Altered IL-7 signaling in CD4+ T cells from patients with visceral leishmaniasis

Abstract

Background: CD4+ T cells play a central role in control of L. donovani infection, through IFN-γ production required for activation of macrophages and killing of intracellular parasites. Impaired control of parasites can in part be explained by hampered CD4+ T cells effector functions in visceral leishmaniasis (VL) patients. In a recent studies that defined transcriptional signatures for CD4+ T cells from active VL patients, we found that expression of the IL-7 receptor alpha chain (IL-7Rα; CD127) was downregulated, compared to CD4+ T cells from endemic controls (ECs). Since IL-7 signaling is critical for the survival and homeostatic maintenance of CD4+ T cells, we investigated this signaling pathway in VL patients, relative to ECs.

Methods: CD4+ T cells were enriched from peripheral blood collected from VL patients and EC subjects and expression of IL7 and IL7RA mRNA was measured by real time qPCR. IL-7 signaling potential and surface expression of CD127 and CD132 on CD4+ T cell was analyzed by multicolor flow cytometry. Plasma levels of soluble IL-7 and sIL-7Rα were measured by ELISA.

Result: Transcriptional profiling data sets generated previously from our group showed lower IL7RA mRNA expression in VL CD4+ T cells as compared to EC. A significant reduction was, however not seen when assessing IL7RA mRNA by RT-qPCR. Yet, the levels of soluble IL-7Rα (sIL-7Rα) were reduced in plasma of VL patients compared to ECs. Furthermore, the levels of soluble IL-7 were higher in plasma from VL patients compared to ECs. Interestingly, expression of the IL-7Rα protein was higher on VL patient CD4+ T cells as compared to EC, with activated CD38+ CD4+ T cells showing higher surface expression of IL-7Rα compared to CD38- CD4+ T cells in VL patients. CD4+ T cells from VL patients had higher signaling potential baseline and after stimulation with recombinant human IL-7 (rhIL-7) compared to EC, as measured by phosphorylation of STAT5 (pSTAT5). Interestingly, it was the CD38 negative cells that had the highest level of pSTAT5 in VL patient CD4+ T cells after IL-7 stimulation. Thus, despite unaltered or potentially lowered IL7RA mRNA expression by CD4+ T cells from VL patients, the surface expression of the IL-7Rα was higher compared to EC and increased pSTAT5 was seen following exposure to rhIL-7. Accordingly, IL-7 signaling appears to be functional and even enhanced in VL CD4+ T cells and cannot explain the impaired effector function of VL CD4+ T cells. The enhanced plasma IL-7 may serve as part of homeostatic feedback mechanism regulating IL7RA expression in CD4+ T cells.

Fig 1. IL7RA expression in VL.

A. Volcano plot of immune-related genes in peripheral blood CD4⁺ T cell data extracted from a previously published dataset [17]. Analysis of differentially expressed immune-related genes in peripheral blood CD4⁺ T cells between visceral leishmaniasis (VL, n = 12) patients prior to treatment (D0) and endemic controls (EC, n = 12) shows downregulation of IL7RA. B. Relative expression of IL7RA determined by RT-qPCR in PBMC and CD4⁺ T cells, as indicated, each dot represents one sample PBMC (EC n = 11, VL n = 13), CD4 (EC n = 11, VL n = 15). C. Soluble IL-7Rα plasma levels in EC (n = 8) and VL before (D-0, n = 13) and 30 days (D-30 n = 13) after initiation of drug treatment. Statistical significance was determined by Kruskal-Wallis with multiple comparison follow-up test for Fig 1C and are indicated as *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig 2. IL-7 expression and secretion following Leishmania donovani infection.

A. Relative expression of IL7RA determined by RT-qPCR in PBMC and CD4⁺ T cells, as indicated, each dot represents one sample, PBMC (EC n = 11, VL n = 13), CD4 (EC n = 12, VL n = 13). Median range is depicted. B. IL-7 levels in the plasma of VL patients (n = 10), ECs (n = 10), as determined by ELISA. Statistical significance was determined by Mann-Whitney U-test for Fig 2A, and Kruskal-Wallis with multiple comparison follow-up test for Fig 2B and are indicated as *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig 3. Surface protein expression of CD127 and CD132 on CD4⁺ T cells from endemic controls (ECs) and visceral leishmaniasis (VL) patients.

A. Gating strategy for CD4⁺ T cell flow cytometry analysis. B. Percentage (top) and mean fluorescent intensity (MFI) (bottom) of CD127 on CD4⁺ T cells. C. Percentage (top) and MFI (bottom) of CD132 on CD4⁺ T cells. D. Percentage (top) and MFI (bottom) of CD38 on CD4⁺ T cells. Statistical significance between ECs (n = 11) and VL patients (n = 12) was determined by Mann-Whitney U-test in Fig 3B-D, and 3F are indicated as *p<0.05; **p<0.01. E. Boolean-Gating (FlowJo), was used to define complex cell sub-population, and Simplified presentation of incredibly complex evaluations (SPICE) polyfunctionality analysis. SPICE was used to establish overlap in expression of CD38, CD127 and CD132 on CD4⁺ T cells. Pie charts represent the entire CD4⁺ T cell population expressing either CD38, CD127, CD132 or none of them. Data was generated from EC (n = 7) and VL patients (n = 7). F. Percentage and MFI of CD127, and CD132, expression on CD38+/- CD4 T⁺ cells of VL (n = 7) and EC (n = 7).

Fig 4. Surface protein expression of CD127 and CD132 on CD4⁺ T cell subsets from endemic controls (ECs) and visceral leishmaniasis (VL) patients.

A. Gating strategy for CD4⁺ T cell subsets. B-H. Boolean-Gating and Simplified presentation of incredibly complex evaluations (SPICE) polyfunctionality analysis. SPICE was used to establish overlap in expression of CD38, CD127 and CD132 on CD4⁺ T cell subsets in endemic controls (ECs) and visceral leishmaniasis (VL) patients; EC (n = 7), VL (n = 7). B. Treg cells C. Tem D. Th E. Th17_Th22 F. Th9 G. Th1 H. Th2.

Fig 5. IL-7-mediated activation of intracellular pSTAT5.

A. Gating strategy for CD4⁺ T cells. B. Representative histograms of pSTAT5 in endemic controls (ECs) and visceral leishmaniasis (VL) patients following rhIL-7 treatment. C. Frequency of CD4⁺ T cells expressing pSTAT5 and D. Mean fluorescence intensity (MFI) of pSTAT5 in CD4⁺ T cell at baseline without (-) and after rhIL-7 stimulation (+) in EC (n = 8) and VL patients (n = 12). Statistical significance was determined by the Wilcoxon matched-pairs signed rank test between control and rhIL-7 stimulation in figure C-D, or Mann-Whitney U-test to compare EC (n = 8) and VL patients (n = 12) in Fig D. Statistically significant differences are indicated as *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 6. pSTAT5 by CD38⁺ and CD38^- CD4⁺ T cells.

A-B. Gating Strategy for CD38⁺ and CD38^- CD4⁺ T cells. C. Frequency of pSTAT5 expressing CD38⁺ and CD38^- CD4⁺ T cells from endemic controls (EC) and visceral leishmaniasis (VL) patients after rhIL-7 stimulation. D. pSTAT5 mean fluorescence intensity (MFI) in CD38⁺ and CD38^- CD4⁺ T cells upon rhIL-7 stimulation. Data was generated from EC (n = 8) and VL (n = 12) donor samples. Statistical differences were determined using comparison between two groups with a Wilcoxon matched-pairs signed rank test between control and rIL-7 stimulation and significant differences are indicated as *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.