Dominique Zeliszewski

Extensive study of DRB, DQA, and DQB gene polymorphism in 23 DR2-positive, insulin-dependent diabetes mellitus patients

To gain insight into the HLA subregions involved in protection against insulin-dependent diabetes mellitus (IDDM) we investigated the polymorphism of HLA-DR and -DQ genes in 23 DR2 IDDM patients. Results show the following. (1) Fourteen patients (61%) possess the DRB1, DRB5, and DQB1 alleles found in DRw16/DQw5 healthy people. These data contrast with the 5% of DRw16 normally found in DR2 populations and are in agreement with former observations supporting that the DRw16 haplotype is not protective. (2) Nine DR2 patients, i.e., 39% versus 95% in published DR2 controls, possess the DRB alleles found in DRw15 unaffected people. Among them, six patients have also DQA1 and DQB1 alleles identical to those found in DRw15/DQw6 healthy individuals. These data confirm that the DRw15/DQw6 haplotype is protective but indicate that none of the DR or DQ alleles, alone or in association, confers an absolute protection. (3) Our most striking results concern the very high frequency of recombinant haplotypes among the DRw15 patients: 3 of 9. In these three patients recombinations led to the elimination of both DQB1 and DQA1 alleles usually associated with DRw15. This strongly suggests that the occurrence of IDDM in these DRw15 patients is due to the absence of the usual DQ product and thus reinforces the assumption that DQ rather than DR region is involved in the protection conferred by the DRw15/DQw6 haplotype. Finally, analysis of the non-DRw15 haplotypes in heterozygous patients showed that IDDM can occur in the absence of any DQ alpha beta heterodimer of susceptibility.

Molecular basis for degenerate T-cell recognition of one peptide in the context of several DR molecules

We report the study of one CD4+ T-cell clone that recognizes peptide HA306-320 in the context of autologous DR1101 molecules as well as of allogeneic DR1301, DR0402, DR1501, and DR1601 molecules. This degenerate T-cell recognition is mediated by a single T-cell receptor (TCR) as judged by both TCR-V beta sequencing and cold-target competition assays. Restriction analysis shows that substitutions of DR residues within the third hypervariable region result in a loss of T-cell reactivity, which is restored by additional substitutions in the first and/or second hypervariable regions. Thus, there is no correlation between antigen presentation abilities of the different allelic DR products and the degree of sequence homology between these products. DR residues whose substitution is compatible with T-cell recognition potentially interact with peptides rather than with TCRs by virtue of their location in the floor of the groove or as previously documented for residues of the alpha-helix. Furthermore, antigen presentation by allogeneic DR molecules occurs independently of their affinity for the peptide, as determined in cell surface-binding assays using biotinylated HA306-320. Altogether these data suggest that degenerate T-cell recognition mainly depends on an influence of polymorphic DR residues on the configuration adopted by the peptide in the DR groove so that the epitope is left intact.

Binding analysis of 95 HIV gp120 peptides to HLA-DR1101 and -DR0401 evidenced many HLA-class II binding regions on gp120 and suggested several promiscuous regions.

To identify HLA-DR-binding peptides within the human immunodeficiency virus (HIV)-1 proteins. 95 overlapping synthetic peptides representing the entire sequence of gp120-LAI were screened for their capacity to bind to two HLA-DR molecules with distant sequences (DR0401 and DR1101). By using a cell surface competitive binding assay, 56 DR-binding peptides were identified, of which 35 bound to both DR1101 and DR0401. A highly significant concordance was evidenced by statistical analysis between binding of peptides to one and to the other DR molecule, suggesting a high proportion of promiscuity among gp120 peptides, even though no clear sequence pattern accounting for such promiscuity was found. DR-binding peptides were located along the entire gp120 sequence. Yet, the majority of them (42 among 56) were concentrated in seven multiagretopic regions that were arbitrarily defined as regions containing four or more overlapping continuous peptides binding to DR1011 and/or DR0401. A good correlation was found between DR-binding regions or DR-binding peptides defined in this study and promiscuous T helper gp120 epitopes previously described in seropositive individuals. All these results suggest that the identification of multiagretopic DR-binding regions may be a great help for the predicition of protein determinants that have the likelihood of being promiscuous T helper epitopes in humans.

