Treg Cell Population Research Articles

Toll-like receptor-4 null mutation causes fetal loss and fetal growth restriction associated with impaired maternal immune tolerance in mice

Maternal immune adaptation to accommodate pregnancy depends on sufficient availability of regulatory T (Treg) cells to enable embryo implantation. Toll-like receptor 4 is implicated as a key upstream driver of a controlled inflammatory response, elicited by signals in male partner seminal fluid, to initiate expansion of the maternal Treg cell pool after mating. Here, we report that mice with null mutation in Tlr4 (Tlr4−/−) exhibit impaired reproductive outcomes after allogeneic mating, with reduced pregnancy rate, elevated mid-gestation fetal loss, and fetal growth restriction, compared to Tlr4+/+ wild-type controls. To investigate the effects of TLR4 deficiency on early events of maternal immune adaptation, TLR4-regulated cytokines and immune regulatory microRNAs were measured in the uterus at 8 h post-mating by qPCR, and Treg cells in uterus-draining lymph nodes were evaluated by flow cytometry on day 3.5 post-coitum. Ptgs2 encoding prostaglandin-endoperoxide synthase 2, cytokines Csf2, Il6, Lif, and Tnf, chemokines Ccl2, Cxcl1, Cxcl2, and Cxcl10, and microRNAs miR-155, miR-146a, and miR-223 were induced by mating in wild-type mice, but not, or to a lesser extent, in Tlr4−/− mice. CD4+ T cells were expanded after mating in Tlr4+/+ but not Tlr4−/− mice, with failure to expand peripheral CD25+FOXP3+ NRP1− or thymic CD25+FOXP3+ NRP1+ Treg cell populations, and fewer Treg cells expressed Ki67 proliferation marker and suppressive function marker CTLA4. We conclude that TLR4 is an essential mediator of the inflammation-like response in the pre-implantation uterus that induces generation of Treg cells to support robust pregnancy tolerance and ensure optimal fetal growth and survival.

Interferon-α-producing plasmacytoid dendritic cells drive the loss of adipose tissue regulatory T cells during obesity

The visceral adipose tissue (VAT) of lean mice hosts a unique population of regulatory Tcells (Tregs) that have a distinct transcriptome and Tcell receptor (TCR) repertoire and regulate local and systemic inflammation and metabolism. Perplexingly, this population disappears in obese mice, limiting the promise of Treg-based therapies for metabolic disorders. We exploited the power of a VAT-Treg TCR-transgenic mouse model to follow the dynamics of, and phenotypic changes in, the VAT-Treg population throughout the development of diet-induced obesity. Our results show that VAT-Tregs are lost under obesogenic conditions due to downregulation of their defining transcription factor, PPARγ, coupled with their strikingly enhanced responses to pro-inflammatory cytokines. In particular, the VAT from obese mice (and reportedly humans) was strongly enriched in plasmacytoid dendritic cells that actively express interferon-alpha. These cells were directly toxic to PPARγ+ VAT-Tregs. Blocking this pathway in obese mice by multiple approaches substantially restored the VAT-Treg population and enhanced insulin sensitivity.

