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CD4+IL-21+T cells are correlated with regulatory T cells and IL-21 promotes regulatory T cells survival during HIV infection
Cytokine 91 (2017) 110–117
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CD4+IL-21+T cells are correlated with regulatory T cells and IL-21 promotes regulatory T cells survival during HIV infection Zi-Ning Zhang a,b, Li-Xin Bai a, Ya-Jing Fu a,b, Yong-jun Jiang a,b, Hong Shang a,b,⇑ a Key Laboratory of AIDS Immunology of National Health and Family Planning Commission, Department of Laboratory Medicine, The First Affiliated Hospital, China Medical University, Shenyang, Liaoning 110001, China b Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, Zhejiang, China
a r t i c l e
i n f o
Article history: Received 19 August 2016 Received in revised form 20 December 2016 Accepted 20 December 2016
Keywords: HIV IL-21 Regulatory T cell
a b s t r a c t Introduction: IL-21 enhances T and natural killer cells survival and antiviral functions without promoting T cell activation during HIV infection, which makes it a better adjuvant in anti-HIV immunotherapy. Due to the pleiotropy and redundancy of cytokines, it is vital to have a comprehensive knowledge of the role of IL-21 in the regulation of immune responses. Regulatory T cells (Tregs) play an important role in immune regulation and are a determinant of immune therapeutic efficacy in certain circumstances. In this study, we explored the direct effect of IL-21 on Tregs during HIV infection, which has not been addressed before. Methods: Thirty-four HIV treatment-naïve patients were enrolled and the relationship between CD4+IL21+T cells and Tregs were studied. The effects of IL-21 on CD4+CD25+CD127low Tregs’ apoptosis, proliferation, and CTLA-4 and TGF-b expression in HIV-infected patients was investigated and compared with the effect of other common c-chain cytokines. Results: We found the percentage and absolute numbers of CD4+IL-21+T cells were positively related to the frequency or absolute numbers of CD4+CD25+ or CD4+CD25+CD127low Tregs. Compared with the media-alone control, IL-21, IL-7, and IL-15 could significantly reduce apoptosis of Tregs (p < 0.05). IL21 did not promote the proliferation of Tregs as compared with media alone, while IL-2, IL-7, and IL15 could significantly increase the proliferation of Tregs (p < 0.05). IL-21 enhanced CTLA-4 expression by Tregs (p < 0.05), but could not induce TGF-b secretion of Tregs from HIV infected patients. There were no significant differences of the fold induction of apoptosis, proliferation, or CTLA-4 and TGF-b expression by Tregs from HIV-infected patients and normal controls after IL-21 treatment. In vitro experiment showed that pretreatment with IL-21 significantly enhanced the suppressive effect of Tregs on CD8+ T cells’ IFN-c expression. Conclusion: We conclude that IL-21 promotes the survival and CTLA-4 expression of Tregs and enhanced the suppressive capacity of Tregs during HIV infection. These results broaden the understanding of HIV pathogenesis and provide critical information for HIV interventions. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction HIV infection is associated with a progressive decline in circulating CD4+ T cells and a concomitant loss of immune function [1,2]. Although antiretroviral therapy (ART) regimens have proven to be effective in controlling active HIV replication, complete recovery of CD4+ T-cell counts does not always occur, even among
⇑ Corresponding author at: Key Laboratory of AIDS Immunology of National Health and Family Planning Commission, Department of Laboratory Medicine, The First Affiliated Hospital, China Medical University, No. 155, Nanjingbei Street, Heping District, Shenyang, Liaoning Province 110001, China. E-mail address: [email protected] (H. Shang). http://dx.doi.org/10.1016/j.cyto.2016.12.012 1043-4666/Ó 2016 Elsevier Ltd. All rights reserved.
patients who display high levels of virologic control [3]. Furthermore, prolonged treatment comes with other problems such as drug resistance, side effects, and reduced adherence to the medication regimen. Accordingly, different adjuvant therapies, including immune modulation, are being tested in clinical trials or are under consideration in hopes of addressing these remaining challenges [4]. Common c-chain (cc) cytokines – including interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15 and IL-21 – regulate a variety of cellular responses such as proliferation, differentiation, and survival [5]. Of these, interleukin-21 was discovered relatively recently [6]. Mainly produced by CD4+ T cells [7–9], IL-21 affects an extremely broad set of target cells including T cells, B cells, NK cells, NKT cells,
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and DCs [10]. During HIV infection, IL-21 promotes the survival of CD4+ and CD8+ T cells as well as NK cells; IL-21 also enhances CD8+ T cell and NK cell antiviral function [11–13]. In addition, IL-21 directly suppresses HIV-1 replication through the promotion of microRNA-29 transcription [14]. Unlike other cc cytokines, IL-21 does not promote T cell activation or proliferation when it enhances the function of cytotoxic cells [11–13,15–19]. This prominent characteristic of IL-21 led to the hypothesis that IL-21, when used as an immunotherapeutic agent, was potentially capable of improving immune reconstruction without promoting immune activation and virus replication [11,12,15]. However, due to the pleiotropy and redundancy of cytokines [20], it is vital to have a comprehensive knowledge of the role of IL-21 in the regulation of immune responses. Regulatory T cells (Tregs) play a prominent role in chronic viral infections by limiting viral-specific immune responses and immune activation [21]. Tregs constitutively express the highaffinity IL-2Rabc complex, therefore IL-2 plays a very important role in regulating the Treg population [22]. Two large clinical trials have revealed that IL-2 administration in HIV-infected patients induced a newly expanded population of immunosuppressive Tregs [23,24]. The induction of Tregs may be the most plausible explanation why IL-2 administration plus ART yielded no clinical benefit in either study when compared to ART alone [23,24]. These results highlight the importance of considering the effects of Tregs in potential immune interventions. Tregs express the IL-21 receptor [25,26], indicating that IL-21 has the potential to affect Tregs. The reported direct effects of IL-21 on the expansion, survival, and function of Tregs have been inconsistent [27–30]. Some reports indicate that IL-21 renders T cells resistant to Tregmediated suppression, but has no direct effect on Treg viability, activation, or function [25,26,31–34]. By contrast, previous studies revealed that IL-21 was able to affect Tregs, either by decreasing the number and function of Tregs [35,36] or by increasing Tregs’ frequency and expression of Foxp3 [37–40] during viral infection or tumor development. The inconsistent results suggest the effect of IL-21 on Tregs varies depending on the disease, the anatomic site, or the physiological condition of the patient. To our knowledge, the effect of IL-21 on Tregs’ survival and function has not yet been clarified in HIV infection. In this study, we investigated the relationship between CD4+IL21+T cells and Tregs in HIV-infected patients. We then analyzed the effects of IL-21 on Treg apoptosis, proliferation, the expression of the key functional molecules, CTLA-4 and TGF-b and Treg’s suppression activity. We found a positive relationship between CD4+IL-21+T cells and Tregs in HIV-infected patients. Although IL-21 did not promote the proliferation of Tregs of HIV-infected patients in contrast to other cc cytokines, IL-21 could decrease Treg apoptosis and CTLA-4 expression and enhance Tregs’ suppressive activity in vitro. Our results expand the knowledge of the function of IL21 in HIV infection, which is helpful in the development of new immunotherapeutic strategies.
2. Materials and methods 2.1. Subjects Thirty-four treatment-naïve HIV-infected patients, including 33 males and 1 female (median age: 36 years) were enrolled in this study. The absolute CD4+ T cell numbers and Log10 viral loads of these patients were 405 ± 202 cells/lL and 4.11 ± 0.90 copies/mL (mean ± SD), respectively. Ethical approval for this study was obtained from the local ethical review committee and written informed consent for participation in the study was obtained from all patients.
