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Correlation of specialized CD16+ γδ T cells with disease course and severity in multiple sclerosis
Journal of Neuroimmunology 194 (2008) 147 – 152 www.elsevier.com/locate/jneuroim
Correlation of specialized CD16 + γδ T cells with disease course and severity in multiple sclerosis Zhihong Chen a,b , Mark S. Freedman a,b,c,⁎ a
Department of Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada b Ottawa Health Research Institute, Ottawa, ON, Canada c The Ottawa Hospital, General Campus, Ottawa, ON, Canada
Received 5 July 2007; received in revised form 6 November 2007; accepted 12 November 2007
Abstract γδ T cells may be important innate immune system contributors to the immunopathogenesis of multiple sclerosis (MS), though the mechanisms are not yet fully understood. CD16 is a low affinity Fcγ receptor, an activation receptor for γδ T cells, and a mediator of cytotoxicity. In this study, we found that the percentage of CD16+ γδ T cells is elevated in MS patients compared with healthy controls. The increase is especially pronounced in patients with a progressive course of the disease, and the extent of this elevation shows a positive correlation with the time of disease progression and severity. In vitro cultured γδ T cells can be shown to upregulate the expression of CD16 in response to inflammatory cytokines such as IL-2 and -15, that have been shown to be elevated in progressive disease. These results suggest that CD16 expressing γδ T cells are somehow involved in the process of disease progression. Understanding more about these cells and their particular function in progressive vs. non-progressive disease could provide important clues to the mechanism of immune-mediated MS disease progression. Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved. Keywords: Multiple sclerosis; γδ T cells; Fcγ receptor III (CD16); Progression; Proinflammatory cytokine; IL-15
1. Introduction Multiple sclerosis (MS) is an autoimmune central nervous system (CNS) disease marked by the immune-mediated destruction of myelin, the ensheathed axons, and the neurons (Pouly and Antel, 1999; Wegner et al., 2006). Heterogeneity, both patho-immunologically and clinically, is a hallmark of the disease (Lucchinetti et al., 2000). Clinically most patients start out with a relapsing–remitting (RR) disease process, but virtually all eventually enter a more progressive phase (secondary progressive, SP) that tends to be chronic and usually unrelenting; a smaller subset starts out and continues with a primarily progressive course in the absence of relapses (primary progressive, PP) (Lublin and Reingold, 1996). Though the exact ⁎ Corresponding author. Division of Neurology, The Ottawa Hospital-General Campus, 501 Smyth Road, Ottawa, Canada K1H 8L6. Tel.: +1 613 737 8917; fax: +1 613 737 8857. E-mail address: [email protected] (M.S. Freedman).
immunopathogenesis of MS remains to be fully elucidated, both innate and adaptive immune responses have been implicated (Raine, 1994). γδ T cells are an important component of the innate immune response. Though they are one of the two lineages of T lymphocytes, γδ T cells are quite different from the conventional αβ T cells in that they are able to recognize unprocessed, nonMHC restricted antigens and that their antigen specificities are not limited only to peptides of certain length (Fisch et al., 1990; Tanaka et al., 1995). γδ T cells are also shown to express various natural killer cell (NK) receptors associated with cytotoxicity, cytokine release, and trans-endothelial migration (Halary et al., 1997; Poggi et al., 2002). Due to the lack of variability in their T cell receptor (TCR) and being close resemblance to NK cells in terms of phenotype, one may conclude that γδ T cells are a more homogeneous population. However, recent studies have demonstrated that γδ T cells could be further subdivided into highly differentiated subsets of central memory, effector memory, and terminal effector cells
0165-5728/$ - see front matter. Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2007.11.010
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based on their physiobiological functions, which coincide with the expressions of several surface molecules (Dieli et al., 2003; Shen et al., 2002). CD16, in combination with CD45RA and CD27, identify a highly cytotoxic subset of γδ T cells lethal to target cells (Angelini et al., 2004). It has also been demonstrated that CD16 expressing cells are required in inducing Experimental Autoimmune Encephalomyelitis (EAE) in animal models (Abdul-Majid et al., 2002; Szalai et al., 2005). CD16 polymorphisms may also be correlated with MS disease course (Myhr et al., 1999). CD16 (FcγRIII) is the low affinity Type III receptor that specifically binds the Fc portion of immunoglobulin G (IgG). It is expressed to varying degrees by several types of leukocytes (macrophages, NK cells, granulocytes, and γδ T cells). Engagement of CD16 on these cells by immune complexes induces one or more biological functions including phagocytosis, antibody-dependent cell cytotoxicity (ADCC), and release of cytokines (Jefferis and Lund, 2002). CD16 is upregulated on cultured resting γδ T cells upon activation through their TCR– CD3 complex (Lafont et al., 2001). These CD16+ γδ T cells are shown to be highly cytotoxic (Angelini et al., 2004), and can release cytokines (e.g. TNF-α) upon stimulation through their CD16 molecules (Lafont et al., 2001). The exact way in which γδ T cells are involved in disease has not yet been discerned. In EAE they have been shown to both contribute and protect from disease (Kobayashi et al., 1997; Ponomarev et al., 2004; Rajan et al., 1996). However, in human studies their role seems more consistent with contributing to disease: they are more numerous in the cerebrospinal fluid (CSF) (Stinissen et al., 1995), concentrated around active demyelinating lesions (Wucherpfennig et al., 1992), and are potent producers of inflammatory cytokines (Hintzen and Polman, 1997). We have demonstrated that γδ T cells cytolyse freshly isolated human oligodendrocytes (ODCs) (Freedman et al., 1991), possibly through the recognition of ODC expression of heat shock proteins (Freedman et al., 1997), death (Fas) receptors (Zeine et al., 1998), or MHC-I related stress molecules (Saikali et al., 2007) by corresponding ligands on the surface of γδ T cells. These studies all suggested that subsets of γδ T cells may be particularly involved in mediating deleterious effects.
Given the particular functions attributed to CD16 expressing γδ T cells and their implication in other autoimmune diseases (Bodman-Smith et al., 2000; Lamour et al., 1995), we investigated this subset in MS. 2. Materials and methods 2.1. Patients and healthy controls Blood and cells were obtained from 64 patients undergoing diagnostic assessment and clinical follow-up at the MS Centre of the Ottawa Hospital — General Campus (their demographic profile shown in Table 1). Among them, 49 met the diagnosis of MS according to the new criteria (Polman et al., 2005); the other 15 patients, suffering initial neurological attacks, were diagnosed with clinical isolated syndromes (CIS) (O'Riordan et al., 1998). None of the patients had experienced an exacerbation or received any disease modifying drugs (INF-β or Glatiramer Acetate) or steroids at least three months before the time of blood sampling. Blood and cells were also obtained from healthy volunteers who were recruited from laboratory staff or hospital personnel. This study was approved by Research Ethics Board of the Ottawa Health Research Institute and all samples were obtained with written informed consent. 2.2. Ex vivo flow cytometric analysis Peripheral blood mononuclear cells (PBMC) were isolated from each subject by Ficoll-Hypaque (Pharmacia Biotech AB, Uppsala, Sweden) density gradient centrifugation following standard procedure. 1 × 106 cells were set aside for ex vivo staining and the rest were used for expansion and purification of γδ T cells as described below in Section 2.3. Cells were stained with a cocktail of FITC-γδ TCR (TRCγδ1, BD Biosciences, Mississauga, ON), PE-CD16 (3G8, Beckman Coulter, Mississauga, ON), and PC5-CD3 (UCHT-1, Beckman Coulter) monoclonal antibodies (mAb) according to standard procedures and analyzed by flow cytometry on an FC500 flow cytometer (Beckman Coulter). Typically 2 × 105 events were acquired for analysis using the supplied Coulter CXP software.
Table 1 Patient demographics
Number Age (years) b Sex (male/female) e Disease duration (years) b EDSS score b, g
Healthy controls a
CIS
MS patients RR
SP
PP
Younger
Older
15 41.4 ± 14.3 3/12 3.33 ± 3.53 1.0 ± 0.9
13 46.9 ± 9.4 6/7 8.5 ± 3.6 2.2 ± 1.0
19 53.3 ± 10.7 7/12 19.4 ± 7.1 6.2 ± 1.0
17 55.7 ± 8.1 8/9 13.6 ± 9.5 5.7 ± 1.7
15 39.2 ± 9.1 c 4/11 N/A f N/A
18 47.3 ± 9.8 d 5/13 N/A N/A
CIS: clinical isolated syndromes; RR: relapsing–remitting MS; SP: secondary progressive MS; PP: primary progressive MS. a Healthy controls were divided into two groups according to their age. b Data were shown as mean ± S.D. c Not statistically different from CIS. d Not statistically different from MS subgroups. e Not statistically different among all the groups. f N/A: non applicable. g EDSS: Expanded Disability Status Scale.
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2.3. γδ T cells expansion and isolation Peripheral blood γδ T cell lines from both patients and healthy controls were expanded and purified as previously described (Zeine et al., 1998). Briefly, 24-well culture plates (Fischer Scientific, Ottawa, Ontario) were sequentially treated with 350 μl (5 μg/ml) sheep anti-mouse IgG1 antibody, 350 μl cRPMI (RPMI-1640 supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin G and 100 μg/ml streptomycin, Life Technologies, Burlington, ON), and monoclonal anti-TCR γδ antibody (Courtesy of Dr. M. Brenner, Harvard University) in cRPMI medium, which had been titrated to yield maximal stimulation. PBMCs generated from step Section 2.2 were seeded in the plate at density of 3–3.5 × 106 cells per well in 3 ml of cRPMI. Cells were kept at 37 °C with 5% CO2 for culturing. 50 U/ml IL-2 was added the next day. On day 5, cRPMI was replaced with serum free AIM-V media and cultured for another 4 or 5 days with fresh IL-2. The αβ T cells were then eliminated by complement lysis using anti-CD4 and anti-CD8 monoclonal antibodies. T cell preparations consisting of N90% γδ T cells were used in all experiments.
