CD25high Treg Cells Research Articles

Distribution and clinical significance of circulating CD8+CD28− regulatory T cells in the peripheral blood of patients with pulmonary tuberculosis

BackgroundRegulatory T cells (Treg cells) in the peripheral blood of patients with pulmonary tuberculosis (PTB) may be closely related to the progression of PTB. In this study, the distribution characteristics and clinical importance of CD8+CD28− Treg cells in patients with tuberculosis were systematically analyzed, and the role and importance of CD8+CD28− Treg cells in influencing the immune response and progression of tuberculosis were discussed, which will provide immunological indices and reference values for the clinical diagnosis of tuberculosis.MethodsFlow cytometry, sputum smears and computed tomography imaging were used to analyze the distribution characteristics of CD8+CD28− Treg cells in the peripheral blood of patients with PTB and the correlation between CD8+CD28−Treg cells and clinical and immune indices.ResultsThe percentages of CD4+CD25high and CD8+CD28− Treg cells in the peripheral blood of patients with PTB were significantly higher than those in the healthy control (HC) group. Further analysis showed that the percentage of CD4+CD25highTreg cells in the Stage II group was significantly higher than that in the HC group. The percentages of CD4+CD25high and CD8+CD28− Treg cells increased significantly in patients in the Stage II group. The proportion of CD8+CD28− Treg cells was directly proportional to the degree of positivity in sputum smears, while CD4+CD25highTreg cells did not exhibit this trend.The correlations between the percentage of CD4+CD25high and CD8+CD28− Treg cells and the percentage of lymphocyte subsets were examined. The percentage of CD8+CD28− Treg cells was negatively correlated with the percentage of CD4+T cells and positively correlated with the CD8+T cell percentage in the HC and PTB groups. The percentage of CD4 + CD25highTreg cells was positively correlated with the percentage of CD4+T cells only in the PTB group.ConclusionsThis study was the first to show that the proportion of CD8+CD28− Treg cells in the peripheral blood of patients with PTB was significantly increased, and the increase in CD8+CD28− Treg cells was related to the progression of PTB, which may affect the proportion of immune cell subsets by inhibiting the immune response, resulting in the progression of PTB.

Stability and inhibitory function of Treg cells under inflammatory conditions in vitro.

Immunotherapy with transplanted T-regulatory (Treg) cells is currently in use. However, patients have complex internal environments with confounding factors, including the presence of inflammatory cytokines. The present study aimed to detect Treg cell function under simulated inflammatory conditions to provide a foundation for Treg cell-based immunotherapy. CD4+CD25high Treg cells were sorted from peripheral blood mononuclear cells and cultured for 14 days in the presence of recombinant human interleukin-2 (rhIL-2) and anti-CD3/CD28 beads, with or without 25 ng/ml rhIL-6. Next, the absolute count of Treg cells was determined, the stability and activity were detected by measuring the expression levels of forkhead box (Fox)P3 and CD39, and the suppressive function of Treg cells was investigated by assessing the suppression of T-effector cell proliferation by Treg cells after co-culture for 5 days. The number of Treg cells cultured in the presence of 25 ng/ml rhIL-6 for 14 days was reduced by 49.7% when compared with that of cells cultured without rhIL-6. Of the Treg cells continually cultured for 14 days without or with 25 ng/ml rhIL-6, 56.15 and 24.7% expressed FoxP3, respectively. There was no difference in the activity of the FoxP3+ Treg cells after culture for 14 days without or with 25 ng/ml rhIL-6. The suppressive function of Treg cells tended to deteriorate in the presence of rhIL-6. In conclusion, IL-6 inhibited the proliferation and stability of Treg cells, suggesting that administration of increased numbers of Treg cells may be required during Treg cell-based immunotherapy.

Effects of allergen-specific immunotherapy on peripheral blood regulatory T cells and serum concentrations of cytokines and immunoglobulins in horses with allergic dermatitis

