Published papers

Objective: To clarify relevant proteins and clinical characteristics of a phenotype of IgG4-related disease (IgG4-RD) with lymphadenopathy. Methods: We enrolled patients newly diagnosed with IgG4-RD in our department between January 2000 and June 2018 and performed proteomic analysis to measure serum concentrations of 1305 proteins. We extracted proteins overexpressed in patients with IgG4-RD with lymphadenopathy by comparing between those with lymphadenopathy, those without lymphadenopathy and healthy controls. We further reviewed all the patients with IgG4-RD in our institution and investigated the characteristics and prognosis of the patients with IgG4-RD with lymphadenopathy. Results: Eighty-five patients with IgG4-RD were enrolled, of which, 55% had lymphadenopathy. Proteomic analysis in 31 patients with IgG4-RD and 6 healthy controls revealed that eotaxin-3 was a potential serum biomarker in the patients with lymphadenopathy versus those without lymphadenopathy and healthy controls. A cohort of 85 patients with IgG4-RD demonstrated that patients with lymphadenopathy showed a significantly higher serum IgG4, IgG4:IgG ratio, IgG4-RD responder index and eosinophilia (P < 0.001 for all), irrelevant of the extent to which organ involvement developed. Patients with lymphadenopathy treated with glucocorticoid alone relapsed with significantly higher rates than those without lymphadenopathy (P = 0.03). Conclusion: Lymphadenopathy in IgG4-RD represents a phenotype associated with high disease activities, eosinophilia and relapsing disease. Eotaxin-3 is a novel biomarker related to IgG4-RD with lymphadenopathy.

T cells play important roles in autoimmune diseases, but it remains unclear how to optimally manipulate them. We focused on the T cell immunoreceptor with Ig and ITIM domains (TIGIT), a coinhibitory molecule that regulates and is expressed in T cells. In autoimmune diseases, the association between TIGIT-expressing cells and pathogenesis and the function of human-TIGIT (hu-TIGIT) signalling modification have not been fully elucidated. Here we generated anti-hu-TIGIT agonistic monoclonal antibodies (mAbs) and generated hu-TIGIT knock-in mice to accurately evaluate the efficacy of mAb function. Our mAb suppressed the activation of CD4+ T cells, especially follicular helper T and peripheral helper T cells that highly expressed TIGIT, and enhanced the suppressive function of naïve regulatory T cells. These results indicate that our mAb has advantages in restoring the imbalance of T cells that are activated in autoimmune diseases and suggest potential clinical applications for anti-hu-TIGIT agonistic mAbs as therapeutic agents.

T peripheral helper (Tph) cells are thought to contribute to extra-follicular B cell activation and play a pathogenic role in autoimmune diseases. However, the role of Tph subsets is not fully elucidated. Here, we investigate the immunological functions of Tph subsets and their involvement in systemic lupus erythematosus (SLE). We have defined four Tph subsets (Tph1: CXCR3+CCR6−, Tph2: CXCR3−CCR6−, Tph17: CXCR3−CCR6+, and Tph1-17: CXCR3+CCR6+) and performed RNA sequencing after cell sorting. Tph1 and Tph17 subsets express substantial levels of IL21, indicating B cell helper functions. However, Tph2 and Tph1-17 subsets express low IL21. Interestingly, we have found Tph2 subset express high levels of CX3CR1, GZMB, PRF1, GLNY, S1PR5, TBX21, EOMES, ZNF863, and RUNX3, indicating a feature of CD4+ cytotoxic T lymphocytes. In SLE patients, the frequency of Tph1 and Tph2 subsets are significantly increased and positively correlated with SLE disease activity indexes. Tph1 cells expansion has been observed in patients with cutaneous and musculoskeletal manifestations. On the other hand, Tph2 cell expansion has been found in patients with lupus nephritis in addition to the above manifestations. Our findings imply that Tph1 and Tph2 subsets exert distinct immunological functions and are contributed to the complexity of clinical manifestations in SLE.

Objective To assess differences in the strength of inhibition of IL-6/STAT3 signalling induced by subcutaneously (sc) administered tocilizumab (TCZ) and sarilumab (SAR). Methods Data were collected on patients with rheumatoid arthritis (RA) who achieved low disease activity (Clinical Disease Activity Index [CDAI]≤10) following treatment with weekly or bi-weekly administration of 162 mg sc of TCZ (TCZ qw group, n=8; TCZ q2w group, n=8), bi-weekly doses of 200 mg sc of SAR (SAR q2w group, n=7), or MTX (n=8) as a control. The clinical characteristics of each group were collected, and the serum concentrations of IL-6 and soluble IL-6 receptor (sIL-6R) were measured using ELISA. Whole blood samples from each group were stimulated with 100 ng/ml of IL-6. The proportion of phosphorylated (p)STAT3-positive CD4+ T cells was measured using phosflow cytometric analysis. Results The proportion of pSTAT3-positive CD4+ T cells following stimulation with 100 ng/ml of recombinant human IL-6 was significantly different among the groups (median 1.8% [0.9–3.0] vs. 7.7% [2.9–8.0] vs. 12.5% [11.4–16.6] vs. 71.5% [68.0-78.5] for the TCZ qw, SAR q2w, TCZ q2w, and MTX control groups, respectively; p<0.01 for all comparisons). Conclusion SAR 200 mg q2w showed significantly stronger inhibition of IL-6/STAT3 signalling than TCZ sc q2w but weaker inhibition than TCZ sc qw. The results of this study may be useful for adjusting the IL-6 blockade treatment for patients with RA.

Objectives: T cells adhere to enthesis fibrocartilage via integrins and intrinsically require IL-17RA-mediated signals to maintain their effector function. We analysed CD29+IL-17RA+ T cells in inflamed lesions and peripheral blood in patients with SpA and investigated their association with disease activity and therapeutic response. Methods: Transcriptome analysis of synovial fluid T cells from PsA was performed using publicly available bulk cell RNA sequencing data. Blood samples were obtained from healthy controls (n = 37), RA (n = 12), IgG4-related disease (IgG4-RD; n = 12), large vessel vasculitis (LVV; n = 12) and SpA (n = 28) and were analysed by flow cytometry. Results: T cells in the inflamed joints of PsA showed CD29 and IL-17RA expression. CD29+IL-17RA+ T cells showed enriched CXCR3+CD45RA+ effector cells and activation of spleen tyrosine kinase (Syk), nuclear factor κB (NF-κB) and Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathways. The proportion of peripheral blood CD29+IL-17RA+ T cells was significantly increased in patients with SpA compared with patients with RA, IgG4-RD or LVV and in healthy controls. Based on the ASDAS-CRP scores, the proportion of CD29+IL-17RA+ T cells was positively correlated with disease activity in treatment-naïve patients with active SpA. Anti-IL-17 but not anti-TNF monoclonal antibodies reduced CD29+IL-17RA+ T cells. Conclusions: CD29+IL-17RA+ T effector cells with enhanced Syk, NF-κB and JAK-STAT pathways were specifically increased in SpA and were correlated with disease activity, implicating a role of this newly identified T cell population in the pathogenesis. Anti-IL-17 monoclonal antibodies may be effective for patients by reducing this pathogenic T cell population.

