Frequency Of Peripheral Tregs Research Articles

STAT activation in regulatory CD4+ T cells of patients with primary sclerosing cholangitis.

Regulatory CD4+ T cells (Tregs) are pivotal for inhibition of autoimmunity. Primary sclerosing cholangitis (PSC) is an autoimmune cholestatic liver disease of unknown etiology where contribution of Tregs is still unclear. Activation of the JAK-STAT pathway critically modifies functions of Tregs. In PSC, we studied activation of STAT proteins and Treg functions in response to cytokines. In 51 patients with PSC, 10 disease controls (chronic replicative hepatitis C), and 36 healthy controls we analyzed frequencies of Foxp3+CD25+CD127lowCD4+ Tregs, their expression of ectonucleotidase CD39, and cytokine-induced phosphorylation of STAT1, 3, 5, and 6 using phospho-flow cytometry. In parallel, we measured cytokines IFN-gamma, interleukin (IL)-6, IL-2, and IL-4 in serum via bead-based immunoassays. In patients with PSC, ex vivo frequencies of peripheral Tregs and their expression of CD39 were significantly reduced (p < .05 each). Furthermore, serum levels of IFN-gamma, IL-6, IL-2, and IL-4 were markedly higher in PSC (p < .05 each). Unlike activation of STAT1, STAT5, and STAT6, IL-6 induced increased phosphorylation of STAT3 in Tregs of PSC-patients (p = .0434). Finally, STAT3 activation in Tregs correlated with leukocyte counts. In PSC, we observed enhanced STAT3 responsiveness of CD4+ Tregs together with reduced CD39 expression probably reflecting inflammatory activity of the disease.

Abstract 1739: Detection of regulatory T cells, a comparison of flow cytometry and epigenetic qPCR immunophenotyping using healthy donor and oncology patient whole blood

Abstract Human regulatory T cells (Tregs) are part of the adaptive immune response and play a pivotal role in suppressing both the immune and anti-tumor responses. Infiltration of Tregs into the tumor microenvironment has been associated with poor clinical prognosis. In addition, increased peripheral Treg frequency is associated with disease progression. Monitoring Tregs in both the blood and in the tumor of patients is being used to evaluate clinical prognosis and response to treatment in immuno-oncology studies. The key challenge is the ability to detect Tregs with high accuracy and precision. Here, we have used epigenetic qPCR (Epiontis ID®) as a method to detect and quantify Tregs in peripheral whole blood and compared it with flow cytometry. Flow cytometry is the current gold standard for immunophenotyping. For method comparison, we analyzed 113 whole blood samples from healthy donors. We have demonstrated equivalence and shown a strong correlation between epigenetic qPCR and flow cytometry-based data, indicating that Epiontis ID® is a robust and sensitive DNA-based method for Treg quantification. This epigenetic qPCR method does not require fresh samples or intact cells, since DNA can be extracted from frozen blood. We then evaluated Treg levels in blood samples from solid tumor cancer patients by epigenetic qPCR to demonstrate the clinical application and relevance for immune monitoring. Here, Treg frequencies were reported as relative (%) and absolute counts (cells/µL) values. Epigenetic qPCR for immune monitoring mitigates the operational and technical challenges of monitoring Tregs in the periphery for immuno-oncology studies which traditionally been performed by flow cytometry. This Epiontis ID® method provides the sensitivity and accuracy needed for determining patient response in progressive disease. Citation Format: Udo Baron, Laura Lozza, Angelina Bisconte, Eva Raschke, Deborah Phippard, Sven Olek. Detection of regulatory T cells, a comparison of flow cytometry and epigenetic qPCR immunophenotyping using healthy donor and oncology patient whole blood [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1739.

H2-O deficiency promotes regulatory T cell differentiation and CD4 T cell hyperactivity.

Regulatory T cells (Treg) are crucial immune modulators, yet the exact mechanism of thymic Treg development remains controversial. Here, we present the first direct evidence for H2-O, an MHC class II peptide editing molecular chaperon, on selection of thymic Tregs. We identified that lack of H2-O in the thymic medulla promotes thymic Treg development and leads to an increased peripheral Treg frequency. Single-cell RNA-sequencing (scRNA-seq) analysis of splenic CD4 T cells revealed not only an enrichment of effector-like Tregs, but also activated CD4 T cells in the absence of H2-O. Our data support two concepts; a) lack of H2-O expression in the thymic medulla creates an environment permissive to Treg development and, b) that loss of H2-O drives increased basal auto-stimulation of CD4 T cells. These findings can help in better understanding of predispositions to autoimmunity and design of therapeutics for treatment of autoimmune diseases.

