH oxidase subunits
Without functional NADPH oxidase = no respiratory burst
Phagosome is too acidic to activate antimicrobial peptides
Numbers of commensal cannot be controlled so they persist as chronic infections
Infected macrophages are concentrated into nodules called granulomas which are unable to digest infected neutrophils

Fever

Effect of cytokines IL-1B, IL-6 and TNF-a
- Called pyrogens
Acts on temperature-control sites in hypothalamus and directly on muscle and fat cells = generate heat
Inhibits the growth and replication of bacterial and viral pathogens
Tissue cells become more resistant to damaging effects of TNF-a (capacity to kill tumor cells)
- Lethargy, anorexia = impedes use of energy to fight infection

Acute-Phase Response

Plasma proteins made by the liver are changed by IL-6
Proteins that increase or decrease are called acute-phase proteins
- C-reactive protein (CRP) and serum amyloid A increases hundredfold
- CRP concentration – diagnostic test for infection and tissue damage
CRP
- Targets phosphorylcholine of LPS
- Acts as opsonin and trigger classical pathway
Serum Amyloid A
- Interacts with TLRs and SRs
- Activate cells to produce inflammatory cytokines

Lectin Pathway

Induced by infection and requires time
Contributes to innate immune response
Initiated by acute-phase protein mannose-binding lectin (MBL) binding to pathogen surface
- Also an opsonin that facilitates uptake of pathogen by monocytes
- Circulates in plasma as complex
  - MBL-associated serine protease (MASP)1 and 2

- when MBL binds to mannose-containing carbs, MASP-2 becomes active and cuts itself
  - MASP-2 cuts C4 into C4a and C4b
  - C4b attaches to pathogen surface
  - C2 binds to MASP-2 and cleaves into C2a (larger) and C2b (smaller)
  - C2a Forms complex with C4b = C4bC2a
  - Forms Classical C3 convertase
  - Cleaves C3 to assemble alternative C3 convertase

Classical Pathway

Contributes to innate and adaptive
Requires binding of antibody or C-reactive protein to pathogen surface
CRP binds to phosphorylcholine on pathogen surface
CRP binds with complement component 1 q (C1q) = cleavage and activation of C1r and C1s
Activated C1s cleaves C4 and C2
Forms classical C3 convertase
C3b attaches to surface
C3b is recognized by CR1 = engulfment
C3b forms complex with C5-C9 = membrane destruction

Lymphoid Cells

NK cells
- Cytotoxic innate lymphoid cells
- Circulate in blood and enter infected tissues
- Type I immunity
Helper Innate Lymphoid cells (ILCs)
- Similar function to adaptive CD4 T cells
- Secretes cytokines to activate effector cells = macrophages and granulocytes
- Reside in tissues and make immediate response
NK cells and Helper ILCs have no antigen receptor
- Instead expresses PRRs
ILC1
- Intracellular (viral)
- Stops infection or limits spread until arrival of NK cells
- Type I immunity
ILC2
- Mucosal surfaces
- Extracellular parasites
- Type 2 immunity
ILC3
- Mucosal tissues
- Extracellular pathogens
- Participates in the containment of commensal microorganisms in the gut
- Tyupe 3 immunity
Lymphoid-tissue inducer (LTi)
- Facilitates development of secondary lymphoid tissue

Innate Lymphocyte Precursor

Common lymphocyte precursor cell (CLp)
- Gives rise to common precursor of all innate lymphocytes CILP
- Which then gives rise to precursor of NK cells NKp and precursor of all innate helper lymphocytes CHILp
- Which then gives rise to precursor of LTi cells LTIp and to common precursor of ILC1-3 ILCp

Circulating Lymphocytes

NK = innate
B and T cells = adaptive
NK cells expresses CD56 but not CD3 that marks T cells
No NK = persistent viral infections even with adaptive immune system
NK importance in containing viral infections until cytotoxic T cells become effective

Subpopulation of NK cells

CD56dim
- 90% of blood NK
- Less CD56 expression
- Differentiated cytotoxic cells
CD56bright
- Gives rise to CD56dim during NK development
- Weak cytotoxic effector cells and more committed to making cytokines
- Most are taken up in tissues
  - 80% of NK in lungs are CD56bright
- Uterine NK cells (uNK cells)
  - Abundant in uterine
  - Numbers fluctuate during menstrual cycle
  - Contributes to embryo implantation and placenta formation
  - Cannot kill cells or create inflammation
  - Cooperates with fetal trophoblast nourishment to ensure mother can supply fetus with oxygen

NK Cytotoxicity

Activated at viral infection sites
To prevent NK from killing healthy human cells, cytotoxicity is regulated
- NK can only discharge weapons after contact
  - Can only kill once cell at a time
- Decision to kill depends on sum of interactions between different NKRs and target cell ligands
- Inhibitory receptors prevent NK from killing healthy cells
NK Differentiation
- Infected cells secrete Type I interferons
- NK have IFN receptors that bind to IFN
  - Aided by adhesion molecules CR3 and LFA-1
- Forms an immunological synapse that holds the cells together and forms channel for exchanging info = NK-cell synapse
- IFN activates NK to proliferate and differentiate into cytotoxic effector cells
- Effector NK kills infected cells by inducing apoptosis

NK expresses TLR3 (ds vRNA), TLR7 and 8 (ss vRNA)
TLR7 and TLR8
- MyD88-dependent pathway that leads to IRF7 activation and production of IFN-a and IFN-B
TLR3
- No MyD88 adaptor
- Signals by IRF3 pathway and leads to production of IFN-B
- Uses TRIF adaptor

NK and Macrophages Activate Each Other

Activated macrophages secrete CXCL8 and IL-12
- Activates and recruits NK
- NK and macrophage forms conjugate pair bound by NK-cell synapse
- Macrophage in synapse secretes IL-12 (for cytokine-secreting effector) and IL-15
  - Activates NK
- NK proliferates and differentiates into effector NK secreting IFN-y
- IFN-y binds to macrophage and activates it
- Improved phagocytosis of virus particles and cells killed by NK
IFN-y
- Potent inflammatory cytokine produced by NK
- Aka Type II IFN

Dendritic Cells and NK cells

DCs may take up pathogens or be infected by pathogens
This causes changes on surface proteins monitored by NK receptors
If infected, DC forms synapse with NK
DC expresses IL-15 = cytotoxic NK proliferation
If cytotoxic NK is high and innate immunity overcomes infection = NK kills DCs and prevent adaptive response
If NK is low and innate cannot control infection = NK induces DCs to differentiate, migrate to lymphoid tissue and initiate adaptive response

NK Memory

All nucleated cells express MHC class I = Human Leukocyte Antigen (HLA)
Loss of HLA class I = marks disease
- Tumors and virus-infected cells escape T cell by reducing or losing HLA class I expression
NK has receptors that detect low HLA class I and kills them

ANTIBODY FUNCTION

Immunoglobulin
- General term for proteins produced by B cells that recognizes antigens (plasma cells)
- Attached or free-floating
  - Antibody = free-floating
- Very specific
- Can recognize proteins and carbohydrates
- Their production is stimulated by vaccines or exposure to the antigen in question
- Antibody repertoire
  - Mixing and matching of proteins to form immunoglobulins
  - 10^16 but usually just 10^9

Antibodies have different functions:

Neutralization
- Antibodies fully cover the antigen to recognize them
- Prevent adherence to cells
Opsonization
- Antibodies fully cover antigen
- Makes it easier for phagocytes to engulf them
Complement Action
- Attach to a certain antigen
- Activates a complement which enhances opsonization and lysis

Clonal Selection

Transformation and proliferation of plasma cells that produce most specific antibodies from B cells
Once a B cell recognizes an antigen, it will undergo further mutation of their antibodies
- Becomes more specific

ANTIBODY COMPONENTS

Held together by a flexible hinge region composed of disulfide bonds

Fab = fragment antigen binding
Fc = fragment crystallizable
- Once it is done, they tend to precipitate and crystallize

ANTIBODY VARIABE REGION

Epitope
- Antigens recognized by antibodies
- Antigenic determinant
- Can be linear or continuous depending on the structure
Variable region light chain and variable region heavy chain
- Tend to vary to suit different shapes and sizes of all possible types of antigens

Hinge region
- Can recognize different types of antigens no matter their positions

HYPERVARIABLE REGION

Hypervariable region protein loops
- Found at the tip of both the light and heavy chain
- Aka complementary determining regions (CDR)
- CDR1, CDR2, CDR3
- Each variable region has three CDRs
Framework regions
- Less variable
- Supports the CDR
Affinity
- Binding strength of a single antigen binding site to the antigen
Avidity
- Bing strength of multiple antigen binding sites to an antigen
Binding to antigen depends on four covalent bonds
- Van der Waals forces
- Hydrophobic bonds
- Electrostatic bonds
- Hydrogen bonds

ANTIBODY ISOTYPES

Isotypes
- Depends on structure of constant region
- Has diverse functions
- Named after the Greek letters they correspond to
- IgG = gamma
- IgM = mu
- IgD = delta
- IgA = alpha
- IgE = epsilon

GENETIC BASIS OF ANTIBODY DIVERSITY

Variable region gene segment combinations within heavy or light chain gene segments (combinatorial diversity)
Junctional diversity between the gene segments
Heavy and light chain V region combinations
Somatic hypermutation
- Occurs after antibody recognizes an antigen
- Undergo further mutation/changes to become more specific

HEAVY AND LIGHT CHAIN DIVERISTY

Recombination of antibody genes determine the specificity and diversity of the CDR and the antibody produced
Light Chain
- Lambda locus gene segment from chromosome 22
- Kapa locus gene segment from chromosome 2
Heavy chain
- Locus from chromosome 14
Gene segments:
- V = variable
- L = Leader
- D = Diversity
- J = Junction
- C = Constant

ANTIBODY DIVERSITY

Has a germline DNA origin
- From both sperm and egg
Somatic recombination
- Antibody variable region diversity comes from the combination of the V, D, and J gene segments
- Light chain : V & J (variable) and C (constant)
- Heavy chain: V, D & J (variable) and 3 C gene segments (constant)

RSS

Recombination signal sequences
- How a B cell know which g segments goes where
- Gene sequences that direct recombination
- Ensures gene segments are always in their proper orders
  - V and J or V, D and J
- Have certain signals where the genes are supposed to join
12/23 Rule
- Segments with a 12 bp spacer will combine with a segment with a 23 bp space

RECOMBINATION

Since the genes are in one linear chromosome, these gene segments are brought together closer first by your RAG complexes
Recombination-activating genes (RAGs)
RAG Complexes
- RAG complexes hold the RSS in place while the genes are recombined
- proteins coded by RAGs
- serve as a clip to place the V and J gene segments closer to each other before cleaving
V(D)J Recombinase
- Set of enzymes needed to recombine the gene segments

JUNCTIONAL DIVERSITY

Since these are always random gene segments, how can they combine next to each other when they don’t have complementary ends?

RAG complex cleaves the gene segments to yield DNA hairpins
RAG complex opens hairpins by nicking one strand of the DNA = palindromic P-nucleotides
Terminal deoxynucleotidyl transferase (TdT) = adds random n- nucleotides
Pairing of strands occur and non-complementary/unpaired parts are removed
Gaps are filled by DNA synthesis and ligation to form coding joint
This adds to the CDR3 diversity

WHICH GENE SEGMENTS CODE FOR WHICH CDR

V segment = CDR 1 and CDR2
V and J junction (and D) = CDR3

CONSTANT REGION DIVERSITY

Free-floating = has different sets of proteins (hydrophilic) at the terminus end compared to transmembrane antibodies
Transmembrane = protein ends help with hooking onto the surface of the B cell membrane

IgM STRUCTURE

IgA and IgB accompany transmembrane IgM since these immunoglobulins do not have their own internal receptors

B CELLS TO PLASMA CELLS

B Cells have their membrane-bound IgM
Once it encounters an antigen, they will undergo somatic hypermutation to further produce more specialized antibodies that would better recognize antigens
Eventually will transform into plasma cells which now solely produce free-floating antibodies specifically recognizing this specific antigen
Allelic exclusion
- One allele is only expressed for producing antibodies while the other is silent
- Different alleles tend to code for antibody production but only one is expressed at a time

SOMATIC HYPERMUTATION

After an antibody recognizes an antigen, the activation-induced cytidine deaminase (AID) will replace all the cytosine in the CDR3 with uracil
Uracil-DNA glycosylase (UNG) will then remove the uracil and DNA polymerase will replace them with random nucleotides

ISOTYPE SWITCHING

AID is also responsible for isotype switching
It has a tendency to fold similar to Recombination of gene segments
They put gene segments specific to an isotype closer to each other
C mu and C delta gene segments are closest to the V, D, and J segments by default
- This is why IgM and IgD are produced first
The rest of the C gene segments are next to each other
Switch regions or switch genes are signals for isotype switching
IgM antibodies exist as bulky pentamers
Antigen activated T cells also mediate isotype switching
Successive switching can occur (ex: IgM to IgG to IgA)

Ig SPECIALIZATION

IgM

Produced in the spleen, bone marrow, and lymph nodes
Low affinity
Multiple binding sites of bulky pentamer free-floating form

IgD

Mostly found in the respiratory tract
Triggers local immune response when bound to basophils

IgA

Monomeric form from spleen and bone marrow and circulates in blood
Dimeric form from lymph nodes and circulate in lymph and other mucosal secretions (ex: tears, saliva, breast milk) and the digestive lumen
May even target resident microbial microfauna to keep their populations in check

IgE

Specialized toward recruiting effector functions of mast cells in epithelium, activated eosinophils in mucosal surfaces, and basophils in blood
Targets parasites
Triggers asthma and allergic reactions if parasites are not very common in the area

IgG

Produced from the bone marrow, spleen, and lymph nodes
Circulated blood and lymph
Most commonly circulating antibody in bloodstream
More flexible hinges compared to other antibodies for hard to reach antigens
Also allows to bind to Fc receptors of phagocytes
Different kinds based on flexibility, function, and targets
Activates complements

IgG SPECIALIZATION

IgG1

Most abundant and versatile
Mostly binds to protein antigens

IgG2

Second most abundant and less flexible
Targets carbohydrates (bacterial capsules)

IgG3

Most flexible with longest hinges
Most susceptible to proteases
Best in activating complement

IgG4

Least abundant and does not activate complement unlike other 3
Produced as monovalent together with IgE in case of allergic ractions
Reduces allergic reaction by competing with IgE when binding
Therapeutic uses

Caused by defects in immunoglobulin class switching and somatic hypermutation

Ig cannot adapt and patients become susceptible to infections

Defects in cd40 and cd40 ligand, AID, and UNG

Very low levels of other ig types but normal levels of igm

Cd40 ligand defects in t cells or cd40 defects in b cells

Cd40 activates isotype switching

AID mutation which disables somatic hypermutation and isotype switching

UNG mutation which disables somatic hypermutation

Effects:

Enlarged spleen or lymph nodes due to accumulation f immature b cells

Severe microbial infections and re-infections such as pneumocystis and cryptosporidium

Respiratory tract infections most common

Gastro are second most common

Includes entamoeba histolyca and salmonella enterica and giardia

Hindi naaalala ng immune system yung infections kaya nagkakaroon ng recurrent infections

Antibodies have Chlorophora or flurophore

Direct: recognize specific epitope

Indirect: gagawa muna ng serum or sample tapos sila marerecognize ng antibody

Polyclonal: antigen injected in animals tapos multiple antibodies are used targeting diff epitopes

Monoclonal: very specific using hybridoma and target the same epitope

Antigen coat (glass slides
Add blocking solution = hindi mag non-specific interactions with other artifcats
Antibody incubation from human sample
Fluorescent antibody incubation and visualization

Names of antibodies:

Source of the antbody, target antibody, conjygate chromo or fluorophore

Goat anti-human IgG alkaline phosphatase

Blocking solution: 1% BSA

For eliza chromophores

Horseradish peroxidase: brown or dark orange; stopping solution: hydrogen peroxide

Alkaline phosphatase: yellow to orange; no need for stopping solution

Immunofluorescent antibody technique

Fluorescein isothiocyanate: green

Western Blot

Transfer proteins from SDS PAGE gel to nitrocellulose membrane

Stain with antibody conjugated with horseradish peroxidase to determine the exact peptide detected by the antibodies

Flow cytometry

Monoclonal antibodies

Hybridoma grow and produce antibodies indefinitely

Separate hybridomas that produce certain antibodies specific for a protein

These are used in therapy as long as same source para marecognize as part of the body

Ex: Rituximab:

Binds to CD20 which is present in both malignant and normal B cells

Fc is the attachment for NK cells which cell-killing machinery

Plasma cells are still present to produce antibodies even if all B cells are being targeted

Non-hodgkin B cell lymphoma

LECTURE 5

CELL MEDIATED IMMUNE RESPONSE

Divided into two:

MHC Class I = Cytotoxic or CD8 T Cells
- Deals with intracellular pathogens
- Requires CD8 receptors
- Found in all cells except RBCs
- Pair of protein segments
MHC Class II = Helper or CD4 T Cells
- Deals with extracellular pathogens
- Requires CD4 receptors
- Found in macrophages, dendritic cells and B cells
- a chain of 4 protein segments

CMIR

Uses both T Cell receptors and Major Histocompatibility Complex (MHC)
- unlike B Cells which produce antibodies that recognizes pathogens

T Cell Receptors

attached to the cell membrane of T cells
function like antibodies of B cells or immunoglobulins
have alpha and beta chains = the light and heavy chains of antibodies
they also have a variable and constant region
Differences
- Can only recognize short peptides
  - Unlike antibodies which can recognize larger proteins as well as carbohydrates
short peptides are presented by an MHC
no free-floating form
no differentiation or somatic hypermutation after recognizing an epitope

T Cell Receptor Diversity

Also dependent on the germline DNA
Alpha chain: chromosome 14
- V and J
Beta chain: chromosome 7
- V, D, and J
Assembly of gene segments involve RAGs
Relies on DNA recombination
Only 1 constant alpha region gene and 2 constant beta region gene
- Both are functionally identical

Transposons

Theorized origin of diversity
Short gene segments that code for transposase
- Enzyme that cuts transposon and copy and paste it to the other parts of the DNA
- Recognizes the repetitive DNA sequences in the transposons
Transposase and RAG recombinase are similar
- Led to the hypothesis that RAGs originated from transposons-transposase interaction
- A transposon was inserted into a gene encoding for a receptor of innate immunity
- The transposon separated the gene into two segments, each flanked by a piece of repetitive transposon DNA
- Chromosomal rearrangements placed the transposase gene onto a different chromosome from the primordial rearranging gene
- The repetitive DNAs became the RSSs of a primordial rearranging gene and the transposase genes became the ancestral RAG-1 and RAG-2 genes (third panel).
- the family of rearranging genes expanded and eventually spread to five human chromosomes
  - TCR: chromosomes 14 and 7
  - Ig: chromosomes 14, 2, and 22

T Cell Receptor Complex

TCRs do not penetrate through the transmembrane of the T cells
Rely on support proteins and proteins that would serve as intracellular signal transmitter
- CD3 delta
- CD3 gamma
- CD3 epsilon
Zeta chain functions in transducing signals

Two Classes of TCR

Most studied are alpha and beta chains since these are similar in jawed vertebrates
Meanwhile, gamma and delta chains are not similar in mice and humans

Overview of TCR and MHC Relationship

Any pathogen that is engulf by the cell for the MHC II will have their proteins broken down
presented by the MHC on the surface of that cell
will be recognized by the respective T cell and T cell receptor

MHC I

if a cell is infected by an intracellular pathogen, its proteins will be presented by the MHC I on the cell surface
this will be identified by the CD8 T cells and CD8 TCRs
signals apoptosis of the infected cell to prevent further infection of other cells

MHC II

break down the segments of extracellular pathogen
will be presented on the cell surface and be recognized by CD4 T cells and CD4 TCR
Promotes production of cytokines or cell signals to
- activate macrophages to signal that there is an extracellular pathogen present
- signal B cells to transform into plasma cells and produce antibodies

Structure of MHC

MHC I
- 3 alpha domains (alpha 1, alpha 2, alpha 3)
- Beta 2 macroglobulin
  - Not encoded in MHC gene
- Has grooves and pockets in its structure
  - Allows only small peptides (8-10 AA) to be presented
MHC II
- Alpha 1, alpha 2
- Beta 1, beta 2
- No grooves or pockets
  - Wider space for larger peptides (13-25 AA)
MHCs exhibit promiscuous binding specificity
- Potential to bind to different peptides
- Not necessarily for a certain peptide

Self VS Non-Self Proteins

Cells are programmed to distinguish their “self” proteins produced by the cell
If they recognize them as “non-self”, they will be immediately presented by the MHCs
MHC I
- foreign proteins are loaded in the ER
- If a pathogen enters the cell, the cell will trigger production of proteins for that pathogen
- These proteins will be recognized as “non self” by MHC I
- Triggers its presentation to CD8 T cells
- Proteosome breakdowns misfolded proteins and recycle the peptides
- Once they recognize non self proteins, interferon-gamma triggers their transformation into immunoproteosome and consequently, the breakdown of these cells
- In a normal setting, the broken down peptides pass through transporter associated proteins into the ER where they will be reused

- In the case of foreign protein, the immunoproteosome will trigger the assembly of MHC I
- The chaperone protein calnexin attaches the beta-2 microglobulin to the 3 alpha domain proteins
  - Forms the peptide-loading complex
- Tapasin: brings the MHC I closer to TAP = easier for foreign peptides to be loaded into MHC I
- ERp57 and Calreticulin: help in peptide bonding
  - Foreign peptide is enclosed in a vacuole and transported to the surface of the cell
- Sometimes, protein segments are too long = ERAP (ER Aminopeptidase) protein
  - Shortens the peptides to fit into MHC I
  - Sometimes the peptide may not fit too well that it just falls off
  - MHC I will then assist them back to the ER and try / load again
MHC II