Background: Efficacy of combination therapy with methotrexate and biological disease-modifying antirheumatic drugs is well established in the management of patients with rheumatoid arthritis; however, the optimal dose of methotrexate to administer with a tumour necrosis factor inhibitor remains unclear. We aimed to clarify the efficacy and safety of adalimumab combined with reduced methotrexate dose compared with the maximum tolerated methotrexate dose in patients with rheumatoid arthritis and an inadequate response to methotrexate monotherapy. Methods: In this open-label, randomised controlled trial, we recruited methotrexate-naive patients with rheumatoid arthritis and a disease duration of less than 2 years across 24 secondary or tertiary care hospitals across Japan, South Korea, and Taiwan. At initiation, methotrexate was given orally and increased to the maximum tolerated dose by week 12. Patients who did not achieve remission on the basis of the Simplified Disease Activity Index (SDAI) at week 24 were randomly assigned (1:1) to receive adalimumab (40 mg biweekly) combined with a continued maximum tolerated dose of methotrexate or adalimumab combined with a reduced dose of methotrexate. The primary endpoint was non-inferiority of adalimumab plus reduced-dose methotrexate to adalimumab plus maximal-dose methotrexate based on SDAI remission at week 48, assessed in the modified full-analysis set with a pre-specified non-inferiority margin of −15%, based on a two-sided 90% CI. Adverse events were assessed in the safety analysis set. This trial is registered with ClinicalTrials.gov, NCT03505008 and has been completed. Findings: From April 18, 2018, to June 2, 2020, from 323 patients screened, 300 were enrolled, and 291 patients were included in the full analysis set. The mean age was 57·7 years (SD 15·2), 217 (75%) were female, 74 (25%) were male, and all patients were of Asian ethnicity. The mean SDAI at study enrolment was 26·5 (SD 12·4). 52 patients discontinued the study before week 24 or at week 24 before randomisation. At week 24, 105 (36%) of 291 patients achieved remission and continued methotrexate monotherapy through week 48. 134 (46%) did not achieve remission at week 24 and were randomly assigned to receive adalimumab plus the maximum tolerated dose of methotrexate (n=68) or adalimumab plus reduced-dose methotrexate (n=66). Remission at week 48 was achieved in 25 (38%) of 66 and 27 (44%) of 61 patients, respectively, with an adjusted risk difference of 6·4% (90% CI −7·0 to 19·8), which met the non-inferiority margin of −15%. Adverse events after week 24 tended to be more frequent in the maximum tolerated dose group than in the reduced-dose group (24 [35%] vs 13 [20%], p=0·054). Between week 24 and 48, there were 14 serious adverse events (6 in the methotrexate monotherapy group, 5 in the adalimumab plus maximal-dose methotrexate, and 3 in the adalimumab plus reduced-dose methotrexate group), and no deaths. Interpretation: The MIRACLE study showed that the efficacy of adalimumab combined with reduced methotrexate dose was not inferior to that with the maximum tolerated methotrexate dose, with a tendency to a better safety profile. Funding: Eisai.

Objectives: Anti-IL-6 receptor antibodies are clinically efficacious in the management of RA with an associated increase in Tregs; however, the role of functional Treg subsets has yet to be clarified. This study aimed to evaluate how functional Treg subsets are altered by IL-6 receptor blockade and to analyse the relationship between these Treg subsets and the clinical outcome of RA. Methods: We collected frozen peripheral blood mononuclear cells (PBMCs) from 40 patients with RA who started tocilizumab (TCZ) with or without MTX and 11 healthy controls (HCs). We fractionated Tregs with flow cytometry based on markers of phenotype and function and measured the proportions of detailed Treg subsets sequentially from baseline to week 52. Results: The proportions of resting Tregs (rTregs) and rTregs+activated Tregs (aTregs) were significantly lower in RA patients at baseline than in HCs. The proportions of all those CD127low Tregs, rTregs, aTregs and rTregs+aTregs were significantly increased with TCZ treatment. In patients treated with TCZ without MTX, rTreg were increased. Patients with an increase in the proportion of rTregs at week 12 had significantly less arthritis flares during the observation period. Conclusions: Blocking the IL-6 receptor with TCZ increased the proportion of rTregs, a functional Treg subpopulation. Patients with an early increase in rTregs showed a favourable treatment course and this increase in rTregs may reflect molecular remission induced by IL-6 signal inhibition.

