Shuji Sumitomo

CD4+CD25−LAG3+ regulatory T cells controlled by the transcription factor Egr-2

Regulatory T cells (Tregs) are engaged in the maintenance of immunological self-tolerance and immune homeostasis. IL-10 has an important role in maintaining the normal immune state. Here, we show that IL-10-secreting Tregs can be delineated in normal mice as CD4+CD25−Foxp3− T cells that express lymphocyte activation gene 3 (LAG-3), an MHC-class-II-binding CD4 homolog. Although ≈2% of the CD4+CD25− T cell population consisted of CD4+CD25−LAG3+ T cells in the spleen, CD4+CD25−LAG3+ T cells are enriched to ≈8% in the Peyers patch. They are hypoproliferative upon in vitro antigenic stimulation and suppress in vivo development of colitis. Gene expression analysis reveals that CD4+CD25−LAG3+ Tregs characteristically express early growth response gene 2 (Egr-2), a key molecule for anergy induction. Retroviral gene transfer of Egr-2 converts naïve CD4+ T cells into the IL-10-secreting and LAG-3-expressing phenotype, and Egr-2-transduced CD4+ T cells exhibit antigen-specific immunosuppressive capacity in vivo. Unlike Foxp3+ natural Tregs, high-affinity interactions with selecting peptide/MHC ligands expressed in the thymus do not induce the development of CD4+CD25−LAG3+ Tregs. In contrast, the number of CD4+CD25−LAG3+ Tregs is influenced by the presence of environmental microbiota. Thus, IL-10-secreting Egr-2+LAG3+CD4+ Tregs can be exploited for the control of peripheral immunity.

Egr‐2 transcription factor is required for Blimp‐1‐mediated IL‐10 production in IL‐27‐stimulated CD4+ T cells

Interleukin‐27 (IL‐27) suppresses immune responses through inhibition of the development of IL‐17 producing Th17 cells and induction of IL‐10 production. We previously showed that forced expression of early growth response gene 2 (Egr‐2), a transcription factor required for T‐cell anergy induction, induces IL‐10 and lymphocyte activation gene 3 expression and confers regulatory activity on CD4+ T cells in vivo. Here, we evaluated the role of Egr‐2 in IL‐27‐induced IL‐10 production. Among various IL‐10‐inducing factors, only IL‐27 induced high levels of Egr‐2 and lymphocyte activation gene 3 expression. Intriguingly, IL‐27 failed to induce IL‐10 in Egr‐2‐deficient T cells. IL‐27‐mediated induction of Prdm1 that codes B lymphocyte induced maturation protein‐1, a transcriptional regulator important for IL‐10 production in CD4+ T cells, was also impaired in the absence of Egr‐2. Although IL‐27‐mediated IL‐10 induction was dependent on both STAT1 and STAT3, only STAT3 was required for IL‐27‐mediated Egr‐2 induction. These results suggest that IL‐27 signal transduction through Egr‐2 and B lymphocyte induced maturation protein‐1 plays an important role in IL‐10 production. Furthermore, Egr‐2‐deficient CD4+ T cells showed dysregulated production of IFN‐γ and IL‐17 in response to IL‐27 stimulation. Therefore, Egr‐2 may play key roles in controlling the balance between regulatory and effector cytokines.

Detection of autoantibodies to citrullinated BiP in rheumatoid arthritis patients and pro-inflammatory role of citrullinated BiP in collagen-induced arthritis

IntroductionAnti-citrullinated protein/peptide antibodies (ACPAs) are highly specific to rheumatoid arthritis (RA) patients and are thought to have a close relationship with the pathogenesis of arthritis. Several proteins, including fibrinogen, vimentin, and alpha-enolase, were reported as ACPA-target antigens, and their importance in RA pathogenesis was widely proposed. We identified citrullinated immunoglobulin binding protein (citBiP) as another ACPA target in RA patients and examined its pro-inflammatory role in arthritis.MethodsWe measured the levels of anti-citBiP, anti-BiP, and anti-cyclic citrullinated peptide (CCP) antibodies in the serum of RA patients (n = 100), systemic lupus erythematosus (SLE) patients (n = 60), and healthy controls (n = 30) using ELISA and immunoblotting. Epitope mapping was performed using 27 citBiP-derived peptides. In the mouse study, after DBA/1J mice were immunized with BiP or citBiP, serum titers of ACPAs were measured by ELISA and immunohistochemistry. The development of collagen-induced arthritis (CIA) was observed in BiP- or citBiP-pre-immunized mice.ResultsThe serum levels of anti-BiP and anti-citBiP antibodies were significantly increased in RA patients, although only anti-BiP antibodies were slightly increased in SLE patients. Interestingly, anti-citBiP antibody levels were higher than anti-BiP antibody levels in 72% of RA patients, whereas no significant increase in anti-citBiP antibody levels was detected in SLE patients and healthy controls. The serum levels of anti-CCP antibodies were correlated with those of anti-citBiP antibodies in RA patients (R2 = 0.41). Several citrulline residues of citBiP were determined to be major epitopes of anti-citBiP antibodies, one of which showed cross-reactivity with CCP. Immunization of DBA/1J mice with citBiP induced several kinds of ACPAs, including anti-CCP and anti-citrullinated fibrinogen antibodies. Pre-immunization with citBiP exacerbated CIA, and anti-CCP antibody levels were increased in citBiP-pre-immunized CIA mice.ConclusionsCitBiP is a newly described ACPA target that may play a pro-inflammatory role in arthritis.

