Discrete PLT clones detecting HLA-D and new HLA-non-D encoded cell surface determinants

Clones of alloreactive T-lymphocytes were isolated from 6-day-old mixed leukocyte cultures (MLCs) by limiting dilution in the presence of filler cells (either pooled irradiated peripheral blood mononuclear cells (PBMCx) or autologous PBMCx) followed by expansion of clonal progeny with Interleukin 2 (IL 2). Using allogeneic IL 2, produced by stimulating pooled PBMC with phytohaemagglutinin (PHA), in conjunction with pooled filler cells for the limiting dilution cloning, over half the clones obtained had primed lymphocyte typing (PLT) function. The majority of these discriminated priming cell HLA-D type with fine specificity. However, clones which were restimulated by cells of specificities other than the priming HLA-D type were also found, and, moreover, some ("wild" clones) were not restimulated by the original priming cells. Antigens responsible for restimulating these clones included hitherto undetected HLA-encoded products (different from HLA-A, B, C, D, DR, MT, MB or SB). Such "wild" clones were also obtained after cloning procedures in which autologous filler cells were used. Autologous IL 2, depleted of mitogenic PHA, and used in conjunction with autologous filler cells, also failed to prevent generation of such clones. These results indicate that although the PLT-functional lymphocyte population alloactivated by a single HLA-DR/Dw specificity expressed by HLA-D homozygous stimulating cells consists of a majority of clones exclusively recognising specific priming-type HLA-DR/Dw products, it also contains clones recognising different HLA-DR/Dw specificities, as well as clones recognising lymphocyte stimulating determinants hitherto undetected by serological or cellular methods.