Germinal centers provide a microenvironment that promotes and regulates the interactions of B-cells with follicular T-helper cells (TFH). Here we show that there are significantly higher frequencies of CXCR5+ICOS+TFH cells in autoimmune BXD2 mice, and these cells express both interleukin (IL)-21R and IL-17RA. Although IL-17 and IL-21 are both important for the formation of spontaneous GCs and development of pathogenic autoantibodies, IL-21, but not IL-17, is required for the proper development of TFH cells in BXD2 mice. The total numbers of TFH cells and their ability to induce B cell responses in vitro were not affected by a deficiency of IL-17RA in BXD2-Il17ra−/− mice, the majority of CXCR5+ TFH cells from BXD2-Il17ra−/− mice were, however, not localized in the GC light zone (LZ). Interruption of IL-17 signaling, either acutely by AdIL-17R:Fc or chronically by Il17ra−/−, disrupted TFH–B interactions and abrogated the generation of autoantibody-forming B cells in BXD2 mice. IL-17 upregulated the expression of regulator of G-protein signaling (RGS)16 to promote the ability of TFH to form conjugates with B cells which was abolished in TFH cells from BXD2-Rgs16−/− mice. The results suggests that IL-17 is an extrinsic stop signal that it acts on post-differentiated IL-17RA+ TFH to enable its interaction with responder B cells in the LZ niche. These data suggest a novel concept that TFH differentiation and its stabilization in the LZ are two separate checkpoints and that IL-21 and IL-17 act at each checkpoint to enable pathogenic GC development.
Objective Follicular regulatory T (Tfr) cells act as the regulatory counterpart of follicular T helper (Tfh) cells to suppress germinal center (GC) B cell differentiation. We recently identified that interleukin 21 (IL-21) promoted Tfh differentiation in autoimmune BXD2 mice that develop spontaneous GCs. The objective of this study was to determine the modulatory effects of IL-21 on Tfr and the Tfr/Tfh balance in BXD2 mice. Methods The percentage and phenotype of Tfr were determined in BXD2 and BXD2-Il21−/− mice. The effects of IL-21 on Tfr and the ratio of Tfr/Tfh were evaluated. Sorted Tfr cells from BXD2-Il21−/− mice were co-cultured with Tfh and B cells, or transferred into BXD2 mice to determine their function. Results GC B cells and Tfh cells were significantly reduced, but the percentage of Tfr cells was 2-fold higher in BXD2-Il21−/− mice than in WT-BXD2. Adenovirus-IL-21 administration to BXD2-Il21−/− mice decreased Tfr and the ratio of Tfr/Tfh but increased GC B cells in the spleen. rmIL-21 suppressed Foxp3 and significantly reduced Tgfb1, Il2 and Gitr but enhanced Il21, Il6, Pd1, Cxcr5 and Icos in Tfr cells. IL-21 also counteracted Tfr-mediated inhibition of antibody secretion in the Tfh-B cell co-culture system. Transfer of Tfr cells into young BXD2 mice reduced GC size and decreased autoantibody-producing B cells. Conclusion High levels of IL-21 selectively enhanced Tfh differentiation but inhibited Tfr commitment and their suppressive function on Tfh and B cells, suggesting that IL-21 skews the balance from Tfr to Tfh to promote autoreactive GC reactions in BXD2 mice.
Purpose: As an E2-conjugating enzyme for sumoylation, Ubc9 plays a critical role in sumoylation-mediated cellular pathways, ultimately impacting cell growth and cancer development. The aim of this study was to investigate the regulation of Ubc9 in cancer cells. Experimental Design: Immunohistochemistry and Western blot were used to determine Ubc9 expression in paraffin-embedded tumor tissue and frozen specimens of the matched tumors from the same patient, respectively. To establish the causal relationship between miR-30e and Ubc9 expression, we overexpressed miR-30e and then determined the resultant effects on Ubc9 expression. To determine whether miR-30e directly targets Ubc9, we did luciferase assays using luciferase reporters carrying the 3 ¶-untranslated region (3 ¶-UTR) of the Ubc9 gene. Results: We found that Ubc9 is up-regulated in breast, head and neck, and lung cancer specimens. In addition, an examination of eight pairs of matched breast tumor specimens by Western blot analysis revealed that, on average, the level of Ubc9 is 5.7-fold higher in tumor than in the matched normal breast tissue. Of interest, we present evidence that Ubc9 is subjected to posttranscriptional regulation by microRNA, and the miR-30 family, such as miR-30e, negatively regulates Ubc9 expression. In contrast to Ubc9, miR-30e is underexpressed in tumors. Moreover, ectopic expression of miR-30e suppresses cell growth, which can be partially reversed by Ubc9. Finally, using luciferase-Ubc9-3 ¶-UTR reporters, we show that Ubc9 is a direct target for miR-30e by interactions with the putative miR-30e binding sites. Conclusion: These results provide new insight into regulation of Ubc9 in cancer cells.
