Skip to main content
NCBI home page
The Journal of Infectious Diseases logoLink to The Journal of Infectious Diseases
. 2016 Oct 26;215(3):420–430. doi: 10.1093/infdis/jiw511

Selective Destruction of Interleukin 23–Induced Expansion of a Major Antigen–Specific γδ T-Cell Subset in Patients With Tuberculosis

Hongbo Shen 1,a,✉, Jin Gu 2,a, Heping Xiao 2,a, Shanshan Liang 1, Enzhuo Yang 1, Rui Yang 1, Dan Huang 4,5, Crystal Chen 4,5, Feifei Wang 3, Ling Shen 4,5, Zheng W Chen 4,5,6
PMCID: PMC5853380  PMID: 27789724

Abstract

A loss of antigen-specific T-cell responses due to defective cytokine signaling during infections has not been reported. We hypothesize that tuberculosis can destroy signaling effects of selective cytokine(s) and induce exhaustion of antigen-specific T cells. To test this hypothesis, mechanistic studies were performed to examine whether and how tuberculosis blocked interleukin 23 (IL-23) and interleukin 2 (IL-2) signaling effects on a major human γδ T-cell subpopulation, phosphoantigen HMBPP–specific Vγ2Vδ2 T cells. IL-23 and IL-2 significantly expanded HMBPP-stimulated Vγ2Vδ2 T cells from subjects with latent tuberculosis infection, and IL-2 synergized the effect of IL-23. IL-23–induced expansion of Vγ2Vδ2 T cells involved STAT3. Surprisingly, patients with tuberculosis exhibited a selective destruction of IL-23–induced expansion of these cells. The tuberculosis-driven destruction of IL-23 signaling coincided with decreases of expression and phosphorylation of STAT3. Interestingly, impairing of STAT3 was linked to marked increases in the microRNAs (miRNAs) hsa-miR-337-3p and hsa-miR-125b-5p in Vγ2Vδ2 T cells from patients with tuberculosis. Downregulation of hsa-miR-337-3p and hsa-miR-125b-5p by miRNA sponges improved IL-23–mediated expansion of Vγ2Vδ2 T cells and restored the ability of these cells to produce anti–tuberculosis cytokines. These results support our hypothesis that tuberculosis can selectively impair a cytokine effect while sparing another and can induce exhaustion of T cells in response to the respective cytokine.

Keywords: tuberculosis, T-cell exhaustion, Vγ2Vδ2 T cells, cytokine signaling, JAK2/STAT3, miRNA

While T cells play important roles in immunity against infections with intracellular pathogens, chronic antigen exposures in certain infections have been shown to induce exhaustion of antigen-specific T cells [1–3]. Immune interventions for reversing the exhaustion pathway, such as suppression of programmed cell death 1 (PD-1), have been proven somehow successful in certain cancers [4] and infections [5]. However, it is still unclear whether impaired cytokine signaling can cause a loss of antigen-specific T-cell responses.

Tuberculosis has recently become the top killer among infectious diseases, owing to the epidemic of human immunodeficiency virus infection and the emergence of multidrug-resistant M. tuberculosis [6]. It is important to note that the majority of humans exposed to M. tuberculosis can develop protective immunity against primary infection and that only a small portion of remaining bacilli are needed to cause latent tuberculosis infection (LTBI) [7]. While cell-mediated immunity appears to be important for antituberculosis immunity, the protection may be linked to αβ T-cell populations and unconventional T cells, including the phosphoantigen-specific primate γδ T-cell subset [8, 9]. It has recently been postulated that M. tuberculosis infection may exhaust protective antigen-specific T-cell populations during the development of tuberculosis. While data for tuberculosis exhaustion of antigen-specific T cells are inconsistent [3, 10–12], we hypothesize that tuberculosis can impair signaling effects of selective cytokine(s) and blunt or exhaust antigen-specific T-cell responses to the respective cytokine.

The Vγ2Vδ2 T-cell subpopulation exists only in primates and constitutes 60%–95% of the total number of circulating human γδ T cells [13, 14]. Vγ2Vδ2 T cells are the sole γδ T-cell subset capable of recognizing phosphoantigens, such as isoprenoid pyrophosphate and (E)-4-hydroxy-3-methyl-but-enyl pyrophosphate (HMBPP), produced by M. tuberculosis and other selected pathogens [15]. Expansion and differentiation of Vγ2Vδ2 T cells by phosphoantigen plus interleukin 2 (IL-2) therapy administered during the early phase of M. tuberculosis infection can confer resistance against tuberculosis in nonhuman primates [16]. Immunologically, while IL-2, interleukin 15, or interleukin 21 (IL-21) can expand HMBPP-stimulated Vγ2Vδ2 T cells [17], T-helper type 17 (Th17)–related cytokines, especially interleukin 23 (IL-23), can help induce recall-like expansion and the effector function of HMBPP-specific Vγ2Vδ2 T cells [18]. Given the possibility that the frequency of cells in the HMBPP-specific Vγ2Vδ2 T-cell subset is higher during mycobacterial infection than the frequencies of single M. tuberculosis peptide–specific CD4+ or CD8+ T-cell subpopulations [15], we presume that tuberculosis-induced exhaustion of Vγ2Vδ2 T cells at the cytokine level would be more readily seen than in other cells in patients with tuberculosis [19–21]. Since IL-2 or IL-23 could expand HMBPP-specific Vγ2Vδ2 T cells, we sought to determine whether and how tuberculosis could destroy the effects of IL-2 and IL-23 signaling and induce exhaustion of this T-cell subpopulation.

MATERIALS AND METHODS

Ethics Statement

The protocols for use of human blood samples for in vitro experimental procedures were evaluated and approved by the institutional review boards for human subjects research and institutional biosafety committees at Institut Pasteur of Shanghai, Shanghai Pulmonary Hospital, and the University of Illinois–Chicago College of Medicine. All studies were consistent with guidelines of Office for Human Research Protections. All subjects are adults and signed written informed consents.

Human Subjects

Patients with tuberculosis were recruited at Shanghai Pulmonary Hospital (Shanghai, China; Table 1). Age- and sex-matched volunteer healthy controls (HCs) and subject with LTBI were recruited into this study (Table 1).

Table 1.

Demographic and Clinical Characteristics of Patients With Tuberculosis, Patients With Latent Mycobacterium tuberculosis (LTBI) Infections and Healthy Controls

Characteristic Patients With TB (n = 51) Subjects With LTBI (n = 46) Healthy Controls (n = 48)
Age, y, mean ± SD 37.6 ± 14.1 38.5 ± 12.7 41.7 ± 14.5
Sex
 Female 25 21 23
 Male 26 25 25
T.SPOT.TB test result
 Positive 51 46 0
 Negative 0 0 48
Pulmonary tuberculosis 51 … …
Treatment time <1 mo 40 … …
Short-term–treated tuberculosis (≥2 mo) 10 … …
Sputum AFB test result
 Positive 14 … …
 Negative 37 … …
Pulmonary cavity
 Yes 13 … …
 No 38 … …
Body mass index,a mean ± SD 19.92 ± 1.55 22.69 ± 3.23 21.89 ± 2.43
Open in a new tab

Healthy controls had records of BCG vaccination at birth, without any history and evidence of tuberculosis or LTBI. LTBI was confirmed on the basis of a positive result of a T.Spot.TB test (Oxford Immunotec; Oxford, United Kingdom) and negative radiographic findings, without any clinical symptoms/signs of tuberculosis. Among all participants, there was no evidence for acute infection with hepatitis B virus, hepatitis C virus, human immunodeficiency virus, and other infectious diseases or cancers.

