Keishi Fujio

IL-17B and IL-17C Are Associated with TNF-α Production and Contribute to the Exacerbation of Inflammatory Arthritis

IL-17A is a T cell-derived proinflammatory cytokine that contributes to the pathogenesis of rheumatoid arthritis. Recently, six related molecules have been identified to form the IL-17 family, as follows: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. Whereas IL-17A and IL-17F up-regulate IL-6 in synovial fibroblasts, IL-17B and IL-17C are reported to stimulate the release of TNF-α and IL-1β from the monocytic cell line, THP-1 cell. However, their detailed function remains to be elucidated. We report in this study the effects of IL-17 family on the collagen-induced arthritis (CIA) progression by T cell gene transfer and bone marrow chimeric mice. The mRNA expressions of IL-17 family (IL-17A, IL-17B, IL-17C, and IL-17F) and their receptor (IL-17R and IL-17Rh1) genes in the arthritic paws of CIA mice were elevated compared with controls. Although IL-17A and IL-17F were expressed in CD4+ T cells, IL-17B and IL-17C were expressed in the cartilage and in various cell populations in the CIA arthritic paws, respectively. In vitro, IL-17A, IL-17B, IL-17C, and IL-17F induced TNF-α production in mouse peritoneal exudate cells. In vivo, adoptive transfer of IL-17B- and IL-17C-transduced CD4+ T cells evidently exacerbated arthritis. Bone marrow chimeric mice of IL-17B and IL-17C exhibited elevated serum TNF-α concentration and the high arthritis score upon CIA induction. Moreover, neutralization of IL-17B significantly suppressed the progression of arthritis and bone destruction in CIA mice. Therefore, not only IL-17A, but also IL-17B and IL-17C play an important role in the pathogenesis of inflammatory arthritis.

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.

Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases

IL-32 is a newly described cytokine in the human found to be an in vitro inducer of tumor necrosis factor alpha (TNFα). We examined the in vivo relationship between IL-32 and TNFα, and the pathologic role of IL-32 in the TNFα-related diseases – arthritis and colitis. We demonstrated by quantitative PCR assay that IL-32 mRNA was expressed in the lymphoid tissues, and in stimulated peripheral T cells, monocytes, and B cells. Activated T cells were important for IL-32 mRNA expression in monocytes and B cells. Interestingly, TNFα reciprocally induced IL-32 mRNA expression in T cells, monocyte-derived dendritic cells, and synovial fibroblasts. Moreover, IL-32 mRNA expression was prominent in the synovial tissues of rheumatoid arthritis patients, especially in synovial-infiltrated lymphocytes by in situ hybridization. To examine the in vivo relationship of IL-32 and TNFα, we prepared an overexpression model mouse of human IL-32β (BM-hIL-32) by bone marrow transplantation. Splenocytes of BM-hIL-32 mice showed increased expression and secretion of TNFα, IL-1β, and IL-6 especially in response to lipopolysaccharide stimulation. Moreover, serum TNFα concentration showed a clear increase in BM-hIL-32 mice. Cell-sorting analysis of splenocytes showed that the expression of TNFα was increased in resting F4/80+ macrophages, and the expression of TNFα, IL-1β and IL-6 was increased in lipopolysaccharide-stimulated F4/80+ macrophages and CD11c+ dendritic cells. In fact, BM-hIL-32 mice showed exacerbation of collagen-antibody-induced arthritis and trinitrobenzen sulfonic acid-induced colitis. In addition, the transfer of hIL-32β-producing CD4+ T cells significantly exacerbated collagen-induced arthritis, and a TNFα blockade cancelled the exacerbating effects of hIL-32β. We therefore conclude that IL-32 is closely associated with TNFα, and contributes to the exacerbation of TNFα-related inflammatory arthritis and colitis.

Functional Reconstitution of Class II MHC-Restricted T Cell Immunity Mediated by Retroviral Transfer of the αβ TCR Complex

Transfer of the αβ TCR genes into T lymphocytes will provide a means to enhance Ag-specific immunity by increasing the frequency of tumor- or pathogen-specific T lymphocytes. We generated an efficient αβ TCR gene transfer system using two independent monocistronic retrovirus vectors harboring either of the class II MHC-restricted α or β TCR genes specific for chicken OVA. The system enabled us to express the clonotypic TCR in 44% of the CD4+ T cells. The transduced cells showed a remarkable response to OVA323–339 peptide in the in vitro culture system, and the response to the Ag was comparable with those of the T lymphocytes derived from transgenic mice harboring OVA-specific TCR. Adoptive transfer of the TCR-transduced cells in mice induced the Ag-specific delayed-type hypersensitivity in response to OVA323–339 challenge. These results indicate that αβ TCR gene transfer into peripheral T lymphocytes can reconstitute Ag-specific immunity. We here propose that this method provides a basis for a new approach to manipulation of immune reactions and immunotherapy.