- foreign proteins are loaded in the endosomes
- extracellular pathogen will be engulfed by macrophages / dendritic cells and be enclosed in endosomes
- endosomes produce enzymes that will breakdown or inactivate proteins
- endosomes will then bind to specialized vacuoles containing MHC II
- the class II-associated invariant chain peptide (CLIP) will cover the groove or pocket on the MHC II while it has not encountered a foreign peptide yet
  - for loading
- Human leukocyte antigen-DM (HLA-DM) removes the CLIP for proper loading of foreign peptide
- Human leukocyte antigen-DO (HLA-DO) retains the CLIP while there is no foreign peptide yet

Cross-Presentation

Dendritic cells, B cells, and macrophages have MHC I and MHC II
Sometimes, the peptides may enter through the MHC I pathway but end up being presented by MHC II or vice versa

MHC Diversity

Human leukocyte antigen complex
- HLA I types: MHC I
  - A, B, C, E, F, G
- HLA II types: MHC II
  - DM, DO, DP, DQ, DR
HLA diversity comes from
- MHC II alpha chains gene families
- MHC II beta chains gene families
- MHC I heavy chains gene families
- Genetic polymorphism

Genetics Review

Gene = gene segments that codes for a certain protein
- In this case, HLA
Alleles = variations (single or multiple nucleotide changes) that would alter the protein structure
- Each allele would code for a different allotype (of HLA)
Can either be
- Monomorphic: there’s only 1 type
- Oligomorphic: dozen or less
- Polymorphic: more than a dozen
From germ line
- Heterozygous: different allele from parents
- Homozygous: same allele from parents
- Haplotype: every set of different alleles represent a different kind of haplotype

MHC I and HLA-I Diversity

HLA-A, -B, -C
- Highly polymorphic
- Present antigen to CD8 T Cells and ligands of natural killer cells
HLA-E, -G
- Oligomorphic
- Present antigen to ligands of natural killer cells
HLA-F
- Monomorphic
- Chaperone to MHC class I molecules that lose their antigen while travelling to cell membrane

MHC II and HLA-II Diversity

HLA-DP, -DQ, -DR
- Highly polymorphic
- Present antigen to CD4 T Cells
HLA-DM, -DO
- Oligomorphic
- Aids in loading antigen onto HLA-DP, -DQ, and -DR

Chromosome 6

Class I Region for HLA-I
- Mostly MHC-related or antigen presentation-related genes
- Proteins of other functions
Class II Region for HLA-II
- mostly MHC-related or antigen presentation-related genes
- TAPs
- Tapasins
- Related to immunoproteosomes
Class III Region
- Proteins not related to MHC

HLA Gene Diversity

MHC I
- Allotype diversity in both alpha-1 and alpha-2 domains
- Beta-2 immunoglobulin gene comes from another chromosome (chromosome 15)
MHC II
- Allotype diversity in either alpha-1 or beta-1 domains
- The other domain is always non-functional
- Depends on MHC II isotype

MHC Activation

Cell signals that activate MHC antigen presentation
- Interferon-alpha
- Interferon-beta
- Interferon-gamma
  - Functions in both MHC I and II responses
MHC I
- IFN-gamma activates proteosomes into immunoproteosomes
- For better breakdown of foreign peptides of intracellular pathogens
MHC II
- IFN-gamma activates MHC class II transactivator (CIITA)
- Activates MHC II

MHC I and MHC II

HLA-I most likely evolved first
Most of HLA II genes code for proteins that are strictly for antigen presentation to T Cells while HLA-I genes contain proteins with other functions
HLA I-related genes are found in other chromosomes
HLA-II is more compact compared to HLA-I
Some organisms such as Atlantic cod can survive with only HLA-I

MHC Restriction

proper order of your MHC, TCR, and antigen for recognition
different sides of the antigen bind to MHC and TCR
peptide binding sites or set of anchor residues
anchor residues or complementary pockets in the MHC binding sites
TCR also recognize the residues on the surface of the alpha helices
The part of the antigen recognized by HLA is different from that recognized by the TCR
- If same peptide but presented by a different HLA = no recognition
- If HLA carries a different antigen but same TCR = no recognition

Natural Selection

MHC and TCR diversity are controlled by natural selection
Balancing selection always ensure that highly polymorphic heterozygotes are always present
- Intermingling of populations = better immunity
- Ensure HLA are sufficiently heterozygous
Ensures survival against new disease
- Meiotic recombination occurs at 2% frequency which increases with succeeding generation
- New alleles from point mutations and recombinations
- Interallelic conversion or segment exchange occurs when recombination combines segments with point mutations to homologous segments

GVHD occurs during hematopoietic stem cell transplants such as bone marrow

HSC ransplants are performed fo patients with damaged or malfunctioning immune system or bone arrow

Autologous = self MHC isoforms

Allogenic = non-self MHC

The donor tissue are recognized as allogeneic or different by the new T cells

Alloreactions

Alloreactive t cells will react against cells from another indiv

About 1-10% circulatnb T cells in an indiv are alloreactive

Alloantibodies of one indiv will atact the allotypic proteins of another

These mismatches can occur between mother and child

HLA types

- - Combinations of HLA alleles in a person
  - Should matcj in order for transplants to proceed
  - Hla types testing by molecula methods
  - Most likely matched at HLA-A B C and DRB1

Symptoms

Skin as the most likely first organ to be affected with presence of rashes

gI disease such as diarrhea

liver disease

chronic GVHD occurs after 100 days

acute GVHD within 100 days

organ transplant rejection

the recipient immune system attack the donor organ

all cels in immune cells possibly involved

can occur months after the transplant

Topic 6

B Cell Development

Pro-B cell

main event in the pro-B-cell stage is the rearrangement of the heavy- chain genes
Early: D and J segments
Late: DJ and V segments
- The rearranged gene is transcribed through to the μ C-region gene, the nearest C gene to the rearranged V region
- The RNA transcript is spliced to produce mRNA for the μ heavy chain, the first type of immunoglobulin chain made by a developing B cell

Pre-B cell

B cell expresses mu chain
Large pre-B cell: less mature
- Heavy-chain rearrangement is done
- Makes mu heavy chain
Small pre-B cell: mature
- Rearrangement of light chain gene
- First is kappa
- If successful, no lambda
  - Light chain is synthesized and assembled sa ER
  - Form IgM
- If fail, lambda
  - Same as above
- If fail both = apoptosis

Immature B cell

Heavy and light done rearranging

Stromal

Environmental cells sa marrow for maturation
Interacts as adhesion molecules to ligands
Has growth factors that attach to b cells
- SCF = Kit receptor on maturing B cell
- IL-7 acts on late pro and pre
Moves from subendosteum to marrow cavity
Later stages are less dependent on stromal cells = leaves BM

Pro-B Heavy Rearrangement

Inefficient and imprecise = due to addition random N and P nucleotides in V, D, J segments
- Limited material tapos random
- Parang sandwich na sakto yung parts
- One in three chances of maintaining correct frame
Nonproductive rearrangements = not useful
Productive = useful
B cell has two copies of heavy chain locus (one from each parent)
- Can still produce heavy chain if one chromosome locus is productive
Express RAG1 and RAG2 = rearrangement starts
- Activated by E2A and EBF which expresses Pax5
Apoptosis is default unless there are survival signals

Pre-B Cell Receptors

Pro-B needs to make mu chain to survive and mu chain must be able to combine with Ig light chain
E2A and EBF roduce surrogate light chain
To test kung functional heavy chain, surrogate light chain ay parang substitute since light chain wont be formed unless functional ang heavy chain
- Synthesizes VpreB (mock variable region) and lambda5 (mock constant region)
- In the ER of pro-B, mu chain binds with surrogate and IgB = pre-B cell receptor = sends signals okay/functional ang heavy chain = interacts with BM ligands = stop RAG = pro-B cell div = large pre-B
- Pre-B cell interacts with BM ligands heparin sulphate and galectin-1 to stop RAG
Unlike the B-cell receptor (IgM), the pre-B-cell receptor does not have an antigen-binding site, nor is it well represented at the cell surface

Allelic Exclusion

Pre-B receptor
- Eliminates nonfunctional mu chains
- Prevents making more than one functional mu chain
In a pro-B
- rearrangement of the first immunoglobu- lin locus succeeds
- synthesis of μ chain
- assembly of the pre-B-cell receptor
- stop RAG transcription
Alleles from each parent, As long as one is working, ignore the other

SMALL Pre-B Light Chain Rearrangement

Large pre-B undergoes several rounds of division = CLONING
- Yields resting pre-B cells that express identical μ chains
- no longer make the surrogate light chain and the pre-B-cell receptor
RAG is reactivated = light chain rearrangement
One locus at a time: kappa locus first before lambda
One recombination event V and J: several attempts can be made in kappa before lambda rearrangement
The organization of the light-chain loci allows initial nonproductive rearrangements at one locus to be followed by further rearrangements of that same locus that can lead to the production of a functional light chain
If IgM is self-reactive
- Receptor editing = mature B cells (for light chain lang)
- Repeated fail = apoptosis
- Bind to monovalent self-antigens = anergy
  - Arrest of B cells
  - Prduce IgM and IgD but IGD allowed to develop
  - Lifespan is 1-5 days
- Bawal na mag-undergo ng receptor editing pag nasa bloodstream na
Peripheral tolerance = apoptosis or anergic lang
Central tolerance = receptor editing, apoptosis or anergic

Functional light chain + mu chain in ER = IgM
IgM + IgA + IgB = B-cell receptor in surface = stop light chain rearrangement = immature B cell

Two Check Points in Bone Marrow

First: late pro-B if may pre-B receptor or wala
Second: small pre-B kung may B-cell receptor or wala

Protein Expression

RAG1 and RAG2: on during heavy/light rearrangement, off during checkpoints
TdT: on during heavy rearrangement, off during light
IgA and IgB: forms receptors so on from pro-B stage until end
Pax5: on early pro-B

B-Cell Translocation

Genes that cause cancer when their function or expression is perturbed are collectively called proto-oncogenes

B Cell CD5 Expression

B1/CD5 B Cells: little to no IgD
Produced pre-natally
Lack N nucleotides cuz no TdT during prenatal
Polyspecificity = binds to many diff antigens

Summary

self-reactive immature B cells that encounter their self antigens, either in the bone mar- row or in the peripheral circulation, are prevented from advancing from the immature B-cell stage to the mature B-cell stage.

This repertoire of immature B-cell receptors includes many with affinity for self antigens that are components of healthy human tissue.

Activation of mature B cells carrying such receptors by these antigens would produce self-reactive antibodies

To prevent this from happening, the B-cell receptors of immature B cells are wired to generate negative intracellular signals when they bind to antigen. These signals cause the B cell either to die by apoptosis or to become inactivated.

In this way, the immature B-cell population is subject to a process of negative selection that prevents the maturation of self-reactive B cells. As a consequence, the resulting population of mature B cells in a healthy individual does not respond to self antigens and is said to be self-tolerant.

Bone marrow = blood stream = lymph organs = plasma cell to BM or glial cells remain in lymph organs

Stromal cells = stem cell development into B cells

Pro BCell

Early: DJ

Late: VDJ

CCL21 CCL19 = from lymph cortec xcells

CXCL13 = from follicular DC

Lymphotoxin preserve integridity of FDC

B cell activating factor BAFF to promote B cell survival

Mature Naïve B Cells

Have IgM not self reactive but yet to biun to antigen

B cells pass thru T cell zone into lymphoid follicles

Anergic B cells pass thru lymphoid follicles but stay outside T cell zone and later die by apoptosis

Plasma Cells

Activated by antigen and CD4 T cells

Terminal differentiation = stop proliferation and differntioation

Return to bone marrow to produce more antibodies = meaning effective na

Memory B cells

Resting quiescent b cells

Reserve in case similar infection occurs

Lasts for a lifetime and more quickly activated

CHAPTER 7

T-CELL DEVELOPMENT

Origin: bone marrow
Maturation: thymus
- Before they can undergo gene rearrangements
- “Thymus-dependent lymphocytes”
Positive Selection
- T cells that can recognize self MHC class I and II isoforms survive
Negative Selection
- T cells that bind too strongly to self MHC proteins (autoreactive) die by apoptosis
a:B Lineage is more dominant than y:d Lineage
a:B has two sublineages distinguished by CD4 and CD8 receptors
- can recognize MHC class II and I respectively

DEVELOPMENT IN THE THYMUS

Thymocytes: immature T cells
- Embedded in epithelial network thymic stroma
- Cortex: outer, close-packed, ectodermal
- Medulla: inner, less dense, endodermal
Thymus is a primary lymphoid organ
- Only concerned with production of useful lymphocytes
- Not concerned with application to problems with infection
- Not involved in lymphocyte recirculation
- Does not receive lymph from tissues
Involution of thymus
- Human thymus is fully developed at birth but degenerates one year after birth
- gradually replaced by fatty tissue
- does not impair T-cell immunity
- same case with thymectomy (removal of thymus)
- T cell repertoire are either long-lived, self-renewing, or both
Thymic anlage: rudimentary thymus of cortex + medulla cells
- Colonized by progenitor cells
- Gives rise to thymocytes and DCs
  - DCs and macrophages populate the medulla
T cell progenitors enter the thymus at the junction between cortex and medulla: cortico-medullary junction
- Thymocytes move out through the cortex > subcapsular region > outer cortex > inner cortex > medulla
- Hassall’s corpuscles: sites of cell destruction
DiGeorge Syndrome
- Thymus fails to develop = T cells are absent
- Suffers from opportunistic infections
- No adaptive immune system

COMMITMENT TO T-CELL LINEAGE

Progenitor cells are not committed to the T-cell lineage when they enter the thymus
- Expresses stem cell markers CD34
CD3: generic marker of T cells
- CD19: generic marker of B cells
- CD56: NK cells
Interacts with thymic stromal cells = signals to divide and differentiate
lose stem cell markers = become thymocytes committed to T-cell lineage
- expresses CD2 adhesion molecule and CD5
double-negative thymocytes (DN thymocytes)
- Thymocytes still lack T-cell receptor complex but begins to rearrange T-cell receptor genes
- Express neither CD4 or CD8
Critical cytokines
- IL-7
  - Secreted by stromal cells and binds to receptors on CD34-expressing progenitor cells
  - Defective IL-7 receptors = T cells absent
- Notch1
  - Cell-surface receptor on thymocytes
  - That’s why they need to go to the thymus because andoon ang Notch ligand transcription factor that it needs to interact with
  - Binds to transmembrane ligands on thymic epithelial cells
  - Drives cells along the pathway of T-cell differentiation and away from B-cell differentiation
  - Parallels that of Pax5 in B cell development

Notch1 Protein
- Has intracellular and extracellular domains
- Extracellular domain binds to ligand on thymic epithelium = cleavage of the two domains
- Intracellular domain translocate to nucleus
- Initiates gene transcription

COMMON THYMOCYTE PROGENITOR

Both lineages derive from DN thymocyte precursors
- Where T-cell receptor gene rearrangement is initiated
Rearrangement of y, d, and B loci occur at the same time
- If thymocyte makes a functional y:d receptor before a functional B = committed y:d T cell
- If thymocyte makes a functional B chain first = incorporated into pre-T cell receptor
  - Still not committed to a:B lineage
double-positive thymocytes (DP thymocytes)
- gene arrangement stops when functional B chain is incorporated into pre-T cell receptor
- cell proliferates and expresses CD4 and CD8 co-receptors
- allows a-chain rearrangement
- continues y and d-chain rearrangement
- If functional a:B receptor first: committed a:B lineage
- If functional y:d receptor first: committed y:d lineage

DN THYMOCYTE GENE REARRANGEMENT

B and d chain : V+(D+J) segments
a and y chain: V+J segments
Functional y:d receptor gene arrangement is made first
- y:d heterodimer is formed
- assembles CD3 signaling complex and moves to surface
- signals to stop B chain rearrangement
- y:d T cell receptors are not subjected to positive and negative selection
Functional B chain gene arrangement is made first
- More frequent outcome
- Translocate to ER
- Binds to surrogate a chain pTa to test capacity
Pre-T cell Receptor
- If functional B chain successfully binds to pTa
  - Forms heterodimer with CD3 complex and ζ chain
  - pTA interacts with C and V regions of the B chain
  - generates signals through the CD3 complex and Lck cytoplasmic protein
- Assembly of pre-T cell receptor signals to stop rearrangement of B, y, and d chain genes
  - First checkpoint to ensure B chain can bind to a chain
  - Cells become pre-T cells

B CHAIN REARRANGEMENT

a:B lineage is favored because it only requires one productive gene rearrangement while y:d requires at least two
this increases the potential to rescue nonproductive B chain rearrangements
- two C genes and their associated D and J segments
- if rearrangement is nonproductive: thymocyte can attempt rearrangement at the other chromosome or same locus
Four attempts to rearrange B chain gene
- 80% of T cells make productive B chains

A CHAIN REARRANGEMENT

Successful rearrangement of B chain signals via pre-T cell receptors to stop gene rearrangement
- Express RAG1 and RAG2 genes
- Ensures that only one productive B chain is expressed = allelic exclusion
Pre-T cell is induced to proliferate and create clones expressing the same B chain
- Accompanied by expression of CD4 first and followed by CD8 = DP thymocytes
- Constitute majority of thymocytes and are found in the inner cortex
- Interacts with epithelial cells
Once Pre-T cells stop proliferating, they become small DP thymocytes
- Reactivation of recombination machinery and targeted to the a, y, and d loci
T-cell receptor a chain genes
- Similar to immunoglobulin kappa and lambda light chain genes
- No D segments
- Rearranged only after B chain has been expressed
- Repeated rearranging attempts possible
  - Multiple V segments and J segments
  - Continues until productive rearrangement occurs or until the supply of V and J segments is exhausted
Rearrangement of a chain
- d locus within the a locus will be deleted irrespective of whether the rearrangement is productive or not
  - reduces the probability that committed a:B lineage expresses y:d receptors and a:B receptors
when DP thymocytes make a productive a chain rearrangement
- gene transcription starts to form a chain
- translocate to ER
- tested to bind to B chain and form T-cell receptor
  - second checkpoint
  - if successful: proceed to positive selection

GENE EXPRESSION CHANGES DURING DEVELOPMENT

Notch transcription factor

RAG1, RAG2: may stop bc of allelic exclusion (checkpoint testing)

TdT: junctional diversity

pTa: surrogate alpha chain

RAG1, RAG2: expressed at the two stages where B and a gene rearrangements are made respectively
TdT, pTa: also expressed throughout this phase of development
pTa: expressed throughout the period when rearrangements occur
CD4, CD8: relay signals from pre-T cell receptors
Tyrosine kinase Lck: signaling from pre-T cell receptor and co-receptor
CD2: adhesion molecule on T cells that interact with cell-surface CD58 on other cells
Ikaros, GATA-3: transcription factors in early progenitors
Th-POK: expressed late in development and is for development of CD4 T cells from DP thymocytes

POSITIVE SELECTION

First phase of T cell dev: produce T cells irrespective of their antigenic specificity
Second phase: examination of receptors produced and selection of those that can work effectively with self MHC molecules
- Only involves a:B T cells
- y:d cells do not depend on MHC molecules for detecting infection
primary T-cell receptor repertoire has a bias towards MHC molecules but T cell receptors are not tailored specifically
- only 2% of total can interact with MHC I or II
Positive Selection
- Small population that can interact with MHC molecules are signaled to mature further
- Takes place in cortex of thymus where thymic cortical epithelium expresses both MHC I and II molecules
  - Forms a web of cell processes that envelop and make contact with DP thymocytes
  - Potential interactions are tested
- If peptide:MHC is bound within 3-4 days after the thymocyte first expresses a functional receptor = further maturation

CONTINUATION OF GENE REARRANGEMENT

If first a chain rearrangement of pre-T cell is productive = positive selection occurs within a few hours
- Turns off recombination machinery
- Degrades RAG proteins
- Stops transcription of RAG genes
- Proliferates
If not = rearrangement continues
- Can continue throughout the 3-4 days of positive selection
After successful B chain = developing T cell turns off recombination machinery = allelic exclusion
- a chain is not subject to allelic exclusion
- rearrangements can occur at both copies of a chain locus
- DP thymocytes can express 2 a chains = 2 T cell receptors
Receptor editing
- Trying out different a chains in order to become reactive with self MHC molecules
- Opposite to B cells = receptor editing is to get rid of reactivity

CD4 CD8 DETERMINATION

Single-positive thymocytes (SP thymocytes)
- result of positive selection
- DP thymocytes mature into SP thymocytes
If peptide interacts with MHC I = CD8 is recruited
- Any cell that is nucleated should be able to present it via MHC I in case infected
If peptide interacts with MHC II = CD4 is recruited
- CD4 coordinating cell in innate and adaptive immune system
- HIV kills CD4 = acquired immue deficiency syndrome
- ACPs magnify signals of infection and is needed sa MHC II since CD4 is what coordinates everything
Bare lymphocyte syndromes
- Lack of expression of either MHC I or II molecules by lymphocytes and thymic epithelial cells
- Patients who lack MHC I have CD4 cells but no CD8 ; vice versa

NEGATIVE SELECTION

T cells whose antigen receptors bind too strongly to self-MHC molecules ( potential autoreactive) are deleted
- To avoid tissue damage and autoimmune diseases
Mediators
- Positive selection: epithelial cells
- Negative selection: dendritic cells, macrophages, thymocytes
  - T cells that bind too strongly to MHC I molecules on DCs or macrophages are signaled to die
To extend negative selection to proteins that are specific to one or a few cell types, such as the insulin made only by Beta cells of the Langerhans cell of the pancreas, transcription factor autoimmune regulator (AIRE) causes several hundred of these tissue-specific genes to be transcribed by a subpopulation of thymic epithelial cells
- Activation of AIRE allows thymus to express those specific proteins na di makikita sa iba, para ma-practice ng T-cell kung madedetect and kung magiging autoreactive ba o hindi
- Peptides derived from these proteins can be bound by MHC class I molecules to form complexes that participate in negative selection
- Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) or autoimmune polyglandular syndrome type 1
  - T cells specific for tissue-specific antigens are not eliminated by negative selection and thus attack variety of tissues

