Age-related accumulation of T cells with markers of relatively stronger autoreactivity leads to functional erosion of T cells

Background

Although it is known that regulatory T cells (Tregs) can suppress the function of effector T cells, and may contribute to impaired immune response, the precise role of Tregs during the course of hepatitis B virus (HBV) infection remains to be elucidated. A newly identified subset of the CD4⁺Foxp3⁺ Tregs, the CD39⁺ Tregs, has been associated with viral infections and autoimmune diseases. Therefore, we hypothesized that this discrete Treg subset may contribute to the chronic infection of HBV.

Results

Initial characterization studies of healthy peripheral CD39⁺FoxP3⁺CD4⁺ T cells revealed that the majority were CD45RA^- Treg cells. Subsequent analysis of HBV-infected patients (38 asymptomatic HBV carriers (AsCs), 37 chronic active hepatitis B (CAH), 29 HBV-associated acute-on-chronic liver failure (ACLF)) and healthy individuals (25 controls) was conducted to assess association with HBV copy number and the liver injury marker alanine aminotransferase (ALT). A higher percentage of CD39⁺ Tregs was detected within the population of FoxP3⁺CD4⁺ T cells in peripheral blood of AsCs patients. Moreover, the percentage of CD39⁺ Tregs was significantly less in CAH and ACLF patients. The increased proportions of circulating CD39⁺ Tregs were positively correlated with serum viral load, but inversely correlated with serum ALT level.

Conclusion

These findings not only suggest that CD39⁺ Treg cells may be involved in HBV disease progression but also identify CD39⁺ Tregs as a dynamic immune regulatory cell population that may represent a new target of immunomodulatory therapeutic interventions.     

Human amylin induces CD4+Foxp3+ regulatory T cells in the protection from autoimmune diabetes

Autoimmune diabetes is a disorder of immune homeostasis that leads to targeted insulin-secreting islet β cell destruction characterized by insulitis. Human amylin (hA) is an important neuroendocrine hormone co-secreted with insulin by pancreatic β cells. Here, we report hA immune-modulatory action through inducing regulatory T cells. We ex vivo-treated human peripheral blood mononuclear cells (hPBMCs) with hA for 24 h and counted CD4+Foxp3+ regulatory T cells (Treg) using flow cytometry. Diabetic status was monitored and splenic Treg were measured in non-obese diabetic (NOD) male mice. NOD mice were intraperitoneally injected once daily with hA (n = 25) or solvent for control (n = 25) for 7 months continuously. Spleen tissues were collected at the end of intervention and processed for flow cytometry and Western blot. We found a 2.9-fold (p < 0.05) increase of CD4+Foxp3+ Treg in hPBMCs treated with 10 nmol/L hA compared with negative control. Incidence of diabetes in hA-treated NOD mice decreased 44% (p = 0.045) in the 6th month and 57% (p = 0.0002) in the 7th month. Meanwhile, the hA treatment induced a 1.5-fold increase of CD4+Foxp3+ Treg from mouse splenocytes (p = 0.0013). Expression of transforming growth factor-β (TGF-β) and toll-like receptor-4 (TLR-4) were upregulated in hA-treated mice. Human amylin might protect against autoimmune diabetes via the induction of CD4+Foxp3+ Treg, which suggests a novel approach to improve autoimmune conditions.     

Immune activation and regulatory T cells in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> infected lymph nodes

Background

Lymph node tuberculosis (LNTB) is the most frequent extrapulmonary form of tuberculosis (TB). Studies of human tuberculosis at sites of disease are limited. LNTB provides a unique opportunity to compare local in situ and peripheral blood immune response in active Mycobacterium tuberculosis (Mtb) disease. The present study analysed T regulatory cells (Treg) frequency and activation along with CD4+ T cell function in lymph nodes from LNTB patients.

