Figure 1.

Enrichment of regulatory T cells (T_reg) cells in bone marrow of visceral leishmaniasis (VL) patients correlates with parasite load. (a) Enumeration of T_reg [forkhead box protein 3 (FoxP3⁺)] frequency: bone marrow mononuclear cells (BM MNCs) from VL patients were used for immunophenotyping of T_reg cells. CD4⁺CD25⁺ population of T cells was gated on pregated lymphocytes (based on forward‐ versus side‐scatter); further, the dual population of CD4⁺CD25⁺ T cells was divided into CD4⁺CD25^dim and CD4⁺CD25^br (Fig. 1a, ii: inset). Intranuclear expression of FoxP3 was observed in these two populations. Histogram plot of FoxP3 expressing CD4⁺CD25^dim or CD4⁺CD25^br (thin red dotted line) was overlaid on FoxP3 fluorescence minus one (FMO) (solid dark field). CD4⁺CD25^br were found to be the major population of activated T cells expressing FoxP3 (br = bright). (b) Increased percentage frequency of activated (CD4⁺CD25⁺) T cells in BM of VL patients with high parasitic load (HPL). The percentage frequency of activated T cells (CD4⁺CD25⁺) was analysed from BM‐derived MNCs of VL patients (n = 26). We observed a significantly higher percentage frequency of CD4⁺CD25⁺T cells in VL patients with HPL [(2⁺and 3⁺: n = 12), mean ± standard deviation (s.d.): 2·76 ± 0·15%] compared with low parasitic load [(1⁺: n = 14) mean ± s.d.: 1·23 ± 0·21%; P = 0·002, unpaired t‐test]. Horizontal lines in scatter‐plot depicted as the mean value (br = bright). (c) Increased T_reg frequency (CD4⁺CD25⁺FoxP3⁺) in BM of VL patients with HPL: significantly increased percentage frequency of FoxP3⁺ T cells on pregated CD4⁺CD25⁺ were observed in BM MNCs of VL patients with HPL (n = 12: mean ± s.d.: 53·84 ± 4·06%) compared to the patients with low parasitic load (LPL) (n = 14: mean ± s.d.: 24·89 ± 2·70%) (P = 0·0001: unpaired t‐test). Horizontal lines in scatter‐plot depicted as mean value. (d) Significantly decreased frequency of T effector cells (CD4⁺CD25⁺FoxP3^–) in bone marrow of VL patients with HPL: significantly decreased percentage frequency of T effector cells [(CD4⁺CD25⁺FoxP3^–): i.e. 100%: % frequency of CD4⁺CD25⁺FoxP3⁺] in BM MNCs of VL patients with HPL (n = 12: mean ± s.d.: 46·16 ± 4·06%) compared to the patients with LPL (n = 14: mean ± s.d.: 75·12 ± 2·70%) (P = 0·0001: unpaired t‐test). Horizontal lines in scatter‐plot depicted as mean value. (e) Increased frequency of T_reg (CD4⁺CD25⁺FoxP3⁺) cells in BM of VL patients was correlated positively with higher percentage frequency of activated T cells (CD4⁺CD25⁺): further, we observed a significant positive correlation between percentage frequency of T_reg (CD4⁺CD25⁺FoxP3⁺) cells with percentage frequency of activated cells (CD4⁺CD25⁺) in VL patients (P = 0·004: Pearson's correlation coefficient r ² = 0·535). (f) Increased frequency of T_reg (CD4⁺CD25⁺FoxP3⁺) cells in BM of VL patients was correlated positively with parasitic load: we observed significant positive correlation between percentage of T_reg frequency (CD4⁺CD25⁺FoxP3⁺) with parasitic load in VL patients (P < 0·0001: Pearson's correlation coefficient r ² = 0·583). [Colour figure can be viewed at wileyonlinelibrary.com]