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Clinical significance of Tim3-positive T cell subsets in patients with multiple sclerosis
Journal of Clinical Neuroscience xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Clinical Study
Clinical significance of Tim3-positive T cell subsets in patients with multiple sclerosis Xuemei Feng a, Juan Feng b,⇑ a b
Department of Nerve Function, Shengjing Hospital of China Medical University, Shenyang, China Department of Neurology, Shengjing Hospital of China Medical University, No. 36, Sanhao Street, Heping District, Shenyang 110004, China
a r t i c l e
i n f o
Article history: Received 14 September 2014 Accepted 3 July 2016 Available online xxxx Keywords: Tim3 Flow cytometry CD4+CD25(high) T cell Galectin-9
a b s t r a c t The present study evaluated associations between the percentages of T cell immunoglobulin and mucin domain 3 (Tim3)-positive T cells and related cytokines and multiple sclerosis (MS). We collected peripheral blood samples from 30 MS patients and 30 healthy controls. Flow cytometry was used to determine the proportions of CD3+Tim3+, CD4+Tim3+, and CD4+CD25+Tim3+ in peripheral blood mononuclear cells (PBMCs) and related cell subsets. The serum concentrations of galectin-9, IL-17, and IFN-c also were determined using enzyme-linked immunosorbent assays (ELISA). The percentages of Tim3-positive T cells in CD4+ and CD4+CD25+ T cell subsets were significantly lower among MS patients than among controls. This difference was particularly evident in the CD4+CD25(high) T cell subset. The proportions of CD4+Tim3+ and CD4+CD25+Tim3+ cells in PBMCs were significantly lower in the MS group than in the control group, whereas no significant differences were detected regarding the percentages of CD3+Tim3+ in PBMCs and T cell subsets. The serum concentrations of galectin-9, IL-17, and IFN-c all were increased in MS patients compared with healthy controls. Our results support that Tim3 and related cytokines may be involved in the onset of MS. Ó 2016 Published by Elsevier Ltd.
1. Introduction Multiple sclerosis (MS) is an autoimmune disease characterized by demyelination within the white matter of the central nervous system. Although the specific pathogenesis of MS is not fully understood, it is widely accepted that a T-cell-mediated immune response is involved [1]. The identification of T cell immunoglobulin and mucin domain 3 (Tim3) served as a breakthrough in the understanding and treatment of autoimmune diseases [2]. Tim3 is preferentially expressed on the surfaces of Th17 and Th1 cells. Tim3 contributes to an important immunoregulatory pathway involving CD4+ T cells and can down-regulate Th1 responses [2–5]. The function of Tim3 as a negative regulator of Th1 cellmediated immune responses has been explained by Monney et al. (2002) [5] using an experimental autoimmune encephalomyelitis (EAE) mouse model with an induced Th1-dominant response and by Huey-Kang Sytwu 2009 in a study evaluating the Th1-dominant response in an autoimmune non-obese diabetic (NOD) mouse model [6]. Several reports have
⇑ Corresponding author. Tel.: +86 024 96615 28815, +86 18940251959. E-mail addresses: [email protected], [email protected] (J. Feng).
described the up-regulation of Tim3 in patients with rheumatoid arthritis or nephrotoxic nephritis, yet it remains unclear in MS [7–9]. Therefore, the specific mechanism of action of Tim3 in these diseases requires further investigation. Few studies have evaluated the involvement of Tim3 in MS, although one report suggests that Tim3 mRNA expression is decreased in peripheral blood CD4+ cells of MS patients [10]. Tim3 functions by binding to galectin-9, its natural ligand [11,12]. Galectin-9 may improve rheumatoid arthritis [7] by indirectly increasing the production of CD4+CD25+ regulatory T cells (Tregs); however, its role in MS is unknown. Tregs impose negative regulatory effects on the proliferation of effector T cells, which exert immunoregulatory effects in MS and other autoimmune diseases by down-regulating the interferon (IFN)-c pathway. Previous studies suggest that CD4+CD25(high) T cells suppress T cell function and protect against autoimmune disease in healthy individuals [13–16]. The pathogenesis of MS may be related to functional deficiencies among CD4+CD25(high) cells [17]. The regulatory functions of this cell population may correlate with changes in the expression or function of Tim3 [18]. To date, potential associations between the functionality or number of CD4+CD25(high) cells and various autoimmune diseases have not been established.
http://dx.doi.org/10.1016/j.jocn.2016.07.007 0967-5868/Ó 2016 Published by Elsevier Ltd.
Please cite this article in press as: Feng X, Feng J. Clinical significance of Tim3-positive T cell subsets in patients with multiple sclerosis. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j.jocn.2016.07.007
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X. Feng, J. Feng / Journal of Clinical Neuroscience xxx (2016) xxx–xxx
2. Materials and methods
3. Results
2.1. Patient and control groups
3.1. Flow cytometry
The patient group consisted of 30 patients (13 men, 17 women; mean age 37 ± 2.92 years) who had been admitted to ShengJing Hospital of China Medical University between January 2008 and October 2013. All 30 patients were in acute relapse phase, excluding three patients who already had secondary progressive multiple sclerosis (SPMS). The mean disease duration is 7.17 ± 4.9 years and the mean Expanded Disability Status Scale (EDSS) is 4.3 ± 2.0. The control group included 30 sex-matched healthy individuals (mean age 35 ± 1.08 years) with no autoimmune system diseases who came to the hospital for physical examinations. The 30 patients all had been clinically diagnosed with MS with relapse and all met the McDonald diagnostic criteria.
The percentage of CD3+Tim3+ cells in the MS patient group did not differ significantly from the control group (Table 1, Fig. 1). The percentage of CD4+Tim3+ cells in the MS group was significantly lower than the percentage of these cells in the control group (P < 0.05) (Table 2, Fig. 2). The percentage of Tim3+ cells among the regulatory T cells in the MS group was significantly lower than the percentage of Tim3+ cells in the control group (P < 0.05) (Table 3, Fig. 3).
2.2. Cells and sample preparation Peripheral venous blood samples were obtained from MS patients at 06:00 on the second day of admission. All patients had avoided immunosuppressants and corticosteroids for at least 1 month at the time of blood sample collection. Peripheral blood samples also were obtained from the control group. Blood samples were collected into anticoagulant tubes containing 0.2 ml heparin and inverted gently. Serum then was obtained by removing supernatants after Ficoll-Hypaque density gradient centrifugation at 4°C and was stored at 70°C. Peripheral blood lymphocytes were isolated by density gradient centrifugation and were diluted with phosphate-buffered saline solution to a cell suspension of 2 106 cells/ml. 2.3. Cell-surface staining and flow cytometry The following antibodies were used according to manufacturer’s Instructions:phycoerythrin conjugated anti-Tim3 monoclonal antibody (Tim3-PE), peridinin chlorophyll protein conjugated anti-CD3 monoclonal antibody (CD3-PerCP), peridinin chlorophyll protein conjugated anti-CD4 monoclonal antibody (CD4-PerCP), fluorescein isothiocyanate-conjugated anti-CD25 monoclonal antibody (CD25-FITC). All antibodies were purchased from BD (San Diego, CA, USA), except for the Tim3-PE (eBioscience, San Diego, CA, USA). Cell suspensions derived from patient and control blood samples were stained with the following antibody combinations: (1) CD4-PerCP, CD25-FITC, Tim3-PE; (2) CD3-PERCP, Tim3-PE. Mixtures were incubated at 4°C for 25 min in the dark. After incubation, excess antibodies were washed off, and flow cytometric analysis was performed on a BD FACSCalibur flow cytometer using FCS ExpressV3 software. 2.4. Enzyme-linked immunosorbent assay The plasma concentrations of galectin-9, IL-17, and IFN-c were determined using enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN, USA). 2.5. Statistics The percentages of cell subsets identified by flow cytometry were analyzed using the Mann–Whitney U-test. All data were represented as means ± standard deviations. P < 0.05 was considered statistically significant.
3.2. Enzyme-linked immunosorbent assay The plasma concentrations of galectin-9 and IFN-c in the MS group was significantly higher than the control group (P < 0.05), and the concentrations of IL-17 in the MS group was lower than the control group (P < 0.05) (Table 4). 4. Discussion Tim3 is a recently identified molecule that is preferentially expressed on the surfaces of Th17 and Th1 cells. Administration of anti-Tim3 monoclonal antibodies accelerates the onset and severity of autoimmune cerebrospinal meningitis in mice [5]. When the interaction between Tim3 and its ligand, galectin-9, is blocked, the result is a predominant Th1-type response with induction of T cell proliferation and secretion of Th1-type cytokines [18,19]. These findings suggest that Tim3 plays a negative regulatory role in the Th1 cell-mediated immune response. In this study, we demonstrated that MS patients do not significantly differ from healthy controls regarding the percentages of Tim3+ T cells in peripheral blood or among all T cells. However, among CD4+ cells, the percentage of Tim3+ T cells was decreased in MS patients. We suggest that the decreased expression of Tim3 in CD4+ cells of MS patients could reduce the inhibitory effects of Tim3 on Th1 and Th17 cells. This reduced negative regulatory effect could contribute to the pathogenesis of MS. Several studies have reported that Tim3 can also induce apoptosis of Th1 cells [18,19]. We speculate that the onset of MS also may be facilitated by a reduction in targeted Th1 cell apoptosis and Th17 cell inhibition caused by the decreased expression of Tim3 in CD4+ cells. Tregs can be summarized as a group of CD4+ T cells that exert potential inhibitory activities against harmful reactions. The immunoregulatory activities of CD4+CD25+ T cells of natural phenotype depend on their surface markers (e.g., CTLA-4, FOXP3, CD127, CD25[high]). Previous studies have indicated that in CD4+CD25+ T cells of natural phenotype, the subset of CD4+CD25(low) cells do not exhibit immunoregulatory functions, whereas the CD4+CD25(high) cells are the true regulators [20]. The results of our study indicate that the percentage of Tim3-positive CD4+CD25+cells among all CD4+CD25+cells is significantly reduced in MS patients compared with controls. Moreover, the percentage of Tim3-positive CD4+CD25(high) cells among all CD4+CD25(high) cells is significantly reduced in MS patients, with
Table 1 Percentages of CD3+Tim3+ T cell subsets in peripheral blood and T cells in the multiple sclerosis (MS) group and the control group. Groups
n
CD3+Tim3+
CD3+Tim3+/CD3
MS Group Control Group
30 30
3.79 ± 0.81 3.70 ± 0.72
6.03 ± 0.91 6.62 ± 1.27
p > 0.05, compare to control: Mann–Whitney U test.
Please cite this article in press as: Feng X, Feng J. Clinical significance of Tim3-positive T cell subsets in patients with multiple sclerosis. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j.jocn.2016.07.007
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X. Feng, J. Feng / Journal of Clinical Neuroscience xxx (2016) xxx–xxx
Fig. 1. Percentages of CD3+Tim3+ cells in the peripheral blood of multiple sclerosis (MS) patients and healthy controls. (a) Among MS patients, CD3+Tim3+ T cells accounted for 3.89% of peripheral blood T cells. (b) Among controls, CD3+Tim3+ cells accounted for 3.77% of peripheral blood T cells. No significant difference was detected between the two groups.
Table 2 Percentages of CD4+Tim3+ T cell subsets in peripheral blood and T helper cells in multiple sclerosis (MS) patients and controls. Groups
n
CD4+Tim3+
CD4+Tim3+/CD4
MS Group Control Group
30 30
1.35 ± 0.35 1.64 ± 0.36*
3.30 ± 0.62 4.20 ± 0.63*
Compare to control: Mann–Whitney U test. * p < 0.05.
the reduction in CD4+CD25(high) cells being more pronounced. Hafler et al. reported that purified CD4+CD25(high) cells from MS patients do not inhibit the proliferation of effector T cells in vitro, suggesting that the normal regulatory functions of CD4+CD25 (high) cells are absent in MS. The development of MS is related not only to the functional deficiency of CD4+CD25(high) cells via unknown mechanisms [21], but also to changes in the expression or function of Tim3 [22,23]. We speculate that the reduced expression of Tim3 in CD4+CD25 (high) cells is involved in the development of MS. The reduced expression or the altered function of CD4+CD25+Tim3(high) cells affects the regulation of CD4+CD25(high) cells, leading to the development of MS. Further investigations should evaluate the level of CD4+CD25+Tim3(high) cell functioning in patients with MS, the potential of these cells to exert immunoregulatory activity, and whether Tim3 can serve as a novel marker of regulatory T cells. Galectin-9 is a natural ligand of Tim3 [24,25] that has been detected on the surfaces of Treg cells and in serum [26,27]. The
Table 3 Percentages of CD4+CD25+Tim3+ T cell subsets in peripheral blood and regulatory T cells in multiple sclerosis (MS) patients and healthy controls.
CD4+CD25+Tim3+ CD4+CD25(high)Tim3+ CD4+CD25+(low)Tim3+ CD4+CD25+Tim3+/CD4+CD25+ CD4+CD25(high)Tim3+/CD4+CD25(high) CD4+CD25+(low)Tim3+/CD4+CD25+(low)
MS
Controls
0.76 ± 0.21 0.20 ± 0.07 0.55 ± 0.16 11.84 ± 2.45 30.68 ± 6.71 13.12 ± 2.74
1.03 ± 0.14* 0.35 ± 0.06* 0.67 ± 0.16* 13.87 ± 1.93* 43.39 ± 6.15* 13.28 ± 2.83
Compare to control: Mann–Whitney U test * p < 0.05.
Table 4 ELISA tests to compare the plasma concentration of Galectin-9, Interleukin-17 (IL-17) and Interferon-c (IFN-c) between multiple sclerosis (MS) patients and healthy controls.
MS CONTROL
Galectin-9 (ng/L)
IL-17 (ng/L)
IFN-c (ng/L)
37.11 ± 6.71 26.56 ± 3.86*
14.1 ± 3.34 8.49 ± 1.50*
339.00 ± 89.45 196.97 ± 53.44*
Compare to control: Mann–Whitney U test. * P < 0.05.
secretion of galectin-9 can be stimulated by IFN-c [28,29], and galectin-9 functions as an anti-inflammatory molecule in autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, and diabetes [30,31]. The binding of galectin-9 to Tim3 can induce T cell apoptosis, down-regulate the Th1 response, and
Fig. 2. Percentages of CD4+Tim3+ cells in the peripheral blood of multiple sclerosis (MS) patients and controls. (a) In the MS patient group, CD4+Tim3+ T cells comprised 1.52% of the peripheral blood T cells. (b) In the control group, CD4+Tim3+ cells accounted for 2.08% of peripheral blood T cells, a significantly higher percentage than that of the MS group (p < 0.05).
