Tregs In Peripheral Blood Mononuclear Cells Research Articles

Up‐regulation of proliferative and migratory genes in regulatory T cells from patients with metastatic castration‐resistant prostate cancer

A higher frequency of regulatory T cells (Tregs) has been observed in peripheral blood mononuclear cells (PBMC) of patients with different types of solid tumors and hematological malignancies as compared to healthy donors. In prostate cancer patients, Tregs in PBMC have been shown to have increased suppressive function. Tumor-induced biological changes in Tregs may enable tumor cells to escape immunosurveillance. We performed genome-wide expression analyses comparing the expression levels of more than 38,500 genes in Tregs with similar suppressive activity, isolated from the peripheral blood of healthy donors and patients with metastatic castration-resistant prostate cancer (mCRPC). The differentially expressed genes in mCRPC Tregs are involved in cell cycle processes, cellular growth and proliferation, immune responses, hematological system development and function and the interleukin-2 (IL-2) and transforming growth factor-β (TGF-β) pathways. Studies revealed that the levels of expression of genes responsible for T-cell proliferation (C-FOS, C-JUN and DUSP1) and cellular migration (RGS1) were greater in Tregs from mCRPC patients as compared to values observed in healthy donors. Increased RGS1 expression in Tregs from mCRPC patients suggests a decrease in these Tregs' migratory ability. In addition, the higher frequency of CD4(+) CD25(high) CD127(-) Tregs in the peripheral blood of mCRPC patients may be the result of an increase in Treg proliferation capacity. Results also suggest that the alterations observed in gene expression profiles of Tregs in mCRPC patients may be part of the mechanism of tumor escape from host immune surveillance.

Targeting Histone Deacetylases As a New Strategy for Graft Versus Host Disease Prevention

AbstractAbstract ▪740▪This icon denotes a clinically relevant abstract Introduction:Graft-versus-host disease (GVHD) precludes an effective utilization of allogeneic hematopoietic stem cell transplantation (HSCT). Histone deacetylase inhibitors (HDACi) are an important class of anti-cancer agent and have recently been shown by us and others to attenuate experimental GVHD in multiple murine models. They suppress proinflammatory cytokine production, modulate antigen presenting cells (APCs), and enhance T regulatory cell (Treg) numbers and functions. We have translated our experimental observations from murine models in a first-in-human clinical trial to test the hypothesis that HDAC inhibition will be safe and reduce the severity of GVHD in patients undergoing matched related donor (MRD) reduced intensity conditioning (RIC) allogeneic HSCT. Methods:From March 2009 through June 2012, we enrolled 45 patients at two centers, the University of Michigan and Washington University, in a phase I/II trial that combined the HDACi, vorinostat, with standard GVHD prophylaxis regimen consisting of tacrolimus and mycophenolate mofetil (MMF) and compared them with historical controls. The primary endpoint was the cumulative incidence of grade 2–4 acute GVHD (aGVHD) with a target risk of 25%, which would represent a statistically significant improvement over the 42% risk experienced by historical controls, assuming a type I error rate of 5%. The controls comprised of patients who underwent MRD RIC allogeneic HSCT and received standard GVHD prophylaxis with tacrolimus and MMF. Study eligibility included adult patients with a hematological malignancy for which RIC HSCT was considered appropriate and had an available 7/8 or 8/8-HLA-MRD. Specifically, patients with CLL and lymphoma must have been in CR, PR or had stable disease. Patients with myelodysplasia, acute leukemia or CML must have had < 20% blasts on the bone marrow examination. Study subjects received fludarabine 40 mg/m2 for 4 days, busulfan 3.2 mg/kg for 2 days, MMF for 29 days, and tacrolimus levels were maintained between 8–12 ng/mL through day (D) 56, and then tapered off by D180 (in the absence of GVHD). The investigational agent, vorinostat, was administered orally from D-10 to D100. Results:The median age of the study patients (vorinostat-treated) was 58 years (range, 43–69 years). There have been no cases of graft failure or excessive toxicity or death attributable to vorinostat. The median time to neutrophil and platelet engraftment was similar between study and controls (12 vs 11 days, respectively, for both groups). Bone marrow chimerism analyses for study patients showed the percent of myeloid (CD33+) engraftment to be 94%, 100% and 100%, and whole T cell (CD3+) engraftment to be 74.5%, 75%, and 100% at D30, D100 and D365, respectively. In the 45 study patients, the cumulative incidence of D100 grade 2–4 aGVHD was significantly lower compared to controls (22% vs 42%, respectively, P=0.04). Severe grade 3–4 aGVHD at D100 (4% vs 19% controls) and transplant-related mortality at 1 year (13% vs 19% controls) were also reduced. The incidence of aGVHD was globally diminished in all target organs, and all but one patient responded rapidly to standard steroid therapy. The incidence of relapse was similar between the study and controls (17% vs 20%, respectively). Infectious complications were not different between the groups. We performed several correlative analyses with patient samples to understand the putative mechanisms based on murine studies. Peripheral blood mononuclear cells (PBMCs) from study patients demonstrated enhanced histone (H3) acetylation at D30 compared with PMBCs from controls (P<0.03). Consistent with experimental data, there was greater acetylation of STAT-3 and increased percent and total numbers of CD4+CD25+CD127− Tregs in PBMCs in study patients compared to control or normal healthy patients (P=0.03 and P=0.04, respectively). Ex vivo stimulation of PBMCs from the study patients with TLR agonist (LPS) demonstrated reduced secretion of proinflammatory cytokines (TNF-α and IL-6) when compared with controls or healthy volunteers. Conclusions:The data herein demonstrate translation of our experimental observations into a proof of concept first-in-human phase I/II clinical trial in prevention of GVHD. More importantly, the data suggest that administration of vorinostat in MRD RIC allogeneic HSCT may be a safe and effective strategy to reduce aGVHD. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Chronically HIV-1 Infected Patients Exhibit Low Frequencies of CD25+ Regulatory T Cells

