Assessment of immunocompetence by limiting dilution analysis in long–term T cell depletion chimeras transplanted across the MHC barrier

Daniel A. Vallera, Christine C.B. Soderling, Charles G. Orosz

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


Chimeras were generated in a system in which donor C57BL/6 bone marrow plus spleen cells were T-cell-depleted prior to transplantation into lethally irradiated DBA/2 recipients. This protocol permits donor lymphohematopoietic engraftment and protects transplanted mice from development of lethal GVHD. The frequencies of alloantigen-specific cytotoxic T cells (CTL) and/or CTL precursors (CTL-P) in the chimera spleens were determined by limiting dilution analysis. This identified a small population of host-reactive CTL-P. The presence of host-reactive CTL-P in the absence of detectable anti-host immune response raises questions concerning the maintenance of the tolerant state in chimeras. Using mixtures of chimera and normal C57BL/6 splenocytes we found no evidence by limiting-dilution analysis for regulatory cells capable of dampening antihost immune reactivity in chimera spleens. We next measured the frequency of third-party-reactive CTL-P in chimeras. Chimeras displayed low CTL-P frequency by the 30th day posttransplant, which increased 15–21-fold over a five-month interval. Interestingly, both chimeric and irradiated syngeneic reconstituted control mice recovered anti-third-party CTL-P at a similar rate, but CTL-P levels never reached those measured in normal unirradiated control mice, suggesting that the radiation regimen has a long-lasting influence on host immunocompetence. In concomitant experiments we measured third-party CTL generation in MLC. Our findings suggest that measurement of CTL generation in MLC may be a less sensitive assessment of immunocompetence than LDA analysis. Our data also suggest that irradiated T-cell-depleted chimeras may suffer prolonged immunologic deficiencies based on reduced frequencies of alloreactive CTL-P.

Original languageEnglish (US)
Pages (from-to)311-316
Number of pages6
Issue number3
StatePublished - Sep 1985


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