Objective: We focused to analyze the time-course changes at pre- and post-flare of T peripheral helper (Tph) cells and circulating T follicular helper (Tfh) cells in the blood of patients with systemic lupus erythematosus (SLE) with lupus low disease activity state (LLDAS) before flare. Methods: This study included inactive (n = 29) and active (n = 55) patients with SLE. Tph subsets, Tfh subsets, CD11chi B cells, and plasma cells in the blood were determined by flow cytometry. The blood levels of cytokines including interferons (IFNs) were measured by electrochemiluminescence assay or cytokine beads array. Results: Active SLE patients exhibited the increased frequency of Tph1, Tph2, Tfh1, and Tfh2 subsets when compared to inactive patients, but no clear changes in the other subsets. During the treatment with medications, Tph1, Tph2, and Tfh2 subsets were significantly reduced along with disease activity and Tph1 and Tph2 subsets were positively correlated with SLE disease activity index (SLEDAI). The time course analysis of patients at pre- and post-flare revealed that in the patients at LLDAS before flare, Tph subsets and Tfh subsets were relatively low levels. At the flare, Tph cells, particularly Tph1 and Tph2 subsets, were increased and correlated with SLEDAI. Furthermore, the blood levels of IFN-α2a, IFN-γ, and IFN-λ1 were low in the patients with LLDAS before flare but these IFNs, particularly IFN-λ1, were increased along with flare. Conclusion: Increased frequency of Tph1 and Tph2 subsets and elevated levels of serum IFN-λ1 are presumably critical for triggering of flare in SLE.

Objectives: The usefulness of methotrexate-polyglutamates (MTX-PGs) concentration for management of rheumatoid arthritis has been debated. We aimed to clarify the association of MTX-PGs concentration with efficacy and safety in MTX-naïve patients initiating MTX in a prospective interventional clinical trial. Methods: The MIRACLE trial enrolled 300 MTX-naïve patients. Oral MTX was initiated and increased to the maximum tolerated dose by week 12. Patients who did not achieve remission according to the Simplified Disease Activity Index at week 24 were randomised to either the continued dose or reduced dose group and were started on subcutaneous adalimumab. We measured the concentrations of MTX-PGs in erythrocytes using liquid chromatography-tandem mass spectrometry and analysed the association of these concentrations with efficacy and safety. Results: The mean concentration of total MTX-PGs increased with an increasing dose of MTX and continued to elevate for another 12 weeks after the dose was fixed. At week 24, the total MTX-PGs concentration was 110.5 (SD 43.8) nmol/L with MTX dose of 12.6 (3.0) mg/week (0.23 (0.07) mg/kg/week). During MTX monotherapy, the higher MTX-PGs concentration was an independent factor for lower disease activity; however, this association disappeared after adalimumab initiation in patients with continued MTX dose. Hepatotoxicity was related to the higher MTX-PGs concentration regardless of adalimumab use. The total MTX-PGs concentration was significantly elevated by lower estimated glomerular filtration rate, serum albumin and body mass index. Conclusions: The MIRACLE trial demonstrated that higher total MTX-PGs concentration in erythrocytes is related to the higher efficacy and lower safety of MTX. Trial registration number: NCT03505008.

Objective Human T follicular helper (Tfh) cells are classified into three subsets: Tfh1, Tfh2, and Tfh17 cells. Among them, Tfh2 cells are defined as CXCR3-negative and CCR6-negative, and may contain diverse cell populations. We examined whether CCR4 serves as a marker for identifying Tfh2 cells that produce interleukin (IL)-4 and its involvement in IgG4-related disease (IgG4-RD). Methods Single cell analysis of IL-4-producing Tfh subset was performed using multi-colour flow cytometry and t-SNE method. Blood samples were obtained from 23 treatment-naïve patients with active IgG4-RD. CCR4+Tfh2 cells were also assessed in affected tissues of IgG4-RD by flow cytometry and immunohistochemical staining. Results Tfh2 cells expressing CCR4 were identified as Tfh cells that specifically produce IL-4. CCR4+Tfh2 cells showed higher expression of GATA-3 and ICOS than CCR4-Tfh2 cells, while there was no difference in the expression of BCL-6 and FOXP3. The proportion of CCR4+Tfh2 cells in peripheral blood was increased in IgG4-RD compared to healthy controls, and even more CCR4+Tfh2 cells infiltrated into the affected lesions. CCR4+GATA-3+Tfh2 cells diffusely infiltrated tertiary lymphoid tissues and storiform fibrosis lesions. The proportion of CCR4+Tfh2 cells showed a significant correlation specifically with serum IgG4 levels among clinical indicators. Glucocorticoid therapy did not correct the increased proportion of CCR4+Tfh2 cells. Conclusion CCR4 serves as a marker for identifying Tfh2 cells that specifically produce IL-4. CCR4+Tfh2 cells are a widely present T cell population that infiltrates tertiary lymphoid tissues and storiform fibrosis of IgG4-RD. Glucocorticoid fails to effectively target CCR4+Tfh2 cells that may contribute to a high relapse rate during glucocorticoid tapering in this disease.

