TRACKING THE FATE OF DONOR REACTIVE CELLS POSTTRANSPLANT BY FLUORESCENCE-BASED PRECURSOR FREQUENCY METHOD.
S Pop, A DeOliveira and NL Reinsmoen, Department of Pathology, Duke University Medical Center & Health System, Durham, NC.
The study of alloimmune responses has been limited by cumbersome methods such as the MLC and limiting dilution precursor frequency analysis that cannot determine the absolute number of daughter cells generated with each division in an allogeneic response. We have developed a quantitative flow cytometry-based alloantigen precursor frequency assay using the intracellular fluorescent label, carboxyfluorescein diacetate succinimidyl ester (CFSE), to tag proliferating cells. CFSE is cell-permeant and remains nonfluorescent until cellular esterases cleave the carboxyl groups from the molecule rendering it both nonpermeant and fluorescent; the succinimidyl moiety covalently attaches to amine groups contributing to the longevity of staining. The covalently bound CFSE is divided equally between daughter cells thereby reducing the fluorescence intensity by half and allowing discrimination of successive rounds of cells division (~ 8 max). Peripheral blood mononuclear responder cells are stained with CFSE and cultured with appropriate stimulator cells for 5 days. The cells are harvested, labeled with anti-CD3, -CD4, -CD8 and analyzed on FACSCalibur with CellQuest and ModFit programs. The precursor frequency is calculated using the absolute number of CD3+/CD4+ or CD3+/CD8+ daughter cells that can be determined at each cell division and the number of undivided cells. Pretransplant donor antigen-specific precursor frequencies generally range from 20 to 30% for one HLA-DR mismatched donor/recipient pairs. This frequency range is higher than those published previously (1-10%); however, this technique detects all CD3+/CD4+ cells responding including class II-directed cytotoxic cells and proliferating cells using the salvage pathway of DNA synthesis (detected by MLC) as well as those using de novo synthesis (not detected by MLC). We have observed that the posttransplant donor antigen-specific precursor frequencies are dynamic. Some recipients have precursor frequencies that remain the same or increase posttransplant (R#1: pretx = 30.9%; 12 mo posttx = 31.1%; R#2: pretx = 24.5%, 12 mo posttx = 35.9%). Other recipients have donor antigen-specific frequencies that decrease with time posttransplant and eventually fall to <10% (ex., 8.2, 5.9, 6.3, 5.1%), while frequencies to third party cells remain unchanged and, thus, these recips may be candidates for steroid withdrawal. In conclusion, this quantitative method provides the ability to accurately follow changes in the donor antigen-specific response, thereby allowing the potential to individualize and optimize immunosuppression.