Dhruv K. Sethi

Deconstructing the Peptide-MHC Specificity of T Cell Recognition.

In order to survey a universe of major histocompatibility complex (MHC)-presented peptide antigens whose numbers greatly exceed the diversity of the T cell repertoire, T cell receptors (TCRs) are thought to be cross-reactive. However, the nature and extent of TCR cross-reactivity has not been conclusively measured experimentally. We developed a system to identify MHC-presented peptide ligands by combining TCR selection of highly diverse yeast-displayed peptide-MHC libraries with deep sequencing. Although we identified hundreds of peptides reactive with each of five different mouse and human TCRs, the selected peptides possessed TCR recognition motifs that bore a close resemblance to their known antigens. This structural conservation of the TCR interaction surface allowed us to exploit deep-sequencing information to computationally identify activating microbial and self-ligands for human autoimmune TCRs. The mechanistic basis of TCR cross-reactivity described here enables effective surveillance of diverse self and foreign antigens without necessitating degenerate recognition of nonhomologous peptides.

A highly tilted binding mode by a self-reactive T cell receptor results in altered engagement of peptide and MHC.

A TCR derived from a patient with relapsing-remitting multiple sclerosis engages the self-peptide myelin basic protein in the context of HLA-DQ1 in a very unusual way.

T cell Receptor Recognition of Self and Foreign Antigens in the Induction of Autoimmunity

The major histocompatibility complex (MHC) on human chromosome 6 represents the most important genetic locus for a number of common human autoimmune diseases. Specific alleles that differ from closely related alleles by only one or a few amino acids in the peptide binding groove are frequently strongly associated with disease susceptibility, raising the important question of which peptide presentation events are critical in disease initiation and progression. This review will cover a number of topics pertinent to this fundamental question, including MHC linked disease susceptibility to autoimmune diseases, molecular mechanisms for the role of MHC molecules in autoimmune diseases as well as the recognition of self and microbial peptides by self-reactive T cell receptors (TCRs).

Self-reactive human CD4 T cell clones form unusual immunological synapses.

Compared with influenza-specific T cells, self-reactive T cells from patients with multiple sclerosis or type 1 diabetes fail to slow down and do not form normal immunological synapses upon encounter with cognate self-peptide presented by MHC.

Mechanisms of peptide repertoire selection by HLA-DM

Recently, crystal structures of key complexes in antigen presentation have been reported. HLA-DM functions in antigen presentation by catalyzing dissociation of an invariant chain remnant from the peptide binding groove and stabilizing empty MHC class II proteins in a peptide-receptive conformation. The crystal structure of a MHC class II-HLA-DM complex explains how HLA-DM stabilizes an otherwise short-lived transition state and promotes a rapid peptide exchange process that favors the highest-affinity ligands. HLA-DO has sequence similarity with MHC class II molecules yet inhibits antigen presentation. The structure of the HLA-DO-HLA-DM complex shows that it blocks HLA-DM activity as a substrate mimic. Alterations in the efficiency of DM-mediated peptide selection may contribute to autoimmune pathologies, which will be an exciting area for future investigation.

Crossreactivity of a human autoimmune TCR is dominated by a single TCR loop.

Self-reactive CD4 T cells are thought to have a central role in the pathogenesis of many chronic inflammatory human diseases. Microbial peptides can activate self-reactive T cells, but the structural basis for such crossreactivity is not well understood. The Hy.1B11 T cell receptor (TCR) originates from a patient with multiple sclerosis and recognizes the self-antigen myelin basic protein. Here we report the structural mechanism of TCR crossreactivity with two distinct peptides from human pathogens. The structures show that a single TCR residue (CDR3α F95) makes the majority of contacts with the self-peptide and both microbial peptides (66.7-80.6%) due to a highly tilted TCR-binding topology on the peptide-MHC surface. Further, a neighbouring residue located on the same TCR loop (CDR3α E98) forms an energetically critical interaction with the MHC molecule. These data show how binding by a self-reactive TCR favors crossreactivity between self and microbial antigens.

Cell surface display of functional human MHC class II proteins: yeast display versus insect cell display

Reliable and robust systems for engineering functional major histocompatibility complex class II (MHCII) proteins have proved elusive. Availability of such systems would enable the engineering of peptide-MHCII (pMHCII) complexes for therapeutic and diagnostic applications. In this paper, we have developed a system based on insect cell surface display that allows functional expression of heterodimeric DR2 molecules with or without a covalently bound human myelin basic protein (MBP) peptide, which is amenable to directed evolution of DR2-MBP variants with improved T cell receptor (TCR)-binding affinity. This study represents the first example of functional display of human pMHCII complexes on insect cell surface. In the process of developing this pMHCII engineering system, we have also explored the potential of using yeast surface display for the same application. Our data suggest that yeast display is a useful system for analysis and engineering of peptide binding of MHCII proteins, but not suitable for directed evolution of pMHC complexes that bind with low affinity to self-reactive TCRs.

Disruption of hydrogen bonds between major histocompatibility complex class II and the peptide N-terminus is not sufficient to form a human leukocyte antigen-DM receptive state of major histocompatibility complex class II.

Peptide presentation by MHC class II is of critical importance to the function of CD4+ T cells. HLA-DM resides in the endosomal pathway and edits the peptide repertoire of newly synthesized MHC class II molecules before they are exported to the cell surface. HLA-DM ensures MHC class II molecules bind high affinity peptides by targeting unstable MHC class II:peptide complexes for peptide exchange. Research over the past decade has implicated the peptide N-terminus in modulating the ability of HLA-DM to target a given MHC class II:peptide combination. In particular, attention has been focused on both the hydrogen bonds between MHC class II and peptide, and the occupancy of the P1 anchor pocket. We sought to solve the crystal structure of a HLA-DR1 molecule containing a truncated hemagglutinin peptide missing three N-terminal residues compared to the full-length sequence (residues 306–318) to determine the nature of the MHC class II:peptide species that binds HLA-DM. Here we present structural evidence that HLA-DR1 that is loaded with a peptide truncated to the P1 anchor residue such that it cannot make select hydrogen bonds with the peptide N-terminus, adopts the same conformation as molecules loaded with full-length peptide. HLA-DR1:peptide combinations that were unable to engage up to four key hydrogen bonds were also unable to bind HLA-DM, while those truncated to the P2 residue bound well. These results indicate that the conformational changes in MHC class II molecules that are recognized by HLA-DM occur after disengagement of the P1 anchor residue.

Structure and Function of TCRαβ Receptors

The T cell receptor (TCR) plays a central role in T cell biology. Each critical step in T cell development requires TCR engagement, and TCR triggering is essential for T cell proliferation, cytokine production, and effector differentiation in response to microbial or tumor antigens. The TCR heterodimer is responsible for ligand recognition, while three associated CD3 dimers carry immunoreceptor tyrosine-based activation motifs (ITAM) that are phosphorylated by Lck following TCR engagement. T cell recognition is highly sensitive and engagement of a single agonist peptide–MHC ligand can be sufficient to induce killing of a target cell. The high sensitivity of TCRs is attributable to the large number of ITAMs in a TCR–CD3 complex and a contribution of endogenous peptide–MHC ligands to T cell activation. TCR triggering occurs in the immunological synapse where TCR microclusters represent the active sites of signaling. These microclusters continuously form in the periphery of the cell–cell interface and are then transported to the center of the synapse where TCRs are dephosphorylated. The opposing membranes are prealigned, enabling TCR binding to peptide–MHC ligands with very fast kinetics. However, TCRs also dissociate very rapidly, and signaling is therefore the cumulative result of many repetitive interactions, a concept referred to as serial triggering.