CLINICAL INVESTIGATION
Decreased Programmed Death-1 Expression on the T Cells of Patients With Ankylosing Spondylitis Lianlian Zhou, MD, Yanhong Zhang, MD, Huiying Xu, MD, Liping Hu, MD, Chunwu Zhang, MD, Li Sun, MD, Yaosheng Xie, MD, Hong Lu, MD, Zhuo Zhang, PhD, Wangqiang Hu, MD and Xiangyang Lin, MD
Abstract: Background: Programmed death-1 (PD-1) plays a vital role in down-modulating immune responses and maintaining peripheral tolerance. Methods: The authors have investigated the inducible expression of PD-1 on activated T cells from patients with ankylosing spondylitis (AS). Thirty patients with AS and 31 unrelated healthy controls (HCs) were recruited in this study. The expression of PD-1 on T cells harvested from nonstimulated (t0) or stimulated cultures with phytohemagglutinin for 24 hours (t24) was determined by flow cytometry. The multiple levels of the PD-1 expression on stimulated and nonstimulated cells from each individual’s sample (t24/t0) represented as the degree of the inducible effect on PD-1 expression. Results: The expression of PD-1 on nonstimulated T cells presented no significant difference between AS group and HC group (P . 0.05). After stimulation, the degree of effect on PD-1 expression of CD4+, CD4+CD25+, CD4+CD25high, CD4+CD25low and CD4+CD252 T cells were significantly lower in patients with AS than those in HC group (1.9 6 0.9 versus 3.6 6 2.3, 9.7 6 7.4 versus 17.8 6 12.6, 87.8 6 48.6 versus 157.3 6 117.0, 3.7 6 1.4 versus 7.3 6 2.4, 0.5 6 0.3 versus 1.1 6 0.6, respectively, P , 0.05). However, there was no significant difference of the effect on all lymphocytes and CD8+ T cells between patients with AS and HCs (P . 0.05). Conclusions: The decreased inducible expression of PD-1 on active T lymphocytes, especially on CD4+CD25high and CD4+CD25+ T cells, may be one of important factors involved in the development of AS. Key Indexing Terms: Ankylosing spondylitis; Programmed cell death-1; T cells; Regulatory T cells. [Am J Med Sci 2015;349(6):488–492.]
A
nkylosing spondylitis (AS) is one of the seronegative spondyloarthropathies characterized by the inflammation mainly in the axial skeleton and sacroiliac joints.1 Because of the late appearance of definite sacroiliitis on radiographs, it is still difficult to diagnose at the onset of the diseases, resulting in a delay of up to 10 years between the onset of 1st symptoms and diagnosis of AS.2 The etiology and pathogenesis of AS remain unclear; however, it is associated with the human leukocyte antigen (HLA)–B27.3 Recent studies have demonstrated that HLA-B27 can be recognized by CD4+ T cells, and From the Department of Clinical Laboratory (LZ, HX, LH, YX, HL, ZZ, WH, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China; Department of Clinical Laboratory (YZ), Yantai Yuhuangding Hospital, Yantai, China; and Departments of Orthpaedics Trauma (CZ) and Rheumatology (LS), The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China. Submitted May 21, 2014; accepted in revised form March 5, 2015. The authors have no conflicts of interest to disclose. Supported by Technology Foundation of Zhejiang Province (No. 2013C33173). L.Z. and Y.Z. contributed equally to this work and should be considered as co-first authors. Correspondence: Xiangyang Lin, MD, Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China (E-mail: [email protected]). XL),
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HLA-B27–reactive CD4+ T cells may be pathogenic in spondyloarthropathies.4,5 Much work has concentrated on the alternations of T cells in patients with AS, such as increased frequency of peripheral Th2 lymphocytes6 and Th17 lymphocytes7 in AS. All above are evidence that the T cells may play an important role in the pathology of AS. Programmed death-1 (PD-1) is an immunoglobulin (Ig) superfamily member, which can be inducibly expressed on the surface of peripheral T cells, B cells, NK T cells, monocytes and dendritic cells and interact with its ligand (PD-L) to maintain peripheral tolerance and protect tissues from autoimmune attack.8,9 The role of PD-1 in regulating T-cell tolerance and autoimmunity was 1st observed among PD-12/2 mice with spontaneous autoimmune diseases.10,11 C57BL/6 mice without PD-1 developed a lupus such as glomerulonephritis and arthritis, with the deposition of IgG3 and C3 in the glomeruli.12 Some studies have revealed that PD-1 pathway was not only important in the initial phase of activation and expansion of self-reactive T cells but also influenced self-reactive T-cell effector function on antigen reencounter in mouse models of autoimmunity and tolerance, indicating that PD-1 was important in the induction and/or the maintenance of peripheral tolerance. Regulatory T cells (Tregs) played a vital role in the pathogenesis of many autoimmune diseases, which resulted from the abnormal number or function of Tregs. Francisco et al13 have summarized that PD-1 pathway may assist Tregs in maintaining immune homeostasis. Recently, some studies demonstrated that CD4+CD25high T cells expressing PD-1 exhibited immunosuppressive functions.13,14 To date, the exploration of the function of PD-1 and its pathway in the tolerance and autoimmunity is a population area; however, the levels of PD-1 on T lymphocytes, specifically Tregs in AS, are still unclear. Therefore, this investigation aimed to determine the cellular expression of PD-1 receptor on T lymphocytes with AS in a Chinese population.
MATERIALS AND METHODS Participants Sixty-one Chinese individuals including 30 patients with AS and 31 age- and gender-matched healthy volunteers were recruited from The First Affiliated Hospital of Wenzhou Medical University from July 2011 to July 2012. Clinical diagnosis of AS was in accordance with the criteria modified in New York in 1984.15 Healthy volunteers consisted of students and staff from above institution were designed as the healthy control group (HC). These volunteers had no abnormal laboratory results, without a family history of autoimmune diseases. The demographic and clinical characteristics of the patients and HCs were shown in Table 1. Peripheral blood samples were obtained from the participants after they provided written informed consent. This study protocol and informed consent forms have been approved by the Ethics Review Committee of the First Affiliated Hospital of Wenzhou Medical University.
