The role of non-H-2 alloantigens, specifically Mls locus products, in secondary in vitro T-cell-mediated cytotoxicity has been studied. Splenic T lymphocytes, activated against Mls locus alloantigens in primary-mixed cultures and isolated by velocity sedimentation gradient separation techniques, were used as responding populations in secondary mixed leukocyte cultures (MLCs) and cell-mediated lympholysis (CML). Such T-cell clones could be shown to exhibit either "self"-H-2-restricted or anti-Mls locus-specific reactivity, with this dichotomy of reactivity depending only on the primary culture conditions. Mls locus-activated T lymphocytes generated in cultures supplemented with homologous serum exhibited specific memory responses in MLC, yet remained incapable of effecting target cell destruction against Mls locus antigens or against "self"-H-2-structures in CML. In contrast, activated T-cell clones generated in the presence of heterologous serum displayed H-2-restricted reactivity in both secondary MLC and CML. H-2-restricted MLC activation was controlled by products of the H-2 serologically defined regions. Although heterologous serum was a necessary (and sufficient) entity for development of H-2-restricted responses, evidence argues against the possibility that heterologous serum acts via modification of cell surface components.

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