&get_box_var;
ORIGINAL ARTICLE
Mucosal-associated Invariant T-Cell Function Is Modulated by Programmed Death-1 Signaling in Patients with Active Tuberculosis Jing Jiang, Xinjing Wang, Hongjuan An, Bingfen Yang, Zhihong Cao, Yanhua Liu, Jinwen Su, Fei Zhai, Ruo Wang, Guangyu Zhang, and Xiaoxing Cheng Key Laboratory of Tuberculosis Prevention and Treatment, Division of Research, Institute of Tuberculosis, 309th Hospital, Beijing, China
Abstract Rationale: Mucosal-associated invariant T (MAIT) cells have
been proven to play an important role in host defense against mycobacterial infection in animal infection models; however, the functional role of MAIT cells in patients with active tuberculosis (TB) is still largely unknown. Objectives: To understand the clinical features and functions of MAIT cells in patients with active TB. Methods: MAIT cells were analyzed in patients with pulmonary TB,
tuberculous pleurisy, and tuberculous peritonitis by flow cytometry. The functions of MAIT cells were compared between patients with active TB and healthy control subjects.
fluids from patients with tuberculous peritonitis. A comparison of bacillus Calmette-Gu´erin (BCG)–stimulated cytokine production showed that patients with active TB had significantly higher production of IFN-g (P = 0.0034) and tumor necrosis factor (TNF)-a (P = 0.0399) compared with healthy control subjects. In contrast, when MAIT cells were stimulated with Escherichia coli, patients with active TB had significantly lower production of IFN-g (P = 0.0007) and TNF-a (P = 0.0032). MAIT cells in patients with active TB exhibited elevated expression of programmed death-1 (PD-1) (P = 0.0015), and blockade of PD-1 signaling resulted in a significantly higher frequency of BCG-stimulated IFN-g production in MAIT cells (P = 0.0178). Conclusions: MAIT-cell immune response to antigen stimulation
Measurements and Main Results: The frequency of MAIT cells
in patients with active TB is regulated by PD-1, which could be a potential target for TB immunotherapy.
was significantly reduced both in peripheral blood from patients with active pulmonary TB (P , 0.0001) and in tuberculous pleural effusions compared with healthy control subjects but not in ascitic
Keywords: tuberculosis; immunity; mucosal-associated invariant
Tuberculosis (TB) is the second most common cause of death from infectious disease (1). It is estimated that 8.7 million cases of TB occurred in 2011 and that 1.4 million died of TB (1). Despite the high rate of Mycobacterium tuberculosis infection in humans, especially in developing countries, only 5–10% of infected people develop active TB in their lifetime (2, 3).
T cells
Interactions between M. tuberculosis and the host largely determine the development and outcome of TB infection (2, 4–6). Mucosal-associated invariant T (MAIT) cells are innate-like T cells that display an invariant T-cell receptor (TCR) Va7.2–Ja33 chain in humans and Va19–Ja33 chain in mice, which is paired with a limited number of TCR b chains, and can be identified by
TCR Va7.2 and CD161 expression in humans (7, 8). They are restricted by the evolutionarily conserved nonclassical MHC-Ib molecule, MR1 (major histocompatibility complex class I–related protein-1; 9, 10). MAIT cells are enriched in the intestinal lamina propria and are abundant in human blood and liver, comprising 1–10% of peripheral blood T cells and up to 50% of liver T cells (11). Unlike
