Tregs In Peripheral Blood Research Articles

Blood transfusion practices affect CD4+ CD25+ FOXP3+ regulatory T cells/T helper-17 cells and the clinical outcome of geriatric patients with hip fracture.

Hip fracture (HF) is common among older individuals and associated with high mortality, poor vitality and functional impairment. HF patients suffer whole body immunological changes and that lead to severe consequences, including immobilization, physical impairment and a high risk of complications. The objective of this study was to decipher the pattern of dynamic immunological changes, especially in two major T cell subsets, CD4+ CD25+ FOXP3+ regulatory T (Treg) cells and T helper-17 (Th17) cells, and their balance, during the hospital stay and to observe whether blood transfusion could influence these cells and clinical patietns’ prognosis. In this study, ninety-eight consecutive HF patients were initially enrolled, and finally fifty-one patients qualified for the study, and correlation analysis of their clinical parameters was carried out to predict the meaning of their distribution in clinical practice. Our results showed that the frequency of Tregs gradually decreased, while the frequency of Th17 cells slowly increased in HF patients who received blood transfusion. The Treg frequency was inversely correlated with the level of hemoglobin (Hb), and Th17 cell frequency was positively related to fluctuations in Hb levels in HF patients after trauma. HF patients with a better prognosis and survival time showed decreased a Treg frequency and a decreased Treg/Th17 ratio. Transfusion helped reverse the imbalance in the frequencies of Tregs and Th17 cells and the Treg/Th17 ratio and especially contributed to a better outcome in HF patients with moderate-to-severe anemia. In conclusion, a higher frequency of peripheral blood Tregs and a higher Treg/Th17 ratio may be associated with unfavorable outcomes in HF patients, and blood transfusion may benefit moderate-to-severe HF patients rebalance their immune response.

Expression and Significance of BLIMP-1 in Regulatory T Cells of Children with Aplastic Anemia

To study the expression of B lymphocyte-induced mature protein-1 (BLIMP-1) in regulatory T cells (Tregs) of children with aplastic anemia (AA), and analyze its correlation with the number of Tregs and the levels of inhibitory cytokines interleukin (IL)-10 and transforming growth factor (TGF)-β in plasma. The peripheral blood samples of 10 newly diagnosed AA children and 10 healthy children were collected for experiment. qPCR was used to detect FOXP3 and PRDM1 mRNA expression levels. Flow cytometry was used to detect the proportion of Tregs, the expression of BLIMP-1 in Tregs, and the levels of cytokines such as IL-2, IL-17A, IL-6, interferon (IFN)-γ, IL-10 and TGF-β in plasma. Pearson correlation model was used to evaluate the relationship between the expression of BLIMP-1 in Treg and the number of Tregs, as well as the levels of IL-10 and TGF-β in plasma. Compared with control group, the proportion of Tregs in peripheral blood of AA children was decreased significantly (P<0.001); The plasma levels of proinflammatory cytokines IL-2, IL-6 and IFN-γ in AA children were increased significantly (P=0.033, P=0.031, P=0.006), and IL-17A also was increased but the difference was not statistically significant (P=0.052), while anti-inflammatory cytokines IL-10 and TGF-β were significantly reduced (P=0.048, P=0.002). The relative expressions level of FOXP3 and PRDM1 mRNA in AA children were significantly lower than those in control group (P=0.037, P=0.016). The expression of BLIMP-1 protein in Tregs of AA children was significantly lower than that in control group (P<0.001). The expression level of BLIMP-1 protein in Tregs was positively correlated with the percentage of Tregs in lymphocytes (r=0.671, P=0.001), and was also positively correlated with the levels of IL-10 and TGF-β in plasma (r=0.500, P=0.029; r=0.486, P=0.030). The expression of BLIMP-1 in Tregs of AA children is impaired, and the low expression of BLIMP-1 is related to the decrease of the number in Tregs and IL-10 and TGF-β expressions.

Accumulation of Regulatory T Cells in Triple Negative Breast Cancer Can Boost Immune Disruption.

Background and AimThe present study was conducted to evaluate the number of Tregs in triple negative breast cancer (TNBC), in normal breast parenchyma and in the peripheral blood of these patients and controls, in addition to their correlations with the clinico-pathologic features and the outcomes of TNBC.MethodsThirty adult treatment-naïve women with non-metastatic TNBC were recruited. In addition, 20 ages matched healthy females participated as a control group. Peripheral blood samples were collected from all participants in tubes containing heparin, fresh tumor tissues were also obtained from all patients undergoing surgery, and 20 normal breast tissue samples were obtained from the same patients’ areas adjacent to the safety margins; all these samples were taken for flow cytometric detection of Tregs.ResultsThe mean percentages of CD4+CD25+highT cells and Tregs were higher in TNBC peripheral blood than healthy controls and in malignant tissue than normal tissue. Moreover, the frequencies of tumor-infiltrating CD4+T cells and Tregs were exceeding those in the peripheral blood of cancer patients. Only tumor-infiltrating Tregs have shown increasing levels with the increase in the tumor size and were significantly higher in patients with local recurrences than those without recurrence. In addition, Tregs showed significant inverse relation with DFS and direct relation with the level of the peripheral Tregs.ConclusionThe findings of the current study support the possibility that TNBC microenvironment conveys specific characteristics on Tregs distinguishing them from those in normal breast tissue or Tregs in peripheral blood, improving the capabilities of tumor-infiltrating Tregs to enhance tumor growth, local recurrence and reduce the DFS.

