Foxp3 Expression In Tregs Research Articles

Increased Frequency of Circulating Activated FOXP3+ Regulatory T Cell Subset in Patients with Chronic Lymphocytic Leukemia Is Associated with the Estimate of the Size of the Tumor Mass, STAT5 Signaling and Disease Course during Follow-Up of Patients on Therapy.

Advanced chronic lymphocytic leukemia (CLL) is accompanied by increased circulating regulatory T cells (Tregs) and increased susceptibility to severe infections, which were also shown to entail a striking induction of FOXP3 expression in Tregs. As homeostasis of the most suppressive CD45RA-FOXP3high activated Treg (aTreg) subset differs, it is critical to analyse homeostatic signalling in Treg subsets. In this study, by using conventional and imaging flow cytometry, we monitored STAT5 signalling/phosphorylation (pSTAT5) and investigated Treg subsets in relation to the Binet stage, the total tumor mass score (TTM) and the disease course during a follow-up of 37 patients with CLL. The aTreg percentage was significantly increased among CD4+ T cells from patients with advanced disease and significantly correlated with the TTM. A subgroup of patients with higher aTreg percentages among CD4+FOXP3+ T cells at the start of therapy was characterised by more frequent episodes of severe infections during follow-up. The results suggesting that an aTreg fraction could represent a possible marker of a severe disease course with infectious complications. Augmented homeostatic STAT5 signalling could support aTreg expansion, as higher pSTAT5 levels were significantly correlated with an increased aTreg frequency among CD4+FOXP3+ T cells during the follow-up of patients on therapy, as well as following SARS-CoV-2 antigen-specific stimulation in vitro.

The effects of combustible cigarettes and electronic nicotine delivery systems on immune cell-driven inflammation and mucosal healing in ulcerative colitis.

The effects of combustible cigarettes (CCs) and electronic nicotine delivery systems (ENDS) on immune cell-driven colon inflammation and intestinal healing of patients with ulcerative colitis (UC) are still unknown and, therefore, were examined in this study. Intracellular staining and flow cytometry analysis of immune cells isolated from UC patients who used ENDS (UCENDS), CCs (UCCC) and who were non-smokers (UCAIR) were performed to elucidate cellular mechanisms which were responsible for CCs and ENDS-dependent modulation of immune response during UC progression. Additionally, dextran sulfate sodium (DSS)-colitis was induced in ENDS/CC/air-exposed mice (DSSENDS/ DSSCC/DSSAIR groups) to support clinical findings. Significantly increased number of immunosuppressive, IL-10, TGF-β and IL-35-producing, FoxP3-expressing CD3+CD4+T regulatory cells (Tregs) was observed in the blood of UCENDS patients while reduced presence of inflammatory, TNF-α and IFN-γ-producing, Tbx21-expressing CD3+CD4+ Th1, IL-4-producing Gata3-expresing Th2 and IL-17, IL-22-producing, RORγT, IL-23R-expressing Th17 cells were noticed in the blood of UCCC patients. Exposure to either CCs or ENDS was associated with enhanced mucosal healing, ameliorated spontaneous recovery and improved survival of DSS-treated mice. An expansion of immunosuppressive cells (IL-10-producing tolerogenic CD11c+ dendritic cells, alternatively activated CD206, Arginase 1-expressing, IL-10-producing F4/80+macrophages, IL-10-producing FoxP3-expressing Tregs) was noticed in the colons of DSSENDS-treated mice, while reduced number of inflammatory, IL-17- and IL-4-producing T lymphocytes was observed in the colons of DSSCC-compared to DSSAIR-treated mice. Despite different mechanisms of action, both ENDS and CCs attenuated on-going colon inflammation, enhanced healing and ameliorated recovery of injured intestines of DSS-treated mice and UC patients. This is the first study that compared the effects of CCs and ENDS on immune cells of patients suffering from ulcerative colitis, providing new information about molecular and cellular mechanisms which were responsible for ENDS and CCs-dependent modulation of immune cell-driven colon injury and inflammation. Obtained results showed that both ENDS and CCs had capacity to attenuate detrimental immune response, enhanced healing and ameliorated recovery of injured intestines.

