High frequencies of activated B cells and T follicular helper cells are correlated with disease activity in patients with new‐onset rheumatoid arthritis

This study aimed to examine the frequency of different subsets of circulating B and T follicular helper (Tfh) cells in patients with new-onset rheumatoid arthritis (RA) and following standard therapies. Twenty-five RA patients and 15 healthy controls (HC) were recruited for characterizing the frequency of CD27⁺, immunoglobulin (Ig)D⁺, CD86⁺, CD95⁺, Toll-like receptor (TLR)-9⁺ B cells and inducible T cell co-stimulator (ICOS) and programmed death 1 (PD-1)-positive Tfh cells and the level of serum interleukin (IL)-21. The potential correlation between the frequency of different subsets of B and Tfh cells and the values of clinical measures in RA patients was analysed. In comparison with HC, significantly higher percentages of circulating IgD⁺CD27⁻CD19⁺ naive B, CD86⁺CD19⁺ and CD95⁺CD19⁺ activated B, CD3⁺CD4⁺CXCR5⁺, CD3⁺CD4⁺CXCR5⁺ICOS⁺, CD3⁺CD4⁺CXCR5⁺PD-1⁺ and CD3⁺CD4⁺CXCR5⁺ICOS⁺PD-1⁺ Tfh cells but lower IgD⁺CD27⁺CD19⁺ preswitch memory B cells were detected, accompanied by significantly higher levels of serum IL-21 in the RA patients. Furthermore, the percentages of CD95⁺ B cells were correlated positively with the frequency of PD-1⁺ Tfh cells, but negatively with ICOS⁺ Tfh cells. The percentages of CD86⁺ B cells and ICOS⁺ Tfh cells were correlated positively with the values of disease activity score 28 (DAS28). Following the drug therapies for 1 month, the percentages of CD86⁺ B and PD-1⁺ Tfh cells were reduced significantly in the drug-responding patients. Our data suggest that activated B and Tfh cells may contribute to the pathogenesis of RA and the frequency of activated B and Tfh cells may be used as biomarkers for evaluating the therapeutic responses of individual patients with RA.     

Rheumatoid arthritis fibroblast‐like synoviocytes co‐cultured with PBMC increased peripheral CD4+CXCR5+ICOS+ T cell numbers

‘Circulating’ T follicular helper cells (Tfh), characterized by their surface phenotypes CD4⁺chemokine receptor 5 (CXCR5)⁺ inducible co‐stimulatory molecule (ICOS)⁺, have been identified as the CD4⁺ T cell subset specialized in supporting the activation, expansion and differentiation of B cells. Fibroblast‐like synoviocytes (FLS) are critical in promoting inflammation and cartilage destruction in rheumatoid arthritis (RA), and the interaction between FLS and T cells is considered to facilitate FLS activation and T cell recruitment. However, it remains unknown whether RA‐FLS co‐cultured with activated peripheral blood mononuclear cells (PBMC) has immunoregulatory effects on peripheral Tfh. In the present study, we co‐cultured RA‐FLS with or without anti‐CD3/CD28‐stimulated PBMC. The results showed that RA‐FLS co‐cultured with stimulated PBMC could increase the numbers of CD4⁺CXCR5⁺ICOS⁺ T cells of RA PBMC possibly via the production of interleukin (IL)‐6, a critical cytokine involved in the differentiation of Tfh cells. We also observed increased reactive oxygen species (ROS) levels in the co‐culture system of RA‐FLS and PBMC. The percentage of CD4⁺CXCR5⁺ICOS⁺ T cells was decreased when ROS production was inhibited by N‐acetyl‐L‐cysteine (NAC), a specific inhibitor which can decrease ROS production. In addition, we showed that the higher levels of tumour necrosis factor (TNF)‐α and IL‐1β in the co‐culture system and the blocking of TNF receptor 2 (TNF‐R2) and IL‐1β receptor (IL‐1βR) both decreased the numbers of CD4⁺CXCR5⁺ICOS⁺ T cells. Our study reveals a novel mechanistic insight into how the interaction of RA‐FLS and PBMC participates in the RA pathogenesis, and also provides support for the biologicals application for RA.     

