Migration Of Treg Cells Research Articles

Ischemic stroke outcome after promoting CD4+CD25+ Treg cell migration through CCR4 overexpression in a tMCAO animal model

The importance of neuroinflammation during the ischemic stroke has been extensively studied. The role of CD4+CD25+ regulatory T (Treg) cells during the recovery phase have shown infarct size reduction and functional improvement, possibly through the mitigation of inflammatory immune responses. We aimed to investigate the molecular factors involved in microglia-Treg cell communication that result in Treg trafficking. First, we observed the migration patterns of CD8+ (cytotoxic) T cells and Treg cells and then searched for chemokines released by activated microglia in an oxygen–glucose deprivation (OGD) model. The transwell migration assay showed increased migration into OGD media for both cell types, in agreement with the increase in chemokines involved in immune cell trafficking from the mouse chemokine profiling array. MSCV retrovirus was transduced to overexpress CCR4 in Treg cells. CCR4-overexpressed Treg cells were injected into the mouse transient middle cerebral artery occlusion (tMCAO) model to evaluate the therapeutic potential via the tetrazolium chloride (TTC) assay and behavioral tests. A general improvement in the prognosis of animals after tMCAO was observed. Our results suggest the increased mobility of CCR4-overexpressed Treg cells in response to microglia-derived chemokines in vitro and the therapeutic potential of Treg cells with increased mobility in cellular therapy.

CD177 drives the transendothelial migration of Treg cells enriched in human colorectal cancer.

Regulatory T (Treg) cells regulate immunity in autoimmune diseases and cancers. However, immunotherapies that target tumor-infiltrating Treg cells often induce unwanted immune responses and tissue inflammation. Our research focussed on exploring the expression pattern of CD177 in tumor-infiltrating Treg cells with the aim of identifying a potential target that can enhance immunotherapy effectiveness. Single-cell RNA sequencing (scRNA-seq) data and survival data were obtained from public databases. Twenty-one colorectal cancer patient samples, including fresh tumor tissues, peritumoral tissues and peripheral blood mononuclear cells (PBMCs), were analysed using flow cytometry. The transendothelial activity of CD177+ Treg cells was substantiated using in vitro experiments. ScRNA-seq and flow cytometry results indicated that CD177 was exclusively expressed in intratumoral Treg cells. CD177+ Treg cells exhibited greater activation status and expressed elevated Treg cell canonical markers and immune checkpoint molecules than CD177- Treg cells. We further discovered that both intratumoral CD177+ Treg cells and CD177-overexpressing induced Treg (iTreg) cells had lower levels of PD-1 than their CD177- counterparts. Moreover, CD177 overexpression significantly enhanced the transendothelial migration of Treg cells in vitro. These results demonstrated that Treg cells with higher CD177 levels exhibited an enhanced activation status and transendothelial migration capacity. Our findings suggest that CD177 may serve as an immunotherapeutic target and that overexpression of CD177 may improve the efficacy of chimeric antigen receptor T (CAR-T) cell therapy.

Treatment of rheumatoid arthritis with baricitinib or upadacitinib is associated with reduced scaffold protein NEDD9 levels in CD4+ T cells.

The JAK/STAT pathway plays a crucial role in the pathogenesis of rheumatoid arthritis (RA) and JAK inhibitors have emerged as a new group of effective drugs for RA treatment. Recently, high STAT3 levels have been associated with the upregulation of the scaffold protein NEDD9, which is a regulator of T-cell trafficking and promotes collagen-induced arthritis (CIA). In this study, we aimed to reveal how treatment with JAK inhibitors affects NEDD9 in CD4+ T cells from RA patients. We analyzed NEDD9 expression in CD4+ T cells from 50 patients treated with either baricitinib, tofacitinib, or upadacitinib and performed cell migration assays to assess the potential influence of JAK inhibitor treatment on CD4+ T-cell migration. We observed that treatment with baricitinib and upadacitinib is associated with reduced NEDD9 expression in CD4+ T cells. In contrast, NEDD9 levels were not altered during treatment with tofacitinib. Moreover, treatment with baricitinib was associated with a significantly reduced migratory capacity of effector CD4+ T cells but not with impaired migration of Treg cells. This study reveals previously unknown associations between JAK inhibitor treatment and NEDD9 expression and indicates that JAK inhibitors could reduce effector T-cell migration.