Alisol B 23-acetate, a new promoter for cholesterol efflux from dendritic cells, alleviates dyslipidemia and inflammation in advanced atherosclerotic mice

Atherosclerosis (AS) is characterized by dyslipidemia and chronic inflammation. In the high-fat environment, the lipid metabolism of dendritic cells (DCs) is abnormal, which leads to abnormal immune function, promotes the occurrence of immune inflammatory reactions, and promotes the development of AS. Alisol B 23-acetate (23B) is a triterpenoid in the rhizomes of Alisma, which is a traditional Chinese medicine. Here, we identified cholesterol metabolism-related targets of 23B through a virtual screen, and further transcriptome analysis revealed that 23B can change antigen presentation and cholesterol metabolism pathways in cholesterol-loaded DCs. In vitro experiments confirmed that 23B promoted cholesterol efflux from ApoE-/- DCs, reduced the expression of MHC II, CD80, and CD86, and inhibited the activation of CD4+ T cells and the production of inflammatory cytokines IL-12 and IFN-γ. In advanced AS mice, 23B can decrease triacylglycerol (TG) levels and increase high-density lipoprotein-cholesterol (HDL-C) levels in plasma and the expression of cholesterol efflux genes in the aorta. Neither helper T cells 1 (Th1) nor regulatory T cells (Tregs) in peripheral blood changed significantly in the presence of 23B, but 23B reduced the levels of IL-12 and IFN-γ in serum. However, 23B did not change the total cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-C) levels in serum or lipid accumulation in the aorta. Moreover, 23B did not increase the production of IL-10 and TGF-β1 in vivo or in vitro. These results indicate that 23B promotes cholesterol efflux from DCs, which can improve the immune inflammatory response and contribute to controlling the inflammatory status of AS.

Lettuce Plants Expressing High Levels of Factor VIII Antigen in the Chloroplast for Oral Tolerance in Hemophilia a

Inhibitor formation is a serious complication in factor VIII (FVIII) replacement therapy for the X-linked bleeding disorder hemophilia A and occurs in 20-30% of patients. No prophylactic tolerance protocols exist. In previous proof-of-principle work, we demonstrated oral tolerance induction to FVIII in hemophilia A mice using frozen tobacco leaf cells expressing the heavy chain (HC) and C2 domain of human B domain-deleted (BDD) FVIII (Blood 124:1659-68, 2014). This approach is based on expression of heterologous proteins in the chloroplasts of green leaves and natural bioencapsulation provided by plant cell wall, so that the antigen is protected until its release in the small intestine. The antigens are expressed as fusions with the transmucosal carrier CTB proteins for effective targeting of the gut epithelium and translocation to the immune system. However, significant hurdles toward clinical feasibility remained that we now successfully addressed. Until now, methods to express large human proteins in chloroplasts of edible crop plants did not exist. Building on our recently established gene transfer technology for lettuce chloroplasts and identification of powerful posttranscriptional regulatory elements, we focused on the development of chloroplast genomics tools (e.g. bioinformatics tools for codon optimization and assays that identify sites of ribosome stalling), which ultimately allowed us to express high levels of FVIII antigen in edible crop plants that are suitable for clinical application. Remarkably, codon-optimization improved expression of FVIII heavy chain (HC) and light chain (LC) by15- to 42-fold. Hence, the entire BDD-FVIII sequence can be orally delivered, thereby covering the entire patient population regardless of individual CD4+ T cell epitope usage. A clinical grade manufacturing process is in place, and lyophilization of leaf cells generates material that is stable at ambient temperature for at least 2 years. Testing in hemophilia A mice showed effectiveness of low FVIII antigen doses upon delivery of lyophilized lettuce cells. Repeated oral gavage (twice/week for 2 months) of a mixture of CTB-HC and CTB-C2 suppressed subsequent inhibitor formation against intravenous FVIII (1IU, once/ week for 4 weeks) in hemophilia A mice as well as the original tobacco material, using very low doses of 0.15-0.5 ug/antigen/mouse. When using CTB-HC/CTB-LC combination, a dose of 1.5 ug was most optimal and suppressed inhibitor titers by ~1 log (avg. 4.7 BU/ml vs 44 BU/ml in controls; 50% of tolerized mice had 0-3 BU/ml, while 86% of controls developed 6-126 BU/ml, n=12-14, P <0.05). Translational oral tolerance studies in humans would benefit from cellular biomarkers. Our prior studies showed induction of two subsets of Treg that suppress antibody formation against FVIII, namely CD4+CD25+FoxP3+ Treg and LAP+CD4+CD25-FoxP3- Treg. The latter stain positive on the cell surface for LAP (latency-associated peptide) because of overexpression of TGF-b. Although we found increases in the frequencies of LAP+CD4+CD25-FoxP3- Treg in peripheral blood, a statistical significance was not always observed when compared to the blood of control mice that received intravenous FVIII but not oral tolerance. However, we found significant and consistent increases in the frequencies of circulating LAP-expressing CD4+CD25+FoxP3+ Treg in tolerized mice, suggesting that LAP may be a suitable cellular biomarker for plant-based oral tolerance induction. In conclusion, clinical candidates for oral tolerance to FVIII are now available in form of lettuce plants expressing immunogenic domains or the entire antigen. DisclosuresHerzog:Novo Nordisk: Research Funding; Applied Genetic Technologies Corporation: Consultancy; Spark Therapeutics: Patents & Royalties. Daniell:Novo Nordisk: Research Funding.

Exploring the Cord Blood T Cell Compartment with Mass Cytometry Reveals the Existence of Memory CD4, CD8 and Regulatory T Cell Pools in the Fetal Period

It has been long perceived that cord blood (CB) T cells are exclusively composed of antigen-inexperienced ‘naïve’ T cells under Th2 trophic and/or Th1 suppressive conditions, and that this is important for the maintenance of tolerance during pregnancy. However, a recent study reported the existence of a small fraction of memory CD4+ T cells with inflammatory cytokine profile within the sterile CB environment. This is in contrast to the common dogma challenging the presence of inflammatory T cells in the CB environment. To produce a more in-depth picture of the T cell compartment in CB, we assembled a panel of 40 antibodies to characterize the phenotypic and functional complexity of CB T cells. We interrogated the immune landscape of 12 CB donors vs 12 healthy adult peripheral blood (PB) donors to obtain single-cell profiles of the T-cell compartment. We used t-SNE algorithm to visualize the high dimensional data and the nearest-neighbor based clustering algorithm, PhenoGraph, to identify the potential clusters in high dimensional space. We observed significant segregation between PB and CB T cells. As expected, the majority of CD4+ and CD8+ T cells in CB were composed of naïve cells, while PB CD4+ and CD8+ T cells were characterized by a dominantly memory T cell phenotype. Within the CB CD4+ T cell compartment, in addition to the CD127+CD25- naïve T cell subset, we observed two more populations, including CD127+CD25int memory T cells (median 1.92%; range 0.69%-4.14%) and CD127lowCD25hi regulatory T-cells (Tregs) (median 1.9%; range 1.36%-2.98%). Similarly, a small subset of memory T cells, defined as CD45RA-CCR7+/-CD95+ (median 3.23%; range:1.53%-7.45%) was present in the CD8 T cell compartment in CB.Each T-cell presents a diversity of surface phenotypes defined by distinct combinations of marker expression. Thus, we sought an overview of the similarities and differences between CB and PB T-cell subsets. Analysis of CB Tregs revealed a repertoire composed mainly of naïve and central memory subsets, the latter expressed CD95, a marker of activation and memory, suggesting the generation of memory Tregs during fetal life. Both CB and PB Tregs were characterized by expression of PD-1, CD39, TIGIT, ICOS, CD161, CCR4, DNAM-1, CD49f and CD26. Expression of TIGIT, PD-1, CD39, CD49f, ICOS and CCR4 was exclusively confined to the memory Treg subset. In contrast to PB Tregs, CB Tregs lacked CCR10 and CXCR5 expression, suggesting differential migratory properties for CB and PB Tregs. We next focused on the differences between the naïve and memory CD4+ T cell subsets in CB. Chemokine receptors can be used to classify T-cell subsets with distinct migratory capacities and effector function. Naïve CD4 T cells do not express chemokine receptors, while memory T cells express different combinations of chemokine receptors that give clues to their lineage commitment. We discovered CCR6, CCR4, CXCR3, CD161, DNAM-1, CD49f, KLRG1, Beta7, PD-1, and TIGIT as the set of combinatorial markers to identify the memory CD4+ T cell pool in CB. Notably, a significant proportion of memory CD4+ T cells in CB expressed CCR4, supporting the notion that the CB environment drives a Th2 rather than a Th1 or Th17 functional phenotype. Moreover, in CB, expression of CCR6, CXCR3 and CD161 was confined to the memory CD4 T cell pool, suggesting that Th1, Th2 and Th17 lineage commitment starts in the fetal period, although the exact mechanism leading to memory T cell formation is not known.Taken together, interrogation of the T cell compartment in CB and PB in high dimensional space reveals well-defined and distinct memory signatures. Acquisition of memory phenotype instructs expression of unique sets of markers for each memory subset and varies among CD4, CD8 and Treg. Our data indicate the presence of a seemingly full-blown immune response, resulting in the generation of committed memory T cells and changes in the immune landscape of CB, although the initiating factor for this is yet to be determined. Mass cytometry proves to be a valuable tool in the characterization and discovery of novel T cell subsets, even if present at low frequencies. Studies to track the differentiation and diversification of T cells in CB transplant recipients and to link the impact of the T cell repertoire on clinical outcomes are under way. DisclosuresNo relevant conflicts of interest to declare.

Transfusion-induced platelet antibodies and regulatory T cells in multiply transfused patients.

BackgroundPlatelet transfusion refractoriness (PTR) remains a difficult problem in patients requiring long‐term platelet supportive care. However, there are little data on the frequency of platelet antibodies in multiply transfused Chinese patients. Moreover, the relationship between peripheral regulatory T cells (Tregs) and PTR remains unclear.MethodsWe retrospectively studied the frequency of alloimmunization against platelet antigens in patients receiving multiple transfusions between 2013 and 2017. Monoclonal antibody solid‐phase platelet antibody test (MASPAT) kits were used to screen for platelet antibodies before each platelet transfusion. Peripheral Tregs and CD4+CD25+CD127− T cells were detected by flow cytometry, while transforming growth factor‐beta (TGF‐β) and interleukin (IL)‐17 cytokines were detected by enzyme‐linked immunosorbent assay.ResultsA total of 399 patients who met the inclusion criteria were enrolled for the analysis of platelet antibodies and refractoriness. Among these patients, 10 (2.5%) were positive for platelet antibodies before transfusion and 47 (11.8%) became antibody‐positive during the study period. The number of alloimmunized patients was significantly higher in patients with hematological disease as compared with other disease groups (p < 0.05). Refractoriness and alloimmunization occurred in 77 (19.3%) and 22 (28.6%) patients, respectively. There were no significant differences in CD4+, CD8+, and CD4+CD25+CD127− T cell numbers and plasma levels of TGF‐β1 and IL‐17 between patients with PTR and the control group.ConclusionsRefractoriness was common in patients undergoing multiple platelet transfusions (19.3%), with alloimmunization observed in 28.6% of patients. However, Tregs in peripheral blood may not play a key role in PTR.

Randomized phase Ib study to evaluate safety, pharmacokinetics and therapeutic activity of simlukafusp α in combination with atezolizumab ± bevacizumab in patients with unresectable advanced/ metastatic renal cell carcinoma (RCC) (NCT03063762).

4556 Background: Simlukafusp α ([SIM], FAP-IL2v) is a novel IL-2v immunocytokine engineered to preferentially activate effector CD8 T and NK cells, but not regulatory T cells (Tregs), due to abolished binding to Interleukin-2 receptor α (IL-2Rα) and retained affinity to IL-2Rβγ. High affinity binding of SIM to fibroblast activation protein (FAP), expressed on cancer-associated fibroblasts, mediates its accumulation in malignant lesions. Methods: The Dose-Escalation (DE) consisted of: Arm A: SIM 5-25 mg weekly for 4 weeks, and every 2 weeks (Q2W) thereafter in combination with atezolizumab [ATZ] 840mg Q2W; and Arm B: same as Arm A + bevacizumab [BEV] 10 mg/kg Q2W. Patients (pts) not previously treated were evaluated in the Extension Part: Arm C (n=3): SIM + ATZ every 3 weeks (Q3W); or Arm D (n=25): SIM + ATZ + BEV (“triplet”) Q3W. Primary objectives were: finding the recommended dose of SIM and assessment of objective response rate (ORR) by RECIST v1.1. Results: We enrolled 69 pts with unresectable advanced/ metastatic clear-cell and/or sarcomatoid RCC. Median age of patients was 57 years (range: 35-78). The recommended dose for extension of SIM was 10 mg. Median treatment duration in days in each arm were: A: 106 (range: 1-877); B: 324 (8-940); C: 659 (71-768); D: 437 (1-682). Twenty-five pts are evaluable for therapeutic activity in Arm A [ORR: 24% (6 PR; 90% CI 12.95, 40.12)]; 15 in Arm B [46.7% (1 CR, 6PR; 90% CI 27.67, 66.68)]; 3 in Arm C [33.3% (1PR; 90% CI 7.83, 74.65)]; and 23 in Arm D [47.8% (2 CR, 9 PR; 90% CI 35.74, 68.15)]. Twelve patients are ongoing on study treatment. Treatment related grade 3 and 4 adverse events (AE) occurred respectively in 69.7% and 9.1% patients. The most common serious AEs were pyrexia (10.6 %) and infusion-related reactions (9.1%). 65.2% Of the patients reported at least one AE of elevations in liver transaminases/GGT/ alkaline phosphatase/bilirubin. Drug-related AEs led to dose modification/interruption in 37.9 % of the pts, and treatment discontinuation in 3% of the patients. SIM led to preferential expansion and activation of NK and CD8 T cells (but not Tregs) in peripheral blood and augmented tumor infiltration and tumor inflammation. Intriguingly responses were observed not only in pts with PD-L1 positive or inflamed tumors, but also in pts with PD-L1 negative tumors (n=13) or poorly infiltrated tumors classified as immune deserts (n=2). Conclusions: The combination of SIM with ATZ ± BEV was feasible with an acceptable safety profile. Clinical activity was more favorable for the triplet among the study Arms, but comparable to the ATZ + BEV combination in the IMmotion151 (Rini B, et al 2019). Observed pharmacodynamic findings were consistent with the expected effects. Clinical trial information: NCT03063762.