( Received in original form January 21, 2014; accepted in final form June 19, 2014 ) Supported by grants from National Natural Science Foundation of China (81201258, 81273225, 81061120518, 81071318, and 81071319) and by an infectious diseases grant from the National Health and Family Planning Commission, China to X.C. (2013ZX10003006-003-001). Author Contributions: Conception and design: X.C. and J.J.; performance of experiments: J.J., X.W., H.A., B.Y., Z.C., Y.L., J.S., F.Z., R.W., and G.Z.; analysis and interpretation: J.J. and X.C.; drafting the manuscript for important intellectual content: X.C. and J.J. Correspondence and requests for reprints should be addressed to Xiaoxing Cheng, M.B., Ph.D., Division of Research, Institute of Tuberculosis, 309th Hospital, 17 Hei Shan Hu Road, Haidian, Beijing 100091, China. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents online at www.atsjournals.org Am J Respir Crit Care Med Vol 190, Iss 3, pp 329–339, Aug 1, 2014 Copyright © 2014 by the American Thoracic Society Originally Published in Press as DOI: 10.1164/rccm.201401-0106OC on June 30, 2014 Internet address: www.atsjournals.org
Jiang, Wang, An, et al.: MAIT Cells in Patients with TB
329
ORIGINAL ARTICLE M. tuberculosis, Escherichia coli, and Shigella flexneri (14, 18). They not only produce cytokines, such as IFN-g, tumor necrosis factor (TNF)-a, and IL-17, but also have a cytotoxic effect (18). In patients with TB, MAIT cells are decreased in peripheral blood (14, 15) and are enriched in human lungs (15). MAIT cells can potently inhibit the intracellular growth of Mycobacterium bovis bacillus Calmette-Gu´erin (BCG) in macrophages through the production of IFN-g (19). MAIT cell–deficient mice have higher bacterial loads at early times after a low dose of M. bovis BCG administered via the aerosol route (19). In the mouse model of infection, MAIT cells protect against infection with Mycobacterium abscessus (14). Although it has been established that MAIT cells play an important role in host defense against mycobacterial infection in animal infection models, the functional role of MAIT cells in patients with TB is still largely unknown. In this study, we investigated the clinical features and functions of MAIT cells in a relatively large cohort of patients with active TB and healthy control subjects.
At a Glance Commentary Scientific Knowledge on the Subject:
Mucosal-associated invariant T (MAIT) cells have been demonstrated to play an important role in host defense against mycobacterial infection in animal infection models; however, the functional role of MAIT cells in patients with active tuberculosis (TB) is still largely unknown. What This Study Adds to the Field: In this study, we investigated
the clinical features and functions of MAIT cells in well-defined groups of patients with active TB and control subjects. The frequency of MAIT cells was significantly reduced both in peripheral blood from patients with active pulmonary TB and in tuberculous pleural effusions compared with healthy control subjects but not in ascitic fluids from patients with tuberculous peritonitis. MAIT cells in patients with active TB exhibited elevated expression of programmed death-1 (PD-1), and blockade of the PD-1 signaling led to significantly enhanced IFN-g production.
Methods
conventional CD41 and CD81 T cells that react with peptide antigens and CD1-restricted T cells that recognize lipid-based antigens, MAIT cells represent a new class of T cells that recognize vitamin B metabolites that are common in bacteria and yeast (9, 12, 13). The functional role of MAIT cells in antimicrobial infection has been reported (7, 14–18). MAIT cells are activated by cells infected with bacteria and yeast, such as
Human Subjects
One hundred and twenty-three patients with active TB, including 93 patients with pulmonary TB, 26 with tuberculous pleurisy, and 4 with tuberculous peritonitis, were recruited (Table 1 and online supplement). They were diagnosed according to the 1990 edition of the Diagnostic Standards and Classification of Tuberculosis (20). All patients were HIV-negative.