Elevated circulating Th17 and follicular helper CD4+ T cells in patients with rheumatoid arthritis

It remains not fully elucidated the potential functions of Th17 cells and follicular helper T (Tfh) cells and secreting cytokines in the pathogenesis of rheumatoid arthritis (RA) and their association with disease activity. In this study, the frequencies of Th17 and Tfh cells were determined by flow cytometry, and the levels of interleukin (IL)‐17, IL‐21, and IL‐22 were measured by ELISA in RA patients with different disease activities. The dynamic changes of cell subsets were also detected in response to disease‐modify antirheumatic drugs (DMARDs) therapy. The percentages of CD3⁺CD4⁺IL‐17A⁺ (Th17) cells and CD3⁺CD4⁺CXCR5⁺ICOS^high (Tfh) cells, as well as the concentrations of IL‐17, IL‐21, and IL‐22 were significantly elevated in RA patients than those in healthy individuals. Furthermore, Tfh cells, IL‐21, and IL‐22 in the serum was positively correlated with the values of disease activity score. Concentrations of IL‐21 and IL‐22 in the serum were remarkably reduced following the DMARDs therapies. Our data suggested that Th17 cells, Tfh cells as well as the secreting cytokines may be involved in the pathogenesis of RA. The frequency of circulating Tfh cells and the productions of IL‐21 and IL‐22 were associated with the disease activity of RA patients, and might be potential therapeutic targets for treatment of RA.     

Immunolocalization of MMP-2 and MMP-9 in human rheumatoid synovium

Matrix metalloproteinase (MMP)-2 and MMP-9, two important members of the matrix metalloproteinase family, have been shown critical contributions in intra-tumor angiogenesis and invasion of tumor progression, and they might also play important roles in the angiogenesis as well as the pannus formation of rheumatoid arthritis (RA). In the present study, we used the immunohistochemistry, the immunofluorescence staining and the con-focal scanning methods to characterize the immunolocalization of MMP-2 and MMP-9 in RA synovium tissues. Our results showed that both MMP-2 and MMP-9 immunostaining could be found in synoviocytes and vascular endothelial cells. Moreover, our con-focal scanning also showed that MMP-2 could be found in infiltrating CD14⁺ monocytes and CD68⁺ macrophages, and MMP-9 could be found in infiltrating CD68⁺ macrophages in RA synovium tissues, while weak or negative staining of these two MMPs could be found in infiltrating CD20⁺B cells and CD3⁺T cells in RA synovium. Thus, our finding suggests that both MMP-2 and MMP-9 expressed by synoviocytes as well as certain infiltrating immune cells role importantly in the angiogenesis in RA progression.     

Increased CD4+CXCR5+T follicular helper cells in diabetic nephropathy

Background: T follicular helper (Tfh) cells are known to regulate humoral immune response. In this study we examined the correlation of different subsets of peripheral blood Tfh cells in patients with diabetic nephropathy (DN). Methods: A total of 23 DN patients and 15 healthy controls (HC) were investigated for various subsets of Tfh cells by flow cytometry. The molecules ICOS⁺, PD-1⁺, CD28⁺, CD154⁺, IL-21⁺, IFN-γ⁺, IL-4⁺, IL-17⁺ Tfh cells were examined. The subsets of B cells were investigated by flow cytometry. The levels of 24?h urinary protein and estimated glomerular filtration rate (eGFR) were calculated. A potential correlation between the number of different subsets of Tfh cells, B cells and DN, was assessed. Results: The circulating CD4⁺CXCR5⁺PD-1⁺, PD-1⁺CD154⁺, PD-1⁺CD28⁺, PD-1⁺IL-21⁺, PD-1⁺IL-4⁺, PD-1⁺-IL-17⁺-Tfh cell counts, CD38⁺CD19⁺, CD38⁺CD19⁺CD40⁺ B cells and plasma levels of IL-21 were significantly increased in DN patients (p?+CXCR5⁺PD-1⁺ Tfh cell counts negatively correlated with eGFR; Tfh cell counts positively correlated with 24?h urinary protein concentration in DN patients. Post-treatment, there was a significant reduction in the CD4⁺CXCR5⁺PD-1⁺ Tfh cell counts and its subsets, with a corresponding decrease in plasma levels of IL-6 and IL-17A (p?Conclusion: An increased number of CD4⁺CXCR5⁺PD-1⁺ Tfh cells were observed in DN patients, which may be new targets for intervention in DN.     