The Expression of Forkhead Box P3 T Regulatory Lymphocytes as a Prognostic Factor in Malignant Melanomas.

Since transcription factor Forkhead Box P3 (FoxP3) was identified as a specific regulatory T cell (Treg) marker, researchers have scrutinized its value as a potential novel therapeutic target or a prognostic factor in various types of cancer with inconsistent results. The present analysis was performed to assess the influence of Treg FoxP3 expression on the prognosis of primary melanoma and to evaluate the correlations with various clinicopathological prognostic factors. We analyzed all eligible patients with stage pT3 primary malignant melanomas treated in a tertiary cancer center. Immunohistochemical staining for Treg FoxP3 expression was performed on retrospectively identified paraffin blocks and subsequently correlated with the outcomes of the patients. A total of 81% of the patients presented a positive Treg FoxP3 expression, being correlated with a higher risk of lymph node metastasis, tumor relapse, and death. Moreover, positive expression was statistically associated with a shorter OS. The tumor relapse rate was estimated at 36.7%. A positive expression of Treg FoxP3 and lymph node metastasis were associated with a higher risk of death based on multivariate analysis. Treg FoxP3 expression may be used as an independent prognostic factor in patients with malignant melanoma to evaluate tumor progression and survival.

Peptidase inhibitor 16 promotes inflammatory arthritis by suppressing Foxp3 expression via regulating K48-linked ubiquitin degradation Bmi-1 in regulatory T cells

Abnormalities of regulatory T cells (Tregs) has been suggested in rheumatoid arthritis (RA), and Forkhead box P3 (Foxp3) is the key transcriptional factor of Tregs expression. However, the underlying molecular mechanism remains unclear. Here, we demonstrated peptidase inhibitor 16 (PI16) was significantly increased in the peripheral blood, synovial fluid, and synovial tissue from RA patients. PI16 transgenic mice (PI16Tg) aggravated arthritis severity partly through suppressing Foxp3 expression. Mechanistically, PI16 could interact with and stabilize Bmi-1 in Tregs via inhibiting K48-linked polyubiquitin of Bmi-1, which promotes the enrichment of repressive histone mark in Foxp3 promoter. Furthermore, Bmi-1 specific inhibitor PTC209 could restore Foxp3 expression and alleviate arthritis progression in PI16Tg mice, accompanied by increased recruitment of active histone mark in the promoter of Tregs. Our results suggest that PI16-Bmi-1 axis plays an important role in RA and other autoimmune diseases by suppressing Foxp3 expression in Tregs via Bmi-1-mediated histone modification.

Strengthening bonds via RyR2 inhibition helps immune suppression.

Foxp3-expressing Tregs employ multiple suppressive mechanisms to curtail conventional T cell (Tconv) responses and establish tissue homeostasis. How Foxp3 coordinates Treg contact-dependent suppressive function is not fully resolved. In this issue of the JCI, Wang and colleagues revealed that Foxp3-mediated inhibition of ryanodine receptor 2 (RyR2) led to strong Treg-DC interactions and enhanced immunosuppression. RyR2 depletion in Tconvs phenocopied this effect and equipped Tconvs with Treg-like suppressive function in multiple inflammatory or autoimmune contexts. This study provides molecular and therapeutic insights underlying how cell-cell contact limits immune reactivity.

Abstract B038: Targeting a Treg-specific deubiquitinase module for antitumor immune therapy