REGULATORY CD4 T CELLS

Functions of CD4 T cells
- Helper T cells: activates macrophages and B cells
- Regulatory T cells
  - Suppress response of self-reactive CD4 to their specific antigens
  - Despite AIRE activity, self-reactive CD4 T cells are still present in healthy people
  - Have T cell receptors specific for self-antigens
  - distinguished from other CD4 T cells by expression of CD25 on cell surface and transcriptional repressor protein FoxP3
On contacting self-antigens presented by MHC II
- Treg do not proliferate
- Suppress the proliferation of naïve T cells responding to self-antigens presented on the same antigen-presenting cell

FURTHER DIFFERENTIATION IN SECONDARY LYMPHOID TISSUES

Only a small fraction of a:B T cells survive positive and negative selection and leave the thymus as mature naïve T cells
Secondary lymphoid tissues provide specialized sites where naïve T cells are activated by their specific antigens
Encounter with antigen provokes the final phases of T cell development and differentiation: the mature T cells divide and differentiate into effector T cells
- Some stay in lymphoid tissues while others migrate to sites of infection

Discussion Notes

B cells (Humoral)

have role in viruses
Already circulating antibodies can neutralization before virus can enter cells

T cells (Cell-Mediated)

Antigen presentation by MHC before T cells can act
Necrosis: cell death due to external factors
MHC I: all nucleated cells (kaya hindi kasama RBCs)
MHC II: professional antigen-presenting cells
- Dendritic
- Macrophage: recruitment
- Mature B cells

Similarity: Somatic recombination (gene rearrangement)

Diversity of receptors
B cell receptors are identical
Allelic exclusion: ano na-arrange, ayun lang ie-express nila
- More effective as an immune cell kasi isang target lang ang need i-focus
The more diverse MHC = the more diverse receptors you need to have
B cells need input from cell-mediated immunity before activation
- Mas derived ang humoral since it cannot function without pre-existing xdsfunction of cell-mediated

Rubor: redness : increased blood flow to recruit more immune cells

Tumor: inflammation due to vasodilation which looses tight junctions

Dolor: pain because of triggering pain receptors

Calor: heat to impede the propagation of pathogens

B cells leave bone marrow as immature B cells

They mature upon meeting follicular dendritic cell (different from APC dendritic cell)

“follicular” = lymph node

Will give B cell maturation factor

Then start looking for infection to complete its maturation

Kaya called lymphocytes kasi paikot ikot sa lymph nodes

B cells only recognize antigen

For t cells, they need to recognize both MHC and antigen

After being able to attach to MHC molecules (positive selection), they must be able to let go para iwas autoreactivity (negative selection)

TLRs= receptors of the innate system

PAMPs= pathogen associated molecular patterns

NK cells are non specific but fast acting

Pero since nonspecific, if a pathogen dominates, di na kaya labanan kaya saka need ng adaptive

Cytokine storm = nagpapanic kasi di macontrol ang infection/ mapatay ang pathogen so release lang ng release ng cytokines = over inflammation

Immune system is aware that inflammation can go overboard

T regulatory = anti-inflammatory cytokines to regulate/

IgG4 is anti-inflammatory sa innate and acts as regulator

CHAPTER 8

T-CELL MEDIATED IMMUNITY

Remember: Adaptive immunity is initiated through interactions of NK cells and dendritic cells (refer to ch. 3)

8-1: DENDRITIC CELLS CARRY ANTIGENS FROM SITES OF INFECTION TO SECONDARY LYMPHOID TISSUES

Adaptive immune responses are initiated by capturing some of the pathogen and sequestering them in the secondary lymphoid tissue
Myeloid dendritic cells capture antigens and present them to naïve T cells
T-cell response to infections is made in the draining lymph node
- Blood infections: spleen
- Mucosal tissues: mucosal secondary lymphoid organs

Dendritic Cells

Immature DCs
- In the skin and peripheral tissues
- Active in capture, uptake, and processing of antigens
Mature/activated DCs
- No longer active in capture, uptake, and processing of antigens
- Upon moving to secondary lymphoid tissues
- Gains capacity to activate naïve T cells
Confined to the outermost part of the cortex where T cells congregate

Macrophage

Present in the cortex and medulla
Removes pathogens and their breakdown products from the afferent lymph that arrives from the site of infection
- Macrophage-mediated lymph filtration
- Prevents infectious microorganisms from passing through the node and gaining access to the blood via efferent lymph
- Prevents systemic infections
Eliminate lymphocytes that are signaled to die by apoptosis

8-2 DCs PROCESS ANTIGENS

DCs present peptides on MHC II molecules to activate naïve T cells
DCs have various endocytic and signaling receptors
Receptor-mediated endocytosis
- Capture bacteria/virus particles from EC fluid and process in lysosomes
- Aka micropinocytosis
- Micropinocytosis involves nonspecific ingestion of larger volumes of ECF and is used to capture pathogens not recognized by any endocytic receptor
DCs can become infected and make viral proteins that enter the endosomal ER and presented on MHC I molecules
- If infection not lethal, DCs carry the virus to the draining lymph node and activate naïve CD8 T cells
- If infection is lethal, DCs reach secondary lymphoid tissue but too sick to activate naïve T cells
  - The virus released by dying DCs can infect healthy DCs which then present the antigens on their MHC I molecules and activate CD8
If virus do not infect DCs, cross-presentation is used to stimulate a CD8 response
- DCs take up antigens by the endocytic pathway
- Transfer the virus degraded peptides to the exocytic pathway for cross-presentation by MHC I

DCs carry all TLRs except TLR9
- Highly sensitive to the presence of all manner of pathogens
- Signals from TLRs lead to DC activation
  - Increase efficiency in uptake, processing, and presenting
  - Appearance of CCR7 on the cell surface which is the receptor for the CCL21 chemokine in the secondary lymphoid tissue
    - Leave lymph and enter the tissue of the draining node
    - Maturation of DCs to focus on presentation

8-3 NAÏVE T CELLS FIRST ENCOUNTER WITH PEPTIDE:MHC COMPLEX

T cells bind to the endothelial cells of the thin-walled high endothelial venules (HEV)
They squeeze through the vessel wall and enter the T-cell area
- They encounter mature DCs
- T-cell receptors examine the peptide:MHC complex on DC surfaces
- If it binds successfully, T cell is activated and retained in LN
T cells can arrive via the afferent lymph
- Entered an “upstream” LN from the blood but did not encounter specific antigen, leave the LN via the efferent lymph and carried “downstream”
- T cells arrive at the node in a different afferent vessel from that carrying pathogens and antigens and DCs from the infected tissue

If T cell does not find its specific antigen, it leaves the medulla in the efferent lymph to continue recirculation
If T cell is activated, it takes several days for the cell to proliferate and differentiate into effector cells
- Accounts for much of the delay between the onset of an infection and the appearance of the adaptive immune response

8-4 HOMING OF NAÏVE T CELLS

Homing: naïve T cells leave bloodstream and enter T-cell area of LN
- Guided by CCL21 and CCL19 chemokines
- Secreted by stromal cells and DCs in T cell area
- Establish a concentration gradient along the endothelial surface
Naïve T cells express CCR7 chemokine receptor which binds to CCL21 and CCL19
- Guides T cells up the chemokine gradient
Exit route: cortex > medulla > efferent lymph
- Controlled by T cell receptor that can recognize sphingosine 1-phosphate (S1P) that establishes gradient on the LN
- Lowest in T-cell area and increases in the direction toward the efferent
For T-cells that recognized antigen on DCs
- Express CD69
  - Moves S1P receptors inside the cell to prevent external sensing
  - Prevents leaving LN during nurturing
  - Upon maturation into effector T cells, they stop expressing CD69

Adhesion Molecules

L-selectin
- On the T cell surface
- Binds to CD34 and GlyCAM-1 on endothelium
- Causes naïve T cell to slow down and attach to endothelial surface
LFA-1
- On T cell surface
- Binds with ICAM-1 and ICAM-2 on endothelium
- Activated by chemokines secreted by LN
- Strengthens hold on the ICAM
- Transient binding with DCs
- Upon encountering specific peptide:MHC complex, LFA-1 molecules increases affinity for ICAMs = conjugate
ICAM-3
- On T cell surface
- Binds to LFA-1 and DC-SIGN (unique to activated DCs)
- Transient binding with DCs
CD2
- On T cell surface
- Binds with LFA-3
- Strengthens adhesion

8-5 ACTIVATION OF NAÏVE T-CELLS

Co-stimulatory signal
- Required because signals generated by T cell receptor and co-receptor (CD4/8) with peptde:MHC complex is not enough for activation
- Without this, T cells cannot divide nor survive
- CD28 T cell co-stimulatory receptor which binds to B7 co-stimulatory molecule on DCs
Only professional APCs express B7 in the presence of infection through presence of inflammation and not constitutively
- When DCs take up pathogens/antigens, signals are generated to induce B7 expression
- DCs arrive at the draining lymph node already expressing B7
- Inflammation is needed or else anergy = pag isang e.coli lang, hindi dapat ma-activate adaptive immune system
  - Hindi enough ang pathogen population to induce inflammation = not a concern = anergy
Effector T cells activates macrophage and naïve pathogen-specific B cells to express co-stimulatory molecules
Once T cells are activated, they express an additional complementary B7 receptor CTLA4
- Binds B7 twentyfold
- Functions as brake on CD28 to inhibit activation and proliferation of T cells

8-6 RECEPTOR SIGNALS FOR ACTIVATION

Steroid signals can pass through to bilayer and into the nucleus kasi andoon ang receptor
Protein signals cant pass through the bilayer kaya receptor niya ay membrane-bound to start signal cascade towards nucleus
T-cell synapse: region of contact and communication between TCs and DCs
- Central supramolecular activation complex (c-SMAC): TC receptors, co-receptors, co-stimulatory receptors, and CD2 adhesion molecule
- Peripheral supramolecular activation complex (p-SMAC): LFA-1, ICAM-1, and talin which forms a tight sea, around the SMAC
Interactions of MHC ligands with TC receptors activate cytoplasmic protein tyrosine kinases
- Phosphorylate tyrosine residues (immunoreceptor tyrosine-based activation motifs or ITAMs) in the CD3 tails and the associated zeta chain CD247
- Extracellular binding of antigen to TC receptor initiates pathways of intracellular signaling that leads to TC differentiation
Cytoplasmic tails of co-receptors associate with Lck
- Phosphorylate the CD3 ITAMS and ZAP-70 upon synapse formation
- Binds to the phosphorylated tyrosines of the zeta chain
Activated ZAP-70
- Leads to activation of nuclear factor of activated T cells (NFAT)
- Leads to activation of protein kinase C-theta which induces NFkB
- Leads to activation of nuclear protein Fos, one of the component of transcription factor AP-1

8-7 IL-2 FOR PROLIFERATION AND DIFFERENTIATION

Synthesized and secreted by activated T cells
- Autocrine action
- Paracrine action: cytokine acts on a different type of cell from the one that made it
Requires signals from TC receptor:co-receptor complex and the co-stimulatory signal delivered by CD28 and activation of NFAT
Co-stimulatory signal stabilizes cytokine mRNA to increase TC production of IL-2 and rate of transcription from the IL-2 gene
Naïve TC has B and Y chain of IL-2 receptor
- Activated TCs: alpha chain is synthesized
- Triggers TCs to progress through cell division

8-8 T CELL ANERGY

Some T cells with specificity for self-antigens escape negative selection in the thymus because the self-antigens they recognize are not expressed in the thymus
- These cells will not express B7
Absence of B7 and co-stimulation, engagement of peptide:MHC complex by TC receptor and co-receptor = TC anergy
- State in which it cannot respond to any external signal
- Cannot be revived
- Fails to make IL-2
- Irreversible mechanism of self-tolerance to render harmless those self-reactive T cells

8-9-10 CD4 T CELL POPULATIONS

CD4 TCs do not directly attack the pathogens
Form a cognate pair with target cell

TH1

Help macrophages
For intracellular bacterial and viral infections
Stimulated by IL-12 (by DCs) and IFN-y (by NK cells)
Controlled by T-bet transcription factor
- Causes TC’s own IFN-y gene to be turned on
- Local production and secretion of IFN-y in the secondary lymphoid tissue
Increases inflammation

TH2

Help eosinophils, basophils, mast cells, and B cells
For parasites
Do not generate inflammation but promote repair and recovery of damaged tissues
Favors production of pathogen-specific IgE antibodies to eliminate parasites
Stimulated by IL-4 which binding induces GATA3 to commit to the TH2 pathway
- Turns on IL-4 and IL-5 which are secreted by TH2
Histamines = expulsion of allergen
- Sneeze, diarrhea, scratching
IgE is found on surface of eosinophil and mast cells

TH17

Secrete IL-17 and CXCL8
Recruits neutrophils to infected tissues
Stimulated by IL-16 and TGF-B
- Induces TC to make IL-21 which activates STAT3
Controlled by RORyT which turns on IL-17

TFH

Cooperate with naïve B cells
Initiate antibody response
Induced by IL-6 and controlled by Bcl6
Co-stimualtion involves the Inducible TC co-stimulator (ICOS)
Bcl6 express CXCR5 the receptor for CXCL13 produced by stromal cells of B cell follicles and causes exit from T cell area into follicles of the B cell areas
Guides switching of immunoglobulin isotypes in B cells

Treg

Keeps immune response under control
Limits tissue damage and facilitate healing
Reduce chance of secondary infections
Natural regulatory cells: commit to regulatory function during thymic development
Induced Treg: emerge in the course of the immune response to infection
- activated by TGF-B but in the absence of IL-6 and other pro-inflammatory cytokines
both expresses FoxP3 and CD4 and CD25
Induced Treg produces TGF-B and IL-10 to inhibit inflammation

8-11 POSITIVE FEEDBACK

For both TH1 and TH2 cells, the cytokine that drives their differentiation is central to their effector function and secreted in quantity
- TH1: IFN-y
- TH2: IL-4
Positive feedback: in which the functioning effector TCs drive further differentiation
- Polarized: if population is dominated by either TH1 or TH2
Cell-mediated immunity
- Polarized TH1
Humoral immunity
- Polarized TH2
Tuberculoid Leprosy
- TH1 bias
- Help macrophages to suppress growth and dissemination of bacteria
- Chronic inflammation damages skin and peripheral nerves
- Disease progress slowly
Lepromatous Leprosy
- TH2 bias
- Large pathogen-antibodies are made but ineffective against bacteria hidden inside macrophages
- Bacteria become disseminated to other sites in body
- Causes gross tissue destruction which is fatal

8-12 CD8 TCs REQUIRE STRONGER ACTIVATION

CD8 TCs are functionally homogenous but must interact with a wide range of target cells
For intracellular infections, mostly viral
Recognize antigens presented on MHC I molecules of DCs
For some viral infections, the interaction of a naïve CD8 with DC that presents antigen via MHC I is enough for activation and differentiation
- Synthesize IL-2 and receptor
- Induce CD8 TCs to proliferate
For other viral infections, DCs solicit help from virus-specific effector CD4
- Gives IL-2 to jump-start activation
- Forms viral peptide:MHC I complex and viral-peptide:MHC II complex
The effector CD4 is activated to make and secrete IL-2 which then binds to the receptor on CD8
- Drives the naïve CD8 to proliferate and differentiate
CD8 are only activated when the evidence of infection is unambiguous
- Cytotoxic TCs inflict tissue damage

8-13 CD8 TCs and Effector CD4 TCs

CD8 cytotoxic TCs and CD4 TH1, TH2, and TH17 TCs travel to infected tissue after differentiation
Adhesion molecules on effector T cells allow them to leave the secondary lymphoid organ
- Differentiates them from naïve TCs
L-selectin is replaced by VLA-4
- Binds to adhesion molecule VCAM-1 on activated endothelial cells at inflammation sites
- Halts passing effector TCs and direct them to enter the infected tissue
Effector TCs express more CD2 and LFA-1 than naïve TCs
- Makes them more sensitive to ICAM-1 and LFA-3 than APCs
Signals from TC receptor and co-receptor are sufficient to activate effector TCs
- Whereas induces anergy in naïve TCs
- Co-stimulation through CD28 and B7 is not needed for activation
- Allows effector TCs to kill any type of cell infected with virus

CD4 effector TCs are activated by antigen recognition on all cells that express MHC II
- Including vascular endothelial cells induced to express MHC II by IFN-y secreted by NK cells and effector TCs at sites of infection
- Naïve TCs are activated by antigen recognition of DCs only
Co-stimulation restricts initiation of TC responses
- Relaxing this requirement allows effector TCs to work quickly and efficiently

8-14 CYTOKINES AND CYTOTOXINS

Cytokines: alter behavior of target cells
- Interleukins by TCs
- Made only after the effector TC has formed a conjugate
- Never made and stored in CD4 TCs for future use
Cytotoxins: kill target cells
- Fewer than cytokines
- Effector CD8 manufacture and store them in lytic granules before an encounter with a target cell
- Comprised of granzymes, serglycin, and granulysin

8-15 CYTOKINES

Janus kinases (JAKs)
- Dimerization of receptor polypeptides = activates JAKs into enzymes
- Phosphorylates STATs (signal tranducers and activators of transcription)
- Phosphorylated STAT dimerize = allows them to move from cytoplasm to nucleus
- Change gene expression in target cell

Effects of cytokines can be turned off
- Phosphatases remove phosphate groups from JAKs, STATs, and cytokine receptors
- Suppressors of cytokine signaling (SOCS) proteins inhibits signaling pathway

8-16 SELECTIVE CYTOTOXINS

CD8 synthesizes cytotoxins in inactive forms and package them into lytic granules
Antigen specificity allows TCs to pick out only infected cells
- Cytoskeleton directs them to go towards T cell receptor with antigen kaya specific
TC focuses granule secretion at the small, localized area of the synapse
- Granules only kill the infected cell
CD8 also secretes cytokines
- IFN-y to inhibit viral replication in infected cells, increase viral antigen presentation, and activate macrophages

One cytotoxic TC can kill many infected cells

8-17 APOPTOSIS

Cytotoxic CD8 kill cells by apoptosis aka programmed cell death
- prevents pathogen replication
- prevents release of infectious pathogen particles from infected cell
cytotoxins make pores in target cell membrane
phosphatidyl serine = marker of apoptosis because PS is normally in the intracellular side of the cell membrane
- if induced to undergo apoptosis, it will flip and move to the extracellular side
- anexinfide stains PS

8-18 MACROPHAGE ACTIVATION BY TH1 CD4

TH1 CD4 help macrophages at infection sites to be more proficient in killing pathogens by secreting cytokines
- Macrophages containing captured pathogens fuse more efficiently with lysosomes
- Increased synthesis of potent microbicidal agents such as nitric oxide, oxygen radicals and proteases
Overall enhancement of macrophage function by effector TCs is called macrophage activation
Macrophages require two signals for activation from TH1
- Delivered by IFN-y and CD40 ligand
- Induces changes in gene expression for activation
After forming conjugate with macrophage, TH1 takes several hours to initiate cytokine gene transcription
- Cytokines are translocated to the ER of TH1
- Delivered by secretory vesicles to synapse
- Only cytokine receptors on the macrophage become loaded with cytokines and subject to activation
activation is antigen-specific
- only macrophages that present pathogen-derived antigens are selected for activation
- prevents unnecessary damage to healthy tissue
regulatory mechanism: TGF-B, IL-4, IL-10, IL-13
- inhibit macrophage activation
- counter effects of TH1 cells
- evident in lepromatous leprosy whose macrophages become overwhelmed by proliferating bacteria

8-19 TFH CELLS AND NAÏVE B CELLS

TFH cells help B cells make antibodies
- TFH cells move from the T cell area of secondary lymphoid organ to near B cell area
- Naïve B cells enter lymph node from blood because of chemokines CCL21 and CCL19
- Allows for interaction of TFH with naïve B cells
once TFH and B cell forms synapse, TFH synthesizes CD40 ligand
- binds with CD40 on B cell
- causes proliferation, differentiation, and maturation of B cell into plasma cell
Linked recognition
- Bounded B cells and T cells are specific for epitopes of the same antigen that was first bound and internalized by B cell receptor

8-19 REGULATORY TCs

Treg expresses high levels of CD25
Treg make immunosuppressive and anti-inflammatory cytokines
- IL-4, IL-10, TGF-B
Suppression depends on physical contact between Treg and target cell
- Treg interacts with DCs to prevent them from interacting and activating naïve T cells
- Treg may also interact directly with effector TCs
Human Treg has FoxP3
- Infants with no functional FoxP3 have no Treg
- fatal because it permits immune responses that are directed at self-antigens

NK cells similar to CD8 cytotoxic

No specific receptor for antigen

Nonspecific PAMPs triggers TLRs of NK cells

Activated dendritic (pag na-outnumber niya ang NK cells) detach from tissue and get washed by lymphatic fluid

MHC I magaling sa intracellular bc proteosome ang naglload ng peptide sa MHC I

Mas efficient ang cross presentation kasi it triggers both CD8 and CD4 (so cytotoxic and b cells are activated)

Kaya sa sars-cov-2 antibodies are needed kasi they play a role both in innate and adaptive

Cytokines that induces inflammation can loosen the tight junctions = recruitment of both neutrophils and T cells

Chemokines parang bread crumbs to direct t cells where to go

Chapter 9

Humoral Immunity

TFH stays in the lymph node to activate B-cell arm of adaptive immunity
Mature B cells has IgM molecules that become cross-linked by the antigen
- Sends signal from receptors to inside of cell
Interaction of antigen and immunoglobulin is communicated by IgA and IgB (B cell receptor together with IgM)
- Not enough to activate naïve B cell

Additional signals: B cell receptor:co-receptor
- CR2/CD21: recognizes iC3b and C3d derivatives deposited on the pathogen
- CD19: signaling chain
- CD81: binds to CD19 to bring it to the surface
Generation of the iC3b and C3d ligands
- CR1 + C3b with ligand
- Becomes susceptible to cleavage Factor I
- Gives iC3b fragment and C3d fragment
- CR1 increases abundance of ligands for B cell co-receptor on pathogen surface
CR2 component of co-receptor
- Upon binding to antigen, binds to C3d
- Causes kinase cascade
- Simultaneous ligation of the B cell receptor:co-receptor increases the B cell sensitivity to antigen
TFH recognizes peptides presented by B cell MHC II
- Sends cytokines and signals to induce division and differentiation

