microbiology 204 tcr structure art weiss october 19, 2015
TRANSCRIPT
![Page 1: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/1.jpg)
Microbiology 204
TCR Structure
Art Weiss
October 19, 2015
![Page 2: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/2.jpg)
T cells and B cells use Distinct Antigen Receptorsto Recognize Fundamentally Different Forms of Antigen
B cells can recognize either linear or conformational epitopes on cell surfaces, of proteins, of carbohydrates or of lipids. The B cell antigen receptor is a form of membrane Ig.
T cells generally recognize “only” linear peptide fragments that are bound to MHC class I or class II molecules.
![Page 3: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/3.jpg)
MHC Restricted Recognition of Antigen
Zinkernagel and Dougherty
Bevan
Mid -1970’s
T cells only recognize specific peptide antigen in the context of self: MHC restriction.Specificity for self recognition is encoded in the MHC (Major Histocompatibility Complex).
![Page 4: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/4.jpg)
MHC Restriction: How does the TCR simultaneouslyrecognize MHC specificity and antigen specificity?
• One receptor or two receptors?• Structure of the MHC provides the immediate insight.• MHC molecules are designed to present peptides.
So, T cells simultaneously recognize a single peptide and MHC molecular complex!
![Page 5: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/5.jpg)
Binding of Class I and Class II MHC Molecules to Peptide Ags
![Page 6: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/6.jpg)
Identification of the TCR Protein
Generation of T cell clone-specific monoclonal antibodies (Allison, Reinherz, Kappler and Marrack, ‘82-’83)
![Page 7: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/7.jpg)
Biochemical Characterization of the TCR
Biochemical Characterization:1. Disulfide-linked heterodimer2. Transmembrane protein3. Constant and variable regions4. Both chains are glycoproteins
Non-reducing (first)
Reducing (second)
* *
![Page 8: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/8.jpg)
Cloning the TCR -chain cDNAHedrick and Davis, 1984Yanagi and Mak, 1984
Predictions:
1. T cell specific
2. Transmembrane protein
3. Genes should be rearranged in T cell but not in non-T cells
4. cDNA should encode Constant and Variable domains
![Page 9: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/9.jpg)
Isolation of TCR -chain cDNA(Hedrick and Davis, 1984)
T Cell B CellPolysome
mRNA mRNA
cDNA
Hybridize T cell cDNA withExcess B cell RNA
Isolate single stranded T cell specific cDNA (flow through - Hydroxyapatite columns)
Prepare labeled T cell specific cDNA probe *
Hybridize to T cell minus B cell cDNA library
![Page 10: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/10.jpg)
TCR -chain cDNA
L V D J C TM
Southern Blots: evidence for rearrangement (J-region probe)
Liver T cell T cell Kidney clone 1 clone 2
Liver T cell T cell Kidney clone 1 clone 2
EcoR1 Digest BamH1 Digest
![Page 11: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/11.jpg)
Both Chains of the TCR Heterodimer are Involved in Antigen and MHC Recognition
![Page 12: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/12.jpg)
Anti-HY
H-2b
Isolate
cDNA
cDNA
Transfect aloneor together
Anti-NP
H-2d
Yes: Anti-HY / H-2b
Yes: Anti-NP / H-2d
No: Anti-HY / H-2d
No: Anti-NP / H-2b
and chains of the TCR do not separately encodeMHC or antigen specificity
![Page 13: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/13.jpg)
![Page 14: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/14.jpg)
T Cells
• Express TCR heterodimer instead of the TCR heterodimer • Distinct lineage of T cells • Most resting T cells lack CD4 and CD8 coreceptors • Activated T cells can express CD8 • Minor subset in mouse and man (2-5%). Epithelial localization predominates. • Expressed early in ontogeny • MHC Restriction/recognition – little good evidence for “MHC restriction”, reactivity to some non-classical MHC molecule is well-documented, but there is no evidence for requirement
• Function:- Secrete lymphokines and mediate cytotoxicity- Role in bacterial infections (mycobacterial, and others)- Respond to non-peptidic ligands i.e. bacterial phospholipids, alkylamines, heat shock proteins,
![Page 15: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/15.jpg)
V segments 2 exons 80-250 nucleotides CDR1 and CDR2 (V encodes CDR4)
J segments 1 exon 47-76 nucleotidesCDR3
D segments 1 exon 9-16 nucleotides
Human TCR Gene Loci
![Page 16: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/16.jpg)
TCR Gene Rearrangement Occurs SequentiallyDuring T cell Ontogeny
![Page 17: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/17.jpg)
Unusual Organization of TCR Gamma/Delta Genes
Enormous Potential of Diversity in Delta Rearrangements
![Page 18: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/18.jpg)
Generating a Diverse TCR Repertoire
1. Recombination of different gene segments (V, D and J segments)
2. Recombination of different numbers of gene segments ( locus)
3. Imprecise joining of gene segments
4. “P” and “N” nucleotide addition (TdT)
5. Assembly of different combinations of rearranged and chains
However, unlike immunoglobulin genes, somatic mutation of TCR genes does not take place.