Epigenetic Control of Foxp3, CAMTA1 and FUT7 Genes in Human Regulatory T Cells

Regulatory T cells (Treg) are essential for immune tolerance and play a key role in autoimmune diseases, allergy and cancer. Foxp3 (Forkhead Box Transcription factor), encoded on Xp11.23 chromosome, is critical for the development, function and lineage commitment of Tregs, and has been widely used as a Treg specific marker. However, human CD4+CD25+Foxp3+ T cells are heterogeneous and include resting and activated Tregs, as well as non-suppressive Treg-like cells. The ability to distinguish between these subtypes may be critical in the development of Treg-based cell therapies. CD15s (sialyl Lewis x) was identified as a highly specific marker of activated and most suppressive effector Treg (eTreg), and the use of both CD45RA and CD15s enables the isolation of several CD4+CD25+Foxp3+ T cell subtypes with diverse functional profiles.Foxp3 is mainly regulated by epigenetic processes such as histone acetylation and methylation of CpG islands. Three regions on Foxp3 locus - enhancer, promoter and CNS 2 (also known as Treg Specific Demethylated Region, TSDR) contribute to Foxp3 expression via demethylation of their CpG islands and can be used as molecular markers that distinguish Treg from conventional CD4+ T lymphocytes (Tcon) and different Treg maturation stages.In this study, we characterised the epigenetic profile of four populations of CD4+CD25+ T cells: CD45RA+ CD15s- Foxp3low (naïve nTregs), CD45RA-15s-Foxp3low (non-suppressive Treg-like cells), CD45RA-CD15s+Foxp3high (eTregs) and Tcons, and CD34+ cells, all isolated from the peripheral blood of healthy male and female donors. Methylation patterns of Foxp3 locus enhancer, promoter, pre-TSDR (located upstream of TSDR) and TSDR were investigated together with those of calmodulin binding transcription activator (CAMTA 1), and fucosyltransferase 7 (FUT 7) promoters. CAMTA 1 was previously identified as a potential second molecular marker that could differentiate Treg from Tcon as this gene is encoded on chromosome 1 and is not affected by X-chromosome inactivation. FUT 7 promoter was chosen as FUT 7 mediates synthesis of CD15s expressed in both eTregs and CD34+ cells and, therefore, it was expected to be demethylated in these cell populations to allow for protein expression.Genomic DNA, isolated from the 5 cell populations, was subjected to bisulphite conversion followed by PCR-amplification of each gene region with bisulphite DNA specific primers and cloning into pGEM-T vector. Up to 24 clones (20 sequences fully covering each amplicon) were analysed and methylation status of each CpG position within all 6 gene regions was determined. As these gene regions were amplified from the same DNA sample, the methylation differences reflect the average status of the cell population.We demonstrate the intricate methylation patterns of FUT7 and CAMTA1 promoters and Foxp3 enhancer, promoter, pre-TSDR and TSDR for 10 male and female donors. Overall, Treg cell populations from donors of both genders displayed high demethylation levels in Foxp3 promoter and TSDR while only certain CpGs (4, 9, 11, 20-30 in Foxp3 enhancer and 1, 5, 7-9 in pre-TSDR) were more demethylated in Treg subtypes compared to Tcon. CD34+, CD45RA-CD15s- Foxp3low and eTreg populations had high demethylation levels in CpGs 5-16 of FUT7 promoter (highest in CpGs 5, 8-10) compared to nTreg and Tcon, suggesting that this regulatory element can be used as an additional molecular marker to distinguish eTreg from nTreg and Tcon. CAMTA 1 promoter (especially CpGs 2 and 11) was more demethylated in CD34+ and Treg cell populations compared to Tcon, with CD45RA-CD15s- Foxp3low cells displaying the highest demethylation levels. In conclusion, the findings in this study suggest that Treg-specific epigenetic changes are evident not only in several regions of Foxp3 locus, but also in CAMTA1 and FUT7 promoters. Interestingly, certain CpG positions in these regions are consistently more demethylated in Treg subtypes, which suggests their importance in epigenetic control, most probably as part of the recognition sequence for methylation-sensitive transcription factors. DisclosuresNo relevant conflicts of interest to declare.

Immune Profile of Patients (pts) with Relapsed and/or Refractory Multiple Myeloma (RRMM) Treated with Pomalidomide (POM) + Low-Dose Dexamethasone (LoDEX) + Daratumumab (DARA) in the Second Line Immediately after Lenalidomide (LEN)

Immune Profile of Patients (pts) with Relapsed and/or Refractory Multiple Myeloma (RRMM) Treated with Pomalidomide (POM) + Low-Dose Dexamethasone (LoDEX) + Daratumumab (DARA) in the Second Line Immediately after Lenalidomide (LEN)

High-fat Diet Alters Male Seminal Plasma Composition to Impair Female Immune Adaptation for Pregnancy in Mice.