The aim of this study was to assess the effect of allergen-specific immunotherapy (ASIT) on the immunological responses of horses. Blood samples were taken from thirty-two horses with allergic dermatitis treated with ASIT and 10 healthy control horses at 0, 3, 6, 9 and 12 months to investigate the evolution of the percentage of regulatory T cells (Treg) in the peripheral blood and the serum levels of cytokines and immunoglobulins. Clinical improvement was appreciated by the majority of the horses' owners (56.6%). No effect of ASIT on CD4+CD25High Treg cells was found during the one year treatment period. No differences in the percentage of CD4+ T cells were observed between the groups, and no effects of ASIT over time were observed. The percentage of CD25+ T cells was always higher in the ASIT group (17.9 ± 11.3%) than in the control group (7.3 ± 4.4%, p < 0.001). We did not detect any effect of ASIT on the serum levels of TGF-β, IL-10 and IFN-γ or on the serum concentrations of IgA and IgG4. A reduction in the serum levels of total IgE in the horses with allergic dermatitis was observed at the 6th month (p < 0.05), but increased again at the end of the study. The results indicate that immunotherapy was insufficient to induce significant changes that could indicate T cell tolerance, a shift in cytokine production to more protective Th1 cells. More studies are needed with new vaccine compositions and administration protocols to improve the immunological responses of the horses with allergic dermatitis.

Cytokine Expression and Alloreactivity in a Humanized Mouse Transplant Arteriosclerosis Model Reflects Primary Graft Dysfunction in Lung Transplant Recipients

Purpose An important cause of early mortality after lung transplantation is primary graft dysfunction (PGD). In a humanized mouse model, we studied the impact of the baseline condition of the endothelium of lung donors in combination with recipient PBMC with or without PGD on the development of luminal occlusion. Methods Segments of arteries were implanted into the aorta of immunodeficient mice. Mice were divided into six treatment groups: group A mice with aortic vessels from PGD grade ≥ 2 (PGD+) patients were reconstituted with the respective allogeneic recipient PBMC, group B mice additionally received recipient CD4+CD25high Treg cells. Group C mice with aortic vessels from PGD grade ≤ 1 (PGD-) patients received recipient PBMC and group D mice additionally CD4+CD25high Treg cells. Control group E mice received arteries from PGD+, group F mice from PGD- patients, both without PBMC reconstitution. Luminal occlusion was quantified histologically and systemic immune responses by quantification of plasma cytokine and chemokines etc. Results Luminal occlusion of aortic vessels as sign of TA was significantly more severe in PGD+ group E compared to those from PGD- group F control mice in the absence of PBMC which was paralleled by higher IFN-γ (p Conclusion We conclude that a pre-existing Th1 response of the donor endothelial system contributes to PGD development, which is further enhanced by alloreactive T cells.

Role of cluster of differentiation 39 (CD39) in the immunosuppressive status associated Acute myeloid leukemia (AML)

Background: CD39 is an integral membrane protein (ectoenzyeme) that phosphohydrolyzes ATP in a Ca2+- and Mg2+-dependent fashion to yield AMP, Can be viewed as immunological switch, and is expressed in spleen, thymus, lung, and placenta, and in these organs it is associated primarily with endothelial cells and immune cell populations, such as B cells, NK cells, dendritic cells, Langerhans cells, monocytes, macrophages, mesangial cells, neutrophils, and regulatory T cells. Aim of the study is to assess the expression of CD39 on CD4+ CD25high T-reg cells in patients with acute myeloid leukemia to identify its role in immunosuppressive status of AML patients. Methods: Flow cytometric analysis of T-reg cells using CD4 CD25 and CD39 in 60 AML patients and 15 apparently healthy controls with matched age and sex was done. Results: There was a significant difference between cases and controls as regards expression of CD39 on T-helper cells. A highly significant elevation of expression of CD39 has been obtained on T-reg cells in AML patients compared with Controls with increased expression of CD39 on T-reg cells than on helper T cells. Conclusion: CD39 plays a significant role in immunosuppressive status of AML patients particularly with expressing the receptor of α chain of IL-2 (CD25)

Astragalus Oral Solution Ameliorates Allergic Asthma in Children by Regulating Relative Contents of CD4+CD25highCD127low Treg Cells.

Objective: To explore the effects of Astragalus oral solution (AOS) on allergic asthma in children by investigating relative contents of CD4+CD25highCD127low Treg cells.Methods: The contents of Astragaloside A in AOS were detected by using HPLC. Eighty children with allergic asthma were recruited from February 2016 to June 2017, and randomly assigned into the control group (received placebo, 0.1% quinine chloride in deionized water, daily) and the AOS group (received 10 mL AOS daily). After 6-month treatment, therapeutic results were compared between the two groups. Serum levels of IL-10 and TGF-beta, Th1 cytokines (IL-2 and IFN-γ), and Th2 cytokines (IL-4 and IL-6) were measured by using ELISA kits. Relative contents of CD4+CD25highCD127low Treg cells were determined by using flow cytometry.Results: Astragaloside A was the main ingredient of AOS with 0.216 ± 0.027 mg/mL from six-batch samples. After 6-month therapy, the AOS group showed improved forced expiratory volume in 1 s (FEV1) and the Pediatric Asthma Quality of Life Questionnaire (PAQLQ) scores compared with the control group (P < 0.05). Serum level of IL-10 was higher and the levels of TGF-beta, Th1 cytokines (IL-2 and IFN-γ), and Th2 cytokines (IL-4 and IL-6) were lower in the AOS group than in the control group (P < 0.05). AOS treatment increased the percentage of gated CD4+ T cells, CD4+CD25+ T cells, CD4+CD25high Treg cells, CD4+CD25+FoxP3+ Treg cells and CD4+CD25highCD127low Treg cells when compared with the control group (P < 0.05).Conclusions: Astragaloside A was the main component of AOS, and AOS ameliorated allergic asthma in children by regulating relative contents of CD4+CD25highCD127low Treg cells.

Novel Effector Phenotype of Tim-3+ Regulatory T Cells Leads to Enhanced Suppressive Function in Head and Neck Cancer Patients.

Purpose: Regulatory T (Treg) cells are important suppressive cells among tumor-infiltrating lymphocytes (TIL). Treg cells express the well-known immune checkpoint receptor PD-1, which is reported to mark "exhausted" Treg with lower suppressive function. T-cell immunoglobulin mucin (Tim)-3, a negative regulator of Th1 immunity, is expressed by a sizeable fraction of TIL Tregs, but the functional status of Tim-3+ Tregs remains unclear.Experimental Design: CD4+CTLA-4+CD25high Treg cells were sorted from freshly excised head and neck squamous cell carcinoma (HNSCC) TIL based on Tim-3 expression. Functional and phenotypic features of these Tim-3+ and Tim-3- TIL Tregs were tested by in vitro suppression assays and multi-color flow cytometry. Gene-expression profiling and NanoString analysis of Tim-3+ TIL Treg were performed. A murine HNSCC tumor model was used to test the effect of anti-PD-1 immunotherapy on Tim-3+ Treg.Results: Despite high PD-1 expression, Tim-3+ TIL Treg displayed a greater capacity to inhibit naïve T-cell proliferation than Tim-3- Treg. Tim-3+ Treg from human HNSCC TIL also displayed an effector-like phenotype, with more robust expression of CTLA-4, PD-1, CD39, and IFN-γ receptor. Exogenous IFN-γ treatment could partially reverse the suppressive function of Tim-3+ TIL Treg. Anti-PD-1 immunotherapy downregulated Tim-3 expression on Tregs isolated from murine HNSCC tumors, and this treatment reversed the suppressive function of HNSCC TIL Tregs.Conclusions: Tim-3+ Treg are functionally and phenotypically distinct in HNSCC TIL, and are highly effective at inhibiting T-cell proliferation despite high PD-1 expression. IFN-γ induced by anti-PD-1 immunotherapy may be beneficial by reversing Tim-3+ Treg suppression. Clin Cancer Res; 24(18); 4529-38. ©2018 AACR.

CD4+ Foxp3+ regulatory T cell-mediated immunomodulation by anti-depressants inhibiting acid sphingomyelinase.

Acid sphingomyelinase (ASM) is the rate-limiting enzyme cleaving sphingomyelin into ceramide and phosphorylcholin. CD4+ Foxp3+ regulatory T (Treg) cells depend on CD28 signaling for their survival and function, a receptor that activates the ASM. Both, basal and CD28-induced ASM activities are higher in Treg cells than in conventional CD4+ T (Tconv) cells. In ASM-deficient (Smpd1-/-) as compared to wt mice, membranes of T cells contain 7-10-fold more sphingomyelin and two- to three-fold more ceramide, and are in a state of higher order than membranes of T cells from wt mice, which may facilitate their activation. Indeed, the frequency of Treg cells among CD4+ T cells in ASM-deficient mice and their suppressive activity in vitro are increased. Moreover, in vitro stimulation of ASM-deficient T cells in the presence of TGF-β and IL-2 leads to higher numbers of induced Treg cells. Pharmacological inhibition of the ASM with a clinically used tricyclic antidepressant such as amitriptyline in mice or in tissue culture of murine or human T cells induces higher frequencies of Treg cells among CD4+ T cells within a few days. This fast alteration of the balance between T cell populations in vitro is due to the elevated cell death of Tconv cells and protection of the CD25high Treg cells by IL-2. Together, these findings suggest that ASM-inhibiting antidepressants, including a fraction of the serotonin re-uptake inhibitors (SSRIs), are moderately immunosuppressive and should be considered for the therapy of inflammatory and autoimmune disorders.