A Genome-Wide Association Study Identified AFF1 as a Susceptibility Locus for Systemic Lupus Eyrthematosus in Japanese

Systemic lupus erythematosus (SLE) is an autoimmune disease that causes multiple organ damage. Although recent genome-wide association studies (GWAS) have contributed to discovery of SLE susceptibility genes, few studies has been performed in Asian populations. Here, we report a GWAS for SLE examining 891 SLE cases and 3,384 controls and multi-stage replication studies examining 1,387 SLE cases and 28,564 controls in Japanese subjects. Considering that expression quantitative trait loci (eQTLs) have been implicated in genetic risks for autoimmune diseases, we integrated an eQTL study into the results of the GWAS. We observed enrichments of cis-eQTL positive loci among the known SLE susceptibility loci (30.8%) compared to the genome-wide SNPs (6.9%). In addition, we identified a novel association of a variant in the AF4/FMR2 family, member 1 (AFF1) gene at 4q21 with SLE susceptibility (rs340630; P = 8.3×10−9, odds ratio = 1.21). The risk A allele of rs340630 demonstrated a cis-eQTL effect on the AFF1 transcript with enhanced expression levels (P<0.05). As AFF1 transcripts were prominently expressed in CD4+ and CD19+ peripheral blood lymphocytes, up-regulation of AFF1 may cause the abnormality in these lymphocytes, leading to disease onset.

Potentiation of Tumor Eradication by Adoptive Immunotherapy with T-cell Receptor Gene-Transduced T-Helper Type 1 Cells

Adoptive immunotherapy using antigen-specific T-helper type 1 (Th1) cells has been considered as a potential strategy for tumor immunotherapy. However, its application to tumor immunotherapy has been hampered by difficulties in expanding tumor-specific Th1 cells from tumor-bearing hosts. Here, we have developed an efficient protocol for preparing mouse antigen-specific Th1 cells from nonspecifically activated Th cells after retroviral transfer of T-cell receptor (TCR)-α and TCR-β genes. We demonstrate that Th1 cells transduced with the TCR-α and -β genes from the I-Ad-restricted ovalbumin (OVA)323–339-specific T-cell clone DO11.10 produce IFN-γ but not interleukin-4 in response to stimulation with OVA323–339 peptides or A20 B lymphoma (A20-OVA) cells expressing OVA as a model tumor antigen. TCR-transduced Th1 cells also exhibited cytotoxicity against tumor cells in an antigen-specific manner. Moreover, adoptive transfer of TCR-transduced Th1 cells, but not mock-transduced Th1 cells, exhibited potent antitumor activity in vivo and, when combined with cyclophosphamide treatment, completely eradicated established tumor masses. Thus, TCR-transduced Th1 cells are a promising alternative for the development of effective adoptive immunotherapies.

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.

Antigen-Specific T Cells Transduced with IL-10 Ameliorate Experimentally Induced Arthritis Without Impairing the Systemic Immune Response to the Antigen

For the treatment of rheumatoid arthritis, efficient drug delivery methods to the inflamed joints need to be developed. Because T cells expressing an appropriate autoantigen-specific receptor can migrate to inflamed lesions, it has been reasoned that they can be employed to deliver therapeutic agents. To examine the ability and efficiency of such T cells as a vehicle, we employed an experimentally induced model of arthritis. Splenic T cells from DO11.10 TCR transgenic mice specific for OVA were transduced with murine IL-10. Adoptive transfer of the IL-10-transduced DO11.10 splenocytes ameliorated OVA-induced arthritis despite the presence of around 95% nontransduced cells. Using green fluorescent protein as a marker for selection, the number of transferred cells needed to ameliorate the disease was able to be reduced to 104. Preferential accumulation of the transferred T cells was observed in the inflamed joint, and the improvement in the disease was not accompanied by impairment of the systemic immune response to the Ag, suggesting that the transferred T cells exert their anti-inflammatory task locally, mainly in the joints where the Ag exists. In addition, IL-10-transduced DO11.10 T cells ameliorated methylated BSA-induced arthritis when the arthritic joint was coinjected with OVA in addition to methylated BSA. These results suggest that T cells specific for a joint-specific Ag would be useful as a therapeutic vehicle in rheumatoid arthritis for which the arthritic autoantigen is still unknown.

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.