Objective. This study was undertaken to identify characteristics of follicular regulatory T (Tfr) cells and elucidate the mechanisms by which follicular helper T (Tfh) cells convert to Tfr cells. We probed the phenotype of T helper cells in patients with systemic lupus erythematosus (SLE) and underlying transcriptional regulation using cytokine-induced STAT family factors. Methods. Peripheral blood mononuclear cells from 41 patients with SLE and 26 healthy donors were used to sort out the memory Tfh cell subset, and Tfh cells were cultured under various conditions. The phenotype of T helper cells and underlying mechanisms of transcriptional regulation were probed using flow cytometry and quantitative polymerase chain reaction analyses. These analyses evaluated the expression of characteristic markers and phosphorylation of STATs. Chromatin immunoprecipitation was used to evaluate histone modifications. Results. In patients with SLE, the proportion of CD4+CXCR5+FoxP3-PD-1 high Tfh cells was increased (P < 0.01), whereas the proportion of CD4+CXCR5+CD45RA-FoxP3 high activated Tfr cells was decreased (P < 0.05). Serum interleukin-2 (IL-2) levels were also reduced in patients with SLE. IL-2 induced conversion of memory Tfh cells to functional Tfr cells, which was characterized by CXCR5+Bcl-6+FoxP3 high pSTAT3+pSTAT5+ cells. The loci of FOXP3 and BCL6 at STAT binding sites were marked by bivalent histone modifications. Following IL-2 stimulation, STAT3 and STAT5 selectively bound to FOXP3 and BCL6 gene loci accompanied by suppression of H3K27me3. Finally, IL-2 stimulation suppressed the generation of CD38+CD27 high plasmablasts in Tfh and B cell coculture assays ex vivo. Conclusion. Impaired function of Tfr cells might be attributed to defective IL-2 production. Exogenous IL-2 restores the function of Tfr cells through the conversion of Tfh cells to Tfr cells in patients with SLE. Thus, restoring balance between Tfh and Tfr cells may provide new therapeutic approaches in SLE.
Objective Peripheral helper T (Tph) cells interact with B cells and promote immune responses at sites of ectopic lymphoid structures (ELSs). To assess the characteristics of Tph cells, we investigated the phenotype of T helper (Th) cells in patients with systemic lupus erythematosus (SLE) and the underlying competitive binding mechanisms using cytokines-mediated signal transducer and activator of transcription (STAT) factors. Methods Peripheral blood mononuclear cells from SLE patients and healthy controls were analyzed for phenotype identifying. Serum cytokine levels were detected using Luminex assays. In vitro culture was performed to assess cytokine-induced conversion of phenotypes and transcriptional regulation using flow cytometry and PCR. Chromatin immunoprecipitation was used to evaluate STATs binding and histone modifications. Results CXCR5-PD-1+Tph-like cells were increased in SLE patients and showed strong association with disease activity and renal involvement. Serum IFN-α levels were increased and associated with Tph frequency. IFN-α promoted the differentiation of IL-10-producing CXCR5-PD-1+Tph-like cells, increased the responsiveness of IL-2, and induced the conversion of Tfh-like cells to Tph-like cells. STAT5 gained advantage and bound to BCL6 locus at expense of STAT1, accompanied by suppression of H3K4me3. Finally, anti-IFNAR1 decreased the differentiation of Tph-like cells, thereby suppressing the generation of CD38highCD27highplasmablasts. Conclusion Tph cells might be crucial makers to effectively reflect disease activity level in SLE patients. The finding that synergy of IFN-α and IL-2 increases Tph cells through competitive transcriptional regulation, could be one of the mechanisms responsible for pathologic formation of ELSs and helpful for selection of individualized therapeutic approaches for SLE.
Objectives T peripheral helper (Tph) cells have major roles in pathological processes in systemic lupus erythematosus (SLE). We sought to clarify the mechanisms of Tph cell differentiation and their relevance to clinical features in patients with SLE. Method Phenotypes and functions of Tph cell–related markers in human CD4+ T cells purified from volunteers or patients were analyzed using flow cytometry and quantitative PCR. Renal biopsy specimens from patients with lupus nephritis (LN) were probed by multicolor immunofluorescence staining. Results Among multiple cytokines, transforming growth factor (TGF)-β3 characteristically induced programmed cell death protein 1 (PD-1) hi musculoaponeurotic fibrosarcoma (MAF)+, interleukin (IL)-21+IL-10+ Tph-like cells with a marked upregulation of related genes including PDCD-1, MAF, SOX4, and CXCL13. The induction of Tph-like cells by TGF-β3 was suppressed by the neutralization of TGF-β type II receptor (TGF-βR2). TGF-β3-induced Tph-like cells efficiently promoted the differentiation of class-switch memory B cells into plasmocytes, resulting in enhanced antibody production. The proportion of Tph cells in the peripheral blood was significantly increased in patients with SLE than in healthy volunteers in concordance with disease activity and severity of organ manifestations such as LN. TGF-β3 was strongly expressed on macrophages, which was associated with the accumulation of CD4+ C-X-C chemokine receptor (CXCR5)-PD-1+ Tph cells, in the renal tissue of patients with active LN. Conclusion The induction of Tph-like cells by TGF-β3 mainly produced from tissue macrophages plays a pivotal role in the pathological processes of active LN by enhancing B cell differentiation in patients with SLE.
Background The single nucleotide polymorphism (SNP) rs62324212, located in IL21 antisense RNA 1 (IL21-AS1), has been identified as a genetic risk variant associated with systemic lupus erythematosus (SLE). We aimed to probe the characteristics of IL21-AS1 and explore its clinical relevance focusing on T helper subsets and disease activity in patients with SLE. Methods rs62324212 genotyping was determined using allelic discrimination by quantitative PCR. Gene expression in peripheral blood mononuclear cells and cell surface markers in CD4+ T cells were analyzed using PCR and flow cytometry. The association among IL21-AS1, CD4+ T cell subsets, and SLE disease activity was accessed. Results Ensembl Genome Browser analysis revealed that rs62324212 (C>A) was located in the predicting enhancer region of IL21-AS1. IL21-AS1 was expressed in the nucleus of CD4+ T and B cells, but its expression was decreased in patients with SLE. IL21-AS1 expression was positively correlated with mRNA levels of IL-2 but not IL-21, and it was associated with the proportion of activated T follicular regulatory (Tfr) cells. Furthermore, we observed a significant negative correlation between IL21-AS1 expression and disease activity in patients with SLE (n = 53, p < 0.05). Conclusion IL21-AS1 has an effect on disease activity through an involvement of IL-2-mediated activation of Tfr cells in SLE. Thus, targeting the IL21-AS1 may provide therapeutic approaches for SLE.
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