Carboxy-terminal residues of major histocompatibility complex class II-associated peptides control the presentation of the bacterial superantigen toxic shock syndrome toxin-1 to T cells

Previous studies have shown that the presentation of some bacterial superantigens by major histocompatibility complex (MHC) class II molecules is strongly influenced by class II-associated peptides. For example, presentation of the toxic shock syndrome toxin-1 (TSST-1) superantigen by antigen-processing-defective T2-I-A^b cells (which expresses I-A^b that is either empty or associated with invariant chain-derived peptides) can be strongly enhanced by some, but not other, I-A^b-binding peptides. Here we investigate the contribution of I-A^b-associated peptides in the presentation of TSST-1 to T cells. The data show that overlapping peptides expressing the same core I-A^b-restricted epitope, but with various N and C termini, can differ profoundly in their ability to promote TSST-1 presentation to T cells. Analysis of altered and truncated peptides indicates that residues at the C-terminal end of the peptide have a dramatic effect on TSST-1 presentation. This effect does not involve a cognate interaction between the peptide and the TSST-1 molecule, but appears to depend on the length of the C-terminal region. These data are consistent with crystallographic studies suggesting that TSST-1 may interact with the C-terminal residues of MHC class II-associated peptides. We also examined the capacity of naturally processed peptides to promote TSST-1 binding using a superantigen blocking assay. The data demonstrated that a naturally processed epitope is dominated by peptides that do not promote strong TSST-1 binding to I-A^b. Taken together, these data suggest that TSST-1 binding to MHC class II molecules is controlled by the C-terminal residues of the associated peptide, and that many naturally processed peptide/class II complexes do not present TSST-1 to T cells. Thus, the peptide dependence of TSST-1 binding to class II molecules may significantly reduce the capacity of TSST-1 to stimulate T cells.     

Efficacy of HLA-DRB1∗03:01 and H2E transgenic mouse strains to correlate pathogenic thyroglobulin epitopes for autoimmune thyroiditis

Thyroglobulin (Tg), a homodimer of 660 kD comprising 2748 amino acids, is the largest autoantigen known. The prevalence of autoimmune thyroid disease, including Hashimoto’s thyroiditis and Graves’ disease, has provided the impetus for identifying pathogenic T cell epitopes from human Tg over two decades. With no known dominant epitopes, the search has long been a challenge for investigators. After identifying HLA-DRB1∗03:01 (HLA-DR3) and H2E^b as susceptibility alleles for Tg-induced experimental autoimmune thyroiditis in transgenic mouse strains, we searched for naturally processed T cell epitopes with MHC class II-binding motif anchors and tested the selected peptides for pathogenicity in these mice. The thyroiditogenicity of one peptide, hTg2079, was confirmed in DR3 transgenic mice and corroborated in clinical studies. In H2E^b-expressing transgenic mice, we identified three T cell epitopes from mouse Tg, mTg179, mTg409 and mTg2342, based on homology to epitopes hTg179, hTg410 and hTg2344, respectively, which we and others have found stimulatory or pathogenic in both DR3- and H2E-expressing mice. The high homology among these peptides with shared presentation by DR3, H2E^b and H2E^k molecules led us to examine the binding pocket residues of these class II molecules. Their similar binding characteristics help explain the pathogenic capacity of these T cell epitopes. Our approach of using appropriate human and murine MHC class II transgenic mice, combined with the synthesis and testing of potential pathogenic Tg peptides predicted from computational models of MHC-binding motifs, should continue to provide insights into human autoimmune thyroid disease.     

