IL-7 induces sCD127 release and mCD127 downregulation in human CD8+ T cells by distinct yet overlapping mechanisms, both of which are impaired in HIV infection

Abstract

The IL-7 receptor specific α chain, CD127, can be expressed both as a membrane-associated (mCD127) and a soluble form (sCD127), however, the mechanisms involved in their regulation remain to be defined. We first demonstrated in primary human CD8⁺ T cells that IL-7-induced downregulation of mCD127 expression is dependent on JAK and PI3K signaling, whereas IL-7-induced sCD127 release is also mediated by STAT5. Following stimulation with IL-7, expression of alternatively spliced variants of the CD127 gene, sCD127 mRNA, is reduced, but to a lesser degree than the full-length gene. Evaluation of the role of proteases revealed that MMP-9 was involved in sCD127 release, without affecting the expression of mCD127, suggesting it does not induce direct shedding from the cell surface. Since defects in the IL-7/CD127 pathway occur in various diseases, including HIV, we evaluated CD8⁺ T cells derived from HAART-treated HIV-infected individuals and found that IL-7-induced (1) downregulation of mCD127, (2) release of sCD127, and (3) expression of the sCD127 mRNA were all impaired. Expression of mCD127 and sCD127 is, therefore, regulated by distinct, but overlapping, mechanisms and their impairment in HIV infection contributes to our understanding of the CD8⁺ T cell dysfunction that persists despite effective antiretroviral therapy.

Keywords: CD127; CD8+ T cells; HAART; HIV; IL-7.

Figure 1

IL‐7‐induced downregulation of mCD127 is dependent on JAK and PI3K but not STAT5. CD8⁺ T cells were magnetically isolated from PBMCs derived from healthy individuals and cultured with or without IL‐7. For some experiments, inhibitors were used as described. Flow cytometry was performed to evaluate expression of CD127. (A) Representative flow cytometry histogram showing the CD127 median fluorescence intensity (MFI) and the percentage of CD127⁺ cells among unstained cells (light gray), medium control (dark gray), and the IL‐7‐ (10 ng/mL) treated cells (black unfilled). The gating strategy is shown in Supporting Information Fig. S6. (B) Time course of CD127 expression on CD8⁺ cells with media control (dotted line), 1 ng/mL IL‐7 (gray line), and 10 ng/mL IL‐7 (black line). Significance was calculated between each IL‐7 concentration in relation to the medium control (*p < 0.05, **p < 0.001, two‐way ANOVA with Bonferroni posttest), n = 8. Data are shown as mean ± SEM of eight samples from four independent experiments. (C) The effect of JAK inhibitor (10 μM) (n = 8), (D) STAT5 inhibitor (250 μM) (n = 8), and € PI3K inhibitor (25 μM) (n = 10) in the IL‐7‐induced reduction in the proportion of CD8⁺ T cells that express CD127 was evaluated at 48 h. Significance was calculated between IL‐7 alone in relation to IL‐7 plus each inhibitor (paired t test). Graphs show the distribution of samples from five independent experiments.

Figure 2

IL‐7‐induced sCD127 release from CD8⁺ T cells involves JAK, STAT5, and PI3K. CD8⁺ T cells were magnetically isolated from PBMCs derived from healthy individuals and cultured with or without IL‐7. For some experiments, inhibitors were used as described. ELISA assays were performed to evaluate secretion of sCD127. (A) sCD127 release with media alone (dotted line), 1 ng/mL IL‐7 (gray line), and 10 ng/mL IL‐7 (black line) over time. sCD127 release was compared in the different time points between media alone and 1 ng/mL IL‐7 or media alone and 10 ng/mL IL‐7 (two‐way ANOVA with Bonferroni posttest); between 1 ng/mL IL‐7 and 10 ng/mL IL‐7 on each time point (two‐way ANOVA with Bonferroni posttest); and between the time points in the media alone (one‐way ANOVA with Bonferroni post‐test), n = 4. Data are shown as mean ± SEM of four samples from two independent experiments. (B) sCD127 release was normalized to the control values (values obtained in the media alone cell cultures were subtracted) and compared between cells treated with 1 ng/mL IL‐7 (grey line) or 10 ng/mL IL‐7 (black line) over time (two‐way ANOVA with Bonferroni posttest), n = 4. Data are shown as mean ± SEM of four samples from two independent experiments. (C) The impact of JAK inhibitor (10 μM), (D) STAT5 inhibitor (250 μM), and (E) PI3K (25 μM) inhibitor in the IL‐7‐induced sCD127 secretion in 48 h (paired t test), n = 7. Graphs show the distribution of seven samples from four independent experiments. The dotted line represents the limit of detection for this assay (125 pg/mL) (*p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant).

