Autoreactive CD4+ T cells are involved in the pathogenesis of many autoimmune diseases, but the antigens that stimulate their responses have been difficult to identify and in most cases are not well defined. In the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D), we have identified a peptide WE14 from chromogranin A (ChgA) as the antigen for highly diabetogenic CD4+ T cell clones. Truncation and extension analysis showed that WE14 binds to the NOD mouse MHCII molecule, I-Ag7, in an atypical manner, occupying only the C-terminal half of the I-Ag7 peptide-binding groove. This finding extends the list of T cell antigens in T1D and supports the idea that autoreactive T cells respond to unusually presented self-peptides.

A peptide derived from the insulin B chain contains a major epitope for diabetogenic CD4 + T cells in the NOD mouse model of type 1 diabetes (T1D). This peptide can fill the binding groove of the NOD MHCII molecule, IA g7 , in a number of ways or "registers." We show here that a diverse set of NOD anti-insulin T cells all recognize this peptide bound in the same register. Surprisingly, this register results in the poorest binding of peptide to IA g7 . The poor binding is due to an incompatibility between the p9 amino acid of the peptide and the unique IA g7 p9 pocket polymorphisms that are strongly associated with susceptibility to T1D. Our findings suggest that the association of autoimmunity with particular MHCII alleles may be do to poorer, rather than more favorable, binding of the critical self-epitopes, allowing T-cell escape from thymic deletion.T he development of type 1 diabetes (T1D) in NOD mice and humans is associated with certain alleles of MHCII (1-3). These alleles present sets of peptides distinct from those presented by other MHCII alleles (4, 5), thus their association with T1D may be because they are better than other MHCII alleles at presentation of peptides derived from pancreatic islet β-cell proteins (6). However, tests of this and other hypotheses about the role of MHCII in T1D require a precise identification of the peptides recognized by pathogenic CD4 + T cells.Peptides bind in an extended conformation to a groove of MHCII. The binding involves a nonamer of the peptide (p), in which the side chains of "anchor" amino acids at positions p1, p4, p6, and p9 interact with corresponding pockets in the groove (7). Polymorphic MHCII residues lining the binding groove influence the preference of each pocket for particular peptide side chains, providing each MHCII allele with a unique preferred peptidebinding motif that dictates the position or "register" of the peptide in the groove. However, the specificities of these pockets are not absolute, and so a peptide may bind in more than one register, with the hierarchy of registers determined by how well the pockets accept the anchor residues. The register of the peptide affects which of its side chains are pointing out of the MHCII groove and hence interaction with T cell receptors. Thus individual peptide-responsive T cells will recognize the peptide bound to MHCII in only one of the possible registers.The NOD mouse has a single MHCII molecule, IA g7 , which is essential for the development of disease (8). Polymorphisms in the IA g7 β-chain (9) shape the p9 binding pocket (10, 11) and contribute to a side chain preference distinct from that of other IA alleles (6). In particular, an Asp-to-Ser alteration at IA g7 β57 disrupts a conserved salt bridge to an Arg at α76. This allows the positively charged Arg to interact with the peptide amino acid side chain in the p9 pocket (11) and confers a unique preference for binding peptide registers with acidic residues at this position (6).The insulin B-chain peptide (B:12-23) is recognized by diabetogenic ...

In the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D), an insulin peptide (B:9-23) is a major target for pathogenic CD4 + T cells. However, there is no consensus on the relative importance of the various positions or "registers" this peptide can take when bound in the groove of the NOD MHCII molecule, IA g7 . This has hindered structural studies and the tracking of the relevant T cells in vivo with fluorescent peptide-MHCII tetramers. Using mutated B:9-23 peptides and methods for trapping the peptide in particular registers, we show that most, if not all, NOD CD4 + T cells react to B:9-23 bound in low-affinity register 3. However, these T cells can be divided into two types depending on whether their response is improved or inhibited by substituting a glycine for the B:21 glutamic acid at the p8 position of the peptide. On the basis of these findings, we constructed a set of fluorescent insulin-IA g7 tetramers that bind to most insulin-specific Tcell clones tested. A mixture of these tetramers detected a high frequency of B:9-23-reactive CD4 + T cells in the pancreases of prediabetic NOD mice. Our data are consistent with the idea that, within the pancreas, unique processing of insulin generates truncated peptides that lack or contain the B:21 glutamic acid. In the thymus, the absence of this type of processing combined with the low affinity of B:9-23 binding to IA g7 in register 3 may explain the escape of insulin-specific CD4 + T cells from the mechanisms that usually eliminate self-reactive T cells.antigen processing | autoimmunity | T cell receptor | self tolerance I n human type 1 diabetes (T1D) and in the nonobese diabetic (NOD) mouse model of the disease, insulin is a major autoantigen for both B cells and T cells (reviewed in refs. 1, 2). A peptide from the insulin beta chain (B:9-23) has been known for many years to be the major target of insulin-reactive CD4 + T cells in NOD T1D . However, the data suggest that this peptide can bind to the NOD class II major histocompatibility (MHCII), IA g7 , in multiple positions or "registers" within the peptide binding groove (3-7). These registers are defined by the peptide amino acids occupying positions p1-p9 in the groove, which include the "anchor" amino acids at p1, p4, p6, and p9, whose side chains interact with compatible pockets in the MHC groove (8, 9). For an individual peptide, each shift in register puts a new set of peptide amino acids into these anchor positions and brings a different set of peptide amino acid side chains to the surface for potential T-cell recognition, generating a unique ligand. Defining which of the possible B:9-23 binding register(s) in the IA g7 groove create the ligand(s) for diabetogenic insulin-reactive T cells has been difficult, leading to uncertainty in exactly how this peptide is processed and presented to T cells in the pancreas and the inability to construct the relevant fluorescent insulin-IA g7 multimers for in vivo tracking the autoimmune B:9-23-specific T cells.Recently, using techniques to trap versio...