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Decreased PD-1 Expression on the T Cells
TABLE 1. Baseline clinical status of the patients with AS and HCs AS (n 5 30) HC (n 5 31) Age, yr Range Mean 6 SD Gender: male, n (%) Disease duration, yr Laboratory parameters HLA-B27+, n (%) CRP, mg/L ESR, mm/hr Clinical features, n (%) Low back pain .3 mo Limitation of back flexion Sacroiliitis Chest expansion decreased Heel pain Iritis
16–46 30.6 6 7.7 20 (66.7) 3.12 6 3.18
21–48 27.1 6 6.4 24 (60.0) NA
30 (100) 8.10 6 8.04 26.1 6 19.2
1 (3.2) NA NA
30 (100) 11 (36.7) 10 (33.3) 8 (26.7) 4 (13.3) 4 (13.3)
NA NA NA NA NA NA
Values are mean 6 SD for quantitative variables or absolute frequency/total measures. CRP, C-reaction protein; ESR, erythrocyte sedimentation rate; NA, not available.
Stimulation and Culture of Peripheral Blood The peripheral blood was treated by the heparin. RPMI1640 medium was supplemented by heat-inactivated fetal bovine serum at a ratio of 4:1, streptomycin/penicillin with the final concentration of 100 U/mL and the phytohemagglutinin (PHA) with final concentration of 0.02 mg/mL. The pH of RPMI1640 medium was adjusted to between 7.2 and 7.4 with 5% NaHCO3. Under aseptic conditions, 0.5 mL of peripheral blood treated by heparin was added into 5 mL of complete RPMI medium. Cells were cultured at 37°C in 5% CO2 for 24 hours (t24). The cultured peripheral blood cells were harvested by low-speed centrifugation at 1,500 rpm for 5 minutes. The supernatant was discarded, and the precipitates were stored for further experiments. Flow Cytometry At baseline (t0) and 24 hours (t24), processed cells were stained with fluorescent monoclonal antibodies (Beckman Coulter, Brea, CA) for flow cytometry analysis. Cell staining was performed for detecting cell surface markers including PD1, CD4, CD8 and CD25 using corresponding monoclonal antibodies according to the manufacturer’s instructions. Mouse IgG isotype was used as controls. Processed cells were harvested using a Cytomics FC 500 system (Beckman Coulter, Brea, CA), and approximate 100,000 cells were acquired for each stained sample. Fluorescence of activated cell sorting data was analyzed using CXP 5.52 software. The lymphocytes were gated by setting appropriate forward and side scatter. The frequency of cells was calculated as the percentage of cells within the histogram gate. Statistical Analysis All statistical analyses were performed using the SPSS 15.0 software (SPSS Inc, Chicago, IL). The data were presented as mean 6 SD. Comparisons of the data between AS group and HC group were made by the Student’s t test, and the differences Copyright © 2015 by the Southern Society for Clinical Investigation.
between the inducible expression and baseline of PD-1 were analyzed by the paired t test. P values of less than 0.05 were considered statistically significant.
RESULTS PD-1 was inducibly expressed on T lymphocytes stimulated by PHA. The frequency of PD-1 expression was analyzed at 2 time points including nonstimulated cells (t0) and stimulated cells for 24 hours (t24). The multiple levels of PD-1 expression at t24 and t0 were calculated as the degree of the inducible effect on the PD-1 expression. PD-1 Expression Levels on Nonstimulated Cells (t0) Compared with HCs, within the CD4+ T cell populations, the frequency of PD-1 expression on CD4+CD25high Tregs was not significantly different in patients with AS (0.9 6 0.5% versus 0.8 6 0.3%, P . 0.05; Figure 1C). The frequency of PD-1 expression on CD4+CD25+, CD4+CD25low and CD4+CD252 T cells in patients with AS were 15.6 6 4.2%, 15.1 6 4.2% and 37.1 6 5.8%, respectively, as similar to those in the HCs, which were 12.2 6 1.6%, 11.6 6 1.4% and 34.1 6 5.9%, respectively (P 5 0.103, P 5 0.142 and P 5 0.263, respectively; Figure 1C). The percentages of CD4+PD-1+ and CD8+PD-1+ among patients with AS and healthy individuals were as follows: 15.7 6 3.9% versus 12.0 6 2.9% and 14.5 6 5.9% versus 15.5 6 6.4% (P 5 0.187, P 5 0.417, respectively; Figures 1A and 1B). The frequency of these markers in patients with AS were similar to those in healthy individuals (P . 0.05). Inducible Expression of PD-1 on T Lymphocytes No significant differences were found in the frequency of PD-1 expression on nonstimulated lymphocytes between 2 groups. The authors detected the PD-1 expression on stimulated T lymphocytes to evaluate whether PD-1 expression on lymphocytes may be affected by activated T cell. In this study, the authors found that the expression of PD-1 was inducible on lymphocytes stimulated by PHA for 24 hours, especially on the CD4+, CD4+CD25+, CD4+CD25high and CD4+CD252 T cells (data not shown, Figure 2). Then, the authors confirmed the ratio of PD-1 expression on CD4+ T cells and CD8+ T cells after stimulated with PHA for 24 hours (t24) compared with nonstimulated cells (CD4+ T cells and CD8+ T cells) (t0). The multiple levels of PD-1 expression on CD4+ T cells in patients with AS were 1.9-folds, which was significantly lower than 3.6folds in HCs (P 5 0.017, Figure 3C). However, for CD8+ T cells, the multiple levels of PD-1 expression in patients were 2.1-folds similar to that in HCs with 1.9-folds (P . 0.05, Figure 3B). CD4+ T cells were gated on a dot plot and analyzed for multiple levels of PD-1 expression on CD4+CD25+ T cells and CD4+CD252 T cells in each histogram gate. The multiple levels of PD-1 expression on stimulated CD4+CD25+ T cells at t24 compared with nonstimulated cells at t0 in patients with AS and HCs were 9.7-folds and 17.8-folds, respectively. However, those on CD4+CD252 T cells were only 0.5-folds and 1.13folds in patients with AS and HCs. Compared with HCs, the multiple levels of PD-1 expression on CD4+CD25+ T cells and CD4+CD252 T cells were significantly lower in patients with AS (P 5 0.034 and P 5 0.001, respectively; Figures 4A and 4D). CD25+ T cells were grouped into CD25high and CD25low cells. The multiple levels of PD-1 expression on CD4+CD25high T cells and CD4+CD25low T cells were significantly lower in
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FIGURE 1. Expression of PD-1 on nonstimulated cells was determined on nonstimulated lymphocytes (t0) by the flow cytometry from patients with AS (n 5 30) compared with HCs (n 5 31). (A) The percentage of PD-1 expression on CD4+ T cells (F2 gate). (B) The percentage of PD-1 expression on CD8+ T cells (H2 gate). (C) CD4+ T cells were gated on a dot plot analysis of PD-1 expression on T cells in each histogram gate. CD25+ cells were grouped as CD25low and CD25high T cells (I4 and I2 gates, respectively); the percentage of PD-1 expression on CD4+CD252, CD4+CD25low, CD4+CD25high and CD4+CD25+ cells. AS, ankylosing spondylitis; HC, healthy control; PD-1, programmed death-1.
patients with AS than HCs (P 5 0.044 and P 5 0.031, respectively; Figures 4B and 4C).
