Interleukin 7 up-regulates CD95 protein on CD4+ T cells by affecting mRNA alternative splicing: priming for a synergistic effect on HIV-1 reservoir maintenance

Abstract

Interleukin-7 (IL-7) has been used as an immunoregulatory and latency-reversing agent in human immunodeficiency virus type 1 (HIV-1) infection. Although IL-7 can restore circulating CD4(+) T cell counts in HIV-1-infected patients, the anti-apoptotic and proliferative effects of IL-7 appear to benefit survival and expansion of HIV-1-latently infected memory CD4(+) T lymphocytes. IL-7 has been shown to elevate CD95 on CD4(+) T cells in HIV-1-infected individuals and prime CD4(+) T lymphocytes to CD95-mediated proliferative or apoptotic signals. Here we observed that through increasing microRNA-124, IL-7 down-regulates the splicing regulator polypyrimidine tract binding protein (PTB), leading to inclusion of the transmembrane domain-encoding exon 6 of CD95 mRNA and, subsequently, elevation of CD95 on memory CD4(+) T cells. Moreover, IL-7 up-regulates cellular FLICE-like inhibitory protein (c-FLIP) and stimulates c-Jun N-terminal kinase (JNK) phosphorylation, which switches CD95 signaling to survival mode in memory CD4(+) T lymphocytes. As a result, co-stimulation through IL-7/IL-7R and FasL/CD95 signal pathways augments IL-7-mediated survival and expansion of HIV-1-latently infected memory CD4(+) T lymphocytes. Collectively, we have demonstrated a novel mechanism for IL-7-mediated maintenance of HIV-1 reservoir.

Keywords: Alternative Splicing; CD95; Human Immunodeficiency Virus (HIV); Interleukin; MicroRNA (miRNA); PTB; Post-transcriptional Regulation.

FIGURE 1.

IL-7 up-regulates CD95 expression on the surface of resting memory CD4⁺ T cells by promoting exon 6 inclusion. A, CD95 expression on memory CD4⁺ T cells from HIV-1-infected donors cultured for 2 days with or without IL-7 (25 ng/ml) was measured by FCM. MFI is shown with error bars representing S.D. calculated from six independent experiments. B, C, E, and F, memory CD4⁺ T cells from HIV-negative controls were isolated and cultured with or without 25 ng/ml IL-7 for 2 days. B, CD95 mRNA was measured by real-time PCR. Error bars represent S.D. calculated from three independent experiments. C, CD95 protein expression was measured by Western blot. A representative result out of eight independent experiments was shown. Values represent -fold changes of CD95 normalized against GAPDH and compared with control. D, a schematic map for two of splice variants of CD95 is shown. DD, death domain; TM, transmembrane domain. E, mRNAs of CD95 splice variants were measured by RT-PCR. Results of six independent experiments are shown. The column graph shows a statistical analysis of the percentage of mCD95/sCD95 (measured by relative density) after IL-7 treatment. mCD95, membrane-bound CD95; sCD95, soluble CD95. F, CD95 expression of permeabilized or non-permeabilized memory CD4⁺ T cells was measured by FCM. MFI is shown with error bars representing S.D. calculated from 10 independent experiments. A, C, and F, paired, two-tailed Student's t test: ***, p < 0.001. NS, non-significant.

FIGURE 2.

IL-7 down-regulates CD95 splicing regulator PTB. A and B, memory CD4⁺ T cells from HIV-negative controls were isolated and cultured with or without 25 ng/ml IL-7 for 2 days. A, cells were harvested for RNA extraction, and PTB mRNA was measured by real-time PCR. Error bars represent S.D. calculated from six independent experiments. B, cells were collected for Western blot analysis of PTB protein expression. A representative sample of five independent experiments is shown. Values represent -fold changes of PTB normalized against β-actin and compared with control. C and D, memory CD4⁺ T cells from HIV-negative controls transfected with PTB-specific siRNA or control siRNA were cultured with or without 25 ng/ml IL-7 for 2 days. C, top panel, mRNAs of CD95 splice variants were measured by RT-PCR. A representative sample of three independent experiments is shown. Bottom panel, silencing of PTB by PTB-specific siRNAs was confirmed by Western blot. mCD95, membrane-bound CD95; sCD95, soluble CD95. Values represent the ratios of the relative density of mCD95/sCD95. D, CD95 expression was measured by FCM. MFI is shown with error bars representing S.D. calculated from three independent experiments. NC, negative control. **, p < 0.01 in a paired t test. NS, non-significant.

