Immunosuppressive Function Of Tregs Research Articles

TNF Receptor Type II as an Emerging Drug Target for the Treatment of Cancer, Autoimmune Diseases, and Graft-Versus-Host Disease: Current Perspectives and In Silico Search for Small Molecule Binders.

There is now compelling evidence that TNF receptor type II (TNFR2) is predominantly expressed on CD4+Foxp3+ regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), and plays a major role in the expansion and function of Tregs and MDSCs. Consequently, targeting of TNFR2 by either antagonists or agonists may represent a novel strategy in the treatment of cancer and autoimmune diseases, by downregulating or upregulating suppressor cell activity. The advance in the understanding of complex structure of TNFR2 and its binding with TNF at molecular levels offers opportunity for structure-guided drug discovery. This article reviews the current evidences regarding the decisive role of TNFR2 in immunosuppressive function of Tregs and MDSCs, and the current effort to develop novel TNFR2-targeting therapeutic agents in the treatment of cancer, autoimmune diseases, and graft-versus-host disease. To shed light on the potential TNFR2-targeting small molecules, we for the first time performed virtual screening of 400,000 natural compounds against the two TNF-binding sites, regions 3 and 4, of TNFR2. Our result showed that the top hits at region 4 had slightly higher docking energies than those at region 3. Nevertheless, free energy calculation from the TNF–TNFR2 molecular dynamics simulation revealed that the binding strength of TNF in region 3 is only one-tenth of that in region 4. This suggests that region 3 is a potentially more viable binding site to be targeted by small molecules than region 4. Therefore, the effectiveness in targeting region 3 of TNFR2 deserves further investigation.

Blocking IFNAR1 inhibits multiple myeloma-driven Treg expansion and immunosuppression.

Despite significant advances in the treatment of multiple myeloma (MM), most patients succumb to disease progression. One of the major immunosuppressive mechanisms that is believed to play a role in myeloma progression is the expansion of regulatory T cells (Tregs). In this study, we demonstrate that myeloma cells drive Treg expansion and activation by secreting type 1 interferon (IFN). Blocking IFN α and β receptor 1 (IFNAR1) on Tregs significantly decreases both myeloma-associated Treg immunosuppressive function and myeloma progression. Using syngeneic transplantable murine myeloma models and bone marrow (BM) aspirates of MM patients, we found that Tregs were expanded and activated in the BM microenvironment at early stages of myeloma development. Selective depletion of Tregs led to a complete remission and prolonged survival in mice injected with myeloma cells. Further analysis of the interaction between myeloma cells and Tregs using gene sequencing and enrichment analysis uncovered a feedback loop, wherein myeloma-cell-secreted type 1 IFN induced proliferation and expansion of Tregs. By using IFNAR1-blocking antibody treatment and IFNAR1-knockout Tregs, we demonstrated a significant decrease in myeloma-associated Treg proliferation, which was associated with longer survival of myeloma-injected mice. Our results thus suggest that blocking type 1 IFN signaling represents a potential strategy to target immunosuppressive Treg function in MM.

Schistosoma japonicum heat shock protein 60 enhances regulatory T cell immunosuppressive function by promoting the expressions of IL-10 and TGF-β

To explore the possible mechanisms by which Schistosoma japonicum heat shock protein 60 (SjH-SP60) enhances CD4+CD25+ regulatory T cell (Treg) immunosuppressive function. An in vitro method was used to investigate the effect of SjHSP60 on Treg immunosuppressive activity. Co-cultures in transwells and in vitro suppression assay were performed to investigate how SjHSP60 enhanced the immunosuppressive function of Tregs. Intracellular cytokine staining combined with flow cytometry was used to detect Treg-expressing IL-10 and TGF-β, and flow cytometry was also used to analyze the expressions of Foxp3 and CTLA-4 in Tregs. SjHSP60 enhanced the immunosuppressive function of Tregs. Soluble cytokines IL-10 and TGF-β mediated inhibitory activity of SjHSP60-triggered Tregs. SjHSP60 induced significant increases in both IL-10 and TGF-β expressions of Tregs. Further investigation showed significant increased Foxp3 and CTLA-4 in SjHSP60-trggered Tregs. SjHSP60 enhances Treg immunosuppressive function by promoting the expressions of IL-10 and TGF-β, possibly due to SjHSP60-mediated induction of Foxp3 and CTLA-4 in Tregs.