DiGeorge Syndrome
- No thymus
- Little to no T cells
- Normal B cell levels
- B1 cells produce low-affinity IgM
  - Does not require T cell activation
  - Polyspecific and no isotype switching
  - Carb or protein epitopes can cross-link B cell receptors and co-receptors and sufficient to activate B cell even without additional signals
  - Aka thymus-independent antigens (TI antigens)
- Highlights importance of B2 cell and T cell activation

Follicular DCs

FDCs provide signals to allow B cells to mature and survive
- Diff from myeloid DCs that present antigens to T cells
- Diff from plasmatycoid DCs that make type I IFN
FDCs serve as a depository of intact antigens
- Available for interaction with antigen receptors of B cells
- They lack phagocytic activity = preserves antigens intact

FCs take up antigens from lymph
- C3b and C3d fragment binds to pathogen surface
- Those tagged with C3b and C3d are taken up by FCs
Subcapsular sinus macrophage
- Little phagocytic activity
- Has CR1 and CR2 to take up antigens tagged with C3d or C3b
- Holds them on surface to be screened by naïve B cells
Medullary sinus macrophage
- Highly phagocytic
- Filters lymph before leaving node

B cell and T cell interaction

Naïve B cells from blood will be attracted to T cell area by chemokines CCL21 CCL19 and to B cell follicle by CXCL13
B cells enter node at subcapsular sinus to screen antigens
- If found, b cell enters B cell area of follicle to interact with TFH cells
- Expresses CD69 which prevents SIP receptor expression
- Stays in the lymphoid tissue to complete differentiation
Activated B cell begin to endocytose and process the antigen and present peptides on MHC II
- Expression of CCR7 binds to CCL21 and CCL19
- Draws the activated B cell to the boundary between T cell and B cell areas to interact with newly differentiated TFH cells

TFH activated by DCs presenting antigen reduce expression of CCR7
- Moves towards boundary of primary follicle
- Screens antigens presented by activated B cells
- If found, forms conjugate and expresses CD40 ligand
- Activates B cell NFkB and enhances adhesion molecules
- Facilitates delivery of cytokines

Primary Focus of Clonal Expansion

Conjugate pair T cells and B cells move together to medullary cords
- They begin to divide to form primary focus
- Gives rise to dividing B lymphoblasts secreting IgM
Some B cells stay in medullary cords
- Differentiate into palsma cells under the influence of cytokines IL5 and IL6 secreted TFH
- Determined by B-lymphocyte-induced maturation protein 1 (BLIMP-1) which stops transcription for proliferation
- Cells then increase expression of immunoglobulin chains

Somatic Hypermutation and Isotype Switching

Other B lymphocytes leave the primary focus and move into primary follicles of the B area while still attached to TFH
- Proliferates and forms germinal center
- FDCs secrete IL6 IL15 8D6 and BAFF to start rapid division of B cells to form centroblasts
TFH also divides and produce more cytokines
- induces B cells to produce activation-induced cytidine deaminase (AID) essential for somatic hypermutation and isotype switching
during somatic hypermutation and isptype switching, centroblasts no longer express immunoglobulins because focus is on expansion of large population of B cells with switched isotype and V-region mutations not antigen selection
- proliferation of antigen-specific B cells in primary follicles give rise to secondary follicle
- GC no has rapidly dividing B cells and T cells in the center with naïve B cells at the periphery screening for their specific antigens and now forms mantle zone

If B cells produce antibodies, what happens next? What are antibodies exactly for?

Can be simplified into 3 effector functions: neutralization, opsonization, complement actication

Neutralization: antibodies attach to target = pathogen cannot replicate or attach to surface

Opsonization: antibodies has Fc or constant regions that promote recognition to phagocytes

Complement fixation/activation: classical complement pathway; pentameric IgM will bind to C1

Endpoint of classical: MAC to perforate target cell to lead to their death

Humoral is like the highest tech level/latter nag-evolve, para magfunction, need ng innate and t cell immunity

For b cells, soluble antigens are enough while t cells need MHC presentation

Once activated > clonal expansion > differentiation

Memory cells and plasma cells may or may not have the same receptors

B cells need maturation factors from follicular DCs in lymph nodes

Myeloid DCs are APCs

FDCs are fixed in lymph nodes

T cells go to lymph node for activation/ screen antigens while B cells look for maturation factors from FDCs

B cells with maturation factor = Mature B cells

Next step is activation

Soluble antigen attaches to B cell = cross-linking of B cell receptors

Cross-linking: same receptors due to allelic exclusion; more receptors are activated by 1 antigen

ITAMs: needed for signaling by receptors

Pag nag cross-link, mas malakas ang signal

CD3 gen marker of T cells while CD19 for b cells which is required for activation

CR2 complement receptor binds with C3d ligand from alternative pathway cleaved by factor I

B cells cannot function without complement system of innate system: supplementary ang humoral

DAF and MHC stops C3 from proceeding with complement

B1 doesn’t need T cells for activation

Antigens can trigger B1

Kaya mas madali dumami ang B1 kasi cross-linking lang ang need

Drawback: hindi magm-mature ang antibody response

IgD part of development of B cells but effector functions is not known; marker lang for what stage na ang B cell development

IgM is referred as primary antibody kasi siya unang pwedeng isecrete ng lahat ng B cells

Kung mature na B cell, FDCs can hold antigens on their dendrites

Subcapsular sinus macrophage:

Primary follicle: kumpulan ng FDCs waving soluble antigens trying to attract B cells

B cells anergy pag walang infection kahit mag match

B2 cells are not fully activated even after finding target pathogen = they are instructed to go to the boundary of T cell area and present antigens to T cells via MHC II = cognate pairing if match sa T cell receptor

Once nag cognate pair, ITAMs will reorganize intracellular vesicles towards synapse and release of cytokines dictates b cells what to do next

After na-encounter ang antigen and naactivate sila, maturation into plasma cells producing IgM

Kaka-encounter lang ng antigen

Mas malaki ang centroblasts kasi may somatic hypermutation and isotype switching; main enzyme is AID

Somatic recombination and junctional diversity occur during development whereas somatic hypermutation and isotype switching occur during maturation

Activated-induced cytidine deaminase:

Hypervariable regions are on the antigen-binding site

Affinity maturation: sobrang improved ang affinity ng antibody

Antibody-response maturation: improves IgM affinity via somatic hypermutation which is changing hypervariable region which leads to affinity maturation to improve specificity of variable region of antibody

FDCs secreting cytokines has an effect on how the b cell will respond on its antibody maturation

Secondary antibodies: IgG (swiss knife ng antibodies), IgA, IgE

Centrocytes tapos na mag isotype switch and som hypermutation

How can we be sure affinity is improved by som hypermutation? Centrocytes will interact with FDCs

Centrocytes need to returm sa same antigen that activated it sa primary follicle = if som hypermutation is successful, dapat mauna siya bumalik sa antigen sa FDCs kasi may time limit ang centrocytes

Need mag compete kasi nag proliferate nga so only those with the highest affinity will reach the antigen first

This leads to further differentiation

Sa Hyper IgM patients, walang germinal centers sa lymph node bc walang isotype switching na nagaganap

Further maturation of b cells are still guided by FDCs

IL4 and IL10 secreted by FDCs

If IL10 dominant, they become plasma cells but the secreted antibodies are isotype switched

If IL4 dominant, they become memory B cells that retain B cell receptors

Memory b cells: activation process is skipped; theyre targeting the same antigen and has higher affinity; they’ll just secrete secondary antibodies upon infection

Primary immune response: IgM

Secondary immune response: memory B cells

High mutation rates ng pathogens kaya babalik ulit sa primary response

Shingles sa nerve nagtatago at nagfoformat

Narereactivate dahil immunocompromised (i.e. nagka flu prior)

Centrocytes stop dividing but may maititrang memory b cells pero may half life so to make sure na enough pa rin = boosters

Some memory B cells can undergo clonal expansion

IgG can cross the placenta and dominant in circulation

IgA dominant and dimeric on mucosal surfaces but monomeric in circulation

IgE allergic response

Transcytosis: pagtawid sa epithelial tissues

Fc receptors bind to Fc of antibodies = transcytose and reach the tissues where they will let go of the antibodies

Poly-Ig receptors binds to IgA for transcytosis

Passive transfer or passive immunization = binigyan ka ng antibodies na hindi immune system mo ang gumawa

Mothers need to be vaccinated or booster vaccines: IgG will pass thru placenta and received by fetus

IgA can be passed thru breast milk = colostrum highly proteinaceous bc it has IgA

Rabies is 99% mortality

Two types of vaccines are given

Active: promote immune response to make antibodies but this takes time

Passive: contains antibodies and injected near the bite mark

Plasma therapy is also an example of passive immunity

Antibodies are still effective for viruse bc it can neutralize their receptors

Clostridium tetani lives naturally in the soil and anaerobic and will secrete toxin that will cause tetanus

Tetanus can thrive pag lagi binabalot nang masikip gamit gauze

IgM and IgG3 can activate complement

Soluble immune complex pwede tulungan ng complkement receptors

Phagocytes has FcG on their surface

Antibodies can be recognized by FcG receptors to help macrophage to be more specific on their targets

Di lagi for activation ang Fc receptors

Bc of FcG3 which is unique to NK cells, they can have a certain defree of specificity kasi didikit sa kanila ang antibodies which is responsible for specific response

Nabigyan ng specificifty si NK cell kasi may FcG3 receptor to use antibodies

Immunosurveillance:

Lateral immunoflow assay

It might take time to produce antibodies kaya IgG- and IgM- still requires precaution

Maraming blood type classifications pero mas ginagamit lang ang ABO

If we want to test if blood will react, we do cross-matching

The ability of igg to cross placenta now becomes detrimental

Maganda sana kasi it was supposed to give immunity to the fetus

Antibody production

Aseptic technique

Wash hands
Proper attire
- Lab gown
- Gloves
- Foot wear
Sterile materials
Autoclave sterilization
- 20 mins, 120 C, 15 psi
Filter sterilization
- For filtering bacteria

Eukaryotic cells are more complex

May TLRs that can recognize PAMPs

Even if the pathogen is dead, their mere presence of their component can trigger a response in the much complex eukaryotic cells

Kaya makakaapekto kung may dead e.coli sa agar plate kahit after ma-autoclave

Clean bench
- Horizontal flow: laminar flowhood
  - Only for protecting workspace
- Vertical flow: biosafety cabinet
  - For protecting worker
70% alcohol
UV light
- UV will only work with direct contact
- It cannot penetrate glasses

How can you tell if contaminated ang culture

Color change ng media
- May pH indicator ang media
- Respiration of live organisms = co2 product = carbonic acid = acidic (yellow) color change ang expected if contaminated
- Okay lang pag it took days before nag yellow ang culture kasi it takes days for eukaryotic to multiply

Multiple b cells can recognize a single antigen if it has multiple epitopes

Polyclonal- diff populations of b cells but their targeting the same antigen just different epitopes

It’s the natural way how our immune system produces antibodies

Choice of animal depends kung gaano karami kailangan na antibodies

Why do we need manufactured antibodies?

Fluorescent microscopy staining

Flow cytometry

We use monoclonal as default cuz we are sure bawat antibody target ay same epitope so same results

Hybridoma: joining plasma cell and a multiple myeloma (type of cancer of b cells)

Mas mabilis ang half life ng b cells so they join it with a cancer cell

May hayflick limit ang isang cell that would render is to stop dividing

So need ng cancer cell para tuloy tuloy ang production

They fuse in polyethylene glycol

The cells will open then rejoin

De novo pathway: cells are making nucleic acids out of nothing

Salvage pathway: the cells recycle

In multiple myeloma cells, they cannot acces the salvage pathway

The culture medium used to separate: HAT medium

Adjuvant: signals that tell ur immune system sige mag inflammation ka para masignal mi sa rest na may ongoing infection para mag manifest yung mga APCs ng B7

Can we use manufactured antibodies on ourselves?

Need muna i-modify

ICR mice is outbred: genetically varied

BalbC is inbred: immunodeficient so mas madalas ginagamit kung need patubuan ng cancer

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BIO 151 Notes.docx

BIO 151

CHAPTER 1

Skin

first line of defense
epithelium = layers of keratinized cells
lines organs and inner cavities
- Respiratory tract
- GI tract
- Urogenital tract

Mucosal surface

Secretes mucus
Beating cilia removes mucus and unwanted material

Sebaceous glands

In hair follicles
Secretes sebum to inhibit bacterial growth

Antimicrobial peptides

Produced by all epithelia
Perturbs pathogen membranes

Tears and saliva

Contains lysozyme that degrades bacterial cell walls

Acidic environments

Stomach, vagina, skin

Innate Immune System

If skin is breached, infection remains localized and are extinguished within a few days without illness
Genetically programmed
In place even before infection
Respond in the same way to repeated exposures
Specific to structures shared by groups of microbes = cannot distinguish microbes
Recognition
- soluble proteins and cell-surface receptors bind to pathogens or to altered cells and serum
- peptides, proteins, glycoproteins, proteoglycans, peptidoglycans, nucleic acids
Recruitment
- effector mechanisms provided by effector cells to kill and eliminate pathogen
- complement serum proteins to mark pathogens

Serum proteins of complement system recognizes pathogen
Activation of serum proteins = fragment binds to pathogen and acts as marker
Fragment recruits leukocytes with surface receptors to site
Leukocyte binds to fragment and engulfs it

Cytokines

Soluble secreted proteins
Trigger and regulate innate immune response
Induces vasodilation of the endothelium
- Leakage of blood plasma
- Expansion of fluid volume = edema or swelling = pressure on nerve endings causes pain
Alters adhesive properties of endothelium
- WBCs attach to it to enter the inflamed tissue = inflammatory cells
- Increases swelling and contribute to pain
Enables cells and molecules of the immune system to be brought rapidly and in large numbers into the site of infection

Adaptive Immune System

Adds/ enhances innate immune response
- Innate immune response slows spread of infection and recruits lymphocytes
Slow response (days to weeks)
Adapts to the nuances of the infecting pathogen
Specificity and diversity
- cell-surface receptors recognize specific antigens and different portions (epitopes) of polysaccharide and macromolecules
- huge lymphocyte repertoire due to variability in structures of antigen-binding sites
  - different clones differ in their receptors and therefore specificity for antigens
Clonal selection
- Selection of lymphocytes by antigens for activation to produce specific antibody
  - specific antigen receptors exist on lymphocytes before they are presented with an antigen due to random mutations during initial maturation and proliferation
Clonal expansion
- Lymphocyte specific for an antigen undergoes proliferation after exposure
- Increase in number of cells that express identical receptors for the antigen
- Keeps pace with rapidly dividing pathogens
Immunological memory
- To elicit a stronger and faster adaptive immune response
- Exposure to antigen generates long-lived memory cells specific for the antigen
- Also called acquired or protective immunity
- Primary immune response: first exposure to pathogen
- Secondary immune response: subsequent exposures with more rapid, larger, and qualitatively different from primary response

Adaptive Immunity is better understood than innate immunity

Infections are overcome before they cause any symptoms
Inherited deficiencies and impairment of function are rare
Clinicians work together with adaptive response for a cure = more favored than innate

Hematopoiesis

Leukocyte are derived from pluripotent hematopoietic stem cell
Gives rise to precursors of lymphoid, myeloid, and erythroid lineages
Sources: yolk sac > fetal liver > spleen > bone marrow
Occurs throughput life due to short-lived nature of blood cells

Erythroid Lineage

Gives rise to erythrocytes and megakaryocytes
Megakaryocyte
- Fusion of multiple precursor cells
- Permanent residents of the bone marrow
- has small, non-nucleated fragments = platelets
  - maintain integrity of vessels
  - blood clotting

Myeloid Lineage

granulocytes
- kill pathogens and enhance inflammation
- aka polymorphonuclear leukocytes
- neutrophil
  - capture, engulfment and killing via phagocytosis
  - effector cells of innate
  - shortly-lived = pus
- eosinophil
  - against parasites and worms
- basophil
  - regulates response to parasites
Monocytes
- Circulates in blood
- Recruited by macrophages into infected tissues = matures into macrophages
  - In the presence of infection and extensive tissue damage, macrophages may be overwhelmed and die due to the number of dead pathogens and dead neutrophils
Macrophage
- Derived from embryonic stem cell
- Becomes resident tissue macrophages
- First detects infections
- Becomes activated and recruit neutrophils and other leukocytes to the site of infection for the innate response by secreting cytokines
- General scavenger cells
Dendritic cells
- Determines whether and when the innate response needs support from adaptive response
- Carry intact and degraded pathogen to lymphoid organs = initiates adaptive response
Mast Cells
- Resident in all connective tissue
- Plays a role in inflammation
- Violent spasms of smooth muscle to eject parasites from respiratory and GI tracts

Lymphoid Lineage

Natural killer cells
- Defense against viral infections
- Recruited by macrophages
- Secretes cytokines that impede viral replication
Small lymphocytes circulate in inactive forms
B cells
- Cell surface receptors: immunoglobulins
- Effector cells: plasma cells = soluble antibodies
- Can recognize antigen in epitopes
T cells
- T-cell receptors
- Cannot recognize antigen alone
- Antigen must be bound to major histocompatibility complex molecule
  - “presents” antigen to the T cell receptor

Effector Cells of Adaptive

Plasma cells
- Production of antibodies
- Circulate in blood
- Enters tissue
- Binds to antigen
Cytotoxic T cells
- Expresses CD8 receptors on its surface
- Kill cells infected with intracellular pathogen
Helper cells
- Expresses CD4 receptors
- Secrete cytokines to help activate other cells
Regulatory T cells
- Inhibit immune response by closing CD4 and CD8 responses
- Prevents unnecessary tissue damage

Humoral Immunity

Immunity due to antibodies
For extracellular microbes
Facilitate engulfment and destruction of microbes and toxins by phagocytes
Neutralization: binds to pathogen and inhibit growth, replication, or interaction with human cell receptors
Phagocytes can bind to antibody molecules
Opsonization: Antibody can coat bacteria to enhance phagocytosis

Lymphoid Tissues

Major lymphoid organs: bone marrow, thymus, spleen, adenoids, tonsils, appendix, lymph nodes, and Peyer’s patches
Primary lymphoid tissues: where lymphocytes develop and mature
Secondary lymphoid tissues: where mature lymphocytes become stimulated
Forms a network of lymphatics and collect the leaked plasma from the blood and returns it to the circulation as lymph via the thoracic duct and empties into the left subclavian vein
No pump = sluggish flow = away from peripheral tissues = driven by continual movements
- Or else edema
B cells and T cells move through both blood and lymph
- If they become activated by a pathogen, they remain in the lymph node
- Otherwise they leave in the efferent lymph and return to the blood
- Continual state of flux = lymphocyte recirculation
- To monitor the secondary lymphoid tissues for infection

Secondary Lymphoid Tissues

To establish infection, a microorganism must colonize a tissue and overwhelm the local innate immune response
Dendritic cells that are either pathogen-infected or loaded with pathogen antigens are carried in the lymph through lymphatics to the nearest lymph node (draining lymph node)
- Prevents potentially dangerous materials from entering circulation

Lymphoid follicles
- Where lymphocytes are segregated into B cell areas and T cell areas
- Pathogens and antigen-laden DC in lymph enter via afferent lymphatic vessel
- Lymph is drained via the efferent lymphatic vessel
- Dendritic cells stay in the node
  - Allows pathogens and other extraneous materials to be extracted by the resident macrophages
- Activated B cells proliferate in the node
  - Forms germinal center
- T cells are activated by antigen-bearing DC
  - Some become Helper T cells and help B cells activate into plasma cells in the node
- Lymphocyte activation, proliferation, and differentiation lead to swelling of lymph node

ADAPTIVE IMMUNE SYSTEM

Antibody Responses
- B lymphocytes or B cells
- Plasma cells
  - When B cells encounter an antigen, they become plasma cells
- Antibodies/ immunoglobulins
Cell-mediated immune responses
- T lymphocytes or t cells found in all cells except RBCs
- Major histocompatibility complex and T-Cell receptors

Spleen

Filters blood to remove damaged and senescent RBCs
Also a secondary lymphoid tissue that defends against blood-borne pathogens
- Splenic macrophages and DCs
- Stimulation of B cells and T cells from the blood
Red pulp: blood cells are monitored and elderly or damaged ones are removed
White pulp: similar to lymph node but without lymphatics

Mucosa-associated Lymphoid Tissue (MALT)

Secondary LT of mucosal tissues
Pathogens arrive by direct delivery mediated by M cells of the mucosal epithelium

Gut-associated LT (GALT)

Tonsils, adenoids, appendix, Peyer’s patches of the GI tract

Bronchial-associated LT (BALT)

Lines the respiratory epithelium

CHAPTER 2

IMMEDIATE INNATE RESPONSE

First line of defenses
- Physical barriers
- Molecular mechanisms of innate immunity
Second line of defense
- Induced mechanisms of innate
- mobilization of cells upon detection of infection
Third line of defense
- Adaptive immune response

Barriers

Skin and mucosal epithelia
Commensal microorganism = microbiota
- Deters pathogen invasion
- Invading pathogen must compete with the resident commensals for nutrients and space
Extracellular pathogens
- Accessible to soluble secreted molecules of immune system
Intracellular pathogens
- to combat, human cells are killed to interfere with the pathogen’s life cycle
- expose any pathogen released from dead cells to soluble molecules

Complement System

plasma proteins made in the liver, present in blood
induced when tissue becomes infected
opsonizes bacteria and extracellular virus particles
has proteases that circulate in the blood, lymph, and tissues
- inactive form = zymogens
Protease cleaves and activates the next protease
C3 most important proteins of the complement
- Those lacking C3 are prone to severe infections
- Has thioester bond with glycoprotein

Activation of the complement system by infection = cleavage of C3 into C3a and C3b
- C3b attaches to the pathogen’s surface = complement fixation
- Tags the pathogen for phagocyte-mediated destruction
- Organize the formation of protein complexes that damage the pathogen’s membrane
- C3a = acts as a chemoattractant to recruit phagocytes and other effector cells from the blood
- The thioester bond is exposed to the hydrophilic environment upon cleavage
  - Most will hydrolyze but a minority will react with the hydroxyl or amino group on the pathogen’s surface

