HST 175 Cellular and Molecular Immunology Midterm Review Sheet (October 2002) Overview: Four fundamental themes of mammalian immunology: W_ generation of diversity (recognition of an enormous variety of foreign antigens); VDJ recombination and somatic mutation discrimination of self vs. non-self: negative and positive selection during development, anergy in the periphery @ protection by neutralization or destruction of non-self @ memory ‘Some dichotomies of immunity: innate (agglutinins, complement, phagocytes, NK cells) vs. specific (antibodies, MIC, TCR) Within specific immunity: humoral (B cell produced antibodies) vs cell- mediated (TCR, cytotoxic T lymphocytes) 1M Within the T cell repertoire: MHC I/CD8 (CTL, intracellular pathogens) vs. MHC I/CD4 (Helper T, involved in most immunological responses) Within Helper T: Thi vs Th2 Generative (“central”) lymphoid organs: bone marrow and thymus peripheral (“secondary”) lymphoid organs: lymph nodes, spleen, Peyer's patches (mucosal) Basic morphology of lymphoid organs: structure is meant to optimize contact and interactions between cells (as well as with antigen) to generate an immune response | thymus: cortex, medulla (structure related to T cell development) m1 lymph node: collection, sampling, and response to antigen in the lymph; consists of a cortex of primary follicles (naive B cells) and secondary follicles or “germinal centers” (where response to antigen stimulation occurs; proliferation, affinity maturation, memory); medulla (with fully differentiated plasma cells); T cells lie in between follicles in high endothelial venules; lymph enters subcapsular sinus, percolates through cortex and sinus, and exits through the efferent lymphatic in hilum of node. B cells enter node from blood and traverse helper T cell zones en route to the follicles (B-T cell cooperation is critical for the immune response) W spleen: the immunologically relevant area is the white pupl; arterioles and periarteriolar lymphoid sheaths (T cell zone) with lymphoid follicles and germinal centers (B cell zone). These are surrounded by a marginal zone (marginal B cells) peripheral lymphoid tissues (GI and respiratory: Peyer's patches, tonsils, appendix)Innate vs. Specific Immunity Innate Immunity: (invertebrates and vertebrates) ~ self/non-self discrimination and protection by neutralization and destruction (pathologic consequence = inflammation) Secretions and physical barriers: like mucus and skin, lysozyme (removes bacterial coats) W Agglutinins: neutralize, opsonize, fix complement, discriminate self and non- self; major examples are lectins (bind to sugars on cell surface, i.e., mannose binding protein) or receptors: (1) Toll-like receptors: bind to LPS (“endotoxin”) on gram-negative bacteria or bacterial lipoproteins (2) £Met-Leu-Phe Receptor: binds to Met peptides (3) CpG DNA receptor ~ recognizes unmethylated CpG DNA (4) Mannose Receptor ~ non-self sugars at microbial surface Macrophages and other phagocytes (i.e., neutrophils): recognition of microbes either through specific receptors ro host agglutinins coated onto microbes % Complement: Especially the alternative pathway (requires no antibody), Functions of complement system: (1) bacterial lysis; (2) induce phagocytosis; (3) mast cell activation, neutrophil recruitment, and inflammation; (4) clearance of immune complexes through binding to complement receptors "NK cells: viruses can cause reduced expression of MHC I in infected cells; NK cells (with similar mechanism of CTL) can kill cells lacking MHC I expression in a non-specific manner. 4 B-1 cells: make natural antibodies of limited diversity; recognize common polysaccharide and lipid antigens of many bacteria like LPS; found in intraepithelial cavities. M1 y6T cells: recognize heat shock proteins, lipoglycans, and other common bacterial antigens; are not MHC restricted; some recognize antigen presented by CDI; found most commonly at epithelial boundaries Specific Immunity: (vertebrates) self/non-self discrimination, generation of diversity, and specificity for destruction ™ Band T cells Antibodies and effector T cells (i.e., CTLs) % MHC molecules (1) In the central lymphoid organs, rearrangerhent of the immune receptor (i.e,, BCR and TCR) occurs as well as the elimination of self-reactive cells. (2) In the periphery, these naive cells see antigen and proliferate/activate into mature lymphocytes.B cell development Setting: Bone Marrow 1. Start with hemopoetic stem cells (HSC). 