Treg Cell Differentiation Research Articles

Inhibition of ROCK activity regulates the balance of Th1, Th17 and Treg cells in myasthenia gravis

Aberrant ROCK activation has been found in patients with several autoimmune diseases, but the role of ROCK in myasthenia gravis (MG) has not yet been clearly investigated. Here, we demonstrated that ROCK activity was significantly higher in peripheral blood mononuclear cells (PBMCs) from MG patients. ROCK inhibitor Fasudil down-regulated the proportions of Th1 and Th17 cells in PBMCs of MG patients in vitro. Intraperitoneal injection of Fasudil ameliorated the severity of experimental autoimmune myasthenia gravis (EAMG) rats and restored the balance of Th1/Th2/Th17/Treg subsets. Furthermore, Fasudil inhibited the proliferation of antigen-specific Th1 and Th17 cells, and inhibited CD4 + T cells differentiated into Th1 and Th17 through decreasing phosphorylated Stat1 and Stat3, but promoted Treg cell differentiation through increasing phosphorylated Stat5. We conclude that dysregulated ROCK activity may be involved in the pathogenic immune response of MG and inhibition of ROCK activity might serve as a novel treatment strategy for MG.

Effect of IL-21 on the Balance of Th17 Cells/Treg Cells in the Pathogenesis of Graves’ Disease

ABSTRACTGraves’ disease (GD) is a common organ-specific autoimmune disease, and its pathogenesis is still unclear. The aim of this study is to investigate the role of interleukin (IL)-21 in the regulation of Th17/Treg cells in GD. We recruited 28 newly diagnosed GD patients, 27 GD patients in remission (eGD), and 24 normal controls (NC). Thyroid function and autoantibodies were evaluated by electrochemical luminescence. Peripheral blood mononuclear cells (PBMCs) were isolated and cultured with or without recombinant human interleukin-21 (rhIL-21), and mRNA and protein levels were quantified by real-time PCR and ELISA, respectively. Compared with those in the eGD and control groups, the thyroid function indexes and autoantibodies levels were significantly different in the GD group (P < 0.05). Without rhIL-21 stimulation, the expression levels of retinoid-related orphan gamma t (RORγt), IL-17, IL-22, forkhead box protein P3 (Foxp3) and IL-10 mRNA and the IL-10 and IL-22 proteins were significantly higher in the GD group than those in the eGD and control groups (P < 0.05). rhIL-21 stimulation increased the RORγt, IL-17, and IL-22 mRNA levels and IL-22 protein levels and decreased the Foxp3 and IL-10 mRNA levels and IL-10 protein levels (P < 0.05) in the GD group. In conclusion, our analyses demonstrated that IL-21 might induce the differentiation of CD4+ T cells to Th17 cells and reduce Treg cell differentiation, which could contribute to activation of the downstream immune response and the pathogenesis of GD.

Bach2 attenuates T cell receptor-dependent transcription to fine-tune regulatory T cell differentiation

Abstract The differentiation of regulatory T (Treg) cells is strictly controlled by T cell receptor (TCR) signals. However, the downstream regulators of this process are incompletely understood. Here we have found that Bach2 blocks the genomic binding of the TCR-induced transcription factor IRF4, attenuating TCR-dependent transcriptional programs to fine-tune Treg cell development and differentiation. In the absence of Bach2 we observed an increase in TCR-driven Treg cell outcomes, including the enhanced differentiation of effector Treg cells and thymic Treg precursor cells, and a reduction in peripheral Treg cell differentiation. Additional loss of the TCR-responsive transcription factor IRF4 was sufficient to normalise frequencies of each of these Treg cell populations in the absence of Bach2. Transcriptomic analysis identified significant deregulation of gene expression in Bach2-deficient cells which was dependent upon IRF4 expression. Assessing genome-wide occupancy of these transcription factors, we found Bach2 to restrict access of IRF4 to most of its genomic binding sites. Together, these data indicate that Bach2 inhibits IRF4-dependent transcription by blocking its access to their shared binding sites. Our work reveals Bach2 and IRF4 to drive opposing programs in Treg cell development and differentiation. Bach2 maintains the functional quiescence of Treg cells, in large part by blocking the genomic binding of IRF4 to attenuate the transcriptional program of TCR signalling.

MicroRNA-31 Regulates Immunosuppression in Ang II (Angiotensin II)-induced Hypertension by Targeting Ppp6C (Protein Phosphatase 6c).

