Measurement of Candida-specific blastogenesis: comparison of carboxyfluorescein succinimidyl ester labelling of T cells, thymidine incorporation, and CD69 expression.
Measurement of the T cell blastogenic response to Candida may be useful in the evaluation of patients with suspected immunodeficiency. The classic blastogenesis assay is based on uptake of [3H]thymidine by peripheral blood lymphocytes stimulated with Candida antigens for 5 days. An alternative approach involves staining peripheral blood lymphocytes with the intracellular fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) and measuring mitotic activity by the successive twofold reductions in fluorescent intensity using flow cytometry (FCM). The two approaches were compared in 16 subjects who demonstrated various proliferative responses to Candida. FCM-derived indices all involved initial gating on CD3+ T cells and included 1) blastic transformation as measured by changes in light scatter, 2) cell division, measured by CFSE fluorescence, and 3) CD69 expression. A good correlation was found between [3H]thymidine uptake and CFSE-derived indices, irrespective of the analysis algorithm used to interpret CFSE division profiles. Furthermore, significant T cell proliferation occurred only in subjects who had had one or more symptomatic episodes of vaginal candidiasis whereas controls with no such history, and patients with chronic vaginal infection, showed minimal proliferation. The increase in proportion of CD69+ T cells in culture also correlated with the blastogenic response to Candida, but less well than mitotic indices. CFSE-derived indices of T cell blastogenesis to Candida are equivalent to [3H]thymidine-based assays and may allow useful laboratory distinction between subjects who have been exposed to and recovered from vaginal Candida infection, who have a strong proliferative response, from those with no exposure or chronic infection who demonstrate a poor response.
Angulo
R
R
Department of Immunopathology, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Sydney, Australia.
Fulcher
D A
DA
D015703:Antigens, CD / Q000378:metabolism*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000378:metabolism*; D000946:Antigens, Fungal / Q000032:analysis; D002181:Candidiasis, Vulvovaginal / Q000175:diagnosis / Q000276:immunology*; D002478:Cells, Cultured; D005260:Female; D005434:Flow Cytometry; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D016867:Immunocompromised Host; D037181:Lectins, C-Type; D008213:Lymphocyte Activation; D009000:Monocytes / Q000276:immunology / Q000378:metabolism; D013388:Succinimides; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism*; D000099192:CD69 Antigens
D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D000946:Antigens, Fungal; D005452:Fluoresceins; D005456:Fluorescent Dyes; D037181:Lectins, C-Type; D013388:Succinimides; D013936:Thymidine; D000099192:CD69 Antigens; C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester