HUMAN IMMUNOLOGY doi: 10.1111/sji.12139 ..................................................................................................................................................................
Peripheral and Site-Specific CD4+CD28null T Cells from Rheumatoid Arthritis Patients Show Distinct Characteristics J. Pieper*, S. Johansson*, O. Snir*,†, L. Linton‡, M. Rieck§, J. H. Buckner§, O. Winqvist‡, R. van Vollenhoven– & V. Malmstro¨m*
Abstract *Rheumatology Unit, Department of Medicine at Karolinska University Hospital, Karolinska Institute, Solna, Stockholm, Sweden; †Centre for Immune Regulation, Department of Immunology, Oslo University HospitalRikshospitalet, University of Oslo, Norway; ‡Translational Immunology Unit, Department of Medicine at Karolinska University Hospital, Karolinska Institute, Solna, Stockholm, Sweden; §Translational Research Program, Benaroya Research Institute at Virginia Mason, Seattle, WA, USA; and –Unit for Clinical Therapy Research, Inflammatory Diseases, Karolinska Institute, Solna, Stockholm, Sweden
Received 18 October 2013; Accepted in revised form 19 November 2013 Correspondence to: V. Malmstro¨m, Rheumatology Unit, Department of Medicine at Karolinska University Hospital, Karolinska Institute, CMM, L8:04, 17 176 Stockholm, Sweden. E-mail: [email protected]
Proinflammatory CD4+CD28null T cells are frequently found in the circulation of patients with rheumatoid arthritis (RA), but are less common in the rheumatic joint. In the present study, we sought to identify functional differences between CD4+CD28null T cells from blood and synovial fluid in comparison with conventional CD28-expressing CD4+ T cells. Forty-four patients with RA, displaying a distinct CD4+CD28null T cell population in blood, were recruited for this study; the methylation status of the IFNG locus was examined in isolated T cell subsets, and intracellular cytokine production (IFN-c, TNF, IL-17) and chemokine receptor expression (CXCR3, CCR6 and CCR7) were assessed by flow cytometry on T cells from the two compartments. Circulating CD4+CD28null T cells were significantly more hypomethylated in the CNS-1 region of the IFNG locus than conventional CD4+CD28+ T cells and produced higher levels of both IFN-c and TNF after TCR cross-linking. CD4+CD28null T cells from the site of inflammation expressed significantly more CXCR3 and CCR6 compared to their counterparts in blood. While IL-17A production could hardly be detected in CD4+CD28null cells from the blood, a significant production was observed in CD4+CD28null T cells from synovial fluid. CD4+CD28null T cells were not only found to differ from conventional CD4+CD28+ T cells in the circulation, but we could also demonstrate that synovial CD4+CD28null T cells showed additional effector functions (IL-17 coproduction) as compared to the same subset in peripheral blood, suggesting an active role for these cells in the perpetuation of inflammation in the subset of patients having a CD28null population.
