Treg Cell Counts Research Articles

IκB kinase β inhibitor, IMD-0354, prevents allergic asthma in a mouse model through inhibition of CD4+ effector T cell responses in the lung-draining mediastinal lymph nodes

IκB kinase (IKK) is important for nuclear factor (NF)-κB activation under inflammatory conditions. It has been demonstrated that IMD-0354, i.e. a selective inhibitor of IKKβ, inhibited allergic inflammation in a mouse model of ovalbumin (OVA)-induced asthma. The present study attempts to shed light on the involvement of CD4+ effector (Teff) and regulatory (Treg) T cells in the anti-asthmatic action of IMD-0354. The animals were divided into three groups: vehicle treated, PBS-sensitized/challenged mice (PBS group); vehicle treated, OVA-sensitized/challenged mice (OVA group); and IMD-0354-treated, OVA-sensitized/challenged mice. The analyzed parameters included the absolute counts of Treg cells (Foxp3+CD25+CD4+), activated Teff cells (Foxp3−CD25+CD4+) and resting T cells (CD25−CD4+) in the mediastinal lymph nodes (MLNs), lungs and peripheral blood. Moreover, lung histopathology was performed to evaluate lung inflammation. It was found that the absolute number of cells in all studied subsets was considerably increased in the MLNs and lungs of mice from OVA group as compared to PBS group. All of these effects were fully prevented by treatment with IMD-0354. Histopathological examination showed that treatment with IMD-0354 protected the lungs from OVA-induced allergic airway inflammation. Our results indicate that IMD-0354 exerts anti-asthmatic action, at least partially, by blocking the activation and clonal expansion of CD4+ Teff cells in the MLNs, which, consequently, prevents infiltration of the lungs with activated CD4+ Teff cells. The beneficial effects of IMD-0354 in a mouse model of asthma are not mediated through increased recruitment of Treg cells into the MLNs and lungs and/or local generation of inducible Treg cells.

Circulating level of regulatory T cells in rheumatic heart disease: An observational study

BackgroundThe regulatory T cell (Treg) is essential for prevention of autoimmunity. In a preliminary study, we showed significant deficiency of Tregs (CD4CD25 T cells) in rheumatic heart disease (RHD) patients (an autoimmune disease), but the markers used could not reliably differentiate Treg from nonregulatory conventional T cells (Tcon). The study aim was to reassess the level of circulatory Tregs by using more specific markers. Methods70 adults of RHD and 35 controls were studied. Patients were subdivided according to the extent of left-sided valvular involvement. 35 patients with significant mitral-valve disease only were enrolled in the univalvular group while 35 patents with significant involvement of both mitral and aortic-valves in the multivalvular group. Circulating Treg cell level was determined by flow-cytometry. ResultsLevel of Tregs (CD4+CD25med–highCD127low Foxp3high) in CD4+ T lymphocyte was significantly lower in RHD patients compared to controls (median 0.6% versus 3.2%; p=0.001) with no significant difference in Tcon cells (p=0.94). Within the study group Treg count was significantly lower in patients with multivalvular-disease only (median 0.1% versus 3.2%; p=0.001) with no significant difference in Treg cell count between the univalvular group and control (median 1.9% versus 3.2%, p=0.10). ConclusionThere is significant deficiency of circulating Tregs in patients of chronic RHD and the deficiency is greater in patients with multivalvular than univalvular involvement.

Simultaneous inhibition of JAK and SYK kinases ameliorates chronic and destructive arthritis in mice.