Increased circulating Tfh17 and PD-1+Tfh cells are associated with autoantibodies in Hashimoto’s thyroiditis

Abstract

Objective: Hashimoto’s thyroiditis (HT) is characterized by autoantibodies targeting the thyroid. Abnormal CD4⁺CXCR5⁺T cell levels were previously shown to be associated with HT. However, Tfh cells consist of heterogeneous subpopulations, and which T follicular helper (Tfh) cell subpopulation participates in the pathogenesis of HT remains poorly understood.

Methods: Thirty healthy controls (HCs) and 52 HT patients were enrolled in the study. The percentages of Tfh, ICOS⁺Tfh, PD1⁺Tfh, Tfh1, Tfh2, Tfh17, effector Tfh, resting Tfh, effector memory Tfh, central memory Tfh, and naïve Tfh cells in the peripheral blood were all determined via flow cytometry, and the associations between the percentages of these cells and thyroid function indices were also investigated.

Results: The percentage of Tfh cells was significantly higher in HT patients than in HCs. Examination of the Tfh cell subsets revealed that the percentages of Tfh1, Tfh2, and resting Tfh cells were significantly decreased, while those of the ICOS⁺Tfh, PD1⁺Tfh, Tfh17, and effector Tfh cells were significantly increased in HT patients. No significant differences in effector memory, central memory or naïve Tfh cell percentages were noted between the HC and HT groups. Furthermore, the percentage of PD1⁺Tfh cells was positively correlated with anti-thyroglobulin antibody levels. Most importantly, only Tfh17 cell percentages were positively correlated with anti-thyroglobulin and anti-thyroid peroxidase antibody levels and were negatively correlated serum free T3 and free T4 levels in HT patients.

Conclusions: Increased circulating Tfh17 cell and PD1⁺Tfh percentages are associated with higher autoantibody levels in HT patients, which imply that Tfh17 or PD1⁺Tfh cells may play a pathogenic role in the development of HT.     

Activated inducible co-stimulator-positive programmed cell death 1-positive follicular helper T cells indicate disease activity and severity in ulcerative colitis patients

Inducible co-stimulator-positive (ICOS) and programmed cell death 1-positive (PD-1) are important markers for follicular helper T cells (Tfh); however, their roles and clinical values in ulcerative colitis (UC) remain unknown. In this study, we recruited 68 UC patients and 34 healthy controls. Circulating ICOS⁺, PD-1⁺ and ICOS⁺PD-1⁺ Tfh subsets were analyzed by flow cytometry. Twelve active UC patients achieving remission after treatment with 5-aminosalicylic acid were followed-up and Tfh subset changes were analyzed. Serum immunoglobulin (Ig)G, C-reactive protein (CRP), interleukin (IL)-4 and IL-21 levels and B cell subsets were analyzed and Mayo scores were calculated. Correlation analyses were performed between Tfh subsets and the clinical indicators. Receiver operating characteristic (ROC) curves were generated to evaluate the efficiency of Tfh subsets for disease monitoring. We found that levels of ICOS⁺, PD-1⁺ and ICOS⁺PD-1⁺ Tfh cells were significantly increased in active UC and significantly decreased when achieving clinical remission. Activated ICOS⁺PD-1⁺Tfh cells were positively correlated with serum CRP and Mayo scores. Furthermore, ICOS⁺PD-1⁺ Tfh cells were significantly correlated with circulating new memory B cells and plasmablasts, as well as serum IgG, IL-4 and IL-21. ROC analyses showed that when ICOS⁺PD-1⁺ Tfh cells were used in combination with PD-1⁺ Tfh cells, the diagnostic efficacy in distinguishing active UC from stable remission patients was higher than that of any one used alone, with area under curve (AUC) value 0·931. Our findings suggest that increased ICOS⁺PD-1⁺ Tfh cells are associated with the activation of B cells in the pathogenesis of UC, and may be a potential biomarker for UC disease monitoring.     