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2.2. Flow cytometric analysis Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll centrifugation. The following monoclonal antibodies (mAbs) and reagents were used in this study: FITC-conjugated anti-CD8, APC-Cy7-conjugated anti-CD4, PerCP-conjugated anti-CD3, PE-Cy7-conjugated anti-CD25, PE-conjugated anti-TGF-b, APC-conjugated anti-CD127, PEconjugated anti-CTLA-4, PE-conjugated anti-IFN-c, PE-conjugated anti-Annexin V, 7-AAD and purified anti-CD3 mAb (BD Biosciences); PE-conjugated anti-IL-21 (eBioscience); Recombinant hIL-21 (Biosource), recombinant hIL-2, hIL-7, and hIL-15 (R&D Systems). For the detection of CD4+IL-21+T cells, 0.5 106 PBMCs were co-cultured for 6 h with phorbol 12-myristate 13-acetate (PMA, 50 ng/mL, Sigma-Aldrich, St Louis, MI, USA) and ionomycin (0.75 lg/mL, Sigma-Aldrich) at 37 °C, 5% CO2; GolgiStop (500 lg/ mL, BD Biosciences) was added after the first 1 h of culture. The cells were stained with anti-CD3-PerCP and anti-CD8-FITC followed by intracellular staining using Cytofix/Cytoperm Kit (BD Biosciences) with PE-conjugated IL-21. For the detection of Tregs, 1 106 cells were incubated with anti-CD3-PerCP, anti-CD4-APCCy7, anti-CD25-PE-Cy7, and anti-CD127-APC for 45 min on ice. The cells were washed, fixed in 1% paraformaldehyde in PBS prior to acquisition on LSR II (Becton Dickinson, San Jose, CA). CD4+CD25+ Tregs or CD4+CD25+CD127low Tregs were identified. The absolute numbers of Tregs were calculated by multiplying the frequencies of Tregs by the absolute numbers of CD4+T cells. Because PMA induced the down-regulation of the CD4 molecule, CD4+IL21+T cells in our study were identified as CD3+CD8-IL-21+ cells. The absolute numbers of CD4+IL-21+T cells were calculated by multiply the frequencies of CD4+IL-21+T by the absolute numbers of CD4+T cells. 2.3. Proliferation and apoptosis of Tregs For proliferation detection, PBMCs were labeled with CFSE at a concentration of 4 lM/5 106 cells for 10 min at 37 °C. After washing 1640 complete media supplemented with 10% FBS, cells were cultured in 96-well plates (200 lL) at 37 °C, 5% CO2 with IL21 (100 ng/mL), IL-2 (200 U/mL), IL-7 (50 ng/mL), IL-15 (50 ng/ mL) or anti-CD3 (0.5 lg/mL) for 5 days. After culture, the cells were washed once and then stained with anti-CD3-PerCP, anti-CD4APC-Cy7, anti-CD25-PE-Cy7, and anti-CD127-APC. For apoptosis detection, PBMCs were cultured for 3 days with IL-21 (100 ng/ mL), IL-2 (200 U/mL), IL-7 (50 ng/mL), IL-15 (50 ng/mL), and antiCD3 (0.5 lg/mL). After culture, the cells were stained with antiCD3-APC-Cy7, anti-CD4-FITC, anti-CD25-PE-Cy7, and CD127-APC for 30 min followed by staining with 5 lL 7-AAD and antiAnnexin V-PE for 15 min before data acquisition. Cells were acquired on LSR II (Becton Dickinson) and analyzed using FACSDiva software. 2.4. Intracellular detection of the expression of CTLA-4 and TGF-b within Tregs PBMCs were cultured with IL-21 (100 ng/mL), IL-2 (200 U/mL), IL-7 (50 ng/mL), IL-15 (50 ng/mL), and anti-CD3 (0.5 lg/mL). For the detection of CTLA-4 expression, the cells were stained with anti-CD3-PerCP, anti-CD4-APC-Cy7, anti-CD25-PE-Cy7, and CD127-APC after 3 days of culture followed by intracellular staining with anti-CTLA-4-PE. For TGF-b detection, PBMCs were cultured for 5 days with the cytokines mentioned above and GolgiStop was added during the last 10 h. The cells were then stained with anti-CD3-APC-Cy7, anti-CD4-FITC, anti-CD25PE-Cy7, and CD127-APC followed by intracellular anti-PE-TGF-b
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staining. Cells were fixed in 1% paraformaldehyde in PBS and analyzed using a BD LSRII and FACSDiva software. 2.5. In vitro Treg-cell-suppression activity Freshly isolated PBMCs from HIV infected patients were stained with anti-CD3-PerCP, anti-CD4-APC-Cy7, anti-CD25-PE-Cy7, antiCD127-APC. To examine whether the pretreatment of IL-21 altered the suppressive capability of Tregs, CD4+CD25+CD127low Tregs or CD8+ (CD3+CD4 ) T cells were sorted with a FACSAria flow cytometer (BD Biosciences). CD4+CD25+CD127low Tregs were cultured with or without IL-21 (100 ng/mL) for 24 h. CD8+T cells were rested, incubated in fresh medium for 24 h. Then CD8+T cells were stimulated for two days with soluble anti-CD3/anti-CD28 antibodies (2.5 lg/mL; BD Biosciences) and co-cultured with either IL-21treated or untreated Tregs at a responder: suppressor ratio of 3:1. During the last 5 h of culture, Golgistop was added to the culture. At the end of culturing, cells were intra-cellularly stained with PEconjugated anti-IFN-c and analyzed on an LSRII flow cytometer. 2.6. Statistical analysis Analyses were carried out using SPSS 17.0 software. The MannWhitney U test was employed to compare the expression levels of Tregs and CD4+IL-21+ T cells of HIV infected patients with different viral loads. Correlation analysis was performed using the Spearman’s rank correlation test. The Wilcoxon matched-pairs signedranks test was used to compare the parameters before and after cytokine stimulation. P values less than 0.05 were considered to be statistically significant. 3. Results 3.1. Higher expression level of CD4+IL-21+T cells correlated with lower viral loads and higher numbers of Tregs Previous studies have confirmed HIV-specific IL-21 producing CD4+ T cells are induced during HIV infection and are associated with relative viral control [6,41]. Consistent with their findings, we found that the percentage and absolute counts of CD4+IL-21+T cells (1.27 ± 1.55%, 5.30 ± 7.81 cells/lL, respectively) in HIVinfected patients with relatively low plasma viral loads VLs (<20,000 copies/mL) were significantly higher than in those with plasma VLs > 20,000 copies/mL (0.55 ± 1.45%, 2.06 ± 5.50 cells/lL; p = 0.021, p = 0.017, Fig. 1A–C). We then focused on the relationship between CD4+IL-21+ T cells with Tregs in HIV infection, which has not been addressed. First, we divided the HIV-infected patients into two groups according to the presence of CD4+IL-21+ T cells (either detectable or undetectable numbers of cells). We found the percentage of CD4+CD25+ Tregs in patients with detectable CD4+IL-21+T cells (17.00 ± 4.86%) was significantly higher than those with undetectable CD4+IL-21+T cells (13.49 ± 2.63%, p = 0.020, Fig. 1D). We further studied the relationship of CD4+IL-21+T cells and Tregs in HIV infected patients. We found the percentage of CD4+IL-21+T cells was positively related to the percentage of CD4+CD25+T cells (r = 0.463, p = 0.006, Fig. 1E). The absolute counts of CD4+IL-21+T cells positively correlated with the absolute numbers of CD4+CD25+T cells and CD4+CD25+CD127low Tregs (r = 0.360, p = 0.037; r = 0.465, p = 0.001, respectively, Fig. 1F & G). These results indicate that elevated expression of CD4+IL-21+ T cells correlated with increased Tregs in HIV infection. 3.2. IL-21 increases survival but not proliferation of Tregs Next, we explored the mechanisms of the correlation of CD4+IL21 T cells with Tregs in HIV infection. We first studied the effects +