Abstract Checkpoint immunotherapies have transformed the treatment landscape for multiple human malignancies, but still with very limited success in treatment of many human cancers. A major hurdle is mediated by regulatory T (Treg) cells, which suppress the function of antitumor effector T cells. However, current approaches in targeting Treg to activate antitumor immune responses have shown only transient efficacy and are highly unspecific. Importantly, our recent studies, through unbiased CRISPR screening, identified the death from cancer cell signature gene USP22 required for optimal FoxP3 expression. USP22 genetic inhibition resulted in an approximately 15-25% reduction of FoxP3 expression in Tregs and partially diminishes Treg suppressive functions, which consequently results in elevated antitumor immunity against a broad spectrum of cancer types in mice. Therefore, a 20% reduction in FoxP3 protein expression, in this case, by USP22 inhibition, creates an ideal therapeutic window to boost antitumor immunity without triggering extensive autoimmune inflammatory responses (Nature, 2020). More recently, we further demonstrated that tumor microenvironment factors, including TGF-β, hypoxia, and tumor metabolites, induce Treg fitness/adaptation through selectively upregulating a deubiquitinase module, including USP21 and USP22 (Science Advances, 2022). Our ongoing study has identified USP22-specific small molecule inhibitors that largely diminish Treg fitness and dramatically inhibit tumor growth at a clinically relevant setting. In addition, our recent unpublished single-cell RNA-seq characterization revealed a novel function of the Treg-specific deubiquitination module in Treg terminal differentiation into a unique immune suppressive subpopulation, specifically in the tumor microenvironment. Collectively, our study defines a novel Treg deubiquitination module in tumor immunosurveillance and provides a rationale for the first Treg-specific targeting in antitumor immune therapy. Citation Format: Deyu Fang. Targeting a Treg-specific deubiquitinase module for antitumor immune therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B038.

Salidroside regulates tumor microenvironment of non-small cell lung cancer via Hsp70/Stub1/Foxp3 pathway in Tregs

BackgroundThe treatment of non-small cell lung cancer (NSCLC) is challenging due to immune tolerance and evasion. Salidroside (SAL) is an extract in traditional Chinese medicine and has a potential antitumor effect. However, the mechanism of SAL in regulating the immunological microenvironment of NSCLC is yet to be clarified.MethodsThe mouse model with Lewis lung cancer cell line (3LL) in C57BL/6 mice was established. And then, the percentage of tumor-infiltrating T cell subsets including Treg was detected in tumor-bearing mice with or without SAL treatment. In vitro, the effect of SAL on the expression of IL-10, Foxp3 and Stub1 and the function of Treg were detected by flow cytometry. Network pharmacology prediction and molecular docking software were used to predict the target of SAL and intermolecular interaction. Furthermore, the effect of SAL on the expression of Hsp70 and the co-localization of Stub1-Foxp3 in Treg was confirmed by flow cytometry and confocal laser microscopy. Finally, Hsp70 inhibitor was used to verify the above molecular expression.ResultsWe discovered that SAL treatment inhibits the growth of tumor cells by decreasing the percentage of tumor-infiltrated CD4+Foxp3+T cells. SAL treatment downregulates the expression of Foxp3 in Tregs, but increases the expression of Stub1, an E3 ubiquitination ligase upstream of Foxp3, and the expression of Hsp70. Inhibiting the expression of Hsp70 reverses the inhibition of SAL on Foxp3 and disrupts the colocalization of Stub1 and Foxp3 in the nucleus of Tregs.ConclusionsSAL inhibits tumor growth by regulating the Hsp70/stub1/Foxp3 pathway in Treg to suppress the function of Treg. It is a new mechanism of SAL for antitumor therapy.

Supraphysiological FOXP3 expression in human CAR-Tregs results in improved stability, efficacy, and safety of CAR-Treg products for clinical application