MUC1 peptide epitopes associated with five different H-2 class I molecules

We have previously described the induction of murine CD8⁺ major histocompatibility complex (MHC) class I-restricted cytotoxic T cells (CTL) recognizing the 20-amino acid repeat region of the human mucin 1 (MUC1) variable number of tandem repeats region (VNTR), a mucin greatly increased in expression in breast cancer and proposed as a target for immunotherapy. In that study, CTL could detect MUC1 peptides associated with the MHC of all nine strains examined, and we now report the different epitopes presented by five different MHC class I molecules. The epitopes were defined in CTL assays using peptide-pulsed phytohemagglutinin blasts or MHC class I-transfected L cells as targets; in addition, peptide binding assays and T cell proliferation studies were performed. Within the 20-amino acid VNTR, nine potential epitopes could be defined. The epitopes for the four MHC class I molecules [K^b (three epitopes), D^d, L^d and K^k] were closely related, all containing the amino acids PDTRPAP. For D^b, three epitopes were identified, all containing APGSTAP. Most of the epitopes did not contain a consensus motif for the particular MHC class I allele, and bound with low ‘affinity’, compared with known high-affinity peptides. CD8⁺ T cell proliferation also occurred to the same MHC class I-presented epitopes. Finally, when conventional anchor residues were introduced into the peptides, peptide binding increased, whereas CTL recognition was either retained (K^b) or lost (D^b) depending on the epitope.     

Cross-reactive trinitrophenylated peptides as antigens for class II major histocompatibility complex-restricted T cells and inducers of contact sensitivity in mice. Limited T cell receptor repertoire

The induction of contact sensitivity in mice by hapten reagents such as trinitrochlorobenzene (TNCB) involves the activation of class II major histocompatibility complex (MHC)-restricted, hapten-specific, CD4⁺ T cells. Reports from different laboratories have indicated that the relevant antigenic epitopes in such reactions might include hapten-conjugated, MHC class II-associated peptides. This study for the first time directly demonstrates that hapten-peptides account for the majority of determinants recognized by trinitrophenyl (TNP)-specific CD4⁺ T lymphocytes. The sequences of those TNP carrier peptides do not have to be related to mouse proteins. Thus, we show that TNP-modified peptides derived from mouse IgG, pigeon cytochrome c or staphylococcal nuclease known to bind to I-A^b or from λ represser with specificity to I-A^d as well as TNP-proteins such as bovine serum albumin, ovalbumin or keyhole limpet hemocyanin all create class II-restricted hapten determinants for a number of TNP-specific T cell clones and hybridomas. All of these cells were induced with cells modified by trinitrobenzene sulfonic acid (TNBS). In addition, we present arguments indicating that individual TNP-specific helper T cells may cross-react with different TNP-peptides bound to identical class II molecules. Chemical treatment of antigen-presenting cells with TNCB or TNBS may thus result in a limited number of particularly repetitive immunodominant hapten epitopes. Immunodominant epitopes were also indicated by an overrepresentation of the TCR elements Vβ2 and Vα10 in I-A^b/TNP-specific T cells. Most importantly, however, we demonstrate that TNP attached to lysine 97 in the staphylococcal nuclease peptide 93–105 (i.e. a clearly “non-self” sequence) is able to prime mice for subsequent elicitation of contact sensitivity by TNCB in the absence of foreign protein. We take this to indicate that those TNP-peptide determinants defined by us as immuno-dominant are responsible for the induction of contact sensitivity to haptens.     

N-terminal elongation of a peptide determinant beyond the first primary anchor improves binding to H-2 I-Ad and HLA-DR1 by backbone-dependent and aromatic side chain-dependent interactions,respectively

The IgG2a^b heavy chain allopeptide determinant γ2a^b 436 – 451 (Kabat numbering) presented by the major histocompatibility complex (MHC) class II molecule I-A^d is recognized by T cells which cross-react with a corneal self antigen and with the UL6 protein of the herpes simplex virus which induce autoimmune keratitis, and is the target of Th1 clones that suppress IgG2a^b production in vivo. In the γ2a^b peptide/I-A^d complex, tyrosine⁴³⁸ is the first primary anchor (P1) and residues 440 – 445 encompass the T cell receptor contact residues. Amino-terminal elongation of γ2a^b 437– 451 by a single residue (P-2) augmented the I-A^d binding capacity 10-fold and the antigenicity 55 –195-fold. This was a function of the peptide main chain, since non-conservative substitutions were accepted. The γ2a^b peptide also bound HLA-DR1, and amino-terminal extension by a single aromatic amino acid at P-3 augmented binding 15-fold. The interaction between HLA-DR1 and P-3 specifically required an aromatic peptide side chain, and computer simulations indicated that the aromatic ring at P-3 engaged conserved HLA-DR1 phenylalanine residues at the edge of the peptide binding groove. Thus, these data demonstrate that residues amino terminal to P1 may substantially increase peptide affinity for MHC class II by main chain-dependent as well as side chain-dependent interactions, and imply that the HLA-DR1 motif should be extended to include an aromatic amino acid at P-3.     

