Based on previous work on this project, the apparent Lyt 2 plus suppressor cells associated with long-term survival of primarily vascularized B10.BR cardiac allografts in B10.A mice will be further investigated. To facilitate analysis of a large number of mice and provide sufficient cell numbers for extensive analysis, in vivo preparation will be employed. Treatment with a single dose of anti-L3T4 monoclonal antibody abolishes the L3T4-positive (helper phenotype) spenocytes nearly completely, leaving a predominant Lyt 2 plus cell effect. As an initial simple assessment of whether the suppressor cells are Lyt 2 plus, splenocytes from long-term recipients that have been treated by anti-L3T4 monoclonal antibody in vivo will be tested for suppression as third-party cells in mixed lymphocyte culture-lymphocyte-mediated cytoxicity (MLC-LMC), with vs. without flow cytometric sorts to remove Lyt 2 plus cells. Next, the time after grafting that the suppressor cells achieve maximum effect will be found by testing splenocytes from L3T4 treated animals at sequential times after grafting. Then, to determine more clearly whether the Lyt 2 plus cells are responsible for the suppression, they will be removed by flow cytometric sorting from splenocytes at the time of maximum suppressor effect and directly tested for suppression as third party cells in MLC-LMC. The specificity of the action of these cells will be assessed by testing their effect on MLC-LMC using other stimulator or responder strains. The correlation with degree of rejection will be assessed by studying other strain pairs the produce strong or weak rejection, and by comparing occasional B10.A recipients that strongly reject their heart grafts with those that undergo the usual transient weak rejection. The effect of these cells in vivo will be further assessed by testing whether they specifically suppress skin graft rejection, and, in other tests, priming for MLC-LMC. The possibility that the suppressor cells may express Ij will be studied by determining whether MLC-LMC suppression, as assessed above, is abolished by removal of subsets bearing this antigen.
