Exam 3 - MCB 442, ANSC - 450

Central vs Peripheral Tolerance

Central Tolerance - In primary or central lymphoid organs

• Positive Selection - Able to weakly recognize MHC or functional

• Negative Selection - Must not bind with high affinity to self peptides, induces clonal deletion
  

Peripheral Tolerance - Acquired by mature lymphocytes in peripheral tissues

• Anergy - State of unresponsiveness, T cells that bind antigen in absence of co-stimulation

• Immunoregulation - Inhibition by products of other cells

• Ignorance - Sequestration of antigen(T cell is physically blocked from antigen)

B Cell Maturation

Generation of receptors(bone marrow) - Negative selection(bone marrow) - Migration to lymphoid organs and activation - Antibody secretion and memory cells(bone marrow + lymphoid tissue)

• FLT3 - Drives commitment to CLP

• CXCL12 - Keeps B cells in contact with bone marrow stromal cell

• IL-7 receptor needed for proliferation

• E2A - Transcription factor for lineage commitment, stimulates early B-cell factor(EBF)

Rearrangement of Heavy Chain Locus(Start of B cell development)

Stem cell - Early pro-B(DJ rearranging) - Late pro-B(V-DJ) - Large pre-B(VDJ)

• E2A + EBF = VpreB, lambda5, RAG1/2, Pax5 = CD19,Iga

• Rearranged VDJ segments combined with VpreB and lambda5 segments(surrogate light chain), Tests functionality

• VpreB - Variable region substitute

• Lambda5 = Constant region sub

• Signaling stops rearrangement - two heavy chains could = two different receptors, Pre-B cell receptor signaling enforces allelic exclusion

• Allelic exclusion = Reduces RAG1/2 expression, phosphorylation of RAG-2 for degradation, decreases access to heavy chain locus

4 possible fates for autoreactive B cells

Apoptosis(clonal deletion)

Receptor Editing - Make new receptor, stim of IgM in an immature B cell induces RAG expression = generation of new light chain

Anergy - Permanent unresponsiveness, cross-linking self antigen with low valence

Ignorance - Remain in circulation but does not see antig

Thymus

Site of T-cell maturation

• Pathway: Precursor cells enter in corticomedullary junction - Migrate to the cortex, proliferate then migrate to the medulla - Migrate out of thymus to secondary lymphoid tissue

• Cortex - Cortical epithelial cells, thymoctes(bone marrow origin)

• Medulla - Hassal's corpsucle, dendritic cell, macrophage
  

Importance

• T cells interact with thymic stromal cells(essential for inducing tolerance)

• Mice that have undergone thymectomy are immunodeficient

• Only 2-4% of cells in thymus leave, rest are dying/phagocytosed

• T cell production rate is greatest before puberty and slows in adults

• Removal of thymus after puberty is not associated with reduced T cell numbers
  

Bone Marrow Chimera studies importance of RAG and thymus

• Lack ability to undergo recombination when no thymus

T cell Development

Requires Notch signaling and TXN factors

• Notch Receptor Signaling - Needed for T-cell lineage commitment = TCF1 and GATA3 = RAG1 and CD3

• Bcl11b induces lineage commitment

• Notch Signaling = IL-7R

Similar to B cell maturation - DN stages characterized by expression of kit, CD44 and CD25

T Cell Positive Selection

CORTEX - Able to weakly recognize self MHC and peptide

• Thymic cortical epithelial cells are responsible

• Express both MHC 1/2 antigens

• Binds with right affinity to MHC 1 = CD8 cell, MHC 2 = CD4 cell

Flow Cytometry

Determines surface expression of protein on cell, cell proliferation and cytokine production, cell viability, percent of positive cells

TXN Factors

Determine co-receptor fate

• ThPOK(CD4) - Prepresses actions of Runx3

• Runx3(CD8) - Represses CD4 transcription

• Some CD4 cells up-regulate CD25 and express FoxP3, are natural Treg cells - Induced by binding self antigens with higher affinity than normal but not strong enough to cause deletion