IntroductionDespite the broad spectrum of antirheumatic drugs, RA is still not well controlled in up to 30-50 % of patients. Inhibition of JAK kinases by means of the pan-JAK inhibitor tofacitinib has demonstrated to be effective even in difficult-to-treat patients. Here, we discuss whether the efficacy of JAK inhibition can be improved by simultaneously inhibiting SYK kinase, since both kinases mediate complementary and non-redundant pathways in RA.MethodsEfficacy of dual JAK + SYK inhibition with selective small molecule inhibitors was evaluated in chronic G6PI-induced arthritis, a non-self-remitting and destructive arthritis model in mice. Clinical and histopathological scores, as well as cytokine and anti-G6PI antibody production were assessed in both preventive and curative protocols. Potential immunotoxicity was also evaluated in G6PI-induced arthritis and in a 28-day TDAR model, by analysing the effects of JAK + SYK inhibition on hematological parameters, lymphoid organs, leukocyte subsets and cell function.ResultsSimultaneous JAK + SYK inhibition completely prevented mice from developing arthritis. This therapeutic strategy was also very effective in ameliorating already established arthritis. Dual kinase inhibition immediately resulted in greatly decreased clinical and histopathological scores and led to disease remission in over 70 % of the animals. In contrast, single JAK inhibition and anti-TNF therapy (etanercept) were able to stop disease progression but not to revert it. Dual kinase inhibition decreased Treg and NK cell counts to the same extent as single JAK inhibition but overall cytotoxicity remained intact. Interestingly, treatment discontinuation rapidly reversed such immune cell reduction without compromising clinical efficacy, suggesting long-lasting curative effects. Dual kinase inhibition reduced the Th1/Th17 cytokine cascade and the differentiation and function of joint cells, in particular osteoclasts and fibroblast-like synoviocytes.ConclusionsConcurrent JAK + SYK inhibition resulted in higher efficacy than single kinase inhibition and TNF blockade in a chronic and severe arthritis model. Thus, blockade of multiple immune signals with dual JAK + SYK inhibition represents a reasonable therapeutic strategy for RA, in particular in patients with inadequate responses to current treatments. Our data supports the multiplicity of events underlying this heterogeneous and complex disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0866-0) contains supplementary material, which is available to authorized users.

Postoperative Regulatory T-Cells and Natural Killer Cells in Stage I Nonsmall Cell Lung Cancer Underwent Video-assisted Thoracoscopic Lobectomy or Thoracotomy.

Background:Regulatory T-cells (Treg) play key roles in suppressing cell-mediated immunity in cancer patients. Little is known about perioperative Treg fluctuations in nonsmall cell lung cancer (NSCLC). Video-assisted thoracoscopic (VATS) lobectomy, as a minimal invasive procedure for treating NSCLC, may have relatively less impact on the patient's immune system. This study aimed to observe perioperative dynamics of circulating Treg and natural killer (NK) cell levels in NSCLC patients who underwent major lobectomy by VATS or thoracotomy.Methods:Totally, 98 consecutive patients with stage I NSCLC were recruited and assigned into VATS or thoracotomy groups. Peripheral blood samples were taken on 1-day prior to operation, postoperative days (PODs) 1, 3, 7, 30, and 90. Circulating Treg and NK cell counts were assayed by flow cytometry, defined as CD4+CD25+CD127low cells in CD4+ lymphocytes and CD56+16+CD3− cells within CD45+ leukocytes respectively. With SPSS software version 21.0 (SPSS Inc., USA), differences between VATS and thoracotomy groups were determined by one-way analysis of variance (ANOVA), and differences between preoperative baseline and PODs in each group were evaluated by one-way ANOVA Dunnett t-test.Results:In both groups, postoperative Treg percentages were lower than preoperative status. No statistical difference was found between VATS and thoracotomy groups on PODs 1, 3, 7, and 30. On POD 90, Treg percentage in VATS group was significantly lower than in thoracotomy group (5.26 ± 2.75 vs. 6.99 ± 3.60, P = 0.012). However, a higher level of NK was found on all PODs except on POD 90 in VATS group, comparing to thoracotomy group.Conclusions:Lower Treg level on POD 90 and higher NK levels on PODs 1, 3, 7, 30 in VATS group might imply better preserved cell-mediated immune function in NSCLC patients, than those in thoracotomy group.

In MCA-induced sarcoma models, booster vaccination with GM-CSF-secreting whole-cell tumor vaccines maintain or improve efficacy but may alter trafficking of tumor-specific T cells (VAC13P.1128)