2. Through various signals, send some HSCs to become erythrocytes, megakaryocytes, eosinophils, analytes, monocytes. Now, the leukocyte precursor (common lymphoid progenitor) is all that’s left. Once a common lymphoid progenitor commits to the B cell lineage (IL-7 helps), it is dubbed pre-pro-B. D-J recombination of the variable region of heavy chain u. This is pro-B. V joins DJ to form VDJq in heavy chain p. Transcribe. Pro-B2/pre-B. Process RNA to splice out region between VDJ and most proximal C segment (Ci). add poly-A tail. Translate to produce p-protein and express on cell surface (with surrogate light chain). This is large pre-B, Itis the product of positive selection (as mediated by BTK, sre family kinases). Roughly, it checks ifthe receptor heavy chain is properly formed. If'it is, signals for survival, proliferation, allelic exclusion, and light chain rearrangement take place. 9. IfV joined DJ out of frame (2/3 chance), or if anything else went wrong on the way, go back to step 5 with 2 allele. If both alleles have been tried, enter cell death sequence. 10. Allelic exclusion (prohibits non-transcribed allele (if it exists) from réarranging) Pre-B receptor signalling is thought to mediate RAG-2 degradation to achieve such exclusion. 11, Start light chain rearrangement (x). V recombines with J, then transcribe. 12. Splice out region between VJ and Cx. smalll pre-B. 13. As in step 9, if this processed RNA is non-productive, try the other allele. If both are unproductive, try the @ light chain alleles. If both alleles for both « and A fail, enter cell death. 14, Translate and express complete IgM molecule on cell surface. Immature B cell. 15. Receptor editing: If the IgM receptor recognizes self antigen, it continues to express Rag genes and undergoes further x and/or 2 rearrangements. If it exhausts all possibilities, default is death (negative selection). 16. If non self-reactive B cell is achieved (successful positive selection 11) cell undergoes light chain isotype and allelic exclusion (prohibits generation of multiple light chains). 17, Through alternative splicing of original heavy chain transcript, IgD is now expressed along with the IgM. (VDJy joins the next C segment (C8).) 18. B cell leaves bone marrow and travels to periphery (to the lymphoid follicles in lymph nodes and spleen.) Now called Mature B cell. 19. If stimulated by protein antigen (T-cell dependent, requiring CD40-CD40ligand interaction plus cytokines (IL-2, 4, 5), this antigen is likely foreign (due to previous selection steps by both B and T cells). The stimulated B cell proliferates and the progeny begin making secreted antibodies. Alternative RNA processing allows for secreted vs. membrane form (tailpiece vs transmembrane and cytoplasmic tail). 20. The cells now undergo isotype (or class) switching to make y, ot, and ¢ heavy chains (6 lacks a switch region). This is different from alternative splicing. The class it chooses depends on the cytokine signal it receives (by T ceils and other cells). Ex. IL-4 yields secreted IgE, TGF-B yields secreted IgA. IFN-y yields IgG. Isotype switching relies on the switching regions 5° of each C segment. But the C5 has no switching region (so no switching to IgD). 21. The cells undergo proliferation and somatic mutation (or somatic hypermutation or affinity maturation) @: the germinal centers (dark zone). These are point mutations in the V region of both heavy and light chains; the number of mutations gradually increases with each generation of the clonal progeny of the originally stimulated B cell, and the resulting cells (that survive) have BCRs and make antibodies that bind with higher SN DAYaffinity to the original antigen. The new B cells use the antigen bound to follicular dendritic cells (in germinal enter light zone) to make sure the antigen is still cognizable (and with higher affinity). . 22. Some of the cells become antibody-seereting plasma cells, while others become memory B cells. 