The forkhead family transcription factor (FOXP3) is an essential regulator for the development of regulatory T cells (Tregs) and orchestrates both suppressive function and Treg lineage identity. Stable expression of FOXP3 enables Tregs to maintain immune homeostasis and prevent autoimmunity. However, under pro-inflammatory conditions, FOXP3 expression in Tregs can become unstable, leading to loss of suppressive function and conversion into pathogenic T effector cells. Therefore, the success of adoptive cell therapy with chimeric antigen receptor (CAR) Tregs is highly dependent on the stability of FOXP3 expression to ensure the safety of the cell product. To warrant the stable expression of FOXP3 in CAR-Treg products, we have developed an HLA-A2-specific CAR vector that co-expresses FOXP3. The transduction of isolated human Tregs with the FOXP3-CAR led to an increase in the safety and efficacy of the CAR-Treg product. In a hostile microenvironment, under pro-inflammatory and IL-2-deficient conditions, FOXP3-CAR-Tregs showed a stable expression of FOXP3 compared to Control-CAR-Tregs. Furthermore, additional exogenous expression of FOXP3 did not induce phenotypic alterations and dysfunctions such as cell exhaustion, loss of functional Treg characteristics or abnormal cytokine secretion. In a humanized mouse model, FOXP3-CAR-Tregs displayed an excellent ability to prevent allograft rejection. Furthermore, FOXP3-CAR-Tregs revealed coherent Treg niche-filling capabilities. Overexpression of FOXP3 in CAR-Tregs has thereby the potential to increase the efficacy and reliability of cellular products, promoting their clinical use in organ transplantation and autoimmune diseases.

Stimulation of antitumor immunity by FoxP3-targeting PROTAC

CD4 + regulatory T cells (Tregs) play a central role in regulating and suppressing anti-tumor immune responses. FoxP3 is a transcription factor and master regulator of the Treg lineage. We developed and characterized a proteolysis targeting chimeric (PROTAC) drug that targets FoxP3 (PF). PF was created by linking the FoxP3 binding peptide P60 to pomalidomide, a ligand for E3 ligase. Ternary complex formation between PF, FoxP3, and cereblon (component of an E3 ligase) was confirmed using surface plasmon resonance assay (cooperativity factor of 2.27). PF decreased mouse and human FoxP3 expression in vitro in a proteasome-dependent manner. In mice, PF decreased FoxP3 in both the spleen and peripheral lymphocytes. PF-treated lymphocytes (human or mice) were better at stimulating CD8 + lymphocyte proliferation and activation. PF treatment decreased RENCA tumor growth in mice. PF enhanced antitumor immunity associated with αPD1 or mTOR inhibitor (mTORi). Lymphocytes from mice treated with PF and mTORi showed reduced metastatic tumor growth in untreated mice, providing further evidence for an adaptive immune response as the mechanism of action. We showed that PF binds FoxP3 and decreases FoxP3 expression in Tregs, reducing Treg function and generating antitumor immunity.

Purine metabolic pathway regulates regulatory T cell stability and function

Abstract Regulatory T cell (Treg) lymphatic migration and maintenance of transcription factor Foxp3 expression are required for suppressor function and allograft protection, and migrated Tregs express ectonucleotidase CD39 hi. Purine metabolism is implicated in Treg phenotype but the precise functions of purine metabolites on Treg function and stability have remained unclear. Sorted Foxp3+ Tregs were used to test Treg stability, migration, and cellular viability in a transendothelial migration (TEM) in vitro model. Intracellular metabolome of Tregs was analyzed by mass spectrometry, and differentially expressed metabolites between Tregs and exTregs were identified and tested. Foxp3 hiTregs displayed higher suppressive function and migration across LECs compared to Foxp3 loexTregs, but cellular viability was similar. Tregs showed differential metabolic profiles compared to exTregs, and the top 16 most differentially expressed metabolites involved the tricarboxylic acid cycle, polyamines, and purine metabolism. Ten metabolites including nicotinamide and inosine monophosphate (IMP) were downregulated, while six metabolites including putrescine, cadaverine, and N-acetylglycine were upregulated in Tregs compared to exTregs. A purine ectonucleotidase CD73 inhibitor decreased Treg conversion to exTregs and adenosine increased Treg migration and suppressive function concomitant with decreased exTreg conversion. Polyamine metabolites showed only subtle effects on exTreg conversion and migration. These results suggest that purine metabolism is a potent regulator that maintains Treg Foxp3 expression and suppressive function during Treg TEM and thus, could be a promising target for therapeutic interventions. NIH grant RO1 A1062765

The Maresin 1–LGR6 axis decreases respiratory syncytial virus-induced lung inflammation

