Immunology Notes Week 5 - T Cells (September 2024)

Week 5 - T cells Saturday, September 28, 2024 2:19 PM Wednesday, September 25, 2024 9:02 AM Antigen recognition T cell development - Comes from common lymphoid progenitor What is a T cell - Charateristic marchers - thy1, cd4 or cd8, etc - Regulatory and effector cell type What d...

Week 5 - T cells Saturday, September 28, 2024 2:19 PM Wednesday, September 25, 2024 9:02 AM Antigen recognition T cell development - Comes from common lymphoid progenitor What is a T cell - Charateristic marchers - thy1, cd4 or cd8, etc - Regulatory and effector cell type What do t cells do - Recognize antigen on MHC - Help B cells produce antibodies - Help other leukocytes - Directly kill virally infected cells Flavors of t cells - Alpha/beta vs gamma/delta ○ a/b (nearly?????) universally recognize only peptide/MHC WHAT § CD4 vs CD8 ○ g/d also recognize lipid/MHC or even whole protein - Treg and NK Inherited deficiencies - SCID - severe combined immunodeficiency - Athymic (mice) Acquired immmunodeficiency - ex. AIDS - Loss of T cells What do T cells do? - Making contact with other cells ○ Cd8 to MHCI --> cell dies ○ CD4 to macrophage --> more activated CD4 to B cell --> activated plasma cell!!! T DOES HE MEAN NEARLY  ○ ○ CD4 to B cell --> activated plasma cell!!! Understanding MHC molecule - MHC restriction ○ Recognize peptide AND MHC for activation T cell receptor - Clonality Germ line ○ Each cell has a unique receptor - Diversity of TCR ○ Germ line configuration ○ Alpha chain doesn’t have D, only VJ ○ Beta chain is VDJ § CAN HAVE MORE THAN ONE D SEGMENT ○ N region of addition in all TCR genes (compared to Only H of Ig) ○ Most of the diversity is at the junction regions (between the joined regions) ○ NO SOMATIC HYPER MUTATION OR CLASS SWITCHING § You get what you get - Co-crystal structure of TCRs and MHC ○ CD4 interacts with MHCII while the TCR joins business end to stabilize interaction ○ CD8 also does this - CDR1 through 4 make the contact with the peptide ○ Most diverse region ○ MOSTLY CDR3 ○ Complementarity determining region - TCR/CD3 - fun facts ○ TCR a/b non-covalently to CD3 gamma, epsilon, delta and zeta ○ WHAT ELSE - TCR vs BCR ○ T cells recognize linear epitopes ○ Unique specificity ○ TCR DOES NOT PERFORM EFFECTOR FUNCTIONS § Like it stays anchored to membrane (compared to Ig can be secreted and d T cell development - THYMUS ! - Early development ○ T cell precursors travel from bone marrow to thymus ○ Mature cells leave thymus and go to secondary lymphoid tissue  homodim do stuff Cortex - cortical epithelial cells Medulla - medullary epithelial cell  ○ Mature cells leave thymus and go to secondary lymphoid tissue - First ○ Rearrange BETA chain ○ Then ALPHA chain - The experiment that determined thymus imparts MHC restriction ○ Heterozygous (a/b) MHC mouse § Thymectomy § Lethal irradiation § Strain B thymus graft (MHC b only) § Bone marrow a/b graft § Infect with LCMV § Take spleen cells Strain A cells Strain B cells No killing Yes killing ○ The control: § Same experiment but leave the thymus, get A and B type killing - Thymic development is catagorized by expression of CD4 and CD8 ○ Checkpoint 1 Only 2% § preTCR CD4- CD8- □ TCR B and preT alpha □ Or they form a gamma delta chain and that stops other rearrangeme □ WHATS THE PRE T ALPHA ® Supports TCRB to get to surface and signal to move to next ste ® Purpose is to shuttle Beta chain □ Unclear is preTCR requires a ligand or if it being on the surface is eno signal to move on § DIVIDE § Become double positive □ (CD4+ and CD8+) If they don’t make an AB receptor, elimina § And have TCRA and TCRB ○ Checkpoint 2 § Can it interact with MHC § Too high avidity --> ELIMINATED RIP § Low avidity MHC I --> become CD8 single pos □ RUNX3 § Low avidity MHC II --> become CD4 single pos □ ThPOK % of cells make it out of the thymus ent ep ough