PD-1/PDL-1 Interaction Suppress IFN-γ and IL-17 Effector cytokines in BL/LL.

<p>A) Percentage frequency of PD-1 expression on CD4+FoxP3+ Treg cells vs. B) non Treg cells, (CD4+FoxP3-) in BT/TT vs. BL/LL (n = 10) patients and healthy contacts (n = 10). Percentage frequency of PDL-1 expression on APCs, C) on CD14+ Monocytes D) on CD19+ B cells in BT/TT vs. BL/LL (n = 10) patients and healthy contacts (n = 10) in PBMCs isolated from peripheral blood. Each bar represents a single category. Error bars are shown in each set while P value< 0.05 was considered to be significant. Bar graph representation showing the increased production of E) IFN-γ and F) IL-17 on total gated CD4+ T lymphocytes in BL/LL patients of leprosy (n = 4). PBMCs isolated from peripheral blood were blocked with α-PD-1 or with α-PDL-1 monoclonal blocking antibodies separately or in combination and kept in culture for 72 hours with or without <i>M</i>. <i>leprae</i> WCL antigen. From cultured cells, T cells were analyzed for effector cytokine production by flow cytometry. Total number of cells analyzed by flow cytometry were 500,000. Each bar represents a single category. Mean with SEM are shown in each bar while P value< 0.05 was considered to be significant. Data analysis was performed with flowjo software. Statistical analysis was done using Student’s t test for unpaired samples.</p

Effect of Th17 cytokines on FoxP3+Treg cells in BL/LL.

<p>A) PBMCs were cultured for 72 h in different conditions with or without the presence of cytokines inducing Th17 population such as TGF-β, IL-6, IL-23 and those secreted by Th17, IL-17 and IL-22 in different wells and readout was measured in terms of FoxP3+ on CD4+CD25+ cells on one hand and simultaneous IL-17 production by CD4+T cells. B) Bar graphs show the cumulative data in BL/LL (n = 5) of FoxP3+ expression on CD4+ CD25+ cells as against C) IL-17 production by CD4+T cells under the same conditions as mentioned above. Mean with SEM are shown in each bar while P value< 0.05 was considered to be significant. Total number of cells analyzed by flow cytometry were 500,000. Data analysis was performed with flowjo software. Statistical analysis was done using Student’s t test for unpaired samples.</p

Inverse Correlation between IL-10 and IL-17 in BL/LL.

<p>A) Treg derived high IL-10 negatively correlates with low IL-17+ CD4 T cells in BL/LL (n = 13) signifying polarized immunity in leprosy. B) No correlation was found in BT/TT (n = 13). Correlation was done using Spearman rank correlation coefficient; r value was equal to -0.65 while p value was significant at 0.01 in case of BL/LL.</p

Identification of Th17 cells in Leprosy.

<p>A) Representative flow cytometric analysis showing IL-17 production on gated CD4+ CD45RO+ memory T cells following <i>in vitro</i> stimulation with <i>M</i>. <i>leprae</i> (WCL) vs. unstimulated for 48 h in a Leprosy patient with BT. 6 h PMA stimulation was used as positive control. B) Scatter plots are showing cumulative IL-17 production in gated CD4+ CD45RO+ cells in different groups of Leprosy patients (BT/TT vs. BL/LL) and Healthy Contacts (n = 10). Each dot represents a single individual of BT/TT (n = 15) and BL/LL (n = 15) patient. C) Scatter graphs are showing CCR6 surface expression on gated CD4+CD45RO+ cells in different groups of Leprosy patients (BT/TT vs. BL/LL) (n = 10). Median values are shown in each set while P value< 0.05 was considered to be significant. Total number of cells analyzed by flow cytometry were 500,000. Data analysis was performed with flowjo software. Statistical analysis was done using Student’s t test for unpaired samples.</p

IL-10/TGF-β suppresses Th17 in BL/LL.

<p>A) Flow cytometric analysis showing the increased production of IL-17 on total gated CD4+ T lymphocytes in a leprosy patient with LL under different conditions, Unstimulated vs. Stimulated with leprosy antigen only vs. Stimulated with antigen and blocked with antibodies against IL-10/TGF-β alone or in combination. B) Cumulative data of BL/LL (n = 5) patients are shown in the bar graphs with the same conditions as above. Readout was taken in terms of IL-17 by CD4+ T cells at the end of 72 h. Mean with SEM are shown in each bar while P value< 0.05 was considered to be significant. Total number of cells analyzed by flow cytometry were 500,000. Data analysis was performed with flowjo software. Statistical analysis was done using Student’s t test for unpaired samples.</p

T regulatory cells in Leprosy.

<p>A) Representative FACS plot showing enumeration of Treg cell frequency (CD4+, CD25+ and FoxP3+) in BT/TT vs. BL/LL patient B) Total frequency of circulating Treg (CD4+, CD25+ and FoxP3+) cells in PBMCs isolated from peripheral blood of BT/TT vs. BL/LL patients (n = 20) vs. Healthy Contacts (n = 10). C) Representative flow cytometric analysis showing IL-10 production following <i>in vitro</i> stimulation with <i>M</i>. <i>leprae</i> (WCL) vs. unstimulated for 48 h in a Leprosy patient with LL. D) Scatter Plots are showing total IL-10 production in gated CD4+ FoxP3+ cells in BT/TT vs. BL/LL (n = 15) vs. Healthy Contacts (n = 10). E) CCR4 surface expression on gated CD4+ FoxP3+ cells in different groups of Leprosy patients (BT/TT vs. BL/LL), (n = 10). Each dot represents a single individual. Median values are shown in each set while P value< 0.05 was considered to be significant. Total number of cells analyzed by flow cytometry were 500,000. Data analysis was performed with flowjo software. Statistical analysis was done using Student’s t test for unpaired samples.</p