of IL-21 on the apoptosis and proliferation of CD4+CD25+CD127low Tregs in HIV-infected individuals and compared the effect of IL-21 with other cc cytokines including IL-2, IL-7, and IL-15. We found that treatment with IL-7 and IL-15 for three days significantly decreased apoptosis of Tregs (Annexin V+) from HIV-infected patients as compared with control (p < 0.001, p < 0.001, respectively, Fig. 2A & B). We found that IL-21 could also decrease apoptosis of Tregs, although to a lesser extent when compared with IL-7 and IL-15 treatment (p = 0.032, Fig. 2A & B). We further studied the effect of IL-21 on the proliferation of Tregs. We found that, unlike the other cc cytokines, five days of culture with IL-21 did not promote proliferation of Tregs from HIV-infected patients (p > 0.05, Fig. 2C & D); however, co-culture with IL-2, IL-7, and IL-15 increased the proliferation of Tregs (p = 0.041, p = 0.002, p = 0.006, respectively, Fig. 2C & D). Our results suggest that the positive relationship between CD4+IL-21+T cells and Tregs may be partially due to IL-21-mediated decrease in Treg apoptosis. 3.3. IL-21 increases CTLA-4 but not TGF-b expression in Tregs CTLA-4 and TGF-b are key factors that contribute to Tregs’ suppression activity. Next, we looked at whether IL-21 was able to modify the CTLA-4 expression and TGF-b secretion by Tregs from HIV-infected individuals. PBMCs were treated with the appropriate cc cytokines for 3 days for CTLA-4 detection or 5 days for TGF-b detection. We found that IL-21 treatment significantly increased CTLA-4 expression within CD4+CD25+CD127low Tregs in HIVinfected patients (p = 0.011, Fig. 3A & B), but can’t increase the secretion of TGF-b (p > 0.05, Fig. 3C & D). Unlike IL-21, other cc cytokines such as IL-2, IL-7, and IL-15 treatment did not increase CTLA-4 expression within Tregs (Fig. 3A & B). IL-2 and IL-15 treatment significantly enhanced Treg secretion of TGF-b (p = 0.007, p = 0.005, respectively, Fig. 3C & D). These results indicate that IL21 can increase the CTLA-4 expression in Tregs and thus may contributing to Treg’s suppression capacity. 3.4. Comparison of the effect of IL-21 on Tregs from HIV-infected patients versus normal controls A previous report showed that T cells became less sensitive to IL-2, IL-7, and IL-12 during HIV infection [42]. Whether IL-21 is able to affect Tregs that have been altered during HIV infection is unknown; thus, we studied the effect of IL-21 on Tregs from normal controls. We found that IL-21 induced the expression of CTLA-4 in Tregs from normal controls (p = 0.046), but did not affect the apoptosis, proliferation, or TGF-b secretion of Tregs from normal controls (Fig. 4A). We then compared the response of Tregs from HIV-infected patients to normal controls after IL-21 stimulation. The fold induction relative to the medium control was calculated as the ‘‘expression level of the parameters induced by IL-21/ expression level of the parameters induced by media alone”. We found there were no significant differences in the induction of apoptosis, proliferation, CTLA-4 expression and TGF-b secretion of Tregs between HIV-infected patients and normal controls (Fig. 4B). These results suggest that HIV infection did not reduce the Treg response to IL-21. 3.5. Suppressive ability of IL-21-treated Tregs from HIV-infected subjects Next, we studied whether IL-21 treatment could affect the suppressive activity of regulatory T cells. CD4+CD25+CD127low Tregs were either treated with IL-21 or left untreated and then cocultured with autologous CD8+T cells from HIV-infected patients. We found that CD4+CD25+CD127low Tregs significantly suppressed the CD3/CD28 stimulated IFN-r expression of CD8+ T cells
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Fig. 1. CD4+IL-21+T cells correlated with regulatory T cells. PBMCs from HIV-infected patients were co-cultured for 6 h with PMA and ionomycin; GolgiStop was added after the first 1 h of culture. (A) Gating strategy of CD4+IL-21+T cells. (B) The percent of CD4+IL-21+T cells in HIV-infected patients with different viral loads. (C) Absolute numbers of CD4+IL-21+T cells from HIV-infected patients with different viral loads. (D) The percent of CD4+CD25+ regulatory T cells in HIV-infected patients with different amounts of CD4+IL-21+T cells (either detectable or undetectable). (E) The relationship between the percentage of CD4+CD25+T cells and the percentage of CD4+IL-21+T cells. The relationship between the absolute count of CD4+IL-21+T cells with the absolute count of the CD4+CD25+ regulatory T cells (F) and the absolute number of CD4+CD25+CD127low regulatory T cells (G). The r and p values are indicated in the figures.
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Fig. 2. Apoptosis and proliferation of Tregs by IL-21, IL-2, IL-7, and IL-15. For apoptosis detection, PBMCs were cultured for 3 days with IL-21, IL-2, IL-7, and IL-15 or anti-CD3. For proliferation detection, PBMCs were labeled with CFSE and were cultured with the cytokines mentioned above for 5 days. Apoptosis (Annexin V+) and proliferation (CFSE+) of CD4+CD25+CD127low regulatory T cells were detected. Representative flow cytometry data (A) and statistical analysis (B) of the number of CD4+CD25+CD127low regulatory T cells undergoing apoptosis is shown. Figure (C and D) show representative flow cytometry and statistical data of the proliferation of Tregs. The p values are indicated in the figure.
(P = 0.024, Fig. 5A & B). Furthermore, pre-treatment with IL-21 increased the suppressive activity of Tregs, reflecting by the decreased expression of IFN-r by CD8+ T cells compared with untreated Tregs (P = 0.011, Fig. 5A & B). The results indicate that IL-21 could increase the suppressive activity of Tregs in HIV infection. 4. Discussion The multifaceted biological applications of IL-21 made it crucial in immunotherapy [22]. Recent studies highlighted the role of IL21 as a better adjuvant in eliciting antiviral immunity in anti-HIV vaccine strategies and immunotherapy [11–13,15–19]. In this study, we focused on the effect of IL-21 on Tregs, because the failure of IL-2 therapy during HIV infection has been linked to its ability to induce the proliferation of Tregs [23,24]. In addition, while Tregs express IL-21 receptor, conflicting results of the effect of IL-21 on Tregs have been reported [27–30]. Finally, the effect of IL-21 on Tregs in the context of HIV-infection has not yet been addressed.