Nfkbid Overexpression in Nonobese Diabetic Mice Elicits Complete Type 1 Diabetes Resistance in Part Associated with Enhanced Thymic Deletion of Pathogenic CD8 T Cells and Increased Numbers and Activity of Regulatory T Cells.

Type 1 diabetes (T1D) in both humans and NOD mice is caused by T cell-mediated autoimmune destruction of pancreatic β cells. Increased frequency or activity of autoreactive T cells and failures of regulatory T cells (Tregs) to control these pathogenic effectors have both been implicated in T1D etiology. Due to the expression of MHC class I molecules on β cells, CD8 T cells represent the ultimate effector population mediating T1D. Developing autoreactive CD8 T cells normally undergo extensive thymic negative selection, but this process is impaired in NOD mice and also likely T1D patients. Previous studies identified an allelic variant of Nfkbid, a NF-κB signal modulator, as a gene strongly contributing to defective thymic deletion of autoreactive CD8 T cells in NOD mice. These previous studies found ablation of Nfkbid in NOD mice using the clustered regularly interspaced short palindromic repeats system resulted in greater thymic deletion of pathogenic CD8 AI4 and NY8.3 TCR transgenic T cells but an unexpected acceleration of T1D onset. This acceleration was associated with reductions in the frequency of peripheral Tregs. In this article, we report transgenic overexpression of Nfkbid in NOD mice also paradoxically results in enhanced thymic deletion of autoreactive CD8 AI4 T cells. However, transgenic elevation of Nfkbid expression also increased the frequency and functional capacity of peripheral Tregs, in part contributing to the induction of complete T1D resistance. Thus, future identification of a pharmaceutical means to enhance Nfkbid expression might ultimately provide an effective T1D intervention approach.

Glucose Metabolism Reprogramming of Regulatory T Cells in Concanavalin A-Induced Hepatitis.

Autoimmune hepatitis (AIH) is an inflammatory liver disease caused by a dysregulated immune response. Although the pathogenesis of AIH remains unclear, impaired regulatory T cells (Tregs) have been considered a driver of AIH development. Unlike autoreactive T cells, Tregs mainly utilize oxidative phosphorylation (OXPHOS) as their energy supply. Elevated glycolysis has been reported to limit the suppressive functions of Tregs. However, whether glucose metabolism reprogramming in Tregs is involved in AIH etiology remains unknown. The aim of this study was to examine alternations in Treg numbers and functions in AIH patients and concanavalin A (Con A)-induced hepatitis, while exploring associations between impaired Tregs and glucose metabolism. The frequency of Tregs was decreased in the peripheral blood but increased in liver biopsies of AIH patients. Moreover, immunosuppressive therapy rescued circulating Tregs in AIH. In Con A-induced immune hepatitis, enhanced intrahepatic Treg accumulation was observed over time, accompanied by reduced splenic Treg numbers. To investigate whether functional impairment of Tregs occurs in AIH, Tregs were isolated from experimental AIH (EAH) model mice and normal controls and the former displayed downregulated mRNA levels of FOXP3, CTLA4, CD103, TIGIT, CD39, and CD73. EAH model-derived Tregs also produced fewer anti-inflammatory mediators (TGF-β and IL-35) than control Tregs. Moreover, enhanced glycolysis and reduced OXPHOS were found in Tregs from EAH model mice, as reflected by elevated levels of key glycolytic enzymes (HK2, PK-M2, and LDH-A) and a decreased ATP concentration. This study revealed a decreased peripheral Treg frequency and abnormal intrahepatic Treg infiltration in AIH. It is first reported that glucose metabolism reprogramming is associated with decreases and functional impairments in the Treg population, promoting AIH development. Targeting glucose metabolism may provide novel insights for the treatment of AIH.

Regulatory T Cells and IL35 in Chronic Hepatitis C Related Cirrhosis and Hepatocellular Carcinoma

Regulatory T cells (Tregs) play a fundamental role in maintaining immune homeostasis to balance between the tissue-damaging and protective effects of the immune response. There are strong evidences that Treg cells and their cytokines may play an important role in the induction of tolerance in the liver and progression of HCV infection. Herein, we investigated the frequency of Treg cells and interleukin 35 (IL-35) level in blood and their potential relationship to the various chronic hepatitis C (CHC) complications and outcomes. A total of 36 HCV infected patients subdivided into, CHC complicated with cirrhosis (HCV LC; n = 18), CHC complicated with hepatocellular carcinoma (HCV- HCC; n=18) and apparently healthy control group (n=18) were enrolled in this study. Treg cells percentages were determined by flow cytometric analysis and ELISA was used to measure IL35 serum levels. A significant increase in the frequency of peripheral Tregs and serum IL35 level was found in HCV HCC, and HCV LC groups compared with the control group. The frequency of peripheral Tregs and plasma (IL-35) levels were significantly positively correlated with viral load along with disease progression. We conclude that the higher percentage of Tregs and IL35 level in peripheral blood of HCV HCC and HCV LC groups compared to the control group may suggest their contribution to viral persistence and progression of HCV infection.