Role of non-anchor residues of D-restricted peptides in class I binding and TCR triggering

To understand better, the role of non-anchor residues of class I restricted T cell epitopes in class I binding and TCR stimulation, a panel of peptides was synthesized in which each of the nonanchor positions of the D^b-restricted influenza peptide, ASNENMETM, was changed to each of the 20 natural amino acids (AAs). The relative affinity of all the peptides for D^b was determined and their ability to stimulate anti-ASNENMETM cytotoxic T cell hybridomas was also assessed. The results illustrated that for D^b binding, the AAs with the most solvent exposure had the smallest effect on binding. Changes at other positions affected binding to different degrees. Results for the recognition by the T cell hybridomas indicated that a peptide-MHC complex represents a multitude of epitopes, as each hybridoma recognized a different subset of peptides. Most changes in the highly solvent-exposed exposed residues negatively affected recognition by all hybridomas while changes in other positions affected each hybridoma differently, independent of the direction of the side chain of the AA at that position. Furthermore, the use of saturating concentrations of low and high binding peptides showed that, as long as the class I-peptide complex is formed, the T-cell receptor does not differentiate between high and low binding peptides. This indicates that, although the stability of the class I-peptide complex is highly dependent on peptide affinity, the class I MHC conformation induced by low affinity peptides does not necessarily differ significantly from that induced by high affinity peptides. The results of peptide-class I recognition by one ASNENMETM-specific hybridoma was used to construct a peptide that differed from ASNENMETM at four of the nine residues, yet stimulated the hybridoma to a level comparable to ASNENMETM. In addition, peptides bearing the canonical D^b-binding motif but unable to bind to the class I molecule with high affinity could be made to bind D^b, by changing unfavorable AAs to favorable ones at appropriate positions. The extended motif determined was used to identify more accurately the peptides derived from Coxsakie b3 virus that would bind D^b. It was also shown that some of the canonical characteristics of the peptide motif could be obviated and still obtain high affinity binding, provided optimal AAs were present at secondary anchor positions.     

A retro-inverso analog mimicks the cognate peptide epitope of a CD4+ T cell clone

Synthetic analogs of peptide epitopes may activate specific T helper cells, antagonize their antigen receptors, or block recognition by competing for major histocompatibility complex (MHC) class II binding sites. Rationally designed peptides may therefore prove useful as vaccines and for treatment of autoimmune diseases and allergies mediated by CD4⁺ T cells. However, their susceptibility to proteolytic degradation limits the applicability of conventional peptides in vivo. By contrast, retro-inverso analogs, in which a native sequence is substituted with D -amino acids linked with a reversed backbone, resist proteolysis and still maintain the side chain topology of the corresponding natural peptide. We report here that an end group-modified retro-inverso analog of the IgG2a^b heavy chain allopeptide determinant γ2a^b 435–447 was recognized by an I-A^d-restricted, γ2a^b 435–447-reactive T cell clone. The pseudopeptide elicited near-maximal interleukin-2 responses, although 300-fold higher concentrations were needed than the native determinant. The weaker antigenicity of the retro-inverso analog could be fully accounted for by an impaired I-A^d binding capacity, which might reflect reduced ability of the distorted main chain to form hydrogen bonds with I-A^d. Glycine substitution at the residue corresponding to the first primary anchor (P1) of the native peptide abrogated I-A^d binding and antigenicity of the retro-inverso analog. Thus, the pseudopeptide resembled the native determinant with respect to orientation in the class II binding site, configuration of the epitopic side chains, and the constraints that governed the interactions between a major anchoring side chain and I-A^d. In conclusion, proteolytically resistant compounds with predefined capacity to interact with MHC class II allelic products and T cell antigen receptors may be designed by retro-inverso modification of native determinants.     