Abstract Previously, we reported that while a single vaccination effectively generated therapeutic tumor-specific T cells for adoptive immunotherapy (AIT), repeated vaccination with the poorly immunogenic B16BL6 melanoma, modified to secrete GM-CSF, induced high levels of CD4+FoxP3+ T regulatory (Treg) cells and AIT with spleen cells was non-therapeutic. The effect could be overcome by transient depletion of CD4+ T cells, confirming the effect of Treg cells. Here experiments were performed to test whether this suppressive effect was observed in active-specific protection in 2 sarcoma models, MCA-304 and MCA-310. Mice were vaccinated once or thrice with either irradiated parental sarcoma or that sarcoma expressing GM-CSF. We did not observe significant changes in percentage or total count of splenic Treg cells under any conditions, however, tumor-specific IFN-γ release decreased in mice thrice vaccinated with GM-CSF-secreting tumors compared to parental (p < 0.001). Importantly, multiple vaccination offered tumor protection that was as good or better than single vaccination. In the most strongly immunogenic tumor, MCA-304, both single and triple vaccination with parental or GM-CSF-secreting tumors provided 100% protection. However, in the weakly immunogenic, MCA-310 sarcoma, multiple vaccinations improved effectiveness with the greatest effect seen with the GM-CSF-secreting MCA-310. Our results provide a rationale for the continued use of GM-CSF as a cancer vaccine adjuvant.

Immune Reconstitution of CD4+T Cells after Allogeneic Hematopoietic Stem Cell Transplantation and its Correlation with Invasive Fungal Infection in Patients with Hematological Malignancies

To explore the immune reconstitution of CD4+T cells after allogeneic hematopoietic stem cell transplantation (Allo-HSCT) and its relationship with invasive fungal infection (IFI) in patients with hematological malignancies. Forty-seven patients with hematological malignancies undergoing Allo- HSCT in Binzhou Medical University Hospital from February, 2010 to October, 2014 were selected. At 1, 2 and 3 months after transplantation, the immune subpopulations and concentration of cytokines were assessed respectively using flow cytometry (FCM) and enzyme linked immunosorbent assay (ELISA). The incidence of IFI after transplantation and its correlation with immune reconstitution of CD4+T cells were investigated. The number of CD4+T cells and immune subpopulations increased progressively after transplantation as time went on, but the subpopulation cell count 3 months after transplantation was still significantly lower than in the control group (p<0.01). In comparison to the control group, the levels of interleukin-6 (IL-6) and IL-10 after transplantation rose evidently (p<0.01), while that of transforming growth factor-β (TGF-β) was decreased (p<0.01). There was no statistically significant difference level of interferon-γ (IFN-γ) (p>0.05). The incidence of IFI was 19.2% (9/47), and multivariate logistic regression revealed that IFI might be related to Th17 cell count (p<0.05), instead of Th1, Th2 and Treg cell counts as well as IL-6, IL-10, TGF-β and IFN-γ levels (p>0.05). After Allo-HSCT, the immune reconstitution of CD4+T cells is delayed and Th17 cell count decreases obviously, which may be related to occurrence of IFI.

FoxP3+ regulatory T cells in the peripheral blood and synovial fluid of patients with rheumatoid arthritis

Human regulatory T (Treg) cells expressing the transcription factor forkhead box P3 (FoxP3) are indispensable for self-tolerance and immune homeostasis. Abnormal Treg cell counts and impaired function have been implicated in the development of various autoimmune diseases. The Treg cell counts in the peripheral blood of subjects with rheumatoid arthritis (RA) vary, probably due to heterogeneity of the Treg cells. Recent studies demonstrated that human FoxP3+ Treg cell populations actually include three functionally distinct subpopulations, and that CD45RA-FoxP3high effector Treg cells are significantly decreased in the peripheral blood of patients with RA. On the other hand, it is generally agreed that the frequency of FoxP3+ Treg cells in synovial fluid is higher in patients with RA than in healthy controls. Although these findings were initially considered paradoxical, a recent study revealed that in RA patients, most of the cells in the FoxP3+ Treg population of the synovial fluid are actually non-suppressive cytokine-producing CD45RA-FoxP3low non-Treg cells. The imbalance between FoxP3+ Treg cell subpopulations in the circulation and at inflammation sites may contribute to the pathogenesis of RA.

Changes of circulating CD4(+)CD25(+)CD127(low) regulatory T cells in patients with acute coronary syndrome and its significance.