CD4+CD25+CD127low regulatory T cells associated with the effect of CD19 CAR-T therapy for relapsed/refractory B-cell acute lymphoblastic leukemia

BackgroundCD19-specific chimeric antigen receptor T-cell (CAR-T) therapy has shown promising clinical outcomes in relapsed/refractory acute B-cell lymphoblastic leukemia (R/R B-ALL) patients. However, some patients did not respond to this therapy or relapsed after remission. Regulatory T cells (Tregs) have shown great importance in promoting tumor escape, but little is known about their role in R/R B-ALL patients with CAR-T therapy. Our previous study has proved that higher Tregs before infusion was an independent high-risk factor for relapse-free survival (RFS). To further clarify the relationship between Tregs and the efficacy of CAR-T therapy, the present study tested the levels of CD4+CD25+CD127low Tregs in peripheral blood (PB) of R/R B-ALL patients at different stages of CD19 CAR-T therapy, and evaluate their impact on the efficacy and prognosis of CAR-T therapy. MethodsFrom November 2015 to May 2019, 47 R/R B-ALL patients successfully received CD19 CAR-T therapy at our institution and followed up for at least 1 month. Among them, one patient did not tested for Tregs, so 46 patients enrolled in this study. We collected clinical information of them and dynamically detected the frequency of CD4+CD25+CD127low Tregs within CD4 + T cells at different time points (before infusion and at 1 week after infusion) by flow cytometry, and validated the relationship of circulating Tregs with clinical efficacy, OS, and recurrence of CAR-T therapy. ResultsCirculating Tregs of R/R B-ALL patients in pre-infusion group (median 6.67%) and in 1 week after infusion group (median 6.80%) were all higher than that of the healthy control group (median 5.04%), with statistical significance (P < 0.05). The frequencies of Tregs in not remission (NR) group at baseline (pre-infusion) and at 1 week after infusion were all significantly higher than those in remission group.With cut-off values of 11.54% (before infusion) and 13.56% (1 week after infusion), the specificity for Tregs were 94.6% and 100% , respectively. In remission group, 11 patients underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) after achieving remission by Sino 19 cell therapy. No significant differences of Tregs expression were found between transplantation and non-transplantation groups. Time-dependent Cox model showed that transplantation group had lower risk of relapse and death when compared with non-transplantation group (HR = 0.664 for RFS and HR = 0.364 for OS), however, no statistical significances were found (P = 0.403 and 0.106, respectively). Higher Tregs before infusion and at 1 week after infusion were significant associated with shorter RFS and OS by Kaplan-Meier analysis. Multivariate analysis showed that higher Tregs at 1 week after infusion was the independently factor for poor RFS (P = 0.032) and shorter OS (P = 0.025) in R/R B-ALL patients with CD19 CAR-T therapy. Besides, Tregs levels before and at 1 week after infusion were negatively correlated with the persistence time of Sino 19 cell. ConclusionHigher circulating Tregs, especially 1 week after CD19 CAR-T cell infusion, was a poor predict indicator for CD19 CAR-T therapy in R/R B-ALL patients.

Increased Dysfunctional and Plastic Regulatory T Cells in Idiopathic Orbital Inflammation.