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flow cytometry. γδ T cells were identified and gated by double positive staining of CD3 and γδ TCR. The gated cells were further analyzed for surface expression of CD16 and the data were expressed as percentage of CD16 positive γδ T cells over total γδ T cells (%CD16+) (Supplementary figure). CIS tends to present in younger patients than those already with disease (by ANOVA test). In order to insure that the percent of CD16+ cells is not just an age-related phenomenon, we divided the healthy controls into two age groups with the younger groups (HC/Y) matching the age of CIS and the older (HC/O) matching the rest of MS groups. When compared with the healthy controls (HC/O: 24.3 ± 14.2% and HC/Y: 21.0 ± 10.2%), the percentages of CD16+ γδ T cells were significantly elevated in patients diagnosed with MS (35.6 ± 24.0%, p = 0.0429) or CIS (30.1 ± 12.6%, p = 0.0387) (Fig. 1A). Looking particularly within MS subgroups (PP-, RR-, and SP-MS) the variations of the means of %CD16+ were compared across each subgroup as well as the HC/O using one-way ANOVA test. A significant difference (p = 0.0132) was noticed among the groups (Fig. 1B). Tukey's post-hoc inter-group comparison identified that it was the SPMS group that had significantly higher %CD16+ than the healthy controls, while the differences between other patient
2.4. Cytokine responsive assay Purified γδ T cells were harvested, washed, and re-plated for another 24 h without IL-2 in culture media cRPMI. Cells were then seeded in U-bottomed 96-well Petri-dish at 2 × 105 cells per well. IL-2 (a gift from Hoffmann-La Roche, Nutley, NJ), IL-12, and IL-15 (both from Serotec, Oxford, UK) were then added to the wells at the optimized concentrations, topping up each well to 200 μl with culture media. Dishes were cultured for 72 h, 37 °C/5% CO2 and the surface expression of CD16 was measured with flow cytometry. 2.5. Statistics Differences between two groups were compared with paired or non-paired two-tailed Student's t test, where appropriate. Differences between three or more groups were evaluated with one way ANOVA. If statistical significance was noted, Tukey's post-hoc test was applied for inter-group comparisons. Normal distribution of the variables was assessed with the D'Agostino and Pearson test. Disease duration and EDSS scores were not following a normal distribution, therefore non-parametric Spearman's rank correlation was used for the correlation analyses. In all assays, p b 0.05 was considered statistically significant. Statistical analyses were performed with Prism (GraphPad Inc., San Diego, CA) or Kyplot (KyensLab Inc. Japan) software. 3. Results 3.1. CD16+ γδ T cells in patients and healthy controls The percentage of CD16 expressing γδ T cells derived from peripheral blood of MS patients or healthy controls was assessed on immediately ex vivo cells using multiple parameter
Fig. 1. CD16 expressing γδ T cells are elevated in patients compared with healthy controls. (A) Healthy controls were divided into two age groups (HC/O and HC/Y). The mean %CD16+ was significantly increased in MS patients when compared with HC/O and in CIS compared with HC/Y. Box and whiskers indicated the median, 25th percentile rage, and highest/lowest values. The means were shown with a crosses. (B) Means of %CD16+ of the subgroups of MS were significantly different from that of the healthy controls (p = 0.0132). SPMS patients had significantly higher %CD16+ than the healthy controls. A cut-off value (dotted line) indicating the mean of the HC/O + 2SD was shown, above which %CD16+ was considered abnormally high. The SPMS group also had the highest percentage (36.8%) of abnormally high CD16+ γδ T cells. The percentages of patients with abnormally high values in RRMS and PPMS were 7.7% and 17.6% respectively. Closed dots represented %CD16+ value of each individual and the means and SD were shown with bars. ⁎ Denotes statistical significance.
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Fig. 4. Increase in %CD16+ in response to in vitro cytokine stimulation. Proinflammatory cytokines IL-2, -12 and -15 significantly elevated the %CD16+ γδ T cells derived from MS patients (n = 10), the most robust stimulation seen with IL-15. IL-2 and IL-15 also increased %CD16+ γδ T cells in healthy controls (n = 6). For each of the cytokines, there was no difference noticed in their ability to increase the %CD16+ γδ T cells between healthy controls and MS patients. ⁎ p b 0.05. Fig. 2. Relationship of %CD16+ to patient clinical characteristics. %CD16+ correlated significantly with both disease duration (years since onset of MS, in A) and severity (expressed by EDSS, in B). Total n = 49, p b 0.05, Spearman correlation test.
groups and the healthy controls did not reach statistical significance. We defined a cut-off value of 52.8% (mean %CD16+ + 2SD) observed in the HC/O; values higher than this were considered to be elevated. We found that more SPMS patients had elevated %CD16+, with 7 out of the 19 (36.8%) exceeding the upper limit. About 17.6% of PPMS patients were found with elevated percentage of CD16+ γδ T cells; while only 1 of 13 patients with RRMS (7.7%) had high %CD16+ (Fig. 1B). 3.2. CD16+ percentage correlates with disease progression and EDSS
There was no correlation found with either age (p = 0.055 by Pearson test) or sex (p = 0.56 by t-test). However, a significant correlation was noted between %CD16+ and either disease duration (r = 0.40, p = 0.0042) or disability (EDSS) (r = 0.31, p = 0.031) (Fig. 2). These correlations remained true when patients were subdivided into four groups (four quadrants in Fig. 3) according to their disease duration and EDSS scores: patients with short disease duration (b = 10 years) and low EDSS (b = 3.5) had the lowest average %CD16+ (25.5 ± 15.5%); patients with both long duration (N10 years) and high EDSS (N 3.5) showed the highest percentage of CD16+ γδ T cells (45.6 ± 26.4%); while the other two groups with different combinations (i.e. short disease duration/high EDSS or long disease duration/low EDSS) demonstrated %CD16+ in between (26.7 ± 18.8% and 27.9 ± 21.6% respectively) (Fig. 3). 3.3. CD16 is upregulated by IL-15 in vitro
We next examined the relationships between the %CD16+ and the clinical characteristics of the patients (see Table 1).
Fig. 3. %CD16+ corresponds to disease duration and EDSS. MS patients were subdivided into 4 quadrants according to their disease duration (separated by 10 years) and EDSS scores (separated by 3.5 points), indicated by the dotted lines. Each rectangle represented one patient and was colorized based on their %CD16+ value converted against the color map on the right. The mean %CD16+ of each quadrant was shown at the corners in a bigger closed rectangle.
In an effort to explain the elevated %CD16+ in SPMS, we postulated that chronic inflammation may be the driving force. In particular, cytokines such as IL-2, IL-12, IL-15, and IFN-γ, have been found to be elevated in the blood of SPMS patients (Killestein et al., 2001; Rentzos et al., 2006; van Boxel-Dezaire et al., 2001). To address this possibility, we cultured the γδ T cells in the presence of cytokines IL-2, -12, and -15 to confirm if %CD16+ could be increased. Purified γδ T cells were cultured with or without IL-2, -12, and -15 at previously optimized concentrations of 100 U/ml, 10 ng/ml, and 10 ng/ml respectively for 72 h. %CD16+ was measured with flow cytometry as described above. We observed that culturing in the presence of all 3 cytokines significantly elevated %CD16+ on γδ T cells derived from MS patients, the most robust stimulation seen with IL-15. However, for each of the cytokines, there was no difference noticed of their ability to increase %CD16+ between healthy controls and MS patients (Fig. 4).