Effect of TGF-β1 on blood CD4+CD25high regulatory T cell proliferation and Foxp3 expression during non-small cell lung cancer blood metastasis.

Metastatic circulating tumor cells in non-small cell lung cancer (NSCLC) metastasis have been reported to be associated with an immune response. The present study aimed to provide a theoretical basis for the immunomodulatory processes during NSCLC blood metastasis. NSCLC blood and normal peripheral blood mononuclear cells (PBMCs) were collected. The quantity of cluster of differentiation (CD)4+CD25high regulatory T (Treg) cells and the intracellular forkhead box protein 3 (Foxp3) expression in CD4+CD25high Treg cells were determined by flow cytometry. Furthermore, the effect of transforming growth factor β1 (TGF-β1) on NSCLC blood CD4+CD25+ Treg cell proliferation was explored by activating blood mononuclear cells with an anti-CD3 monoclonal antibody, interleukin-2 and different doses of TGF-β1. Reverse transcription-quantitative polymerase chain reaction assays were used to detect the mRNA expression of Foxp3. Carboxyfluorescein succinimidyl ester staining was used to analyze the proliferation dynamics of lymphocyte subsets. Results indicate that the proportion of CD4+ T cells in the blood of patients with NSCLC was significantly higher compared with normal peripheral blood (P<0.01). Foxp3 expression in NSCLC blood Treg cells was significantly decreased compared with normal peripheral blood (P<0.01). NSCLC blood mononuclear cells treated with TGF-β1 at 1, 5 and 25 ng/ml significantly induced Foxp3 expression in CD4+CD25+ Treg cells compared with the control group (P<0.05). The proportion of CD4+CD25+ Treg and CD8+ T cells were elevated in generation 6, 7, 8 after 6 days of TGF-β1 treatment compared with untreated cells. The proportion of CD4+CD25+ Treg and CD8+ T cells were elevated in generation 8, 9 and with TGF-β1 treatment after 8 days compared with untreated cells. These results indicate that CD4+CD25+ Treg cells proliferate at a greater rate compared with CD8+ T cells after 4, 6 or 8 days of treatment. The proportion of CD4+CD25high Treg cells in NSCLC blood was significantly higher (P<0.05) compared with normal peripheral blood. The number of Foxp3+ T cells was significantly lower (P<0.05) compared with normal peripheral blood. The data presented in this study suggest that NSCLC blood CD4+CD25high Treg cells are functionally immature and that TGF-β1 may promote maturation.

Novel IL-2Ralpha (CD25)-Binding RNA Aptamer to Target T Regulatory Cells

Background:RNA aptamers are short RNA molecules that bind to antigens and ligands in a manner analogous to antibodies. RNA aptamers are being evaluated as clinical therapeutic agents based on their advantages and flexibility as cell targeting agents. Here, we report development and evaluation of a novel human IL-2Ralpha (CD25)-binding RNA aptamer that can be used to target T regulatory (Treg) cells.Methods:A. RNA aptamers:A whole cell SELEX strategy was designed to enrich for RNA aptamers that specifically bind to CD4+CD25high Tregs cells, but not to lineage-related CD4+CD25-T effector (Teff) cells. Selection began with a library of random RNA aptamers (SEL2-N20) with an expected structural diversity of ~1012. The aptamer pool was pre-cleared of aptamers that bound to common T cell antigens using Teff cells before aptamers were positively selected that bound to Tregs from the same donor. This process was repeated for eight successive enrichment rounds, each round using T cells from different donor. Enriched aptamers were sequenced and the top enriched aptamer (Tr-1) was characterized for its antigen specificity.B. Target antigen identification:The specificity of Tr-1 aptamer and its structural mutants was evaluated by testing its binding to recombinant CD25 by RT-qPCR.C. Targeting Tregs with chimeric Tr-1:Chimeric Treg-targeting reagents were created by linking Tr-1 aptamers with cytotoxic Saporin (Tr-1-Sap) or with Treg-inhibiting Foxp3 silencing RNA (Tr-1-Foxp3 siRNA). Unfractionated CD4+ T cells or Tregs were treated with Tr-1-Sap or Tr-1-Foxp3 siRNA. Reduction in Treg percentage (for Tr-1-Sap treatment) or Foxp3 mRNA levels (for Tr-1-siRNA treatment) was determined by flow cytometry or RT-qPCR, respectively.Results:A. Treg-binding RNA aptamers:A large panel of Treg-binding RNA aptamers was identified using whole cell SELEX and were synthesized. Most of the selected aptamers, particularly Tr-1, bound to CD25high Treg cells to a greater degree than to CD25low Teff cells (Figure 1).B. Target antigen identification:Tr-1 bound to human CD25 (IL-2Ralpha) protein while a control aptamer (C-248) did not. Structural mutants of Tr-1 that had lost Treg binding ability showed significantly reduced binding to CD25 protein (Figure 2).C. Treg targeting with chimeric Tr-1:Treg-targeting agents created with Tr-1 aptamers successfully delivered toxic Saporin and Foxp3 siRNA into Tregs.a. CD4+ T cells treated with Tr-1-Sap had a decrease in the percentage of CD25+ Treg population as determined by flow cytometry.b. Enriched human Tregs treated with Tr-1-Foxp3 siRNA chimera showed reduction in their Foxp3 mRNA levels.Conclusion and significance:RNA aptamers that target human Tregs were identified. The most predominant Treg-binding aptamer, Tr-1, binds to human IL-2Ralpha/CD25, a clinically-targeted molecule expressed by Tregs. Chimeric reagents based on Tr-1 aptamer effectively targeted Tregs signifying their potential use as novel immunomodulators. Ongoing studies are further exploring the significance of Tr-1 aptamer as a diagnostic agent and as a therapeutic modulator of Treg activity. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CD4(+)CD25(High) Treg cells in HIV/HTLV co-infected patients with neuropathy: high expression of Alpha4 integrin and lower expression of Foxp3 transcription factor.