Obesity-associated T-cell and macrophage activation improve partly after a lifestyle intervention.

The relation between low-grade inflammation and metabolic dysfunction in obesity is not fully explored. To evaluate immune parameters in the obese state and after a lifestyle intervention program. Patients with obesity (n = 87) from an academic obesity clinic were compared with controls with regard to macrophage and T-cell activation (reflected by serum levels of soluble CD163 (sCD163) and soluble IL-2 receptor (sIL-2R), respectively), and an array of cytokines, chemokines, and growth factors. In addition, these parameters and regulatory T-cells (Treg), were studied in 27 patients who followed a 75-week lifestyle intervention (dietary advice, exercise, and psychoeducation). Mean sIL-2R and sCD163 levels were higher in patients than controls (sIL-2R:2884 ± 936 pg/ml vs. 2207 ± 813 pg/ml, p = 0.001; sCD163:1279 ± 580 pg/ml vs. 661 ± 271 pg/ml, p < 0.0001 respectively). Patients with metabolic syndrome (MetS) had higher sCD163 than those without (1467 ± 656 pg/ml vs. 1103 ± 438 pg/ml). Patients had higher IL-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-15, IL-17A, MCP-1/CCL2, MIP-1α/CCL3, MIP-1β/CCL4, G-CSF, GM-CSF, FGF, IFN-γ, and TNF-α than controls, whereas VEGF-A, PDGF-BB, and eotaxin were lower. Upon intervention, sIL-2R decreased while peripheral Treg frequencies increased within the reference range (p = 0.042 and p = 0.005 respectively). The sIL-2R decrease correlated to a decrease in waist circumference (rho = 0.388, p = 0.045) and in trend to a decrease in MetS components (rho = 0.345, p = 0.078). The Treg increase was unrelated to weight loss or metabolic improvement. Mean sCD163 did not change significantly upon intervention, nor did the cytokines, chemokines, and growth factors (except IP-10/CXCL10). In obesity, T-cell homeostasis improves after a lifestyle intervention. Immunologic alterations can occur independently of metabolic improvement.

Pharmacological Inhibition of MALT1 Protease Leads to a Progressive IPEX-Like Pathology.

Genetic disruption or short-term pharmacological inhibition of MALT1 protease is effective in several preclinical models of autoimmunity and B cell malignancies. Despite these protective effects, the severe reduction in regulatory T cells (Tregs) and the associated IPEX-like pathology occurring upon congenital disruption of the MALT1 protease in mice has raised concerns about the long-term safety of MALT1 inhibition. Here we describe the results of a series of toxicology studies in rat and dog species using MLT-943, a novel potent and selective MALT1 protease inhibitor. While MLT-943 effectively prevented T cell-dependent B cell immune responses and reduced joint inflammation in the collagen-induced arthritis rat pharmacology model, in both preclinical species, pharmacological inhibition of MALT1 was associated with a rapid and dose-dependent reduction in Tregs and resulted in the progressive appearance of immune abnormalities and clinical signs of an IPEX-like pathology. At the 13-week time point, rats displayed severe intestinal inflammation associated with mast cell activation, high serum IgE levels, systemic T cell activation and mononuclear cell infiltration in multiple tissues. Importantly, using thymectomized rats we demonstrated that MALT1 protease inhibition affects peripheral Treg frequency independently of effects on thymic Treg output and development. Our data confirm the therapeutic potential of MALT1 protease inhibitors but highlight the safety risks and challenges to consider before potential application of such inhibitors into the clinic.

The Proportion of Regulatory T Cells in Patients with Ankylosing Spondylitis: A Meta-Analysis.

