Foxp3 Treg Research Articles

PD-1 and ICOS counter-regulate tissue resident regulatory T cell development and IL-10 production during flu.

Regulatory T cells that express the transcription factor Foxp3 (Treg cells) are a highly heterogenous population of immunoregulatory cells critical for maintaining immune homeostasis and preventing immunopathology during infections. Tissue resident Treg (TR-Treg) cells are maintained within nonlymphoid tissues and have been shown to suppress proinflammatory tissue resident T cell responses and promote tissue repair. Human populations are repetitively exposed to influenza infections and lung tissue resident effector T cell responses are associated with flu-induced long-term pulmonary sequelae. The kinetics of TR-Treg cell development and molecular features of TR-Treg cells during repeated and/or long-term flu infections are unclear. Utilizing a Foxp3RFP/IL-10GFP dual reporter mouse model along with intravascular fluorescent in vivo labeling, we characterized the TR-Treg cell responses to repetitive heterosubtypic influenza infections. We found lung tissue resident Treg cells accumulated and expressed high levels of co-inhibitory and co-stimulatory receptors post primary and secondary infections. Blockade of PD-1 or ICOS signaling reveals that PD-1 and ICOS signaling pathways counter-regulate TR-Treg cell expansion and IL-10 production, during secondary influenza infection. Furthermore, the virus-specific TR-Treg cell response displayed distinct kinetics, when compared to conventional CD4+ tissue resident memory T cells, during secondary flu infection. Our results provide insight into the tissue resident Foxp3+ regulatory T cell response during repetitive flu infections, which may be applicable to other respiratory infectious diseases such as tuberculosis and COVID.

Depletion of Foxp3+ regulatory T cells but not the absence of CD19+IL-10+ regulatory B cells hinders tumor growth in a para-orthotopic neuroblastoma mouse model.

Adaptive immune cells with regulatory function reportedly mediate immune escape in a variety of tumors. Little is known regarding the relevance of the most prominent regulatory cell populations, namely Foxp3+ T regulatory cells (Tregs) and CD19+IL-10+ B regulatory cells (Bregs), for neuroblastoma (NB) survival. After establishing a novel immunocompetent syngeneic NB mouse model where orthotopic tumors can be generated after intrarenal injection of NB975A cells, we studied the importance of Tregs and Bregs in Foxp3-DTR mice whose Tregs can be depleted by diphtheria toxin (DT) application as well as in CD19-specific IL-10 deficient mice that lack IL-10+ Bregs (CD19cre+/- × IL-10fl/fl mice). We observed Foxp3 Treg cells in tumors from wild type mice. On the contrary, Bregs or B cells were scarce. Specific depletion of Tregs in Foxp3-DTR mice resulted in an 85% reduction of tumor volume and weight compared to DT-treated wild type mice and untreated Foxp3-DTR mice. In contrast, NB tumor growth was not affected in CD19-specific IL-10 deficient mice. Similarly, mice lacking mature B cells (μMT mice) and CD19 deficient mice (CD19cre mice) showed no change in growth pattern of NB tumors. In Treg-depleted mice, reduced tumor growth was associated with an increased concentration of IFN-gamma, TNF-alpha, IL-4, IL-6, and IL-10 in isolated splenocytes. In summary, transient ablation of Tregs but not absence of Bregs hindered the growth of NB, strongly suggesting the therapeutic potential of targeting Tregs for this aggressive childhood tumor.

Low-dose IL-2 prevents murine chronic cardiac allograft rejection: Role for IL-2-induced T regulatory cells and exosomes with PD-L1 and CD73

To determine the effects and immunological mechanisms of low-dose interleukin-2 (IL-2) in a murine model of chronic cardiac allograft rejection (BALB/c to C57BL/6) after costimulatory blockade consisting of MR1 (250 μg/ip day 0) and CTLA4-Ig (200 μg/ip day 2), we administered low-dose IL-2 (2000 IU/day) starting on posttransplant day 14 for 3 weeks. T regulatory (Treg) cell infiltration of the grafts was determined by immunohistochemistry; circulating exosomes by western blot and aldehyde bead flow cytometry; antibodies to donor MHC by immunofluorescent staining of donor cells; and antibodies to cardiac self-antigens (myosin, vimentin) by ELISA. We demonstrated that costimulation blockade after allogeneic heart transplantation induced circulating exosomes containing cardiac self-antigens and antibodies to both donor MHC and self-antigens, leading to chronic rejection by day 45. Treatment with low-dose IL-2 prolonged allograft survival (>100 days), prevented chronic rejection, and induced splenic and graft-infiltrating CD4+ CD25+ Foxp3 Treg cells by day 45 and circulating exosomes (Foxp3+) with PD-L1 and CD73. MicroRNA 142, associated with the TGFβ pathway, was significantly downregulated in exosomes from IL-2-treated mice. In conclusion, low-dose IL-2 delays rejection in a murine model of chronic cardiac allograft rejection and also induces graft-infiltrating Tregs and circulating exosomes with immunoregulatory molecules.

Max interacting protein 1 induces IL-17-producing T helper/regulatory T imbalance in osteoarthritis by upregulating tectonic family member 2

Background/AimOsteoarthritis (OA) is a common total joint disorder associated with regulatory T cell (Treg)/IL-17-producing T helper (Th17) cell imbalance. This study elucidated the mechanism underlying Th17/Treg imbalance during OA progression. MethodsCD4+ T cells were isolated and induced to differentiate and obtain Th17 and Treg cells, and an OA mouse model was established by anterior cruciate ligament transection surgery, followed by loss- and gain-of-function assays. Max interacting protein 1 (MXI1), tectonic family member 2 (TCTN2), Forkhead Box Protein P3 (Foxp3), signal transducer and activator of transcription 3 (STAT3), and retinoic acid receptor-related orphan nuclear receptor gamma t (RORγt) expression was determined in cells and mice, accompanied by the measurement of the proportion of Th17 and Treg cells and the levels of interleukin (IL)− 1β, tumor necrosis factor (TNF)-α, and interferon (INF)-γ. Articular cartilage histopathology was observed by hematoxylin and eosin staining and Safranin O-Fast Green staining. Relationship between MXI1 and TCTN2 was assessed. ResultsBioinformatics analysis identified MXI1 and TCTN2 upregulation in OA patients. Mechanistically, MXI1 bound to TCTN2 promoter to promote its transcription. Upregulated MXI1 boosted INF-γ, STAT3, IL-1β, TNF-α, and RORγt levels and Th17 cell differentiation, but restricted Foxp3 expression and Treg cell differentiation in CD4+ T cells. Effects caused by overexpressed MXI1 were negated by silenced TCTN2. Also, the impacts of MXI1 overexpression on Th17/Treg imbalance and IL-1β, STAT3, TNF-α, Foxp3, INF-γ, and RORγt expression were further validated in OA mice, accompanied by aggravated articular cartilage degeneration. ConclusionConclusively, MXI1 facilitated Th17/Treg imbalance to accelerate OA progression.

Nature vs. nurture: FOXP3, genetics, and tissue environment shape Treg function.

The importance of regulatory T cells (Tregs) in preventing autoimmunity has been well established; however, the precise alterations in Treg function in autoimmune individuals and how underlying genetic associations impact the development and function of Tregs is still not well understood. Polygenetic susceptibly is a key driving factor in the development of autoimmunity, and many of the pathways implicated in genetic association studies point to a potential alteration or defect in regulatory T cell function. In this review transcriptomic control of Treg development and function is highlighted with a focus on how these pathways are altered during autoimmunity. In combination, observations from autoimmune mouse models and human patients now provide insights into epigenetic control of Treg function and stability. How tissue microenvironment influences Treg function, lineage stability, and functional plasticity is also explored. In conclusion, the current efficacy and future direction of Treg-based therapies for Type 1 Diabetes and other autoimmune diseases is discussed. In total, this review examines Treg function with focuses on genetic, epigenetic, and environmental mechanisms and how Treg functions are altered within the context of autoimmunity.

A Paradoxical Role for Regulatory T Cells in the Tumor Microenvironment of Pancreatic Cancer.

Simple SummaryPancreatic cancer is one of the most lethal cancer types and its high refractoriness to therapies, including immunotherapy, has often been associated with the predominantly immune suppressive tumor microenvironment that characterizes pancreatic tumors. Regulatory T cells (Tregs) are generally considered as drivers of immune suppression in cancers. However, an increasing number of reports suggest a paradoxical association between tumor infiltration by Tregs and improved patient prognosis, in particular in gastrointestinal cancers. Here we show that Treg infiltration in pancreatic ductal adenocarcinomas (PDAC) is associated with better overall survival of patients.Pancreatic ductal adenocarcinoma (PDAC) is considered to be a poorly immunogenic cancer type that combines a low mutation burden with a strong immunosuppressive tumor microenvironment. Regulatory T cells (Tregs) are major drivers of immune suppression but their prognostic role, particularly in gastrointestinal malignancies, remains controversial. Lymphocytic infiltration in 122 PDAC samples was assessed by multispectral immunofluorescence with anti-Keratin, -CD3, -CD8, -FOXP3 and -CD163 antibodies. Differential infiltration by Tregs was analyzed in the context of transcriptomic profiles that were available for 65 tumors. High infiltration of CD3+CD8− (mainly CD4+) T cells and, especially, of the subset expressing FOXP3 (Tregs) was associated with improved patient survival, whilst cytotoxic CD3+CD8+ T cell infiltration did not have an impact on overall survival. Transcriptomic analysis revealed three signatures in PDAC tumors comprising of epithelial-mesenchymal transition (EMT)/stromal, metabolic, and secretory/pancreatic signature. However, none of these signatures explained differences in Treg infiltration. We show that Tregs associate with improved overall survival in PDAC patients. This effect was independent of cytotoxic T cell infiltration and the transcriptomic profiles of their respective tumors. These findings provide a new layer of complexity in the study of PDAC tumor microenvironment that must be considered when developing immunotherapeutic interventions for this disease.

Prostate Infiltration by Treg and Th17 Cells as an Immune Response to Propionibacterium acnes Infection in the Course of Benign Prostatic Hyperplasia and Prostate Cancer.

Benign prostatic hyperplasia (BPH) and prostate cancer (PCa) belong to the most frequent diseases in ageing men. It has been proposed that prostate chronic inflammation is a risk factor for the development of both BPH and PCa. However, potential stimuli that cause or maintain inflammation in the prostate gland are still poorly characterized. Bacterial infections seems to be one of the potential sources of prostatitis. Recent studies show that Propionibacterium acnes (P. acnes) is the most prevalent microorganism in the prostate gland and may be a predisposing factor for inflammation of prostatic tissue. It indicates that P. acnes may contribute to cancer development by enhancing proinflammatory responses, as well as by modifying the prostate extracellular environment. In this review, we discuss the potential role of P. acnes in the development of BPH and PCa and highlight the importance of regulatory T CD4(+)FoxP3(+) (Treg) and Th17 cells in response to P. acnes infection in the context of both prostate diseases.

A pan-tumor-siRNA aptamer chimera to block nonsense-mediated mRNA decay inflames and suppresses tumor progression

Immune-checkpoint blockade (ICB) therapy has changed the clinical outcome of many types of aggressive tumors, but there still remain many cancer patients that do not respond to these treatments. There is an unmet need to develop a feasible clinical therapeutic platform to increase the rate of response to ICB. Here we use a previously described clinically tested aptamer (AS1411) conjugated with SMG1 RNAi (AS1411-SMG1 aptamer-linked siRNA chimeras [AsiCs]) to inhibit the nonsense-mediated RNA decay pathway inducing tumor inflammation and improving response to ICB. The aptamer AS1411 shows binding to numerous mouse and human tumor cell lines tested. AS1411 induces tumor cytotoxicity in long incubation times, which allows for the use of the aptamer as a carrier to target the RNAi inhibition to the tumor. The AS1411-SMG1 AsiCs induce a strong antitumor response in local and systemic treatment in different types of tumors. Finally, AS1411-SMG1 AsiCs are well tolerated with no detected side effects.

The Mechanisms of Effector Th Cell Responses Contribute to Treg Cell Function: New Insights into Pathogenesis and Therapy of Asthma.