Results

Lymph node mononuclear cells (LNMC) were compared to autologous peripheral blood mononuclear cells (PBMC). LNMC were enriched for CD4+ T cells with a late differentiated effector memory phenotype. No differences were noted in the frequency and mutifunctional profile of memory CD4+ T cells specific for Mtb. The proportion of activated CD4+ and Tregs in LNMC was increased compared to PBMC. The correlation between Tregs and activated CD4+ T cells was stronger in LNMC than PBMC. Tregs in LNMC showed a strong positive correlation with Th1 cytokine production (IL2, IFNγ and TNFα) as well as MIP-1α after Mtb antigen stimulation. A subset of Tregs in LNMC co-expressed HLA-DR and CD38, markers of activation.

Conclusion

Further research will determine the functional relationship between Treg and activated CD4+ T cells at lymph node sites of Mtb infection.

     

Decreased Levels of Circulating CD4+CD25+Foxp3+ Regulatory T Cells in Patients with Primary Antiphospholipid Syndrome

Introduction

CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cell dysfunction has been documented in various autoimmune disorders, but not in antiphospholipid syndrome (APS) so far.

Methods

In this cross-sectional study, we aim to investigate CD4⁺CD25⁺Foxp3⁺ Treg cells, CD3⁺CD19^? T cells and CD3^?CD19⁺ B cells in patients with primary APS and healthy controls. Cell subtypes were immunophenotyped using specific monoclonal antibodies (anti-CD3 CY5, anti-CD4 FITC, anti-CD25, anti-Foxp3, anti-CD19 PE) and flow cytometry.

Results

Twenty patients with APS and 20 age- and sex-matched controls were studied. The percentage of total lymphocytes, activated Th cells (CD4+CD25+), Treg cells and CD3^?CD19⁺ B cells were found significantly lower in APS patients as compared to controls (all p?<?0.05).

Conclusion

A dysfunction in CD4⁺CD25⁺Foxp3⁺ Treg cells may represent one of the mechanisms leading to autoimmunity in APS patients. The decreased number of CD3^?CD19⁺ B cells of APS patients warrants further elucidation.     

Iloprost modulates the immune response in systemic sclerosis

Background

Recent evidence shows that allograft survival rates show a positive correlation with the number of circulating T regulatory cells (Tregs). This study investigated both the number and the cytokine profiles exhibited by Foxp3⁺ Tregs in blood, spleen and lymph nodes of Lewis rat recipients of BN rat cardiac allografts after a single-dose of Rapamycin (RAPA).

Results

Rats were divided into three groups: control group (containing healthy control and acute rejection group), and recipients treated with a single dose of RAPA on either Day 1 (1D group)or Day 3 (3D group) post-transplant. We analyzed the number of Foxp3+Tregs and the expression of Foxp3 and cytokines in the peripheral blood and the peripheral lymphoid tissues. No difference was found in the numbers of circulating Foxp3+ Tregs between these three groups. RAPA administration significantly increased Foxp3 expression in peripheral lymphoid tissues after a single dose of RAPA on Day 3 post-transplant. Foxp3+Tregs inhibited the activity of effector T cells (T_eff) via the secretion of TGF-β1.

Conclusion

The number of Tregs in the recipient's blood may not be a good predictor of transplant rejection. Foxp3+Tregs inhibit the activity of T_eff cells mainly in the peripheral lymphoid tissues.     

Broadened T-cell Repertoire Diversity in ivIg-treated SLE Patients is Also Related to the Individual Status of Regulatory T-cells

Purpose

Intravenous IgG (ivIg) is a therapeutic alternative for lupus erythematosus, the mechanism of which remains to be fully understood. Here we investigated whether ivIg affects two established sub-phenotypes of SLE, namely relative oligoclonality of circulating T-cells and reduced activity of CD4?+?Foxp3+ regulatory T-cells (Tregs) reflected by lower CD25 surface density.

Methods

We conducted a longitudinal study of 15 lupus patients (14 with SLE and one with discoid LE) treated with ivIg in cycles of 2–6 consecutive monthly infusions. Among these 15 patients, 10 responded to ivIg therapy with clear clinical improvement. We characterized Tregs and determined TCR spectratypes of four Vβ families with reported oligoclonality. Cell counts, cytometry and TCR spectratypes were obtained from peripheral blood at various time points before, during and after ivIg treatment. T-cell oligoclonality was assessed as Vβ-familywise repertoire perturbation, calculated for each patient in respect to an individual reference profile averaged over all available time points.