Background: Recent evidence suggests that clonally expanded cytotoxic T cells play a role in various autoimmune diseases. Late-onset rheumatoid arthritis (LORA) exhibits unique characteristics compared to other RA forms, suggesting distinct immunological mechanisms. This study aimed to examine the involvement of cytotoxic T cells in LORA. Methods: Fresh peripheral blood samples were collected from 78 treatment-naïve active RA patients, 12 with difficult-to-treat RA, and 16 healthy controls. Flow cytometry was employed to measure the proportions of CX3CR1+cytotoxic CD4+ and CD8+ T cells in these samples. Additionally, immunohistochemical staining was performed on lymphoid node and synovial biopsy samples from patients with RA. Results: CX3CR1+cytotoxic CD4+ T cells were specifically increased in untreated, active patients with LORA, displaying features of CXCR3mid age-associated T helper cells known as “ThA”. CX3CR1⁺CD4⁺ T cells were identified as a cytotoxic ThA subset, as nearly all of these cells specifically expressed granzyme B. These cells were observed in enlarged lymph nodes and were found to infiltrate synovial tissues from patients with LORA. The proportions of CX3CR1+CD4+ T cells positively correlated with arthritis activity in LORA. The number of cells decreased after treatment with methotrexate, tumor necrosis factor inhibitors, and interleukin-6 inhibitors, whereas T-cell activation modulators did not affect them. Moreover, PD-1+CD38+CX3CR1+CD4+ T cells were identified as a treatment-resistant T cell subset that was characteristically increased in difficult-to-treat RA. CX3CR1+CD8+ T cells showed no significant difference between RA patients and healthy individuals, and no correlation with disease activity was observed. However, a correlation with age was observed in RA patients. Conclusions: Our findings suggest that the immunopathogenesis of RA differs by age of onset, with CX3CR1+ age-associated cytotoxic CD4+ T cells playing a significant role in LORA. Additionally, the presence of a specific CX3CR1+ T cell subset may be linked to treatment resistance.

Objective To compare the involvement of cytotoxic CX3CR1+ T cell subsets between giant cell arteritis (GCA) and Takayasu’s arteritis (TAK). Methods We examined the proportions of CX3CR1+ CD4+ and CD8+ T cells in whole blood freshly obtained from 30 treatment-naive patients with active large-vessel vasculitis (GCA, n=22 and TAK, n=8) and 16 healthy controls (HC). Infiltration of CX3CR1+ T cells into the affected arteries was assessed using immunohistochemical staining. Furthermore, CX3CR1+ CD4+ and CD8+ T cells were followed up after glucocorticoid treatment for longitudinal assessment of both diseases. Results The proportion of CX3CR1+ CD4+ T cells was significantly higher in GCA than in HC but not in TAK. No differences were observed in the proportions of CX3CR1+ CD8+ T cells among the GCA, TAK, and HC groups. The increased proportion of CX3CR1+ CD4+ T cells in GCA strongly correlated with the severity of systemic inflammation, whereas no significant correlation was found in TAK. Compared to TAK, CX3CR1+ CD4+ T cells from GCA patients showed significantly higher expression of granzyme B and perforin. The inflamed temporal arterial tissues of the GCA were infiltrated by numerous CX3CR1+ T cells, contributing to inflammation, disruption of the elastic lamina, and intimal hyperplasia. In contrast, no infiltration of CX3CR1+ T cells was observed in the aortitis lesions of TAK. Longitudinal analysis of post-glucocorticoid treatment showed a reduction in CX3CR1+ T cells in GCA, whereas no significant change was observed in TAK. Conclusion Differences in immune mechanisms between GCA and TAK highlight cytotoxic CX3CR1+ T cells as potential drivers for GCA-related inflammation and vessel damage but not for TAK.

