The peptide binding specificity of the MHC class II I-A molecule of the Lewis rat, RT1.B 1 (original) (raw)

Direct binding of autoimmune disease related T cell epitopes to purified Lewis rat MHC class II molecules

International Immunology, 1994

New strategies applied In the treatment of experimental autoimmune disease models Involve blocking or modulation of MHC-peptide-TCR Interactions either at the level of peptide-MHC interaction or, alternatively, at the level of T cell recognition. In order to Identify useful competitor peptldes one must be able to assess peptide-MHC Interactions. Several well described autoimmune disease models exist In the Lewis rat and thus this particular rat strain provides a good model system to study the effect of competitor peptldes. So far no Information has been available on the peptide binding characteristics of the Lewis rat MHC class II RT1.B 1 molecule. We have now developed a biochemical binding assay which enables competition studies In which the relative MHC binding affinity of a set of non-labelled peptldes can be assessed while employing detection of blotlnylated marker peptides by chemllumlnescence. The assay Is sensitive and specific. We have used this assay to determine the binding characteristics of several disease associated T cell determinants and their sequence analogues In the Lewis rat. Notably, most of the autoimmune disease associated peptide sequences tested were found to be Intermediate to poor binders. Single amino acid substitutions at defined positions were sufficient to turn certain peptldes Into good binders. These results are relevant to the design of competitor peptldes In the treatment of experimental autoimmune diseases.

Peptide motif for the rat MHC class II molecule RT1. Da

Experimental autoimmune encephalomyelitis induced with myelin proteins in DA and LEW.1AV1 rats is a model of multiple sclerosis (MS). It reproduces major aspects of this detrimental disease of the central nervous system. MS is associated with the HLA-DRB1*1501, DRB5*0101, and DQB1*0602 haplotype. DA and LEW. 1AV1 rats share the RT1 av1 haplotype. So far, no MHC class II peptide motif of RT1.D a molecules has been described. Sequence alignment of the β chain of the rat MHC class II molecule RT1.D a with human HLA class II molecules revealed strong similarity in the peptide-binding groove of RT1.D a and HLA-DRB1*1501. According to the putative peptide-binding pockets of RT1.D a , after comparison with the pockets of HLA-DRB1*1501, we predicted the peptide motif of RT1.D a . To verify the predicted motif, naturally processed peptides were eluted by acidic treatment from immunoaffinity-purified RT1.D a molecules of lymphoid tissue of DA rats and subsequently analyzed by ESI tandem mass spectrometry. In addition, we performed binding studies with combinatorial nonapeptide libraries to purified RT1.D a molecules. Based on these studies we could define a peptide-binding motif for RT1.D a characterized by aliphatic amino acid residues (L, I, V, M) and of F for the peptide pocket P1, aromatic residues (F, Y, W) for P4, basic residues (K, R) for P6, aliphatic residues (I, L, V) for P7, and aromatic residues (F, Y, W) and L for P9. Both methods revealed similar binding characteristics for peptides to RT1.D a . This data will allow epitope predictions for analysis of peptides, relevant for experimental autoimmune diseases.

Peptide motif for the rat MHC class II molecule RT1.Da: similarities to the multiple sclerosis-associated HLA-DRB1*1501 molecule

Immunogenetics, 2005

Structure of an Autoimmune T Cell Receptor Complexed with Class II Peptide-MHCInsights into MHC Bias and Antigen Specificity