Tissue factor expression in rheumatoid synovium: a potential role in pannus invasion of rheumatoid arthritis

Angiogenesis, as well as pannus formation within the joint, plays an important role in the erosion of articular cartilage and bone in the pathological process of rheumatoid arthritis (RA). Tissue factor (TF), an essential initiator of the extrinsic pathway of blood coagulation, is also involved in the angiogenesis and the pannus formation of RA progression. In the present study, we used immunofluorescence and confocal scanning methods to characterize TF immunolocalization in RA synovium. We showed that positive staining of TF could be immunolocalized in synoviocytes, CD19⁺ B cells and CD68⁺ macrophages, whereas weak or negative staining of tissue factor could be found in CD34⁺ endothelial cells of neo-vessels, CD3⁺ T cells and CD14⁺ monocytes in RA synovium tissues. Our study demonstrates a detailed local expression of TF in the rheumatoid synovium, and supports the notion that TF, expressed not only by the synoviocytes themselves, but also the infiltrating CD19⁺ B cells and CD68⁺ macrophages, is involved in the pannus invasion in the progression of rheumatoid arthritis.     

CD4+CXCR5+ follicular helper T cells in salivary gland promote B cells maturation in patients with primary Sjogren’s syndrome

Objective: To examine amount of CD4⁺CXCR5⁺Tfh cells and B cells subsets in salivary gland and peripheral blood from patients with primary Sjogren’s syndrome (pSS) and to analyze whether the frequency of CD4⁺CXCR5⁺Tfh cells is associated with pSS pathologic process. Methods: The percentages of CD4⁺CXCR5⁺Tfh cells and B cell subsets were examined by flow cytometry. B-lymphocyte chemoattraetant (BLC; also called CXCL13), IL-21, IL-6 from the serum of pSS patients was assessed by polymerase chain reaction–enzyme-linked immunosorbent assay (ELISA). Results: The percentages of CD4⁺CXCR5⁺Tfh cells in peripheral blood were increased in pSS patients, but decreased after treatment with glucocorticoid and/or immunosuppressive drugs. Abnormal B cell subsets appeared in salivary and peripheral blood of pSS patients. The frequency of salivary CD4⁺CXCR5⁺Tfh cells was positively correlated with CD19⁺CD27⁺ memory B cells and CD19⁺CD27^high plasma cells. Also increase of salivary CD19⁺CD27^high plasma cells was positively associated with serum ANA titer of pSS patients. Conclusions: CD4⁺CXCR5⁺Tfh cells are significantly increased in salivary and peripheral blood in pSS patients with aberrant CD19⁺CD27⁺ memory B cells and CD19⁺CD27^high plasma cells, suggesting that CD4⁺CXCR5⁺Tfh cells may contribute to the pathogenesis of pSS by promoting the maturation of B cells.     

Contribution of FoxP3+ Tfr cells to overall human blood CXCR5+ T cells

The identification that T follicular helper (Tfh) cells is critical for the emergence of germinal centre responses prompted the study of CXCR5-expressing CD4⁺ T cell subsets in autoimmunity. However, circulating CXCR5-expressing T cells are heterogeneous by containing Forkhead box protein 3 (FoxP3)⁺ T follicular regulatory (Tfr) cells in addition to bona fide Tfh cells. Such heterogeneity may hamper the analysis of the contribution of specific follicular T cell subsets for autoimmune pathogenesis. Therefore, separate assessment of Tfh and Tfr populations offer greater opportunities for stratification of autoimmune patients, such as Sjögren’s syndrome patients.     

