Limiting Dilution Analysis of Alloantigen-Reactive T Lymphocytes

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Abstract
A sensitive mixed leukocyte microculture (micro MLC) system has been developed in which both proliferative and cytolytic responses can be detected from small numbers of responding alloantigen-reactive T lymphocytes. Cytolysis and proliferation in micro MLC were assessed by conventional 51Cr release and 3H-thymidine uptake assays, respectively. Optimal sensitivity of the system was dependent on several parameters, the most important of which was the presence of supernatant fluid derived from secondary MLC (2° MLC SN). Under optimal conditions, formal limiting dilution analysis could be achieved since excellent discrimination between positive and negative microcultures was obtained for both the cytolytic and proliferative assays. The system was therefore used to obtain simultaneously minimal estimates of the frequency of cytolytic T lymphocyte precursors (CTL-P) and of the precursors of proliferating T lymphocytes (PTL-P). The results of a series of experiments involving C57BL/6 spleen cells responding against DBA/2 alloantigens indicated that the minimal frequency of CTL-P was approximately 2-fold lower than the frequency of PTL-P in the same responding cell population (average of 1:570 and 1:290, respectively). Since analysis of individual micro MLC further demonstrated that essentially all CTL-P were contained within the PTL-P population, the simplest interpretation of these findings would be that approximately 50% of alloantigen-reactive T cells in this strain combination have the capacity to give rise to specific CTL. Some applications of this model system are briefly discussed.

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