Paternal experiences and exposures before conception can influence fetal development and offspring phenotype. The composition of seminal plasma contributes to paternal programming effects through modulating the female reproductive tract immune response after mating. To investigate whether paternal obesity affects seminal plasma immune-regulatory activity, C57Bl/6 male mice were fed an obesogenic high-fat diet (HFD) or control diet (CD) for 14 weeks. Although HFD consumption caused only minor changes to parameters of sperm quality, the volume of seminal vesicle fluid secretions was increased by 65%, and the concentrations and total content of immune-regulatory TGF-β isoforms were decreased by 75% to 80% and 43% to 55%, respectively. Mating with BALB/c females revealed differences in the strength and properties of the postmating immune response elicited. Transcriptional analysis showed >300 inflammatory genes were similarly regulated in the uterine endometrium by mating independently of paternal diet, and 13 were dysregulated by HFD-fed compared with CD-fed males. Seminal vesicle fluid factors reduced in HFD-fed males, including TGF-β1, IL-10, and TNF, were among the predicted upstream regulators of differentially regulated genes. Additionally, the T-cell response induced by mating with CD-fed males was blunted after mating with HFD-fed males, with 27% fewer CD4+ T cells, 26% fewer FOXP3+CD4+ regulatory T cells (Treg) cells, and 19% fewer CTLA4+ Treg cells, particularly within the NRP1+ thymic Treg cell population. These findings demonstrate that an obesogenic HFD alters the composition of seminal vesicle fluid and impairs seminal plasma capacity to elicit a favorable pro-tolerogenic immune response in females at conception.

B-cell Deficiency Attenuates Transplant Glomerulopathy in a Rat Model of Chronic Active Antibody-mediated Rejection.

Transplant glomerulopathy (TG) is a pathological feature of chronic active antibody-mediated rejection (cAMR) and is associated with renal allograft failure. The specific role of B cells in the pathogenesis of TG is unclear. We used a minor mismatched rat kidney transplant model with B cell-deficient recipients, generated by clustered regularly interspaced short palindromic repeats/Cas9 technology, to investigate the impact of B-cell depletion on the pathogenesis of TG. We hypothesized that B-cell deficiency would prevent TG in the rat kidney transplant model of cAMR. Treatment groups included syngeneic, allogeneic, sensitized allogeneic, and B cell-deficient allogeneic transplant recipients. B cell-deficient recipients demonstrated reduced TG lesions, decreased microvascular inflammation, reduced allograft infiltrating macrophages, and reduced interferon gamma transcripts within the allograft. Allograft transcript levels of interferon gamma, monocyte chemoattractant protein-1, and interleukin-1β correlated with numbers of intragraft macrophages. B cell-deficient recipients lacked circulating donor-specific antibodies and had an increased splenic regulatory T-cell population. In this model of cAMR, B-cell depletion attenuated the development of TG with effects on T cell and innate immunity.

Taurine promotes the production of CD4+CD25+FOXP3+ Treg cells through regulating IL-35/STAT1 pathway in a mouse allergic rhinitis model

BackgroundAllergic rhinitis (AR) is one of the most widespread immune conditions worldwide. However, common treatments often present with significant side effects or are cost-prohibitive for much of the population. A plethora of treatments have been used for the treatment of AR including antihistamines, steroids, and immune modulators. Among the treatments which have shown potential for efficacy in treating AR with a minimum of side effects but remains understudied is the conditionally essential amino acid taurine. Taurine has been previously shown to reduce AR symptoms. Here, we examine the role of taurine in modulating T regulatory cells, modulating the cytokine response in AR, and restoring healthy nasal mucosa.MethodsBlood samples from 20 healthy donors and 20 AR patients were compared for CD4+CD25+FoxP3+ T regulatory (Treg) cell population percentage, cytokine release, and STAT1 signaling with and without taurine treatment or IL-35 neutralization. An OVA-induced AR mouse model was administered vehicle, taurine, or taurine plus an IL-35 neutralizing antibody and assayed for sneezing frequency, inflammatory cytokine response, nasal mucosa goblet cell density, and T regulatory cell percentage. CD4+ cells were further examined for cytokine release, STAT1 phosphorylation, and response to an anti-IL-35 antibody with and without a STAT1 inhibitor.ResultsComparison of blood from normal donors and AR patients showed a reduction in CD4+CD25+FoxP3+ Treg cells in AR patients and a strong correlation between Treg percentage and IL-35 release. A similar pattern of Treg suppression was found in untreated AR mice when compared to normal control mice wherein there was a reduction in Treg percentage and a corresponding decrease in IL-35 release. AR mice also demonstrated increased sneezing frequency, an infiltration of goblet cell in nasal mucosa, and a reduction in IL-35 release from CD4+ cells. Conversely, IL-4, IL-5, and IL-13 secretion from CD4+ cells were increased in AR model mice, as was STAT1 phosphorylation. When AR mice were treated with taurine, sneezing frequency and nasal mucosa goblet cell content were reduced while Treg abundance was increased to that of normal mice. Accordingly, IL-35 release was restored, while IL-4, IL-5, and IL-13 secretion from CD4+ cells were suppressed. Likewise, STAT1 phosphorylation was inhibited with taurine treatment. Taurine-treated mice also given an IL-35 neutralizing antibody exhibited AR pathology including frequent sneezing and high nasal goblet cell content while retaining a restoration of Tregs. Furthermore, murine AR model CD4+ cells exposed to recombinant IL-35 responded with a reduction in inflammatory cytokine release and a decrease in STAT1 phosphorylation, mimicking the effect of taurine treatment.ConclusionsTaurine induces release of IL-35 in AR; IL-35 promotes the production of CD4+CD25+FoxP3+ Treg cells via a STAT1-dependent pathway. The restoration of Treg populations by taurine normalizes the inflammatory response, reduces AR symptomology, and reduces histopathologic signs of AR.