Identification of an HLA-DQ2 peptide binding motif and HLA-DPw3-bound self-peptide by pool sequencing

Molecules of the major histocompatibility complex (MHC) present antigenic peptides to T cells. Sequencing peptide pools eluted from MHC class I molecules has established allele-specific peptide binding motifs. We applied pool sequencing to analyze human MHC class II-bound peptides and found that HLA-DQ2-eluted peptides predominantly contained lysine, isoleucine, and phenylalanine at relative position i, i + 3 and i + 8, respectively. These residues putatively represent anchor residues for MHC binding. Analysis of a heterogeneous HLA-DPw3/DPw4-eluted peptide pool yielded a sequence matching an epitope from the endogeneous enzyme glyceraldehyde-3-phosphate dehydrogenase. This self-peptide and a partially identical, known allo-epitope bound specifically to DPw3 and DR13 molecules, suggesting the sharing of a binding motif. In particular, the presence of an arginine at relative position 4 appeared important for binding to these HLA class II specificities. Thus, pool sequencing is applicable for the analysis of MHC class II-eluted peptides.     

Major histocompatibility complex class I allele-specific peptide libraries: Identification of peptides that mimic an H-Y T cell epitope

We describe a novel method for screening large libraries of random peptides for T cell antigens. Two libraries were constructed, containing fixed amino acids representing the major histocompatibility complex (MHC) class I anchor residues for H-2K^b-restricted octamers and H-2D^b-restricted nonamers. Peptides from the K^b-restricted library (K^bL: SXIXFXXL) and the D^b-restricted library (D^bL: XXXXNXXX^I_M) specifically stabilize empty K^b and D^b molecules, respectively. The libraries contain peptides that mimic several H-2^b-restricted cytotoxic T lymphocyte epitopes, and 21 mimotopes for a D^b-restricted H-Y epitope were isolated. A degenerate synthetic peptide of limited complexity containing the identified H-Y sequence motif was found to be similar to the natural H-Y epitope by reverse-phase high performance liquid chromatography analysis. This peptide is also capable of immunizing female mice against male splenocytes. Several applications for MHC-restricted peptide libraries are discussed.     

Steric recognition of T-cell receptor contact residues is required to map mutant epitopes by immunoinformatical programmes

MHC class I-restricted CD8 T-lymphocyte epitopes comprise anchor motifs, T-cell receptor (TCR) contact residues and the peptide backbone. Serial variant epitopes with substitution of amino acids at either anchor motifs or TCR contact residues have been synthesized for specific interferon-γ responses to clarify the TCR recognition mechanism as well as to assess the epitope prediction capacity of immunoinformatical programmes. CD8 T lymphocytes recognise the steric configuration of functional groups at the TCR contact side chain with a parallel observation that peptide backbones of various epitopes adapt to the conserved conformation upon binding to the same MHC class I molecule. Variant epitopes with amino acid substitutions at the TCR contact site are not recognised by specific CD8 T lymphocytes without compromising their binding capacity to MHC class I molecules, which demonstrates two discrete antigen presentation events for the binding of peptides to MHC class I molecules and for TCR recognition. The predicted outcome of immunoinformatical programmes is not consistent with the results of epitope identification by laboratory experiments in the absence of information on the interaction with TCR contact residues. Immunoinformatical programmes based on the binding affinity to MHC class I molecules are not sufficient for the accurate prediction of CD8 T-lymphocyte epitopes. The predictive capacity is further improved to distinguish mutant epitopes from the non-mutated epitopes if the peptide-TCR interface is integrated into the computing simulation programme.     

Biochemical characterization of the human diabetes-associated HLA-DQ8 allelic product: Similarity to the major histocompatibility complex class II I-Ag7 protein of non-obese diabetic mice

