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: ΔESE2 and ΔESS3 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.