Objective Accumulating evidence indicates that regulatory T cells (Tregs) may be involved in the pathogenesis of ankylosing spondylitis (AS). As different markers have been used to identify Tregs, some studies on the proportions of Tregs in AS patients have generated considerable controversy. To clarify the status of Tregs in such patients, we determine the proportion changes of peripheral Tregs during development of the disease, with different cellular markers. Methods We systematically searched Embase, PubMed, Cochrane, Web of Knowledge, FDA.gov, and Clinical Trials.gov for the studies reporting the proportion of Tregs in AS patients. Using the PRISMA guidelines, we performed a random-effects meta-analysis of the frequencies of peripheral Tregs defined in different ways. Inconsistency was evaluated using the I-squared index (I2), and publication bias was assessed by examining funnel plot asymmetry using the Begger and Egger tests. Results A total 29 studies involving 1732 participants were included in the meta-analysis. Their conclusions of using the diversity of Tregs surface markers were inconsistent with each other. No significant difference in the proportions of Tregs was evident regardless of the definitions used [−0.709, (−1.455, 0.037, p = 0.063), I2 = 97.3%]. Six studies used “single CD25-positive” cells as Tregs, which revealed a significant increase in AS patients compared with healthy blood donors [0.736, (0.138, 1.334), p = 0.016, I2 = 80.7%]. Notably, the proportions of “CD4+CD25+FOXP3+,” “CD4+CD25highCD127low/−,” or “CD4+CD25+CD127low” T cells were lower in AS patients [−2.856, (−4.645, −1.066), p = 0.002; −1.812, (−2.648, −0.977), p < 0.001; −1.12, (−1.605, −0.635), p < 0.001]. Tregs defined as “CD25high,” “CD25bright,” “CD25bright/highCD127low/−,” “CD4+FOXP3+,” “CD4+CD25highFOXP3+,” and “CD4+CD25+CD127−” did not differ in proportion between AS patients and healthy blood donors. Conclusions The levels of Tregs varied based on the cellular identification markers used. The proportions of CD4+CD25+FOXP3+Tregs, CD4+CD25highCD127low/−, or CD4+CD25+CD127low in blood of AS patients were significantly decreased as compared with those in healthy blood donors, and our findings lend support to the idea that the Treg status of AS patients is important. And we recommend the above as the best definition of Tregs when evaluating the status of such patients.

Recipient-Derived Allo-iTregs Induced by Donor DCs Effectively Inhibit the Proliferation of Donor T Cells and Reduce GVHD.

To compare the potency of recipient-derived, antigen-specific regulatory T cells induced by different dendritic cells (DCs; iTregs) and freshly isolated natural regulatory T cells (nTregs) in preventing mouse graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). CD4+ T cells from recipient BALB/c mice were stimulated with DCs from recipient BALB/c (syn-DCs), donor B6 (allo-DCs), and third-party C3H (third-party-DCs) mice to induce different iTregs. In parallel, nTregs were isolated from spleen cells of recipient BALB/c (syn-nTregs) and donor B6 (allo-nTregs) mice using magnetic-activated cell sorting. Mixed lymphocyte reaction (MLR) assays were performed to evaluate the suppressive ability of these various regulatory T cells (Tregs). Both the iTregs and nTregs were transfused to GVHD mice on Days 0, 1, 3, and 5. Body weight, GVHD score, and survival time were monitored. Peripheral Tregs were subsequently examined on Days 7, 14, 21, and 28 after BMT, while chimerism was evaluated on Days 14 and 60. Histopathology of colon, liver, and spleen were also performed. DCs markedly induced CD25+ and Foxp3+ expression on CD4+ T cells. The allo-DC-induced Tregs (allo-iTregs) suppressed the proliferation of alloreactive T cells better than the other iTregs/nTregs in MLR assays (P < 0.05). Meanwhile, transfusion of the allo-iTregs reduced the severity of GVHD (P < 0.05), increased survival time compared with the GVHD group (P < 0.05), and enhanced the chimerism proportion. On Day 28 after BMT, the allo-iTregs group had the highest frequency of peripheral Tregs (P < 0.05). Recipient-derived allo-iTregs induced by donor DCs included predominant clones that specifically recognized donor antigens. These allo-iTregs not only prevented GVHD by suppressing the proliferation of donor-alloreactive T cells, but also promoted engraftment, and prolonged the survival of GVHD mice. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc. Anat Rec, 302:825-836, 2019. © 2018 Wiley Periodicals, Inc.

Low-dose IL-2 expands CD4+ regulatory T cells with a suppressive function in vitro via the STAT5-dependent pathway in patients with chronic kidney diseases