23. Non-protein anti ide ic acids, etc. reach T cells, so isotype switching, somatic mutation, and memory B responses are not possible. Instead, only B cell receptor eross-linking will stimulate IgM secretion only. This is T-cell independent. AID (Activation Induced Cytidine Deaminase) seems to be required for both class-switching and somatic mutation. It perhaps edits the RNA for a key protein involved in these Processes. note; Table 9-3 of Abbas Ed.4 ts great for summarizing steps 19-23. VDL recombination and VJ recombination (as in steps 5, 6, and 11 above) Mediated by “recombinases” (like RAG-1 and RAG-2) specific to lymphocytes plus DNA repair enzymes. For recombination by looping out and deletion, recombinases find the recognition signal sequences (RSS) 3° of V segments, flanking D segments, and 5’ of J segments, Ifthe RSS is on the other side of these segments, recombination by inversion would be necessary instead of recombination by deletion. Each recognition sequence consists of a conserved heptamer (Top) and nonamer (9bp) separated by a spacer of either 12 or 23 nonconserved base pairs. The recognition sequences are oriented such that the @ heptamer is always closer to the segment it is marking (so the heptamer is immediately 3° of all V and D segments and immediately 5’ of all D and J segments), 12/23 RULE: RSSs with 12 bp spacers only recombine with RSSs with 23 bp spacers. 1) Cleavage and double stranded break. RAG-1 and RAG-2 recognize RSSs and nicks (single strand cut) between heptamer and coding sequence, the coding sequence free end displaces the other strand, forming a hairpin at the 3° end of coding sequence. 2) The hairpin ends are opened up and nucleotides are added or removed (generates greater diversity), Junctional diversity — generated by imprecise joining of hairpin ends and subsequent addition of P nucleotides (joining is mediated by double strand break repair proteins like Ku70, Ku80, DNA dependent protein kinase (DNA-PK). Also generated by N region addition between the cut ends (random addition of extranucleotides directed by terminal deoxynucleotidyl transferase (TdT). GENERATION OF DIVERSITY: VDJNV3 recombination, Junctional diversity (P nucs, and N region), Heavy chain-light chain combos, and somatic mutation, BCR signaling, very roughly: Once antigen binds to BCR: - Signaling is NOT through cytoplasmic tail of BCR proper (that is only 3 amino acids long -- KVK). Itis insted) transduced by Tgo. and IgB (components of the BCR complex). In their cytoplasmic domains, Iga: and IgB contain immunoreceptor tyrosine-based activation motifs (ITAMS).1. BCR is cross-linked, the tyrosines in the ITAMS of Iga: and Igf are phosphorylated by src family kinases (lyn, blk, fyn). @: Syk or Btk then binds to these phosphorylated sites, and subsequently gets phosphorylated itself. 3. This activation can trigger a number of cascades IP3, DAG, PKC, Ras, ¢-Fos, JunB, NFKB, Myc. This will be a focus in the second half of the course. 4. Can trigger phagocytosis of the antigen on the BCR, the B cell, acting as an APC, can then present the antigen fragments to T cells (CD4) via MHC. In development, yields the signals for selection (see above). Bik deficiency (X-linked agammaglobunemia) ~ yields reduction in B cells and all Ig isotypes because of poor B cell maturation (positive selection signaling is impaired). Similar to syk knockouts in mice, Other proteins on B cell surface: CRI (involved in comp. med. opsonization) and CR2 (positive reg. of B cell signaling), CD45R (ID of lymphocyte subset), FeyRITI (feedback inhibition), MHC II (antigen presentation), CD40 (B-T cell interaction (B-cell costimulation, req. for somatic mutation, and isotype switching), B7-1 and 2 (for T cell costimulation). rete rt Secreted antibodies produced as a result of alternative splicing. IgA~dimer IgM ~ pentamer Both are formed by interaction of the tailpieces of the monomers with a completely separate protein, the J- @ais via cisuisise tonds (Fis fr joining). IgE, IgGs ~-monomers ‘Structure of Antibodies: 2 heavy, 2 light chains Variable region (Vi and V.) composed of highly variable complementarity determining regions (CDRI, 2, and 3). CDR 3 is most variable (due to VDJ recombination, junctional diversity). Constant regions, which contains hinge region. ‘The Fab (antigen binding) part can be cleaved from Fe part by papain (yields 2 Fab halves) or pepsin (yields fully associated Fab). Functions of Antibodies: 1, The B-cell receptor (membrane-bound form). Recognition of antigen by membrane bound antibodies initiates proliferation and secretion of antibody as described in the B-cell development process. Requires, at least, [get and IgB to generate signal. IgM and IgD are key players in initial B cell activation. 