HLA-DR Binding Research Articles

In Silico Prediction of Peptides Binding to Multiple HLA-DR Molecules Accurately Identifies Immunodominant Epitopes from gp43 of Paracoccidioides brasiliensis Frequently Recognized in Primary Peripheral Blood Mononuclear Cell Responses from Sensitized Individuals

One of the major drawbacks limiting the use of synthetic peptide vaccines in genetically distinct populations is the fact that different epitopes are recognized by T cells from individuals displaying distinct major histocompatibility complex molecules. Immunization of mice with peptide (181-195) from the immunodominant 43 kDa glycoprotein of Paracoccidioides brasiliensis (gp43), the causative agent of Paracoccidioidomycosis (PCM), conferred protection against infectious challenge by the fungus. To identify immunodominant and potentially protective human T-cell epitopes in gp43, we used the TEPITOPE algorithm to select peptide sequences that would most likely bind multiple HLA-DR molecules and tested their recognition by T cells from sensitized individuals. The 5 most promiscuous peptides were selected from the gp43 sequence and the actual promiscuity of HLA binding was assessed by direct binding assays to 9 prevalent HLA-DR molecules. Synthetic peptides were tested in proliferation assays with peripheral blood mononuclear cells (PBMC) from PCM patients after chemotherapy and healthy controls. PBMC from 14 of 19 patients recognized at least one of the promiscuous peptides, whereas none of the healthy controls recognized the gp43 promiscuous peptides. Peptide gp43(180-194) was recognized by 53% of patients, whereas the other promiscuous gp43 peptides were recognized by 32% to 47% of patients. The frequency of peptide binding and peptide recognition correlated with the promiscuity of HLA-DR binding, as determined by TEPITOPE analysis. In silico prediction of promiscuous epitopes led to the identification of naturally immunodominant epitopes recognized by PBMC from a significant proportion of a genetically heterogeneous patient population exposed to P. brasiliensis. The combination of several such epitopes may increase the frequency of positive responses and allow the immunization of genetically distinct populations.

Selecting peptides to optimize Th1 responses to an equine lentivirus using HLA-DR binding motifs and defined HIV-1 Th peptides.

Three moderately to broadly recognized equine infectious anemia virus (EIAV) peptides that contained helper T-lymphocyte (Th) 1 epitopes were previously identified. Although lipopeptide immunization was only weakly immunostimulatory in a preliminary study, as measured by T-lymphocyte proliferation responses, it was of interest to define additional broadly recognized Th1 epitopes to include in future immunization trials. Using broadly cross-reactive and conserved Th epitopes known in the related human immunodeficiency virus-1 (HIV-1) and binding motifs defined in human leukocyte antigen DR molecules as guides, this work identified three new peptides containing Th1 epitopes recognized by 60-75% of EIAV infected horses. The observed similarity across species of major histocompatibility complex (MHC) class II binding motifs and the conservation of Th peptides between related viruses should allow easier targeting of Th epitope regions in less well characterized pathogens and/or in species whose MHC class II molecules are poorly defined.

Hidden Markov model-based approach as the first screening of binding peptides that interact with MHC class II molecules

The immune system is initiated and regulated through a process starting from the binding of antigenic peptides to major histocompatibility complex (MHC) molecules. Detailed understanding of such interactions would lead to the development of vaccine design for infectious diseases, and immunotherapies for autoimmune diseases and cancer. Since MHC class II genes are highly polymorphic, a computational prediction tool for the first screening of antigenic peptides that bind to MHC class II molecules, is highly desirable. In the present study, hidden Markov model (HMM) was applied for the screening of peptides that interact with nine MHC class II molecules, specifically, human leukocyte antigen (HLA)-DR1, -DR2, -DR4, -DR7, -DR11, -DR15, -DR17, -DR51, and -DQ2. When high-binding peptides interacting with each MHC molecule were subjected to the constructed HMM model, significantly high likelihood values were obtained, as compared to the non-binding peptides as negative control. With the receiver-operating characteristic analysis for the prediction evaluation, our model showed high prediction accuracy, with an average AUC value of 0.87 for all molecules. The HMM model that was trained by HLA-DQ2 showed significantly low likelihood values to peptides that bind to eight HLA-DR molecules. This suggests the high potency of our HMM model for discriminating HLA-DQ binding peptides from HLA-DR binding peptides.