DISCUSSION PD-1 pathway regulates T-cell activation and tolerance by providing 2nd signals that can influence T-cell proliferation, cytokine production and cytolytic function.16 This investigation focused on the expression of PD-1 on T lymphocytes. The data indicated an aberrant expression of PD-1 on activated T cells in patients with AS. There are accumulating evidence indicating a vital role of coinhibitory receptors PD-1 in regulating T-cell response and maintenance of peripheral tolerance.17 These findings showed
that the multiple levels of PD-1–expressed CD4+ T cells decreased in patients with AS (P 5 0.017). Chen et al18 suggested that the percentage of CD4+PD-1+ T cells was significantly lower in patients with AS with higher modified Stokes Ankylosing Spondylitis Spinal Scores (mSASSS $30) than in those with lower mSASSS. Meng et al19 demonstrated that CD4+PD-1+ T cells from anterior chamber–associated immune deviation mice exhibited antigen-specific suppressive activity in association with enhanced expression of IL-10. The data provided a support for the hypothesis that the decreased expression of PD-1 on CD4+ T cells may continuously stimulate T-cell activity and increase product of cytokines, contributing to spinal inflammation and destruction in patients with AS.
FIGURE 2. Expression of PD-1 on total lymphocytes, CD4+, CD8+, CD4+CD25+, CD4+CD25high, CD4+CD25low and CD4+CD252 T cells at t0 and t24. The expression of CD4+PD-1+, CD8+PD-1+, CD4+CD25+PD-1+, CD4+CD25highPD-1+, CD4+CD25lowPD-1+ and CD4+CD252PD-1+ were determined on nonstimulated cells (t0) and stimulated cells (t24) with PHA by flow cytometry. Except total lymphocytes, CD8+ and CD4+CD252 T cells, the expression of PD-1 on stimulated cells was significantly higher than that on corresponding nonstimulated cells in both patients with AS and HCs. The PD-1 was decreased, expressed in the CD4+CD252 T cells from patients with AS after stimulation, which was opposed to the increased expression in CD4+CD252 T cells from patients with AS. NS, not significant; AS-0 and HC-0, the expression of PD-1 on nonstimulated cells (t0) in patients with AS and HCs, respectively; AS-24 and HC-24, the expression of PD-1 on stimulated cells (t24) in patients with AS and HCs; *P , 0.05, AS t0 versus t24, HC t0 versus t24; AS, ankylosing spondylitis; HC, healthy control; PD-1, programmed death-1; PHA, phytohemagglutinin.
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FIGURE 3. Multiple levels of PD-1 expression on lymphocytes, CD4+ T and CD8+ T cells. Multiple levels of PD-1 expression on lymphocytes from patients with AS (n 5 30) compared with the HCs (n 5 31). (A) Compared with HCs, multiple levels of PD-1 receptor expression on lymphocytes were not significantly different in patients with AS (NS). (B) Compared with HCs, multiple levels of PD-1 receptor expression on CD8+ T cells were not significantly different in patients with AS (NS). (C) Compared with HCs, multiple levels of PD-1 receptor expression on CD4+ T cells were lower significantly in patients with AS (P 5 0.017). P . 0.05, not significant (NS). P , 0.05, significant, AS t24/t0 versus HC t24/t0. AS, ankylosing spondylitis; HC, healthy control; PD-1, programmed death-1.
The data of this study indicated that the expression of PD-1 on the CD4+CD25+ T cells and CD4+CD25high T cells was significantly lower on activated cells from systemic lupus
erythematosus patients than those in HCs (P , 0.05).20 The study may suggest that CD4+CD25+ T effector T cells may be crucial in the pathogenesis. It has also been documented that the CD4+CD252PD-1+ T cells exhibited a suppressor activity and contained substantial amounts of Tregs.21 Fife et al22 demonstrated that PD-1 could inhibit T-cell receptor-mediated stop signal and that blocking of PD-1 or PD-L1 inhibited T-cell migration, enhanced T-cell dendritic cells engagement and T-cell receptor signaling, which abrogated peripheral tolerance. Tregs are implicated in maintaining peripheral tolerance by inhibiting the activation and proliferation of autoreactive T cells.23 A recent publication suggested that PD-1 expression played a pivotal role in the development and function of Treg cells.14,24 This study demonstrated that the expression of PD-1 on primary CD4+CD25 high cells was not significantly different between patients with AS and HCs. But after stimulation, the inducibility of PD-1 expression on CD4+CD25high T cells in patients with AS was far less than that in HC group. Wang et al25 demonstrated that the blockade of PD-1 not only augmented melanoma antigen-specific cytotoxic lymphocyte responses by promoting the proliferation, enhancing cytolytic function and strengthening resistance to inhibition by Treg but also directly limited the suppressive ability of Treg. These CD4+CD25+ T cells isolated from the lungs of a PD-1 antibody–treated mice that were Foxp3-deficient lacked immunosuppressive ability, as demonstrated by the elevated airway hyper-responsiveness and severely remodeled airways.26 These findings showed that the decreased expression of PD-1 on Tregs in patients with AS was similar to recent studies.27,28 Thus, the authors thought that the abnormal expression of PD-1 resulted in proliferation of autoreactive T cells, which increased production of inflammatory cytokine and immune-mediated tissue damages. However, the absence of the comprehensive parameters including disease and medication status of patients was a limitation in this study. It could be more beneficial to add the elucidation about the relationship between PD-1 levels
FIGURE 4. Multiple levels of PD-1 receptor expression on CD4+CD25+, CD4+CD25high, CD4+CD25low and CD4+CD252 T cells in patients with AS (n 5 30) compared with the HCs (n 5 31). (A) Compared with HCs, multiple levels of PD-1 receptor expression on CD4+CD25+ T cells were lower in patients with AS (P 5 0.034). (B) Compared with HCs, multiple levels of PD-1 receptor expression on CD4+CD25high T cells were lower in patients with AS (P 5 0.044). (C) Compared with HCs, multiple levels of PD-1 receptor expression on CD4+CD25low T cells were lower in patients with AS (P 5 0.031). (D) Compared with HCs, multiple levels of PD-1 receptor expression on CD4+CD252 T cells were lower significantly in patients with AS (P 5 0.001). P , 0.05, significant, AS t24/t0 versus HC t24/t0. AS, ankylosing spondylitis; HC, healthy control; PD-1, programmed death-1.
