Dampening of IL-2 Function in Infants With Severe Respiratory Syncytial Virus Disease

Dampening of IL-2 Function in Infants With Severe Respiratory Syncytial Virus Disease Abstract Background FOXP3+ regulatory T cells (Tregs) restrain the destructive potential of the immune system. We have previously reported a pronounced reduction in circulating Tregs in infants with severe respiratory syncytial virus (RSV) disease. Because interleukin-2 (IL-2) is critical for Treg growth, survival, and activity, we here analyzed IL-2 production and function in RSV-infected infants. Methods Phenotype, proliferation, IL-2 production, and IL-2 signaling in CD4+ T cells were analyzed by flow cytometry. Serum soluble CD25 levels were quantified by ELISA. Results CD4+ T cells from RSV-infected infants produced lower amounts of IL-2 and showed a reduced proliferative response compared with healthy infants. IL-2 increased CD4+ T-cell proliferation and FOXP3 expression in both healthy and RSV-infected infants. However, although IL-2 induced a similar pattern of STAT5 phosphorylation, the proliferative response of CD4+ T cells and the expression of FOXP3+ remained significantly lower in RSV-infected infants. Interestingly, we found a negative correlation between disease severity and both the production of IL-2 by CD4+ T cells and the ability of exogenous IL-2 to restore the pool of FOXP3+CD4+ T cells. Conclusions A reduced ability to produce IL-2 and a limited response to this cytokine may affect the function of CD4+ T cells in RSV-infected infants. respiratory syncytial virus, infants, severe bronchiolitis, CD4+ T cells, FOXP3, IL-2, CD25 Respiratory syncytial virus (RSV) infection is the commonest cause of bronchiolitis and hospitalization in infants, accounting for about 100000–200000 deaths annually [1]. Pathogenesis of RSV infection involves both cytopathic effects induced by the virus and an exacerbated inflammatory response, which explain, at least partially, damage to the airway [2, 3]. FOXP3+ regulatory T cells (Tregs) not only prevent autoimmune diseases and maintain immune homeostasis, but also regulate the immune response in the course of infectious diseases, controlling tissue injury [4, 5]. In fact, experimental models of RSV infection have shown that depletion of Tregs leads to more severe disease [6–8]. The role of Tregs during human RSV infection has not yet been clarified. However, the Treg cell compartment undergoes important changes in the course of severe RSV infection. In this regard, we have previously reported that severe RSV infection in infants is associated with a marked reduction in the frequency of peripheral blood Tregs [9]. Most studies analyzing the role of Tregs have focused on chronic conditions, such as autoimmunity, cancer, and persistent infectious diseases [10–13]. By contrast, very little is known about the function of Tregs in acute viral infections. Our previous findings showing the depletion of Tregs in RSV-infected infants contrast with the increased frequencies of Tregs found in other acute viral diseases such as dengue [14] and influenza A virus (IAV) [15], suggesting a particular signature of RSV infection. Because Treg survival and function are dependent on interleukin-2 (IL-2) receptor signaling [16], we speculated that severe RSV disease could be associated with a dysregulation of the IL-2/IL-2 receptor system. This hypothesis may also explain another key feature of RSV infection, that is its inability to induce long-lasting immunity, a response strongly dependent on IL-2 function [17]. Here, we found that the production and function of IL-2 are compromised in infants with severe RSV infection. CD4+ T cells from RSV-infected patients produced lower amounts of IL-2 and showed a limited proliferative response compared with cells from healthy donors. We also found that the addition of IL-2 failed to fully restore IL-2–dependent functions such as FOXP3 expression and CD4+ T-cell proliferation. Interestingly, a negative correlation was observed between disease severity and both IL-2 production by CD4+ T cells and the ability of IL-2 to increase the pool of FOXP3+CD4+ T cells. MATERIALS AND METHODS Ethics Statement Our study was approved by the Ethics Committee of the Hospital de Pediatría Pedro de Elizalde, Buenos Aires, Argentina, in accordance with the Declaration of Helsinki (Fortaleza 2013). Written informed consent was obtained from all donors or legal guardians. Study Population We recruited 85 infants <18 months old, hospitalized at the Hospital de Pediatría Pedro de Elizalde (Buenos Aires, Argentina), with a severe episode of RSV bronchiolitis during the 2016–2017 respiratory seasons. We excluded children with history of prematurity, immunodeficiency, congenital heart disease, and chronic conditions. All of the infants required hospitalization. RSV infection was confirmed by direct immunofluorescence of nasopharyngeal aspirates. Disease severity was assessed by applying a clinical disease severity score (CDSS) based on the modified Tal score, which classifies patients as having mild (0–4), moderate (5–8), or severe (9–12) RSV bronchiolitis at the time of sampling [18, 19]. The control group consisted of 40 infants admitted to the hospital for scheduled surgery (healthy donors, HD). They had no identifiable airway infections for a 4-week period before the study. Characteristics of the patients are shown in Table 1. Table 1. Baseline Characteristics of Children Infected With Respiratory Syncytial Virus   RSV Patients  Healthy Children    N = 85  N = 40  Demographic characteristics  Age, mo, mean ± SD  7.5 ± 7.1  12.6 ± 3.8  Male sex, n (%)  57 (67%)  22 (55%)  Disease severity  CDSS, n (%)a   0–6  0 (0%)  NA   7–8  48 (57%)  NA   9–12  37 (43%)  NA  O2 requirement, n (%)  85 (100%)  NA  Hospital stay, days, mean ± SD  7.3 ± 0.2  NA  PICU admission, n (%)  3 (3.6%)  NA  Laboratory parameters  WBC, counts/mm3, mean ± SD  10559 ± 4021  8952 ± 1250  Lymphocytes, %, mean ± SD  37.4 ± 15.4  53.1 ± 4.0  CD4+, %, mean ± SDb  28.0 ± 11.0  32.3 ± 5.7  CD8+, %, mean ± SDb  12.4 ± 6.8  20.0 ± 7.5  CD19+, %, mean ± SDb  36.4 ± 10.2  16.5 ± 7.2    RSV Patients  Healthy Children    N = 85  N = 40  Demographic characteristics  Age, mo, mean ± SD  7.5 ± 7.1  12.6 ± 3.8  Male sex, n (%)  57 (67%)  22 (55%)  Disease severity  CDSS, n (%)a   0–6  0 (0%)  NA   7–8  48 (57%)  NA   9–12  37 (43%)  NA  O2 requirement, n (%)  85 (100%)  NA  Hospital stay, days, mean ± SD  7.3 ± 0.2  NA  PICU admission, n (%)  3 (3.6%)  NA  Laboratory parameters  WBC, counts/mm3, mean ± SD  10559 ± 4021  8952 ± 1250  Lymphocytes, %, mean ± SD  37.4 ± 15.4  53.1 ± 4.0  CD4+, %, mean ± SDb  28.0 ± 11.0  32.3 ± 5.7  CD8+, %, mean ± SDb  12.4 ± 6.8  20.0 ± 7.5  CD19+, %, mean ± SDb  36.4 ± 10.2  16.5 ± 7.2  Abbreviations: CDSS, clinical disease severity score; NA, not applicable; PICU, pediatric intensive care unit; RSV, respiratory syncytial virus; WBC, white blood cells. aCDSS was calculated using the modified Tal score. bPercentages of cell subsets in the gate of lymphocytes (n = 12 in each group). View Large Table 1. Baseline Characteristics of Children Infected With Respiratory Syncytial Virus   RSV Patients  Healthy Children    N = 85  N = 40  Demographic characteristics  Age, mo, mean ± SD  7.5 ± 7.1  12.6 ± 3.8  Male sex, n (%)  57 (67%)  22 (55%)  Disease severity  CDSS, n (%)a   0–6  0 (0%)  NA   7–8  48 (57%)  NA   9–12  37 (43%)  NA  O2 requirement, n (%)  85 (100%)  NA  Hospital stay, days, mean ± SD  7.3 ± 0.2  NA  PICU admission, n (%)  3 (3.6%)  NA  Laboratory parameters  WBC, counts/mm3, mean ± SD  10559 ± 4021  8952 ± 1250  Lymphocytes, %, mean ± SD  37.4 ± 15.4  53.1 ± 4.0  CD4+, %, mean ± SDb  28.0 ± 11.0  32.3 ± 5.7  CD8+, %, mean ± SDb  12.4 ± 6.8  20.0 ± 7.5  CD19+, %, mean ± SDb  36.4 ± 10.2  16.5 ± 7.2    RSV Patients  Healthy Children    N = 85  N = 40  Demographic characteristics  Age, mo, mean ± SD  7.5 ± 7.1  12.6 ± 3.8  Male sex, n (%)  57 (67%)  22 (55%)  Disease severity  CDSS, n (%)a   0–6  0 (0%)  NA   7–8  48 (57%)  NA   9–12  37 (43%)  NA  O2 requirement, n (%)  85 (100%)  NA  Hospital stay, days, mean ± SD  7.3 ± 0.2  NA  PICU admission, n (%)  3 (3.6%)  NA  Laboratory parameters  WBC, counts/mm3, mean ± SD  10559 ± 4021  8952 ± 1250  Lymphocytes, %, mean ± SD  37.4 ± 15.4  53.1 ± 4.0  CD4+, %, mean ± SDb  28.0 ± 11.0  32.3 ± 5.7  CD8+, %, mean ± SDb  12.4 ± 6.8  20.0 ± 7.5  CD19+, %, mean ± SDb  36.4 ± 10.2  16.5 ± 7.2  Abbreviations: CDSS, clinical disease severity score; NA, not applicable; PICU, pediatric intensive care unit; RSV, respiratory syncytial virus; WBC, white blood cells. aCDSS was calculated using the modified Tal score. bPercentages of cell subsets in the gate of lymphocytes (n = 12 in each group). View Large Isolation of Peripheral Blood Mononuclear Cells Peripheral blood mononuclear cells (PBMCs) were obtained from blood samples (0.3–0.4 mL) from RSV-infected infants or HD, by Ficoll-Hypaque gradient centrifugation (GE Healthcare Life Sciences, Uppsala, Sweden). PBMCs were washed twice and suspended in complete culture medium: RPMI 1640 (Hyclone, Logan, Utah) supplemented with 10% heat-inactivated fetal calf serum (FCS; Natocor, Córdoba, Argentina), 200 mM l-glutamine, and 50 μg/mL gentamicin (Sigma-Aldrich, St. Louis, MO). Flow Cytometry Labeled monoclonal antibodies (mAb) directed to CD3, CD4, CD8, CD14, CD19, CD25, CD39, FOXP3, IL-2, STAT5, and Ki-67 were obtained from BD Biosciences (San José, CA). In all cases, isotype-matched mAb were used as controls. For intracellular IL-2 detection, cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 µg/mL ionomycin (Sigma-Aldrich) for 5 hours in the presence of monensin (Golgi-Stop, BD Biosciences), and stained with anti-CD4 and anti-IL-2 mAbs, after cell fixation and permeabilization (BD Biosciences). To analyze STAT5 phosphorylation in CD4+ T cells, PBMCs were treated, or not, with IL-2 (20 ng/mL; Peprotech, Rocky Hill, NJ) for 30 minutes at 37°C. Cells were then stained with a mAb directed to phosphorylated STAT5 after cell fixation and permeabilization. Data were acquired using a FACS Canto (Becton Dickinson) and analyzed with FlowJo software. Statistical analyses were based on at least 100000 events gated on the population of interest. Culture of PBMCs in the Presence of IL-2 To test the proliferative response of CD4+ T cells, PBMCs from RSV or HD (1 × 106 cells/mL) were activated with phytohemagglutinin (PHA, 4 µg/mL, Sigma-Aldrich) and cultured in the absence or presence of IL-2 (20 ng/mL) for 3 days. Then, the expression of the proliferation marker Ki-67 was assessed by flow cytometry. The concentration of IL-2 used in these assays was selected on the basis of preliminary experiments performed with PBMCs from healthy children. These experiments showed that the proliferation of PHA-activated CD4+ T cells reached similar values with either 20 or 100 ng/mL of IL-2. To test the ability of IL-2 to increase the expression of FOXP3, resting PBMCs (1 × 106 cells/mL) were cultured without or with IL-2 (20 ng/mL) for 24 hours. In some experiments, resting PBMCs were cultured with IL-2 alone or IL-2 plus recombinant soluble CD25 (sCD25, 200 ng/mL, Peprotech), for 30 minutes or 24 hours, and phosphorylation of STAT5 and FOXP3 expression was evaluated. Culture of PBMCs in the Presence of Serum From RSV-Infected or Healthy Children Serum was obtained from HD and RSV-infected children, while PBMCs were obtained from unrelated healthy adult donors. Briefly, PBMCs (1 × 106/mL) were incubated for 24 hours in complete culture medium supplemented with heat-inactivated serum from healthy or RSV-infected children (final dilution 1:10), in the absence or presence of IL-2 (20 ng/mL). Then, the frequency of FOXP3+CD4+ T cells was analyzed. Flow cytometry crossmatch was used to detect the presence or lack of IgG antibodies