The human HLA-DQ8 (A1*0301/B1*0302) allelic product manifests a strong association with insulin-dependent diabetes mellitus (IDDM). Previous biochemical studies of the major histocompatibility complex (MHC) class II I-A^g7 protein of IDDM-prone non-obese diabetic mice produced controversial results. To better define the biochemical properties of IDDM-associated MHC class II molecules, we analyzed DQ8 proteins, in comparison to other DQ allelic products, by partially denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We now report that DQ8 proteins have a normal peptide occupancy and lifespan in cells. Similar to I-A^g7, DQ8 proteins formed only a minor fraction of SDS-stable complexes with peptides. Although this phenotype was not unique to DQ8, some DQ allelic products such as IDDM-protective DQ6 proteins were SDS resistant. The DQ9 allelic product, differing from DQ8 only at position (P) β57, was SDS stable, suggesting that non-Asp residues at β57 might decrease the SDS stability of DQ proteins. We identified a single peptide which specifically induced an SDS-stable conformation in DQ8 as well as in I-A^g7 molecules. The residues at anchor P1 in this peptide were found to influence the SDS stability of both molecules. Together with our previous observation of similar binding motifs of I-A^g7 and DQ8, these results demonstrate an overall biochemical similarity of mouse and human diabetes-associated MHC class II molecules. This similarity might contribute to a common immunological mechanism of IDDM in both species.     

Presentation of a horse cytochrome c peptide by multiple H-2b class I major histocompatibility complex (MHC) molecules to C57BL/6- and bm1-derived cytotoxic T lymphocytes: Presence of a single MHC anchor residue may confer efficient peptide-specific CTL recognition

In this study the immunogenic tryptic fragment from a horse cytochrome c (cyt c) digest recognized by cytotoxic T lymphocytes (CTL), induced by in vitro peptide stimulation from C57BL/6 (B6) and mutant B6.C-H-2^bm1 (bm1) mice is identified. An identical sequence, p40—53, is recognized by CTL from both B6 and bm1 mice. In addition, both B6 and bm1 cloned CTL lines display unusual major histocompatibility complex (MHC) class I-restricted recognition of this peptide in that they respond to it in the context of H-2K^b, H-2D^b, and H-2K^bm1 class I molecules, although the sequence lacks the usual structural K^b and D^b peptide-binding motifs. Truncated analogues which resemble the lengths of naturally processed MHC class I-presented peptides, confer reactivity for B6 and bm1 CTL against EL4 (H-2^b) targets as well as the L cell transfectants, L + K^b, L + D^b, and L + K^bm1. The antigenic peptide with the greatest potency is p41—49, which appears to be generated by angiotensin converting enzyme cleavage of the full-length p40—53 tryptic peptide. The minimum antigenic peptide recognized by both B6 and bm1 CTL, and which targets lysis on each of the transfectants, is the hexamer p43—48 peptide from horse cyt c. Residues Pro⁴⁴ and Thr⁴⁷, which occupy polymorphic positions with respect to other species-variant cyt c molecules, influence recognition of these peptides differently for the B6 and bm1 CTL. The ability of H-2K^b, H-2D^b, and mutant H-2K^bm1 class I molecules to present the same peptide to a single cloned CTL is discussed in the context of current knowledge of peptide anchor residues and side chain-specific binding pockets in the MHC class I peptide-binding site.     

Exact prediction of a natural T cell epitope.

T lymphocytes recognize their antigen as peptides associated with major histocompatibility complex (MHC) molecules. Peptides naturally presented by MHC class I molecules are uniform in length and have a specific motif, both defined by the respective MHC allele (Falk, K. et al. Nature 1991. 351:290). These allele-specific motifs should allow exact prediction of natural T cell epitopes. H-2Kb-restricted epitopes, for example, have a length of eight amino acid residues and conserved anchor residues at positions 5 and 8. According to this information, we predicted the natural Kb-restricted epitope of ovalbumin, thought to be contained in the 19-mer IINFEKLTEWTSSNVMEER, to be SIINFEKL. Here we show that this prediction is correct. Thus, exact prediction of natural T cell epitopes is possible.     

Characterizing immunodominant and protective influenza hemagglutinin epitopes by functional activity and relative binding to major histocompatibility complex class II sites