Immunity, 2005

the helical surface ‫)%57-%07ف(‬ is conserved and evolved for biased recognition by the TCR, suggests that TCR/pMHC interactions are tuned for the sampling of different antigens (Daniel et al.). and Structural Biology In support of this hypothesis, examples exist of struc-Stanford University School of Medicine turally diverse, crossreactive peptides to a single T cell Stanford, California 94305 clone (Bhardwaj et al., 1993; Crawford et al., 2004; Hag-2 Torrey Pines Institute for Molecular Studies erty and Allen, 1995; Hemmer et al., 1998a; Ignatowicz San Diego, California 92121 et al., 1997; Kersh and Allen, 1996b; Krogsgaard et al., 2003; Loftus et al., 1999; Reiser et al., 2003; Sykulev et al., 1994; Wucherpfennig and Strominger, 1995). While some of these peptides retain recognizable sequence Summary similarities with the cognate peptides (Crawford et al., 2004; Krogsgaard et al., 2003; Sykulev et al., 1998), other T cell receptor crossreactivity with different peptide crossreactive peptides have been shown to be minimally ligands and biased recognition of MHC are coupled homologous in sequence and therefore presumably enfeatures of antigen recognition that are necessary for gaging the TCR though a unique structural solution the T cell's diverse functional repertoire. In the crystal (Lang et al., 2002; Loftus et al., 1999; Reiser et al., 2003; structure between an autoreactive, EAE T cell clone Wucherpfennig, 2004). 172.10 and myelin basic protein (1-11) presented by In contradiction to the notion of a promiscuous TCR, class II MHC I-A u , recognition of the MHC is dominated most T cell clones are exquisitely sensitive to mutations by the V␤ domain of the TCR, which interacts with the in the peptide (Shih and Allen, 2004). Some of the most MHC ␣ chain in a manner suggestive of a germlineextensively studied TCR/MHC systems, such as 2B4/ encoded TCR/MHC "anchor point." Strikingly, there I-E k (Krogsgaard et al., 2003), 3L.2/I-E k (Kersh et al., are few specific contacts between the TCR CDR3 1998; Shih and Allen, 2004), KRN/I-A g7 (Basu et al., 2000), loops and the MBP peptide. We also find that over and 2C/H-2K b (Degano et al., 2000; Sykulev et al., 1996), 1,000,000 different peptides derived from combinatoexhibit extremely specific peptide recognition and are rial libraries can activate 172.10, yet the TCR strongly largely intolerant of amino acid changes in the TCR conprefers the native MBP contact residues. We suggest tacts. In most reported cases of degenerate TCR recogthat while TCR scanning of pMHC may be degenerate nition, the TCR contact residues of the crossreactive due to the TCR germline bias for MHC, recognition of peptides are similar (Basu et al., 2000; Crawford et al., structurally distinct agonist peptides is not indicative 2004; Grogan et al., 1999; Sykulev et al., 1998; Wilson of TCR promiscuity, but rather highly specific alternaet al., 1999). Indeed, a single centrally located peptide tive solutions to TCR engagement. residue is sufficient to produce tight selectivity by a TCR (Degano et al., 2000; Ding et al., 1998; Krogsgaard et al., Introduction 2003; Shih and Allen, 2004). Thus, the idea of degenerate T cell recognition is difficult to reconcile with experimen-The engagement of the T cell receptor by peptide-MHC tal observations of T cell specificity. is the central antigen-specific event mediating the cellu-To address these questions, we have been studying lar immune response. The concept of an inherent TCR TCR/pMHC interactions in murine experimental allergic degeneracy has emerged to explain how a TCR is able encephalomyelitis (EAE), an intensively studied model to recognize the diverse peptide antigens it encounters system, to understand autoimmunity to neural self-antiduring the processes of thymic education and peripheral gens, such as myelin basic protein (MBP) (Zamvil and surveillance (Ignatowicz et al., 1997; Nikolic-Zugic and Steinman, 1990). The immunodominant encephalito-Bevan, 1990; Hemmer et al., 1998b; Holler and Kranz, genic T cell epitope of MBP, recognized by T cells in 2004; Kersh and Allen, 1996a; Mason, 1998; Wuchermice of the H-2 u haplotype (PL/J or B10.PL), is the acetpfennig, 2004). This concept has been buttressed by ylated N-terminal 11-mer (Ac1-11) (Zamvil et al., 1987). biophysical studies of TCR/MHC interactions (Rudolph The Ac1-11 epitope in the context of class II MHC I-A u and Wilson, 2002), which indicate that flexibility in the has a number of unusual features such as a very short central CDR3 loops of the TCR may serve as an adaptahalf-life (Ͻ15 min.) and a requirement for an N-terminal tion mechanism to "read out" different peptide antigens acetylation, and MBP peptides as short as Ac1-6 can during TCR "scanning" of the universe of peptide-MHC still activate EAE