Valproic Acid Suppresses Autoimmune Recurrence and Allograft Rejection in Islet Transplantation through Induction of the Differentiation of Regulatory T Cells and Can Be Used in Cell Therapy for Type 1 Diabetes.

Type 1 diabetes mellitus (T1D) results from the destruction of insulin-producing β cells in the islet of the pancreas by lymphocytes. Non-obese diabetic (NOD) mouse is an animal model frequently used for this disease. It has been considered that T1D is a T cell-mediated autoimmune disease. Both CD4+ and CD8+ T cells are highly responsible for the destruction of β cells within the pancreatic islets of Langerhans. Previous studies have revealed that regulatory T (Treg) cells play a critical role in the homeostasis of the immune system as well as immune tolerance to autoantigens, thereby preventing autoimmunity. Valproic acid (VPA), a branched short-chain fatty acid, is widely used as an antiepileptic drug and a mood stabilizer. Previous reports have demonstrated that VPA treatment decreases the incidence and severity of collagen-induced arthritis and experimental autoimmune neuritis by increasing the population of Treg cells in these mouse disease models. Given the effect of VPA in the induction of Treg cells’ population, we evaluated the therapeutic potential and the protective mechanism of VPA treatment in the suppression of graft autoimmune rejection and immune recurrence in syngeneic or allogenic islet transplantation mouse models. In our study, we found that the treatment of VPA increased the expression of forkhead box P3 (FOXP3), which is a critical transcription factor that controls Treg cells’ development and function. Our data revealed that 400 mg/kg VPA treatment in recipients effectively prolonged the survival of syngeneic and allogenic islet grafts. The percentage of Treg cells in splenocytes increased in VPA-treated recipients. We also proved that adoptive transfer of VPA-induced Tregs to the transplanted recipients effectively prolonged the survival of islet grafts. The results of this study provide evidence of the therapeutic potential and the underlying mechanism of VPA treatment in syngeneic islet transplantation for T1D. It also provides experimental evidence for cell therapy by adoptive transferring of in vitro VPA-induced Tregs for the suppression of autoimmune recurrence.

Augmenting ATG14 alleviates atherosclerosis and inhibits inflammation via promotion of autophagosome-lysosome fusion in macrophages