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Involvement of zinc in the binding of Mycoplasma arthritidis-derived mitogen to the proximity of the HLA-DR binding groove regardless of histidine 81 of the beta chain.

Although our recent studies have provided the first evidence demonstrating the direct binding of Mycoplasma arthritidis-derived mitogen (MAM) to MHC class II molecules, it is not yet established how MAM interacts with these molecules. Herein, we demonstrate that MAM binds preferentially and with high affinity to HLA-DR molecules in a zinc-dependent manner. MAM's affinity (25 nM) for HLA-DR molecules is comparable to that of staphylococcal superantigens, and is slightly higher than that for murine MHC class II molecules expressed on the A20 B cell line (111 nM). The amino acid residues located between 14 - 31 and 76 - 90 of the MAM N-terminus play a critical role in MAM / HLA-DR interactions. Histidine at position 81 of the HLA-DR beta-chain, known to be critical for binding of zinc-coordinated superantigens, is not necessary for MAM / HLA-DR interactions. The HLA-DR residues involved in MAM binding are located in the proximal binding groove of the HLA-DR molecule, where the nature of the peptide of the binding groove plays an important role in MAM / HLA-DR interaction. This is the first detailed characterization of MAM's interactions with MHC class II molecules showing a mode of interaction with HLA-DR distinct from that of other superantigens.

ProPred: prediction of HLA-DR binding sites.

ProPred is a graphical web tool for predicting MHC class II binding regions in antigenic protein sequences. The server implement matrix based prediction algorithm, employing amino-acid/position coefficient table deduced from literature. The predicted binders can be visualized either as peaks in graphical interface or as colored residues in HTML interface. This server might be a useful tool in locating the promiscuous binding regions that can bind to several HLA-DR alleles. The server is available at http://www.imtech.res.in/raghava/propred/ raghava@imtech.res.in http://www.imtech.res.in/raghava/propred/page3.html

T-Helper Cell-Response to MHC Class II-Binding Peptides of the Renal Cell Carcinoma-Associated Antigen RAGE-1

Recently, epitope prediction software for HLA-DR binding sequences has become available. In view of the importance of T helper (Th) cell activation in immunotherapy of cancer and evidences supporting immunogenicity of renal cell carcinoma (RCC), we have tested 4 peptides of RAGE-1 binding promiscuously to HLA-DR molecules for induction of an immune response. The peptides predicted by the TEPITOPE program using a stringent threshold were derived from the open reading frame 2 and 5 of RAGE-1. Induction of response was evaluated by culturing peripheral blood mononuclear cells (PBMC) in the presence of peptide-loaded dendritic cells (DC) to determine proliferative activity and cytokine expression. Two out of 5 donors did not respond to any of the 4 peptides, 2 donors responded to one peptide and one donor responded to two other peptides. Notably, as revealed by blocking studies and T cell subtype definition, peptides bound to MHC class II molecules and peptide pulsed DC exclusively activated CD4 + T cells, which were of the Th1 subtype. With respect to clinical application it is important that (un)responsiveness of individual donors' PBMC was a very consistent feature. Though we have not tested explicitly whether these peptides correspond to naturally processed peptides, the possibility to define those patients whose Th might respond to in silico predicted peptides of RAGE-1, by an in vitro assay, could well be a helpful step towards setting up a RAGE-1 based immunotherapeutic protocol.

Identification and antigenicity of broadly cross-reactive and conserved human immunodeficiency virus type 1-derived helper T-lymphocyte epitopes.

