Ghislaine Sterkers

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.

Fine specificity analysis of human influenza-specific cloned cell lines

Influenza-specific human-T-cell clones, proliferating in the presence of virus-infected cells with restriction by class II molecules and displaying class II-restricted CTL activity or specific helper activity in antibody synthesis, have been analyzed for antigenic specificities. All of them were obtained by in vitro stimulation against influenza A/Texas virus. In all cases the virus specificity appeared identical in cytolytic and proliferative responses. Three of the clones were broadly cross-reactive, recognizing all or almost all type A influenza strains. The three remaining clones were subtype specific when tested with human strains and recognized the surface glycoproteins of influenza virus. One of these lines reacted with an epitope of the neuraminidase N2 while the other two recognized the hemagglutinin H3. By using a large panel of mammalian and avian influenza strains, it can be demonstrated that hemagglutinin-specific human T cells can recognize a cross-reacting determinant shared by H3 and H4 subtypes of hemagglutinin which has never been detected with antibodies.

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.

DR-restricted T-cell reactivities associated with the Dw19 specificity can be directed against the products of either locus DRB3 (DRw52c) or locus DRB1

HLA-Dw 19 antigen presenting cells express two different DR beta chains encoded respectively by DRB1 and DRB3 genes. In the present study we determined which of these two DR beta chains is recognized by DR-restricted T-cell clones. First we selected influenza-specific, DR-restricted T-cell clones of which restriction is strictly associated with the Dw19 specificity. Then we characterized by oligonucleotide typing one antigen presenting cell (HC12M) which exhibits a new haplotype associating a DRB1 gene highly related or identical to that from Dw 18 haplotypes with a DRB3 gene highly related or identical to that from Dw19 haplotypes. Finally, by testing the reactivity of the selected T-cell clones against Dw18, Dw19, and HC12M antigen presenting cells, we show that these DR-restricted Dw19-specific effectors can recognize either the DRB1-encoded chain present only on Dw19 antigen presenting cell or the DRB3-encoded chain shared by Dw19 and HC12M antigen presenting cells. Interestingly, our results show that DRB1 chains from Dw19 and Dw18 which differ by a single amino acid substitution at position 86 may be distinguished by T cells, implicating that this residue plays a role in T-cell recognition of HLA-DR-antigen complex. The implication of our results with regard to the new nomenclature of HLA specificities defined by T-cell clones will be discussed.

The two-dimensional migration pattern of the single HLA-DR beta chain expressed in DRw8 haplotypes is not fully predictive of its activity in antigen presentation

By the use of MLR with homozygous typing cells, three subtypes in the HLA-DRw8 haplotype have been described: Dw8.1, Dw8.2, and Dw8.3 (Grosse-Wilde et al. 1984). The heterogeneity of this haplotype has been also suggested by serology (Betuel et al. 1984), cellular typing (Mickelson et al. 1983, Herrera et al. 1985), and biochemical analysis (Baldwin et al. 1985). Here we report results of a biochemical analysis of 12 HLA-DRw8 molecules from Dw8.1, Dw8.2, or Dw8.blank cells. The results have been correlated with the ability of these cells to present influenza viral antigens to two HLA-DRw8, Dw8.1-restricted T-cell clones, TA4 and TA10 (Henin et al. 1985). The EBV-transformed cell lines issued from six Dw8.1 donors were labeled at 37 °C for 5 h with 35Smethionine (18.5 Mbq for 10-20 x 106 cells); after lysis of cells in 1% Nonidet P-40, the DR molecules were immunoprecipitated with a monomorphic HLA-DR-specific mAb, L243 (Lampson and Levy 1980). The invariant (Ii) chain (M r 31000-33000) and the alpha chain ( M r 32 000-34 000) migrated to their known constant positions in the 2D-PAGE pattern. Using the homozygous DRw8. DwS. 1 MAD cell (Mickelson et al. 1983) as a reference cell, we were able to identify four spots characteristic of the DRw8 beta chain (Fig. 1A). As illustrated in Figure 1B, C, and D for HC12, GAL4, and 267 cells, all six Dw8.1 cells presented the same pattern of four heterogeneous spots (arrowheads) which were associated with the classical spots of other HLA-DR specificities

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.

