Detection of specific T lymphocytes in systemic abnormal delayed type hypersensitivity to Candida albicans.

Abstract

In humans, Candida albicans (C.a.) is a commensal organism of the gastrointestinal tract and vagina. It is a major opportunistic fungal pathogen. Cell-mediated immunity plays an important defensive role against C.a. infections [14]. In immunocompetent subjects intradermal injection of C.a. antigen induces a local delayed-type hypersensitivity (DTH) reaction. Abnormal DTH to C.a. is suspected in certain patients presenting with cutaneous, mucosal or general symptoms, but the role of C.a. in these disorders is difficult to ascertain. Abnormal reactions to intradermal injection of C.a. have been described as ocular or systemic reactions 2‐4 days after skin testing [5], but no simple biological tests have been established to investigate these patients. Cellular immunity to specific antigens can be detected in flow cytometry after in vitro activation [6, 7]. The present study was undertaken to detect C.a.-specific activated lymphocytes in the peripheral blood of patients with abnormal DTH reactions. Abnormal DTH reactions were associated with the detection of activated CD3-positive lymphocytes after in vitro stimulation with C.a. antigen. Patients and methods Eighty-four patients (52 females) suspected of C. a. hypersensitivity were enrolled in this study after informed consent. The study was approved by the local Ethics Committee. A routine intradermal injection of 30 ml of a C. a. extract at 100 mg/ml (kindly provided by Dr. De Montclos, Institut Pasteur Lyon) was performed. Patients with specific IgE to C. a. were excluded from the present study. The intensity of the local DTH reactions and systemic reactions were noted 48 h later: + 0.5 ‐ 1 cm, ++ 1 ‐ 2 cm, +++ 2 ‐ 2.5 cm, ++++ >2.5 cm. Specific activation of heparinised whole blood was detected in vitro as previously described [6, 7]. Briefly 50 ml of blood were incubated at 37 °C in CO2 (5 %) either with 50 ml of phosphate-buffered saline (PBS) (Sigma, St. Quentin Fallavier, France) or 50 ml of different concentrations of C. a. (20 and 10 mg/ml). Blood cells were collected after 24 h culture for determination of CD69 expression and after 7 days for determination of CD25 expression. The 7-day cultures were supplemented at 24 h with 500 ml of RPMI 1640 medium. After ammonium chloride lysis of erythrocytes, leukocytes were stained using either a commercial antibody combination including CD4-fluorescein isothiocyanate (FITC), CD3-peridin chlorophyll protein and CD69-phycoerythrin (PE) (Becton Dickinson, Pont de Claix, France), or an antibody combination including CD25-FITC, CD3-cyanin-5/PE and CD4-PE (Dako, Trappes, France). After washing in PBS, the cells were analysed with a Facscan flow cytometer (Becton Dickinson). Lysis II software was used in acquisistion, with triggering in the fl3 channel to gate on CD3+ lymphocyte populations as previously described [7]. In healthy controls, less than 1 % of unstimulated lymphocytes spontaneously expressed CD69 (data not shown): patients with more than 1 % spontaneously CD69 positive T cells were excluded from the study. Activation was considered positive when more than 2 % of C. a. activated CD3 + lymphocytes expressed CD69. Results and Discussion