Human immunodeficiency virus (HIV)-specific helper T lymphocytes (HTL) play a key role in the immune control of HIV type 1 (HIV-1) infection, and as such are an important target of potential HIV-1 vaccines. In order to identify HTL epitopes in HIV-1 that might serve as vaccine targets, conserved HIV-1-derived peptides bearing an HLA-DR binding supermotif were tested for binding to a panel of the most representative HLA-DR molecules. Eleven highly cross-reactive binding peptides were identified: three in Gag and eight in Pol. Lymphoproliferative responses to this panel of peptides, as well as to the HIV-1 p24 and p66 proteins, were evaluated with a cohort of 31 HIV-1-infected patients. All 11 peptides were recognized by peripheral blood mononuclear cells from multiple HIV-infected donors. Many of the responsive HIV-infected subjects showed recognition of multiple peptides, indicating that HIV-1-specific T-helper responses may be broadly directed in certain individuals. A strong association existed between recognition of the parental recombinant HIV-1 protein and the corresponding HTL peptides, suggesting that these peptides represent epitopes that are processed and presented during the course of HIV-1 infection. Lastly, responses to the supermotif peptides were mediated by CD4(+) T cells and were restricted by major histocompatibility complex class II molecules. The epitopes described herein are potentially important components of HIV-1 therapeutic and prophylactic vaccines.

T細胞株MT2のHLA-DR分子結合ペプチドの解析

HLA分子は抗原ペプチドを結合し, これをT細胞レセプターに提示することによりT細胞を分化, 活性化する免疫制御分子である. HLA分子に結合する抗原ペプチドの結合選択性は, HLA分子から溶出した結合ペプチドのアミノ酸配列から決められるアリル特異的モチーフの検討によって解析されてきている. これ迄に解析されてきたHLA分子結合ペプチド, 特にHLA-DR分子結合ペプチドは主にトランスフォームされたB細胞由来の分子を用いて明らかにされてきた. 今回我々はT細胞由来のHLA-DR分子に結合している抗原ペプチドを検討するため, HLA-DR分子を発現しているヒトT細胞のモデルとして, ヒトリンパ球向性ウイルス1型感染細胞MT2細胞株を用い, そのHLA-DR分子に結合しているペプチドを分離しアミノ酸配列を決定した. 決定した配列を基に合成したペプチドとHLA-DR分子を発現させたL細胞との結合性を解析した.

A postgenomic approach to identification of Mycobacterium leprae-specific peptides as T-cell reagents.

To identify Mycobacterium leprae-specific human T-cell epitopes, which could be used to distinguish exposure to M. leprae from exposure to Mycobacterium tuberculosis or to environmental mycobacteria or from immune responses following Mycobacterium bovis BCG vaccination, 15-mer synthetic peptides were synthesized based on data from the M. leprae genome, each peptide containing three or more predicted HLA-DR binding motifs. Eighty-one peptides from 33 genes were tested for their ability to induce T-cell responses, using peripheral blood mononuclear cells (PBMC) from tuberculoid leprosy patients (n = 59) and healthy leprosy contacts (n = 53) from Brazil, Ethiopia, Nepal, and Pakistan and 20 United Kingdom blood bank donors. Gamma interferon (IFN-gamma) secretion proved more sensitive for detection of PBMC responses to peptides than did lymphocyte proliferation. Many of the peptides giving the strongest responses in leprosy donors compared to subjects from the United Kingdom, where leprosy is not endemic, have identical, or almost identical, sequences in M. leprae and M. tuberculosis and would not be suitable as diagnostic tools. Most of the peptides recognized by United Kingdom donors showed promiscuous recognition by subjects expressing differing HLA-DR types. The majority of the novel T-cell epitopes identified came from proteins not previously recognized as immune targets, many of which are cytosolic enzymes. Fifteen of the tested peptides had > or =5 of 15 amino acid mismatches between the equivalent M. leprae and M. tuberculosis sequences; of these, eight gave specificities of > or =90% (percentage of United Kingdom donors who were nonresponders for IFN-gamma secretion), with sensitivities (percentage of responders) ranging from 19 to 47% for tuberculoid leprosy patients and 21 to 64% for healthy leprosy contacts. A pool of such peptides, formulated as a skin test reagent, could be used to monitor exposure to leprosy or as an aid to early diagnosis.