Background: Patients with chronic kidney disease (CKD) often have CD4+ regulatory T cells (Tregs) dysfunction and chronic inflammation. We aim to investigate the effect, function, and related mechanism of low-dose IL-2 on CD4+ regulatory T cells expansion in vitro from patients with CKD.Methods: A total of 148 newly diagnosed patients with CKD at Stage III and 35 healthy volunteer subjects were recruited into our studies. The number of peripheral Tregs in peripheral blood mononuclear cells isolated from CKD patients, which were characterized by FACS as CD4+CD25hi and CD4+CD25+FoxP3+. The effect of low-dose IL-2 on expansion of Tregs, and the suppressive function of expanded Tregs were also analyzed by FACS. The levels of FoxP3 mRNA were detected by qRT-PCR. The activation of IL-2 induced Stat5 and blocking experiments were assessed by Western Blotting and FACS.Results: We found that the frequency of peripheral Tregs from CKD patients was significantly lower than that in healthy volunteer subjects. We also showed that IL-2 selectively expanded CD4+CD25hi and CD4+CD25+FoxP3+ regulatory T cells, and also upregulated the expression of FoxP3 mRNA. Our in vitro studies demonstrated that expanded CD4+ regulatory T cells from CKD patients suppressed proinflammatory Th1 and Th17 cell response. Furthermore, STAT5 activation is required for IL-2-induced expansion of regulatory T cells and expression of FoxP3 mRNA from CKD patients.Conclusions: Our findings support the clinical Treg defects in CKD patients with glomerular diseases, and the rationale of evaluating low-dose IL-2 treatment for selectively modulating CD4+ Tregs.

Pertuzumab, trastuzumab and eribulin mesylate therapy for previously treated advanced HER2-positive breast cancer: a feasibility study with analysis of biomarkers.

The standard treatment for advanced human epidermal growth factor receptor 2 (HER2)-positive breast cancer is the triple combination of pertuzumab, trastuzumab and docetaxel, but some patients cannot tolerate taxane. To explore a non-taxane triple therapy, we conducted a feasibility study of pertuzumab, trastuzumab and eribulin mesylate (PTE) therapy for previously treated advanced HER2-positive breast cancer with analyses of quality of life and biomarkers. Ten patients were enrolled, two of whom had a history of docetaxel allergy. The median number of prior regimens was 3. The most common Grade 3 toxicities were leukopenia (70%) and neutropenia (70%). Grade 4 or 5 adverse events were not observed. An improving trend for the Functional Assessment of Cancer Therapy-Breast (FACT-B) score at 3 months was observed. Eight cases were included in the biomarker analysis. The peripheral CD8+ T cell/ CD4+Foxp3+ regulatory T cells (Tregs) ratio was significantly increased (p = 0.039). The frequency of peripheral Tregs was associated with the trastuzumab trough concentration (p = 0.019). In a non-clinical analysis, Eribulin mesylate significantly inhibited Ser473 Akt phosphorylation in PIK3CA wild-type cells and mutated cells. These results suggest that PTE therapy is a feasible and promising option for advanced HER2-positive breast cancer. Further investigation is warranted.

Role of Regulatory T Cells in Tubercular Uveitis

ABSTRACTPurpose: To study the role of regulatory T cells (Tregs) in patients with tubercular uveitis.Methods: Frequencies of peripheral Tregs, Th1, Th17 cells, and intracellular cytokines were determined in 17 tubercular uveitis patients and 18 disease controls. Function of Tregs, Th1, and Th17 cells was assessed in vitro. Simultaneously, ocular levels of IFN-γ, IL-17A, IL-4, and IL-10 were also measured.Results: Frequencies of peripheral Tregs in tubercular uveitis subjects were significantly lower compared with disease controls. Furthermore, expression of TGF-β and IL-2Rα, but not CTLA4, was reduced in Tregs of the tubercular uveitis group. The tubercular uveitis group demonstrated heightened Th1, Th17 responses following in vitro stimulation with phorbol myristate acetate (PMA)/ionomycin. Interestingly, Treg suppression assay did not show a significant difference between the two groups. Ocular levels of IFN-γ, IL-17A, and IL-10 were also elevated in tubercular uveitis group.Conclusions: Low Treg frequency and hyporesponsive function contribute to proinflammatory responses manifesting at ocular level in tubercular uveitis.

Presence of circulating Her2-reactive CD8 + T-cells is associated with lower frequencies of myeloid-derived suppressor cells and regulatory T cells, and better survival in older breast cancer patients.