Please cite this article in press as: Feng X, Feng J. Clinical significance of Tim3-positive T cell subsets in patients with multiple sclerosis. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j.jocn.2016.07.007
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Fig. 3. Percentages of CD4+CD25+Tim3+ T cell subsets in peripheral blood and regulatory T cells. (a) We subdivided the CD4+CD25+ T cell population into CD4+CD25(high) and CD4+CD25(low) cells according to the CD25 expression level on CD4+ T cells. (b) The percentage of Tim3+ cells among CD4+CD25+(high) T cells. (c) The percentage of Tim3+ cells among CD4+CD25+(low) T cells. The main method is to observe where the population naturally appears to ‘pinch in’ from the continuum of low CD25 staining cells. This is more easily seen if a greater number of events are shown on the histogram.
inhibit Th17 cell generation in vitro [9,32]. The Tim3–galectin-9 pathway also is implicated in rheumatoid arthritis [33]. We speculate that the development of MS may result from reduced functioning of the Tim3–galectin-9 pathway caused by a reduction in Tim3 expression. Although studies have reported an elevation of galectin-9 in cervical cancer patients [25], no studies to date have measured the serum level of galectin-9 in patients with MS. In the present study, the serum level of galectin-9 was elevated in the MS group versus the control group, which likely resulted from stimulation by increased IFN-c [34]. Elevated galectin-9 could in turn bind additional Tim3, which would induce the apoptosis of mature T cells, leading to reductions in Th1 and Th17 cells. We speculate that when Tim3 is low, galectin-9 may be up-regulated as the result of a feedback loop; this could improve the patient’s condition. Therefore, the therapeutic effect of galectin-9 should be studied further. The development of MS with immune dysfunction is considered primarily to be associated with Th1 cells and their reaction product, IFN-c. A more recent proposal suggests that Th17 cells play a considerable, even dominant, role in the development of MS [35]. Th17 cells are a Th-cell subset that produce IL-17 and are involved in the development and evolution of many inflammatory responses and autoimmune diseases. In mice, Th17 cells are responsible for the induction of EAE [36]. We observed elevated concentrations of IFN-c and IL-17 in the peripheral blood of MS patients compared with controls as measured by ELISA. Although elevated IFN-c and galectin-9 can down-regulate IL-17, the serum level of IL-17 remained high in MS patients. We speculate that increased IL-17 may be more closely related to the development of MS than previously suggested. In lupus nephritis, increased Th17 is accompanied by a decline in Treg cells [37]. In addition,
the development of rheumatoid arthritis can be caused by disturbances in the Th17/Treg balance [33,34]. Our results suggest that an increased secretion of IL-17 in MS is associated with the development and progression of the disease. Further study is required to determine whether Tim3 induces MS by affecting the function of Tregs, which consequently disturb the Th17/Treg balance.
Conflicts of Interest/Disclosures The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication.
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Please cite this article in press as: Feng X, Feng J. Clinical significance of Tim3-positive T cell subsets in patients with multiple sclerosis. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j.jocn.2016.07.007
X. Feng, J. Feng / Journal of Clinical Neuroscience xxx (2016) xxx–xxx [9] Seki M, Oomizu S, Sakata KM, et al. Galectin-9 suppresses the generation of Th17, promotes the induction of regulatory T cells, and regulates experimental autoimmune arthritis. Clin Immunol 2008;127:78–88. [10] Koguchi K, Anderson DE, Yang L, et al. Dysregulated T cell expression of Tim3 in multiple sclerosis. J Exp Med 2006;203:1413–8. [11] Matsumoto R, Matsumoto H, Seki M, et al. Human ecalectin, a variant of human galectin-9, is a novel eosinophil chemoattractant produced by T lymphocytes. J Biol Chem 1998;273:16976–84. [12] Wang F, He WT, Yuan J, et al. Activation of Tim-3-Galectin-9 pathway improves survival of fully allogeneic skin grafts. Transpl Immunol 2008;19:12–9. [13] Baecher-Allan C, Brown JA, Freeman GJ, et al. CD4+CD25high regulatory cells in human peripheral blood. J Immunol 2001;167:1245–53. [14] Shevach EM. Certified professionals: CD4(+)CD25(+) suppressor T cells. J Exp Med 2001;193:F41–6. [15] Valencia X, Yarboro C, Illei G, et al. Deficient CD4+CD25high T regulatory cell function in patients with active systemic lupus erythematosus. J Immunol 2007;178:2579–88. [16] Sugiyama H, Gyulai R, Toichi E, et al. Dysfunctional blood and target tissue CD4 +CD25high regulatory T cells in psoriasis: mechanism underlying unrestrained pathogenic effector T cell proliferation. J Immunol 2005;174:164–73. [17] Viglietta V, Baecher-Allan C, Weiner HL, et al. Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis. J Exp Med 2004;199:971–9. [18] Sanchez-Fueyo A, Tian J, Picarella D, et al. Tim-3 inhibits T helper type 1– mediated auto- and alloim-mune responses and promotes immunological tolerance. Nat Immunol Adv 2003. [19] Sabatos CA, Chakravarti S, Cha E, et al. Tim-3/Tim-3- ligand interaction regulates Th1 responses and induction of peripheral tolerance. Nat Immunol 2003;4:1102–10. [20] Baecher-Allan C, Viglietta V, Hafler DA. Inhibition of human CD4(+)CD25 (+high) regulatory T cell function. J Immunol 2002;169:6210–7. [21] Costantino Cristina Maria, Baecher-Allan Clare, David A. Hafler multiple sclerosis and regulatory T cells. J Clin Immunol 2008;28:697–706. [22] Wang F, Wan L, Zhang C, et al. Tim-3-galectin-9 pathway involves the suppression induced by CD4+CD25+ regulatory T cells. Immunobiology 2009;214:342–9. [23] Kuchroo Juhi, Hafler DavidA. Lack of TIM-3 immunoregulation in multiple sclerosis. J Immunol 2008;180:4409–14.
