Tuberculosis 94 (2014) 131e139
Contents lists available at ScienceDirect
Tuberculosis journal homepage: http://intl.elsevierhealth.com/journals/tube
IMMUNOLOGICAL ASPECTS
PD-1/PD-Ls pathways between CD4þ T cells and pleural mesothelial cells in human tuberculous pleurisy Wen Yin a, b, Zhao-Hui Tong a, c, Ai Cui a, Jian-Chu Zhang b, Zhi-Jian Ye b, Ming-Li Yuan b, Qiong Zhou b, Huan-Zhong Shi a, b, c, * a
Department of Respiratory and Critical Care Medicine, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China Department of Respiratory and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China c Center of Medical Research, Beijing Institute of Respiratory Diseases, Beijing, China b
a r t i c l e i n f o
s u m m a r y
Article history: Received 4 June 2013 Received in revised form 24 October 2013 Accepted 27 October 2013
Programmed death 1 (PD-1), PD-ligand 1 (PD-L1), and PD-L2 have been demonstrated to be involved in tuberculosis immunity, however, the expression and regulation of PD-1/PD-Ls pathways in pleural mesothelial cells (PMCs) and CD4þ T cells in tuberculous pleural effusion (TPE) have not been investigated. Expression of PD-1 on CD4þ T cells and expressions of PD-L1 and PD-L2 on PMCs in TPE were determined. The impacts of PD-1/PD-Ls pathways on proliferation, apoptosis, adhesion, and migration of CD4þ T cells were explored. Concentrations of soluble PD-l, but not of soluble PD-Ls, were much higher in TPE than in serum. Expressions of PD-1 on CD4þ T cells in TPE were significantly higher than those in blood. Expressions of PD-Ls were much higher on PMCs from TPE when compared with those from transudative effusion. Interferon-g not only upregulated the expression of PD-1 on CD4þ T cells, but also upregulated the expressions of PD-Ls on PMCs. Blockage PD-1/PD-Ls pathways abolished the inhibitory effects on proliferation and adhesion activity of CD4þ T cells induced by PMCs. PD-1/PD-Ls pathways on PMCs inhibited proliferation and adhesion activity of CD4þ T cells, suggesting that Mycobacterium tuberculosis might exploit PD-1/PD-Ls pathways to evade host cell immune response in human. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Pleural effusion Programmed death-1 Tuberculosis
1. Introduction Tuberculosis remains a major health problem, with an estimated one third of the world’s population infected with Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis, causing about 1.4 million deaths in 2011. Tuberculous pleural effusion (TPE) is the second most frequent manifestation of extrapulmonary tuberculosis after lymph node tuberculosis, and is restricted to the pleural cavity, which contains protein-enriched fluid and numerous immunocompetent cells, especially CD4þ T cells [1]. Pleural mesothelial cells (PMCs), presented in a single layer covering each pleural membrane, are the most common cells of the pleural space
* Corresponding author. Department of Respiratory and Critical Care Medicine, Beijing Chaoyang Hospital, Capital Medical University, 8 Gongren Tiyuchang Nanlu, Chaoyang District, Beijing 100020, China. Tel./fax: þ86 10 85231412. E-mail address: [email protected] (H.-Z. Shi). 1472-9792/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tube.2013.10.007
which not only encounter invading microbes, but also subsequently initiate and propagate an inflammatory reaction by coordinating the other kinds of inflammatory cells [2]. Early studies have demonstrated that PMCs facilitate monocyte transmigration across pleural mesothelium during MTB infection [3]. Our recent data have demonstrated that PMCs were able to function as antigenpresenting cells to stimulate proliferation of CD4þ T cells and differentiation of Th22, Th9, Th17 and Th1 cells in response to MTB antigens in CD80- and CD86-dependent means [4,5]. Programmed death 1 (PD-1) is a key immune checkpoint receptor expressed by activated T cells, and it mediates immunosuppression. PD-1 has two known ligands, PD-L1 (B7-H1) and PDL2 (B7-DC) [6e9]. In vitro studies have shown that engagement of PD-1 by PD-L1 and PD-L2 inhibited T cell proliferation and cytokine production, indicating that the crosslinking of PD-1 by its PD-Ls leads to downregulation of T cell responses [7,8], although not all the studies support the inhibitory role for the PD-1/PD-Ls pathways [9]. In vivo animal studies have demonstrated that MTB infection
132
W. Yin et al. / Tuberculosis 94 (2014) 131e139
resulted in upregulation of PD-1, PD-L1 and PD-L2 [10e12], and PD1 deficient mice were extremely sensitive to MTB infection when compared to their wild type littermates, showing dramatically reduced survival and higher bacterial loads [12,13], whereas MTB numbers in spleen of PD-1 deficient mice were decreased compared with wild type mice [11]. In human studies, PD-1/PD-Ls pathways have also been shown to inhibit T cell effector functions during active pulmonary tuberculosis [14]. We therefore designed the present study to evaluate the regulation and immune function of PD-1/PD-Ls pathways of PMCs in TPE. 2. Methods 2.1. Subjects The study protocol was approved by our Institutional Review Boards for human studies of Capital Medical University, Beijing, China; and Tongji Medical College, Wuhan, China; and informed written consent was obtained from all subjects. Twenty anti-HIV Ab negative patients were proven to have TPE, as evidenced by growth of MTB from pleural fluid or by demonstration of granulomatous pleurisy on pleural biopsy specimen in the absence of any evidence of other granulomatous diseases (Table 1). Another 10 anti-HIV Ab negative patients suffering from heart failure or hypoalbuminemia were diagnosed to have pleural transudative effusion according to Light’s criteria [15]. 2.2. Collecting and processing samples Five-hundred to 1000 mL of pleural fluid samples from each patient were collected in heparin-treated tubes, through a standard thoracocentesis technique within 24 h after hospitalization. Twenty milliliters of peripheral blood were drawn simultaneously. TPE specimens were immersed in ice immediately and were then centrifuged at 1200 g for 5 min before isolating PMCs. In some experiments, the cell pellets of TPE were resuspended in HBSS, and mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation (Pharmacia, Uppsala, Sweden). 2.3. Measurement of soluble (s) PD-1/PD-Ls The concentrations of sPD-1, sPD-L1, and sPD-L2 in TPE, serum and PMC culture supernatants were measured by ELISA kits (sPD-1 and sPD-L2 kits from R&D systems, Minneapolis, MN; sPD-L1 kits from MyBioSource, San Diego, CA) according to the instructions provided by the manufacturers.
2.4. Flow cytometry Expression of PD-1 on T cells from TPE and blood were determined by flow cytometry after surface staining with anti-human CD3-PerCP-cy5.5, CD8-FITC, PD-1-APC mAbs, which were purchased from BD Biosciences (Franklin Lakes, NJ) or eBioscience (San Diego, CA). CD11a-PE and CD29-APC mAbs were used for the detection of ligands of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1). Fixed and permeabilized PMCs were primarily stained with mouse anti-alretinin mAb (BD Biosciences) to identify PMCs, and then stained with FITClabeled goat anti-mouse Igs (BD Biosciences). Appropriate species matched Abs served as isotype controls. To explore the expression of molecules on PMCs, anti-PD-L1, -PD-L2, -PD-1, -ICAM-1, -VCAM1, -CD80 or -CD86 (all from eBioscience), conjugated with PE or APC were used. Flow cytometry was performed on a FACS Canto II (BD Biosciences) and analyzed using BD FCSDiva Software and FCS Epress 4 software (De Novo Software, Los Angeles, CA). 2.5. Isolation and culture of PMCs and CD4þ T cells For isolating PMCs, the cell pellets of TPE were resuspended in RPMI-1640 containing 20% heat-inactivated FBS, and 50 mg/mL gentamycin. The cells were seeded into 25-cm [2] flasks at a density of 1 l04 cells/cm2 and placed in an incubator at 37 C in 5% CO2. After 24 h the monolayers were washed with HBSS to remove nonadherent cells and fresh media was added. The monolayers were monitored until confluent (7e10 d), then trypsinized, and subcultured. To prepare PMC culture supernatants, the second generation PMCs were seeded and cultured in complete medium at a density of 106 cells per well in the absence or presence of designated cytokines, and the culture supernatants were harvested 72 h later. CD4þ T cells were isolated from blood and TPE by MACS based on negative selection using CD4þ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The purity of CD4þ T cells was >97%, as measured by flow cytometry. To investigate the effect of MTB-specific peptide of early secretory antigenic target-6kDa/culture filtrate protein-10 (ESAT-6/CFP10) and cytokine IFN-g on regulation of membrane PD-1 expression on CD4þ T cells, mononuclear cells isolated from blood were cultured in 24-well plates at a density of 5 105/well with or without ESAT/CFP10 (10 mg/mL, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China) and/or IFN-g (50 ng/mL) for 3 d in RPMI-1640 (Gibco, Invitrogen, Carlsbad, CA) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco), and 50 mg/mL gentamycin. 2.6. Effects of cytokines on expressions of PD-Ls on PMCs
Table 1 Characteristics of the study population. Pleural effusions
Patients, No. Sex, male/female, No. Age, yr Protein, g/L Lactate dehydrogenase, IU/L Adenosine deaminase, U/L Total cell counts, 109/L Differential cell counts, % Lymphocytes Neutrophils Macrophages Mesothelial cells
Tuberculous
Transudative
20 14/6 42.3 3.6 41.2 7.6 468.9 50.7 55.5 6.8 3.43 1.2
10 6/4 47.6 8.2 17.8 9.8 123.6 56.3 9.2 4.5 0.56 0.08
75.5 5.8 8.4 1.2 13.7 1.3 2.4 0.4
32.5 3.9 7.4 1.3 48.2 3.7 11.9 1.9
To investigate the effects of cytokines such as IFN-g (50 ng/mL), TNF-a (50 ng/mL), IL-1b (10 ng/mL), IL-6 (50 ng/mL), IL-12 (20 ng/ mL), IL-27 (100 ng/mL) and IL-32 (10 ng/mL) (all from R&D systems) on regulation of membrane PD-Ls expression on PMCs, nonmalignant transformed mesothelial cell line (Met5A cells) were cultured in 12-well plates at a density of 5 105/well with or without the above cytokines for 3 d in RPMI-1640 containing 20% heat-inactivated FBS and 50 mg/mL gentamycin. 2.7. Proliferation and apoptosis of CD4þ T cells Confluent PMC monolayers in 24-well plates (1 105/well) were activated by adding IFN-g (100 ng/mL) for 24 h and then treated by adding blocking mAbs specific for PD-L1 (10 mg/mL) and/or PD-L2
W. Yin et al. / Tuberculosis 94 (2014) 131e139
(10 mg/mL) or isotype control Igs for another 24 h. Before cocultured with purified CD4þ T (5 105) cells isolated from TPE in the presence of anti-CD3 (1.0 mg/mL, clone OKT3), PMCs were gently washed by RPMI-1640 to remove the uncombined mAbs. To determine whether other soluble factors contributed to the proliferation or apoptosis of T cells, we separated the activated PMCs and CD4þ T cell by semipermeable transwell system with 0.4-mm pore in noncontact group. 4 d later, the suspension cells were harvested and stained intracellularly for Ki-67-APC (eBioscience) or with APC conjugated Annexin V and propidium iodide (Annexin V Apoptosis Detection Kit APC; eBioscience) after surface staining with anti-CD4-FITC and then tested by flow cytometry. 2.8. Adhesion and migration of CD4þ T cells to PMCs Before the adhesion assays, confluent PMC monolayers in 24well plates (1 105/well) were incubated with anti-PD-L1 and -PD-L2 mAbs or control Igs, while C2925 (4 mM; Invitrogen, Carlsbad, CA)-labeled CD4þ T cells (1 106/well) isolated from blood were stimulated with 10 ng/mL phorbol myristate acetate (PMA, SigmaeAldrich St. Louis, MO) in the presence of anti-PD-1 (10 mg/ mL) blocking Ab or control Ig for 4 h. Then, both types of cells were washed twice with medium and pretreated CD4þ T cells (1 106/ well) were added to PMC monolayers in a final volume of 1 mL. 2 d later, non-adherent cells were washed off and cells adhering to PMC monolayers were counted in 25 different fields under a fluorescence microscope at low magnification. The adhesion indexes were calculated by dividing cell counts by those binding to the control Igs treated group. For migration assay, the 8-mm pore polycarbonate filters in 24well transwell chambers were used. When PMCs (1 105) cultured on transwell chambers reached confluence, they were stimulated with IFN-g (100 ng/mL) for 24 h and then treated with anti-PD-L1 and anti-PD-L2 or irrelevant control Igs for another 4 h before adding CD4þ T cells (5 105) isolated from blood to the top chambers in the final volume of 100 mL. Corresponding TPE were added to the bottom chambers to induce CD4þ T cell migration in the volume of 600 mL. RPMI 1640 containing 10% FBS served as control group to assess the gravity action. The chambers were incubated at 37 C in 5% CO2 atmosphere. 24 h later, CD4þ T cells migrated into the bottom chambers were harvested and counted. 2.9. Statistics Data are expressed as means SEM. For variables in TPE and in corresponding blood, paired data comparisons were made using a
133
Wilcoxon signed-rank test, while unpaired data comparisons were made using a Mann Whitney U test. Comparisons of the data between different groups were performed using a KruskaleWallis one-way analysis of variance on ranks. Analysis was completed with SPSS version 16.0 Statistical Software (Chicago, IL), and p values of less than 0.05 were considered to indicate statistical significance. 3. Results 3.1. PD-1, PD-L1 and PD-L2 in TPE Biochemical and cytological characteristics in pleural fluid are illustrated in Table 1. Subjects with TPE showed a significant infiltration on total cell counts, and a large proportion of these cells were lymphocytes, with some neutrophils, macrophages and PMCs. As shown in Figure 1, sPD-1 concentrations were much higher in TPE than in the corresponding serum (261.5 24.3 ng/L versus 64.1 15.3 ng/L, n ¼ 20, p < 0.001, Wilcoxon signed-rank test); on the other hand, concentrations of sPD-L1 (36.1 3.2 ng/L versus 34.2 2.4 ng/L) and sPD-L2 (3159.2 29.9 ng/L versus 3220.1 34.6 ng/L) in TPE were not different from those in serum (both p > 0.05, Wilcoxon signed-rank test). We performed flow cytometry to determine PD-1 expression on CD4þ T cells, and found that percentages of PD-1þCD4þ T cells represented the higher values in TPE (22.2 2.2%), showing a significant increase in comparison with those in blood (10.4 1.5%, n ¼ 10, p ¼ 0.001) (Figures 2A and 2B). We noted that PD-1þCD4þ T cells in TPE and blood expressed higher levels of CD45RO than CD45RA, and that percentages of CD45RAþPD-1þCD4þ T cells in TPE and blood were not different (28.5 1.5% versus 31.4 1.5%, n ¼ 10, p ¼ 0.136) (Figure 2C); in contrast, percentages of CD45ROþPD1þCD4þ T cells in TPE were much higher than those in blood (79.4 2.9% versus 40.9 2.6%, n ¼ 10, p < 0.001) (Figure 2D), indicating that most PD-1þCD4þ T cells in TPE were memory cells. We next isolated mononuclear cells from blood of patient with TPE and cultured them in vitro in the absence or presence of ESAT6/ CFP10 or/and IFN-g for 3 d. It was found that either ESAT6/CFP10 or IFN-g promoted the expression of PD-1 on CD4þ T cells compared with medium alone (Figures 2E and 2F). As shown in Figure 3A, PMCs isolated from TPE and transudative effusion expressed more PD-L1 (51.0 4.0% and 23.2 2.7%, respectively; both n ¼ 10) than PD-L2 (18.0 2.8% and 3.7 0.7%, respectively; both n ¼ 10); and similar results were observed with non-malignant transformed mesothelial cells (Met5A cells) (36.4 1.2% versus 8.9 2.6%, n ¼ 5). Notably, expressions of PD-L1
Figure 1. Concentrations of soluble programmed death-1 (PD-1) (A), soluble PD-ligand 1 (PD-L1) (B) and soluble PD-L2 (C) in tuberculous pleural effusion (TPE) and serum (n ¼ 20). Horizontal bars indicate means, and the comparisons were made by Wilcoxon signed-rank test.
134
W. Yin et al. / Tuberculosis 94 (2014) 131e139
Figure 2. Expression of programmed death-1 (PD-1) on CD4þ T cells from tuberculous pleural effusion (TPE). (A) The representative flow cytometric dot-plots of PD-1 expression on CD4þ T cells isolated from TPE and blood gated on CD3þ and CD8e T cells. Comparisons of percentages of PD-1þCD4þ T cells (B), CD45RAþPD-1þCD4þ T cells (C) and CD45RAþPD1þCD4þ T cells (D) in TPE and blood are shown; the results are presented as mean SEM (n ¼ 10). Horizontal bars indicate means, and the comparisons were made by Wilcoxon signed-rank test. (E) The representative flow cytometric dot-plots of PD-1 expression on CD4þ T cells in blood from patient with TPE in the absence or presence of Mycobacterium tuberculosis antigens ESAT6/CFP10 or/and IFN-g. (F) Summary data on percentages of PD-1þCD4þ T cells of each group. The results are presented as mean SEM from 5 independent experiments. Comparisons were made by KruskaleWallis one-way analysis of variance on ranks. *p < 0.05 compared with medium control.
and PD-L2 were much higher on PMCs isolated from TPE when compared with those from transudative effusion (both n ¼ 10, p < 0.01, Mann Whitney U test) (Figure 3B). On the other hand, concentrations of sPD-L1 and sPD-L2 in the supernatants of cultured PMCs were not different between TPE and transudative effusion (both p > 0.05) (Figure 3C). Neither primary PMCs isolated from pleural effusions nor Met5A cells expressed the receptor PD-1 (data not show).
3.2. Expressions of PD-L1 and PD-L2 on PMCs enhanced by IFN-g We cultured Met5A cells in the presence of designated cytokines for 3 d, and noted that IFN-g was the only one of the cytokines tested that could increase the expression of both ligands on Met5A cells (Figure 4A). As expected, IFN-g enhanced the expressions of PD-Ls in timee (Figure 4B) and dose-dependent (Figure 4C) manners. In addition, when Met5A cells were cultured in medium
W. Yin et al. / Tuberculosis 94 (2014) 131e139
135
Figure 3. Pleural mesothelial cells (PMCs) expressed programmed death ligand 1 (PD-L1) and PD-L2. (A) PMCs was identified by expression of calretinin and sideward scatter (SSC); and the representative flow cytometric histograms show expressions of PD-L1 and PD-L2 on PMCs isolated from transudative effusion (TE) and tuberculous pleural effusion (TPE), as well as on non-malignant transformed mesothelial cell line (Met5A cells) compared with isotype control (solid). (B) Comparisons of percentages of PD-L1 (left panel) and PD-L2 (right panel) positive PMCs from TE (n ¼ 10) and TPE (n ¼ 10). (C) Concentrations of soluble PD-L1 (left panels) and soluble PD-L2 (right panels) in PMC culture supernatants (n ¼ 10). In both (B) and (C), horizontal bars indicate means, and the comparisons were made by Mann Whitney U test.
supplemented with half volume of TPE, the numbers of PD-L1- and PD-L2-positive Met5A cells were significantly elevated compared with medium, and anti-IFN-g mAb abrogated this elevation induced by TPE (Figure 4D).
We also detected the concentrations of sPD-L1 and sPD-L2 in the supernatants of Met5A cells cultured for 3 d in the presence of the above cytokines, and noted that none of cytokines tested could stimulate release of sPD-L1 when compared with medium control
Figure 4. IFN-g enhanced expressions of programmed death ligand 1 (PD-L1) and PD-L2 on pleural mesothelial cells. (A) Non-malignant transformed mesothelial cell line (Met5A cells) were cultured and stimulated by designated cytokines for 3 d and harvested for flow cytometry to determine expressions of PD-L1 and PD-L2. IFN-g upregulated the expressions of PD-L1 and PD-L2 on Met5A cells in timee (B) and dose-dependent (C) manners. Met5A cells were cultured in medium or tuberculous pleural effusion (TPE) in the absence or presence of anti-IFN-g mAb, expressions of PD-L1 (open bars) and PD-L2 (solid bars) were determined by flow cytometry (D). Met5A cells were cultured and stimulated by designated cytokines for 3 d, the supernatants were collected for determining concentrations of soluble PD-L1 (E) and soluble PD-L2 (F) by ELISA. The results are presented as mean SEM from 5 independent experiments. Comparisons were made by KruskaleWallis one-way analysis of variance on ranks. *p < 0.05 compared with medium control; yp < 0.05 compared with TPE group.
136
W. Yin et al. / Tuberculosis 94 (2014) 131e139
(Figure 4E), whereas IL-1b, IL-12, or IL-32 each promoted release of sPD-L2 (Figure 4F).
3.4. PD-1/PD-Ls pathways downregulated adhesion, but not migration of CD4þ T cells
3.3. PD-1/PD-Ls pathways inhibited proliferation, but not apoptosis of CD4þ T cells
Since T cell adhesion and migration are critical for an effective immune response in lesion, we performed T cell adhesion and migration assays by treating CD4þ T cells from blood or PMCs with blocking Abs or irrelevant isotype control. Anti-PD-1 mAb promoted PMA activated CD4þ T cell adhesion (Figure 6A) without upregulating the expressions of CD11a and CD29, the ligands for ICAM-1 and VCAM-1 (Figure 6B), respectively. As shown in Figure 6C, TPE induced CD4þ T cell migration but PD-1/PD-Ls pathways did not contribute to this process.
PMCs isolated from TPE were preincubated with or without anti-PD-L1 or/and PD-L2 mAbs and then were co-cultured with non-autologous CD4þ T cells from TPE in the presence of anti-CD3 mAb for 4 d. As shown in Figures 5A and 5B, compared with CD4þ T cells alone, PMCs were found to be able to inhibit proliferation of CD4þ T cells. This inhibition was dependent on cellecell contact, since such an inhibition disappeared when PMCs and CD4þ T cells were separated by using transwell system (right top panel of Figure 5A). It was also noted that the addition of anti-PD-L1 or/and -PD-L2 mAbs into the co-culture abolished the inhibition induced by PMCs. On the other hand, PD-1/PD-Ls pathways did not affect the apoptosis of CD4þ T cells under the same culture conditions (Figures 5C and 5D).
4. Discussion In the past few years, mounting animal studies have demonstrated that PD-1/PD-Ls pathways play important roles in immune responses during MTB infection [10e13,16,17]. In humans, blockage of PD-1/PD-Ls pathways enhanced the specific degranulation of
Figure 5. Pleural mesothelial cells (PMCs) influenced proliferation, but not apoptosis, of CD4þ T cells via PD-1/PD-Ls pathways. (A) Confluent PMC monolayers preincubated with anti-PD-L1 or/and -PD-L2 mAbs for 24 h were cocultured with CD4þ T cells isolated from tuberculous pleural effusion at a ratio of 1:5 for 4 d in the presence of anti-CD3 mAb. In some experiments, 0.4-mm pore size transwells were used to separate the PMC monolayers and CD4þ T cells (right top panel). Ki-67þ cells were determined gating on CD4þ T cells, and the representative flow cytometric dot-plots are from one of five independent experiments. (B) Summary data of percentages of Ki-67þCD4þ T cells from each group. (C) Annexin Vþ cells were determined gating on CD4þ T cells, and the representative flow cytometric dot-plots are from one of five independent experiments. (D) Summary data of percentages of Annexin Vþ T cells from each group. In both (B) and (D), the results are presented as means SEM. Comparisons were made by KruskaleWallis one-way analysis of variance on ranks. *p < 0.05 compared with coculture of PMCs and CD4þ T cells without mAb treatment.
W. Yin et al. / Tuberculosis 94 (2014) 131e139
137
Figure 6. PD-1/PD-Ls pathways downregulated CD4þ T cell adhesion. (A) C2925-labled CD4þ T cells purified from blood were added to PMC monolayers. In some experiments, phorbol myristate acetate stimulated CD4þ T cells were pretreated with anti-PD-1 mAb whereas PMCs were pretreated with anti-PD-L1 and -PD-L2 mAbs (n ¼ 5). (B) Anti-PD-1 mAb did not influence the expression of CD11a and CD29 on PMA activated CD4þ T cells. The representative flow cytometric dot-plots are from one of five independent experiments. (C) Activated CD4þ T cells migrated through IFN-g treated PMC monolayers in the absence or presence of tuberculous pleural effusion with or without anti-PD-L1 and -PD-L2 mAbs (n ¼ 5). All results are presented as means SEM. Comparisons were made by KruskaleWallis one-way analysis of variance on ranks. *p < 0.05 compared with medium control.
CD8þ T cells and numbers of specific IFN-g-producing lymphocytes against MTB [14]. Moreover, blockade of the PD-1/PD-Ls pathways significantly augmented lytic degranulation and IFN-g production of NK cells against MTB [18]. To our knowledge, the present study was the first one to investigate the expressions of PD-Ls on PMCs, and to explore the roles played by PD-1/PD-Ls pathways between CD4þ T cells and PMCs in TPE. PD-1 was expressed on activated T cell, B cells, and mesenchymal stem cells [19e21]. PD-L1 and PD-L2 were expressed on various cell types including T cell, B cell, NK cell and antigenpresenting cells [8,14,18,22,23]. PD-L1 expression was significantly overrepresented in whole blood from patients with active tuberculosis as compared with patients with latent tuberculosis infection and healthy controls; whereas levels of PD-1 and PD-L2 overall were not different in all three groups [24]. It has been demonstrated that MTB antigen stimulation could induce a significant increase in PD-1 expression on pleural T cells from TPE patients with high T cell response to MTB antigens [14]. In another human study [18], Alvarez and colleagues have observed that PD-1 basal levels were significantly higher on blood NK cells compared TPE; however, when stimulated with MTB, PD-1 expression levels on NK cells were markedly higher in TPE compared to blood. In the
present study, our data clearly indicated that the concentration of sPD-1 in TPE and surface expression of PD-1 in CD4þ T cells in TPE were much higher than their compartments in the corresponding blood. We also noted that the percentages of CD45ROþPD-1þCD4þ T cells in TPE were much higher than those in blood, indicating that most PD-1þCD4þ T cells in TPE were memory T cells. The most novel findings in our study were that the membrane expressions of two ligands for PD-1, PD-L1 and PD-L2, on PMCs from TPE were much higher than those from transudative effusion. Surprisingly, although PMCs from TPE expressed higher levels of PD-Ls than those from transudative effusion did, they did not release more sPD-L1 and sPD-L2 into the supernatants when cultured in vitro. Taken together, the above data suggested that TPE microenvironment results in the upregulation of PD-1 and PD-Ls, and that PD-1/ PD-Ls pathways might be involved in the pathogenesis of TPE. It has been reported that TPE was enriched with CD4þCDw29þ T cells, which are thought to represent “memory” T cells, and these pleural CD4þCDw29þ cells, but not CD4þCDw29e cells, proliferated vigorously and produced high levels of IFN-g when stimulated with purified protein derivative of MTB [25]. High concentration of IFN-g could always be found in TPE and served as a reasonable diagnostic biomarker for TPE [26]. Our current data showed that exogenous
138
W. Yin et al. / Tuberculosis 94 (2014) 131e139
IFN-g notably enhanced expression of PD-1 on CD4þ T cells, just like MTB-specific antigen ESAT-6/CFP-10 did. Furthermore, consistent with previous findings with endothelial cells [27,28], we also noted that IFN-g dramatically induced the expressions of PD-Ls on PMCs in time- and dose-dependent manners. The observation that IL-1b, IL-12 and IL-32 increased soluble PD-L2 in culture supernatants of Met5A cells indicated that these proinflammatory cytokines might contribute to the immune response against MTB. It should be mentioned that although high concentration of IFN-g is always found in TPE [26], adding anti-IFN-g mAb did not abolish the upregulation of PD-L1 in response to TPE, we believed that other cytokines enriched in TPE could be responsible for this upregulation. Many previous studies revealed that PD-1/PD-Ls pathways negatively regulated T cell proliferation and cytokine production [7,8,29] and positively regulated T cell apoptosis [30]; controversially, Tseng et al. reported positive regulation on T cell activation [9]. Human studies revealed that these co-stimulatory molecules negatively regulated the effector function of CD3þ, CD8þ T and NK cells in MTB infection [14,18], which were not inconsistent with the findings obtained from animal studies [11e13]. Our previous data have demonstrated that PMCs expressed co-stimulatory molecules, CD80 and CD86, and functioned as antigen-presenting cells to stimulate the differentiation of Th22, Th9, Th17, and Th1 cells from autologous naive CD4þ T cells [4,5], we therefore investigated whether PD-Ls expressed on PMCs could serve as negative costimulator signals for regulation of CD4þ T cells. We noted in the current study that when PMCs were co-cultured with pleural CD4þ T cells from unrelated donors, CD4þ T cell proliferation could be inhibited, and preincubation with anti-PD-L1 or/and -PD-L2 mAbs abolished the inhibition induced by PMCs, indicating that PD-1/PDLs pathways inhibited CD4þ T cell proliferation. In addition, we did not observe PD-1/PD-Ls pathways could affect apoptosis of CD4þ T cells. Different disease and different effector cells might explain the difference between our observations and the findings reported by Dong et al. [30]. Given that ICAM-1 and VCAM-1 participate in cell adhesion and migration, and mesothelial cells express adhesion molecules ICAM1 and VCAM-1 [3,31], and that PD-1/PD-L2 influence adhesion activity of T cells [29], we investigated the effects of PD-1/PD-Ls pathways on activated T cell adhesion to PMC monolayer. Our data revealed that anti-PD-1 mAb promoted CD4þ T cell adhesion without upregulating the corresponding receptor of ICAM-1 and VCAM-1. As blocking PD-Ls on human brain endothelial cells increased migration of CD8þ and CD4þ T cells [32], we developed a similar migration assay by building a model for pleural cavity but failed to obtain the similar results. The above observation demonstrated that PD-1/PD-Ls pathways on PMCs negatively regulated adhesion, but not migration, of CD4þ T cells. In summary, we have demonstrated that concentration of sPD-1, expression of PD-1 on CD4þ T cells, and expressions of PD-Ls on PMCs were increased in TPE, and that MTB-specific antigen stimulation and/or cytokine micro-environment contributed to the upregulation. We confirmed that PMCs regulated proliferation and adhesion activity of CD4þ T cells via PD-1/PD-Ls pathways. Our present study suggested that MTB might exploit PD-1/PD-Ls pathways to evade host cell immune response in human. Acknowledgments This work was supported in part by grants from National Natural Science Foundation of China (No. 81270149 and No. 81272591); in part by a grant from National Science Fund for Distinguished Young Scholars of China (No. 30925032).
Funding:
None.
Competing interests:
None.
