CD4+CD25+LAG3+ T Cells With a Feature of Th17 Cells Associated With Systemic Lupus Erythematosus Disease Activity.

Systemic lupus erythematosus (SLE) is an autoimmune disease that involves multiple immune cell subsets. We analyzed immune cell subsets in human peripheral blood mononuclear cells (PBMC) in order to identify the cells that are significantly associated with SLE disease activity and treatment. The frequencies of various subsets of CD4+ T cells, B cells, monocytes and NK cells in PBMC were assessed in 30 healthy controls (HC), 30 rheumatoid arthritis (RA) patients and 26 SLE patients using flow cytometry. The correlations between subset frequencies in SLE and clinical traits including Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) were examined. Changes in subset frequencies after the treatment in SLE patients were investigated. We focused on CD25+LAG3+ T cells and investigated their characteristics, including cytokine secretion, mRNA expression and suppression capacity. We assessed correlations between CD25+LAG3+ T cells and SLEDAI by Spearman's rank correlation coefficient. CD25+LAG3+ T cells were significantly increased in SLE whereas there were few in RA and HC groups. CD25+LAG3+ T cell frequencies were significantly correlated with SLEDAI and were increased in patients with a high SLEDAI score (> 10). CD25+LAG3+ T cells produced both IL-17 and FOXP3, expressed mRNA of both FOXP3 and RORC and lacked suppressive capacity. CD25+LAG3+ T cells were associated with disease activity of SLE. CD25+LAG3+ T cells had features of both CD25+FOXP3+ regulatory T cells (CD25+ Treg) and Th17. CD25+LAG3+ T cells could be associated with the inflammatory pathophysiology of SLE.

A gene module associated with dysregulated TCR signaling pathways in CD4+ T cell subsets in rheumatoid arthritis.

We analyzed the transcriptome of detailed CD4+ T cell subsets including them after abatacept treatment, and examined the difference among CD4+ T cell subsets and identified gene sets that are closely associated disease activity and abatacept treatment. Seven CD4+ T cell subsets (naive, Th1, Th17, Th1/17, nonTh1/17, Tfh and Treg) were sorted from PBMCs taken from 10 RA patients and 10 healthy controls, and three RA patients donated samples before and 6 months after abatacept treatment. Paired-end RNA sequencing was performed using HiSeq 2500. A total of 149 samples except for 12 outliers were analyzed. Overview of expression pattern of RA revealed that administration of abatacept exerts a large shift toward the expression pattern of HC. Most of differentially expressed gene (DEG) upregulated in RA (n = 1776) were downregulated with abatacept treatment (n = 1349). Inversely, most of DEG downregulated in RA (n = 1860) were upregulated with abatacept treatment (n = 1294). This DEG-based analysis revealed shared pathway changes in RA CD4+ T cell subsets. Knowledge-based pathway analysis revealed the upregulation of activation-related pathways in RA that was substantially ameliorated by abatacept. Weighted gene co-expression network analysis (WGCNA) evaluated CD4+ T cells collectively and identified a gene module that consisted of 227 genes and was correlated with DAS28-CRP (Spearman's rho = 0.46, p = 4 × 10-9) and abatacept administration (Spearman's rho = -0.91, p = 5 × 10-57). The most highly connected 30 genes of this module included ZAP70 and JAK3, and pathway analysis of this module revealed dysregulation of the TCR signaling pathway network, which was ameliorated by abatacept.

Interleukin-10-producing LAG3+ regulatory T cells are associated with disease activity and abatacept treatment in rheumatoid arthritis.

