Toshihiko Komai

TGF-β3-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-β3 in an Egr2- and Fas-dependent manner. LAG3+ Treg require TGF-β3 to suppress B-cell responses in a murine model of lupus. Moreover, TGF-β3- and LAG3+ Treg-mediated suppression requires PD-1 expression on B cells. We also show that TGF-β3-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.

Revisiting the regulatory roles of the TGF-β family of cytokines

TGF-β family members are multipotent cytokines that are involved in many cellular processes, including cell differentiation, organ development, wound healing and immune regulation. TGF-β has pleiotropic effects on adaptive immunity, especially in the regulation of CD4(+) T cell and B cell responses. Furthermore, identification of CD4(+) T cell subsets that produce TGF-β3 revealed unexpected roles of TGF-β3 in the control of adaptive immunity. In contrast to TGF-β1, which induces extensive fibrosis, TGF-β3 induces non-scarring wound healing and counteracts tissue fibrosis. Recent progress in the understanding of the activation mechanism of TGF-β may enable us to develop novel biologic therapies based on advanced protein engineering.

Egr2 and Egr3 in regulatory T cells cooperatively control systemic autoimmunity through Ltbp3-mediated TGF-β3 production

Significance Transcription factors early growth response gene 2 (Egr2) and Egr3 have long been regarded as negative regulators of T-cell activation. Egr2 is also known as a susceptibility gene for systemic lupus erythematosus characterized by dysregulated humoral immune responses to autoantigens. Previously, we reported that Egr2-expressing CD4+CD25-LAG3+ regulatory T cells regulate lupus pathogenesis via production of TGF-β3. However, the role of Egr2 and Egr3 in the regulation of humoral immunity is unclear. Here we report that Egr2 and Egr3 regulate germinal center reactions by promoting TGF-β3 production from regulatory T cells. Egr2 and Egr3 induce the expression of latent TGF-β binding protein 3 (Ltbp3), which is required for TGF-β3 secretion. These findings suggest that Egr2 and Egr3 in T cells may be potential novel therapeutic targets for autoantibody-mediated autoimmune diseases. Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by multiorgan inflammation induced by autoantibodies. Early growth response gene 2 (Egr2), a transcription factor essential for T-cell anergy induction, controls systemic autoimmunity in mice and humans. We have previously identified a subpopulation of CD4+ regulatory T cells, CD4+CD25−LAG3+ cells, that characteristically express both Egr2 and LAG3 and control mice model of lupus via TGF-β3 production. However, due to the mild phenotype of lymphocyte-specific Egr2-deficient mice, the presence of an additional regulator has been speculated. Here, we show that Egr2 and Egr3 expressed in T cells cooperatively prevent humoral immune responses by supporting TGF-β3 secretion. T cell-specific Egr2/Egr3 double-deficient (Egr2/3DKO) mice spontaneously developed an early onset lupus-like disease that was more severe than in T cell-specific Egr2-deficient mice. In accordance with the observation that CD4+CD25−LAG3+ cells from Egr2/3DKO mice completely lost the capacity to produce TGF-β3, the excessive germinal center reaction in Egr2/3DKO mice was suppressed by the adoptive transfer of WT CD4+CD25−LAG3+ cells or treatment with a TGF-β3–expressing vector. Intriguingly, latent TGF-β binding protein (Ltbp)3 expression maintained by Egr2 and Egr3 was required for TGF-β3 production from CD4+CD25−LAG3+ cells. Because Egr2 and Egr3 did not demonstrate cell intrinsic suppression of the development of follicular helper T cells, Egr2- and Egr3-dependent TGF-β3 production by CD4+CD25−LAG3+ cells is critical for controlling excessive B-cell responses. The unique attributes of Egr2/Egr3 in T cells may provide an opportunity for developing novel therapeutics for autoantibody-mediated diseases including SLE.

Neuromyelitis optica spectrum disorder complicated with Sjogren syndrome successfully treated with tocilizumab: A case report.