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[24] Zhu C, Anderson AC, Schubart A, et al. The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity. Nat Immunol 2005;6:1245–52. [25] Wada J, Kanwar YS. Identification and characterization of galectin-9, a novel bgalactoside-binding mammalian lectin. J Biol Chem 1997;272:6078–86. [26] Wada J, Ota K, Kumar A, et al. Developmental regulation, expression, and apoptotic potential of galectin-9, a b-galactoside binding lectin. J Clin Invest 1997;99:2452–61. [27] Liang MY, Lu YM, Zhang Y, et al. Serum galectin-9 in cervical cancer. Zhonghua Yi Xue Za Zhi 2008;88:2783–5. [28] Imaizumi Tadaatsu, Kumagai Mika, Sasaki Naoko, et al. Interferon-g stimulates the expression of galectin-9 in cultured human endothelial cells. J Leukoc Biol 2002;72:486–91. [29] Asakura Hirofumi, Kashio Yumiko, Nakamura Kazuhiro, et al. Selective eosinophil adhesion to fibroblast via IFN-g-induced galectin-9. J Immunol 2002;169:5912–8. [30] Lee J, Park EJ, Noh JW, et al. Underexpression of TIM-3 and blunted galectin-9induced apoptosis of CD4+ T cells in rheumatoid arthritis. Inflammation 2011. [31] Bianco GA, Toscano MA, Ilarregui JM, et al. Impact of proteinglycaninteractions in the regulation of autoimmunity and chronic inflammation. Autoimmun Rev 2006;5:349–56. [32] Nakae S, Iwakura Y, Suto H, et al. Phenotypic differences between Th1 and Th17 cells and negative regulation of Th1 cell differentiation by IL-17. J Leukoc Biol 2007;81:1258–68. [33] Lee J, Oh JM, Hwang JW, et al. Expression of human TIM-3 and its correlation with disease activity in rheumatoid arthritis. Scand J Rheumatol 2011. [34] Mengshol John A, Golden-Mason Lucy, Arikawa Tomohiro, et al. A crucial role for kupffer cell-derived galectin-9 in regulation of t cell immunity in hepatitis C infection. PLoS One 2010;5:e9504. [35] Tzartos JS et al. Interleukin-17 production in central nervous systeminfiltrating T cells and glial cells is associated with active disease in multiple sclerosis. Am J Pathol 2008;172:146–55. [36] Bernhard Hemmer, Thomas Korn. Th17 lymphocytes traffic to the central nervous system independently of a4 integrin expression during EAE. J Exp Med 2011;208:12. [37] Xing Q, Wang B, Su H, et al. Elevated Th17 cells are accompanied by FoxP3+ Treg cells decrease in patients with lupus nephritis. Rheumatol Int 2011.
Please cite this article in press as: Feng X, Feng J. Clinical significance of Tim3-positive T cell subsets in patients with multiple sclerosis. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j.jocn.2016.07.007
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Clinical Study
Clinical significance of Tim3-positive T cell subsets in patients with multiple sclerosis Xuemei Feng a, Juan Feng b,⇑ a b
Department of Nerve Function, Shengjing Hospital of China Medical University, Shenyang, China Department of Neurology, Shengjing Hospital of China Medical University, No. 36, Sanhao Street, Heping District, Shenyang 110004, China
a r t i c l e
i n f o
Article history: Received 14 September 2014 Accepted 3 July 2016 Available online xxxx Keywords: Tim3 Flow cytometry CD4+CD25(high) T cell Galectin-9
a b s t r a c t The present study evaluated associations between the percentages of T cell immunoglobulin and mucin domain 3 (Tim3)-positive T cells and related cytokines and multiple sclerosis (MS). We collected peripheral blood samples from 30 MS patients and 30 healthy controls. Flow cytometry was used to determine the proportions of CD3+Tim3+, CD4+Tim3+, and CD4+CD25+Tim3+ in peripheral blood mononuclear cells (PBMCs) and related cell subsets. The serum concentrations of galectin-9, IL-17, and IFN-c also were determined using enzyme-linked immunosorbent assays (ELISA). The percentages of Tim3-positive T cells in CD4+ and CD4+CD25+ T cell subsets were significantly lower among MS patients than among controls. This difference was particularly evident in the CD4+CD25(high) T cell subset. The proportions of CD4+Tim3+ and CD4+CD25+Tim3+ cells in PBMCs were significantly lower in the MS group than in the control group, whereas no significant differences were detected regarding the percentages of CD3+Tim3+ in PBMCs and T cell subsets. The serum concentrations of galectin-9, IL-17, and IFN-c all were increased in MS patients compared with healthy controls. Our results support that Tim3 and related cytokines may be involved in the onset of MS. Ó 2016 Published by Elsevier Ltd.