Regulatory T cells (Treg cells) play important roles in hypertension and organ damages. MicroRNA-31 (miR-31) is a critical regulator for Treg cell generation. However, the role of miR-31 in hypertension has not been elucidated. We aim to study the functionality of miR-31 and the detailed mechanism in Ang II (Angiotensin II)-induced hypertensive mouse model. We found: In vitro, miR-31 expression was higher in T helper 17 cells and lower in Treg cells than that of naïve T cells. The genetic deficiency of miR-31 promoted Treg cell differentiation, whereas no impact on T helper 17 cells differentiation. Ang II-induced hypertension resulted in increased expression of miR-31 in the aorta, splenic CD4+ T cells, and kidney leukocytes. MiR-31 deficiency strikingly decreased systolic blood pressure and diastolic blood pressure and attenuated renal and vascular damage. MiR-31 deletion altered the accumulation of Treg cells and macrophages and expression of inflammatory cytokines in kidneys in Ang II-induced hypertensive mice. Ang II treatment reduced the levels of anti-inflammatory cytokines and increased proinflammatory cytokines in plasma that were blunted by the miR-31 deletion. Ppp6C (protein phosphatase 6c; a direct target of miR-31) specific deletion in Treg cells led to marked impairment of Treg cell induction, increased Ang II-induced blood pressure elevation, and organ damage in mice. In conclusion, we provided novel evidence of miR-31 as an emerging key posttranscriptional regulator of hypertension-associated immunosuppression through targeting ppp6C which is a critical regulator in the differentiation of Treg cells. This study offers new perspectives on miRNA-based therapeutic approaches.

T-cell signaling in Th17-mediated inflammation

Abstract MAP kinase kinase kinase kinases (MAP4Ks) are a subfamily of mammalian Ste20-like serine/threonine kinases that activate the JNK kinase cascade. We have cloned and characterized three MAP4Ks, namely HPK1 (MAP4K1), GLK (MAP4K3) and HGK (MAP4K4). Epigenetic downregulation of HGK (MAP4K4) in T cell results in differentiation of IL-6+ Th17 cells, which play important roles in the pathogenesis of Asia-prevalent non-obese T2D. HPK1 (MAP4K1) is a negative regulator of T-cell signaling through phosphorylation and sequential ubiquitination of the T-cell adaptor SLP-76. GLK (MAP4K3) also interacts with SLP-76 but does not negatively regulate SLP-76. GLK activates PKC-θ during TCR signaling; GLK-deficient mice showed impaired Th17 differentiation. T-cell specific GLK transgenic mice spontaneously developed autoimmune diseases with an induction of systemic inflammation. We found that GLK signaling specifically induced IL-17A transcription in the T cells of GLK transgenic mice. We will present the data on a novel signal transduction mechanism of IL-17A transcriptional activation of AhR and RORγt by GLK in inflammatory T cells and activated T cells. In addition, we found that GLK negatively regulated differentiation and activity of Treg cells through IKKβ. Collectively, MAP4K3/GLK plays a critical role in IL-17A-induced inflammation.

Helios predisposes human fetal CD4+ naive T cells towards regulatory T cell differentiation

Abstract Activation of naïve CD4+ T cells by T cell receptor (TCR) stimulation and cytokine cues lead to differentiation into effector T cell populations with distinct pro-inflammatory or regulatory functions. Unlike adult naïve T cells, human fetal naïve CD4+ T cells uniquely differentiate into FOXP3+ regulatory T (Treg) cells upon TCR activation independent of exogenous cytokine signalling. This facility for Treg differentiation is crucial for generating tolerance in utero; however, the mechanisms underlying this ability in fetal naïve cells are largely unknown. Here, we reveal FOXP3-independent transcriptional and epigenetic programs shared between fetal naive T cells and committed adult Treg cells that are inactive in adult naive T cells. We show that a subset of adult Treg-specific super-enhancers is active within fetal naive T cells, including two super-enhancers at Helios, a thymic Treg gene. Helios is expressed in fetal naive T cells, but not in adult naïve T cells, and only fetal-derived induced Treg (iTreg) cells continue to express Helios. Fetal, but not adult iTreg cells, have suppressed IL-2 production, which is regulated by Helios in committed Treg cells. CRISPR-Cas9 ablation of Helios in fetal naive T cells then resulted in increased IL-2 production in fetal iTreg cells. Crucially, the loss of Helios expression in fetal naïve T cells impaired their differentiation into Treg cells upon TCR stimulation, indicating Helios as a critical contributor to the cell-intrinsic predisposition of fetal naive T cells for Treg cell differentiation. Treg-biased transcriptional and epigenetic programs within fetal naive T cells identified here could be utilized to engineer enhanced adult iTreg populations for adoptive cellular therapies.