Modulation of CD4+ CD25+ CD127dim/- regulatory T cells by interleukin-35 in the pathogenesis of ventilator-associated pneumonia

Objective To investigate the changes in CD4+ CD25+ CD127dim/- regulatory T cells (Tregs) and interleukin (IL)-35 in peripheral blood and bronchoalveolar lavage fluid (BALF) samples from patients with ventilator-associated pneumonia (VAP). Methods A total of 23 patients with VAP and 11 normal controls (NCs) were enrolled in this study. Peripheral blood mononuclear cells, serum and BALF samples were isolated and collected. Levels of IL-35 were measured by enzyme linked-immunosorbent assay. Percentages of CD4+ CD25+ CD127dim/- Tregs were measured by flow cytometry. CD4+ CD25+ CD127dim/- Tregs in BALF samples were isolated and purified, which were then stimulated with recombinant human IL-35 and co-cultured with autologous CD4+ CD25-T cells. Cells and supernatants were harvested for analysis of cell proliferation and cytokine secretion. Results No significant difference in peripheral Tregs and serum IL-35 was found between patients with VAP and NCs. The percentages of Tregs and the levels of IL-35 in BALF samples collected from infectious sites were remarkably higher than those collected from non-infectious sites in patients with VAP. Moreover, there was a positive correlation between Tregs and IL-35 in BALF (r=0.441, P=0.035). Tregs and IL-35 in BALF samples as well as Treg-secreted IL-35 were significantly reduced in patients who had good response to therapy. However, no significant change in these parameters was observed in patients who had poor response to therapy. Besides, suppression of cell proliferation and IL-10 secretion that were related to Tregs were inhibited in patients whose condition was improved as compared with those in patients who had no response to therapy. Stimulation with recombinant human IL-35 enhanced the immunosuppressive function of purified Tregs that were separated from BALF of treatment-naive patients with VAP, which was mainly marked by suppressed cell proliferation, increased secretion of inhibitory cytokines (IL-35 and IL-10), and decreased secretion of proinflammatory cytokines (IFN-γ and TNF-α). However, IL-35 had little effect on the activities of Tregs that were separated from patients with VAP who responded to therapy. Conclusion Both IL-35 and Tregs are increased in BALF of patients with VAP and IL-35 enhances the immunosuppressive function of Tregs, which indicates that IL-35-mediated modulation of Tregs might take part in the pathogenesis of VAP. Key words: Ventilator-associated pneumonia; Interleukin-35; Regulatory T cell; Immunomodulation; Pathogenesis

Forsythoside A exerts an anti-endotoxin effect by blocking the LPS/TLR4 signaling pathway and inhibiting Tregs in vitro.

Endotoxins, also referred to as lipopolysaccharides(LPS), are powerful immunostimulators involved in a number of severe diseases. ForsythosideA (FTA), a monomer of phenethyl alcohol glycosides extracted from Forsythia suspensa, has been shown to possess anti-bacterial and immunomodulatory properties. However, it is currently not known whether FTA can counter the adverse effects of endotoxins. We investigated the effect of FTA on LPS-stimulated RAW264.7 cells and primary lymphocytes to determine its molecular mechanism of action. RAW264.7 cells and primary lymphocytes were incubated with or without LPS (100ng/ml) in the presence or absence of FTA or polymyxinB. We found that FTA increased the viability of LPS-treated RAW264.7 cells and primary lymphocytes suggesting that FTA effectively counters the adverse effects of endotoxins. FTA decreased the percentage of regulatory Tcells (Tregs) and inhibited the TLR4/MyD88/NF-κB signaling pathway, downregulating Foxp3, IL-10 and TGF-β1, molecules involved in the immunosuppressive function of Tregs. These findings elucidate the molecular mechanism underlying the anti-endotoxin effects of FTA and suggest its use as a new treatment for LPS-induced diseases.

