Interleukin-21 combined with ART reduces inflammation and viral reservoir in SIV-infected macaques

Abstract

Despite successful control of viremia, many HIV-infected individuals given antiretroviral therapy (ART) exhibit residual inflammation, which is associated with non-AIDS-related morbidity and mortality and may contribute to virus persistence during ART. Here, we investigated the effects of IL-21 administration on both inflammation and virus persistence in ART-treated, SIV-infected rhesus macaques (RMs). Compared with SIV-infected animals only given ART, SIV-infected RMs given both ART and IL-21 showed improved restoration of intestinal Th17 and Th22 cells and a more effective reduction of immune activation in blood and intestinal mucosa, with the latter maintained through 8 months after ART interruption. Additionally, IL-21, in combination with ART, was associated with reduced levels of SIV RNA in plasma and decreased CD4(+) T cell levels harboring replication-competent virus during ART. At the latest experimental time points, which were up to 8 months after ART interruption, plasma viremia and cell-associated SIV DNA levels remained substantially lower than those before ART initiation in IL-21-treated animals but not in controls. Together, these data suggest that IL-21 supplementation of ART reduces residual inflammation and virus persistence in a relevant model of lentiviral disease and warrants further investigation as a potential intervention for HIV infection.

Figure 7. Effects of IL-21 treatment on immune activation and viral replication following ART interruption.

(A) Longitudinal assessment of plasma SIVmac₂₃₉ RNA copies (presented as the mean ± SEM) up to 8 months after ART interruption. Starting from day 90 off ART, viremia levels were lower (average of a 0.7-log reduction) in IL-21–treated RMs up to day 240 off ART. (B) Plasma viral loads before ART (day 58 p.i.) were compared with those at days 140, 180, and 240 off ART in IL-21–treated (n = 7) and control (n = 8) RMs. At all 3 experimental time points off ART (as well as at day 90 off ART, not shown), viral loads remained significantly lower than before ART in IL-21–treated RMs but not in controls. (C) Cell-associated SIV DNA content (total) in purified blood CD4⁺ T cells was longitudinally determined before ART initiation (day 58 p.i.), during ART (days 50 and 200 on ART), and after ART interruption (days 90 and 180 off ART) in IL-21–treated and control RMs. At day 180 off ART, SIVmac₂₃₉ DNA levels were significantly higher than levels during ART in control animals, but not in IL-21–supplemented animals. (D) Differences were even more pronounced when only Mamu-A*01^– animals were analyzed. (E) Percentages of memory CD4⁺ (left panels) and CD8⁺ (right panels) T cells coexpressing the activation markers HLA-DR and CD38 were longitudinally determined at different experimental time points off ART in PB (top panels) and RB (bottom panels) biopsies. T cell activation levels were significantly lower in IL-21–treated RMs compared with levels in control RMs. IL-21–treated animals are indicated with orange circles and controls with black squares. Averaged data are presented as the mean ± SEM. Repeated-measures analyses were performed with a means model (SAS PROC MIXED, version 9) to generate statistical outcomes.

Figure 6. IL-21 supplementation reduces the frequency of CD4⁺ T cells harboring replication-competent SIV.

(A) Representative staining of flow cytometric analyses of intracellular SIV-Gag p27 on days 9, 16, and 25 in LN CD4⁺ T cells cocultured with CEMx174 cells. Top 2 rows show positive (CEMx174 cells infected with SIVmac₂₃₉) and negative (CEMx174 cells, uninfected) controls. The lower 4 rows show results for 2 control (ART-only) and 2 IL-21–treated RMs. (B) Longitudinal quantification of intracellular SIV-Gag p27 and (C) supernatant SIV-Gag RNA on days 9, 16, and 25 in IL-21–treated (n = 7) (orange bars) and control (n = 8) (black bars) RMs. Both measurements were significantly lower (P < 0.01) in IL-21–treated RMs when compared with those for controls. *P < 0.05. (D) The frequency of latently infected cells, expressed as IUPM CD4⁺ T cells, was significantly lower in IL-21–treated RMs (orange circles) than in control RMs (black squares) (P = 0.009). Averaged data are presented as the median with an interquartile range. Repeated-measures analyses were performed with a means model (SAS PROC MIXED, version 9) to generate statistical outcomes.

Figure 5. IL-21 supplementation reduces residual plasma viremia and SIV DNA content in ART-treated, SIV-infected RMs.

(A) Residual levels (copies/ml) of plasma SIVmac₂₃₉ RNA were assessed by ultrasensitive viral load assay (LOD, 3 copies per ml; dashed lines) at 3 experimental time points on ART: day 75 (day 135 p.i.), day 104 (day 164 p.i.), and day 200 (day 256 p.i.). Percentages at the top of the graphs indicate the fraction of RMs with undetectable viremia at the different time points. When all 3 time points were considered, the decrease in residual viremia was significantly higher in IL-21–treated (n = 7) (orange circles) RMs compared with viremia levels in controls (n = 8) (black squares) (P = 0.03). When a single experimental time point was considered, SIVmac₂₃₉ RNA copies were significantly lower in IL-21–treated RMs at day 200 on ART (P = 0.0203). (B) Copies of total SIVmac₂₃₉ DNA per 10⁸ intestinal cell equivalents were longitudinally quantified at days 50 and 200 on ART. SIVmac₂₃₉ DNA content significantly decreased between the 2 experimental points only in IL-21–treated animals (P = 0.0156). (C) Correlations between the levels of activated (HLA-DR⁺CD38⁺) CD8⁺ T cells at days 50 and 200 on ART and the content of SIV DNA in the rectum at day 200 on ART (top panels). Correlations between plasma viremia before ART initiation and the content of SIV DNA in PBMCs and in the rectum at day 200 on ART (bottom panels). Averaged data are presented as the geometric mean. Repeated-measures and GEE analyses were used (SAS PROC MIXED and GENMOD, version 9) to generate statistical outcomes. VL, viral load.

