Control of type 1 diabetes by CD4+Foxp3+ regulatory T cells: lessons from mouse models and implications for human disease

In recent years, there has been a revival of the concept of CD4⁺ regulatory T (T_reg) cells as being a central control point in various immune responses, including autoimmune responses and immunity to transplants, allergens, tumours and infectious microbes. The current literature suggests that T_reg cells are diverse in their phenotype and mechanism(s) of action, and as such, may constitute a myriad of naturally occurring and induced T cell precursors with variable degrees of regulatory potential. In this review, we summarize research from various laboratories, including our own, showing that CD4⁺Foxp3⁺ T_reg cells are critical in the control of type 1 diabetes (T1D) in mouse models and humans. In this review, we also discuss cellular and molecular determinants that impact CD4⁺Foxp3⁺ T_reg cell development and function and consequential resistance to organ‐specific autoimmune disease. Recent advances in the use of CD4⁺Foxp3⁺ T_reg cellular therapy to promote immunological tolerance in the absence of long‐term generalized immunosuppression are also presented. Copyright © 2009 John Wiley & Sons, Ltd.     

Function of Treg Cells Decreased in Patients With Systemic Lupus Erythematosus Due To the Effect of Prolactin

Prolactin has different functions, including cytokine secretion and inhibition of the suppressor effect of regulatory T (T_reg) cells in healthy individuals. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by defects in the functions of B, T, and T_reg cells. Prolactin plays an important role in the physiopathology of SLE. Our objective was to establish the participation of prolactin in the regulation of the immune response mediated by T_reg cells from patients with SLE. CD4⁺CD25^hiCD127^−/low cells were purified using magnetic beads and the relative expression of prolactin receptor was measured. The functional activity was evaluated by proliferation assay and cytokine secretion in activated cells, in the presence and absence of prolactin. We found that both percentage and function of T_reg cells decrease in SLE patients compared to healthy individuals with statistical significance. The prolactin receptor is constitutively expressed on T_reg and effector T (T_eff) cells in SLE patients, and this expression is higher than in healthy individuals. The expression of this receptor differs in inactive and active patients: in the former, the expression is higher in T_reg cells than in T_eff cells, similar to healthy individuals, whereas there is no difference in the expression between T_reg and T_eff cells from active patients. In T_reg:T_eff cell cocultures, addition of prolactin decreases the suppressor effect exerted by T_reg cells and increases IFNγ secretion. Our results suggest that prolactin plays an important role in the activation of the disease in inactive patients by decreasing the suppressor function exerted by T_reg cells over T_eff cells, thereby favoring an inflammatory microenvironment.     

IL-9 induces differentiation of TH17 cells and enhances function of FoxP3+ natural regulatory T cells

The development of T helper (T_H)17 and regulatory T (T_reg) cells is reciprocally regulated by cytokines. Transforming growth factor (TGF)-β alone induces FoxP3⁺ T_reg cells, but together with IL-6 or IL-21 induces T_H17 cells. Here we demonstrate that IL-9 is a key molecule that affects differentiation of T_H17 cells and T_reg function. IL-9 predominantly produced by T_H17 cells, synergizes with TGF-β1 to differentiate naïve CD4⁺ T cells into T_H17 cells, while IL-9 secretion by T_H17 cells is regulated by IL-23. Interestingly, IL-9 enhances the suppressive functions of FoxP3⁺ CD4⁺ T_reg cells in vitro, and absence of IL-9 signaling weakens the suppressive activity of nT_regs in vivo, leading to an increase in effector cells and worsening of experimental autoimmune encephalomyelitis. The mechanism of IL-9 effects on T_H17 and T_regs is through activation of STAT3 and STAT5 signaling. Our findings highlight a role of IL-9 as a regulator of pathogenic versus protective mechanisms of immune responses.     