BACKGROUND: Regulatory T cells (Tregs) play a role in the suppression of inflammation in autoimmune diseases, and lymphocyte activation gene 3 (LAG3) was reported as a marker of interleukin (IL)-10-producing Tregs. We aimed to clarify the function of human IL-10-producing CD4+CD25-LAG3+ T cells (LAG3+ Tregs) and their association with rheumatoid arthritis (RA). METHODS: LAG3+ Tregs of human peripheral blood mononuclear cells (PBMCs) were cultured with B cells and follicular helper T cells to examine antibody suppression effects. The frequency of LAG3+ Tregs was evaluated in peripheral blood samples from 101 healthy donors and 85 patients with RA. In patients treated with abatacept, PBMC samples were analyzed before and after treatment. Naive CD4+ T cells were sorted and cultured in the presence of abatacept, followed by flow cytometric analysis and function assays. RESULTS: LAG3+ Tregs produced high amounts of IL-10 and interferon-γ, and they suppressed B-cell antibody production more strongly than CD25+ Tregs. Cell-to-cell contact was required for the suppressive function of LAG3+ Tregs. The frequency of LAG3+ Tregs was lower in patients with RA, especially those with higher Clinical Disease Activity Index scores. LAG3+ Tregs significantly increased after 6 months of abatacept treatment, whereas CD25+ Tregs generally decreased. Abatacept treatment in vitro conferred LAG3 and EGR2 expression on naive CD4+ T cells, and abatacept-treated CD4+ T cells exhibited suppressive activity. CONCLUSIONS: IL-10-producing LAG3+ Tregs are associated with the immunopathology and therapeutic response in RA. LAG3+ Tregs may participate in a mechanism for the anti-inflammatory and immune-modulating effects of targeted therapy for costimulation.

Polygenic burdens on cell-specific pathways underlie the risk of rheumatoid arthritis.

Recent evidence suggests that a substantial portion of complex disease risk alleles modify gene expression in a cell-specific manner. To identify candidate causal genes and biological pathways of immune-related complex diseases, we conducted expression quantitative trait loci (eQTL) analysis on five subsets of immune cells (CD4+ T cells, CD8+ T cells, B cells, natural killer (NK) cells and monocytes) and unfractionated peripheral blood from 105 healthy Japanese volunteers. We developed a three-step analytical pipeline comprising (i) prediction of individual gene expression using our eQTL database and public epigenomic data, (ii) gene-level association analysis and (iii) prediction of cell-specific pathway activity by integrating the direction of eQTL effects. By applying this pipeline to rheumatoid arthritis data sets, we identified candidate causal genes and a cytokine pathway (upregulation of tumor necrosis factor (TNF) in CD4+ T cells). Our approach is an efficient way to characterize the polygenic contributions and potential biological mechanisms of complex diseases.

Enhanced gut homing receptor expression of unswitched memory B cells in rheumatoid arthritis.

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Identification of tonsillar CD4+CD25-LAG3+ T cells as naturally occurring IL-10-producing regulatory T cells in human lymphoid tissue.

IL-10-producing regulatory T cells (IL-10-producing Tregs) are one of the regulatory T cell subsets characterized by the production of high amounts of IL-10, the lack of FOXP3 expression and the strong immunosuppressive capabilities. IL-10-producing Tregs have been primarily reported as induced populations thus far, in part because identifying naturally occurring IL-10-producing Tregs was difficult due to the lack of definitive surface markers. Lymphocyte-activation gene 3 (LAG3) is a CD4 homologue that we have identified as being expressed on IL-10 producing Tregs. In human PBMC, LAG3 combined with CD49b efficiently identifies IL-10-producing Tregs. However, naturally occurring IL-10-producing Tregs in human secondary lymphoid tissue have not been described. In this report, we identified CD4+CD25-LAG3+ T cells in human tonsil. This T cell subset produced high amounts of IL-10 and expressed low levels of FOXP3. Surface markers and microarray analysis revealed that this is a distinct tonsillar CD4+ T cell subset. CD4+CD25-LAG3+ T cells expressed interleukin 10 (IL10), PR/SET domain 1 (PRDM1), and CD274 at high levels and chemokine receptor 5 (CXCR5) at low levels. CD4+CD25-LAG3+ T cells suppressed antibody production more efficiently than CD4+CD25+ T cells, and CD4+CD25-LAG3+ T cells induced B cell apoptosis. Moreover, analysis of humanized mice revealed that this cell subset suppressed a graft-versus-host disease (GVHD) reaction in vivo. Our study reveals the existence of naturally occurring IL-10-producing Tregs in human secondary lymphoid tissue and their function in immune regulation.

Efficacy of intensive immunosuppression in exacerbated rheumatoid arthritis-associated interstitial lung disease.