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4. Discussion The exact contribution of γδ T cells to the pathogenesis of MS remains to be elucidated. In this study we report that MS patients have a significant increase in a particular subset of γδ T cells, those bearing the NK-type marker CD16. In addition, the percentage of CD16 expressing γδ T cells (%CD16+) appears to correlate with disease duration and severity suggesting they may be an important component of the immune system damage that contributes to disease progression. This finding of elevated %CD16 + is in parallel with observations in other autoimmune diseases such as rheumatoid arthritis (RA) or Sjogren's syndromes, albeit the extent of this elevation in RA is relatively moderate (Bodman-Smith et al., 2000; Lamour et al., 1995). Increased %CD16+ may reflect their activation status in these diseases as it has been shown that CD16+ γδ T cells represent terminally differentiated memory cells (Angelini et al., 2004). Previous studies had implied that there might be an age related expression of CD16 molecule on NK cells or neutrophils (CD16 expression on neutrophils decreases with aging) (Butcher et al., 2001; Solana and Mariani, 2000). We did not observe any correlation of age and CD16 positivity on γδ T cells from our studies (p = 0.055 for all MS patients and p = 0.196 for healthy controls). We did notice a tendency that the older controls may have higher %CD16 compared with younger controls on a population level, but this could be caused by a single outlier in our HC/O subset. Increased levels of various proinflammatory cytokines were observed in the systemic circulation at different stages of MS (Merrill and Benveniste, 1996); in particular, at the SPMS stage heightened levels of IL-2, -12 and -15 were reported (Killestein et al., 2001; Rentzos et al., 2006; van Boxel-Dezaire et al., 2001). We observed that these same cytokines significantly increased %CD16+ suggesting that chronic dysregulation and an inflammatory milieu might be the cause for the observed increased %CD16+ in our SPMS patients. Although IL-2 and IL-15 are structurally and functionally related cytokines and their receptors share two of the three heterotrimer subunits, IL-2 is produced by activated CD4+T cells and functions on αβ T lymphocytes; while IL-15 is generated by a large variety of cell types, but preferentially works on γδ T cells (Leclercq et al., 1996). We found the most robust increase in %CD16+ in response to IL-15. The ability of IL-15 to increase CD16 expressing γδ T cells was also noted in other studies (Eberl et al., 2002). Increased % CD16+ may not solely be due to the presence of IL-15 selectively expanding the CD16+ population, since NK cells, both CD16− and CD16+, proliferate equally in response to IL15 (Dunne et al., 2001). CD16+ cells might also come from the CD16− pool in response to antigen-stimulation, such that the increase in CD16+ γδ T cells that we observed could actually derive from CD16− cells in response to unknown antigen stimulation together with the proinflammatory cytokine milieu seen in progressive MS (Caccamo et al., 2005). Studies have shown that immunomodulatory therapies have little effect once patients enter a progressive phase of their
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illness, reflecting probably more the neurodegenerative disease component (Bjartmar et al., 2003). However, evidence has also shown that the immune system itself is much more dysregulated in the same progressive stage of MS and that it carries a different, diffusive form of inflammation in the brain (VakninDembinsky and Weiner, 2007). This prompts Vaknin-Dembinsky and Weiner to propose that while abnormalities in adaptive immunity play an important role in RRMS, innate immunity may be a more important contributor to the disease process in SPMS (Vaknin-Dembinsky and Weiner, 2007). γδ T cells are a very important and prominent T cell subset involved in innate immune responses and could be postulated to contribute to disease progression by releasing proinflammatory cytokines, or via cytotoxic mechanisms. CD16 expressing γδ T cells may play a very particular role via ADCC in conjunction possibly with antibodies to myelin proteins that are seen in MS patients (Ziemssen and Ziemssen, 2005), and this is an area that we are currently investigating (manuscript under review). Our results showing elevated percentages of CD16 expressing γδ T cells that correlate with disease progression is a further demonstration that innate immune processes may be particularly involved in mediating the chronicity of MS. Acknowledgements We thank Ms. Iva Stonebridge for her excellence in technical assistances. ZC is a recipient of scholarships from Natural Science and Engineering Research Council of Canada (NSERC, 2004–06) and Multiple Sclerosis Society of Canada (MSSC, 2006–08). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jneuroim.2007.11.010. References Abdul-Majid, K.B., Stefferl, A., Bourquin, C., Lassmann, H., Linington, C., Olsson, T., Kleinau, S., Harris, R.A., 2002. Fc receptors are critical for autoimmune inflammatory damage to the central nervous system in experimental autoimmune encephalomyelitis. Scand. J. Immunol. 55, 70–81. Angelini, D.F., Borsellino, G., Poupot, M., Diamantini, A., Poupot, R., Bernardi, G., Poccia, F., Fournie, J.J., Battistini, L., 2004. FcgammaRIII discriminates between 2 subsets of Vgamma9Vdelta2 effector cells with different responses and activation pathways. Blood 104, 1801–1807. Bjartmar, C., Wujek, J.R., Trapp, B.D., 2003. Axonal loss in the pathology of MS: consequences for understanding the progressive phase of the disease. J. Neurol. Sci. 206, 165–171. Bodman-Smith, M.D., Anand, A., Durand, V., Youinou, P.Y., Lydyard, P.M., 2000. Decreased expression of FcgammaRIII (CD16) by gammadelta T cells in patients with rheumatoid arthritis. Immunology 99, 498–503. Butcher, S.K., Chahal, H., Nayak, L., Sinclair, A., Henriquez, N.V., Sapey, E., O'Mahony, D., Lord, J.M., 2001. Senescence in innate immune responses: reduced neutrophil phagocytic capacity and CD16 expression in elderly humans. J. Leukoc. Biol. 70, 881–886. Caccamo, N., Meraviglia, S., Ferlazzo, V., Angelini, D., Borsellino, G., Poccia, F., Battistini, L., Dieli, F., Salerno, A., 2005. Differential requirements for antigen or homeostatic cytokines for proliferation and differentiation of human Vgamma9Vdelta2 naive, memory and effector T cell subsets. Eur. J. Immunol. 35, 1764–1772.