2, Neutralization of toxic or infectious antigenic process, Secreted antibody binds antigen to sterically prevent it from injuring cells, 3. Isotype specific functions (remember isotypes come from isotype switching which is directed by Qproxines):Activation of Complement (secreted IgG, IgM): the classical complement pathway, initiated by Clq binding to constant domains of IgG, IgG3 or IgM that are part of immune complexes or on the cell surface. Opsonization for Easy Phagocytosis (secreted IgG): Secreted IgG coats free antigen. Phagocytes have receptors that recognize the Fe portion of IgGl or 1gG3, called FeyR. The phagocytes find this coated antigen casily and ingest it. (Also C3b, a product of the complement pathway can also opsonize and be recognized by C3b receptors on phagocytes.) Cell-Mediated Cytotoxicity [ADCC] (secreted IgG, IgE, IgA): A cell to be killed has a foreign antigen on its surface. The antibody binds to this antigen. A cell that kills has Fe receptors, finds the antibody-coated cell, and destroys it. Ex. NK cells have FeyRIII, so they will find IgG coated cells and lyse them. Immediate Hypersensitivity (secreted IgE): Mast cells and basophils have FceRI which binds IgE with such high affinity, IgE need not be bound to an antigen (this is unlike ADCC). Subsequently introducing an antigen will cluster these activated Fe receptors on the cell, which causes them to release histamine from granules and synthesize leukotrienes, prostaglandins, cytokine, all of which causes an inflammatory response. ‘Mucosal Immunity (secreted IgA): Epithelial cells in mucosal areas have Fe receptors (poly-lg receptor) that bind IgA dimer on blood side, and transport it, via vesicles, to the mucosal surface (Iumen). There, the IgA (bound to a cleaved portion of the poly-Ig receptor known as the secretory component) can direct neutralization of harmful antigens, Neonatal Immunity (maternal IgG2, IgG4, IgA):matemal IgG goes across placenta via FeRn to fetus and |; and IgG is in breast milk. Feedback inhibition (IgG): ex. IgG can bind to FeyRIIB receptors on B cells and inhibit their activation. Using antibodies in lab: ELISA, immunofluorescence histology, immunoprecipitation, analyzing Western blots, FACS staining. It might be good to understand the basics of how these work. Key cytokines: IFNy stimulates IgG production; IL-4 stimulates IgE; TGF and maybe ILS - IgA. lopm Thymocyte precursors develop in the bone marrow and migrate to thymus during fetal and adult life. TCR gene rearrangement takes place after arriving in thymus. Positive selection occurs in the cortex; negative selection in the medulla, Importance of non-lymphoid stromal cells in thymus: (1) express MHC essential in selection of mature T cells; (2) secrete thymic hormones and cytokines eg thymosin, thymopoietin, IL-7 important in T cell survival. Steps in development: 1, HSC diffentiates into oligopotent pregenitor (CD44+, CD25-); progenitor travels to thymus. 2. Progenitor commites to T lineage (CD44+, CD25+); but still double negative (CD4-/CD8-) 3, VDJ recombination of B chain gene (requires RAG, Ku80, DNA-PK, TdT), transcribe, and express pre-TCR, receptor with surrogate a: chain (pTa:) 4. If this recombination is productive, pre-TCR will provide proliferation signals (positive selection D, ® chain allelic exclusion, pTo: shut-off, and induce a recombination; if not, the cell dies5. Double negative cell now converts to double positive (CD4+/CD8+) 6. VJ Recombine a. chain (requires RAG, etc); express membrane o/B TCR (eventually with CD3 as well) TCR is presented self-MHC + self peptide from thymic epithelium (can be MHC I or II): a. IF TCR is triggered strongly (by self MHC + self antigen) cell is eliminated (negative selection) b. If TCR sees nothing that even closely resembles MHC, cell undergoes death by neglect. ©. If TCR recognized the MHC/peptide complex, but with