Copyright © 2015 by the Southern Society for Clinical Investigation.
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and disease status of patients. Although these results are just preliminary, it will be of importance to determine the function of the PD-1–expressed Tregs in AS for subsequent research.
12. Nishimura H, Nose M, Hiai H, et al. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity 1999;11:141–51.
CONCLUSIONS
14. Kitazawa Y, Fujino M, Wang Q, et al. Involvement of the programmed death-1/programmed death-1 ligand pathway in CD4+CD25+ regulatory T-cell activity to suppress alloimmune responses. Transplantation 2007; 83:774–82.
Above all, these findings showed that aberrant PD-1 expression on T lymphocytes in patients with AS provided a support for the importance of PD-1 receptor expression in immune responses and maintenance of peripheral tolerance. Furthermore, the low inducible expression of PD-1 on T lymphocytes, especially on CD4+CD25high and CD4+CD25+ T cells, maybe one of important factors involved in the development of AS. ACKNOWLEDGMENTS The authors are grateful to all participants included in this study, Dr. Wu Zhou for analyzing the data and Dr. Fangyou Yu for careful reading the article. REFERENCES 1. Braun J, Sieper J. Ankylosing spondylitis. Lancet 2007;369:1379–90. 2. Feldtkeller E, Erlendsson J. Definition of disease duration in ankylosing spondylitis. Rheumatol Int 2008;28:693–6.
13. Francisco LM, Sage PT, Sharpe AH. The PD-1 pathway in tolerance and autoimmunity. Immunol Rev 2010;236:219–42.
15. van der Linden S, Valkenburg HA, Cats A. Evaluation of diagnostic criteria for ankylosing spondylitis. A proposal for modification of the New York criteria. Arthritis Rheum 1984;27:361–8. 16. Riley JL. PD-1 signaling in primary T cells. Immunol Rev 2009;229: 114–25. 17. Riella LV, Paterson AM, Sharpe AH, et al. Role of the PD-1 pathway in the immune response. Am J Transplant 2012;12:2575–87. 18. Chen MH, Chen WS, Lee HT, et al. Inverse correlation of programmed death 1 (PD-1) expression in T cells to the spinal radiologic changes in Taiwanese patients with ankylosing spondylitis. Clin Rheumatol 2011;30:1181–7. 19. Meng Q, Yang P, Li B, et al. CD4+PD-1+ T cells acting as regulatory cells during the induction of anterior chamber-associated immune deviation. Invest Ophthalmol Vis Sci 2006;47:4444–52.
3. Colbert RA, DeLay ML, Klenk EI, et al. From HLA-B27 to spondyloarthritis: a journey through the ER. Immunol Rev 2010;233:181–202.
20. Kristjansdottir H, Steinsson K, Gunnarsson I, et al. Lower expression levels of the programmed death 1 receptor on CD4+CD25+ T cells and correlation with the PD-1.3A genotype in patients with systemic lupus erythematosus. Arthritis Rheum 2010;62:1702–11.
4. Azizi E, Massoud A, Amirzargar AA, et al. Association of CTLA4 gene polymorphism in Iranian patients with ankylosing spondylitis. J Clin Immunol 2010;30:268–71.
21. Totsuka T, Kanai T, Makita S, et al. Regulation of murine chronic colitis by CD4+CD25-programmed death-1+ T cells. Eur J Immunol 2005;35:1773–85.
5. Brionez TF, Reveille JD. The contribution of genes outside the major histocompatibility complex to susceptibility to ankylosing spondylitis. Curr Opin Rheumatol 2008;20:384–91.
22. Fife BT, Pauken KE, Eagar TN, et al. Interactions between PD-1 and PD-L1 promote tolerance by blocking the TCR-induced stop signal. Nat Immunol 2009;10:1185–92.
6. Yang PT, Kasai H, Zhao LJ, et al. Increased CCR4 expression on circulating CD4(+) T cells in ankylosing spondylitis, rheumatoid arthritis and systemic lupus erythematosus. Clin Exp Immunol 2004;138: 342–7.
23. Sakaguchi S, Yamaguchi T, Nomura T, et al. Regulatory T cells and immune tolerance. Cell 2008;133:775–87.
7. Jandus C, Bioley G, Rivals JP, et al. Increased numbers of circulating polyfunctional Th17 memory cells in patients with seronegative spondylarthritides. Arthritis Rheum 2008;58:2307–17. 8. Keir ME, Butte MJ, Freeman GJ, et al. PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol 2008;26:677–704.
24. Yogev N, Frommer F, Lukas D, et al. Dendritic cells ameliorate autoimmunity in the CNS by controlling the homeostasis of PD-1 receptor(+) regulatory T cells. Immunity 2012;37:264–75. 25. Wang W, Lau R, Yu D, et al. PD1 blockade reverses the suppression of melanoma antigen-specific CTL by CD4+ CD25(Hi) regulatory T cells. Int Immunol 2009;21:1065–77.
9. Shinohara T, Taniwaki M, Ishida Y, et al. Structure and chromosomal localization of the human PD-1 gene (PDCD1). Genomics 1994; 23:704–6.
26. McGee HS, Yagita H, Shao Z, et al. Programmed death-1 antibody blocks therapeutic effects of T-regulatory cells in cockroach antigen-induced allergic asthma. Am J Respir Cell Mol Biol 2010; 43:432–42.
10. Ansari MJ, Salama AD, Chitnis T, et al. The programmed death-1 (PD-1) pathway regulates autoimmune diabetes in nonobese diabetic (NOD) mice. J Exp Med 2003;198:63–9.
27. Qiao G, Yang L, Li Z, et al. Program death-1 regulates peripheral T cell tolerance via an anergy-independent mechanism. Clin Immunol 2012;143:128–33.
11. Salama AD, Chitnis T, Imitola J, et al. Critical role of the programmed death-1 (PD-1) pathway in regulation of experimental autoimmune encephalomyelitis. J Exp Med 2003;198:71–8.
28. Francisco LM, Salinas VH, Brown KE, et al. PD-L1 regulates the development, maintenance, and function of induced regulatory T cells. J Exp Med 2009;206:3015–29.