CD4 + helper T (Th) cell subsets are critically involved in the pathogenesis of asthma. Naive Th cells differentiate into different subsets under the stimulation of different sets of cytokines, and the differentiation process is dominantly driven by lineage specific transcription factors, such as T-bet (Th1), GATA3 (Th2), RORγt (Th17) and Foxp3 (Treg). The differentiation mechanisms driven by these transcription factors are mutually exclusive, resulting in functional inhibition of these Th subsets to each other, particularly prominent between effector Th cells and Treg cells, such as Th2 versus Treg cells and Th17 versus Treg cells. Being of significance in maintaining immune homeostasis, the balance between effector Th cell response and Treg cell immunosuppression provides an immunological theoretical basis for us to understand the immunopathological mechanism and develop the therapy strategies of asthma. However, recent studies have found that certain factors involved in effector Th cells response, such as cytokines and master transcription factors (IL-12 and T-bet of Th1, IL-4 and GATA3 of Th2, IL-6 and RORγt of Th17), not only contribute to immune response of effector Th cells, but also promote the development and function of Treg cells, therefore bridging the interplay between effector Th cell immune responses and Treg cell immunosuppression. Although we have an abundant knowledge concerning the role of these cytokines and transcription factors in effector Th cell responses, our understanding on their role in Treg cell development and function is scattered thus need to be summarized. This review summarized the role of these cytokines and transcription factors involved in effector Th cell responses in the development and function of Treg cells, in the hope of providing new insights of understanding the immunopathological mechanism and seeking potential therapy strategies of asthma.

Gallic acid induces T-helper-1-like Treg cells and strengthens immune checkpoint blockade efficacy

BackgroundFoxp3+ regulatory T (Treg) cells facilitate tumor immune evasion by forming a suppressive tumor microenvironment. Therefore, immune therapies promoting Treg fragility may greatly enhance immune checkpoint blockade (ICB) efficacy in...

P-392 Peripheral blood naïve Treg (nTreg) cell subsets and implantation success in patients undergoing in vitro fertilization (IVF) treatment

Abstract Study question Which populations of circulating Treg cells, based on immunophenotyping with thirteen color flow cytometry and analysis with clustering algorithms play a role in implantation? Summary answer The phenotype of subpopulation within Treg cells that was most significantly increased in women who became pregnant was nTreg-like phenotype, which lacked CXCR5 expression. What is known already There are indications that Treg lymphocytes mediated immuno-regulatory mechanisms contribute to the occurrence of idiopathic infertility. Findings of studies indicate that higher nTreg cell subsets were associated with successful IVF treatment. These findings suggest that changes in the composition of the nTreg subset may allow increased tolerance induction during implantation. More detailed characterization of the Tregs in blood samples from women undergoing IVF/ICSI treatment has not been performed and the degree of heterogeneity of among these cells has not been studied to date. Using 13-color flow cytometry Tregs cells can be divided into multiple complex subsets based on different markers. Study design, size, duration This prospective observational study included 36 patients undergoing IVF/ICSI treatment. Blood samples for 13-color flow cytometry, UMAP and FlowSOM cell clustering analyses were obtained from 19 patients undergoing controlled ovarian hyperstimulation and 17 patients undergoing vitrified-warmed blastocysts transfer (FET) in the natural cycle on the day of optimal blastocysts transfer, for data analysis GraphPad Prism was used. The study was performed between March 2020 and October 2021. Participants/materials, setting, methods In this study peripheral blood Treg cells composition was determined. We analyzed the phenotype of populations over or under-represented in samples from women who became pregnant compared to the women who did not within Treg cells. Treg cells were analyzed by both standard gating flow cytometry analysis and clustered by algorithms according to their level of expression of cell surface markers: CD25 (IL-2Rα), CD127 (IL-7Rα), CCR7, CXCR5, CD28, CD38, CD161, CD31, CD45RA, HLA-DR and CD15s. Main results and the role of chance Thirteen color flow cytometry and dimensionality reduction analyses were carried out, using the UMAP and FlowSOM cell clustering algorithms. Combining these tools, we were able to analyze the phenotype of populations in samples from women who became pregnant ( n = 23) compared to the women who did not (n = 13) within Treg cells. FlowSOM identified 15 phenotypically distinct populations within Treg cells. The most prevalent population that was increased in women who became pregnant was nTreg-like phenotype, which lacked CXCR5 expression. This related to our results by standard gating, where we observed that the percentage of CXCR5-nTreg cells and CD31+RTEnTreg cells within CD25+CD127low Treg cells was significantly higher in pregnant women (p = 0.003; p = 0.0162, respectively). Pregnant females had significantly higher percentage of CD45RA+nTreg cells within CD25hiCD127low Treg cells (p = 0.0162) which was in good correlation with the percentage of CD45RA+FOXP3low nTreg cells within CD25+FOXP3+ Treg cells (p < 0.0001). Furthermore, the percentage of CD45RA+FOXP3+low nTreg cells within CD25+FOXP3+ Treg cells was significantly higher in pregnant women (p = 0.045). Meanwhile, the percentage of CD45RA-FOXP3hi effector (eTreg) Treg cells within CD25+FOXP3 Treg cells was lower in pregnant women (p = 0.0238). Finally, women who achieve a pregnancy had significantly lower percentage of CD45RA-FOXP3hiCD15s+eTreg cells within CD25+FOXP3+ Treg cells (p = 0.0233). Limitations, reasons for caution This study has some limitations. The sample size is too small. Standard gating in flow cytometry has the limitation of analysing only few markers at a time in a flow plot, the levels of expression of the markers are not considered, as an alternative, dimensionality reduction analyses were carried out. Wider implications of the findings Our findings imply that the composition of distinct populations within nTreg cells can influence the women’s chance of becoming pregnant, especially nTreg cells, lacking CXCR5 expression, which distingushes T-follicular regulatory cells. Findings help us to understand the role of nTreg subpopulations in process of implantation in women undergoing IVF/ICSI treatment. Trial registration number not applicable