ated  Low avidity MHC II --> become CD4 single pos □ ThPOK § Low/no binding, death by neglect § Positive selection □ Can you bind MHC □ Epithelial cells □ How do T cells decide CD4/8 fate? ® Models: ® New hypothesis: maybe it’s the regularoty regions of cd4 and c determine the lineage § Negative selection □ Dendritic cells □ T cell can still bind MHC but not too strong, goes on forward ○ Spatial restriction of thymocyte development § See picture ○ What about the peptides § The MHCs have peptides in them!!! In the thymus □ One mechanism is that the thymic APCs express tissue specific antige Mediated by AIRE (autoimmune regulator) ® Not really a TF but modulates transcription ® Inherited mutations in AIRE --> ◊ Failure to delete autoreactive T cells in the thymus □ Regulatory T cells ® Recognize antigen with high affinity, not deleted, turned into T ® CD4 pos TCR Tg mice as a tool to study T cell dev - Remember allelic exclusion - Force expression of a known TCR a/b of known specificity in all T cells (MOSTLY) preven endogenous TCR chains ○ Allows study of T cell activation and dev in a less complex (and less normal ") se ○ Provides a way to track/isolate endogenous antigen specific T cells ○ One problem: TCR interactions with peptide/MHC are low affinity § Solve with tetramer of MHC/peptide complex on streptavidin beads T cell Activation - Get much bigger with activation - Sequence of events - First signalling event is the phosphorylation of CD3 and zeta chain by LCK on the CD4 /c ○ CD3 and zeta chains have ITAM regions that have tyrosines that are phosphorylat LCK (also sometimes FYN) § KICKS OFF DOWNSTREAM EVENTS cd8 that Approach: - Express CD8 from the CD4 regulatory region - What happens to development ○ They could switch development ○ The CD8 and CD4 mice switched their TFs (CD8 mice had ThPOK and CD4 - Conclusion - not determined by TCR strength but by TCR signaling duration ens --> T regs nts etting cD8 ted by  § KICKS OFF DOWNSTREAM EVENTS □ Phosphorylated ITAMs recruite ZAP70 which binds the phosphorylate tyrosines and signals downstream □ Zap70 phosphorylates PLCgamma1 ® Leads to NF-AT and NF-KB □ and LAT? □ § NF-AT pathway □ IP3 helps intracellular calcium be released from stores □ Calcuym combines with calmodulin □ Activates caciner=yrin □ Dephosphorylates NFAT so it can enter the nucleus and transcribe ac ® Ex. IL-2 □ DRUGGING THIS PATHWAY ® To stop NFAT from being dephosphorylated and thereby reduc activation □ There are analogous pathways in B cells ® Still get activation of PLCgammaTWO - Secondary signals ○ Need CD4 and CD8 ○ CD28 (signal 2) binds B7 on APC § Constituatively expressed in T cells § Needed for full activation § (if it sees antigen only on another cell, induces anergy unless/until activated § Function is a little unknown, □ Apparently modulates parts of the signaling pathway ® Nfat, nfkb, ap1 - CTLA4 comes up with activation ○ Homologous with CD28 ○ Stronger interaction with B7 than CD28 § Drug - CTLA4-IG --> B7 blockade ○ Important for maintaining T cell homeostasis Interactions with other cell types - Different classes of costim inhibitory and adhesion responses ○ Ex. Adhesion is LFA-1 to ICAM-1 § Aids stability of T cell/APC contact § This is the initial binding to allow interaction between the cells § TCR causes APC to have higher affinity at the LFA1/ICAM1 interaction to all ed ctivation ce d by APC) low Saturday, September 28, 2024 2:19 PM Week 4 - B cell development and Ab generation - Dr. Singh 9/18 BCR/Ig because of duality of function Central and peripheral tolerance Hallmarks of adaptive immunity ○ Clonality § Each cell expresses receptor of unique specificity ○ Specificity ○ Diversity ○ Repertoire § Public (similar receptor emerges across people experiencing the same disease) and private (specific to an individual) clones specificities TCR is monovalent while BCR is bivalent