- Email: [email protected]
Defective CD55 costimulation in multiple sclerosis — A preliminary study
Abstracts
MOG-EAE was induced in NR1D1−/− mice that were obtained from the European Mutant Mouse Archive (EMMA). Results: While confirming the known role of RORalpha and RORgammaT, we found that additionally, the two nuclear receptors NR1D1 and vitamin D receptor (VDR) were upregulated in Th17 cells. Upregulation of VDR was more prominent in Th17 cells than in Th1 cells, whereas no significant regulation of NR1D1 could be detected in Th1 cells. In vitro, 1,25 dihydroxyvitamin D3 inhibited Th17 cell development and induction of NR1D1 in a dose-dependent manner. Incubation of naïve Th0 cells with heme, a presumed natural ligand of NR1D1, did not significantly alter Th17 cell differentiation. NR1D1 deficiency did not have a significant impact on EAE incidence and severity. Conclusions: Our study supports a prominent role for nuclear receptors in Th17 biology and their possible link to vitamin D metabolism. Despite being upregulated in Th17 cells in vitro, we could not find a major role of NR1D1 in EAE pathogenesis. This contrasts with a recent report of NR1D1 being involved into the pathogenesis of chronic IBD in mice. As has been suggested by recent genome-wide screens, this finding is in line with the current view that despite striking similarities, different autoimmune diseases share some but not all immunological features. doi:10.1016/j.jneuroim.2014.08.533
199
IFN-γ producing cells were 47.5% in CD56+ T cells and 40.3% in CD56− T cells on fingolimod, and 23.0% in CD56+ T cells and 8.3% in CD56− T cells in fingolimod-unexperienced. In each relapsed patients, the frequencies of IFN-γ producing cells in CD56+ T cells were 31.1% and 49.6% each, those in CD56− T cells were 28.6% and 58.4%. The mean frequencies of IL17-A producing cells were 13.2% in CD56+ CD4+ T cells and 0.5% in CD56− CD4+ T cells on fingolimod in remission, and 2.6% in CD56+ CD4+ T cells and 0.3% in CD56− CD4+ T cells in fingolimod-unexperienced. The proportions of IL-17A producing cells in two relapsed patients were 0.1% and 65.4% among CD4+ CD56+ population, and 0.1% and 7.1% among CD4+ CD56− population. Conclusions: Increase of Th1-biased T cells with cytotoxicity-related molecule was observed under fingolimod treatment. T cell mediated cytotoxicity may play a key role in the development of relapses on fingolimod. doi:10.1016/j.jneuroim.2014.08.534
583 Defective CD55 costimulation in multiple sclerosis — A preliminary study Ruhcha Sutavania, Bruno Granb, Ian Spendlovec
626 a
High expression of cytotoxic molecules and Th1 bias of circulating T cells in patients with multiple sclerosis under fingolimod treatment Chihiro Fujiia, Yoichiro Okadab, Kimitoshi Kimurab, Masanori Nakagawaa, Sadayuki Matsumotoc, Ryosuke Takahashib, Hirofumi Ochid, Takayuki Kondob, Toshiki Mizunoa a
Department of Neurology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan; bDepartment of Neurology, Kyoto University Graduate School of Medicine, Kyoto, Japan; cDepartment of Neurology, Tazuke Kofukai Foundation, Medical Research Institute, Kitano Hospital, Osaka, Japan; dDepartment of Neurology and Geriatric Medicine, Ehime University Graduate School of Medicine, Toon, Japan Background: Fingolimod is highly effective in preventing multiple sclerosis (MS) relapses. However, relapses can occur in fingolimodtreated patients by unknown mechanisms. Objectives: To study several features of circulating T cells under fingolimod treatment. Methods: Enrolled were totally 12 MS patients with informed consent, five fingolimod-unexperienced patients in remission, five fingolimod-treated patients in remission and two in relapse. Following molecules were analyzed using a BD FACSCantoII flow cytometer (BD Biosciences, USA) according to the manufacturers’ protocols: CD3, CD4, CD56, Fas ligand (FasL), granzyme B, and perfolin. Interferon-γ (IFN-γ) and interleukin 17A (IL-17A) were measured after four hours of stimulation by PMA/ionomycine. Results: The mean frequency of CD56+ T cells was more elevated up to 12.7% in remission on fingolimod than that in fingolimod-unexperienced (2.6%, P b 0.0001). The frequency of this subset markedly increased in relapsed patients on fingolimod, 61.1% in one subject and 47.8% in the other. Granzyme B, FasL and perfolin were more expressed on fingolimod (fingolimod-treated vs. fingolimod-unexperienced: granzyme B, 81.1% vs. 38.1% in CD56+ T cells, 66.7% vs. 8.9% in CD56− T cells; FasL, 1.23% vs. 0.54% in CD56+ T cells, 0.29% vs. 0.17% in CD56− T cells; perfolin 19.6% vs. 15.8% in CD56+ T cells, 9.8% vs. 2.6% in CD56− T cells). The frequency of granzyme B reached 96.8% in CD56+ T cells and 92.0% in CD56− T cells in one relapsed patient. The mean frequencies of