low affinity (tickling), it is positively selected 8. Positive selection yields a single positive cell (CD4+ if it saw MHC Il; CD8+ if it saw MHC 1) Positive selection > leads to self MHC restricted T cell repertoire:: T cells that bind MHC ~ rescued from cell death. CD4 and CD8 also required Due to weak recognition of self-peptide-MHC complexes presented by thymic epithelium. Leads to self-MHC restriction because only thymocytes which express TCRs that recognize self-MHC are selected. Thus the thymus is critical host organ in MHC restriction! ‘Negative selection > leads to development of self tolerance (central tolerance not peripheral). T cells that bind self-MHC plus self peptide are killed by an apoptotic mechanism — Due to high avidity/affinity interaction between thymocyte and peptide-MHC complexes on APC. Fate of thymocyte depends on: (1) number of TCR molecules it expresses; (2) number of peptide- MHC molecules on thymic stromal cells it contacts; (3) affinity of TCR for peptide- MHC complexes; (4) density of CD4 or CD8 on thymocytes can influence strength of signals generated by TCR. Basics of TCR signal transduction: ITAMs on CD3 (esp. ¢) are phosphoylated by sre family kinases (Ick and fyn), then recognized by syk (zap70) family kinases. Downstream yields: (1) survival and proliferation signals; (Qe) atetc extusion at TCR B locus; (3) induction of TCR c-rearrangement; (4) shut off pTo. expression, From all the above description, itis clear that T cell development and TCR rearrangement are similar to B cell development and BCR (Ig) rearrangment, But TCR rearrangement differs from B cell processes in following respects > (1) no somatic mutation (2) no affinity maturation; (3) random use of constant regions rather than isotype switching as in Ig; (4) no functional association with C region; (5) fewer V genes than Ig but more J genes — thus greater junctional diversity MHC and Antigen P ‘ou: MHC = gene structure and functions Physiologic relevance: 1. Determines immune response to foreign protein; 2. Restricts T cell activation to other cells bearing MHC-antigen (no T cell reaction with soluble antigen); 3. Determines which type of T cell different antigens will stimulate. Expression of class I and II largely transcriptionally regulated. Class I widely expressed. Class II differential expression on different cell types eg mononuclear phagocytes induced to express class II by cytokines (IFN-y) whereas B cells and dendritic cells express class II constitutively. Class I MHC (human = HLA-A, B, C; Mouse = H2K, D, L) @ —- secognition by cDe+ 7 cells © structure: a chain + 8-2 microglobulin chain © cytosol generated (‘endogenous’) proteins ['eytosolic space’)© generation of peptide-class I MHC complex is continuous normal function of cells -- does not distinguish between foreign and self antigens ‘Class I] MHC (Human = HLA-DR, DP, DQ; Mouse = 1-A and 1-E) @ © recognition by CD4+ T cells structure: o: chain + B chain endosome/lysosome generated peptide exogenous’) antigens [extracellular space’) ‘The most polymorphic group of genes in all species! Most alleleic variation at amino terminal peptide binding domains. Class 1 heterozygous individual = 6 different alleles (3 each parent); class II heterozygous = 6 alleles but each cell can express 10-20 different class products due to different combinations of o and B chains (and use of more than one fichain). Expressed codominantly (ie offspring expresses alleles from both parents). Each MHC can bind a number of different peptides (interaction not very specific; slow on-off rate = conformation changes?) but only one at a time and specific T cell recognizes only one peptide-MHC complex. Other genes in MHC region : (1) genes for biosynthesis and loading of MHC: TAP 1 and 2 (transporter in antigen processing - mainly for MHC 1); (2) genes for subunits of proteasome (LMP-2, LMP-7) involved in generating peptides from cytosolic proteins incorporated into class I MHC; (3) HLA-DM for peptide binding to class II intigen pri Forms of antigens recognized by T cells: T cells recognize mostly peptides vs B cells which recognize verted proteins, nucleic acids, polysaccharides, lipids, haptens. Thus T cell-mediated response induced only by protein antigen. Uses basic cellular proteolytic mechanisms which also operate independent of immune system — both self and foreign antigens displayed Extracellular proteins (MHC I): © specialized APC’s (macrophages, B cells, dendritic cells) © recognition by CD4+ helper T cells Cytosolic proteins(MHC D): © proteins synthesized inside all cells eg viral, cancer cells, mycobacteria * recognition by CD8+ T cells (CTLs) Assembly/surface expression of MHC I complexes: 1. Gand B2 microglobulin synthesized on rough ER > transported into smooth ER as separate polypeptide chains. a chain associated with chaperones BiP and ealnexin to prevent degradation and promote folding. 