Dysregulation of Circulating Tfr/Tfh Ratio in Primary biliary cholangitis

Follicular helper T (Tfh) cells and follicular regulatory T (Tfr) cells are critical for the development and maintenance of germinal centre (GC) and humoral immune responses. Accumulating evidence has demonstrated that the dysregulation of either Tfh cells or Tfr cells contributes to the pathogenesis of autoimmune diseases. We aim to investigate the roles of circulating Tfh cells and circulating Tfr cells in the pathogenesis of primary biliary cholangitis (PBC). A total of 34 patients with PBC and 27 health individuals were enrolled in this study. Flow cytometry revealed that circulating Tfh (CD4⁺CXCR5⁺CD127^hiCD25^lo) cells were increased, but Tfr (CD4⁺CXCR5⁺CD127^loCD25^hi) cells and ratio of Tfr/Tfh were dramatically decreased in PBC patients compared with healthy controls. The Tfr/Tfh ratio was negatively correlated with level of serum IgM. Meanwhile, we also observed effector memory (CCR7^loPD‐1^hi) Tfh cells and Tfr cells were dramatically increased, but central memory (CCR7^hiPD‐1^lo) Tfh cells and Tfr cells were decreased in PBC patients compared with healthy controls. Effector memory Tfr cells were positively correlated with level of serum ALP. These results indicate that an imbalance of circulating Tfr cells and Tfh cells may be involved in the immunopathogenesis of PBC and may provide novel insight for the development of PBC therapies.     

Higher frequencies of circulating ICOS+, IL-21+ T follicular helper cells and plasma cells in patients with new-onset membranous nephropathy

Background: T follicular helper (TFH) cells and B cells are known to regulate humoral immune responses. This study is aimed at examining the putative contribution of different subsets of circulating of TFH cells and B cells to membranous nephropathy (MN).

Methods: A total of 45?MN patients and 19 healthy controls (HCs) were examined for the number of TFH cells and B cells by flow cytometry. The level of 24-h urinary protein and eGFR were calculated, and the level of serum cytokines was examined. The potential association among these measures was analyzed.

Results: Compared to the HCs, MN patients had significantly higher numbers of circulating CD4⁺CXCR5⁺, CD4⁺CXCR5⁺ICOS⁺, CD4⁺CXCR5⁺CD154⁺, CD4⁺CXCR5⁺IL-21⁺, and CD4⁺CXCR5⁺CD28⁺ TFH cells, as well as IgD⁺CD27^?CD19⁺ and CD138⁺CD19⁺ B cells. However, the number of IgD⁺CD27⁺CD19⁺ B cells was significantly lower in MN patients than in the HC. The levels of serum IL-21, IL-2, IL-4, IL-10, IL-17A, and IFN-γ were significantly higher in MN patients than in the HC. Furthermore, the numbers of CD4⁺CXCR5⁺, CD4⁺CXCR5⁺ICOS⁺, CD4⁺CXCR5⁺CD154⁺, CD4⁺CXCR5⁺IL-21⁺, CD4⁺CXCR5⁺CD28⁺ TFH cells, CD138⁺CD19⁺ B cells, and the level of sera IL-21 were negatively correlated with the values of eGFR, but positively correlated with the levels of 24-h urinary proteins. Following treatment, the numbers of CD4⁺CXCR5⁺, CD4⁺CXCR5⁺ICOS⁺, CD4⁺CXCR5⁺CD154⁺, CD4⁺CXCR5⁺IL-21⁺, CD4⁺CXCR5⁺CD28⁺ TFH cells, CD138⁺CD19⁺ B cells, and the levels of IL-21 were significantly reduced. In contrast, IL-4 and IL-10 levels were noticeably increased after treatment.

Conclusions: Data suggest that activated TFH and plasma cells may contribute to the pathogenesis of MN.     

