Recognition of core and flanking amino acids of MHC class II-bound peptides by the T cell receptor

CD4 T cells recognize peptides bound to major histocompatibility complex (MHC) class II molecules. Most MHC class II molecules have four binding pockets occupied by amino acids 1, 4, 6, and 9 of the minimal peptide epitope, while the residues at positions 2, 3, 5, 7, and 8 are available to interact with the T cell receptor (TCR). In addition MHC class II bound peptides have flanking residues situated outside of this peptide core. Here we demonstrate that the flanking residues of the conalbumin peptide bound to I-A(k) have no effect on recognition by the D10 TCR. To study the role of peptide flanks for recognition by a second TCR, we determined the MHC and TCR contacting amino acids of the I-A(b) bound Ealpha peptide. The Ealpha peptide is shown to bind I-A(b) using four alanines as anchor residues. TCR recognition of Ealpha peptides with altered flanking residues again suggested that, in general, no specific interactions occurred with the peptide flanks. However, using an HLA-DM-mediated technique to measure peptide binding to MHC class II molecules, we found that the peptide flanking residues contribute substantially to MHC binding.     

The human major histocompatibility complex class Ib molecule HLA-E binds signal sequence-derived peptides with primary anchor residues at positions 2 and 9

Human histocompatibility leukocyte antigen E (HLA-E) and mouse major histocompatibility complex (MHC) class Ib antigen, Qa-1, share the same substitutions at two normally conserved positions 143 and 147, which are likely to affect binding of the C terminus of peptides. Qa-1 is able to bind a peptide derived from the leader sequence of H-2 D and H-2 L molecules. We developed a peptide binding assay in vitro to compare the binding specificity of HLA-E with the mouse MHC class Ib molecule Qa-1. We demonstrate that HLA-E binds, although poorly, the peptide which binds to Qa-1 and that it also binds nonamer signal sequence-derived peptides from human MHC class I molecules. Using alanine and glycine substitutions, we could define primary anchor residues at positions 2 and 9 and secondary anchor residues at position 7 and possibly 3.     

The peptide binding motif of the disease associated HLA-DQ (α 1* 0501, β 1* 0201) molecule

To identify the binding motifs of peptides which bind to the celiac disease and insulin-dependent-diabetes-mellitus (IDDM)-associated DQ2 molecule, peptides were eluted from affinity-purified DQ2 molecules. The eluted peptides were separated by reverse-phase HPLC. Prominent peptide peaks and the remaining pool of peptides were sequenced by Edman degradation. Truncated variants of eight different peptides with a length of 9–19 amino acids were identified; among them class II-associated invariant chain peptides (CLIP) and peptides that stem from HLA class I α, HLA-DQα1*0501, Ig and CD20 molecules. Data from the pool sequencing and the biochemical binding analyses of synthetic variants of an eluted high-affinity ligand (HLA class I α 46–60), indicate that the side chains of amino acid residues at relative position P1 (bulky hydrophobic), P4 (negatively charged or aliphatic), P6 (Pro or negatively charged), P7 (negatively charged) and P9 (bulky hydrophobic) are important for binding of peptides to DQ2. Computer modeling of the DQ2 with variants of the high-affinity ligand in the groove suggests that peptides bind to DQ2 through the primary anchors P1, P7 and P9 and making additional advantageous interactions using the P4 and P6 positions.     

Interactions of peptide side chains with structurally complementary pockets in DQ molecules are critical for allele-specific peptide binding and T cell reactivity.

Peptides derived from glutamate decarboxylase, λ-repressor and VP-16 of HSV-2 were used to study peptide binding to different DQ3 alleles. Four residues at relative positions n, n+3, n+5 and n+8 of the peptide were found to be important for binding to the DQ3.1 and DQ3.2 molecules, presumably by anchoring into pockets designated 1, 4, 6 and 9 of the DQ molecules. Preferential binding of peptides with specific amino acid substitutions defined distinct binding motifs for each allelic molecule. The DQ3.1 molecule differs from DQ3.2 in its motif for position 4 and position 9, and both of these differences in the binding motif correlate with the allelic polymorphisms between the DQ3.1 and DQ3.2 β chains polymorphisms in pocket 4 and 9 of the class II peptide binding groove. The polymorphism at residue 57 of DQ was of particular interest: single amino acid substitutions at position 57 of the DQ3.2 molecule or at position n+8 of the peptide reciprocally regulated DQ-peptide complex formation. These binding studies were paralleled by specific T cell recognition, in which the substituted peptide abolished T cell reactivity which was directed to the DQ3.2-peptide complex, while the same T cell clone recognized the substituted peptide presented by DQ3.3, a class II restriction element differing from DQ3.2 only at residue 57.     