A simple screening method for detecting bindings between oligopeptides and HLA-DR molecules on filter papers: possible application for mapping of putative helper T-cell epitopes on MSP1 of Plasmodium falciparum.

Binding capacities of synthetic peptides to HLA-DR molecules were tested on filter papers to identify putative helper T-cell epitopes on a malarial protein. The antigen tested was the merozoite surface glycoprotein 1 (MSP1) of Plasmodium falciparum, a vaccine candidate targeting the asexual erythrocytic stage. Bindings between synthetic oligopeptides and HLA-DR molecules were tested. Such bindings were not non-specific, and a known helper T-cell epitope peptide showed positive binding to the restricting HLA-DR molecule. By using this screening system, we observed the unequal distribution of HLA-DR-binding peptides in 10 out of 17 MSP1 blocks tested. Block #6 of MSP1 seemed to show the highest frequency in the positive binding; on the other hand, blocks #1 and #17, both of which were thought to be vaccine candidate regions, contained fewer HLA-DR binding peptides. This was not inconsistent with the results that block #17 was less stimulatory to peripheral T cells than block #6. The peptides with positive binding to HLA-DR showed actual epitope activities when we tested peptide-driven proliferation of human bulk T-cell lines, and association between the two parameters was statistically significant (P<0.001). For more detailed information for vaccine development, peptides with both IgG- and HLA-DR binding activities were mapped in block #17 of MSP1. Together with these results, we demonstrate that our simple screening system seems to provide essential information for vaccine development through uncovering locations of putative epitopes for human helper T cells.

Specific and general HLA-DR binding motifs: comparison of algorithms

Using panels of peptides well characterized for their ability to bind to HLA DR1, DRB1∗1101, or DRB1∗0401 molecules, algorithms were deduced to predict binding to these molecules. These algorithms consist of blocks of 8 amino acids containing an amino acid anchor (Tyr, Phe, Trp, Leu, Ile, or Val) at position i and different amino acid combinations at positions i+2 to i+7 depending on the class II molecule. The sensitivity (% of correctly predicted binder peptides) and specificity (% of correctly predicted non-binder peptides) of these algorithms, were tested against different independent panels of peptides and compared to other algorithms reported in the literature. Similarly, using a panel of 232 peptides able to bind to one or more HLA molecules as well as 43 non-binder peptides, we deduced a general motif for the prediction of binding to HLA-DR molecules. The sensitivity and specificity of this general motif was dependent on the threshold score used for the predictions. For a score of 0.1, the sensitivity and specificity were 84.7% and 69.8%, respectively. This motif was validated against several panels of binder and non-binder peptides reported in the literature, as well as against 35, 15-mer peptides from hepatitis C virus core protein, that were synthesized and tested in a binding assay against a panel of 19 HLA-DR molecules. The sensitivities and specificities against these panels of peptides were similar to those attained against the panels used to deduce the algorithm. These results show that comparison of binder and non-binder peptides, as well as correcting for the relative abundance of amino acids in proteins, is a useful approach to deduce performing algorithms to predict binding to HLA molecules.

Major peptide autoepitopes for nucleosome-specific T cells of human lupus.