T Cell Negative Selection

Deleting clones of T cells that bind too tightly to self peptide/MHC

• In cortex + medulla

• Likely dependent on bone-marrow APCs(more DCs than macrophage)

• Thymic medullary stromal cells express tissue specific proteins that are not expressed elsewhere(AIRE - autoimmune regulator)

Affinity Hypothesis

No selection = death(low affinity)

Medium affinity = positive selection

High = Treg cells

Too high = negative selection(clonal deletion

Tregs

Naturally occurring(nTregs) = from thymus, CD25+, FoxP3+

• FoxP3 - Transcription factor needed for Treg polarization

• CD25 - IL-2R, on all activated T cells

• Inducible Tregs are made from TGFbeta stimulation in periphery, function to maintain peripheral tolerance
  

Function of Suppression

• CTLA-4 - Negative regulator of T-cell activation, binds CD80/86, inhibits DC and macrophage activation

• Consumption of cytokines needed for T cell viability

• Production of cytokines(IL-10, TGFB, IL-35) - Anti-inflammatory

Lymphocyte entry into 2nd lymphoid tissues is dependent on expression of chemokines/adhesion molecules

CD62L - Marker on naive T-cells, is needed for homing to the LN

L-selection - Binds to vascular addressins on HEV

Entry into LN parenchyma requires expression of integrins(LFA-1, ect) and chemokine receptors(CCR7)

Integrins bind to adhesion molecules(ICAM) - where T cells are needed

IFN-y, IL-2, LT-a/B

Th1 Cytokines(pro-inflammatory/Intracellular defense)

• IFN-y - Macrophage activator, boosts MHC expression and helps B cells switch of IgG classes

• IL-2 - T cell growth factor, needed for proliferation after antigen-T cell interaction

• LT-a/B - Lymphotoxin, kills target cells during lymphoid organ development(similar to TNF)

IL-4, IL-5, IL-13

Th 2 Cytokines(Anti-parasitic/Allergy)

• IL-4 - B cell switcher, tells B cells to make IgE, also helps naive T cells = Th2

• Il-5 - Eosinophil activator

• IL-13 - Similar to IL-4, increases mucus production in gut and lungs

IL-17A

Th 17 Cytokine

• IL-17A - Recruiter of neutrophils, vital for fighting extracellular bacteria and fungi, big role in autoimmune inflammation

IL-10, TGF-Beta

Treg Cytokines

• IL-10 - Brakes, inhibits Th1 cells and macrophages to prevent immune system from overreacting and damaging tissue

• TGF-B - Generally anti-inflammatory but is also required to tell a T cell to become a Th17 or Treg

TNF, GM-CSF

Generally inflammatory + Growth Factors

• TNF - Tumor necrosis factor, master of acute inflammation, activates vascular endothelium(leaky blood vessels, so cells can move)) and can induce fever

• GM-CSF - Recruitment factor, stimulates bone marrow to produce more monocytes and granulocytes

Conventional DC vs Plasmacytoid DC

Conventional DC - T cell priming
Plasmacytoid DC - Interferon production

L-2R(CD25) alpha

On activated T cell surface, makes receptor high affinity, naive only have gamma + beta subunits

• Stimulates proliferation, IL-2 acts in an autocrine + paracrine fashion

• Instability sequence at 3' end makes RNA unstable

T Helper 1 Cell

Fights Intracellular infection (viral/bacterial)