ABSTRACT Dysfunction of macroautophagy/autophagy in macrophages contributes to atherosclerosis. Impaired autophagy-lysosomal degradation system leads to lipid accumulation, facilitating atherosclerotic plaque. ATG14 is an essential regulator for the fusion of autophagosomes with lysosomes. Whether ATG14 plays a role in macrophage autophagy dysfunction in atherosclerosis is unknown. To investigate the effects of ATG14 on macrophage autophagy, human atherosclerotic plaque, apoe−/- mice and cultured mouse macrophages were evaluated. Overexpression of ATG14 by adenovirus was used to reveal its function in autophagy, inflammation and atherosclerotic plaque formation. Results showed that impaired autophagy function with reduction of ATG14 expression existed in macrophages of human and mouse atherosclerotic plaques. Ox-LDL impaired autophagosome-lysosome fusion with reduction of ATG14 expression in macrophages. Overexpression of ATG14 in macrophages enhanced fusion of autophagosomes with lysosomes and promoted lipid degradation, decreasing Ox-LDL-induced apoptosis and inflammatory response. Augmenting ATG14 expression reversed the autophagy dysfunction in macrophages of apoe−/- mice plaque, blunted SQSTM1/p62 accumulation, inhibited inflammation, and upregulated the population of Treg cells, resulting in alleviating atherosclerotic lesions. Abbreviations: ABCC1: ATP-binding cassette, sub-family C (CFTR/MRP), member 1; ABCA1: ATP-binding cassette, sub-family A (ABC1), member 1; Ad-Atg14: adenovirus vector carrying the mouse Atg14 gene; Ad-LacZ: adenovirus vector carrying the gene for bacterial β-galactosidase; apoe−/- : apolipoprotein E knockout; ATG14: autophagy-related 14; CD68: CD68 antigen; DAPI: 4ʹ,6-diamidino-2-phenylindole; Dil-ox-LDL: Dil-oxidized low density lipoprotein; ELISA: enzyme-linked immunosorbent assay; HFD: high-fat diet (an atherogenic diet); IL: interleukin; LAMP2: lysosomal-associated membrane protein 2; LDL-C: low density lipoprotrein cholesterol; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; ND: normal diet; Ox-LDL: oxidized low density lipoprotein; PBMC: peripheral blood mononuclear cells; SQSTM1/p62: sequestosome 1; SREBF1/SREBP1c: sterol regulatory element binding transcription factor 1; SREBF2/SREBP2: sterol regulatory element binding factor 2; STX17: syntaxin 17; TC: serum total cholesterol; TG: triglyceride; TNF: tumor necrosis factor; IFN: interferon; Treg cell: regulatory T cell

Microbial metabolite Urolithin A induces expansion of Regulatory T Cells in aryl hydrocarbon receptor (AhR)-dependent manner

Abstract Intestinal inflammation and loss of gut barrier integrity contribute to severity of inflammatory bowel diseases (IBD). Gut microbiota and their metabolites play an important role in maintenance of immunological balance within the gut. Previously, our group demonstrated that treatment with Urolithin A enhanced gut barrier function by inducing junctional proteins and blocked LPS-induced inflammation. Moreover, treatment with this microbial metabolite mitigated colitis in pre-clinical models. In the current study, we identified novel function for Urolithin A, where treatment with Urolithin A significantly expanded FoxP3+ T regulatory cells (Tregs) both ex vivo and in vivo models. Urolithin A failed to induce Treg expansion in AhR−/− mice suggesting Urolithin A mediates its activities through AhR. Moreover, treatment with Urolithin A mitigated murine model of dextran sulfate sodium (DSS)-colitis and enhanced Treg cell population compared to vehicle treatment. These results suggest that Urolithin A offers multi-pronged beneficial activities on gut epithelial layer (enhanced barrier function), macrophages (down regulation of inflammatory cytokines) and expansion of Treg cells to protect against colitis.

Epigenetic and transcriptional programs of CD4+ T cell anergy

Abstract T cell tolerance is critical to avoid autoimmunity and resolve inflammation. CD4+ T cells that recognize self-antigens in the periphery can exert active suppression as regulatory T cells (Treg) or enter an inactive state known as anergy. Recent evidence suggests that anergy occurs naturally within a subpopulation of polyclonal CD4+ T cells and can generate precursors to Foxp3-expressing Treg cells. We aimed to identify transcription factors underlying T cell anergy induction and its potential plasticity. Polyclonal anergic CD4+ T cells from untreated wildtype mice were sorted based on previously reported markers and compared to naïve, effector-memory, and Treg cell populations identified within the same samples. Paired epigenome and transcriptome analysis revealed a unique anergy signature driven by bZIP family (Batf, Nfil3), NFAT family (Nfatc1), and EGR family (Egr3, Egr4) transcription factors. Following tolerization by repeated peptide infusions, antigen-specific CD4+ T cells from Batf-deficient mice demonstrated exaggerated expansion and minimal Foxp3+ Treg cell differentiation. Our data suggest that a unique transcription factor network, including BATF, generates and maintains peripheral CD4+ T cell anergy.