Abbreviation: AFB, acid-fast bacilli.

a Calculated as the weight in kilograms divided by the height in meters squared.

Peripheral Blood Mononuclear Cell (PBMC) Isolation and Expansion of Vγ2Vδ2 T Cells by HMBPP Plus IL-2 or IL-23

PBMCs were isolated from ethylenediaminetetraacetic acid–treated blood, using a technique previously described by us [22]. Cells were cultured in the absence or presence of 80 ng/mL of HMBPP with 5 ng/mL hIL-2 (Sigma-Aldrich) or 100 ng/mL hIL-23 (R&D Systems, Minneapolis, MN). In special treatments, the following antibodies were used: anti-human IL-2 (clone MQ1-17H12; BD), mouse anti-human PD-1 (clone EH12.2H7; Biolegend), mouse immunoglobulin G2a (IgG2a), κ chain (Isotype, BD); goat anti-human PD-1 (R&D Systems); and goat IgG (Isotype, R&D Systems). The PD-1 antibodies can restore PD-1–associated exhaustion of T cells [23, 24]. Cucurbitacin I (CUI; Sigma-Aldrich) was used for STAT3-targeted blockade experiments.

Flow Cytometry

Flow cytometry was performed as we previously described [18, 22]. The following antibodies were used: anti-CD3–PB (clone SP34-2; BD), anti-Vγ2–FITC (clone 7A5; Pierce), anti-interferon γ (IFN-γ)–Brilliant Violet 711ۛ (clone 4S.B3; Biolegend), anti-IL-23R–PE (clone 218213; R&D Systems), and anti-interleukin 17A (IL-17A)–allophycocyanin (clone eBio64DEC17; eBioscience). After staining, cells were fixed and subjected to flow cytometry. Lymphocytes were gated on the basis of forward and side scatter, and at least 40 000 gated events were analyzed using Summit Data Acquisition and Analysis Software (Dako Cytomation).

Two antibodies specific for STAT3 phosphorylation sites pS727 and pY705 (mouse anti-Stat3 [pS727]–PE [clone 49/p-Stat3; BD] and mouse anti-Stat3 [pY705]–Alexa Fluor 647 [clone 4/p-Stat3; BD], respectively) were used to determine the expression of phosphorylated STAT3, in accordance with the manufacturer's instructions.

Measurement of Cytokine Production

PBMCs from patients with tuberculosis, patients with LTBI, or HCs were cultured for 7 days with medium, HMBPP, HMBPP plus IL-2, or HMBPP plus IL-23. The supernatants were collected on day 7 to measure the level of IFN-γ, tumor necrosis factor α (TNF-α), IL-22, or IL-17A by enzyme-linked immunosorbent assays (ELISA; R&D Systems), using a protocol we previously described [25].

Real-Time Quantitative Polymerase Chain Reaction (qPCR) Analysis for Quantification of Gene Expression

Total RNA was extracted from cultured PBMCs or freshly isolated Vδ2+ T cells, using RNA column enrichment procedures according to the manufacturer's protocol (Zymo Research, CA). Vδ2+ T cells were isolated and enriched from fresh PBMC using undirected MACS method according to the manufacturer's instructions. Purified cell population contained >95% of viable Vδ2+ T cells as assessed by flow cytometry (data not shown). RNA were reverse-transcribed into complementary DNA (cDNA) using the kit from Vazyme Biotech (Nanjing, China). The cDNA was added into 2X Syber Green Mastermix (Toyobo, Shanghai, China) to amplify target gene fragment in triplicate reactions for each gene. Negative controls (no cDNA) were prepared in parallel with each run. Sequences of primers were as follows: IL23R, 5′-CTGGCAGCCTTGGAGTT-3′ and 5′-CCCTGTAGAGATGGAAGCAACTG-3′; STAT3, 5′-TTTGAGACCGAGGTGTATCACC-3′ and 5′-GGTCAGCATGTTGTACCACAGG-3′; SOCS3, 5′-TTCTGATCCGCGACAGCTC-3′ and 5′-TGCAGAGAGAAGCTGCCCC-3′; Vγ2, 5′-TCAACTGGTACAGGAAGACCCAAG-3′ and 5′-TGACAGCATTGTACTTCCCACTGG-3′, IFNG, 5′-GCAGGTCATTCAGATGTAGCGG-3′ and 5′-TGTCTT CCTTGATGGTCTCCACAC-3′; IL17A, 5′-CAATCCCACGAAATCCAGGATG-3′ and 5′-GGTGGAGATTCCAAGGTGAGG-3′; and β-actin, 5′-GCCCTGAGGCACTCTTCCA-3′ and 5′-TGTTGGCGTAC AGGTCTTTGC-3′.

The β-actin was used as internal control gene for normalization.

Real-Time qPCR for Quantification of microRNA (miRNA) Expression

Total miRNA was extracted from freshly isolated Vδ2 T cells, using the Quick-RNA MicroPrep kit according to the manufacturer's instructions (Zymo Research, CA). Relevant complementary DNA (cDNA) was synthesized with miRcute miRNA First-Strand cDNA Synthesis Kit (Tiangen, Beijing, China). qPCR primers of miR-223-3p, miR-106a-5p, miR-93-5, miR-124-3p, miR-17-5p, miR-223-3p, miR-17-5p, miR-337-3p, miR-125b-5p, U6, and 5s were also purchased from Tiangen. Real-time qPCR was performed by use of the miRcute miRNA q-PCR Detection kit (SYBR Green). The relative expression level of miRNA was normalized by U6 or 5s, and the data from HCs were used as calibrator.

Lentiviral Transduction of Human PBMCs for Silencing miRNA and Restoring IL-23–Associated Expansion of Vγ2Vδ2+ T Cells

Lentiviral vectors containing a gene encoding enhanced green fluorescence protein (EGFP) were used to develop miRNA constructs to achieve high-efficiency introduction of miRNA into cells. Recombined pCMV-LV-GV234-GFP vectors bearing the has-miR-125b-5p (MIMAT0000754) gene (LV-has-125b-5p), the has-miR-337-3p (MIMAT0000423) gene (LV-has-337-3p), or a scrambled control sequence (LV-GV234) were constructed by Genechem (Shanghai). Lentivirus vectors were transduced into cells according to the company's instructions. Briefly, PBMCs isolated from patients with tuberculosis were seeded in plates and transduced with 5 μg/mL Polybrene (Cruz Biotechnology, CA). After incubation for 12 hours, medium was replaced by fresh medium supplemented with 80 ng/mL HMBPP and 100 ng/mL IL-23, and then cells were cultured for 7 days. Cultured cells were harvested and stained and then analyzed by flow cytometry. A fraction of cultured cells was collected and subjected to real-time qPCR for quantitation of Vδ2, IFN-γ, and IL-17A expression.