Ethical approval: The study protocol was approved by our Institutional Review Boards for human studies of Capital Medical University, Beijing, China; and Tongji Medical College, Wuhan, China; and informed written consent was obtained from all subjects. Authors’ contributions: HZS designed the study and drafted the manuscript. WY, ZHT, and AC isolated, processed and cultured cells, and performed flow cytometry. JCZ and ZJY were responsible for sample collection and ELISA. MLY and QZ recruited subjects and analyzed the data. All authors read, critically revised, and approved the final manuscript. References [1] Jafari C, Lange C. Suttons’s law: local immunodiagnosis of tuberculosis. Infection 2008;36:510e4. http://dx.doi.org/10.1007/s15010-008-8089-9. [2] Jantz MA, Antony VB. Pathophysiology of the pleura. Respiration 2008;75: 121e33. http://dx.doi.org/10.1159/000113629. 000113629 [pii]. [3] Nasreen N, Mohammed KA, Ward MJ, Antony VB. Mycobacterium-induced transmesothelial migration of monocytes into pleural space: role of intercellular adhesion molecule-1 in tuberculous pleurisy. J Infect Dis 1999;180: 1616e23. http://dx.doi.org/10.1086/315057. JID981576 [pii]. [4] Ye ZJ, Zhou Q, Yuan ML, Du RH, Yang WB, Xiong XZ, Huang B, Shi HZ. Differentiation and recruitment of IL-22-producing helper T cells stimulated by pleural mesothelial cells in tuberculous pleurisy. Am J Respir Crit Care Med 2012;185:660e9. http://dx.doi.org/10.1164/rccm.201107-1198OC. rccm.201107-1198OC [pii]. [5] Ye ZJ, Yuan ML, Zhou Q, Du RH, Yang WB, Xiong XZ, Zhang JC, Wu C, Qin SM, Shi HZ. Differentiation and recruitment of Th9 cells stimulated by pleural mesothelial cells in human Mycobacterium tuberculosis infection. PLoS ONE 2012;7:e31710. http://dx.doi.org/10.1371/journal.pone.0031710. PONE-D-1119196 [pii]. [6] Dong H, Zhu G, Tamada K, Chen L. B7-H1, a third member of the B7 family, costimulates T-cell proliferation and interleukin-10 secretion. Nat Med 1999;5: 1365e9. http://dx.doi.org/10.1038/70932. [7] Freeman GJ, Long AJ, Iwai Y, Bourque K, Chernova T, Nishimura H, Fitz LJ, Malenkovich N, Okazaki T, Byrne MC, Horton HF, Fouser L, Carter L, Ling V, Bowman MR, Carreno BM, Collins M, Wood CR, Honjo T. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med 2000;192:1027e34. [8] Latchman Y, Wood CR, Chernova T, Chaudhary D, Borde M, Chernova I, Iwai Y, Long AJ, Brown JA, Nunes R, Greenfield EA, Bourque K, Boussiotis VA, Carter LL, Carreno BM, Malenkovich N, Nishimura H, Okazaki T, Honjo T, Sharpe AH, Freeman GJ. PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat Immunol 2001;2:261e8. http://dx.doi.org/10.1038/85330. [9] Tseng SY, Otsuji M, Gorski K, Huang X, Slansky JE, Pai SI, Shalabi A, Shin T, Pardoll DM, Tsuchiya H. B7-DC, a new dendritic cell molecule with potent costimulatory properties for T cells. J Exp Med 2001;193:839e46. [10] Reiley WW, Shafiani S, Wittmer ST, Tucker-Heard G, Moon JJ, Jenkins MK, Urdahl KB, Winslow GM, Woodland DL. Distinct functions of antigen-specific CD4 T cells during murine Mycobacterium tuberculosis infection. Proc Natl Acad Sci USA 2010;107:19408e13. http://dx.doi.org/10.1073/ pnas.1006298107. 1006298107 [pii]. [11] Sakai S, Kawamura I, Okazaki T, Tsuchiya K, Uchiyama R, Mitsuyama M. PD-1PD-L1 pathway impairs T(h)1 immune response in the late stage of infection with Mycobacterium bovis bacillus Calmette-Guerin. Int Immunol 2010;22: 915e25. http://dx.doi.org/10.1093/intimm/dxq446. dxq446 [pii]. [12] Tousif S, Singh Y, Prasad DV, Sharma P, Van Kaer L, Das G. T cells from programmed death-1 deficient mice respond poorly to Mycobacterium tuberculosis infection. PLoS ONE 2011;6:e19864. http://dx.doi.org/10.1371/ journal.pone.0019864. PONE-D-11-02781 [pii]. [13] Lazar-Molnar E, Chen B, Sweeney KA, Wang EJ, Liu W, Lin J, Porcelli SA, Almo SC, Nathenson SG, Jacobs Jr WR. Programmed death-1 (PD-1)-deficient mice are extraordinarily sensitive to tuberculosis. Proc Natl Acad Sci USA 2010;107:13402e7. http://dx.doi.org/10.1073/pnas.1007394107 [pii]. [14] Jurado JO, Alvarez IB, Pasquinelli V, Martinez GJ, Quiroga MF, Abbate E, Musella RM, Chuluyan HE, Garcia VE. Programmed death (PD)-1:PD-ligand 1/ PD-ligand 2 pathway inhibits T cell effector functions during human tuberculosis. J Immunol 2008;181:116e25. doi: 181/1/116 [pii]. [15] Light RW, Macgregor MI, Luchsinger PC, Ball Jr WC. Pleural effusions: the diagnostic separation of transudates and exudates. Ann Intern Med 1972;77: 507e13. [16] Qiu L, Huang D, Chen CY, Wang R, Shen L, Shen Y, Hunt R, Estep J, Haynes BF, Jacobs Jr WR, Letvin N, Du G, Chen ZW. Severe tuberculosis induces
W. Yin et al. / Tuberculosis 94 (2014) 131e139
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
unbalanced up-regulation of gene networks and overexpression of IL-22, MIP1alpha, 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;198:1514e 9. http://dx.doi.org/10.1086/592448. Henao-Tamayo M, Irwin SM, Shang S, Ordway D, Orme IM. T lymphocyte surface expression of exhaustion markers as biomarkers of the efficacy of chemotherapy for tuberculosis. Tuberculosis (Edinb) 2011;91:308e13. http:// dx.doi.org/10.1016/j.tube.2011.04.001. S1472-9792(11)00064-3 [pii]. Alvarez IB, Pasquinelli V, Jurado JO, Abbate E, Musella RM, de la Barrera SS, Garcia VE. Role played by the programmed death-1-programmed death ligand pathway during innate immunity against Mycobacterium tuberculosis. J Infect Dis 2010;202:524e32. http://dx.doi.org/10.1086/654932. Keir ME, Latchman YE, Freeman GJ, Sharpe AH. Programmed death-1 (PD-1): PD-ligand 1 interactions inhibit TCR-mediated positive selection of thymocytes. J Immunol 2005;175:7372e9. doi: 175/11/7372 [pii]. Keir ME, Francisco LM, Sharpe AH. PD-1 and its ligands in T-cell immunity. Curr Opin Immunol 2007;19:309e14. http://dx.doi.org/10.1016/ j.coi.2007.04.012. S0952-7915(07)00064-7 [pii]. Keir ME, Butte MJ, Freeman GJ, Sharpe AH. PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol 2008;26:677e704. http://dx.doi.org/ 10.1146/annurev.immunol.26.021607.090331. Brown JA, Dorfman DM, Ma FR, Sullivan EL, Munoz O, Wood CR, Greenfield EA, Freeman GJ. Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production. J Immunol 2003;170:1257e66. Karakhanova S, Bedke T, Enk AH, Mahnke K. IL-27 renders DC immunosuppressive by induction of B7-H1. J Leukoc Biol 2011;89:837e45. http:// dx.doi.org/10.1189/jlb.1209788. jlb.1209788 [pii]. McNab FW, Berry MP, Graham CM, Bloch SA, Oni T, Wilkinson KA, Wilkinson RJ, Kon OM, Banchereau J, Chaussabel D, O’Garra A. Programmed death ligand 1 is over-expressed by neutrophils in the blood of patients with
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
139
active tuberculosis. Eur J Immunol 2011;41:1941e7. http://dx.doi.org/ 10.1002/eji.201141421. Barnes PF, Mistry SD, Cooper CL, Pirmez C, Rea TH, Modlin RL. Compartmentalization of a CD4þ T lymphocyte subpopulation in tuberculous pleuritis. J Immunol 1989;142:1114e9. Jiang J, Shi HZ, Liang QL, Qin SM, Qin XJ. Diagnostic value of interferon-gamma in tuberculous pleurisy: a metaanalysis. Chest 2007;131:1133e41. http:// dx.doi.org/10.1378/chest.06-2273. Eppihimer MJ, Gunn J, Freeman GJ, Greenfield EA, Chernova T, Erickson J, Leonard JP. Expression and regulation of the PD-L1 immunoinhibitory molecule on microvascular endothelial cells. Microcirculation 2002;9:133e45. http://dx.doi.org/10.1038/sj/mn/7800123. Rodig N, Ryan T, Allen JA, Pang H, Grabie N, Chernova T, Greenfield EA, Liang SC, Sharpe AH, Lichtman AH, Freeman GJ. Endothelial expression of PDL1 and PD-L2 down-regulates CD8þ T cell activation and cytolysis. Eur J Immunol 2003;33:3117e26. http://dx.doi.org/10.1002/eji.200324270. Saunders PA, Hendrycks VR, Lidinsky WA, Woods ML. PD-L2:PD-1 involvement in T cell proliferation, cytokine production, and integrin-mediated adhesion. Eur J Immunol 2005;35:3561e9. http://dx.doi.org/10.1002/ eji.200526347. Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, Roche PC, Lu J, Zhu G, Tamada K, Lennon VA, Celis E, Chen L. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med 2002;8:793e800. http://dx.doi.org/10.1038/nm730. nm730 [pii]. Jonjic N, Peri G, Bernasconi S, Sciacca FL, Colotta F, Pelicci P, Lanfrancone L, Mantovani A. Expression of adhesion molecules and chemotactic cytokines in cultured human mesothelial cells. J Exp Med 1992;176:1165e74. Pittet CL, Newcombe J, Prat A, Arbour N. Human brain endothelial cells endeavor to immunoregulate CD8 T cells via PD-1 ligand expression in multiple sclerosis. J Neuroinflammation 2011;8:155. http://dx.doi.org/10.1186/ 1742-2094-8-155. 1742-2094-8-155 [pii].