T cell clones (Fairchild et al.Mason et al., The fact that peptide comprises a fraction ‫)%03-%52ف(‬ 1995; Wraith et al., 1992). A crystal structure of I-A u of the composite pMHC surface, while the majority of complexed with MBP1-11 provided a rationale for these properties by finding that the peptide sits in an unusual shifted register in the groove, which results in empty p1 *Correspondence: kcgarcia@stanford.edu 3 These authors contributed equally to this work. and p2 pockets, the MBP N terminus in the p3 pocket, Immunity 82 model included A2-A116 (the ␣ chain of TCR), B3-B117 (the ␤ chain of TCR), C1-C181 (the ␣ chain of I-A u ), D1-D190 (the ␤ chain of I-A u ), Received: August 11, 2004 and P-3-P8 (p-3-P0 is part of the linker and P1-P8 is the MBP Revised: October 7, 2004 peptide). Repeated iterations between manual rebuilding and mini-Accepted: November 17, 2004 mization as well as B factor refinement resulted in a model with R Published: January 25, 2005 factors of 24.3% and R free of 27.4%. The stereochemistry of the structure was analyzed with PROCHECK (Laskowski et al., 1993). References Details of the refinement statistics are given in Supplemental Table S1. Libraries and Peptides Steinman, L. (1988). Limited heterogeneity of T cell receptors from Libraries and biased sublibrary mixtures were prepared at Mixture lymphocytes mediating autoimmune encephalomyelitis allows spe-Sciences, Inc. (San Diego, CA) as described previously (Pinilla et cific immune intervention. Cell 54, 263-273. al., 1994). PCL 97-4 is a synthetic N-acetylated, C-terminal amide, L-amino acid combinatorial decapeptide library arrayed in a posi-Anderton, S.M., Manickasingham, S.P., Burkhart, C., Luckcuck, T.A., Holland, S.J., Lamont, A.G., and Wraith, D.C. (1998). Fine specificity tional scanning format. It consists of 200 mixtures in the OX 9 format, Structure of an Autoimmune TCR/Peptide-MHC Complex 91 of the myelin-reactive T cell repertoire: implications for TCR antago-peptide can induce clinical signs of experimental autoimmune encephalomyelitis. J. Immunol. 161, 60-64. nism in autoimmunity. . Negative selection during the peripheral immune response to antigen. J. Exp. Med. 193, 1-11. Goverman, J. (1999). Tolerance and autoimmunity in TCR transgenic mice specific for myelin basic protein. Immunol. Rev. 169, 147-159. Bankovich, A.J., and Garcia, K.C. (2003). Not just any T cell receptor will do. Immunity 18, 7-11. Goverman, J., Woods, A., Larson, L., Weiner, L.P., Hood, L., and Zaller, D.M. (1993). Transgenic mice that express a myelin basic Basu, D., Horvath, S., Matsumoto, I., Fremont, D.H., and Allen, P.M. protein-specific T cell receptor develop spontaneous autoimmunity. (2000). Molecular basis for recognition of an arthritic peptide and a Cell 72, 551-560. foreign epitope on distinct MHC molecules by a single TCR. software suite for macromolecular structure determination. Acta (2002). Structural snapshot of aberrant antigen presentation linked Crystallogr. D Biol. Crystallogr. 54, 905-921. to autoimmunity: the immunodominant epitope of MBP complexed Buslepp, J., Wang, H., Biddison, W.E., Appella, E., and Collins, E.J. with I-Au. Immunity 17, 83-94. (2003). A correlation between TCR Valpha docking on MHC and CD8 Hemmer, B., Vergelli, M., Gran, B., Ling, N., Conlon, P., Pinilla, C., dependence: implications for T cell selection. Immunity 19, 595-606. Houghten, R., McFarland, H.F., and Martin, R. (1998a). Predictable CCP4 (Collaborative Computational Project, Number 4) (1994). The TCR antigen recognition based on peptide scans leads to the identi-CCP4 suite: programs for protein crystallography. Acta Crystallogr. fication of agonist ligands with no sequence homology. . A basis for alloreactivtions up close. Cell 104, 1-4. ity: MHC helical residues broaden peptide recognition by the TCR. Hennecke, J., Carfi, A., and Wiley, D.C. (2000). Structure of a cova-Immunity 8, 543-552. lently stabilized complex of a human alphabeta T-cell receptor, influ-Davis, M.M., and Bjorkman, P.J. (1988). T-cell antigen receptor enza HA peptide and MHC class II molecule, HLA-DR1. EMBO J. genes and T-cell recognition. Nature 334, 395-402. 19, 5611-5624. Degano, M., Garcia, K.C., Apostolopoulos, V., Rudolph, M.G., Tey-Holler, P.D., and Kranz, D.M. (2004). T cell receptors: affinities, crosston, L., and Wilson, I.A. (2000). A functional hot spot for antigen reactivities, and a conformer model. Mol. Immunol. 40, 1027-1031. recognition in a superagonist TCR/MHC complex. Immunity 12, Houghten, R.A. (1985). General method for the rapid solid-phase 251-261. synthesis of large numbers of peptides: specificity of antigen-anti-Delano, W.L. (2002). The PyMOL Molecular Graphics System (San body interaction at the level of individual amino acids. Proc. Natl. Carlos, CA: DeLano Scientific).

