Cytotoxic Tph subset with low B-cell helper functions and its involvement in systemic lupus erythematosus.

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.

Th17/IL-17A axis is critical for pulmonary arterial hypertension (PAH) in systemic sclerosis (SSc): SSc patients with high levels of serum IL-17A exhibit reduced lung functions and increased prevalence of PAH.

BACKGROUND: It is thought that systemic sclerosis (SSc) might be a T helper 17 (Th17) cell-driven autoimmune disease. Noticeably, pulmonary arterial hypertension (PAH) is a leading cause of death in patients with SSc. Here, we investigated the association between serum Th17-related cytokines and prevalence of PAH in SSc patients. METHODS: This study included 72 SSc patients and 51 healthy controls (HC). We determined clinical manifestations, immunophenotypes including Th subsets in peripheral blood lymphocytes, and the serum levels of interleukin (IL)-17A, IL-17A/F, IL-17B. IL-17C, IL-17D. IL-1ß, IL-6, IL-21, IL-22, and IL-23. RESULTS: The frequency of Th17 cells was significantly increased in SSc patients compared to HC and was positively correlated with the modified Rodnan skin scores. Furthermore, the serum levels of IL-17A, IL-17D, IL-1ß, and IL-6 were significantly increased in SSc patients compared to HC. SSc patients with detected IL-17A showed high levels of IL-17A/F, IL-1ß, IL-6, and IL-22, and high frequency of Th17 cells. Interestingly, these patients exhibited the reduced lung functions and increased prevalence of PAH significantly compared to patients with undetected IL-17A. Similarly, SSc patients with detected IL-17A and high IL-6 (≥1.2 pg/mL) exhibited the decreased lung functions and increased prevalence of PAH compared to patients with undetected IL-17A and low IL-6. CONCLUSION: We found that SSc patients with high levels of serum IL-17A or both IL-17A and IL-6 show reduced lung functions and high prevalence of PAH. Consequently, it is highly probable that Th17/IL-17A axis is critical for the prevalence of PAH in SSc patients.

Distinct impact of malignancy and allergy on the clinical and immunological features of IgG4-related disease.

OBJECTIVES: 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 among 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. CONCLUSIONS: The clinical and immunological phenotypes of IgG4-RD differ among those with underlying diseases.

Role of interferons (IFNs) in the differentiation of T peripheral helper (Tph) cells.

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-γ and 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.

Role of interferons (IFNs) in the differentiation of T peripheral helper (Tph) cells.

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)-ß. However, it remains unclear what cytokines are required for Tph differentiation. In this study, we found that interferon (IFN)-α and IFN-ß reduce the frequency of Tfh-like cells under the IL-12 plus TGF-ß condition, whereas they promote generation of PD-1+CXCR5-CD4+ T cells and secretion of IL-21, IFN-γ and 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.

Proton Pump Inhibitor and Tacrolimus Uses are Associated With Hypomagnesemia in Connective Tissue Disease: a Potential Link With Renal Dysfunction and Recurrent Infection.

Background: Low levels of serum magnesium perturb renal tubular cell function and lymphocytes, resulting in renal deterioration and an imbalance in mononuclear cells. This study investigated the mechanism and influence of hypomagnesemia in patients with connective tissue disease. Methods: We retrospectively evaluated patients with connective tissue disease and available serum magnesium data who visited Keio University Hospital in 2019. Patients were divided into two groups: those with (serum magnesium < 1.8 mg/dl) and those without hypomagnesemia; their rates of hospitalization for severe infection and cumulative renal deterioration were compared. Patients' fractions of lymphocytes and natural killer and dendritic cell subsets, as measured by fluorescence-activated cell sorting (FACS) analysis, were also compared. Results: Among 284 patients, hypomagnesemia was detected in 63 (22.2%). Multivariate analysis revealed that the use of proton pump inhibitors [odds ratio (OR), 1.48; p = 0.01] and tacrolimus (OR, 6.14; p < 0.01) was independently associated with hypomagnesemia. In addition, the renal deterioration rate was significantly higher in tacrolimus and/or proton pump inhibitor users with hypomagnesemia (p = 0.01). The hospitalization rate for severe infection was also higher in patients with hypomagnesemia (p = 0.04). FACS analysis showed lower CD8+ T cell, CD19+ B cell, natural killer cell, and dendritic cell counts in patients with hypomagnesemia (p = 0.03, p = 0.02, p = 0.02, and p = 0.03, respectively). Conclusion: The use of tacrolimus and proton pump inhibitors may be associated with hypomagnesemia and lead to poor renal outcomes and severe infection in patients with connective tissue disease.

Distinct features between HLA-DR+ and HLA-DR- PD-1hi CXCR5- T peripheral helper cells in seropositive rheumatoid arthritis.

OBJECTIVES: PD-1hi CXCR5- T peripheral helper (Tph) cells are newly identified pathogenic CD4 helper T cells in RA. We evaluated the usefulness of Tph cell subsets as biomarkers of RA. METHODS: RA patients who visited our rheumatology department between May 2015 and September 2017 and met the 2010 ACR/EULAR classification criteria were included. We compared the correlation of DAS28-ESR between Tph cell subsets and 40 immune cell subsets. We also explored which subsets reflected the chronological changes in the disease activity after treatment. RESULTS: Thirty-four seropositive RA patients, 11 seronegative RA patients and 34 healthy controls were included. Tph cell subsets that correlated with the DAS28-ESR were HLA-DR+ Tph cells (rs = 0.50, P = 0.002), HLA-DR- Tph cells (rs = 0.39, P = 0.03) and Tph1 cells (rs = 0.41, P = 0.02). Among the other 40 immune cell subsets, HLA-DR+ Th1-17 cells (rs = 0.38, P = 0.03), activated B cells (rs = -0.35, P = 0.04), plasma cells (rs = 0.43, P = 0.01) and CD14++ CD16+ monocytes (rs = 0.36, P = 0.04) correlated, but not strongly as HLA-DR+ Tph cells. However, MTX treatment reduced the proportion of HLA-DR+ Tph cells independently of the disease activity. In contrast, HLA-DR- Tph cells accurately reflected the change in the DAS28-ESR during MTX treatment. CONCLUSION: HLA-DR+ Tph cells were decreased with MTX treatment, independent of the disease activity, while HLA-DR- Tph cells reflected the disease activity accurately during the treatment.

Lymphadenopathy in IgG4-related disease: a phenotype of severe activity and poor prognosis, with eotaxin-3 as a new biomarker.

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.

Milk fat globule epidermal growth factor 8 (MFG-E8) on monocytes is a novel biomarker of disease activity in systemic lupus erythematosus.

BACKGROUND: Milk fat globule epidermal growth factor (MFG-E8) is related secreted protein which links phosphatidylserine on apoptotic cells and integrin αvß3/5 on phagocytes. To clarify the clinical significance of MFG-E8 in SLE, we analyzed the correlation between expression level of MFG-E8 in circulating phagocytic leukocytes and clinical parameters of patients. METHODS: The study was conducted under a multi-center, prospective cohort design. Patients with one or both BILAG A or B, or SLEDAI- 2 K ≥ 4 with clinical symptoms were defined as the active SLE group. Expression of MFG-E8 on monocytes and concentration in serum were measured by FACS and ELISA, respectively. RESULTS: 96 subjects were enrolled. The absolute number and proportion of MFG-E8-positive monocytes to total monocytes were significantly higher in the active SLE group (p < 0.01). Importantly, the proportion was also significantly correlated with SLEDAI-2K, clinical SLEDAI, as well as serum levels of anti-ds-DNA antibody and complement and C1q. In addition, the proportion of MFG-E8-positive monocytes to total monocytes was significantly decreased from baseline in active SLE patients after 6 months' treatment and increased concordantly with disease activity in 6 refractory cases. Further, in receiver operating characteristic curve analysis for discrimination between active and inactive SLE, the AUC of the proportion of MFG-E8 was 0.854, which was equivalent to classical activity markers such as anti-ds DNA antibody (0.776), complement (0.897) and C1q (0.815). CONCLUSIONS: The proportion of MFG-E8-positive monocytes to total monocytes in peripheral blood was positively associated with disease activity in SLE and may be a novel biomarker of disease activity.

Longitudinal immune cell monitoring identified CD14++ CD16+ intermediate monocyte as a marker of relapse in patients with ANCA-associated vasculitis.

BACKGROUND: Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is an autoimmune disease that affects small- to medium-sized blood vessels. Despite treatments having been improved, patients often experience disease relapses. It remains unclear how the immune cells involve in the development of vasculitis and how they fluctuate over the course of treatment. In this study, we aimed to identify the immune subsets and serum cytokines associated with disease relapse by comprehensive immuno-phenotyping in AAV patients. METHODS: We reviewed consecutive patients (n = 29) from Keio University Hospital who had been newly diagnosed with AAV from January 2015 to February 2019 and chronologically followed until 52 weeks. Numbers of circulating T cells, B cells, monocytes, and granulocytes were analyzed by flow cytometry (FACS). Serum levels of cytokines were measured by electrochemiluminescence enzyme immunoassay. Clinical information was obtained from patients' records and association with time-course changes in immuno-phenotypes and serum levels of cytokines were assessed. RESULTS: Comprehensive immuno-phenotyping data from 161 samples from 29 AAV patients at diagnosis; at weeks 4, 12, 24, and 52 of treatment; and at time of major relapse were examined. FACS analysis from patients with relapse revealed that CD14++ CD16+ intermediate monocytes and plasma cells concomitantly changed associated with disease relapse, which were independent from treatment regimen, ANCA status, or disease phenotype. In particular, the number of CD14++ CD16+ intermediate monocytes at relapse was significantly higher than that in remission or in healthy controls. Serum cytokine measurement revealed that changes of monocyte-derived proinflammatory cytokines such as IL-1ß, IL-6, IL-8, and TNF-α were associated with disease status. CONCLUSIONS: Chronological changes in CD14++ CD16+ intermediate monocyte counts can be a marker of disease relapse in AAV patients.

Significant association between clinical characteristics and immuno-phenotypes in patients with ANCA-associated vasculitis.

OBJECTIVES: To elucidate the association between clinical characteristics and immuno-phenotypes in patients with ANCA-associated vasculitis (AAV). METHODS: Peripheral blood from 36 patients with active AAV and 18 healthy controls was examined for numbers of circulating T cells, B cells, NK cells, dendritic cells, monocytes and granulocytes using flow cytometry. These immuno-phenotyping data were subjected to cluster analysis and principal components analysis to divide AAV patients into subgroups. Associated organ involvement or therapeutic prognosis were assessed for each subgroup. RESULTS: AAV patients had higher proportions of plasma cells, plasmablasts, activated T cells, CD14++ CD16+ monocytes, eosinophils and neutrophils than healthy controls. Immuno-phenotyping findings were similar between patients with microscopic polyangiitis, granulomatosis with polyangiitis and eosinophilic granulomatosis with polyangiitis. Cluster analysis indicated that AAV patients could be divided into three subgroups according to peripheral immune cell numbers: antibody production-related (n = 9), cytotoxic activity-related (n = 4) and neutrocytosis/lymphocytopenia-related (n = 23). The antibody production-related or cytotoxic activity-related group was associated with CNS involvement, and the neutrocytosis/lymphocytopenia-related group was associated with high incidence of kidney involvement. Incidence of severe infection was markedly higher in the neutrocytosis/lymphocytopenia-related group than the other two groups. Incidence of disease relapse was comparable among the three groups. CONCLUSION: Patients with active AAV can be divided into three subgroups based on immuno-phenotyping. These results may provide a hint to understanding disease pathophysiology and prognosis, and determining appropriate treatment.

Significant association between clinical characteristics and changes in peripheral immuno-phenotype in large vessel vasculitis.

