The epitope recognized by pan-HLA class I-reactive monoclonal antibody W6/32 and its relationship to unusual stability of the HLA-B27/beta2-microglobulin complex.

A broadly used pan-HLA class I-reactive monoclonal antibody W6/32 is believed to recognize a conformational epitope dependent on association between heavy chains and beta2-microglobulin (beta2m). However, in the present study we report that W6/32 does recognize at least some free HLA class I heavy chains under the partially denaturating conditions of nonreducing Western blotting, namely nearly all HLA-B allelic products. Furthermore, we confirm and largely extend our previous observation that complexes of beta2m with heavy chains of a few HLA class I allelic forms (most notably HLA-B27) exhibit unusual resistance to dissociation by SDS, which is reminiscent of MHC class II molecules. In addition, our data indicate the existence of covalent (disulfide-linked) heterodimers of certain HLA class I heavy chains (namely Cw1 and Cw4) and beta2m.

Strong association of HLA-B27 heavy chain with beta 2-microglobulin.

Monoclonal antibody TG1 recognizes specifically antigens HLA-B27, B7, B22 and B17 on cell surface in cytotoxicity and cytofluorometry tests. When cell detergent extracts were subjected to SDS PAGE under mild conditions (no heating and no reduction of the sample) followed by Western blotting, TG1 detected exclusively a complex of B27 heavy chains with beta(2)-microglobulin (as a 50 kDa band) whereas the other B-locus antigens (B7, B22, B17) were detected as free 43 kDa heavy chains under the same conditions. When the samples were boiled prior to SDS PAGE, TG1 detected again the 43 kDa free heavy chains of B7, B22 and B17 but no zone corresponding to B27 could be detected indicating that the epitope in free B27 chains is more sensitive to denaturation by SDS. Thus, our main finding is that the interaction of HLA-B27 heavy chain with beta(2)-microglobulin appears to be stronger than that of the other HLA-B chains. The resistance of the HLA-B27/beta(2)-microglobulin complex to the SDS dissociation is strikingly similar to the behavior of MHC class II molecules under similar conditions. Thus, it may be speculated that HLA-B27 complexes can be also more stable than other MHC class I molecules under more physiological dissociative conditions (e.g. in endosomal compartments). This feature might potentially influence antigen presentation by HLA-B27 and contribute to the well known disease linkage of HLA-B27.

Blind confirmation in Leiden of Geczy factor on the cells of Dutch patients with ankylosing spondylitis.

A follow-up blind study, of the ability of "cross-reactive" antisera to distinguish between the cells of Dutch patients with ankylosing spondylitis (AS) and normal controls, was performed in Leiden. Of the 45 cell samples tested, 29 were fresh peripheral blood mononuclear (PBM) cells while 15 were cryopreserved PBM. No false positives but one false negative was identified among the 45 samples, and the "negative" was confirmed after the recoded cryopreserved cells from this patient were retested. It is concluded that the "cross-reactive" antisera raised in Sydney give good discrimination between patients and normals. Factors affecting the success of the 51Cr-release cytotoxicity assay, and possible reasons for the failure of others to confirm these observations, are briefly discussed.

Activation of cytotoxic T lymphocytes in HLA-A, -B and -C-identical responder-stimulator pairs. I. Variations in generation of anti-class-II CTL in primary mixed lymphocyte cultures.

Cytotoxic T lymphocytes were activated in primary one-way mixed lymphocyte cultures of cells matched for serologically defined HLA-A, -B and -C antigens. In 16 out of the 29 combinations mismatched for the HLA-D/DR antigens, cell-mediated lympholysis of the stimulator cells occurred. The specificity of 5 selected cytotoxic T lymphocytes was studied in detail. Three of these cytotoxic T lymphocytes recognize antigenic determinants associated with HLA-Bw35 (Breuning et al. 1984, II). The 2 other cytotoxic T lymphocytes failed to lyse T-target cells enriched by rosetting with sheep red blood cells, whereas target cells from the 'non-T' fraction were strongly lysed, indicating that antigenic determinants associated with Class-II HLA molecules were the targets recognized by these cytotoxic T lymphocytes. This notion was supported by a study of a panel of HLA-typed third-party target cells. One cytotoxic T-lymphocyte population preferentially lysed HLA-DR2-positive target cells. Family studies, including a family with a recombination between HLA-B and -D, showed that the target antigen recognized by the latter cytotoxic T lymphocyte segregated with DR2. The second cytotoxic T-lymphocyte population recognized a determinant associated with DRw8. However, in 13 of the 29 HLA-A-, -B- and -C-identical, D/DR-different combinations, cell-mediated lympholysis of stimulator target cells could not be detected, not even on enriched 'non-T' target cells. Thus, after primary mixed lymphocyte culture of HLA-A-, -B- and C-identical, HLA-D/DR-non-identical cells, cytotoxic T lymphocytes directed against sensitizing Class-II molecules can be detected in some combinations, but not in others.

Activation of cytotoxic T lymphocytes in HLA-A, -B and -C-identical responder-stimulator pairs. II. New subtypes of HLA-Bw35.

We compared five cytotoxic T lymphocytes raised by primary mixed lymphocyte cultures of HLA-A, -B and -C serologically identical Bw35-positive responder-stimulator combinations. When tested on a panel of third-party target cells, the reactivity pattern of these cytotoxic T lymphocytes allowed the distinction of three subtypes of HLA-Bw35. Cold-target inhibition experiments and analysis of CTL activity at the clonal level showed the existence of subsets of CTLs directed against distinct antigenic determinants associated with HLA-Bw35.

Specificity of anti-HLA-B27 cytotoxic T lymphocytes.

Sub-types of HLA-B27 were detected by cytotoxic T lymphocytes (CTL) generated between HLA-A, -B- and -C-identical B27-positive individuals. We now report the specificity of six independent CTL's generated by mixed lymphocyte culture (MLC) of HLA-A, -B and -C serologically identical B27-positive responder and stimulator cells. Three CTL's recognize one sub-type, and three the other. The combined reactivity of all CTL's allows unequivocal "typing" of B27-positive cells for the two different sub-types B27K and B27W. The specificity of two CTL's was analysed by cold-target inhibition. The results indicate that (1) no further sub-types of HLA-B27 can be detected by the CTL's raised in these combinations; (2) the majority of the CTL's is directed against the B27 antigens; and (3) "extra reactions" on B27-negative cells are caused by a subset(s) of CTL's recognizing unknown antigens shared between stimulator and target cells. CTL's raised by stimulation of HLA-B27-negative responder cells with B27-positive cells of either sub-type lysed all B27-positive target cells indiscriminately. In cold-target inhibition, however, B27-positive cells, carrying the sub-type of B27 different from that of the stimulator, could not inhibit the lysis of cells bearing the stimulator sub-type of B27. This indicates the activation, in B27-negative responders, of at least two different groups of CTL clones, one directed against shared determinants of HLA-B27, and one against the HLA-B27 sub-type. Heterogeneity of the HLA-B27 antigen may have implications for studies on the well-known association between this antigen and various diseases.

Subtypes of HLA-B27 detected by cytotoxic T lymphocytes and their role in self-recognition.

In the present study cytotoxic T lymphocytes were generated in MLC of lymphocytes from two unrelated HLA-A, B, C-identical, B27-positive, but D/DR-different, individuals. These CTL were shown to detect subtypes of HLA-B27. CTL specific for influenza virus lysed infected target cells matched for HLA-B27 only when they shared the same subtype. This indicates that the two subtypes of HLA-B27 detected by CTL function also as distinct elements in a self-restricted CTL response. Both subtypes were found among patients with ankylosing spondylitis.

Identification of human CML target. HLA-B locus (B12) antigen variants defined by CTL generated between B locus-identical (B12) responder-stimulator pairs.

The genetics of human CML targets were studied by seven CTL generated in combinations iun which the responder/stimulator difference was limited to one (or two) HLA-A, -B, or -C antigens. Unstimulated peripheral blood lymphocytes were used as targets. CTL sensitized against antigens A11, Aw31, or B17 lysed all cells bearing the respective target from a large panel of cells from unrelated individuals. Hence, at least one CTL clone was directed against the HLA antigen molecule. However, all CTL also exerted cross-kill to cells not sharing the stimulating HLA antigen. For two CTL, the target of the cross-kill was not clarified. Five CTL were found where the cross-kill was directed against antigen HLA-B12 (Bw44 and Bw45). All these cTL were generated in R/S pairs identical for B locus antigens (Bw44/Bw35 heterozygotes). The individual CTL lysed different parts of the panel of B12-positive target cells. The interpretation is that these CTL detect subtypes of HLA antigens, but alternative possibilities are also considered. Four B12 subtypes are described, tentatively designated as B12-related CML targets. Identification of HLA-B-related CML targets represent CML "typing" of HLA-antigen differences that were not detected serologically. The subtypes can now be tested for their possible functional significance.