Seventy-nine healthy control subjects were randomly recruited from among individuals undergoing annual health check-ups at the clinics of the 309th Hospital (Beijing, China) (online supplement). Ten individuals with latent TB infection were identified by screening with a T-SPOT.TB kit (Oxford Immunotec, Abingdon, UK), as described previously (21) and were defined as T-SPOT.TB positive (Table 1). This study was approved by the Ethics Committee of the 309th Hospital. The institutional review board for human studies approved the protocols, and written consent was obtained from the subjects. Flow Cytometry
For the identification of MAIT cells, appropriate fluorochrome-labeled antibodies were mixed with 100 ml of whole blood and incubated at room temperature for 15 minutes in the dark. OptiLyse C buffer (Beckman Coulter, Brea, CA) was added to the blood and incubated at room temperature for 10 minutes to lyse red blood cells. For all other experiments, peripheral blood mononuclear cells (PBMCs) were purified from anticoagulated peripheral whole blood by density gradient centrifugation, using Ficoll-Paque PLUS (GE Healthcare Life Sciences, Pittsburgh, PE) as described previously (21–23). PBMCs (1 3 106) were washed with GIBCO RPMI 1640 (Life Technologies, Grand Island, NY) and then resuspended in GIBCO AIM V serum-free medium (Life Technologies). The cells were used for antibody staining; or added to 96well tissue culture plates (Corning, New York, NY) for further treatment. Pleural effusions and ascitic fluids were obtained from patients with tuberculous
Table 1. Demographic and Clinical Characteristics of Patients with Tuberculosis, Subjects with Latent Tuberculosis Infection, and Healthy Control Subjects
n (male/female) Age (yr), mean 6 SD Pulmonary TB Sputum smear/culture positive New pulmonary TB Failed treatment of pulmonary TB Tuberculous pleurisy Tuberculous peritonitis
Patients with TB
Subjects with Latent TB Infection
123 (79/44) 36.92 6 17.00 93 49 74 19 26 4
10 (7/3) 28.64 6 3.75 No ND N/A N/A No No
Healthy Control Subjects 79 (41/38) 32.42 6 8.847 No ND N/A N/A No No
Definition of abbreviations: N/A = not applicable; ND = not determined; TB = tuberculosis.
330
American Journal of Respiratory and Critical Care Medicine Volume 190 Number 3 | August 1 2014
ORIGINAL ARTICLE
Blood
Pleural effusion
103
103 36.6%
88.5%
102 CD3
CD3
102 101 100
Antigen and Nonspecific Stimulation
101 100
100
101
102
103
100
TCR γδ
101
102
103
TCR γδ
Blood
B Healthy control
TB patient
103
Pleural effusion
103 6.4%
103 0.4%
101 100
102 CD161
102 CD161
102 CD161
cytometer (Beckman Coulter). Appropriate isotype-matched control antibodies were used to determine background levels of staining. Lymphocytes were gated in the FSC/SSC dot plot, and at least 100,000 events were collected.
Gated on CD3+ and TCR γδ – cells
A
101 100
100 101 102 TCR Va7.2
C
103
101 100
100 101 102 TCR Va7.2
103
100 101 102 TCR Va7.2
D
p
ORIGINAL ARTICLE
Mucosal-associated Invariant T-Cell Function Is Modulated by Programmed Death-1 Signaling in Patients with Active Tuberculosis Jing Jiang, Xinjing Wang, Hongjuan An, Bingfen Yang, Zhihong Cao, Yanhua Liu, Jinwen Su, Fei Zhai, Ruo Wang, Guangyu Zhang, and Xiaoxing Cheng Key Laboratory of Tuberculosis Prevention and Treatment, Division of Research, Institute of Tuberculosis, 309th Hospital, Beijing, China
Abstract Rationale: Mucosal-associated invariant T (MAIT) cells have
been proven to play an important role in host defense against mycobacterial infection in animal infection models; however, the functional role of MAIT cells in patients with active tuberculosis (TB) is still largely unknown. Objectives: To understand the clinical features and functions of MAIT cells in patients with active TB. Methods: MAIT cells were analyzed in patients with pulmonary TB,
tuberculous pleurisy, and tuberculous peritonitis by flow cytometry. The functions of MAIT cells were compared between patients with active TB and healthy control subjects.
fluids from patients with tuberculous peritonitis. A comparison of bacillus Calmette-Gu´erin (BCG)–stimulated cytokine production showed that patients with active TB had significantly higher production of IFN-g (P = 0.0034) and tumor necrosis factor (TNF)-a (P = 0.0399) compared with healthy control subjects. In contrast, when MAIT cells were stimulated with Escherichia coli, patients with active TB had significantly lower production of IFN-g (P = 0.0007) and TNF-a (P = 0.0032). MAIT cells in patients with active TB exhibited elevated expression of programmed death-1 (PD-1) (P = 0.0015), and blockade of PD-1 signaling resulted in a significantly higher frequency of BCG-stimulated IFN-g production in MAIT cells (P = 0.0178). Conclusions: MAIT-cell immune response to antigen stimulation
Measurements and Main Results: The frequency of MAIT cells
in patients with active TB is regulated by PD-1, which could be a potential target for TB immunotherapy.
was significantly reduced both in peripheral blood from patients with active pulmonary TB (P , 0.0001) and in tuberculous pleural effusions compared with healthy control subjects but not in ascitic
Keywords: tuberculosis; immunity; mucosal-associated invariant
Tuberculosis (TB) is the second most common cause of death from infectious disease (1). It is estimated that 8.7 million cases of TB occurred in 2011 and that 1.4 million died of TB (1). Despite the high rate of Mycobacterium tuberculosis infection in humans, especially in developing countries, only 5–10% of infected people develop active TB in their lifetime (2, 3).