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.

Roles of LAG3 and EGR2 in regulatory T cells

Regulatory T cells (Tregs) participate in the maintenance of tolerance to self-antigens and suppressive control of excessive immune responses to exogenous antigens. A lack or dysfunction of these cells is responsible for the pathogenesis and development of many autoimmune diseases. It is well known that CD4 Tregs play a major role in controlling immune responses and can be classified into two main populations: thymus-derived naturally occurring Tregs (nTregs) and induced Tregs (iTregs) generated from CD4+CD25− precursors in the peripheral lymphoid organs. The most extensively studied Tregs are the nTregs, which express the interleukin 2 (IL-2) receptor CD25 and the transcription factor Forkhead box P3 (Foxp3). On the other hand, iTregs contain multiple heterogeneous subsets, including interleukin (IL)-10-producing CD4 type I Tregs (Tr1 cells) and transforming growth factor -β-producing Th3 cells, and so on. However, the extent of the contribution of iTregs to immunoregulation in normal animals has been difficult to evaluate because of the lack of suitable cell surface markers. It has been found recently that IL-10-secreting iTregs can be delineated as CD4+CD25−Foxp3− T cells that characteristically express both the lymphocyte activation gene-3 (LAG3) and the early growth response gene-2 (EGR2). In this review, opinions about the roles of LAG3 and EGR2 in Tregs are presented.

Regulatory cell subsets in the control of autoantibody production related to systemic autoimmunity

Dysregulated autoantibody production is responsible for the severe organ damage that occurs in systemic autoimmune diseases. Immune complexes play important roles in the pathogenesis of these diseases as they initiate and maintain the inflammatory cascade, which leads to tissue destruction. Conventional therapy for autoimmune diseases suppresses immunological accelerator in the absence of knowledge of the immunological brake. Application of a physiological regulatory system could be a rational strategy to treat autoimmune diseases. Accumulating evidence has suggested that specialised subsets of B cells and T cells could control autoantibody production. A significant decrease and impaired function of regulatory B cells (Breg) was recently reported in patients with systemic lupus erythematosus and a mice model of lupus. In T cells, follicular regulatory T cells and Qa-1 restricted CD8 regulatory T cells (Treg) were identified as the populations that control follicular T helper cell-mediated antibody production. Moreover, other Treg populations might also be involved in the control of autoantibody production. Elucidating the roles of Breg and Treg in the control of antibody production might lead to the development of a new therapeutic approach to antibody-mediated autoimmune disease.

Autoantigen BiP-Derived HLA–DR4 Epitopes Differentially Recognized by Effector and Regulatory T Cells in Rheumatoid Arthritis

The balance between effector and regulatory CD4+ T cells plays a key role in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to examine whether the RA autoantigen BiP has epitopes for both effector and regulatory immunities.

Regulatory T Cell-Mediated Control of Autoantibody-Induced Inflammation

Autoimmune inflammation including autoantibody-induced inflammation is responsible for the lethal organ damage. Autoantibody-induced inflammation can be separated in two components, autoantibody production, and local inflammatory responses. Accumulating evidence has suggested that regulatory T cells (Treg) control both antibody production and the numbers and functions of effector cells such as innate cells and T helper cells. Autoantibodies are produced by both the follicular and extrafollicular pathways. Recently, follicular regulatory T cells (TFR) and Qa-1 restricted CD8+ Treg were identified as populations that are capable of suppressing follicular T helper cell (TFH)-mediated antibody production. In local inflammation, CD4+CD25+Foxp3+ Treg have the capacity to control inflammation by suppressing cytokine production in T helper cells. Although complement proteins contribute to autoantibody-induced local inflammation by activating innate cells, Treg including CD4+CD25+Foxp3+ Treg are able to suppress innate cells, chiefly via IL-10 production. IL-10-secreting T cells such as T regulatory type I (Tr1) and Tr1-like cells might also play roles in the control of Th17 and innate cells. Therefore, several kinds of Tregs have the potential to control autoimmune inflammation by suppressing both autoantibody production and the local inflammatory responses induced by autoantibodies.

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.

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.