How to generate diversity of repertoire ○ Throws away evolutionary knowledge??? WHY would they do that?? § The innate system reflects the history though pamps etc § B and T cells have no (very low) evolutionary knowledge, its an anticipatory system they don't know what you might encounter Antibody structure ○ Heavy chain to light chain held by (INSERT KIND OF BOND) ○ Heavy chains connected by two disulfide bonds ○ Fc (constant portion) (stands for fragment crystallizable) to Fab (light chain to heavy chain branch region) (cleaved by protease) ○ Backbone is similar across BCR and TCR Complementarity determining regions Ig and TCR, key unique structural features ○ Ig § Membrane bound § Secreted for humoral immunity □ Different structural types (IgM, IgG, etc) § New B cells are all membrane IgM+, dont secrete until after antigen encounter § Higher ag affinity than TCR generally speaking ○ TCR § Only membrane bound Antibody versatility of structural format ○ Heavy chain and light chain both have three highly variable regions  § Membrane bound § Secreted for humoral immunity □ Different structural types (IgM, IgG, etc) § New B cells are all membrane IgM+, dont secrete until after antigen encounter § Higher ag affinity than TCR generally speaking ○ TCR § Only membrane bound Antibody versatility of structural format ○ Heavy chain and light chain both have three highly variable regions ○ V(D)J recombination in the CDRs and then SHM (Somatic Hyper Mutation) ○ Class Switch Recombination (CSR) switching of the Fc region Signaling upon antigen contact ○ BCR also internalizes whole complex (receptor mediated endocytosis) § Proteolysis § Loaded onto class II molecules § Activate CD4 T cells ○ BCR is associated with CD79 A and B, these molecules do the signaling, Receptor is signaling inert (same as TCR, CD3 signals there) How are B cells activated? ○ Look this up what does it take to move to plasma cell and undergo SHM Functions of antibodies ○ Neutralization ○ Fix (activate) complement to lyse pathogen ○ Signaling/activating other immune cells by binding Fc receptors on macrophages, NK cells, etc V(D)J recombination ○ Each locus has multiple versions (many many V, 5-10 of D, 10ish J segments) and each encode functionally different protein segments ○ DJ get combined first (removes intervening DNA) ○ And then brings in one of the V segments so they are all spliced together and not all spread out ○ LIGHT CHAIN ONLY HAS VJ (NO D SEGMENTS) § And there are two light chain options (kappa or lambda if kappa is no good) ○ All of these are mediated by RAG 1 and 2 (site specific recombinase) § All other cells have germ line configuration of VDJ locus § RAG genes only expressed in developing B and T cells ○ AID only expressed in activated B cells, § class switch recombination and somatic hypermutation while keeping antigen specificity ○ Heavy chain is first, followed by light chain rearrangement  recombinase) § All other cells have germ line configuration of VDJ locus § RAG genes only expressed in developing B and T cells ○ AID only expressed in activated B cells, § class switch recombination and somatic hypermutation while keeping antigen specificity ○ Heavy chain is first, followed by light chain rearrangement ○ Recombination step by step § rag1/2 recognizes recombination signal sequences (RSS) § rag1/2 nicks one strand of DNA § Free 3’ OH group attacks second strand producing double strand DNA break § Hairpin opened by artemis § Terminal deoxynucleotidly transferase TdT adds/deletes nucleotides § Relegation of broken DNA ends by DNA repair enzymes § Some rules □ Spacers/RSS have to be different lengths (12 and 23) (cant be two 12 or two 23) □ Usually deletional joining, sometimes inversional joining ® Head to head vs head to tail ◊ Head to tail does not remove intervening DNA ○ Recombination errors - clinical features § Severe deficiency - failure to initiate or complete VDJ § Hematological malignancy - recombination recognition mistakes as drivers or secondary hits in leukemogenesis (recombines areas it shouldnt) (sometimes interchromosomal recombination) B cell development in fetus and adult ○ Fetal liver ○ Adult bone marrow § From HSC → pro B (rags for the first time) (rearranged heavy chain) → pre B (functional heavy chain expressed with surrogate light chain function is to test quality of heavy chian) → immature B (successful light chain rearrangement and express permanent, signaling competent BCR) → spleen transitional B-2 B cell, marginal zone or follicular B-2 ell Quality control checkpoints ○ Is the antibody competent for signaling? § Test heavy chain first then rearrange and test light chain ○ Is the antibody self reactive § If it is self reactive, 3 possible fates: □ Receptor editing, new Ig light chain rearrangement □ Deletion via apoptosis □ Reduced receptor expression or signaling leading to anergic B cell ® Why would you want this Saturday, September 28, 2024 2:17 PM Week 3 - MHC - Dr. Binder MHC gene complex ○ Chr 6 (chr17 in mice) ○ Set of related genes (listed names for people)(gene names are different in mice) § Class 1 locus - u get 6 versions, ABC from mom and ABC from dad □ A ® Alpha chain, transmembrane domain, and makes peptide binding domain ® Supported by B2 macroglobulin (not part of MHC locus)(doesnt engage peptide, only for structure □ B □ C § class 2 - same here u get 6 versions but the Alpha and Beta can scramble and pair with each other □ DP - can only pair with itself, cant pair with DQ etc ® Alpha chain and B chain, both have transmembrane regions ® Both chains form peptide cleft ® Both part of MHC locus □ DQ □ DR § and class 3 locus □ Complement and cytokines (LTB, TNF, LT) ○ Both parental alleles of each MHC genes are expressed § This increases the number of different MHC that can present peptides to T cells § The most polymorphic gene And polygeny ○ More than one copy per chromosome Both these qualities add to the diversity Class I ○ All cells with a nucleus (not red blood cells)  And polygeny ○ More than one copy per chromosome Both these qualities add to the diversity Class I ○ All cells with a nucleus (not red blood cells) ○ Regular trash ○ CD8 CTLs can kill any virus infected cells ○ 8-10 amino acids Class II ○ Professional APCs, DCs, Macrophages, B cells ○ 11-20 amino acids How peptides bind MHC ○ Peptide binds between two alpha helicies, nice and cozy hotdog style ○ Structure of the peptide § Has one or two anchor residues that bind to the floor of MHC molecule § Has residues that stick out, more likely to bind T cells ○ Anchor residues consensus have more common positions and residues Features and significance ○ Displays one peptide at a time § T cell only responds to one at a time ○ Low affinity, broad specificity § Many peptides can bind it ○ Very slow off-rate § Has to display long enough for T cells to look at it § Can stay up to a day ○ Peptides are acquired during intracellular assembly § Class I and class II MHC molecule display peptides from different cellular compartments § Class I and class II are assembled in different physical space ○ Stable expression requires peptide § Only those with peptide are expressed for recognition by T cells ○ MHC binds only peptides MHC II pathway - pathogen derived peptides ○ ER makes MHC 2 and invariant chain (to plug the binding pocket) § MHC II gets its own vesicle § Vesicle fuses with endocytic vesicle so the variant chain is removed and MHC II binds endocytosed extracellular peptides instead § MHC I pathway ○ Cytosolic microbe proteins degraded in cytosol, make their way to the ER  removed and MHC II binds endocytosed extracellular peptides instead § MHC I pathway ○ Cytosolic microbe proteins degraded in cytosol, make their way to the ER ○ MHC I made in the