We tested 154 peptides spanning the entire length of core histones of nucleosomes for the ability to stimulate an anti-DNA autoantibody-inducing T helper (Th) clone, as well as CD4(+) T-cell lines and T cells, in fresh PBMCs from 23 patients with lupus erythematosus. In contrast to normal T cells, lupus T cells responded strongly to certain histone peptides, irrespective of the patient's disease status. Nucleosomal peptides in histone regions H2B(10-33), H4(16-39) (and overlapping H4(14-28)), H4(71-94), and H3(91-105) (and overlapping H3(100-114)) were recurrently recognized by CD4 T cells from the patients with lupus. Remarkably, these same peptides overlap with major epitopes for the Th cells that induce anti-DNA autoantibodies and nephritis in lupus-prone mice. We localized 2 other recurrent epitopes for human lupus T cells in H2A(34-48) and H4(49-63). All the T-cell autoepitopes have multiple HLA-DR binding motifs, and the epitopes are located in histone regions recognized by lupus autoantibodies, suggesting a basis for their immunodominance. Native nucleosomes and their peptides H4(16-39), H4(71-94), and H3(91-105) induced a stronger IFN-gamma response, whereas others, particularly, H2A(34-48), favored an IL-10- and/or IL-4-positive T-cell response. The major autoepitopes may reveal the mechanism of autoimmune T-cell expansion and lead to antigen-specific therapy of human lupus.

Binding Motifs of Copolymer 1 to Multiple Sclerosis- and Rheumatoid Arthritis-Associated HLA-DR Molecules

Copolymer 1 (Cop 1, poly (Y, E, A, K)) is a random synthetic amino acid copolymer effective in the treatment of relapsing forms of multiple sclerosis (MS). Cop 1 binds promiscuously, with high affinity and in a peptide-specific manner to purified MS-associated HLA-DR2 (DRB1*1501) and rheumatoid arthritis-associated HLA-DR1 (DRB1*0101) or HLA-DR4 (DRB1*0401) molecules. In the present work at least 95% of added Cop 1 could be bound to recombinant "empty" HLA-DR1 and -DR4, and 80% could be bound to HLA-DR2 proteins. Amino acid composition, HPLC profiles, and sequencing patterns of Cop 1 eluted by acid extraction from HLA-DR molecules were similar to those of the unseparated Cop 1. Protruding N-terminal ends of Cop 1 bound to HLA-DR1, -DR2, or -DR4 molecules were then treated with aminopeptidase I, followed by elution, HPLC, and pool sequencing. In contrast to untreated or unbound Cop 1, this material exhibited distinct motifs at some positions with increases in levels of E at the first and second cycles, of K at the second and third cycles, and of Y (presumably at P1 of the bound peptide) at the third to fifth cycles, regardless of the HLA-DR molecule employed. No preference was seen at the following cycles that were mainly A. These first pooled HLA-DR binding epitopes provide clues to the components of Cop 1 that are biologically active in suppressing MS and possibly rheumatoid arthritis.

Naturally processed peptides from HLA-DQ7 (alpha1*0501-beta1*0301): influence of both alpha and beta chain polymorphism in the HLA-DQ peptide binding specificity.

Self peptides bound to HLA-DQ7 (alpha1*0501-beta1*0301), one of the HLA molecules associated with protection against insulin-dependent diabetes mellitus, were characterized after their acid elution from immunoaffinity-purified HLA-DQ7 (alpha1*0501-beta1*0301) molecules. The majority of these self peptides derived from membrane-associated proteins including HLA class I, class II, class II-associated invariant chain peptide and the transferrin-receptor (TfR). By in vitro binding assays, the specificity of these endogenous peptides for HLA-DQ7 (alpha1*0501-beta1*0301) molecules was confirmed. Among these peptides, the binding specificity of the TfR 215-230 self peptide was further examined on a variety of HLA-DQ and DR dimers. Several findings emerged from this analysis: (1) this peptide displayed HLA-DQ allelic specificity, binding only to HLA-DQ7 (alpha1*0501-beta1*0301); (2) when either the DQalpha or DQbeta chain was exchanged, little or no binding was observed, indicating that specificity of HLA-DQ peptide binding was determined by polymorphic residues of both the alpha and beta chains. (3) Unexpectedly, the TfR 215-230 self peptide, eluted from DQ, was promiscuous with regard to HLA-DR binding. This distinct DR and DQ binding pattern could reflect the structure of these two molecules as recently evidenced by crystallography.