Functional definition of HLA-DR and -DQ epitopes specific for DR2-short, DwFJO haplotype

T-lymphocyte clones specific for the influenza A/Texas virus were obtained by limiting dilution of activated T cells from an HLA A2/3, B7/39, Cw -/-, DR2-short/2 short, DQw1/w1, DwFJO/FJO donor. Among the proliferating clones studied, and irrespective of their antigenic specificities, most of them were restricted by epitope(s) on HLA-DR molecules present only on DR2-short/DwFJO cells but not on DR2-negative or DR2-long positive (Dw2, Dw12, Dw-) cells. Two clones were restricted by epitopes borne by DQ products. Here again, these epitopes were present on DR2-short/DwFJO but not on DR2-long, DQw1 (Dw2, Dw12) cells, indicating that the DQwl molecules of DR2-long and DR2-short haplotypes are different. Taken together, these results indicate that the DR2-short, DwFJO haplotype is characterized by both HLA-DR- and DQ-specific molecules. Finally, one clone was restricted by an epitope shared by DR products from DR2 short/DwFJO, DRw11, and DRw13 haplotypes. This latter functional determinant has never been described until now.

Both HLA-DR and HLA-DQ determinants contribute to HLA-Dw typing

The respective contribution of HLA-DR and HLA-DQ gene products in the induction of allogeneic proliferative responses in primary mixed lymphocyte reaction and, therefore, in HLA-Dw typing, is still unclear or controversial. This is in part due to a strong linkage disequilibrium between HLA-DR and -DQ genes. We used DR- or DQ-restricted influenza-specific T-cell clones to define DR and DQ products on a large panel of allogeneic antigen presenting cells. With this functional screening assay, we identified two haplotypes with unusual DR/DQ associations. Cells of these haplotypes were then used as responder cells in mixed lymphocyte culture and stimulated by homozygous typing cells displaying DR or DQ incompatibilities. Our results indicate that DR or DQ incompatibilities alone can give rise, in both cases, to strong T-cell proliferation in a mixed lymphocyte reaction. This was further verified by blocking experiments of secondary mixed lymphocyte reactions by HLA-specific monoclonal antibodies. Anti-DQ, but not anti-DR, antibodies inhibited DQ-incompatible responses. Conversely, anti-DR, but not anti-DQ, antibodies could block DR-incompatible mixed lymphocyte reactions. Together, the results suggest that both HLA-DR and DQ gene products can be involved in HLA-Dw typing. Finally, in dual DR- and DQ-incompatible mixed lymphocyte reaction combinations, HLA-DR molecules seem to have an immunodominant effect, because the response is mostly inhibited by anti-DR antibodies. Immunodominance of HLA-DR allodeterminants may, at least in part, explain some of the controversial conclusions reported by others concerning the role of HLA-DQ molecules in HLA-Dw typing.

Requirements for lysis of activated T cells by class-II-restricted cytolytic T-lymphocytes.