on the surface of CD4+ lymphocytes, after incubation of PBMCs with serum samples. Cell-associated IgG antibodies were detected in no samples. Cytometric Bead Array PHA (4 µg/mL)-activated PBMCs (1 × 106/mL) were cultured for 3 days and the supernatants obtained were frozen until use. Measurement of IL-2, IL-4, IL-6, IL-10, IL-17, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ) was carried out according to the manufacturer’s protocol (BD Biosciences). Data were acquired using a FACS Canto. ELISA Serum levels of sCD25 were determined by enzyme-linked immunosorbent assay (ELISA; Thermo Fisher, Fredrick, MD). Statistical Analysis Statistical analyses were performed using GraphPad Prism software. Data normality was evaluated by Shapiro Wilk test. Groups were compared using the Χ2 test, or Wilcoxon signed rank test, and/or Mann-Whitney test as appropriate. Correlations were assessed using Spearman correlation test. A P value < .05 was considered statistically significant. RESULTS Clinical Characteristics of Study Population The characteristics of RSV-infected and healthy infants enrolled in this study are summarized in Table 1. The CDSS of all admitted patients was equal to or higher than 7. All admitted patients needed O2 supplementation. Those admitted to the intensive care unit (n = 3, 3.6%) required invasive mechanical ventilation for a median of 4.5 days (range 3.6–5.9 days) and received broad-spectrum antibiotic therapy. RSV-infected children showed a higher frequency of B cells (CD19+ cells) and a lower frequency of CD8+ T cells compared with HD. The frequency of CD4+ T cells was similar in both groups. CD4+ T Cells From RSV-Infected Infants Produce Low Amounts of IL-2 and Show a Decreased Proliferative Response We first analyzed whether the production of IL-2, which is mainly mediated by conventional CD4+ T cells [20], is reduced during severe RSV infection. PBMCs from RSV-infected infants and HD were stimulated for 5 hours with PMA/ionomycin in the presence of monensin, and IL-2 production was analyzed by intracellular staining and flow cytometry in gated CD4+ T cells. We observed a similar frequency of IL-2+CD4+ T cells in RSV-infected infants and HD; however, the mean fluorescence intensity (MFI) of IL-2 staining was significantly lower in infected patients (Figure 1A). Interestingly, an inverse correlation was found between disease severity (CDSS) and the MFI of IL-2 staining in activated CD4+ T cells from RSV-infected patients. Moreover, because young age represents one of the most important risk factors for the development of severe RSV disease [1], we reanalyzed our data by dividing patients into 2 groups; ≤6 months and >7 months. Both groups of patients showed a lower production of IL-2 compared with HD (Figure 1A, right). To confirm that CD4+ T cells from RSV-infected children actually produced low amounts of IL-2, we analyzed IL-2 production in the supernatants of PBMCs stimulated with PHA for 3 days, using a cytometric bead array. IL-2 levels were markedly lower in RSV-infected children compared with HD. Moreover, we found that cells from RSV-infected children produced lower amounts of IL-10 and IL-17. No significant differences were observed regarding the production of IL-4, IL-6, TNF-α, and IFN-γ (Figure 1B). Figure 1. View largeDownload slide CD4+ T cells from respiratory syncytial virus (RSV)-infected infants produce low amounts of interleukin-2 (IL-2) and show a limited proliferative response. A, Peripheral blood mononuclear cells (PBMCs) from healthy donors (HD; n = 8) and RSV-infected infants (n = 20) were stimulated with phorbol 12-myristate 13-acetate/ionomycin in the presence of monensin for 5 hours. Percentage of IL-2+CD4+ T cells and the mean fluorescence intensity (MFI) of IL-2 staining in the gate of IL-2+CD4+ T cells were analyzed by flow cytometry. The correlation between clinical disease severity score (CDSS) and the MFI of IL-2 staining is also shown (Spearman rank correlation test). Right, MFI of IL-2 staining in PBMCs from HD (n = 8) and RSV-infected infants >7 months (n = 6) and ≤6 months (n = 13). B, Levels of cytokines in the supernatant of phytohemagglutinin (PHA)-activated PBMCs after 3 days of culture, quantified by cytometric bead array (n = 12 in each group). C, PHA-activated PBMCs were cultured in the absence or presence of IL-2 (20 ng/mL) for 3 days (n = 12 for each group). Frequency of Ki-67+CD4+ T cells in the gate of CD4+ T cells was evaluated by flow cytometry. D, PBMCs were treated, or not, with IL-2 (20 ng/mL) for 30 minutes at 37°C (n = 10 for each group). Frequency of pSTAT5+CD4+ T cells was analyzed by flow cytometry. A (middle), C (right), and D (right), show representative experiments. Mean ± SEM of n donors; *P < .05, **P < .01, ***P < .001, ****P < .0001. Abbreviations: INF-γ, interferon-gamma; TNF-α, tumor necrosis factor-alpha. Figure 1. View largeDownload slide CD4+ T cells from respiratory syncytial virus (RSV)-infected infants produce low amounts of interleukin-2 (IL-2) and show a limited proliferative response. A, Peripheral blood mononuclear cells (PBMCs) from healthy donors (HD; n = 8) and RSV-infected infants (n = 20) were stimulated with phorbol 12-myristate 13-acetate/ionomycin in the presence of monensin for 5 hours. Percentage of IL-2+CD4+ T cells and the mean fluorescence intensity (MFI) of IL-2 staining in the gate of IL-2+CD4+ T cells were analyzed by flow cytometry. The correlation between clinical disease severity score (CDSS) and the MFI of IL-2 staining is also shown (Spearman rank correlation test). Right, MFI of IL-2 staining in PBMCs from HD (n = 8) and RSV-infected infants >7 months (n = 6) and ≤6 months (n = 13). B, Levels of cytokines in the supernatant of phytohemagglutinin (PHA)-activated PBMCs after 3 days of culture, quantified by cytometric bead array (n = 12 in each group). C, PHA-activated PBMCs were cultured in the absence or presence of IL-2 (20 ng/mL) for 3 days (n = 12 for each group). Frequency of Ki-67+CD4+ T cells in the gate of CD4+ T cells was evaluated by flow cytometry. D, PBMCs were treated, or not, with IL-2 (20 ng/mL) for 30 minutes at 37°C (n = 10 for each group). Frequency of pSTAT5+CD4+ T cells was analyzed by flow cytometry. A (middle), C (right), and D (right), show representative experiments. Mean ± SEM of n donors; *P < .05, **P < .01, ***P < .001, ****P < .0001. Abbreviations: INF-γ, interferon-gamma; TNF-α, tumor necrosis factor-alpha. Consistent with our observations indicating a deficient production of IL-2 in RSV-infected patients, we found that the proliferative response of CD4+ T cells induced by PHA, assessed by detecting Ki-67 antigen expression, was substantially lower in RSV-infected infants compared with HD. The addition of IL-2 significantly increased the proliferative response of CD4+ T cells; however, even in the presence of exogenous IL-2, the proliferative response of CD4+ T cells from RSV-infected infants was markedly lower compared with HD (Figure 1C). This suggests that factors other than IL-2 production may also compromise the expansion of T cells in RSV-infected infants. Because the activation of STAT5 is one of the earliest events in IL-2 signaling through the high affinity IL-2 receptor [21], we analyzed STAT5 phosphorylation in response to IL-2 stimulation. We found a similar pattern of phosphorylation in CD4+ T cells from both RSV-infected infants and HD (Figure 1D), suggesting that the IL-2-STAT5 pathway is preserved during infection. IL-2 Increases the Frequency of FOXP3+ Tregs in PBMCs From RSV-Infected Infants We have previously reported that severe RSV infection in infants is associated with a pronounced reduction in the frequency of circulating Tregs [9]. To explore whether exogenous IL-2 was able to restore the pool of Tregs, PBMCs were cultured with or without IL-2 for 24 hours, and the frequency of Tregs was then analyzed. IL-2 significantly increased the frequency of FOXP3+CD4+ T cells in both HD and RSV-infected infants. However, even after IL-2 treatment, the frequency of FOXP3+CD4+ T cells was shown to be significantly lower in RSV-infected infants compared with HD (Figure 2A). Reanalysis of the data by dividing patients according to their age into 2 groups (≤6 months and >7 months), revealed that IL-2 failed to normalize the frequency of FOXP3+CD4+ T cells in both groups of patients (Figure 2A, right). Moreover, as shown in Figure 2B, IL-2 treatment also increased Treg expression of the ectonucleotidase CD39, which hydrolyzes ATP into the immunosuppressive agent adenosine [22]. This suggests that the increased expression of FOXP3 in CD4+ T cells from RSV-infected infants induced by IL-2 was actually associated with a regulatory signature. Interestingly, we found a negative correlation between CDSS values and the frequency of FOXP3+CD4+ T cells in IL-2–treated PBMCs, suggesting that more severe disease is associated with a limited response to IL-2 (Figure 2C). Further confirming the compromise in the compartment of FOXP3+CD4+ T cells in RSV-infected infants, we found that the proliferative response of this cell subset in response to PHA stimulation, assessed either in the absence or presence of IL-2, was severely reduced (Figure 2D). Figure 2. View largeDownload slide Interleukin-2 (IL-2) increases the frequency of FOXP3+CD4+ T cells in peripheral blood mononuclear cells (PBMCs) from respiratory syncytial virus (RSV)-infected infants. A, PBMCs from healthy donors (HD; n = 8) and RSV-infected infants (n = 41) were stimulated, or not, with IL-2 (20 ng/mL) for 24 hours. Frequency of FOXP3+CD4+ T cells was analyzed by flow cytometry. Left, analysis of all patients; right, analysis of patients divided into 2 groups: >7 months (n = 19) and ≤6 months (n = 22). B, Frequency of CD39+FOXP3+CD4+ T cells in PBMCs from RSV-infected children cultured for 24 hours in the absence or presence of IL-2 (20 ng/mL, n = 18). C, Correlation between the frequency of FOXP3+CD4+ T cells in PBMCs from RSV-infected infants cultured for 24 hours with IL-2 (20 ng/mL) and clinical disease severity score (CDSS), analyzed by Spearman rank correlation test. D, PBMCs from HD and RSV-infected infants were activated with phytohemagglutinin (PHA; 4 µg/mL) and cultured in the absence or presence of IL-2 (20 ng/mL) for 3 days (n = 12 for each group). Frequency of Ki-67+FOXP3+CD4+ T cells was evaluated by flow cytometry. A (middle) and B (right), show representative experiments. A (left and right), B (left), and D, mean ± SEM of n donors; *P < .05, **P < .01, ***P < .001, ****P < .0001. Figure 2. View largeDownload slide Interleukin-2 (IL-2) increases the frequency of FOXP3+CD4+ T cells in peripheral blood mononuclear cells (PBMCs) from respiratory syncytial virus (RSV)-infected infants. A, PBMCs from healthy donors (HD; n = 8) and RSV-infected infants (n = 41) were stimulated, or not, with IL-2 (20 ng/mL) for 24 hours. Frequency of FOXP3+CD4+ T cells was analyzed by flow cytometry. Left, analysis of all patients; right, analysis of patients divided into 2 groups: >7 months (n = 19) and ≤6 months (n = 22). B, Frequency of CD39+FOXP3+CD4+ T cells in PBMCs from RSV-infected children cultured for 24 hours in the absence or presence of IL-2 (20 ng/mL, n = 18). C, Correlation between the frequency of FOXP3+CD4+ T cells in PBMCs from RSV-infected infants cultured for 24 hours with IL-2 (20 ng/mL) and clinical disease severity score (CDSS), analyzed by Spearman rank correlation test. D, PBMCs from HD and RSV-infected infants were activated with phytohemagglutinin (PHA; 4 µg/mL) and cultured in the absence or presence of IL-2 (20 ng/mL) for 3 days (n = 12 for each group). Frequency of Ki-67+FOXP3+CD4+ T cells was evaluated by flow cytometry. A (middle) and B (right), show representative experiments. A (left and right), B (left), and D, mean ± SEM of n donors; *P < .05, **P < .01, ***P < .001, ****P < .0001. Soluble CD25 Limits IL-2 Function During Severe RSV Infection Elevated concentrations of sCD25 are found in autoimmunity, cancer, and inflammatory conditions [20, 23]. Looking for factors that might affect the function of IL-2 in the course of RSV infection, we evaluated serum levels of sCD25 (IL-2 receptor α chain soluble form). It has been reported that serum from RSV-infected infants has increased amounts of sCD25 [24, 25]. In agreement with this observation, we found higher amounts of sCD25 in the serum from RSV-infected infants compared with HD (Figure 3A). However, no correlation was found between disease severity and sCD25 levels (not shown). Because the ability of sCD25 to inhibit IL-2 activity remains controversial [20, 26, 27], we evaluated the effect of recombinant sCD25 on the function of IL-2 in CD4+ T cells from RSV-infected infants. We found that both IL-2–dependant STAT5 phosphorylation and FOXP3 expression were significantly inhibited by the addition of recombinant sCD25 (Figure 3B and C). Consistent with this observation, we found that serum from RSV-infected infants, but not from HD, diminished the frequency of FOXP3+CD4+ T cells in PBMCs isolated from healthy adult donors, cultured with or without IL-2 (Figure 3D). Moreover, we found a significant positive correlation between the ability of serum from RSV-infected children to reduce the frequency of FOXP3+CD4+ T cells and the concentration of sCD25 in the serum (Figure 3D, right). Figure 3. View largeDownload slide Soluble CD25 (sCD25) interferes with interleukin-2 (IL-2) function in respiratory syncytial virus (RSV)-infected children. A, Levels of sCD25 in the serum from RSV-infected (n = 24) and healthy donors (HD; n = 21) were quantified by enzyme-linked immunosorbent assay (ELISA). B, C, Peripheral blood mononuclear cells (PBMCs) from RSV-infected infants were cultured with IL-2 (20 ng/mL) or IL-2 plus sCD25 (200 ng/mL) for 30 minutes (B) or 24 hours (C). The frequencies of pSTAT5+CD4+ T cells (B; n = 12) or FOXP3+CD4+ T cells (C; n = 7) were then analyzed by flow cytometry. D, PBMCs from healthy donors were incubated for 24 hours with or without serum from healthy (n = 9) or RSV-infected children (final dilution 1:10; n = 9), in the absence or presence of IL-2 (20 ng/mL). The frequency of FOXP3+CD4+ T cells was then analyzed by flow cytometry (left and middle). Right, correlation between the ability of serum from RSV-infected infants to reduce the frequency of FOXP3+CD4+ T cells and serum levels of sCD25 (Spearman rank correlation test). Fold decrease of FOXP3+CD4+ T-cell frequency was calculated as the ratio between the frequencies of FOXP3+CD4+ T cells cultured in the absence and presence of serum from RSV-infected children. B (right) and D (middle), show representative experiments. A, B (left), C, and D (left), mean ± SEM of n donors;*P < .05, ***P < .001. Figure 3. View largeDownload slide Soluble CD25 (sCD25) interferes with interleukin-2 (IL-2) function in respiratory syncytial virus (RSV)-infected children. A, Levels of sCD25 in the serum from RSV-infected (n = 24) and healthy donors (HD; n = 21) were quantified by enzyme-linked immunosorbent assay (ELISA). B, C, Peripheral blood mononuclear cells (PBMCs) from RSV-infected infants were cultured with IL-2 (20 ng/mL) or IL-2 plus sCD25 (200 ng/mL) for 30 minutes (B) or 24 hours (C). The frequencies of pSTAT5+CD4+ T cells (B; n = 12) or FOXP3+CD4+ T cells (C; n = 7) were then analyzed by flow cytometry. D, PBMCs from healthy donors were incubated for 24 hours with or without serum from healthy (n = 9) or RSV-infected children (final dilution 1:10; n = 9), in the absence or presence of IL-2 (20 ng/mL). The frequency of FOXP3+CD4+ T cells was then analyzed by flow cytometry (left and middle). Right, correlation between the ability of serum from RSV-infected infants to reduce the frequency of FOXP3+CD4+ T cells and serum levels of sCD25 (Spearman rank correlation test). Fold decrease of FOXP3+CD4+ T-cell frequency was calculated as the ratio between the frequencies of FOXP3+CD4+ T cells cultured in the absence and presence of serum from RSV-infected children. B (right) and D (middle), show representative experiments. A, B (left), C, and D (left), mean ± SEM of n donors;*P < .05, ***P < .001. Activated conventional CD4+ T cells and Tregs are the main sources of sCD25 [20]. Because severe RSV infection is associated with a marked depletion of Tregs [9], we speculated that conventional CD4+ T cells may be the primary source of sCD25. In fact, we found that FOXP3− CD4+ T cells from RSV-infected infants express not only a higher frequency of CD25+ cells compared with HD, but also a great expression of CD25 measured as MFI (Figure 4A). A very low or negligible percentage of CD25+ cells was detected in CD8+ T cells, B cells, and monocytes (Figure 4B). Interestingly, we observed an inverse correlation between the frequency of peripheral blood FOXP3+ Tregs and the levels of serum sCD25 when HD and RSV-infected infants were analyzed collectively (Figure 4C), suggesting that sCD25 may actually act as a decoy receptor for IL-2 during severe RSV infection. Figure 4. View largeDownload slide High expression of CD25 in conventional CD4+ T cells from respiratory syncytial virus (RSV)-infected infants. A, Frequency and mean fluorescence intensity (MFI) of CD25 on gated FOXP3− CD4+ T cells from healthy donor (HD; n = 9) and RSV-infected infants (n = 21), evaluated by flow cytometry. B, Percentage of CD25 on gated CD4+, CD8+, CD19+, and CD14+ cells from RSV-infected infants (n = 15). C, Correlation between the frequency of FOXP3+CD4+ T cells and the serum levels of sCD25 in the group of healthy and RSV-infected children analyzed together by Spearman rank correlation coefficient test. A (right), representative experiment. A (left and middle) and B, mean ± SEM of n donors; **P < .01, ****P < .0001. Figure 4. View largeDownload slide High expression of CD25 in conventional CD4+ T cells from respiratory syncytial virus (RSV)-infected infants. A, Frequency and mean fluorescence intensity (MFI) of CD25 on gated FOXP3− CD4+ T cells from healthy donor (HD; n = 9) and RSV-infected infants (n = 21), evaluated by flow cytometry. B, Percentage of CD25 on gated CD4+, CD8+, CD19+, and CD14+ cells from RSV-infected infants (n = 15). C, Correlation between the frequency of FOXP3+CD4+ T cells and the serum levels of sCD25 in the group of healthy and RSV-infected children analyzed together by Spearman rank correlation coefficient test. A (right), representative experiment. A (left and middle) and B, mean ± SEM of n donors; **P < .01, ****P < .0001. DISCUSSION Our results suggest that both the production and function of IL-2 is compromised in the course of severe RSV infection in infants. We found that conventional CD4+ T cells from RSV-infected infants produce low amounts of IL-2. Moreover, we observed that exogenous IL-2 was unable to fully restore IL-2–dependent functions such as the proliferative response of CD4+ T cells and the expansion of Tregs. These observations suggest that different mechanisms contribute to limiting the function of IL-2 in the scenario of RSV disease. Interestingly, this phenomenon may contribute to an explanation of not only the depletion of circulating Tregs, but also an essential feature of RSV infection, that is its inability to promote a robust memory T-cell response [2, 3, 28, 29]. In this regard, it should be noted that IL-2 is required for the effective generation of effector and memory CD8+ T cells [30]. Moreover, it has been reported that the impaired effector and memory function of CD8+ T cells, as well as the antibody response observed in RSV-infected animals, were markedly improved by administration of IL-2, and these effects were associated with reduced weight loss and illness in challenged mice [31]. A reduced capacity of conventional CD4+ T cells to produce IL-2 has been previously described in autoimmunity and infectious diseases. Autoimmune diseases such as type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosus are associated with defective ability of CD4+ T cells to secrete IL-2 [32–35]. This defect appears to explain the decrease in Tregs, favoring the expansion of autoreactive T cells [32]. On the other hand, observations made in experimental models of infections induced by Toxoplasma gondi, Listeria monocytogenes, and vaccinia virus have shown that the acute infection phase is associated with a limited production of IL-2, resulting in a diminished Treg frequency [36, 37]. Regarding RSV infection, previous studies suggested that the production of IL-2 by CD4+ T cells may be limited. Using PBMCs from adults who have been naturally and recurrently exposed to RSV and IAV, Fleming and coworkers reported that in vitro exposure to RSV resulted in reduced production of IL-2 and a low lymphocyte proliferative response, compared to IAV-stimulated cells, although PBMCs from adult donors expressed a similar frequency of specific T lymphocytes in response to both viruses [38]. Moreover, we previously reported that activated CD4+ T cells are permissive to RSV infection, and also that infection promotes a marked inhibition of IL-2 production [39]. IL-2 availability during RSV infection may be limited, not only by decreased production but also by the high systemic levels of sCD25. Consistent with previous studies [24, 25], we found high levels of sCD25 in the serum from RSV-infected infants. Elevated serum concentrations of sCD25 have been reported in inflammatory conditions [20, 23]. Activated conventional T cells, Tregs, and dendritic cells can release sCD25 by proteolytic cleavage of surface CD25 [20]. Our observations show a great expression of CD25 on conventional CD4+ T cells from RSV-infected infants, suggesting that they may be the main source of sCD25. Regarding the biological significance of sCD25, it appears to compete with activated T cells for IL-2 binding, thereby reducing T-cell proliferation [20, 26]. However, it has also been reported that sCD25 may enhance the biological activity of IL-2. In fact, by forming a complex with IL-2, sCD25 has been shown to enhance IL-2–mediated phosphorylation of STAT5 in CD4+ T cells, promoting their differentiation into inducible FOXP3+ Tregs [27]. These observations suggest that sCD25 may either decrease or enhance the biological activity of IL-2, depending on the experimental setting. Our observations that sCD25 partially inhibits the ability of IL-2 to induce both STAT5 phosphorylation and FOXP3 expression in CD4+ T cells from RSV-infected infants, suggest that sCD25 acts as a decoy receptor for IL-2 during severe RSV disease. We observed that IL-2 significantly increased the proliferative response and the expression of FOXP3 in CD4+ T cells from RSV-infected infants. However, even in the presence of IL-2, both the proliferation rate of CD4+ T cells and the expression of FOXP3 remained substantially lower in CD4+ T cells from RSV-infected infants compared with HD. This suggests that factors other than IL-2 availability may compromise the activity of IL-2 during severe RSV infection. In contrast to other acute viral infections such as those produced by measles virus and cytomegalovirus, RSV infection is not associated with a generalized immunologic hyporesponsiveness [2, 40]. On the other hand, our results showing that IL-2 induced a similar pattern of STAT5 phosphorylation in CD4+ T cells from RSV-infected infants and HD suggest that signaling through the IL-2 receptor is preserved. Several factors may account for the limited activity of IL-2 in the course of RSV infection. Mediators such as IL-4, TGF-β, type I interferons, and prostaglandin E2 are produced at high levels in the context of RSV infection, all of them being capable of damping the function of IL-2 [41–46]. All patients recruited in our study required hospitalization and supplemental oxygen. We analyzed the impact of disease severity on IL-2 production and IL-2 ability to restore the pool of FOXP3+CD4+ T cells. This analysis revealed a significant negative correlation for both parameters, suggesting that patients with more severe disease produce lower levels of IL-2 and show a limited response to IL-2. It should be noted that although IL-2 failed in vitro to fully restore the function of CD4+ T cells from RSV-infected infants, it significantly improved both the proliferative response of CD4+ T cells and the frequency of FOXP3+CD4+ T cells. Interestingly, observations made in experimental models of RSV infection have shown that IL-2 administration improves disease outcome in RSV-challenged mice [31]. Proof-of-concept clinical trials have shown that, at low doses, IL-2 improves autoimmune and inflammatory conditions [32]. Further studies are needed to define whether IL-2 could represent a useful therapeutic tool in severe RSV infection. Notes Acknowledgments. We thank all the team members of the Hospital General de Niños Pedro de Elizalde. Most of all, we are indebted to all the participating children and their families. Financial support. This work was supported by grants from the Fondo Nacional para la Investigacion Cientifica y Tecnologica de Argentina (PIDC 0010-2015 to J. G., PMO BID PICT 2014-1578 and PMO BID PICT 2016-0444 to L. A.), and Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 2015-2017-0223 to L. A.). Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Presented in part: RSV Vaccines For The World Conference 2017–4th RESVINET Meeting, Málaga, Spain, 29 November–1 December 2017. References 1. Shi T, McAllister DA, O’Brien KL, et al.   Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study. Lancet  2017; 390: 946– 58. Google Scholar CrossRef Search ADS PubMed  2. Openshaw PJM, Chiu C, Culley FJ, Johansson C. Protective and harmful immunity to RSV infection. Annu Rev Immunol  2017; 35: 501– 32. Google Scholar CrossRef Search ADS PubMed  3. Arruvito L, Raiden S, Geffner J. Host response to respiratory syncytial virus infection. Curr Opin Infect Dis  2015; 28: 259– 66. Google Scholar CrossRef Search ADS PubMed  4. Campbell DJ, Koch MA. Phenotypical and functional specialization of FOXP3+ regulatory T cells. Nat Rev Immunol  2011; 11: 119– 30. Google Scholar CrossRef Search ADS PubMed  5. Belkaid Y, Rouse BT. Natural regulatory T cells in infectious disease. Nat Immunol  2005; 6: 353– 60. Google Scholar CrossRef Search ADS PubMed  6. Lee DC, Harker JA, Tregoning JS, et al.   CD25+ natural regulatory T cells are critical in limiting innate and adaptive immunity and resolving disease following respiratory syncytial virus infection. J Virol  2010; 84: 8790– 8. Google Scholar CrossRef Search ADS PubMed  7. Loebbermann J, Thornton H, Durant L, et al.   Regulatory T cells expressing granzyme B play a critical role in controlling lung inflammation during acute viral infection. Mucosal Immunol  2012; 5: 161– 72. Google Scholar CrossRef Search ADS PubMed  8. Krishnamoorthy N, Khare A, Oriss TB, et al.   Early infection with respiratory syncytial virus impairs regulatory T cell function and increases susceptibility to allergic asthma. Nat Med  2012; 18: 1525– 30. Google Scholar CrossRef Search ADS PubMed  9. Raiden S, Pandolfi J, Payasliàn F, et al.   Depletion of circulating regulatory T cells during severe respiratory syncytial virus infection in young children. Am J Respir Crit Care Med  2014; 189: 865– 8. Google Scholar CrossRef Search ADS PubMed  10. Sakaguchi S, Miyara M, Costantino CM, Hafler DA. FOXP3+ regulatory T cells in the human immune system. Nat Rev Immunol  2010; 10: 490– 500. Google Scholar CrossRef Search ADS PubMed  11. Nishikawa H, Sakaguchi S. Regulatory T cells in tumor immunity. Int J Cancer  2010; 127: 759– 67. Google Scholar PubMed  12. Wing K, Sakaguchi S. Regulatory T cells exert checks and balances on self tolerance and autoimmunity. Nat Immunol  2010; 11: 7– 13. Google Scholar CrossRef Search ADS PubMed  13. Wohlfert E, Belkaid Y. Plasticity of T reg at infected sites. Mucosal Immunol  2010; 3: 213– 5. Google Scholar CrossRef Search ADS PubMed  14. Lühn K, Simmons CP, Moran E, et al.   Increased frequencies of CD4+ CD25(high) regulatory T cells in acute dengue infection. J Exp Med  2007; 204: 979– 85. Google Scholar CrossRef Search ADS PubMed  15. Giamarellos-Bourboulis EJ, Raftogiannis M, Antonopoulou A, et al.   Effect of the novel influenza A (H1N1) virus in the human immune system. PLoS One  2009; 4: e8393. Google Scholar CrossRef Search ADS PubMed  16. Chinen T, Kannan AK, Levine AG, et al.   An essential role for the IL-2 receptor in Tregcell function. Nat Immunol  2016; 17: 1322– 33. Google Scholar CrossRef Search ADS PubMed  17. Hall CB, Walsh EE, Long CE, Schnabel KC. Immunity to and frequency of reinfection with respiratory syncytial virus. J Infect Dis  1991; 163: 693– 8. Google Scholar CrossRef Search ADS PubMed  18. Mella C, Suarez-Arrabal MC, Lopez S, et al.   Innate immune dysfunction is associated with enhanced disease severity in infants with severe respiratory syncytial virus bronchiolitis. J Infect Dis  2013; 207: 564– 73. Google Scholar CrossRef Search ADS PubMed  19. Mejias A, Dimo B, Suarez NM, et al.   Whole blood gene expression profiles to assess pathogenesis and disease severity in infants with respiratory syncytial virus infection. PLoS Med  2013; 10: e1001549. Google Scholar CrossRef Search ADS PubMed  20. Boyman O, Sprent J. The role of interleukin-2 during homeostasis and activation of the immune system. Nat Rev Immunol  2012; 12: 180– 90. Google Scholar CrossRef Search ADS PubMed  21. Burchill MA, Yang J, Vogtenhuber C, Blazar BR, Farrar MA. IL-2 receptor beta-dependent STAT5 activation is required for the development of Foxp3+ regulatory T cells. J Immunol  2007; 178: 280– 90. Google Scholar CrossRef Search ADS PubMed  22. Cook L, Munier CML, Seddiki N, et al.   Circulating gluten-specific FOXP3+CD39+ regulatory T cells have impaired suppressive function in patients with celiac disease. J Allergy Clin Immunol  2017; 140: 1592– 603.e8. Google Scholar CrossRef Search ADS PubMed  23. Rubin LA, Nelson DL. The soluble interleukin-2 receptor: biology, function, and clinical application. Ann Intern Med  1990; 113: 619– 27. Google Scholar CrossRef Search ADS PubMed  24. Smyth RL, Fletcher JN, Thomas HM, Hart CA, Openshaw PJ. Respiratory syncytial virus and wheeze. Lancet  1999; 354: 1997– 8. Google Scholar CrossRef Search ADS PubMed  25. Alonso Fernández J, Roine I, Vasquez A, Cáneo M. Soluble interleukin-2 receptor (sCD25) and interleukin-10 plasma concentrations are associated with severity of primary respiratory syncytial virus (RSV) infection. Eur Cytokine Netw  2005; 16: 81– 90. Google Scholar PubMed  26. von Bergwelt-Baildon MS, Popov A, Saric T, et al.   CD25 and indoleamine 2,3-dioxygenase are up-regulated by prostaglandin E2 and expressed by tumor-associated dendritic cells in vivo: additional mechanisms of T-cell inhibition. Blood  2006; 108: 228– 37. Google Scholar CrossRef Search ADS PubMed  27. Yang ZZ, Grote DM, Ziesmer SC, et al.   Soluble IL-2Rα facilitates IL-2-mediated immune responses and predicts reduced survival in follicular B-cell non-Hodgkin lymphoma. Blood  2011; 118: 2809– 20. Google Scholar CrossRef Search ADS PubMed  28. Meng J, Stobart CC, Hotard AL, Moore ML. An overview of respiratory syncytial virus. PLoS Pathog  2014; 10: e1004016. Google Scholar CrossRef Search ADS PubMed  29. Christiaansen AF, Knudson CJ, Weiss KA, Varga SM. The CD4 T cell response to respiratory syncytial virus infection. Immunol Res  2014; 59: 109– 17. Google Scholar CrossRef Search ADS PubMed  30. Williams MA, Tyznik AJ, Bevan MJ. Interleukin-2 signals during priming are required for secondary expansion of CD8+ memory T cells. Nature  2006; 441: 890– 3. Google Scholar CrossRef Search ADS PubMed  31. Chang J, Choi SY, Jin HT, Sung YC, Braciale TJ. Improved effector activity and memory CD8 T cell development by IL-2 expression during experimental respiratory syncytial virus infection. J Immunol  2004; 172: 503– 8. Google Scholar CrossRef Search ADS PubMed  32. Klatzmann D, Abbas AK. The promise of low-dose interleukin-2 therapy for autoimmune and inflammatory diseases. Nat Rev Immunol  2015; 15: 283– 94. Google Scholar CrossRef Search ADS PubMed  33. Kammer GM. Altered regulation of IL-2 production in systemic lupus erythematosus: an evolving paradigm. J Clin Invest  2005; 115: 836– 40. Google Scholar CrossRef Search ADS PubMed  34. Zier KS, Leo MM, Spielman RS, Baker L. Decreased synthesis of interleukin-2 (IL-2) in insulin-dependent diabetes mellitus. Diabetes  1984; 33: 552– 5. Google Scholar CrossRef Search ADS PubMed  35. Kitas GD, Salmon M, Farr M, Gaston JS, Bacon PA. Deficient interleukin 2 production in rheumatoid arthritis: association with active disease and systemic complications. Clin Exp Immunol  1988; 73: 242– 9. Google Scholar PubMed  36. Benson A, Murray S, Divakar P, et al.   Microbial infection-induced expansion of effector T cells overcomes the suppressive effects of regulatory T cells via an IL-2 deprivation mechanism. J Immunol  2012; 188: 800– 10. Google Scholar CrossRef Search ADS PubMed  37. Oldenhove G, Bouladoux N, Wohlfert EA, et al.   Decrease of FOXP3+ Treg cell number and acquisition of effector cell phenotype during lethal infection. Immunity  2009; 31: 772– 86. Google Scholar CrossRef Search ADS PubMed  38. Fleming EH, Ochoa EE, Nichols JE, O’Banion MK, Salkind AR, Roberts NJJr. Reduced activation and proliferation of human lymphocytes exposed to respiratory syncytial virus compared to cells exposed to influenza virus. J Med Virol  2018; 90: 26– 33. Google Scholar CrossRef Search ADS PubMed  39. Raiden S, Sananez I, Remes-Lenicov F, et al.   Respiratory syncytial virus (RSV) infects CD4+ T cells: frequency of circulating CD4+ RSV+ T cells as a marker of disease severity in young children. J Infect Dis  2017; 215: 1049– 58. Google Scholar CrossRef Search ADS PubMed  40. Bakaletz LO. Viral-bacterial co-infections in the respiratory tract. Curr Opin Microbiol  2017; 35: 30– 5. Google Scholar CrossRef Search ADS PubMed  41. Golding A, Rosen A, Petri M, Akhter E, Andrade F. Interferon-alpha regulates the dynamic balance between human activated regulatory and effector T cells: implications for antiviral and autoimmune responses. Immunology  2010; 131: 107– 17. Google Scholar PubMed  42. Martinez OM, Gibbons RS, Garovoy MR, Aronson FR. IL-4 inhibits IL-2 receptor expression and IL-2-dependent proliferation of human T cells. J Immunol  1990; 144: 2211– 5. Google Scholar PubMed  43. Das L, Levine AD. TGF-beta inhibits IL-2 production and promotes cell cycle arrest in TCR-activated effector/memory T cells in the presence of sustained TCR signal transduction. J Immunol  2008; 180: 1490– 8. Google Scholar CrossRef Search ADS PubMed  44. Katamura K, Shintaku N, Yamauchi Y, et al.   Prostaglandin E2 at priming of naive CD4+ T cells inhibits acquisition of ability to produce IFN-gamma and IL-2, but not IL-4 and IL-5. J Immunol  1995; 155: 4604– 12. Google Scholar PubMed  45. Wang L, van Panhuys N, Hu-Li J, Kim S, Le Gros G, Min B. Blimp-1 induced by IL-4 plays a critical role in suppressing IL-2 production in activated CD4 T cells. J Immunol  2008; 181: 5249– 56. Google Scholar CrossRef Search ADS PubMed  46. McKarns SC, Schwartz RH, Kaminski NE. Smad3 is essential for TGF-beta 1 to suppress IL-2 production and TCR-induced proliferation, but not IL-2-induced proliferation. J Immunol  2004; 172: 4275– 84. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Infectious Diseases Oxford University Press

Dampening of IL-2 Function in Infants With Severe Respiratory Syncytial Virus Disease

Loading next page...
