Flow cytometric analysis of leishmania reactive CD4[+]/CD8[+] lymphocyte proliferation in cutaneous leishmaniasis

ABSTRACT

Determination of the division histoty of T cells in vitro is helpful in the study of effector mechanisms against infections. Technique described here uses the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester [CFSE] to monitor the proliferation. In a cross sectional study, blood samples were collected from 7 volunteers with history of cutaneous leishmaniasis [CL] and one healthy control from endemic areas in Isfahan province who referred to the Center for Research and Training in Skin Diseases and Leprosy [CRTSDL], then CD4[+]/CD8[+] lymphocytes and CD14[+] monocytes were isolated from peripheral blood mononuclear cells [PBMC] using mAbs and magnetic nanoparticles. CFSE labeled CD4[+] or CD8[+] lymphocytes cultured with autologous monocytes in the presence of PHA, SLA, live Leishmania major or as control without stimulation. Cells were harvested after 7 days and were analyzed using flow cytometry. Five consecutive divisions were monitored separately. Stimulation of CD4[+] or CD8[+] lymphocytes from CL subjects with SLA showed a significant difference in proliferation comparing with unstimulated cells [P< 0.05]. The significant difference in the percentages of CD4[+] cells stimulated with SLA was revealed at different divisions for each subject. In CD8[+] lymphocyte, significant stronger stimulation of SLA was evident later in the proliferation process. The mean number of divisions in both CD4[+]/CD8[+] lymphocytes stimulated with SLA was significantly greater than when stimulated with live L. major [P=0.007 / P=0.012, respectively]. The percentage of divided cells might be calculated separately in each division. The cells remained active following CFSE staining and there is possibility of functional analysis simultaneously