• Macrophage activating effector molecules, IFN-y, GM-CSF, TNF-a, IL-2

• IL-12 induced STAT4 phosphorylation is required for TH1 polarization

T Helper 2 Cell

Fights Worms, part of allergic response

• Barrier immunity activating effector molecules: IL-4,5,13 + IL-3,10

• STAT6 - TH2 polarization

T Helper 17 Cell

Fights extracellular bacteria/fungi

• Barrier immunity, neutrophil recruitment: IL-17A, 17F

• IL-17A,F, IL-22, drives production of G-CSF + KC/CXCL1

• G-CSF = neutrophil hematopoietic factor

• KC = Neutrophil chemoattractant

• IL-17 = Causes neutrophilia

• Signaling for IL-17 requires Act1 and NFkB pathway

T Regulatory Cells

Immunoregulation

• Suppressive cytokines: IL-10, TGFbeta

• iTreg cells: TGF-β; STAT5; FoxP3; CTLA-4 expressio

Cytotoxic T cell Mediated Cell Death

CD8 attaches to target via LFA/ICAM

• Recognition of MHC 1 = cytoskeletal rearrangement, directs cytotoxic granules toward cell

• Release of granules induces cell death

• Granzyme/perforin/serglycin complex ensures granzyme mediated cleavage of caspase 3

B-cell co-receptor Complex

CD19, 21, 81

Binding of both BCR to an antigen and CD21 or CR2 binding to complement proteins(C3d) on cell surface = B-cell proliferation and activation
Linked Recognition - B cell can only be activated by its cognate T cell that recognizes the same antigen, ensures tolerance

Because B cell activation requires linked recognition for activation, this means that both T and B-cells need to display a receptor for same antigen

• B cell must come in contact with T-cells in order to receive help(otherwise they die)

• Trafficking of activated B-cells toward T-cells = Upregulation of EBI2, CCR7, and continued expression of CXCR5

EBI2, CCR7, CXCR5

EBI2 - Recognizes 7a, 25-dihydroxycholesterol
CCR7 - Receptor for CCL21, CCL19(Tfh)
CXCR5 - Receptor for CXCL13(fDC)

All contribute to trafficking of activated B-cells towards T cells

Germinal Centers

Important for somatic hypermutation, affinity maturation, class switching, and formation of memory cells

• Three zones(Mantle, light, dark)

• Follicular dendritic cells CXCL13 to CXCR5(In light zone)

• Stromal cell CXCL12 to CXCR4(in dark zone)

• Dark zone is filled with proliferating B cells(stained green with (Ki67+)

• Both light and dark is where somatic hypermutation occurs

• Centrocytes - Reduced proliferation and CXCR4 expression, increased surface Ig

• B cells can only enter dark zone if they are CXCR4 positive

ICOS and CD40 expression by Tfh cells are needed for germinal center maintenance
B-cell Pax5 and Bcl6 inhibit expression of transcription factors needed for differentiation into plasma cells

• These factors are decreased prior to differentiation
  Expression of BLIMP-1, transcriptional repressor is increased - Decreased CXCR5 and upregulates CXCR4 and a4B1 integrins
  Some B cells differentiate into memory cells which divide slowly but maintain BCR specificity

Activation Induced Cytidine Deaminase(AID)

Expressed by germinal center B-cells, causes random mutations in V genes

• B cells with successful mutations can move to dark zone and undergo rounds of division

• If mutations weakens affinity for antigen it will no longer receive T-cell help(Dies)

• If mutation strengthens affinity, it receives T cell help and will proliferation and differentiate

• Is positive selection, ensures only a few B-cell clones are highly responsive to the antigen

AID Mechanism:

• Switch regions = G-rich, as transcription occurs G-rich RNA invades DNA duplex and binds to C-rich template strand creating an R-loop, this R loop provides a binding site

• AID = C to U mutations, processed by Uracil DNA Glycosylase(UNG) and APE1 creating DNA nicks on both strands

• DNA is cut and sewed to different antibody constant regions = Class switching

• Just AID or just UNG mechanism = somatic hypermutation

Class Switching(B cell)

Directed process as the isotype that the antibody switches to is determined by the type of Th-cell cytokines are made

• Cytokine-specific receptor = which isotype is created by dictating which C gene segments are transcriptionally expressed