The aim of this study was to investigate the changes of circulating CD4(+)CD25(+)CD127(low) regulatory T cells (Treg) in patients with acute coronary syndrome (ACS) and its significance. The experiment was divided into three groups: ACS (48 patients), stable angina pectoris (SAP) (24 patients), and normal controls (24 subjects). The CD4(+)CD25(+)CD127(low) Treg cell counts were tested by flow cytometry, and the levels of high-sensitivity C-reactive protein (hs-CRP) and peripheral blood leukocytes (PWBCs) were determined in the peripheral blood of each group; comparisons were made among groups. The frequency of CD4(+)CD25(+)CD127(low) to CD4(+) cell in the ACS group (3.18 ± 1.76%) was significantly lower than those observed in control (5.64 ± 1.63%) and SAP (5.60 ± 1.56%) groups (F = 25.247, P < 0.01), while the hs-CRP and PWBC levels in the ACS group were significantly higher than those in the control group (P < 0.05). In addition, the reduced frequency of CD4(+)CD25(+)CD127(low) to CD4(+) cells was negatively correlated with the increased hs-CRP and PWBC counts by correlation analysis, and the related coefficients (r) were -0.518 and -0.311, respectively (P < 0.01). These findings indicate that the decrease of the frequency of Treg cells in the peripheral blood of patients with ACS might destroy the balance of tolerance of the peripheral immune system and might activate inflammation, thus participating in the occurrence and development of the pathological processes of atherosclerosis.

Abstract A24: Investigation of the spatial heterogeneity of specific immune cell phenotypes in the tumor microenvironment of follicular lymphoma

Abstract Background. Tumor-infiltrating lymphocytes (TILs) are present in the tumor microenvironment of many cancers, with consequent tumor immunogenicity and association with survival. In particular, increased levels of regulatory T cells (Tregs) are associated with poorer prognosis in some cancers. However, given the complexity of TIL phenotype their visualization in situ is difficult, and impossible without multiplex immunophenotyping. An understanding of both the phenotype and spatial distribution of TILs in situ within the tumor microenvironment would be advantageous to understanding their role in tumour immunobiology, especially given growing interest in tumour immunotherapy. Here we present a multi-marker, computer-aided method for analysing the distributions of CD3/FOXP3 (Treg) and CD3/CD69 (Tact) T cells in follicular lymphoma sections using a multispectral imaging (MSI) and automated analysis approach. An hypothesized interaction distance (HID) analysis was used to determine whether the spatial patterns of Tregs and Tacts were prognostically significant Design. A single section of a tissue microarray containing triplex follicular lymphoma cores from 40 subjects [24 male, 16 female, age 35 to 75 years at diagnosis, median 55 years , 2- 171 months follow-up] was stained for CD3, FOXP3, CD69 and hematoxylin. Each core was imaged using MSI and the individual staining of each marker separated from each other using spectral unmixing. CD3+ TILs were located using automated image analysis. The FOXP3 and CD69 status of each CD3+ TIL was then determined and the spatial distributions of the CD3/FOXP3 and CD3/CD69 cells were used as input into the HID analysis. Results. Multiplexed IHC staining, MSI and automated per-cell quantitative analysis was successful. Kaplan-Meier analysis demonstrated favourable outcome with higher numbers of CD3+, CD3+/FOXP3+ and CD3+/CD69+ cells. HID analysis demonstrated the association of favourable outcome with a high entropy , representative of a diffuse spatial pattern, of FOXP3+ and CD69+ positive T cells. Conclusion. In this study we report that higher Treg cell counts in a diffuse pattern was associated with favorable prognosis. This supports the importance of Tregs in the tumour microenvironment. It is pertinent to mention that contradictory findings are routinely reported from studies investigating the role of Tregs in solid and haematological malignancies. This is due to the complex interactions between pro-/anti- tumour immune factors present in the tumour microenvironment. The resultant effects are due to the summation of the activities of these factors. It is therefore even more relevant that a method such as exhibited here, capable of defining and measuring the effect on patient outcome of the spatial patterns of multiple cellular phenotypes in the tumor microenvironment is available. Citation Format: Lilli Nelson, James Mansfield, Roslyn Lloyd, Chris van der Loos, Chris Rose, Richard Byers. Investigation of the spatial heterogeneity of specific immune cell phenotypes in the tumor microenvironment of follicular lymphoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A24. doi:10.1158/1538-7445.CHTME14-A24