Contents lists available at ScienceDirect
Tuberculosis journal homepage: http://intl.elsevierhealth.com/journals/tube
IMMUNOLOGICAL ASPECTS
PD-1/PD-Ls pathways between CD4þ T cells and pleural mesothelial cells in human tuberculous pleurisy Wen Yin a, b, Zhao-Hui Tong a, c, Ai Cui a, Jian-Chu Zhang b, Zhi-Jian Ye b, Ming-Li Yuan b, Qiong Zhou b, Huan-Zhong Shi a, b, c, * a
Department of Respiratory and Critical Care Medicine, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China Department of Respiratory and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China c Center of Medical Research, Beijing Institute of Respiratory Diseases, Beijing, China b
a r t i c l e i n f o
s u m m a r y
Article history: Received 4 June 2013 Received in revised form 24 October 2013 Accepted 27 October 2013
Programmed death 1 (PD-1), PD-ligand 1 (PD-L1), and PD-L2 have been demonstrated to be involved in tuberculosis immunity, however, the expression and regulation of PD-1/PD-Ls pathways in pleural mesothelial cells (PMCs) and CD4þ T cells in tuberculous pleural effusion (TPE) have not been investigated. Expression of PD-1 on CD4þ T cells and expressions of PD-L1 and PD-L2 on PMCs in TPE were determined. The impacts of PD-1/PD-Ls pathways on proliferation, apoptosis, adhesion, and migration of CD4þ T cells were explored. Concentrations of soluble PD-l, but not of soluble PD-Ls, were much higher in TPE than in serum. Expressions of PD-1 on CD4þ T cells in TPE were significantly higher than those in blood. Expressions of PD-Ls were much higher on PMCs from TPE when compared with those from transudative effusion. Interferon-g not only upregulated the expression of PD-1 on CD4þ T cells, but also upregulated the expressions of PD-Ls on PMCs. Blockage PD-1/PD-Ls pathways abolished the inhibitory effects on proliferation and adhesion activity of CD4þ T cells induced by PMCs. PD-1/PD-Ls pathways on PMCs inhibited proliferation and adhesion activity of CD4þ T cells, suggesting that Mycobacterium tuberculosis might exploit PD-1/PD-Ls pathways to evade host cell immune response in human. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Pleural effusion Programmed death-1 Tuberculosis
1. Introduction Tuberculosis remains a major health problem, with an estimated one third of the world’s population infected with Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis, causing about 1.4 million deaths in 2011. Tuberculous pleural effusion (TPE) is the second most frequent manifestation of extrapulmonary tuberculosis after lymph node tuberculosis, and is restricted to the pleural cavity, which contains protein-enriched fluid and numerous immunocompetent cells, especially CD4þ T cells [1]. Pleural mesothelial cells (PMCs), presented in a single layer covering each pleural membrane, are the most common cells of the pleural space
* Corresponding author. Department of Respiratory and Critical Care Medicine, Beijing Chaoyang Hospital, Capital Medical University, 8 Gongren Tiyuchang Nanlu, Chaoyang District, Beijing 100020, China. Tel./fax: þ86 10 85231412. E-mail address: [email protected] (H.-Z. Shi). 1472-9792/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tube.2013.10.007
which not only encounter invading microbes, but also subsequently initiate and propagate an inflammatory reaction by coordinating the other kinds of inflammatory cells [2]. Early studies have demonstrated that PMCs facilitate monocyte transmigration across pleural mesothelium during MTB infection [3]. Our recent data have demonstrated that PMCs were able to function as antigenpresenting cells to stimulate proliferation of CD4þ T cells and differentiation of Th22, Th9, Th17 and Th1 cells in response to MTB antigens in CD80- and CD86-dependent means [4,5]. Programmed death 1 (PD-1) is a key immune checkpoint receptor expressed by activated T cells, and it mediates immunosuppression. PD-1 has two known ligands, PD-L1 (B7-H1) and PDL2 (B7-DC) [6e9]. In vitro studies have shown that engagement of PD-1 by PD-L1 and PD-L2 inhibited T cell proliferation and cytokine production, indicating that the crosslinking of PD-1 by its PD-Ls leads to downregulation of T cell responses [7,8], although not all the studies support the inhibitory role for the PD-1/PD-Ls pathways [9]. In vivo animal studies have demonstrated that MTB infection
132
W. Yin et al. / Tuberculosis 94 (2014) 131e139
resulted in upregulation of PD-1, PD-L1 and PD-L2 [10e12], and PD1 deficient mice were extremely sensitive to MTB infection when compared to their wild type littermates, showing dramatically reduced survival and higher bacterial loads [12,13], whereas MTB numbers in spleen of PD-1 deficient mice were decreased compared with wild type mice [11]. In human studies, PD-1/PD-Ls pathways have also been shown to inhibit T cell effector functions during active pulmonary tuberculosis [14]. We therefore designed the present study to evaluate the regulation and immune function of PD-1/PD-Ls pathways of PMCs in TPE. 2. Methods 2.1. Subjects The study protocol was approved by our Institutional Review Boards for human studies of Capital Medical University, Beijing, China; and Tongji Medical College, Wuhan, China; and informed written consent was obtained from all subjects. Twenty anti-HIV Ab negative patients were proven to have TPE, as evidenced by growth of MTB from pleural fluid or by demonstration of granulomatous pleurisy on pleural biopsy specimen in the absence of any evidence of other granulomatous diseases (Table 1). Another 10 anti-HIV Ab negative patients suffering from heart failure or hypoalbuminemia were diagnosed to have pleural transudative effusion according to Light’s criteria [15]. 2.2. Collecting and processing samples Five-hundred to 1000 mL of pleural fluid samples from each patient were collected in heparin-treated tubes, through a standard thoracocentesis technique within 24 h after hospitalization. Twenty milliliters of peripheral blood were drawn simultaneously. TPE specimens were immersed in ice immediately and were then centrifuged at 1200 g for 5 min before isolating PMCs. In some experiments, the cell pellets of TPE were resuspended in HBSS, and mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation (Pharmacia, Uppsala, Sweden). 2.3. Measurement of soluble (s) PD-1/PD-Ls The concentrations of sPD-1, sPD-L1, and sPD-L2 in TPE, serum and PMC culture supernatants were measured by ELISA kits (sPD-1 and sPD-L2 kits from R&D systems, Minneapolis, MN; sPD-L1 kits from MyBioSource, San Diego, CA) according to the instructions provided by the manufacturers.
2.4. Flow cytometry Expression of PD-1 on T cells from TPE and blood were determined by flow cytometry after surface staining with anti-human CD3-PerCP-cy5.5, CD8-FITC, PD-1-APC mAbs, which were purchased from BD Biosciences (Franklin Lakes, NJ) or eBioscience (San Diego, CA). CD11a-PE and CD29-APC mAbs were used for the detection of ligands of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1). Fixed and permeabilized PMCs were primarily stained with mouse anti-alretinin mAb (BD Biosciences) to identify PMCs, and then stained with FITClabeled goat anti-mouse Igs (BD Biosciences). Appropriate species matched Abs served as isotype controls. To explore the expression of molecules on PMCs, anti-PD-L1, -PD-L2, -PD-1, -ICAM-1, -VCAM1, -CD80 or -CD86 (all from eBioscience), conjugated with PE or APC were used. Flow cytometry was performed on a FACS Canto II (BD Biosciences) and analyzed using BD FCSDiva Software and FCS Epress 4 software (De Novo Software, Los Angeles, CA). 2.5. Isolation and culture of PMCs and CD4þ T cells For isolating PMCs, the cell pellets of TPE were resuspended in RPMI-1640 containing 20% heat-inactivated FBS, and 50 mg/mL gentamycin. The cells were seeded into 25-cm [2] flasks at a density of 1 l04 cells/cm2 and placed in an incubator at 37 C in 5% CO2. After 24 h the monolayers were washed with HBSS to remove nonadherent cells and fresh media was added. The monolayers were monitored until confluent (7e10 d), then trypsinized, and subcultured. To prepare PMC culture supernatants, the second generation PMCs were seeded and cultured in complete medium at a density of 106 cells per well in the absence or presence of designated cytokines, and the culture supernatants were harvested 72 h later. CD4þ T cells were isolated from blood and TPE by MACS based on negative selection using CD4þ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The purity of CD4þ T cells was >97%, as measured by flow cytometry. To investigate the effect of MTB-specific peptide of early secretory antigenic target-6kDa/culture filtrate protein-10 (ESAT-6/CFP10) and cytokine IFN-g on regulation of membrane PD-1 expression on CD4þ T cells, mononuclear cells isolated from blood were cultured in 24-well plates at a density of 5 105/well with or without ESAT/CFP10 (10 mg/mL, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China) and/or IFN-g (50 ng/mL) for 3 d in RPMI-1640 (Gibco, Invitrogen, Carlsbad, CA) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco), and 50 mg/mL gentamycin. 2.6. Effects of cytokines on expressions of PD-Ls on PMCs
Table 1 Characteristics of the study population. Pleural effusions
Patients, No. Sex, male/female, No. Age, yr Protein, g/L Lactate dehydrogenase, IU/L Adenosine deaminase, U/L Total cell counts, 109/L Differential cell counts, % Lymphocytes Neutrophils Macrophages Mesothelial cells
Tuberculous
Transudative
20 14/6 42.3 3.6 41.2 7.6 468.9 50.7 55.5 6.8 3.43 1.2
10 6/4 47.6 8.2 17.8 9.8 123.6 56.3 9.2 4.5 0.56 0.08
75.5 5.8 8.4 1.2 13.7 1.3 2.4 0.4
32.5 3.9 7.4 1.3 48.2 3.7 11.9 1.9
To investigate the effects of cytokines such as IFN-g (50 ng/mL), TNF-a (50 ng/mL), IL-1b (10 ng/mL), IL-6 (50 ng/mL), IL-12 (20 ng/ mL), IL-27 (100 ng/mL) and IL-32 (10 ng/mL) (all from R&D systems) on regulation of membrane PD-Ls expression on PMCs, nonmalignant transformed mesothelial cell line (Met5A cells) were cultured in 12-well plates at a density of 5 105/well with or without the above cytokines for 3 d in RPMI-1640 containing 20% heat-inactivated FBS and 50 mg/mL gentamycin. 2.7. Proliferation and apoptosis of CD4þ T cells Confluent PMC monolayers in 24-well plates (1 105/well) were activated by adding IFN-g (100 ng/mL) for 24 h and then treated by adding blocking mAbs specific for PD-L1 (10 mg/mL) and/or PD-L2
