Bertrand Georges

HLA-DP4, the Most Frequent HLA II Molecule, Defines a New Supertype of Peptide-Binding Specificity

Among HLA-DP specificities, HLA-DP4 specificity involves at least two molecules, HLA-DPA1*0103/DPB1*0401 (DP401) and HLA-DPA1*0103/DPB1*0402 (DP402), which differ from each other by only three residues. Together, they are present worldwide at an allelic frequency of 20–60% and are the most abundant human HLA II alleles. Strikingly, the peptide-binding specificities of these molecules have never been investigated. Hence, in this study, we report the peptide-binding motifs of both molecules. We first set up a binding assay specific for the immunopurified HLA-DP4 molecules. Using multiple sets of synthetic peptides, we successfully defined the amino acid preferences of the anchor residues. With these assays, we were also able to identify new peptide ligands from allergens and viral and tumor Ags. DP401 and DP402 exhibit very similar patterns of recognition in agreement with molecular modeling of the complexes. Pockets P1 and P6 accommodate the main anchor residues and interestingly contain only two polymorphic residues, β86 and β11, respectively. Both positions are almost dimorphic and thus produce a limited number of pocket combinations. Taken together, our results support the existence of three main binding supertypes among HLA-DP molecules and should significantly contribute to the identification of universal epitopes to be used in peptide-based vaccines for cancer, as well as for allergic or infectious diseases.

Determination of a HLA II promiscuous peptide cocktail as potential vaccine against EBV latency II malignancies

The Epstein-Barr virus (EBV) is associated with several malignant diseases, which can be distinguished by their patterns of viral latent gene expression. The latency II program is limited to the expression of the nonimmunodominant antigens EBNA1, LMP1 and LMP2 and is seen in EBV-positive Hodgkin disease, nasopharyngeal carcinomas, and peripheral T/NK-cell lymphomas. CD4+ T cells may play a crucial role in controlling these EBV latency II malignancies. In this study, we used the prediction software TEPITOPE to predict promiscuous major histocompatibility complex class II epitopes derived from the latency II antigens EBNA1, LMP1, and LMP2. The predicted peptides were then submitted to peptide-binding assays on HLA II purified molecules, which allowed the selection of 6 peptides (EBNA1: 3; LMP1: 1; and LMP2: 2) with a highly promiscuous capability of binding. This peptide cocktail was immunogenic in a model of HLA-DR1 transgenic mice, leading to a specific cellular and humoral TH1 response. The peptides were also recognized by human CD4+ T cells from individuals expressing various HLA II genotypes. This promiscuous peptide cocktail could be immunogenic in the majority of the population and may be used as a peptide-based vaccine in EBV latency II malignancies.

Differential capacity of T cell priming in naive donors of promiscuous CD4+ T cell epitopes of HCV NS3 and Core proteins

To understand the inter‐individual and virus‐independent variability of CD4+ T cell responses to HCV components, we evaluated the effect on these responses of HLA II molecules in uninfected healthy donors. Using HLA II‐specific binding assays, we identified, in the Core and NS3 proteins, 21 long fragments and 24 15‐mer peptides that bound to four to eight of the most preponderant HLA II molecules. We then evaluated the priming capacity of eight long promiscuous peptides in 12 HLA‐unrelated healthy donors. The NS3 1250–1264 peptide primed T cells in all the naive donors, while five others were stimulating in at least half of the individuals. We also report sequences that bind to multiple HLA II molecules but are weakly immunogenic. We therefore conclude that (i) broad HLA II specificity is only a prerequisite for a peptide to be stimulating in multiple individuals, and (ii) promiscuous peptides widely differ in their capacity to prime CD4+ T cells from uninfected healthy donors. We suggest that these priming differences result from inter‐individual variations in the peptide‐specific T cell repertoire. Interestingly, five of the most immunogenic peptides we identified correspond to frequently targeted T cell epitopes in infected patients.

Evidences of conformational changes in class II Major Histocompatibility Complex molecules that affect the immunogenicity.

The N-terminal part of class II-associated invariant chain peptide (CLIP) is assumed to interact with an accessory peptide-binding site on the class II Major Histocompatibility Complex (MHC) molecule, and promote a conformational modification. We have linked this immunoregulatory segment (residues 81-88) to the N-terminus of the influenza hemagglutinin (HA) 307-319 epitope in order to evaluate relationships between the MHC conformational changes and their implication in immune responses. Our chimeric peptide, named CLIP-HA, bind with the same affinity to class II HLA-DR1 molecules as the HA peptide, and is normally recognized by HA-specific T cells. Interestingly, the presence of the N-terminal CLIP region enhances the rate of association to soluble DR1 molecules but prevents the formation of SDS-resistant complexes. These features suggest the existence of HLA-DR1 conformational changes induced by the chimeric peptide. Furthermore, while in vitro HA and CLIP-HA peptides associated to DR1 could not be differentiated based on T-cell recognition, in vivo the CLIP residues strongly impaired the immunogenicity of HA epitope as assessed in HLA-DR1 transgenic mice. Our study demonstrates for the first time that MHC conformational changes, revealed at molecular level, may influence the immunogenicity.

Structural diversity of human class II histocompatibility molecules induced by peptide ligands

SDS–PAGE analyses of stable HLA‐DR1 complexes indicate that the binding of T cell epitopes can lead to multiple conformational variants. Whereas short T epitopes (<14‐mer) induce complexes with apparent MW ranging from 47 to 57 kDa, longer peptides form generally high mobility complexes (44–45 kDa). The generation of HLA‐DR1 conformational variants appears dependent on core peptide residues fitting inside the groove but can additionally be attributed to the presence of N‐ and C‐terminal flanking residues (PFRs) acting as a complementary mechanism. These PFRs can jointly affect major histocompatibility complex class II conformation and stability, supporting the existence of alternative contacts at a distance from the classical binding site.