IN-VITRO CONSTRUCTION OF GRADED THYMUS CHIMERAS

A simple in vitro approach is described for constructing chimeric thym i in which the degree of chimeric contribution can be tightly controll ed. This is achieved by protease-assisted dissociation of fresh fetal thymic tissue, mixing the resulting cell suspensions at the desired ra tios, and subsequent reaggregation. Analysis of these graded chimeras shows that stromal elements become evenly interspersed and form a micr oenvironment able to support the maturation of endogenous T cell precu rsors. Chimeras between normal and MHC-deficient thymi have been used to demonstrate the rescue of mutant, allotype-marked thymocytes by wil d-type stromal cells. Other potential applications of the method inclu de quantitative studies on positive and negative thymic selection, the functional role of defined stromal cell types in these processes, and the analysis of mutants (either natural or engineered) by complementa tion.