A DeOliveira, S Pop and NL Reinsmoen, Department of Pathology, Duke University Medical Center & Health System, Durham, NC.
The immune parameter, donor antigen-specific hyporeactivity, as determined by a significantly decreased post- vs pretransplant (pretx) donor antigen-specific proliferative (MLC) response is a useful predictor of a chronic rejection-free state in kidney, heart and lung recipients (recips) (predictive values = 91 to 100%). We are currently using this MLC assay to identify hyporesponsive (hypo) recips who are candidates for steroid withdrawal; however, the assay is cumbersome and not quantitative. We developed an assay using the fluorescent base intracellular dye 5-(and 6)-carboxyflourescein diacetate succinimidyl ester (CFSE) and flow cytometry analysis to measure directly the donor antigen specific CD4+ precursor frequency. Peripheral blood mononuclear responder cells were stained with CFSE and cultured together with appropriate stimulator cells for a period of 5 days. The cells were harvested, labeled with anti-CD3 and CD4, and analyzed on FACSCalibur with CellQuest and ModFit programs. The CFSE intensity is reduced by half in sequential cell divisions; the precursor frequency is calculated using the number of undivided cells (peak at far right in histograms), the absolute number of CD4+ daughter cells that can be determined at each cell division (successive peaks toward left represent each daughter cell population). In general, we have observed that recips defined as donor antigen responsive by MLC have precursor frequencies that remain the same or increase posttx (ex., Fig 1). Recips defined as hypo by MLC have a concomitant posttx donor antigen-specific frequency generally <10% (ex., Fig 2), while frequencies to third party cells remain unchanged. We anticipate this quantitative method will identify additional recips with significantly decreased donor antigen-specific CD4+ frequencies who were not identified by the MLC assay and may be candidates for withdrawal. In conclusion, the CFSE flow cytometry assay is a reproducible, accurate, quantitative method to determine donor antigen-specific precursor frequencies and to follow precisely the posttx immune response of solid organ recips, thereby allowing for individualization of immunosuppression.
