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 studie
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