Intrathymic Plasmablasts Are Affected in Patients With Myasthenia Gravis With Active Disease

Background and ObjectivesTo investigate intrathymic B lymphopoiesis in patients with myasthenia gravis (MG) and explore thymus pathology associated with clinical impact.MethodsThymic lymphocytes from 15 young patients without MG, 22 adult patients without MG, 14 patients with MG without thymoma, and 11 patients with MG with thymoma were subjected to flow cytometry analysis of T follicular helper (Tfh), naive B, memory B, plasmablasts, CD19+B220high thymic B cells, B-cell activating factor receptor, and C-X-C chemokine receptor 5 (CXCR5). Peripheral blood mononuclear cells of 16 healthy subjects and 21 untreated patients with MG were also analyzed. Immunologic values were compared, and correlations between relevant values and clinical parameters were evaluated.ResultsThe frequencies of circulating and intrathymic plasmablasts were significantly higher in patients with MG than controls. On the other hand, the frequency of CD19+B220high thymic B cells was not increased in MG thymus. We observed a significant increase in CXCR5 expression on plasmablasts in MG thymus and an increased frequency of intrathymic plasmablasts that was correlated with preoperative disease activity. The frequency of intrathymic Tfh cells was significantly lower in patients who received immunosuppressive (IS) therapy than those without IS therapy. However, there was no significant difference in the frequency of intrathymic plasmablasts irrespective of IS therapy.DiscussionOur findings confirmed a correlation between increased frequency of intrathymic plasmablasts and disease activity before thymectomy. We postulate that activated intrathymic plasmablasts endow pathogenic capacity in MG.

CD40 Signaling Promotes CXCR5 Expression in B Cells via Noncanonical NF-κB Pathway Activation

Chemokine receptor CXCR5-mediated control of B cell trafficking in the lymphoid tissues plays a central role in orchestrating the B cell function, which not only guides the colocalization of B cells with follicular helper T cells in the follicular mantle zone but also determines the position of germinal center dark and light zones. However, the mechanisms that regulate the expression of CXCR5 in B cells remain unclear. Here, we show that the expression level of CXCR5 in B cells was substantially reduced in vitro culture conditions, while being maintained in the presence of CD40 signals. Furthermore, CD40 signaling promotes CXCR5 expression in B cells at least partially through noncanonical NF-κB signaling pathway activation. However, other non-B cells also contributed to the optimal expression of CXCR5 in B cells through cell-cell contact and cytokine secretion. Our findings suggest that CD40 signaling-mediated activation of the noncanonical NF-κB pathway promotes the expression of CXCR5 in a B cell-intrinsic way to orchestrate the trafficking of B cells.

CXCR5 gene expression in human lymph node CD8+ T cells is regulated by DNA methylation and nucleosomal occupancy

CD8+ T cells play an important role in viral and tumour control. However, in human lymph nodes (LNs), only a small subset of CD8+ T cells called follicular CD8+ T cells (fCD8s) expresses CXCR5, the chemokine receptor required for cell migration into B cell follicles, thought to promote immune evasion. Here we obtained LNs from HIV infected persons to investigate regulation of CXCR5 expression in lymphoid CD8+ T cells, and compared this to the more abundant CXCR5 expressing T follicular CD4+ helper cells (GCTfh). Our results show that DNA hypermethylation and closed chromatin at the transcriptional start site (TSS) prevent CXCR5 expression in non-fCD8s. We also found that greater nucleosomal density at the CXCR5 TSS could be responsible for reduced CXCR5 expression in fCD8s relative to GCTfh. Together, these data provide critical insights into both the underlying molecular mechanisms that repress CXCR5 expression in non-fCD8s and the plausible mechanism responsible for the low CXCR5 expression in fCD8s, with implications for HIV cure strategies.Author SummaryA paucity of CD8+ T cells that express CXCR5, the chemokine receptor critical for entering the B cell follicles of secondary lymphoid tissues have recently been described. Animal studies have revealed transcriptional networks that govern the expression of CXCR5 in CD8+ T cells. However, it is not known if similar or additional networks regulate the expression of CXCR5 in human CD8+ T cells. In this study, we demonstrated that DNA methylation coupled with chromatin compaction at the transcriptional start site (TSS) of CXCR5 gene prevent the expression CXCR5 in human CD8+ T cells. In addition, we observed greater nucleosomal occupancy at the TSS of CXCR5 gene which could impact expression levels of CXCR5 in human CXCR5+CD8+ T cells. This study revealed multitiered epigenetic mechanisms that repress CXCR5 expression in human CD8+ T cells, with implications for HIV cure strategy or eradication of B cell-derived tumours.