Tangeretin promotes regulatory T cell differentiation by inhibiting Notch1/Jagged1 signaling in allergic rhinitis

Background and objectiveTangeretin demonstrates broad anti-inflammatory effects. The present study aimed to assess whether tangeretin functions in regulating T-regulatory cells (Tregs) and alleviating allergic rhinitis (AR). MethodsAn ovalbumin (OVA)-induced AR animal model was constructed to monitor the changes in the allergic symptom score, OVA-specific IgE titers, histopathological characteristics and T-helper cell (Th1, Th2, and Th17)-related cytokine levels under tangeretin or dexamethasone (DXM) administration. The expression levels of Notch1/Jagged1 and FOXP3, and the proportion of Tregs in the spleens of these animals, were also detected. Furthermore, purified naive CD4 + T cells were utilized to assess the effects of tangeretin on Notch1 expression and their differentiation in vitro. ResultsBoth tangeretin and DXM administration alleviated airway inflammation, decreased the production of serum OVA-induced IgE, but only tangeretin administration restored the balance of cytokine profiles compared with those in the AR group. The abundance of splenic CD4 + CD25 + FOXP3 + Treg cells and the transcription factor FOXP3 were significantly increased under tangeretin treatment, either in AR mice or in naïve CD4 + T-cell differentiation, followed by a concomitant reduction in Notch1/Jagged1 expression. However, as a positive control, the treatment of allergic rhinitis with dexamethasone was not related to the expression of Notch1/Jagged1 or the differentiation of Treg cells. ConclusionTangeretin could promote regulatory T cell responses by inhibiting Notch1/Jagged1 expression, followed by promoting FOXP3/Treg cell differentiation and thus could serve as a novel curative therapeutic for AR.

Long noncoding RNA NEAT1 modulates immune cell functions and is suppressed in early onset myocardial infarction patients.

Inflammation is a key driver of atherosclerosis and myocardial infarction (MI), and beyond proteins and microRNAs (miRs), long noncoding RNAs (lncRNAs) have been implicated in inflammation control. To obtain further information on the possible role of lncRNAs in the context of atherosclerosis, we obtained comprehensive transcriptome maps of circulating immune cells (peripheral blood mononuclear cells, PBMCs) of early onset MI patients. One lncRNA significantly suppressed in post-MI patients was further investigated in a murine knockout model. Individual RNA-sequencing (RNA-seq) was conducted on PBMCs from 28 post-MI patients with a history of MI at age ≤50 years and stable disease ≥3 months before study participation, and from 31 healthy individuals without manifest cardiovascular disease or family history of MI as controls. RNA-seq revealed deregulated protein-coding transcripts and lncRNAs in post-MI PBMCs, among which nuclear enriched abundant transcript (NEAT1) was the most highly expressed lncRNA, and the only one significantly suppressed in patients. Multivariate statistical analysis of validation cohorts of 106 post-MI patients and 85 controls indicated that the PBMC NEAT1 levels were influenced (P = 0.001) by post-MI status independent of statin intake, left ventricular ejection fraction, low-density lipoprotein or high-density lipoprotein cholesterol, or age. We investigated NEAT1-/- mice as a model of NEAT1 deficiency to evaluate if NEAT1 depletion may directly and causally alter immune regulation. RNA-seq of NEAT1-/- splenocytes identified disturbed expression and regulation of chemokines/receptors, innate immunity genes, tumour necrosis factor (TNF) and caspases, and increased production of reactive oxygen species (ROS) under baseline conditions. NEAT1-/- spleen displayed anomalous Treg and TH cell differentiation. NEAT1-/- bone marrow-derived macrophages (BMDMs) displayed altered transcriptomes with disturbed chemokine/chemokine receptor expression, increased baseline phagocytosis (P < 0.0001), and attenuated proliferation (P = 0.0013). NEAT1-/- BMDMs responded to LPS with increased (P < 0.0001) ROS production and disturbed phagocytic activity (P = 0.0318). Monocyte-macrophage differentiation was deregulated in NEAT1-/- bone marrow and blood. NEAT1-/- mice displayed aortic wall CD68+ cell infiltration, and there was evidence of myocardial inflammation which could lead to severe and potentially life-threatening structural damage in some of these animals. The study indicates distinctive alterations of lncRNA expression in post-MI patient PBMCs. Regarding the monocyte-enriched NEAT1 suppressed in post-MI patients, the data from NEAT1-/- mice identify NEAT1 as a novel lncRNA-type immunoregulator affecting monocyte-macrophage functions and T cell differentiation. NEAT1 is part of a molecular circuit also involving several chemokines and interleukins persistently deregulated post-MI. Individual profiling of this circuit may contribute to identify high-risk patients likely to benefit from immunomodulatory therapies. It also appears reasonable to look for new therapeutic targets within this circuit.