Statistical Analysis

Statistical analysis was done by using GraphPad Prism software (GraphPad Software, CA). Data were analyzed by the Student t test (parametric method) or by the Mann–Whitney test (nonparametric method). P values of < .05 were considered statistically significant.

RESULTS

IL-23 and IL-2 Signals Significantly Expanded HMBPP-Stimulated Vγ2Vδ2 T Cells From Subjects With LTBI, and IL-2 Facilitated IL-23–Mediated Expansion

In the current study, we hypothesize that tuberculosis can destroy a selected cytokine signal but spare another, leading to nonexpansion or exhaustion of antigen-stimulated T cells. To test this hypothesis, we used the HMBPP-specific Vγ2Vδ2 T-cell subset as a model system. We investigated whether tuberculosis could impair IL-23 or IL-2 signaling and lead to exhaustion of Vγ2Vδ2 T cells at the cytokine level. These 2 cytokines were selected for evaluation on the basis of the observations that IL-23 is involved in recall-like expansion of the primate Vγ2Vδ2 T-cell subset after infections or vaccination [18] and that IL-2 can act as a master T-cell growth factor [25–27]. As an initial effort, Vγ2Vδ2 T cells from subjects with LTBI were assessed for the ability to expand in coculture with IL-23 plus HMBPP (IL-23/HMBPP) or IL-2/HMBPP. While IL-23 and IL-2 signals each expanded HMBPP-stimulated Vγ2Vδ2 T cells in PBMC from subjects with LTBI (Figure 1A), endogenous IL-2 produced by IL-23/HMBPP–activated Vγ2Vδ2 T cells sustained the IL-23–induced expansion of the Vγ2Vδ2 T-cell subset (Figure 1B). Notably, blockade experiments revealed that anti-IL-2 neutralizing antibodies but not controls clearly reduced the magnitude of IL-23–induced expansion of HMBPP-stimulated Vγ2Vδ2 T cells (Figure 1B). Consistently, when these γδ T cells among PBMCs were transiently stimulated by HMBPP for only 3 hours to induce a suboptimal T-cell receptor activation signal, exogenous IL-2 synergized or enhanced IL-23–induced expansion of HMBPP-specific Vγ2Vδ2 T cells (Figure 1C). In contrast, IL-6 [28] and/or IL-1B [28] did not stimulate or synergize IL-23–induced expansion of Vγ2Vδ2 T cells (data not shown). Thus, these results suggest that IL-23 and IL-2 signals significantly expanded HMBPP-stimulated Vγ2Vδ2 T cells from subjects with LTBI and that IL-2 could facilitate IL-23–induced expansion of the phosphoantigen-specific γδ T-cell subset.

Figure 1.

Figure 1.

Open in a new tab

Interleukin 23 (IL-23) and interleukin 2 (IL-2) signals significantly expanded HMBPP-stimulated Vγ2Vδ2 T cells from subjects with latent tuberculosis infection (LTBI), and IL-2 facilitates the IL-23–mediated expansion. A, Representative flow cytometry histograms and bar graph of pooled data show that IL-23, as well as IL-2, reproducibly expanded Vγ2Vδ2 T cells from subjects with LTBI, on coculture with HMBPP. Peripheral blood mononuclear cells (PBMCs) from subjects with LTBI were cultured for 7 days with medium, HMBPP, HMBPP plus IL-2, and HMBPP plus IL-23 and then subjected to surface staining and flow cytometry. B, Endogenous IL-2 produced by IL-23/HMBPP-activated Vγ2Vδ2 T cells sustained IL-23–induced expansion of the Vγ2Vδ2 T-cell subset. Note that blockade experiments using anti-IL-2 neutralizing antibody but not those using controls clearly reduced the magnitude of IL-23–induced expansion of HMBPP-stimulated Vγ2Vδ2 T cells. C, The transient first signaling model of T-cell receptor stimulation revealed that exogenous IL-2 synergized or enhanced IL-23–induced expansion of HMBPP-specific Vγ2Vδ2 T cells. In the transient first signaling model, PBMCs were incubated with HMBPP for 3 hours, HMBPP was washed out, and IL-2 alone, IL-23 alone, or IL-23 with different concentrations of IL-2 (0.5 ng/mL and 0.05 ng/mL) were added in the culture. IL-23 could not effectively expand Vγ2Vδ2 T cells after transient HMBPP stimulation, but a low concentration of IL-2 (0.5 ng/mL) helped IL-23 expand transiently stimulated Vγ2Vδ2 T cells. Data are means ± standard errors of the mean derived from 10 subjects with LTBI in 3 independent experiments. *P < .05 and **P < .01.

Patients With Tuberculosis Exhibited Selective Impairment of the Signal Effect of IL-23 and Showed Exhaustion of the HMBPP-Specific Vγ2Vδ2 T-Cell Subpopulation in Response to IL-23

Since both IL-23 and IL-2 signals could expand the HMBPP-specific Vγ2Vδ2 T-cell subset during LTBI, we examined whether tuberculosis could selectively impair one of these two cytokine signals and lead to nonexpansion of Vγ2Vδ2 T cells in response to the respective cytokine plus HMBPP. Interestingly, patients with tuberculosis exhibited selective impairment of the effect of IL-23 on the HMBPP-specific Vγ2Vδ2 T-cell subset, since IL-23 but not IL-2 was unable to induce significant expansion of HMBPP-stimulated Vγ2Vδ2 T cells from patients with tuberculosis, compared with subjects with LTBI and BCG-vaccinated HCs (Figure 2A). In addition, IL-23/HMBPP costimulation of Vγ2Vδ2 T cells from patients with tuberculosis but not HCs failed to induce effector function for production of the antituberculosis cytokines IFN-γ, TNF-α, IL-17A, and IL-22 (Figure 2B). Furthermore, short-term treatment of patients with tuberculosis with tuberculosis drugs could partially restore the ability of Vγ2Vδ2 T cells to mount an effector function for cytokine production in response to IL-23, although such initial therapy did not effectively reconstitute the expansion capability of Vγ2Vδ2 T cells in response to IL-23/HMBPP cocultures (Figure 2C). These results suggest that chronic tuberculosis could impair the signaling effect of one single cytokine but spare another, leading to nonexpansion or exhaustion of the HMBPP-specific Vγ2Vδ2 T-cell subpopulation in response to IL-23.

Figure 2.

Figure 2.