![Page 19: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/19.jpg)
Comparison of Diversity Generated in TCRand BCR Assembly
Ig TCR TCR H L
Variable (V) segments 45 35 45 50 5 2
Diversity (D) segments 23 0 0 2 0 3
D’s in all frames rarely - - often - often
N-region addition V-D, D-J None V-J V-D, D-J V-J V-D1, D1-D2, D1-J
Joining segments 6 5 55 12 5 4
Total potential diversity ~ 1011 ~ 1016 ~ 1018
![Page 20: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/20.jpg)
Unusual Features of TCR Recognition of MHC molecule/peptide Complex
Simultaneous recognition of MHC specificity and peptide specificity
TCR affinity for peptide and MHC is very weak relative to antibodies:Kd of 10-5 to 10-7 M for TCRKd of 10-7 to 10-11 M for Ig(Based on solution binding of monomers – flawed analysis)Main determinant is off rate
Cell-Cell interaction context (avidity issues, coreceptors, particles/diffusion)
Tetramers of MHC/peptide can bind with high avidity
Exquisite specificity despite low affinity:agonist peptidesaltered peptide ligandsantagonist peptides
![Page 21: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/21.jpg)
Crystal Structure of an TCR - Class I MHC/peptideGarcia, et al., Science, 274:176, 1996
![Page 22: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/22.jpg)
Are there Different Orientations of and TCRs in Antigen Recognition?
Adams et al, Science, 2005
![Page 23: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/23.jpg)
Is the TCR and Class II MHC/peptide InteractionOriented Differently?
Reinherz, et al., Science, 286:1867, 1999
I-A alpha chain
I-A beta chain
TCR-Class II peptide/MHC complex
TCR-Class I peptide/MHC complex
![Page 24: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/24.jpg)
Distinct Orientations of Different TCR/MHC-peptideComplexes
Hennecke and Wiley, Cell, 104:1, 2001
![Page 25: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/25.jpg)
CDR Loops Are Involved in Distinct Recognition Functions
Garcia, Trends Immunol., 2012
![Page 26: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/26.jpg)
TCR Germline Bias for MHC recognitionGarcia, Trends Immunol., 2012
40 TCR-Class I MHC structures
Structures of limited V or V containingTCRs with different Peptide antigen specificitiesshow similar TCR/MHC interactions
![Page 27: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/27.jpg)
The TCR Can Interact with MHC/peptide ComplexVia Many Different Biochemical Interactions
Hennecke and Wiley, Cell, 104:1, 2001
![Page 28: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/28.jpg)
Distinct Structural and Energetic Ways that the TCR Uses for Antigen Recognition
Godfrey, et al, Immunity 2008
![Page 29: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/29.jpg)
Distinct Conformations of the TCR CDR3 Loops in theLigand-Unbound and Bound States
![Page 30: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/30.jpg)
Model of a Flexibility in CDR3 During Peptide/MHC Docking
![Page 31: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/31.jpg)
Two Step Model for TCR/MHC-peptide RecognitionWu et al, Nature 418:552, 2002
![Page 32: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/32.jpg)
The Same TCR can Adopt Distinct Conformations to be Polyspecific
Allo-MHCplus peptide
Self-MHCplus peptide
Colf, et al., Cell, 2007
![Page 33: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/33.jpg)
YAe62 TCR bound to IAb-p3K
YAe62 TCR bound to Kb-pWM
A Single T Cell Receptor Bound to Major Histocompatibility Complex Class I and Class II Molecules Reveals Different
Ways CDR Loops Can Interact with pMHCYin et al, Immunity, 2011
![Page 34: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/34.jpg)
Michael E. Birnbaum, et al., Cell, 2014
A Single TCR can Recognize Hundreds of Different Peptides Which Share Some Common Features
![Page 35: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/35.jpg)
Superantigens
Bacterial enterotoxinsStaphylococcal, Streptococcal and Mycobacterial
Minor lymphocyte stimulating (Mls) antigenEndogenous mouse retroviral products
Unidentified endogenous antigens