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

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.     

Preferred size of peptides that bind to H-2 Kb is sequence dependent.

The identification of naturally processed viral peptides reveals that major histocompatibility complex (MHC) class I epitopes are composed of nine or eight amino acid residues. Peptides eluted from H-2 Kb MHC class I molecules have been suggested, as a class, to be eight amino acid residues long. To assay for peptide-class I interactions, a stabilization assay described for surface labeled "empty" class I molecules was employed, but on biosynthetically labeled class I molecules. The Sendai virus nucleoprotein-derived octapeptide APGNYPAL does not bind and stabilize Kb molecules, whereas other octameric Kb-restricted peptides and the nonameric peptide FAPGNYPAL interact stably. We attribute the failure of Sendai octamer binding to the presence of proline in position two: replacement of proline renders the resulting octamers as efficient as FAPGNYPAL for binding and stabilization of H-2 Kb. Substitution of glycine in position three of APGNYPAL slightly improves its Kb stabilizing capacity. Iodination of the tyrosine residue significantly alters the binding properties of the nonamer peptide. We conclude that the length of epitopes as selected by the class I Kb molecule is influenced by their sequence and suggest that proper positioning of the NH2 terminus of peptides is essential for class I stabilizing properties. The ability to stabilize newly synthesized "empty" class I molecules with peptide argues against an involvement of beta 2 microglobulin exchange in the experiments described here.     

Peptide length preferences for rat and mouse MHC class I molecules using random peptide libraries

MHC class I molecules bind short peptides for presentation to CD8⁺ T cells. The determination of the three-dimensional structure of various MHC class I complexes has revealed that both ends of the peptide binding site are composed of polar residues conserved among all human and murine MHC class I sequences, which act to lock the ends of the peptide into the groove. In the rat, however, differences in these important residues occur, suggesting the possibility that certain rat MHC class I molecules may be able to bind and present longer peptides. Here we have studied the peptide length preferences of two rat MHC class I a molecules expressed in the TAP2-deficient mouse cell line RMA-S: RT1-A1^c, which carries unusual key residues at both ends of the groove, and RT1.A^a which carries the canonical residues. Temperature-dependent peptide stabilization assays were performed using synthetic random peptide libraries of different lengths (7 – 15 amino acids) and successful stabilization was determined by FACS analysis. Results for two naturally expressed mouse MHC class I molecules revealed different length preferences (H2-K^b, 8 – 13-mer and H2-D^b, 9 – 15-mer peptides). The rat MHC class Ia molecule, RT1-A^a, revealed a preference for 9 – 15-mer peptides, whereas RT1-A1^c showed a more stringent preference for 9 – 12-mer peptides, thereby ruling out the hypothesis that unusual residues in rat MHC molecules allow binding of longer peptides.     

MHC II and the Endocytic Pathway: Regulation by Invariant Chain

The major histocompatibility complex (MHC) class I and II molecules perform vital functions in innate and adaptive immune responses towards invading pathogens. MHC class I molecules load peptides in the endoplasmatic reticulum (ER) and display them to the T cell receptors (TcR) on CD8⁺ T lymphocytes. MHC class II molecules (MHC II) acquire their peptides in endosomes and present these to the TcR on CD4+ T lymphocytes. They are vital for the generation of humoral immune responses. MHC II assembly in the ER and trafficking to endosomes is guided by a specialized MHC II chaperone termed the invariant chain (Ii). Ii self-associates into a trimer in the ER, this provides a scaffold for the assembly of three MHC II heterodimers and blocks their peptide binding grooves, thereby avoiding premature peptide binding. Ii then transports the nascent MHC II to more or less specialized compartment where they can load peptides derived from internalized pathogens.     

Universally immunogenic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells

To understand the effect of human MHC class II polymorphism on antigen recognition, we analyzed the memory T cell response to three tetanus toxin epitopes defined by three short synthetic peptides (p2, p4 and p30). We found that p2 and p30 are universally immunogenic, since they are recognized by all primed donors, irrespective of their MHC haplotypes. The analysis of specific clones indicates that both peptides are very promiscuous in their capacity to bind to class II. p30 can be recognized in association with DRw11(5), 7, 9 and with DPw2 and DPw4, while p2 can be recognized in association with DR1, DRw15(2), DRw18 (3), DR4Dw4, DRw11(5), DRw13(w6), DR7, DRw8, DR9, DRw52a and DRw52b. On the contrary, the third peptide, p4, can be recognized by only half of the donors in association with only DRw52a and DRw52c. Analysis of truncated peptides shows that p30 contains three distinct epitopes, each recognized in association with different class II molecules. Therefore, the restriction specificity is already set at the level of the peptide-MHC complex and, in all cases, T cells discriminate p30 bound to different class II molecules. On the contrary, p2 contains only one epitope, which is recognized in association with all DR molecules. In this case we found two different restriction patterns. Some clones are monogamous, since they recognize the peptide in association with one DR allele, while others are promiscuous, since they recognize by peptide in association with several different DR molecules. Thus, in this case, the restriction specificity is also set at the level of the T cell receptor. We suggest that both the promiscuous binding of peptides and the promiscuous recognition by T cells are dependent on the particular structure of the DR molecules, having a monomorphic alpha chain associated with a polymorphic beta chain.     