Open in a new tab

Patients with tuberculosis exhibited selective impairment of the effect of interleukin 23 (IL-23) signaling but not interleukin 2 (IL-2) signaling and showed exhaustion of the HMBPP-specific Vγ2Vδ2 T-cell subpopulation in response to IL-23. A, IL-23 could not expand HMBPP-stimulated Vγ2Vδ2 T cells from patients with tuberculosis, compared with those from subjects with latent tuberculosis infection (LTBI) and BCG-vaccinated healthy controls (HCs). Note that the effect of IL-2 signaling on γδ T cells from patients with tuberculosis was not impaired. Data are means ± standard errors of the mean (SEM), pooled from 40 subjects with LTBI, 40 HCs, and 40 patients with tuberculosis in 5 independent experiments. **P < .01. B, IL-23/HMBPP costimulation of Vγ2Vδ2 T cells from patients with tuberculosis but not HCs failed to induce an effector function for production of the anti-tuberculosis cytokines interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin 17A (IL-17A), and interleukin 22 (IL-22). Enzyme-linked immunosorbent assays were used to measure cytokines in culture supernatants of Vγ2Vδ2 T cells after stimulation for 3 days in peripheral blood mononuclear cells (PBMCs) treated with medium, HMBPP, HMBPP + IL-2, or HMBPP + IL-23. Data are means ± SEM, pooled from 43 subjects with LTBI, 40 HCs, and 45 patients with tuberculosis in 7 independent experiments. *P < .05 and **P < .01. C, Short-term (ie, 1-month) treatment of patients with tuberculosis with antituberculosis antibiotics did not effectively reconstitute the expansion capability of Vγ2Vδ2 T cells in response to IL-23/HMBPP in vitro (top panels), but such transient therapy partially restored the ability of HMBPP-specific Vγ2Vδ2 T cells to mount an effector function for cytokine production in response to IL-23 (lower panels). Data are means ± SEM, pooled from 20 patients with tuberculosis in 3 independent experiments. *P < .05, **P < .01, and ***P < .001. Abbreviation: NS, not significant.

IL-23/HMBPP–Induced Expansion of Vγ2Vδ2 T Cells Involves STAT3: Vγ2Vδ2 T Cells in Patients With Tuberculosis Showed Decreased Expression and Phosphorylation of STAT3 but Displayed Overexpression of Antagonizing SOCS3

We then sought to explore potential mechanisms underlying the destruction of IL-23–induced expansion of the HMBPP-specific Vγ2Vδ2 T-cell subset. We found that IL-23R expression in Vγ2Vδ2 T cells from patients with tuberculosis was detectable but lower than that for subjects with LTBI or HCs (Figure 3A and 3B). These levels of IL-23R expression did not appear to fully explain the impairment of the effects of IL-23 signaling on Vγ2Vδ2 T cells during tuberculosis. Moreover, exchange of antigen-presenting cells between subjects with LTBI and patients with tuberculosis could not reconstitute the ability of Vγ2Vδ2 T cells from patients with tuberculosis to undergo expansion in response to the IL-23/HMBPP costimulation (Figure 3C). Concurrently, we also found that anti-PD-1 antibodies from 2 resources capable of reversing αβ T-cell exhaustion [29, 30] could not rescue the expansion of Vγ2Vδ2 T cells from patients with tuberculosis in response to IL-23/HMBPP costimulation (Figure 3D).

Figure 3.

Figure 3.

Open in a new tab

Selective impairment of the effects of interleukin 23 (IL-23) signaling during tuberculosis could not be attributed to nonexpression of IL-23R, dysfunction of antigen-presenting cells (APCs), or regulation of the programmed cell death 1 (PD-1) pathway. A, IL-23R expression was detectable in the absence or presence of HMBPP/IL-23 costimulation of Vγ2Vδ2 T cells in peripheral blood mononuclear cells (PBMCs) from patients with tuberculosis, although the HMBPP/IL-23–driven expression of IL-23R in patients with tuberculosis was lower than that in subjects with latent tuberculosis infection (LTBI) or healthy controls (HCs). Messenger RNA (mRNA) extracted from treated PBMCs was used for complementary DNA synthesis and real-time quantitative polymerase chain reaction (qPCR) analysis of IL-23R gene expression, with β-actin as a control. Expression levels are shown as fold expression, verified by β-actin. Data are means ± standard errors of the mean (SEM), pooled from 10 subjects with LTBI, 10 HCs, and 10 patients with tuberculosis in 3 independent experiments. *P < .05. B, Representative flow cytometry–determined histograms and bar graph show that the HMBPP/IL-23–driven expression of IL-23R in patients with tuberculosis was lower than that in HCs (P = .0286). *P < .05. C, APCs from HCs could not reconstitute the ability of Vγ2Vδ2 T cells from patients with tuberculosis to undergo expansion in response to HMBPP/IL-23 costimulation. In contrast, APCs from patients with tuberculosis (TB) could still induce HMBPP/IL-23–induced expansion of Vγ2Vδ2 T cells from subjects with LTBI (LTBI), which was similar to findings in the setting of autologous APCs. Data are means ± SEM, pooled from 10 patients with tuberculosis and 10 subjects with LTBI in 3 independent experiments. **P < .01. D, Blockade experiments using anti-PD-1 antibody showed that PD-1 antibody could not rescue the nonexpansion of Vγ2Vδ2 T cells from patients with tuberculosis in response to IL-23/HMBPP costimulation. Two sources of anti-human PD-1 antibodies (αPD-1_1 [clone EH12.2H7; Biolegend] and αPD-1_2 [R&D]) or isotype control were added to PBMC culture in the presence of IL-23 plus HMBPP. These 2 anti-PD-1 antibodies were shown to blockade PD-1–mediated exhaustion of αβ T cells (data not shown) [23, 24, 43]. Data are means ± SEM, derived from 18 patients with tuberculosis in 3 independent experiments. **P < .01.

Next, we examined which activation molecules were involved in IL-23–induced expansion of γδ T cells and determined whether their dysfunction could be linked to the impairment of IL-23–induced expansion of HMBPP-stimulated Vγ2Vδ2 T cells. It was recently reported that IL-23–mediated expansion of CD4+ T cells mainly uses the JAK2 and Tyk2 pathway for phosphorylating STAT3 [31–33] and that the phosphorylated STAT3 dimer subsequently activates transcription of cytokine-responsive genes in the nucleus [31, 32] (Figure 4A).

Figure 4.

Figure 4.