IntroductionBreast cancer is one of the most common cancers among women. Its incidence is increasing in many countries and a higher number of older women are now being diagnosed with the disease. Immune parameters are implicated in disease progression, and the frequencies of both myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), associated with tumour burden, have been suggested to be indicators of poor prognosis in cases of metastatic breast cancer.MethodsHere, we have assessed the frequency of peripheral Tregs and MDSCs in relation to in vitro T cell responses to Her2 antigen in 40 untreated breast cancer patients 65 to 87 years of age at diagnosis.ResultsThe five-year survival rate of patients who mounted a CD8+ T cell response to Her2 peptides and had a lower frequency of Lin−CD14+HLA-DR−MDSCs was 100% compared to only 38% in patients without Her2-reactive CD8+ T cells and with higher frequencies of MDSCs (P = 0.03). Patients who lacked a CD8 response to Her2 tended to have higher frequencies of MDSCs. Similarly, patients who lacked a CD8 response to Her2 and had higher frequencies of CD4+Foxp3+CD127lowCD25+ Tregs had only 50% survival compared to the 100% survival of patients who did mount a CD8 response and had lower frequencies of Tregs (P = 0.03). A similar trend was observed for activated (CD4+CD45RA−Foxp3hi) but not resting Tregs (CD4+CD45RA+FoxP3+). This survival advantage was observed in both metastatic and non-metastatic patients.ConclusionsOur data demonstrate a negative role of both MDSCs and Tregs in the prognosis of breast cancer patients, the mechanism of which might be through dampening favourable CD8+ T cell immune responses to tumour-associated antigens.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0541-z) contains supplementary material, which is available to authorized users.

Enhanced frequency and potential mechanism of B regulatory cells in patients with lung cancer.

BackgroundRegulatory T cells (Tregs) and B cells (Bregs) play an important role in the development of lung cancer. The present study aimed to investigate the phenotype of circulating Tregs and Bregs in patients with lung cancer and explore potential mechanism by which lung cancer cells act on the development of both.MethodsPatients with lung cancer (n = 268) and healthy donors (n = 65) were enrolled in the study. Frequencies of Tregs and Bregs were measured by flow cytometry with antibodies against CD4, CD25, CD127, CD45RA, CD19, CD24, CD27 and IL-10 before and after co-cultures. qRT-PCR was performed to evaluate the mRNA levels of RANTES, MIP-1α, TGF-β, IFN-γ and IL-4.ResultsWe found a lower frequency of Tregs and a higher frequency of Bregs in patients with lung cancer compared to healthy donors. Co-culture of lung cancer cells with peripheral blood mononuclear cells could polarize the lymphocyte phenotype in the similar pattern. Lipopolysaccharide (LPS)-stimulated lung cancer cells significantly modulated regulatory cell number and function in an in vitro model.ConclusionWe provide initial evidence that frequencies of peripheral Tregs decreased or Bregs increased in patients with lung cancer, which may be modulated directly by lung cancer cells. It seems cancer cells per se plays a crucial role in the development of tumor immunity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0304-0) contains supplementary material, which is available to authorized users.

In vitro TNF blockade enhances ex vivo expansion of regulatory T cells in patients with immune thrombocytopenia.

Tumour necrosis factor-α (TNF) is an inflammatory cytokine that is elevated in a number of autoimmune diseases including immune thrombocytopenia (ITP), a bleeding disorder characterized by low platelet counts. In vitro TNF blockade increases expansion of the regulatory T cell (Treg) IKZF2 (also termed Helios) subset in T cell-monocyte cocultures from healthy donors, but its role on proliferative responses of Tregs in ITP patients, who have altered immunoregulatory compartment, remains unclear. TNF in CD4+ T cells from patients with chronic ITP were elevated and negatively correlated with peripheral Treg frequencies, suggesting a possible inhibitory effect of TNF on ITP Tregs. In vitro antibody neutralization with anti-TNF in T cell-monocyte cocultures resulted in a robust expansion of pre-existing ITP Tregs, higher than in healthy controls. Similar to the effects of anti-TNF antibodies, TNF blockade with antibodies against TNFRSF1B (anti-TNFRSF1B, previously termed anti-TNFRII) almost doubled ITP Treg expansion whereas neutralization with anti-TNFRSF1A (anti-TNFRI) antibodies had no effect on proliferative responses of Tregs. In addition, TNFRSF1B levels on ITP Tregs were significantly elevated, which may explain the increased susceptibility of patient Tregs to the actions of TNF blockade. Altogether, these data raise the possibility that TNF blockers, through their ability to increase Treg proliferation, may be efficacious in ITP patients.