W. Yin et al. / Tuberculosis 94 (2014) 131e139
(10 mg/mL) or isotype control Igs for another 24 h. Before cocultured with purified CD4þ T (5 105) cells isolated from TPE in the presence of anti-CD3 (1.0 mg/mL, clone OKT3), PMCs were gently washed by RPMI-1640 to remove the uncombined mAbs. To determine whether other soluble factors contributed to the proliferation or apoptosis of T cells, we separated the activated PMCs and CD4þ T cell by semipermeable transwell system with 0.4-mm pore in noncontact group. 4 d later, the suspension cells were harvested and stained intracellularly for Ki-67-APC (eBioscience) or with APC conjugated Annexin V and propidium iodide (Annexin V Apoptosis Detection Kit APC; eBioscience) after surface staining with anti-CD4-FITC and then tested by flow cytometry. 2.8. Adhesion and migration of CD4þ T cells to PMCs Before the adhesion assays, confluent PMC monolayers in 24well plates (1 105/well) were incubated with anti-PD-L1 and -PD-L2 mAbs or control Igs, while C2925 (4 mM; Invitrogen, Carlsbad, CA)-labeled CD4þ T cells (1 106/well) isolated from blood were stimulated with 10 ng/mL phorbol myristate acetate (PMA, SigmaeAldrich St. Louis, MO) in the presence of anti-PD-1 (10 mg/ mL) blocking Ab or control Ig for 4 h. Then, both types of cells were washed twice with medium and pretreated CD4þ T cells (1 106/ well) were added to PMC monolayers in a final volume of 1 mL. 2 d later, non-adherent cells were washed off and cells adhering to PMC monolayers were counted in 25 different fields under a fluorescence microscope at low magnification. The adhesion indexes were calculated by dividing cell counts by those binding to the control Igs treated group. For migration assay, the 8-mm pore polycarbonate filters in 24well transwell chambers were used. When PMCs (1 105) cultured on transwell chambers reached confluence, they were stimulated with IFN-g (100 ng/mL) for 24 h and then treated with anti-PD-L1 and anti-PD-L2 or irrelevant control Igs for another 4 h before adding CD4þ T cells (5 105) isolated from blood to the top chambers in the final volume of 100 mL. Corresponding TPE were added to the bottom chambers to induce CD4þ T cell migration in the volume of 600 mL. RPMI 1640 containing 10% FBS served as control group to assess the gravity action. The chambers were incubated at 37 C in 5% CO2 atmosphere. 24 h later, CD4þ T cells migrated into the bottom chambers were harvested and counted. 2.9. Statistics Data are expressed as means SEM. For variables in TPE and in corresponding blood, paired data comparisons were made using a
133
Wilcoxon signed-rank test, while unpaired data comparisons were made using a Mann Whitney U test. Comparisons of the data between different groups were performed using a KruskaleWallis one-way analysis of variance on ranks. Analysis was completed with SPSS version 16.0 Statistical Software (Chicago, IL), and p values of less than 0.05 were considered to indicate statistical significance. 3. Results 3.1. PD-1, PD-L1 and PD-L2 in TPE Biochemical and cytological characteristics in pleural fluid are illustrated in Table 1. Subjects with TPE showed a significant infiltration on total cell counts, and a large proportion of these cells were lymphocytes, with some neutrophils, macrophages and PMCs. As shown in Figure 1, sPD-1 concentrations were much higher in TPE than in the corresponding serum (261.5 24.3 ng/L versus 64.1 15.3 ng/L, n ¼ 20, p < 0.001, Wilcoxon signed-rank test); on the other hand, concentrations of sPD-L1 (36.1 3.2 ng/L versus 34.2 2.4 ng/L) and sPD-L2 (3159.2 29.9 ng/L versus 3220.1 34.6 ng/L) in TPE were not different from those in serum (both p > 0.05, Wilcoxon signed-rank test). We performed flow cytometry to determine PD-1 expression on CD4þ T cells, and found that percentages of PD-1þCD4þ T cells represented the higher values in TPE (22.2 2.2%), showing a significant increase in comparison with those in blood (10.4 1.5%, n ¼ 10, p ¼ 0.001) (Figures 2A and 2B). We noted that PD-1þCD4þ T cells in TPE and blood expressed higher levels of CD45RO than CD45RA, and that percentages of CD45RAþPD-1þCD4þ T cells in TPE and blood were not different (28.5 1.5% versus 31.4 1.5%, n ¼ 10, p ¼ 0.136) (Figure 2C); in contrast, percentages of CD45ROþPD1þCD4þ T cells in TPE were much higher than those in blood (79.4 2.9% versus 40.9 2.6%, n ¼ 10, p < 0.001) (Figure 2D), indicating that most PD-1þCD4þ T cells in TPE were memory cells. We next isolated mononuclear cells from blood of patient with TPE and cultured them in vitro in the absence or presence of ESAT6/ CFP10 or/and IFN-g for 3 d. It was found that either ESAT6/CFP10 or IFN-g promoted the expression of PD-1 on CD4þ T cells compared with medium alone (Figures 2E and 2F). As shown in Figure 3A, PMCs isolated from TPE and transudative effusion expressed more PD-L1 (51.0 4.0% and 23.2 2.7%, respectively; both n ¼ 10) than PD-L2 (18.0 2.8% and 3.7 0.7%, respectively; both n ¼ 10); and similar results were observed with non-malignant transformed mesothelial cells (Met5A cells) (36.4 1.2% versus 8.9 2.6%, n ¼ 5). Notably, expressions of PD-L1
Figure 1. Concentrations of soluble programmed death-1 (PD-1) (A), soluble PD-ligand 1 (PD-L1) (B) and soluble PD-L2 (C) in tuberculous pleural effusion (TPE) and serum (n ¼ 20). Horizontal bars indicate means, and the comparisons were made by Wilcoxon signed-rank test.
134
W. Yin et al. / Tuberculosis 94 (2014) 131e139
Figure 2. Expression of programmed death-1 (PD-1) on CD4þ T cells from tuberculous pleural effusion (TPE). (A) The representative flow cytometric dot-plots of PD-1 expression on CD4þ T cells isolated from TPE and blood gated on CD3þ and CD8e T cells. Comparisons of percentages of PD-1þCD4þ T cells (B), CD45RAþPD-1þCD4þ T cells (C) and CD45RAþPD1þCD4þ T cells (D) in TPE and blood are shown; the results are presented as mean SEM (n ¼ 10). Horizontal bars indicate means, and the comparisons were made by Wilcoxon signed-rank test. (E) The representative flow cytometric dot-plots of PD-1 expression on CD4þ T cells in blood from patient with TPE in the absence or presence of Mycobacterium tuberculosis antigens ESAT6/CFP10 or/and IFN-g. (F) Summary data on percentages of PD-1þCD4þ T cells of each group. The results are presented as mean SEM from 5 independent experiments. Comparisons were made by KruskaleWallis one-way analysis of variance on ranks. *p < 0.05 compared with medium control.
and PD-L2 were much higher on PMCs isolated from TPE when compared with those from transudative effusion (both n ¼ 10, p < 0.01, Mann Whitney U test) (Figure 3B). On the other hand, concentrations of sPD-L1 and sPD-L2 in the supernatants of cultured PMCs were not different between TPE and transudative effusion (both p > 0.05) (Figure 3C). Neither primary PMCs isolated from pleural effusions nor Met5A cells expressed the receptor PD-1 (data not show).
3.2. Expressions of PD-L1 and PD-L2 on PMCs enhanced by IFN-g We cultured Met5A cells in the presence of designated cytokines for 3 d, and noted that IFN-g was the only one of the cytokines tested that could increase the expression of both ligands on Met5A cells (Figure 4A). As expected, IFN-g enhanced the expressions of PD-Ls in timee (Figure 4B) and dose-dependent (Figure 4C) manners. In addition, when Met5A cells were cultured in medium
W. Yin et al. / Tuberculosis 94 (2014) 131e139
135
Figure 3. Pleural mesothelial cells (PMCs) expressed programmed death ligand 1 (PD-L1) and PD-L2. (A) PMCs was identified by expression of calretinin and sideward scatter (SSC); and the representative flow cytometric histograms show expressions of PD-L1 and PD-L2 on PMCs isolated from transudative effusion (TE) and tuberculous pleural effusion (TPE), as well as on non-malignant transformed mesothelial cell line (Met5A cells) compared with isotype control (solid). (B) Comparisons of percentages of PD-L1 (left panel) and PD-L2 (right panel) positive PMCs from TE (n ¼ 10) and TPE (n ¼ 10). (C) Concentrations of soluble PD-L1 (left panels) and soluble PD-L2 (right panels) in PMC culture supernatants (n ¼ 10). In both (B) and (C), horizontal bars indicate means, and the comparisons were made by Mann Whitney U test.
supplemented with half volume of TPE, the numbers of PD-L1- and PD-L2-positive Met5A cells were significantly elevated compared with medium, and anti-IFN-g mAb abrogated this elevation induced by TPE (Figure 4D).
We also detected the concentrations of sPD-L1 and sPD-L2 in the supernatants of Met5A cells cultured for 3 d in the presence of the above cytokines, and noted that none of cytokines tested could stimulate release of sPD-L1 when compared with medium control
Figure 4. IFN-g enhanced expressions of programmed death ligand 1 (PD-L1) and PD-L2 on pleural mesothelial cells. (A) Non-malignant transformed mesothelial cell line (Met5A cells) were cultured and stimulated by designated cytokines for 3 d and harvested for flow cytometry to determine expressions of PD-L1 and PD-L2. IFN-g upregulated the expressions of PD-L1 and PD-L2 on Met5A cells in timee (B) and dose-dependent (C) manners. Met5A cells were cultured in medium or tuberculous pleural effusion (TPE) in the absence or presence of anti-IFN-g mAb, expressions of PD-L1 (open bars) and PD-L2 (solid bars) were determined by flow cytometry (D). Met5A cells were cultured and stimulated by designated cytokines for 3 d, the supernatants were collected for determining concentrations of soluble PD-L1 (E) and soluble PD-L2 (F) by ELISA. The results are presented as mean SEM from 5 independent experiments. Comparisons were made by KruskaleWallis one-way analysis of variance on ranks. *p < 0.05 compared with medium control; yp < 0.05 compared with TPE group.