Interleukin-1, a pro-inflammatory cytokine, plays a crucial role in inflammatory disease pathogenesis. Interleukin-1 receptor antagonist knockout (IL-1Ra KO) mice spontaneously develop aortitis, arthritis and dermatitis, and are employed as a model for human inflammatory diseases. Previous studies have shown that transferring total T cells from IL-1Ra KO mice into nude mice induces aortitis and arthritis; however, the roles of specific T cell subsets in these inflammatory responses remain unclear. In this study, we aimed to investigate the T cell subsets in IL-1Ra KO mice. We found that the proportion of PD-1+CD44+CD62L-CD4+ T cells in the spleen and lymph nodes of IL-1Ra KO mice was significantly higher than that of wild type mice. RNA sequencing revealed elevated expression of basic helix-loop-helix family member e40 and granulocyte macrophage colony stimulating factor (GM-CSF) in splenic CD44+CD62L-CD4+ T cells from IL-1Ra KO mice. In addition, GM-CSF production from splenic CD4+ T cells of IL-1Ra KO mice was significantly higher than that of wild type mice when stimulated with PMA and ionomycin in vitro. Notably, immunohistochemical staining showed infiltration of GM-CSF+CD4+ T cells at inflammatory sites in IL-1Ra KO mice. Our results suggest that a subset of GM-CSF+CD4 + T cells emerges under IL-1 signal-enhanced inflammatory conditions.

We explored effective therapeutic targets for systemic sclerosis (SSc) patients with high risk for pulmonary arterial hypertension (PAH) by plasma proteomics analysis. A total of fifty-seven patients with SSc were enrolled in the study and the prevalence of PAH was 19.3%. On the other hand, 75.4% of SSc patients showed the ratio of forced vital capacity percentage/diffusing capacity of the lungs for carbon monoxide percentage> 1.6 and met criteria for high risk of PAH. Identification of elevated plasma proteins in SSc patients with high risk of PAH, followed by upstream regulator analysis, predicted interleukin (IL)-17A as a major upstream molecule. Furthermore, we performed in vitro neutralization study using MT-6194, a bispecific antibody targeting both IL-17A and IL-6 receptor. We found that MT-6194 broadly suppressed the increased expression of downstream molecules of IL-17A including IL-17A-related cytokines/chemokines, IL-17A-driven NFB pathway and IL-6-driven JAK/STAT pathway which were shown to be increased in SSc patients with high risk of PAH by the proteomics. Consequently, it is revealed that IL-17A is a promising target for early intervention in SSc patients with high risk for PAH.

Objectives: To investigate the efficacy and safety of hydroxychloroquine (HCQ) in patients with rheumatoid arthritis (RA). Methods: Patients with active RA, despite conventional synthetic disease-modifying antirheumatic drugs (csDMARDs), were recruited. HCQ was administered for 24 weeks, in addition to prior treatment. The primary end-point was the proportion of patients achieving American College of Rheumatology (ACR) 20 at Week 24, compared to that of a propensity score–matched historical control group. Results: Sixty patients were enrolled and administered HCQ. We also identified 276 patients as candidates for the historical control group. Propensity score matching yielded 46 patients in each group. The proportion of patients achieving ACR20 at Week 24 was significantly higher in the HCQ group than in the control group (54.4% vs. 28.3%, P = .007). The proportion of patients achieving ACR50 and ACR70 at Week 24 were also higher in the HCQ group than in the control group (ACR50, 30.4% vs. 4.3%, P = .006; ACR70, 17.4% vs. 0%, P = .005). Neither HCQ retinopathy nor any new safety signal was observed during the study. Conclusion: The addition of HCQ to csDMARDs was effective, with no new safety signal in patients with RA.