CD45RA-CD25highCD127-CD4+ activated regulatory T cells are correlated with de novo donor-specific anti-HLA antibody formation after kidney transplantation in standard immunosuppression

Although de novo donor-specific anti-HLA antibodies (dnDSA) remain a barrier for human kidney transplantation (KTx), the role of regulatory T (Treg) cells in dnDSA formation remains unknown. To address this question, we evaluated Treg cell subsets in peripheral blood mononuclear cells in 15 healthy volunteers and 59 KTx recipients using flow cytometric analysis. The post-transplant CD25highCD127-CD4+ Treg cells in KTx recipients were down-regulated compared with those of healthy volunteers (P < .001). Among them, 11 KTx recipients showed dnDSA formation, which was associated with lower frequencies of CD25highCD127-CD4+ Treg cells (P = .040). Furthermore, of the total Treg cell population, CD45RA-CD25highCD127-CD4+ activated Treg (aTreg) cells were significantly dominant in patients with dnDSA (P = .038), but not CD45RA+CD25highCD127-CD4+ resting Treg cells (P = .961). In contrast, non-donor-specific anti-HLA antibody formation was not associated with CD45RA- aTreg cells (P = .772). Multivariate logistic regression analyses revealed that CD45RA- aTreg cells were independently associated with dnDSA formation (Odds ratio = 6.69, P = .040). These findings indicate that CD45RA- aTreg cells are strongly associated with dnDSA formation in KTx recipients and might be an important risk factor of antibody-mediated rejection before clinical diagnosis.

Paeoniflorin protects against dextran sulfate sodium (DSS)-induced colitis in mice through inhibition of inflammation and eosinophil infiltration

Ulcerative colitis (UC) is a type of inflammatory bowel disease (IBD) that causes inflammation and ulcers in the digestive tract. The treatment commonly includes anti-inflammatory agents like 5-aminosalicylic acid or corticosteroids or biologics for people with UC who are no longer responding to corticosteroids. The radices of Paeonia lactiflora Pall. or similar plants of the Paeonia genus have been used in Chinese medicine to treat certain diseases that resemble the symptoms of UC. Paeoniflorin, a terpenoid glycoside, is a major active component for the anti-inflammatory and antitumor activity. In this study, we evaluated the therapeutic effect of paeoniflorin (PF) against dextran sulfate sodium (DSS)-induced colitis in mice and found that PF exhibited protective activity against colitis. PF treatment suppressed NF-κB pathway activation, resulting down regulation of pro-inflammatory factor expression. In addition, we detected reduction in eosinophil-related chemokine gene expression and eosinophil infiltration. The treatment also reversed Treg cell population suppression. Although PF treatment did not block COX2 induction, the compound weakly inhibited COX2 activity in an enzymatic assay. Taken together, PF exerts its therapeutic activity against UC through inhibition of inflammation and eosinophil infiltration.

Interleukin-33-Dependent Accumulation of Regulatory T Cells Mediates Pulmonary Epithelial Regeneration During Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) triggered mostly by infection, is a syndrome that involves respiratory failure. ARDS induces strong local infiltration of regulatory T cells (Treg cells) in the lungs, and Treg cells were recently highlighted as being related to the repair of various tissue. However, at present, there is still a lack of adequate evidence showing the impact of Treg cells on pulmonary regeneration during ARDS. Here, we verified that Treg cells are strongly induced in ARDS mice and Treg depletion results in impaired lung repair. Moreover, Treg cells show high expression of ST2, a cellular receptor for the tissue alarmin IL-33, which is strongly upregulated in the lung during ARDS. In addition, we demonstrated that IL-33 signaling is crucial for Treg cell accumulation, and ST2-blocked mice show a decrease in the Treg cell population. Critically, transfer of exogenous IL-33 into Treg depleted mice restored Treg cells and facilitated lung regeneration by promoting alveolar type II cell (AEC2) recovery in ARDS, with elevated neutrophils infiltration and upregulated TGF-β1 release. These results emphasized the importance of IL-33 in accelerating the expansion of pulmonary Treg cells and promoting their activity to mediate pulmonary epithelial regeneration during ARDS in a TGF-β1-dependent manner.

Activated Regulatory T-Cells, Dysfunctional and Senescent T-Cells Hinder the Immunity in Pancreatic Cancer.