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Decreased Programmed Death-1 Expression on the T Cells of Patients With Ankylosing Spondylitis Lianlian Zhou, MD, Yanhong Zhang, MD, Huiying Xu, MD, Liping Hu, MD, Chunwu Zhang, MD, Li Sun, MD, Yaosheng Xie, MD, Hong Lu, MD, Zhuo Zhang, PhD, Wangqiang Hu, MD and Xiangyang Lin, MD
Abstract: Background: Programmed death-1 (PD-1) plays a vital role in down-modulating immune responses and maintaining peripheral tolerance. Methods: The authors have investigated the inducible expression of PD-1 on activated T cells from patients with ankylosing spondylitis (AS). Thirty patients with AS and 31 unrelated healthy controls (HCs) were recruited in this study. The expression of PD-1 on T cells harvested from nonstimulated (t0) or stimulated cultures with phytohemagglutinin for 24 hours (t24) was determined by flow cytometry. The multiple levels of the PD-1 expression on stimulated and nonstimulated cells from each individual’s sample (t24/t0) represented as the degree of the inducible effect on PD-1 expression. Results: The expression of PD-1 on nonstimulated T cells presented no significant difference between AS group and HC group (P . 0.05). After stimulation, the degree of effect on PD-1 expression of CD4+, CD4+CD25+, CD4+CD25high, CD4+CD25low and CD4+CD252 T cells were significantly lower in patients with AS than those in HC group (1.9 6 0.9 versus 3.6 6 2.3, 9.7 6 7.4 versus 17.8 6 12.6, 87.8 6 48.6 versus 157.3 6 117.0, 3.7 6 1.4 versus 7.3 6 2.4, 0.5 6 0.3 versus 1.1 6 0.6, respectively, P , 0.05). However, there was no significant difference of the effect on all lymphocytes and CD8+ T cells between patients with AS and HCs (P . 0.05). Conclusions: The decreased inducible expression of PD-1 on active T lymphocytes, especially on CD4+CD25high and CD4+CD25+ T cells, may be one of important factors involved in the development of AS. Key Indexing Terms: Ankylosing spondylitis; Programmed cell death-1; T cells; Regulatory T cells. [Am J Med Sci 2015;349(6):488–492.]
A
nkylosing spondylitis (AS) is one of the seronegative spondyloarthropathies characterized by the inflammation mainly in the axial skeleton and sacroiliac joints.1 Because of the late appearance of definite sacroiliitis on radiographs, it is still difficult to diagnose at the onset of the diseases, resulting in a delay of up to 10 years between the onset of 1st symptoms and diagnosis of AS.2 The etiology and pathogenesis of AS remain unclear; however, it is associated with the human leukocyte antigen (HLA)–B27.3 Recent studies have demonstrated that HLA-B27 can be recognized by CD4+ T cells, and From the Department of Clinical Laboratory (LZ, HX, LH, YX, HL, ZZ, WH, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China; Department of Clinical Laboratory (YZ), Yantai Yuhuangding Hospital, Yantai, China; and Departments of Orthpaedics Trauma (CZ) and Rheumatology (LS), The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China. Submitted May 21, 2014; accepted in revised form March 5, 2015. The authors have no conflicts of interest to disclose. Supported by Technology Foundation of Zhejiang Province (No. 2013C33173). L.Z. and Y.Z. contributed equally to this work and should be considered as co-first authors. Correspondence: Xiangyang Lin, MD, Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China (E-mail: [email protected]). XL),
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HLA-B27–reactive CD4+ T cells may be pathogenic in spondyloarthropathies.4,5 Much work has concentrated on the alternations of T cells in patients with AS, such as increased frequency of peripheral Th2 lymphocytes6 and Th17 lymphocytes7 in AS. All above are evidence that the T cells may play an important role in the pathology of AS. Programmed death-1 (PD-1) is an immunoglobulin (Ig) superfamily member, which can be inducibly expressed on the surface of peripheral T cells, B cells, NK T cells, monocytes and dendritic cells and interact with its ligand (PD-L) to maintain peripheral tolerance and protect tissues from autoimmune attack.8,9 The role of PD-1 in regulating T-cell tolerance and autoimmunity was 1st observed among PD-12/2 mice with spontaneous autoimmune diseases.10,11 C57BL/6 mice without PD-1 developed a lupus such as glomerulonephritis and arthritis, with the deposition of IgG3 and C3 in the glomeruli.12 Some studies have revealed that PD-1 pathway was not only important in the initial phase of activation and expansion of self-reactive T cells but also influenced self-reactive T-cell effector function on antigen reencounter in mouse models of autoimmunity and tolerance, indicating that PD-1 was important in the induction and/or the maintenance of peripheral tolerance. Regulatory T cells (Tregs) played a vital role in the pathogenesis of many autoimmune diseases, which resulted from the abnormal number or function of Tregs. Francisco et al13 have summarized that PD-1 pathway may assist Tregs in maintaining immune homeostasis. Recently, some studies demonstrated that CD4+CD25high T cells expressing PD-1 exhibited immunosuppressive functions.13,14 To date, the exploration of the function of PD-1 and its pathway in the tolerance and autoimmunity is a population area; however, the levels of PD-1 on T lymphocytes, specifically Tregs in AS, are still unclear. Therefore, this investigation aimed to determine the cellular expression of PD-1 receptor on T lymphocytes with AS in a Chinese population.
MATERIALS AND METHODS Participants Sixty-one Chinese individuals including 30 patients with AS and 31 age- and gender-matched healthy volunteers were recruited from The First Affiliated Hospital of Wenzhou Medical University from July 2011 to July 2012. Clinical diagnosis of AS was in accordance with the criteria modified in New York in 1984.15 Healthy volunteers consisted of students and staff from above institution were designed as the healthy control group (HC). These volunteers had no abnormal laboratory results, without a family history of autoimmune diseases. The demographic and clinical characteristics of the patients and HCs were shown in Table 1. Peripheral blood samples were obtained from the participants after they provided written informed consent. This study protocol and informed consent forms have been approved by the Ethics Review Committee of the First Affiliated Hospital of Wenzhou Medical University.