2. B2 microglobulin binds partially folded a chain and chaperones dissociate — these dimers are unstable thus associate with the luminal portion TAP (MHC I specific) for transport to ER (and further folding); 3. Meanwhile, cytosolic proteins are ubiquitinated and thus targeted for proteasome degradation,4, Proteasome (barrel shaped with 7 subunits, 3 active subunits) degrades the ubiquitinated protein. Note that «special "plug-in" catalytic subunits LMP-2 and LMP-7 are synthesized in response to IFN-y. The proteasome, when equipped with these subunits, is converted from a general housekeeping proteasome to one that's tuned to reice peptides of proper length and residues for MHC I binding. 5. TAP (transporter for antigen processing) then transports peptides from cytosol to ER, and since TAP is also associated with MHC I Iuminally, the peptide can readily associate with MHC I. 6. Peptide binds and enhances stability causing release of TAP; move through Golgi for carbohydrate modification; transported to cell surface in exocytic vesicles — recognition by CD8+ T cells Assembly/surface expression of MHC II complexes: 1, and B chains coordinately synthesized and associate with each other in ER; heterodimers temporarily associate with ealnexin (folding chaperone) and later the invariant chain (Ii) 2. The invariant chain binds of complex, releases calnexin and complex moves out of ER. It prevents peptides or nascent proteins in ER associating with newly formed class II MHC heterodimers, and it directs newly formed MHC molecules to specialized endosomal/lysosomal organelles where internalized proteins are proteolytically cleaved — thes compartments are called MIIC/CIIV (class II compartment/vesicle) 3. Meanwhile, proteins have been endocytosed and digested by proteases that work optimally in aciding environment of endosomes/lysosomes. These vesicles then fuse with the MIIC/CIIV. 4, In the MIIC, proteases digest a part of the invariant chain, leaving only CLAP (class IT invariant chain peptide) in the peptide binding cleft of MHC Il. 5. CLIP is removed by HLA-DM in MIIC compartment; after CLIP is removed, complexes of antigen peptide 3d class II MHC can form — peptide binding to class II MHC stabilizes a8 heterodimer — so only properly loaded peptide-MHC complexes will survive long enough to reach cell surface by membrane fusion with exocytic vesicles > displayed for CD4+ T cells. Lymphocyte Homing inf a 1. Granulocytes, Lymphocytes, and Monocytes express L-selectin constitutively. Endothelial cells express E- seleetin (and also P-selectin) in response to inflammation (TNF, IL1, LPS). L-selectin binds Mambomin, so Lymphocytes will tend to ROLL along endothelium of venules in inflamed regions. fe ao 2. Chemoattractants (like chemokines) upregulate receptors (usually a cocktail of chemokines specific for each type of cell.) 3, Leukocytes express integrins like LFA-1 whose ligand is ICAM-1 (along with other -CAMS) on endothelial cells. Lymphocytes will tend to STICK to endothelium due to this interaction (upregulatable by LeukotrieneB4). 4, Emigration out of vessel into inflammed tissue. Denroeyie Adhesion Defects: LAD I - CD18 (integrin) defect ~ no sticking LAD IT- fucose metabolism defect ~- required for selectins - no rolling (also no blood group antigens)ial ve 2. TCA-4 is made by HEV which activates CCR7 only on Naive T cell to attract it further (PMN pass 1, HEV cells express PNAd (ligand for L-selectin) so leukocytes will roll. @ through). 