• Class switching only occurs after stimulation with antigen
  IL-4 = IgG1, IgE
  IFN-y = IgG3, IgG2a
  TGF-B = IgG2b, IgA
  IL-21 = IgG3, IgG1, IgA
  IL-5 = IgG1, IgA

Humoral Response: TI antigens

BCR-activation is first signal for antibody production

For thymus independent antibody generation costimulation is from activation of PRRs by PAMPs
Thymus Independent Antigens

• TI-1 - Activate B-cells without antigen specificity

• Polyclonal Activation - Activation of more than one B-cell clone - leads to B-cell mitogens: Induce mitotic division; LPS
  

TI-2 - Activate only mature B-cells, have highly repetitive structures

• Infants do not have mature B-cells so cannot elicit an antibody response to polysaccharides

• Made predominantly by B-1 cells

• Provide a prompt antibody response against pathogens(IgM) but can undergo class switching

Follicle Periphery

Antigen recognition - Antigen presentation - Proliferation/effector function, cytokines circle back and activate B cells(same as antigen recognition)

• IL-21 promotes B cell proliferation

• IL-4 + IFN-y will influence class switching

IgM

First antibody that is made

• Largest antibody

• Highest avidity but lowest affinity for antigen(10 binding sites)

• Most effective at complement activation

• Largely made by B-1 cells in spleen

• Effective at complement activation(C1q binds to one Fc region)

IgA

Made after class switching, IL-5 enhances IgA production

• In secretions, two subclasses(IgA1,2)

• Has neutralizing capacity(IgA1)

• Not a good opsonin or good at complement activation

• Can form dimers(IgA2)

• Dimeric IgA can bind to polymeric immunoglobulin receptor(plgR)

Transcytosis - Process of transporting IgA(and IgM to lesser extent) across the epithelial barrier

IgG

Made after class switching

• 5 subclasses(IgG1, IgG2a, IgG2b, IgG3, IgG4)

• Has neutralizing capacity

• Principal antibody in blood and extracellular fluid

• Can activate complement(worse than IgM)

IgE

Made after class switching(IL-4)

• Potent inducer of Mast cell activation

• Also binds FceRII on eosinophils and basophils

• Is monomeric
  

Mast Cells

• Cross linking two IgE molecules on surface causes degranulation

• 3 Functions

• Histamine = vasodilation

• Immune cell attraction - Lipid inflammatory mediators, prostaglandin D2, leukotriene C4, cytokine production(TNF)

• Muscle contraction - Physical expulsion

Active vs Passive Immunization

Active immunization

• Require generation of adaptive immune responses

• Long lived = memory
  

Passive Immunization

• Do not need to generate an immune response

• Short lived

• Some toxins are too toxic to vaccinate against

  • Inject small amounts into larger animals, generate antibody

  • Injection of antivenin(purified IgG) into person that has been bitten

  • Referred to passive immunization

B cell and natural killer cell positive feedback loop

Binding of Fc receptors on NK cells promotes perforin and granzyme secretion

• B cell secrete IgG3 = activates NK cells = IFN-y = activates B-cell

Innate Lymphoid Cells

Shape effector functions of adaptive immunity

• 4 Groups(found mostly at epithelial barriers) - Cytotoxic ILC, group 1, group 2, group 3

• Cytotoxic ILC(NK) - Viruses, activated by IL-12,15, makes IFN-y

• ILCs 1 - Type 1 immunity(intracellular bacteria) - IFN-y, Il-2, TNF, Macrophage activation, granuloma formation, IgG2a, IgG3

• ILCs 2 - Type 2 immunity(Helminths) - IL-4,5,13, Mucous production, SM contraction, IGE, IgG1, Eosinophil and mast cell activation, granuloma formation

• ILCs 3 - Type 3 immunity(Extracellular bacteria) - IL-17A, IL-17F, IL-22, Neutrophil attraction

