Failure of in vitro assays to predict accurately the existence of neonatally induced H-2 tolerance.

Tolerogen-specific alloreactivity, in the form of mixed lymphocyte responses and cell mediated lympholysis, was measured in vitro using lymphocytes from mice that received neonatal inoculations of H-2 semiallogeneic hematopoietic cells. It was found that depletion of specific alloreactivity, as detected by these assays, was discernable within the thymus glands within 24 to 48 hr of injection. Reduced in vitro alloreactivity was also apparent among spleen cells obtained from neonatally injected mice that had matured immunologically (8 weeks of life). In mice that accepted their test skin allografts (in vivo evidence of tolerance), tolerogen-specific activity was essentially undetectable in vitro. Unexpectedly, markedly attenuated MLR and CML activity was the characteristic finding in neonatally injected mice that rejected their test grafts (in vivo evidence of lack of tolerance). Thus, in vitro assays failed to be predictive of the in vivo tolerant state. These results indicate that clonal reduction of tolerogen-reactive cells is an expected and persistent consequence of the injection of tolerance conferring inocula into neonatal mice. Moreover, we believe that the state of tolerance is maintained in adult tolerant mice by mechanisms that cannot easily be studied or predicted by the results of in vitro assays.