Experimental cardiac allograft survival across major histocompatibility complex barriers in the rhesus monkey following T lymphocyte-depleted autologous marrow transplantation. I. In vitro T lymphocyte depletion studies.

We have developed a rhesus monkey model consisting of myeloablative total-body irradiation and T lymphocyte-depleted autologous bone marrow transplantation followed by MHC-mismatched heterotopic cardiac allograft implantation that has provided an opportunity to study the role of marrow T cells in cardiac allograft rejection. In order to assess quantitatively the effects of low numbers of residual marrow T cells following depletion, methods to deplete rhesus marrow extensively and to detect residual T cells following depletion at levels below the sensitivity of standard assays have been developed. A rhesus marrow limiting dilution assay has been developed that quantifies less than 1 T cell in 10(5) marrow cells and is superior to traditional detection methods by at least 3 logs. In a direct comparison of four T cell depletion methods, effective depletion has been achieved with complement-mediated cytotoxicity (C'MC), erythrocyte rosetting, and counterflow centrifugal elutriation (CCE), the latter with a simplified single-flow rate protocol. Median marrow T cell depletions of 2.1, 1.1, and 3.1 logs, and total nucleated cell losses of 40%, 61%, and 42% respectively, have been observed. A reported use of ricin A-chain-like toxins for the enhancement of C'MC was of low efficacy with rhesus peripheral blood T cell targets. CCE followed by C'MC has resulted in a median 4.8 logs depletion with residual marrow T cell contents less than 0.001%. Thus, C'MC, E-rosetting, and particularly CCE are effective methods of T cell depletion--and, when used in combination, extensively eliminate marrow T cells. A rhesus marrow limiting dilution assay detects residual T cells at these low levels. These techniques provide a basis for the quantitative study of the role of T cells in organ graft rejection following T lymphocyte-depleted autologous marrow transplantation.