Pathways

All lead to C3 activation, deposition of C3b and recruitment of effector mechanisms

Alternative Pathway

Start of infection
Binding of C3 with water = iC3
iC3 binds to factor B and is susceptible to cleavage by factor D
fragment Ba is released while fragment Bb with protease activity remains bound to iC3 = iC3Bb complex
iC3Bb cleaves C3 into C3a and C3b
high concentration of C3 in blood = activation and cleavage of C3 in large quantity = some C3b will bind to pathogen’s surface
C3 convertase = proteases that cleave and activate C3
- iC3Bb is an example

C3b fragments bound to a pathogen also binds to factor B and cleaved by factor D
- Forms C3Bb complex
- The C3 convertase of the alternative pathway
- Works right at the pathogen surface
- Binds C3 and cleaves into C3a and C3b
- Because this convertase is present on the pathogen surface, it is more efficient in fixing C3b to the pathogen surface
- Rapid formation of additional C3Bb molecules

Regulatory Proteins

Plasma protein properdin (factor P) increases complement activation
- Binds to C3Bb on pathogen surface and prevent its degradation by proteases
- Factor H plasma protein counters factor P
  - Binds C3b and facilitates further cleavage to iC3b by factor I plasma serine protease
- Combined effects of factors H and I is to decrease the amount of C3 convertase on the pathogen surface
- Those who lack factor I = immunodeficiency
  - Formation of C3Bb remains unregulated until the C3 reservoir in blood, extracellular fluid, and lymph is exhausted
  - When faced with bacterial infections, individuals with factor I deficiency fix very little C3b to bacterial surfaces, causing inadequate clearance of bacteria by phagocytes
  - More susceptible to ear infections and abscesses caused by encapsulated polysaccharide bacteria
Decay-accelerating factor (DAF)
- Binds to C3b component of the alternative C3 convertase
- Causes its dissociation and inactivation
membrane cofactor protein (MCP)
- Binding of MCP to C3b makes C3b susceptible to cleavage and inactivation by factor I
Complement control protein (CCP) modules
- DAF, MCP, factor H
- Proteins made up of CCP modules = regulators of complement activation
The combined effect of the reactions that promote and regulate C3 activation is to ensure that C3b is deposited only on pathogen surface and not on human cells
- Effective way of distinguishing human cells from microbial cells

Macrophage

First effector cell to encounter invaded tissue
Prevalent in connective tissues, linings of GI and respiratory tracts, liver (Kupffer cells), and alveoli of lungs
Macrophage cell surface receptors enhances phagocytosis

Complement receptor 1 (CR1)
- Binds to C3b fragments on pathogen surface
- Facilitates engulfment of the pathogen into endosome or phagosome
  - Fuses with lysosome which delivers toxins and enzymes to degrade the pathogen
- Opsonization
- Protects the surface of cells on which it is expressed
  - Disrupts C3 convertase by making C3b susceptible to cleavage by factor I
- Made up of CCP modules
CR3 and CR4
- Examples of integrins which contributes to adhesive interactions
- Bind iC3b fragments on microbial surfaces
- Although the iC3b fragment has no C3 convertase activity, it facilitates phagocytosis and pathogen destruction as a ligand for CR3 and CR4
- Combination of CR1, CR3 and CR4 > single complement receptor

Terminal Components

C5, C6, C7, C8, C9 proteins
Cooperate to form the membrane-attack complex (MAC)
- Large pore assembled in the pathogen membrane to disrupt its integrity

C5
- Similar to C3 but lacks a thioester bond
- Activated by the alternative C5 convertase
- Has 2 fragments of C3b and one Bb fragment = C3b2Bb
- Cleaves C5 into C5a and C5b

- C5b
  - Initiates MAC formation
  - C6 and C7 binds to C5b = forms complex to to expose hydrophobic region of C7
  - C7 inserts into the lipid bilayer
- C8 joins complex
  - C8 binds to C5b to expose its hydrophobic site and inserted into the membrane
- These events lead to the polymerization of 18 C9 molecules and MAC formation
  - Final complex: one molecule each of C5b, C6, and C7, three molecules of C8, and 18 molecules of C9

Soluble Proteins

S protein, clusterin and factor J
Prevent association of the C5b, C6 and C7 complex with the cell membranes
Homologous restriction factor (HRF) and CD59 (protectin)
- Prevents C9 recruitment to the complex by binding to C5b678 complex
- Impaired synthesis of the glycosylphosphatidylinositol tail CD59 = human disease paroxysmal nocturnal hemoglobinuria (PNH) = complement-mediated lysis of RBCs which have no DAF, HRF, or CD59 = not protected from actions of the complement
- Treatment: monoclonal antibody specific for C5 and prevent its cleavage and activation by C5 convertase

Inflammatory Peptides

C3a and C5a fragments are ligands for receptors on phagocytes, endothelial cells, and mast cells
Increases inflammation at the site of complement activation
Can induce anaphylactic shock = C3a and C5a are anaphylatoxins
- C5a more potent and stable than C3a
- Induces contraction of smooth muscle and degranulation of mast cells and basophils
  - Release of histamine and other vasoactive substances that increase capillary permeability
  - Increases exit of plasma and cells from the blood
  - C5a induces neutrophils and monocytes to adhere to vessel walls and recruit phagocytes
  - C5a increases CR1 and CR3 on surfaces

Plasma Proteins

Vessels damaged by pathogens activate the coagulation system
- Enzymes in plasma that cooperates with platelets to form blood clots
- Pathogens are immobilized in the clots and cannot enter the blood and lymph
- During clotting, platelets degranulate and release active agents to recruit immune cells, antimicrobial defenses, and tissue repair
Kinin system
- Enzymatic cascade of plasma proteins induced by damaged tissue
- Leads to the production of bradykinin
- Reduces hypertension and promotes vasodilation and smooth muscle relaxation

Protease inhibitors
- Contained in human secretions and plasma
- For pathogens that have acquired proteases (i.e., plasmin) to hide from the immune system
- Ex: alpha2-macroglobulin
- Lures microbial protease with its “bait” region to be cleaved by the protease
- Activates the thioester in a2-macroglobulin = binds and envelops the protease
- Immediately bound by specific receptors on macrophages, hepatocytes, and fibroblasts

Defensins

Antimicrobial peptide that can neutralize broad range of structurally diverse toxins
Alpha-defensins and beta-defensins
- Amphipathic (has hydrophobic and hydrophilic regions to penetrate membranes) = pore formation
When binding to microbial toxin, it promotes unfolding of the toxin = susceptible to proteases = anti-chaperones

Pentraxins

Plasma proteins that bind microorganisms and deliver them to phagocytes
Same role as antibodies

CHAPTER 3

INDUCED INNATE RESPONSES

Inflammation

Induced phase involves soluble and cellular receptors that detect the presence of a pathogen and recruit leukocytes to make an inflammatory response
Inflammation will help induce a subsequent adaptive immune response
Macrophages activated by the presence of an infection will release inflammatory cytokines

Self vs Non-Self vs Altered Self

Receptors can recognize structural features that distinguish microbial macromolecules from human macromolecules
Non-self: microbes
Altered-self: infected cells and cancerous cells
Individual cells express different combinations of receptors = functional diversity = increases likelihood that at least some of the cells will be able to mount an effective response to any given pathogen

Lectins

Carbohydrate-specific receptors found in macrophages

Surface Receptors of Resident Macrophages

Receptor-mediated endocytosis = macrophages surface receptors capture pathogens and deliver them to endosomes where the pathogen is killed and partly degraded
Endosome fuses with lysosome where extreme acidity and concentration of degradative enzymes completely degrades the pathogen
Pattern-recognition receptors (PRRs): receptors that recognize a structural feature common to many different types of pathogen
- Structural feature on microbe is called pathogen-associated molecular pattern (PAMP)
- Damage to cells or tissue that does not involve an infection: Damage-associated molecular pattern (DAMP)
Scavenger receptors: most of the PRRs of macrophages
- Trigger phagocytosis, cell adhesion and intracellular signaling to identify microbes and molecules and cause their elimination
- SR classes A-L (11 classes)
- Bind to many different ligands including surface components of microbes
- In the absence of infection, SRs remove dead and dying cells and unwanted macromolecules and cells that have died by apoptosis (damaged or infected cells)

- Some SRs are lectins
  - Mannose receptor (SR-E3) and Dectin-1 (SR-E2)
    - MR recognizes mannose and other sugars present in the glycans of bacterial surfaces and internalized by receptor-mediated endocytosis
    - Dectin recognizes beta-glucans (glucose polymers) on fungal and bacterial surfaces
  - Belongs to C-type lectin domain (CTLD)
  - Calcium ion coordinates the interaction of the carbohydrate ligand with the protein receptor

SR-A Class

Macrophage receptor with collagenous structure (MARCO)
- Recognizes bacterial lipopolysaccharide (LPS) of the outer membrane of Gram-negative bacteria
- Also binds to Gram-positive bacteria
SR-A1
- Recognizes LPS and lipoteichoic acid cell wall component of Gram-positive bacteria
- Additional ligands: hepatitis C virus, beta-amyloid, and heat shock proteins

CR3 and CR4

Binds to iC3b
Part of integrins that contribute to adhesive interactions between cells
Recognizes LPS of E.coli, Salmonella, N. gonorrhoeae, and other Gram-negative bacteria
Recognizes lipophosphoglycan of protozoan parasite

Toll-like Receptors

Toll-like receptors (TLRs) = signaling receptors present on immune cells
- Responds to a range of microbial and viral products
- Main macrophage receptor for LPS = TLR4 which recognizes the lipid A component of LPS

TLR4
- Extracellular domain binds LPS
  - Pathogen-recognition domain: has repeated leucine residues called leucine rich repeat region (LRR)
  - Variation in the number of LRRs give each type of TLR a different ligand specificity
- Cytoplasmic domain signals macrophage to transcribe genes encoding proteins needed for innate immune response
  - Signaling domain: Toll/interleukin-1 receptor (TIR) domain
- Can bind two molecules of LPS

Gram-negative Infection

Macrophages use mannose receptor to internalize and degrade bacteria and release LPS from the bacterial surface
LPS is bound by LPS-binding protein and delivers it to CD14 at the macrophage surface
TLR4 and myeloid differentiation factor 2 (MD2) forms a complex with CD14 and LPS
- MD2 only associates with extracellular domains of TLR4 but not other TLRs
The TIR domain engages with the domain of MyD88 which acts as an adaptor protein
The second domain of MyD88 engages with protein kinase IRAK4 (Interleukin-1 receptor-associated kinase 4)
- Induces IRAK4 to self-phosphorylate and dissociate from the complex and phosphorylate TRAF6 (tumor necrosis factor receptor-associated factor 6)
- Leads to the activation of inhibitor of kappa-B kinase (IKK) kinase complex
- IKK phosphorylates inhibitor of kappa-B (IkB)
- releases nuclear factor kappa-B (NFkB) to enter nucleus
  - Initiates transcriptions of genes encoding cytokines and adhesion molecules
  - When not needed, NFkB is held in the cytoplasm by IkB
- IkB degrades

NFkB Essential Modulator Deficiency (NEMO Deficiency)

Loss of y subunit (NEMO) of IKK
Impairs the activation of NFkB = impairs activation of macrophages by TLR4 signaling
X-linked = males are more susceptible

TLR Sensing

Receptors on plasma membrane
- Recognizes carbs, lipid, and protein ligands on outer surface of pathogen
Receptors within endosomal vesicle membranes
- Recognizes features of nuceic acids of pathogens and distinguish from human DNA and RNA
TLR4 and TLR1:TLR2 sense bacterial infection
TLR3 in endosomes sense viral infection

TLR4 Polymorphism

Allotype: proteins encoded by different alleles of the same gene
Causes genetic polymorphism
- More common in TLRs that recognize surface epitopes than nucleic acids due to greater diversity of carbs, lipids and proteins
Septic shock
- Bacterial infection spreads from tissue to blood and becomes systemic
- Potent activation of the innate causes vasodilation and therefore leakage of fluid throughout the body
  - Produces septic shock in which blood supply is perturbed and vital organs fail
- Caused by Gram-negative

NOD Proteins

Intracellular
Recognizes bacterial degradation products in cytoplasm
Has three different regions
- Carboxy-terminal region = pathogen-recognition domain with binding site for degraded products
- Central region: nucleotide-binding oligomerization domain (NOD domain) which enables receptors to form oligomers
- Amino-terminal region = caspase-recruitment domain (CARD)
  - contains binding site for RIPK2 (receptor-interacting serine-threonine protein kinase 2) that initiates signaling from NODs and acts as an adaptor molecule
  - 1 in NOD1 and 2 in NOD2
  - Mediates RIPK2 and NODs interaction
    - Phosphorylation of RIPK2
    - Activation of kinase TAK1
    - Phosphorylation of IKK
    - IKK activates NFkB
    - Macrophage activation
    - normally recruits caspases protease to protein complexes

Interferon Viral Response

Cytoplasmic proteins that can detect viral nucleic acids and produce Type I Interferons
- Interferes with viral replication in the infected cell
- Instructs nearby uninfected cells to fight infection
- Alerts immune system that infection is present and causes virus-infected cells to be more vulnerable to NK cells
In response to infection, one cell secretes cytokine that binds to a cytokine receptor on another cell
- Induces intracellular signals that change the behavior if second cell
- Makes contact with target cell before releasing cytokines to avoid unnecessary immune response damage to tissues
- When the cytokine and its receptor are both from same type of cell = give autocrine signals
- Cytokine and receptor are products of different cells: paracrine signals

RIG-I-like receptors (RLRs)
- Detects cytoplasmic viral RNAs
- Comprised of RIG-I and MDA-5
  - Recognizes ds vRNA
- Two CARDs
  - Interacts with mitochondrial antiviral signaling protein (MAVS)
  - Forms a dimer on mitochondrial membrane
  - MAVS engages TRAF6 which initiates signaling pathway to activation of IRF3 and IRF7
    - IRF3 initiates IFN-beta gene granscription
    - IRF7 initiates IFN-alpha gene transcription
  - secretion of Type I interferons

Secreted IFN-Beta maintains activation of infected cells (autocrine action) and binds to receptors on nearby uninfected cells (paracrine action)
Infection induces phosphorylation of IRF3 to enter nucleus
NFkB and IRF3 activates transcription of INF-beta gene followed by secretion of IFN-B
Some IFN-B are bound by IFN-B receptors (autocrine)
IRF7 is mobilized and induces production and secretion of IFN-a
IFN-B receptors of uninfected cells bind to IFN-B secreted by infected cell (paracrine)
- Induces uninfected cell to secrete IFN-B and contribute to interferon response

Plasmacytoid Dendritic Cells

Produce Type I IFNs
Present in blood and lymph tissues
Expresses TLR7
- Detects ss vRNA
Expresses TLR9
- Detects presence of unmethylated CpG motifs in ds DNA

Inflammasomes

Enable activated macrophages to release IL-1beta
Interleukin-1B
- Master regulator of inflammation
- Inactive pro-form is stored in cytoplasm by macrophages
- Activated by cleavage and secreted in response to infection
- Macrophage assembles inflammasome to cleave pro-IL-1B into active IL-1B
  - Inflammasome has NOD-like receptors (NLR) that detects infection
  - NLR protein 3 basis for NLRP3 inflammasome
    - same domains as NODs
    - recruits molecules for oligomerization of inflammasome upon sensing infection
    - procaspase 1 self cleaveage forms caspase 1 which cleaves pro-IL-1B
- Allows macrophage to respond to infection with large burst of IL-1B
- Gasdermin D
  - To allow IL-1B to escape macrophage
  - Upon sensing infection, and when pro-IL-1B is cleaved, caspase 4 cleaves gasdermin D
  - Forms pores in PM where IL-1B leaves the dying macrophafe
  - Pyroptosis = cytokine release, pore formation, death of macrophage
IL-1a
- For homeostasis
- Always expressed by healthy cells

Autoinflammatory Diseases

Chronic and recurrent bouts of systemic inflammation mediated by cells of innate immunity and do not involve adaptive immunity

Inflammation of Infected Tissue

Attracts blood-borne immune effector cells
Macrophage and neutrophils have distinct complementary properties
Macrophage
- Long-lived
- Reside in tissues
- Work from the start of infection
- Raise the alarm
Neutrophils
- Short-lived
- Dedicated killers that circulate in blood
- Awaiting call from macrophage and defend infected tissue
- First population of effector cells recruited to infected tissue

Release of IL-1B by Inflammasome

Activates macrophages
Two main effects
- Improve speed and efficiency in capturing and digesting pathogens
- Secretion of cytokines to recruit other effector cells
  - Tumor necrosis factor-a (TNF-a) = vasodilation
  - IL-6 = heat generation via metabolism of fat and muscle cells
  - CXCL8 = chemokine to attract neutrophils
  - CCL2 = chemokine to attract monocytes
  - IL-12 = recruits and activates NK cells to secrete cytokines to enhance and maintain macrophage response

Recruitment of Neutrophils

Macrophages release CXCL8 which guides neutrophils into site of infection
Adhesion molecules on leukocyte surface and tissue-cell surface drives movement from blood and tissue
In absence of infection, neutrophils move rapidly through capillaries and do not interact with vascular endothelium
Within infected tissue, vasodilation and adhesion molecules of endothelial cells allow contact with endothelium
- Rolling adhesion

TNF-a induces endothelium to express intercellular adhesion molecules 1 and 2 (ICAM-1 and ICAM-2) and neutrophils to express leukocyte function associated antigen-1 (LFA-1)
ICAMs bind CR3 and LFA-1
CXCL8 binds to neutrophil’s chemokine receptors CXCR1 or CXCR2
- Tight binding to immobilize neutrophil on endothelium
CXCL8 binding with receptor
- Allows binding with G protein
- GDP is replaced by GTP
- Activates and releases G protein from receptor
- Neutrophil leaves blood by squeezing between cells = diapedesis
- Overall process of leaving the blood = extravasation
Neutrophil migration is directed by gradient of CXCL8 bound to cell surface and ECM
- Neutrophil moves towards CXCL8 concentration at actuvated macrophage
Pyogenic bacteria = pus-forming bacteria

Neutrophil Programmed Death

They devote all their resources to storage and delivery of antimicrobial weaponry
Has receptors for recognition: CR4 and CD14

Neutrophil Granules

Order of loading of granules
Azurophilic/ primary granules
Specific/ secondary granules
Gelatinase/ tertiary granules
Secretory vesicles
Order of degranulation/ releasing of granules is reversed
- Controlled by calcium concentration
- Low levels = sufficient for secretory vesicles
- Higher level in tissue = specific and gelatinase degranulation

Bacterium is engulfed to form endosome that fuses with azurophilic, specific and gelatinase granules
Components of NADPH oxidase by specific granules facilitate a respiratory burst
- Raises pH = becomes phagosome and kills pathogen
Fuses with lysosomes
- Lowers pH and activates hydrolases to degrade pathogen
Respiratory burst
- Increase in oxygen consumption
After neutrophils have deliverd all granules, they die
- Apoptosis
- NETosis which produces neutrophil extracellular traps (NETs) that capture and kill pathogens
  - Nucleus swell and burst

Chronic Granulomatous Disease (CGD)

Genetic syndrome caused by defective forms of genes encoding NADPH oxidase subunits
Without functional NADPH oxidase = no respiratory burst
Phagosome is too acidic to activate antimicrobial peptides
Numbers of commensal cannot be controlled so they persist as chronic infections
Infected macrophages are concentrated into nodules called granulomas which are unable to digest infected neutrophils

Fever

Effect of cytokines IL-1B, IL-6 and TNF-a
- Called pyrogens
Acts on temperature-control sites in hypothalamus and directly on muscle and fat cells = generate heat
Inhibits the growth and replication of bacterial and viral pathogens
Tissue cells become more resistant to damaging effects of TNF-a (capacity to kill tumor cells)
- Lethargy, anorexia = impedes use of energy to fight infection

Acute-Phase Response

Plasma proteins made by the liver are changed by IL-6
Proteins that increase or decrease are called acute-phase proteins
- C-reactive protein (CRP) and serum amyloid A increases hundredfold
- CRP concentration – diagnostic test for infection and tissue damage
CRP
- Targets phosphorylcholine of LPS
- Acts as opsonin and trigger classical pathway
Serum Amyloid A
- Interacts with TLRs and SRs
- Activate cells to produce inflammatory cytokines

Lectin Pathway

Induced by infection and requires time
Contributes to innate immune response
Initiated by acute-phase protein mannose-binding lectin (MBL) binding to pathogen surface
- Also an opsonin that facilitates uptake of pathogen by monocytes
- Circulates in plasma as complex
  - MBL-associated serine protease (MASP)1 and 2

- when MBL binds to mannose-containing carbs, MASP-2 becomes active and cuts itself
  - MASP-2 cuts C4 into C4a and C4b
  - C4b attaches to pathogen surface
  - C2 binds to MASP-2 and cleaves into C2a (larger) and C2b (smaller)
  - C2a Forms complex with C4b = C4bC2a
  - Forms Classical C3 convertase
  - Cleaves C3 to assemble alternative C3 convertase

Classical Pathway

Contributes to innate and adaptive
Requires binding of antibody or C-reactive protein to pathogen surface
CRP binds to phosphorylcholine on pathogen surface
CRP binds with complement component 1 q (C1q) = cleavage and activation of C1r and C1s
Activated C1s cleaves C4 and C2
Forms classical C3 convertase
C3b attaches to surface
C3b is recognized by CR1 = engulfment
C3b forms complex with C5-C9 = membrane destruction

Lymphoid Cells

NK cells
- Cytotoxic innate lymphoid cells
- Circulate in blood and enter infected tissues
- Type I immunity
Helper Innate Lymphoid cells (ILCs)
- Similar function to adaptive CD4 T cells
- Secretes cytokines to activate effector cells = macrophages and granulocytes
- Reside in tissues and make immediate response
NK cells and Helper ILCs have no antigen receptor
- Instead expresses PRRs
ILC1
- Intracellular (viral)
- Stops infection or limits spread until arrival of NK cells
- Type I immunity
ILC2
- Mucosal surfaces
- Extracellular parasites
- Type 2 immunity
ILC3
- Mucosal tissues
- Extracellular pathogens
- Participates in the containment of commensal microorganisms in the gut
- Tyupe 3 immunity
Lymphoid-tissue inducer (LTi)
- Facilitates development of secondary lymphoid tissue