Naturally processed peptides from HLA-DQ7 (α1*0501-β1*0301): influence of both α and β chain polymorphism in the HLA-DQ peptide binding specificity

Self peptides bound to HLA-DQ7 (α1*0501-β1*0301), one of the HLA molecules associated with protection against insulin-dependent diabetes mellitus, were characterized after their acid elution from immunoaffinity-purified HLA-DQ7 (α1*0501-β1*0301) molecules. The majority of these self peptides derived from membrane-associated proteins including HLA class I, class II, class II-associated invariant chain peptide and the transferrin-receptor (TfR). By in vitro binding assays, the specificity of these endogenous peptides for HLA-DQ7 (α1*0501-β1*0301) molecules was confirmed. Among these peptides, the binding specificity of the TfR 215 – 230 self peptide was further examined on a variety of HLA-DQ and DR dimers. Several findings emerged from this analysis: (1) this peptide displayed HLA-DQ allelic specificity, binding only to HLA-DQ7 (α1*0501-β1*0301); (2) when either the DQα or DQβ chain was exchanged, little or no binding was observed, indicating that specificity of HLA-DQ peptide binding was determined by polymorphic residues of both the α and β chains. (3) Unexpectedly, the TfR 215 – 230 self peptide, eluted from DQ, was promiscuous with regard to HLA-DR binding. This distinct DR and DQ binding pattern could reflect the structure of these two molecules as recently evidenced by crystallography.

HLA-DR binding analysis of peptides from islet antigens in IDDM.

HLA molecules are essential for thymic education and HLA restriction of T-cell responses. We therefore analyzed the HLA-DR binding affinities of synthetic peptides covering the entire sequences of GAD65, islet cell antigen 69 (ICA69), and (pro)insulin, which are candidate antigens in the autoimmune process of T-cell-mediated destruction of the pancreatic beta-cells. Subsequently, peptide HLA-DR binding was correlated to peptide antigenicity by comparing known T-cell epitopes with their HLA-binding affinities defined in this study. The results demonstrate the following. 1) (Pro)insulin peptides display a strong binding affinity for HLA-DR2, which is associated with negative genetic predisposition to IDDM, whereas poor binding was observed for HLA-DR molecules neutrally or positively associated with IDDM. This suggests that the absence of insulin-reactive T-cells in DR2+ individuals may be explained by negative selection on high-affinity DR2 binding insulin peptides. 2) Most autoantigenic peptides display promiscuous HLA-DR binding patterns. This promiscuity in itself is not sufficient to explain the genetic association of HLA-DR with development of IDDM. 3) HLA-DR3 binding of autoantigenic GAD65 peptides is relatively weak compared with that of other known T-cell epitopes. 4) All peptide epitopes recognized by HLA-DR-restricted T-cells from either IDDM patients or GAD65-immunized HLA-DR transgenic mice bind with high affinity to their HLA-DR restriction molecule (P < 0.0006). In contrast, T-cell epitopes recognized by nondiabetic controls bind DR molecules with weak or undetectable affinity. These results thus indicate a strong correlation between antigenicity and HLA-DR binding affinity of GAD65 peptides in IDDM. Furthermore, negative thymic selection of insulin peptides in low-risk (HLA-DR2 expressing) subjects may explain the lack of autoreactivity to insulin in such individuals.

Altered Peptide Ligands of Islet Autoantigen Imogen 38 Inhibit Antigen Specific T Cell Reactivity in Human Type-1 Diabetes