A novel first primary anchor extends the MHC class II I-Ad binding motif to encompass nine amino acids

International Immunology, 1997

The MHC class II molecule I-A d has been reported to bind peptides containing a motif of six consecutive amino acids. We demonstrate that binding of the murine IgG2a b heavy chain allopeptide γ2a b 435-451 (Kabat numbering) to I-A d is strongly enhanced by a novel first primary anchor (P1) three residues N-terminal to this hexamer. This is based on flow cytometric assessment of the I-A d binding capacity of γ2a b peptide analogues, their antigenicity for I-A drestricted T cell clones and molecular modelling. The P1 pocket is broadly specific since aliphatic, aromatic, acidic, the basic histidine and small polar side chains all allowed good binding. By contrast, asparagine, arginine and glycine reduced the binding capacity 10-, 16-and >100-fold respectively. Truncation or glycine substitution at P1 decreased antigenicity by a factor >1000.

Characterisation of RT1-E2, a multigenic family of highly conserved rat non-classical MHC class I molecules initially identified in cells from immunoprivileged sites

BMC Immunology, 2003

Background So-called "immunoprivileged sites" are tissues or organs where slow allograft rejection correlates with low levels of expression of MHC class I molecules. Whilst classical class I molecules are recognised by cytotoxic T lymphocytes (CTL), some MHC class I molecules are called "non-classical" because they exhibit low polymorphism and are not widely expressed. These last years, several studies have shown that these can play different, more specialised roles than their classical counterparts. In the course of efforts to characterise MHC class I expression in rat cells obtained from immunoprivileged sites such as the central nervous system or the placenta, a new family of non-classical MHC class I molecules, which we have named RT1-E2, has been uncovered. Results Members of the RT1-E2 family are all highly homologous to one another, and the number of RT1-E2 loci varies from one to four per MHC haplotype among the six rat strains studied so far, with some loci predicted to give rise to soluble molecules. The RT1 n MHC haplotype (found in BN rats) carries a single RT1-E2 locus, which lies in the RT1-C/E region of the MHC and displays the typical exon-intron organisation and promoter features seen in other rat MHC class I genes. We present evidence that: i) RT1-E2 molecules can be detected at the surface of transfected mouse L cells and simian COS-7 cells, albeit at low levels; ii) their transport to the cell surface is dependent on a functional TAP transporter. In L cells, their transport is also hindered by protease inhibitors, brefeldin A and monensin. Conclusions These findings suggest that RT1-E2 molecules probably associate with ligands of peptidic nature. The high homology between the RT1-E2 molecules isolated from divergent rat MHC haplotypes is particularly striking at the level of their extra-cellular portions. Compared to other class I molecules, this suggests that RT1-E2 molecules may associate with well defined sets of ligands. Several characteristics point to a certain similarity to the mouse H2-Qa2 and human HLA-G molecules.

Poor correspondence between predicted and experimental binding of peptides to class I MHC molecules

Tissue Antigens, 2000

Naturally processed peptides presented by class I major histocompatibility complex (MHC) molecules display a characteristic allele specific motif of two or more essential amino acid side chains, the so-called peptide anchor residues, in the context of an 8-10 amino acid long peptide. Knowledge of the peptide binding motif of individual class I MHC molecules permits the selection of potential peptide antigens from proteins of infectious organisms that could induce protective T-cell-mediated immunity. Several methods have been developed for the prediction of potential class I MHC binding peptides. One is based on a simple scanning for the presence of primary peptide anchor residues in the sequence of interest. A more sophisticated technology is the utilization of predictive computer algorithms. Here, we have analyzed the experimental binding of 84 peptides selected on the basis of the presence of peptide binding motifs for individual class I MHC molecules. The actual binding was compared with the results obtained when analyzing the same peptides by two well-known, publicly available computer algorithms. We conclude that there is no strong correlation between actual and predicted binding when using predictive computer algorithms. Furthermore, we found a high number of false-negatives when using a predictive algorithm compared to simple scanning for the presence of primary anchor residues. We conclude that the peptide binding assay remains an important step in the identification of cytotoxic T lymphocyte (CTL) epitopes which can not be substituted by predictive algorithms.