We found a positive relationship between CD4+IL-21+T cells and Tregs in HIV-infected patients. Because CD4+IL-21+T cells are the predominant source of IL-21, we speculated that the relationship may due to the direct effects of IL-21 on Tregs. Our in vitro results indicated that IL-21 inhibited apoptosis of Tregs from HIV-infected patients. Our results are consistent with the reports by Liu et al. and Piao et al., which found that blockade of IL-21 caused a reduction in the number of Tregs [35,36]. These results suggest that IL21 may increase the numbers of Tregs during HIV infection through enhancing their survival. Whether IL-21 has a direct effect on Tregs’ function remains a matter of debate [32,36,43,44]. Some studies have reported that IL-21 reduced the susceptibility of conventional T cells to Treg-mediated suppression rather than inhibiting Tregs’ activity [32,43], while other studies revealed that IL-21 governs the expression of the master regulators of both the development and function of Tregs [36,44]. CTLA-4 and TGF-b are two well-known molecules important to Tregs’ contact-dependent or cytokine-mediated suppression [45]. Whether the expression of CTLA-4 and TGF-b in Tregs was affected by IL-21 in HIV-infection was unclear prior to this study. We found that IL-21 significantly
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Fig. 3. The CTLA-4 and TGF-b expression of Tregs by IL-21, IL-2, IL-7 and IL-15. PBMCs were cultured for 3 days (CTLA-4 expression) or 5 days (TGF-b expression, GolgiStop was added during the last 10 h) with IL-21, IL-2, IL-7, IL-15, or anti-CD3. Representative flow cytometry data (A) and statistical data (B) of CTLA-4 expression by CD4+CD25+CD127low regulatory T cells is shown. Figures (C and D) present representative flow cytometry and statistical data of the TGF-b expression within CD4+CD25+CD127low regulatory T cells.
Fig. 4. Comparison of the effects of IL-21 on Tregs from HIV-infected patients and normal controls (A) Apoptosis, proliferation, and CTLA-4 and TGF-b expression by CD4+CD25+CD127low regulatory T cells from normal controls. (B) Comparison of the fold induction relative to control, which was calculated as ‘‘expression level of the parameters induced by IL-21/expression level of the parameters induced by media alone” between HIV-infected patients and normal controls.
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Fig. 5. Suppressive ability of IL-21-treated Tregs from HIV-infected subjects. CD4+CD25+CD127low Tregs and CD8+T cells were sorted. Tregs were cultured with or without IL21 (100 ng/mL) for 24 h. CD8+ T cells were stimulated for two days with soluble anti-CD3/anti-CD28 antibodies and co-cultured with either IL-21-treated or untreated Tregs at a responder: suppressor ratio of 3:1. During the last 5 h of culture, Golgistop was added to the culture. At the end of culturing, cells were intra-cellularly stained with IFN-c. Representative flow cytometry data (A) and statistical data (B) of IFN-c expression by CD8+ T cells, CD8+ T cells with untreated Tregs and CD8+ T cells with IL-21 treated Tregs were shown.
induced expression of CTLA-4, but not the secretion of TGF-b by Tregs. CTLA-4 is critically required by Tregs to suppress immune responses by affecting the potency of antigen-presenting cells to activate other T cells [46]. Our in vitro experiments further confirmed that pre-treatment with IL-21 augmented the suppressive activity of Treg on CD8+ T cells, indicating that IL-21 enhanced Tregs’ inhibitory function during HIV infection. We also studied the effect of other cc cytokines, including IL-2, IL-7, and IL-15, on Tregs in the context of HIV infection. The IL-2 signaling pathway is likely to be critical to Treg function, either by influencing the development and homeostasis of Tregs or by inducing Treg inhibitory function. Our results showed that IL-2 promoted proliferation and TGF-b secretion by Tregs from HIV-infected patients, which was consistent with other published studies [24,47,48]. IL-7 and IL-15 are presumed to have multiple therapeutic applications in HIV infection [47,49], but their reported effects on Tregs have not been consistent. Our results indicated that IL-7 and IL-15 promoted proliferation and survival of Tregs from HIV-infected patients, but didn’t increase Treg effector molecule expression. Recent studies have reported the IL-7 enhanced Treg survival and expansion [50,51]; Wuest et al. also found that IL-15 could increase Treg proliferation, but not Treg inhibitory function [27]. Our results were inconsistent with their studies. However, Sereti et al. reported that Treg counts remained stable in IL-7-treated HIV patients [52]. Julie et al. indicated that IL-15 did not appreciably increase the frequency of Tregs in HIVinfected individuals after ART treatment [47]. We speculated that their result may be due to selection of patients that were treated with ART, while the patients enrolled in our study were treatment naïve. Because HIV can promote Treg survival and function [53], HIV viral production in treatment-naïve patients may cooperate with these cytokines to enhance the survival of Tregs. Compared with IL-2, IL-7, and IL-15, IL-21 promoted the survival of Tregs, although to a lesser extent, and induced expression of CTLA-4 in Tregs, indicating that IL-21 had a direct effect to Tregs in the context of HIV infection. It has been reported that T cells become less sensitive to IL-2, IL-7, and IL-12 during HIV infection, which contributes to HIV-related immune dysfunction [42]. We compared the responses of Tregs to IL-21 stimulation between HIV-infected patients and normal controls. There were no significant difference in the fold induction of Tregs to IL-21 between HIV-infected patients and normal controls. However, a tendency toward higher responses was observed in HIV-infected patients. It has been reported that HIV-1 binding enhances expression of Tregassociated functional markers sCTLA-4 and FoxP3 [53]. Moreover, HIV-1 binding extended the survival of CD4+CD25+ Treg cells [53]. We postulated that in HIV infection, cc cytokines might
cooperate with HIV to promote Tregs’ proliferation, survival and inhibition [53]. In conclusion, we found that IL-21 promotes Treg survival and CTLA-4 expression, but not the proliferation and secretion of TGF-b during HIV infection. Our results broaden the understanding of HIV pathogenesis and indicate that the induction of Tregs should not be overlooked during IL-21-related interventions for HIV infection. 5. Conclusion IL-21 enhances T and natural killer cells survival and antiviral functions without promoting T cell activation during HIV infection, which makes IL-21 a better adjuvant in anti-HIV immunotherapy. In this study, we found the percentage or absolute counts of CD4+IL-21+T cells were positively related to both the frequency and absolute numbers of CD4+CD25+ and CD4+CD25+CD127low Tregs. IL-21 could significantly reduce the apoptosis of Tregs, but did not promote their proliferation. IL-21 also enhanced CTLA-4 expression by Tregs, but could not induce TGF-b secretion by Tregs from HIV-infected patients. IL-21 could augment the suppressive capacity of HIV infected patients. Our results broadened the understanding of HIV pathogenesis. Because Tregs have a dominant role in immune regulation and play an important role in immune therapeutic efficacy, the induction of Tregs should not be overlooked in IL-21 related interventions for HIV infection. Acknowledgements The authors wish to express their gratitude for the generosity of patients who participated in this study. This study was supported by grants from the Mega-Projects of National Science Research for the 12th Five-Year Plan (2012ZX10001-006), National Natural Science Foundation of China (81371884), and the Fundamental and Clinical Medicine Closed Union Platform-Clinical Evaluation Technology Platform of Anti-HIV and Anti-neoplastic New Drugs (YDFZ-2013-5). References [1] M. Fevrier, K. Dorgham, A. Rebollo, CD4+ T cell depletion in human immunodeficiency virus (HIV) infection: role of apoptosis, Viruses 3 (5) (2011) 586–612. [2] J.C. Gaardbo et al., Incomplete immune recovery in HIV infection: mechanisms, relevance for clinical care, and possible solutions, Clin. Develop. Immunol. (2012). [3] A. Leone, L.J. Picker, D.L. Sodora, IL-2, IL-7 and IL-15 as immuno-modulators during SIV/HIV vaccination and treatment, Curr. HIV Res. 7 (1) (2009) 83–90.