Amino acid substitutions at position 97 in HLA-A2 segregate cytolysis from cytokine release in MART-1/Melan-A peptide AAGIGILTV-specific cytotoxic T lymphocytes

CD8⁺ T lymphocytes recognize antigenic peptides presented by major histocompatibility complex (MHC) class I molecules. Individual peptide termini appear to be fixed at the C- and N-terminal ends. In contrast, central peptide side chains residues may point in different directions and exhibit limited flexibility, dependent on the MHC class I structural variation. For instance, position 97 in HLA-A201 has been shown to shift individual peptide species into different coordinations, one oriented towards the peptide N terminus, or more towards the C-terminal end. The conformational shape of such non-anchor peptide residues may affect the affinity of MHC/peptide / TCR interaction, resulting in quantitative, or qualitative different T cell effector functions. To characterize the impact of different amino acid residues occupying position 97 in HLA-A2 on peptide binding and presentation to CTL, we generated a panel of mutated HLA-A2 molecules containing either M, K, T, V, G, Q, W, P or H at position 97. The HLA-A0201 presented melanoma-associated MART-1/Melan-A derived peptide AAGIGILTV was employed to assess the impact of such position-97 mutations on HLA-A2 in peptide binding measured in an HLA-A2 reconstitution assay and presentation to AAGIGILTV-specific polyclonal or clonal T lymphocytes as measured by cytotoxicity, or interferon (IFN)-γ and granulocyte/macrophage colony-stimulating factor (GM-CSF) secretion. The high-affinity AAGIGILTV peptide bound to all position-97 mutants, albeit with differential efficiencies, and elicited specific release of IFN-γ and GM-CSF by CTL. CTL responses were triggered only by the HLA-A2 wild type, by HLA-A2-H97 (histidine position 97 mutant), and HLA-A2-W97. The HLA-A2-M97 presenting molecule elicited enhanced cytokine release and CTL effector functions by polyclonal and by clonal effector T cells. These results indicate that MHC class I-bound peptides can trigger specific cytokine release by effector T cells independently of their ability to induce cytolysis. We conclude that relatively minor changes in the MHC class I peptide binding groove, including substitutions at position 97, can affect recognition by antigen-specific T cells. Mutant MHC class I molecules, such as those described here, may act as partial peptide antagonists and could be usefol for inducing T lymphocytes with qualitatively different effector functions.     

Peptide analysis,stability studies,and structural modeling explain contradictory peptide motifs and unique properties of the NOD mouse MHC class II molecule H2-A(g7)

The MHC class II molecule H2-A(g7) is the chief genetic determinant in insulin-dependent diabetes mellitus of the non-obese diabetic (NOD) mice. Poor peptide binding ability, as well as presentation of a unique subset of peptides by this molecule was suggested to promote autoimmunity in this strain. However, several laboratories have presented results in favor of an H2-A(g7) molecule that can avidly bind many different peptides. The crystal structures of H2-A(g7) in complex with two different peptides did not completely resolve this issue. To analyze the peptide binding capacity and the motif requirements of H2-A(g7), we eluted natural ligands from purified H2-A(g7) molecules isolated from the H2-A(g7)-transfected M12-C3 cells. A low peptide yield dominated by a few peptide ligands was found. Pool sequencing and alignment of individual ligands on the basis of molecular modeling revealed a peptide-binding motif with basic/aliphatic/small hydrophilic amino acids at relative position 1 (p1), aliphatic amino acids at p4, Ala at p6, and acidic amino acids and Ser/Gly at p9, as well as acidic residues at p10/11. Though weak, the binding of individual ligands, as well as the importance of an acidic C-terminal residue was confirmed by peptide binding studies to isolated H2-A(g7) molecules. Furthermore, the H2-A(g7) molecule incompletely dissociated into its constituent chains in SDS-electrophoresis under nonreducing conditions. This provides additional evidence of its weak affinity for peptides, which probably arises from the combination of beta56His/beta57Ser/beta78Ala and other unique H2-A(g7) residues in contact with the antigenic peptide. These results allow a better understanding of the role of this molecule in the development of autoimmunity and the identification of epitopes relevant to diabetes.     

