Structural Basis for Self-Peptide/MHC Recognition by Autoimmune T Cell Receptors in Multiple...

Structural Basis for Self-Peptide/MHC Recognition by Structural Basis for Self-Peptide/MHC Recognition by Autoimmune T Cell Receptors in Multiple SclerosisAutoimmune T Cell Receptors in Multiple Sclerosis

(recognition of self)(recognition of self)

Structural Basis for Self-Peptide/MHC Recognition by Structural Basis for Self-Peptide/MHC Recognition by Autoimmune T Cell Receptors in Multiple SclerosisAutoimmune T Cell Receptors in Multiple Sclerosis

(recognition of self)(recognition of self)

Structural Basis for Tumor Antigen Recognition by Structural Basis for Tumor Antigen Recognition by CD4CD4++ T Cells T Cells

(recognition of mutated self)(recognition of mutated self)

PLP and MBPPLP and MBPMAGMAGCNPaseCNPaseMOGMOG

• Proteolipid protein (PLP)Proteolipid protein (PLP)• Myelin basic protein (MBP)Myelin basic protein (MBP)• Myelin oligodendroglia glycoprotein Myelin oligodendroglia glycoprotein (MOG)(MOG)• 2’3’ Cyclic nucleotide 3’ 2’3’ Cyclic nucleotide 3’ phosphodiesterase (CNPase) phosphodiesterase (CNPase) • Myelin-associated glycoprotein Myelin-associated glycoprotein (MAG) (MAG)

All the above self-antigens are able to All the above self-antigens are able to induce EAE, an animal model of MS.induce EAE, an animal model of MS.

Myelin proteins as targets for CD4Myelin proteins as targets for CD4++ T cells T cells

•Susceptibility to MS is associated with certain MHC class II Susceptibility to MS is associated with certain MHC class II haplotypes, most notably HLA-DR2.haplotypes, most notably HLA-DR2.

•HLA-DR2 haplotype comprises two alleles (HLA-DR2a and HLA-HLA-DR2 haplotype comprises two alleles (HLA-DR2a and HLA-DR2b) that are co-expressed on the cell surface and are thought to act DR2b) that are co-expressed on the cell surface and are thought to act in concert in conferring susceptibility to MS. in concert in conferring susceptibility to MS.

•In humans, an immunodominant T cell epitope has been identified in In humans, an immunodominant T cell epitope has been identified in MBP, corresponding to residues 84MBP, corresponding to residues 84––102. 102.

•MBP 84MBP 84–102 can be presented by both DR2a and DR2b to T cells –102 can be presented by both DR2a and DR2b to T cells from MS patients, but in completely different ways.from MS patients, but in completely different ways.

Immunology of Multiple SclerosisImmunology of Multiple Sclerosis

•TCR 3A6 was isolated from an HLA-DR2TCR 3A6 was isolated from an HLA-DR2++ MS patient. MS patient.

•TCR 3A6 recognizes MBP 84TCR 3A6 recognizes MBP 84–102 in the context of HLA-DR2a. –102 in the context of HLA-DR2a.

•Mice transgenic for TCR 3A6 and HLA-DR2a develop spontaneous Mice transgenic for TCR 3A6 and HLA-DR2a develop spontaneous CNS inflammation, demonstrating the pathogenic potential of this TCR. CNS inflammation, demonstrating the pathogenic potential of this TCR.

•TCR 3A6 displays considerable degeneracy in peptide recognition, as TCR 3A6 displays considerable degeneracy in peptide recognition, as determined using combinatorial peptide libraries.determined using combinatorial peptide libraries.

Properties of T Cell Receptor 3A6Properties of T Cell Receptor 3A6

• Do autoimmune TCRs engage peptide/MHC in the same manner as Do autoimmune TCRs engage peptide/MHC in the same manner as anti-microbial TCRs, or do fundamental differences in binding mode anti-microbial TCRs, or do fundamental differences in binding mode exist that may allow autoreactive T cells to escape negative exist that may allow autoreactive T cells to escape negative selection?selection?

• What is the structural basis for TCR degeneracy? TCR degeneracy What is the structural basis for TCR degeneracy? TCR degeneracy underlies the “molecular mimicry” hypothesis for autoimmune underlies the “molecular mimicry” hypothesis for autoimmune disease.disease.

QuestionsQuestions

Binding of TCR 3A6 tetramers to MBP/DR2a and superagonist/DR2a complexes

MBP/DR2a immobilized self-peptide

HSP/DR2a immobilized irrelevant peptide

K38/DR2a immobilized

superagonist peptide

•TCR 3A6 binds peptide/MHC with low affinity (Kd>100 M) but high specificity.•Nevertheless, 3A6 T cells are activated by nanomolar concentrations of MBP peptide.•Superagonist peptide stimulates 3A6 T cells ~10,000-fold more efficiently than MBP peptide.

