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CBA (H-2k) responder spleen cells have been cultured at limit dilution with trinitrophenyl (TNP)-modified BALB/c (H-2d) stimulator cells and a source of T-cell growth factor in order to generate cytotoxic effector clones. After culture, such clones were split into two to four replicates and each was assayed against a different target. This allowed identification of clones capable of lysing TNP-modified P815 (H-2d) targets but not unmodified P815 targets. Thus, clones specific for TNP and allogeneic restriction elements were detected without the need to use techniques that deplete the responder population of alloreactive cells. Cytotoxic T lymphocyte precursors (CTL-P) specific for TNP-modified P815 (major histocompatibility complex-nonidentical) targets were identified, at a low frequency (28.2 x 10(-6)) compared to CTL-P for TNP-modified C1.18 (H-2k) (identical) targets (224 x 10(-6)). The hapten specificity, H-2 restriction specificity, and Thy-1 status of these clones have been examined. Fourteen percent of CBA CTL-P reactive to TNP-modified P815 targets also showed reactivity to NIP-modified P815 targets, and 86% of CBA clones reactive to TNP-modified P815 targets ("allo-TNP-reactive" clones) failed to show reactivity to TNP-modified C1.18 targets--i.e., showed a restriction preference for allo rather than self. All such H-2d-restricted, TNP-specific clones were uniformly sensitive to anti-Thy-1 antibody and complement. Among the H-2k responders studied, we have not demonstrated CTL-P reactive to TNP-modified syngeneic cells which also react with H-2d cells or NIP-modified H-2d cells among 168 clones analyzed. This suggests that such clones, if present, are relatively rare.

proceedings of the national academy of sciences of the united states of america

Clones of cytotoxic T lymphocytes reactive to haptenated allogeneic cells: precursor frequency and characteristics as determined by a split-culture approach.