The American Journal of the Medical Sciences
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TABLE 1. Baseline clinical status of the patients with AS and HCs AS (n 5 30) HC (n 5 31) Age, yr Range Mean 6 SD Gender: male, n (%) Disease duration, yr Laboratory parameters HLA-B27+, n (%) CRP, mg/L ESR, mm/hr Clinical features, n (%) Low back pain .3 mo Limitation of back flexion Sacroiliitis Chest expansion decreased Heel pain Iritis
16–46 30.6 6 7.7 20 (66.7) 3.12 6 3.18
21–48 27.1 6 6.4 24 (60.0) NA
30 (100) 8.10 6 8.04 26.1 6 19.2
1 (3.2) NA NA
30 (100) 11 (36.7) 10 (33.3) 8 (26.7) 4 (13.3) 4 (13.3)
NA NA NA NA NA NA
Values are mean 6 SD for quantitative variables or absolute frequency/total measures. CRP, C-reaction protein; ESR, erythrocyte sedimentation rate; NA, not available.
Stimulation and Culture of Peripheral Blood The peripheral blood was treated by the heparin. RPMI1640 medium was supplemented by heat-inactivated fetal bovine serum at a ratio of 4:1, streptomycin/penicillin with the final concentration of 100 U/mL and the phytohemagglutinin (PHA) with final concentration of 0.02 mg/mL. The pH of RPMI1640 medium was adjusted to between 7.2 and 7.4 with 5% NaHCO3. Under aseptic conditions, 0.5 mL of peripheral blood treated by heparin was added into 5 mL of complete RPMI medium. Cells were cultured at 37°C in 5% CO2 for 24 hours (t24). The cultured peripheral blood cells were harvested by low-speed centrifugation at 1,500 rpm for 5 minutes. The supernatant was discarded, and the precipitates were stored for further experiments. Flow Cytometry At baseline (t0) and 24 hours (t24), processed cells were stained with fluorescent monoclonal antibodies (Beckman Coulter, Brea, CA) for flow cytometry analysis. Cell staining was performed for detecting cell surface markers including PD1, CD4, CD8 and CD25 using corresponding monoclonal antibodies according to the manufacturer’s instructions. Mouse IgG isotype was used as controls. Processed cells were harvested using a Cytomics FC 500 system (Beckman Coulter, Brea, CA), and approximate 100,000 cells were acquired for each stained sample. Fluorescence of activated cell sorting data was analyzed using CXP 5.52 software. The lymphocytes were gated by setting appropriate forward and side scatter. The frequency of cells was calculated as the percentage of cells within the histogram gate. Statistical Analysis All statistical analyses were performed using the SPSS 15.0 software (SPSS Inc, Chicago, IL). The data were presented as mean 6 SD. Comparisons of the data between AS group and HC group were made by the Student’s t test, and the differences Copyright © 2015 by the Southern Society for Clinical Investigation.
between the inducible expression and baseline of PD-1 were analyzed by the paired t test. P values of less than 0.05 were considered statistically significant.
RESULTS PD-1 was inducibly expressed on T lymphocytes stimulated by PHA. The frequency of PD-1 expression was analyzed at 2 time points including nonstimulated cells (t0) and stimulated cells for 24 hours (t24). The multiple levels of PD-1 expression at t24 and t0 were calculated as the degree of the inducible effect on the PD-1 expression. PD-1 Expression Levels on Nonstimulated Cells (t0) Compared with HCs, within the CD4+ T cell populations, the frequency of PD-1 expression on CD4+CD25high Tregs was not significantly different in patients with AS (0.9 6 0.5% versus 0.8 6 0.3%, P . 0.05; Figure 1C). The frequency of PD-1 expression on CD4+CD25+, CD4+CD25low and CD4+CD252 T cells in patients with AS were 15.6 6 4.2%, 15.1 6 4.2% and 37.1 6 5.8%, respectively, as similar to those in the HCs, which were 12.2 6 1.6%, 11.6 6 1.4% and 34.1 6 5.9%, respectively (P 5 0.103, P 5 0.142 and P 5 0.263, respectively; Figure 1C). The percentages of CD4+PD-1+ and CD8+PD-1+ among patients with AS and healthy individuals were as follows: 15.7 6 3.9% versus 12.0 6 2.9% and 14.5 6 5.9% versus 15.5 6 6.4% (P 5 0.187, P 5 0.417, respectively; Figures 1A and 1B). The frequency of these markers in patients with AS were similar to those in healthy individuals (P . 0.05). Inducible Expression of PD-1 on T Lymphocytes No significant differences were found in the frequency of PD-1 expression on nonstimulated lymphocytes between 2 groups. The authors detected the PD-1 expression on stimulated T lymphocytes to evaluate whether PD-1 expression on lymphocytes may be affected by activated T cell. In this study, the authors found that the expression of PD-1 was inducible on lymphocytes stimulated by PHA for 24 hours, especially on the CD4+, CD4+CD25+, CD4+CD25high and CD4+CD252 T cells (data not shown, Figure 2). Then, the authors confirmed the ratio of PD-1 expression on CD4+ T cells and CD8+ T cells after stimulated with PHA for 24 hours (t24) compared with nonstimulated cells (CD4+ T cells and CD8+ T cells) (t0). The multiple levels of PD-1 expression on CD4+ T cells in patients with AS were 1.9-folds, which was significantly lower than 3.6folds in HCs (P 5 0.017, Figure 3C). However, for CD8+ T cells, the multiple levels of PD-1 expression in patients were 2.1-folds similar to that in HCs with 1.9-folds (P . 0.05, Figure 3B). CD4+ T cells were gated on a dot plot and analyzed for multiple levels of PD-1 expression on CD4+CD25+ T cells and CD4+CD252 T cells in each histogram gate. The multiple levels of PD-1 expression on stimulated CD4+CD25+ T cells at t24 compared with nonstimulated cells at t0 in patients with AS and HCs were 9.7-folds and 17.8-folds, respectively. However, those on CD4+CD252 T cells were only 0.5-folds and 1.13folds in patients with AS and HCs. Compared with HCs, the multiple levels of PD-1 expression on CD4+CD25+ T cells and CD4+CD252 T cells were significantly lower in patients with AS (P 5 0.034 and P 5 0.001, respectively; Figures 4A and 4D). CD25+ T cells were grouped into CD25high and CD25low cells. The multiple levels of PD-1 expression on CD4+CD25high T cells and CD4+CD25low T cells were significantly lower in
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FIGURE 1. Expression of PD-1 on nonstimulated cells was determined on nonstimulated lymphocytes (t0) by the flow cytometry from patients with AS (n 5 30) compared with HCs (n 5 31). (A) The percentage of PD-1 expression on CD4+ T cells (F2 gate). (B) The percentage of PD-1 expression on CD8+ T cells (H2 gate). (C) CD4+ T cells were gated on a dot plot analysis of PD-1 expression on T cells in each histogram gate. CD25+ cells were grouped as CD25low and CD25high T cells (I4 and I2 gates, respectively); the percentage of PD-1 expression on CD4+CD252, CD4+CD25low, CD4+CD25high and CD4+CD25+ cells. AS, ankylosing spondylitis; HC, healthy control; PD-1, programmed death-1.
patients with AS than HCs (P 5 0.044 and P 5 0.031, respectively; Figures 4B and 4C).