The expression and anatomical distribution of BTLA and its ligand HVEM in rheumatoid synovium

Co-inhibitory signaling from B and T lymphocyte attenuator (BTLA) can suppress lymphocyte activation and maintain peripheral tolerance. However, the expression and anatomical distribution of BTLA and its ligand, herpesvirus entry mediator (HVEM), in rheumatoid arthritis (RA) synovium have not been reported. In this study, we analyzed the expression of HVEM and BTLA in RA synovium by immunohistochemistry, and our results showed that both factors were observed in all four cases of RA samples. At the cellular level, both HVEM and BTLA were found on the cell membrane and in the cytoplasm. Fluorescence dual staining demonstrated that HVEM was chiefly on CD3⁺ T cells, CD68⁺ macrophages, and to a lesser extent was found on CD31⁺ endothelial cells. Similarly, the expression of BTLA was observed on infiltrated CD3⁺ T cells and CD68⁺ macrophages. The co-expression of HVEM and BTLA with some members of the B7 family in these sections was also analyzed, and the results showed that HVEM antigen was also found on B7-H3⁺ capillaries, while it was absent on B7-H1⁺, B7-DC⁺, B7-H4⁺, and Z39Ig⁺ cells. Interestingly, BTLA was observed on B7-H1⁺, B7-H4⁺, and HVEM⁺ cells in the synovium. The characteristic expression and distribution of BTLA/HVEM in the synovium indicated that their signaling probably affects the pathogenesis of RA, and a clear understanding of their functional roles may further elucidate the pathogenesis of this disease.     

End‐stage renal disease,dialysis, kidney transplantation and their impact on CD4+ T‐cell differentiation

Premature aging of both CD4⁺ regulatory T (Treg) and CD4⁺ responder‐T (Tresp) cells in patients with end‐stage renal disease (ESRD) is expected to affect the success of later kidney transplantation. Both T‐cell populations are released from the thymus as inducible T‐cell co‐stimulator‐positive (ICOS⁺) and ICOS^? recent thymic emigrant (RTE) Treg/Tresp cells, which differ primarily in their proliferative capacities. In this study, we analysed the effect of ESRD and subsequent renal replacement therapies on the differentiation of ICOS⁺ and ICOS^? RTE Treg/Tresp cells into ICOS⁺ CD31^? or ICOS^? CD31^? memory Treg/Tresp cells and examined whether diverging pathways affected the suppressive activity of ICOS⁺ and ICOS^? Treg cells in co‐culture with autologous Tresp cells. Compared with healthy controls, we found an increased differentiation of ICOS⁺ RTE Treg/Tresp cells and ICOS^? RTE Treg cells through CD31⁺ memory Treg/Tresp cells into CD31^? memory Treg/Tresp cells in ESRD and dialysis patients. In contrast, ICOS^? RTE Tresp cells showed an increased differentiation via ICOS^? mature naive (MN) Tresp cells into CD31^? memory Tresp cells. Thereby, the ratio of ICOS⁺ Treg/ICOS⁺ Tresp cells was not changed, whereas that of ICOS^? Treg/ICOS^? Tresp cells was significantly increased. This differentiation preserved the suppressive activity of both Treg populations in ESRD and partly in dialysis patients. After transplantation, the increased differentiation of ICOS⁺ and ICOS^? RTE Tresp cells proceeded, whereas that of ICOS⁺ RTE Treg cells ceased and that of ICOS^? RTE Treg cells switched to an increased differentiation via ICOS^? MN Treg cells. Consequently, the ratios of ICOS⁺ Treg/ICOS⁺ Tresp cells and of ICOS^? Treg/ICOS^? Tresp cells decreased significantly, reducing the suppressive activity of Treg cells markedly. Our data reveal that an increased tolerance‐inducing differentiation of ICOS⁺ and ICOS^? Treg cells preserves the functional activity of Treg cells in ESRD patients, but this cannot be maintained during long‐term renal replacement therapy.     