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.

CXCL5 inhibits tumor immune of lung cancer via modulating PD1/PD-L1 signaling

Objective: To investigate the role of CXCL5 in tumor immune of lung cancer and to explore the potential molecular mechanisms. Methods: A total of 62 cases of patients with lung cancer admitted in the First Affiliated Hospital of Henan University from May 2018 to December 2019 were recruited as study object. Another 20 cases of patients with pulmonary infectious diseases and 20 cases of healthy control were selected as control. Enzyme-linked immunosorbent assay (ELISA) was used to determine serum levels of CXCL5 in patients with lung cancer, pulmonary infectious diseases and healthy control. Immunohistochemical staining (IHC) was used to detect the expressions of CXCL5 and PD-1/PD-L1 in tumor and paracarcinoma tissues of patients with lung cancer. Pearson correlation analysis was used to evaluate the correlation between CXCL5 and PD-1 in tumor and paracarcinoma tissues of patients with lung cancer. Lewis cells either expressing CXCL5 or vector plasmids were used to establish C57BL/6J mice model of lung cancer, and all mice were then divided into vehicle and PD-1 antibody treatment groups, 10 mice for each group. The mice survival and tumor growth curves were recorded. IHC was used to evaluate the expressions of CXCL5, PD-1 as well as the proportions of CD8(+) T and Treg cells in xenograft tumor tissues. Results: In patients with lung cancer, the serum level of CXCL5 [(351.7±51.5) ng/L] was significant higher than that in patients with pulmonary infectious diseases and healthy control [(124.7±23.4) ng/L, P<0.001]. The expression levels of CXCL5 (0.136±0.034), CXCR2 (0.255±0.050), PD-1 (0.054±0.012) and PD-L1 (0.350±0.084) in tumor were significant higher than those in paracarcinoma normal tissues [(0.074±0.022), (0.112±0.023), (0.041±0.007) and (0.270±0.043) respectively, P<0.001]. CXCL5 was significant positively correlated with PD-1 in tumor tissues of lung cancer (r=0.643, P<0.001), but not correlated with PD-1 in paracarcinoma tissues(r=0.088, P=0.496). The vector control group, CXCL5 overexpression group, vector control + anti-PD-1 antibody treatment group and CXCL5 overexpression + anti-PD-1 antibody treatment group all successfully formed tumors in mice, while CXCL5 overexpression increased the tumor growth significantly (P<0.01), which was abrogated by the treatment of anti-PD-1 antibody. CXCL5 overexpression decreased the mice survival time significantly (P<0.01), this effect was also abrogated by the treatment of anti-PD-1 antibody. The proportion of CD8(+) T cells in CXCL5 overexpression group [(10.40±2.00)%] was significant lower than that in vector control group [(21.20±3.30)%, P=0.002]. The proportion of CD4(+) Foxp3(+) Treg cells in CXCL5 overexpression group [(38.40±3.70)%] was significant higher than that in vector control group [(23.30±2.25)%, P<0.001]. After the treatment of anti-PD-1 antibody, no significant difference were observed for the proportion of CD8(+) T cells [(34.10±5.00)% and (33.40±4.00)% respectively] and Treg cells [(14.70±3.50)% and (14.50±3.30)% respectively] in xenograft tumor tissues between CXCL5 overexpression+ anti-PD-1 antibody treatment group and vector control + anti-PD-1 antibody treatment group (P>0.05). Conclusion: The expressions of CXCL5 and PD-1/PD-L1 are all increased significantly in the tumor tissues of patients with lung cancer, CXCL5 may inhibit tumor immune of lung cancer via modulating PD-1/PD-L1 signaling.

