Fig 2: 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.

Fig 3: 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.

Fig 4: 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.

Fig 5: Screen of ASOs targeting functional cis-acting splicing elements within IL7R exon 6. (A) Schematics of the GFP-IL7R splicing reporter, wherein GFP is only expressed by exclusion of IL7R exon 6. (B) Schematics of approach comparing the effects of mutagenesis of functional cis-acting splicing elements to steric blocking of the corresponding elements with ASOs using the GFP-IL7R reporter. The blue dotted lines expand the exon 6 sequence (gray box), and the relevant cis-acting elements are color-coded: enhancer ESE2 (green), silencers ESS2 and ESS3 (red), and 5'ss (yellow). The SNP rs6897932 is indicated by the red C letter (nt 25). Mutations to ESE2, ESS3, and the 5'SS are shown underneath each element: ?ESE2 and ?ESS3 represent mutations to ESE2 and ESS3, respectively, and 5'Cons and 5'Mut represent consensus and inactivating mutations to the 5'ss, respectively. The sequence of ASOs targeting these cis-elements (IL7R-001–IL7R-005) is shown above the exon sequence: IL7R-001 blocks the 5'ss of exon 6, IL7R-002 blocks ESE2, IL7R-003 blocks ESS2 and ESS3, IL7R-004 blocks the sequence in between ESS2 and ESS3, and IL7R-005 blocks ESS3. (C,E) Percentage (%) of exon 6 exclusion (mean ± S.D.) in transcripts from the reporter in HeLa cells stably expressing wild-type or mutant versions of the reporter (C), or cells stably expressing the wild-type reporter and transfected with control (ASO-Ctrl) or experimental (IL7R-001–IL7R-005) morpholino ASOs (E) are shown under the gel images. The top band in the gels represents the exon 6 included product, whereas the lower band represents the exon 6 excluded product. (D,F) Mean fluorescence intensity (MFI) of GFP expression in the cells from panel C stably expressing the different versions of the reporter (D), or in cells from panel E transfected with ASOs (F). The left panels show representative GFP MFI histograms for selected mutants (wild-type, 5'Cons and 5'Mut) (D) and selected ASOs (ASO-Ctrl, IL7R-001, and IL7R-005) (F), with quantification shown on the right as log2(fold-change) [log2(FC)] relative to wild-type for all mutants (D) and relative to control for all ASOs (F). The data are shown for one representative experiment in triplicate wells (n = 3 per condition) out of three independent experiments with similar results. (***) P < 0.001.