Multifunctional CD4⁺ T cells in patients with American cutaneous leishmaniasis

Abstract

Leishmaniasis is a group of important parasitic diseases affecting millions worldwide. To understand more clearly the quality of T helper type 1 (Th1) response stimulated after Leishmania infection, we applied a multiparametric flow cytometry protocol to evaluate multifunctional T cells induced by crude antigen extracts obtained from promastigotes of Leishmania braziliensis (LbAg) and Leishmania amazonensis (LaAg) in peripheral blood mononuclear cells from healed cutaneous leishmaniasis patients. Although no significant difference was detected in the percentage of total interferon (IFN)-γ-producing CD4(+) T cells induced by both antigens, multiparametric flow cytometry analysis revealed clear differences in the quality of Th1 responses. LbAg induced an important proportion of multifunctional CD4(+) T cells (28% of the total Th1 response evaluated), whereas LaAg induced predominantly single-positive cells (68%), and 57% of those were IFN-γ single-positives. Multifunctional CD4(+) T cells showed the highest mean fluorescence intensity (MFI) for the three Th1 cytokines assessed and MFIs for IFN-γ and interleukin-2 from those cells stimulated with LbAg were significantly higher than those obtained after LaAg stimulation. These major differences observed in the generation of multifunctional CD4(+) T cells suggest that the quality of the Th1 response induced by L. amazonensis antigens can be involved in the mechanisms responsible for the high susceptibility observed in L. amazonensis-infected individuals. Ultimately, our results call attention to the importance of studying a Th1 response regarding its quality, not just its magnitude, and indicate that this kind of evaluation might help understanding of the complex and diverse immunopathogenesis of American tegumentary leishmaniasis.

Fig. 1

Response of cytokine-producing CD4⁺T cells analysed by the frequency of cytokine positive cells (a) or by the integrated mean fluorescence intensity (iMFI) of interferon (IFN)-γ, tumour necrosis factor (TNF)-α and interleukin (IL)-2 (b) after stimulation with Leishmaniasis braziliensis (LbAg) and L. amazonensis (LaAg) total antigen extracts of peripheral blood mononuclear cells (PBMCs) obtained from healed cutaneous leishmanaisis (CL) patients and uninfected controls (Ctrl). #Different from LaAg CL Wilcoxon matched-pairs signed-ranks test, P < 0·05; *different from Ctrl group; Mann–Whitney U-test; *P < 0·05; **P < 0·005; ***P < 0·0005.

Fig. 2

Distinct quality of CD4 T helper type 1 (Th1) cells induced by LbAg and LaAg. Multiparametric flow cytometry was used to determine (a) the frequency of cells expressing each of the seven possible combinations of interferon (IFN)-γ, interleukin (IL)-2 and tumour necrosis factor (TNF)-α and (b) the fraction of the total response comprising cells expressing all three cytokines (3+), any two cytokines (2+) or any one cytokine (1+). Detached from the pie charts are the fraction that consists of IFN-γ single-positive cells. (c) The IFN-γ, IL-2 and TNF-α median fluorescence intensities (MFIs) of antigen-specific 3+, 2+, and 1+ cells for Leishmaniasis braziliensis (LbAg) and L. amazonensis (LaAg). Shown is the mean ± standard deviation of the mean. *Different from the Ctrl group. Mann–Whitney U-test; *P < 0·05; **P < 0·005. #Different from LaAg Wilcoxon matched-pairs signed-ranks test; #P < 0·05;##P < 0·005. ^§Mann–Whitney test, P < 0·05; ^§different from single-positive cells; ^§§different from all other phenotypes.