A study for proposal of use of regulatory T cells as a prognostic marker and establishing an optimal threshold level for their expression in chronic lymphocytic leukemia

Although regulatory T cells (Tregs) have been extensively studied in chronic lymphocytic leukemia, there is no uniform guideline or consensus regarding their use as a prognostic marker. This study describes the methodology used to develop an optimal threshold level for Tregs in these patients. Treg levels were assessed in the peripheral blood of 130 patients and 150 controls. Treg frequencies were linked to established prognostic markers as well as overall survival and time to first treatment. The cut-offs for Treg positivity were assessed by receiver operating characteristic (ROC) analysis. A cut-off of 5.7% for Treg cell percentage and of 35 cells/μL for absolute Treg cell count were determined as optimal in patients with CLL along with a median Treg percentage of 15.5% used to separate patients with low- and high-risk disease. The experiments presented here will possibly aid in the use of Treg frequencies as a potential prognostic marker in CLL.

Abstract 1647: Favorable diffuse prognostic pattern of FOXP3+ and CD69+ T cells in follicular lymphoma demonstrated using automated imaging and analysis

Abstract Background. In many cancers, tumor-infiltrating lymphocytes (TILs) indicate levels of tumor immunogenicity and are a strong predictor of survival. In particular, increased levels of regulatory T cells (Tregs) are associated with poorer prognosis in some cancers. However, visual TIL assessment cannot easily determine the phenotype of lymphocyte in situ. An understanding of both the phenotype and spatial distribution of TILs in situ within tumor regions would be advantageous. Here we present a multi-marker, computer-aided prognostication method for analysing the distributions of CD3/FOXP3 (Treg) and CD3/CD69 (Tact) T cells in follicular lymphoma sections using a multispectral imaging (MSI) and automated analysis approach. An hypothesized interaction distance (HID) analysis was used to determine whether the spatial patterns of Tregs and Tacts was prognostically significant. Design. A single section of a tissue microarray containing triplex follicular lymphoma cores from 40 subjects [24 male, 16 female, age 35 to 75 years at diagnosis, median 55 years , 2- 171 months follow-up] was stained for CD3, FOXP3, CD69 and hematoxylin. Each core was imaged using MSI and the individual staining of each marker separated from each other using spectral unmixing. CD3+ TILs were located using automated image analysis. The FOXP3 and CD69 status of each CD3+ TIL was then determined and the spatial distributions of the CD3/FOXP3 and CD3/CD69 cells were used as input into the HID analysis. Results. Multiplexed IHC staining, MSI and automated per-cell quantitative analysis was successful. Kaplan-Meier analysis demonstrated favorable outcome with higher numbers of CD3+, CD3+/FOXP3+ and CD3+/CD69+ cells. HID analysis demonstrated the association of favorable outcome with a high entropy / diffuse pattern of FOXP3+ and CD69+ positive T cells. Conclusion. In this study we report that higher Treg cell counts in a diffuse pattern was associated with favorable prognosis. This supports the importance of Tregs in the tumor microenvironment. It is pertinent to mention that contradictory findings are routinely reported from studies investigating the role of Tregs in solid and haematological malignancies. This is due to the complex interactions between pro-/anti- tumor immune factors present in the tumor microenvironment. The resultant effects are due to the summation of the activities of these factors. It is therefore even more relevant that a method such as exhibited here, capable of defining, and measuring the effect on biological behaviour, in this case patient outcome, of cellular pattern is available, as demonstrated for the HID method in the present study. Citation Format: James Mansfield, Lilli Nelson, Roslyn Lloyd, Chris van der Loos, Ken Oguejiofor, Lia Menasce, Kim Linton, Chris Rose, Richard J. Byers. Favorable diffuse prognostic pattern of FOXP3+ and CD69+ T cells in follicular lymphoma demonstrated using automated imaging and analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1647. doi:10.1158/1538-7445.AM2014-1647

Over-expression of Roquin aggravates T cell mediated hepatitis in transgenic mice using T cell specific promoter