BackgroundRegulatory CD4 T cells (Tregs) are critical in maintaining the homeostasis of the immune system. Quantitative or phenotypic alterations and functional impairment of Tregs have been associated with the development of pathologies including those of the central nervous system. Individuals with HIV-1/HTLV-1 co-infection are more prone to develop neurological complications. The aim of this study was to characterize phenotypically Treg cells in HIV-1/HTLV-1 co-infected Mozambican individuals presenting neurological symptoms.MethodsA cross-sectional study was conducted among HIV-infected individuals presentingneurological symptoms, with and without HTLV co-infection, and blood donors. Peripheral bloodmononuclear cells were stained with monoclonal antibodies conjugated with fluorochromes to quantifyTregs and activated T cells by four colors flow cytometry.ResultsHigher Treg cell frequency (10.6 %) was noted in HIV-1/HTLV-1 co-infected group with neurological symptoms when compared to HIV-1 mono-infected group with neurological symptoms (0.38 %, p = 0.003) and control group (0.9 %, p = 0.0105). An inverse correlation between Foxp3 and CD49d expression was observed in all study groups (rh = −0.71, p = 0.001). In addition, increased levels of Treg cells in co-infected patients were strongly associated with total activated CD4 T cells (rh = 0.8, p = 0.01).ConclusionTreg cells in co-infected patients present phenotypic alterations and might have dysfunction marked by low expression of Foxp3 and increased expression of molecules not frequently seen on Treg cells, such as CD49d. These alterations may be related to (1) changes in Treg cell trafficking and migration, possibly making those cells susceptible to HIV infection, and (2) inability to control the activation and proliferation of effector T lymphocytes.

Recombinant IL-21 and anti-CD4 antibodies cooperate in syngeneic neuroblastoma immunotherapy and mediate long-lasting immunity.

IL-21 is an immune-enhancing cytokine, which showed promising results in cancer immunotherapy. We previously observed that the administration of anti-CD4 cell-depleting antibody strongly enhanced the anti-tumor effects of an IL-21-engineered neuroblastoma (NB) cell vaccine. Here, we studied the therapeutic effects of a combination of recombinant (r) IL-21 and anti-CD4 monoclonal antibodies (mAb) in a syngeneic model of disseminated NB. Subcutaneous rIL-21 therapy at 0.5 or 1 μg/dose (at days 2, 6, 9, 13 and 15 after NB induction) had a limited effect on NB development. However, coadministration of rIL-21 at the two dose levels and a cell-depleting anti-CD4 mAb cured 28 and 70 % of mice, respectively. Combined immunotherapy was also effective if started 7 days after NB implant, resulting in a 30 % cure rate. Anti-CD4 antibody treatment efficiently depleted CD4(+) CD25(high) Treg cells, but alone had limited impact on NB. Combination immunotherapy by anti-CD4 mAb and rIL-21 induced a CD8(+) cytotoxic T lymphocyte response, which resulted in tumor eradication and long-lasting immunity. CD4(+) T cells, which re-populated mice after combination immunotherapy, were required for immunity to NB antigens as indicated by CD4(+) T cell depletion and re-challenge experiments. In conclusion, these data support a role for regulatory CD4(+) T cells in a syngeneic NB model and suggest that rIL-21 combined with CD4(+) T cell depletion reprograms CD4(+) T cells from immune regulatory to anti-tumor functions. These observations open new perspectives for the use of IL-21-based immunotherapy in conjunction with transient CD4(+) T cell depletion, in human metastatic NB.