The characterization of regulatory T cells (Treg) during HIV infection has become of particular interest considering their potential role in the pathogenesis of the acquired immunodeficiency syndrome. Different reports on Tregs in HIV-infected patients vary greatly, depending on the state of disease progression, anatomical compartment, and the phenotypic markers used to define this cell subpopulation. To determine the frequency of Tregs we included paired samples from peripheral blood and rectal biopsies from controls and chronic HIV patients with or without detectable viral load. Tregs were determined by flow cytometry using three different protocols: CD4+Foxp3+; CD4+Foxp3+CD127Low/-, and CD4+CD25+CD127Low/-. In addition, and with the purpose to compare the different protocols we also characterized Tregs in peripheral blood of HIV negative individuals with influenza like symptoms. Here, we report that Treg characterization in HIV-infected patients as CD4+Foxp3+ and CD4+Foxp3+CD127Low/- cells was similar, indicating that both protocols represent a suitable method to determine the frequency of Tregs in peripheral blood mononuclear cells (PBMC) and gut associated lymphoid tissue (GALT). In contrast, in HIV but not in flu-like patients, detection of Tregs as CD4+CD25+CD127Low/- cells resulted in a significantly lower percentage of these cells. In both, HIV patients and controls the frequency of Treg was significantly higher in GALT compared to PBMC. The frequency of Tregs in PBMC and GALT using CD4+Foxp3+ and CD4+Foxp3+CD127Low/- was higher in HIV patients than in controls. Similarly, the frequency of Treg using any protocol was higher in flu-like patients compared to controls. The results suggest that relying on the expression of CD25 could be unsuitable to characterize Tregs in PBMC and GALT samples from a chronic infection such as HIV.

Abstract B51: Circulating regulatory T-cell function and overall survival in metastatic castration-resistant prostate cancer (mCRPC) patients treated with vaccine.