Antigen-specific induced Foxp3+ regulatory T cells are generated following CD40/CD154 blockade

Blockade of the CD40/CD154 pathway potently attenuates T-cell responses in models of autoimmunity, inflammation, and transplantation. Indeed, CD40 pathway blockade remains one of the most powerful methods of prolonging graft survival in models of transplantation. But despite this effectiveness, the cellular and molecular mechanisms underlying the protective effects of CD40 pathway blockade are incompletely understood. Furthermore, the relative contributions of deletion, anergy, and regulation have not been measured in a model in which donor-reactive CD4⁺ and CD8⁺ T-cell responses can be assessed simultaneously. To investigate the impact of CD40/CD154 pathway blockade on graft-specific T-cell responses, a transgenic mouse model was used in which recipients containing ovalbumin-specific CD4⁺ and CD8⁺ TCR transgenic T cells were grafted with skin expressing ovalbumin in the presence or absence of anti-CD154 and donor-specific transfusion. The results indicated that CD154 blockade altered the kinetics of donor-reactive CD8⁺ T-cell expansion, delaying differentiation into IFN-γ⁺ TNF⁺ multifunctional cytokine producers. The eventual differentiation of cytokine-producing effectors in tolerant animals coincided with the emergence of an antigen-specific CD4⁺ CD25^hi Foxp3⁺ T-cell population, which did not arise from endogenous natural T_reg but rather were peripherally generated from naïve Foxp3⁻ precursors.     

A commensal symbiotic factor derived from Bacteroides fragilis promotes human CD39+Foxp3+ T cells and Treg function

Polysaccharide A (PSA) derived from the human commensal Bacteroides fragilis is a symbiosis factor that stimulates immunologic development within mammalian hosts. PSA rebalances skewed systemic T helper responses and promotes T regulatory cells (T_regs). However, PSA-mediated induction of Foxp3 in humans has not been reported. In mice, PSA-generated Foxp3⁺ T_regs dampen Th17 activity thereby facilitating bacterial intestinal colonization while the increased presence and function of these regulatory cells may guard against pathological organ-specific inflammation in hosts. We herein demonstrate that PSA induces expression of Foxp3 along with CD39 among naïve CD4 T cells in vitro while promoting IL-10 secretion. PSA-activated dendritic cells are essential for the mediation of this regulatory response. When cultured with isolated Foxp3⁺ T_regs, PSA enriched Foxp3 expression, enhanced the frequency of CD39⁺HLA-DR⁺ cells, and increased suppressive function as measured by decreased TNFα expression by LPS-stimulated monocytes. Our findings are the first to demonstrate in vitro induction of human CD4⁺Foxp3⁺ T cells and enhanced suppressive function of circulating Foxp3⁺ T_regs by a human commensal bacterial symbiotic factor. Use of PSA for the treatment of human autoimmune diseases, in particular multiple sclerosis and inflammatory bowel disease, may represent a new paradigm in the approach to treating autoimmune disease.     

A commensal symbiotic factor derived from Bacteroides fragilis promotes human CD39+Foxp3+ T cells and Treg function

Polysaccharide A (PSA) derived from the human commensal Bacteroides fragilis is a symbiosis factor that stimulates immunologic development within mammalian hosts. PSA rebalances skewed systemic T helper responses and promotes T regulatory cells (T_regs). However, PSA-mediated induction of Foxp3 in humans has not been reported. In mice, PSA-generated Foxp3⁺ T_regs dampen Th17 activity thereby facilitating bacterial intestinal colonization while the increased presence and function of these regulatory cells may guard against pathological organ-specific inflammation in hosts. We herein demonstrate that PSA induces expression of Foxp3 along with CD39 among naïve CD4 T cells in vitro while promoting IL-10 secretion. PSA-activated dendritic cells are essential for the mediation of this regulatory response. When cultured with isolated Foxp3⁺ T_regs, PSA enriched Foxp3 expression, enhanced the frequency of CD39⁺HLA-DR⁺ cells, and increased suppressive function as measured by decreased TNFα expression by LPS-stimulated monocytes. Our findings are the first to demonstrate in vitro induction of human CD4⁺Foxp3⁺ T cells and enhanced suppressive function of circulating Foxp3⁺ T_regs by a human commensal bacterial symbiotic factor. Use of PSA for the treatment of human autoimmune diseases, in particular multiple sclerosis and inflammatory bowel disease, may represent a new paradigm in the approach to treating autoimmune disease.     