Innate Lymphocyte Precursor

Common lymphocyte precursor cell (CLp)
- Gives rise to common precursor of all innate lymphocytes CILP
- Which then gives rise to precursor of NK cells NKp and precursor of all innate helper lymphocytes CHILp
- Which then gives rise to precursor of LTi cells LTIp and to common precursor of ILC1-3 ILCp

Circulating Lymphocytes

NK = innate
B and T cells = adaptive
NK cells expresses CD56 but not CD3 that marks T cells
No NK = persistent viral infections even with adaptive immune system
NK importance in containing viral infections until cytotoxic T cells become effective

Subpopulation of NK cells

CD56dim
- 90% of blood NK
- Less CD56 expression
- Differentiated cytotoxic cells
CD56bright
- Gives rise to CD56dim during NK development
- Weak cytotoxic effector cells and more committed to making cytokines
- Most are taken up in tissues
  - 80% of NK in lungs are CD56bright
- Uterine NK cells (uNK cells)
  - Abundant in uterine
  - Numbers fluctuate during menstrual cycle
  - Contributes to embryo implantation and placenta formation
  - Cannot kill cells or create inflammation
  - Cooperates with fetal trophoblast nourishment to ensure mother can supply fetus with oxygen

NK Cytotoxicity

Activated at viral infection sites
To prevent NK from killing healthy human cells, cytotoxicity is regulated
- NK can only discharge weapons after contact
  - Can only kill once cell at a time
- Decision to kill depends on sum of interactions between different NKRs and target cell ligands
- Inhibitory receptors prevent NK from killing healthy cells
NK Differentiation
- Infected cells secrete Type I interferons
- NK have IFN receptors that bind to IFN
  - Aided by adhesion molecules CR3 and LFA-1
- Forms an immunological synapse that holds the cells together and forms channel for exchanging info = NK-cell synapse
- IFN activates NK to proliferate and differentiate into cytotoxic effector cells
- Effector NK kills infected cells by inducing apoptosis

NK expresses TLR3 (ds vRNA), TLR7 and 8 (ss vRNA)
TLR7 and TLR8
- MyD88-dependent pathway that leads to IRF7 activation and production of IFN-a and IFN-B
TLR3
- No MyD88 adaptor
- Signals by IRF3 pathway and leads to production of IFN-B
- Uses TRIF adaptor

NK and Macrophages Activate Each Other

Activated macrophages secrete CXCL8 and IL-12
- Activates and recruits NK
- NK and macrophage forms conjugate pair bound by NK-cell synapse
- Macrophage in synapse secretes IL-12 (for cytokine-secreting effector) and IL-15
  - Activates NK
- NK proliferates and differentiates into effector NK secreting IFN-y
- IFN-y binds to macrophage and activates it
- Improved phagocytosis of virus particles and cells killed by NK
IFN-y
- Potent inflammatory cytokine produced by NK
- Aka Type II IFN

Dendritic Cells and NK cells

DCs may take up pathogens or be infected by pathogens
This causes changes on surface proteins monitored by NK receptors
If infected, DC forms synapse with NK
DC expresses IL-15 = cytotoxic NK proliferation
If cytotoxic NK is high and innate immunity overcomes infection = NK kills DCs and prevent adaptive response
If NK is low and innate cannot control infection = NK induces DCs to differentiate, migrate to lymphoid tissue and initiate adaptive response

NK Memory

All nucleated cells express MHC class I = Human Leukocyte Antigen (HLA)
Loss of HLA class I = marks disease
- Tumors and virus-infected cells escape T cell by reducing or losing HLA class I expression
NK has receptors that detect low HLA class I and kills them

ANTIBODY FUNCTION

Immunoglobulin
- General term for proteins produced by B cells that recognizes antigens (plasma cells)
- Attached or free-floating
  - Antibody = free-floating
- Very specific
- Can recognize proteins and carbohydrates
- Their production is stimulated by vaccines or exposure to the antigen in question
- Antibody repertoire
  - Mixing and matching of proteins to form immunoglobulins
  - 10^16 but usually just 10^9

Antibodies have different functions:

Neutralization
- Antibodies fully cover the antigen to recognize them
- Prevent adherence to cells
Opsonization
- Antibodies fully cover antigen
- Makes it easier for phagocytes to engulf them
Complement Action
- Attach to a certain antigen
- Activates a complement which enhances opsonization and lysis

Clonal Selection

Transformation and proliferation of plasma cells that produce most specific antibodies from B cells
Once a B cell recognizes an antigen, it will undergo further mutation of their antibodies
- Becomes more specific

ANTIBODY COMPONENTS

Held together by a flexible hinge region composed of disulfide bonds

Fab = fragment antigen binding
Fc = fragment crystallizable
- Once it is done, they tend to precipitate and crystallize

ANTIBODY VARIABE REGION

Epitope
- Antigens recognized by antibodies
- Antigenic determinant
- Can be linear or continuous depending on the structure
Variable region light chain and variable region heavy chain
- Tend to vary to suit different shapes and sizes of all possible types of antigens

Hinge region
- Can recognize different types of antigens no matter their positions

HYPERVARIABLE REGION

Hypervariable region protein loops
- Found at the tip of both the light and heavy chain
- Aka complementary determining regions (CDR)
- CDR1, CDR2, CDR3
- Each variable region has three CDRs
Framework regions
- Less variable
- Supports the CDR
Affinity
- Binding strength of a single antigen binding site to the antigen
Avidity
- Bing strength of multiple antigen binding sites to an antigen
Binding to antigen depends on four covalent bonds
- Van der Waals forces
- Hydrophobic bonds
- Electrostatic bonds
- Hydrogen bonds

ANTIBODY ISOTYPES

Isotypes
- Depends on structure of constant region
- Has diverse functions
- Named after the Greek letters they correspond to
- IgG = gamma
- IgM = mu
- IgD = delta
- IgA = alpha
- IgE = epsilon

GENETIC BASIS OF ANTIBODY DIVERSITY

Variable region gene segment combinations within heavy or light chain gene segments (combinatorial diversity)
Junctional diversity between the gene segments
Heavy and light chain V region combinations
Somatic hypermutation
- Occurs after antibody recognizes an antigen
- Undergo further mutation/changes to become more specific

HEAVY AND LIGHT CHAIN DIVERISTY

Recombination of antibody genes determine the specificity and diversity of the CDR and the antibody produced
Light Chain
- Lambda locus gene segment from chromosome 22
- Kapa locus gene segment from chromosome 2
Heavy chain
- Locus from chromosome 14
Gene segments:
- V = variable
- L = Leader
- D = Diversity
- J = Junction
- C = Constant

ANTIBODY DIVERSITY

Has a germline DNA origin
- From both sperm and egg
Somatic recombination
- Antibody variable region diversity comes from the combination of the V, D, and J gene segments
- Light chain : V & J (variable) and C (constant)
- Heavy chain: V, D & J (variable) and 3 C gene segments (constant)

RSS

Recombination signal sequences
- How a B cell know which g segments goes where
- Gene sequences that direct recombination
- Ensures gene segments are always in their proper orders
  - V and J or V, D and J
- Have certain signals where the genes are supposed to join
12/23 Rule
- Segments with a 12 bp spacer will combine with a segment with a 23 bp space

RECOMBINATION

Since the genes are in one linear chromosome, these gene segments are brought together closer first by your RAG complexes
Recombination-activating genes (RAGs)
RAG Complexes
- RAG complexes hold the RSS in place while the genes are recombined
- proteins coded by RAGs
- serve as a clip to place the V and J gene segments closer to each other before cleaving
V(D)J Recombinase
- Set of enzymes needed to recombine the gene segments

JUNCTIONAL DIVERSITY

Since these are always random gene segments, how can they combine next to each other when they don’t have complementary ends?

RAG complex cleaves the gene segments to yield DNA hairpins
RAG complex opens hairpins by nicking one strand of the DNA = palindromic P-nucleotides
Terminal deoxynucleotidyl transferase (TdT) = adds random n- nucleotides
Pairing of strands occur and non-complementary/unpaired parts are removed
Gaps are filled by DNA synthesis and ligation to form coding joint
This adds to the CDR3 diversity

WHICH GENE SEGMENTS CODE FOR WHICH CDR

V segment = CDR 1 and CDR2
V and J junction (and D) = CDR3

CONSTANT REGION DIVERSITY

Free-floating = has different sets of proteins (hydrophilic) at the terminus end compared to transmembrane antibodies
Transmembrane = protein ends help with hooking onto the surface of the B cell membrane

IgM STRUCTURE

IgA and IgB accompany transmembrane IgM since these immunoglobulins do not have their own internal receptors

B CELLS TO PLASMA CELLS

B Cells have their membrane-bound IgM
Once it encounters an antigen, they will undergo somatic hypermutation to further produce more specialized antibodies that would better recognize antigens
Eventually will transform into plasma cells which now solely produce free-floating antibodies specifically recognizing this specific antigen
Allelic exclusion
- One allele is only expressed for producing antibodies while the other is silent
- Different alleles tend to code for antibody production but only one is expressed at a time

SOMATIC HYPERMUTATION

After an antibody recognizes an antigen, the activation-induced cytidine deaminase (AID) will replace all the cytosine in the CDR3 with uracil
Uracil-DNA glycosylase (UNG) will then remove the uracil and DNA polymerase will replace them with random nucleotides

ISOTYPE SWITCHING

AID is also responsible for isotype switching
It has a tendency to fold similar to Recombination of gene segments
They put gene segments specific to an isotype closer to each other
C mu and C delta gene segments are closest to the V, D, and J segments by default
- This is why IgM and IgD are produced first
The rest of the C gene segments are next to each other
Switch regions or switch genes are signals for isotype switching
IgM antibodies exist as bulky pentamers
Antigen activated T cells also mediate isotype switching
Successive switching can occur (ex: IgM to IgG to IgA)

Ig SPECIALIZATION

IgM

Produced in the spleen, bone marrow, and lymph nodes
Low affinity
Multiple binding sites of bulky pentamer free-floating form

IgD

Mostly found in the respiratory tract
Triggers local immune response when bound to basophils

IgA

Monomeric form from spleen and bone marrow and circulates in blood
Dimeric form from lymph nodes and circulate in lymph and other mucosal secretions (ex: tears, saliva, breast milk) and the digestive lumen
May even target resident microbial microfauna to keep their populations in check

IgE

Specialized toward recruiting effector functions of mast cells in epithelium, activated eosinophils in mucosal surfaces, and basophils in blood
Targets parasites
Triggers asthma and allergic reactions if parasites are not very common in the area

IgG

Produced from the bone marrow, spleen, and lymph nodes
Circulated blood and lymph
Most commonly circulating antibody in bloodstream
More flexible hinges compared to other antibodies for hard to reach antigens
Also allows to bind to Fc receptors of phagocytes
Different kinds based on flexibility, function, and targets
Activates complements

IgG SPECIALIZATION

IgG1

Most abundant and versatile
Mostly binds to protein antigens

IgG2

Second most abundant and less flexible
Targets carbohydrates (bacterial capsules)

IgG3

Most flexible with longest hinges
Most susceptible to proteases
Best in activating complement

IgG4

Least abundant and does not activate complement unlike other 3
Produced as monovalent together with IgE in case of allergic ractions
Reduces allergic reaction by competing with IgE when binding
Therapeutic uses

Caused by defects in immunoglobulin class switching and somatic hypermutation

Ig cannot adapt and patients become susceptible to infections

Defects in cd40 and cd40 ligand, AID, and UNG

Very low levels of other ig types but normal levels of igm

Cd40 ligand defects in t cells or cd40 defects in b cells

Cd40 activates isotype switching

AID mutation which disables somatic hypermutation and isotype switching

UNG mutation which disables somatic hypermutation

Effects:

Enlarged spleen or lymph nodes due to accumulation f immature b cells

Severe microbial infections and re-infections such as pneumocystis and cryptosporidium

Respiratory tract infections most common

Gastro are second most common

Includes entamoeba histolyca and salmonella enterica and giardia

Hindi naaalala ng immune system yung infections kaya nagkakaroon ng recurrent infections

Antibodies have Chlorophora or flurophore

Direct: recognize specific epitope

Indirect: gagawa muna ng serum or sample tapos sila marerecognize ng antibody

Polyclonal: antigen injected in animals tapos multiple antibodies are used targeting diff epitopes

Monoclonal: very specific using hybridoma and target the same epitope

Antigen coat (glass slides
Add blocking solution = hindi mag non-specific interactions with other artifcats
Antibody incubation from human sample
Fluorescent antibody incubation and visualization

Names of antibodies:

Source of the antbody, target antibody, conjygate chromo or fluorophore

Goat anti-human IgG alkaline phosphatase

Blocking solution: 1% BSA

For eliza chromophores

Horseradish peroxidase: brown or dark orange; stopping solution: hydrogen peroxide

Alkaline phosphatase: yellow to orange; no need for stopping solution

Immunofluorescent antibody technique

Fluorescein isothiocyanate: green

Western Blot

Transfer proteins from SDS PAGE gel to nitrocellulose membrane

Stain with antibody conjugated with horseradish peroxidase to determine the exact peptide detected by the antibodies

Flow cytometry

Monoclonal antibodies

Hybridoma grow and produce antibodies indefinitely

Separate hybridomas that produce certain antibodies specific for a protein

These are used in therapy as long as same source para marecognize as part of the body

Ex: Rituximab:

Binds to CD20 which is present in both malignant and normal B cells

Fc is the attachment for NK cells which cell-killing machinery

Plasma cells are still present to produce antibodies even if all B cells are being targeted

Non-hodgkin B cell lymphoma

LECTURE 5

CELL MEDIATED IMMUNE RESPONSE

Divided into two:

MHC Class I = Cytotoxic or CD8 T Cells
- Deals with intracellular pathogens
- Requires CD8 receptors
- Found in all cells except RBCs
- Pair of protein segments
MHC Class II = Helper or CD4 T Cells
- Deals with extracellular pathogens
- Requires CD4 receptors
- Found in macrophages, dendritic cells and B cells
- a chain of 4 protein segments

CMIR

Uses both T Cell receptors and Major Histocompatibility Complex (MHC)
- unlike B Cells which produce antibodies that recognizes pathogens

T Cell Receptors

attached to the cell membrane of T cells
function like antibodies of B cells or immunoglobulins
have alpha and beta chains = the light and heavy chains of antibodies
they also have a variable and constant region
Differences
- Can only recognize short peptides
  - Unlike antibodies which can recognize larger proteins as well as carbohydrates
short peptides are presented by an MHC
no free-floating form
no differentiation or somatic hypermutation after recognizing an epitope

T Cell Receptor Diversity

Also dependent on the germline DNA
Alpha chain: chromosome 14
- V and J
Beta chain: chromosome 7
- V, D, and J
Assembly of gene segments involve RAGs
Relies on DNA recombination
Only 1 constant alpha region gene and 2 constant beta region gene
- Both are functionally identical

Transposons

Theorized origin of diversity
Short gene segments that code for transposase
- Enzyme that cuts transposon and copy and paste it to the other parts of the DNA
- Recognizes the repetitive DNA sequences in the transposons
Transposase and RAG recombinase are similar
- Led to the hypothesis that RAGs originated from transposons-transposase interaction
- A transposon was inserted into a gene encoding for a receptor of innate immunity
- The transposon separated the gene into two segments, each flanked by a piece of repetitive transposon DNA
- Chromosomal rearrangements placed the transposase gene onto a different chromosome from the primordial rearranging gene
- The repetitive DNAs became the RSSs of a primordial rearranging gene and the transposase genes became the ancestral RAG-1 and RAG-2 genes (third panel).
- the family of rearranging genes expanded and eventually spread to five human chromosomes
  - TCR: chromosomes 14 and 7
  - Ig: chromosomes 14, 2, and 22

T Cell Receptor Complex

TCRs do not penetrate through the transmembrane of the T cells
Rely on support proteins and proteins that would serve as intracellular signal transmitter
- CD3 delta
- CD3 gamma
- CD3 epsilon
Zeta chain functions in transducing signals

Two Classes of TCR

Most studied are alpha and beta chains since these are similar in jawed vertebrates
Meanwhile, gamma and delta chains are not similar in mice and humans

Overview of TCR and MHC Relationship

Any pathogen that is engulf by the cell for the MHC II will have their proteins broken down
presented by the MHC on the surface of that cell
will be recognized by the respective T cell and T cell receptor

MHC I

if a cell is infected by an intracellular pathogen, its proteins will be presented by the MHC I on the cell surface
this will be identified by the CD8 T cells and CD8 TCRs
signals apoptosis of the infected cell to prevent further infection of other cells

MHC II

break down the segments of extracellular pathogen
will be presented on the cell surface and be recognized by CD4 T cells and CD4 TCR
Promotes production of cytokines or cell signals to
- activate macrophages to signal that there is an extracellular pathogen present
- signal B cells to transform into plasma cells and produce antibodies

Structure of MHC

MHC I
- 3 alpha domains (alpha 1, alpha 2, alpha 3)
- Beta 2 macroglobulin
  - Not encoded in MHC gene
- Has grooves and pockets in its structure
  - Allows only small peptides (8-10 AA) to be presented
MHC II
- Alpha 1, alpha 2
- Beta 1, beta 2
- No grooves or pockets
  - Wider space for larger peptides (13-25 AA)
MHCs exhibit promiscuous binding specificity
- Potential to bind to different peptides
- Not necessarily for a certain peptide

Self VS Non-Self Proteins

Cells are programmed to distinguish their “self” proteins produced by the cell
If they recognize them as “non-self”, they will be immediately presented by the MHCs
MHC I
- foreign proteins are loaded in the ER
- If a pathogen enters the cell, the cell will trigger production of proteins for that pathogen
- These proteins will be recognized as “non self” by MHC I
- Triggers its presentation to CD8 T cells
- Proteosome breakdowns misfolded proteins and recycle the peptides
- Once they recognize non self proteins, interferon-gamma triggers their transformation into immunoproteosome and consequently, the breakdown of these cells
- In a normal setting, the broken down peptides pass through transporter associated proteins into the ER where they will be reused

- In the case of foreign protein, the immunoproteosome will trigger the assembly of MHC I
- The chaperone protein calnexin attaches the beta-2 microglobulin to the 3 alpha domain proteins
  - Forms the peptide-loading complex
- Tapasin: brings the MHC I closer to TAP = easier for foreign peptides to be loaded into MHC I
- ERp57 and Calreticulin: help in peptide bonding
  - Foreign peptide is enclosed in a vacuole and transported to the surface of the cell
- Sometimes, protein segments are too long = ERAP (ER Aminopeptidase) protein
  - Shortens the peptides to fit into MHC I
  - Sometimes the peptide may not fit too well that it just falls off
  - MHC I will then assist them back to the ER and try / load again
MHC II

- foreign proteins are loaded in the endosomes
- extracellular pathogen will be engulfed by macrophages / dendritic cells and be enclosed in endosomes
- endosomes produce enzymes that will breakdown or inactivate proteins
- endosomes will then bind to specialized vacuoles containing MHC II
- the class II-associated invariant chain peptide (CLIP) will cover the groove or pocket on the MHC II while it has not encountered a foreign peptide yet
  - for loading
- Human leukocyte antigen-DM (HLA-DM) removes the CLIP for proper loading of foreign peptide
- Human leukocyte antigen-DO (HLA-DO) retains the CLIP while there is no foreign peptide yet

Cross-Presentation

Dendritic cells, B cells, and macrophages have MHC I and MHC II
Sometimes, the peptides may enter through the MHC I pathway but end up being presented by MHC II or vice versa

MHC Diversity

Human leukocyte antigen complex
- HLA I types: MHC I
  - A, B, C, E, F, G
- HLA II types: MHC II
  - DM, DO, DP, DQ, DR
HLA diversity comes from
- MHC II alpha chains gene families
- MHC II beta chains gene families
- MHC I heavy chains gene families
- Genetic polymorphism

Genetics Review

Gene = gene segments that codes for a certain protein
- In this case, HLA
Alleles = variations (single or multiple nucleotide changes) that would alter the protein structure
- Each allele would code for a different allotype (of HLA)
Can either be
- Monomorphic: there’s only 1 type
- Oligomorphic: dozen or less
- Polymorphic: more than a dozen
From germ line
- Heterozygous: different allele from parents
- Homozygous: same allele from parents
- Haplotype: every set of different alleles represent a different kind of haplotype

MHC I and HLA-I Diversity

HLA-A, -B, -C
- Highly polymorphic
- Present antigen to CD8 T Cells and ligands of natural killer cells
HLA-E, -G
- Oligomorphic
- Present antigen to ligands of natural killer cells
HLA-F
- Monomorphic
- Chaperone to MHC class I molecules that lose their antigen while travelling to cell membrane

MHC II and HLA-II Diversity

HLA-DP, -DQ, -DR
- Highly polymorphic
- Present antigen to CD4 T Cells
HLA-DM, -DO
- Oligomorphic
- Aids in loading antigen onto HLA-DP, -DQ, and -DR

Chromosome 6

Class I Region for HLA-I
- Mostly MHC-related or antigen presentation-related genes
- Proteins of other functions
Class II Region for HLA-II
- mostly MHC-related or antigen presentation-related genes
- TAPs
- Tapasins
- Related to immunoproteosomes
Class III Region
- Proteins not related to MHC

HLA Gene Diversity

MHC I
- Allotype diversity in both alpha-1 and alpha-2 domains
- Beta-2 immunoglobulin gene comes from another chromosome (chromosome 15)
MHC II
- Allotype diversity in either alpha-1 or beta-1 domains
- The other domain is always non-functional
- Depends on MHC II isotype