Abstract P2153: A Long Noncoding RNA Plentiful In Cardiosphere-derived Cell Extracellular Vesicles (CDC-EVs), BCYRN1, And Its Short Synthetic Derivatives Enhance Regulatory T Cell (Treg) Proliferation, Migration, And Activation

Background: By modulating inflammation, Treg cells influence outcomes in myocardial infarction (MI). Here, we test the hypothesis that BCYRN1 (Brain Cytoplasmic RNA 1) and BCYRN1-derived binding small sequences (BDSSs) are cardioprotective by activating Tregs. Methods: RNA sequencing identified BCYRN1 as plentiful in cardiosphere-derived cell extracellular vesicles (CDC-EVs), validated by qPCR. RNA pulldown and luciferase assays probed BCYRN1's potential function as a miRNA sponge. We assessed BCYRN1 and BDSS effects on Treg proliferation (Cell Counting Kit-8), migration (trans-well migration), and IL-10 (qPCR and ELISA). In vivo , MI was induced in C57BL/6 mouse by left coronary artery ligation for 45 minutes followed by 15 minutes reperfusion. Afterwards, mice received (via tail vein) CDC-EV-BCYRN1 (CDC-EVs overexpressing BCYRN1), BDSSs, mut-BDSSs (scrambled inactive versions of BDSS) or vehicle (PBS). MI size (by TTC staining) and infiltrating Treg (by flow cytometry) were assessed 72 hrs post-MI. Findings: BCYRN1 was found to contain putative binding sites for miR-138, miR-150 and miR-98; these miRs target ATG7, CCR6 and IL-10 mRNAs, respectively. BCYRN1 serves as a miRNA sponge, leading to Treg cell ATG7/autophagy dependent proliferation (3.0± 0.1 fold increase, N=5), CCR-6 dependent migration (1.8 ± 0.1 fold increase, N=5) and increased IL-10 production (fold increase, qPCR: 2.6 ± 0.4, N=6 and ELISA: 6.1 ± 0.8, N=5). Lipid nanoparticles containing a cocktail of 3BDSSs likewise induce Treg cell proliferation (1.9± 0.6 fold increase, N=6), migration (1.3± 0.2 fold increase, N=6), and IL-10 induction (6.8± 0.5 fold increase, N=6). In vivo (N=5/group), CDC-EV-BCYRN1 (but not vehicle) and BDSS cocktail (but not mut-BDSS) induced Treg infiltration, proliferation and IL10 production in the heart with a concomitant decrease of infarct size. Conclusions: A single LncRNA (BCYRN1) and shorter synthetic derivatives mimic the effects of CDC-EV on Treg in vitro and in vivo . Our study provides a novel approach, targeting Treg, to treat MI. This approach could be useful for other diseases where increased Treg activity is a therapeutic goal (e.g. autoimmune disorders).

Inhibition of the CCR6-CCL20 axis prevents regulatory T cell recruitment and sensitizes head and neck squamous cell carcinoma to radiation therapy.

Radioresistance of HNSCCs remains a major challenge for effective tumor control. Combined radiotherapy (RT) and immunotherapy (IT) treatment improved survival for a subset of patients with inflamed tumors or tumors susceptible to RT-induced inflammation. To overcome radioresistance and improve treatment outcomes, an understanding of factors that suppress anti-tumor immunity is necessary. In this regard, regulatory T cells (Tregs) are critical mediators of immune suppression in HNSCCs. In this study, we investigated how radiation modulates Treg infiltration in tumors through the chemokine CCL20. We hypothesized that radiation induces CCL20 secretion resulting in Treg infiltration and suppression of anti-tumor immunity. Human and mouse HNSCC cell lines with different immune phenotypes were irradiated at doses of 2 or 10Gy. Conditioned media, RNA and protein were collected for assessment of CCL20. qPCR was used to determine CCL20 gene expression.In vivo, MOC2 cells were implanted into the buccal cavity of mice and the effect of neutralizing CCL20 antibody was determined alone and in combination with RT. Blood samples were collected before and after RT for analysis of CCL20. Tumor samples were analyzed by flow cytometry to determine immune infiltrates, including CD8 T cells and Tregs. Mass-spectrometry was performed to analyze proteomic changes in the tumor microenvironment after anti-CCL20 treatment. Cal27 and MOC2 HNSCCs had a gene signature associated with Treg infiltration, whereas SCC9 and MOC1 tumors displayed a gene signature associated with an inflamed TME.In vitro, tumor irradiation at 10Gy significantly induced CCL20 in Cal27 and MOC2 cells relative to control. The increase in CCL20 was associated with increased Treg migration. Neutralization of CCL20 reversed radiation-induced migration of Treg cells in vitro and decreased intratumoral Tregs in vivo. Furthermore, inhibition of CCL20 resulted in a significant decrease in tumor growth compared to control in MOC2 tumors. This effect was further enhanced after combination with RT compared to either treatment alone. Our results suggest that radiation promotes CCL20 secretion by tumor cells which is responsible for the attraction of Tregs. Inhibition of the CCR6-CCL20 axis prevents infiltration of Tregs in tumors and suppresses tumor growth resulting in improved response to radiation.