 
/lp/ou_press/dampening-of-il-2-function-in-infants-with-severe-respiratory-0ADyayDgp0
Publisher
Oxford University Press
Copyright
© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
ISSN
0022-1899
eISSN
1537-6613
D.O.I.
10.1093/infdis/jiy180
Publisher site
See Article on Publisher Site

Abstract

Abstract Background FOXP3+ regulatory T cells (Tregs) restrain the destructive potential of the immune system. We have previously reported a pronounced reduction in circulating Tregs in infants with severe respiratory syncytial virus (RSV) disease. Because interleukin-2 (IL-2) is critical for Treg growth, survival, and activity, we here analyzed IL-2 production and function in RSV-infected infants. Methods Phenotype, proliferation, IL-2 production, and IL-2 signaling in CD4+ T cells were analyzed by flow cytometry. Serum soluble CD25 levels were quantified by ELISA. Results CD4+ T cells from RSV-infected infants produced lower amounts of IL-2 and showed a reduced proliferative response compared with healthy infants. IL-2 increased CD4+ T-cell proliferation and FOXP3 expression in both healthy and RSV-infected infants. However, although IL-2 induced a similar pattern of STAT5 phosphorylation, the proliferative response of CD4+ T cells and the expression of FOXP3+ remained significantly lower in RSV-infected infants. Interestingly, we found a negative correlation between disease severity and both the production of IL-2 by CD4+ T cells and the ability of exogenous IL-2 to restore the pool of FOXP3+CD4+ T cells. Conclusions A reduced ability to produce IL-2 and a limited response to this cytokine may affect the function of CD4+ T cells in RSV-infected infants. respiratory syncytial virus, infants, severe bronchiolitis, CD4+ T cells, FOXP3, IL-2, CD25 Respiratory syncytial virus (RSV) infection is the commonest cause of bronchiolitis and hospitalization in infants, accounting for about 100000–200000 deaths annually [1]. Pathogenesis of RSV infection involves both cytopathic effects induced by the virus and an exacerbated inflammatory response, which explain, at least partially, damage to the airway [2, 3]. FOXP3+ regulatory T cells (Tregs) not only prevent autoimmune diseases and maintain immune homeostasis, but also regulate the immune response in the course of infectious diseases, controlling tissue injury [4, 5]. In fact, experimental models of RSV infection have shown that depletion of Tregs leads to more severe disease [6–8]. The role of Tregs during human RSV infection has not yet been clarified. However, the Treg cell compartment undergoes important changes in the course of severe RSV infection. In this regard, we have previously reported that severe RSV infection in infants is associated with a marked reduction in the frequency of peripheral blood Tregs [9]. Most studies analyzing the role of Tregs have focused on chronic conditions, such as autoimmunity, cancer, and persistent infectious diseases [10–13]. By contrast, very little is known about the function of Tregs in acute viral infections. Our previous findings showing the depletion of Tregs in RSV-infected infants contrast with the increased frequencies of Tregs found in other acute viral diseases such as dengue [14] and influenza A virus (IAV) [15], suggesting a particular signature of RSV infection. Because Treg survival and function are dependent on interleukin-2 (IL-2) receptor signaling [16], we speculated that severe RSV disease could be associated with a dysregulation of the IL-2/IL-2 receptor system. This hypothesis may also explain another key feature of RSV infection, that is its inability to induce long-lasting immunity, a response strongly dependent on IL-2 function [17]. Here, we found that the production and function of IL-2 are compromised in infants with severe RSV infection. CD4+ T cells from RSV-infected patients produced lower amounts of IL-2 and showed a limited proliferative response compared with cells from healthy donors. We also found that the addition of IL-2 failed to fully restore IL-2–dependent functions such as FOXP3 expression and CD4+ T-cell proliferation. Interestingly, a negative correlation was observed between disease severity and both IL-2 production by CD4+ T cells and the ability of IL-2 to increase the pool of FOXP3+CD4+ T cells. MATERIALS AND METHODS Ethics Statement Our study was approved by the Ethics Committee of the Hospital de Pediatría Pedro de Elizalde, Buenos Aires, Argentina, in accordance with the Declaration of Helsinki (Fortaleza 2013). Written informed consent was obtained from all donors or legal guardians. Study Population We recruited 85 infants <18 months old, hospitalized at the Hospital de Pediatría Pedro de Elizalde (Buenos Aires, Argentina), with a severe episode of RSV bronchiolitis during the 2016–2017 respiratory seasons. We excluded children with history of prematurity, immunodeficiency, congenital heart disease, and chronic conditions. All of the infants required hospitalization. RSV infection was confirmed by direct immunofluorescence of nasopharyngeal aspirates. Disease severity was assessed by applying a clinical disease severity score (CDSS) based on the modified Tal score, which classifies patients as having mild (0–4), moderate (5–8), or severe (9–12) RSV bronchiolitis at the time of sampling [18, 19]. The control group consisted of 40 infants admitted to the hospital for scheduled surgery (healthy donors, HD). They had no identifiable airway infections for a 4-week period before the study. Characteristics of the patients are shown in Table 1. Table 1. Baseline Characteristics of Children Infected With Respiratory Syncytial Virus   RSV Patients  Healthy Children    N = 85  N = 40  Demographic characteristics  Age, mo, mean ± SD  7.5 ± 7.1  12.6 ± 3.8  Male sex, n (%)  57 (67%)  22 (55%)  Disease severity  CDSS, n (%)a   0–6  0 (0%)  NA   7–8  48 (57%)  NA   9–12  37 (43%)  NA  O2 requirement, n (%)  85 (100%)  NA  Hospital stay, days, mean ± SD  7.3 ± 0.2  NA  PICU admission, n (%)  3 (3.6%)  NA  Laboratory parameters  WBC, counts/mm3, mean ± SD  10559 ± 4021  8952 ± 1250  Lymphocytes, %, mean ± SD  37.4 ± 15.4  53.1 ± 4.0  CD4+, %, mean ± SDb  28.0 ± 11.0  32.3 ± 5.7  CD8+, %, mean ± SDb  12.4 ± 6.8  20.0 ± 7.5  CD19+, %, mean ± SDb  36.4 ± 10.2  16.5 ± 7.2    RSV Patients  Healthy Children    N = 85  N = 40  Demographic characteristics  Age, mo, mean ± SD  7.5 ± 7.1  12.6 ± 3.8  Male sex, n (%)  57 (67%)  22 (55%)  Disease severity  CDSS, n (%)a   0–6  0 (0%)  NA   7–8  48 (57%)  NA   9–12  37 (43%)  NA  O2 requirement, n (%)  85 (100%)  NA  Hospital stay, days, mean ± SD  7.3 ± 0.2  NA  PICU admission, n (%)  3 (3.6%)  NA  Laboratory parameters  WBC, counts/mm3, mean ± SD  10559 ± 4021  8952 ± 1250  Lymphocytes, %, mean ± SD  37.4 ± 15.4  53.1 ± 4.0  CD4+, %, mean ± SDb  28.0 ± 11.0  32.3 ± 5.7  CD8+, %, mean ± SDb  12.4 ± 6.8  20.0 ± 7.5  CD19+, %, mean ± SDb  36.4 ± 10.2  16.5 ± 7.2  Abbreviations: CDSS, clinical disease severity score; NA, not applicable; PICU, pediatric intensive care unit; RSV, respiratory syncytial virus; WBC, white blood cells. aCDSS was calculated using the modified Tal score. bPercentages of cell subsets in the gate of lymphocytes (n = 12 in each group). View Large Table 1. Baseline Characteristics of Children Infected With Respiratory Syncytial Virus   RSV Patients  Healthy Children    N = 85  N = 40  Demographic characteristics  Age, mo, mean ± SD  7.5 ± 7.1  12.6 ± 3.8  Male sex, n (%)  57 (67%)  22 (55%)  Disease severity  CDSS, n (%)a   0–6  0 (0%)  NA   7–8  48 (57%)  NA   9–12  37 (43%)  NA  O2 requirement, n (%)  85 (100%)  NA  Hospital stay, days, mean ± SD  7.3 ± 0.2  NA  PICU admission, n (%)  3 (3.6%)  NA  Laboratory parameters  WBC, counts/mm3, mean ± SD  10559 ± 4021  8952 ± 1250  Lymphocytes, %, mean ± SD  37.4 ± 15.4  53.1 ± 4.0  CD4+, %, mean ± SDb  28.0 ± 11.0  32.3 ± 5.7  CD8+, %, mean ± SDb  12.4 ± 6.8  20.0 ± 7.5  CD19+, %, mean ± SDb  36.4 ± 10.2  16.5 ± 7.2    RSV Patients  Healthy Children    N = 85  N = 40  Demographic characteristics  Age, mo, mean ± SD  7.5 ± 7.1  12.6 ± 3.8  Male sex, n (%)  57 (67%)  22 (55%)  Disease severity  CDSS, n (%)a   0–6  0 (0%)  NA   7–8  48 (57%)  NA   9–12  37 (43%)  NA  O2 requirement, n (%)  85 (100%)  NA  Hospital stay, days, mean ± SD  7.3 ± 0.2  NA  PICU admission, n (%)  3 (3.6%)  NA  Laboratory parameters  WBC, counts/mm3, mean ± SD  10559 ± 4021  8952 ± 1250  Lymphocytes, %, mean ± SD  37.4 ± 15.4  53.1 ± 4.0  CD4+, %, mean ± SDb  28.0 ± 11.0  32.3 ± 5.7  CD8+, %, mean ± SDb  12.4 ± 6.8  20.0 ± 7.5  CD19+, %, mean ± SDb  36.4 ± 10.2  16.5 ± 7.2  Abbreviations: CDSS, clinical disease severity score; NA, not applicable; PICU, pediatric intensive care unit; RSV, respiratory syncytial virus; WBC, white blood cells. aCDSS was calculated using the modified Tal score. bPercentages of cell subsets in the gate of lymphocytes (n = 12 in each group). View Large Isolation of Peripheral Blood Mononuclear Cells Peripheral blood mononuclear cells (PBMCs) were obtained from blood samples (0.3–0.4 mL) from RSV-infected infants or HD, by Ficoll-Hypaque gradient centrifugation (GE Healthcare Life Sciences, Uppsala, Sweden). PBMCs were washed twice and suspended in complete culture medium: RPMI 1640 (Hyclone, Logan, Utah) supplemented with 10% heat-inactivated fetal calf serum (FCS; Natocor, Córdoba, Argentina), 200 mM l-glutamine, and 50 μg/mL gentamicin (Sigma-Aldrich, St. Louis, MO). Flow Cytometry Labeled monoclonal antibodies (mAb) directed to CD3, CD4, CD8, CD14, CD19, CD25, CD39, FOXP3, IL-2, STAT5, and Ki-67 were obtained from BD Biosciences (San José, CA). In all cases, isotype-matched mAb were used as controls. For intracellular IL-2 detection, cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 µg/mL ionomycin (Sigma-Aldrich) for 5 hours in the presence of monensin (Golgi-Stop, BD Biosciences), and stained with anti-CD4 and anti-IL-2 mAbs, after cell fixation and permeabilization (BD Biosciences). To analyze STAT5 phosphorylation in CD4+ T cells, PBMCs were treated, or not, with IL-2 (20 ng/mL; Peprotech, Rocky Hill, NJ) for 30 minutes at 37°C. Cells were then stained with a mAb directed to phosphorylated STAT5 after cell fixation and permeabilization. Data were acquired using a FACS Canto (Becton Dickinson) and analyzed with FlowJo software. Statistical analyses were based on at least 100000 events gated on the population of interest. Culture of PBMCs in the Presence of IL-2 To test the proliferative response of CD4+ T cells, PBMCs from RSV or HD (1 × 106 cells/mL) were activated with phytohemagglutinin (PHA, 4 µg/mL, Sigma-Aldrich) and cultured in the absence or presence of IL-2 (20 ng/mL) for 3 days. Then, the expression of the proliferation marker Ki-67 was assessed by flow cytometry. The concentration of IL-2 used in these assays was selected on the basis of preliminary experiments performed with PBMCs from healthy children. These experiments showed that the proliferation of PHA-activated CD4+ T cells reached similar values with either 20 or 100 ng/mL of IL-2. To test the ability of IL-2 to increase the expression of FOXP3, resting PBMCs (1 × 106 cells/mL) were cultured without or with IL-2 (20 ng/mL) for 24 hours. In some experiments, resting PBMCs were cultured with IL-2 alone or IL-2 plus recombinant soluble CD25 (sCD25, 200 ng/mL, Peprotech), for 30 minutes or 24 hours, and phosphorylation of STAT5 and FOXP3 expression was evaluated. Culture of PBMCs in the Presence of Serum From RSV-Infected or Healthy Children Serum was obtained from HD and RSV-infected children, while PBMCs were obtained from unrelated healthy adult donors. Briefly, PBMCs (1 × 106/mL) were incubated for 24 hours in complete culture medium supplemented with heat-inactivated serum from healthy or RSV-infected children (final dilution 1:10), in the absence or presence of IL-2 (20 ng/mL). Then, the frequency of FOXP3+CD4+ T cells was analyzed. Flow cytometry crossmatch was used to detect the presence or lack of IgG antibodies on the surface of CD4+ lymphocytes, after incubation of PBMCs with serum samples. Cell-associated IgG antibodies were detected in no samples. Cytometric Bead Array PHA (4 µg/mL)-activated PBMCs (1 × 106/mL) were cultured for 3 days and the supernatants obtained were frozen until use. Measurement of IL-2, IL-4, IL-6, IL-10, IL-17, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ) was carried out according to the manufacturer’s protocol (BD Biosciences). Data were acquired using a FACS Canto. ELISA Serum levels of sCD25 were determined by enzyme-linked immunosorbent assay (ELISA; Thermo Fisher, Fredrick, MD). Statistical Analysis Statistical analyses were performed using GraphPad Prism software. Data normality was evaluated by Shapiro Wilk test. Groups were compared using the Χ2 test, or Wilcoxon signed rank test, and/or Mann-Whitney test as appropriate. Correlations were assessed using Spearman correlation test. A P value < .05 was considered statistically significant. RESULTS Clinical Characteristics of Study Population The characteristics of RSV-infected and healthy infants enrolled in this study are summarized in Table 1. The CDSS of all admitted patients was equal to or higher than 7. All admitted patients needed O2 supplementation. Those admitted to the intensive care unit (n = 3, 3.6%) required invasive mechanical ventilation for a median of 4.5 days (range 3.6–5.9 days) and received broad-spectrum antibiotic therapy. RSV-infected children showed a higher frequency of B cells (CD19+ cells) and a lower frequency of CD8+ T cells compared with HD. The frequency of CD4+ T cells was similar in both groups. CD4+ T Cells From RSV-Infected Infants Produce Low Amounts of IL-2 and Show a Decreased Proliferative Response We first analyzed whether the production of IL-2, which is mainly mediated by conventional CD4+ T cells [20], is reduced during severe RSV infection. PBMCs from RSV-infected infants and HD were stimulated for 5 hours with PMA/ionomycin in the presence of monensin, and IL-2 production was analyzed by intracellular staining and flow cytometry in gated CD4+ T cells. We observed a similar frequency of IL-2+CD4+ T cells in RSV-infected infants and HD; however, the mean fluorescence intensity (MFI) of IL-2 staining was significantly lower in infected patients (Figure 1A). Interestingly, an inverse correlation was found between disease severity (CDSS) and the MFI of IL-2 staining in activated CD4+ T cells from RSV-infected patients. Moreover, because young age represents one of the most important risk factors for the development of severe RSV disease [1], we reanalyzed our data by dividing patients into 2 groups; ≤6 months and >7 months. Both groups of patients showed a lower production of IL-2 compared with HD (Figure 1A, right). To confirm that CD4+ T cells from RSV-infected children actually produced low amounts of IL-2, we analyzed IL-2 production in the supernatants of PBMCs stimulated with PHA for 3 days, using a cytometric bead array. IL-2 levels were markedly lower in RSV-infected children compared with HD. Moreover, we found that cells from RSV-infected children produced lower amounts of IL-10 and IL-17. No significant differences were observed regarding the production of IL-4, IL-6, TNF-α, and IFN-γ (Figure 1B). Figure 1. View largeDownload slide CD4+ T cells from respiratory syncytial virus (RSV)-infected infants produce low amounts of interleukin-2 (IL-2) and show a limited proliferative response. A, Peripheral blood mononuclear cells (PBMCs) from healthy donors (HD; n = 8) and RSV-infected infants (n = 20) were stimulated with phorbol 12-myristate 13-acetate/ionomycin in the presence of monensin for 5 hours. Percentage of IL-2+CD4+ T cells and the mean fluorescence intensity (MFI) of IL-2 staining in the gate of IL-2+CD4+ T cells were analyzed by flow cytometry. The correlation between clinical disease severity score (CDSS) and the MFI of IL-2 staining is also shown (Spearman rank correlation test). Right, MFI of IL-2 staining in PBMCs from HD (n = 8) and RSV-infected infants >7 months (n = 6) and ≤6 months (n = 13). B, Levels of cytokines in the supernatant of phytohemagglutinin (PHA)-activated PBMCs after 3 days of culture, quantified by cytometric bead array (n = 12 in each group). C, PHA-activated PBMCs were cultured in the absence or presence of IL-2 (20 ng/mL) for 3 days (n = 12 for each group). Frequency of Ki-67+CD4+ T cells in the gate of CD4+ T cells was evaluated by flow cytometry. D, PBMCs were treated, or not, with IL-2 (20 ng/mL) for 30 minutes at 37°C (n = 10 for each group). Frequency of pSTAT5+CD4+ T cells was analyzed by flow cytometry. A (middle), C (right), and D (right), show representative experiments. Mean ± SEM of n donors; *P < .05, **P < .01, ***P < .001, ****P < .0001. Abbreviations: INF-γ, interferon-gamma; TNF-α, tumor necrosis factor-alpha. Figure 1. View largeDownload slide CD4+ T cells from respiratory syncytial virus (RSV)-infected infants produce low amounts of interleukin-2 (IL-2) and show a limited proliferative response. A, Peripheral blood mononuclear cells (PBMCs) from healthy donors (HD; n = 8) and RSV-infected infants (n = 20) were stimulated with phorbol 12-myristate 13-acetate/ionomycin in the presence of monensin for 5 hours. Percentage of IL-2+CD4+ T cells and the mean fluorescence intensity (MFI) of IL-2 staining in the gate of IL-2+CD4+ T cells were analyzed by flow cytometry. The correlation between clinical disease severity score (CDSS) and the MFI of IL-2 staining is also shown (Spearman rank correlation test). Right, MFI of IL-2 staining in PBMCs from HD (n = 8) and RSV-infected infants >7 months (n = 6) and ≤6 months (n = 13). B, Levels of cytokines in the supernatant of phytohemagglutinin (PHA)-activated PBMCs after 3 days of culture, quantified by cytometric bead array (n = 12 in each group). C, PHA-activated PBMCs were cultured in the absence or presence of IL-2 (20 ng/mL) for 3 days (n = 12 for each group). Frequency of Ki-67+CD4+ T cells in the gate of CD4+ T cells was evaluated by flow cytometry. D, PBMCs were treated, or not, with IL-2 (20 ng/mL) for 30 minutes at 37°C (n = 10 for each group). Frequency of pSTAT5+CD4+ T cells was analyzed by flow cytometry. A (middle), C (right), and D (right), show representative experiments. Mean ± SEM of n donors; *P < .05, **P < .01, ***P < .001, ****P < .0001. Abbreviations: INF-γ, interferon-gamma; TNF-α, tumor necrosis factor-alpha. Consistent with our observations indicating a deficient production of IL-2 in RSV-infected patients, we found that the proliferative response of CD4+ T cells induced by PHA, assessed by detecting Ki-67 antigen expression, was substantially lower in RSV-infected infants compared with HD. The addition of IL-2 significantly increased the proliferative response of CD4+ T cells; however, even in the presence of exogenous IL-2, the proliferative response of CD4+ T cells from RSV-infected infants was markedly lower compared with HD (Figure 1C). This suggests that factors other than IL-2 production may also compromise the expansion of T cells in RSV-infected infants. Because the activation of STAT5 is one of the earliest events in IL-2 signaling through the high affinity IL-2 receptor [21], we analyzed STAT5 phosphorylation in response to IL-2 stimulation. We found a similar pattern of phosphorylation in CD4+ T cells from both RSV-infected infants and HD (Figure 1D), suggesting that the IL-2-STAT5 pathway is preserved during infection. IL-2 Increases the Frequency of FOXP3+ Tregs in PBMCs From RSV-Infected Infants We have previously reported that severe RSV infection in infants is associated with a pronounced reduction in the frequency of circulating Tregs [9]. To explore whether exogenous IL-2 was able to restore the pool of Tregs, PBMCs were cultured with or without IL-2 for 24 hours, and the frequency of Tregs was then analyzed. IL-2 significantly increased the frequency of FOXP3+CD4+ T cells in both HD and RSV-infected infants. However, even after IL-2 treatment, the frequency of FOXP3+CD4+ T cells was shown to be significantly lower in RSV-infected infants compared with HD (Figure 2A). Reanalysis of the data by dividing patients according to their age into 2 groups (≤6 months and >7 months), revealed that IL-2 failed to normalize the frequency of FOXP3+CD4+ T cells in both groups of patients (Figure 2A, right). Moreover, as shown in Figure 2B, IL-2 treatment also increased Treg expression of the ectonucleotidase CD39, which hydrolyzes ATP into the immunosuppressive agent adenosine [22]. This suggests that the increased expression of FOXP3 in CD4+ T cells from RSV-infected infants induced by IL-2 was actually associated with a regulatory signature. Interestingly, we found a negative correlation between CDSS values and the frequency of FOXP3+CD4+ T cells in IL-2–treated PBMCs, suggesting that more severe disease is associated with a limited response to IL-2 (Figure 2C). Further confirming the compromise in the compartment of FOXP3+CD4+ T cells in RSV-infected infants, we found that the proliferative response of this cell subset in response to PHA stimulation, assessed either in the absence or presence of IL-2, was severely reduced (Figure 2D). Figure 2. View largeDownload slide Interleukin-2 (IL-2) increases the frequency of FOXP3+CD4+ T cells in peripheral blood mononuclear cells (PBMCs) from respiratory syncytial virus (RSV)-infected infants. A, PBMCs from healthy donors (HD; n = 8) and RSV-infected infants (n = 41) were stimulated, or not, with IL-2 (20 ng/mL) for 24 hours. Frequency of FOXP3+CD4+ T cells was analyzed by flow cytometry. Left, analysis of all patients; right, analysis of patients divided into 2 groups: >7 months (n = 19) and ≤6 months (n = 22). B, Frequency of CD39+FOXP3+CD4+ T cells in PBMCs from RSV-infected children cultured for 24 hours in the absence or presence of IL-2 (20 ng/mL, n = 18). C, Correlation between the frequency of FOXP3+CD4+ T cells in PBMCs from RSV-infected infants cultured for 24 hours with IL-2 (20 ng/mL) and clinical disease severity score (CDSS), analyzed by Spearman rank correlation test. D, PBMCs from HD and RSV-infected infants were activated with phytohemagglutinin (PHA; 4 µg/mL) and cultured in the absence or presence of IL-2 (20 ng/mL) for 3 days (n = 12 for each group). Frequency of Ki-67+FOXP3+CD4+ T cells was evaluated by flow cytometry. A (middle) and B (right), show representative experiments. A (left and right), B (left), and D, mean ± SEM of n donors; *P < .05, **P < .01, ***P < .001, ****P < .0001. Figure 2. View largeDownload slide Interleukin-2 (IL-2) increases the frequency of FOXP3+CD4+ T cells in peripheral blood mononuclear cells (PBMCs) from respiratory syncytial virus (RSV)-infected infants. A, PBMCs from healthy donors (HD; n = 8) and RSV-infected infants (n = 41) were stimulated, or not, with IL-2 (20 ng/mL) for 24 hours. Frequency of FOXP3+CD4+ T cells was analyzed by flow cytometry. Left, analysis of all patients; right, analysis of patients divided into 2 groups: >7 months (n = 19) and ≤6 months (n = 22). B, Frequency of CD39+FOXP3+CD4+ T cells in PBMCs from RSV-infected children cultured for 24 hours in the absence or presence of IL-2 (20 ng/mL, n = 18). C, Correlation between the frequency of FOXP3+CD4+ T cells in PBMCs from RSV-infected infants cultured for 24 hours with IL-2 (20 ng/mL) and clinical disease severity score (CDSS), analyzed by Spearman rank correlation test. D, PBMCs from HD and RSV-infected infants were activated with phytohemagglutinin (PHA; 4 µg/mL) and cultured in the absence or presence of IL-2 (20 ng/mL) for 3 days (n = 12 for each group). Frequency of Ki-67+FOXP3+CD4+ T cells was evaluated by flow cytometry. A (middle) and B (right), show representative experiments. A (left and right), B (left), and D, mean ± SEM of n donors; *P < .05, **P < .01, ***P < .001, ****P < .0001. Soluble CD25 Limits IL-2 Function During Severe RSV Infection Elevated concentrations of sCD25 are found in autoimmunity, cancer, and inflammatory conditions [20, 23]. Looking for factors that might affect the function of IL-2 in the course of RSV infection, we evaluated serum levels of sCD25 (IL-2 receptor α chain soluble form). It has been reported that serum from RSV-infected infants has increased amounts of sCD25 [24, 25]. In agreement with this observation, we found higher amounts of sCD25 in the serum from RSV-infected infants compared with HD (Figure 3A). However, no correlation was found between disease severity and sCD25 levels (not shown). Because the ability of sCD25 to inhibit IL-2 activity remains controversial [20, 26, 27], we evaluated the effect of recombinant sCD25 on the function of IL-2 in CD4+ T cells from RSV-infected infants. We found that both IL-2–dependant STAT5 phosphorylation and FOXP3 expression were significantly inhibited by the addition of recombinant sCD25 (Figure 3B and C). Consistent with this observation, we found that serum from RSV-infected infants, but not from HD, diminished the frequency of FOXP3+CD4+ T cells in PBMCs isolated from healthy adult donors, cultured with or without IL-2 (Figure 3D). Moreover, we found a significant positive correlation between the ability of serum from RSV-infected children to reduce the frequency of FOXP3+CD4+ T cells and the concentration of sCD25 in the serum (Figure 3D, right). Figure 3. View largeDownload slide Soluble CD25 (sCD25) interferes with interleukin-2 (IL-2) function in respiratory syncytial virus (RSV)-infected children. A, Levels of sCD25 in the serum from RSV-infected (n = 24) and healthy donors (HD; n = 21) were quantified by enzyme-linked immunosorbent assay (ELISA). B, C, Peripheral blood mononuclear cells (PBMCs) from RSV-infected infants were cultured with IL-2 (20 ng/mL) or IL-2 plus sCD25 (200 ng/mL) for 30 minutes (B) or 24 hours (C). The frequencies of pSTAT5+CD4+ T cells (B; n = 12) or FOXP3+CD4+ T cells (C; n = 7) were then analyzed by flow cytometry. D, PBMCs from healthy donors were incubated for 24 hours with or without serum from healthy (n = 9) or RSV-infected children (final dilution 1:10; n = 9), in the absence or presence of IL-2 (20 ng/mL). The frequency of FOXP3+CD4+ T cells was then analyzed by flow cytometry (left and middle). Right, correlation between the ability of serum from RSV-infected infants to reduce the frequency of FOXP3+CD4+ T cells and serum levels of sCD25 (Spearman rank correlation test). Fold decrease of FOXP3+CD4+ T-cell frequency was calculated as the ratio between the frequencies of FOXP3+CD4+ T cells cultured in the absence and presence of serum from RSV-infected children. B (right) and D (middle), show representative experiments. A, B (left), C, and D (left), mean ± SEM of n donors;*P < .05, ***P < .001. Figure 3. View largeDownload slide Soluble CD25 (sCD25) interferes with interleukin-2 (IL-2) function in respiratory syncytial virus (RSV)-infected children. A, Levels of sCD25 in the serum from RSV-infected (n = 24) and healthy donors (HD; n = 21) were quantified by enzyme-linked immunosorbent assay (ELISA). B, C, Peripheral blood mononuclear cells (PBMCs) from RSV-infected infants were cultured with IL-2 (20 ng/mL) or IL-2 plus sCD25 (200 ng/mL) for 30 minutes (B) or 24 hours (C). The frequencies of pSTAT5+CD4+ T cells (B; n = 12) or FOXP3+CD4+ T cells (C; n = 7) were then analyzed by flow cytometry. D, PBMCs from healthy donors were incubated for 24 hours with or without serum from healthy (n = 9) or RSV-infected children (final dilution 1:10; n = 9), in the absence or presence of IL-2 (20 ng/mL). The frequency of FOXP3+CD4+ T cells was then analyzed by flow cytometry (left and middle). Right, correlation between the ability of serum from RSV-infected infants to reduce the frequency of FOXP3+CD4+ T cells and serum levels of sCD25 (Spearman rank correlation test). Fold decrease of FOXP3+CD4+ T-cell frequency was calculated as the ratio between the frequencies of FOXP3+CD4+ T cells cultured in the absence and presence of serum from RSV-infected children. B (right) and D (middle), show representative experiments. A, B (left), C, and D (left), mean ± SEM of n donors;*P < .05, ***P < .001. Activated conventional CD4+ T cells and Tregs are the main sources of sCD25 [20]. Because severe RSV infection is associated with a marked depletion of Tregs [9], we speculated that conventional CD4+ T cells may be the primary source of sCD25. In fact, we found that FOXP3− CD4+ T cells from RSV-infected infants express not only a higher frequency of CD25+ cells compared with HD, but also a great expression of CD25 measured as MFI (Figure 4A). A very low or negligible percentage of CD25+ cells was detected in CD8+ T cells, B cells, and monocytes (Figure 4B). Interestingly, we observed an inverse correlation between the frequency of peripheral blood FOXP3+ Tregs and the levels of serum sCD25 when HD and RSV-infected infants were analyzed collectively (Figure 4C), suggesting that sCD25 may actually act as a decoy receptor for IL-2 during severe RSV infection. Figure 4. View largeDownload slide High expression of CD25 in conventional CD4+ T cells from respiratory syncytial virus (RSV)-infected infants. A, Frequency and mean fluorescence intensity (MFI) of CD25 on gated FOXP3− CD4+ T cells from healthy donor (HD; n = 9) and RSV-infected infants (n = 21), evaluated by flow cytometry. B, Percentage of CD25 on gated CD4+, CD8+, CD19+, and CD14+ cells from RSV-infected infants (n = 15). C, Correlation between the frequency of FOXP3+CD4+ T cells and the serum levels of sCD25 in the group of healthy and RSV-infected children analyzed together by Spearman rank correlation coefficient test. A (right), representative experiment. A (left and middle) and B, mean ± SEM of n donors; **P < .01, ****P < .0001. Figure 4. View largeDownload slide High expression of CD25 in conventional CD4+ T cells from respiratory syncytial virus (RSV)-infected infants. A, Frequency and mean fluorescence intensity (MFI) of CD25 on gated FOXP3− CD4+ T cells from healthy donor (HD; n = 9) and RSV-infected infants (n = 21), evaluated by flow cytometry. B, Percentage of CD25 on gated CD4+, CD8+, CD19+, and CD14+ cells from RSV-infected infants (n = 15). C, Correlation between the frequency of FOXP3+CD4+ T cells and the serum levels of sCD25 in the group of healthy and RSV-infected children analyzed together by Spearman rank correlation coefficient test. A (right), representative experiment. A (left and middle) and B, mean ± SEM of n donors; **P < .01, ****P < .0001. DISCUSSION Our results suggest that both the production and function of IL-2 is compromised in the course of severe RSV infection in infants. We found that conventional CD4+ T cells from RSV-infected infants produce low amounts of IL-2. Moreover, we observed that exogenous IL-2 was unable to fully restore IL-2–dependent functions such as the proliferative response of CD4+ T cells and the expansion of Tregs. These observations suggest that different mechanisms contribute to limiting the function of IL-2 in the scenario of RSV disease. Interestingly, this phenomenon may contribute to an explanation of not only the depletion of circulating Tregs, but also an essential feature of RSV infection, that is its inability to promote a robust memory T-cell response [2, 3, 28, 29]. In this regard, it should be noted that IL-2 is required for the effective generation of effector and memory CD8+ T cells [30]. Moreover, it has been reported that the impaired effector and memory function of CD8+ T cells, as well as the antibody response observed in RSV-infected animals, were markedly improved by administration of IL-2, and these effects were associated with reduced weight loss and illness in challenged mice [31]. A reduced capacity of conventional CD4+ T cells to produce IL-2 has been previously described in autoimmunity and infectious diseases. Autoimmune diseases such as type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosus are associated with defective ability of CD4+ T cells to secrete IL-2 [32–35]. This defect appears to explain the decrease in Tregs, favoring the expansion of autoreactive T cells [32]. On the other hand, observations made in experimental models of infections induced by Toxoplasma gondi, Listeria monocytogenes, and vaccinia virus have shown that the acute infection phase is associated with a limited production of IL-2, resulting in a diminished Treg frequency [36, 37]. Regarding RSV infection, previous studies suggested that the production of IL-2 by CD4+ T cells may be limited. Using PBMCs from adults who have been naturally and recurrently exposed to RSV and IAV, Fleming and coworkers reported that in vitro exposure to RSV resulted in reduced production of IL-2 and a low lymphocyte proliferative response, compared to IAV-stimulated cells, although PBMCs from adult donors expressed a similar frequency of specific T lymphocytes in response to both viruses [38]. Moreover, we previously reported that activated CD4+ T cells are permissive to RSV infection, and also that infection promotes a marked inhibition of IL-2 production [39]. IL-2 availability during RSV infection may be limited, not only by decreased production but also by the high systemic levels of sCD25. Consistent with previous studies [24, 25], we found high levels of sCD25 in the serum from RSV-infected infants. Elevated serum concentrations of sCD25 have been reported in inflammatory conditions [20, 23]. Activated conventional T cells, Tregs, and dendritic cells can release sCD25 by proteolytic cleavage of surface CD25 [20]. Our observations show a great expression of CD25 on conventional CD4+ T cells from RSV-infected infants, suggesting that they may be the main source of sCD25. Regarding the biological significance of sCD25, it appears to compete with activated T cells for IL-2 binding, thereby reducing T-cell proliferation [20, 26]. However, it has also been reported that sCD25 may enhance the biological activity of IL-2. In fact, by forming a complex with IL-2, sCD25 has been shown to enhance IL-2–mediated phosphorylation of STAT5 in CD4+ T cells, promoting their differentiation into inducible FOXP3+ Tregs [27]. These observations suggest that sCD25 may either decrease or enhance the biological activity of IL-2, depending on the experimental setting. Our observations that sCD25 partially inhibits the ability of IL-2 to induce both STAT5 phosphorylation and FOXP3 expression in CD4+ T cells from RSV-infected infants, suggest that sCD25 acts as a decoy receptor for IL-2 during severe RSV disease. We observed that IL-2 significantly increased the proliferative response and the expression of FOXP3 in CD4+ T cells from RSV-infected infants. However, even in the presence of IL-2, both the proliferation rate of CD4+ T cells and the expression of FOXP3 remained substantially lower in CD4+ T cells from RSV-infected infants compared with HD. This suggests that factors other than IL-2 availability may compromise the activity of IL-2 during severe RSV infection. In contrast to other acute viral infections such as those produced by measles virus and cytomegalovirus, RSV infection is not associated with a generalized immunologic hyporesponsiveness [2, 40]. On the other hand, our results showing that IL-2 induced a similar pattern of STAT5 phosphorylation in CD4+ T cells from RSV-infected infants and HD suggest that signaling through the IL-2 receptor is preserved. Several factors may account for the limited activity of IL-2 in the course of RSV infection. Mediators such as IL-4, TGF-β, type I interferons, and prostaglandin E2 are produced at high levels in the context of RSV infection, all of them being capable of damping the function of IL-2 [41–46]. All patients recruited in our study required hospitalization and supplemental oxygen. We analyzed the impact of disease severity on IL-2 production and IL-2 ability to restore the pool of FOXP3+CD4+ T cells. This analysis revealed a significant negative correlation for both parameters, suggesting that patients with more severe disease produce lower levels of IL-2 and show a limited response to IL-2. It should be noted that although IL-2 failed in vitro to fully restore the function of CD4+ T cells from RSV-infected infants, it significantly improved both the proliferative response of CD4+ T cells and the frequency of FOXP3+CD4+ T cells. Interestingly, observations made in experimental models of RSV infection have shown that IL-2 administration improves disease outcome in RSV-challenged mice [31]. Proof-of-concept clinical trials have shown that, at low doses, IL-2 improves autoimmune and inflammatory conditions [32]. Further studies are needed to define whether IL-2 could represent a useful therapeutic tool in severe RSV infection. Notes Acknowledgments. We thank all the team members of the Hospital General de Niños Pedro de Elizalde. Most of all, we are indebted to all the participating children and their families. Financial support. This work was supported by grants from the Fondo Nacional para la Investigacion Cientifica y Tecnologica de Argentina (PIDC 0010-2015 to J. G., PMO BID PICT 2014-1578 and PMO BID PICT 2016-0444 to L. A.), and Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 2015-2017-0223 to L. A.). Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Presented in part: RSV Vaccines For The World Conference 2017–4th RESVINET Meeting, Málaga, Spain, 29 November–1 December 2017. References 1. Shi T, McAllister DA, O’Brien KL, et al.   Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study. Lancet  2017; 390: 946– 58. Google Scholar CrossRef Search ADS PubMed  2. Openshaw PJM, Chiu C, Culley FJ, Johansson C. Protective and harmful immunity to RSV infection. Annu Rev Immunol  2017; 35: 501– 32. Google Scholar CrossRef Search ADS PubMed  3. Arruvito L, Raiden S, Geffner J. Host response to respiratory syncytial virus infection. Curr Opin Infect Dis  2015; 28: 259– 66. Google Scholar CrossRef Search ADS PubMed  4. Campbell DJ, Koch MA. Phenotypical and functional specialization of FOXP3+ regulatory T cells. Nat Rev Immunol  2011; 11: 119– 30. Google Scholar CrossRef Search ADS PubMed  5. Belkaid Y, Rouse BT. Natural regulatory T cells in infectious disease. Nat Immunol  2005; 6: 353– 60. Google Scholar CrossRef Search ADS PubMed  6. Lee DC, Harker JA, Tregoning JS, et al.   CD25+ natural regulatory T cells are critical in limiting innate and adaptive immunity and resolving disease following respiratory syncytial virus infection. J Virol  2010; 84: 8790– 8. Google Scholar CrossRef Search ADS PubMed  7. Loebbermann J, Thornton H, Durant L, et al.   Regulatory T cells expressing granzyme B play a critical role in controlling lung inflammation during acute viral infection. Mucosal Immunol  2012; 5: 161– 72. Google Scholar CrossRef Search ADS PubMed  8. Krishnamoorthy N, Khare A, Oriss TB, et al.   Early infection with respiratory syncytial virus impairs regulatory T cell function and increases susceptibility to allergic asthma. Nat Med  2012; 18: 1525– 30. Google Scholar CrossRef Search ADS PubMed  9. Raiden S, Pandolfi J, Payasliàn F, et al.   Depletion of circulating regulatory T cells during severe respiratory syncytial virus infection in young children. Am J Respir Crit Care Med  2014; 189: 865– 8. Google Scholar CrossRef Search ADS PubMed  10. Sakaguchi S, Miyara M, Costantino CM, Hafler DA. FOXP3+ regulatory T cells in the human immune system. Nat Rev Immunol  2010; 10: 490– 500. Google Scholar CrossRef Search ADS PubMed  11. Nishikawa H, Sakaguchi S. Regulatory T cells in tumor immunity. Int J Cancer  2010; 127: 759– 67. Google Scholar PubMed  12. Wing K, Sakaguchi S. Regulatory T cells exert checks and balances on self tolerance and autoimmunity. Nat Immunol  2010; 11: 7– 13. Google Scholar CrossRef Search ADS PubMed  13. Wohlfert E, Belkaid Y. Plasticity of T reg at infected sites. Mucosal Immunol  2010; 3: 213– 5. Google Scholar CrossRef Search ADS PubMed  14. Lühn K, Simmons CP, Moran E, et al.   Increased frequencies of CD4+ CD25(high) regulatory T cells in acute dengue infection. J Exp Med  2007; 204: 979– 85. Google Scholar CrossRef Search ADS PubMed  15. Giamarellos-Bourboulis EJ, Raftogiannis M, Antonopoulou A, et al.   Effect of the novel influenza A (H1N1) virus in the human immune system. PLoS One  2009; 4: e8393. Google Scholar CrossRef Search ADS PubMed  16. Chinen T, Kannan AK, Levine AG, et al.   An essential role for the IL-2 receptor in Tregcell function. Nat Immunol  2016; 17: 1322– 33. Google Scholar CrossRef Search ADS PubMed  17. Hall CB, Walsh EE, Long CE, Schnabel KC. Immunity to and frequency of reinfection with respiratory syncytial virus. J Infect Dis  1991; 163: 693– 8. Google Scholar CrossRef Search ADS PubMed  18. Mella C, Suarez-Arrabal MC, Lopez S, et al.   Innate immune dysfunction is associated with enhanced disease severity in infants with severe respiratory syncytial virus bronchiolitis. J Infect Dis  2013; 207: 564– 73. Google Scholar CrossRef Search ADS PubMed  19. Mejias A, Dimo B, Suarez NM, et al.   Whole blood gene expression profiles to assess pathogenesis and disease severity in infants with respiratory syncytial virus infection. PLoS Med  2013; 10: e1001549. Google Scholar CrossRef Search ADS PubMed  20. Boyman O, Sprent J. The role of interleukin-2 during homeostasis and activation of the immune system. Nat Rev Immunol  2012; 12: 180– 90. Google Scholar CrossRef Search ADS PubMed  21. Burchill MA, Yang J, Vogtenhuber C, Blazar BR, Farrar MA. IL-2 receptor beta-dependent STAT5 activation is required for the development of Foxp3+ regulatory T cells. J Immunol  2007; 178: 280– 90. Google Scholar CrossRef Search ADS PubMed  22. Cook L, Munier CML, Seddiki N, et al.   Circulating gluten-specific FOXP3+CD39+ regulatory T cells have impaired suppressive function in patients with celiac disease. J Allergy Clin Immunol  2017; 140: 1592– 603.e8. Google Scholar CrossRef Search ADS PubMed  23. Rubin LA, Nelson DL. The soluble interleukin-2 receptor: biology, function, and clinical application. Ann Intern Med  1990; 113: 619– 27. Google Scholar CrossRef Search ADS PubMed  24. Smyth RL, Fletcher JN, Thomas HM, Hart CA, Openshaw PJ. Respiratory syncytial virus and wheeze. Lancet  1999; 354: 1997– 8. Google Scholar CrossRef Search ADS PubMed  25. Alonso Fernández J, Roine I, Vasquez A, Cáneo M. Soluble interleukin-2 receptor (sCD25) and interleukin-10 plasma concentrations are associated with severity of primary respiratory syncytial virus (RSV) infection. Eur Cytokine Netw  2005; 16: 81– 90. Google Scholar PubMed  26. von Bergwelt-Baildon MS, Popov A, Saric T, et al.   CD25 and indoleamine 2,3-dioxygenase are up-regulated by prostaglandin E2 and expressed by tumor-associated dendritic cells in vivo: additional mechanisms of T-cell inhibition. Blood  2006; 108: 228– 37. Google Scholar CrossRef Search ADS PubMed  27. Yang ZZ, Grote DM, Ziesmer SC, et al.   Soluble IL-2Rα facilitates IL-2-mediated immune responses and predicts reduced survival in follicular B-cell non-Hodgkin lymphoma. Blood  2011; 118: 2809– 20. Google Scholar CrossRef Search ADS PubMed  28. Meng J, Stobart CC, Hotard AL, Moore ML. An overview of respiratory syncytial virus. PLoS Pathog  2014; 10: e1004016. Google Scholar CrossRef Search ADS PubMed  29. Christiaansen AF, Knudson CJ, Weiss KA, Varga SM. The CD4 T cell response to respiratory syncytial virus infection. Immunol Res  2014; 59: 109– 17. Google Scholar CrossRef Search ADS PubMed  30. Williams MA, Tyznik AJ, Bevan MJ. Interleukin-2 signals during priming are required for secondary expansion of CD8+ memory T cells. Nature  2006; 441: 890– 3. Google Scholar CrossRef Search ADS PubMed  31. Chang J, Choi SY, Jin HT, Sung YC, Braciale TJ. Improved effector activity and memory CD8 T cell development by IL-2 expression during experimental respiratory syncytial virus infection. J Immunol  2004; 172: 503– 8. Google Scholar CrossRef Search ADS PubMed  32. Klatzmann D, Abbas AK. The promise of low-dose interleukin-2 therapy for autoimmune and inflammatory diseases. Nat Rev Immunol  2015; 15: 283– 94. Google Scholar CrossRef Search ADS PubMed  33. Kammer GM. Altered regulation of IL-2 production in systemic lupus erythematosus: an evolving paradigm. J Clin Invest  2005; 115: 836– 40. Google Scholar CrossRef Search ADS PubMed  34. Zier KS, Leo MM, Spielman RS, Baker L. Decreased synthesis of interleukin-2 (IL-2) in insulin-dependent diabetes mellitus. Diabetes  1984; 33: 552– 5. Google Scholar CrossRef Search ADS PubMed  35. Kitas GD, Salmon M, Farr M, Gaston JS, Bacon PA. Deficient interleukin 2 production in rheumatoid arthritis: association with active disease and systemic complications. Clin Exp Immunol  1988; 73: 242– 9. Google Scholar PubMed  36. Benson A, Murray S, Divakar P, et al.   Microbial infection-induced expansion of effector T cells overcomes the suppressive effects of regulatory T cells via an IL-2 deprivation mechanism. J Immunol  2012; 188: 800– 10. Google Scholar CrossRef Search ADS PubMed  37. Oldenhove G, Bouladoux N, Wohlfert EA, et al.   Decrease of FOXP3+ Treg cell number and acquisition of effector cell phenotype during lethal infection. Immunity  2009; 31: 772– 86. Google Scholar CrossRef Search ADS PubMed  38. Fleming EH, Ochoa EE, Nichols JE, O’Banion MK, Salkind AR, Roberts NJJr. Reduced activation and proliferation of human lymphocytes exposed to respiratory syncytial virus compared to cells exposed to influenza virus. J Med Virol  2018; 90: 26– 33. Google Scholar CrossRef Search ADS PubMed  39. Raiden S, Sananez I, Remes-Lenicov F, et al.   Respiratory syncytial virus (RSV) infects CD4+ T cells: frequency of circulating CD4+ RSV+ T cells as a marker of disease severity in young children. J Infect Dis  2017; 215: 1049– 58. Google Scholar CrossRef Search ADS PubMed  40. Bakaletz LO. Viral-bacterial co-infections in the respiratory tract. Curr Opin Microbiol  2017; 35: 30– 5. Google Scholar CrossRef Search ADS PubMed  41. Golding A, Rosen A, Petri M, Akhter E, Andrade F. Interferon-alpha regulates the dynamic balance between human activated regulatory and effector T cells: implications for antiviral and autoimmune responses. Immunology  2010; 131: 107– 17. Google Scholar PubMed  42. Martinez OM, Gibbons RS, Garovoy MR, Aronson FR. IL-4 inhibits IL-2 receptor expression and IL-2-dependent proliferation of human T cells. J Immunol  1990; 144: 2211– 5. Google Scholar PubMed  43. Das L, Levine AD. TGF-beta inhibits IL-2 production and promotes cell cycle arrest in TCR-activated effector/memory T cells in the presence of sustained TCR signal transduction. J Immunol  2008; 180: 1490– 8. Google Scholar CrossRef Search ADS PubMed  44. Katamura K, Shintaku N, Yamauchi Y, et al.   Prostaglandin E2 at priming of naive CD4+ T cells inhibits acquisition of ability to produce IFN-gamma and IL-2, but not IL-4 and IL-5. J Immunol  1995; 155: 4604– 12. Google Scholar PubMed  45. Wang L, van Panhuys N, Hu-Li J, Kim S, Le Gros G, Min B. Blimp-1 induced by IL-4 plays a critical role in suppressing IL-2 production in activated CD4 T cells. J Immunol  2008; 181: 5249– 56. Google Scholar CrossRef Search ADS PubMed  46. McKarns SC, Schwartz RH, Kaminski NE. Smad3 is essential for TGF-beta 1 to suppress IL-2 production and TCR-induced proliferation, but not IL-2-induced proliferation. J Immunol  2004; 172: 4275– 84. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

The Journal of Infectious DiseasesOxford University Press

Published: Mar 28, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off