The resolution of infection is an active process with specific molecular and cellular mechanisms that temper inflammation and enhance pathogen clearance. Here, the specialized pro-resolving mediator (SPM)Maresin 1 (MaR1) inhibited respiratory syncytial virus (RSV)-induced inflammation. inlerleukin-13production from type 2 innate lymphoid cells (ILC)and CD4 T helper type 2 cells was decreased by exogenous MaR1. In addition, MaR1 increased amphiregulin production and decreased RSV viral transcripts to promote resolution. MaR1 also promoted interferon-β production in mouse lung tissues and also in pediatric lung slices. MaR1 significantly inhibited the RSV-triggered aberrant inflammatory phenotype in FoxP3-expressing Tregs. The receptor for MaR1, leucine-rich repeat-containing G protein-coupled receptor 6 (LGR6), was constitutively expressed on Tregs. Following RSV infection, mice lacking Lgr6 had exacerbated type 2 immune responses with an increased viral burden and blunted responses to MaR1. Together, these findings have uncovered a multi-pronged protective signaling axis for MaR1-Lgr6, improving Tregs's suppressive function and upregulating host antiviral genes resulting in decreased viral burden and pathogen-mediated inflammation, ultimately promoting restoration of airway mucosal homeostasis.

A deubiquitination module essential for Treg fitness in the tumor microenvironment.

The tumor microenvironment (TME) enhances regulatory T (Treg) cell stability and immunosuppressive functions through up-regulation of lineage transcription factor Foxp3, a phenomenon known as Treg fitness or adaptation. Here, we characterize previously unknown TME-specific cellular and molecular mechanisms underlying Treg fitness. We demonstrate that TME-specific stressors including transforming growth factor-β (TGF-β), hypoxia, and nutrient deprivation selectively induce two Foxp3-specific deubiquitinases, ubiquitin-specific peptidase 22 (Usp22) and Usp21, by regulating TGF-β, HIF, and mTOR signaling, respectively, to maintain Treg fitness. Simultaneous deletion of both USPs in Treg cells largely diminishes TME-induced Foxp3 up-regulation, alters Treg metabolic signatures, impairs Treg-suppressive function, and alleviates Treg suppression on cytotoxic CD8+ T cells. Furthermore, we developed the first Usp22-specific small-molecule inhibitor, which dramatically reduced intratumoral Treg Foxp3 expression and consequently enhanced antitumor immunity. Our findings unveil previously unappreciated mechanisms underlying Treg fitness and identify Usp22 as an antitumor therapeutic target that inhibits Treg adaptability in the TME.

Analysis of FOXP3 DNA Methylation Patterns to Identify Functional FOXP3+ T-Cell Subpopulations.

Human regulatory CD4+CD25+FOXP3+ T cells (Tregs) are involved in the suppression of immune responses and play important roles in the maintenance of self-tolerance and immune homeostasis. Abnormal Treg function may result in disease states of varying severity. As FOXP3-expressing Treg cells are phenotypically and functionally heterogeneous, the success of Treg therapies depends on the ability to reliably distinguish subpopulations of T cells bearing a Treg-like phenotype. Methylation of cytosines within CpG dinucleotides is an important epigenetic mechanism involved in regulation (and suppression) of gene expression. On the other hand, demethylation of regulatory DNA sequences, such as promoters and enhancers, is essential for initiation of gene transcription. This protocol shows that bisulfite sequencing (BS) distinguishes methylated and unmethylated cytosines within DNA and reveals the methylation status of individual CpGs in cells within each population, identifying functionally different FOXP3+ subpopulations.