T cells
Interactions between M. tuberculosis and the host largely determine the development and outcome of TB infection (2, 4–6). Mucosal-associated invariant T (MAIT) cells are innate-like T cells that display an invariant T-cell receptor (TCR) Va7.2–Ja33 chain in humans and Va19–Ja33 chain in mice, which is paired with a limited number of TCR b chains, and can be identified by
TCR Va7.2 and CD161 expression in humans (7, 8). They are restricted by the evolutionarily conserved nonclassical MHC-Ib molecule, MR1 (major histocompatibility complex class I–related protein-1; 9, 10). MAIT cells are enriched in the intestinal lamina propria and are abundant in human blood and liver, comprising 1–10% of peripheral blood T cells and up to 50% of liver T cells (11). Unlike
( Received in original form January 21, 2014; accepted in final form June 19, 2014 ) Supported by grants from National Natural Science Foundation of China (81201258, 81273225, 81061120518, 81071318, and 81071319) and by an infectious diseases grant from the National Health and Family Planning Commission, China to X.C. (2013ZX10003006-003-001). Author Contributions: Conception and design: X.C. and J.J.; performance of experiments: J.J., X.W., H.A., B.Y., Z.C., Y.L., J.S., F.Z., R.W., and G.Z.; analysis and interpretation: J.J. and X.C.; drafting the manuscript for important intellectual content: X.C. and J.J. Correspondence and requests for reprints should be addressed to Xiaoxing Cheng, M.B., Ph.D., Division of Research, Institute of Tuberculosis, 309th Hospital, 17 Hei Shan Hu Road, Haidian, Beijing 100091, China. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents online at www.atsjournals.org Am J Respir Crit Care Med Vol 190, Iss 3, pp 329–339, Aug 1, 2014 Copyright © 2014 by the American Thoracic Society Originally Published in Press as DOI: 10.1164/rccm.201401-0106OC on June 30, 2014 Internet address: www.atsjournals.org
Jiang, Wang, An, et al.: MAIT Cells in Patients with TB
329
ORIGINAL ARTICLE M. tuberculosis, Escherichia coli, and Shigella flexneri (14, 18). They not only produce cytokines, such as IFN-g, tumor necrosis factor (TNF)-a, and IL-17, but also have a cytotoxic effect (18). In patients with TB, MAIT cells are decreased in peripheral blood (14, 15) and are enriched in human lungs (15). MAIT cells can potently inhibit the intracellular growth of Mycobacterium bovis bacillus Calmette-Gu´erin (BCG) in macrophages through the production of IFN-g (19). MAIT cell–deficient mice have higher bacterial loads at early times after a low dose of M. bovis BCG administered via the aerosol route (19). In the mouse model of infection, MAIT cells protect against infection with Mycobacterium abscessus (14). Although it has been established that MAIT cells play an important role in host defense against mycobacterial infection in animal infection models, the functional role of MAIT cells in patients with TB is still largely unknown. In this study, we investigated the clinical features and functions of MAIT cells in a relatively large cohort of patients with active TB and healthy control subjects.
At a Glance Commentary Scientific Knowledge on the Subject:
Mucosal-associated invariant T (MAIT) cells have been demonstrated to play an important role in host defense against mycobacterial infection in animal infection models; however, the functional role of MAIT cells in patients with active tuberculosis (TB) is still largely unknown. What This Study Adds to the Field: In this study, we investigated
the clinical features and functions of MAIT cells in well-defined groups of patients with active TB and control subjects. The frequency of MAIT cells was significantly reduced both in peripheral blood from patients with active pulmonary TB and in tuberculous pleural effusions compared with healthy control subjects but not in ascitic fluids from patients with tuberculous peritonitis. MAIT cells in patients with active TB exhibited elevated expression of programmed death-1 (PD-1), and blockade of the PD-1 signaling led to significantly enhanced IFN-g production.