Results

For 11 out of 15 patients, average Vβ1/Vβ2/Vβ11/Vβ14 repertoires were less perturbed under than outside ivIg therapy. The four exceptions with relatively increased average perturbation during ivIg therapy included three patients who failed to respond clinically to an ivIg therapy cycle. Patients' Treg CD25 surface density (cytometric MFI) was clearly reduced when compared to healthy controls, but not obviously influenced by ivIg. However, patients' average Treg CD25 MFI was found negatively correlated with both Vβ11 and Vβ14 perturbations measured under ivIg therapy.

Conclusions

This indicates a role of active Tregs in the therapeutic effect of ivIg.     

Changes of Treg-Associated Molecules on CD4+CD25+Treg Cells in Myasthenia Gravis and Effects of Immunosuppressants

Objective

Myasthenia gravis (MG) is a CD4⁺ T cell-dependent autoimmune disease, and close attention has been paid to the role of CD4⁺CD25⁺Treg cells (Tregs). Previous results regarding Tregs in MG patients have been conflicting. The discrepancy was partly ascribed to selecting different Treg-associated molecules in defining Tregs. Therefore, we considered it necessary to find a reliable index for assessing the immunologic state in MG patients and explore the effect of IS on them.

Methods

We adopted flow cytometric techniques to measure the numbers and frequencies of Tregs in peripheral blood taken from 57 patients and 91 age-matched healthy donors, and we also analyzed FOXP3 mean fluorescence intensity on Tregs.

Results

The number and frequency of Tregs in peripheral blood of MG patients significantly decreased, together with down-regulation of FOXP3 expression. There was dynamic change of Treg cell level and the inverse relationship with clinical symptom, suggesting that the immunologic disorder in MG patients was related to peripheral Tregs population. Meanwhile, CD4⁺CD25⁺FOXP3⁺Helios⁺T cells might be activated Tregs, rather than nTregs. Moreover, the number and frequency of CD4⁺CD25⁺FOXP3⁺Helios⁺T cells significantly decreased in MG patients, indicating that the reduction of the activated Tregs population might be a critical contributor to the pathogenesis of MG.

Conclusions

The significant reduction of the peripheral Tregs population in MG patients might be responsible for the immunologic disorders in MG patients. IS such as GC took its effect possible by increasing the population size, and the underlying mechanism should be further investigated.     

Immune system: a double-edged sword in cancer

Objective

The objective of the review is to examine the role of innate and adaptive immune cells in cancer.

Introduction

Immune system functions as a host defensive mechanism protecting against invading pathogens and transformed cells, including cancer. However, a body of research carried out over the last few decades has disclosed the unexpected role of immune system in fostering the tumor growth.

Methods

A computer-based online search was performed in the PubMed, Scopus and Web of Science databases for articles published, concerning natural killer (NK) cells, Macrophages, CD4+ and CD8+ T cells with relevance to cancer. After finding relevant articles within these search limits, a manual search was conducted through the references from these articles.

Results and Conclusions

This review summarizes the role of immune system in Immunosurveillance and Immunoediting. It then focused mainly on role of macrophages, regulatory T cells (Treg), T_H17 cells and on the immunosuppressive mechanisms, which facilitate immune evasion of tumor cells. Our results shows that, immune cells, such as CD8+ cytotoxic T lymphocytes (CTL), CD4+ T helper (T_H)1 cells and NK cells along with their characteristic cytokine interferon (IFN)-γ, function as major antitumor effector cells. Whereas CD4+T_H2 cells, myeloid-derived suppressor cells (MDSCs) and their derived cytokines function as dominant tumor-promoting forces. In contrast to these cells, macrophages, Treg, and T_H17 cells show a dual effect in cancer. Thus, it appears that most components of the immune system are potentially endowed with dual functions i.e., promoting tumor development on the one hand and restraining tumor development on the other and hence immune system can be considered as a double-edged sword in cancer.     