Forced Fox-P3 expression can improve the safety and antigen-specific function of engineered regulatory T cells

Regulatory T cells (Treg) are potent inhibitors of autoreactive T cells. The intracellular transcription factor FoxP3 controls the expression levels of a diverse set of genes and plays a critical role in programming functional Tregs. Although, antigen-specific Tregs are more potent than polyclonal Tregs in treating ongoing autoimmunity, phenotype plasticity associated with loss of FoxP3 expression in Tregs can lead to the conversion into antigen-specific effector T cells which might exacerbate autoimmune pathology. In this study, we designed a retroviral vector driving the expression of FoxP3 and a human HLA-DR-restricted TCR from the same promoter. Transduction of purified human Tregs revealed that all TCR-positive cells had elevated levels of FoxP3 expression, increased CD25 and CTLA4 expression and potent suppressive function. Elevated FoxP3 expression did not impair the in vitro expansion of engineered Tregs. Adoptive transfer into HLA-DR transgenic mice revealed that FoxP3+TCR engineered Tregs showed long-term persistence with stable FoxP3 and TCR expression. In contrast, adoptive transfer of Tregs engineered with TCR only resulted in the accumulation of TCR-positive, FoxP3-negative T cells which displayed antigen-specific effector function when stimulated with the TCR-recognised peptides. Our data indicate that forced expression of FoxP3 can prevent accumulation of antigen-specific effector T cells without impairing the engraftment and persistence of engineered Tregs.

Suppression of FOXP3 expression by the AP-1 family transcription factor BATF3 requires partnering with IRF4.

FOXP3 is the lineage-defining transcription factor for Tregs, a cell type critical to immune tolerance, but the mechanisms that control FOXP3 expression in Tregs remain incompletely defined, particularly as it relates to signals downstream of TCR and CD28 signaling. Herein, we studied the role of IRF4 and BATF3, two transcription factors upregulated upon T cell activation, to the conversion of conventional CD4+ T cells to FOXP3+ T cells (iTregs) in vitro. We found that IRF4 must partner with BATF3 to bind to a regulatory region in the Foxp3 locus where they cooperatively repress FOXP3 expression and iTreg induction. In addition, we found that interactions of these transcription factors are necessary for glycolytic reprogramming of activated T cells that is antagonistic to FOXP3 expression and stability. As a result, Irf4 KO iTregs show increased demethylation of the critical CNS2 region in the Foxp3 locus. Together, our findings provide important insights how BATF3 and IRF4 interactions integrate activating signals to control CD4+ cell fate decisions and govern Foxp3 expression.

P1270: IMMUNE REGULATORY CELLS IN B-CELL LYMPHOMAS: SIGNIFICANT HETEROGENEITY ACROSS HISTOLOGIC TYPES.

Background: Immune regulatory cell populations play a fundamental role in tumor development and tumor immune evasion. Myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) considered as dominant suppressor cells which are significantly enriched in blood, bone marrow (BM), or tumor sites of patients with hematologic malignancies and often associate with high tumor burden, adverse clinical features, and outcome. Studies in lymphoma patients however, have generated conflicting results, regarding the function and phenotype of MDSC and Tregs mainly due to large heterogeneity of the disease as well as due to the lack of a unanimous nomenclature of these cell subsets in human samples. Furthermore, the molecular mechanisms via which MDSCs and Tregs contribute to clinical outcomes of disease and how they interfere with response to therapy remain elusive. Aims: To investigate the prevalence of immune suppressive population in B-cell lymphomas according to histologic, subtype providing the basis for further analysis of their biologic properties and association with clinical features and outcome of specific lymphoma subtypes. Methods: Peripheral blood (PB) and BM samples were collected from healthy individuals (n= 22) and lymphoma patients (n=50). Specifically, lymphoma subtypes were as follows:Marginal Zone (MZL, N=19), Diffuse Large B-cell Lymphoma (DLBCL, N=18) and Follicular Lymphoma (FL, N=13). Peripheral blood mononuclear cells (PBMCs) were isolated with ficoll gradient and subjected to flow cytometry analysis. MDSC characterization was based on HLADR-/lowCD11b+CD33- expression with CD14+CD15- for M-MDSCs and CD14-CD15+ for PMN-MDSCs. In addition, Treg cells were denoted as CD4+CD25highCD127-Foxp3+. Expression of PDL1 and PD1 on MDSCs and Tregs respectively, was also determined by flow cytometry. M-MDSCs and PMN-MDSCs were sorted from BM samples, RNA was extracted in order to subjected to transcriptomic analysis (RNAseq). Informed consent was obtained. Results: Frequencies of MDSCs were significantly increased in PB of all lymphoma patients compared to healthy individuals, with MZL subtype exhibiting the most prominent increase of MDSCs (p<.0001). Notably numbers of M-MDSCs were increased in FL and MZL patients and to a lesser extend in DLBCL patients while PMN-MDSCs were elevated in all subtypes with MZL displaying the higher frequencies. Regarding the Treg cells, although all lymphoma patients exhibited increased absolute numbers of CD4+CD25highCD127- Treg cells, Foxp3-expressing Tregs were significantly increased in the PB of DLBCL and MZL patients, but not in FL patients. Of interest, CD4+ and CD8+ T cells in the PB of DLBCL patients exhibited increase expression of PD1 compared to respective cell subsets from healthy individuals. Summary/Conclusion: Our findings comprehensively demonstrate enhanced numbers of MDSC and Treg immune suppressive cells in the PB of B-cell lymphoma patients while an increased heterogeneity was observed among the different histological lymphoma types. Functional characterization of MDSCs and Tregs by transcriptomic (RNAseq) and proteomic analysis are ongoing.