ICOS-Deficient Regulatory T Cells Can Prevent Spontaneous Autoimmunity but Are Impaired in Controlling Acute Inflammation.

ICOS is induced in activated T cells and its main role is to boost differentiation and function of effector T cells. ICOS is also constitutively expressed in a subpopulation of Foxp3+ regulatory T cells under steady-state condition. Studies using ICOS germline knockout mice or ICOS-blocking reagents suggested that ICOS has supportive roles in regulatory T (Treg) cell homeostasis, migration, and function. To avoid any compounding effects that may arise from ICOS-deficient non-Treg cells, we generated a conditional knockout system in which ICOS expression is selectively abrogated in Foxp3-expressing cells (ICOS FC mice). Compared to Foxp3-Cre control mice, ICOS FC mice showed a minor numerical deficit of steady-state Treg cells but did not show any signs of spontaneous autoimmunity, indicating that tissue-protective Treg populations do not heavily rely on ICOS costimulation. However, ICOS FC mice showed more severe inflammation in oxazolone-induced contact hypersensitivity, a model of atopic dermatitis. This correlated with elevated numbers of inflammatory T cells expressing IFN-γ and/or TNF-α in ICOS FC mice compared with the control group. In contrast, elimination of ICOS in all T cell compartments negated the differences, confirming that ICOS has a dual positive role in effector and Treg cells. Single-cell transcriptome analysis suggested that ICOS-deficient Treg cells fail to mature into T-bet+CXCR3+ "Th1-Treg" cells in the draining lymph node. Our results suggest that regimens that preferentially stimulate ICOS pathways in Treg cells might be beneficial for the treatment of Th1-driven inflammation.

Serum amyloid A/anti-CCL20 induced the rebalance of Th17/regulatory T cells in SodA-induced sarcoidosis

Sarcoidosis is a multisystemic granulomatous inflammation associated with Th17/regulatory T cell (Treg) polarization. As a marker of inflammation, serum amyloid A (SAA) could upregulate the expression of chemokine ligand 20 (CCL20), which induces the migration of Treg cells and Th17 cells by binding and activating thechemokine C-C receptor (CCR) 6. Our goal was to determine whether SAA/anti-CCL20 induces Th17/Treg rebalance in pulmonary sarcoidosis. The deposition of SAA- and Th17/Treg-related proteins in SodA-induced granulomas was tested using immunohistochemistry. Mice with SodA-induced sarcoidosis were treated with SAA or SAA + anti-CCL20, and then Th1/Th2 and Th17/Treg cells were detected by fluorescence-activated cell sorting (FACS) analysis. The expression of SAA/CCL20 and IL-23/IL-17A was detected by enzyme-linked immunosorbent assay (ELISA) and multiplex. Key proteins in the TGF-β/Smad signaling pathway were tested by western blot. SAA mainly plays a pro-inflammatory role by promoting the expression of CCL20 and IL-17A in bronchoalveolar lavage fluid (BALF) and serum, exacerbating this elevation of CD4+/CD8+ T cells in both mediastinal lymph nodes (LNs) and BALF, as well as proliferating Th1 in LNs in SodA-induced pulmonary sarcoidosis. In addition, SAA could also promote the proliferation of Tregs in LNs. Intriguingly, blocking of CCL20 could partially reverse the expression of Th17-related cytokine, ameliorate Th1/Th2 and Treg/Th17 bias in mice with SodA-induced pulmonary sarcoidosis, and rescue the overactivation of the TGF-β/Smad2/Smad3 signaling pathway. Anti-CCL20 may have the potential for therapeutic translation, targeting on the immunopathogenesis of pulmonary sarcoidosis.

