Abstract Class-switched antinuclear autoantibodies produced by T follicular helper (TFH) cell–dependent germinal center (GC) B cell response play an essential pathogenic role in lupus nephritis (LN). The role of T follicular regulatory (TFR) cells, an effector subset of CD4+Foxp3+ T regulatory cells (Tregs), which are specialized in suppressing TFH-GC response and Ab production, remains elusive in LN. Contrasting reports have shown increased/reduced circulating TFR cells in human lupus that might not accurately reflect their presence in the GCs of relevant lymphoid organs. In this study, we report a progressive reduction in TFR cells and decreased TFR/TFH ratio despite increased Tregs in the renal lymph nodes of NZBWF1/j mice, which correlated with increased GC-B cells and proteinuria onset. Cotreatment with soluble OX40L and Jagged-1 (JAG1) proteins increased Tregs, TFR cells, and TFR/TFH ratio, with a concomitant reduction in TFH cells, GC B cells, and anti-dsDNA IgG Ab levels, and suppressed LN onset. Mechanistic studies showed attenuated TFH functions and diminished GC events such as somatic hypermutation and isotype class-switching in OX40L-JAG1–treated mice. RNA sequencing studies revealed inhibition of hypoxia-inducible factor 1-α (HIF-1a) and STAT3 signaling in T conventional cells from OX40L-JAG1–treated mice, which are critical for the glycolytic flux and differentiation into TFH cell lineage. Therefore, the increased TFR/TFH ratio seen in OX40L-JAG1–treated mice could involve both impaired differentiation of TFH cells from T conventional cells and expansion of TFR cells. We show a key role for GC-TFR/TFH imbalance in LN pathogenesis and how restoring homeostatic balance can suppress LN.
Abstract Regulatory T-cells (Tregs) play a pivotal role in maintaining peripheral tolerance. Increasing Treg numbers/functions has been shown to ameliorate autoimmune diseases. However, common Treg expansion approaches use T-Cell Receptor (TCR)-mediated stimulation which also causes proliferation of effector T-cells (Teff). To overcome this limitation, purified patient-specific Tregs are expanded ex vivo and transfused. Although promising, this approach is not suitable for routine clinical use. Therefore, an alternative approach to selectively expand functional Tregs in vivo is highly desired. We report a novel TCR-independent strategy for the selective proliferation of Foxp3+Tregs (without Teff proliferation), by co-culturing CD4+ T-cells with OX40 L+Jagged(JAG)-1+ bone marrow-derived DCs differentiated with GM-CSF or treating them with soluble OX40 L and JAG1 in the presence of exogenous IL-2. Tregs expanded using soluble OX40 L and JAG1 were of suppressive phenotype and delayed the onset of diabetes in NOD mice. Ligation of OX40 L and JAG1 with their cognate-receptors OX40 and Notch3, preferentially expressed on Tregs but not on Teff cells, was required for selective Treg proliferation. Soluble OX40L-JAG1-induced NF-κB activation as well as IL-2-induced STAT5 activation were essential for the proliferation of Tregs with sustained Foxp3 expression. Altogether, these findings demonstrate the utility of soluble OX40 L and JAG1 to induce TCR-independent Treg proliferation.
Abstract Previously, we have shown that GM-CSF-induced Bone marrow-derived dendritic cells (G-BMDCs), upon co-culture with splenic CD4+ T cells, have the capability to induce preferential proliferation of regulatory T cells (Tregs) in TCR-independent and OX40L/OX40 dependent manner. In this study we investigated the signaling involved in this Treg proliferation induced by G-BMDCs. Firstly, using splenic CD4+ T cells from OX40 deficient mice and G-BMDCs from WT mice, we confirmed the important role of OX40L/OX40 interaction in Treg expansion. Since OX40 stimulation in the absence of TCR engagement has been reported to induce the assembly of a signalosome involving PKC-θ, we investigated the requirement of PKC-θ in our Treg proliferation mechanism. Interestingly, PKC-θ deficient-CD4+ T cells showed impaired Treg proliferation upon co-culture with WT G-BMDCs. However, this defect in proliferation could be rescued upon IL-2 addition suggesting that PKC-θ was dispensable for Treg proliferation. We further demonstrated that PKC-θ was essential for T-effectors to provide IL-2, which was required for OX40L/OX40-induced Treg proliferation. Furthermore, injecting WT, OX40- deficient, and PKC-θ-deficient mice with soluble OX40L, we established that OX40L/OX40 signaling was sufficient to induce in vivo expansion of Tregs in WT and PKC- deficient mice, but not in OX40-deficient mice. Finally, our data suggested a possible involvement of TRAF1 in Tregs, downstream of OX40 activation.