MHC Activation

Cell signals that activate MHC antigen presentation
- Interferon-alpha
- Interferon-beta
- Interferon-gamma
  - Functions in both MHC I and II responses
MHC I
- IFN-gamma activates proteosomes into immunoproteosomes
- For better breakdown of foreign peptides of intracellular pathogens
MHC II
- IFN-gamma activates MHC class II transactivator (CIITA)
- Activates MHC II

MHC I and MHC II

HLA-I most likely evolved first
Most of HLA II genes code for proteins that are strictly for antigen presentation to T Cells while HLA-I genes contain proteins with other functions
HLA I-related genes are found in other chromosomes
HLA-II is more compact compared to HLA-I
Some organisms such as Atlantic cod can survive with only HLA-I

MHC Restriction

proper order of your MHC, TCR, and antigen for recognition
different sides of the antigen bind to MHC and TCR
peptide binding sites or set of anchor residues
anchor residues or complementary pockets in the MHC binding sites
TCR also recognize the residues on the surface of the alpha helices
The part of the antigen recognized by HLA is different from that recognized by the TCR
- If same peptide but presented by a different HLA = no recognition
- If HLA carries a different antigen but same TCR = no recognition

Natural Selection

MHC and TCR diversity are controlled by natural selection
Balancing selection always ensure that highly polymorphic heterozygotes are always present
- Intermingling of populations = better immunity
- Ensure HLA are sufficiently heterozygous
Ensures survival against new disease
- Meiotic recombination occurs at 2% frequency which increases with succeeding generation
- New alleles from point mutations and recombinations
- Interallelic conversion or segment exchange occurs when recombination combines segments with point mutations to homologous segments

GVHD occurs during hematopoietic stem cell transplants such as bone marrow

HSC ransplants are performed fo patients with damaged or malfunctioning immune system or bone arrow

Autologous = self MHC isoforms

Allogenic = non-self MHC

The donor tissue are recognized as allogeneic or different by the new T cells

Alloreactions

Alloreactive t cells will react against cells from another indiv

About 1-10% circulatnb T cells in an indiv are alloreactive

Alloantibodies of one indiv will atact the allotypic proteins of another

These mismatches can occur between mother and child

HLA types

- - Combinations of HLA alleles in a person
  - Should matcj in order for transplants to proceed
  - Hla types testing by molecula methods
  - Most likely matched at HLA-A B C and DRB1

Symptoms

Skin as the most likely first organ to be affected with presence of rashes

gI disease such as diarrhea

liver disease

chronic GVHD occurs after 100 days

acute GVHD within 100 days

organ transplant rejection

the recipient immune system attack the donor organ

all cels in immune cells possibly involved

can occur months after the transplant

Topic 6

B Cell Development

Pro-B cell

main event in the pro-B-cell stage is the rearrangement of the heavy- chain genes
Early: D and J segments
Late: DJ and V segments
- The rearranged gene is transcribed through to the μ C-region gene, the nearest C gene to the rearranged V region
- The RNA transcript is spliced to produce mRNA for the μ heavy chain, the first type of immunoglobulin chain made by a developing B cell

Pre-B cell

B cell expresses mu chain
Large pre-B cell: less mature
- Heavy-chain rearrangement is done
- Makes mu heavy chain
Small pre-B cell: mature
- Rearrangement of light chain gene
- First is kappa
- If successful, no lambda
  - Light chain is synthesized and assembled sa ER
  - Form IgM
- If fail, lambda
  - Same as above
- If fail both = apoptosis

Immature B cell

Heavy and light done rearranging

Stromal

Environmental cells sa marrow for maturation
Interacts as adhesion molecules to ligands
Has growth factors that attach to b cells
- SCF = Kit receptor on maturing B cell
- IL-7 acts on late pro and pre
Moves from subendosteum to marrow cavity
Later stages are less dependent on stromal cells = leaves BM

Pro-B Heavy Rearrangement

Inefficient and imprecise = due to addition random N and P nucleotides in V, D, J segments
- Limited material tapos random
- Parang sandwich na sakto yung parts
- One in three chances of maintaining correct frame
Nonproductive rearrangements = not useful
Productive = useful
B cell has two copies of heavy chain locus (one from each parent)
- Can still produce heavy chain if one chromosome locus is productive
Express RAG1 and RAG2 = rearrangement starts
- Activated by E2A and EBF which expresses Pax5
Apoptosis is default unless there are survival signals

Pre-B Cell Receptors

Pro-B needs to make mu chain to survive and mu chain must be able to combine with Ig light chain
E2A and EBF roduce surrogate light chain
To test kung functional heavy chain, surrogate light chain ay parang substitute since light chain wont be formed unless functional ang heavy chain
- Synthesizes VpreB (mock variable region) and lambda5 (mock constant region)
- In the ER of pro-B, mu chain binds with surrogate and IgB = pre-B cell receptor = sends signals okay/functional ang heavy chain = interacts with BM ligands = stop RAG = pro-B cell div = large pre-B
- Pre-B cell interacts with BM ligands heparin sulphate and galectin-1 to stop RAG
Unlike the B-cell receptor (IgM), the pre-B-cell receptor does not have an antigen-binding site, nor is it well represented at the cell surface

Allelic Exclusion

Pre-B receptor
- Eliminates nonfunctional mu chains
- Prevents making more than one functional mu chain
In a pro-B
- rearrangement of the first immunoglobu- lin locus succeeds
- synthesis of μ chain
- assembly of the pre-B-cell receptor
- stop RAG transcription
Alleles from each parent, As long as one is working, ignore the other

SMALL Pre-B Light Chain Rearrangement

Large pre-B undergoes several rounds of division = CLONING
- Yields resting pre-B cells that express identical μ chains
- no longer make the surrogate light chain and the pre-B-cell receptor
RAG is reactivated = light chain rearrangement
One locus at a time: kappa locus first before lambda
One recombination event V and J: several attempts can be made in kappa before lambda rearrangement
The organization of the light-chain loci allows initial nonproductive rearrangements at one locus to be followed by further rearrangements of that same locus that can lead to the production of a functional light chain
If IgM is self-reactive
- Receptor editing = mature B cells (for light chain lang)
- Repeated fail = apoptosis
- Bind to monovalent self-antigens = anergy
  - Arrest of B cells
  - Prduce IgM and IgD but IGD allowed to develop
  - Lifespan is 1-5 days
- Bawal na mag-undergo ng receptor editing pag nasa bloodstream na
Peripheral tolerance = apoptosis or anergic lang
Central tolerance = receptor editing, apoptosis or anergic

Functional light chain + mu chain in ER = IgM
IgM + IgA + IgB = B-cell receptor in surface = stop light chain rearrangement = immature B cell

Two Check Points in Bone Marrow

First: late pro-B if may pre-B receptor or wala
Second: small pre-B kung may B-cell receptor or wala

Protein Expression

RAG1 and RAG2: on during heavy/light rearrangement, off during checkpoints
TdT: on during heavy rearrangement, off during light
IgA and IgB: forms receptors so on from pro-B stage until end
Pax5: on early pro-B

B-Cell Translocation

Genes that cause cancer when their function or expression is perturbed are collectively called proto-oncogenes

B Cell CD5 Expression

B1/CD5 B Cells: little to no IgD
Produced pre-natally
Lack N nucleotides cuz no TdT during prenatal
Polyspecificity = binds to many diff antigens

Summary

This repertoire of immature B-cell receptors includes many with affinity for self antigens that are components of healthy human tissue.

Activation of mature B cells carrying such receptors by these antigens would produce self-reactive antibodies

Bone marrow = blood stream = lymph organs = plasma cell to BM or glial cells remain in lymph organs

Stromal cells = stem cell development into B cells

Pro BCell

Early: DJ

Late: VDJ

CCL21 CCL19 = from lymph cortec xcells

CXCL13 = from follicular DC

Lymphotoxin preserve integridity of FDC

B cell activating factor BAFF to promote B cell survival

Mature Naïve B Cells

Have IgM not self reactive but yet to biun to antigen

B cells pass thru T cell zone into lymphoid follicles

Anergic B cells pass thru lymphoid follicles but stay outside T cell zone and later die by apoptosis

Plasma Cells

Activated by antigen and CD4 T cells

Terminal differentiation = stop proliferation and differntioation

Return to bone marrow to produce more antibodies = meaning effective na

Memory B cells

Resting quiescent b cells

Reserve in case similar infection occurs

Lasts for a lifetime and more quickly activated

CHAPTER 7

T-CELL DEVELOPMENT

Origin: bone marrow
Maturation: thymus
- Before they can undergo gene rearrangements
- “Thymus-dependent lymphocytes”
Positive Selection
- T cells that can recognize self MHC class I and II isoforms survive
Negative Selection
- T cells that bind too strongly to self MHC proteins (autoreactive) die by apoptosis
a:B Lineage is more dominant than y:d Lineage
a:B has two sublineages distinguished by CD4 and CD8 receptors
- can recognize MHC class II and I respectively

DEVELOPMENT IN THE THYMUS

Thymocytes: immature T cells
- Embedded in epithelial network thymic stroma
- Cortex: outer, close-packed, ectodermal
- Medulla: inner, less dense, endodermal
Thymus is a primary lymphoid organ
- Only concerned with production of useful lymphocytes
- Not concerned with application to problems with infection
- Not involved in lymphocyte recirculation
- Does not receive lymph from tissues
Involution of thymus
- Human thymus is fully developed at birth but degenerates one year after birth
- gradually replaced by fatty tissue
- does not impair T-cell immunity
- same case with thymectomy (removal of thymus)
- T cell repertoire are either long-lived, self-renewing, or both
Thymic anlage: rudimentary thymus of cortex + medulla cells
- Colonized by progenitor cells
- Gives rise to thymocytes and DCs
  - DCs and macrophages populate the medulla
T cell progenitors enter the thymus at the junction between cortex and medulla: cortico-medullary junction
- Thymocytes move out through the cortex > subcapsular region > outer cortex > inner cortex > medulla
- Hassall’s corpuscles: sites of cell destruction
DiGeorge Syndrome
- Thymus fails to develop = T cells are absent
- Suffers from opportunistic infections
- No adaptive immune system

COMMITMENT TO T-CELL LINEAGE

Progenitor cells are not committed to the T-cell lineage when they enter the thymus
- Expresses stem cell markers CD34
CD3: generic marker of T cells
- CD19: generic marker of B cells
- CD56: NK cells
Interacts with thymic stromal cells = signals to divide and differentiate
lose stem cell markers = become thymocytes committed to T-cell lineage
- expresses CD2 adhesion molecule and CD5
double-negative thymocytes (DN thymocytes)
- Thymocytes still lack T-cell receptor complex but begins to rearrange T-cell receptor genes
- Express neither CD4 or CD8
Critical cytokines
- IL-7
  - Secreted by stromal cells and binds to receptors on CD34-expressing progenitor cells
  - Defective IL-7 receptors = T cells absent
- Notch1
  - Cell-surface receptor on thymocytes
  - That’s why they need to go to the thymus because andoon ang Notch ligand transcription factor that it needs to interact with
  - Binds to transmembrane ligands on thymic epithelial cells
  - Drives cells along the pathway of T-cell differentiation and away from B-cell differentiation
  - Parallels that of Pax5 in B cell development

Notch1 Protein
- Has intracellular and extracellular domains
- Extracellular domain binds to ligand on thymic epithelium = cleavage of the two domains
- Intracellular domain translocate to nucleus
- Initiates gene transcription

COMMON THYMOCYTE PROGENITOR

Both lineages derive from DN thymocyte precursors
- Where T-cell receptor gene rearrangement is initiated
Rearrangement of y, d, and B loci occur at the same time
- If thymocyte makes a functional y:d receptor before a functional B = committed y:d T cell
- If thymocyte makes a functional B chain first = incorporated into pre-T cell receptor
  - Still not committed to a:B lineage
double-positive thymocytes (DP thymocytes)
- gene arrangement stops when functional B chain is incorporated into pre-T cell receptor
- cell proliferates and expresses CD4 and CD8 co-receptors
- allows a-chain rearrangement
- continues y and d-chain rearrangement
- If functional a:B receptor first: committed a:B lineage
- If functional y:d receptor first: committed y:d lineage

DN THYMOCYTE GENE REARRANGEMENT

B and d chain : V+(D+J) segments
a and y chain: V+J segments
Functional y:d receptor gene arrangement is made first
- y:d heterodimer is formed
- assembles CD3 signaling complex and moves to surface
- signals to stop B chain rearrangement
- y:d T cell receptors are not subjected to positive and negative selection
Functional B chain gene arrangement is made first
- More frequent outcome
- Translocate to ER
- Binds to surrogate a chain pTa to test capacity
Pre-T cell Receptor
- If functional B chain successfully binds to pTa
  - Forms heterodimer with CD3 complex and ζ chain
  - pTA interacts with C and V regions of the B chain
  - generates signals through the CD3 complex and Lck cytoplasmic protein
- Assembly of pre-T cell receptor signals to stop rearrangement of B, y, and d chain genes
  - First checkpoint to ensure B chain can bind to a chain
  - Cells become pre-T cells

B CHAIN REARRANGEMENT

a:B lineage is favored because it only requires one productive gene rearrangement while y:d requires at least two
this increases the potential to rescue nonproductive B chain rearrangements
- two C genes and their associated D and J segments
- if rearrangement is nonproductive: thymocyte can attempt rearrangement at the other chromosome or same locus
Four attempts to rearrange B chain gene
- 80% of T cells make productive B chains

A CHAIN REARRANGEMENT

Successful rearrangement of B chain signals via pre-T cell receptors to stop gene rearrangement
- Express RAG1 and RAG2 genes
- Ensures that only one productive B chain is expressed = allelic exclusion
Pre-T cell is induced to proliferate and create clones expressing the same B chain
- Accompanied by expression of CD4 first and followed by CD8 = DP thymocytes
- Constitute majority of thymocytes and are found in the inner cortex
- Interacts with epithelial cells
Once Pre-T cells stop proliferating, they become small DP thymocytes
- Reactivation of recombination machinery and targeted to the a, y, and d loci
T-cell receptor a chain genes
- Similar to immunoglobulin kappa and lambda light chain genes
- No D segments
- Rearranged only after B chain has been expressed
- Repeated rearranging attempts possible
  - Multiple V segments and J segments
  - Continues until productive rearrangement occurs or until the supply of V and J segments is exhausted
Rearrangement of a chain
- d locus within the a locus will be deleted irrespective of whether the rearrangement is productive or not
  - reduces the probability that committed a:B lineage expresses y:d receptors and a:B receptors
when DP thymocytes make a productive a chain rearrangement
- gene transcription starts to form a chain
- translocate to ER
- tested to bind to B chain and form T-cell receptor
  - second checkpoint
  - if successful: proceed to positive selection

GENE EXPRESSION CHANGES DURING DEVELOPMENT

Notch transcription factor

RAG1, RAG2: may stop bc of allelic exclusion (checkpoint testing)

TdT: junctional diversity

pTa: surrogate alpha chain

RAG1, RAG2: expressed at the two stages where B and a gene rearrangements are made respectively
TdT, pTa: also expressed throughout this phase of development
pTa: expressed throughout the period when rearrangements occur
CD4, CD8: relay signals from pre-T cell receptors
Tyrosine kinase Lck: signaling from pre-T cell receptor and co-receptor
CD2: adhesion molecule on T cells that interact with cell-surface CD58 on other cells
Ikaros, GATA-3: transcription factors in early progenitors
Th-POK: expressed late in development and is for development of CD4 T cells from DP thymocytes

POSITIVE SELECTION

First phase of T cell dev: produce T cells irrespective of their antigenic specificity
Second phase: examination of receptors produced and selection of those that can work effectively with self MHC molecules
- Only involves a:B T cells
- y:d cells do not depend on MHC molecules for detecting infection
primary T-cell receptor repertoire has a bias towards MHC molecules but T cell receptors are not tailored specifically
- only 2% of total can interact with MHC I or II
Positive Selection
- Small population that can interact with MHC molecules are signaled to mature further
- Takes place in cortex of thymus where thymic cortical epithelium expresses both MHC I and II molecules
  - Forms a web of cell processes that envelop and make contact with DP thymocytes
  - Potential interactions are tested
- If peptide:MHC is bound within 3-4 days after the thymocyte first expresses a functional receptor = further maturation

CONTINUATION OF GENE REARRANGEMENT

If first a chain rearrangement of pre-T cell is productive = positive selection occurs within a few hours
- Turns off recombination machinery
- Degrades RAG proteins
- Stops transcription of RAG genes
- Proliferates
If not = rearrangement continues
- Can continue throughout the 3-4 days of positive selection
After successful B chain = developing T cell turns off recombination machinery = allelic exclusion
- a chain is not subject to allelic exclusion
- rearrangements can occur at both copies of a chain locus
- DP thymocytes can express 2 a chains = 2 T cell receptors
Receptor editing
- Trying out different a chains in order to become reactive with self MHC molecules
- Opposite to B cells = receptor editing is to get rid of reactivity

CD4 CD8 DETERMINATION

Single-positive thymocytes (SP thymocytes)
- result of positive selection
- DP thymocytes mature into SP thymocytes
If peptide interacts with MHC I = CD8 is recruited
- Any cell that is nucleated should be able to present it via MHC I in case infected
If peptide interacts with MHC II = CD4 is recruited
- CD4 coordinating cell in innate and adaptive immune system
- HIV kills CD4 = acquired immue deficiency syndrome
- ACPs magnify signals of infection and is needed sa MHC II since CD4 is what coordinates everything
Bare lymphocyte syndromes
- Lack of expression of either MHC I or II molecules by lymphocytes and thymic epithelial cells
- Patients who lack MHC I have CD4 cells but no CD8 ; vice versa

NEGATIVE SELECTION

T cells whose antigen receptors bind too strongly to self-MHC molecules ( potential autoreactive) are deleted
- To avoid tissue damage and autoimmune diseases
Mediators
- Positive selection: epithelial cells
- Negative selection: dendritic cells, macrophages, thymocytes
  - T cells that bind too strongly to MHC I molecules on DCs or macrophages are signaled to die
To extend negative selection to proteins that are specific to one or a few cell types, such as the insulin made only by Beta cells of the Langerhans cell of the pancreas, transcription factor autoimmune regulator (AIRE) causes several hundred of these tissue-specific genes to be transcribed by a subpopulation of thymic epithelial cells
- Activation of AIRE allows thymus to express those specific proteins na di makikita sa iba, para ma-practice ng T-cell kung madedetect and kung magiging autoreactive ba o hindi
- Peptides derived from these proteins can be bound by MHC class I molecules to form complexes that participate in negative selection
- Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) or autoimmune polyglandular syndrome type 1
  - T cells specific for tissue-specific antigens are not eliminated by negative selection and thus attack variety of tissues

REGULATORY CD4 T CELLS

Functions of CD4 T cells
- Helper T cells: activates macrophages and B cells
- Regulatory T cells
  - Suppress response of self-reactive CD4 to their specific antigens
  - Despite AIRE activity, self-reactive CD4 T cells are still present in healthy people
  - Have T cell receptors specific for self-antigens
  - distinguished from other CD4 T cells by expression of CD25 on cell surface and transcriptional repressor protein FoxP3
On contacting self-antigens presented by MHC II
- Treg do not proliferate
- Suppress the proliferation of naïve T cells responding to self-antigens presented on the same antigen-presenting cell

FURTHER DIFFERENTIATION IN SECONDARY LYMPHOID TISSUES

Only a small fraction of a:B T cells survive positive and negative selection and leave the thymus as mature naïve T cells
Secondary lymphoid tissues provide specialized sites where naïve T cells are activated by their specific antigens
Encounter with antigen provokes the final phases of T cell development and differentiation: the mature T cells divide and differentiate into effector T cells
- Some stay in lymphoid tissues while others migrate to sites of infection

Discussion Notes

B cells (Humoral)

have role in viruses
Already circulating antibodies can neutralization before virus can enter cells

T cells (Cell-Mediated)

Antigen presentation by MHC before T cells can act
Necrosis: cell death due to external factors
MHC I: all nucleated cells (kaya hindi kasama RBCs)
MHC II: professional antigen-presenting cells
- Dendritic
- Macrophage: recruitment
- Mature B cells

Similarity: Somatic recombination (gene rearrangement)

Diversity of receptors
B cell receptors are identical
Allelic exclusion: ano na-arrange, ayun lang ie-express nila
- More effective as an immune cell kasi isang target lang ang need i-focus
The more diverse MHC = the more diverse receptors you need to have
B cells need input from cell-mediated immunity before activation
- Mas derived ang humoral since it cannot function without pre-existing xdsfunction of cell-mediated

Rubor: redness : increased blood flow to recruit more immune cells

Tumor: inflammation due to vasodilation which looses tight junctions

Dolor: pain because of triggering pain receptors

Calor: heat to impede the propagation of pathogens

B cells leave bone marrow as immature B cells

They mature upon meeting follicular dendritic cell (different from APC dendritic cell)

“follicular” = lymph node

Will give B cell maturation factor

Then start looking for infection to complete its maturation

Kaya called lymphocytes kasi paikot ikot sa lymph nodes

B cells only recognize antigen

For t cells, they need to recognize both MHC and antigen

After being able to attach to MHC molecules (positive selection), they must be able to let go para iwas autoreactivity (negative selection)

TLRs= receptors of the innate system

PAMPs= pathogen associated molecular patterns

NK cells are non specific but fast acting

Pero since nonspecific, if a pathogen dominates, di na kaya labanan kaya saka need ng adaptive

Cytokine storm = nagpapanic kasi di macontrol ang infection/ mapatay ang pathogen so release lang ng release ng cytokines = over inflammation

Immune system is aware that inflammation can go overboard

T regulatory = anti-inflammatory cytokines to regulate/

IgG4 is anti-inflammatory sa innate and acts as regulator

CHAPTER 8

T-CELL MEDIATED IMMUNITY

Remember: Adaptive immunity is initiated through interactions of NK cells and dendritic cells (refer to ch. 3)

8-1: DENDRITIC CELLS CARRY ANTIGENS FROM SITES OF INFECTION TO SECONDARY LYMPHOID TISSUES

Adaptive immune responses are initiated by capturing some of the pathogen and sequestering them in the secondary lymphoid tissue
Myeloid dendritic cells capture antigens and present them to naïve T cells
T-cell response to infections is made in the draining lymph node
- Blood infections: spleen
- Mucosal tissues: mucosal secondary lymphoid organs