Simple SummaryPancreatic cancer has the worst survival of any human cancer. Checkpoint blockade has not yielded much benefit in pancreatic cancer. We explored immune cell phenotypes with this disease to identify new targets for checkpoint blockade therapy. We created a checkpoint-focused panel to analyse immune cells from eight pancreatic cancer patients. This showed us the majority of T-cells are senescent. Further T-cell investigation demonstrated the majority of cytotoxic T-cells have intermediate to low PD1 expression suggesting why PD1 may not work as a pancreatic cancer therapy strategy. Our data has also highlighted a regulatory T-cell population which is highly activated and can mediate immunosuppression. The checkpoints that are highly expressed on these cells are TIGIT, ICOS and CD39, suggesting inhibition of these may be a viable therapeutic strategy. Furthermore, we showed that Tregs were retained amongst the fibroblast stroma of the tumour. Our work suggests there are key checkpoints on Tregs that may help guide therapeutic strategies in pancreatic cancer.Pancreatic cancer has one of the worst prognoses of any human malignancy and leukocyte infiltration is a major prognostic marker of the disease. As current immunotherapies confer negligible survival benefits, there is a need to better characterise leukocytes in pancreatic cancer to identify better therapeutic strategies. In this study, we analysed 32 human pancreatic cancer patients from two independent cohorts. A multi-parameter mass-cytometry analysis was performed on 32,000 T-cells from eight patients. Single-cell RNA sequencing dataset analysis was performed on a cohort of 24 patients. Multiplex immunohistochemistry imaging and spatial analysis were performed to map immune infiltration into the tumour microenvironment. Regulatory T-cell populations demonstrated highly immunosuppressive states with high TIGIT, ICOS and CD39 expression. CD8+ T-cells were found to be either in senescence or an exhausted state. The exhausted CD8 T-cells had low PD-1 expression but high TIGIT and CD39 expression. These findings were corroborated in an independent pancreatic cancer single-cell RNA dataset. These data suggest that T-cells are major players in the suppressive microenvironment of pancreatic cancer. Our work identifies multiple novel therapeutic targets that should form the basis for rational design of a new generation of clinical trials in pancreatic ductal adenocarcinoma.

Immunosuppressive regulatory T cells are unable to suppress T helper 17 cells in post-traumatic osteoarthritis

Immunosuppressive regulatory T cells are unable to suppress T helper 17 cells in post-traumatic osteoarthritis

TGF-β1 overexpressing human MSCs generated using gene editing show robust therapeutic potential for treating collagen-induced arthritis.

Transforming growth factor β (TGF-β) plays a pivotal role in cartilage differentiation and other functions of mesenchymal stem cells (MSCs). In this study, we investigated the therapeutic potential of TGF-β1 overexpressing amniotic MSCs (AMMs) generated using gene editing in a mouse model of damaged cartilage. The TGF-β1 gene was inserted into a safe harbor genomic locus in AMMs using transcription activator-like effector nucleases. The chondrogenic properties of TGF-β1-overexpressing AMMs (AMM/T) were characterized using reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR, and histological analysis, and their therapeutic effects were evaluated in mouse model of collagen-induced arthritis (CIA). AMM/T expressed cartilage-specific genes and showed intense Safranin O and Alcian blue staining. Furthermore, injecting AMM/T attenuated CIA progression compared with AMM injection, and increased the regulatory T (Treg) cell population, while suppressing T helper (Th)17 cell activation in CIA mice. Proinflammatory factors, such as interleukin-1β (IL-1β), IL-6, monocyte chemoattractant protein-1, and tumor necrosis factor-α were significantly decreased in AMM/T injected CIA mice compared with their AMM injected counterparts. In conclusion, genome-edited AMMs overexpressing TGF-β1 may be a novel and alternative therapeutic option for protecting cartilage and treating inflammatory joint arthritis.

Genome Edited Sirt1-Overexpressing Human Mesenchymal Stem Cells Exhibit Therapeutic Effects in Treating Collagen-Induced Arthritis.

Even though mesenchymal stem cells (MSCs) are known for cartilage regeneration, their therapeutic efficacy needs to be enhanced. In the present study, we produced genome-edited silent information regulator 2 type 1 (Sirt1)-overexpressing MSCs, and evaluated their therapeutic potential in a damaged cartilage mouse liver fibrosis model. The Sirt1 gene was successfully inserted into a ‘safe harbor’ genomic locus in amniotic mesenchymal stem cells (AMMs), and the chondrogenic properties of the Sirt1 gene overexpressing AMMs (AMM/S) were characterized using quantitative PCR and histology. Therapeutic potentials were investigated in a collagen-induced arthritis (CIA) mouse model. Chondrocyte-differentiated AMM/S expressed cartilage-specific genes and were positive for Safranin O staining. Transplantation of AMM/S attenuated CIA progression and suppressed T helper (Th)-17 cell activation while increasing the Treg cell population in CIA mice. Pro-inflammatory factors, such as interleukin (IL)-1β, IL-6, monocyte chemoattractant protein (MCP)-1, and tumor necrosis factor (TNF)-α were significantly decreased in AMM/S-injected joint tissues. In conclusion, genome-edited AMM/S may represent a safe and alternative therapeutic option for the treatment and repair of damaged cartilage, or in inflammatory joint arthritis.