Identification of EPZ004777 and FG2216 as inhibitors of TGF-β1 induced Treg cells by screening a library of epigenetic compounds

AimsRegulatory T cells play an essential role in immune tolerance and homeostasis. Their long-term stability depends on epigenetic modifications and identification of small molecule modulators of Treg differentiation therefore has many applications. In this study, we performed a novel functional screen of an epigenetic compound library to identify compounds that can modulate generation of TGF-β1 induced T regulatory cells. Main methodsA screening strategy based on IFN-γ ELISA was designed to screen epigenetic compound library. Effect of hit compounds on Treg phenotype and function was assessed by RT-PCR, flow cytometry and suppression assays. TGF-β signalling proteins were assayed by western blotting. Chromatin Immunoprecipitation (ChIP) assay was used to assess epigenetic modifications at Foxp3 gene locus. Key findingsWe screened 160 compounds to identify hits capable of reversing TGF-β induced inhibition of IFN-γ production in activated spleen cells and CD4+ T cells. Two compounds EPZ004777 and FG-2216 consistently reversed TGF-β1 iTregs in terms of (a) differentiation of naïve T cells into CD4+CD25+Foxp3+Treg cells, (b) Foxp3 target gene expression and (c) Treg suppressive function without affecting TGF-β downstream signalling. ChIP assay revealed that the compounds were able to reverse – TGF- β mediated decrease in epigenetic marks H3K27me3 and 5-mC and an increase in epigenetic marks H3K4me3 and H3K27Ac in the promoter and conserved non coding sequence (CNS1) regions of the Foxp3 gene. SignificanceEPZ004777 and FG-2216 have been identified as potent epigenetic modulators that can reverse TGF-β1 induced T regulatory cells and may be used to treat diverse immune disorders.

The PARP inhibitor ABT-888 impairs the suppressive capacity of regulatory T cells through modulation of FoxP3 and CTLA-4.

Abstract Immune checkpoint blockade (ICB) has achieved long-term survival in melanoma but results remain limited in other solid malignancies including ovarian cancer. Combination treatments are a promising strategy to optimize responses to ICB. We have previously shown that the PARP inhibitor (PARPi) ABT-888 synergizes with CTLA-4 ICB to achieve long-term survival in a preclinical model of ovarian cancer. While the role of PARPi on DNA repair is well established, the impact on T cells is unclear. Published data using PARP1-KO mice suggests that PARP impairs regulatory T cell (Treg) function through modulation of FoxP3. Tregs suppress conventional T cell responses using multiple mechanisms including consumption of co-stimulatory molecules via the inhibitory receptor CTLA-4. Here we sought to define the impact of ABT-888 on the suppressive function of Tregs using FoxP3-eGFP transgenic mice. Using flow cytometry, we examined the levels of FoxP3 and CTLA-4 in Tregs stimulated in the presence of increasing doses of ABT-888 (0 – 50μM). Independent of stimulation, the GMFI of FoxP3 and CTLA-4 were significantly reduced by ABT-888 in a dose-dependent manner (P &amp;lt; 0.05). To test suppressive function, we pre-treated Tregs with increasing doses of ABT-888 prior to use in a standard proliferation assay. Similarly, ABT-888 pre-treatment significantly reduced the suppressive capacity of Tregs in a dose-dependent manner (P &amp;lt; 0.05). Taken together, these data identify a potentially novel area of synergy between PARPi and ICB. Furthermore, these data highlight the immunomodulatory effects of PARPi which are independent of their role in DNA repair. Future studies will focus on the impact of ABT-888 on tumor-associated Tregs and optimization of ICB therapy. This research was supported by NCI R37CA229221 (PI: Adams) and UNM Comprehensive Cancer Center Support Grant NCI P30CA11810. D.F. was supported by the Academic Science Education and Research Training program at the University of New Mexico Health Sciences Center (NIGMS Institutional Research and Academic Career Development Award; K12-GM088021).