Foxp3, Regulatory T Cell, and Autoimmune Diseases.

Regulatory T cells (Tregs) represent a cell type that promotes immune tolerance to autologous components and maintains immune system homeostasis. The abnormal function of Tregs is relevant to the pathogenesis of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and other autoimmune diseases. Therefore, therapeutic modulation of Tregs could be a potent means of treating autoimmune diseases. Human Tregs are diverse, however, and not all of them have immunosuppressive effects. Forkhead box P3 (Foxp3), a pivotal transcription factor of Tregs that is crucial in maintaining Treg immunosuppressive function, can be expressed heterogeneously or unstably across Treg subpopulations. Insights into modulating Treg differentiation on the level of DNA transcription or protein modification may improve the success of Treg modifying immunotherapies. In this review, we will summarize three main prospects: the regulatory mechanism of Foxp3, the influence on Foxp3 and Tregs in autoimmune diseases, then finally, how Tregs can be used to treat autoimmune diseases.

Tumor Progression Locus 2 (Tpl2) Activates the Mammalian Target of Rapamycin (mTOR) Pathway, Inhibits Forkhead Box P3 (FoxP3) Expression, and Limits Regulatory T Cell (Treg) Immunosuppressive Functions

The serine/threonine kinase tumor progression locus 2 (Tpl2, also known as Map3k8/Cot) is a potent inflammatory mediator that drives the production of TNFα, IL-1β, and IFNγ. We previously demonstrated that Tpl2 regulates T cell receptor (TCR) signaling and modulates T helper cell differentiation. However, very little is known about how Tpl2 modulates the development of regulatory T cells (Tregs). Tregs are a specialized subset of T cells that express FoxP3 and possess immunosuppressive properties to limit excess inflammation. Because of the documented role of Tpl2 in promoting inflammation, we hypothesized that Tpl2 antagonizes Treg development and immunosuppressive function. Here we demonstrate that Tpl2 constrains the development of inducible Tregs. Tpl2(-/-) naïve CD4(+) T cells preferentially develop into FoxP3(+) inducible Tregs in vitro as well as in vivo in a murine model of ovalbumin (OVA)-induced systemic tolerance. Treg biasing of Tpl2(-/-) T cells depended on TCR signal strength and corresponded with reduced activation of the mammalian target of rapamycin (mTOR) pathway. Importantly, Tpl2(-/-) Tregs have basally increased expression of FoxP3 and immunosuppressive molecules, IL-10 and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). Furthermore, they were more immunosuppressive in vivo in a T cell transfer model of colitis, as evidenced by reduced effector T cell accumulation, systemic production of inflammatory cytokines, and colonic inflammation. These results demonstrate that Tpl2 promotes inflammation in part by constraining FoxP3 expression and Treg immunosuppressive functions. Overall, these findings suggest that Tpl2 inhibition could be used to preferentially drive Treg induction and thereby limit inflammation in a variety of autoimmune diseases.

Abstract A068: Imbalance in regulatory T (Treg) cells frequency in peripheral circulation of patients with oral squamous cell carcinoma and their effects on growth of oral cancer