ER, binds to these peptides and sent to surface via vesicle Cross presentation pathway ○ Phagocytosed peptides make their way to MHC I somehow The peptide loading complex ○ MHC I held in place by other proteins until B2m and peptide is bound so it can be sent to surface ○ MHC is very unstable until then ERAP - MHC is loaded with peptide thats too long at the N terminus ○ ERAP removes N terminal AAs one at a time to cut it to size How do T cell receptors and MHC molecules dock? ○ Wasnt listening ○ Cell type thats displaying the molecule triggers different responses Thymic selection and MHC ○ Selects T cells with intermediate interaction with MHC and peptide (strong and weak interaction are negatively selected) ○ MHC and transplantation ○ Two mechanisms of graft rejection § Direct □ TCR recognises foreign MHC regardless of peptide and destroys the cell § Indirect □ Foreign MHC is acquired by APC and presented on recipient MHC to recipient T cells as foreign HLA typing and nomenclature ○ HLA-A § Mouse, A means class 1 ○ Theres rules for matching to ensure survival ○ Have to match class I and class II all the genes for it to work § 2*10^18 possible combinations of MHC → more than there are people Immune evasion ○ Many ways to interfere § Prevent loading § Prevent expression Takeaway slide!!!!!!! Saturday, September 28, 2024 2:17 PM Week 2 - innate immunity Dr. Binder 09/04/2024 Innate immune system compared to adaptive ○ Faster (minutes and hours) ○ Specific for patterns associated with pathogens ○ Limited number of germ line encoded receptors ○ Perfect self/nonself discrimination ○ Proteins, complement, ifns Immunity in multicellular organisms ○ plants/invertebrates/etc § Do not have t and b cells Exposure to infectious agents ○ What to do to cause infection § Adhere to epithelium § Breaching of barriers □ First encounter macrophages and dendritic cells ○ How does the immune system change its response depending on the pathogen? §  § ○ Barriers to infection § § ○ Pathogen recognition § PAMP (pathogen associated molecular patterns)  § ○ Pathogen recognition § PAMP (pathogen associated molecular patterns) § PRRs bind PAMPS (pattern recognition receptors) § PRRs located throughout (extracellular, cell surface, intracellular) § Once triggered, elicits response § Neutrophils, dendritic cell, macrophage, NK cells express PRRs ○ Cellular elements of innate immunity § Lineage chart § Originate in Bone Marrow ○ Macrophages and dendritic cells mature from monocytes, leave into tissues ○ Neutrophils are resident in the blood, only go in the tissue during infection Macrophage defenses? ○ Phagocytosis § Bring microbe into a vesicle □ Fuse vesicle with lysosome (contains lysosomes and other degradative enzymes) ○ Respiratory burst § Increase in oxygen consumption by macrophages □ Membrane associated NADPH oxidase converts O2 → O2- □ Superoxide dismutase converts superozide ion into hydrogen peroxide O2- → H2O2 → toxic for pathogens □ What else on this slide ○ Defensins § Small cationic antimicrobial peptides found in plants, insects, fungi, reptiles, birds and mammals, dont know how they work but they are effective at killing bacteria… ○ Lactoferrin § Bind up iron? ○ Cytokines secreted by macrophages and DCs § IL-1, IL-6, IL-8, IL-12, and TNFa ○ Complement system § Two triggers - □ Classical pathway (C1, c4, c2) ® (antibody/antigen complex) □ Alternative pathway (factor B, D, and P ® Microorganisms cell wall and polysaccharides § Both pathways trigger C3 into C3b C5b C6 C7 C8 C9 - creates PORE AND INSERTS INTO MEMBRANE  □ Classical pathway (C1, c4, c2) ® (antibody/antigen complex) □ Alternative pathway (factor B, D, and P ® Microorganisms cell wall and polysaccharides § Both pathways trigger C3 into C3b C5b C6 C7 C8 C9 - creates PORE AND INSERTS INTO MEMBRANE § C3b → opsonization § C3a → inflammation Neutrophils ○ Not present in normal