Effects of chimeric B7 antibodies on mononuclear cells in ankylosing spondlitis

Objective To study the effects of chimeric B7 antibodies on mononuclear cells from patients with ankylosing spondylitis (AS).Methods Chimeric antibodies of ch4E5 and chlD1 against B7 with a final concentration of 5 μg/ml were respectively added into culture media of mononuclear cells from patients with AS.Two other groups including huIgG-treated and untreated culture medium of mononuclear cells from patients with AS were set up as the controls.The culture medium of mononuclear cells from healthy subjects were prepared as normal control group.The disparities of mononuclear cell death in culture among each group were analyzed by using micro-lymphocyte cytotoxicity test (MLCT).The expression of membrane molecules were analyzed by FCM.The concentrations of cytokines in culture media were measured by ELISA.Results Both groups treated with the chimeric antibody showed lower scores for cell death than untreated group as indicated by MLCT (P＜0.01).FCM analysis demonstrated that both CD4/CD8 ratio (P ＜0.05) and membrane expression of CD154 (P＜0.01) decreased in chimeric antibody treated groups,but the number of CD4+CD25high Treg cells (P＜0.01) increased as compared with those in untreated group.The concentrations of IL-2 and TNF-α were down-regulated (P＜0.05) in chimeric antibody treated groups,while IL-4 and TGF-β were up-regulated (P＜0.02) as compared with those in untreated group.Conclusion Chimeric B7 antibodies might affect the expression of T cell membrane molecules and the cytokine production by mononuclear cells through B7/CD28 and CD4O/CD154 signaling pathways in patients with AS. Key words: Chimeric antibody; B7/CD28 signaling pathway; CD40/CD154 signaling pathway; Cytokine; Ankylosing spondlitis

Higher frequency of CD4+CD25high Treg cells in hemophilia patients with factor VIII inhibitor

The production of factor VIII inhibitor antibodies remains the most costly and serious complication in replacement therapy of hemophilia A. We investigated the clinical significance of CD4(+)CD25(high) T regulatory (Treg) cells in hemophilia patients. Our trial included 6 severe hemophilia A patients with factor VIII inhibitors, 6 hemophilia patients without inhibition of factor VIII, and 6 healthy persons (controls). Plasma factor VIII: c was measured by clotting assay. Peripheral blood samples were examined using mutiparameter flow cytometry with fluorescent-labeled monoclonal antibodies. Plasma levels of IFN-γ, IL-2, IL-10, and TGF-β were measured by ELISA. The frequency of CD4(+)CD25(high) Treg cells in CD4(+) cells was 1.07 ± 0.38% in inhibitor patients and 0.57 ± 0.14% in non-inhibitor patients. The proportion of Treg cells in healthy controls was similar to that of the non-inhibitor patients. However, there were significant differences between the inhibitor and non-inhibitor patients in levels of IFN-γ, IL-2, IL-10, and TGF-β. We conclude that the proportions of Treg cells and the concentrations of T cell cytokines in inhibitor patients are higher than those in non-inhibitor patients. The increased number of Treg cells and increased T-cell cytokines may be related to the development and efficiency of the factor VIII inhibitor.

Long acting propranolol and HSP-70 rich tumor lysate reduce tumor growth and enhance immune response against fibrosarcoma in Balb/c mice.