DISCUSSION PD-1 pathway regulates T-cell activation and tolerance by providing 2nd signals that can influence T-cell proliferation, cytokine production and cytolytic function.16 This investigation focused on the expression of PD-1 on T lymphocytes. The data indicated an aberrant expression of PD-1 on activated T cells in patients with AS. There are accumulating evidence indicating a vital role of coinhibitory receptors PD-1 in regulating T-cell response and maintenance of peripheral tolerance.17 These findings showed
that the multiple levels of PD-1–expressed CD4+ T cells decreased in patients with AS (P 5 0.017). Chen et al18 suggested that the percentage of CD4+PD-1+ T cells was significantly lower in patients with AS with higher modified Stokes Ankylosing Spondylitis Spinal Scores (mSASSS $30) than in those with lower mSASSS. Meng et al19 demonstrated that CD4+PD-1+ T cells from anterior chamber–associated immune deviation mice exhibited antigen-specific suppressive activity in association with enhanced expression of IL-10. The data provided a support for the hypothesis that the decreased expression of PD-1 on CD4+ T cells may continuously stimulate T-cell activity and increase product of cytokines, contributing to spinal inflammation and destruction in patients with AS.
FIGURE 2. Expression of PD-1 on total lymphocytes, CD4+, CD8+, CD4+CD25+, CD4+CD25high, CD4+CD25low and CD4+CD252 T cells at t0 and t24. The expression of CD4+PD-1+, CD8+PD-1+, CD4+CD25+PD-1+, CD4+CD25highPD-1+, CD4+CD25lowPD-1+ and CD4+CD252PD-1+ were determined on nonstimulated cells (t0) and stimulated cells (t24) with PHA by flow cytometry. Except total lymphocytes, CD8+ and CD4+CD252 T cells, the expression of PD-1 on stimulated cells was significantly higher than that on corresponding nonstimulated cells in both patients with AS and HCs. The PD-1 was decreased, expressed in the CD4+CD252 T cells from patients with AS after stimulation, which was opposed to the increased expression in CD4+CD252 T cells from patients with AS. NS, not significant; AS-0 and HC-0, the expression of PD-1 on nonstimulated cells (t0) in patients with AS and HCs, respectively; AS-24 and HC-24, the expression of PD-1 on stimulated cells (t24) in patients with AS and HCs; *P , 0.05, AS t0 versus t24, HC t0 versus t24; AS, ankylosing spondylitis; HC, healthy control; PD-1, programmed death-1; PHA, phytohemagglutinin.
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FIGURE 3. Multiple levels of PD-1 expression on lymphocytes, CD4+ T and CD8+ T cells. Multiple levels of PD-1 expression on lymphocytes from patients with AS (n 5 30) compared with the HCs (n 5 31). (A) Compared with HCs, multiple levels of PD-1 receptor expression on lymphocytes were not significantly different in patients with AS (NS). (B) Compared with HCs, multiple levels of PD-1 receptor expression on CD8+ T cells were not significantly different in patients with AS (NS). (C) Compared with HCs, multiple levels of PD-1 receptor expression on CD4+ T cells were lower significantly in patients with AS (P 5 0.017). P . 0.05, not significant (NS). P , 0.05, significant, AS t24/t0 versus HC t24/t0. AS, ankylosing spondylitis; HC, healthy control; PD-1, programmed death-1.
The data of this study indicated that the expression of PD-1 on the CD4+CD25+ T cells and CD4+CD25high T cells was significantly lower on activated cells from systemic lupus
erythematosus patients than those in HCs (P , 0.05).20 The study may suggest that CD4+CD25+ T effector T cells may be crucial in the pathogenesis. It has also been documented that the CD4+CD252PD-1+ T cells exhibited a suppressor activity and contained substantial amounts of Tregs.21 Fife et al22 demonstrated that PD-1 could inhibit T-cell receptor-mediated stop signal and that blocking of PD-1 or PD-L1 inhibited T-cell migration, enhanced T-cell dendritic cells engagement and T-cell receptor signaling, which abrogated peripheral tolerance. Tregs are implicated in maintaining peripheral tolerance by inhibiting the activation and proliferation of autoreactive T cells.23 A recent publication suggested that PD-1 expression played a pivotal role in the development and function of Treg cells.14,24 This study demonstrated that the expression of PD-1 on primary CD4+CD25 high cells was not significantly different between patients with AS and HCs. But after stimulation, the inducibility of PD-1 expression on CD4+CD25high T cells in patients with AS was far less than that in HC group. Wang et al25 demonstrated that the blockade of PD-1 not only augmented melanoma antigen-specific cytotoxic lymphocyte responses by promoting the proliferation, enhancing cytolytic function and strengthening resistance to inhibition by Treg but also directly limited the suppressive ability of Treg. These CD4+CD25+ T cells isolated from the lungs of a PD-1 antibody–treated mice that were Foxp3-deficient lacked immunosuppressive ability, as demonstrated by the elevated airway hyper-responsiveness and severely remodeled airways.26 These findings showed that the decreased expression of PD-1 on Tregs in patients with AS was similar to recent studies.27,28 Thus, the authors thought that the abnormal expression of PD-1 resulted in proliferation of autoreactive T cells, which increased production of inflammatory cytokine and immune-mediated tissue damages. However, the absence of the comprehensive parameters including disease and medication status of patients was a limitation in this study. It could be more beneficial to add the elucidation about the relationship between PD-1 levels
FIGURE 4. Multiple levels of PD-1 receptor expression on CD4+CD25+, CD4+CD25high, CD4+CD25low and CD4+CD252 T cells in patients with AS (n 5 30) compared with the HCs (n 5 31). (A) Compared with HCs, multiple levels of PD-1 receptor expression on CD4+CD25+ T cells were lower in patients with AS (P 5 0.034). (B) Compared with HCs, multiple levels of PD-1 receptor expression on CD4+CD25high T cells were lower in patients with AS (P 5 0.044). (C) Compared with HCs, multiple levels of PD-1 receptor expression on CD4+CD25low T cells were lower in patients with AS (P 5 0.031). (D) Compared with HCs, multiple levels of PD-1 receptor expression on CD4+CD252 T cells were lower significantly in patients with AS (P 5 0.001). P , 0.05, significant, AS t24/t0 versus HC t24/t0. AS, ankylosing spondylitis; HC, healthy control; PD-1, programmed death-1.