Figure 4. IL-21 supplementation upregulates the expression of genes regulating antimicrobial immunity in PBMCs from ART-treated, SIV-infected RMs.

(A and E) Global heatmaps of genes whose expression levels were significantly different (Q < 0.05) between IL-21–treated (n = 7) and control (n = 8) RMs at day 50 (A) or day 200 (E) on ART. The specific genes are indicated on the left side of the heatmaps. (B and F) FPKM values for the selected genes of interest were compared between IL-21–treated (orange circles) and control (black squares) RMs at day 50 (B) and day 200 (F) on ART. (C and G) Gene ranking for IL-21–, IL-17–, and IL-22–related genes at day 50 (C) and day 200 (G) on ART. Dots represent individual genes plotted according to their ranking (x axis) and their addition to the cumulative score (y axis). P values below 0.25 were considered significant according to GSEA guidelines. Leading genes with higher enrichment scores in IL-21–treated RMs (as compared with controls) are highlighted in red and shown as heatmaps in D and H. Statistical analyses were performed with the Partek Genomics Suite, version 6.6, and the GSEA tool.

Figure 3. IL-21 supplementation limits residual T cell activation in ART-treated, SIV-infected RMs.

(A–F) Longitudinal assessment of the activation levels of total memory (A and D), CM (B and E), and EM (C and F) CD4⁺ (A–C) and CD8⁺ (D–F) T cells in blood (PB). (G–L) Longitudinal assessment of the activation levels of total memory (G and J), CM (H and K), and EM (I and L) CD4⁺ (G–I) and CD8⁺ (J–L) T cells in colorectum (RB). T cell activation was assessed by dual expression of the immune activation markers HLA-DR and CD38. IL-21–treated animals (n = 7) are indicated by orange circles and controls (n = 8) by black squares. Shaded area represents the time of ART treatment, and orange arrows represent IL-21 administrations. Averaged data are presented as the mean ± SEM. Repeated-measures analyses were performed with a means model (SAS PROC MIXED, version 9) to generate statistical outcomes. *P < 0.05; **P < 0.01; ***P < 0.001; ^‡P < 0.0001.

Figure 2. IL-21 supplementation improves mucosal immunity in ART-treated, SIV-infected RMs.

(A and B) Representative staining for IL-17 (A) and IL-22 (B) within intestinal CD4⁺ T cells in IL-21–treated (top plots) and control (bottom plots) RMs before infection (day –20), after infection but before ART and IL-21 initiation (day 58 p.i.), and at 3 different experimental time points on ART (days 25, 75, and 200). (C and F) Longitudinal assessment of the frequency of intestinal CD4⁺ T cells producing IL-17 (C), IL-22 (D), IFN-γ (E), and IL-2 (F) in IL-21–treated (n = 7) and control (n = 8) RMs. (G and H) Rectal mucosa tissues were stained for MPO (brown) as a marker for PMNs. (G) Representative MPO staining (original magnification, ×200) in 1 IL-21–treated RM (top panels) and 1 control RM (bottom panels) before ART (day 58 p.i.) and at day 25 (day 85 p.i.) and day 200 (day 256 p.i.) on ART. (H) Random, high-powered (×400) images of gut LP were taken, and the percentage of area staining for MPO was determined in IL-21–treated and control RMs. Shaded area represents the time of ART treatment, and orange arrows represent IL-21 administrations. Averaged data are presented as the mean ± SEM. Repeated-measures analyses were performed with a means model (SAS PROC MIXED, version 9) to generate statistical outcomes. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 1. Longitudinal variations in viremia and CD4⁺ T cell levels in IL-21–treated and control SIV-infected RMs.

(A). Schematic of the study design. Sixteen RMs were infected i.v. with 300 TCID₅₀ SIVmac₂₃₉ (day 0), and starting on day 60 p.i., treated with combination ART (PMPA, FTC, raltegravir, and ritonavir-boosted darunavir) for 7 months. Seven animals (ROc10 died on day 140 p.i. due to post-surgical [LN biopsy] complications) received 2 courses of IL-21 treatment (100 μg/kg s.c.) weekly for 6 weeks at the beginning (from days 67–105 p.i.) and at the end (from days 203–241 p.i.) of ART, as well as 4 additional administrations upon ART interruption (day 271 p.i.). The remaining 8 animals served as ART-treated controls. On day 270 p.i., ART was interrupted, and all the animals were monitored for 8 additional months. PB, RB, and LN biopsies were collected at the indicated time points. (B and C) Plasma SIVmac₂₃₉ RNA levels expressed as copies/ml (LOD, 60 copies/ml, dashed line) are shown for each individual animal (B) and as an average (C) in IL-21–treated (orange circles) versus control (black squares) RMs. (D–G) CD4⁺ T cell levels, expressed as a fraction of live CD3⁺ T cells, were compared between IL-21–treated and control RMs in PB (D), RB (F), and LN (G). In PB, CD4⁺ T cells were also expressed as absolute counts (cells/μl blood; E). Gray shaded area represents the time of ART treatment, and the orange arrows mark IL-21 administrations. Averaged data are presented as the mean ± SEM. cART, combination ART.