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CD4+IL-21+T cells are correlated with regulatory T cells and IL-21 promotes regulatory T cells survival during HIV infection Zi-Ning Zhang a,b, Li-Xin Bai a, Ya-Jing Fu a,b, Yong-jun Jiang a,b, Hong Shang a,b,⇑ a Key Laboratory of AIDS Immunology of National Health and Family Planning Commission, Department of Laboratory Medicine, The First Affiliated Hospital, China Medical University, Shenyang, Liaoning 110001, China b Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, Zhejiang, China
a r t i c l e
i n f o
Article history: Received 19 August 2016 Received in revised form 20 December 2016 Accepted 20 December 2016
Keywords: HIV IL-21 Regulatory T cell
a b s t r a c t Introduction: IL-21 enhances T and natural killer cells survival and antiviral functions without promoting T cell activation during HIV infection, which makes it a better adjuvant in anti-HIV immunotherapy. Due to the pleiotropy and redundancy of cytokines, it is vital to have a comprehensive knowledge of the role of IL-21 in the regulation of immune responses. Regulatory T cells (Tregs) play an important role in immune regulation and are a determinant of immune therapeutic efficacy in certain circumstances. In this study, we explored the direct effect of IL-21 on Tregs during HIV infection, which has not been addressed before. Methods: Thirty-four HIV treatment-naïve patients were enrolled and the relationship between CD4+IL21+T cells and Tregs were studied. The effects of IL-21 on CD4+CD25+CD127low Tregs’ apoptosis, proliferation, and CTLA-4 and TGF-b expression in HIV-infected patients was investigated and compared with the effect of other common c-chain cytokines. Results: We found the percentage and absolute numbers of CD4+IL-21+T cells were positively related to the frequency or absolute numbers of CD4+CD25+ or CD4+CD25+CD127low Tregs. Compared with the media-alone control, IL-21, IL-7, and IL-15 could significantly reduce apoptosis of Tregs (p < 0.05). IL21 did not promote the proliferation of Tregs as compared with media alone, while IL-2, IL-7, and IL15 could significantly increase the proliferation of Tregs (p < 0.05). IL-21 enhanced CTLA-4 expression by Tregs (p < 0.05), but could not induce TGF-b secretion of Tregs from HIV infected patients. There were no significant differences of the fold induction of apoptosis, proliferation, or CTLA-4 and TGF-b expression by Tregs from HIV-infected patients and normal controls after IL-21 treatment. In vitro experiment showed that pretreatment with IL-21 significantly enhanced the suppressive effect of Tregs on CD8+ T cells’ IFN-c expression. Conclusion: We conclude that IL-21 promotes the survival and CTLA-4 expression of Tregs and enhanced the suppressive capacity of Tregs during HIV infection. These results broaden the understanding of HIV pathogenesis and provide critical information for HIV interventions. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction HIV infection is associated with a progressive decline in circulating CD4+ T cells and a concomitant loss of immune function [1,2]. Although antiretroviral therapy (ART) regimens have proven to be effective in controlling active HIV replication, complete recovery of CD4+ T-cell counts does not always occur, even among
⇑ Corresponding author at: Key Laboratory of AIDS Immunology of National Health and Family Planning Commission, Department of Laboratory Medicine, The First Affiliated Hospital, China Medical University, No. 155, Nanjingbei Street, Heping District, Shenyang, Liaoning Province 110001, China. E-mail address: [email protected] (H. Shang). http://dx.doi.org/10.1016/j.cyto.2016.12.012 1043-4666/Ó 2016 Elsevier Ltd. All rights reserved.
patients who display high levels of virologic control [3]. Furthermore, prolonged treatment comes with other problems such as drug resistance, side effects, and reduced adherence to the medication regimen. Accordingly, different adjuvant therapies, including immune modulation, are being tested in clinical trials or are under consideration in hopes of addressing these remaining challenges [4]. Common c-chain (cc) cytokines – including interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15 and IL-21 – regulate a variety of cellular responses such as proliferation, differentiation, and survival [5]. Of these, interleukin-21 was discovered relatively recently [6]. Mainly produced by CD4+ T cells [7–9], IL-21 affects an extremely broad set of target cells including T cells, B cells, NK cells, NKT cells,
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and DCs [10]. During HIV infection, IL-21 promotes the survival of CD4+ and CD8+ T cells as well as NK cells; IL-21 also enhances CD8+ T cell and NK cell antiviral function [11–13]. In addition, IL-21 directly suppresses HIV-1 replication through the promotion of microRNA-29 transcription [14]. Unlike other cc cytokines, IL-21 does not promote T cell activation or proliferation when it enhances the function of cytotoxic cells [11–13,15–19]. This prominent characteristic of IL-21 led to the hypothesis that IL-21, when used as an immunotherapeutic agent, was potentially capable of improving immune reconstruction without promoting immune activation and virus replication [11,12,15]. However, due to the pleiotropy and redundancy of cytokines [20], it is vital to have a comprehensive knowledge of the role of IL-21 in the regulation of immune responses. Regulatory T cells (Tregs) play a prominent role in chronic viral infections by limiting viral-specific immune responses and immune activation [21]. Tregs constitutively express the highaffinity IL-2Rabc complex, therefore IL-2 plays a very important role in regulating the Treg population [22]. Two large clinical trials have revealed that IL-2 administration in HIV-infected patients induced a newly expanded population of immunosuppressive Tregs [23,24]. The induction of Tregs may be the most plausible explanation why IL-2 administration plus ART yielded no clinical benefit in either study when compared to ART alone [23,24]. These results highlight the importance of considering the effects of Tregs in potential immune interventions. Tregs express the IL-21 receptor [25,26], indicating that IL-21 has the potential to affect Tregs. The reported direct effects of IL-21 on the expansion, survival, and function of Tregs have been inconsistent [27–30]. Some reports indicate that IL-21 renders T cells resistant to Tregmediated suppression, but has no direct effect on Treg viability, activation, or function [25,26,31–34]. By contrast, previous studies revealed that IL-21 was able to affect Tregs, either by decreasing the number and function of Tregs [35,36] or by increasing Tregs’ frequency and expression of Foxp3 [37–40] during viral infection or tumor development. The inconsistent results suggest the effect of IL-21 on Tregs varies depending on the disease, the anatomic site, or the physiological condition of the patient. To our knowledge, the effect of IL-21 on Tregs’ survival and function has not yet been clarified in HIV infection. In this study, we investigated the relationship between CD4+IL21+T cells and Tregs in HIV-infected patients. We then analyzed the effects of IL-21 on Treg apoptosis, proliferation, the expression of the key functional molecules, CTLA-4 and TGF-b and Treg’s suppression activity. We found a positive relationship between CD4+IL-21+T cells and Tregs in HIV-infected patients. Although IL-21 did not promote the proliferation of Tregs of HIV-infected patients in contrast to other cc cytokines, IL-21 could decrease Treg apoptosis and CTLA-4 expression and enhance Tregs’ suppressive activity in vitro. Our results expand the knowledge of the function of IL21 in HIV infection, which is helpful in the development of new immunotherapeutic strategies.
2. Materials and methods 2.1. Subjects Thirty-four treatment-naïve HIV-infected patients, including 33 males and 1 female (median age: 36 years) were enrolled in this study. The absolute CD4+ T cell numbers and Log10 viral loads of these patients were 405 ± 202 cells/lL and 4.11 ± 0.90 copies/mL (mean ± SD), respectively. Ethical approval for this study was obtained from the local ethical review committee and written informed consent for participation in the study was obtained from all patients.
