DONOR–SPECIFIC HYPORESPONSIVENESS MEASURED BY CTLP AND CFSE DYE DILUTION

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TITLE: DONOR–SPECIFIC HYPORESPONSIVENESS MEASURED BY CTLP AND CFSE DYE DILUTION

M. Gallagher,¹ J. Pei,² D. Youngs,¹ K. Hamilton,¹ W. Yadock,¹ S. Dieterich,¹ R Lagasca,¹ E. Mickelson,²C. Marsh,³ J. Hansen,² K. Nelson.¹

¹Immunogenetics, Puget Sound Blood Center, Seattle, WA; ²Clinical Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA; ³Div.of Transplantation, University of Washington, Seattle, WA

Limiting dilution culture assays measuring cytotoxic T cells (CTLp) have been the gold standard for quantifying changes in cellular responses. Dye dilution (CFSE) analysis of responding T cells quantitatively measures of responding cells in bulk culture and allows staining for the phenotype and intracellular contents. These two approaches were compared in analysis of lymphocytes collected pre and post–transplant from 8 renal transplant recipients. Recipient cells were cultured with irradiated leukocytes from themselves, the donor or a third party. CTLp frequencies (CTLpf) were compared to CFSE mitotic indices (CFSEmi) or normalized CFSEmi (CFSENmi) where the response to donor was corrected for the response to autologous cells and the response to third party set at 100. Responses of post–transplant cells were compared to those of cells collected pre–transplant. By CTLpf analysis, one patient showed a decreased response to donor and increased response to 3rd party. Another 5 patients showed a reduction in the response to donor and 3rd party. Two showed an increased response to donor. CFSENmi for CD8+ T cells showed donor–specific–hyporesponsiveness (DSH) in 5 patients: the one identified by CTLp and 4 of the 5 where CTLp did not show specificity. Both CTLpf and CFSENmi agreed on 2 patients who showed an increase in the response to donor. The assays disagreed on the response of one patient. Three patients were analysed for intracellular cytokines and perforin. Two patients showing DSH by CFSENmi also showed a decrease in CD8+ perforin+ cells and an increase in CD8+IL–10+ cells. One patient without DSH showed an increase in CD8+ perforin+ cells and in CD8+ IL–10 + cells. In summary, CFSE analysis of CD8 T cells showed strong correlation with identification of DSH using CTLp. CFSE has the advantage of additional staining for perforin, granzymes or cytokines. CTLp has the advantage of measuring cells with cytotoxic function. Both assays quantitate changes in responses to donor alloantigens. The ability to normalize CSFEmi data allowed analysis of the specificity of the response in patients with immunosuppression or disease.