NK cells vs ILC1

Both innate immune cells, both produce IFN-y

• ILC1 progenitor is different

• NK cells present in blood and spleen, ILC reside in tissue

• NK cells express cytotoxic granules

CXCL10, CCL 5,17,22,20

CXCL10, CCL5 will attract Th1 cells
CCL17, CCL22 will attract Th2 cells
CCL20 will attract Th17 cells

Naive Cells express CD62L(LFA) and CCR7

Granuloma Formation

Granuloma formation is dependent on Th1 responses

• Microbes are walled off from surrounding tissue by macrophages and T cells

• IFN-y enhances macrophage killing of pathogen by causing upregulation of iNOS and production of ROS and RNS

• Comes at a cost - Tissue in center is destroyed(caseous necrosis) by intense ongoing immune reaction
  

ILC1 + Th1 = IFN-y = Inducible nitric oxide synthase(M1), arginine reacts with iNOS = NO = Toxic to intracellular microbes

IL2 + Th2, functions

= IL-4,13 = Arginase 1, Arginine + arginase 1 = Ornithine and proline = Toxic to worm, SM contraction, tissue remodeling

Immunological Memory

Maintenance is dependent on IL-7, IL-15

• Memory T cells express many markers of activated T cells but not others

• Important Marker: IL-7Ra(CD127)

• IL-7 needed for CD4/8, IL-15 needed for CD8 maintenance

Memory Cells

Classified by surface markers that control localization

• Naive T cells - CCR7(localization to LN) and CD45RA

• Central Memory T Cells - CD45RO

• Effector T Cells - Cytokines and CD69(Lack CD127)

• Effector Memory T Cells - CD45RO and non-lymphoid homing receptors

• Resident Memory T Cells - CD69 and CD127(IL-7Ra)

B Cell Immunological Memory

Memory B cells respond more rapidly than naive B cells

• Usually have undergone class switching and affinity maturation

• Distinguished by surface markers - Naive = IgM, IgD - Memory = CD27, slightly higher MHC 2 and CD80 able to re-enter germinal center

Original Antigenic Sin

From Thomas Francis, Tendency to make antibodies against epitopes expressed on first exposure to flu even if exposed to different variants with highly immunogenic epitopes

Absorptive Enterocyte

Uptake of nutrients/fluids, microbial and metabolic sensing, transport of secretory immunoglobulins

Microfold(M) cell

Antigen uptake, bacterial translocation

Goblet Cell

Production of mucins, antigen uptake

Paneth Cell

Has lysozyme, secretion of antimicrobial peptides - Support for intestinal stem cells

Tuft Cell

Il-25, sensing of helminthic odorants and succinate, mobilization of ILCs 2 via IL-25 and eicosanoid release

Peyer’s Patch

Surveillance and antigen sampling in intestines

Traffics antigen to mesenteric lymph nodes

• Consists of T,B, M, and dendritic cells

Cryptopatches

Functions like a smaller Peyer’s patch

ILC 3 instead of B and T cells

Function on tissue repair and precursor for larger follicles

Isolated Lymphoid Follicle(ILF)

Antibody factory in intestines

Mainly makes IgA

Ensures that sections far away from Peyer’s Patches still have antibodies

Gut-specific homing by antigen-stimulated T and B cells

Gut lymphocytes express CCR9 which binds CCL25(epithelial cells in small intestine) or CCL28(colon, mammary/salivary glands)

• Integrin a4:B7 on lymphocytes binds MAdCAM-1 on gut endothelium

• Antigen encounter outside gut induces a4:B7 binds VCAM1 and CCR4 expression = licenses cells to skin

• Encountering antigen IN gut induces expression of a4:B7 which directs cells to mucosa

Intestinal Macrophages

Replenished from blood monocytes

• Highly phagocytic

• Do not readily respond to PAMP by making proinflammatory cytokines

• Product large amounts of IL-10 - ACT AS IMMUNE SUPPRESSORS TO PREVENT DAMAGE