![Page 36: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/36.jpg)
Comparison of Superantigens and ConventialPeptide Antigens
Conventional Antigens Superantigens
Frequency of responsive T cells 1 in 104 to 105 1 in 4 to 20
Interaction with the TCR + +
Interaction with MHC + +
MHC restricted recognition + -
Requirement for processing + -
Binding to peptide groove in MHC + -
![Page 37: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/37.jpg)
SEB/TCR/MHC Structural Model
SEBSEB
![Page 38: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/38.jpg)
Superantigens have Relative Specificityfor V Segments
V specificityToxin
Human Mouse
SEA ? 1, 3, 10, 11, 17SEE 5.1, 6.1-3, 8, 18 11, 15, 17SED 5, 12, ? 3, 7, 8.1-3, 11, 17SEB 3, 12, 14, 15, 17, 20 3, 7, 8.1-3, 17TSST1 2 3, 15, 17ExFT 2 3, 10, 11, 15, 17MAM ? 6, 8.1-3
Adapted from Marrack and Kappler, Science, 248:705, 1990
![Page 39: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/39.jpg)
Diseases Caused by Superantigens
Toxin Organism Disease
Staphylococcal enterotoxins (SE) S. aureaus Food poisoning,A, B, C1, C2, C3, D and E Shock
Toxic Shock Syndrome Toxin S. aureus Toxic Shock Syndrome (TSST1)
Exfoliating Toxins A and B S. aureus Scalded Skin Syndrome
Pyrogenic exotoxins A, B, C S. pyogenes Fever, Rash, shock
M. arthritides mitogen M. arthritides Shock
Adapted from Marrack and Kappler, Science, 248:705, 1990
![Page 40: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/40.jpg)
The TCR is an Oligomer
Evidence:
1. Cointernalization of the CD3 and heterodimer
2. Coimmunoprecipitation (very detergent dependent)
3. Chemical cross-linking ( and CD3 )
4. Mutants (high CD3 expression requires , or pre-TCR)
5. In vitro assembly studies
![Page 41: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/41.jpg)
TCR Assembly:Ordered Interactions and Quality Checkpoints
![Page 42: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/42.jpg)
TCR Stochiometry: Models
![Page 43: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/43.jpg)
In Vitro Assembly Favors a Single Heterodimer per TCR and Unusual
Transmembrane Interactions
Call, et al., Cell, 111:967, 2002
![Page 44: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/44.jpg)
TCR complex structureWucherpfennig et al. CSH 2010
![Page 45: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/45.jpg)
Model of TCR Heterodimer - CD3 complexSun, et al, PNAS, 2004
CD4 and CD8would be on thisside - based on TCR and MHCinteractions
![Page 46: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/46.jpg)
Transmembrane Domains Allow Structural and Functional Coupling of the Heterodimer
to CD3 Chains
IL-2 IL-2
Tan and Weiss, J. Exp. Med, 1991
![Page 47: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/47.jpg)
Irving and Weiss, Cell, 1991
![Page 48: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/48.jpg)
![Page 49: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/49.jpg)
ITAM-Containing Receptors
![Page 50: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/50.jpg)
The ITAM as an ConservedSignal Transduction Module
ITAM can confer signal transduction function to heterologous receptors,17 aa are enough
ITAMs are encoded on 2 exons, evolutionary conservation
Tyrosines and Leucines (or Isoleucines) are critical, as is spacing between YXXL residues 7 and 8 aa spacer are OK, 6 is not
Function of redundancy: Signal Amplification vs Distinct FunctionsMultimers signal betterEffector binding differences
Viruses usurp signaling function
![Page 51: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/51.jpg)
An Immature Form of the TCR has a SurrogateFor the Chain, Pre-T
![Page 52: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/52.jpg)
SS Pang et al. Nature 467, 844-848 (2010) doi:10.1038/nature09448
The pre-T structure
![Page 53: Microbiology 204 TCR Structure Art Weiss October 19, 2015](https://reader036.vdocuments.us/reader036/viewer/2022062309/5697bf981a28abf838c912df/html5/thumbnails/53.jpg)
SS Pang et al. Nature 467, 844-848 (2010) doi:10.1038/nature09448
The pre-TCR dimer forms a constitutive dimer