Background: Giant cell arteritis (GCA) is a primary large-vessel vasculitis (LVV) of unknown origin. Its management is a challenge due to the late onset of disease symptoms and frequent relapse; therefore, clarifying the pathophysiology of GCA is essential to improving treatment. This study aimed to identify the transition of molecular signatures in immune cells relevant to GCA pathogenesis by analyzing longitudinal transcriptome data in patients. Methods: We analyzed the whole blood transcriptome of treatment-naive patients with GCA, patients with Takayasu arteritis (TAK), age-matched, old healthy controls (HCs), and young HCs. Characteristic genes for GCA were identified, and the longitudinal transition of those genes was analyzed using cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT). Results: Repeated measures analysis of variance revealed 739 differentially expressed genes among all patients and HCs. Of the 739 genes, 15 were characteristically upregulated and 36 were downregulated in patients with GCA compared to those with TAK and HCs. Pathway enrichment analysis showed that downregulated genes in GCA were associated with B cell activation. CIBERSORT analysis revealed that upregulation of “M0-macrophages” and downregulation of B cells were characteristic of GCA. Upregulation of “M0-macrophages” reflects the activation of monocytes in GCA toward M0-like phenotypes, which persisted under 6 weeks of treatment. Combined treatment with prednisolone and an interleukin-6 receptor antagonist normalized molecular profiles more efficiently than prednisolone monotherapy. Conclusions: Gene signatures of monocyte activation and B cell inactivation were characteristic of GCA and associated with treatment response.

Objective To clarify the clinical and immunological characteristics of IgG4-RD based on the underlying diseases. Methods Consecutive patients with IgG4-RD treated at Keio University Hospital between 2010 and 2021 were divided according to the presence of malignancy or allergy into three groups. The clinical characteristics and 56 immune cell subsets in the peripheral blood were compared between the groups. Results Among 123 patients, 18 (14.6%) had malignancy including 4 with allergy (malignancy group), 57 (46.3%) had allergy alone (allergy group), and 48 (39.0%) had neither (idiopathic group). In the malignancy group, the patients were older (70.1 vs. 54.4 vs. 64.9 years, p<0.001), male-dominant (83.3 vs. 42.1 vs. 54.2%, p=0.008), and had smoking habits (77.8 vs. 42.1 vs. 43.8%, p=0.02). They also had significant involvement of the aorta/large vessels (33.3 vs. 7.0 vs. 20.8%, p=0.02), while the patients in the allergy group tended to have orbital/lacrimal gland involvement. Remission and relapse rates were not different between the groups; however, overall survival was significantly poorer in the malignancy group (p=0.02). Comprehensive immunophenotyping of the peripheral blood revealed that the increase in CXCR5+CD2-double negative T cells and the decrease in naive CD8 T cells were characteristic of the malignancy group. Conclusion The clinical and immunological phenotypes of IgG4-RD differ among those with underlying diseases.

T follicular helper (Tfh) cells and T peripheral helper (Tph) cells produce interleukin (IL)-21 and are thought to contribute to follicular and extra-follicular B-cell activation, respectively, in autoimmune diseases. It is known that programmed cell death-1 (PD-1)-positive CXCR5+ Tfh-like cells are differentiated from human naive CD4+ T cells by IL-12 plus transforming growth factor (TGF)-β. However, it remains unclear what cytokines are required for Tph differentiation. In this study, we found that interferon (IFN)-α and IFN-β reduce the frequency of Tfh-like cells under the IL-12 plus TGF-β condition, whereas they promote generation of PD-1+CXCR5-CD4+ T cells and secretion of IL-21, IFN-γand CXCL13. Intracellular cytokine staining and T-cell-B-cell co-culture studies indicated that IFN-α promotes generation of IL-21+IFN-γ+CXCR5-CD4+ T cells thereby enhancing B-cell helper function. By IFN-α treatment, the mRNA levels of IL21, IFNG, CXCL13, CD244, SLAMF7, GZMB and PRDM1 were significantly up-regulated but BCL6 mRNA expression was down-regulated, suggesting a Tph-related gene expression pattern. On the other hand, IL-2-neutralization increased mRNA levels of IL21, CXCL13 and CXCR5, retained BCL6, but showed no clear effect on IFNG or PRDM1. RNA sequencing analyses revealed that PD-1hiCXCR5-CD4+ T cells prepared from in vitro culture show a Tph-related gene expression pattern similar with that of PD-1hiCXCR5- Tph cells obtained from the blood of patients with systemic lupus erythematosus. From our findings, it is highly probable that type I IFNs play a key role in differentiation of Tph cells and trigger Tph cell expansion in autoimmune diseases.