Expression of IL-7 receptor in human peripheral regulatory T cells

Introduction

Regulatory T cells (Tregs, CD4 + CD25^high Foxp3⁺) play a crucial role in allergy and other inflammatory diseases. However, the isolation of viable Tregs on the basis of intracellular expression of specific Forkhead Box Protein P3 (Foxp3) is difficult. In this study we checked if the expression of IL-7 receptor (CD127) on the Tregs could be a useful marker for isolation of viable Treg Foxp3⁺ cells.

Material and methods

Twenty-five patients sensitized to grass pollen with allergic rhinitis (AR) and ten healthy subjects were included. We compared Foxp3 expression in different CD4⁺ T cell subsets by flow cytometry and we assessed the relationship between the expression of Foxp3 and CD127 within regulatory T cells.

Results

Within the CD⁴+ lymphocytes 3.68 ±2.0% showed expression of Foxp3, 51.82 ±8.03% of CD4⁺CD25^high were Foxp3 positive (Foxp3⁺), whereas 82.12 ±5.4% of CD4⁺CD25^highCD127^low were Foxp3⁺. High intracellular expression of Foxp3 correlated with low superficial CD127 expression (r = 0.42, p = 0.017). There were no significant differences regarding the analysed markers between AR patients and healthy controls.

Conclusions

Regulatory T cells may be purified from the fresh peripheral blood as viable regulatory Foxp3 bright cells using CD4, high expression of CD25 and low expression of CD127 antigen.     

In vitro generation of cytotoxic and regulatory T cells by fusions of human dendritic cells and hepatocellular carcinoma cells

Background

Human hepatocellular carcinoma (HCC) cells express WT1 and/or carcinoembryonic antigen (CEA) as potential targets for the induction of antitumor immunity. In this study, generation of cytotoxic T lymphocytes (CTL) and regulatory T cells (Treg) by fusions of dendritic cells (DCs) and HCC cells was examined.

Methods

HCC cells were fused to DCs either from healthy donors or the HCC patient and investigated whether supernatants derived from the HCC cell culture (HCCsp) influenced on the function of DCs/HCC fusion cells (FCs) and generation of CTL and Treg.

Results

FCs coexpressed the HCC cells-derived WT1 and CEA antigens and DCs-derived MHC class II and costimulatory molecules. In addition, FCs were effective in activating CD4⁺ and CD8⁺ T cells able to produce IFN-γ and inducing cytolysis of autologous tumor or semiallogeneic targets by a MHC class I-restricted mechanism. However, HCCsp induced functional impairment of DCs as demonstrated by the down-regulation of MHC class I and II, CD80, CD86, and CD83 molecules. Moreover, the HCCsp-exposed DCs failed to undergo full maturation upon stimulation with the Toll-like receptor 4 agonist penicillin-inactivated Streptococcus pyogenes. Interestingly, fusions of immature DCs generated in the presence of HCCsp and allogeneic HCC cells promoted the generation of CD4⁺ CD25^high Foxp3⁺ Treg and inhibited CTL induction in the presence of HCCsp. Importantly, up-regulation of MHC class II, CD80, and CD83 on DCs was observed in the patient with advanced HCC after vaccination with autologous FCs. In addition, the FCs induced WT1- and CEA-specific CTL that were able to produce high levels of IFN-γ.

Conclusion

The current study is one of the first demonstrating the induction of antigen-specific CTL and the generation of Treg by fusions of DCs and HCC cells. The local tumor-related factors may favor the generation of Treg through the inhibition of DCs maturation; however, fusion cell vaccination results in recovery of the DCs function and induction of antigen-specific CTL responses in vitro. The present study may shed new light about the mechanisms responsible for the generation of CTL and Treg by FCs.     