Membrane-bound IL-6R is upregulated on Th17 cells and inhibits Treg cell migration by regulating post-translational modification of VASP in autoimmune arthritis

Autoimmune arthritis is characterized by impaired regulatory T (Treg) cell migration into inflamed joint tissue and by dysregulation of the balance between Treg cells and Th17 cells. Interleukin-6 (IL-6) is known to contribute to this dysregulation, but the molecular mechanisms behind impaired Treg cell migration remain largely unknown. In this study, we assessed dynamic changes in membrane-bound IL-6 receptor (IL6R) expression levels on Th17 cells by flow cytometry during the development of collagen-induced arthritis (CIA). In a next step, bioinformatics analysis based on proteomics was performed to evaluate potential pathways affected by altered IL-6R signaling in autoimmune arthritis. Our analysis shows that membrane-bound IL-6R is upregulated on Th17 cells and is inversely correlated with IL-6 serum levels in experimental autoimmune arthritis. Moreover, IL-6R expression is significantly increased on Th17 cells from untreated patients with rheumatoid arthritis (RA). Interestingly, CD4+ T cells from CIA mice and RA patients show reduced phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Bioinformatics analysis based on proteomics of CD4+ T cells with low or high phosphorylation levels of VASP revealed that integrin signaling and related pathways are significantly enriched in cells with low phosphorylation of VASP. Specific inhibition of p-VASP reduces the migratory function of Treg cells but has no influence on effector CD4+ T cells. Importantly, IL-6R blockade restores the phosphorylation level of VASP, thereby improving the migratory function of Treg cells from RA patients. Thus, our results establish a link between IL6R signaling and phosphorylation of VASP, which controls Treg cell migration in autoimmune arthritis.

Kinase activity profiling reveals contribution of G-protein signaling modulator 2 deficiency to impaired regulatory T cell migration in rheumatoid arthritis

The ability of regulatory T (Treg) cells to migrate into inflammatory sites is reduced in autoimmune diseases, including rheumatoid arthritis (RA). The reasons for impaired Treg cell migration remain largely unknown. We performed multiplex human kinase activity arrays to explore possible differences in the post-translational phosphorylation status of kinase related proteins that could account for altered Treg cell migration in RA. Results were verified by migration assays and Western blot analysis of CD4+ T cells from RA patients and from mice with collagen type II induced arthritis. Kinome profiling of CD4+ T cells from RA patients revealed significantly altered post-translational phosphorylation of kinase related proteins, including G-protein-signaling modulator 2 (GPSM2), protein tyrosine kinase 6 (PTK6) and vitronectin precursor (VTNC). These proteins have not been associated with RA until now. We found that GPSM2 expression is reduced in CD4+ T cells from RA patients and is significantly downregulated in experimental autoimmune arthritis following immunization of mice with collagen type II. Interestingly, GPSM2 acts as a promoter of Treg cell migration in healthy individuals. Treatment of RA patients with interleukin-6 receptor (IL-6R) blocking antibodies restores GPSM2 expression, thereby improving Treg cell migration. Our study highlights the potential of multiplex kinase activity arrays as a tool for the identification of RA-related proteins which could serve as targets for novel treatments.

Endometriotic Peritoneal Fluid Stimulates Recruitment of CD4+CD25highFOXP3+ Treg Cells.

Endometriosis is a common gynecological disorder characterized by the presence of endometrial-like tissue outside the uterus. The disease is associated with disturbed local and systemic immunity. It has been reported that the proportion of CD4+CD25highFOXP3+ Treg cells may be significantly increased in the peritoneal fluid of patients with endometriosis. Therefore, the aim of our study was to investigate whether the proportions of Treg cells in the peritoneal cavity of patients with endometriosis are related to the chemotactic and stimulatory activity of the local peritoneal milieu. The peritoneal fluid was collected from 13 women with ovarian endometriosis and 12 control women without the disease. T cell populations were analyzed by flow cytometry, cytokines and chemokines were evaluated using the cytometric bead kit, and cell chemotaxis was studied by cell migration assay. We confirmed that the proportions of Treg cells are increased in the peritoneal fluid of women with endometriosis as compared to the control women. Endometriosis was also associated with elevated concentrations of IL-6, IL-10, and TGF-β1/2 as well as CCL20, CXCL8, CXCL9, and CXCL10. We did not reveal any changes in the proportion of peritoneal Th17 cells and concentrations of IL-17A. Peritoneal Treg cells positively correlated with concentrations of TGF-β, IL-10, and CCL20. Endometriotic peritoneal fluid stimulated chemotaxis of both CD4+ and Treg cells. This chemotactic activity positively correlated with concentrations of CCL20. CCL20 stimulated the migration of Treg cells, and the chemotactic activity of the endometriotic peritoneal fluid was inhibited by neutralizing anti-CCL20 antibodies. These results imply that increased proportions of the peritoneal Treg cells in women with endometriosis may result from attraction and activation by local chemokines and cytokines, especially CCL20 and TGF-β. Since Treg cells contribute to the immunopathogenesis of endometriosis, their chemotaxis and activation may be considered as a target for therapeutic intervention.