healthy tissue ○ Short lived ○ Eat a lot and then they die quicker PRRs ○ These patterns are absent or invisible in the host ○ Surface of macrophages, DCs, neutrophils, secreted, or intracellular ○ Mannose binding lectin (MBL) § Free in the blood § Recognizes mannose (carbohydrate) § It is a 6 part dimer (see picture) § Works because host mannose is not abundant enough to trigger § Facilitates phagocytosis via opsonization § Facilitates complement initiation § Part of the family of Surfactant proteins ○ Macrophage mannose receptor (MMR) § Cell surface receptor § Calcium dependent lectin § Functions as a phagocytic receptor directly ○ Scavenger receptors § Cell surface phagocytic receptor 1-6 § Recognizes anionic polymers and acetylated LDLs § Present in the host but shielded by sialic acid on host cells § Leads to phagocytosis of microbes ○ fMLP receptors § Recognizes fMet-Leu-Phe the start of many bacterial polypeptides § Not a phagocytic receptor!! § Leads to induction of cytokines § Methionine present in host but not formylated as it is in pathogens ○ Toll-like receptors (TLRs) § Engagement leads to cellular response that deal with the specific pathogen type □ dsRNA binds TLR3 → interferon (antiviral) for example § Recognise different microbes, placed accordingly □ 3, 7, 8, 9 ○ Toll-like receptors (TLRs) § Engagement leads to cellular response that deal with the specific pathogen type □ dsRNA binds TLR3 → interferon (antiviral) for example § Recognise different microbes, placed accordingly □ 3, 7, 8, 9 ® Inside cells, recognise viruses (nucleic acids) □ 1,2,4,5,6 ® Recognize bacteria, outside cells ® 2 and 6 heterodimer ® 1 and 2 heterodimer § Macrophages and dendritic cells across the board ○ Cooperation of PRRs - LPS, CD14, and TLR4 § LPS - cell wall gram neg bacteria § LPS is recognised by TLR4 in combination with MD-2 and CD14 □ LBP binds LPS, transfers LPS to CD14 □ MD2 forms complex with TLR4 □ That complex binds with CD14 to trigger signaling ○ TLR signal transduction pathway § Usually the MyD88 pathway § TFs bind DNA and lead to specific cytokine sets depending on the what TLR was activated ○ NFKB signaling in cells § See diagram ○ Sunday, September 22, 2024 4:32 PM Week 1 - 08/28 - basic review Hinterleitner lab - mucosal immunology in hte gut Non self vs self; innocuous vs dangerous 4 classes of pathogens ○ Parasites ○ Viruses ○ Fungi ○ Bacteria Smallpox vaccine + eradication of smallpox 3 big killers ○ TB, malaria, HIV New diseases ○ Ebola, sars, flu Innate mechanisms and adaptive mechanisms ○ Innate mechanisms § Mechanical □ Flow of air or fluid □ Mucus □ Tears and nasal cilia § Chemical □ Fatty acids, enzymes □ Low ph § Microbiological § Within hours ○ Adaptive § Takes longer § Builds upon innate response All immune cells originate in the bone marrow ○ Arise from hematopoietic stem cells ○ See flow chart ○ B cells differentiate in bone marrow, t cells in the thymus ○ Secondary lymphoid organs Innate immune mechanisms ○ Blood proteins § Complement  ○ See flow chart ○ B cells differentiate in bone marrow, t cells in the thymus ○ Secondary lymphoid organs Innate immune mechanisms ○ Blood proteins § Complement § Mannose binding lectins (recognize microbial carbs) § C-reactive protein (recognize microbial membranes) ○ Receptors in all locales § Surface □ Toll like □ Mannose □ scavenger § Cytoplasm □ Card family □ Nod like § Endosomal □ Toll like ○ Destroy § Macrophage and neutrophils § Acidification § Toxic o2 and nitrogen § Etc ○ Some bacteria take up residence in cells → need adaptive response Adaptive basics ○ Clonal selection ○ Different types of CD4 depending on type of infection § 1, 2 17, FH, and tregs Tools ○ Flow § MFI - how much of a receptor is expressed ○ KO mice § Use littermate controls in every experiment § Microbiota matters!!! ○ Spectral flow § Uses prisms to look at more colors - how???