OBJECTIVES: Acute or subacute exacerbations are recognized as a severe complication of rheumatoid arthritis-associated interstitial lung disease (RA-ILD). Nevertheless, the role of intensive immunosuppression in RA-ILD remains elusive. We attempted to evaluate the clinical characteristics and efficacy of immunosuppressive treatment in exacerbated RA-ILD. METHODS: Clinical data, including respiratory function, imaging, treatment, and prognosis, were retrospectively collected for 17 patients with RA-ILD who required hospitalization at the University of Tokyo Hospital due to an acute exacerbation (12 patients) or subacute exacerbation (5 patients). RESULTS: Patients with RA-ILD demonstrated a significantly higher titers of anticyclic citrullinated peptide antibodies compared with RA patients in Japanese Ninja registry, suggesting the role of adaptive immunity. Immunosuppressive treatment suppressed the deterioration of pulmonary functions with improved ground grass opacity and consolidation. In particular, in patients with less fibrosis on computed tomography (CT) images showed a better response to treatment. Although five patients treated with combination therapy, including cyclophosphamide, showed a severely decreased lung volume, these intensive therapies provided a good prognosis without fatalities for the average observation period of 474 days. CONCLUSIONS: Immunosuppressive therapy is effective for exacerbations of RA-ILD. For severe cases with low respiratory function, intensive therapy, including cyclophosphamide, has a potential to improve the prognosis.

Immunophenotyping of rheumatoid arthritis reveals a linkage between HLA-DRB1 genotype, CXCR4 expression on memory CD4(+) T cells, and disease activity.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that leads to destructive arthritis. Although the HLA class II locus is the strongest genetic risk factor for rheumatoid arthritis, the relationship between HLA class II alleles and lymphocyte activation remains unclear. We performed immunophenotyping of peripheral blood mononuclear cells on 91 HLA-DRB1-genotyped RA patients and 110 healthy donors. The frequency of memory CXCR4(+)CD4(+) T cells, and not Th1 and Th17 cells, was significantly associated with disease severity by multiple linear regression analysis. RA patients with one or more susceptible HLA-DR haplotypes (shared epitope: SE) displayed a significantly higher frequency of memory CXCR4(+)CD4(+) T cells. Moreover, the frequency of memory CXCR4(+)CD4(+) T cells significantly correlated with the expression level of HLA-DR on B cells, which was elevated in RA patients with SE. In vitro analysis and transcriptomic pathway analysis suggested that the interaction between HLA-DR and T cell receptors is an important regulator of memory CXCR4(+)CD4(+) T cells. Clinically, a higher frequency of memory CXCR4(+)CD4(+) T cells predicted a better response to CTLA4-Ig. Memory CXCR4(+)CD4(+) T cells may serve as a powerful biomarker for unraveling the linkage between HLA-DRB1 genotype and disease activity in RA.

TGF-ß3-expressing CD4+CD25(-)LAG3+ regulatory T cells control humoral immune responses.

Autoantibodies induce various autoimmune diseases, including systemic lupus erythematosus (SLE). We previously described that CD4(+)CD25(-)LAG3(+) regulatory T cells (LAG3(+) Treg) are regulated by Egr2, a zinc-finger transcription factor required for the induction of T-cell anergy. We herein demonstrate that LAG3(+) Treg produce high amounts of TGF-ß3 in an Egr2- and Fas-dependent manner. LAG3(+) Treg require TGF-ß3 to suppress B-cell responses in a murine model of lupus. Moreover, TGF-ß3- and LAG3(+) Treg-mediated suppression requires PD-1 expression on B cells. We also show that TGF-ß3-expressing human LAG3(+) Treg suppress antibody production and that SLE patients exhibit decreased frequencies of LAG3(+) Treg. These results clarify the mechanism of B-cell regulation and suggest therapeutic strategies.

Serum ß2-microglobulin level is a useful indicator of disease activity and hemophagocytic syndrome complication in systemic lupus erythematosus and adult-onset Still's disease.