Reduced CCR6+IL-17A+Treg Cells in Blood and CCR6-Dependent Accumulation of IL-17A+Treg Cells in Lungs of Patients With Allergic Asthma.

Human regulatory T (Treg) cells play a central role in controlling allergic inflammation in the airways. A reduced number of peripheral Treg cells and decreased suppressive function have been previously reported in the pathogenesis of allergic asthma. However, the characteristic role of specific Treg cell subsets and their mechanisms in the pathogenesis of allergic asthma remain unclear. In this study, we examined the proportion of different Treg cell subsets in both healthy subjects and patients with allergic asthma using flow cytometry and single-cell RNA sequencing. The migration function of the cells was compared using cell sorting and Transwell experiments. Furthermore, two allergen-challenged mouse models and a cell transfer experiment were used to examine the role of these Treg subsets. We found that the proportion of CD25+Foxp3+CD127- Treg cells in the peripheral blood of patients with allergic asthma was lower than in those of healthy subjects. Furthermore, the circulating Treg cells expressed lower levels of CCR6 and IL-17 compared with healthy subjects. The chemokine from the airway mucosa, CCL20, was abundantly expressed, and Transwell experiments further proved that this chemokine promoted CCR6+ Treg cell migration in vitro. A mouse model induced by house dust mite (HDM) revealed that the number of CCR6+ Treg cells in the lung tissue increased remarkably. The incidence of allergic asthma may be related to an increase in Treg cells secreting IL-17 in the lung tissue. Recruited CCR6+ Treg cells are likely to differentiate into Th17-like cells under the Th17 environment present in the lungs. IL-17 derived from Th17-like cells could be associated with the pathology of allergic asthma by promoting Th17 responses, thereby favoring HDM-induced asthma exacerbations.

LncRNA H19 induces immune dysregulation of BMMSCs, at least partly, by inhibiting IL-2 production

BackgroundSystemic lupus erythematosus (SLE) is a representative systemic autoimmune disease. LncRNA H19 has been identified to participate in various biological processes in human diseases. However, the role of H19 in SLE remains unclear.MethodsIn this study, we first examined H19 expression in SLE patients by RT-qPCR and found that H19 expression was significantly upregulated in the serum and bone marrow-derived mesenchymal stem cells (BMMSCs) of SLE patients and positively associated with SLE disease activity index. We then performed gain-of-function and loss-of-function using mimic-H19 (H19-OE) and inhibitor-H19 (H19-KD) to examine the effects of H19 on BMMSC differentiation, proliferation, migration, and apoptosis using flow cytometry, DAPI staining, and migration and apoptosis assays.ResultsThe results showed that H19 inhibited proliferation and migration but promoted apoptosis of BMMSCs, interfered with BMMSCs-mediated Treg cell proliferation and differentiation, and regulated BMMSCs-mediated Tfh/Treg cell balance. Dual-luciferase reporter assay confirmed the in silico prediction of interaction between H19 and IL-2. Furthermore, RT-qPCR showed that H19 directly inhibited IL-2 transcription in BMMSCs. ELISA showed that both active and total IL-2 protein levels were significantly lower in SLE BMMSCs. More importantly, we found that IL-2 significantly enhanced H19-OE-induced Treg cell differentiation and migration of BMMSCs, and these effects were reversed by anti-IL-2 antibody.ConclusionOverall, our study indicates that LncRNA H19 induces immune dysregulation of BMMSCs, at least partly, by inhibiting IL-2 production and might be a novel therapeutic target for SLE.