In the present study the analysis of functional activity and major histocompatibility complex (MHC) binding of two adjacent MHC class II-restricted epitopes, located in the C-terminal 306–329 region of human influenza A virus hemagglutinin 1 subunit (HA1) conserved with subtype sequences and not affected by antigenic drift, was undertaken to explore the hierarchy of local immnodominance. The functional activity of two T cell hybridomas of the memory/effector Th1 phenotype in combination with in vivo immunization studies provided a good tool for investigating the functional characteristics of the T cell resonse. The in vitro binding assays performed with a series of overlapping, N-terminal biotinylated peptides covering the 306–341 sequence enabled us to compare the relative binding efficiency of peptides, comprising two distinct epitopes of this region, to I-E^d expressed on living antigen-presenting cells. Our studies revealed that (i) immunization of BALB/c mice with the 306–329 H1 or H2 peptides resulted in the activation and proliferation of T cells recognizing both the 306–318 and the 317–329 epitopes, while the 306–329 H3 peptide elicits predomonantly 306–318-specific T cells, (ii) the 317–329 HA1 epitope of the H1 and H2 but not the H3 sequence is recognized by T cells and is available for recognition not only in the 317–329 peptide but also in the extended 306–329 or 306–341 peptides, (iii) the 306–318 and the 317–329 hemagglutinin peptides encompassing the H1, H2 but not the H3 sequence bind with an apparently similar affinity to and therefore compete for I-E^d binding sites, and (iv) the 317–341, the 317–329 peptides and their truncated analogs show subtype-dependent differences in MHC binding and those with lower binding capacity represent the H3 subtype sequences. These results demonstrate that differences in the binding capacity of peptides comprising two non-overlapping epitopes located in the C-terminal 306–329 region of HA1 of all three subtype-specific sequences to MHC class II provide a rationale for the local and also for the previously observed in vivo immunodominance of the 306–318 region over the 317–329 epitope in the H3 but not in the H1 or H2 sequences. In good correlation with the results of the binding and functional inhibition assays, these data demonstrate that in the H1 and H2 subtypes both regions are available for T cell recognition, they compete for the same restriction element with an appearently similar binding efficiency and, therefore, function as co-dominant epitopes. Due to the stabilizing effect of the fusion peptide, peptides comprising the 306–341 or 317–341 H1 sequences are highly immunogenic and elicit a protective immune response which involves the production of antibodies and interleukin-2 and tumor necrosis factor producing effector Th1 cells both directed against the 317–329 region. Based on the similarity of the I-E^d and HLA-DR1 peptide binding grooves and motifs, these results suggest that amino acid substitutions inserted to the H3 subtype sequence during viral evolution can modify the relative MHC binding capacity and invert the local hierarchy of immunodominance of two closely situated epitopes that are able to bind to the same MHC class II molecule.     

B and T Cell Epitope‐Based Peptides Predicted from Evolutionarily Conserved and Whole Protein Sequences of Ebola Virus as Vaccine Targets

Ebola virus (EBV) has become a serious threat to public health. Different approaches were applied to predict continuous and discontinuous B cell epitopes as well as T cell epitopes from the sequence‐based and available three‐dimensional structural analyses of each protein of EBV. Peptides ‘⁷⁹VPSATKRWGFRSGVPP⁹⁴’ from GP1 and ‘⁵¹⁵LHYWTTQDEGAAIGLA⁵³⁰’ from GP2 of Ebola were found to be the consensus peptidic sequences predicted as linear B cell epitope of which the latter contains a region ⁵¹⁹TTQDEG⁵²⁴ that fulfilled all the criteria of accessibility, hydrophilicity, flexibility and beta turn region for becoming an ideal B cell epitope. Different nonamers as T cell epitopes were obtained that interacted with different numbers of MHC class I and class II alleles with a binding affinity of <100 nm . Interestingly, these alleles also bound to the MHC class I alleles mostly prevalent in African and South Asian regions. Of these, ‘LANETTQAL’ and ‘FLYDRLAST’ nonamers were predicted to be the most potent T cell epitopes and they, respectively, interacted with eight and twelve class I alleles that covered 63.79% and 54.16% of world population, respectively. These nonamers were found to be the core sequences of 15mer peptides that interacted with the most common class II allele, HLA‐DRB1*01:01. They were further validated for their binding to specific class I alleles using docking technique. Thus, these predicted epitopes may be used as vaccine targets against EBV and can be validated in model hosts to verify their efficacy as vaccine.     

