IL-10 production by the Treg cells derived from visceral leishmaniasis (VL) patients:

<p><b>A</b>) <b>IL-10 producing FoxP3<sup>+</sup> cells in BMMNCs of VLs patients before treatment:</b> (i) Histogram plot depicts gating strategy of CD4<sup>+</sup> cells. Bi-variant plots show IL-10 production by CD4<sup>+</sup>FoxP3<sup>+</sup> Treg cells and CD4<sup>+</sup>FoxP3<sup>−</sup> cells under different <i>in vitro</i> conditions (ii) without stimulation and (iii) with <i>L. donovani</i> antigen stimulation. <b>B</b>) <b>IL-10 production by CD4<sup>+</sup>FoxP3<sup>+</sup> and CD4<sup>+</sup>FoxP3<sup>−</sup> cells:</b> Scatter dot plot shows frequency of IL-10 producing cells within gated CD4<sup>+</sup>FoxP3<sup>+</sup> (Treg) and CD4<sup>+</sup>FoxP3<sup>−</sup> (Teff) cells in blood as well as in BM of VL patients before anti-<i>Leishmania</i> therapy. Data shows that FoxP3<sup>+</sup> cells are one of the important producers of IL-10 along with CD4<sup>+</sup>FoxP3<sup>−</sup> cells. Horizontal lines in dot plot depict median value. Significant differences are indicated with <i>p-values</i> using paired <i>t</i> test. <b>C</b>) <b>CD4<sup>+</sup>CD25<sup>+</sup> (FoxP3 enriched) cells are major producer of IL-10 at disease site (BMA):</b> CD25<sup>+</sup> cells were magnetically sorted out from (<b>i</b>) PBMCs and (<b>ii</b>) BMMNCs. IL-10 was measured in supernatant of cultured cells (unsorted cells and CD25 depleted cells) stimulated with PHA (mitogen) and <i>L. donovani</i> antigen. IL-10 production was dominantly restricted to CD25<sup>+</sup> cells which were enriched with Treg cells. Data are represented in Mean± SD. Significant differences are indicated with <i>p-values</i> using paired <i>t</i> test. <b>D</b>) <b>Effect of IL-10 blocking and depletion of FoxP3<sup>+</sup> enriched cells on T cell activation upon polyclonal stimulation: </b><b>i–v</b>) <i>In vitro</i> PMA stimulation for 24 hrs caused activation of CD4 T cells (CD3<sup>+</sup>CD8<sup>−</sup>) derived from BM-MNCs as measured by the expression of CD69 (% positive cells) on them (v; <i>p = 0.011</i>, <i>paired t test</i>). Significant increase in the frequency of CD69<sup>+</sup> early activated CD4 (CD3<sup>+</sup>CD8<sup>−</sup>) T cells occurred upon PMA stimulation of BM-MNCs for 24 hrs when endogenously produced IL-10 was blocked by monoclonal antibody (v; <i>p = 0.018</i>, <i>paired t test</i>). Similar increase in the frequency of CD69<sup>+</sup> CD4 (CD3<sup>+</sup>CD8<sup>−</sup>) T cells was also observed when CD4<sup>+</sup>CD25<sup>+</sup> (Treg enriched) were sorted out using MACS sorting kit and CD25<sup>−</sup> BMMNCs were cultured with PMA for 24 hrs (v; <i>p = 0.009, paired t test</i>). Data are represented in Mean± SD. <b>E</b>) <b>Treg cells and IL-10 suppress </b><b><i>L.donovani</i></b><b> specific activation of CD4<sup>+</sup> T cells derived from pathologic site</b>: <i>LD</i> antigen caused significant increase in the frequency of CD69<sup>+</sup>CD4<sup>+</sup> T cells (<i>p = 0.036, paired t test</i>) after 72 hrs of stimulation. Upon blocking of soluble IL-10 by anti IL-10 ab, the frequency of CD69<sup>+</sup>CD4<sup>+</sup> T cells were increased as compared to antigen alone (<i>p = 0.065, unpaired t test</i>). Upon stimulation of CD25 depleted BMMNCs with antigen for 72 hrs, the frequency of CD69<sup>+</sup>CD4<sup>+</sup> T cells was increased as compared to antigen stimulated unsorted cells (<i>p = 0.025, paired t test</i>) as well as unstimulated CD25 depleted cells (<i>p = 0.014, paired t test</i>). However, in 24 hrs stimulation experiments, unsorted (with/without blocking of IL-10) and CD25 depleted BMMNCs upon stimulation with <i>LD</i> antigen show no significant increase in the frequency of CD69<sup>+</sup>CD4<sup>+</sup> T cells. {A = unsorted cells (with/without ag stimulation), B = CD25 depleted cells (with/without ag stimulation), C = unsorted cells with IL-10 blocking}. Data are represented in Mean± SD.</p>