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Correlation of specialized CD16 + γδ T cells with disease course and severity in multiple sclerosis Zhihong Chen a,b , Mark S. Freedman a,b,c,⁎ a
Department of Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada b Ottawa Health Research Institute, Ottawa, ON, Canada c The Ottawa Hospital, General Campus, Ottawa, ON, Canada
Received 5 July 2007; received in revised form 6 November 2007; accepted 12 November 2007
Abstract γδ T cells may be important innate immune system contributors to the immunopathogenesis of multiple sclerosis (MS), though the mechanisms are not yet fully understood. CD16 is a low affinity Fcγ receptor, an activation receptor for γδ T cells, and a mediator of cytotoxicity. In this study, we found that the percentage of CD16+ γδ T cells is elevated in MS patients compared with healthy controls. The increase is especially pronounced in patients with a progressive course of the disease, and the extent of this elevation shows a positive correlation with the time of disease progression and severity. In vitro cultured γδ T cells can be shown to upregulate the expression of CD16 in response to inflammatory cytokines such as IL-2 and -15, that have been shown to be elevated in progressive disease. These results suggest that CD16 expressing γδ T cells are somehow involved in the process of disease progression. Understanding more about these cells and their particular function in progressive vs. non-progressive disease could provide important clues to the mechanism of immune-mediated MS disease progression. Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved. Keywords: Multiple sclerosis; γδ T cells; Fcγ receptor III (CD16); Progression; Proinflammatory cytokine; IL-15
1. Introduction Multiple sclerosis (MS) is an autoimmune central nervous system (CNS) disease marked by the immune-mediated destruction of myelin, the ensheathed axons, and the neurons (Pouly and Antel, 1999; Wegner et al., 2006). Heterogeneity, both patho-immunologically and clinically, is a hallmark of the disease (Lucchinetti et al., 2000). Clinically most patients start out with a relapsing–remitting (RR) disease process, but virtually all eventually enter a more progressive phase (secondary progressive, SP) that tends to be chronic and usually unrelenting; a smaller subset starts out and continues with a primarily progressive course in the absence of relapses (primary progressive, PP) (Lublin and Reingold, 1996). Though the exact ⁎ Corresponding author. Division of Neurology, The Ottawa Hospital-General Campus, 501 Smyth Road, Ottawa, Canada K1H 8L6. Tel.: +1 613 737 8917; fax: +1 613 737 8857. E-mail address: [email protected] (M.S. Freedman).
immunopathogenesis of MS remains to be fully elucidated, both innate and adaptive immune responses have been implicated (Raine, 1994). γδ T cells are an important component of the innate immune response. Though they are one of the two lineages of T lymphocytes, γδ T cells are quite different from the conventional αβ T cells in that they are able to recognize unprocessed, nonMHC restricted antigens and that their antigen specificities are not limited only to peptides of certain length (Fisch et al., 1990; Tanaka et al., 1995). γδ T cells are also shown to express various natural killer cell (NK) receptors associated with cytotoxicity, cytokine release, and trans-endothelial migration (Halary et al., 1997; Poggi et al., 2002). Due to the lack of variability in their T cell receptor (TCR) and being close resemblance to NK cells in terms of phenotype, one may conclude that γδ T cells are a more homogeneous population. However, recent studies have demonstrated that γδ T cells could be further subdivided into highly differentiated subsets of central memory, effector memory, and terminal effector cells
0165-5728/$ - see front matter. Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2007.11.010
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based on their physiobiological functions, which coincide with the expressions of several surface molecules (Dieli et al., 2003; Shen et al., 2002). CD16, in combination with CD45RA and CD27, identify a highly cytotoxic subset of γδ T cells lethal to target cells (Angelini et al., 2004). It has also been demonstrated that CD16 expressing cells are required in inducing Experimental Autoimmune Encephalomyelitis (EAE) in animal models (Abdul-Majid et al., 2002; Szalai et al., 2005). CD16 polymorphisms may also be correlated with MS disease course (Myhr et al., 1999). CD16 (FcγRIII) is the low affinity Type III receptor that specifically binds the Fc portion of immunoglobulin G (IgG). It is expressed to varying degrees by several types of leukocytes (macrophages, NK cells, granulocytes, and γδ T cells). Engagement of CD16 on these cells by immune complexes induces one or more biological functions including phagocytosis, antibody-dependent cell cytotoxicity (ADCC), and release of cytokines (Jefferis and Lund, 2002). CD16 is upregulated on cultured resting γδ T cells upon activation through their TCR– CD3 complex (Lafont et al., 2001). These CD16+ γδ T cells are shown to be highly cytotoxic (Angelini et al., 2004), and can release cytokines (e.g. TNF-α) upon stimulation through their CD16 molecules (Lafont et al., 2001). The exact way in which γδ T cells are involved in disease has not yet been discerned. In EAE they have been shown to both contribute and protect from disease (Kobayashi et al., 1997; Ponomarev et al., 2004; Rajan et al., 1996). However, in human studies their role seems more consistent with contributing to disease: they are more numerous in the cerebrospinal fluid (CSF) (Stinissen et al., 1995), concentrated around active demyelinating lesions (Wucherpfennig et al., 1992), and are potent producers of inflammatory cytokines (Hintzen and Polman, 1997). We have demonstrated that γδ T cells cytolyse freshly isolated human oligodendrocytes (ODCs) (Freedman et al., 1991), possibly through the recognition of ODC expression of heat shock proteins (Freedman et al., 1997), death (Fas) receptors (Zeine et al., 1998), or MHC-I related stress molecules (Saikali et al., 2007) by corresponding ligands on the surface of γδ T cells. These studies all suggested that subsets of γδ T cells may be particularly involved in mediating deleterious effects.