Chronic hepatitis is a major cause of liver cancer, so earlier treatment of hepatitis might be reducing liver cancer incidence. Hepatitis can be induced in mice by treatment with Concanavalin A (Con A); the resulting liver injury causes significant CD4+ T cell activation and infiltration. In these T cells, Roquin, a ring-type E3 ubiquitin ligase, is activated. To investigate the role of Roquin, we examined Con A-induced liver injury and T cell infiltration in transgenic (Tg) mice overexpressing Roquin specifically in T cells. In Roquin Tg mice, Con A treatment caused greater increases in both the levels of liver injury enzymes and liver tissue apoptosis, as revealed by TUNEL and H&E staining, than wild type (WT) mice. Further, Roquin Tg mice respond to Con A treatment with greater increases in the T cell population, particularly Th17 cells, though Treg cell counts are lower. Roquin overexpression also enhances increases in pro-inflammatory cytokines, including IFN-γ, TNF-α and IL-6, upon liver injury. Furthermore, Roquin regulates the immune response and apoptosis in Con A induced hepatitis via STATs, Bax and Bcl2. These findings suggest that over-expression of Roquin exacerbates T-cell mediated hepatitis.

Phenotyping Immune Cells In-Situ: an Investigation of the Spatial Heterogeneity of Specific Immune Cell Phenotypes in the Tumor Microenvironment

ABSTRACT Aim: Successes with PD-L1 drugs and adoptive immunotherapy demonstrate the efficacy of leveraging the immune system to fight cancer. However, host-tumor interaction is complex and difficult to characterize with immunohistochemistry or flow cytometry. Capturing spatial relationships of immune phenotypes in and around tumor is enabled by multiplexed immunofluorescence labelling and multispectral imaging, potentially forming the basis of assays to guide therapy and monitor response. Here we present a multi-marker, computer-aided method for analysing the distributions of CD3/FOXP3 (Treg) and CD3/CD69 (Tact) T cells in follicular lymphoma sections using a multispectral imaging (MSI) and automated analysis approach. Methods: A single section of a tissue microarray containing triplex follicular lymphoma cores from 40 subjects was stained for CD3, FOXP3, CD69 and hematoxylin. Each core was imaged using MSI and the individual staining of each marker separated from each other using spectral unmixing. CD3+ TILs were located using automated image analysis. The FOXP3 and CD69 status of each CD3+ TIL was then determined and the spatial distributions of each were used as input into the spatial distribution analysis. Results: Kaplan-Meier analysis demonstrated favourable outcome with higher numbers of CD3 + , CD3 + /FOXP3+ and CD3 + /CD69+ cells. HID analysis demonstrated the association of favourable outcome with high entropy, representative of a diffuse spatial pattern, of FOXP3+ and CD69+ positive T cells. Conclusions: In this study we report that higher Treg cell counts in a diffuse pattern was associated with favorable prognosis. This supports the importance of Tregs in the tumour microenvironment. It is pertinent to mention that contradictory findings are routinely reported from studies investigating the role of Tregs in solid and haematological malignancies. This is due to the complex interactions between pro-/anti- tumour immune factors present in the tumour microenvironment. The resultant effects are due to the summation of the activities of these factors. It is therefore even more relevant that a method such as exhibited here, capable of defining and measuring the effect on patient outcome of the spatial patterns of multiple cellular phenotypes in the tumor microenvironment is available. Disclosure: R. Lloyd and J.R. Mansfield: Employee of PerkinElmer. All other authors have declared no conflicts of interest.

Id1 expression promotes T regulatory cell differentiation by facilitating TCR costimulation.

T regulatory (Treg) cells play crucial roles in the regulation of cellular immunity. The development of Treg cells depends on signals from TCRs and IL-2Rs and is influenced by a variety of transcription factors. The basic helix-loop-helix proteins are known to influence TCR signaling thresholds. Whether this property impacts Treg differentiation is not understood. In this study, we interrogated the role of basic helix-loop-helix proteins in the production of Treg cells using the CD4 promoter-driven Id1 transgene. We found that Treg cells continued to accumulate as Id1 transgenic mice aged, resulting in a significant increase in Treg cell counts in the thymus as well as in the periphery compared with wild-type controls. Data from mixed bone marrow assays suggest that Id1 acts intrinsically on developing Treg cells. We made a connection between Id1 expression and CD28 costimulatory signaling because Id1 transgene expression facilitated the formation of Treg precursors in CD28(-/-) mice and the in vitro differentiation of Treg cells on thymic dendritic cells despite the blockade of costimulation by anti-CD80/CD86. Id1 expression also allowed in vitro Treg differentiation without anti-CD28 costimulation, which was at least in part due to enhanced production of IL-2. Notably, with full strength of costimulatory signals, however, Id1 expression caused modest but significant suppression of Treg induction. Finally, we demonstrate that Id1 transgenic mice were less susceptible to the induction of experimental autoimmune encephalomyelitis, thus illustrating the impact of Id1-mediated augmentation of Treg cell levels on cellular immunity.

