Variation in indirect cell-mediated lympholysis

The ability of an allograft recipient to respond to donor mononuclear cells in an indirect cell-mediated lympholysis (ICML) assay is an in vitro correlate of allograft rejection, but the value of this correlation depends upon the assay's reliability. We had observed inconsistency in the cytotoxic response of normal human mononuclear cells (MNC) to the same allogeneic stimulator MNC when cytotoxicity was measured repeatedly on different occasions by micro-ICML. We, therefore, investigated the extent and reasons for this inconsistency. Method variation, determined by duplicate ICML of 18 stimulator: responder MNC, was not statistically significant. Variation in cytotoxicity over time was greater but still not statistically significant. The contribution to method variation of 51Cr release from 3 different sets of target cells, cultured and labeled in duplicate, was minimal (6.33%). We then asked if in vitro generation of effector MNC under laboratory conditions was a major cause of ICML variation. We tested this using a stable transplant's in vivo sensitized effector cells against donor MNC in a direct CML (DCML) and obtained consistent results. Finally, to gain an understanding of some of the factors which might influence the generation of in vitro cytotoxicity, we measured the frequencies of cell surface antigens (DR, TAC, transferrin, Leu 2 and 3) concomitantly with ICML on day 6 of culture. Statistical analysis of the results led us to conclude that the micro-ICML is reproducible. The magnitude of lysis depends upon activated target cells (TAC- and transferrin-positive) and an increase in the proportion of helper/inducer to cytotoxic/suppressor T-lymphocytes during effector cell generation.