Open in a new tab

Interleukin 23 (IL-23)–induced expansion of Vγ2Vδ2 T cells involves STAT3. Vγ2Vδ2 T cells in patients with tuberculosis showed decreased expression and phosphorylation of STAT3, with overexpression of antagonizing SOCS3. A, At top, hypothetical IL-23 signaling via the JAK2 and Tyk2 pathway resulted in phosphorylation of STAT3 in Vγ2Vδ2 T cells, a modification based on IL-23 engagement of CD4+ T cells [33, 44]. Note that the phosphorylated STAT3 dimer subsequently activates transcription of proliferating/cytokine-responsive genes in the nucleus [31, 32]. Below, the STAT3-targeted inhibitor cucurbitacin I (CUI) blocked IL-23–induced expansion of HMBPP-stimulated Vγ2Vδ2 T cells from subjects with latent tuberculosis infection (LTBI). CUI at a concentration of 2 nM significantly reduced IL-23–mediated expansion of HMBPP-stimulated Vγ2Vδ2 T cells but spared the effect of IL-2 signaling on these cells. B, Flow cytometry–based analysis showed that phosphorylation levels of STAT3 at pS727 and pY705 sites in Vγ2Vδ2 T cells were significantly decreased in fresh, unstimulated peripheral blood mononuclear cells (PBMCs) from patients with tuberculosis as compared to healthy controls (HCs). At left are representative flow cytometry–based histograms displaying percentage numbers of Vγ2+pY705+ cells (upper) and Vγ2+pS727+ cells (middle) among CD3+ T cells, as well as pY705+pS727+ cells among Vγ2+ cells (lower panels) from PBMCs of patients with tuberculosis and HCs. Representative flow panel of the isotype control is shown at top left. At right are bar graphs comparing frequencies of corresponding cell subpopulations between patients with tuberculosis and HCs. In accordance with manufacturer instructions, fresh PBMCs were briefly treated for 15 minutes with PMA and interleukin 6 (IL-6) and then stained for phosphorylated STAT3, using anti-Stat3 (pS727)–PE and anti-Stat3 (pY705)–Alexa Fluor 647 after surface staining for CD3 and Vγ2. C, The bar graph shows that, 72 hours after IL-23/HMBPP costimulation, the frequency of the Vγ2+pY705+pS727+ cell subpopulation among Vγ2+ T cells in patients with tuberculosis was also significantly lower than that in HCs. In contrast, IL-2/HMBPP costimulation did not cause significant changes in phosphorylated STAT3 between patients with TB and HCs. PBMCs were stimulated for 3 days with medium, HMBPP, HMBPP plus IL-2, and HMBPP plus IL-23 and then were assessed for percentages of pY705+pS727+ cells among CD3+Vγ2+ cells, using flow cytometry. D, Expression levels of STAT3 and SOCS3 genes in Vγ2Vδ2 T cells among patients with tuberculosis and HCs. Vγ2Vδ2 T cells were purified from PBMCs by flow cytometry sorting and subjected to RNA extraction and real-time quantitative polymerase chain reaction analysis. Expression of the STAT3 gene in Vγ2Vδ2 T cells from patients with tuberculosis decreased to 16% of the level seen in Vγ2Vδ2 T cells from HCs. Expression of SOCS3 in Vγ2Vδ2 T cells from TB patients is more than 4 times of that in HCs. Data in panels B–D are means ± standard errors of the mean, pooled from 10 HCs and 10 patients with tuberculosis in 3 independent experiments. *P < .05, **P < .01, and ***P < .001. Abbreviation: DMSO, dimethyl sulfoxide.

To test whether STAT3 was also involved in IL-23–induced expansion of Vγ2Vδ2 T cells, the STAT3-targeted inhibitor CUI [34] was assessed for the ability to block IL-23/HMBPP–induced expansion of Vγ2Vδ2 T cells from subjects with LTBI. CUI at a concentration of 2 nM significantly reduced IL-23/HMBPP–induced expansion of Vγ2Vδ2 T cells but spared the effects of IL-2/HMBPP signaling (Figure 4A), perhaps because IL-2 signaling differently involves JAK1, JAK3, LCK, and SYK [35]. This result prompted us to address whether expression and phosphorylation of STAT3 in Vγ2Vδ2 T cells were different between subjects with tuberculosis and HCs.

The flow cytometry–based analysis showed that phosphorylation levels of STAT3 at pS727 and pY705 sites with or without stimulation were significantly decreased in Vγ2Vδ2 T cells from patients with tuberculosis as compared to HCs (Figure 4B). When assessing the STAT3 response to 72-hour costimulation with IL-23/HMBPP, frequencies of the Vγ2+pY705+pS727+ cell subpopulation in patients with tuberculosis were also significantly lower than those in HCs (Figure 4C). However, IL-2/HMBPP costimulation did not uncover any significant difference in phosphorylated STAT3 between patients with tuberculosis and HCs (Figure 4C).

In parallel, we also measured the expression of the gene encoding STAT3 in Vγ2Vδ2 T cells, using real-time qPCR. The level of expression of the STAT3 gene in Vγ2Vδ2 T cells from patients with tuberculosis was only approximately 16% of that seen in Vγ2Vδ2 T cells from HCs (Figure 4D).

Concurrently, we compared SOCS3 gene expression between patients with tuberculosis and HCs, because SOCS3 could inhibit STAT3/JAK pathway via binding to JAK2 and negatively regulate cytokine production [36, 37]. Vγ2Vδ2 T cells from patients with tuberculosis showed >4 times greater expression of SOCS3 than those from HCs (Figure 4D). This represents additional data supporting the notion that tuberculosis might impair the JAK2/STAT3 activation pathway in Vγ2Vδ2 T cells.

Taken together, these results suggest that, while IL-23–induced expansion of Vγ2Vδ2 T cells involves STAT3, tuberculosis might block the STAT3/JAK2 activation pathway in Vγ2Vδ2 T cells, leading to IL-23–targeted exhaustion of these γδ T cells in response to IL-23/HMBPP costimulation.

Impaired STAT3 Pathway Coincided With Marked Increases in hsa-miR-337-3p and hsa-miR-125b-5p Levels in Vγ2Vδ2 T Cells From Patients With Tuberculosis

While miRNAs represent a class of bioactive molecules capable of regulating gene expression [38], some miRNAs have been shown to correlate closely with the STAT3 activation pathway [39]. We therefore sought to determine whether tuberculosis could lead to increased expression of miRNAs that interfere with the STAT3 activation pathway in Vγ2Vδ2 T cells. Interestingly, we found that Vγ2Vδ2 T cells from patients with tuberculosis expressed levels of hsa-miRNA-337-3p and hsa-miRNA-125b-5p that were approximately 6 times higher than those expressed by Vγ2Vδ2 T cells from HCs (Figure 5A). Thus, while hsa-miR-337-3p and hsa-miRNA-125b-5p have been shown to downregulate STAT3 gene expression [39], marked increases in these 2 miRNAs in Vγ2Vδ2 T cells from patients with tuberculosis correlated with low expression or phosphorylation of STAT3.

Figure 5.

Figure 5.

Open in a new tab

Impairment of the STAT3 pathway coincided with marked increases in hsa-miR-337-3p and hsa-miR-125b-5p expression in Vγ2Vδ2 T cells from patients with tuberculosis. A, Among the microRNAs (miRNAs) capable of targeting STAT3 expression, hsa-miR-337-3p and hsa-miRNA-125b-5p were upregulated by approximately 6-fold in Vγ2Vδ2 T cells from patients with tuberculosis, compared with those from healthy controls (HCs). Expression of 2 other STAT3-targeted miRNAs, hsa-miR-124-3p and has-miR-223-3p, was also increased, although individual variations were seen. Total miRNA was extracted from freshly purified Vδ2+ T cells. The relative expression level of miRNAs was measured by real-time quantitative polymerase chain reaction (qPCR) analysis and normalized by U6 or 5s. BCG-vaccinated HCs were used as a calibrator. Data are means ± SEM standard errors of the mean (SEM), pooled from 16 patients with tuberculosis and 16 HCs in 3 independent experiments. **P < .01. B, Mycobacterial infection of human cells significantly increased the expression of hsa-miR-337-3p and hsa-miRNA-125b-5p. Human A549 cells were infected with Mycobacterium bovis BCG and assessed for expression of miRNAs, using real-time qPCR analysis. Data are fold changes expressed as means ± SEM. ***P < .001.