Single-Cell Sequencing Reveals the Transcriptome and TCR Characteristics of pTregs and in vitro Expanded iTregs.

Regulatory T cells (Tregs) play a critical role in the maintenance of immune tolerance and tumor evasion. However, the relative low proportion of these cells in peripheral blood and tissues has hindered many studies. We sought to establish a rapamycin-based in vitro Treg expansion procedure in patients diagnosed with colorectal cancer and perform single-cell sequencing to explore the characteristics of Treg cells. CD25+ cells enriched from peripheral blood mononuclear cells (PBMC) of colorectal tumor patients were cultured in X-VIVO15 medium, supplemented with 5% human AB serum, L-glutamine, rapamycin, interleukin-2 (IL-2), and Dynabeads human Treg expander for 21 days to expand Tregs. Treg cells with satisfactory phenotype and function were successfully expanded from CD4+CD25+ cells in patients with colorectal cancer. The median expansion fold was 75 (range, 20–105-fold), and >90.0% of the harvest cells were CD4+CD25+CD127dim/− cells. The ratio of CD4+CD25+Foxp3+ cells exceeded 60%. Functional assays showed that iTregs significantly inhibited CD8+T cell proliferation in vitro. Single-cell sequencing showed that the transcriptome of pTreg (CD4+CD25+CD127dim/− cells isolated from PBMC of colorectal cancer patients) and iTreg (CD4+CD25+CD127dim/− cells expanded in vitro according to the above regimen) cells were interlaced. pTregs exhibited enhanced suppressive function, whereas iTregs exhibited increased proliferative capacity. TCR repertoire analysis indicated minimal overlap between pTregs and iTregs. Pseudo-time trajectory analysis of Tregs revealed that pTregs were a continuum composed of three main branches: activated/effector, resting and proliferative Tregs. In contrast, in vitro expanded iTregs were a mixture of proliferating and activated/effector cells. The expression of trafficking receptors was also different in pTregs and iTregs. Various chemokine receptors were upregulated in pTregs. Activated effector pTregs overexpressed the chemokine receptor CCR10, which was not expressed in iTregs. The chemokine CCL28 was overexpressed in colorectal cancer and associated with poor prognosis. CCR10 interacted with CCL28 to mediate the recruitment of Treg into tumors and accelerated tumor progression. Depletion of CCR10+Treg cells from tumor microenvironment (TME) could be used as an effective treatment strategy for colorectal cancer patients. Our data distinguished the transcriptomic characteristics of different subsets of Treg cells and revealed the context-dependent functions of different populations of Treg cells, which was crucial to the development of alternative therapeutic strategies for Treg cells in autoimmune disease and cancer.

Single-cell chromatin accessibility landscape identifies tissue repair program in human regulatory T cells

SummaryMurine regulatory T (Treg) cells in tissues promote tissue homeostasis and regeneration. We sought to identify features that characterize human Treg cells with these functions in healthy tissues. Single-cell chromatin accessibility profiles of murine and human tissue Treg cells defined a conserved, microbiota-independent tissue-repair Treg signature with a prevailing footprint of the transcription factor BATF. This signature, combined with gene expression profiling and TCR fate mapping, identified a population of tissue-like Treg cells in human peripheral blood that expressed BATF, chemokine receptor CCR8 and HLA-DR. Human BATF+CCR8+ Treg cells from normal skin and adipose tissue shared features with nonlymphoid T follicular helper-like (Tfh-like) cells, and induction of a Tfh-like differentiation program in naive human Treg cells partially recapitulated tissue Treg regenerative characteristics, including wound healing potential. Human BATF+CCR8+ Treg cells from healthy tissue share features with tumor-resident Treg cells, highlighting the importance of understanding the context-specific functions of these cells.