HLA-dependent variations in human immunodeficiency virus Nef protein alter peptide/HLA binding

In human immunodeficiency virus (HIV) infection, sequence variations within defined cytotoxic T lymphocyte (CTL) epitopes may lead to escape from CTL recognition. In a previous report, we have shown that the variable central region of HIV Nef protein (amino acids 73–144) that contains potential CTL epitopes, can escape the CTL response. We suggested that this non recognition occurs through a variety of mechanisms. In particular, we provided evidence that HIV Nef sequences recovered from HLA-A11-expressing individuals have alterations in the major anchor residues essential for binding of the two Nef epitopes (amino acids 73–82 and 84–92) to the HLA-A11 molecule. Here, we investigate in more detail whether variations in autologous Nef sequences affect HLA binding, leading to CTL escape. Potential epitopes were sought by testing Nef peptides containing the HLA-A11-specific motif or related motifs. We confirmed that only the two previously described epitopes identified in cytolysis tests have optimal reactivity with the HLA-A11 molecule. We then sequenced several viral variants from donors that do not express the HLA-A11 molecule and compared the variability of these epitopes with those obtained from HLA-A11-expressing individuals. One substitution (Leu⁸⁵) found in the sequences isolated from both populations increase the reactivity of the HLA-A11-restricted epitope 84–92, and might explain the difference in immunogenicity observed between the two HLA-A11-restricted epitopes from HLA-A11⁺ individuals. In addition, selective variations were only detected in virus isolated from HLA-A11-expressing individuals. Furthermore, examination of the association of variant peptides with the HLA-A11 molecule demonstrated that a single substitution within the minimal epitope could not always completely abrogate HLA binding, suggesting that multiple alterations within a particular epitope may accumulate during disease progression, allowing the virus to escape CTL recognition.     

Dissection of major histocompatibility complex (MHC) and T cell receptor contact residues in a Kb-restricted ovalbumin peptide and an assessment of the predictive power of MHC-binding motifs.

The effect of alanine substitution on the major histocompatibility complex (MHC) binding and T cell receptor recognition of the Kb-restricted ovalbumin 257-264 peptide was investigated. Positions 3, 5 and 8 of the octamer were important for Kb binding, as predicted from the motifs found in Kb-associated peptides, while mutations at positions 4, 6 and 7 affected cytotoxic T lymphocyte recognition. Substitutions at positions 1 and 2 had very minor effects on T cell recognition. In addition, we tested the capacity of sequence motifs to predict MHC binding by analysis of a series of peptides which all bear the minimal Kb motif. We found that possession of good motifs was not always sufficient to give strong MHC binding, indicating secondary effects of the residues flanking the "MHC anchor" positions.     

Extensive major histocompatibility complex class I binding promiscuity for Mycobacterium tuberculosis TB10.4 peptides and immune dominance of human leucocyte antigen (HLA)‐B*0702 and HLA‐B*0801 alleles in TB10.4 CD8+ T‐cell responses

The molecular definition of major histocompatibility complex (MHC) class I‐presented CD8⁺ T‐cell epitopes from clinically relevant Mycobacterium tuberculosis (Mtb) target proteins will aid in the rational design of T‐cell‐based diagnostics of tuberculosis (TB) and the measurement of TB vaccine‐take. We used an epitope discovery system, based on recombinant MHC class I molecules that cover the most frequent Caucasian alleles [human leucocyte antigen (HLA)‐A*0101, A*0201, A*0301, A*1101, A*2402, B*0702, B*0801 and B*1501], to identify MHC class I‐binding peptides from overlapping 9‐mer peptides representing the Mtb protein TB10.4. A total of 33 MHC class I‐binding epitopes were identified, spread across the entire amino acid sequence, with some clustering at the N‐ and C‐termini of the protein. Binding of individual peptides or closely related peptide species to different MHC class I alleles was frequently observed. For instance, the common motif of xIMYNYPAMx bound to six of eight alleles. Affinity (50% effective dose) and off‐rate (half life) analysis of candidate Mtb peptides will help to define the conditions for CD8⁺ T‐cell interaction with their nominal MHC class I‐peptide ligands. Subsequent construction of tetramers allowed us to confirm the recognition of some of the epitopes by CD8⁺ T cells from patients with active pulmonary TB. HLA‐B alleles served as the dominant MHC class I restricting molecules for anti‐Mtb TB10.4‐specific CD8⁺ T‐cell responses measured in CD8⁺ T cells from patients with pulmonary TB.     

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.     

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.