A 38-year-old woman with relapsing longitudinal extensive transverse myelitis and Sjogrens syndrome (SS) was admitted with lower extremity muscle weakness. Studies showed high serum titer of anti-aquaporin4 antibody and gadolinium-enhanced-MRI T1-weighted lesions within thoracic cord. Clinical findings suggested neuromyelitis optica-spectrum disorder (NMO-SD). High-dose corticosteroids, plasma exchange and cyclophosphamide were not effective. After starting tocilizumab, her neurological findings gradually improved. This report describes the first evidence to show tocilizumab could be effective for NMO-SD with SS.

TGF-β3 Inhibits Antibody Production by Human B Cells

TGF-β is a pleotropic cytokine involved in various biological processes. Of the three isoforms of TGF-β, TGF-β1 has long been recognized as an important inhibitory cytokine in the immune system and has been reported to inhibit B cell function in both mice and humans. Recently, it has been suggested that TGF-β3 may play an important role in the regulation of immune system in mice. Murine CD4+CD25-LAG3+ regulatory T cells suppress B cell function through the production of TGF-β3, and it has been reported that TGF-β3 is therapeutic in a mouse model of systemic lupus erythematosus. The effect of TGF-β3 on human B cells has not been reported, and we herein examined the effect of TGF-β3 on human B cells. TGF-β3 suppressed B cell survival, proliferation, differentiation into plasmablasts, and antibody secretion. Although the suppression of human B cells by TGF-β1 has long been recognized, the precise mechanism for the suppression of B cell function by TGF-β1 remains elusive; therefore, we examined the effect of TGF-β1 and β3 on pathways important in B cell activation and differentiation. TGF-β1 and TGF-β3 inhibited some of the key molecules of the cell cycle, as well as transcription factors important in B cell differentiation into antibody secreting cells such as IRF4, Blimp-1, and XBP1. TGF-β1 and β3 also inhibited B cell receptor signaling. Our results suggest that TGF-β3 modifying therapy might be therapeutic in autoimmune diseases with B cell dysregulation in humans.

A Case of Polymyxin b-Immobilized Fiber Column Treatment for Rapidly Progressive Interstitial Pneumonia Associated with Clinically Amyopathic Dermatomyositis

We report a case of rapidly progressive interstitial pneumonia associated with clinically amyopathic dermatomyositis who responded to single course of polymyxin b-immobilized fiber column treatment. Initial treatment with pulsed corticosteroids and cyclophosphamide, intravenous immunoglobulin, and cyclosporine seemed to suppress the activity of interstitial lung disease temporarily, but signs of relapse were detected such as elevation of serum KL-6 level and progressing pulmonary shadows in chest computed tomography scan. After polymyxin b-immobilized fiber column treatment, the areas of pulmonary shadows drastically decreased. Gradually, arterial partial pressure of oxygen/fraction of inspired oxygen (PaO2/FiO2) ratio recovered, and serum ferritin level and KL-6 level decreased. These findings indicate that polymyxin b-immobilized fiber column treatment could be promising in combination with conventional therapy for rapidly progressive interstitial pneumonia associated with clinically amyopathic dermatomyositis, especially at the early phase of relapse.

The effects of TGF-βs on immune responses.

Transforming growth factor (TGF)-β family is a cytokine family with various biological processes and forms a highly homologous group of three mammalian isoforms, TGF-β1, TGF-β2, and TGF-β3. Most of the attention on TGF-β family in immunology has been mainly focused on TGF-β1 in that TGF-β1 induces anti-inflammatory regulatory T cells (Treg), and inflammatory T helper 17 (Th17) cells in combination with interleukin-6. Although little attention has been focused on the immunological roles of TGF-β2 and TGF-β3, the function of TGF-β3 for maintaining immunological homeostasis has recently been identified such as the induction of Th17 cells and direct regulatory effects on humoral immunity. TGF-β1 and TGF-β3 shares similar anti-inflammatory or pro-inflammatory functions, but exhibits significantly different effects on fibrosis and chondrogenesis. For the clinical application of TGF-βs, the mechanisms by which each TGF-β isoform regulates immunity has to be elucidated. In this review, we provide an overview of the effects, cellular targets, and therapeutic potential of TGF-βs on immune responses and autoimmune diseases.

Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals

Inhibitory cytokines, such as transforming growth factor-β (TGF-β) and interleukin-10 (IL-10), are humoral factors involved in the suppressive function of regulatory T cells and play critical roles in maintaining immune homeostasis. However, TGF-β and IL-10 also have pleiotropic effects and induce humoral immune responses depending on conditions, and thus their therapeutic application to autoimmune diseases remains limited. Here, we show that a combination of TGF-β and IL-10, but not single cytokine, is required to suppress B cell activation induced by toll-like receptor (TLR) stimulation. In in vivo analyses, the simultaneous presence of TGF-β and IL-10 effectively suppressed TLR-mediated antigen-specific immune responses and ameliorated pathologies in imiquimod (TLR7 agonist)-induced lupus model and lupus-prone MRL/lpr mice. Intriguingly, TGF-β and IL-10 synergistically modulated transcriptional programs and suppressed cellular energetics of both glycolysis and oxidative phosphorylation via inhibition of the mammalian target of rapamycin complex 1 (mTORC1)/S6 kinase 1 (S6K1) pathway in TLR-stimulated B cells. On the other hand, enhancement of mTOR signaling and mitochondrial biosynthesis in TLR-stimulated B cells counteracted the synergistic inhibitory effects. The inhibitory cytokine synergy of TGF-β and IL-10 via suppression of energy metabolism was also observed in human TLR-stimulated B cells. There is increasing evidence supporting the importance of adequate metabolic signals in various immune cells to exert their immune function. In this study, we have shown that a previously unrecognized synergy of inhibitory cytokines regulates systemic humoral immune responses via modulating immunometabolism in B cells. Our findings indicate that inhibition of B cell metabolism mediated by two synergistic cytokines contributes to the induction of immune tolerance and could be a new therapeutic strategy for autoimmune diseases such as systemic lupus erythematosus.

Tofacitinib rapidly ameliorated polyarthropathy in a patient with systemic sclerosis