College of Life Sciences, University of Dundee, Dundee, United Kingdom; Division of Clinical Neuroscience, University of Nottingham School of Medicine, Nottingham, United Kingdom; cDivision of Cancer and Stem Cells, University of Nottingham School of Medicine, Nottingham, United Kingdom
b
A healthy immune system is maintained in a state of balance between pro- and anti-inflammatory cells. One population of regulatory cells is the T regulatory type 1 cells (Tr1), whose functions are mediated by the production of IL-10. In diseased states there is an imbalance in this equilibrium which contributes to the condition. In cancer there are known to be tumour resident antigen specific Tr1 cells which not only prevent immune mediated mechanisms of tumour control but are also thought to promote tumour progression. In autoimmune conditions, including rheumatoid arthritis (RA) and Multiple Sclerosis (MS) there is evidence that the levels of IL-10 is reduced and at the same time pro-inflammatory Th1 cells and IFN-g are elevated. However the mechanism controlling this balance remains uncharacterized. We have recently identified that CD55 acts as an alternative co-stimulatory signal in human CD4+ cells via engagement of its cellular ligand CD97. A consequence of CD55 co-stimulation is production of high levels of IL-10. We hypothesise that CD55 costimulation induces a Tr1 regulatory phenotype that suppresses effector cells via IL-10. We show that CD55 co-stimulation results in high IL-10, low IFNg and no IL-4 being produced by the cells. This cytokine profile and the suppression of effector cell function, in an IL-10 dependent manner, indicate a novel Tr1 co-stimulatory pathway. When comparing CD55 co-stimulation to CD28 co-stimulation of naïve CD4+ cells, we show that both stimuli induce differentiation and proliferation, but CD55 induces an IL-10high IFNglow IL4neg Tr1 phenotype while CD28 induced an IFNghigh IL-10low IL4neg Th1 phenotype. However, when carrying out the same assays on naïve T cells of MS patients we can clearly demonstrate a lack of the Tr1 induction in MS patient samples. This absence or Tr1 induction in MS patients may highlight defects in the co-stimulatory pathways used by T cells but also highlight potential mechanisms that can be corrected. doi:10.1016/j.jneuroim.2014.08.535
MOG-EAE was induced in NR1D1−/− mice that were obtained from the European Mutant Mouse Archive (EMMA). Results: While confirming the known role of RORalpha and RORgammaT, we found that additionally, the two nuclear receptors NR1D1 and vitamin D receptor (VDR) were upregulated in Th17 cells. Upregulation of VDR was more prominent in Th17 cells than in Th1 cells, whereas no significant regulation of NR1D1 could be detected in Th1 cells. In vitro, 1,25 dihydroxyvitamin D3 inhibited Th17 cell development and induction of NR1D1 in a dose-dependent manner. Incubation of naïve Th0 cells with heme, a presumed natural ligand of NR1D1, did not significantly alter Th17 cell differentiation. NR1D1 deficiency did not have a significant impact on EAE incidence and severity. Conclusions: Our study supports a prominent role for nuclear receptors in Th17 biology and their possible link to vitamin D metabolism. Despite being upregulated in Th17 cells in vitro, we could not find a major role of NR1D1 in EAE pathogenesis. This contrasts with a recent report of NR1D1 being involved into the pathogenesis of chronic IBD in mice. As has been suggested by recent genome-wide screens, this finding is in line with the current view that despite striking similarities, different autoimmune diseases share some but not all immunological features. doi:10.1016/j.jneuroim.2014.08.533
199
IFN-γ producing cells were 47.5% in CD56+ T cells and 40.3% in CD56− T cells on fingolimod, and 23.0% in CD56+ T cells and 8.3% in CD56− T cells in fingolimod-unexperienced. In each relapsed patients, the frequencies of IFN-γ producing cells in CD56+ T cells were 31.1% and 49.6% each, those in CD56− T cells were 28.6% and 58.4%. The mean frequencies of IL17-A producing cells were 13.2% in CD56+ CD4+ T cells and 0.5% in CD56− CD4+ T cells on fingolimod in remission, and 2.6% in CD56+ CD4+ T cells and 0.3% in CD56− CD4+ T cells in fingolimod-unexperienced. The proportions of IL-17A producing cells in two relapsed patients were 0.1% and 65.4% among CD4+ CD56+ population, and 0.1% and 7.1% among CD4+ CD56− population. Conclusions: Increase of Th1-biased T cells with cytotoxicity-related molecule was observed under fingolimod treatment. T cell mediated cytotoxicity may play a key role in the development of relapses on fingolimod. doi:10.1016/j.jneuroim.2014.08.534