Microbiota Metabolite Butyrate Differentially Regulates Th1 and Th17 Cells' Differentiation and Function in Induction of Colitis.

How the gut microbiota regulates intestinal homeostasis is not completely clear. Gut microbiota metabolite short-chain fatty acids (SCFAs) have been reported to regulate T-cell differentiation. However, the mechanisms underlying SCFA regulation of T-cell differentiation and function remain to be investigated. CBir1, an immunodominant microbiota antigen, transgenic T cells were treated with butyrate under various T-cell polarization conditions to investigate butyrate regulation of T-cell differentiation and the mechanism involved. Transfer of butyrate-treated CBir T cells into Rag1-/- mice was performed to study the in vivo role of such T cells in inducing colitis. Although butyrate promoted Th1 cell development by promoting IFN-γ and T-bet expression, it inhibited Th17 cell development by suppressing IL-17, Rorα, and Rorγt expression. Interestingly, butyrate upregulated IL-10 production in T cells both under Th1 and Th17 cell conditions. Furthermore, butyrate induced T-cell B-lymphocyte-induced maturation protein 1 (Blimp1) expression, and deficiency of Blimp1 in T cells impaired the butyrate upregulation of IL-10 production, indicating that butyrate promotes T-cell IL-10 production at least partially through Blimp1. Rag1-/- mice transferred with butyrate-treated T cells demonstrated less severe colitis, compared with transfer of untreated T cells, and administration of anti-IL-10R antibody exacerbated colitis development in Rag-/- mice that had received butyrate-treated T cells. Mechanistically, the effects of butyrate on the development of Th1 cells was through inhibition of histone deacetylase but was independent of GPR43. These data indicate that butyrate controls the capacity of T cells in the induction of colitis by differentially regulating Th1 and Th17 cell differentiation and promoting IL-10 production, providing insights into butyrate as a potential therapeutic for the treatment of inflammatory bowel disease.

Helicobacter pylori VacA Targets Myeloid Cells in the Gastric Lamina Propria To Promote Peripherally Induced Regulatory T-Cell Differentiation and Persistent Infection.

The gastric bacterium Helicobacter pylori causes a persistent infection that is directly responsible for gastric ulcers and gastric cancer in some patients and protective against allergic and other immunological disorders in others. The two outcomes of the Helicobacter-host interaction can be modeled in mice that are infected as immunocompetent adults and as neonates, respectively. Here, we have investigated the contribution of the Helicobacter immunomodulator VacA to H. pylori-specific local and systemic immune responses in both models. We found that neonatally infected mice are colonized at higher levels than mice infected as adults and fail to generate effector T-cell responses to the bacteria; rather, T-cell responses in neonatally infected mice are skewed toward Foxp3-positive (Foxp3+) regulatory T cells that are neuropilin negative and express RORγt. We found these peripherally induced regulatory T cells (pTregs) to be enriched, in a VacA-dependent manner, not only in the gastric mucosa but also in the lungs of infected mice. Pulmonary pTreg accumulation was observed in mice that have been infected neonatally with wild-type H. pylori but not in mice that have been infected as adults or mice infected with a VacA null mutant. Finally, we traced VacA to gastric lamina propria myeloid cells and show that it suppressed interleukin-23 (IL-23) expression by dendritic cells and induced IL-10 and TGF-β expression in macrophages. Taken together, the results are consistent with the idea that H. pylori creates a tolerogenic environment through its immunomodulator VacA, which skews T-cell responses toward Tregs, favors H. pylori persistence, and affects immunity at distant sites.IMPORTANCEHelicobacter pylori has coexisted with humans for at least 60.000 years and has evolved persistence strategies that allow it to evade host immunity and colonize its host for life. The VacA protein is expressed by all H. pylori strains and is required for high-level persistent infection in experimental mouse models. Here, we show that VacA targets myeloid cells in the gastric mucosa to create a tolerogenic environment that facilitates regulatory T-cell differentiation, while suppressing effector T-cell priming and functionality. Tregs that are induced in the periphery during H. pylori infection can be found not only in the stomach but also in the lungs of infected mice, where they are likely to affect immune responses to allergens.