Next, we conducted a proof-of-concept study to explore a connection between M. tuberculosis infection and increased expression of miRNAs. Interestingly, mycobacterial infection of host cells significantly increased the expression of hsa-miR-337-3p and hsa-miRNA-125b-5p (Figure 5B). These results might help explain the increases in these miRNAs seen in the γδ T-cell subset from patients with tuberculosis.

Silencing of hsa-miR-337-3p and hsa-miR-125b-5p by miRNA Sponges Could Improve IL-23–Mediated Expansion of HMBPP-Stimulated Vγ2Vδ2 T Cells in Patients With Tuberculosis and Restore the Ability of These Cells to Produce Cytokines

Given the tuberculosis-driven increases in hsa-miR-337-3p and hsa-miR-125b-5p expression, we investigated the possibility that these STAT3-targeted miRNAs help to impair the STAT3 activation pathway and selectively destroy IL-23–induced expansion of HMBPP-stimulated Vγ2Vδ2 T cells in patients with tuberculosis. To test this hypothesis, we used the pCMV-LV-GV234-GFP lentiviral vector system and developed LV-hsa-125b-5p and LV-hsa-337-3p sponge constructs to manipulate decreases in hsa-miR-337-3p and/or hsa-miRNA-125b-5p expression in Vγ2Vδ2 T cells. When the transduction of these miRNA sponges proved successful (Figure 6A), we found that a combination of LV-hsa-125b-5p and LV-hsa-337-3p but not controls could significantly improve IL-23–induced expansion of HMBPP-stimulated Vγ2Vδ2 T cells in PBMCs from patients with tuberculosis (P = .013136; Figure 6B). Such improvement or restoration was also seen at the gene-expression level of the Vγ2 T-cell receptor (Figure 6C). Furthermore, LV-hsa-125b-5p and LV-hsa-337-3p sponges but not controls reconstituted the ability of IL-23/HMBPP-stimulated Vγ2Vδ2 T cells to express the antituberculosis cytokines IFN-γ and IL-17A (Figure 6C). Consistently, immunohistochemical staining followed by flow cytometry analyses showed that the combined miRNA sponges significantly increased the production of IL-17A and IFN-γ by IL-23/HMBPP-costimulated Vγ2Vδ2 T cells as compared to controls (Figure 6D). These results therefore support our hypothesis that tuberculosis-driven increases in hsa-125b-5p and hsa-337-3p expression might help to downregulate the STAT3 activation pathway and impair IL-23 signaling for expansion of HMBPP-stimulated Vγ2Vδ2 T cells in patients with tuberculosis.

Figure 6.

Figure 6.

Open in a new tab

Downregulation of hsa-miR-337-3p and hsa-miR-125b-5p by microRNA (miRNA) sponges improved the interleukin 23 (IL-23)–mediated expansion of HMBPP-stimulated Vγ2Vδ2 T cells in patients with tuberculosis and restored the ability of these cells to produce cytokines. The pCMV-LV-GV234-GFP vector system was used to develop LV-has-125b-5p, LV-has-337-3p, and control miRNA sponge constructs to manipulate decreases in hsa-miR-337-3p and/or hsa-miRNA-125b-5p expression in Vγ2Vδ2 T cells. A, Microscopic fluorescence (left) and optical (right) pictures show that most peripheral blood mononuclear cells (PBMCs) containing Vγ2Vδ2 T cells give rise to green fluorescent protein–associated florescence after transduction with the miRNA sponges. B, Representative flow cytometry–based histograms (left panels) and bar graph data (right panel) show that transduction with a combination of LV-has-125b-5p and LV-has-337-3p but not controls significantly improved IL-23–induced expansion of HMBPP-stimulated Vγ2Vδ2 T cells in PBMCs from 12 patients with tuberculosis in 4 independent experiments (mean values [ ± SD] were 10.89% ± 1.17% for LV-has-125b-5p and LV-has-337-3p and 7.86% ± 2.08% for control; P = .013136). C, Transduction with LV-has-125b-5p and LV-has-337-3p combined also restored increases in the expression levels of T-cell receptor Vγ2 and the anti–tuberculosis cytokines interferon γ (IFN-γ) and interleukin 17A (IL-17A). D, Representative flow cytometry–based histograms and bar graph data show that transduction with a combination of LV-has-125b-5p and LV-has-337-3p but not controls significantly improved production of IL-17A and IFN-γ by IL-23–expanded, HMBPP-stimulated Vγ2Vδ2 T cells from patients with tuberculosis. Data are means ± standard errors of the mean, pooled from 12 patients with tuberculosis in 4 independent experiments. *P < .05 and **P < .01.

DISCUSSION

The current study is the first to demonstrate that patients with tuberculosis exhibit selective destruction of the IL-23 signal effect on the HMBPP-specific γδ T-cell subset, with consequent IL-23–targeted exhaustion of Vγ2Vδ2 T-cell responses. Such selective impairment of the effect of IL-23 signaling can be linked to depressed expression and phosphorylation of STAT3, as well as to overexpression of the antagonizing factor SOCS3. Downregulation of the STAT3 signaling pathway in Vγ2Vδ2 T cells correlates with remarkable increases in 2 miRNAs targeting STAT3. Silence of these miRNAs by miRNA sponges can improve the IL-23–induced expansion and function of HMBPP-stimulated Vγ2Vδ2 T cells from patients with tuberculosis. Thus, these findings support the compelling hypothesis that tuberculosis can impair the signaling pathway of a selected cytokine and consequently exhaust antigen-specific T cells.

IL-23–mediated expansion of HMBPP-stimulated Vγ2Vδ2 T cells can clearly be demonstrated in subjects with LTBI or a BCG vaccination history. This result in humans appears to be consistent with our previous report demonstrating recall-like expansion of Vγ2Vδ2 T cells induced by Th17-related cytokines, including IL-23, in macaques after primary M. tuberculosis infection or vaccination [18]. Our new data from humans suggest that IL-23 and IL-2 significantly expand HMBPP-stimulated Vγ2Vδ2 T cells. Results from IL-2–blockade and transient HMBPP activation models demonstrate that IL-2 synergizes or facilitates IL-23–induced expansion of Vγ2Vδ2 T cells but that IL-2 blockade cannot totally abrogate the IL-23–induced expansion. This is also supported by STAT3 blockade data demonstrating that STAT3 is involved in the expansion of Vγ2Vδ2 T cells by IL-23 but not IL-2.