Homeostatic imbalance of regulatory and effector T cells due to IL-2 deprivation amplifies murine lupus

The origins and consequences of a regulatory T cell (Treg) disorder in systemic lupus erythematosus (SLE) are poorly understood. In the (NZBxNZW) F₁ mouse model of lupus, we found that CD4⁺Foxp3⁺ Treg failed to maintain a competitive pool size in the peripheral lymphoid organs resulting in a progressive homeostatic imbalance of CD4⁺Foxp3⁺ Treg and CD4⁺Foxp3⁻ conventional T cells (Tcon). In addition, Treg acquired phenotypic changes that are reminiscent of IL-2 deficiency concomitantly to a progressive decline in IL-2-producing Tcon and an increase in activated, IFN-γ-producing effector Tcon. Nonetheless, Treg from lupus-prone mice were functionally intact and capable to influence the course of disease. Systemic reduction of IL-2 levels early in disease promoted Tcon hyperactivity, induced the imbalance of Treg and effector Tcon, and strongly accelerated disease progression. In contrast, administration of IL-2 partially restored the balance of Treg and effector Tcon by promoting the homeostatic proliferation of endogenous Treg and impeded the progression of established disease. Thus, an acquired and self-amplifying disruption of the Treg-IL-2 axis contributed essentially to Tcon hyperactivity and the development of murine lupus. The reversibility of this homeostatic Treg disorder provides promising approaches for the treatment of SLE.     

Conversion of Th2 memory cells into Foxp3+ regulatory T cells suppressing Th2-mediated allergic asthma

Genetic and epigenetic programming of T helper (Th) cell subsets during their polarization from naive Th cells establishes long-lived memory Th cells that stably maintain their lineage signatures. However, whether memory Th cells can be redifferentiated into another Th lineage is unclear. In this study, we show that Ag-specific memory Th cells were redifferentiated into Foxp3⁺ T cells by TGF-β when stimulated in the presence of all-trans retinoic acid and rapamycin. The “converted” Foxp3⁺ T cells that were derived from Th2 memory cells down-regulated GATA-3 and IRF4 and produced little IL-4, IL-5, and IL-13. Instead, the converted Foxp3⁺ T cells suppressed the proliferation and cytokine production of Th2 memory cells. More importantly, the converted Foxp3⁺ T cells efficiently accumulated in the airways and significantly suppressed Th2 memory cell-mediated airway hyperreactivity, eosinophilia, and allergen-specific IgE production. Our findings reveal the plasticity of Th2 memory cells and provide a strategy for adoptive immunotherapy for the treatment of allergic diseases.     

Clinical significance of Treg cell frequency in acute myeloid leukemia

This study was designed to investigate the clinical significance of peripheral blood CD4⁺ CD25⁺ CD127 low-regulatory T (T_reg) cells in acute myeloid leukemia (AML) patients. T_reg cells in the peripheral blood of 80 AML patients and 35 age-matched healthy controls were counted by flow cytometry. Correlations between the frequency of circulating T_reg cells and disease status, treatment outcome, or prognosis were evaluated. The percentages of T_reg cells in patients at diagnosis and during refractory/relapse were significantly higher than that in healthy controls. There was no significant difference in the percentages of T_reg cells between patients in remission and healthy controls. After six cycles of chemotherapy, the percentage of T_reg cells in patients who achieved complete remission was significantly lower than that in patients at diagnosis, but there was no difference in T_reg frequency between refractory/relapse patients and patients at diagnosis. T_reg cells in the peripheral blood of AML patients may play a suppressive role in host antitumor immune response. The frequency of T_reg cells in peripheral blood may thus be used as a biomarker for predicting sensitivity to chemotherapy and prognosis of AML patients. Additionally, T_reg number in peripheral blood could be used to monitor disease status and evaluate disease progression.     