Tissue resident and follicular Treg cell differentiation is regulated by CRAC channels

T regulatory (Treg) cells maintain immunological tolerance and organ homeostasis. Activated Treg cells differentiate into effector Treg subsets that acquire tissue-specific functions. Ca2+ influx via Ca2+ release-activated Ca2+ (CRAC) channels formed by STIM and ORAI proteins is required for the thymic development of Treg cells, but its function in mature Treg cells remains unclear. Here we show that deletion of Stim1 and Stim2 genes in mature Treg cells abolishes Ca2+ signaling and prevents their differentiation into follicular Treg and tissue-resident Treg cells. Transcriptional profiling of STIM1/STIM2-deficient Treg cells reveals that Ca2+ signaling regulates transcription factors and signaling pathways that control the identity and effector differentiation of Treg cells. In the absence of STIM1/STIM2 in Treg cells, mice develop a broad spectrum of autoantibodies and fatal multiorgan inflammation. Our findings establish a critical role of CRAC channels in controlling lineage identity and effector functions of Treg cells.

Rapamycin Prevents Surgery-Induced Immune Dysfunction in Patients with Bladder Cancer.

The mechanistic target of rapamycin (mTOR) integrates environmental inputs to regulate cellular growth and metabolism in tumors. However, mTOR also regulates T-cell differentiation and activation, rendering applications of mTOR inhibitors toward treating cancer complex. Preclinical data support distinct biphasic effects of rapamycin, with higher doses directly suppressing tumor cell growth and lower doses enhancing T-cell immunity. To address the translational relevance of these findings, the effects of the mTOR complex 1 (mTORC1) inhibitor, rapamycin, on tumor and T cells were monitored in patients undergoing cystectomy for bladder cancer. MB49 syngeneic murine bladder cancer models were tested to gain mechanistic insights. Surgery-induced T-cell exhaustion in humans and mice and was associated with increased pulmonary metastasis and decreased PD-L1 antibody efficacy in mouse bladder cancer. At 3 mg orally daily, rapamycin concentrations were 2-fold higher in bladder tissues than in blood. Rapamycin significantly inhibited tumor mTORC1, shown by decreased rpS6 phosphorylation in treated versus control patients (P = 0.008). Rapamycin reduced surgery-induced T-cell exhaustion in patients, evidenced by a significant decrease in the prevalence of dysfunctional programmed death-1 (PD-1)-expressing T cells. Grade 3 to 4 adverse event rates were similar between groups, but rapamycin-treated patients had a higher rate of wound complications versus controls. In conclusion, surgery promoted bladder cancer metastasis and decreased the efficacy of postoperative bladder cancer immunotherapy. Low-dose (3 mg daily) oral rapamycin has favorable pharmacodynamic and immune modulating activity in surgical patients and has the potential to decrease surgery-induced immune dysfunction.

T cells transduce T-cell receptor signal strength by generating different phosphatidylinositols

T-cell receptor (TCR) signaling strength is a dominant factor regulating T-cell differentiation, thymic development, and cytokine signaling. The molecular mechanisms by which TCR signal strength is transduced to downstream signaling networks remains ill-defined. Using computational modeling, biochemical assays, and imaging flow cytometry, we found here that TCR signal strength differentially generates phosphatidylinositol species. Weak TCR signals generated elevated phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and reduced phosphatidylinositol (3,4,5)-trisphosphate (PIP3) levels, whereas strong TCR signals reduced PI(4,5)P2 and elevated PIP3 levels. A proteomics screen revealed that focal adhesion kinase bound PI(4,5)P2, biochemical assays disclosed that focal adhesion kinase is preferentially activated by weak TCR signals and is required for optimal Treg induction, and further biochemical experiments revealed how TCR signaling strength regulates AKT activation. Low PIP3 levels generated by weak TCR signals were sufficient to activate phosphoinositide-dependent kinase-1 to phosphorylate AKT on Thr-308 but insufficient to activate mTOR complex 2 (mTORC2), whereas elevated PIP3 levels generated by a strong TCR signal were required to activate mTORC2 to phosphorylate Ser-473 on AKT. Our results provide support for a model that links TCR signaling to mTORC2 activation via phosphoinositide 3-kinase signaling. Together, the findings in this work establish that T cells measure TCR signal strength by generating different levels of phosphatidylinositol species that engage alternate signaling networks to control cell fate decisions.