Introduction CD4+CD28null cells are highly differentiated effector memory CD4+ T cells that have downregulated the costimulatory molecule CD28, due to the loss of an CD28-specific initiator complex [1, 2]. CD28 is progressively lost after replicative senescence [3] (continuous viral or autoantigen stimulation) or under proinflammatory conditions, for example, by TNF [4]. CD4+CD28null cells differ from conventional CD4+ T cells with respect to shortened telomeres [5], reduced TCR diversity [6, 7], by displaying cytotoxic capacity [8, 9], expression of NK cell receptors [10] and resistance to apoptosis [11]. While being independent of classical costimulation, CD4+CD28null T cells are not anergic, but respond rapidly to stimulation [12]. Because of their proinflammatory features (cytokine production and cytotoxicity), it is likely that they
Ó 2013 John Wiley & Sons Ltd
contribute to disease progression of several inflammatory disorders. Increased frequencies of CD4+CD28null cells can be found in the peripheral circulation of various immune disorders, such as rheumatoid arthritis (RA) [12], multiple sclerosis [13, 14] and inflammatory bowel disease [15, 16]. CD4+CD28null T cells can be found in about one-third of patients with RA. The frequency in the circulation varies, but can be up to 50% of all CD4+ T cells [17]. Notably, despite their presence in the circulation at significant frequencies, CD4+CD28null cells are infrequent in the joints of patients with RA (synovial fluid and synovial membrane) [17]; the nature behind this observation has remained largely unexplored. Because the distribution of these cells differs between the circulation and the target site of disease, we wanted to examine whether the CD4+CD28null cells that are present in the joint have different features than the CD28null
149
150 CD28null Cells in RA Show Distinct Characteristics J. Pieper et al. .................................................................................................................................................................. population seen in peripheral blood. For this purpose, we examined the ability of CD4+CD28null T cells from blood and synovial fluid of patients with RA to produce cytokines by studying the methylation status of the IFNG locus, because CD4+CD28null T cells are well known to make this cytokine, and their cytokine secretion capacity by comparing IFN-c, TNF and IL-17 production, that is, cytokines implicated in RA pathogenesis. Furthermore, because it is not known why only some of these cells migrate to the joint, we characterized the cells with regard to chemokine receptor expression and compared CD4+CD28null T cells to conventional CD4+CD28+ T cells. We found that cells from the joints of patients with RA differ from those found in the periphery with regard to methylation status, cytokine production and chemokine receptor expression.
Materials and methods Patients and samples. Altogether, 44 patients with RA were enrolled in the study. The diagnosis was made by rheumatologists according to the American College of Rheumatology 1987 revised criteria for the classification of RA [18]. All patients had at least 5% CD28null cells in their circulation. PB and SF samples from 14 patients were used for methylation status. Twenty-three patients with RA were included in cytokine analysis and 12 in the chemokine receptor expression. Some of the patients were included in several experiments. The mean age was 57 years (range: 18–86); 75% were female. Three of the patients had a disease duration of less than a year, seven patients
Peripheral and Site-Specific CD4+CD28null T Cells from Rheumatoid Arthritis Patients Show Distinct Characteristics J. Pieper*, S. Johansson*, O. Snir*,†, L. Linton‡, M. Rieck§, J. H. Buckner§, O. Winqvist‡, R. van Vollenhoven– & V. Malmstro¨m*
Abstract *Rheumatology Unit, Department of Medicine at Karolinska University Hospital, Karolinska Institute, Solna, Stockholm, Sweden; †Centre for Immune Regulation, Department of Immunology, Oslo University HospitalRikshospitalet, University of Oslo, Norway; ‡Translational Immunology Unit, Department of Medicine at Karolinska University Hospital, Karolinska Institute, Solna, Stockholm, Sweden; §Translational Research Program, Benaroya Research Institute at Virginia Mason, Seattle, WA, USA; and –Unit for Clinical Therapy Research, Inflammatory Diseases, Karolinska Institute, Solna, Stockholm, Sweden
Received 18 October 2013; Accepted in revised form 19 November 2013 Correspondence to: V. Malmstro¨m, Rheumatology Unit, Department of Medicine at Karolinska University Hospital, Karolinska Institute, CMM, L8:04, 17 176 Stockholm, Sweden. E-mail: [email protected]
Proinflammatory CD4+CD28null T cells are frequently found in the circulation of patients with rheumatoid arthritis (RA), but are less common in the rheumatic joint. In the present study, we sought to identify functional differences between CD4+CD28null T cells from blood and synovial fluid in comparison with conventional CD28-expressing CD4+ T cells. Forty-four patients with RA, displaying a distinct CD4+CD28null T cell population in blood, were recruited for this study; the methylation status of the IFNG locus was examined in isolated T cell subsets, and intracellular cytokine production (IFN-c, TNF, IL-17) and chemokine receptor expression (CXCR3, CCR6 and CCR7) were assessed by flow cytometry on T cells from the two compartments. Circulating CD4+CD28null T cells were significantly more hypomethylated in the CNS-1 region of the IFNG locus than conventional CD4+CD28+ T cells and produced higher levels of both IFN-c and TNF after TCR cross-linking. CD4+CD28null T cells from the site of inflammation expressed significantly more CXCR3 and CCR6 compared to their counterparts in blood. While IL-17A production could hardly be detected in CD4+CD28null cells from the blood, a significant production was observed in CD4+CD28null T cells from synovial fluid. CD4+CD28null T cells were not only found to differ from conventional CD4+CD28+ T cells in the circulation, but we could also demonstrate that synovial CD4+CD28null T cells showed additional effector functions (IL-17 coproduction) as compared to the same subset in peripheral blood, suggesting an active role for these cells in the perpetuation of inflammation in the subset of patients having a CD28null population.