1. Introduction Multiple sclerosis (MS) is an autoimmune disease characterized by demyelination within the white matter of the central nervous system. Although the specific pathogenesis of MS is not fully understood, it is widely accepted that a T-cell-mediated immune response is involved [1]. The identification of T cell immunoglobulin and mucin domain 3 (Tim3) served as a breakthrough in the understanding and treatment of autoimmune diseases [2]. Tim3 is preferentially expressed on the surfaces of Th17 and Th1 cells. Tim3 contributes to an important immunoregulatory pathway involving CD4+ T cells and can down-regulate Th1 responses [2–5]. The function of Tim3 as a negative regulator of Th1 cellmediated immune responses has been explained by Monney et al. (2002) [5] using an experimental autoimmune encephalomyelitis (EAE) mouse model with an induced Th1-dominant response and by Huey-Kang Sytwu 2009 in a study evaluating the Th1-dominant response in an autoimmune non-obese diabetic (NOD) mouse model [6]. Several reports have
⇑ Corresponding author. Tel.: +86 024 96615 28815, +86 18940251959. E-mail addresses: [email protected], [email protected] (J. Feng).
described the up-regulation of Tim3 in patients with rheumatoid arthritis or nephrotoxic nephritis, yet it remains unclear in MS [7–9]. Therefore, the specific mechanism of action of Tim3 in these diseases requires further investigation. Few studies have evaluated the involvement of Tim3 in MS, although one report suggests that Tim3 mRNA expression is decreased in peripheral blood CD4+ cells of MS patients [10]. Tim3 functions by binding to galectin-9, its natural ligand [11,12]. Galectin-9 may improve rheumatoid arthritis [7] by indirectly increasing the production of CD4+CD25+ regulatory T cells (Tregs); however, its role in MS is unknown. Tregs impose negative regulatory effects on the proliferation of effector T cells, which exert immunoregulatory effects in MS and other autoimmune diseases by down-regulating the interferon (IFN)-c pathway. Previous studies suggest that CD4+CD25(high) T cells suppress T cell function and protect against autoimmune disease in healthy individuals [13–16]. The pathogenesis of MS may be related to functional deficiencies among CD4+CD25(high) cells [17]. The regulatory functions of this cell population may correlate with changes in the expression or function of Tim3 [18]. To date, potential associations between the functionality or number of CD4+CD25(high) cells and various autoimmune diseases have not been established.
http://dx.doi.org/10.1016/j.jocn.2016.07.007 0967-5868/Ó 2016 Published by Elsevier Ltd.
Please cite this article in press as: Feng X, Feng J. Clinical significance of Tim3-positive T cell subsets in patients with multiple sclerosis. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j.jocn.2016.07.007
2
X. Feng, J. Feng / Journal of Clinical Neuroscience xxx (2016) xxx–xxx
2. Materials and methods
3. Results
2.1. Patient and control groups
3.1. Flow cytometry
The patient group consisted of 30 patients (13 men, 17 women; mean age 37 ± 2.92 years) who had been admitted to ShengJing Hospital of China Medical University between January 2008 and October 2013. All 30 patients were in acute relapse phase, excluding three patients who already had secondary progressive multiple sclerosis (SPMS). The mean disease duration is 7.17 ± 4.9 years and the mean Expanded Disability Status Scale (EDSS) is 4.3 ± 2.0. The control group included 30 sex-matched healthy individuals (mean age 35 ± 1.08 years) with no autoimmune system diseases who came to the hospital for physical examinations. The 30 patients all had been clinically diagnosed with MS with relapse and all met the McDonald diagnostic criteria.
The percentage of CD3+Tim3+ cells in the MS patient group did not differ significantly from the control group (Table 1, Fig. 1). The percentage of CD4+Tim3+ cells in the MS group was significantly lower than the percentage of these cells in the control group (P < 0.05) (Table 2, Fig. 2). The percentage of Tim3+ cells among the regulatory T cells in the MS group was significantly lower than the percentage of Tim3+ cells in the control group (P < 0.05) (Table 3, Fig. 3).
2.2. Cells and sample preparation Peripheral venous blood samples were obtained from MS patients at 06:00 on the second day of admission. All patients had avoided immunosuppressants and corticosteroids for at least 1 month at the time of blood sample collection. Peripheral blood samples also were obtained from the control group. Blood samples were collected into anticoagulant tubes containing 0.2 ml heparin and inverted gently. Serum then was obtained by removing supernatants after Ficoll-Hypaque density gradient centrifugation at 4°C and was stored at 70°C. Peripheral blood lymphocytes were isolated by density gradient centrifugation and were diluted with phosphate-buffered saline solution to a cell suspension of 2 106 cells/ml. 2.3. Cell-surface staining and flow cytometry The following antibodies were used according to manufacturer’s Instructions:phycoerythrin conjugated anti-Tim3 monoclonal antibody (Tim3-PE), peridinin chlorophyll protein conjugated anti-CD3 monoclonal antibody (CD3-PerCP), peridinin chlorophyll protein conjugated anti-CD4 monoclonal antibody (CD4-PerCP), fluorescein isothiocyanate-conjugated anti-CD25 monoclonal antibody (CD25-FITC). All antibodies were purchased from BD (San Diego, CA, USA), except for the Tim3-PE (eBioscience, San Diego, CA, USA). Cell suspensions derived from patient and control blood samples were stained with the following antibody combinations: (1) CD4-PerCP, CD25-FITC, Tim3-PE; (2) CD3-PERCP, Tim3-PE. Mixtures were incubated at 4°C for 25 min in the dark. After incubation, excess antibodies were washed off, and flow cytometric analysis was performed on a BD FACSCalibur flow cytometer using FCS ExpressV3 software. 2.4. Enzyme-linked immunosorbent assay The plasma concentrations of galectin-9, IL-17, and IFN-c were determined using enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN, USA). 2.5. Statistics The percentages of cell subsets identified by flow cytometry were analyzed using the Mann–Whitney U-test. All data were represented as means ± standard deviations. P < 0.05 was considered statistically significant.