Abstract Purpose: Targeted therapies using a receptor tyrosine kinase inhibitor Sunitinib has been reported to reduce levels of myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs). MDSCs and Tregs can diminish active immunization against tumor-associated antigens in cancer patients. High levels of Tregs (CD4+CD25high FoxP3+CD127-) have been reported in peripheral blood mononuclear cells (PBMCs) of patients with several types of cancer. Treg numbers are not significantly increased in patients with prostate cancer (PCa) compared to healthy donors; However, Treg suppressive function is enhanced in PCa patients suggesting that Treg function may play an important role in immune modulation. We investigated the number and suppressive function Tregs in PBMCs from patients with mCRPC pre- and post-treatment with PSA-TRICOM vaccine in terms of baseline patient characteristics and overall survival (OS). Experimental Design: PSA-TRICOM is a poxviral-based vaccine that expresses 3 T-cell costimulatory molecules and prostate-specific antigen (PSA). Chemotherapy-naïve patients with mCRPC were treated monthly with PSA-TRICOM and 23 patients were evaluated for OS as well as frequency and suppressive function of Tregs on day 0 and after 3 monthly vaccines (around day 85). Treg function from PBMC was determined by the suppression of CD4+CD25- T-cell proliferation by Tregs. Results: OS for all 23 patients was 8.5 to 50.2 mos. By day 85 post vaccination, 14 of 23 patients (61%) showed decreases in Treg function. We found a significant correlation between OS and a decrease in the suppressive function of Tregs in PBMCs post-vaccination (Spearman's r = −0.454; P = 0.029). Of the 15 patients with an OS longer than the Halabi predicted survival (PS), 12 (80%) had decreased Treg function. Of 8 patients who had an OS less than the Halabi PS, 2 (25%) showed a decrease in Treg function by day 85. The Halabi nomogram is based on seven baseline disease characteristics: visceral disease, Gleason score, performance status, baseline PSA, LDH, alkaline phosphpatase, and hemoglobin. Patients with an OS greater than Halabi PS also had a significant increase in the effector:CTLA-4+ Treg ratio post-vaccination (P = 0.029 and P = 0.024, respectively). Conclusion: These data suggest a possible association between improved OS and a decrease in Treg function after treatment with PSA-TRICOM vaccine. Patients with an OS longer than Halabi PS appeared more likely to have decreased Treg function following vaccine. These results provide the rationale for the use of certain targeted therapies, such as Sunitinab, to facilitate the antitumor immunity in concert with prostate cancer vaccines such as PSA-TRICOM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B51.

Impaired Balance of Th17/Treg in Patients with Nasal Polyposis

Nasal polyposis (NP) is a chronic inflammatory disease of the nasal cavity and sinuses that is regulated by T lymphocyte subsets. Imbalance of Th17/Treg has been considered critical in the development of inflammation and atopic reactions. To assess whether the balance of Th17/Treg is disrupted in patients with NP, we evaluated the distribution of Th17 and Treg cells among peripheral blood mononuclear cells (PBMCs) in atopic patients with NP, non-atopic patients with NP and controls. We then determined mRNA levels of RORc and Foxp3 and protein levels of IL-17, TGF-β and IL-10 in polyp tissue among the three groups. Finally, we investigated the correlation between Th17-, Treg- and Th1-, Th2-related cytokines (INF-γ, IL-4, IL-5). The results demonstrated that both atopic and non-atopic patients with NP revealed significantly increased Th17 proportion and decreased Treg proportion in PBMCs, as well as significantly increased RORc and IL-17 levels and decreased Foxp3 and TGF-β levels in polyp tissue. Furthermore, these differences were significant between atopic and non-atopic groups. The frequency of Treg in PBMCs was found to be negatively correlated with Th1 and Th2 cytokines in polyps. These results indicated that an impaired balance of Th17/Treg existed in patients with NP and was more severe in atopic patients, suggesting that the imbalance of Treg/Th17 may play an important role in the development of NP and that atopy may aggravate NP by promoting the imbalance of Th17/Treg.