Mesenchymal stem cells augment regulatory T cell function via CD80-mediated interactions and promote allograft survival

Mesenchymal stem cells (MSCs) and regulatory T cells (Tregs) both have been shown to modulate the alloimmune response and promote transplant survival. Mounting evidence suggests that MSCs augment Treg function, but the mechanisms underlying this phenomenon have not been fully deciphered. Here, we identified that MSCs express substantial levels of CD80 and evaluated its immunoregulatory function using in vivo and in vitro experiments. Our in vitro culture assays demonstrated that MSCs induce expression of FoxP3 in Tregs in a contact-dependent manner, and the blockade of CD80 abrogates this FoxP3 induction and Treg-mediated suppression of T cell proliferation. Moreover, supplementation of soluble CD80significantly upregulated FoxP3 expression. Using a well-characterized murine model of corneal transplantation, we show that silencing CD80 in MSCs diminishes the capacity of MSCs to promote selective graft infiltration of Tregs, promote FoxP3 expression and upregulate suppressive function of Tregs. Consequently, MSCs, following CD80knockdown, failed to promote corneal allograft survival.

Alcohol Impairs Immunometabolism and Promotes Naïve T Cell Differentiation to Pro-Inflammatory Th1 CD4+ T Cells.

CD4+ T cell differentiation to pro-inflammatory and immunosuppressive subsets depends on immunometabolism. Pro-inflammatory CD4+ subsets rely on glycolysis, while immunosuppressive Treg cells require functional mitochondria for their differentiation and function. Previous pre-clinical studies have shown that ethanol (EtOH) administration increases pro-inflammatory CD4+ T cell subsets; whether this shift in immunophenotype is linked to alterations in CD4+ T cell metabolism had not been previously examined. The objective of this study was to determine whether ethanol alters CD4+ immunometabolism, and whether this affects CD4+ T cell differentiation. Naïve human CD4+ T cells were plated on anti-CD3 coated plates with soluble anti-CD28, and differentiated with IL-12 in the presence of ethanol (0 and 50 mM) for 3 days. Both Tbet-expressing (Th1) and FOXP3-expressing (Treg) CD4+ T cells increased after differentiation. Ethanol dysregulated CD4+ T cell differentiation by increasing Th1 and decreasing Treg CD4+ T cell subsets. Ethanol increased glycolysis and impaired oxidative phosphorylation in differentiated CD4+ T cells. Moreover, the glycolytic inhibitor 2-deoxyglucose (2-DG) prevented the ethanol-mediated increase in Tbet-expressing CD4+ T cells but did not attenuate the decrease in FOXP3 expression in differentiated CD4+ T cells. Ethanol increased Treg mitochondrial volume and altered expression of genes implicated in mitophagy and autophagosome formation (PINK1 and ATG7). These results suggest that ethanol impairs CD4+ T cell immunometabolism and disrupts mitochondrial repair processes as it promotes CD4+ T cell differentiation to a pro-inflammatory phenotype.