Correlation between CXCR5 expression and prognosis in stage T1 non-small cell lung cancer

Background: Lung cancer is the leading cause of cancer-related death. Even if early detection has been applied and proved to be effective, the survival outcomes are still poor. Thus, to find out novel prognostic factors is of great significance to identify high-risk patients and guide individualized precise treatment in order to reduce recurrence and improve survival outcomes.Methods: Tissue samples and clinicopathologic data of 244 stage T1 NSCLC patients were collected. We investigated CXCR5 expression using immunochemical method and analyzed its correlation with pathologic subtypes and prognosis.Results: Elevated level of positive CXCR5 expression was found in tumor tissues (p<0.0001) and patients with positive CXCR5 expression are more likely to have LVIs (p=0.030) and recurrences (p<0.043). Besides, CXCR5 expression is also correlated to histological type and differentiation. Lepidic predominant tumors have significantly lower expression of CXCR5 (p<0.001). Survival analyses showed that patients with positive CXCR5 had a significantly lower DFS (p=0.038) and patients with solid or micropapillary predominant NSCLC had a significantly worse prognosis. CXCR5 expression was proved to be an independent prognostic factor for DFS through multivariate analysis.Conclusions: CXCR5 expression has been proved to be an independent prognostic factor for stage T1 NSCLC patients. In addition, CXCR5 was also found to be relevant to cancer recurrence, and this indicated that CXCR5 may play an important role in lymph node and distant metastasis.

Exosomal miR-214-5p Released from Glioblastoma Cells Modulates Inflammatory Response of Microglia after Lipopolysaccharide Stimulation through Targeting CXCR5

Background and Objective: Exosomes communicate inter-cellularly and miRNAs play critical roles in this scenario. MiR-214-5p was implicated in multiple tumors with diverse functions uncovered. However, whether miR-214-5p is mechanistically involved in glioblastoma, especially via exosomal pathway, is still elusive. Here we sought to comprehensively address the critical role of exosomal miR-214-5p in glioblastoma (GBM) microenvironment.Methods:The relative expression of miR-214-5p was determined by real-time PCR. Cell viability and migration were measured by MTT and transwell chamber assays, respectively. The secretory cytokines were measured with ELISA kits. The regulatory effect of miR-214-5p on CXCR5 expression was interrogated by luciferase reporter assay. Protein level was analyzed by Western blot.Results:We demonstrated that miR-214-5p was aberrantly overexpressed in GBM and associated with poorer clinical prognosis. High level of miR-214-5p significantly contributed to cell proliferation and migration. GBM-derived exosomal miR-214-5p promoted inflammatory response in primary microglia upon lipopolysaccharide challenge. We further identified CXCR5 as the direct target of miR-214- 5p in this setting.Conclusion:Overexpression of miR-214-5p in GBM modulated the inflammatory response in microglia via exosomal transfer.

Mapping the lineage relationship between CXCR5+ and CXCR5- CD4+ T cells in HIV infected human lymph nodes

SummaryCXCR5 is a key surface marker expressed on follicular helper T (TFH) cells. We report here B cell help functionality in a population of CD4+ T cells isolated from primary human lymph nodes (LN) that lacked CXCR5 expression. This CXCR5- subset is distinguished from other CXCR5- CD4+ T cells by high PD-1 expression. Accumulation of CXCR5-PD-1+ T cells correlated with peripheral CD4+ T cell depletion and an increase in T-bet+ B cells in the LN, highlighting these atypical CD4+ T cells as a key component of lymphoid dysregulation during chronic HIV infection. By interrogating the phenotypic heterogeneity, functional capacity, TCR repertoire, transcriptional profile, and epigenetic state of CXCR5-PD-1+ T cells, we showed that CXCR5-PD-1+ T cells are related to CXCR5+PD-1+ T cells and provided evidence for the down regulation of CXCR5 following cell division as one mechanism for the absence of CXCR5 expression. Notably, CXCR5-PD-1+ T cells exhibited a migratory transcriptional program and contributed to circulating CXCR5-PD-1+ T cells with B cell help functionality in the peripheral blood. Thus, these data link LN pathology to circulating T cells and expand the current understanding on T cell diversity in the regulation of B cell responses during chronic inflammation.High dimensional profiling of activated CD4+ T cells in HIV infected lymph nodes revealed an accumulation of a CXCR5 negative subset.CXCR5-PD-1+CD4+ T cells exhibited TFH-like protein expression and function.CXCR5-PD-1+CD4+ T cells are related to TFH cells by clonal lineage and epigenetic similarity.CXCR5-PD-1+CD4+ T cells upregulate a migratory gene program and contribute to circulating T cells with B cell help functionality