Systemic sclerosis (SSc) is a chronic autoimmune-mediated disease affectingmultiple organ systems (1, 2). Polyarthritis is a common manifestation of SSc, and disease-modifying anti-rheumatic drugs, including methotrexate (MTX) and biologic agents for rheumatoid arthritis (RA), have been used to treat refractory polyarthritis in SSc (2). Previous reports have suggested that biological blockade of tumour necrosis factor (3) or interleukin-6 (IL-6) (4) and recombinant cytotoxic T-lymphocyte antigen 4–immunoglobulin (CTLA4-Ig) (4) had therapeutic effects on polyarthritis in SSc. Although tofacitinib, a Janus kinase (JAK) inhibitor, is used as an alternative therapeutic option for RA (5), the clinical application of tofacitinib for SSc has not been reported. Here, we report the first case of SSc arthropathy successfully treated with tofacitinib. Written informed consent for the publication was obtained from the patient. A 53-year-old Japanese female was admitted to our hospital owing to polyarthritis. She had been diagnosed with limited cutaneous SSc 3 years previously. She presented with multiple arthralgias involved in the wrists, metacarpophalangeal joints, and proximal interphalangeal joints, with long-term stiffness. She also had Raynaud’s phenomenon, oesophageal dysmotility, puffy fingers, telangiectasias, and thickened sclerotic lower legs. Her serum anti-centromere antibody was positive, whereas other autoantibodies, including rheumatoid factor, anti-cyclic citrullinated peptide antibody, anti-Scl-70 antibody, and anti-RNA polymerase III antibody, were negative. No other organ involvement, including lung and kidney, was found. Inflammatory markers were negative, including C-reactive protein (CRP) at 0.15 mg/dL (reference value 0–0.3 mg/dL) and erythrocyte sedimentation rate (ESR) at 9 mm/h (reference 3–15 mm/h). Physical and laboratory examination revealed high arthritis activity (Figure 1A), and the functional disability index assessed by the Health Assessment Questionnaire (HAQ) was 1.37. An X-ray study did not detect any bone erosions. Imaging studies using power Doppler ultrasound and magnetic resonance imaging found broad subcutaneous oedema and swelling of the fingers, but detected no active synovitis or tenosynovitis. She also showed skin sclerosis [12 points of skin sclerosis as measured by the modified Rodnan skin score (mRSS)], telangiectasias, and nailfold bleeding. Previous treatment with MTX or abatacept had been discontinued owing to the limited efficacy for arthritis, and tocilizumab had been discontinued owing to recurrent pneumonia, despite its partial efficacy for polyarthritis. To regulate the severe arthritis, tofacitinib 5 mg daily was initiated. Four days after administration of tofacitinib, her articular parameters including 28-joint Disease Activity Score (DAS28) improved (Figure 1A), although inflammatory markers were not significantly changed; CRP and ESR were 0.17 mg/dL and 10 mm/h, respectively. In line with the improvement in arthritis disease activity, her HAQ score improved to 1.125. Her skin sclerosis semi-quantified by mRSS was also rapidly ameliorated with tofacitinib (Figure 1B). Furthermore, her nailfold capillary bleeding improved with tofacitinib (Figure 1C). To date, the therapeutic effects of tofacitinib on polyarthritis and skin sclerosis have been continuously observed for 40 days without any serious adverse events (Figure 1A,B). These observations suggest that tofacitinib could be a safe and efficacious treatment for SSc. Because of the complex pathogenesis of SSc, which links vascular, immune, and mesenchymal components (1, 2), treatment may require targeting multiple pathologies in SSc. Hence, there is a growing interest in intracellular signalling inhibitors for novel therapeutic approaches in systemic rheumatic diseases including SSc (2). Blocking the signal cascade could have a continuous effect of inhibition, in comparison with pulsed administration of anti-cytokine antibody. In addition, not only the therapeutic effects of IL-6 blockade on SSc (4, 6), but also the presence of a type I interferon gene signature in SSc implied that JAK pathways could be one of the intracellular signalling targets for the treatment of SSc (7). Moreover, thymic stromal lymphopoietin (TSLP) is a potential inducer of skin fibrosis in SSc patients (8), and the signalling from TSLP is mediated by JAK1/JAK2. JAK inhibitors could modulate these key signalling cytokines and molecules in the pathogenesis of SSc. Our report implies the therapeutic potential of tofacitinib for polyarthritis, skin sclerosis, and nailfold capillary abnormality in SSc, and these observations are consistent with the expected effects of IL-6 blockade on polyarthritis (4), skin sclerosis, and nailfold capillary changes (6). Thus, our findings Scand J Rheumatol 2018;47:505–506 505

Egr2-independent, Klf1-mediated induction of PD-L1 in CD4 + T cells

Programmed death ligand 1 (PD-L1)-mediated induction of immune tolerance has been vigorously investigated in autoimmunity and anti-tumor immunity. However, details of the mechanism by which PD-L1 is induced in CD4+ T cells are unknown. Here, we revealed the potential function of Klf1 and Egr2-mediated induction of PD-L1 in CD4+ T cells. We focused on the molecules specifically expressed in CD4+CD25−LAG3+ regulatory T cells (LAG3+ Tregs) highly express of PD-L1 and transcription factor Egr2. Although ectopic expression of Egr2 induced PD-L1, a deficiency of Egr2 did not affect its expression, indicating the involvement of another PD-L1 induction mechanism. Comprehensive gene expression analysis of LAG3+ Tregs and in silico binding predictions revealed that Krüppel-like factor 1 (Klf1) is a candidate inducer of the PD-L1 gene (Cd274). Klf1 is a transcription factor that promotes β-globin synthesis in erythroid progenitors, and its role in immunological homeostasis is unknown. Ectopic expression of Klf1 induced PD-L1 in CD4+ T cells through activation of the PI3K-mTOR signaling pathway, independent of STATs signaling and Egr2 expression. Our findings indicate that Klf1 and Egr2 are modulators of PD-L1-mediated immune suppression in CD4+ T cells and might provide new insights into therapeutic targets for autoimmune diseases and malignancies.