Abstract Regulatory T (Treg) cells play a major role in establishment and maintenance of self tolerance of immune system. Deficit in the numbers of Tregs contributes to the development of autoimmune diseases, allergy and graft rejection whereas abundance of Tregs suppresses anti-tumour immune response leading to tumour immuno-evasion. In this study, we have estimated the frequency of distinct immune cells, Tregs and its phenotypic subsets in patients with Oral Squamous Cell Carcinoma (OSCC) by multicolour flowcytometry. Subsequently we investigated the effects of MACS sorted Tregs on growth/proliferation/angiogenesis of OSCC cell lines in vitro. Frequency of all the effector type anti-tumour immune cells such as CD8+ T-cells, NK (CD56+) and NKT (CD3+CD56+) cells was found to be decreased whereas CD3+, CD4+, γδ-T cells were upregulated in OSCC patients. We observed significantly increased (p < 0.05) prevalence of Tregs and their phenotypic subsets (CD4+CD25+, CD4+FoxP3+, CD8+FoxP3+, CD4+CD25+FoxP3+) in peripheral circulation of OSCC patients as compared to that of the normal individuals. Our study further showed a definite association between the OSCC progression and elevated Tregs frequency. An association between reciprocally regulated Treg and Th17 cells was also observed. The increased frequency of CD4+CD8+CD25+FoxP3+, a unique T cells subset was seen in OSCC patients. The CTLA-4+ and GITR+ Tregs also showed higher occurrence in patients indicating their contribution to the immuno-suppressive function of Tregs in OSCC. Introduction of MACS isolated Treg cells to in vitro oral cancer cell culture resulted in increased proliferation and expression of angiogenic marker VEGF by cancer cells showing that these suppressor cells helps in growth and proliferation of OSCC cells. Accumulation of heterogeneous subsets of Tregs in the peripheral circulation indicates their suggestive role in tumour induced immuno-suppression as well as in growth and proliferation of oral cancer cells. Therefore, inhibitory manipulation of Tregs, in terms of their frequency and functional activity, can be added to the therapeutic armamentarium for enhancing anti-tumour immunity in OSCC patients and preventing the establishment and progression of oral cancer. Citation Format: Sadhna Aggarwal, Satya N. Das, Suresh C. Sharma. Imbalance in regulatory T (Treg) cells frequency in peripheral circulation of patients with oral squamous cell carcinoma and their effects on growth of oral cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A068.

Integrative analysis of mRNA and miRNA array data reveals the suppression of retinoic acid pathway in regulatory T cells of Graves' disease.

It is well known that regulatory T cells (Tregs) are abnormal in Graves' disease (GD) and play crucial roles in the breakdown of immune tolerance and GD development. However, there are controversies about whether the quantity and/or function of Tregs is aberrant in GD. The molecular mechanism of Tregs abnormality and its effects on GD development was still unclear, until now. MiRNAs play important roles in the function and development of the immune system including Tregs. To reveal the Tregs abnormality and its molecular mechanism in GD, we systematically studied the quantity and immunosuppressive function as well as the differential expression profiles of miRNA and mRNA of Tregs in newly diagnosed patients with GD using TaqMan miRNA array and mRNA microarray. Our results showed that the quantity and immunosuppressive function of Tregs in initial patients with GD was significantly decreased. More importantly, the retinoic acid (RA) pathway was markedly suppressed and its agonist, all-trans retinoic acid, could notably improve the quantity and immunosuppressive function of Tregs from patients with GD in vitro. In addition, many other pathways including protein ubiquitination and circadian rhythm were also significantly regulated in Tregs of GD. This integrative study first revealed the expression profiles of mRNA/miRNA in Tregs of initial GD and RA pathway might play important roles in GD development. Our results implied that all-trans RA, which had been used for a long time in the clinical setting, had potential value in the treatment of GD and was worthy of additional study.

CpG oligodeoxynucleotide ligand potentiates the activity of the pVAX1-Sj26GST

Schistosomiasis is considered one of the most important neglected tropical diseases and remains a major public health problem in endemic countries. Toll-like receptor (TLR) ligands have been investigated as potential vaccine adjuvants for tumor and virus immunotherapy. However, few TLR ligands affecting schistosoma vaccines have been characterized. In this study, we evaluated a TLR9 ligand (CpG oligodeoxynucleotide 1826, CpG) as an adjuvant for a partially protective DNA vaccine encoding a 26-kDa glutathione S-transferase of Schistosoma japonicum (pVAX1-Sj26GST). Vaccination with pVAX1-Sj26GST in combination with CpG inhibited Treg immunosuppressive function, upregulated the production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-4, IL-10, IL-2 and IL-6, and decreased CD4+CD8+Foxp3+ expression in vitro, which may contribute to the escape from Treg-mediated suppression during vaccination, allowing expansion of antigen-specific T cells against pathogens. In conclusion, our data demonstrated that selective TLR ligand combination may increase protective efficacy against schistosomiasis, which may synergistically antagonize Treg-mediated suppression.