Given the particular functions attributed to CD16 expressing γδ T cells and their implication in other autoimmune diseases (Bodman-Smith et al., 2000; Lamour et al., 1995), we investigated this subset in MS. 2. Materials and methods 2.1. Patients and healthy controls Blood and cells were obtained from 64 patients undergoing diagnostic assessment and clinical follow-up at the MS Centre of the Ottawa Hospital — General Campus (their demographic profile shown in Table 1). Among them, 49 met the diagnosis of MS according to the new criteria (Polman et al., 2005); the other 15 patients, suffering initial neurological attacks, were diagnosed with clinical isolated syndromes (CIS) (O'Riordan et al., 1998). None of the patients had experienced an exacerbation or received any disease modifying drugs (INF-β or Glatiramer Acetate) or steroids at least three months before the time of blood sampling. Blood and cells were also obtained from healthy volunteers who were recruited from laboratory staff or hospital personnel. This study was approved by Research Ethics Board of the Ottawa Health Research Institute and all samples were obtained with written informed consent. 2.2. Ex vivo flow cytometric analysis Peripheral blood mononuclear cells (PBMC) were isolated from each subject by Ficoll-Hypaque (Pharmacia Biotech AB, Uppsala, Sweden) density gradient centrifugation following standard procedure. 1 × 106 cells were set aside for ex vivo staining and the rest were used for expansion and purification of γδ T cells as described below in Section 2.3. Cells were stained with a cocktail of FITC-γδ TCR (TRCγδ1, BD Biosciences, Mississauga, ON), PE-CD16 (3G8, Beckman Coulter, Mississauga, ON), and PC5-CD3 (UCHT-1, Beckman Coulter) monoclonal antibodies (mAb) according to standard procedures and analyzed by flow cytometry on an FC500 flow cytometer (Beckman Coulter). Typically 2 × 105 events were acquired for analysis using the supplied Coulter CXP software.
Table 1 Patient demographics
Number Age (years) b Sex (male/female) e Disease duration (years) b EDSS score b, g
Healthy controls a
CIS
MS patients RR
SP
PP
Younger
Older
15 41.4 ± 14.3 3/12 3.33 ± 3.53 1.0 ± 0.9
13 46.9 ± 9.4 6/7 8.5 ± 3.6 2.2 ± 1.0
19 53.3 ± 10.7 7/12 19.4 ± 7.1 6.2 ± 1.0
17 55.7 ± 8.1 8/9 13.6 ± 9.5 5.7 ± 1.7
15 39.2 ± 9.1 c 4/11 N/A f N/A
18 47.3 ± 9.8 d 5/13 N/A N/A
CIS: clinical isolated syndromes; RR: relapsing–remitting MS; SP: secondary progressive MS; PP: primary progressive MS. a Healthy controls were divided into two groups according to their age. b Data were shown as mean ± S.D. c Not statistically different from CIS. d Not statistically different from MS subgroups. e Not statistically different among all the groups. f N/A: non applicable. g EDSS: Expanded Disability Status Scale.
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2.3. γδ T cells expansion and isolation Peripheral blood γδ T cell lines from both patients and healthy controls were expanded and purified as previously described (Zeine et al., 1998). Briefly, 24-well culture plates (Fischer Scientific, Ottawa, Ontario) were sequentially treated with 350 μl (5 μg/ml) sheep anti-mouse IgG1 antibody, 350 μl cRPMI (RPMI-1640 supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin G and 100 μg/ml streptomycin, Life Technologies, Burlington, ON), and monoclonal anti-TCR γδ antibody (Courtesy of Dr. M. Brenner, Harvard University) in cRPMI medium, which had been titrated to yield maximal stimulation. PBMCs generated from step Section 2.2 were seeded in the plate at density of 3–3.5 × 106 cells per well in 3 ml of cRPMI. Cells were kept at 37 °C with 5% CO2 for culturing. 50 U/ml IL-2 was added the next day. On day 5, cRPMI was replaced with serum free AIM-V media and cultured for another 4 or 5 days with fresh IL-2. The αβ T cells were then eliminated by complement lysis using anti-CD4 and anti-CD8 monoclonal antibodies. T cell preparations consisting of N90% γδ T cells were used in all experiments.
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flow cytometry. γδ T cells were identified and gated by double positive staining of CD3 and γδ TCR. The gated cells were further analyzed for surface expression of CD16 and the data were expressed as percentage of CD16 positive γδ T cells over total γδ T cells (%CD16+) (Supplementary figure). CIS tends to present in younger patients than those already with disease (by ANOVA test). In order to insure that the percent of CD16+ cells is not just an age-related phenomenon, we divided the healthy controls into two age groups with the younger groups (HC/Y) matching the age of CIS and the older (HC/O) matching the rest of MS groups. When compared with the healthy controls (HC/O: 24.3 ± 14.2% and HC/Y: 21.0 ± 10.2%), the percentages of CD16+ γδ T cells were significantly elevated in patients diagnosed with MS (35.6 ± 24.0%, p = 0.0429) or CIS (30.1 ± 12.6%, p = 0.0387) (Fig. 1A). Looking particularly within MS subgroups (PP-, RR-, and SP-MS) the variations of the means of %CD16+ were compared across each subgroup as well as the HC/O using one-way ANOVA test. A significant difference (p = 0.0132) was noticed among the groups (Fig. 1B). Tukey's post-hoc inter-group comparison identified that it was the SPMS group that had significantly higher %CD16+ than the healthy controls, while the differences between other patient