Dendritic Cells

Immature DCs
- In the skin and peripheral tissues
- Active in capture, uptake, and processing of antigens
Mature/activated DCs
- No longer active in capture, uptake, and processing of antigens
- Upon moving to secondary lymphoid tissues
- Gains capacity to activate naïve T cells
Confined to the outermost part of the cortex where T cells congregate

Macrophage

Present in the cortex and medulla
Removes pathogens and their breakdown products from the afferent lymph that arrives from the site of infection
- Macrophage-mediated lymph filtration
- Prevents infectious microorganisms from passing through the node and gaining access to the blood via efferent lymph
- Prevents systemic infections
Eliminate lymphocytes that are signaled to die by apoptosis

8-2 DCs PROCESS ANTIGENS

DCs present peptides on MHC II molecules to activate naïve T cells
DCs have various endocytic and signaling receptors
Receptor-mediated endocytosis
- Capture bacteria/virus particles from EC fluid and process in lysosomes
- Aka micropinocytosis
- Micropinocytosis involves nonspecific ingestion of larger volumes of ECF and is used to capture pathogens not recognized by any endocytic receptor
DCs can become infected and make viral proteins that enter the endosomal ER and presented on MHC I molecules
- If infection not lethal, DCs carry the virus to the draining lymph node and activate naïve CD8 T cells
- If infection is lethal, DCs reach secondary lymphoid tissue but too sick to activate naïve T cells
  - The virus released by dying DCs can infect healthy DCs which then present the antigens on their MHC I molecules and activate CD8
If virus do not infect DCs, cross-presentation is used to stimulate a CD8 response
- DCs take up antigens by the endocytic pathway
- Transfer the virus degraded peptides to the exocytic pathway for cross-presentation by MHC I

DCs carry all TLRs except TLR9
- Highly sensitive to the presence of all manner of pathogens
- Signals from TLRs lead to DC activation
  - Increase efficiency in uptake, processing, and presenting
  - Appearance of CCR7 on the cell surface which is the receptor for the CCL21 chemokine in the secondary lymphoid tissue
    - Leave lymph and enter the tissue of the draining node
    - Maturation of DCs to focus on presentation

8-3 NAÏVE T CELLS FIRST ENCOUNTER WITH PEPTIDE:MHC COMPLEX

T cells bind to the endothelial cells of the thin-walled high endothelial venules (HEV)
They squeeze through the vessel wall and enter the T-cell area
- They encounter mature DCs
- T-cell receptors examine the peptide:MHC complex on DC surfaces
- If it binds successfully, T cell is activated and retained in LN
T cells can arrive via the afferent lymph
- Entered an “upstream” LN from the blood but did not encounter specific antigen, leave the LN via the efferent lymph and carried “downstream”
- T cells arrive at the node in a different afferent vessel from that carrying pathogens and antigens and DCs from the infected tissue

If T cell does not find its specific antigen, it leaves the medulla in the efferent lymph to continue recirculation
If T cell is activated, it takes several days for the cell to proliferate and differentiate into effector cells
- Accounts for much of the delay between the onset of an infection and the appearance of the adaptive immune response

8-4 HOMING OF NAÏVE T CELLS

Homing: naïve T cells leave bloodstream and enter T-cell area of LN
- Guided by CCL21 and CCL19 chemokines
- Secreted by stromal cells and DCs in T cell area
- Establish a concentration gradient along the endothelial surface
Naïve T cells express CCR7 chemokine receptor which binds to CCL21 and CCL19
- Guides T cells up the chemokine gradient
Exit route: cortex > medulla > efferent lymph
- Controlled by T cell receptor that can recognize sphingosine 1-phosphate (S1P) that establishes gradient on the LN
- Lowest in T-cell area and increases in the direction toward the efferent
For T-cells that recognized antigen on DCs
- Express CD69
  - Moves S1P receptors inside the cell to prevent external sensing
  - Prevents leaving LN during nurturing
  - Upon maturation into effector T cells, they stop expressing CD69

Adhesion Molecules

L-selectin
- On the T cell surface
- Binds to CD34 and GlyCAM-1 on endothelium
- Causes naïve T cell to slow down and attach to endothelial surface
LFA-1
- On T cell surface
- Binds with ICAM-1 and ICAM-2 on endothelium
- Activated by chemokines secreted by LN
- Strengthens hold on the ICAM
- Transient binding with DCs
- Upon encountering specific peptide:MHC complex, LFA-1 molecules increases affinity for ICAMs = conjugate
ICAM-3
- On T cell surface
- Binds to LFA-1 and DC-SIGN (unique to activated DCs)
- Transient binding with DCs
CD2
- On T cell surface
- Binds with LFA-3
- Strengthens adhesion

8-5 ACTIVATION OF NAÏVE T-CELLS

Co-stimulatory signal
- Required because signals generated by T cell receptor and co-receptor (CD4/8) with peptde:MHC complex is not enough for activation
- Without this, T cells cannot divide nor survive
- CD28 T cell co-stimulatory receptor which binds to B7 co-stimulatory molecule on DCs
Only professional APCs express B7 in the presence of infection through presence of inflammation and not constitutively
- When DCs take up pathogens/antigens, signals are generated to induce B7 expression
- DCs arrive at the draining lymph node already expressing B7
- Inflammation is needed or else anergy = pag isang e.coli lang, hindi dapat ma-activate adaptive immune system
  - Hindi enough ang pathogen population to induce inflammation = not a concern = anergy
Effector T cells activates macrophage and naïve pathogen-specific B cells to express co-stimulatory molecules
Once T cells are activated, they express an additional complementary B7 receptor CTLA4
- Binds B7 twentyfold
- Functions as brake on CD28 to inhibit activation and proliferation of T cells

8-6 RECEPTOR SIGNALS FOR ACTIVATION

Steroid signals can pass through to bilayer and into the nucleus kasi andoon ang receptor
Protein signals cant pass through the bilayer kaya receptor niya ay membrane-bound to start signal cascade towards nucleus
T-cell synapse: region of contact and communication between TCs and DCs
- Central supramolecular activation complex (c-SMAC): TC receptors, co-receptors, co-stimulatory receptors, and CD2 adhesion molecule
- Peripheral supramolecular activation complex (p-SMAC): LFA-1, ICAM-1, and talin which forms a tight sea, around the SMAC
Interactions of MHC ligands with TC receptors activate cytoplasmic protein tyrosine kinases
- Phosphorylate tyrosine residues (immunoreceptor tyrosine-based activation motifs or ITAMs) in the CD3 tails and the associated zeta chain CD247
- Extracellular binding of antigen to TC receptor initiates pathways of intracellular signaling that leads to TC differentiation
Cytoplasmic tails of co-receptors associate with Lck
- Phosphorylate the CD3 ITAMS and ZAP-70 upon synapse formation
- Binds to the phosphorylated tyrosines of the zeta chain
Activated ZAP-70
- Leads to activation of nuclear factor of activated T cells (NFAT)
- Leads to activation of protein kinase C-theta which induces NFkB
- Leads to activation of nuclear protein Fos, one of the component of transcription factor AP-1

8-7 IL-2 FOR PROLIFERATION AND DIFFERENTIATION

Synthesized and secreted by activated T cells
- Autocrine action
- Paracrine action: cytokine acts on a different type of cell from the one that made it
Requires signals from TC receptor:co-receptor complex and the co-stimulatory signal delivered by CD28 and activation of NFAT
Co-stimulatory signal stabilizes cytokine mRNA to increase TC production of IL-2 and rate of transcription from the IL-2 gene
Naïve TC has B and Y chain of IL-2 receptor
- Activated TCs: alpha chain is synthesized
- Triggers TCs to progress through cell division

8-8 T CELL ANERGY

Some T cells with specificity for self-antigens escape negative selection in the thymus because the self-antigens they recognize are not expressed in the thymus
- These cells will not express B7
Absence of B7 and co-stimulation, engagement of peptide:MHC complex by TC receptor and co-receptor = TC anergy
- State in which it cannot respond to any external signal
- Cannot be revived
- Fails to make IL-2
- Irreversible mechanism of self-tolerance to render harmless those self-reactive T cells

8-9-10 CD4 T CELL POPULATIONS

CD4 TCs do not directly attack the pathogens
Form a cognate pair with target cell

TH1

Help macrophages
For intracellular bacterial and viral infections
Stimulated by IL-12 (by DCs) and IFN-y (by NK cells)
Controlled by T-bet transcription factor
- Causes TC’s own IFN-y gene to be turned on
- Local production and secretion of IFN-y in the secondary lymphoid tissue
Increases inflammation

TH2

Help eosinophils, basophils, mast cells, and B cells
For parasites
Do not generate inflammation but promote repair and recovery of damaged tissues
Favors production of pathogen-specific IgE antibodies to eliminate parasites
Stimulated by IL-4 which binding induces GATA3 to commit to the TH2 pathway
- Turns on IL-4 and IL-5 which are secreted by TH2
Histamines = expulsion of allergen
- Sneeze, diarrhea, scratching
IgE is found on surface of eosinophil and mast cells

TH17

Secrete IL-17 and CXCL8
Recruits neutrophils to infected tissues
Stimulated by IL-16 and TGF-B
- Induces TC to make IL-21 which activates STAT3
Controlled by RORyT which turns on IL-17

TFH

Cooperate with naïve B cells
Initiate antibody response
Induced by IL-6 and controlled by Bcl6
Co-stimualtion involves the Inducible TC co-stimulator (ICOS)
Bcl6 express CXCR5 the receptor for CXCL13 produced by stromal cells of B cell follicles and causes exit from T cell area into follicles of the B cell areas
Guides switching of immunoglobulin isotypes in B cells

Treg

Keeps immune response under control
Limits tissue damage and facilitate healing
Reduce chance of secondary infections
Natural regulatory cells: commit to regulatory function during thymic development
Induced Treg: emerge in the course of the immune response to infection
- activated by TGF-B but in the absence of IL-6 and other pro-inflammatory cytokines
both expresses FoxP3 and CD4 and CD25
Induced Treg produces TGF-B and IL-10 to inhibit inflammation

8-11 POSITIVE FEEDBACK

For both TH1 and TH2 cells, the cytokine that drives their differentiation is central to their effector function and secreted in quantity
- TH1: IFN-y
- TH2: IL-4
Positive feedback: in which the functioning effector TCs drive further differentiation
- Polarized: if population is dominated by either TH1 or TH2
Cell-mediated immunity
- Polarized TH1
Humoral immunity
- Polarized TH2
Tuberculoid Leprosy
- TH1 bias
- Help macrophages to suppress growth and dissemination of bacteria
- Chronic inflammation damages skin and peripheral nerves
- Disease progress slowly
Lepromatous Leprosy
- TH2 bias
- Large pathogen-antibodies are made but ineffective against bacteria hidden inside macrophages
- Bacteria become disseminated to other sites in body
- Causes gross tissue destruction which is fatal

8-12 CD8 TCs REQUIRE STRONGER ACTIVATION

CD8 TCs are functionally homogenous but must interact with a wide range of target cells
For intracellular infections, mostly viral
Recognize antigens presented on MHC I molecules of DCs
For some viral infections, the interaction of a naïve CD8 with DC that presents antigen via MHC I is enough for activation and differentiation
- Synthesize IL-2 and receptor
- Induce CD8 TCs to proliferate
For other viral infections, DCs solicit help from virus-specific effector CD4
- Gives IL-2 to jump-start activation
- Forms viral peptide:MHC I complex and viral-peptide:MHC II complex
The effector CD4 is activated to make and secrete IL-2 which then binds to the receptor on CD8
- Drives the naïve CD8 to proliferate and differentiate
CD8 are only activated when the evidence of infection is unambiguous
- Cytotoxic TCs inflict tissue damage

8-13 CD8 TCs and Effector CD4 TCs

CD8 cytotoxic TCs and CD4 TH1, TH2, and TH17 TCs travel to infected tissue after differentiation
Adhesion molecules on effector T cells allow them to leave the secondary lymphoid organ
- Differentiates them from naïve TCs
L-selectin is replaced by VLA-4
- Binds to adhesion molecule VCAM-1 on activated endothelial cells at inflammation sites
- Halts passing effector TCs and direct them to enter the infected tissue
Effector TCs express more CD2 and LFA-1 than naïve TCs
- Makes them more sensitive to ICAM-1 and LFA-3 than APCs
Signals from TC receptor and co-receptor are sufficient to activate effector TCs
- Whereas induces anergy in naïve TCs
- Co-stimulation through CD28 and B7 is not needed for activation
- Allows effector TCs to kill any type of cell infected with virus

CD4 effector TCs are activated by antigen recognition on all cells that express MHC II
- Including vascular endothelial cells induced to express MHC II by IFN-y secreted by NK cells and effector TCs at sites of infection
- Naïve TCs are activated by antigen recognition of DCs only
Co-stimulation restricts initiation of TC responses
- Relaxing this requirement allows effector TCs to work quickly and efficiently

8-14 CYTOKINES AND CYTOTOXINS

Cytokines: alter behavior of target cells
- Interleukins by TCs
- Made only after the effector TC has formed a conjugate
- Never made and stored in CD4 TCs for future use
Cytotoxins: kill target cells
- Fewer than cytokines
- Effector CD8 manufacture and store them in lytic granules before an encounter with a target cell
- Comprised of granzymes, serglycin, and granulysin

8-15 CYTOKINES

Janus kinases (JAKs)
- Dimerization of receptor polypeptides = activates JAKs into enzymes
- Phosphorylates STATs (signal tranducers and activators of transcription)
- Phosphorylated STAT dimerize = allows them to move from cytoplasm to nucleus
- Change gene expression in target cell

Effects of cytokines can be turned off
- Phosphatases remove phosphate groups from JAKs, STATs, and cytokine receptors
- Suppressors of cytokine signaling (SOCS) proteins inhibits signaling pathway

8-16 SELECTIVE CYTOTOXINS

CD8 synthesizes cytotoxins in inactive forms and package them into lytic granules
Antigen specificity allows TCs to pick out only infected cells
- Cytoskeleton directs them to go towards T cell receptor with antigen kaya specific
TC focuses granule secretion at the small, localized area of the synapse
- Granules only kill the infected cell
CD8 also secretes cytokines
- IFN-y to inhibit viral replication in infected cells, increase viral antigen presentation, and activate macrophages

One cytotoxic TC can kill many infected cells

8-17 APOPTOSIS

Cytotoxic CD8 kill cells by apoptosis aka programmed cell death
- prevents pathogen replication
- prevents release of infectious pathogen particles from infected cell
cytotoxins make pores in target cell membrane
phosphatidyl serine = marker of apoptosis because PS is normally in the intracellular side of the cell membrane
- if induced to undergo apoptosis, it will flip and move to the extracellular side
- anexinfide stains PS

8-18 MACROPHAGE ACTIVATION BY TH1 CD4

TH1 CD4 help macrophages at infection sites to be more proficient in killing pathogens by secreting cytokines
- Macrophages containing captured pathogens fuse more efficiently with lysosomes
- Increased synthesis of potent microbicidal agents such as nitric oxide, oxygen radicals and proteases
Overall enhancement of macrophage function by effector TCs is called macrophage activation
Macrophages require two signals for activation from TH1
- Delivered by IFN-y and CD40 ligand
- Induces changes in gene expression for activation
After forming conjugate with macrophage, TH1 takes several hours to initiate cytokine gene transcription
- Cytokines are translocated to the ER of TH1
- Delivered by secretory vesicles to synapse
- Only cytokine receptors on the macrophage become loaded with cytokines and subject to activation
activation is antigen-specific
- only macrophages that present pathogen-derived antigens are selected for activation
- prevents unnecessary damage to healthy tissue
regulatory mechanism: TGF-B, IL-4, IL-10, IL-13
- inhibit macrophage activation
- counter effects of TH1 cells
- evident in lepromatous leprosy whose macrophages become overwhelmed by proliferating bacteria

8-19 TFH CELLS AND NAÏVE B CELLS

TFH cells help B cells make antibodies
- TFH cells move from the T cell area of secondary lymphoid organ to near B cell area
- Naïve B cells enter lymph node from blood because of chemokines CCL21 and CCL19
- Allows for interaction of TFH with naïve B cells
once TFH and B cell forms synapse, TFH synthesizes CD40 ligand
- binds with CD40 on B cell
- causes proliferation, differentiation, and maturation of B cell into plasma cell
Linked recognition
- Bounded B cells and T cells are specific for epitopes of the same antigen that was first bound and internalized by B cell receptor

8-19 REGULATORY TCs

Treg expresses high levels of CD25
Treg make immunosuppressive and anti-inflammatory cytokines
- IL-4, IL-10, TGF-B
Suppression depends on physical contact between Treg and target cell
- Treg interacts with DCs to prevent them from interacting and activating naïve T cells
- Treg may also interact directly with effector TCs
Human Treg has FoxP3
- Infants with no functional FoxP3 have no Treg
- fatal because it permits immune responses that are directed at self-antigens

NK cells similar to CD8 cytotoxic

No specific receptor for antigen

Nonspecific PAMPs triggers TLRs of NK cells

Activated dendritic (pag na-outnumber niya ang NK cells) detach from tissue and get washed by lymphatic fluid

MHC I magaling sa intracellular bc proteosome ang naglload ng peptide sa MHC I

Mas efficient ang cross presentation kasi it triggers both CD8 and CD4 (so cytotoxic and b cells are activated)

Kaya sa sars-cov-2 antibodies are needed kasi they play a role both in innate and adaptive

Cytokines that induces inflammation can loosen the tight junctions = recruitment of both neutrophils and T cells

Chemokines parang bread crumbs to direct t cells where to go

Chapter 9

Humoral Immunity

TFH stays in the lymph node to activate B-cell arm of adaptive immunity
Mature B cells has IgM molecules that become cross-linked by the antigen
- Sends signal from receptors to inside of cell
Interaction of antigen and immunoglobulin is communicated by IgA and IgB (B cell receptor together with IgM)
- Not enough to activate naïve B cell

Additional signals: B cell receptor:co-receptor
- CR2/CD21: recognizes iC3b and C3d derivatives deposited on the pathogen
- CD19: signaling chain
- CD81: binds to CD19 to bring it to the surface
Generation of the iC3b and C3d ligands
- CR1 + C3b with ligand
- Becomes susceptible to cleavage Factor I
- Gives iC3b fragment and C3d fragment
- CR1 increases abundance of ligands for B cell co-receptor on pathogen surface
CR2 component of co-receptor
- Upon binding to antigen, binds to C3d
- Causes kinase cascade
- Simultaneous ligation of the B cell receptor:co-receptor increases the B cell sensitivity to antigen
TFH recognizes peptides presented by B cell MHC II
- Sends cytokines and signals to induce division and differentiation

DiGeorge Syndrome
- No thymus
- Little to no T cells
- Normal B cell levels
- B1 cells produce low-affinity IgM
  - Does not require T cell activation
  - Polyspecific and no isotype switching
  - Carb or protein epitopes can cross-link B cell receptors and co-receptors and sufficient to activate B cell even without additional signals
  - Aka thymus-independent antigens (TI antigens)
- Highlights importance of B2 cell and T cell activation

Follicular DCs

FDCs provide signals to allow B cells to mature and survive
- Diff from myeloid DCs that present antigens to T cells
- Diff from plasmatycoid DCs that make type I IFN
FDCs serve as a depository of intact antigens
- Available for interaction with antigen receptors of B cells
- They lack phagocytic activity = preserves antigens intact

FCs take up antigens from lymph
- C3b and C3d fragment binds to pathogen surface
- Those tagged with C3b and C3d are taken up by FCs
Subcapsular sinus macrophage
- Little phagocytic activity
- Has CR1 and CR2 to take up antigens tagged with C3d or C3b
- Holds them on surface to be screened by naïve B cells
Medullary sinus macrophage
- Highly phagocytic
- Filters lymph before leaving node

B cell and T cell interaction

Naïve B cells from blood will be attracted to T cell area by chemokines CCL21 CCL19 and to B cell follicle by CXCL13
B cells enter node at subcapsular sinus to screen antigens
- If found, b cell enters B cell area of follicle to interact with TFH cells
- Expresses CD69 which prevents SIP receptor expression
- Stays in the lymphoid tissue to complete differentiation
Activated B cell begin to endocytose and process the antigen and present peptides on MHC II
- Expression of CCR7 binds to CCL21 and CCL19
- Draws the activated B cell to the boundary between T cell and B cell areas to interact with newly differentiated TFH cells

TFH activated by DCs presenting antigen reduce expression of CCR7
- Moves towards boundary of primary follicle
- Screens antigens presented by activated B cells
- If found, forms conjugate and expresses CD40 ligand
- Activates B cell NFkB and enhances adhesion molecules
- Facilitates delivery of cytokines

Primary Focus of Clonal Expansion

Conjugate pair T cells and B cells move together to medullary cords
- They begin to divide to form primary focus
- Gives rise to dividing B lymphoblasts secreting IgM
Some B cells stay in medullary cords
- Differentiate into palsma cells under the influence of cytokines IL5 and IL6 secreted TFH
- Determined by B-lymphocyte-induced maturation protein 1 (BLIMP-1) which stops transcription for proliferation
- Cells then increase expression of immunoglobulin chains

Somatic Hypermutation and Isotype Switching

Other B lymphocytes leave the primary focus and move into primary follicles of the B area while still attached to TFH
- Proliferates and forms germinal center
- FDCs secrete IL6 IL15 8D6 and BAFF to start rapid division of B cells to form centroblasts
TFH also divides and produce more cytokines
- induces B cells to produce activation-induced cytidine deaminase (AID) essential for somatic hypermutation and isotype switching
during somatic hypermutation and isptype switching, centroblasts no longer express immunoglobulins because focus is on expansion of large population of B cells with switched isotype and V-region mutations not antigen selection
- proliferation of antigen-specific B cells in primary follicles give rise to secondary follicle
- GC no has rapidly dividing B cells and T cells in the center with naïve B cells at the periphery screening for their specific antigens and now forms mantle zone