IL-10-Expressing Th2 Cells Contribute to the Elevated Antibody Production in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a common autoimmune disease associated with progressive disability, systemic complications, and early death. Multiple lines of evidence have placed adaptive immune responses in the center of RA pathogenesis. However, the functional roles of T helper cells are insufficiently described. Here, we examined the Th2 cell subsets and their functions in RA patients. A downregulation of IL-4⁺ cells in CD4⁺ T cells were observed in RA patients, indicating a downregulation of Th2 cells, and these results were confirmed by using and CXCR3 and CCR6 surface markers. We then found that CXCR3^-CCR6^? Th2 cells can be separated into IL-4⁺ (single positive), IL-10⁺ (single positive), and IL-4⁺IL-10⁺ (double positive) subsets. Further results showed that CXCR5 only expressed on IL-10+ Th2 cells. The CXCR5⁺ and CXCR5^? Th2 cells each exhibited distinctive features in helping B cell antibody secretion. CXCR5⁺ Th2 cells were more potent at stimulating total Ig and IgM secretion, while CXCR5^? Th2 cells were more potent at stimulating IgE. IL-10 was required for helping B cell total Ig, IgM, and IgE production, while IL-4 was required for total Ig and IgE. The frequencies of IL-10⁺ and IL-4⁺IL-10⁺ Th2 cells were positively correlated with rheumatoid factor titer in vivo. Together, our study demonstrated distinctive subsets within Th2 cells, each with different impacts on antibody production and RA disease.     

T follicular‐like helper cells in the peripheral blood of patients with primary Sjögren's syndrome

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease characterized by exocrine gland dysfunction, mainly causing sicca symptoms. B cells have a prominent role in SS, and the T follicular helper (T_FH) cells provide B cells with survival and specialization signals in germinal centres. Here, we investigate peripheral T_FH cells in pSS. Sixteen pSS patients and healthy controls were enrolled in the study, with 13 women and 3 men in each group. Whole blood was collected and separated into PBMC and plasma, followed by cryopreservation. Plasma samples were analysed for Ro52, Ro60 and La48 autoantibodies by indirect ELISA. For flow cytometric analysis, we defined 4 subsets of TFH‐like cells within the CD3⁺CD4⁺CXCR5⁺ population, namely the ICOS⁻PD‐1⁻, ICOS⁻PD‐1⁺, ICOS⁺PD‐1⁻ and ICOS⁺PD‐1⁺ (“TFH”) cells. We also investigated 4 CD19⁺ B cell subsets, the CD20⁺CD27⁺CD38⁻ memory B cells, CD20⁺CD27⁺CD38⁺ memory B cells, CD20⁻CD27⁺CD38⁺⁺CD138⁻ plasmablasts and CD20⁻CD27⁺CD38⁺⁺CD138⁺ plasma cells. We observed higher fractions of ICOS⁺PD‐1⁻ cells, ICOS⁺PD‐1⁺ (“T_FH”) cells and plasmablasts in pSS patients compared to controls, and lower frequencies of both types of memory B cells. The number of T_FH cells correlated positively with the levels of plasmablasts and plasma cells in the pSS patients, but not in the controls. The pSS patients were stratified according to Ro52/Ro60/La48 serology, and a positive association was found between autoantibody levels and increased level of T_FH cells, plasmablasts and plasma cells and lowered levels of memory B cells. We observed a higher response to Ro/La stimulation in pSS patients compared to controls of the memory B cells, although only significantly for the CD38⁻ memory B cells. Overall, a pathological relation between the ICOS⁺ T follicular‐like helper cells and B cells in pSS was observed, but further work should be conducted to explore their overall impact upon disease progression.     

Peripheral and Site‐Specific CD4+CD28null T Cells from Rheumatoid Arthritis Patients Show Distinct Characteristics

Proinflammatory CD4⁺CD28^null 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⁺CD28^null 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⁺CD28^null 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‐γ, 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⁺CD28^null 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‐γ and TNF after TCR cross‐linking. CD4⁺CD28^null 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⁺CD28^null cells from the blood, a significant production was observed in CD4⁺CD28^null T cells from synovial fluid. CD4⁺CD28^null 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⁺CD28^null 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 CD28^null population.