2.4. Cytokine responsive assay Purified γδ T cells were harvested, washed, and re-plated for another 24 h without IL-2 in culture media cRPMI. Cells were then seeded in U-bottomed 96-well Petri-dish at 2 × 105 cells per well. IL-2 (a gift from Hoffmann-La Roche, Nutley, NJ), IL-12, and IL-15 (both from Serotec, Oxford, UK) were then added to the wells at the optimized concentrations, topping up each well to 200 μl with culture media. Dishes were cultured for 72 h, 37 °C/5% CO2 and the surface expression of CD16 was measured with flow cytometry. 2.5. Statistics Differences between two groups were compared with paired or non-paired two-tailed Student's t test, where appropriate. Differences between three or more groups were evaluated with one way ANOVA. If statistical significance was noted, Tukey's post-hoc test was applied for inter-group comparisons. Normal distribution of the variables was assessed with the D'Agostino and Pearson test. Disease duration and EDSS scores were not following a normal distribution, therefore non-parametric Spearman's rank correlation was used for the correlation analyses. In all assays, p b 0.05 was considered statistically significant. Statistical analyses were performed with Prism (GraphPad Inc., San Diego, CA) or Kyplot (KyensLab Inc. Japan) software. 3. Results 3.1. CD16+ γδ T cells in patients and healthy controls The percentage of CD16 expressing γδ T cells derived from peripheral blood of MS patients or healthy controls was assessed on immediately ex vivo cells using multiple parameter
Fig. 1. CD16 expressing γδ T cells are elevated in patients compared with healthy controls. (A) Healthy controls were divided into two age groups (HC/O and HC/Y). The mean %CD16+ was significantly increased in MS patients when compared with HC/O and in CIS compared with HC/Y. Box and whiskers indicated the median, 25th percentile rage, and highest/lowest values. The means were shown with a crosses. (B) Means of %CD16+ of the subgroups of MS were significantly different from that of the healthy controls (p = 0.0132). SPMS patients had significantly higher %CD16+ than the healthy controls. A cut-off value (dotted line) indicating the mean of the HC/O + 2SD was shown, above which %CD16+ was considered abnormally high. The SPMS group also had the highest percentage (36.8%) of abnormally high CD16+ γδ T cells. The percentages of patients with abnormally high values in RRMS and PPMS were 7.7% and 17.6% respectively. Closed dots represented %CD16+ value of each individual and the means and SD were shown with bars. ⁎ Denotes statistical significance.
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Fig. 4. Increase in %CD16+ in response to in vitro cytokine stimulation. Proinflammatory cytokines IL-2, -12 and -15 significantly elevated the %CD16+ γδ T cells derived from MS patients (n = 10), the most robust stimulation seen with IL-15. IL-2 and IL-15 also increased %CD16+ γδ T cells in healthy controls (n = 6). For each of the cytokines, there was no difference noticed in their ability to increase the %CD16+ γδ T cells between healthy controls and MS patients. ⁎ p b 0.05. Fig. 2. Relationship of %CD16+ to patient clinical characteristics. %CD16+ correlated significantly with both disease duration (years since onset of MS, in A) and severity (expressed by EDSS, in B). Total n = 49, p b 0.05, Spearman correlation test.
groups and the healthy controls did not reach statistical significance. We defined a cut-off value of 52.8% (mean %CD16+ + 2SD) observed in the HC/O; values higher than this were considered to be elevated. We found that more SPMS patients had elevated %CD16+, with 7 out of the 19 (36.8%) exceeding the upper limit. About 17.6% of PPMS patients were found with elevated percentage of CD16+ γδ T cells; while only 1 of 13 patients with RRMS (7.7%) had high %CD16+ (Fig. 1B). 3.2. CD16+ percentage correlates with disease progression and EDSS
There was no correlation found with either age (p = 0.055 by Pearson test) or sex (p = 0.56 by t-test). However, a significant correlation was noted between %CD16+ and either disease duration (r = 0.40, p = 0.0042) or disability (EDSS) (r = 0.31, p = 0.031) (Fig. 2). These correlations remained true when patients were subdivided into four groups (four quadrants in Fig. 3) according to their disease duration and EDSS scores: patients with short disease duration (b = 10 years) and low EDSS (b = 3.5) had the lowest average %CD16+ (25.5 ± 15.5%); patients with both long duration (N10 years) and high EDSS (N 3.5) showed the highest percentage of CD16+ γδ T cells (45.6 ± 26.4%); while the other two groups with different combinations (i.e. short disease duration/high EDSS or long disease duration/low EDSS) demonstrated %CD16+ in between (26.7 ± 18.8% and 27.9 ± 21.6% respectively) (Fig. 3). 3.3. CD16 is upregulated by IL-15 in vitro
We next examined the relationships between the %CD16+ and the clinical characteristics of the patients (see Table 1).
Fig. 3. %CD16+ corresponds to disease duration and EDSS. MS patients were subdivided into 4 quadrants according to their disease duration (separated by 10 years) and EDSS scores (separated by 3.5 points), indicated by the dotted lines. Each rectangle represented one patient and was colorized based on their %CD16+ value converted against the color map on the right. The mean %CD16+ of each quadrant was shown at the corners in a bigger closed rectangle.
In an effort to explain the elevated %CD16+ in SPMS, we postulated that chronic inflammation may be the driving force. In particular, cytokines such as IL-2, IL-12, IL-15, and IFN-γ, have been found to be elevated in the blood of SPMS patients (Killestein et al., 2001; Rentzos et al., 2006; van Boxel-Dezaire et al., 2001). To address this possibility, we cultured the γδ T cells in the presence of cytokines IL-2, -12, and -15 to confirm if %CD16+ could be increased. Purified γδ T cells were cultured with or without IL-2, -12, and -15 at previously optimized concentrations of 100 U/ml, 10 ng/ml, and 10 ng/ml respectively for 72 h. %CD16+ was measured with flow cytometry as described above. We observed that culturing in the presence of all 3 cytokines significantly elevated %CD16+ on γδ T cells derived from MS patients, the most robust stimulation seen with IL-15. However, for each of the cytokines, there was no difference noticed of their ability to increase %CD16+ between healthy controls and MS patients (Fig. 4).