Changes of regulatory T cells related to CCl₄-induced liver fibrosis in mice

To investigate liver fibrosis-related changes of CD4⁺CD25⁺Foxp3+ regulatory T cells (Tregs) in peripheral blood and in liver-infiltrating lymphocytes (LILs) using a mouse model. C57BL/6 mice were randomly divided into a model group and a control group. The model group received intraperitoneal injection of carbon tetrachloride (CC1₄) to induce liver fibrosis, and the control group received an equal volume of physiological saline. Serum was collected for detection of alanine aminotransferase level. Histopathological changes in liver were assessed by microscopic observation of tissues stained by hematoxylineosin and Masson. Frequencies of peripheral and intrahepatic Tregs, NK1.1⁺ cells, CD4⁺ and CD8⁺ cells were analyzed by flow cytometry. Intrahepatic Foxp3+ cells were detected by immunofluorescence. Liver expression of IL-6, IL-10, TGFb and Foxp3 was measured by RT-PCR detection of mRNA .Inter-group differences were evaluated by t-test. The model group showed a significantly higher frequency of intrahepatic CD25⁺Foxp3⁺/CD4⁺ Tregs (10.63% ± 1.50% vs. control group:1.80% ± 0.66%; P less than 0.01) but only slightly higher frequency of peripheral Tregs (6.00% ± 0.62% vs. 5.33% ± 2.86%). The model group also showed significantly higher levels of intrahepatic Foxp3+ cells and of Foxp3 mRNA (both P less than 0.05), but significantly lower frequencies of NK1.1 cells in LILs (9.53% ± 2.25% vs. 19.80% ± 5.97%; P less than 0.05) and in peripheral blood (0.38% ± 0.13% vs. 1.06% ± 0.63%; P less than 0.05). The CD4⁺ cell frequency and the CD4⁺/CD8⁺ ratio were lower in LILs and peripheral blood of the model group, but none differed significantly from the control group. The intrahepatic expression of TGFb mRNA was significantly higher in the model group (P less than 0.01). In liver fibrosis, intrahepatic CD4⁺CD25⁺Foxp3+ Tregs are increased while NK1.1+ cells are decreased. Tregs may suppress NK1.1+ cells through a mechanism involving TGFb.

Increased CD4+CD25+ regulatory T cells correlate with poor short-term outcomes in hepatitis B virus-related acute-on-chronic liver failure patients

The roles of CD4(+)CD25(+) regulatory T cells (Treg) in chronicity of hepatitis B virus (HBV) infection have been confirmed. We aimed to explore alteration of Treg in patients with HBV-related acute-on-chronic liver failure (ACLF). Thirty-two HBV-related ACLF patients, 44 chronic hepatitis B patients, and 41 healthy controls were recruited. We detected frequencies of peripheral Treg and intrahepatic forkhead winged helix transcription factor (Foxp3)(+) cells. Inhibitory activity of Treg was assessed by functional suppression assays. Serum interferon-γ and interleukin-10 were also determined. Peripheral Treg and intrahepatic Foxp3(+) cells were more markedly increased in ACLF than chronic hepatitis B and controls (all p<0.001), and the Foxp3(+) cells located predominantly in the portal areas. The Treg frequency was positively correlated with HBV DNA load, international normalized ratio, model of end stage liver disease score, and serum interleukin-10 level in ACLF patients. Functional assays invitro demonstrated that ACLF patients exhibited higher suppressive effects of Treg on proliferations of autologous CD4(+)CD25(-) T cells than controls. On logistic regression, prolonged international normalized ratio and higher peripheral Treg frequency predicted 30-day survival of ACLF. The patients with HBV-related ACLF exhibit increased amounts of Treg, of which redistribution from periphery to liver seems to modulate liver inflammation. Higher Treg amounts are associated with more severe liver disease in ACLF, and its level in combination with international normalized ratio may assist prediction of short-term outcomes of HBV-related ACLF.

Elevated NKT cell levels in adults with severe chronic immune thrombocytopenia

The aim of this study was to investigate the frequency of circulating natural killer T (NKT) cells and regulatory T cells (Tregs), as well as serum cytokine profiles, in adult chronic primary immune thrombocytopenia (ITP). The frequency of circulating T cell receptor (TCR) Vα24+Vβ11+CD3+ NKT cells and CD4+CD25+CD127−/low Tregs was measured using multi-color flow cytometry. The serum concentrations of 11 cytokines were determined with a cytometric bead assay. The frequency of circulating NKT cells in patients with ITP was 0.13±0.03%, whereas the frequency in healthy controls was 0.07±0.01% of CD3+ (P>0.05). However, the frequency of NKT cells in patients with ITP with platelet counts ≤20×109/l (0.22±0.05%) was significantly higher than that in patients with platelet counts >20×109/l (0.05±0.01%; P<0.05) and that in healthy controls (0.07±0.01%; P<0.05). The frequency of peripheral Tregs was comparable between patients with ITP (3.97±0.44% of CD4+) and healthy controls (3.69±0.31%; P>0.05). No significant differences were observed in the serum concentrations of 11 cytokines between patients with ITP and healthy controls, despite the fact that the serum levels of interleukin (IL)-12p70, IL-8, IL-4, interferon (IFN)-γ and tumor necrosis factor (TNF)-α in patients with ITP were higher than those in the healthy controls. The platelet count was negatively correlated with the frequency of circulating NKT cells in chronic ITP. These results indicate that NKT cells may be involved in ITP with severe thrombocytopenia, and NKT and Tregs may be important in cytokine deregulation in chronic ITP.