BackgroundIdiopathic orbital inflammation (IOI) is a disfiguring and vision-threatening fibroinflammatory disorder. The pathogenesis of IOI has not been elucidated. We sought to clarify the regulatory T cell (Treg) distribution and function in patients with IOI.MethodsThe frequency, phenotype and function of Tregs were identified by multicolor flow cytometry and in vitro cell functional assays. Plasma and tissue samples were obtained to investigate cytokines, chemokines and their receptors of interest by relative real-time polymerase chain reaction (PCR) and Luminex assays.ResultsCompared with healthy subjects, patients with IOI exhibited obvious increases of Tregs in peripheral blood and affected orbital tissues. Circulating Tregs from patients with IOI were significantly more polarized to a Th17-like phenotype with defective regulatory function, whereas orbit-derived Tregs were polarized to a Th2-like phenotype. Furthermore, ST2 expression levels in circulating Tregs and interleukin (IL)-33 mRNA levels in orbital tissues were decreased in IOI. IL-33 restored the suppressive function of Tregs, reduced interferon (IFN)-γ production by Tregs and decreased the activation of orbital fibroblasts (OFs) cocultured with Tregs in IOI.ConclusionIncreased Tregs with proinflammatory and profibrotic polarization were first identified in IOI, suggesting that Treg plasticity and heterogeneity plays an essential role in IOI pathogenesis. Additionally, our study identified a regulatory effect of IL-33 on inflammation and fibrosis in IOI. Reversing the plastic Tregs via IL-33 might be a potential option for IOI patients.

Peroxisome proliferator-activated receptor gamma activation reverses adipose tissue regulatory T cell dysfunction.

Abstract Adipose tissue (AT) regulatory T Cells (Tregs) play an essential role in regulating inflammatory and metabolic responses. Obesity is associated with a decrease in AT Tregs and a corresponding increase in tissue inflammation and systemic insulin resistance in both humans and mice. In human samples, we have found Tregs isolated from visceral AT (VAT) display a dysfunctional phenotype characterized by decreased viability, decreased effector function, and increased expression of inhibitory co-receptors (PD-1 and OX40). VAT Tregs cultured with peripheral blood mononuclear cells (PBMCs) display reduced ability to suppress proliferation, while peripheral blood Tregs suppress proliferation normally. As peroxisome proliferator-activated receptor (PPAR)-gamma has been shown to be a regulator of AT Tregs, we hypothesize that the Treg dysfunction can be reversed by addition of the PPAR-gamma agonist Pioglitazone. We isolated Tregs from VAT and peripheral blood and added cells to CFSE-labelled PBMCs in the presence and absence of Pioglitazone. After 4 days culture, Pioglitazone was found to significantly improve the ability of VAT Tregs to suppress proliferation. This effect was specific to AT Treg populations as Pioglitazone alone did not affect PBMC proliferation. These data suggest that Treg dysfunction contributes to AT inflammation and activation of PPAR-gamma promotes VAT-specific Treg activity providing therapeutic implications for PPAR-gamma agonists. (Partially supported by the OSU Cancer Center Support Grant P30CA016058).

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

Imbalance of Th17 and Tregs in thymoma may be a pathological mechanism of myasthenia gravis

An imbalance in Th17 cells and Tregs may be an important cause of the pathogenesis of thymoma with myasthenia gravis (MG). In this study, 30 patients with simple thymoma and 30 patients with thymoma with MG were analyzed. Flow cytometry analysis of Th17 and Tregs in peripheral blood revealed that the percentages of Th17 in thymoma were lower than those in thymoma with MG, while the percentages of Tregs were higher than those in simple thymoma. Serum cytokine ELISA assays showed that IL-6 levels in simple thymoma were lower than those in MG patients. Further, Th17 and Tregs levels were detected by immunohistochemical double staining of thymoma tissue; the number of positive Th17 cells in thymoma with MG was higher than that in simple thymoma, while positive Tregs showed the opposite results. RORγt protein and mRNA expression in thymoma with MG were both higher than those in simple thymoma. FOXP3 protein and mRNA expression in the thymoma with MG group were lower than those in simple thymoma. The results of coculture of thymoma cells and CD4 + T cells showed that thymoma cells could promote the differentiation of Th17 cells and inhibit the Tregs. Overall, Th17 cells and related transcription factors and cytokines in thymoma with MG patients were higher than those in thymoma patients, whereas, Tregs showed the opposite results, the mechanism may be that thymoma can secrete IL6 and IL21. These findings indicated that imbalances in Th17/Tregs may account for the pathogeny of thymoma with MG.

Common clonal origin of conventional T cells and induced regulatory T cells in breast cancer patients

Regulatory CD4+ T cells (Treg) prevent tumor clearance by conventional T cells (Tconv) comprising a major obstacle of cancer immune-surveillance. Hitherto, the mechanisms of Treg repertoire formation in human cancers remain largely unclear. Here, we analyze Treg clonal origin in breast cancer patients using T-Cell Receptor and single-cell transcriptome sequencing. While Treg in peripheral blood and breast tumors are clonally distinct, Tconv clones, including tumor-antigen reactive effectors (Teff), are detected in both compartments. Tumor-infiltrating CD4+ cells accumulate into distinct transcriptome clusters, including early activated Tconv, uncommitted Teff, Th1 Teff, suppressive Treg and pro-tumorigenic Treg. Trajectory analysis suggests early activated Tconv differentiation either into Th1 Teff or into suppressive and pro-tumorigenic Treg. Importantly, Tconv, activated Tconv and Treg share highly-expanded clones contributing up to 65% of intratumoral Treg. Here we show that Treg in human breast cancer may considerably stem from antigen-experienced Tconv converting into secondary induced Treg through intratumoral activation.