STAT1 signaling shields T cells from NK cell-mediated cytotoxicity

The JAK-STAT pathway critically regulates T-cell differentiation, and STAT1 is postulated to regulate several immune-mediated diseases by inducing proinflammatory subsets. Here we show that STAT1 enables CD4+ T-cell-mediated intestinal inflammation by protecting them from natural killer (NK) cell-mediated elimination. Stat1−/− T cells fail to expand and establish colitis in lymphopenic mice. This defect is not fully recapitulated by the combinatorial loss of type I and II IFN signaling. Mechanistically, Stat1−/− T cells have reduced expression of Nlrc5 and multiple MHC class I molecules that serve to protect cells from NK cell-mediated killing. Consequently, the depletion of NK cells significantly rescues the survival and spontaneous proliferation of Stat1−/− T cells, and restores their ability to induce colitis in adoptive transfer mouse models. Stat1−/− mice however have normal CD4+ T cell numbers as innate STAT1 signaling is required for their elimination. Overall, our findings reveal a critical perspective on JAK-STAT1 signaling that might apply to multiple inflammatory diseases.

E-protein regulatory network links TCR signaling to effector Treg cell differentiation

T cell antigen receptor (TCR) signaling is essential for the differentiation and maintenance of effector regulatory T (Treg) cells. However, the contribution of individual TCR-dependent genes in Treg cells to the maintenance of immunotolerance remains largely unknown. Here we demonstrate that Treg cells lacking E protein undergo further differentiation into effector cells that exhibit high expression of effector Treg signature genes, including IRF4, ICOS, CD103, KLRG-1, and RORγt. E protein-deficient Treg cells displayed increased stability and enhanced suppressive capacity. Transcriptome and ChIP-seq analyses revealed that E protein directly regulates a large proportion of the genes that are specific to effector Treg cell activation, and importantly, most of the up-regulated genes in E protein-deficient Treg cells are also TCR dependent; this indicates that E proteins comprise a critical gene regulatory network that links TCR signaling to the control of effector Treg cell differentiation and function.

The Th17/Treg Cytokine Imbalance in Chronic Obstructive Pulmonary Disease Exacerbation in an Animal Model of Cigarette Smoke Exposure and Lipopolysaccharide Challenge Association

We proposed an experimental model to verify the Th17/Treg cytokine imbalance in COPD exacerbation. Forty C57BL/6 mice were exposed to room air or cigarette smoke (CS) (12 ± 1 cigarettes, twice a day, 30 min/exposure and 5 days/week) and received saline (50 µl) or lipopolysaccharide (LPS) (1 mg/kg in 50 µl of saline) intratracheal instillations. We analyzed the mean linear intercept, epithelial thickness and inflammatory profiles of the bronchoalveolar lavage fluid and lungs. We evaluated macrophages, neutrophils, CD4+ and CD8+ T cells, Treg cells, and IL-10+ and IL-17+ cells, as well as STAT-3, STAT-5, phospho-STAT3 and phospho-STAT5 levels using immunohistochemistry and IL-17, IL-6, IL-10, INF-γ, CXCL1 and CXCL2 levels using ELISA. The study showed that CS exposure and LPS challenge increased the numbers of neutrophils, macrophages, and CD4+ and CD8+ T cells. Simultaneous exposure to CS/LPS intensified this response and lung parenchymal damage. The densities of Tregs and IL-17+ cells and levels of IL-17 and IL-6 were increased in both LPS groups, while IL-10 level was only increased in the Control/LPS group. The increased numbers of STAT-3, phospho-STAT3, STAT-5 and phospho-STAT5+ cells corroborated the increased numbers of IL-17+ and Treg cells. These findings point to simultaneous challenge with CS and LPS exacerbated the inflammatory response and induced diffuse structural changes in the alveolar parenchyma characterized by an increase in Th17 cytokine release. Although the Treg cell differentiation was observed, the lack of IL-10 expression and the decrease in the density of IL-10+ cells observed in the CS/LPS group suggest that a failure to release this cytokine plays a pivotal role in the exacerbated inflammatory response in this proposed model.