In the present study, we explored the specific requirements for lysis of human activated T cells by CD4+ CTLs. This was achieved by using human CD4+ T cell lines or clones specific for a peptidic fragment of influenza virus as both CTL effectors and target T cells (TTCs). Our results further establish that human activated T cells expressing HLA-DR molecules can present Ag to and be lysed by CD4+ HLA-DR restricted CTLs. This killing is Ag specific and HLA-DR restricted. It can be observed whether TTCs are heterologous or autologous, CD4+ or CD8+. However, we find that in our model: (a) TTCs are able to present artificially processed peptidic fragments of Ag, but not the corresponding natural Ag in the context of class II determinants, even if they can process whole virus in the context of class I determinants; (b) TTCs must express high density of HLA-DR molecules on their membrane; (c) preincubation of TTCs with high concentrations of peptide is required; and (d) interestingly enough, addition of free peptide at similar concentration during the cytolytic assay to replace TTC preincubation inhibits TTC lysis by at least two different mechanisms, i.e., cold-target inhibition in which CTLs serve as their own cold targets and inhibition at the effector cell level. From these results, one can conclude that stringent conditions are required for lysis of activated T cells by class-II-restricted CTLs.

An antiviral T-Cell clone defines a functional supertypic specificity shared by different HLA-DR molecules from DR2-short, DRw11, and DRwl3 Haplotypes

An influenza virus-specific HLA class IIrestricted human T4+ clone (Ij) allows us to define a new functional supertypic HLA class II specificity shared by three different haplotypes. Influenza A virus-infected antigen-presenting cells of these three haplotypes, HLA-DR2 short, DRw11, and DRw13, are able to stimulate Ij cells. The same precise viral specificity is seen in all three cases. Proliferation inhibition experiments using HLA-specific monoclonal antibodies demonstrate that HLA-DR products are involved in all cases. However, according to the DR specificity of the antigen-presenting cell, differential blockings by a series of DR-specific monoclonal antibodies suggest that the functional epitope is shared by different HLA DR molecules. This is confirmed by two-dimensional gel analysis of the HLA DRβ chains expressed in the three haplotypes.

BINDING OF ALA-SUBSTITUTED ANALOGS OF HA306-320 TO DR1101, DR1301, AND DR0402 MOLECULES : CORRELATION OF DR-PEPTIDE INTERACTIONS WITH RECOGNITION BY A SINGLE TCR

We tested the hypothesis that a cross-reactive T-cell clone could recognize HA306-320 peptide complexed to autologous HLA-DR1101, and also to allogenic HLA-DR0402 and HLA-DR1301 molecules, because of similar orientations of HA306-320 side chains in the groove of the three DR molecules. To approach peptide orientations in each HLA groove we compared the capacity of Ala-monosubstituted analogs to bind and be presented by DR1101, DR0402, and DR1301. Results indicated that the orientation of HA306-320 in DR1101 was grossly similar to the known orientation of HA307-319 in DR0101. Data suggested many similarities in peptide orientations in DR0402 and DR1301 as well. However, differences in binding were also observed. Ala substitution of Y309 had much less effect on peptide binding to DR1301 and DR0402 than to DR1101 and Ala-substitution of T314 increased affinity for DR1301 but not for DR1101 and DR0402. These alterations of peptide-DR interactions were probably communicated to the upper peptide surface. Indeed, the levels of T-cell clone reactivities against analogs mutated at positions predicted to face the TCR were lower when complexed to allogeneic DR molecules than when complexed to DR1101. Yet these epitopic alterations are likely subtle, since the decreased reactivity of the clone to allogeneic molecules could be compensated by peptide substitution at Y309, predicted to face the MHC.

Functional relationships between HLA-DR2-short/FJO, DB9, and AZH as defined by DR- and DQ-restricted influenza-specific cloned T cells

i Laboratoire Immunologic et Virologic des Tumeurs, groupe INSERM U152, H6pital Cochin, 27, rue du Faubourg Saint-Jacques, 75674 Paris Cedex 14, France 2 TissueTyping Unit, DepartmentofMedical Microbiology andtheLautenbergCenter for Generaland Tumor Immunology, Hadassah, Hebrew University, Jerusalem, Israel 3 Centre de transfusion r6gional de Lyon, 1-3, rue du Vercors, 69342 Lyon Cedex 7, France 4 Instituto Venezolano des investigationes cientificas, Aparto 1827, Caracas 1010 A., Venezuela