583 Defective CD55 costimulation in multiple sclerosis — A preliminary study Ruhcha Sutavania, Bruno Granb, Ian Spendlovec
626 a
High expression of cytotoxic molecules and Th1 bias of circulating T cells in patients with multiple sclerosis under fingolimod treatment Chihiro Fujiia, Yoichiro Okadab, Kimitoshi Kimurab, Masanori Nakagawaa, Sadayuki Matsumotoc, Ryosuke Takahashib, Hirofumi Ochid, Takayuki Kondob, Toshiki Mizunoa a
Department of Neurology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan; bDepartment of Neurology, Kyoto University Graduate School of Medicine, Kyoto, Japan; cDepartment of Neurology, Tazuke Kofukai Foundation, Medical Research Institute, Kitano Hospital, Osaka, Japan; dDepartment of Neurology and Geriatric Medicine, Ehime University Graduate School of Medicine, Toon, Japan Background: Fingolimod is highly effective in preventing multiple sclerosis (MS) relapses. However, relapses can occur in fingolimodtreated patients by unknown mechanisms. Objectives: To study several features of circulating T cells under fingolimod treatment. Methods: Enrolled were totally 12 MS patients with informed consent, five fingolimod-unexperienced patients in remission, five fingolimod-treated patients in remission and two in relapse. Following molecules were analyzed using a BD FACSCantoII flow cytometer (BD Biosciences, USA) according to the manufacturers’ protocols: CD3, CD4, CD56, Fas ligand (FasL), granzyme B, and perfolin. Interferon-γ (IFN-γ) and interleukin 17A (IL-17A) were measured after four hours of stimulation by PMA/ionomycine. Results: The mean frequency of CD56+ T cells was more elevated up to 12.7% in remission on fingolimod than that in fingolimod-unexperienced (2.6%, P b 0.0001). The frequency of this subset markedly increased in relapsed patients on fingolimod, 61.1% in one subject and 47.8% in the other. Granzyme B, FasL and perfolin were more expressed on fingolimod (fingolimod-treated vs. fingolimod-unexperienced: granzyme B, 81.1% vs. 38.1% in CD56+ T cells, 66.7% vs. 8.9% in CD56− T cells; FasL, 1.23% vs. 0.54% in CD56+ T cells, 0.29% vs. 0.17% in CD56− T cells; perfolin 19.6% vs. 15.8% in CD56+ T cells, 9.8% vs. 2.6% in CD56− T cells). The frequency of granzyme B reached 96.8% in CD56+ T cells and 92.0% in CD56− T cells in one relapsed patient. The mean frequencies of
College of Life Sciences, University of Dundee, Dundee, United Kingdom; Division of Clinical Neuroscience, University of Nottingham School of Medicine, Nottingham, United Kingdom; cDivision of Cancer and Stem Cells, University of Nottingham School of Medicine, Nottingham, United Kingdom
b
A healthy immune system is maintained in a state of balance between pro- and anti-inflammatory cells. One population of regulatory cells is the T regulatory type 1 cells (Tr1), whose functions are mediated by the production of IL-10. In diseased states there is an imbalance in this equilibrium which contributes to the condition. In cancer there are known to be tumour resident antigen specific Tr1 cells which not only prevent immune mediated mechanisms of tumour control but are also thought to promote tumour progression. In autoimmune conditions, including rheumatoid arthritis (RA) and Multiple Sclerosis (MS) there is evidence that the levels of IL-10 is reduced and at the same time pro-inflammatory Th1 cells and IFN-g are elevated. However the mechanism controlling this balance remains uncharacterized. We have recently identified that CD55 acts as an alternative co-stimulatory signal in human CD4+ cells via engagement of its cellular ligand CD97. A consequence of CD55 co-stimulation is production of high levels of IL-10. We hypothesise that CD55 costimulation induces a Tr1 regulatory phenotype that suppresses effector cells via IL-10. We show that CD55 co-stimulation results in high IL-10, low IFNg and no IL-4 being produced by the cells. This cytokine profile and the suppression of effector cell function, in an IL-10 dependent manner, indicate a novel Tr1 co-stimulatory pathway. When comparing CD55 co-stimulation to CD28 co-stimulation of naïve CD4+ cells, we show that both stimuli induce differentiation and proliferation, but CD55 induces an IL-10high IFNglow IL4neg Tr1 phenotype while CD28 induced an IFNghigh IL-10low IL4neg Th1 phenotype. However, when carrying out the same assays on naïve T cells of MS patients we can clearly demonstrate a lack of the Tr1 induction in MS patient samples. This absence or Tr1 induction in MS patients may highlight defects in the co-stimulatory pathways used by T cells but also highlight potential mechanisms that can be corrected. doi:10.1016/j.jneuroim.2014.08.535