Alcohol Dysregulates CD4 T Cell Mitochondrial Homeostasis

CD4 T cell differentiation to pro‐inflammatory and immunosuppressive subsets requires distinct metabolic pathways. Pro‐inflammatory CD4 subsets rely on glycolysis, while immunosuppressive (Treg cells) subsets, require functional mitochondria for their differentiation and function. Previous studies have shown that binge alcohol (ethanol, EtOH) administration increased Tbet‐expressing (Th1) and decreased FOXP3‐expressing (Treg) CD4 T cells in the colons of mice. We tested the hypothesis that EtOH dysregulates normal CD4 T cell differentiation, after stimulation, by impairing mitochondrial homeostasis. Human naïve CD4 T cells were isolated from buffy coats from blood bank donors (N = 6) using MACS sorting. Cells were stimulated using anti‐CD3‐coated dishes in the presence of anti‐CD28 and IL‐12, and exposed to EtOH (0 and 50 mM) for 3 days. Mitochondrial content was measured with Mitotracker Deep Red. Gene expression indicative of: autophagosome formation (ATG5, ATG7, ATG13, MAP1LC3B, BECN1, BNIP3L, ULK1), mitophagy (PINK1, PRKN),mitochondrial fusion (MFN1, MFN2, OPA1), mitochondrial fission (MFFand FIS1), and mitochondrial biogenesis (PPARC1A, PPARC1B, TFAM) was determined by RT2 profiler arrays. EtOH‐treated CD4 T cells had increased mitochondrial content (p = 0.0008) with Tregs accounting for the greatest increase in mitochondria (p = 0.04). There was a main effect of stimulation (p &lt; 0.05) to increase ATG5, ATG13, MAP1LC3B, BECN1, BNIP3L, ULK1, MFF, PPARC1B, and TFAM, and a main effect of EtOH (p &lt; 0.05) to increase PINK1 and decrease ATG7. There was a main effect of both EtOH and stimulation (p &lt; 0.05) to increase MFN2, and OPA1.Taken together, these results indicate that EtOH increases mitochondrial content in Treg cells and dysregulates mitochondrial gene expression important for mitochondrial repair and mitophagy. These EtOH‐mediated alterations in gene expression could result in an inability of CD4 T cells to maintain mitochondrial homeostasis and remove damaged mitochondria that is required for normal differentiation and function of anti‐inflammatory Treg cells.

Target the activin receptor 1c on CD4+ T cells to achieve anti-tumor therapeutic effects.

Abstract Activin signaling molecules and their receptors have not been substantially studied in the context of the immune system and cancer. We show here that tumor-bearing mice had elevated Activins levels, which correlated directly with tumor burden. Likewise, cancer patients had elevated plasma Activins compared to healthy controls. Importantly, our in vitro studies suggested that Activins promoted differentiation of naive wild type CD4+ cells into induced FoxP3-expressing Tregs. Database and qRT-PCR analysis of sorted major immune cell subsets in mice revealed that Activin Receptor 1C (ActR1c) was uniquely expressed on Tregs. ActR2b, as paired to ActR1c, was found to be highly upregulated during iTreg differentiation. In humans, these receptors were expressed on CD4+ Foxp3− cells. Thus, while Activins may have many cellular targets, signaling through these receptors on CD4+ cells likely promotes Treg-mediated immunosuppression. In vivo B16 melanoma and MC38 colon tumor studies have demonstrated that mice lacking ActR1c were more resistant to cancer progression compared to wild type mice. This phenotype correlated with reduced expression of the FoxP3 transcription factor in CD4+ cells. The anti-tumor effect was T-cell dependent, as T cell-deficient mice reconstituted with naïve ActR1c knock out CD4+ cells had delayed tumor progression. In vitro, naïve CD4+ cells lacking ActR1c had a defect in their ability to gain FoxP3 expression when Activin A was present and when TGF-β was limited. Thus, blocking Activins signaling through this receptor is a promising and disease-specific strategy for preventing the accumulation of immunosuppressive iTregs in cancer. Supported by grants from NIH (R01AI137046)