The deubiquitinase CYLD controls protective immunity against helminth infection by regulation of Treg cell plasticity

Type 2 immunity can be modulated by regulatory T (Treg) cell activity. It has been suggested that the deubiquitinase cylindromatosis (CYLD) plays a role in the development or function of Treg cells, implying that it could be important for normal protective immunity, where type 2 responses are prevalent. We sought to investigate the role of CYLD in Treg cell function and TH2 cell immune responses under steady-state conditions and during helminth infection. Foxp3-restricted CYLD conditional knockout (KO) mice were examined in mouse models of allergen-induced airway inflammation and Nippostrongylus brasiliensis infection. We performed multiplex magnetic bead assays, flow cytometry, and quantitative PCR to understand how a lack of CYLD affected cytokine production, homing, and suppression in Treg cells. Target genes regulated by CYLD were identified and validated by microarray analysis, coimmunoprecipitation, short hairpin RNA knockdown, and transfection assays. Treg cell-specific CYLD KO mice showed severe spontaneous pulmonary inflammation with increased migration of Treg cells into the lung. CYLD-deficient Treg cells furthermore produced high levels of IL-4 and failed to suppress allergen-induced lung inflammation. Supporting this, the conditional KO mice displayed enhanced protection against N brasiliensis infection by contributing to type 2 immunity. Treg cell conversion into IL-4-producing cells was due to augmented mitogen-activated protein kinase and nuclear factor κB signaling. Moreover, Scinderin, a member of the actin-binding gelsolin family, was highly upregulated in CYLD-deficient Treg cells, and controlled IL-4 production through forming complexes with mitogen-activated protein kinase kinase/extracellular receptor kinase. Correspondingly, both excessive IL-4 production invivo and the protective role of CYLD-deficient Treg cells against N brasiliensis were reversed by Scinderin ablation. Our findings indicate that CYLD controls type 2 immune responses by regulating Treg cell conversion into TH2cell-like effector cells, which potentiates parasite resistance.

EBV-EBNA1 constructs an immunosuppressive microenvironment for nasopharyngeal carcinoma by promoting the chemoattraction of Treg cells

BackgroundNasopharyngeal carcinoma (NPC) is primarily caused by the Epstein-Barr virus (EBV) infection in NPC endemic areas. EBNA1 is an EBV-encoded nuclear antigen, which plays a critical role in the maintenance...

Diet-induced dyslipidemia induces metabolic and migratory adaptations in regulatory T cells.

AimsA hallmark of advanced atherosclerosis is inadequate immunosuppression by regulatory T (Treg) cells inside atherosclerotic lesions. Dyslipidemia has been suggested to alter Treg cell migration by affecting the expression of specific membrane proteins, thereby decreasing Treg cell migration towards atherosclerotic lesions. Besides membrane proteins, cellular metabolism has been shown to be a crucial factor in Treg cell migration. We aimed to determine whether dyslipidemia contributes to altered migration of Treg cells, in part, by affecting cellular metabolism.Methods and resultsDyslipidemia was induced by feeding Ldlr−/− mice a western-type diet for 16–20 weeks and intrinsic changes in Treg cells affecting their migration and metabolism were examined. Dyslipidemia was associated with altered mTORC2 signalling in Treg cells, decreased expression of membrane proteins involved in migration, including CD62L, CCR7, and S1Pr1, and decreased Treg cell migration towards lymph nodes. Furthermore, we discovered that diet-induced dyslipidemia inhibited mTORC1 signalling, induced PPARδ activation and increased fatty acid (FA) oxidation in Treg cells. Moreover, mass-spectrometry analysis of serum from Ldlr−/− mice with normolipidemia or dyslipidemia showed increases in multiple PPARδ ligands during dyslipidemia. Treatment with a synthetic PPARδ agonist increased the migratory capacity of Treg cells in vitro and in vivo in an FA oxidation-dependent manner. Furthermore, diet-induced dyslipidemia actually enhanced Treg cell migration into the inflamed peritoneum and into atherosclerotic lesions in vitro.ConclusionAltogether, our findings implicate that dyslipidemia does not contribute to atherosclerosis by impairing Treg cell migration as dyslipidemia associated with an effector-like migratory phenotype in Treg cells.

Intratumoral regulatory T cells: markers, subsets and their impact on anti-tumor immunity.