Construction of a stabilized TCR–MBP–MHC class II complex

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Peptide (MBP)-TCR Refold chain and pep- chain together

MHC class II (HLA-DR2a)Refold chain, chain, and CLIP (class II associated invariant chainpeptide) together

Purify byAnion Exchange& Gel Filtration


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MBP-TCR/DR2a complexMix MBP-TCR and CLIP/DR2a together in the presence of HLA-DM (peptide exchanger) at pH 5.8 / 37°C


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TCR 3A6/MBP/HLA-DR2a (autoimmune)

TCR HA1.7/HA/HLA-DR1 (anti-microbial)

TCR 3A6/MBP/HLA-DR2a (autoimmune)

TCR HA1.7/HA/HLA-DR1 (anti-microbial)

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3A6/MBP/DR2a HA1.7/HA/DR1

172.1/MBP/I-Au Ob.1A/MBP/DR2b

Comparison of MBP/HLA-DR2a and MBP/HLA-DR2b complexes

MBP 87-104 PVVHFFKNIVTPRTPPPS DR2a (DRB5*0101)

PVVHFFKNIVTPRTPPPS DR2b (DRB1*1501) P1 P4 P6 P9

Peptide register is shifted by three residues.

TCR-contacting residues in the MBP/DR2a and MBP/DR2b complexes

Interactions of TCR 3A6 with HLA-DR2a

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TCR 3A6/MBP/HLA-DR2a TCR HA1.7/HA/HLA-DR1

Interactions of autoimmune vs. anti-microbial TCR with MHC

Interactions of TCR 3A6 with MBP peptide

•No hydrogen bonds or salt bridges between 3A6 and MBP•Interactions are restricted to van der Waals contacts•Suboptimal shape and chemical complementarity•Structural degeneracy is consistent with functional degeneracy

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Denotes a.a. of native MBP ligandDenotes a.a. of native MBP ligand

Proliferative testing of mouse transgenic 3A6 T cells with Proliferative testing of mouse transgenic 3A6 T cells with human DR2a cells using combinatorial peptide librarieshuman DR2a cells using combinatorial peptide libraries

•Agonist peptides were deduced from combinatorial decapeptide libraries•Agonists are at least 10,000-fold more potent than the MBP self-peptide

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FFKNIVTPRT: MBP 89-98WFKLIPTTKL: AGONIST 1WFKLILTPKL: AGONIST 2

Proliferative responses to predicted superagonists of TCR 3A6

Interactions of TCR 3A6 with MBP peptide

Prediction: TCRs that recognize the N-terminal portion of peptides are more degenerate (cross-reactive) than TCRs targeting the central portion. Why?

Superposition of peptides bound to human MHC class II molecules

Is the unconventional binding Is the unconventional binding mode utilized by TCR 3A6 mode utilized by TCR 3A6 broadly characteristic of broadly characteristic of

autoreactive TCRs?autoreactive TCRs?

From Pieper et al. (1999) J. Exp. Med. 189, 757-765

Stimulation of tumor-infiltrating E8 T cells by wild type and mutant TPI 23–37

•TCR E8 was isolated from tumor-infiltrating CD4+ T cells of a melanoma patient.•TCR E8 recognizes a mutated melanoma antigen presented by HLA-DR1.•The tumor-specific antigen is a peptide from triose phosphate isomerase (TPI 23–37) with a single amino acid substitution (Thr28Ile).•TCR E8 therefore recognizes a neo-antigen derived from a self-antigen.•T cell stimulation is enhanced >100,000-fold for mutated TPI 23–37 relative to the wild type peptide.

Properties of TCR E8

P-3Gly23

P-2Glu24

P-1Leu25

P1Ile26

P2Gly27

P3Thr28/ Ile28

P4Leu29

P5Asn30

P6Ala31

P7Ala32

P8Lys33

P9Val34

P10Pro35

P11Ala36

P12Asp37

Conformations of wild type and mutant TPI23-37 bound to HLA-DR1

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MHC class II (HLA-DR1)Refold chain, chain, and CLIP (class II associated invariant chainpeptide) together



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Overall structure of the E8/mutTPI/HLA-DR1 complex

Interactions between TCR E8 and mutant TPI peptide

TCR-induced conformational changes in peptide/MHC

Free TPI/DR1Bound TPI/DR1

Docking topologies of TCRs

HA1.7/HA/DR1anti-foreign

E8/TPI/DR1anti-mutated self

3A6/MBP/DR2aanti-self

Docking topologies of TCRs




Affinity: HA1.7 > E8 > 3A6

HA1.7/HA/DR1anti-microbial

E8/TPI/DR1anti-tumor

3A6/MBP/DR2aautoimmune

CDR3 positions on peptide/MHC

Position of TCRs along peptide antigen

P5

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NINDS/NIHNINDS/NIHRoland MartinRoland MartinJacqueline QuandtJacqueline Quandt

NCI/NIHNCI/NIHSuzanne TopalianSuzanne Topalian

CARBCARBYili LiYili LiLu DengLu DengRies LangleyRies LangleyPatrick BrownPatrick BrownYiyuan YinYiyuan YinGang XuGang XuYuping HuangYuping HuangJessica LueJessica LueLesley TengLesley Teng

Conformational changes in TCR E8

Conformational changes in MBP/HLA-DR2a upon binding TCR 3A6

Free MBP/DR2aBound MBP/DR2a

MBP/DR2a after binding 3A6 MBP/DR2a before binding 3A6

Conformational changes in self-peptide/MHC upon TCR binding

•TCR-induced adjustments in MBP/DR2a increase interfacial shape complementarity.•However, energetic cost of distorting MBP/DR2a from ground state reduces affinity.

Sc = 0.62 Sc = 0.50

Structural Basis for Self-Peptide/MHC Recognition by Autoimmune T Cell Receptors in Multiple...

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