3.2. Enzyme-linked immunosorbent assay The plasma concentrations of galectin-9 and IFN-c in the MS group was significantly higher than the control group (P < 0.05), and the concentrations of IL-17 in the MS group was lower than the control group (P < 0.05) (Table 4). 4. Discussion Tim3 is a recently identified molecule that is preferentially expressed on the surfaces of Th17 and Th1 cells. Administration of anti-Tim3 monoclonal antibodies accelerates the onset and severity of autoimmune cerebrospinal meningitis in mice [5]. When the interaction between Tim3 and its ligand, galectin-9, is blocked, the result is a predominant Th1-type response with induction of T cell proliferation and secretion of Th1-type cytokines [18,19]. These findings suggest that Tim3 plays a negative regulatory role in the Th1 cell-mediated immune response. In this study, we demonstrated that MS patients do not significantly differ from healthy controls regarding the percentages of Tim3+ T cells in peripheral blood or among all T cells. However, among CD4+ cells, the percentage of Tim3+ T cells was decreased in MS patients. We suggest that the decreased expression of Tim3 in CD4+ cells of MS patients could reduce the inhibitory effects of Tim3 on Th1 and Th17 cells. This reduced negative regulatory effect could contribute to the pathogenesis of MS. Several studies have reported that Tim3 can also induce apoptosis of Th1 cells [18,19]. We speculate that the onset of MS also may be facilitated by a reduction in targeted Th1 cell apoptosis and Th17 cell inhibition caused by the decreased expression of Tim3 in CD4+ cells. Tregs can be summarized as a group of CD4+ T cells that exert potential inhibitory activities against harmful reactions. The immunoregulatory activities of CD4+CD25+ T cells of natural phenotype depend on their surface markers (e.g., CTLA-4, FOXP3, CD127, CD25[high]). Previous studies have indicated that in CD4+CD25+ T cells of natural phenotype, the subset of CD4+CD25(low) cells do not exhibit immunoregulatory functions, whereas the CD4+CD25(high) cells are the true regulators [20]. The results of our study indicate that the percentage of Tim3-positive CD4+CD25+cells among all CD4+CD25+cells is significantly reduced in MS patients compared with controls. Moreover, the percentage of Tim3-positive CD4+CD25(high) cells among all CD4+CD25(high) cells is significantly reduced in MS patients, with
Table 1 Percentages of CD3+Tim3+ T cell subsets in peripheral blood and T cells in the multiple sclerosis (MS) group and the control group. Groups
n
CD3+Tim3+
CD3+Tim3+/CD3
MS Group Control Group
30 30
3.79 ± 0.81 3.70 ± 0.72
6.03 ± 0.91 6.62 ± 1.27
p > 0.05, compare to control: Mann–Whitney U test.
Please cite this article in press as: Feng X, Feng J. Clinical significance of Tim3-positive T cell subsets in patients with multiple sclerosis. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j.jocn.2016.07.007
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X. Feng, J. Feng / Journal of Clinical Neuroscience xxx (2016) xxx–xxx
Fig. 1. Percentages of CD3+Tim3+ cells in the peripheral blood of multiple sclerosis (MS) patients and healthy controls. (a) Among MS patients, CD3+Tim3+ T cells accounted for 3.89% of peripheral blood T cells. (b) Among controls, CD3+Tim3+ cells accounted for 3.77% of peripheral blood T cells. No significant difference was detected between the two groups.
Table 2 Percentages of CD4+Tim3+ T cell subsets in peripheral blood and T helper cells in multiple sclerosis (MS) patients and controls. Groups
n
CD4+Tim3+
CD4+Tim3+/CD4
MS Group Control Group
30 30
1.35 ± 0.35 1.64 ± 0.36*
3.30 ± 0.62 4.20 ± 0.63*
Compare to control: Mann–Whitney U test. * p < 0.05.
the reduction in CD4+CD25(high) cells being more pronounced. Hafler et al. reported that purified CD4+CD25(high) cells from MS patients do not inhibit the proliferation of effector T cells in vitro, suggesting that the normal regulatory functions of CD4+CD25 (high) cells are absent in MS. The development of MS is related not only to the functional deficiency of CD4+CD25(high) cells via unknown mechanisms [21], but also to changes in the expression or function of Tim3 [22,23]. We speculate that the reduced expression of Tim3 in CD4+CD25 (high) cells is involved in the development of MS. The reduced expression or the altered function of CD4+CD25+Tim3(high) cells affects the regulation of CD4+CD25(high) cells, leading to the development of MS. Further investigations should evaluate the level of CD4+CD25+Tim3(high) cell functioning in patients with MS, the potential of these cells to exert immunoregulatory activity, and whether Tim3 can serve as a novel marker of regulatory T cells. Galectin-9 is a natural ligand of Tim3 [24,25] that has been detected on the surfaces of Treg cells and in serum [26,27]. The
Table 3 Percentages of CD4+CD25+Tim3+ T cell subsets in peripheral blood and regulatory T cells in multiple sclerosis (MS) patients and healthy controls.