Noradrenaline (NA), the principal neurotransmitter released from sympathetic nerve terminals, influences T-cell maturation, not only directly in developing T cells, but also indirectly, by acting on the thymic nonlymphoid cells. In vitro and in vivo studies have demonstrated the anti-proliferative, anti-migratory, anti-angiogenic and cytotoxic properties of propranolol, β-AR blocker, against various cancers. To evaluate the effect of propranolol on efficacy of HSP-70 rich lysate vaccine in immunotherapy of fibrosarcoma. Mouse fibrosarcoma WEHI-164 cells were used to immunize tumor-bearing mice with or without propranolol and HSP-70. Splenocytes proliferation, cytotoxicity activity of the splenocytes, naturally occurring CD4+ CD25high T-reg cells and IFN-γ and IL-4 secretion as well as tumor size, were assessed to describe the anti-tumor immune response. A significant increase in the level of IFN-γ in the mice vaccinated with WEHI-164 cells enriched with HSP-70 and co-treated with propranolol was observed compared to controls. However, HSP enrichment or propranolol treatment alone did not enhance the immune response as measured by the level of IFN-γ. Likewise, a decrease in tumor growth in the test group (p<0.01) and a significant increase in CTL activity (p<0.05) was observed. HSP enriched vaccine shows anti-tumor activity, probably due to the modulation of immune responses.

Differential Expression of Rac-1, Cxcr4 and CCR5 on CD4 T-Cells at Different Stages of HIV-1 Disease Relate To Its Progression in Therapy Naive Individuals

Background: HIV-1 uses different co-receptors (CCR5/CXCR4) at different stages of disease to enter target cells. Any information in understanding this mechanism has important implication on both, the rate of disease progression and our understanding of the immunopathogenesis of this disease. Methods: This cross-sectional study involved 67 treatment-naive HIV-1 infected individuals. The expression of CCR5 and CXCR4 on T-cell subsets was evaluated in the peripheral blood of patients at different stages of HIV disease, active pulmonary tuberculosis (PTB), HIV/PTB co-infection and healthy controls, to assess their possible role or association with change in Treg frequency during the disease progression. Furthermore, we investigated the impact of Rac1 expression in relation to functionally active CXCR4 and NF-κB expression. Results: Significantly higher CCR5 expression on CD4+CD25high Treg cells in HIV-1 subjects in early stage of the disease correlated well with initial decrease in Treg frequency. On the contrary, higher CXCR4 expression on CD4+CD25low/negative T-cells (non-Treg cells) in advanced stage of disease explained the shift to X4 type with faster progression to AIDS. This shift is further supported by initial significant decrease in Rac1 expression in early disease followed by returning to normal expression with disease progression. Interestingly, PTB co-infection correlated significantly with increase in CCR5 expression on Treg population only, at the same time favoring increase in CXCR4 expression on CD4+CD25low/negative subset. Conclusion: The study indicates novel inter-relationships of co-receptor expression and their regulatory genes in therapy naive individuals and how the virus manipulates the host machinery to its advantage. The Rac-1 seems to regulate not only the functional conformation of CXCR4, but also the expression of both FoxP3 and NF-κB genes there by affecting the disease progression in HIV subjects. These findings need further attention to look at their clinical implication and disease outcome in a larger study.

Inhibiting effect of Astragalus polysaccharides on the functions of CD4+CD25 highTreg cells in the tumor microenvironment of human hepatocellular carcinoma.

Astragalus polysaccharides (APS), the main active extract from Astragalus membranaceus (a traditional Chinese medicinal herb), is associated with a variety of immunomodulatory activities. The purpose of the present study was to examine the effect of APS on the function of Treg cells in the tumor microenvironment of human hepatocellular carcinoma (HCC) and to identify the pharmacologic mechanism of APS responsible for the anti-chemotactic activity in CD4+CD25highTreg cells in tumor site of HCC. The prevalence of Treg in fresh tissue samples from 31 patients with HCC after radicalhepatectomy was detected. CD4, CD25 and CD127 were selected as Treg cell makers to phenotype cell populations. The expression of FOXp3 mRNA was also analyzed. The migration and proliferation of Treg cells were observed. Interleukin (IL)-4, IL-10, IFN-γ and SDF-1 in cell supernatant were detected. For all tests, functions of Treg cells were evaluated after treatment with APS. APS can inhibit the growth and proliferation of CD4+CD25+Treg cells in vitro in a dose- and time-dependent manner. APS may inhibit CD4+CD25+Treg cells through restoring the cytokine imbalance and reducing the expression of FOXp3 in local HCC microenvironments. SDF-1 played an important role in there recruitment of Treg cells into the tumor microenvironment of HCC. APS might have inhibiting effects on Treg cell migration by blocking SDF-1 or its receptor through the CXCR4/CXCL12 pathway. The increase in numbers of tumor associated Treg cells might play a role in modulation of the immune response against HCC. APS can restore the cytokine balance in the tumor micro environment and suppress the expression of FOXp3 mRNA to inhibit the immune suppressive effects of Treg cells. The application of APS in the tumor microenvironment might act to enhance the anti-tumor effects of the immunotherapy-based methods, and consequently to increase the survival rate in HCC.

