Tomohisa Okamura

The Family of IL-10-secreting CD4+ T cells.

Regulatory T cells (Treg cells) play critical roles in the induction of peripheral tolerance to self- and foreign antigens. Naturally occurring CD4(+)CD25(+) Treg cells, which characteristically express the transcription factor forkhead box protein P3 (Foxp3), have been studied intensively because their deficiency abrogates self-tolerance and causes autoimmune disease. However, several lines of evidence suggest that additional important mechanisms other than the Foxp3 system are required to enforce immunological self-tolerance in the periphery. Interleukin-10 (IL-10) is a regulatory cytokine that plays a central role in controlling inflammatory processes, and IL-10-secreting T cells may constitute an additional mechanism that are responsible for peripheral tolerance. Type-1 T regulatory (Tr1) cells, CD46-stimulated IL-10-secreting T cells, and IL-10-secreting T cells induced by vitamin D3 (VitD3) and dexamethasone (Dex) are induced populations with significant regulatory activities. However, assessing the detailed physiological function of these cells is difficult, because of the lack of specific markers that can reliably differentiate the population of IL-10-secreting Treg cells from other T cells. Recently, CD4(+)CD25(-)LAP(+) T cells, CD4(+)NKG2D(+) T cells, CD4(+)IL-7R(-) T cells, and CD4(+)CD25(-)LAG3(+) T cells have been reported as naturally present IL-10-secreting Treg cells. Although the relationship between these induced and naturally present IL-10-secreting Treg cells is unclear, elucidation of their respective roles in modulating immune responses is crucial to understand T cell-mediated tolerance. Furthermore, the identification of specific markers and molecular signatures will enable the purification or induction of IL-10-secreting Treg cells for the treatment of patients having inflammatory diseases.

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.

Regulatory T-Cell-Associated Cytokines in Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody production, complement activation, and immune complex deposition, resulting in tissue and organ damage. An understanding of the mechanisms responsible for homeostatic control of inflammation, which involve both innate and adoptive immune responses, will enable the development of novel therapies for SLE. Regulatory T cells (Treg) play critical roles in the induction of peripheral tolerance to self- and foreign antigens. Naturally occurring CD4+CD25+ Treg, which characteristically express the transcription factor forkhead box protein P3 (Foxp3), have been intensively studied because their deficiency abrogates self-tolerance and causes autoimmune disease. Moreover, regulatory cytokines such as interleukin-10 (IL-10) also play a central role in controlling inflammatory processes. This paper focuses on Tregs and Treg-associated cytokines which might regulate the pathogenesis of SLE and, hence, have clinical applications.

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.

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.

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.

Interleukin-27 in T Cell Immunity

Interleukin (IL)-27, a member of IL-12/IL-23 heterodimeric family of cytokines, has pleiotropic properties that can enhance or limit immune responses. IL-27 acts on various cell types, including T cells, B cells, macrophages, dendritic cells, natural killer (NK) cells and non-hematopoietic cells. Intensive studies have been conducted especially on T cells, revealing that various subsets of T cells respond uniquely to IL-27. IL-27 induces expansion of Th1 cells by activating signal transducer and activator of transcription (STAT) 1-mediated T-bet signaling pathway. On the other hand, IL-27 suppresses immune responses through inhibition of the development of T helper (Th) 17 cells and induction of IL-10 production in a STAT1- and STAT3-dependent manner. IL-27 is a potentially promising cytokine for therapeutic approaches on various human diseases. Here, we provide an overview of the biology of IL-27 related to T cell subsets, its structure, and production mechanism.

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.