Interleukin (IL)-21-producing T peripheral helper (Tph) cells are thought to contribute to extra-follicular B cell activation and play a pathogenic role in autoimmune diseases. In this study, we investigated the relationship between Tph cells and interferons (IFNs) in several autoimmune diseases because our previous study demonstrated that type I IFNs promote the differentiation of IL-21-producing Tph-like cells. The frequency of Tph cells in the blood as well as serum IFN-α2a and IFN-λ1 were markedly elevated in patients with active systemic lupus erythematosus (SLE) compared to other autoimmune diseases or healthy controls. Notably, the frequency of Tph cells was positively correlated with the SLE disease activity index, serum IFN-α and serum IFN-λ1 in SLE patients. Additionally, we found that type III IFNs (IFN-λ1, IFN-λ2 and IFN-λ3) promote the differentiation of programmed cell death-1 (PD-1)+ CXCR5 -CD4+ T cells and enhance the secretion of IL-21, IFN-γand CXCL13. IFN-λ1, like IFN-α, up-regulated the mRNA expression of IL21, IFNG, CXCL13, CD244, SLAMF7, GZMB, PRF1, CCR5 and PRDM1, whereas it down-regulated that of CXCR5 and BCL6, reflecting a Tph-related gene expression pattern. IFN-α in combination with IFN-λ1, IFN-λ2 or IFN-λ3 significantly increased the differentiation of PD-1+CXCR5- Tph-like cells and the secretion of Tph-related cytokines as compared with each IFN alone, suggesting a cooperative interaction. From these findings, it is highly probable that type III IFNs in addition to type I IFNs play a key role in the differentiation of Tph cells and that high levels of IFN-α and IFN-λ1 trigger the differentiation and expansion of Tph cells in SLE.

Introduction: /objectives Several biological disease-modifying anti-rheumatic drugs (bDMARDs) have been widely used for the management of rheumatoid arthritis (RA). These drugs target different molecules important for the pathophysiology of RA; however, only a few studies have compared the effects of these biological drugs on cytokines and bone metabolic markers. The main aim of this study is to clarify the effects of bDMARDs with different modes of action on the cytokine and bone metabolic marker levels in patients with RA. Methods: Patients with RA who were initiated on infliximab, tocilizumab, or abatacept as the first bDMARD were prospectively enrolled in this study. Serum cytokine and bone metabolic marker levels were measured longitudinally, and changes in their levels were compared. Results: A total of 174 patients were enrolled in this study, with 55, 70, and 49 patients in the infliximab, tocilizumab, and abatacept groups, respectively. At six months, despite the similar clinical effectiveness of the three drugs, changes in the cytokine and bone metabolic marker levels were distinct; interferon-γ and tumor necrosis factor-α levels were significantly increased with infliximab, interleukin-6 levels were increased with tocilizumab, and interleukin-1β and interleukin-8 levels were increased with abatacept treatment. Bone-specific alkaline phosphatase and osteocalcin levels increased more significantly with tocilizumab than with infliximab, while osteopontin and osteonectin levels decreased with infliximab treatment. Conclusions: bDMARDs with different modes of action exert different effects on the cytokine and bone metabolic marker levels in patients with RA.