136
W. Yin et al. / Tuberculosis 94 (2014) 131e139
(Figure 4E), whereas IL-1b, IL-12, or IL-32 each promoted release of sPD-L2 (Figure 4F).
3.4. PD-1/PD-Ls pathways downregulated adhesion, but not migration of CD4þ T cells
3.3. PD-1/PD-Ls pathways inhibited proliferation, but not apoptosis of CD4þ T cells
Since T cell adhesion and migration are critical for an effective immune response in lesion, we performed T cell adhesion and migration assays by treating CD4þ T cells from blood or PMCs with blocking Abs or irrelevant isotype control. Anti-PD-1 mAb promoted PMA activated CD4þ T cell adhesion (Figure 6A) without upregulating the expressions of CD11a and CD29, the ligands for ICAM-1 and VCAM-1 (Figure 6B), respectively. As shown in Figure 6C, TPE induced CD4þ T cell migration but PD-1/PD-Ls pathways did not contribute to this process.
PMCs isolated from TPE were preincubated with or without anti-PD-L1 or/and PD-L2 mAbs and then were co-cultured with non-autologous CD4þ T cells from TPE in the presence of anti-CD3 mAb for 4 d. As shown in Figures 5A and 5B, compared with CD4þ T cells alone, PMCs were found to be able to inhibit proliferation of CD4þ T cells. This inhibition was dependent on cellecell contact, since such an inhibition disappeared when PMCs and CD4þ T cells were separated by using transwell system (right top panel of Figure 5A). It was also noted that the addition of anti-PD-L1 or/and -PD-L2 mAbs into the co-culture abolished the inhibition induced by PMCs. On the other hand, PD-1/PD-Ls pathways did not affect the apoptosis of CD4þ T cells under the same culture conditions (Figures 5C and 5D).
4. Discussion In the past few years, mounting animal studies have demonstrated that PD-1/PD-Ls pathways play important roles in immune responses during MTB infection [10e13,16,17]. In humans, blockage of PD-1/PD-Ls pathways enhanced the specific degranulation of
Figure 5. Pleural mesothelial cells (PMCs) influenced proliferation, but not apoptosis, of CD4þ T cells via PD-1/PD-Ls pathways. (A) Confluent PMC monolayers preincubated with anti-PD-L1 or/and -PD-L2 mAbs for 24 h were cocultured with CD4þ T cells isolated from tuberculous pleural effusion at a ratio of 1:5 for 4 d in the presence of anti-CD3 mAb. In some experiments, 0.4-mm pore size transwells were used to separate the PMC monolayers and CD4þ T cells (right top panel). Ki-67þ cells were determined gating on CD4þ T cells, and the representative flow cytometric dot-plots are from one of five independent experiments. (B) Summary data of percentages of Ki-67þCD4þ T cells from each group. (C) Annexin Vþ cells were determined gating on CD4þ T cells, and the representative flow cytometric dot-plots are from one of five independent experiments. (D) Summary data of percentages of Annexin Vþ T cells from each group. In both (B) and (D), the results are presented as means SEM. Comparisons were made by KruskaleWallis one-way analysis of variance on ranks. *p < 0.05 compared with coculture of PMCs and CD4þ T cells without mAb treatment.
W. Yin et al. / Tuberculosis 94 (2014) 131e139
137
Figure 6. PD-1/PD-Ls pathways downregulated CD4þ T cell adhesion. (A) C2925-labled CD4þ T cells purified from blood were added to PMC monolayers. In some experiments, phorbol myristate acetate stimulated CD4þ T cells were pretreated with anti-PD-1 mAb whereas PMCs were pretreated with anti-PD-L1 and -PD-L2 mAbs (n ¼ 5). (B) Anti-PD-1 mAb did not influence the expression of CD11a and CD29 on PMA activated CD4þ T cells. The representative flow cytometric dot-plots are from one of five independent experiments. (C) Activated CD4þ T cells migrated through IFN-g treated PMC monolayers in the absence or presence of tuberculous pleural effusion with or without anti-PD-L1 and -PD-L2 mAbs (n ¼ 5). All results are presented as means SEM. Comparisons were made by KruskaleWallis one-way analysis of variance on ranks. *p < 0.05 compared with medium control.
CD8þ T cells and numbers of specific IFN-g-producing lymphocytes against MTB [14]. Moreover, blockade of the PD-1/PD-Ls pathways significantly augmented lytic degranulation and IFN-g production of NK cells against MTB [18]. To our knowledge, the present study was the first one to investigate the expressions of PD-Ls on PMCs, and to explore the roles played by PD-1/PD-Ls pathways between CD4þ T cells and PMCs in TPE. PD-1 was expressed on activated T cell, B cells, and mesenchymal stem cells [19e21]. PD-L1 and PD-L2 were expressed on various cell types including T cell, B cell, NK cell and antigenpresenting cells [8,14,18,22,23]. PD-L1 expression was significantly overrepresented in whole blood from patients with active tuberculosis as compared with patients with latent tuberculosis infection and healthy controls; whereas levels of PD-1 and PD-L2 overall were not different in all three groups [24]. It has been demonstrated that MTB antigen stimulation could induce a significant increase in PD-1 expression on pleural T cells from TPE patients with high T cell response to MTB antigens [14]. In another human study [18], Alvarez and colleagues have observed that PD-1 basal levels were significantly higher on blood NK cells compared TPE; however, when stimulated with MTB, PD-1 expression levels on NK cells were markedly higher in TPE compared to blood. In the
present study, our data clearly indicated that the concentration of sPD-1 in TPE and surface expression of PD-1 in CD4þ T cells in TPE were much higher than their compartments in the corresponding blood. We also noted that the percentages of CD45ROþPD-1þCD4þ T cells in TPE were much higher than those in blood, indicating that most PD-1þCD4þ T cells in TPE were memory T cells. The most novel findings in our study were that the membrane expressions of two ligands for PD-1, PD-L1 and PD-L2, on PMCs from TPE were much higher than those from transudative effusion. Surprisingly, although PMCs from TPE expressed higher levels of PD-Ls than those from transudative effusion did, they did not release more sPD-L1 and sPD-L2 into the supernatants when cultured in vitro. Taken together, the above data suggested that TPE microenvironment results in the upregulation of PD-1 and PD-Ls, and that PD-1/ PD-Ls pathways might be involved in the pathogenesis of TPE. It has been reported that TPE was enriched with CD4þCDw29þ T cells, which are thought to represent “memory” T cells, and these pleural CD4þCDw29þ cells, but not CD4þCDw29e cells, proliferated vigorously and produced high levels of IFN-g when stimulated with purified protein derivative of MTB [25]. High concentration of IFN-g could always be found in TPE and served as a reasonable diagnostic biomarker for TPE [26]. Our current data showed that exogenous
138
W. Yin et al. / Tuberculosis 94 (2014) 131e139
IFN-g notably enhanced expression of PD-1 on CD4þ T cells, just like MTB-specific antigen ESAT-6/CFP-10 did. Furthermore, consistent with previous findings with endothelial cells [27,28], we also noted that IFN-g dramatically induced the expressions of PD-Ls on PMCs in time- and dose-dependent manners. The observation that IL-1b, IL-12 and IL-32 increased soluble PD-L2 in culture supernatants of Met5A cells indicated that these proinflammatory cytokines might contribute to the immune response against MTB. It should be mentioned that although high concentration of IFN-g is always found in TPE [26], adding anti-IFN-g mAb did not abolish the upregulation of PD-L1 in response to TPE, we believed that other cytokines enriched in TPE could be responsible for this upregulation. Many previous studies revealed that PD-1/PD-Ls pathways negatively regulated T cell proliferation and cytokine production [7,8,29] and positively regulated T cell apoptosis [30]; controversially, Tseng et al. reported positive regulation on T cell activation [9]. Human studies revealed that these co-stimulatory molecules negatively regulated the effector function of CD3þ, CD8þ T and NK cells in MTB infection [14,18], which were not inconsistent with the findings obtained from animal studies [11e13]. Our previous data have demonstrated that PMCs expressed co-stimulatory molecules, CD80 and CD86, and functioned as antigen-presenting cells to stimulate the differentiation of Th22, Th9, Th17, and Th1 cells from autologous naive CD4þ T cells [4,5], we therefore investigated whether PD-Ls expressed on PMCs could serve as negative costimulator signals for regulation of CD4þ T cells. We noted in the current study that when PMCs were co-cultured with pleural CD4þ T cells from unrelated donors, CD4þ T cell proliferation could be inhibited, and preincubation with anti-PD-L1 or/and -PD-L2 mAbs abolished the inhibition induced by PMCs, indicating that PD-1/PDLs pathways inhibited CD4þ T cell proliferation. In addition, we did not observe PD-1/PD-Ls pathways could affect apoptosis of CD4þ T cells. Different disease and different effector cells might explain the difference between our observations and the findings reported by Dong et al. [30]. Given that ICAM-1 and VCAM-1 participate in cell adhesion and migration, and mesothelial cells express adhesion molecules ICAM1 and VCAM-1 [3,31], and that PD-1/PD-L2 influence adhesion activity of T cells [29], we investigated the effects of PD-1/PD-Ls pathways on activated T cell adhesion to PMC monolayer. Our data revealed that anti-PD-1 mAb promoted CD4þ T cell adhesion without upregulating the corresponding receptor of ICAM-1 and VCAM-1. As blocking PD-Ls on human brain endothelial cells increased migration of CD8þ and CD4þ T cells [32], we developed a similar migration assay by building a model for pleural cavity but failed to obtain the similar results. The above observation demonstrated that PD-1/PD-Ls pathways on PMCs negatively regulated adhesion, but not migration, of CD4þ T cells. In summary, we have demonstrated that concentration of sPD-1, expression of PD-1 on CD4þ T cells, and expressions of PD-Ls on PMCs were increased in TPE, and that MTB-specific antigen stimulation and/or cytokine micro-environment contributed to the upregulation. We confirmed that PMCs regulated proliferation and adhesion activity of CD4þ T cells via PD-1/PD-Ls pathways. Our present study suggested that MTB might exploit PD-1/PD-Ls pathways to evade host cell immune response in human. Acknowledgments This work was supported in part by grants from National Natural Science Foundation of China (No. 81270149 and No. 81272591); in part by a grant from National Science Fund for Distinguished Young Scholars of China (No. 30925032).
Funding:
None.
Competing interests:
None.