Methods
conventional CD41 and CD81 T cells that react with peptide antigens and CD1-restricted T cells that recognize lipid-based antigens, MAIT cells represent a new class of T cells that recognize vitamin B metabolites that are common in bacteria and yeast (9, 12, 13). The functional role of MAIT cells in antimicrobial infection has been reported (7, 14–18). MAIT cells are activated by cells infected with bacteria and yeast, such as
Human Subjects
One hundred and twenty-three patients with active TB, including 93 patients with pulmonary TB, 26 with tuberculous pleurisy, and 4 with tuberculous peritonitis, were recruited (Table 1 and online supplement). They were diagnosed according to the 1990 edition of the Diagnostic Standards and Classification of Tuberculosis (20). All patients were HIV-negative.
Seventy-nine healthy control subjects were randomly recruited from among individuals undergoing annual health check-ups at the clinics of the 309th Hospital (Beijing, China) (online supplement). Ten individuals with latent TB infection were identified by screening with a T-SPOT.TB kit (Oxford Immunotec, Abingdon, UK), as described previously (21) and were defined as T-SPOT.TB positive (Table 1). This study was approved by the Ethics Committee of the 309th Hospital. The institutional review board for human studies approved the protocols, and written consent was obtained from the subjects. Flow Cytometry
For the identification of MAIT cells, appropriate fluorochrome-labeled antibodies were mixed with 100 ml of whole blood and incubated at room temperature for 15 minutes in the dark. OptiLyse C buffer (Beckman Coulter, Brea, CA) was added to the blood and incubated at room temperature for 10 minutes to lyse red blood cells. For all other experiments, peripheral blood mononuclear cells (PBMCs) were purified from anticoagulated peripheral whole blood by density gradient centrifugation, using Ficoll-Paque PLUS (GE Healthcare Life Sciences, Pittsburgh, PE) as described previously (21–23). PBMCs (1 3 106) were washed with GIBCO RPMI 1640 (Life Technologies, Grand Island, NY) and then resuspended in GIBCO AIM V serum-free medium (Life Technologies). The cells were used for antibody staining; or added to 96well tissue culture plates (Corning, New York, NY) for further treatment. Pleural effusions and ascitic fluids were obtained from patients with tuberculous
Table 1. Demographic and Clinical Characteristics of Patients with Tuberculosis, Subjects with Latent Tuberculosis Infection, and Healthy Control Subjects
n (male/female) Age (yr), mean 6 SD Pulmonary TB Sputum smear/culture positive New pulmonary TB Failed treatment of pulmonary TB Tuberculous pleurisy Tuberculous peritonitis
Patients with TB
Subjects with Latent TB Infection
123 (79/44) 36.92 6 17.00 93 49 74 19 26 4
10 (7/3) 28.64 6 3.75 No ND N/A N/A No No
Healthy Control Subjects 79 (41/38) 32.42 6 8.847 No ND N/A N/A No No
Definition of abbreviations: N/A = not applicable; ND = not determined; TB = tuberculosis.
330
American Journal of Respiratory and Critical Care Medicine Volume 190 Number 3 | August 1 2014
ORIGINAL ARTICLE
Blood
Pleural effusion
103
103 36.6%
88.5%
102 CD3
CD3
102 101 100
Antigen and Nonspecific Stimulation
101 100
100
101
102
103
100
TCR γδ
101
102
103
TCR γδ
Blood
B Healthy control
TB patient
103
Pleural effusion
103 6.4%
103 0.4%
101 100
102 CD161
102 CD161
102 CD161
cytometer (Beckman Coulter). Appropriate isotype-matched control antibodies were used to determine background levels of staining. Lymphocytes were gated in the FSC/SSC dot plot, and at least 100,000 events were collected.
Gated on CD3+ and TCR γδ – cells
A
101 100
100 101 102 TCR Va7.2
C
103
101 100
100 101 102 TCR Va7.2
103
100 101 102 TCR Va7.2
D
p