Cytomegalovirus-Specific Regulatory and Effector T Cells Share TCR Clonality—Possible Relation to Repetitive CMV Infections

Cytomegalovirus (CMV) infections have a major impact on morbidity and mortality of transplant patients. Among the complex antiviral T-cell response, CMV-IE-1 antigen-specific CD8+ cells are crucial for preventing CMV disease but do not protect from recurring/lasting CMV reactivation. Recently, we confirmed that adoptive transfer of autologous IE-1/pp65-specific T-cell lines was able to combat severe CMV disease; however, the control of CMV infection was only temporary. We hypothesized that CMV-induced regulatory T cells (iTreg) might be related to recurring/lasting CMV infection. In fact, kidney transplant patients with recurring CMV infections expressed enhanced suppression on CMV response. Analysis of in vitro expanded CD4+ epitope-specific cells revealed that CMV-specific CD4+CD25(high) Treg cells functionally suppress CD25(low) effector T cells (Teff) upon epitope-specific reactivation. Their phenotype is similar to iTreg - CD39(high) /Helios-/IL-2(low) /IFNγ(high) /IL-10±/TGFß-LAP±/FOXP3+ and methylated foxp3 locus. Remarkably, in vitro expanded CD4+CD25(high) iTreg share the same dominant TCR-Vβ-CDR3 clones with functionally distinct CD4+CD25(low) Teff. Moreover, the same clones were present in freshly isolated CD4+CD25(high) and CD4+CD25(low) T cells suggesting their in vivo generation. These findings directly demonstrate that Teff and iTreg can differentiate from one "mother" clone with specificity to the same viral epitope and indicate that peripheral iTreg generation is related to frequent antigen appearance.

Cord Blood Derived CD4+CD25high T Cells Become Functional Regulatory T Cells upon Antigen Encounter

Background: Upon antigen exposure, cord blood derived T cells respond to ubiquitous environmental antigens by high proliferation. To date it remains unclear whether these “excessive” responses relate to different regulatory properties of the putative T regulatory cell (Treg) compartment or even expansion of the Treg compartment itself.Methods: Cord blood (>37 week of gestation) and peripheral blood (healthy controls) were obtained and different Treg cell subsets were isolated. The suppressive potential of Treg populations after antigen exposure was evaluated via functional inhibition assays ([3H]thymidine incorporation assay and CFSE staining) with or without allergen stimulation. The frequency and markers of CD4+CD25highFoxP3+ T cells were characterized by mRNA analysis and flow cytometry.Results: Cord blood derived CD4+CD25high cells did not show substantial suppressor capacity upon TCR activation, in contrast to CD4+CD25high cells freshly purified from adult blood. This could not be explained by a lower frequency of FoxP3+CD4+CD25highcells or FOXP3 mRNA expression. However, after antigen-specific stimulation in vitro, these cells showed strong proliferation and expansion and gained potent suppressive properties. The efficiency of their suppressive capacity can be enhanced in the presence of endotoxins. If T-cells were sorted according to their CD127 expression, a tiny subset of Treg cells (CD4+CD25+CD127low) is highly suppressive even without prior antigen exposure.Conclusion: Cord blood harbors a very small subset of CD4+CD25high Treg cells that requires antigen-stimulation to show expansion and become functional suppressive Tregs.

CD4+CD25high Treg cells in peripheral blood during remission and exacerbation of allergic asthma in children

To determine the percentage of CD4+CD25(high) Treg cells in peripheral bloodCD4+ T cells of allergic asthmatic children during disease remission and exacerbation. Peripheral blood mononuclear cells (PBMC) and serum samples were collected from 6- to 11-year-old children with mild-to-moderate allergic asthma (n = 34)and from healthy controls (n = 15). CD4+CD25(high) T cells in PBMC were detected by flow cytometry. Total and specific IgE in serum were analysed by enzyme-amplified chemiluminescence, and IL-2 was measured by ELISA. There was no significant difference in CD4+CD25(high) T-cell proportions between asthmatic children in exacerbation and remission as compared with controls.CD4+CD25(high) T-cell percentages were not correlated with total and specific IgE. IL-2 was elevated in both disease remission and exacerbation but did not correlate significantly with CD4+CD25(high) T-cell percentages. CD4+CD25(high) T-cell proportion in the peripheral blood of total CD4+T cells is not reduced in children with allergic IgE-mediated asthma and does not differ between disease remission and exacerbation.