CD6 – a costimulatory receptor at the nexus of Teff / Treg development

Abstract The balance of proinflammatory effector T cells (Teff) and anti-inflammatory regulatory T cells (Treg) is associated with autoimmune and inflammatory diseases. CD6 is a costimulatory receptor expressed on T cells with its greatest expression on pathogenic Teff cells, while Tregs are CD6low. Notably T cells from CD6knockout mice fail to differentiate to Th17 cells but were seen to skew to a Treg phenotype more rapidly. Although the role of CD6 in Teff development is well characterized, here we sought to investigate the role of CD6 in the development of Treg cells. Itolizumab, a humanized anti-CD6 monoclonal antibody that modulates cell surface levels of CD6, was used to generate CD6low naïve T cells, after which itolizumab was removed and Tregs were differentiated using a standard protocol. Tregs derived from CD6low cells had greater co-expression of FOXP3 and HELIOS (~2-fold) vs. Tregs derived from isotype-treated CD6high. These Tregs possessed greater suppressive function, with increased inhibition of proliferation and cytokine release by stimulated T responder cells by at least 50% more than Tregs derived from CD6high cells; this was verified in three different donors. Similar observations were made when measuring levels of cytokines. CD6low Tregs were able to suppress T responder’s production of proinflammatory cytokines by 60–90% compared to CD6high Tregs. This is the first study to directly characterize the role of CD6 in the development and activity of Treg cells. These data suggest that reduced levels of cell surface CD6 facilitate the development of Treg cells with greater stability and suppressive activity and that modulating the levels of CD6 may decrease the Teff/Treg ratio in patients with autoimmune and inflammatory diseases.

ITK tunes the Th17/Treg switch response by controlling calcium dependent signaling.

Abstract Naïve CD4+ T cells can differentiate into pro inflammatory T helper type 17 (Th17) and anti inflammatory T regulatory (Treg) lineages following T cell receptor (TCR) activation. The strength of the TCR signal is tuned by Interleukin-2 inducible T cell Kinase (ITK) and ITK can tune the development of Th17 and Treg cells. ITK signals can also tune a Th17/Treg switch under Th17 conditions. How ITK mediated tuning of TCR signal control naïve CD4+ T cell commitment into Th17/Treg lineages is unclear. Here we used ITK inhibitors, conventional ITK and allele specific ITK (ITKas) IL17 GFP/Foxp3 RFP reporter mice, to track naïve CD4+ T cell commitment into Th17/Treg lineages. Under Th17 conditions, reduced TCR signal strength in absence of ITK activity/expression, tuned naïve CD4+ T cell fate by reducing Th17 differentiation and inducing switch to cells resembling Tregs expressing Foxp3. The ITKas model showed this switch is due to specific ITK inhibition. The switched Treg like cells resemble Tregs by expression of Treg markers (CD25, CTLA4, PD1) and suppression of effector T cell proliferation. Transcriptomic analysis of switched Treg like cells by RNA Seq indicate they resemble Tregs, with reduced Th17 (Rorc, Il17) and increased Treg (Foxp3, Nrp1, Ikzf2) gene expression. Analysis of chromatin accessibility by ATAC Seq indicated closed loci for Th17 (Rorc, Il17) genes. Activating intracellular calcium dependent pathways led to a loss of the Th17/Treg switch response in naïve CD4+ T cell commitment, effectively bypassing ITK mediated tuning of the TCR signal critical for the switch response. In conclusion, we show here ITK tunes a Th17/Treg switch response in naïve CD4+ T cell commitment under Th17 conditions by controlling calcium dependent signaling. Supported by grants from NIH (NIH AI129422).