Abstract We previously reported granulocyte macrophage colony-stimulating factor (GM-CSF) prevented type 1 diabetes in NOD mice through CD11c+CD8− dendritic cell (DCs) mobilization, precipitating regulatory T-cell (Treg) expansion. Furthermore, we found GM-CSF derived bone-marrow dendritic cells (G-BMDCs) can drive Treg expansion in ex-vivo CD4+splenic T-cell co-cultures through surface-bound molecule OX40L. To determine whether GM-CSF may lead to the formation of a distinct, novel CD11c+OX40L+ expressing DC subset, we generated OX40L+G-BMDCs (CD11c+OX40L+) and analyzed these cells through flow cytometry for various cell lineage markers. We determined OX40L+G-BMDCs expressed increased levels of co-stimulatory molecules CD80, CD86, PD-L1 and MHC-II in comparison to the OX40L− counterpart. Additionally, OX40L+G-BMDCs lacked expression of Ly6G, Ly6C, FceR1, surface markers indicative of granulocyte and mast cell lineage, but to our surprise, highly expressed macrophage markers F4/80, MerTK, and CD200R. To elucidate an in vivo physiological component of these OX40L+ G-BMDCs, we performed an unsupervised hierarchical clustering comparing the transcriptome of OX40L+G-BMDCs to that of all myeloid and lymphoid lineages from the Immgen database. We found that OX40L+G-BMDCs clustered most closely with macrophages and dendritic cells. Furthermore, a principal component analysis with the transcriptome of OX40L+G-BMDCs and that of macrophages and dendritic cells from the Immgen database revealed OX40L+G-BMDCs most closely clustered with macrophages. Collectively, these results suggest OX40L+ G-BMDCs may represent a distinct GM-CSF dependent, macrophage-like cell subset involved in physiological Treg homeostasis.
Abstract Disclosure: K. Xu: None. M. Priyadarshini: None. P. Kumar: None. H.M. Electricwala: None. B.S. Prabhakar: None. B.T. Layden: None. Gut microbiome is recently recognized environmental factor in pathogenesis of type 1 diabetes (T1D). Reduced short chain fatty acid (SCFA) producing gut bacteria and also SCFA levels correlate with T1D risk (TEDDY study). We have previously shown that FFA2 is one of the main SCFA receptors, which is expressed on the β cell and mediates gut microbiome-β cell crosstalk. Based on these data, we hypothesized that β cell FFA2 may play a role in T1D β cell pathology. To examine this, we followed our novel tamoxifen induced β cell FFA2 knockout mice (FFA2 fl/fl; MIP CreERT+, FFA2 BKO) and wildtype littermates (FFA2 fl/fl) after multiple low dose streptozotocin (MLDS) induced T1D. Comparing to wildtype littermates, FFA2 BKO were significantly protected from MLDS insult and exhibited normal glycemic control owing to higher β cell mass. To understand the mechanism of this protective effect, we mined islet transcriptome data from our FFA2 global knockout mice and identified markedly upregulated type 1 interferon pathway in FFA2 global knockout. Accordingly, wildtype islets treated with high dose IFNα (mimicking T1D like conditions) showed significant upregulation of IFNαtargets while FFA2 BKO islets were unresponsive. Using pharmacological inhibition in isolated islets, we identified FFA2 mediated inhibition of NF-κB activation as a critical event in FFA2 mediated regulation of IFNαsignaling. In vivo studies with IFNAR1 antibody showed partial rescue of wildtype mice in the early stages of MLDS induced T1D. Thus, we propose a key role of β cell FFA2 in preserving β cell mass from IFNα signaling mediated damage in T1D. Further studies are needed to test this model. Presentation: Saturday, June 17, 2023
Earlier, we have shown that GM-CSF derived bone marrow (BM) dendritic cells (G-BMDCs) can expand Foxp3+ regulatory T-cells (Tregs) through a TCR-independent, but IL-2 dependent mechanism that required OX40L/OX40 interaction. While some reports have shown suppression of autoimmunity upon treatment with an OX40 agonist, others have shown exacerbation of autoimmune disease instead. To better understand the basis for these differing outcomes, we compared the effects of OX40L treatment in 6-week-old pre-diabetic and 12-week-old near diabetic NOD mice. Upon treatment with OX40L, 6-week-old NOD mice remained normoglycemic and showed a significant increase in Tregs in their spleen and lymph nodes, while 12-week-old NOD mice very rapidly developed hyperglycemia and failed to show Treg increase in spleen or LN. Interestingly, OX40L treatment increased Tregs in the thymus of both age groups. However, it induced Foxp3+CD103+CD38− stable-phenotype Tregs in the thymus and reduced the frequency of autoreactive Teff cells in 6-week-old mice; while it induced Foxp3+CD103−CD38+ labile-phenotype Tregs in the thymus and increased autoreactive CD4+ T cells in the periphery of 12-week-old mice. This increase in autoreactive CD4+ T cells was likely due to either a poor suppressive function or conversion of labile Tregs into Teff cells. Using ex vivo cultures, we found that the reduction in Treg numbers in 12-week-old mice was likely due to IL-2 deficit, and their numbers could be increased upon addition of exogenous IL-2. The observed divergent effects of OX40L treatment were likely due to differences in the ability of 6- and 12-week-old NOD mice to produce IL-2.
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