Regulatory T cells in chronic lymphocytic leukemia

Controversial results concerning the prognostic value of the regulatory T (Treg) cell percentage in peripheral blood of chronic lymphocytic leukemia (CLL) patients have been reported in the previous studies. This study aimed to estimate the prognostic relevance of Treg cells in untreated CLL patients at diagnosis. CLL patients showed significantly higher Treg cell percentage and Treg cell counts as compared to that identified in healthy normal controls (P < 0.01 for both). Furthermore, CLL patients with high LDH, β2 microglobulin levels, and those positive for CD38% express significantly higher Treg cell percentage as compared to those patients with normal LDH, β2 microglobulin levels, and negative CD38%. Also, the percentage of Treg cells was significantly higher among CLL patients having autoimmune cytopenias. In conclusion, Treg cell percentage is higher in CLL patients as compared to normal healthy controls and related to advanced stages as well as poor prognostic markers. Treg cell manipulation may represent a future strategy for management of CLL patients.

Th17/Treg ratio derived using DNA methylation analysis is associated with the late phase asthmatic response.

BackgroundThe imbalance between Th17 and Treg cells has been studied in various diseases including allergic asthma but their roles have not been fully understood in the development of the late phase asthmatic response.ObjectivesTo determine changes in Th17 and Treg cell numbers between isolated early responders (ERs) and dual responders (DRs) undergoing allergen inhalation challenge. To identify gene expression profiles associated with Th17 and Treg cells.Methods14 participants (8 ERs and 6 DRs) with mild allergic asthma underwent allergen inhalation challenge. Peripheral blood was collected prior to and 2 hours post allergen challenge. DNA methylation analysis was used to quantifiy the relative frequencies of Th17, Tregs, total B cells, and total T cells. Gene expression from whole blood was measured using microarrays. Technical replication of selected genes was performed using nanoString nCounter Elements.ResultsThe Th17/Treg ratio significantly increased in DRs compared to ERs post allergen challenge compared to pre-challenge. Genes significantly correlated to Th17 and Treg cell counts were inversely correlated with each other. Genes significantly correlated with Th17/Treg ratio included the cluster of genes of the leukocyte receptor complex located on chromosome 19q 13.4.ConclusionsTh17/Treg imbalance post-challenge may contribute to the development of the late phase inflammatory phenotype.

The Role of Anti-TNFα Therapy in the Amelioration of Disease Burden in Patients with Refractory Rheumatoid Arthritis

To evaluate the changes in anti cyclic citrullinated peptide (Anti-CCP) antibodies,rheumatoid factor (RF), interleukins 17 and 10 (IL-17 and IL-10), high sensitivity C- reactiveprotein (hs CRP) and T regulatory cells (Treg) count following an anti TNF-α biological agent(etanercept) therapy in patients with rheumatoid arthritis.Refractory rheumatoid arthritis patients who failed treatment with DMARDs (diseasemodifying anti rheumatic drugs) were treated with etanercept for three months. Serum and bloodsamples were tested before and after therapy for six markers including ACCP, RF, IL- 10, IL 17,hs CRP, and Treg cell. A significant dropping down in the serum level of ACCP, RF and hs CRPwas documented 3 months after etanercept therapy. On the other hand a non- significant elevationin IL-10 serum level and peripheral blood Treg cell count, as well as a non significant droppingdown in the serum level of IL-17 were also reported. Etanercept is a successful anti-TNF-αtherapy in refractory RA patients for the first three months of the course therapy as indicated byclinical improvement and amelioration in 6 disease activity markers.