Type 1 diabetes, insulin-dependent diabetes mellitus (IDDM) results from autoimmune T cell-dependent destruction of insulin producing β-cells in the pancreatic islets of Langerhans. T cells from recent-onset IDDM patients specifically proliferate to β cell membrane Ag enriched fractions, containing the mitochondrial 38kD islet antigen (Imogen). Recently, we identified a peptide epitope (Imogen p55-70) that is recognized by a 38kD-specific, Th1 clone from an IDDM patient. In animal models of autoimmune diseases, altered self peptide ligands (APL) have been used effectively in peptide-based immune prevention or therapy. No such APL, however, have been reported so far that can modulate autoreactive T-cell responses in IDDM. Here, we have designed APL of p55-70. These APL efficiently downregulatein vitroactivation of the 38kD-specific Th1 clone induced by either p55-70 or by native β cell autoantigens. Self peptide reactive T-cell proliferation could be inhibited only when APL and the self peptide were present on the same APC. Unrelated peptides with equal HLA-DR binding affinity were not effective, excluding simple MHC competition as the mechanism for T-cell modulation. APL triggered upregulation of CD69 and CD25 expression, but not T-cell proliferation, TCR down-modulation or T-cell anergy. Thus, the p55-70 APL inhibit β cell autoantigen-induced activation of an Imogen-reactive T-cell clone derived from an IDDM patient, by acting as partial TCR agonists that inhibit TCR down-modulation.

Six unrelated HLA-DR-matched adults recognize identical CD4+ T cell epitopes from influenza A haemagglutinin that are not simply peptides with high HLA-DR binding affinities.

Influenza A/Beijing/32/92 (H3N2) haemagglutinin (HA)-specific short-term CD4+ T cell lines were generated from six unrelated HLA-DR0701, 1501 positive adults (aged 27-60 years) 3 months following administration of an influenza subunit vaccine containing HA A/Beijing/32/92. Epitope recognition was examined using 118 HA A/Beijing/32/92-specific 16mer peptides which overlapped by 11 residues and which spanned the entire molecule. Following influenza vaccination the donors recognized identical HA peptides. The selected peptides represented HA regions which have been free from extensive drift mutation since the emergence of human H3N2 influenza A strains. Using DAP DR7.0701 cells (a murine cell line expressing HLA-DR0701) as antigen-presenting cells the majority of CD4+ T cell responses were shown to be HLA-DR0701 restricted. The relationship between HA peptide recognition and relative strength of HA peptide-HLA-DR0701 binding was then explored in a competition assay with biotinylated CLIP peptide. Although peptides representing dominant HA epitopes bound to DR0701, the relationship between relative strength of binding and immunodominance was complex, and many strongly binding peptides, particularly those with glycosylation sites and showing inter-strain variation, were not recognized. These results illustrate the control HLA class II exerts over CD4+ T cell HA epitope selection in unrelated adult humans. Immunodominance appears not to be directly related to the relative strength of HA peptide-HLA class II binding, and thus reflects complex interactions between antigen processing, intracellular competition for HLA binding, TCR repertoires and repeated exposure to different strains of influenza A viruses.

Distinct conformations of a peptide bound to HLA-DR1 or DRB5*0101 suggested by molecular modelling.

The conformation of peptides when bound to different HLA class II molecules is of interest in the study of specificity and function of responding T cells. Here, we report the investigation of the HLA-DR binding profiles of an immunodominant and HLA-promiscuous mycobacterial peptide, p38G. Its binding affinities were found to be high for DR1, moderate for DR2, DR7 and DR8, low for DR4, DR5, DR6 and DR9, and below detection for DR3. The minimum peptide length required for binding was, in the majority of cases, nine residues and 11 in two instances (DR2 and DR4). Peptide binding to DR2 was attributed to the DRB5*0101 and not to the DRB1*1501 gene product. Substitution analysis of the amino acid residues involved in binding to DR1 and DRB5*0101 identified F-354 as the common primary contact residue (P1), while allele-specific differences were found in positions P4, P6 and in the C-terminal anchor residue (valine at P9 for DR1 or lysine at P10 for DRB5*0101). Computer-assisted evaluation of these empirical data produced a molecular model, suggesting that the peptide binds to DR1 in an elongated conformation, similar to that of other peptide MHC class II complexes. In contrast, the DRB5*0101 bound peptide is likely to be kinked, which so far was considered characteristic only for peptides within MHC class I complexes. The different conformations imposed on the same peptide by distinct HLA alleles may represent an important mechanism for the control of T cell responses.