BACKGROUND: Large vessel vasculitis (LVV) is a type of vasculitis characterized by granulomatous inflammation of medium- and large-sized arteries. Clinical assessment of acute phase reactants has been conventionally used to diagnose and monitor diseases; however, accurate assessment of vascular disease activity status can be difficult. In this study, we investigated comprehensive immuno-phenotyping to explore useful biomarkers associated with clinical characteristics. METHODS: Consecutive patients with newly diagnosed LVV who visited our institution between May 2016 and May 2019 were enrolled. The number of circulating T cells, B cells, natural killer cells, dendritic cells, monocytes, and granulocytes was examined and chronologically followed. Baseline and time-course changes in immuno-phenotyping associated with disease activity were assessed. RESULTS: Comprehensive immuno-phenotyping data from 90 samples from each of 20 patients with LVV were compared with those from healthy controls (HCs). The number of helper T (Th), follicular helper T (Tfh), CD8+ T, CD14++ CD16+ monocytes, and neutrophils were higher in patients with giant cell arteritis (GCA) and/or Takayasu arteritis (TAK) than in HCs. Among them, the number of CD8+ T and CD8+ Tem were higher in patients with TAK than in GCA. Notably, memory CD4+ and CD8+ T cells in patients with TAK remained high even in the remission phase. Further analysis revealed that the number of Th1, Th17, and Tfh cells was associated with disease relapse in GCA and TAK and that the number of CD8+ T cells was associated with relapse in TAK. Th1, Th17, and Tfh cells decreased after treatment with biologic agents, while CD8+ T cells did not. CONCLUSIONS: Our results from peripheral immuno-phenotyping analysis indicate that the numbers of Th and Tfh cells changed along with the disease condition in both GCA and TAK, while that of CD8+ T cells did not, especially in TAK. Treatment with biologic agents decreased the proportion of Th and Tfh cells, but not CD8+ T cells, in the patients. Chronological immuno-phenotyping data explained the difference in therapeutic response, such as reactivities against biologics, between GCA and TAK.

IL-17-Producing Vγ4+ γδ T Cells Require Sphingosine 1-Phosphate Receptor 1 for Their Egress from the Lymph Nodes under Homeostatic and Inflammatory Conditions.

Conventional αß T cells require sphingosine 1-phosphate (S1P) receptor 1 (S1P1) for circulation through the lymph nodes (LN); however, it is unclear whether γδ T cells use similar mechanisms. In this study, we found that treatment with fingolimod (FTY720, 1 mg/kg, orally) markedly reduced not only conventional CD4 T cells but also circulating γδ T cells (Vγ4(+) and Vγ4(-) subsets) in the blood of mice. In contrast, IL-17(+)Vγ4(+), IL-17(+)Vγ4(-), and IL-17(-)Vγ4(-) subsets were significantly accumulated in the LN after 6 h of FTY720 treatment. By skin application of a synthetic TLR7/8 agonist, Vγ4(+) γδ T cells (IL-17(+) and IL-17(-) subsets) were accumulated and expanded in the draining LN (DLN), whereas the IL-17(+) subset predominantly migrated to the inflamed skin. FTY720 induced a marked sequestration of IL-17-producing Vγ4(+) γδ T cells in the DLN and inhibited their infiltration into the inflamed skin. Similarly, FTY720 inhibited infiltration of Vγ4(+) γδ T cells into the CNS by their sequestration into the DLN in experimental autoimmune encephalomyelitis. Vγ4(+) γδ T cells expressed a significant level of S1P1 and showed a migratory response toward S1P. FTY720 treatment induced almost complete downregulation of S1P1 expression and S1P responsiveness in Vγ4(+) γδ T cells. Our findings strongly suggest that IL-17-producing Vγ4(+) γδ T cells require S1P1 for their egress from the LN under homeostatic and inflammatory conditions. Consequently, inhibition of S1P1-dependent egress of pathogenic IL-17-producing Vγ4(+) γδ T cells from the DLN may partly contribute the clinical therapeutic effects of FTY720 in relapsing multiple sclerosis.

A new phenylpyrazoleanilide, y-320, inhibits interleukin 17 production and ameliorates collagen-induced arthritis in mice and cynomolgus monkeys.

Interleukin (IL)-15 and IL-17 are thought to play an important role in the pathogenesis of rheumatoid arthritis (RA) because both pro-inflammatory cytokines are found in synovial fluid of RA patients. In this study, we examined the pharmacological profiles of Y-320, a new phenylpyrazoleanilide immunomodulator. Y-320 inhibited IL-17 production by CD4 T cells stimulated with IL-15 with IC50 values of 20 to 60 nM. Oral administration of Y-320 (0.3 to 3 mg/kg) significantly inhibited the development and progression of arthritis and joint destruction with reduction of IL-17 mRNA expression in arthritic joints of type II collagen-induced arthritis (CIA) in DBA/1J mice. Y-320 in combination with anti-murine tumor necrosis factor-α monoclonal antibody showed a synergistic effect on mouse CIA. Moreover, therapeutic treatment with Y-320 (0.3 and 1 mg/kg orally) ameliorated CIA in cynomolgus monkeys. Our results suggest that Y-320, an orally active inhibitor for IL-17 production, provides a useful therapy for RA.