Abstract A067: Cognate interaction with CD4+ T-cells instructs M2-like macrophages to acquire M1-like phenotype

Abstract Current tumor immunotherapy approaches are based on application of checkpoint inhibitors, such as monoclonal antibodies against PD-1/PD-L1 and CTLA-4 or adoptive T-cell therapy using ex vivo expanded TILs or genetically modified autologous T-cells expressing recombinant T-cell receptors (TCRs) or chimeric antigen receptors (CARs). However, the success of these therapeutic strategies is often limited by various inhibitory immune cell types accumulating in the tumor. In particular, tumor-associated macrophages (TAMs) contribute to the immune suppressive tumor micro-milieu, since so-called M2-like macrophages suppress the function of T effector cells and promote the differentiation of regulatory T-cells (Treg) through secretion of inhibitory cytokines such as TGF-β and IL10. Tumor antigen-specific CD4+ T effector cells, on the other hand, can essentially sustain antitumoral immune responses as shown for various tumor entities. In fact, using peritoneal exudate cells (PECs) as source for macrophages we demonstrate that MHC II restricted interaction between ovalbumin (OVA) specific CD4+ T-cells and M2-like macrophages drives M1 polarization. This was confirmed by detailed gene and protein expression analyses as well as functional assays testing phagocytic and pinocytic activities of repolarized macrophages. Moreover, in a set of preclinical experiments, adoptive transfer of CD4+, OVA-specific OT-II cells into C57BL/6 mice bearing OVA expressing IAb-/- tumors resulted in increased intratumoral number of M1-like TAMs as determined by gene expression analysis and flow cytometry. Furthermore, we observed a significant survival benefit of mice treated with a combination of OVA-specific CD4+ (OT-II) and CD8+ (OT-I) cells after transplantation of B16F10-Ova tumors and a complete response in some mice that rejected the tumor cells also upon a later re-challenge. While the antitumoral effect of this adoptive transfer experiment has been already described, we now offer a possible explanation for the supportive effect of specific CD4+ cells on co-transferred CD8+ cells. Taken together, the instructive impact of CD4+ T-cells on M2-like macrophages described in this presentation points towards a so far underestimated function of the CD4+ T-cell / TAM axis in reconditioning the immunosuppressive tumor micro-milieu. Citation Format: David Eisel, Wolfram Osen, Krishna Das, Franziska Marie-Claire Hoerhold, Rainer König, Stefan B. Eichmüller. Cognate interaction with CD4+ T-cells instructs M2-like macrophages to acquire M1-like phenotype [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A067.

Abstract A190: Mechanisms governing Foxp3-dependent and -independent gene expression in regulatory T-cells in evolutionary distant mice

Abstract T regulatory (Treg) cells expressing the forkhead box transcription factor (Foxp3) are important regulators of immunity and play a key role in cancer and metastasis by controlling the activity of effector T-cells. Lineage-specifying transcription factors (TFs) establish cell identity and maintain cell specific transcriptional programs. Foxp3 is a key master TF controlling various Treg cell developmental and functional modalities. Foxp3 deficiency in humans and mice causes a life threatening autoimmune disease, and the enforced expression of Foxp3 in CD4+ T-cells confers suppressor functions. However, our understanding of Treg cell regulation during differentiation in the thymus, and in mature Treg cells in the periphery, whether Foxp3 dependent or independent, is still lacking. Single-nucleotide polymorphism (SNPs) may occur within coding genes or noncoding genomic regulatory areas and can have severe phenotypic consequences. We have recently attempted to link SNPs with differential Treg transcriptomic and epigenetic features in humans. However, such studies are limited by their intrinsic correlative nature, and by the limited number of SNPs. Here we use evolutionally distant mice strains C57Bl/6 (B6), CAST/EiJ (Cast) and SPRET/EiJ (Spret), to identify allele-specific imbalances and functional differences in the transcriptional and epigenetic programs during differentiation and in various conditions of Treg cells and their precursors. Using Foxp3 reporter and conditional knockout (KO) systems combined with interbreeding of the evolutionary distant mice strains in vivo, we explore the genetic and molecular mechanisms of Treg cell differentiation and function, employing high-throughput genomic methods including RNA-seq, ATAC-seq and Chip-seq. Such studies may pave a way for the development of novel therapeutic approaches to treat cancer and metastasis. Citation Format: Ariella Glasner, Yi Zhong, Joris Van Der Veeken, Alexander Rudensky. Mechanisms governing Foxp3-dependent and -independent gene expression in regulatory T-cells in evolutionary distant mice [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A190.

