Week 3 Quiz Section Antigen Presentation/Processing MHC Diversity Antigen Receptor Structure

Week 3 Outline Exam Reminder MHCI and MHCII Antigen processing Immunoevasions Antigen cross presentation Natural Killer cell activation MHC Diversity MHC Restriction Lymphocytes antigen receptor structure and function Affinity vs Avidity Questions

REMINDER! Exam 1: This Friday, October 21 Exam Covers Lectures 1-8 50 minutes, 50 questions: mostly multiple choice, few T/F, and some matching Starts at 9:30 sharp, ends at 10:20 sharp We will supply scantrons, you bring #2 PENCILS Exam Review session: Thursday, October 20, 2 PM - 4PM, T-733 You bring questions! old test online Inflammasome ?? Extra OH??

MHC molecules activate T cells with effector function appropriate to the type of infection MHC Class I + peptide activates CD8 Cytotoxic T cells MHC Class II + peptide activates CD4 Helper T cells Causes killing of cell Activates macrophage to kill intracellular pathogen Induces B cell class switching and somatic hypermutation

1 X 8 = 8 2 X 4 = 8

MHC Class II MHC Class I Intracellular antigen Extracellular antigen Endocytosis Proteosome Phagolysosome ER Bind MHC II Bind MHC I Present on surface Present on surface Extracellular vs. intracellular source

MHC I Processing Require chaperones for stabilization in the ER. MHC Class I molecules are “unstable” until complexed with 2M and peptide 2M – Beta 2 Microglobulin

MHC I Processing These chaperones also hold MHC Class I molecules in an “open” conformation until the binding of high affinity peptides delivered by TAP. The TAP proteins form a peptide transporter in the ER membrane Peptides from old proteins OR intracellular pathogens are translocated from cytoplasm to the ER Process is ATP Dependent TAP1/2 expression is induced by interferons, which signal the presence of viral infection Self antigen presentation Transporters associated with Antigen Processing (TAP)

MHC I Processing The proteasome is a cytoplasmic multi-protein complex that degrades proteins to peptides in the cytoplasm both for normal protein turnover and for MHC Class I binding Ubiquitinated proteins are substrates for the proteasome The proteasome degrades properly folded proteins in the normal process of protein turnover and degrades misfolded proteins or mistranslated protein

MHC I Processing

Immunoevasins for MHC Class I Processing (Adenovirus) (CMV) (HSV) HSV-1 HCMV Adenovirus HCMV (g herpes virus)

MHC II Processing Extracellular antigen Endocytosis Phagolysosome Bind MHC II How do MHC Class II molecules get from the ER to the endosomal network? Why don’t they bind peptides in the ER? Present on surface Invariant chain blocks peptide loading in the ER Cytoplasmic tail of Ii shuttles MHC into endosome Ii digested by cathepsins leaving CLIP CLIP acts as peptide placeholder until displaced by peptide via HLA-DM MHC then expressed at cell surface

MHC II Processing Endocytosed proteins are cleaved into peptides HLA-DM releases CLIP. Peptides can now bind to MHC II Invariant chain blocks MHC II binding spot Ii is cleaved by activated cathepsins

Antigen Cross-Presentation This is an exception to the rule that MHC class I presents peptides generated in the cytoplasm…Dendritic cells, but not other cells, have a specialized mechanism for shuttling peptides taken up by endocytosis or phagocytosis into the MHC class I pathway. If DC phagocytose dying infected cells (or bits of infected cells), they have a specialized machinery to bring peptides from these endocytic compartments out of the endosomal system and into the MHC I presentation pathway.

Natural Killer cell activation Normal, healthy cells express MHC Class I ligands for NK inhibitory receptors and are not killed by NK cells. Virally-infected cells can reduce levels of class I MHC on their surface and also express ligands for NK cell activating receptors. This allows the cells to escape CD8 T-cell detection but can be killed by NK cells.

Polygenic vs Polymorphic Both polymorphism and polygeny contribute to the diversity of MHC molecules expressed by an individual Co-dominance allows for the potential to present a greater variety of peptides

MHC Class I Molecules MHC Class II Molecules Pairing of MHC class II proteins from different chromosomes increases the potential number of MHC molecules expressed Ca Cb Bb Aa MHC Class I Molecules Ba Ab Maternal MHC MHC class I: 3 genes x 2 alleles = 6 different MHC I proteins DPaa DQaa DRaa Ba Ca Aa DPbb DQbb DRbb Bb Cb Ab MHC class II: 3 genes x 2 alleles x 2 (pairing of a and b genes from different chromosomes) = 12 different MHC II proteins DRaa DQaa Paternal MHC DRbb DQbb MHC Class II Molecules DRab DQab DRba DQba

New alleles arise by point mutation and gene conversion MHC allelic variation within peptide-binding pocket and TCR contact residues New alleles arise by point mutation and gene conversion Different MHC alleles bind different pathogen-derived peptides. There is diversity in antigen presentation, not just in antigen recognition (T cell receptor). Diversity in the MHC (as well as TCR) contributes to variety of peptides presented/recognized