Introduction CD4+CD28null cells are highly differentiated effector memory CD4+ T cells that have downregulated the costimulatory molecule CD28, due to the loss of an CD28-specific initiator complex [1, 2]. CD28 is progressively lost after replicative senescence [3] (continuous viral or autoantigen stimulation) or under proinflammatory conditions, for example, by TNF [4]. CD4+CD28null cells differ from conventional CD4+ T cells with respect to shortened telomeres [5], reduced TCR diversity [6, 7], by displaying cytotoxic capacity [8, 9], expression of NK cell receptors [10] and resistance to apoptosis [11]. While being independent of classical costimulation, CD4+CD28null T cells are not anergic, but respond rapidly to stimulation [12]. Because of their proinflammatory features (cytokine production and cytotoxicity), it is likely that they
Ó 2013 John Wiley & Sons Ltd
contribute to disease progression of several inflammatory disorders. Increased frequencies of CD4+CD28null cells can be found in the peripheral circulation of various immune disorders, such as rheumatoid arthritis (RA) [12], multiple sclerosis [13, 14] and inflammatory bowel disease [15, 16]. CD4+CD28null T cells can be found in about one-third of patients with RA. The frequency in the circulation varies, but can be up to 50% of all CD4+ T cells [17]. Notably, despite their presence in the circulation at significant frequencies, CD4+CD28null cells are infrequent in the joints of patients with RA (synovial fluid and synovial membrane) [17]; the nature behind this observation has remained largely unexplored. Because the distribution of these cells differs between the circulation and the target site of disease, we wanted to examine whether the CD4+CD28null cells that are present in the joint have different features than the CD28null
149
150 CD28null Cells in RA Show Distinct Characteristics J. Pieper et al. .................................................................................................................................................................. population seen in peripheral blood. For this purpose, we examined the ability of CD4+CD28null T cells from blood and synovial fluid of patients with RA to produce cytokines by studying the methylation status of the IFNG locus, because CD4+CD28null T cells are well known to make this cytokine, and their cytokine secretion capacity by comparing IFN-c, TNF and IL-17 production, that is, cytokines implicated in RA pathogenesis. Furthermore, because it is not known why only some of these cells migrate to the joint, we characterized the cells with regard to chemokine receptor expression and compared CD4+CD28null T cells to conventional CD4+CD28+ T cells. We found that cells from the joints of patients with RA differ from those found in the periphery with regard to methylation status, cytokine production and chemokine receptor expression.
Materials and methods Patients and samples. Altogether, 44 patients with RA were enrolled in the study. The diagnosis was made by rheumatologists according to the American College of Rheumatology 1987 revised criteria for the classification of RA [18]. All patients had at least 5% CD28null cells in their circulation. PB and SF samples from 14 patients were used for methylation status. Twenty-three patients with RA were included in cytokine analysis and 12 in the chemokine receptor expression. Some of the patients were included in several experiments. The mean age was 57 years (range: 18–86); 75% were female. Three of the patients had a disease duration of less than a year, seven patients