Fingolimod (FTY720), sphingosine 1-phosphate receptor modulator, shows superior efficacy as compared with interferon-ß in mouse experimental autoimmune encephalomyelitis.

Fingolimod (FTY720), a sphingosine 1-phosphate (S1P) receptor modulator, inhibits S1P-dependent lymphocyte egress from secondary lymphoid organs and is highly effective in experimental autoimmune encephalomyelitis (EAE) in mice. In this study, we directly compared the therapeutic effects of FTY720 and recombinant mouse interferon (rm-IFN)-ß on relapse and progression of EAE in mice. When FTY720 at oral dose of 0.03 to 1 mg/kg was administered daily after establishment of EAE induced by myelin proteolipid protein (PLP) in SJL/J mice, relapse of EAE was significantly inhibited during administration period. Subcutaneous injection of rm-IFN-ß (10,000 IU/mouse) also inhibited the relapse of EAE at early period; however EAE was relapsed in all the mice within administration period. Therapeutic administration of FTY720 (0.03 to 1 mg/kg) significantly improved the symptoms of chronic EAE induced by myelin oligodendrocyte glycoprotein in C57BL/6 mice whereas rm-IFN-ß (10,000 IU/mouse) showed no clear effect. These results indicate that FTY720 is more efficacious in mouse EAE as compared with rm-IFN-ß. FTY720 markedly reduced the frequency of PLP-specific Th17 and Th1 cells in the spinal cord of EAE mice. On the contrary, FTY720 increased the frequency of PLP-specific Th17 and Th1 cells in the inguinal lymph nodes, suggesting inhibition of egress of myelin antigen-specific Th cells from draining lymph nodes. From these results, the ameliorating effects of FTY720 on EAE are likely due to reduction of infiltration of myelin antigen-specific Th17 and Th1 cells into the central nervous system.

Sphingosine 1-phosphate receptor type 1 regulates egress of mature T cells from mouse bone marrow.

Sphingosine 1-phosphate (S1P) and its receptor, S1P receptor type 1 (S1P(1)), are essential for lymphocyte egress from secondary lymphoid organs (SLO). Fingolimod (FTY720), the S1P receptor modulator, inhibits lymphocyte egress from SLO and decreases circulating lymphocytes; however, it also induces a significant decrease in the number of peripheral blood lymphocytes in alymphoplasia (aly/aly) mice lacking SLO. In this study, we demonstrated that the administration of FTY720 induced sequestration of mature lymphocytes, particularly T cells, into the bone marrow (BM) in aly/aly mice, implying that the reduction of circulating lymphocytes in these mice by FTY720 was due to inhibition of lymphocyte egress from the BM. Since sequestration of mature T cells into the BM was also induced in normal mice by selective S1P(1) agonist or S1P lyase inhibitor, it is suggested that S1P(1) expression and the S1P gradient play an important role in egress of mature T cells from the BM. Prophylactic administration of FTY720 to ovalbumin (OVA)-immunized mice significantly inhibited footpad swelling induced by OVA challenging with a marked reduction of OVA-specific T(h) cells in the BM, indicating that immunomodulation by FTY720 is likely due to reduced circulation of antigen-specific T(h) cells. On the other hand, OVA-specific T(h) cells, like naive T cells, were also sequestered into the BM and SLO of OVA-immunized mice by a short exposure of FTY720 after OVA challenging. These results suggest that the S1P-S1P(1) axis plays a regulatory role in egress of mature T cells including antigen-specific T(h) cells from the BM.

Carbachol-induced membrane ruffling and its desensitization in rat basophilic leukemia (RBL-2H3) cells transfected with m3 muscarinic acetylcholine receptors.