Influence of hemodialysis on circulating CD4lowCD25high regulatory T cells in end-stage renal disease patients

Objective

Immunodeficiency of end-stage renal disease (ESRD) is caused by several factors including uremic toxins and biocompatibility reactions due to the repeated hemodialysis (HD) procedure. It has also been suggested that poor T cell responses could be associated with the increased number of regulatory T cells (Tregs) which are necessary to limit the function of activated T cells. The aim of the study was to determine the proportion of CD4⁺CD25⁺ cells (activated T cells) to CD4^lowCD25^high cells (Tregs) within the CD4⁺ population in ESRD patients.

Patients and methods

Two groups of ESRD patients, predialysis patients treated conservatively and patients undergoing hemodialysis (HD), as well as healthy controls were included in the study. Percentages of activated and regulatory T cells were determined ex vivo with flow cytometry based on the expression of CD4 and CD25 antigens.

Results and conclusions

HD patients showed an increased percentage of CD4⁺CD25⁺ cells when compared with healthy controls, while there was no difference in the percentage of CD4^lowCD25^high cells between the patient groups. In our opinion, the repeated hemodialysis procedure significantly disturbs the balance between activated T cells and regulatory T cells in ESRD patients. Lack of Treg mobilization and chronic stimulation of T cells may contribute to the immune deficiency observed in these patients.     

Epstein–Barr virus induces the differentiation of semi-mature dendritic cells from cord blood monocytes

Background

Epstein–Barr virus (EBV) is a tumorigenic virus which has effectively infected nearly all human beings with over 95% adult being seropositive. The persistence of latent EBV infection is not fully understood. Recent studies point towards a hypothesis of immune suppression and immune evasion involving regulatory T cells (Tregs) and dendritic cells (DCs). We sought to explore the mechanism of EBV suppression and immune evasion.

Methods

We compared the effects of EBV on cord blood (CB) and adult DCs differentiation and maturation including phenotype by flow cytometry, cytokine by ELISA and RT-PCR. And we evaluated the function of DC by co-culture DC and Treg by detection the expression of Foxp3, the phenotype and the cytokine profile of Tregs by flow cytometry.

Results

CB DCs derived from EBV-infected CB monocytes or from EBV-infected CB immature DCs (iDCs) displayed distinct phenotypes of “semi-mature” DCs with high expression of co-stimulatory molecules, such as CD40, CD80 and CD86 but low cytokine production, related to immune tolerance and homeostasis. While the EBV-infected adult iDCs resemble that of “pathogen-driven regulatory mature DCs” with high expression of co-stimulatory molecules, down-regulation of IL-12 secretion and up-regulation of IL-10 secretion, related to protection of host and immune evasion of pathogens. EBV infected cord blood monocytes-derived DCs drived Tregs development by driving the expression of Foxp3, increasing the expression of CTLA-4, decreasing the expression of GITR and promoted the generation of intracellular IL-2 and IL-10 by Tregs.

Conclusion

Epstein–Barr virus induces the differentiation of semi-mature dendritic cells from cord blood monocytes. The differences between CB and adult DCs suggested that the developmental maturity of the cells may affect their immune responses to EBV infection.     

The association between CD4+ CD25+ regulatory T cells and non-small cell lung carcinoma

Lung cancer is the second most common malignancy in the USA, and it is the leading cause of cancer-related deaths among men and women. Regulatory T cells (Tregs) in peripheral blood play crucial roles in suppressing antitumor immune responses in cancer patients. A defect in the immune response may contribute to tumor growth. This study was conducted to test the hypothesis that regulatory T cell lymphocytes might contribute to immune dysfunction in non-small cell lung carcinoma (NSCLC) patients, with a specific aim of determining its role in the development and progression of NSCLC. CD4+ CD25+ Treg cell enumeration by flow cytometry was performed for 30 NSCLC patients both preoperatively and postoperatively, together with 20 controls. Higher percentages of CD4+ CD25+ Treg cells were found in NSCLC patients both pre- (11.24?±?5.34) and postoperatively (8.75?±?4.66) in comparison to the control group (2.16?±?0.97); this difference was statistically significant (p?NSCLC patients: healthy donor < stage I < stage II < stage III whether preoperatively (r?=?0.5, p?=?0.005) or postoperatively (r?=?0.4, p?=?0.008). Treg cells decreased significantly after surgical removal of the tumors, from 11.24?±?5.34 to 8.75?±?4.66; the difference was statistically significant (p?NSCLC patients; their percentage correlates with tumor staging, and it decreases following surgical removal of the tumor. Thus, manipulation of these regulatory T cells could lead to new strategies for the treatment of cancer.     