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2.2. Flow cytometric analysis Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll centrifugation. The following monoclonal antibodies (mAbs) and reagents were used in this study: FITC-conjugated anti-CD8, APC-Cy7-conjugated anti-CD4, PerCP-conjugated anti-CD3, PE-Cy7-conjugated anti-CD25, PE-conjugated anti-TGF-b, APC-conjugated anti-CD127, PEconjugated anti-CTLA-4, PE-conjugated anti-IFN-c, PE-conjugated anti-Annexin V, 7-AAD and purified anti-CD3 mAb (BD Biosciences); PE-conjugated anti-IL-21 (eBioscience); Recombinant hIL-21 (Biosource), recombinant hIL-2, hIL-7, and hIL-15 (R&D Systems). For the detection of CD4+IL-21+T cells, 0.5 106 PBMCs were co-cultured for 6 h with phorbol 12-myristate 13-acetate (PMA, 50 ng/mL, Sigma-Aldrich, St Louis, MI, USA) and ionomycin (0.75 lg/mL, Sigma-Aldrich) at 37 °C, 5% CO2; GolgiStop (500 lg/ mL, BD Biosciences) was added after the first 1 h of culture. The cells were stained with anti-CD3-PerCP and anti-CD8-FITC followed by intracellular staining using Cytofix/Cytoperm Kit (BD Biosciences) with PE-conjugated IL-21. For the detection of Tregs, 1 106 cells were incubated with anti-CD3-PerCP, anti-CD4-APCCy7, anti-CD25-PE-Cy7, and anti-CD127-APC for 45 min on ice. The cells were washed, fixed in 1% paraformaldehyde in PBS prior to acquisition on LSR II (Becton Dickinson, San Jose, CA). CD4+CD25+ Tregs or CD4+CD25+CD127low Tregs were identified. The absolute numbers of Tregs were calculated by multiplying the frequencies of Tregs by the absolute numbers of CD4+T cells. Because PMA induced the down-regulation of the CD4 molecule, CD4+IL21+T cells in our study were identified as CD3+CD8-IL-21+ cells. The absolute numbers of CD4+IL-21+T cells were calculated by multiply the frequencies of CD4+IL-21+T by the absolute numbers of CD4+T cells. 2.3. Proliferation and apoptosis of Tregs For proliferation detection, PBMCs were labeled with CFSE at a concentration of 4 lM/5 106 cells for 10 min at 37 °C. After washing 1640 complete media supplemented with 10% FBS, cells were cultured in 96-well plates (200 lL) at 37 °C, 5% CO2 with IL21 (100 ng/mL), IL-2 (200 U/mL), IL-7 (50 ng/mL), IL-15 (50 ng/ mL) or anti-CD3 (0.5 lg/mL) for 5 days. After culture, the cells were washed once and then stained with anti-CD3-PerCP, anti-CD4APC-Cy7, anti-CD25-PE-Cy7, and anti-CD127-APC. For apoptosis detection, PBMCs were cultured for 3 days with IL-21 (100 ng/ mL), IL-2 (200 U/mL), IL-7 (50 ng/mL), IL-15 (50 ng/mL), and antiCD3 (0.5 lg/mL). After culture, the cells were stained with antiCD3-APC-Cy7, anti-CD4-FITC, anti-CD25-PE-Cy7, and CD127-APC for 30 min followed by staining with 5 lL 7-AAD and antiAnnexin V-PE for 15 min before data acquisition. Cells were acquired on LSR II (Becton Dickinson) and analyzed using FACSDiva software. 2.4. Intracellular detection of the expression of CTLA-4 and TGF-b within Tregs PBMCs were cultured with IL-21 (100 ng/mL), IL-2 (200 U/mL), IL-7 (50 ng/mL), IL-15 (50 ng/mL), and anti-CD3 (0.5 lg/mL). For the detection of CTLA-4 expression, the cells were stained with anti-CD3-PerCP, anti-CD4-APC-Cy7, anti-CD25-PE-Cy7, and CD127-APC after 3 days of culture followed by intracellular staining with anti-CTLA-4-PE. For TGF-b detection, PBMCs were cultured for 5 days with the cytokines mentioned above and GolgiStop was added during the last 10 h. The cells were then stained with anti-CD3-APC-Cy7, anti-CD4-FITC, anti-CD25PE-Cy7, and CD127-APC followed by intracellular anti-PE-TGF-b
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staining. Cells were fixed in 1% paraformaldehyde in PBS and analyzed using a BD LSRII and FACSDiva software. 2.5. In vitro Treg-cell-suppression activity Freshly isolated PBMCs from HIV infected patients were stained with anti-CD3-PerCP, anti-CD4-APC-Cy7, anti-CD25-PE-Cy7, antiCD127-APC. To examine whether the pretreatment of IL-21 altered the suppressive capability of Tregs, CD4+CD25+CD127low Tregs or CD8+ (CD3+CD4 ) T cells were sorted with a FACSAria flow cytometer (BD Biosciences). CD4+CD25+CD127low Tregs were cultured with or without IL-21 (100 ng/mL) for 24 h. CD8+T cells were rested, incubated in fresh medium for 24 h. Then CD8+T cells were stimulated for two days with soluble anti-CD3/anti-CD28 antibodies (2.5 lg/mL; BD Biosciences) and co-cultured with either IL-21treated or untreated Tregs at a responder: suppressor ratio of 3:1. During the last 5 h of culture, Golgistop was added to the culture. At the end of culturing, cells were intra-cellularly stained with PEconjugated anti-IFN-c and analyzed on an LSRII flow cytometer. 2.6. Statistical analysis Analyses were carried out using SPSS 17.0 software. The MannWhitney U test was employed to compare the expression levels of Tregs and CD4+IL-21+ T cells of HIV infected patients with different viral loads. Correlation analysis was performed using the Spearman’s rank correlation test. The Wilcoxon matched-pairs signedranks test was used to compare the parameters before and after cytokine stimulation. P values less than 0.05 were considered to be statistically significant. 3. Results 3.1. Higher expression level of CD4+IL-21+T cells correlated with lower viral loads and higher numbers of Tregs Previous studies have confirmed HIV-specific IL-21 producing CD4+ T cells are induced during HIV infection and are associated with relative viral control [6,41]. Consistent with their findings, we found that the percentage and absolute counts of CD4+IL-21+T cells (1.27 ± 1.55%, 5.30 ± 7.81 cells/lL, respectively) in HIVinfected patients with relatively low plasma viral loads VLs (<20,000 copies/mL) were significantly higher than in those with plasma VLs > 20,000 copies/mL (0.55 ± 1.45%, 2.06 ± 5.50 cells/lL; p = 0.021, p = 0.017, Fig. 1A–C). We then focused on the relationship between CD4+IL-21+ T cells with Tregs in HIV infection, which has not been addressed. First, we divided the HIV-infected patients into two groups according to the presence of CD4+IL-21+ T cells (either detectable or undetectable numbers of cells). We found the percentage of CD4+CD25+ Tregs in patients with detectable CD4+IL-21+T cells (17.00 ± 4.86%) was significantly higher than those with undetectable CD4+IL-21+T cells (13.49 ± 2.63%, p = 0.020, Fig. 1D). We further studied the relationship of CD4+IL-21+T cells and Tregs in HIV infected patients. We found the percentage of CD4+IL-21+T cells was positively related to the percentage of CD4+CD25+T cells (r = 0.463, p = 0.006, Fig. 1E). The absolute counts of CD4+IL-21+T cells positively correlated with the absolute numbers of CD4+CD25+T cells and CD4+CD25+CD127low Tregs (r = 0.360, p = 0.037; r = 0.465, p = 0.001, respectively, Fig. 1F & G). These results indicate that elevated expression of CD4+IL-21+ T cells correlated with increased Tregs in HIV infection. 3.2. IL-21 increases survival but not proliferation of Tregs Next, we explored the mechanisms of the correlation of CD4+IL21 T cells with Tregs in HIV infection. We first studied the effects +