It is interesting that tuberculosis can selectively impair IL-23 signaling but spare the effect of IL-2 on Vγ2Vδ2 T cells. Such selective impairment of the effect of IL-23 is relative to IL-2 sparing, and further studies may uncover broad destruction of other cytokines′ signals, involving other immune cells, during tuberculosis or other infections. Selective impairment of the effect of IL-23 signaling is implicative because Vγ2Vδ2 T cells in patients with tuberculosis exhibit lower levels of STAT3 expression and phosphorylation [40]. These changes may be interrelated with the impairment of the effect of IL-23, although a precise cause and effect associated with these changes is currently unclear. Such selective impairment of IL-23 effect may occur as a result of persistent exposure of Vγ2Vδ2 T cells to HMBPP or IL-23 during chronic M. tuberculosis infection [41]. Since STAT3 is involved in IL-23–induced expansion of Vγ2Vδ2 T cells, it is not surprising that inadequate levels of STAT3 expression and phosphorylation would impair the IL-23–mediated expansion of these γδ T cells. It is also reasonable that IL-2 can still adequately expand HMBPP-stimulated Vγ2Vδ2 T cells, because IL-2 uses signaling pathways other than STAT3 [42].

Tuberculosis-driven impairing of IL-23–induced expansion of Vγ2Vδ2 T cells appears to be different from T-cell exhaustion linked to PD-1 signaling. Upregulation of PD-1 has been shown to correlate with T-cell exhaustion in cancers and viral infection [5], although functional aspects of PD-1 expression in M. tuberculosis infection remain to be solved [3]. Here, we show that tuberculosis impairs the effect of IL-23/STAT3 signaling on Vγ2Vδ2 T cells. While blockade of the PD-1 pathway by antibody can reverse the exhaustion of αβ T cells linked to PD-1 expression [43], such a blockade cannot restore IL-23–induced expansion of Vγ2Vδ2 T cells, despite our use of 2 different sources of PD-1 antibodies capable of recovering from T-cell exhaustion [23, 24].

Remarkable increases in STAT3-targeted miRNAs in Vγ2Vδ2 T cells may help to explain the selective impairment of IL-23–mediated expansion of Vγ2Vδ2 T cells in patients with tuberculosis. Vγ2Vδ2 T cells in patients with tuberculosis display greater expression of 4 miRNAs that have been shown to negatively regulate STAT3 activation or phosphorylation [39]. Among them, hsa-miR-337-3p and hsa-miRNA-125b-5p are expressed significantly higher during tuberculosis than in BCG-vaccinated HCs. Most strikingly, downregulation of hsa-miR-337-3p and hsa-miRNA-125b-5p by using an miRNA sponge allows for detectable recovery of IL-23–induced expansion of Vγ2Vδ2 T cells in patients with tuberculosis, as well as the effector function of these cells for producing the antituberculosis cytokines IFN-γ and IL-17A.

Thus, we demonstrate that tuberculosis can selectively destroy IL-23–induced expansion of Vγ2Vδ2 T cells. Such selective destruction appears to be linked to the following interrelated mechanistic changes in Vγ2Vδ2 T cells: (1) remarkable upregulation of STAT3-targeted miRNAs, (2) lower expression and phosphorylation of STAT3, and (3) reciprocal overexpression of the regulatory factor SOCS3, thereby inhibiting STAT3 phosphorylation. Tuberculosis-driven upregulation of STAT3-targeted miRNAs may be one of the driving factors, since silencing of them can restore IL-23–mediated expansion and cytokine production.

Notes

Acknowledgments. We thank the staff in the laboratory of Z. W. C., for technical support; and Dr Hassan Jomaa (Justus Liebig University of Giessen, Germany), for providing HMBPP.

Financial support. This work was supported by the Chinese National Major Projects (2013ZX10003009-002 to Z. W. C.) and the National Institutes of Health (grant R01 HL64560/OD015092/HL129887 to Z. W. C.).

Potential conflicts of interest. All authors: No reported conflicts. 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.

