Carboxyfluorescein succinimidyl ester-based proliferative assays for assessment of T cell function in the diagnostic laboratory

Immune deficiency diseases are often accompanied by abnormalities in one or both arms of the specific immune system. Impairment can often be detected as a decrease in the number of T or B lymphocytes or their products in the circulation, but questions are often asked as to the functional capabilitie s of T lymphocytes in patients with recurrent infections. Function of T cel ls has traditionally been measured by their uptake of [H-3]-thymidine follo wing stimulation with antigen or mitogen in vitro. However, the ability of carboxyfluorescein succinimidyl ester (CFSE) to label lymphocytes intracell ularly and track their mitotic activity by progressive two-fold reduction i n fluorescence intensity prompted an alternative methodology based on flow cytometry, an approach which has the advantage of allowing specific gating on particular T cell subsets and simultaneous assessment of activation mark ers. This method was therefore evaluated for T cell responses to mitogen an d antigen. Phytohaemagglutinin-induced blast transformation of CFSE-labelle d T cells was reflected by an increase in forward and orthogonal light scat ter and a progressive two-fold decrease in CFSE fluorescence intensity. The se changes allowed the derivation of various measures of mitotic activity, which correlated well with [H-3]-thymidine uptake. Patients with T cell fun ctional deficiencies showed impairment in their responses by both assays, w hereas the CFSE-based assay demonstrated that impaired blastogenesis was no t simply due to depressed T cell numbers. Concomitant measurement of the ac tivation markers CD69 and CD25 showed that CD69 was rapidly expressed on no n-mitotic cells and that this expression was progressively diluted with sub sequent rounds of cell division. In contrast, CD25 expression was unaffecte d by cell cycle, but was expressed in proportion to the PHA dose. Antigen-s pecific responsiveness to Candida was also assessed using a CFSE-based assa y. Initial gating on the relatively minor population of T cells that underw ent blast transformation demonstrated progressive twofold dilutions of CFSE intensity in responsive cells. These normal Candida responses, found in pa tients who had recovered from Candida infection, contrasted with those who had not been infected with Candida or who had chronic recurrent infection, in whom neither blast transformation nor significant mitosis could be detec ted. Again, there was good correlation with [H-3]-thymidine uptake. The CFS E-based assays are equivalent to traditional measures of mitogen-and antige n-specific T cell responsiveness in the diagnostic laboratory and have sign ificant advantages in terms of decreased labour intensiveness, avoidance of radioactivity, the ability to gate on a specific population of lymphocytes and the concomitant measurement of activation markers.