Suppression of Inhibitor Formation in Protein and Gene Therapy for Hemophilia Using Ex Vivo Expanded Treg

Abstract 13Hemophilia A and B result from deficiency in clotting factor VIII (FVIII) or IX (FIX), respectively. In a subset of patients, treatment by factor replacement therapy is limited by formation of inhibitory antibodies to the clotting factors, representing a serious complication that increases risks of morbidity and mortality. Immune responses to the therapeutic coagulation factors are also a concern in newly emerging gene therapies. Regulatory T cells (Treg) offer to be a novel alternative pathway toward immune tolerance. Treg has been identified as a major component of immune tolerance to coagulation factors in pre-clinical studies. Therefore, we hypothesize that ex vivo expanded autologous Treg can suppress inhibitor formation. Our study seeks to test this approach in hemophilic mice. Initially, we optimized in vitro expansion of murine BALB/c-derived Treg. Using flow sorting, GFP+ cells were purified (>98% purity) from spleens of BALB/c knock-in mice containing a GFP reporter linked to FoxP3 expression with an IRES sequence. Sorted cells were stimulated in culture using anti-CD28/anti-CD3 beads in the presence of high-level IL-2 (1000 U/ml). IL-2 was replenished every second day in culture. After ∼1 week, cells were freshly stimulated. At the end of 2 weeks, viability, purity, and FoxP3/GFP expression was confirmed. Greater than 30-fold expansion was repeatedly accomplished. Assumming a dose of 1×106 Treg/mouse, expansion is sufficiently robust to treat >30 mice starting with Treg from 2–3 donor mice. Ex vivo expanded Tregs were adoptively transferred to male hemophilia A mice (BALB/c F8e16 −/−), which were then treated with F.VIII (1 IU human B domain-deleted F.VIII, IV, once per week) for two months. Bethesda assays demonstrated that Treg transplant had effectively suppressed inhibitor formation. Inhibitor titers in control mice were 15–20 BU at 1 month and 30–40 BU at two months. In contrast, Treg treated mice (n=5 per group) formed at most low-titer inhibitors (2–3 BU for both time points). By 2 months, peripheral Treg frequencies had returned to near baseline. To further demonstrate presence of a Treg population capable of suppressing antibody formation against F.VIII, a secondary transfer of sorted CD4+CD25+ splenocytes was performed. Recipient hemophilia A mice were immunized against F.VIII in adjuvant. Compared to mice receiving control Treg, there was significant (P<0.05) suppression of inhibitor formation against F.VIII. In other experiments, Treg therapy was also able to significantly reduce inhibitor titers in hemophilia A mice with pre-existing F.VIII inhibitors. We chose hemophilia A mice (n=6) that developed on average 25 BU from F.VIII replacement therapy. Half of these received Treg transplant, and all mice received 8 more weeks of F.VIII treatment. Inhibitor titers in the control group increased to ∼100 BU. Treg therapy substantially reduced this response (to 15–20 BU, P<0.001). In order to evaluate Treg therapy for hemophilia B, BALB/c F9 −/− × FoxP3-GFP mice were generated. Treg were isolated from these mice as described above, expanded in vitro, and transferred to (BALB/c F9 −/−) hemophilia B mice. Two days later, to test their effectiveness in a gene therapy setting, these mice were treated by intramuscular injection of AAV1 vector expressing human F.IX. By 6 weeks after gene transfer, control mice had formed high-titer antibody against hF.IX (>20 mg IgG/ml plasma, ∼ 10 BU). In contrast, anti-hF.IX formation was undetectable in mice that had received Treg prior to vector administration (n=4/group). While perhaps not as potent as antigen-specific Treg, our data demonstrate the ability of highly purified and ex vivo expanded bulk Treg to control inhibitor formation and thus support their utility for tolerance induction in hemophilia. Their effectiveness may involve emergence of a more specific Treg population after repeated in vivo exposure to antigen. In gene therapy, Treg transplant may be a more desirable alternative to use of immune suppressive drugs. Providing additional immune regulation around the time of vector administration, i.e. when activation signals are provided to the immune system, could be sufficient to prevent immune rejection long-term while inducing antigen-experienced Treg for durable tolerance. Disclosures:Herzog:Genzyme Corp.: Royalties, AAV-FIX technology, Royalties, AAV-FIX technology Patents & Royalties.