of IL-21 on the apoptosis and proliferation of CD4+CD25+CD127low Tregs in HIV-infected individuals and compared the effect of IL-21 with other cc cytokines including IL-2, IL-7, and IL-15. We found that treatment with IL-7 and IL-15 for three days significantly decreased apoptosis of Tregs (Annexin V+) from HIV-infected patients as compared with control (p < 0.001, p < 0.001, respectively, Fig. 2A & B). We found that IL-21 could also decrease apoptosis of Tregs, although to a lesser extent when compared with IL-7 and IL-15 treatment (p = 0.032, Fig. 2A & B). We further studied the effect of IL-21 on the proliferation of Tregs. We found that, unlike the other cc cytokines, five days of culture with IL-21 did not promote proliferation of Tregs from HIV-infected patients (p > 0.05, Fig. 2C & D); however, co-culture with IL-2, IL-7, and IL-15 increased the proliferation of Tregs (p = 0.041, p = 0.002, p = 0.006, respectively, Fig. 2C & D). Our results suggest that the positive relationship between CD4+IL-21+T cells and Tregs may be partially due to IL-21-mediated decrease in Treg apoptosis. 3.3. IL-21 increases CTLA-4 but not TGF-b expression in Tregs CTLA-4 and TGF-b are key factors that contribute to Tregs’ suppression activity. Next, we looked at whether IL-21 was able to modify the CTLA-4 expression and TGF-b secretion by Tregs from HIV-infected individuals. PBMCs were treated with the appropriate cc cytokines for 3 days for CTLA-4 detection or 5 days for TGF-b detection. We found that IL-21 treatment significantly increased CTLA-4 expression within CD4+CD25+CD127low Tregs in HIVinfected patients (p = 0.011, Fig. 3A & B), but can’t increase the secretion of TGF-b (p > 0.05, Fig. 3C & D). Unlike IL-21, other cc cytokines such as IL-2, IL-7, and IL-15 treatment did not increase CTLA-4 expression within Tregs (Fig. 3A & B). IL-2 and IL-15 treatment significantly enhanced Treg secretion of TGF-b (p = 0.007, p = 0.005, respectively, Fig. 3C & D). These results indicate that IL21 can increase the CTLA-4 expression in Tregs and thus may contributing to Treg’s suppression capacity. 3.4. Comparison of the effect of IL-21 on Tregs from HIV-infected patients versus normal controls A previous report showed that T cells became less sensitive to IL-2, IL-7, and IL-12 during HIV infection [42]. Whether IL-21 is able to affect Tregs that have been altered during HIV infection is unknown; thus, we studied the effect of IL-21 on Tregs from normal controls. We found that IL-21 induced the expression of CTLA-4 in Tregs from normal controls (p = 0.046), but did not affect the apoptosis, proliferation, or TGF-b secretion of Tregs from normal controls (Fig. 4A). We then compared the response of Tregs from HIV-infected patients to normal controls after IL-21 stimulation. The fold induction relative to the medium control was calculated as the ‘‘expression level of the parameters induced by IL-21/ expression level of the parameters induced by media alone”. We found there were no significant differences in the induction of apoptosis, proliferation, CTLA-4 expression and TGF-b secretion of Tregs between HIV-infected patients and normal controls (Fig. 4B). These results suggest that HIV infection did not reduce the Treg response to IL-21. 3.5. Suppressive ability of IL-21-treated Tregs from HIV-infected subjects Next, we studied whether IL-21 treatment could affect the suppressive activity of regulatory T cells. CD4+CD25+CD127low Tregs were either treated with IL-21 or left untreated and then cocultured with autologous CD8+T cells from HIV-infected patients. We found that CD4+CD25+CD127low Tregs significantly suppressed the CD3/CD28 stimulated IFN-r expression of CD8+ T cells
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Fig. 1. CD4+IL-21+T cells correlated with regulatory T cells. PBMCs from HIV-infected patients were co-cultured for 6 h with PMA and ionomycin; GolgiStop was added after the first 1 h of culture. (A) Gating strategy of CD4+IL-21+T cells. (B) The percent of CD4+IL-21+T cells in HIV-infected patients with different viral loads. (C) Absolute numbers of CD4+IL-21+T cells from HIV-infected patients with different viral loads. (D) The percent of CD4+CD25+ regulatory T cells in HIV-infected patients with different amounts of CD4+IL-21+T cells (either detectable or undetectable). (E) The relationship between the percentage of CD4+CD25+T cells and the percentage of CD4+IL-21+T cells. The relationship between the absolute count of CD4+IL-21+T cells with the absolute count of the CD4+CD25+ regulatory T cells (F) and the absolute number of CD4+CD25+CD127low regulatory T cells (G). The r and p values are indicated in the figures.
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Fig. 2. Apoptosis and proliferation of Tregs by IL-21, IL-2, IL-7, and IL-15. For apoptosis detection, PBMCs were cultured for 3 days with IL-21, IL-2, IL-7, and IL-15 or anti-CD3. For proliferation detection, PBMCs were labeled with CFSE and were cultured with the cytokines mentioned above for 5 days. Apoptosis (Annexin V+) and proliferation (CFSE+) of CD4+CD25+CD127low regulatory T cells were detected. Representative flow cytometry data (A) and statistical analysis (B) of the number of CD4+CD25+CD127low regulatory T cells undergoing apoptosis is shown. Figure (C and D) show representative flow cytometry and statistical data of the proliferation of Tregs. The p values are indicated in the figure.
(P = 0.024, Fig. 5A & B). Furthermore, pre-treatment with IL-21 increased the suppressive activity of Tregs, reflecting by the decreased expression of IFN-r by CD8+ T cells compared with untreated Tregs (P = 0.011, Fig. 5A & B). The results indicate that IL-21 could increase the suppressive activity of Tregs in HIV infection. 4. Discussion The multifaceted biological applications of IL-21 made it crucial in immunotherapy [22]. Recent studies highlighted the role of IL21 as a better adjuvant in eliciting antiviral immunity in anti-HIV vaccine strategies and immunotherapy [11–13,15–19]. In this study, we focused on the effect of IL-21 on Tregs, because the failure of IL-2 therapy during HIV infection has been linked to its ability to induce the proliferation of Tregs [23,24]. In addition, while Tregs express IL-21 receptor, conflicting results of the effect of IL-21 on Tregs have been reported [27–30]. Finally, the effect of IL-21 on Tregs in the context of HIV-infection has not yet been addressed.