Do endometrial natural killer and regulatory T cells differ in infertile and clinical pregnancy patients? An analysis in patients undergoing frozen embryo transfer cycles.

Clinical significance of endometrial and peripheral blood natural killer (NK) and regulatory T cells (Tregs) during frozen embryo transfer (FET) cycles has not been well characterized. Retrospective cohort study. Endometrial tissue was collected from infertility patients prior to a frozen embryo transfer cycle as part of an endometrial receptivity analysis (ERA® ) biopsy or endometrial scratch test. Uterine NK (uNK) and Treg cell density was compared based on pregnancy status in the subsequent frozen embryo transfer cycle. Peripheral blood was also collected from a separate cohort of patients undergoing frozen embryo transfer. Treg cell density was compared by the presence or the absence of a clinical pregnancy in each phase of the cycle. In the 33 luteal phase biopsies there were more endometrial Tregs, similar uNK and a trend toward lower CD16+ uNK cells in women with a future ongoing clinical pregnancy compared to non-pregnant women. There were no differences in uNK and Treg density in natural scratch cycles vs programmed cycles or in non-receptive vs receptive endometrium (ERA® cycles). In the peripheral blood analysis, the pregnant group had higher peripheral blood Tregs on the day of serum β-hCG time point when compared to the non-pregnant group. Higher levels of endometrial Tregs and lower levels of CD16+ uNK cells are positive prognostic factors for infertile women prior to frozen embryo transfer. Our work on phenotypic and proportional analyses of endometrial immune cells may complement the ERA® in predicting improved pregnancy rates in patients with implantation failure.

A novel potential target of IL-35-regulated JAK/STAT signaling pathway in lupus nephritis.

BackgroundIn this study, we have investigated the potential regulatory mechanisms of IL‐35 to relieve lupus nephritis (LN) through regulating Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway in mesangial cells.ResultsAmong 105 significant differentially expressed proteins (DEPs) between juvenile systemic lupus erythematosus (JSLE) patients with LN and healthy controls, LAIR1, PDGFRβ, VTN, EPHB4, and EPHA4 were downregulated in JSLE‐LN. They consist of an interactive network with PTPN11 and FN1, which involved in IL‐35‐related JAK/STAT signaling pathway. Besides, urinary LAIR1 was significantly correlated with JSLE‐LN clinical parameters such as SLEDAI‐2K, %CD19+ B, and %CD3+ T cells. Through bioinformatics analysis of co‐immunoprecipitation with mass spectrometry results, including GO, KEGG, and STRING, five genes interacted with Lair1 were upregulated by IL‐35, but only Myh10 was downregulated. Therefore, we presumed an interactive network among these DEPs, JAK/STAT, and IL‐35. Moreover, the downregulated phosphorylated (p)‐STAT3, p‐p38 MAPK, and p‐ERK, and the upregulated p‐JAK2/p‐STAT1/4 in IL‐35 overexpressed mesangial cells, and RNA‐sequencing results validated the potential regulatory mechanisms of IL‐35 in alleviating JSLE‐LN disease. Moreover, the relieved histopathological features of nephritis including urine protein and leukocyte scores, a decreased %CD90+αSMA+ mesangial cells and pro‐inflammatory cytokines, the inactivated JAK/STAT signals and the significant upregulated Tregs in spleen, thymus and peripheral blood were validated in Tregs and IL‐35 overexpression plasmid‐treated lupus mice.ConclusionsOur study provided a reference proteomic map of urinary biomarkers for JSLE‐LN and elucidated evidence that IL‐35 may regulate the interactive network of LAIR1‐PTPN11‐JAK‐STAT‐FN1 to affect JAK/STAT and MAPK signaling pathways to alleviate inflammation in JSLE‐LN. This finding may provide a further prospective mechanism for JSLE‐LN clinical treatment.

The Relationship between Reducing of Peripheral Blood Treg in Primary Diffuse Large B-Cell Lymphoma Patients and Poor Prognosis

To explore the relationship between Treg cells level in peripheral blood and prognosis of patients with diffuse large B-cell lymphoma (DLBCL). The percentage and absolute value of Treg cells in peripheral blood of DLBCL patients were detected by flow lytometry, and their correlation to prognosis was analyzed by survival analysis. The absolute count of Treg cells was detected by using maximally selected Log-rank statistic, and it was used as cutoff point to distinguish difference survival. The new group of Treg based on cutoff point was combined with age, sex, pathological subtype, risk stratification, treatment plan, and other indicators to include in the single factor survival analysis of Kaplan-Meier. Finally, the COX proportional risk model was used to verify the effect of the above indicators on progression-free survival. The absolute count of Treg cells in DLBCL patients was significantly lower in the disease progressed group than those in the remission group. The cutoff point of absolute value of the Treg cell was 19 cells /μl. The absolute count of Treg cells was an independent prognostic factor of the risk stratification. At the beginning of diagnosis, the reduction of the absolute count of Treg cells in peripheral blood of DLBCL patients show a poor prognosis.