Inherited variants in regulatory T cell genes and outcome of ovarian cancer.

Although ovarian cancer is the most lethal of gynecologic malignancies, wide variation in outcome following conventional therapy continues to exist. The presence of tumor-infiltrating regulatory T cells (Tregs) has a role in outcome of this disease, and a growing body of data supports the existence of inherited prognostic factors. However, the role of inherited variants in genes encoding Treg-related immune molecules has not been fully explored. We analyzed expression quantitative trait loci (eQTL) and sequence-based tagging single nucleotide polymorphisms (tagSNPs) for 54 genes associated with Tregs in 3,662 invasive ovarian cancer cases. With adjustment for known prognostic factors, suggestive results were observed among rarer histological subtypes; poorer survival was associated with minor alleles at SNPs in RGS1 (clear cell, rs10921202, p = 2.7×10−5), LRRC32 and TNFRSF18/TNFRSF4 (mucinous, rs3781699, p = 4.5×10−4, and rs3753348, p = 9.0×10−4, respectively), and CD80 (endometrioid, rs13071247, p = 8.0×10−4). Fo0r the latter, correlative data support a CD80 rs13071247 genotype association with CD80 tumor RNA expression (p = 0.006). An additional eQTL SNP in CD80 was associated with shorter survival (rs7804190, p = 8.1×10−4) among all cases combined. As the products of these genes are known to affect induction, trafficking, or immunosuppressive function of Tregs, these results suggest the need for follow-up phenotypic studies.

Abstract 3547: Disruption of Galectin1-glycan interaction impairs tumor growth and metastasis in breast cancer by disarming the immunosuppressive capacity of regulatory T cells

Abstract Galectin-1 (Gal1) is an endogenous lectin that emerged as a key regulator of immune cell homeostasis. By binding to surface glycoconjugates, Gal1 modulates diverse physiological processes and also shapes the immune response during cancer progression to a suppressive phenotype. For that reason Gal1 has become a molecule with a profound impact in the tumor microenvironment biology. In the present work we investigated whether tumor Gal1 expression contributes to immunosuppression, tumor growth and metastasis in breast cancer in order to validate the Gal-1-glycan axis as a novel therapeutic target in these tumors. We used the highly metastatic mouse mammary tumor model 4T1 which expresses high levels of Gal1. We generated Gal1-deficient 4T1 cell line (4T1 KD) using a retrovirus encoding a shRNA specific for mouse Gal1. After injecting Balb/c mice with either 4T1 WT or KD cells we studied tumor growth, metastasis formation and analyzed tumor-associated immune compartments. Silencing of Gal-1 induced a marked reduction in both tumor growth and the number of lung metastases. This effect was abrogated if a 4T1 WT tumor was inoculated in the contralateral flank of these mice, suggesting that the antitumor effect involves modulation of the immune system. In regard to the latter we observed that 4T1 WT tumor-bearing mice exhibited a higher frequency of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in the lungs, spleen, tumor draining lymph nodes (TDLN) and tumor microenvironment. Remarkably, knocking down Gal1 reverted the systemic immune tolerance that characterizes tumor progression, and decreased the frequency and immunosuppressive function of Tregs as evidenced by a suppression assay and expression of Tregs tolerogenic molecular markers as Foxp3, TGF-β and LAT. In this sense, mice bearing Gal-1 KD tumors were able to reject an allogeneic B16 tumor similarly to tumor free mice. In addition, antigen-specific proliferation of lymphocytes purified from 4T1 KD-bearing mice was increased when reestimulated ex vivo. Concomitantly, blockade of tumor Gal1 was accompanied by a reduction of the IL-10/IFNγ cytokines ratio. Finally, therapeutic administration of a Gal1 neutralizing mAb (F8.G7) to 4T1 WT tumor bearing mice not only induced a reduction in tumor growth and lung metastasis formation but also reverted tumor associated immunosuppression. These results are strengthened by the observation that in human breast cancer biopsies Gal1 expression correlates with tumor grade (Bloom-Richardson histopathological grade I vs II-III). In conclusion our results indicate that disruption of Gal1 impairs tumor growth and metastasis via mechanisms involving reversal of systemic immunosuppression and further validate the Gal1-glycan axis as an attractive target in the therapeutic treatment of metastasic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3547. doi:1538-7445.AM2012-3547