The changes in the reorganization of actin filaments during desensitization of secretion were investigated by transfecting RBL-2H3 cells with cDNA encoding the human m3 muscarinic acetylcholine receptors (RBL-m3 cells). Incubation of RBL-m3 cells with 10-100 microM carbachol in Ca2+ -free medium developed membrane ruffling. When the cells were desensitized under the condition where desensitization of carbachol-induced secretion occurred, desensitized cells failed to develop membrane ruffling with the subsequent addition of carbachol. These results suggest that m3 muscarinic receptor-mediated desensitization of secretion involves negative regulation of actin reorganization leading to membrane ruffling.

Carbachol-induced secretion and homologous desensitization in rat basophilic leukemia (RBL-2H3) cells transfected with human m2 muscarinic acetylcholine receptors.

Carbachol (CCh) caused a dose-dependent release of beta-hexosaminidase and an increase in the production of inositol 1,4,5-trisphosphate (IP3) in RBL-2H3 cells transfected with m2 mAChR cDNA (RBL-m2 cells). The secretion was completely inhibited by LaCl3 and pertussis toxin. The secretion was dependent on extracellular Ca2+ and mediated through the pertussis toxin-sensitive G protein. Exposing RBL-m2 cells to 100 microM CCh for 30 min in Ca2+ -free medium (desensitizing treatment) inhibited the secretion induced by the subsequent addition of 10 microM CCh plus Ca2+, but not by stimulating the high affinity IgE receptor (FcepsilonRI). Desensitizing treatment of RBL-m2 cells reduced the affinity of the lipophilic ligand [3H]quinuclidinyl benzilate to m2 mAChR without a reduction of the total m2 mAChR number. The treatment also decreased the cell surface mAChR number to 14% with a slight reduction in its affinity. Desensitizing treatment of RBL-m2 cells inhibited the CCh-induced transient increase in levels of IP3 and intracellular Ca2+ concentration. The results suggested that the CCh-induced desensitization of m2 mAChR-mediated secretion is due to the receptor sequestration followed by blocking the increase in [Ca2+]i and that this desensitizing mechanism is receptor-subtype-specific.

IL-4-induced GATA-3 expression is a time-restricted instruction switch for Th2 cell differentiation.

An initial activation signal via the TCR in a restricted cytokine environment is critical for the onset of Th cell development. Cytokines regulate the expression of key transcriptional factors, T-bet and GATA-3, which instruct the direction of Th1 and Th2 differentiation, through changes in chromatin conformation. In this study, we investigated the kinetics of IL-4-mediated signaling in a transgenic mouse, expressing human IL-4R on a mouse IL-4alphaR-deficient background. These experiments, allowing induction with human IL-4 at defined times, demonstrated that an IL-4 signal was required at the early stage of TCR-mediated T cell activation for lineage commitment to Th2, along with structural changes in chromatin, which take place in the conserved noncoding sequence-1 and -2 within the IL-4 locus. At later times, however, IL-4 failed to promote efficient Th2 differentiation and decondensation of chromatin, even though GATA-3 was clearly induced in the nuclei by IL-4 stimulation. Moreover, IL-4-mediated Th2 instruction was independent from cell division mediated by initial TCR stimulation. The role of IL-4 signaling may have a time restriction during Th2 differentiation. In late stages of initial T cell activation, the chromatin structure of the IL-4 locus retains condensation state. These results demonstrate that IL-4-induced GATA-3 expression is time-restriction switch for Th2 differentiation.

The Runx1 transcription factor inhibits the differentiation of naive CD4+ T cells into the Th2 lineage by repressing GATA3 expression.

Differentiation of naive CD4+ T cells into helper T (Th) cells is controlled by a combination of several transcriptional factors. In this study, we examined the functional role of the Runx1 transcription factor in Th cell differentiation. Naive T cells from transgenic mice expressing a dominant interfering form of Runx1 exhibited enhanced interleukin 4 production and efficient Th2 differentiation. In contrast, transduction of Runx1 into wild-type T cells caused a complete attenuation of Th2 differentiation and was accompanied by the cessation of GATA3 expression. Furthermore, endogenous expression of Runx1 in naive T cells declined after T cell receptor stimulation, at the same time that expression of GATA3 increased. We conclude that Runx1 plays a novel role as a negative regulator of GATA3 expression, thereby inhibiting the Th2 cell differentiation.