FIGURE 3.

IL-7 up-regulates PTB-targeting miR-124. A, top panel, memory CD4⁺ T cells from HIV-negative controls were transfected with AGO1- and AGO2-specific siRNAs or control oligonucleotides and then cultured with or without the presence of 25 ng/ml IL-7 for 2 days. CD95 expression was measured by FCM. MFI is shown with error bars representing S.D. calculated from four independent experiments. Bottom panel, silencing of AGO1/2 by AGO1/2-specific siRNAs was confirmed by Western blot. B, predicted target site of miR-124 within the 3′-UTR of PTB is shown. C, memory CD4⁺ T cells from HIV-negative controls were transfected with miR-124 mimic or control oligonucleotides and harvested for Western blot of PTB protein expression. A representative sample of three independent experiments is shown. Values represent fold changes of PTB normalized against β-actin and values compared with control. D, memory CD4⁺ T cells from HIV-negative controls were isolated and cultured with or without 25 ng/ml IL-7 for 2 days. Cells were harvested for miR-124 quantification by stem-loop PCR. Error bars represent S.D. calculated from three independent experiments. NC, negative control. Paired, two-tailed Student's t test: *, p < 0.05; **, p < 0.01.

FIGURE 4.

miR-124 up-regulates CD95 expression on memory CD4⁺ T cells. A and B, memory CD4⁺ T cells from HIV-negative controls were transfected with miR-124 mimic, miR-124 inhibitor, or their respective control oligonucleotides. IL-7 was added to the cell cultures 12 h after miR-124 inhibitor transfection. A, the mRNAs of CD95 splice variants were measured by RT-PCR. A representative sample of three independent experiments is shown. mCD95, membrane-bound CD95; sCD95, soluble CD95. Values represent the ratios of the relative density of mCD95/sCD95. B, CD95 expression was measured by FCM. MFI is shown with error bars representing S.D. calculated from three independent experiments. NC, negative control. *, p < 0.05 in a paired t test.

FIGURE 5.

IL-7 and CD95 synergistically promote survival and proliferation in memory CD4⁺ T cells. A, B, C, and D, memory CD4⁺ T cells from HIV-negative controls were treated with or without 25 ng/ml IL-7 for 48 h. Cells were then stimulated with 1 μg/ml anti-CD95 or control IgG. Human anti-CD3 was added into culture along with anti-CD95 or control IgG in activated cultures (B). A, apoptosis of T cells was measured after 24 h of anti-CD95 activation using annexin-V staining. B. proliferation of T cells was measured at day 4 of anti-CD95 activation using carboxyfluorescein diacetate succinimidyl ester labeling. A and B consist of results from 12 independent experiments. C and D, protein levels of c-FLIP (C) and JNK/p-JNK (D) were detected by Western blot. A representative sample of three independent experiments is shown. Values in C and D represent -fold changes of c-FLIP (C) and JNK/p-JNK (D) normalized against GAPDH and compared with control. ***, p < 0.001 in a paired t test.

FIGURE 6.

IL-7 and CD95 synergistically promote the maintenance of HIV-1 reservoir in vitro. A, B, C, and D, CD4⁺ T cells from HIV-1-infected donors on suppressive ART were cultured in the presence of 10 μm azidothymidine (AZT) and treated with or without 25 ng/ml IL-7 for 48 h. Cells were then stimulated with 1 μg/ml anti-CD95 or control IgG. Human anti-CD3 were added into culture along with anti-CD95 or control IgG in activated cultures (B, C, and D). A, apoptosis of T cells was measured after 24 h of anti-CD95 activation using annexin-V staining. B, proliferation of T cells was measured at day 4 of anti-CD95 activation using carboxyfluorescein diacetate succinimidyl ester labeling (left) and cell counting (right). C. integrated HIV-1 DNA copy numbers per well after IL-7 and anti-CD95 treatment were quantified by Alu-gag PCR. D, HIV-1 RNA copy numbers per milliliter after IL-7 and anti-CD95 treatment were measured by real-time PCR. These data consist of results from different donors indicated in each graph. Empty squares in D represent negative results (<10 HIV-1 copies/ml). Wilcoxon matched-pairs signed rank test: **, p < 0.01; ***, p < 0.001.