Qa-1 interaction and T cell recognition of the Qa-1 determinant modifier peptide

The peptide-binding properties of the nonclassical major histocompatibility complex (MHC) class 1b molecule Qa-1 were investigated using a transfected hybrid molecule composed of the α1 and α2 domains of Qa-1^b and the α3 domain of H-2D^b. This allowed the use of a monoclonal antibody directed against H-2D^b whilst retaining the peptide-binding groove of Qa-1^b. By comparison with classical MHC class I molecules, intracellular maturation of the chimeric molecule was inefficient with weak intracellular association with β2-microglobulin. However, at the cell surface the hybrid molecules were stably associated with β2-microglobulin and were recognized by cytotoxic T lymphocyte (CTL) clones specific for the Qa-1^b -presented peptide Qdm (AMAPRTLLL). A whole-cell binding assay was used to determine which residues of Qdm were important for binding to Qa-1^b and CTL clones served to identify residues important for T cell recognition. Substitutions at position 1 and 5 did not reduce the efficiency of binding and had little effect on CTL recognition. In contrast, substitutions at position 9 resulted in loss of MHC class I binding. Mass spectrometric analysis of peptides eluted from immunopurified Qa-1^b/D^b molecules indicated that Qdm was the dominant peptide. The closely related peptide, AMVPRTLLL, which is derived from the signal sequence of H-2D^k, was also present, although it was considerably less abundant. The mass profile suggested the presence of additional peptides the majority of which consisted of eight to ten amino acid residues. Finally, the finding that a peptide derived from Klebsiella pneumoniae can bind raises the possibility that this non-classical MHC class I molecule may play a role in the presentation of peptides of microorganisms.     

DR4Dw4/DR53 molecules contain a peptide from the autoantigen calreticulin

Abstract: Rheumatoid arthritis (RA) occurs more frequently in HLA-DR4⁺ individuals than in those who do not express this MHC class II molecule. Although the role of this genetic factor in the immunopathology of this autoimmune disease is unclear, the association of RA with HLA-DR4 may indicate that DR4 molecules present autoantigen(s) to T cells. Here we report the analysis of naturally processed peptides, eluted from a mixture of HLA-DR4Dw4 (DRB1*0401) and DR53 (DRB4* 0101) molecules isolated from an RA patient-derived EBV-transformed B cell line. Several (size variants of) self-peptides originating from the autologous molecules HLA-A2, HLA-Cw9, HLA-B62, HLA-DR4Dw4 and HLA-DR53, were identified. We also found a sequence that has no homology to any protein in the SwissProt protein sequence databank, and a peptide identical to an internal fragment of the autoantigen calreticulin. The association of the identified peptides with cells expressing HLA-DR4Dw4/DR53 was confirmed by peptide binding analysis. In agreement with previously described peptide binding motifs for DR4Dw4, most peptides contained an aromatic residue (Phe, Tyr, Trp) at relative position i and a small hydroxyl-containing residue (Ser, Thr) at i+5. Our findings indicate that in RA patient-derived EBV-transformed B cells DR4Dw4/ DR53 molecules present a peptide from the autoantigen calreticulin. Interestingly, autoantibodies against calreticulin have been found in various rheumatic diseases, including rheumatoid arthritis. Thus, the analysis of HLA class II-bound peptides can lead to the identification of putative T helper epitopes, which might be involved in the immunopathology of autoimmune diseases.     

Use of eluted peptide sequence data to identify the binding characteristics of peptides to the insulin-dependent diabetes susceptibility allele HLA-DQ8 (DQ 3.2)