Studies of individual T cells receptors (TCRs) have shed some light on structural features that underlie self-reactivity. However, general rules that predict whether TCRs are self-reactive have not been fully elucidated. Analyses of thymocytes expressing all Vβ family members show that the interfacial hydrophobicity of amino acids at positions 6 and 7 of the CDR3β segment robustly promotes the development of self-reactive TCRs. An index based on these findings distinguishes Vβ2+, Vβ6+ and Vβ8.2+ regulatory T cells from conventional T cells, as well as T cells selected on a major histocompatibility complex (MHC) allele associated with mouse type-1 diabetes from those selected on a non-autoimmune promoting MHC. These results provide a means for distinguishing normal and autoimmune-prone T cell repertoires.

SUMMARY A limited set of T cell receptor (TCR) variable (V) gene segments are used to create a repertoire of TCRs that recognize all major histocompatibility complex (MHC) ligands within a species. How individual αβTCRs are constructed to specifically recognize a limited set of MHC ligands is unclear. Here we have identified a role for the differential paring of particular V gene segments in creating TCRs that recognized MHC class II ligands exclusively, or cross-reacted with classical and non-classical MHC class I ligands. Biophysical and structural experiments indicated TCR specificity for MHC ligands is not driven by germline encoded pairwise interactions. Rather, identical TCRβ chains can have altered peptide-MHC (pMHC) binding modes when paired with different TCRα chains. The ability of TCR chain pairing to modify how V region residues interact with pMHC helps to explain how the same V genes are used to create TCRs specific for unique MHC ligands.

How regulatory T cells (Treg cell) control lymphocyte homeostasis is not fully understood. Here we identify two Treg cell populations with differing degrees of self-reactivity and distinct regulatory functions. Triplehi (GITRhiPD-1hiCD25hi) Treg cell are highly self-reactive and control lympho-proliferation in peripheral lymph nodes. Triplelo (GITRloPD-1loCD25lo) Treg cells are less self-reactive and limit development of colitis by promoting conversion of CD4+ Tconv cells into induced Treg cells (iTreg cells). Although Foxp3-deficient (scurfy) mice lack Treg cells, they contain Triplehi-like and Triplelo-like CD4+ T cells with distinct pathological properties. Scurfy TriplehiCD4+T cells infiltrate the skin whereas scurfy TripleloCD4+T cells induce colitis and wasting disease. These findings indicate that T cell receptor affinity for self-antigens drives the differentiation of Tregs into distinct subsets with non-overlapping regulatory activities.

Peptide/protein display libraries are powerful tools for identifying and manipulating receptor/ligand pairs. While the large size of bacterial phage display libraries has made them the platform of choice in many applications, often considerable engineering has been required to achieve display of properly folded and active eukaryotic proteins, such as antibodies. This problem has been partially solved in several eukaryotic display systems, e.g. using yeast or retroviruses, but these systems have their own limitations. Recently, baculovirus has been developed as a display system using the virus itself or infected insect cells as the display platform. Here, we review the development and use of baculovirus-infected cells as a platform for display libraries of peptides bound to major histocompatibility complex (MHC) class I (MHCI) or class II (MHCII). We have used fluorescent multimeric soluble T-cell receptors (TCRs) to screen these libraries and to identify peptide antigen mimotopes. We also present some improvements to this system that allow very large libraries to be constructed and screened. We have used these libraries to examine the role of MHCII-bound peptides in the presentation of the staphylococcal enterotoxin A (SEA) and to manipulate an MHCI tumor-associated antigen.

A growing body of evidence suggests that autoreactive CD8 T cells contribute to the disease process in multiple sclerosis (MS). Lymphocytes in MS plaques are biased toward the CD8 lineage, and MS patients harbor CD8 T cells specific for multiple central nervous system (CNS) antigens. Currently, there are relatively few experimental model systems available to study these pathogenic CD8 T cells in vivo. However, the few studies that have been done characterizing the mechanisms used by CD8 T cells to induce CNS autoimmunity indicate that several of the paradigms of how CD4 T cells mediate CNS autoimmunity do not hold true for CD8 T cells or for patients with MS. Thus, myelin-specific CD4 T cells are likely to be one of several important mechanisms that drive CNS disease in MS patients. The focus of this review is to highlight the current models of pathogenic CNS-reactive CD8 T cells and the molecular mechanisms these lymphocytes use when causing CNS inflammation and damage. Understanding how CNS-reactive CD8 T cells escape tolerance induction and induce CNS autoimmunity is critical to our ability to propose and test new therapies for MS.