Sandra Pouvelle-Moratille

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.

Complementarity and redundancy of the binding specificity of HLA-DRB1, -DRB3, -DRB4 and -DRB5 molecules

The second HLA‐DR molecules, which are encoded by loci different from HLA‐DRB1 are weakly polymorphic. Predominant alleles such as HLA‐DRB3*0101, HLA‐DRB4*0101 and HLA‐DRB5*0101 are therefore interesting targets to define antigenic peptides with major impact for the entire population. Strikingly, they have been poorly investigated. Thus we have characterized peptides from the major bee venom allergen that bind efficiently to these molecules and compared them to peptides specific for preponderant HLA‐DRB1 molecules. Interestingly, DRB5*0101 and DRB1*0701 molecules share four bindingpeptides and use some identical anchor residues. Similarities are also found between DRB3*0101 and its haplotype‐associated molecules DRB1*0301 and DRB1*1301. In sharp contrast, DRB4*0101 exhibits a unique binding specificity, which results from particular structural features of its peptide binding site. Yβ81 seems to alter the amino acid preferences of the P1 pocket, while Rβ71, Eβ74, Nβ26 and Cβ13 confer to the P4 pocket a unique topology. Our results show that the two HLA‐DR molecules expressed in most haplotypes studied here have mostly complementary binding patterns. Only haplotype HLA‐DR52 exhibits peptide binding redundancies. Finally our results document functional similarities among HLA‐DR molecules and allow us to propose peptide sequences that might be useful for bee venom immunotherapy.

Selective identification of HLA-DP4 binding T cell epitopes encoded by the MAGE-A gene family

Because of the high frequency of HLA-DP4 in the Caucasian population, we have selectively delineated HLA-DP4 restricted T cell epitopes in the MAGE-A tumor antigens. We identified 12 good binders to HLA-DP4 and investigated the capacity of the seven best binders to induce in vitro specific CD4+ T cell lines from HLA-DP4 healthy donors. We found that the MAGE-A1 90–104 peptide exhibited a high and constant frequency of CD4+ T cell precursors in all the six tested donors. The MAGE-A1 268–282 peptide was found immunogenic in only two donors but with a high precursor frequency. The MAGE-A12 127–141 peptide was T cell stimulating in six different donors and induced fewer T cell lines. The peptide-specific T cell lines were stimulated by DC loaded with the lysates of cells transfected with MAGE-A1 or MAGE-A12, or loaded with the recombinant protein. We also show that the immunoreactivity of CD4+ T cell epitopes restricted to the same HLA II molecule may vary from one individual to another, as a result of inter-individual variations in the CD4+ T cell repertoire.

Alteration of the tertiary structure of the major bee venom allergen Api m 1 by multiple mutations is concomitant with low IgE reactivity

We have engineered a recombinant form of the major bee venom allergen (Api m 1) with the final goal of reducing its IgE reactivity. This molecule (Api mut) contains 24 mutations and one deletion of 10 amino acids. The successive introduction of these sequence modifications led to a progressive loss of specific IgE and IgG reactivity and did not reveal any immunodominant epitopes. However, Api mut exhibited a clear loss of reactivity for Api m 1‐specific IgE and IgG. Injection of Api mut into mice induced specific antibody production. This humoral response was as high as that induced by the Api m 1 but the cross‐reactivity of the antibodies was weak. As inferred by far UV circular dichroism, this mutant was correctly folded. However, near UV circular dichroism and denaturation curves of Api mut showed that it exhibits a dynamic tertiary structure and that it is a highly flexible molecule. Finally, as all the sequence modifications have been introduced outside the human and murine T cell epitope regions, we investigated its T cell properties in mice. We showed that Api mut‐specific T lymphocytes induced in vivo were stimulated in vitro by both proteins. These data provide new insights in the design of hypoallergenic molecules.

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.

Emerging principles for the design of promiscuous HLA-DR-restricted peptides: an example from the major bee venom allergen

Mechanisms underlying successful immunotherapy of allergic patients operate at the level of CD4+ helper T cells. T cell epitopes from allergens may thus constitute interesting molecules for immunotherapy, provided they are efficient for all patients and are not recognized by IgE. In an attempt to define such peptides for allergy to bee venom, we have investigated the capacity ofpeptides encompassing the sequence of the major bee venom allergen to stimulate PBMC from allergic patients and to react specifically with their IgE. The region 77–110 emerged as the most frequently T cell stimulating. We then analyzed the binding modes of the sequence 81–97 for ten different HLA‐DR molecules and introduced punctual mutations to enhance the peptide affinity for these molecules. Six different modes have been identified on the sequence 81–97, one mode being common to eight HLA‐DR molecules. Four HLA‐DR molecules can bind the P85–97 peptide by two different modes with an equivalent affinity. The peptide N89L has a higher affinity for DRB1*0301 and DRB3*0101 and remains as active as the native peptide towards the other HLA‐DR molecules.