Rapamycin alleviates inflammation by up-regulating TGF-β/Smad signaling in a mouse model of autoimmune encephalomyelitis

To evaluate the efficacy of rapmycin for treatment of experimental autoimmune encephalomyelitis (EAE) in mice and explore the underlying mechanism. An EAE model was established in C57BL/6 mice. After immunization, the mice were divided into model group and rapamycin groups treated daily with low-dose (0.3 mg/kg) or high-dose (1 mg/kg) rapamycin. The clinical scores of the mice were observed using Knoz score, the infiltration of IL-17 cells in the central nervous system (CNS) was determined using immunohistochemistry; the differentiation of peripheral Treg cells was analyzed using flow cytometry, and the changes in the levels of cytokines were detected with ELISA; the changes in the expressions of p-Smad2 and p- smad3 were investigated using Western blotting. High-dose rapamycin significantly improved the neurological deficits scores of EAE mice. In high-dose rapamycin group, the scores in the onset stage, peak stage and remission stage were 0.14±0.38, 0.43±1.13 and 0.14±0.37, respectively, as compared with 1.14±0.69, 2.14±1.06 and 2.2±0.75 in the model group. The infiltration of inflammatory IL-17 cells was significantly lower in high-dose rapamycin group than in the model group (43±1.83 vs 153.5±7.02). High-dose rapamycin obviously inhibited the production of IL-12, IFN-γ, IL-17 and IL-23 and induced the anti-inflammatory cytokines IL-10 and TGF-β. The percentage of Treg in CD4+ T cells was significantly higher in high- dose rapamycin group than in the model group (10.17 ± 0.68 vs 3.52 ± 0.32). In the in vitro experiment, combined treatments of the lymphocytes isolated from the mice with rapamycin and TGF-β induced a significant increase in the number of Treg cells (13.66±1.89) compared with the treatment with rapamycin (6.23±0.80) or TGF-β (4.87±0.85) alone. Rapamycin also obviously up-regulated the expression of p-Smad2 and p-Smad3 in the lymphocytes. Rapamycin can promote the differentiation of Treg cells by up-regulating the expression of p-Smad2 and p-smad3 to improve neurological deficits in mice with EAE.

Butyrate inhibit collagen-induced arthritis via Treg/IL-10/Th17 axis

Rheumatoid arthritis (RA) is a chronic autoimmune disorder demanding the development of novel therapeutic strategy. Butyrate is a functional short-chain fatty acid produced by the anaerobic intestinal microbiota. This study aimed to investigate the attenuation of butyrate on RA. The collagen-induced arthritis (CIA) mouse model was established and butyrate was administered in drinking water along with the collagen immunization. The histopathological features, clinical score, paw swelling, as well as the production of pro-inflammatory cytokines including interleukin (IL)-1β, IL-6 and IL-17A were measured to determine the amelioration of butyrate on arthritis. The differentiation of Treg cells and Th17 cells in the splenic cells was assessed by flow cytometry. The expression of Foxp3, IL-10, Rorγt and IL-17A were detected by RT-PCR and FACS immunostaining. Anti-IL10R antibody was used in the CIA and CD4+ cell cultures to mediate the effects of butyrate. Butyrate significantly inhibited expressions of IL-1β, IL-6 and IL-17A, but promoted the expression of IL-10. Butyrate also increased systematical Treg cells and reduced Th17 cells. Mechanism study revealed that butyrate directly enhanced the polarization of Treg cells but not Th17 cells. All effects of butyrate on RA were inversed by the co-administered anti-IL10R antibody. This study showed that butyrate administration inhibited arthritis in CIA mice model, suppressed the expression of inflammatory cytokines. The modulation may be mediated the differentiation of CD4 T cells towards Treg cells, which produce anti-inflammatory cytokine IL-10, and thus influenced the function of Th17 cells.