Increased Frequency of CD4+CD25highFoxP3+ Regulatory T Cells in Patients with Hepatocellular Carcinoma

Accumulating evidence suggests regulatory T cells (Tregs) are associated with impaired antitumor responses. However, the relationship between the CD4(+)CD25(high)FoxP3(+) Treg and hepatocellular carcinoma (HCC) has not been well investigated. Levels of CD4(+)CD25(high)FoxP3(+) Tregs in peripheral blood mononuclear cells (PBMCs) from HCC patients and healthy donors, tumor infiltrating lymphocytes (TILs) extracted from HCC, and hepatic lymphocytes extracted from resected liver were measured by flow cytometry, and their effects on T-cell proliferation was determined by (3)H-thymidine incorporation. Serum levels of interleukin (IL)-10 and transforming growth factor (TGF)-β1 were measured by enzyme linked immunosorbent assay. The frequency of Tregs in PBMCs from HCC patients was higher than that from healthy donors. Similarly, the frequency of Tregs in TILs was higher than that of hepatic lymphocytes. On the other hand, the (3)H-thymidine uptake by TILs and PBMCs from HCC patients was decreased drastically when compared to the counterparts from normal controls. Furthermore, serum IL-10 and TGF-β1 levels increased significantly in HCC patients when compared to the healthy donors. This study identified an increased frequency of CD4(+)CD25(high)FoxP3(+) Tregs in patients with HCC. The elevated serum IL-10, TGF-β1 levels also correlated with impaired antitumor responses in these patients. Further effort is needed to establish new immunotherapeutic strategies designed to modulate Tregs to promote a competent antitumor response.

Induction of regulatory T cells by probiotics: potential for treatment of allergy?

Induction of regulatory T cells by probiotics: potential for treatment of allergy?

Correlation of dynamic alteration of regulatory T cells with the genesis and development of intrahepatic cholangiocarcinoma in a rat model

Objective To investigate the correlation of dynamic alteration of regulatory T cells(Tregs) with the genesis and development of intrahepatic cholangiocarcinoma in a rat model. Methods A total of 40 male SD rats were administered orally with drinking water containing 300 mg/L thioacetamide, and 40 rats administered normally as control group. Peripheral blood mononuclear cells(PBMCs) ,spleen, tumor and liver were harvested and detected at a two-weeks interval from 10 weeks after initiation of thioacetamide. Tregs in PBMCs and spleen were examined by flow cytometry, and those in tumor microenvironment by immunohistochemistry. Results(1) The main pathologic features in different phases were observed as follows:hyperplasia of duct gland from 12th to 14th week;atypical hyperplasia at 16th week;the visible tumor after 18th week.(2) In PBMCs,an increasing proportion of Tregs was observed from the 14th week [(4.25±0. 64) % vs(3. 76±0. 60) % , P<0.01 ]. The proportion of Tregs reached the peak at 18th week [(6.18±0.64)% ]. Tregs in the spleen began to increase at 18th week,reached the peak at 20th week [(8.54±0.36)% ].(3) A significant increase of Tregs was examined at 18th week in tumor microenvironment(P<0.05). With the progress of tumor,the number of Tregs in tumor microenvironment was increased. Conclusion The increased Tregs in PBMCs and the spleen were related to tumor genesis. But they did not show any correlation with tumor progression. The proportion of Tregs in tumor microenvironment was correlated with tumor progression. Key words: Cholangiocarcinoma; Model,animal; T cells

Regulatory T cells contribute to the immunoregulatory effect on graft versus host reaction after liver transplantation in donor-dominant one-way MHC matching rats