References

  • 1. Penaloza-MacMaster P, Kamphorst AO, Wieland A et al. Interplay between regulatory T cells and PD-1 in modulating T cell exhaustion and viral control during chronic LCMV infection. J Exp Med 2014; 211:1905–18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. Severson JJ, Serracino HS, Mateescu V et al. PD-1+ Tim-3+ CD8+ T lymphocytes display varied degrees of functional exhaustion in patients with regionally metastatic differentiated thyroid cancer. Cancer Immunol Res 2015; 3:620–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Qiu Y, Chen J, Liao H et al. Tim-3-Expressing CD4+ and CD8+ T cells in human tuberculosis (TB) exhibit polarized effector memory phenotypes and stronger anti-TB effector functions. PLoS Pathog 2012; 8:e1002984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Posch SKC, Barthel SR, Mueller H et al. Melanoma cell-intrinsic PD-1 receptor functions promote tumor growth. Cell 2015; 162:1242–56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Li Q, Skinner PJ, Duan S-JHL et al. Visualizing antigen-specific and infected cells in situ predicts outcomes in early viral infection. Science 2009; 323:1726–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. World Health Organization (WHO). Global tuberculosis report. Geneva: WHO, 2015. http://www.who.int/tb.
  • 7. Dutta NK, Karakousis PC. Latent tuberculosis infection: myths, models, and molecular mechanisms. Microbiol Mol Biol Rev 2014; 78:343–71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Kaufmann SHE. Future vaccination strategies against tuberculosis: thinking outside the box. Immunity 2010; 33:567–77. [DOI] [PubMed] [Google Scholar]
  • 9. Chen ZW. Multifunctional immune responses of HMBPP-specific Vγ2Vδ2T cells in M. tuberculosis and other infections. Cell Mol Immunol 2013; 10:58–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Lázár-Molnár E, Chen B, Sweeney KA et al. Programmed death-1 (PD-1)-deficient mice are extraordinarily sensitive to tuberculosis. Proc Natl Acad Sci U S A 2010; 107:13402–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Mujib S, Jones RB, Aidarus CLN et al. Antigen-independent induction of Tim-3 expression on human T cells by the common γ-chain cytokines IL-2, IL-7, IL-15, and IL-21 is associated with proliferation and is dependent on the phosphoinositide 3-kinase pathway. J Immunol 2012; 188:3745–56. [DOI] [PubMed] [Google Scholar]
  • 12. Singh A, Mohan A, Dey AB, Mitra DK. Inhibiting the programmed death 1 pathway rescues Mycobacterium tuberculosis-specific interferon γ-producing T cells from apoptosis in patients with pulmonary tuberculosis. J Infect Dis 2013; 208:603–15. [DOI] [PubMed] [Google Scholar]
  • 13. Chen ZW, Letvin NL. Adaptive immune response of Vγ2Vδ2T cells: a new paradigm. Trends Immunol 2003; 24:213–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Born WK, Reardon CL, O'Brien RL. The function of γδ T cells in innate immunity. Curr Opin Immunol 2006; 18:31–8. [DOI] [PubMed] [Google Scholar]
  • 15. Shen Y, Zhou D, Qiu L et al. Adaptive immune response of Vγ2Vδ2+ T cells during mycobacterial infections. Science 2002; 295:2255–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Bonneville M, Chen ZW, Déchanet-Merville J et al. Chicago 2014—30 years of γδ T cells. Cell Immunol 2015; 296:3–9. [DOI] [PubMed] [Google Scholar]
  • 17. Vermijlen D, Ellis P, Langford C et al. Distinct cytokine-driven responses of activated blood γδ T Cells: insights into unconventional T cell pleiotropy. J Immunol 2007; 178:4304–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Shen H, Wang Y, Chen CY et al. Th17-related cytokines contribute to recall-like expansion/effector function of HMBPP-specific Vγ2Vδ2T cells after M. tuberculosis infection or vaccination . Eur J Immunol 2015; 45:442–51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Schwander SK, Sada E, Torres M et al. T lymphocytic and immature macrophage alveolitis in active pulmonary tuberculosis. J Infect Dis 1996; 173:1267–72. [DOI] [PubMed] [Google Scholar]
  • 20. Daker SE, Sacchi A, Montesano C et al. An abnormal phenotype of lung Vγ9Vδ2 T cells impairs their responsiveness in tuberculosis patients. Cell Immunol 2013; 282:106–12. [DOI] [PubMed] [Google Scholar]
  • 21. Dieli F, Sireci G, Caccamo N et al. Selective depression of interferon-gamma and granulysin production with increase of proliferative response by Vgamma9/Vdelta2 T cells in children with tuberculosis. J Infect Dis 2002; 186:1835–9. [DOI] [PubMed] [Google Scholar]
  • 22. Chen CY, Huang D, Yao S et al. IL-2 simultaneously expands Foxp3+ T regulatory and T effector cells and confers resistance to severe tuberculosis (TB): implicative Treg-T effector cooperation in immunity to TB. J Immunol 2012; 199:4278–88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Nattawat Onlamoon KR, Mayne AE, Pattanapanyasat K, Mori K, Villinger F, Ansari AA. Soluble PD-1 rescues the proliferative response of simian immunodeficiency virus-specific CD4 and CD8 T cells during chronic infection. Immunology 2008; 124:277–93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Conrad JA, Ramalingam RK, Duncan CB et al. , RM Antiretroviral therapy reduces the magnitude and T cell receptor repertoire diversity of HIV-Specific T cell responses without changing T cell clonotype dominance. J Virol 2012; 86:4213–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Gong G, Shao L, Wang Y et al. Phosphoantigen-activated V gamma 2 V delta 2 T cells antagonize IL-2-induced CD4+ CD25+ Foxp3+ T regulatory cells in mycobacterial infection. Blood 2009; 113:837–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Huang D, Chen CY, Ali Z et al. Antigen-specific Vgamma2Vdelta2 T effector cells confer homeostatic protection against pneumonic plaque lesions. Proc Natl Acad Sci U S A 2009; 106:7553–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Zhang J, Scordi I, Smyth MJ, Lichtenheld MG. Interleukin 2 receptor signaling regulates the perforin gene through signal transducer and activator of transcription (Stat)5 activation of two enhancers. J Exp Med 1999; 190:1297–308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Yang XO, Pappu B, Nurieva R et al. TH17 lineage differentiation is programmed by orphan nuclear receptors RORα and RORγ. Immunity 2008; 28:29–39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Bandaru A, Devalraju KP et al. Phosphorylated STAT3 and PD-1 regulate IL-17 production and IL-23 receptor expression in Mycobacterium tuberculosis infection. Eur J Immunol 2014; 44. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Bengsch B, Martin B, Thimme R. Restoration of HBV-specific CD8+ T cell function by PD-1 blockade in inactive carrier patients is linked to T cell differentiation. J Hepatol 2014; 61:1212–9. [DOI] [PubMed] [Google Scholar]
  • 31. Huang G, Yan H, Ye S, Tong C, Ying Q-L. STAT3 phosphorylation at tyrosine 705 and serine 727 differentially regulates mouse ESC fates. Stem Cells 2014; 32:1149–60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Zhong Z, Wen Z, Darnell J Jr. Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6. Science 1994; 264:95–8. [DOI] [PubMed] [Google Scholar]
  • 33. Polgar N, Csongei V, Szabo M et al. Investigation of JAK2, STAT3 and CCR6 polymorphisms and their gene-gene interactions in inflammatory bowel disease. Int J Immunogenet 2012; 39:247–52. [DOI] [PubMed] [Google Scholar]
  • 34. Seo C-R, Yang DK, Song N-J et al. Cucurbitacin B and cucurbitacin I suppress adipocyte differentiation through inhibition of STAT3 signaling. Food Chem Toxicol 2014; 64:217–24. [DOI] [PubMed] [Google Scholar]
  • 35. Motegi H, Shimo Y, Akiyama T, Inoue J-I. TRAF6 negatively regulates the Jak1-Erk pathway in interleukin-2 signaling. Genes Cells 2011; 16:179–89. [DOI] [PubMed] [Google Scholar]
  • 36. Liu Z, Gan L, Zhou Z, Jin W, Sun C. SOCS3 promotes inflammation and apoptosis via inhibiting JAK2/STAT3 signaling pathway in 3T3-L1 adipocyte. Immunobiology 2015; S0171-2985:00025–X. [DOI] [PubMed] [Google Scholar]
  • 37. Elliott J, Suessmuth Y, Scott LM et al. SOCS3 tyrosine phosphorylation as a potential bio-marker for myeloproliferative neoplasms associated with mutant JAK2 kinases. Haematologica 2009; 94:576–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Ambros V. microRNAs: tiny regulators with great potential. Cell 2001; 107:823–6. [DOI] [PubMed] [Google Scholar]
  • 39. Cao Q, Li Y-Y, He W-F et al. Interplay between microRNAs and the STAT3 signaling pathway in human cancers. Physiol Genomics 2013; 45:1206–14. [DOI] [PubMed] [Google Scholar]
  • 40. Babon JJ, Kershaw NJ, Murphy JM et al. Suppression of cytokine signaling by SOCS3: characterization of the mode of inhibition and the basis of its specificity. Immunity 2012; 36:239–50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Qiu L, Huang D, Chen CY et al. Severe tuberculosis induces unbalanced up-regulation of gene networks and overexpression of IL-22, MIP-1alpha, CCL27, IP-10, CCR4, CCR5, CXCR3, PD1, PDL2, IL-3, IFN-beta, TIM1, and TLR2 but low antigen-specific cellular responses. J Infect Dis 2008; 198:1514–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42. Valle-Mendiola A, Weiss-Steider B, Rocha-Zavaleta L, Soto-Cruz I. IL-2 enhances cervical cancer cells proliferation and JAK3/STAT5 phosphorylation at low doses, while at high doses IL-2 has opposite effects. Cancer Invest 2014; 32:115–25. [DOI] [PubMed] [Google Scholar]
  • 43. Quan L, Chen X, Liu A et al. PD-1 blockade can restore functions of T-cells in Epstein-Barr virus-positive diffuse large B-cell lymphoma in vitro. PLoS One 2015; 10:e0136476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Ma NFC, Zhang X, Guzzo C, Gee K. Interleukin-23-Induced interleukin-23 receptor subunit expression is mediated by the Janus kinase/signal transducer and activation of transcription pathway in human CD4 T cells. J Interferon Cytokine Res 2011; 31:363–71. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Infectious Diseases are provided here courtesy of Oxford University Press

RESOURCES