We found a positive relationship between CD4+IL-21+T cells and Tregs in HIV-infected patients. Because CD4+IL-21+T cells are the predominant source of IL-21, we speculated that the relationship may due to the direct effects of IL-21 on Tregs. Our in vitro results indicated that IL-21 inhibited apoptosis of Tregs from HIV-infected patients. Our results are consistent with the reports by Liu et al. and Piao et al., which found that blockade of IL-21 caused a reduction in the number of Tregs [35,36]. These results suggest that IL21 may increase the numbers of Tregs during HIV infection through enhancing their survival. Whether IL-21 has a direct effect on Tregs’ function remains a matter of debate [32,36,43,44]. Some studies have reported that IL-21 reduced the susceptibility of conventional T cells to Treg-mediated suppression rather than inhibiting Tregs’ activity [32,43], while other studies revealed that IL-21 governs the expression of the master regulators of both the development and function of Tregs [36,44]. CTLA-4 and TGF-b are two well-known molecules important to Tregs’ contact-dependent or cytokine-mediated suppression [45]. Whether the expression of CTLA-4 and TGF-b in Tregs was affected by IL-21 in HIV-infection was unclear prior to this study. We found that IL-21 significantly
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Fig. 3. The CTLA-4 and TGF-b expression of Tregs by IL-21, IL-2, IL-7 and IL-15. PBMCs were cultured for 3 days (CTLA-4 expression) or 5 days (TGF-b expression, GolgiStop was added during the last 10 h) with IL-21, IL-2, IL-7, IL-15, or anti-CD3. Representative flow cytometry data (A) and statistical data (B) of CTLA-4 expression by CD4+CD25+CD127low regulatory T cells is shown. Figures (C and D) present representative flow cytometry and statistical data of the TGF-b expression within CD4+CD25+CD127low regulatory T cells.
Fig. 4. Comparison of the effects of IL-21 on Tregs from HIV-infected patients and normal controls (A) Apoptosis, proliferation, and CTLA-4 and TGF-b expression by CD4+CD25+CD127low regulatory T cells from normal controls. (B) Comparison of the fold induction relative to control, which was calculated as ‘‘expression level of the parameters induced by IL-21/expression level of the parameters induced by media alone” between HIV-infected patients and normal controls.
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Fig. 5. Suppressive ability of IL-21-treated Tregs from HIV-infected subjects. CD4+CD25+CD127low Tregs and CD8+T cells were sorted. Tregs were cultured with or without IL21 (100 ng/mL) for 24 h. CD8+ T cells were stimulated for two days with soluble anti-CD3/anti-CD28 antibodies and co-cultured with either IL-21-treated or untreated Tregs at a responder: suppressor ratio of 3:1. During the last 5 h of culture, Golgistop was added to the culture. At the end of culturing, cells were intra-cellularly stained with IFN-c. Representative flow cytometry data (A) and statistical data (B) of IFN-c expression by CD8+ T cells, CD8+ T cells with untreated Tregs and CD8+ T cells with IL-21 treated Tregs were shown.
induced expression of CTLA-4, but not the secretion of TGF-b by Tregs. CTLA-4 is critically required by Tregs to suppress immune responses by affecting the potency of antigen-presenting cells to activate other T cells [46]. Our in vitro experiments further confirmed that pre-treatment with IL-21 augmented the suppressive activity of Treg on CD8+ T cells, indicating that IL-21 enhanced Tregs’ inhibitory function during HIV infection. We also studied the effect of other cc cytokines, including IL-2, IL-7, and IL-15, on Tregs in the context of HIV infection. The IL-2 signaling pathway is likely to be critical to Treg function, either by influencing the development and homeostasis of Tregs or by inducing Treg inhibitory function. Our results showed that IL-2 promoted proliferation and TGF-b secretion by Tregs from HIV-infected patients, which was consistent with other published studies [24,47,48]. IL-7 and IL-15 are presumed to have multiple therapeutic applications in HIV infection [47,49], but their reported effects on Tregs have not been consistent. Our results indicated that IL-7 and IL-15 promoted proliferation and survival of Tregs from HIV-infected patients, but didn’t increase Treg effector molecule expression. Recent studies have reported the IL-7 enhanced Treg survival and expansion [50,51]; Wuest et al. also found that IL-15 could increase Treg proliferation, but not Treg inhibitory function [27]. Our results were inconsistent with their studies. However, Sereti et al. reported that Treg counts remained stable in IL-7-treated HIV patients [52]. Julie et al. indicated that IL-15 did not appreciably increase the frequency of Tregs in HIVinfected individuals after ART treatment [47]. We speculated that their result may be due to selection of patients that were treated with ART, while the patients enrolled in our study were treatment naïve. Because HIV can promote Treg survival and function [53], HIV viral production in treatment-naïve patients may cooperate with these cytokines to enhance the survival of Tregs. Compared with IL-2, IL-7, and IL-15, IL-21 promoted the survival of Tregs, although to a lesser extent, and induced expression of CTLA-4 in Tregs, indicating that IL-21 had a direct effect to Tregs in the context of HIV infection. It has been reported that T cells become less sensitive to IL-2, IL-7, and IL-12 during HIV infection, which contributes to HIV-related immune dysfunction [42]. We compared the responses of Tregs to IL-21 stimulation between HIV-infected patients and normal controls. There were no significant difference in the fold induction of Tregs to IL-21 between HIV-infected patients and normal controls. However, a tendency toward higher responses was observed in HIV-infected patients. It has been reported that HIV-1 binding enhances expression of Tregassociated functional markers sCTLA-4 and FoxP3 [53]. Moreover, HIV-1 binding extended the survival of CD4+CD25+ Treg cells [53]. We postulated that in HIV infection, cc cytokines might
cooperate with HIV to promote Tregs’ proliferation, survival and inhibition [53]. In conclusion, we found that IL-21 promotes Treg survival and CTLA-4 expression, but not the proliferation and secretion of TGF-b during HIV infection. Our results broaden the understanding of HIV pathogenesis and indicate that the induction of Tregs should not be overlooked during IL-21-related interventions for HIV infection. 5. Conclusion IL-21 enhances T and natural killer cells survival and antiviral functions without promoting T cell activation during HIV infection, which makes IL-21 a better adjuvant in anti-HIV immunotherapy. In this study, we found the percentage or absolute counts of CD4+IL-21+T cells were positively related to both the frequency and absolute numbers of CD4+CD25+ and CD4+CD25+CD127low Tregs. IL-21 could significantly reduce the apoptosis of Tregs, but did not promote their proliferation. IL-21 also enhanced CTLA-4 expression by Tregs, but could not induce TGF-b secretion by Tregs from HIV-infected patients. IL-21 could augment the suppressive capacity of HIV infected patients. Our results broadened the understanding of HIV pathogenesis. Because Tregs have a dominant role in immune regulation and play an important role in immune therapeutic efficacy, the induction of Tregs should not be overlooked in IL-21 related interventions for HIV infection. Acknowledgements The authors wish to express their gratitude for the generosity of patients who participated in this study. This study was supported by grants from the Mega-Projects of National Science Research for the 12th Five-Year Plan (2012ZX10001-006), National Natural Science Foundation of China (81371884), and the Fundamental and Clinical Medicine Closed Union Platform-Clinical Evaluation Technology Platform of Anti-HIV and Anti-neoplastic New Drugs (YDFZ-2013-5). References [1] M. Fevrier, K. Dorgham, A. Rebollo, CD4+ T cell depletion in human immunodeficiency virus (HIV) infection: role of apoptosis, Viruses 3 (5) (2011) 586–612. [2] J.C. Gaardbo et al., Incomplete immune recovery in HIV infection: mechanisms, relevance for clinical care, and possible solutions, Clin. Develop. Immunol. (2012). [3] A. Leone, L.J. Picker, D.L. Sodora, IL-2, IL-7 and IL-15 as immuno-modulators during SIV/HIV vaccination and treatment, Curr. HIV Res. 7 (1) (2009) 83–90.
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