Treg donation of sIL-6R to promote intestinal integrity and repair

Abstract IBD affects roughly 1.6 million US adults, with its prevalence increasing since the late 1990s. Characterized by unchecked intestinal inflammation, multiple proinflammatory cytokines including IL6 have been implicated as critical factors in this pathology. It has been established that EZH2 is a gene involved in the production of specific histone methyltransferase enzymes, as well as in the differentiation of Treg cells. The association between the Treg master transcription factor, Foxp3 and EZH2 results in the suppressive function of Tregs, with our previous work revealing impairment of this interaction under the influence of IL6 signaling in an inflamed intestinal setting. We investigated this further in a murine preventative T cell transfer model of colitis that involved injecting donor Treg cells that lacked IL6Ra, into immunocompromised mice. Histological scoring of the mice receiving the IL6Rko Tregs revealed a resolved inflammatory milieu and flow cytometric analysis of cells isolated from the spleens and MLNs showed increased Foxp3+ expression when compared to mice that received WT Tregs. Foxp3IL6Rako mice were subjected to a DSS model of colitis to further characterize the absence of the IL6R on Treg function in the intestinal milieu. Exacerbated signs of colitis were observed through histology and colon shortening of the conditional knockout mice when compared to their WT counterparts. We postulate that Tregs could be acting as a donor of sILR to promote intestinal integrity and healing. Further experiments will be conducted to define a role for the IL6R in the development of Foxp3+ cells, along with outlining the instrumental process of sIL6R donation in the intestinal milieu. Supported by Mayo Clinic Graduate School for Biomedical Sciences

A Review of the Effects of Cervical Cancer Standard Treatment on Immune Parameters in Peripheral Blood, Tumor Draining Lymph Nodes, and Local Tumor Microenvironment.

Cervical cancer remains a public health concern despite all the efforts to implement vaccination and screening programs. Conventional treatment for locally advanced cervical cancer consists of surgery, radiotherapy (with concurrent brachytherapy), combined with chemotherapy, or hyperthermia. The response rate to combination approaches involving immunomodulatory agents and conventional treatment modalities have been explored but remain dismal in patients with locally advanced disease. Studies exploring the immunological effects exerted by combination treatment modalities at the different levels of the immune system (peripheral blood (PB), tumor-draining lymph nodes (TDLN), and the local tumor microenvironment (TME)) are scarce. In this systemic review, we aim to define immunomodulatory and immunosuppressive effects induced by conventional treatment in cervical cancer patients to identify the optimal time point for immunotherapy administration. Radiotherapy (RT) and chemoradiation (CRT) induce an immunosuppressive state characterized by a long-lasting reduction in peripheral CD3, CD4, CD8 T cells and NK cells. At the TDLN level, CRT induced a reduction in Nrp1+Treg stability and number, naïve CD4 and CD8 T cell numbers, and an accompanying increase in IFNγ-producing CD4 helper T cells, CD8 T cells, and NK cells. Potentiation of the T-cell anti-tumor response was particularly observed in patients receiving low irradiation dosage. At the level of the TME, CRT induced a rebound effect characterized by a reduction of the T-cell anti-tumor response followed by stable radioresistant OX40 and FoxP3 Treg cell numbers. However, the effects induced by CRT were very heterogeneous across studies. Neoadjuvant chemotherapy (NACT) containing both paclitaxel and cisplatin induced a reduction in stromal FoxP3 Treg numbers and an increase in stromal and intratumoral CD8 T cells. Both CRT and NACT induced an increase in PD-L1 expression. Although there was no association between pre-treatment PD-L1 expression and treatment outcome, the data hint at an association with pro-inflammatory immune signatures, overall and disease-specific survival (OS, DSS). When considering NACT, we propose that posterior immunotherapy might further reduce immunosuppression and chemoresistance. This review points at differential effects induced by conventional treatment modalities at different immune compartments, thus, the compartmentalization of the immune responses as well as individual patient’s treatment plans should be carefully considered when designing immunotherapy treatment regimens.

Direct targeting of FOXP3 in Tregs with AZD8701, a novel antisense oligonucleotide to relieve immunosuppression in cancer

BackgroundThe Regulatory T cell (Treg) lineage is defined by the transcription factor FOXP3, which controls immune-suppressive gene expression profiles. Tregs are often recruited in high frequencies to the tumor microenvironment...