CD4+CD25+Tim3+ CD4+CD25(high)Tim3+ CD4+CD25+(low)Tim3+ CD4+CD25+Tim3+/CD4+CD25+ CD4+CD25(high)Tim3+/CD4+CD25(high) CD4+CD25+(low)Tim3+/CD4+CD25+(low)
MS
Controls
0.76 ± 0.21 0.20 ± 0.07 0.55 ± 0.16 11.84 ± 2.45 30.68 ± 6.71 13.12 ± 2.74
1.03 ± 0.14* 0.35 ± 0.06* 0.67 ± 0.16* 13.87 ± 1.93* 43.39 ± 6.15* 13.28 ± 2.83
Compare to control: Mann–Whitney U test * p < 0.05.
Table 4 ELISA tests to compare the plasma concentration of Galectin-9, Interleukin-17 (IL-17) and Interferon-c (IFN-c) between multiple sclerosis (MS) patients and healthy controls.
MS CONTROL
Galectin-9 (ng/L)
IL-17 (ng/L)
IFN-c (ng/L)
37.11 ± 6.71 26.56 ± 3.86*
14.1 ± 3.34 8.49 ± 1.50*
339.00 ± 89.45 196.97 ± 53.44*
Compare to control: Mann–Whitney U test. * P < 0.05.
secretion of galectin-9 can be stimulated by IFN-c [28,29], and galectin-9 functions as an anti-inflammatory molecule in autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, and diabetes [30,31]. The binding of galectin-9 to Tim3 can induce T cell apoptosis, down-regulate the Th1 response, and
Fig. 2. Percentages of CD4+Tim3+ cells in the peripheral blood of multiple sclerosis (MS) patients and controls. (a) In the MS patient group, CD4+Tim3+ T cells comprised 1.52% of the peripheral blood T cells. (b) In the control group, CD4+Tim3+ cells accounted for 2.08% of peripheral blood T cells, a significantly higher percentage than that of the MS group (p < 0.05).
Please cite this article in press as: Feng X, Feng J. Clinical significance of Tim3-positive T cell subsets in patients with multiple sclerosis. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j.jocn.2016.07.007
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X. Feng, J. Feng / Journal of Clinical Neuroscience xxx (2016) xxx–xxx
Fig. 3. Percentages of CD4+CD25+Tim3+ T cell subsets in peripheral blood and regulatory T cells. (a) We subdivided the CD4+CD25+ T cell population into CD4+CD25(high) and CD4+CD25(low) cells according to the CD25 expression level on CD4+ T cells. (b) The percentage of Tim3+ cells among CD4+CD25+(high) T cells. (c) The percentage of Tim3+ cells among CD4+CD25+(low) T cells. The main method is to observe where the population naturally appears to ‘pinch in’ from the continuum of low CD25 staining cells. This is more easily seen if a greater number of events are shown on the histogram.
inhibit Th17 cell generation in vitro [9,32]. The Tim3–galectin-9 pathway also is implicated in rheumatoid arthritis [33]. We speculate that the development of MS may result from reduced functioning of the Tim3–galectin-9 pathway caused by a reduction in Tim3 expression. Although studies have reported an elevation of galectin-9 in cervical cancer patients [25], no studies to date have measured the serum level of galectin-9 in patients with MS. In the present study, the serum level of galectin-9 was elevated in the MS group versus the control group, which likely resulted from stimulation by increased IFN-c [34]. Elevated galectin-9 could in turn bind additional Tim3, which would induce the apoptosis of mature T cells, leading to reductions in Th1 and Th17 cells. We speculate that when Tim3 is low, galectin-9 may be up-regulated as the result of a feedback loop; this could improve the patient’s condition. Therefore, the therapeutic effect of galectin-9 should be studied further. The development of MS with immune dysfunction is considered primarily to be associated with Th1 cells and their reaction product, IFN-c. A more recent proposal suggests that Th17 cells play a considerable, even dominant, role in the development of MS [35]. Th17 cells are a Th-cell subset that produce IL-17 and are involved in the development and evolution of many inflammatory responses and autoimmune diseases. In mice, Th17 cells are responsible for the induction of EAE [36]. We observed elevated concentrations of IFN-c and IL-17 in the peripheral blood of MS patients compared with controls as measured by ELISA. Although elevated IFN-c and galectin-9 can down-regulate IL-17, the serum level of IL-17 remained high in MS patients. We speculate that increased IL-17 may be more closely related to the development of MS than previously suggested. In lupus nephritis, increased Th17 is accompanied by a decline in Treg cells [37]. In addition,
the development of rheumatoid arthritis can be caused by disturbances in the Th17/Treg balance [33,34]. Our results suggest that an increased secretion of IL-17 in MS is associated with the development and progression of the disease. Further study is required to determine whether Tim3 induces MS by affecting the function of Tregs, which consequently disturb the Th17/Treg balance.
Conflicts of Interest/Disclosures The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication.
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Please cite this article in press as: Feng X, Feng J. Clinical significance of Tim3-positive T cell subsets in patients with multiple sclerosis. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j.jocn.2016.07.007