Flow Cytometric Analysis of Leishmania Reactive CD4+/CD8+ Lymphocyte Proliferation in Cutaneous Leishmaniasis

Abstract

Background: Determination of the division history 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.

Methods: 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.

Results: 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)

Conclusion: 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.