Maturation of CD4+ Regulatory T Lymphocytes and of Cytokine Secretions in Infants Born Prematurely

Purpose

To characterize the maturation of CD4⁺ regulatory T lymphocytes (Treg) and of cytokine productions in preterm infants during their first 16 months of life.

Methods

The proportions of CD4⁺ Treg cells, their phenotypic characteristics, and the mitogen-induced cytokine productions by peripheral blood mononuclear cells (PBMC) were analysed in 26 very-preterm infants from 2 to 16 months of age, and compared to results obtained for 17 cord blood mononuclear cells (CBMC) from very-preterm infants, 12 from term infants and to blood samples from 40 adults.

Results

High proportion of CD25^+/highCD4⁺ Treg cells was found at birth in preterm CB with a gradual decreased afterwards. However, their percentage at 16 months of age was still higher than in term CB. In contrast to adults, preterm infants were characterized by excellent linear correlations between the proportions of CD25^+/highCD4⁺ and CD25^+/highFoxP3⁺ CD4⁺ or CD25^+/highCD127^low CD4⁺ or CD25^+/highFoxP3⁺CD127^low CD4⁺T lymphocytes. CD45RO⁺ and HLA-DR⁺ expressions were very low on preterm Treg and progressively increased with age. Functionally, preterm compared to term CBMC secreted in response to phytohaemagglutinin lower IFN-γ, higher IL-5 and similar IL-12p70, IL-10, IL-2 and IL-13 concentrations. IFN-γ, IL-12p70 and IL-10 productions were at 16 months still lower than those obtained for adults

Conclusion

Preterm differed from term CBMC both by their proportion and phenotype of CD4⁺ Treg lymphocytes and by their cytokine secretions. Maturation occurred during infancy with similar IFN-γ secretion but with persistently higher proportion of CD4⁺ Treg cells in 1 year preterm infants compared to term neonates.     

Baseline Immune Phenotypes and CD4+ T Lymphocyte Responses to Antiretroviral Therapy in Younger versus Older HIV-infected Individuals

Objective

The purpose of the study was to determine associations between pre-antiretroviral therapy (ART) senescent CD8+ T lymphocytes and naïve versus non-naive CD8+ and CD4+ T lymphocyte subpopulations and CD4+ responses after initiation of ART in younger versus older individuals.

Methods

Retrospective analysis of 100 subjects with pre-ART cryopreserved peripheral blood mononuclear cells samples was performed with flow cytometry. Subjects were divided into four groups by age (30–50 years or?>?50 years) and 96-week CD4+ response (<100 or >200 cells/mm³). All subjects had 96-week viral suppression to <50 copies/mm³. Regression was utilized to investigate associations between pre-ART CD8+ and CD4+ T cell phenotypes with age and CD4+ response categories.

Results

Individuals <50 years had a lower frequency of senescent CD8+ T lymphocytes of the CD56?+?57+, CD56+, and CD28? phenotypes (95%CI ?3.6 to ?0.02; 95%CI ?4.2 to ?0.03; 95%CI ?12.5 to ?1.4, respectively) and a higher frequency of naïve (CD45RA?+?CD28+) CD8+ T lymphocytes (95%CI 2.6 to 10.9). Younger age and good CD4+ response were associated with a higher frequency of pre-ART naïve CD4+ T cells (95%CI 2.0 to 16.4 and 95%CI 1.5 to 15.6, respectively).

Conclusions

Prior to ART, younger HIV-infected individuals have a higher frequency of naïve CD4+ and CD8+ T cells and lower frequency of senescent CD8+ T cell phenotypes.