Figure 3

IL‐7 induces a preference for the alternatively spliced CD127 mRNA. CD8⁺ T cells were magnetically isolated from PBMCs derived from healthy individuals and cultured with or without IL‐7. RNA was extracted, converted to cDNA, and a quantitative probe‐based PCR was performed. (A) Time course of total CD127 mRNA (i.e., including RNA encoding both mCD127 and sCD127 variants) detected following IL‐7 stimulation of CD8⁺ T cells, n = 8. (B) Time course of alternatively spliced CD127 mRNA (sCD127 mRNA) expression after stimulation with IL‐7, n = 8. (C) The sCD127:total CD127 mRNA ratio over time, n = 8. *p < 0.01, **p < 0.001 by two‐way ANOVA with Bonferroni posttest. Significance was calculated in relation to the medium control. Data are shown as mean ± SEM of eight samples per group from four independent experiments.

Figure 4

Role of MMP‐2, MMP‐9, and brefeldin on IL‐7‐induced sCD127 release and impact of MMP‐9 inhibition on IL‐7‐induced reduction of mCD127 expression on CD8⁺ T cells. CD8⁺ T cells were magnetically isolated from PBMCs derived from healthy individuals and cultured with or without IL‐7. For some experiments, inhibitors were used as described. Release of sCD127 was evaluated through ELISA. Expression of CD127 was evaluated through flow cytometry. (A) Effect of the addition of MMP‐2 inhibitor (20 μM) (n = 3) and (B) MMP‐9 inhibitor (20 μM) (n = 9) in the IL‐7‐induced production of sCD127 (paired t test). (C) Effect of the addition of MMP‐9 inhibitor in the IL‐7‐induced reduction in the proportion of CD8⁺ cells expressing CD127 (paired t test), n = 9. (D) Effect of brefeldin A (1 μg/mL) in the IL‐7‐induced sCD127 expression at 24 h (paired t test), n = 6. The dotted line represents the limit of detection for this assay (125 pg/mL). Significance was calculated between IL‐7 alone in relation to IL‐7 plus each inhibitor. Graphs show the distribution of samples from four independent experiments.

Figure 5

CD8⁺ T cells isolated from HIV+ HAART‐treated individuals are less responsive to IL‐7 than those from healthy individuals. CD8⁺ T cells were magnetically isolated from PBMCs derived from healthy controls and HIV+ HAART‐treated individuals and cultured with or without IL‐7. Expression of CD127 was evaluated through flow cytometry. Release of sCD127 was evaluated through ELISA. RNA was extracted, converted to cDNA, and a quantitative probe‐based PCR was performed to quantify mRNA as described. (A) The percentage of CD127⁺ cells within the CD8⁺ cell population in HIV+ individuals compared to healthy individuals (unpaired t test), n = 8 for both cohorts. (B, C) Relative change in the surface CD127 expression over time with 1 ng/mL and 10 ng/mL IL‐7 stimulation (two‐way ANOVA), n = 8. (D, E) IL‐7 induced sCD127 release in HIV+ and healthy individuals. Upon stimulation with IL‐7, sCD127 release was quantified over time in the HIV+ individuals and compared to the healthy individuals (two‐way ANOVA) (1 ng/mL IL‐7 HIV+, n = 6, healthy individuals, n = 7; 10 ng/mL IL‐7 HIV+, n = 9, healthy individuals, n = 7; controls: HIV+, n = 9, healthy individuals, n = 7). (F) Total CD127 mRNA (i.e., includes mCD127 and sCD127 variants) detected following 24 h of IL‐7 stimulation in HIV+ and healthy individuals (HIV+, n = 9, healthy individuals, n = 8) (two‐way ANOVA with Bonferroni posttest). (G) sCD127 mRNA expression following 24 h of 1 ng/mL and 10 ng/mL IL‐7 stimulation in HIV+ and healthy individuals (two‐way ANOVA with Bonferroni posttest) (controls: HIV+, n = 9, healthy, n = 10; IL‐7, 1 ng/mL: HIV+, n = 10, healthy, n = 10; IL‐7, 10 ng/mL: HIV+, n = 10, healthy, n = 4). (H) Ratio of sCD127:mCD127 mRNA induced by 24 h incubation with 1 ng/mL and 10 ng/mL IL‐7 in HIV+ and healthy individuals (two‐way ANOVA with Bonferroni posttests) (controls: HIV+, n = 9, healthy, n = 8; IL‐7, 1 ng/mL and 10 ng/mL: HIV+, n = 10, healthy, n = 8). Data are shown as mean ± SEM from four (A–C) and five (D–H) independent experiments.