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4. Discussion The exact contribution of γδ T cells to the pathogenesis of MS remains to be elucidated. In this study we report that MS patients have a significant increase in a particular subset of γδ T cells, those bearing the NK-type marker CD16. In addition, the percentage of CD16 expressing γδ T cells (%CD16+) appears to correlate with disease duration and severity suggesting they may be an important component of the immune system damage that contributes to disease progression. This finding of elevated %CD16 + is in parallel with observations in other autoimmune diseases such as rheumatoid arthritis (RA) or Sjogren's syndromes, albeit the extent of this elevation in RA is relatively moderate (Bodman-Smith et al., 2000; Lamour et al., 1995). Increased %CD16+ may reflect their activation status in these diseases as it has been shown that CD16+ γδ T cells represent terminally differentiated memory cells (Angelini et al., 2004). Previous studies had implied that there might be an age related expression of CD16 molecule on NK cells or neutrophils (CD16 expression on neutrophils decreases with aging) (Butcher et al., 2001; Solana and Mariani, 2000). We did not observe any correlation of age and CD16 positivity on γδ T cells from our studies (p = 0.055 for all MS patients and p = 0.196 for healthy controls). We did notice a tendency that the older controls may have higher %CD16 compared with younger controls on a population level, but this could be caused by a single outlier in our HC/O subset. Increased levels of various proinflammatory cytokines were observed in the systemic circulation at different stages of MS (Merrill and Benveniste, 1996); in particular, at the SPMS stage heightened levels of IL-2, -12 and -15 were reported (Killestein et al., 2001; Rentzos et al., 2006; van Boxel-Dezaire et al., 2001). We observed that these same cytokines significantly increased %CD16+ suggesting that chronic dysregulation and an inflammatory milieu might be the cause for the observed increased %CD16+ in our SPMS patients. Although IL-2 and IL-15 are structurally and functionally related cytokines and their receptors share two of the three heterotrimer subunits, IL-2 is produced by activated CD4+T cells and functions on αβ T lymphocytes; while IL-15 is generated by a large variety of cell types, but preferentially works on γδ T cells (Leclercq et al., 1996). We found the most robust increase in %CD16+ in response to IL-15. The ability of IL-15 to increase CD16 expressing γδ T cells was also noted in other studies (Eberl et al., 2002). Increased % CD16+ may not solely be due to the presence of IL-15 selectively expanding the CD16+ population, since NK cells, both CD16− and CD16+, proliferate equally in response to IL15 (Dunne et al., 2001). CD16+ cells might also come from the CD16− pool in response to antigen-stimulation, such that the increase in CD16+ γδ T cells that we observed could actually derive from CD16− cells in response to unknown antigen stimulation together with the proinflammatory cytokine milieu seen in progressive MS (Caccamo et al., 2005). Studies have shown that immunomodulatory therapies have little effect once patients enter a progressive phase of their
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illness, reflecting probably more the neurodegenerative disease component (Bjartmar et al., 2003). However, evidence has also shown that the immune system itself is much more dysregulated in the same progressive stage of MS and that it carries a different, diffusive form of inflammation in the brain (VakninDembinsky and Weiner, 2007). This prompts Vaknin-Dembinsky and Weiner to propose that while abnormalities in adaptive immunity play an important role in RRMS, innate immunity may be a more important contributor to the disease process in SPMS (Vaknin-Dembinsky and Weiner, 2007). γδ T cells are a very important and prominent T cell subset involved in innate immune responses and could be postulated to contribute to disease progression by releasing proinflammatory cytokines, or via cytotoxic mechanisms. CD16 expressing γδ T cells may play a very particular role via ADCC in conjunction possibly with antibodies to myelin proteins that are seen in MS patients (Ziemssen and Ziemssen, 2005), and this is an area that we are currently investigating (manuscript under review). Our results showing elevated percentages of CD16 expressing γδ T cells that correlate with disease progression is a further demonstration that innate immune processes may be particularly involved in mediating the chronicity of MS. Acknowledgements We thank Ms. Iva Stonebridge for her excellence in technical assistances. ZC is a recipient of scholarships from Natural Science and Engineering Research Council of Canada (NSERC, 2004–06) and Multiple Sclerosis Society of Canada (MSSC, 2006–08). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jneuroim.2007.11.010. References Abdul-Majid, K.B., Stefferl, A., Bourquin, C., Lassmann, H., Linington, C., Olsson, T., Kleinau, S., Harris, R.A., 2002. Fc receptors are critical for autoimmune inflammatory damage to the central nervous system in experimental autoimmune encephalomyelitis. Scand. J. Immunol. 55, 70–81. Angelini, D.F., Borsellino, G., Poupot, M., Diamantini, A., Poupot, R., Bernardi, G., Poccia, F., Fournie, J.J., Battistini, L., 2004. FcgammaRIII discriminates between 2 subsets of Vgamma9Vdelta2 effector cells with different responses and activation pathways. Blood 104, 1801–1807. Bjartmar, C., Wujek, J.R., Trapp, B.D., 2003. Axonal loss in the pathology of MS: consequences for understanding the progressive phase of the disease. J. Neurol. Sci. 206, 165–171. Bodman-Smith, M.D., Anand, A., Durand, V., Youinou, P.Y., Lydyard, P.M., 2000. Decreased expression of FcgammaRIII (CD16) by gammadelta T cells in patients with rheumatoid arthritis. Immunology 99, 498–503. Butcher, S.K., Chahal, H., Nayak, L., Sinclair, A., Henriquez, N.V., Sapey, E., O'Mahony, D., Lord, J.M., 2001. Senescence in innate immune responses: reduced neutrophil phagocytic capacity and CD16 expression in elderly humans. J. Leukoc. Biol. 70, 881–886. Caccamo, N., Meraviglia, S., Ferlazzo, V., Angelini, D., Borsellino, G., Poccia, F., Battistini, L., Dieli, F., Salerno, A., 2005. Differential requirements for antigen or homeostatic cytokines for proliferation and differentiation of human Vgamma9Vdelta2 naive, memory and effector T cell subsets. Eur. J. Immunol. 35, 1764–1772.
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