Regulatory T (Treg) cells play a crucial role in maintaining self-tolerance and resolution of immune responses by employing multifaceted immunoregulatory mechanisms. However, Treg cells readily infiltrate into the tumor microenvironment (TME) and dampen anti-tumor immune responses, thereby becoming a barrier to effective cancer immunotherapy. There has been a substantial expansion in the development of novel immunotherapies targeting various inhibitory receptors (IRs), such as CTLA4, PD1 and LAG3, but these approaches have mechanistically focused on the elicitation of anti-tumor responses. However, enhanced inflammation in the TME could also play a detrimental role by facilitating the recruitment, stability and function of Treg cells by up-regulating chemokines that promote Treg cell migration, and/or increasing inhibitory cytokine production. Furthermore, IR blockade may enhance Treg cell function and survival, thereby serving as a resistance mechanism against effective immunotherapy. Given that Treg cells are comprised of functionally and phenotypically heterogeneous sub-populations that may alter their characteristics in a context-dependent manner, it is critical to identify unique molecular pathways that are preferentially used by intratumoral Treg cells. In this review, we discuss markers that serve to identify certain Treg cell subsets, distinguished by chemokine receptors, IRs and cytokines that facilitate their migration, stability and function in the TME. We also discuss how these Treg cell subsets correlate with the clinical outcome of patients with various types of cancer and how they may serve as potential TME-specific targets for novel cancer immunotherapies.

Deubiquitinase CYLD controls the plasticity of Treg cells by regulation of Scinderin expression

Abstract Regulatory T (Treg) cells are indispensable for the maintenance of immune tolerance and the prevention of autoimmunity; however, how Treg cells retain their homeostasis with immune suppressive function remains largely unclear. Here we report that deubiquitinase CYLD plays a critical role in the maintenance of Treg cells. Foxp3− specific CYLD knockout mice showed severe pulmonary inflammation due to preferential migration of Treg cells into the lung and increased interleukin-4 (IL-4) production compared to control mice, which was reversed by the deletion of IL-4. Genome-wide microarray analysis unveiled that Scinderin, a member of the actin-binding gelsolin family, was highly upregulated in CYLD-deficient Treg cells, which contributed to IL-4 production through complex formation with MEK/ERK. Increased IL-4 production by CYLD-deficient Treg cells was significantly rectified by Scinderin ablation. Our findings indicate that CYLD is essential to maintain Treg cell function through regulating Scinderin expression.

SAT-196 Maternal immunity: Preimplantation Preparation

The maternal uterus in mammals undergoes extensive remodeling in preparation for implantation of the semi-allogeneic embryo. Activation of the T cell homing receptor, CCR7, regulates multiple aspects of adaptive immunity. Genetic deletion of CCR7 reduces T regulatory (Treg) cell migration into mouse uteri and decreases embryo implantation. CCL19 and CCL21 are the sole ligands for CCR7. We hypothesized that CCL19 and/or CCL21 expression could attract Treg cells into the pre-implantation uterus and provide local immune suppression prior to implantation. Sprague Dawley rat uteri were removed from pregnant rats at Days 3-6. RNA was isolated and real time PCR was used to measure CCL19 and CCL21 expression. To determine the spatial distribution of these ligands, rat uteri were fixed, embedded in paraffin and uterine sections were analyzed by immunocytochemistry. The influence of sex steroids on the spatial distribution of CCL19 was evaluated in uterine sections from steroid treated rats. Hormonal control of CCL19 and CCL21 expression was investigated in a rat uterine stromal cell line treated with female sex steroids. Expression of CCL19 and CCL21 peaked at Day 4 of pregnancy compared with Days 3, 5 or 6 (Mann Whitney, p <0.05). Interestingly, expression of both ligands was significantly higher (p < 0.05) in the inter-site regions compared with the implantation sites at Day 6 of pregnancy. At Day 3 of pregnancy, CCL21 expression was limited to the glandular epithelium. Expression appeared in the antimesometrial uterine stroma at Day 4 of pregnancy. The distribution of CCL21 was similar between Days 4 and 5 of pregnancy but expression was less robust at Day 5. CCL21 expression was lost from the glands at Day 6 but was maintained in the antimesometrial stroma of the inter-site regions. CCL19 was expressed in the luminal and glandular epithelia of ovariectomized rat uteri. Progesterone pretreatment (2 mg daily, 3 days) stimulated expression of CCL19 in the periluminal stroma, which extended into the antimesometrial regions. Estradiol (0.2 µg) administration to progesterone pretreated rats, increased the expression of CCL19 but did not change its spatial distribution. Both ligands were detected in a rat uterine stromal cell line. CCL19 expression increased in response to estradiol (10 nM) at 6 h after hormone addition. Taken together, these results suggest that the maternal preparation for implantation includes a peak in the expression of CCL19 and CCL21 at Day 4 of pregnancy, one day prior to implantation in the rat. Unlike the constitutive expression of these ligands in primary and secondary lymphoid tissue, the spatial distribution and expression of CCL19 and CCL21 is regulated by female sex steroids in the uterus. The data support the concept that immune-mediated events, necessary for successful implantation and pregnancy, are induced in females prior to implantation. Funded by NIH.