Acute graft-versus-host disease following liver transplantation (LTx-aGVHD) poses a major diagnostic and therapeutic challenge, and the mortality rate is as high as 85%. Even the liver contains large numbers of lymphoid cells in the parenchyma and surgical damage of liver transplantation would induce an immunosuppressive, the incidence of LTx-aGVHD is just approximately 1–2%. CD4 +CD25 +regulatory T (Treg) cells have recently been shown to suppress proliferative responses of CD4 +CD25 −T cells to alloantigenic stimulation in vitro and are required for ex vivo tolerization of donor T cells, which results in their reduced potential to induce aGVHD after bone marrow transplantation. To investigate the role of Treg in LTx-aGVHD, we compared the proportional frequency of Treg in syngeneic liver transplantation recipients (sLT group), semiallogeneic liver transplantation recipients (Semi-LT group), LTx-aGVHD induced recipients (LTx-aGVHD group) and healthy controls. Here we show that replacement of (LewisXBN)F1 liver by Lewis liver alone in Semi-LT group was not sufficient to induce aGVHD, and all recipients grew in a normal pattern as the syngeneic LT from Lewis to Lewis rat. However, when 4 × 10 8 of donor splenocytes were transferred simultaneously with LT, the morbidity of lethal aGVHD were 100%. A relative stable percentage of Treg, defined as CD4, CD25 and Foxp3, was detected in peripheral blood mononuclear cells (PBMCs) of sLT group compared with healthy controls. In early time after transplantation, no significantly change of Treg population was observed in these recipients after semiallogeneic liver transplantation in comparison with healthy controls ( P > 0.05). However, Treg levels showed a relative increase 4 days after transplantation. Especially on 12th and 16th day after transplantation, there was significantly increased proportion of Treg cells compared with healthy controls ( P < 0.05). The present of Treg decreased progressively in LTx-aGVHD group, which was significantly lower than group 1 and group 2 on the 12th and 16th day after liver transplantation ( p < 0.05). In conclusion, recipients in semi-LT group harbor an increased percentage of Treg in peripheral blood compared with controls. Treg have an immunoregulatory effect on graft versus host reaction after liver transplantation. Additional donor splenocyte transplanted with the liver provoke the development of aGVHD after liver transplantation, and reversed the change of Treg in PBMCs as in Semi-LT group, which destroy the balance between Treg and conventional effector T cells which determine the outcome of aGVHD after liver transplantation.

A Unique Subset of CD4+CD25highFoxp3+ T Cells Secreting Interleukin-10 and Transforming Growth Factor-β1 Mediates Suppression in the Tumor Microenvironment

Immunosuppression, including that mediated by CD4(+)CD25(high)Foxp3(+) regulatory T cells (Treg), is a characteristic feature of head and neck squamous cell carcinoma (HNSCC). Tregs with a distinct phenotype in tumor-infiltrating lymphocytes (TIL) contribute to local immune suppression. The frequency and phenotype of Treg in TIL and/or peripheral blood mononuclear cells (PBMC) in 15 HNSCC patients and PBMC in 15 normal controls were compared. Single-cell sorted CD4(+)CD25(high) T cells were tested for regulatory function by coculture with carboxyfluorescein diacetate succinimidyl ester-labeled and activated autologous CD4(+)CD25(-) responder T cells. Transwell inserts separating Treg from responders and neutralizing interleukin-10 (IL-10) or transforming growth factor-beta1 (TGF-beta1) antibodies were used to evaluate the mechanisms used by Treg to suppress responder cell proliferation. In TIL, CD25(+) cells were enriched in the CD3(+)CD4(+) subset (13 +/- 3%) relative to circulating CD3(+)CD4(+) T cells (3 +/- 0.7%) in HNSCC patients (P < or = 0.01) or normal controls (2 +/- 1.5%; P < or = 0.001). Among the CD3(+)CD4(+) subset, CD25(high) Treg represented 3 +/- 0.5% in TIL, 1 +/- 0.3% in PBMC, and 0.4 +/- 0.2% in normal controls. Tregs in TIL were GITR(+), IL-10(+), and TGF-beta1(+), although circulating Treg up-regulated CD62L and CCR7 but not GITR, IL-10, or TGF-beta1. Treg in TIL mediated stronger suppression (P < or = 0.001) than Treg in PBMC of HNSCC patients. The addition of neutralizing IL-10 and TGF-beta antibodies almost completely abrogated suppression (5 +/- 2.51%). Transwell inserts partly prevented suppression (60 +/- 5% versus 95 +/- 5%). Suppression in the tumor microenvironment is mediated by a unique subset of Treg, which produce IL-10 and TGF-beta1 and do not require cell-to-cell contact between Treg and responder cells for inhibition.