Low levels of circulating T-regulatory lymphocytes and short cervical length are associated with preterm labor

Recent discoveries suggest that T-regulatory lymphocytes (Treg) might play an important role in the pathophysiology of preterm labor. The aim of this study was to assess the relationship among the levels of maternal circulating Treg cells, uterine cervical length, and the risk of preterm labor. Sixty women with regular contractions and/or cervical incompetence at 24–32 weeks’ gestation were recruited into a prospective study. Each patient underwent transvaginal ultrasound examination of the cervical length, and regulatory T cells were quantified in peripheral blood samples by flow cytometry. Patients with cervical incompetence were prescribed vaginal progesterone until birth. Measurements of Treg levels and cervical length correlated with the timing of labor. The risk of preterm labor happening within 48h of testing was demonstrated to be almost 35 times higher (OR=35.21, CI 13.3; 214, p<0.001) in the group with simultaneously low Treg values (<0.031×109/L) and a shortened uterine cervix (<17.5mm), compared with the situation where both of these values were normal. Similar results were found in predicting preterm delivery before 34 weeks, or between 34 and 37 weeks. A statistically nonsignificant trend toward increased cervical length and increased Treg count was noted in the women on progesterone treatment. We show for the first time that the combined assessment of Treg cell count and cervical length is a much better predictor of preterm delivery than either parameter used on its own. This combined approach may offer clinical application in patients who present with risk factors for preterm labor.

Th17 and Treg cells in adolescents with Graves’ disease. Impact of treatment with methimazole on these cell subsets

The role of T helper 17 (Th17) and T regulatory cells (Treg) in the pathogenesis of Graves’ disease (GD) remains uncertain. The influence of methimazole (MMI) on the human immune system is still poorly understood. The aim of the present research was to assess changes in the frequencies of peripheral blood Th17 and Treg cells during GD treatment in the group of teenagers. The frequencies of Th17 and Treg were measured by flow cytometry in 60 adolescents at the time of GD diagnosis and after achieving MMI-induced euthyreosis. The control group consisted of 20 healthy volunteers. Lower percentages and absolute counts of Treg cells were found in the study group before the treatment in comparison with healthy controls (p = 0.032 and p = 0.006, respectively). Treatment with MMI caused an increase in the percentages and absolute counts of Treg lymphocytes (p = 0.037 and p = 0.007). After the treatment, no clinically significant differences in Treg cells between GD patients and controls were found. Higher absolute counts of Th17 lymphocytes were found in hyperthyroid adolescents before the treatment initiation and after achieving euthyreosis than in healthy individuals (p = 0.0001 and p = 0.047). Treatment with MMI caused a significant decrease in the percentages and absolute counts of Th17 lymphocytes (p = 0.047 and p = 0.043). The present study demonstrates that both Th17 and Treg cells might play a role in the pathogenesis of GD. Increased percentage of Treg after MMI therapy seems a predictor of response to anti-hypertensive treatment as it is associated with the normalization of thyroid hormone levels.

Effect of Th17 and Treg axis disorder on outcomes of pulmonary arterial hypertension in connective tissue diseases.

This prospective cohort study is to verify the hypothesis that the balance of Th17 and Treg cells frequencies in the peripheral circulation is disturbed in patients with varying degrees of connective tissue diseases-associated pulmonary arterial hypertension (CTD-aPAH) and to prove the influence of Th17/Treg imbalance on prognosis. We detected the frequencies and absolute counts of Th17 and Treg cells and related serum cytokines secretion and expressions of key transcription factors in 117 patients with connective tissue diseases (CTD), 53 patients with CTD-aPAH, and 48 healthy volunteers. Moreover, the median value according to levels of Th17/Treg ratios in patients with CTD-aPAH was chosen as basis of group division for survival analysis. CTD-aPAH patients revealed significant increase in peripheral Th17 cells, Th17-related cytokines, and ROR γt mRNA levels. They also presented a significant decrease in Treg cells, Treg-related cytokines, and Foxp3 mRNA levels as compared with CTD patients and healthy controls. More importantly, the Th17/Treg ratio was significantly related to the severity and prognosis of CTD-aPAH. This study indicated that the Th17/Treg axis disorder plays a critical role in CTD-aPAH. Furthermore, the dynamic balance between Th17 and Treg cells was likely to influence prognosis of patients with CTD-aPAH.