Hippo Kinases Mst1 and Mst2 Sense and Amplify IL-2R-STAT5 Signaling in Regulatory T Cells to Establish Stable Regulatory Activity

Interleukin-2 (IL-2) and downstream transcription factor STAT5 are important for maintaining regulatory T(Treg) cell homeostasis and function. Treg cells can respond to low IL-2 levels, but the mechanisms of STAT5 activation during partial IL-2 deficiency remain uncertain. We identified the serine-threonine kinase Mst1 as a signal-dependent amplifier of IL-2-STAT5 activity in Treg cells. High Mst1 and Mst2 (Mst1-Mst2) activity in Treg cells was crucial to prevent tumor resistance and autoimmunity. Mechanistically, Mst1-Mst2 sensed IL-2 signals to promote the STAT5 activation necessary for Treg cell homeostasis and lineage stability and to maintain the highly suppressive phosphorylated-STAT5+ Treg cell subpopulation. Unbiased quantitative proteomics revealed association of Mst1 with the cytoskeletal DOCK8-LRCHs module. Mst1 deficiency limited Treg cell migration and access to IL-2 and activity of the small GTPase Rac, which mediated downstream STAT5 activation. Collectively, IL-2-STAT5 signaling depends upon Mst1-Mst2 functions to maintain a stable Treg cell pool and immune tolerance.

The Foxp3+ regulatory T-cell population requires IL-4Rα signaling to control inflammation during helminth infections.

Forkhead box P3 (Foxp3+) regulatory T (Treg)-cell function is controlled by environmental cues of which cytokine-mediated signaling is a dominant component. In vivo, interleukin-4 (IL-4)-mediated signaling via IL-4 receptor alpha (IL-4Rα) mediates Treg cell transdifferentiation into ex-Foxp3 T helper 2 (Th2) or T helper 17 (Th17) cells. However, IL-4-mediated signaling also reinforces the Foxp3 Treg compartment in vitro. We generated Foxp3-specific IL-4Rα-deficient mice and demonstrated differential efficiency of IL-4Rα deletion in male (approximately 90%) and female (approximately 40%) animals, because of cyclic recombinase (Cre)-mediated X-linked foxp3 inactivation. Irrespective of the degree of IL-4Rα deletion within the Foxp3+ Treg cell population, mice showed exacerbation of immune effector responses with aggravated tissue pathology in tissue-dwelling helminth infections (Schistosoma mansoni or Nippostrongylus brasiliensis). Mechanistically, IL-4Rα deletion in males and females led to a reduced expression of Foxp3 and subsequently an impaired accumulation of Foxp3+ Treg cells to inflamed tissues. In-depth cellular typing by flow cytometry revealed that the impairment of IL-4Rα-mediated signaling during helminth infections decreased the ability of central Treg cells to convert into effector Treg (eTreg) cells and caused a significant down-regulation of markers associated with Treg cell migration (C-X-C motif chemokine receptor 3 [CXCR3]) and accumulation in inflamed tissues (GATA binding protein 3 [GATA3]) as well as survival (B cell lymphoma 2 [Bcl-2]). These findings unprecedentedly, to our knowledge, uncover a role for IL-4Rα signaling in the positive regulation of Foxp3+ Treg cell function in vivo. Complementing our past knowledge on a widely reported role for IL-4Rα signaling in the negative regulation and transdifferentiation of Foxp3+ Treg cells in vivo, our present findings reveal the host requirement for an intact, but not reduced or potentiated, IL-4Rα-mediated signaling on Foxp3+ Treg cells to optimally control inflammation during helminth infections.