Copyright © 2015 by the Southern Society for Clinical Investigation.
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and disease status of patients. Although these results are just preliminary, it will be of importance to determine the function of the PD-1–expressed Tregs in AS for subsequent research.
12. Nishimura H, Nose M, Hiai H, et al. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity 1999;11:141–51.
CONCLUSIONS
14. Kitazawa Y, Fujino M, Wang Q, et al. Involvement of the programmed death-1/programmed death-1 ligand pathway in CD4+CD25+ regulatory T-cell activity to suppress alloimmune responses. Transplantation 2007; 83:774–82.
Above all, these findings showed that aberrant PD-1 expression on T lymphocytes in patients with AS provided a support for the importance of PD-1 receptor expression in immune responses and maintenance of peripheral tolerance. Furthermore, the low inducible expression of PD-1 on T lymphocytes, especially on CD4+CD25high and CD4+CD25+ T cells, maybe one of important factors involved in the development of AS. ACKNOWLEDGMENTS The authors are grateful to all participants included in this study, Dr. Wu Zhou for analyzing the data and Dr. Fangyou Yu for careful reading the article. REFERENCES 1. Braun J, Sieper J. Ankylosing spondylitis. Lancet 2007;369:1379–90. 2. Feldtkeller E, Erlendsson J. Definition of disease duration in ankylosing spondylitis. Rheumatol Int 2008;28:693–6.
13. Francisco LM, Sage PT, Sharpe AH. The PD-1 pathway in tolerance and autoimmunity. Immunol Rev 2010;236:219–42.
15. van der Linden S, Valkenburg HA, Cats A. Evaluation of diagnostic criteria for ankylosing spondylitis. A proposal for modification of the New York criteria. Arthritis Rheum 1984;27:361–8. 16. Riley JL. PD-1 signaling in primary T cells. Immunol Rev 2009;229: 114–25. 17. Riella LV, Paterson AM, Sharpe AH, et al. Role of the PD-1 pathway in the immune response. Am J Transplant 2012;12:2575–87. 18. Chen MH, Chen WS, Lee HT, et al. Inverse correlation of programmed death 1 (PD-1) expression in T cells to the spinal radiologic changes in Taiwanese patients with ankylosing spondylitis. Clin Rheumatol 2011;30:1181–7. 19. Meng Q, Yang P, Li B, et al. CD4+PD-1+ T cells acting as regulatory cells during the induction of anterior chamber-associated immune deviation. Invest Ophthalmol Vis Sci 2006;47:4444–52.
3. Colbert RA, DeLay ML, Klenk EI, et al. From HLA-B27 to spondyloarthritis: a journey through the ER. Immunol Rev 2010;233:181–202.
20. Kristjansdottir H, Steinsson K, Gunnarsson I, et al. Lower expression levels of the programmed death 1 receptor on CD4+CD25+ T cells and correlation with the PD-1.3A genotype in patients with systemic lupus erythematosus. Arthritis Rheum 2010;62:1702–11.
4. Azizi E, Massoud A, Amirzargar AA, et al. Association of CTLA4 gene polymorphism in Iranian patients with ankylosing spondylitis. J Clin Immunol 2010;30:268–71.
21. Totsuka T, Kanai T, Makita S, et al. Regulation of murine chronic colitis by CD4+CD25-programmed death-1+ T cells. Eur J Immunol 2005;35:1773–85.
5. Brionez TF, Reveille JD. The contribution of genes outside the major histocompatibility complex to susceptibility to ankylosing spondylitis. Curr Opin Rheumatol 2008;20:384–91.
22. Fife BT, Pauken KE, Eagar TN, et al. Interactions between PD-1 and PD-L1 promote tolerance by blocking the TCR-induced stop signal. Nat Immunol 2009;10:1185–92.
6. Yang PT, Kasai H, Zhao LJ, et al. Increased CCR4 expression on circulating CD4(+) T cells in ankylosing spondylitis, rheumatoid arthritis and systemic lupus erythematosus. Clin Exp Immunol 2004;138: 342–7.
23. Sakaguchi S, Yamaguchi T, Nomura T, et al. Regulatory T cells and immune tolerance. Cell 2008;133:775–87.
7. Jandus C, Bioley G, Rivals JP, et al. Increased numbers of circulating polyfunctional Th17 memory cells in patients with seronegative spondylarthritides. Arthritis Rheum 2008;58:2307–17. 8. Keir ME, Butte MJ, Freeman GJ, et al. PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol 2008;26:677–704.
24. Yogev N, Frommer F, Lukas D, et al. Dendritic cells ameliorate autoimmunity in the CNS by controlling the homeostasis of PD-1 receptor(+) regulatory T cells. Immunity 2012;37:264–75. 25. Wang W, Lau R, Yu D, et al. PD1 blockade reverses the suppression of melanoma antigen-specific CTL by CD4+ CD25(Hi) regulatory T cells. Int Immunol 2009;21:1065–77.
9. Shinohara T, Taniwaki M, Ishida Y, et al. Structure and chromosomal localization of the human PD-1 gene (PDCD1). Genomics 1994; 23:704–6.
26. McGee HS, Yagita H, Shao Z, et al. Programmed death-1 antibody blocks therapeutic effects of T-regulatory cells in cockroach antigen-induced allergic asthma. Am J Respir Cell Mol Biol 2010; 43:432–42.
10. Ansari MJ, Salama AD, Chitnis T, et al. The programmed death-1 (PD-1) pathway regulates autoimmune diabetes in nonobese diabetic (NOD) mice. J Exp Med 2003;198:63–9.
27. Qiao G, Yang L, Li Z, et al. Program death-1 regulates peripheral T cell tolerance via an anergy-independent mechanism. Clin Immunol 2012;143:128–33.
11. Salama AD, Chitnis T, Imitola J, et al. Critical role of the programmed death-1 (PD-1) pathway in regulation of experimental autoimmune encephalomyelitis. J Exp Med 2003;198:71–8.
28. Francisco LM, Salinas VH, Brown KE, et al. PD-L1 regulates the development, maintenance, and function of induced regulatory T cells. J Exp Med 2009;206:3015–29.
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