Dihydroartemisinin attenuated the symptoms of mice model of systemic lupus erythematosus by restoring the Treg/Th17 balance.

The Treg/Th17 imbalance is associated with the development of systemic lupus erythematosus (SLE). Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, is isolated from the traditional Chinese herb Artemisia annua Artemisia annua L. This study aims to evaluate the effects of DHA alone or in combination with prednisone in immunodeficiency of SLE. In vivo, the therapeutical effect of DHA alone or in combination with prednisone was assessed in the pristane-induced SLE mouse model. Then, the level of serum antibodies, creatinine (Cre), blood urea nitrogen (BUN), urine protein, kidney histopathology, interleukin (IL)-17, IL-6, transforming growth factor (TGF)-β, the expression of RORγt and Foxp3, the percentages of Treg and Th17 in peripheral blood and spleen were assayed. In vitro, the mouse spleen lymphocytes were separated and treated with DHA alone or DHA in combination with prednisone. Then the percentages of Treg and Th17, the concentration of IL-17, IL-6, TGF-β, and the expression of RORγt and Foxp3 were assayed. It was shown that DHA alone or in combination with prednisone treatment significantly alleviated the manifestations of pristane-induced SLE mice, suppressed inflammation and restored the Treg/Th17 balance. DHA alone or in combination with prednisone significantly inhibited Th17 cell differentiation while induced Treg cell differentiation in vitro. DHA alone or in combination with prednisone also reduced the transcription of RORγt and increased Foxp3 in lymphocytes, as well as IL-17 and TGF-β levels. Our data indicated that DHA can produce synergistic effect with prednisone to attenuate the symptoms of SLE by restoring Treg/Th17 balance.

5-Aza-2’-deoxycytidine may enhance the frequency of T regulatory cells from CD4+ naïve T cells isolated from the peripheral blood of patients with chronic HBV infection

ABSTRACT Objectives Methylation pattern of gene modification is essential for the differentiation of T regulatory cells (Tregs) and 5-Aza-2ʹ-deoxycytidine is a common inhibitor of methylation. This study aimed to investigate the potential effects of Treg polarizing conditions and 5-Aza-2ʹ-deoxycytidine treatment in the differentiation of naïve T cells during chronic hepatitis B virus (HBV) infection. Methods The frequency of Tregs in peripheral blood was determined by flow cytometry from patients with chronic hepatitis B (CHB) (n = 51), liver cirrhosis (LC) (n = 47), hepatocellular carcinoma (HCC) (n = 40) and healthy controls (HCs) (n = 17). Gene expression were detected by qRT-PCR and DNA methyltransferases (DNMT) Activity was also determined. Results The frequency of Tregs and Foxp3 expression in peripheral blood from 5-Aza-2ʹ-deoxycytidine-treated groups were higher than that with acetic acid treatment as a control. Foxp3 mRNA and the frequency of Tregs derived from naïve CD4+T cells from peripheral blood of patients with HCC or LC were more pronounced compared with HCs. 5-Aza-2ʹ-deoxycytidine may have induced a more pronounced upward trend of PD-1 expression in HBV patients. Conclusions 5-Aza-2ʹ-deoxycytidine mediated demethylation has potential effects on enhancing the differentiation of naïve T cells to Tregs in chronic HBV infection.

Increased regulatory T cells in peripheral blood of children with eosinophilic esophagitis.

Considering the allergic basis of Eosinophilic esophagitis (EoE), this study was conducted to evaluate peripheral blood Tregs in children with EoE. Eosinophilic esophagitis (EoE) is an allergic inflammatory disease of gastrointestinal tract. Regulatory T cells (Tregs) have a confirmed role in allergic disorders. Children with EoE, gastroesophageal reflux disease (GERD) and healthy controls (HC) (10 subjects in each group) were recruited after diagnosis by a pediatric gastroenterologist and allergist. After obtaining informed written consent, peripheral blood was obtained. Peripheral blood mononuclear cells were isolated by Ficoll gradient centrifugation. Flowcytometry was used to enumerate peripheral blood Tregs (CD4 +CD25 +FOXP3+ gated lymphocytes were considered as Tregs). CD4+ gated lymphocytes significantly increased in EoE and GERD groups compared to HC group (p= 0.018). Tregs also was significantly increased in EoE in comparison to HC group (p=0.016). There were no statistically significant differences in Tregs of EoE as compared to GERD subjects (p=0.085). Peripheral blood Tregs increase in patients with EoE as compared to healthy controls, which may be indicative of a feedback mechanism to regulate inflammatory responses.