Ethical approval: The study protocol was approved by our Institutional Review Boards for human studies of Capital Medical University, Beijing, China; and Tongji Medical College, Wuhan, China; and informed written consent was obtained from all subjects. Authors’ contributions: HZS designed the study and drafted the manuscript. WY, ZHT, and AC isolated, processed and cultured cells, and performed flow cytometry. JCZ and ZJY were responsible for sample collection and ELISA. MLY and QZ recruited subjects and analyzed the data. All authors read, critically revised, and approved the final manuscript. References [1] Jafari C, Lange C. Suttons’s law: local immunodiagnosis of tuberculosis. Infection 2008;36:510e4. http://dx.doi.org/10.1007/s15010-008-8089-9. [2] Jantz MA, Antony VB. Pathophysiology of the pleura. Respiration 2008;75: 121e33. http://dx.doi.org/10.1159/000113629. 000113629 [pii]. [3] Nasreen N, Mohammed KA, Ward MJ, Antony VB. Mycobacterium-induced transmesothelial migration of monocytes into pleural space: role of intercellular adhesion molecule-1 in tuberculous pleurisy. J Infect Dis 1999;180: 1616e23. http://dx.doi.org/10.1086/315057. JID981576 [pii]. [4] Ye ZJ, Zhou Q, Yuan ML, Du RH, Yang WB, Xiong XZ, Huang B, Shi HZ. Differentiation and recruitment of IL-22-producing helper T cells stimulated by pleural mesothelial cells in tuberculous pleurisy. Am J Respir Crit Care Med 2012;185:660e9. http://dx.doi.org/10.1164/rccm.201107-1198OC. rccm.201107-1198OC [pii]. [5] Ye ZJ, Yuan ML, Zhou Q, Du RH, Yang WB, Xiong XZ, Zhang JC, Wu C, Qin SM, Shi HZ. Differentiation and recruitment of Th9 cells stimulated by pleural mesothelial cells in human Mycobacterium tuberculosis infection. PLoS ONE 2012;7:e31710. http://dx.doi.org/10.1371/journal.pone.0031710. PONE-D-1119196 [pii]. [6] Dong H, Zhu G, Tamada K, Chen L. B7-H1, a third member of the B7 family, costimulates T-cell proliferation and interleukin-10 secretion. Nat Med 1999;5: 1365e9. http://dx.doi.org/10.1038/70932. [7] Freeman GJ, Long AJ, Iwai Y, Bourque K, Chernova T, Nishimura H, Fitz LJ, Malenkovich N, Okazaki T, Byrne MC, Horton HF, Fouser L, Carter L, Ling V, Bowman MR, Carreno BM, Collins M, Wood CR, Honjo T. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med 2000;192:1027e34. [8] Latchman Y, Wood CR, Chernova T, Chaudhary D, Borde M, Chernova I, Iwai Y, Long AJ, Brown JA, Nunes R, Greenfield EA, Bourque K, Boussiotis VA, Carter LL, Carreno BM, Malenkovich N, Nishimura H, Okazaki T, Honjo T, Sharpe AH, Freeman GJ. PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat Immunol 2001;2:261e8. http://dx.doi.org/10.1038/85330. [9] Tseng SY, Otsuji M, Gorski K, Huang X, Slansky JE, Pai SI, Shalabi A, Shin T, Pardoll DM, Tsuchiya H. B7-DC, a new dendritic cell molecule with potent costimulatory properties for T cells. J Exp Med 2001;193:839e46. [10] Reiley WW, Shafiani S, Wittmer ST, Tucker-Heard G, Moon JJ, Jenkins MK, Urdahl KB, Winslow GM, Woodland DL. Distinct functions of antigen-specific CD4 T cells during murine Mycobacterium tuberculosis infection. Proc Natl Acad Sci USA 2010;107:19408e13. http://dx.doi.org/10.1073/ pnas.1006298107. 1006298107 [pii]. [11] Sakai S, Kawamura I, Okazaki T, Tsuchiya K, Uchiyama R, Mitsuyama M. PD-1PD-L1 pathway impairs T(h)1 immune response in the late stage of infection with Mycobacterium bovis bacillus Calmette-Guerin. Int Immunol 2010;22: 915e25. http://dx.doi.org/10.1093/intimm/dxq446. dxq446 [pii]. [12] Tousif S, Singh Y, Prasad DV, Sharma P, Van Kaer L, Das G. T cells from programmed death-1 deficient mice respond poorly to Mycobacterium tuberculosis infection. PLoS ONE 2011;6:e19864. http://dx.doi.org/10.1371/ journal.pone.0019864. PONE-D-11-02781 [pii]. [13] Lazar-Molnar E, Chen B, Sweeney KA, Wang EJ, Liu W, Lin J, Porcelli SA, Almo SC, Nathenson SG, Jacobs Jr WR. Programmed death-1 (PD-1)-deficient mice are extraordinarily sensitive to tuberculosis. Proc Natl Acad Sci USA 2010;107:13402e7. http://dx.doi.org/10.1073/pnas.1007394107 [pii]. [14] Jurado JO, Alvarez IB, Pasquinelli V, Martinez GJ, Quiroga MF, Abbate E, Musella RM, Chuluyan HE, Garcia VE. Programmed death (PD)-1:PD-ligand 1/ PD-ligand 2 pathway inhibits T cell effector functions during human tuberculosis. J Immunol 2008;181:116e25. doi: 181/1/116 [pii]. [15] Light RW, Macgregor MI, Luchsinger PC, Ball Jr WC. Pleural effusions: the diagnostic separation of transudates and exudates. Ann Intern Med 1972;77: 507e13. [16] Qiu L, Huang D, Chen CY, Wang R, Shen L, Shen Y, Hunt R, Estep J, Haynes BF, Jacobs Jr WR, Letvin N, Du G, Chen ZW. Severe tuberculosis induces
W. Yin et al. / Tuberculosis 94 (2014) 131e139
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
unbalanced up-regulation of gene networks and overexpression of IL-22, MIP1alpha, 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;198:1514e 9. http://dx.doi.org/10.1086/592448. Henao-Tamayo M, Irwin SM, Shang S, Ordway D, Orme IM. T lymphocyte surface expression of exhaustion markers as biomarkers of the efficacy of chemotherapy for tuberculosis. Tuberculosis (Edinb) 2011;91:308e13. http:// dx.doi.org/10.1016/j.tube.2011.04.001. S1472-9792(11)00064-3 [pii]. Alvarez IB, Pasquinelli V, Jurado JO, Abbate E, Musella RM, de la Barrera SS, Garcia VE. Role played by the programmed death-1-programmed death ligand pathway during innate immunity against Mycobacterium tuberculosis. J Infect Dis 2010;202:524e32. http://dx.doi.org/10.1086/654932. Keir ME, Latchman YE, Freeman GJ, Sharpe AH. Programmed death-1 (PD-1): PD-ligand 1 interactions inhibit TCR-mediated positive selection of thymocytes. J Immunol 2005;175:7372e9. doi: 175/11/7372 [pii]. Keir ME, Francisco LM, Sharpe AH. PD-1 and its ligands in T-cell immunity. Curr Opin Immunol 2007;19:309e14. http://dx.doi.org/10.1016/ j.coi.2007.04.012. S0952-7915(07)00064-7 [pii]. Keir ME, Butte MJ, Freeman GJ, Sharpe AH. PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol 2008;26:677e704. http://dx.doi.org/ 10.1146/annurev.immunol.26.021607.090331. Brown JA, Dorfman DM, Ma FR, Sullivan EL, Munoz O, Wood CR, Greenfield EA, Freeman GJ. Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production. J Immunol 2003;170:1257e66. Karakhanova S, Bedke T, Enk AH, Mahnke K. IL-27 renders DC immunosuppressive by induction of B7-H1. J Leukoc Biol 2011;89:837e45. http:// dx.doi.org/10.1189/jlb.1209788. jlb.1209788 [pii]. McNab FW, Berry MP, Graham CM, Bloch SA, Oni T, Wilkinson KA, Wilkinson RJ, Kon OM, Banchereau J, Chaussabel D, O’Garra A. Programmed death ligand 1 is over-expressed by neutrophils in the blood of patients with
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
139
active tuberculosis. Eur J Immunol 2011;41:1941e7. http://dx.doi.org/ 10.1002/eji.201141421. Barnes PF, Mistry SD, Cooper CL, Pirmez C, Rea TH, Modlin RL. Compartmentalization of a CD4þ T lymphocyte subpopulation in tuberculous pleuritis. J Immunol 1989;142:1114e9. Jiang J, Shi HZ, Liang QL, Qin SM, Qin XJ. Diagnostic value of interferon-gamma in tuberculous pleurisy: a metaanalysis. Chest 2007;131:1133e41. http:// dx.doi.org/10.1378/chest.06-2273. Eppihimer MJ, Gunn J, Freeman GJ, Greenfield EA, Chernova T, Erickson J, Leonard JP. Expression and regulation of the PD-L1 immunoinhibitory molecule on microvascular endothelial cells. Microcirculation 2002;9:133e45. http://dx.doi.org/10.1038/sj/mn/7800123. Rodig N, Ryan T, Allen JA, Pang H, Grabie N, Chernova T, Greenfield EA, Liang SC, Sharpe AH, Lichtman AH, Freeman GJ. Endothelial expression of PDL1 and PD-L2 down-regulates CD8þ T cell activation and cytolysis. Eur J Immunol 2003;33:3117e26. http://dx.doi.org/10.1002/eji.200324270. Saunders PA, Hendrycks VR, Lidinsky WA, Woods ML. PD-L2:PD-1 involvement in T cell proliferation, cytokine production, and integrin-mediated adhesion. Eur J Immunol 2005;35:3561e9. http://dx.doi.org/10.1002/ eji.200526347. Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, Roche PC, Lu J, Zhu G, Tamada K, Lennon VA, Celis E, Chen L. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med 2002;8:793e800. http://dx.doi.org/10.1038/nm730. nm730 [pii]. Jonjic N, Peri G, Bernasconi S, Sciacca FL, Colotta F, Pelicci P, Lanfrancone L, Mantovani A. Expression of adhesion molecules and chemotactic cytokines in cultured human mesothelial cells. J Exp Med 1992;176:1165e74. Pittet CL, Newcombe J, Prat A, Arbour N. Human brain endothelial cells endeavor to immunoregulate CD8 T cells via PD-1 ligand expression in multiple sclerosis. J Neuroinflammation 2011;8:155. http://dx.doi.org/10.1186/ 1742-2094-8-155. 1742-2094-8-155 [pii].