Elevated frequency and function of regulatory T cells in patients with active chronic hepatitis C

Regulatory T cells (Tregs) play a pivotal role in the persistence of hepatitis C virus infection. The aim of this study was to evaluate the frequency and function of Tregs in patients with chronic hepatitis C (CHC). We enrolled 44 CHC patients with elevated alanine aminotransferase (ALT) levels (CH group), 13 CHC patients with persistent normal ALT levels (PNALT group), and 14 age-matched healthy subjects (HS group; controls). Tregs were identified as CD4+, CD25+, and forkhead box P3 (Foxp3)+ T lymphocytes, using three-color fluorescence-activated cell sorting (FACS). The frequency of Tregs was determined by calculating the percentage of CD4+CD25(high) T cells among CD4 T cells. CD127 and CD45RA were also analyzed for subsets of Tregs. The levels of serum transforming growth factor (TGF)-β and interleukin (IL)-10 in immunosuppressive assays were detected by enzyme-linked immunosorbent assay (ELISA). The immunosuppressive abilities of Tregs were evaluated by measuring their ability to inhibit the proliferation of effector cells. Higher proportions of Tregs were found in the CH and PNALT groups compared with the HS group. The populations of CD127 low/negative and CD45RA negative cells were higher in the CH group than in the PNALT group. The expressions of IL-10 and TGF-β in the CH and PNALT groups were significantly higher than those in the HS group. In addition, the immunosuppressive ability of Tregs from the CH group was increased relative to that in the PNALT and the HS group. CHC patients, irrespective of liver function, had higher frequencies of Tregs than healthy subjects; however, only CHC patients with inflammation showed enhanced immunosuppressive function of Tregs.

Regulatory T cells: immune suppression and beyond

Foxp3-expressing regulatory T cells (Tregs) were originally identified as critical in maintaining self-tolerance and immune homeostasis. The immunosuppressive functions of Tregs are widely acknowledged and have been extensively studied. Recent studies have revealed many diverse roles of Tregs in shaping the immune system and the inflammatory response. This review will discuss our efforts as well as the efforts of others towards understanding the multifaceted function of Tregs in immune regulation.

Imatinib Mesylate Inhibits CD4+CD25+ Regulatory T Cell Activity and Enhances Active Immunotherapy against BCR-ABL− Tumors

Imatinib mesylate (Gleevec, STI571), a selective inhibitor of a restricted number of tyrosine kinases, has been effectively used for the treatment of Philadelphia chromosome-positive leukemias and gastrointestinal stromal tumors. Imatinib may also directly influence immune cells. Suppressive as well as stimulating effects of this drug on CD4(+) and CD8(+) T lymphocytes or dendritic cells have been reported. In the current study, we have investigated the influence of imatinib mesylate on CD4(+)CD25(+)FoxP3(+) regulatory T cells (Treg), a critical population of lymphocytes that contributes to peripheral tolerance. Used at concentrations achieved clinically, imatinib impaired Treg immunosuppressive function and FoxP3 expression but not production of IL-10 and TGF-beta in vitro. Imatinib significantly reduced the activation of the transcription factors STAT3 and STAT5 in Treg. Analysis of Treg TCR-induced signaling cascade indicated that imatinib inhibited phosphorylation of ZAP70 and LAT. Substantiating these observations, imatinib treatment of mice decreased Treg frequency and impaired their immunosuppressive function in vivo. Furthermore, imatinib mesylate significantly enhanced antitumor immune responses to dendritic cell-based immunization against an imatinib-resistant BCR-ABL negative lymphoma. The clinical applications of imatinib mesylate might thus be expanded with its use as a potent immunomodulatory agent targeting Treg in cancer immunotherapy.