HLA-DQ8 (A1*0301, B1*0302) and -DQ2 (A1*0501, B1*0201) are both associated with diseases such as insulin-dependent diabetes mellitus and coeliac disease. We used the technique of pool sequencing to look at the requirements of peptides binding to HLA-DQ8, and combined these data with naturally sequenced ligands and in vitro binding assays to describe a novel motif for HLA-DQ8. The motif, which has the same basic format as many HLA-DR molecules, consists of four or five anchor regions, in the positions from the N-terminus of the binding core of n, n + 3, n + 5/6 and n + 8, i.e. P1, P4, P6/7 and P9. P1 and P9 require negative or polar residues, with mainly aliphatic residues at P4 and P6/7. The features of the HLA-DQ8 motif were then compared to a pool sequence of peptides eluted from HLA-DQ2. A consensus motif for the binding of a common peptide which may be involved in disease pathogenesis is described. Neither of the disease-associated alleles HLA-DQ2 and -DQ8 have Asp at position 57 of the beta-chain. This Asp, if present, may form a salt bridge with an Arg at position 79 of the alpha-chain and so alter the binding specificity of P9. HLA-DQ2 and - DQ8 both appear to prefer negatively charged amino acids at P9. In contrast, HLA-DQ7 (A1*0301, B1*0301), which is not associated with diabetes, has Asp at beta 57, allowing positively charged amino acids at P9. This analysis of the sequence features of DQ-binding peptides suggests molecular characteristics which may be useful to predict epitopes involved in disease pathogenesis.      

Analysis of peptide-binding motifs for two disease associated HLA-DR13 alleles using an M13 phage display library.

Major histocompatibility complex (MHC) molecules bind peptides bearing an appropriate ''sequence motif'' for MHC binding. The use of phage display libraries exploits the ability of MHC class II molecules to exchange peptides in solution and thus select out peptide sequences with high-affinity binding from a large array of random peptides. We have analysed the peptide binding motifs of HLA-DRB1*1301 and *1302 using affinity purified HLA-DR13 molecules to purify sequentially HLA-DR13-binding peptides from a large random library of M13 phage containing nonamer inserts in the pIII coat protein. These DR13 alleles differ only at position 86 of the HLA-DR beta chain, where they contain valine and glycine residues respectively. These alleles were chosen because of their association with protection from severe malaria and chronic hepatitis B virus infection in West Africa. Analysis of the phage bound to these DR molecules suggests binding motifs. We compare the results derived from the use of the phage display library with results obtained from analysis of eluted peptides and peptide-binding studies. This analysis shows that although there is a common theme to motifs derived using different methods, there are also subtle variations between them.     

The relative importance of individual DR binding motif positions as defined by peptide anchor analysis of influenza hemagglutinin peptide 306–318 and human myelin basic protein peptide 152–165 binding to several DR molecules: definition of a common extended DR binding motif

Definition of peptide binding motifs for DR molecules has proven difficult as the peptides that bind to a DR molecule have shown extensive variability at putative motif positions. Recent studies suggest that specific peptide anchor residues (motif positions) and specific DR residues can differ in importance for peptide binding to a DR molecule. To assess further the relevance of individual peptide anchor residues, the binding of serial alanine-substituted analogs of influenza virus hemagglutinin (HA) 306–318 and human myelin basic protein (MBP) 152–165 to a panel of transfected wild-type DR molecules was examined. This analysis included DR molecules from a wide range of allelic families and, unlike most earlier studies, multiple members of single DR allelic families. The data show that different peptide residues serve as critical anchors for binding to different DR molecules. For example, MBP binding to DR(α,β1*0303) required peptide residues F154 (i), R159 (i + 5) and R162 (i + 8). In contrast, MBP binding to DR(α,β1*0102) required peptide residues I153 (i) and L156 (i + 3). More importantly, the combination of critical anchor residues in HA and MBP differed for binding to a single DR molecule [e.g. V309 (i) for HA and I153 (i) and L156 (i + 3) for MBP binding to DR(α,β1*0102)]. Although the location of the binding pocket in each DR molecule compared to the DR (α,β1*0101) crystal is expected to be similar and suggests a common extended DR binding motif, the present results suggest that the relative importance of individual peptide anchor residues and of the corresponding DR binding pockets will differ for each DR/peptide complex.     

Short peptide sequences mimic HLA-DM functions

HLA-DM (DM) plays a critical role in Ag presentation to CD4 T cells by catalyzing the exchange of peptides bound to MHC class II molecules. It is known that DM interaction with MHC II involves conformational changes in the MHC II molecule leading to the disturbance of H-bonds formed between the bound peptide and the MHC II groove leading to peptide dissociation. The specific region of the DM molecule that induces this peptide dissociation is not defined. In this study, we describe three short peptides (helper peptides) that accelerate DM-catalyzed peptide exchange. Kinetic studies presented here demonstrate that these peptides act similarly to DM in; (a) enhancing peptide binding to HLA-DR1; (b) dissociation of complexes of peptide-DR1; and (c) maintaining a receptive conformation of empty DR1. We further report that helper peptides are effective in increasing peptide binding to DR1 expressed on B cells in vitro, and, when mixed with peptide and adjuvant, cause enhanced T cell priming in HLA-DR1 Tg mice. We suggest that helper peptides might interact with the same critical residues on MHC class II that is targeted by DM.