Objective. Bidirectional interactions between granulocyte-macrophage colony-stimulating factor–positive (GM-CSF+) T cell and interferon regulatory factor 5–positive (IRF-5+) macrophages play a major role in autoimmunity. In the absence of SH2 domain-containing phosphatase 1 (SHP-1), GM-CSF–stimulated cells are resistant to death receptor (DR)–mediated apoptosis. The objective of this study was to determine whether TRA-8, an anti-DR5 agonistic antibody, can eliminate inflammatory macrophages and CD4 T cells in the SHP-1–defective condition. Methods. Ubiquitous Cre (Ubc.Cre) human/mouse-chimeric DR5-transgenic mice were crossed with viable SHP-1–defective motheaten (mev/mev) mice. TRA-8 was administered weekly for up to 4 weeks. The clinical scores, histopathologic severity, and macrophage and CD4 T cell phenotypes were evaluated. The role of TRA-8 in depleting inflammatory macrophages and CD4 T cells was also evaluated, using synovial fluid obtained from patients with rheumatoid arthritis (RA). Results. The levels of Inflammatory macrophages (interleukine-23–positive [IL-23+] IRF5+) and CD4 T (IL-17+GM-CSF+) cells were elevated in mev/mev mice. In DR5-transgenic mev/mev mice, DR5 expression was up-regulated in these 2 cell populations. TRA-8 treatment depleted these cells and resulted in a significant reduction of inflammation and in the titers of autoantibodies. In synovial cells from patients with RA, the expression of IRF5 and DR5 was negatively correlated with the expression of PTPN6. TRA-8, but not TRAIL, suppressed RA inflammatory macrophages and Th17 cells under conditions in which the expression of SHP-1is low. Conclusion. In contrast with TRAIL, which lacks the capability to counteract the survival signal in the absence of SHP-1, TRA-8 eliminated both IRF5+ IL-23+ M1 macrophages and pathogenic GM-CSF+ IL-17+ CD4 T cells in a SHP-1-independent manner. The results of the current study suggest that TRA-8 can deplete inflammatory cell populations that result from a hyperactive GM-CSF/IRF-5 axis.
Microascus gracilis is a specie of the genus Microascus in the family of Microascaceae and has been isolated from lung. It has never been reported as the cause of disseminated infection in humans. Herein, we report a fatal case of disseminated Microascus gracilis infection in a 65-year-old man with a history of primary idiopathic pulmonary fibrosis, status-post bilateral lung transplant. His course was complicated by donor lung cultures positive for multiple organisms and persistent pleural effusions. Multiple lung biopsy and bronchial lavage specimens were negative for mold. Later, pleural fluid cultures grew M. gracilis confirmed by DNA sequencing. Despite aggressive antifungal treatment, the patient continued to deteriorate with altered mental status. Imaging showed scattered hemorrhagic and hypodense lesions in the brain. The patient eventually succumbed to his infections and a restricted autopsy was performed. Autopsy findings included multiple hemorrhagic foci and abscesses involving the whole brain. Numerous punctuate, tan-white circular lesions were on the endocardium and diffuse tan exudates covered the pericardium and lungs. Histologically, similar fungal organisms with septate branching hyphae and short chains of conidia were identified, along with hemorrhage, neutrophilic inflammation, and necrosis in the brain, pleura, peripheral parenchyma of lungs and heart. This is the first reported case of disseminated M. gracilis infection in an immunosuppressed human, indicating it can cause localized infections and disseminated infections. This case increases our awareness of such fatal opportunistic infections, particularly in lung transplant patients, and urges earlier aggressive prophylaxis, diagnosis, and treatment.
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