Anchor residues are essential for stable peptide binding Different MHC proteins have different anchor residues and therefore bind different peptides MHC I Anchor residues are essential for stable peptide binding Each MHC molecule can bind many peptides, as long as they have the appropriate anchor residues MHC II

Different MHC proteins have different anchor residues and therefore bind different peptides Binds 8-10 aa peptides Green anchor residues define the “peptide-binding motif” of peptides N and C-termini contribute to binding MHC I    Binds peptides 13 aa or longer Green anchor residues define the “peptide-binding motif” of peptides N and C-termini do not contribute to binding and extend past the end of the peptide-binding groove MHC II

MHC Restriction The TCR binds both peptide and MHC MHC restriction: the TCR recognizes foreign antigen only when bound to MHC

MHC Restriction MHC restriction: the TCR recognizes foreign antigen only when bound to MHC Doherty and Zinkernagel, 1974 Nobel Prize, 1996

Lymphocyte Antigen Receptor Structures

Antibody Structure Antibody molecules are made of four polypeptide chains: Two identical Light or L chains Two identical Heavy or H chains Abs have TWO identical antigen binding sites made of variable (V) regions of the H and L chains. The less diverse regions of the H and L chains are called constant (C) regions. variable vs constant: tells you which genes encoded those regions light vs heavy: tells you which regions were combined into a single peptide Fab: fragment, antigen-binding Fc: fragment, crystallizable

Comparing BCRs/Igs and TCRs Membrane-bound or secreted Two antigen-binding sites Binds native antigen (any kind) Heavy chains and Light chains Common Features One antigen specificity per cell Homologous Variable (V) and Constant (C) regions 6 CDR/hypervariable loops per antigen- binding site Antiparallel β sheets “rolled” into barrels (the Ig fold) TCR: Membrane-bound only One antigen-binding site Binds peptide antigens only MHC presentation required ɑ chain and β chain Assisted by co-receptors (CD4 and CD8) Cognate antigen: Describes two biomolecules that normally interact such as an enzyme and its normal substrate or a receptor and its normal ligand.

Hypervariable regions within variable domains determine antigen specificity Remember that the antigen-binding sites are at the ends of the variable regions of these molecules This is where the CDR loops are (complementarity determining regions) FR: framework region Having increased variability at these sites allow for fine tuned specificity in binding antigens The antigen-binding sites are arranged in antiparallel beta sheets

T cell co-receptors bind to invariant regions of MHC I and II conversely, the coreceptors bind the invariant regions on the MHC to stabilize the TCR:pMHC interaction knowing which regions of these molecules are invariable and variable are important for understanding their function

Non-covalent antigen/antibody interactions are defined by affinity and avidity Affinity = strength of one single antigen:antigen receptor interaction Avidity = overall strength of combined interactions Low affinity High affinity **Multiple interactions decrease likelihood of antigen dissociation.** Eraser demonstration! https://www.abdserotec.com/antigen-antibody-interactions.html

Avidity can refer to either antibody multimers or a single antibody engaging all of its antigen-binding sites on a multivalent antigen All antibodies are either bivalent or multivalent (IgM). https://www.abdserotec.com/antigen-antibody-interactions.html

Questions?

Supplementary Slides

MHC molecule expression by cell type Professional antigen-presenting cells

IMMUN 441: Quiz Section AE TCR/BCR Comparison Worksheet 10.22.2015 TCR Localization Membrane-bound Membrane-bound OR secreted # of antigen binding sites/receptor One Two # of antigen specificities/receptor Types of antigen that can be bound Peptide antigens All native antigens (peptide, carbohydrate, lipid) Structural subunits α chain, β chain Heavy chain, light chain # of CDR/hypervariable loops per antigen-binding site 6 (3 HV from α + 3 HV from β) 6 (3 HV from HC + 3 HV from LC) Important co-receptors CD4, CD8 None Composition of antigen binding site 1 variable region of α + 1 variable region of β 1 variable region of HC + 1 variable region of LC Requirement of MHC presentation Yes No (can bind soluble antigens) Presence of Ig fold (antiparallel β sheets that form a rolled β barrel)

IMMUN 441: Quiz Section AE TCR/BCR Comparison Worksheet 10.22.2015 Localization # of antigen binding sites/receptor # of antigen specificities/receptor Types of antigen that can be bound Structural subunits # of CDR/hypervariable loops per antigen-binding site Important co-receptors Composition of antigen binding site Requirement of MHC presentation Presence of Ig fold (antiparallel β sheets that form a rolled β barrel)

proteasome, & proteasome Most cells Professional APCs CD8 CD4 8-10 amino acids 13-? amino acids Yes No Closed Open No No Cytoplasmic proteins Endocytic vesicles proteasome, Invariant chain (Ii), CLIP, HLA-DM TAP, proteasome & proteasome