3. Naive T cells sticks via LFA-1/ICAM-I and then emigrates into lymph node tissue to survey presented antigens. THI/TH2 cells paradigm: THL: develop via IL12/STAT4 activation pathway; ‘TH2: dey [edits makes IL-2, TNE, IFN-y intracellular microbes (pneumocystis, TB, leischmaniasis); ‘macrophage and neutrophil activation; complement binding and opsonizing antibodies; CTLs velop via IL-4/STATS activation pathway : makes 11-4, IL-5, IL-10 IL-4 stimulates IgE production; IL-5 activates eosinophils eosinophil activation; mast cell degranulation; (allergies) suppression of macrophage activation; (antagonize TH1) @ production of neutralizing IgG antibodies Form of immunity transferred by cells not serum (can be adoptively transferred by viable T cells). A.phases: hypersensitivity); Effector cell is activated macrophage that has phagocytosed microbe, eg Listeria. If against soluble proteins>tissue injury (‘hypersensitivity’) * Sensitization phase - initiation of T cell response> antigens transported by lymph to lymph nodes > naive T cells & APCs interact in node > CD4+ T cells recognize antigen > antigens activate macrophages to produce IL-12 > differentiation of naive T cells into Th! cells. © Migration phase - T cells to site of infection > previously activated T cells migrate to infection site > restimulated by local antigens > antigen-stimulated T cells stay in tissues * Effector phase ~ Til cells secrete cytokines >IFN-y activates macrophages to destroy Phagocytose@iy microbes > > TNF and IFN-y lead to local inflammation. (Also, bidirectional activation of T cells and macrophages: B7-1, B7-2 and CD28; CD40/CD40L) 10‘The DTH reaction is a variant of this, where foreign protein, from a previously encountered bug, is injected (like PPD skin test), and T cells activate macrophages to induce inflammation and tissue damage. The 24-48 hours ee” is due to time for effector T cells home to site of injection Role of macrophages in CMI: CD40L and IFN-y from T cells signal to macrophage > induce expression of B7- 1/B7-2, Fe receptors, class II MHC, IL-12, Nitric oxide «Killing of microorganisms (oxygen radicals ete) Stimulation of acute inflammation ‘Antigen presentation and positive feedback to T cell following class Il and B7 expression Changes in local tissue environment (chronic DTH> fibrosis: PDGF, TGF, FGF) ll Medi relevant for intracellular infections, allograft rejection, tumor rejection. CTLs: CD8+ -- recognize MHC1 Maturation of pre-CTL to mature CTL for lysis Microbes (eg viruses) that infect non-phagocytic cells cannot be eliminated by activated macrophages — thus eliminated by CD8+ cytolytic T cells (although antibodies play role in initial neutralization of viruses). CTL killing (1) antigen specific; (2) requires cell contact; (3) CTLs are ‘serial killers’ - not themselves killed but kill multiple targets. e@ Induction of CTLs: © Pre-CTLs (prior to activation) not active in cytolysis © 2 signals for full activation: 1. eytosolic peptide in association with MHC I 2. costimulators/cytokines produced by other simultaneously activated T cells 2. Mechanisms of CTL lysis © Antigen recognition and conjugate formation: TCR-peptide/MHC; CD2/LFA-3, IFA-I/ICAM © Activation of CTL © Lethal hit: Perforin-mediated osmotic hole in membrane; granzymes enter cells > proteases cleave ICE >ICE-proteolytic cascade leads to activation of DNA cleaving enzymes> apoptosis, © Release of CTL fatural Ki Non B/non T cells with perforin and granzymes. Primitive CTL without CTL specificity ‘NK cells - part of innate immune system against intracellular microbes: © Secrete IFN-y - activate macrophages — kill phagocytosed microbes @ © Lyse infected cells (mechanisms similar to CTLs). uNK cells express inhibitory receptors (KIRs = tyrosine phosphatases) that shut off NK cell responses. : Recognize self class I MHC. Replacement of self peptides in MHC cleft with microbial/foreign peptides (or reduction in class I expression) will release this inhibition and lead to NK activation. There's also a lesser @ known second activating signel required (can be an integrin interaction). NK cell response to secretion of cytokines: NK cells express IFN-y, IL-2, IL-15, IL-12 receptors. High dose Il- 12 converts NK into Lymphokine Activated Killer (LAK) cells. Role of NK: (1) lysis of virally infected cells; (2) activated by innate immune system (I-12, I-15); (3) activate macrophages by secreting IFN-y Relax and good luck!