This study demonstrates whether serum ß2-microglobulin (ß2-MG) level can be an indicator of the status of systemic lupus erythematosus (SLE) and adult-onset Still's disease (AOSD), and development of hemophagocytic syndrome (HPS) complication. Serum ß2-MG level was compared between the active and inactive statuses of SLE and AOSD in hospitalized patients. Active status was defined as a state for which a therapy was introduced. Serum ß2-MG level was also compared between patients with and without HPS complication. HPS was diagnosed on the basis of clinical and pathological findings. Laboratory markers of HPS including peripheral blood cell counts and levels of serum lactate dehydrogenase (LDH), serum ferritin, plasma fibrin/fibrinogen degradation product (FDP), and plasma D-dimer were examined to determine their correlations with serum ß2-MG level. Sixteen SLE and seven AOSD patients (all females, aged 39.0 ± 16.4) were included. The serum ß2-MG level was high in the active status of underlying diseases and decreased significantly after the therapy (3.5 ± 1.4 vs. 2.1 ± 0.8 mg/L, p < 0.001). Among patients with active status, the ß2-MG level was higher in patients with HPS (two with SLE and three with AOSD) than in patients without HPS (4.9 ± 1.8 vs. 3.3 ± 1.4 mg/L, p < 0.05). Serum ß2-MG level significantly correlated with the levels of serum LDH (r(s) = 0.42, p < 0.05), plasma FDP (r(s) = 0.58, p < 0.05), and plasma D-dimer (r(s) = 0.77, p < 0.01). Serum ß2-MG level would be a useful indicator of disease activity and development of HPS complication in patients with SLE and AOSD.

Eleven cases of angioedema with eosinophilia treated in a single hospital in Japan.

BACKGROUND: Angioedema with eosinophilia (AE) is mostly reported in Japanese patients, and only as case reports. In this study, we aimed to determine how prevalent AE cases appear, the characteristic features and the course of AE, and to evaluate whether corticosteroid therapy for AE is necessary or not. METHODS: The patients whose blood samples showed an eosinophil count of ≥2,000/µL, among the samples tested for blood cell counts and differential counts between January 2006 and December 2010, in Japanese Red Cross Medical Center, were firstly included. Among these, patients with AE were extracted. RESULTS: All of the 11 patients were Japanese young females. One patient with arthralgia showed radioisotope accumulation in the joints by bone scintigraphy. The peak peripheral blood eosinophil count was 7,839 ± 6,008 (2,130-23,170)/µL after visiting our hospital. An increase in white blood cell count was only due to an increase in eosinophil count. Serum C-reactive protein and IgE levels remained almost normal. Peripheral blood eosinophil count decreased steadily for 8 weeks, regardless of corticosteroid use. Edema in all of the patients and arthralgia in 6 patients improved within 12 weeks. As far as followed, none of the patients had a recurrence of AE. CONCLUSIONS: AE developed in Japanese young females and likely showed a single course. In AE, the count of eosinophil of 104/µL was observed. Only eosinophil count increased among leukocyte series. Serum C-reactive protein and IgE levels remained almost normal. The eosinophil count in AE patients will return to the normal level within 8 weeks even without corticosteroid therapy.

Serum KL-6 level as an indicator of active or inactive interstitial pneumonitis associated with connective tissue diseases.

OBJECTIVE: To elucidate the cut off levels of serum KL-6 indicating patients with interstitial pneumonitis (IP) and patients with active IP associated with connective tissue diseases (CTDs). METHODS: CTD patients whose serum KL-6 level was measured were included. IP was diagnosed on the basis of medical records including XP/CT findings, and active IP was assumed in case that intervention for IP was newly added. The cut off levels were determined by receiver operating characteristic (ROC) curve analysis. RESULTS: Among 240 (174 females) patients, 67 (42) had IP and 15 (9) had active IP. The ages of patients with and without IP, and with active IP and with inactive IP were 70.3±9.5 and 62.8±15.3, and 72.8±8.1 and 69.6±9.8, respectively. IP was significantly more prevalent in males and the elderly. The KL-6 levels were 990±90 and 301±12 U/mL in patients with and without IP, and 1,905±236 and 726±54 U/mL in those with active IP and with inactive IP, respectively. ROC curve analysis showed a cut off level of 509 U/mL for indicating IP, and that of 1,051-1,060 U/mL for indicating active IP. CONCLUSION: A serum KL-6 level of higher than 500 U/mL is a marker of the presence of IP, and a level of higher than 1,000 U/mL is a marker of the presence of active IP associated with CTDs.