Regulation of helper T cell clone proliferation via the CD2 molecule

We have investigated the requirements for CD2-induced proliferation of a CD4+, CD8-, CD3+, CD2+ antigen-specific, class II-restricted proliferating cloned cell line. A combination pair of two monoclonal antibodies (MoAb) recognizing, respectively, TII1 and D66 epitopes on the CD2 molecule was used as a stimulus. The regulatory function of accessory cells and various interleukins in this proliferation was determined. The results show that although this clone was able to proliferate in the absence of accessory cells (AC) or interleukin 1 (IL-1) when stimulated by these MoAb, AC constantly enhanced the response to these MoAb. AC acted by increasing high-affinity IL-2 receptor expression. On the contrary they did not play any role in IL-2 production. This regulation of IL-2 receptor expression by AC was specific of adherent cells, did not involve Fc receptors, was impaired when AC were metabolically inactivated and did not require T cell-AC interaction via LFA1, CD4, or HLA molecules. The AC function was not abrogated by anti-IL-1 antibodies and could not be replaced by exogenous IL-1. These results were compared to previously described AC effects on resting T-cell proliferation when stimulated with the same pair of anti-CD2 MoAb. Clear differences in activation requirements in resting and activated T cells via CD2 molecules were found.

Contribution of p56lck to the upregulation of cytokine production and T cell proliferation by IL-2 in human CD3-stimulated T cell clones

Interaction of the interleukin 2 receptor (IL-2R) beta chain with the lymphocyte-specific protein tyrosine kinase (PTK), p56lck, has led to the speculation that p56lck participates in growth signal transduction. Although activation of T cells with interleukin 2 (IL-2) results in the activation of p56lck, accumulating data support the notion that Lck does not play an essential role in mitogenic signal delivery from the IL-2R. Since this src-related PTK has been shown to enhance TCR/CD3-mediated T cell responsiveness, here we investigated whether activation of Lck by IL-2 could contribute to enhance TCR/CD3-mediated T cell functions. This was achieved by using human CD4(+)-cloned T cells and comparing the effects of IL-2 on p56lck kinase activation and cytokine production. Results show that p56lck kinase activity increased as early as 1 min, reached a maximum within 5 min and decreased within 60 min after IL-2 stimulation. Such treatment with IL-2 also resulted in enhancing T cell responsiveness to CD3+PMA stimulation, as assessed by IL-2 and IFN-gamma secretion and by T cell proliferation. This increase of T cell functions was correlated with IL-2-induced p56lck activation in both dose-response and time-course experiments. Taken together these results strongly suggest that activation of Lck by IL-2 may play a role in regulating CD3-mediated T cell functions.

Structural Mapping of T Cell Clones Restriction Elements Suggests That a DQα-DRβ Hybrid Molecule Could Be Functional in DRw13 Cells

The activation of competent T cells requires the recognition of both an antigen and a major histocompatibility complex (MHC) molecule. The strong polymorphism of HLA-D genes, their homologies, and their complex associations in various haplotypes hindered the localization of functional determinants at the polypeptidic level. Here, using a selected panel of DRw13-DQw1 cells we looked for correlations between the two-dimensional gel profiles of class II antigens and the reactivity of antigen-specific T-cell clones. The polymorphism of DRβ, DQα, and DQβ chains encoded by DRw13-DQw1 haplotypes (Dw18 and Dw19) was studied by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), revealing one DRβ1 and two DRβ2 chains and no electrophoretic polymorphism of DQα and DQβ chains. Eight T-cell clones specific for influenza A-infected DRw13-DQw1 antigen presenting cell (APC) were generated from a Dw19 homozygous individual. Three patterns of reactivity were observed: 1) three clones proliferated with Dw18 and Dw19 APC and were blocked by anti-DR and anti-DRβ1 monoclonal antibody (MAb), showing a restriction by the DRαβ1 molecule; (2) three clones proliferated with Dw19 APC and were blocked by anti-DR MAb, (The restriction by the DRαβ2 unique to Dw 19 is very likely); 3) two clones proliferated with Dw19 APC. Since these clones were blocked by both anti-DQ and anti-DR MAb and since we did not observe any structural difference by 2D gel analysis in DQ products from Dw18 and Dw19 cells, these results suggest a restriction by a hybrid DQα-DRβ2 molecule..