Plasma Exchange Downregulates Activated Monocytes and Restores Regulatory T Cells in Kawasaki Disease

In Kawasaki disease (KD), the effect of plasma exchange (PE) on immune cells has not been fully elucidated. Therefore, we examined the changes in the number of CD14⁺ CD16⁺ activated monocytes, regulatory T (T_reg), and T‐helper type 17 (Th17) cells in KD patients treated with PE. The percentage of total monocytes and subclasses of lymphocytes, including CD4⁺ and CD8⁺ T cells, and CD19⁺ B cells, showed no significant difference before and after PE. However, the percentage of CD14⁺ CD16⁺ monocytes in total leukocytes decreased significantly after PE (1.1% ± 1.5% vs. 2.1% ± 2.3%, P < 0.05). Furthermore, while the percentage of Th17 cells in CD4⁺ T cells did not change, the percentage of T_reg cells in CD4⁺ T cells increased significantly after PE (11.1% ± 5.1% vs. 8.0% ± 4.4%, P < 0.05). Therefore, PE downregulates activated monocytes and upregulates T_reg cells toward normal levels and thus attenuates inflammation in KD.     

Gut microbiota amplifies host-intrinsic conversion from the CD8 T cell lineage to CD4 T cells for induction of mucosal immune tolerance

Microbiota has been shown to promote tolerogenic differentiation of T lymphocytes. It remains unclear to what extent microbiota triggers de novo re-programming or amplify pre-existing plasticity intrinsic to T cells. In a study with mouse models to track the clonal fate of CD4 and CD8 T cells, we discovered that CD8 T cells converted to MHC class I-restricted CD4 T cells without regard to selfness of their antigen specificity. In mesenteric lymph nodes (MLN), CD8 T cells converted to CD4⁺Foxp3⁺ regulatory T (T_reg) cells which were enriched in the large intestine lamina propria (LILP) and suppressed chemical- or immune-mediated inflammatory damage. In germ-free conditions, the converted CD4 populations were present in MLN, but absent in LILP. Therefore, an intrinsic plasticity in the host was amplified by the gut microbiota, leading to selfless tolerance induction in the intestinal mucosa. The findings may be relevant to HIV infection, cancer and autoimmune disorders.     

Inducible Foxp3+ regulatory T-cell development by a commensal bacterium of the intestinal microbiota

To maintain intestinal health, the immune system must faithfully respond to antigens from pathogenic microbes while limiting reactions to self-molecules. The gastrointestinal tract represents a unique challenge to the immune system, as it is permanently colonized by a diverse amalgam of bacterial phylotypes producing multitudes of foreign microbial products. Evidence from human and animal studies indicates that inflammatory bowel disease results from uncontrolled inflammation to the intestinal microbiota. However, molecular mechanisms that actively promote mucosal tolerance to the microbiota remain unknown. We report herein that a prominent human commensal, Bacteroides fragilis, directs the development of Foxp3⁺ regulatory T cells (Tregs) with a unique “inducible” genetic signature. Monocolonization of germ-free animals with B. fragilis increases the suppressive capacity of Tregs and induces anti-inflammatory cytokine production exclusively from Foxp3⁺ T cells in the gut. We show that the immunomodulatory molecule, polysaccharide A (PSA), of B. fragilis mediates the conversion of CD4⁺ T cells into Foxp3⁺ Treg cells that produce IL-10 during commensal colonization. Functional Foxp3⁺ Treg cells are also produced by PSA during intestinal inflammation, and Toll-like receptor 2 signaling is required for both Treg induction and IL-10 expression. Most significantly, we show that PSA is not only able to prevent, but also cure experimental colitis in animals. Our results therefore demonstrate that B. fragilis co-opts the Treg lineage differentiation pathway in the gut to actively induce mucosal tolerance.     

Programmed death 1 ligand signaling regulates the generation of adaptive Foxp3+CD4+ regulatory T cells

Although mature dendritic cells (DCs) are potent initiators of adaptive immune response, immature steady-state DCs contribute to immune tolerance. In this study, we show that ex vivo splenic DCs are capable of inducing conversion of naïve CD4⁺ T cells to adaptive Foxp3⁺CD4⁺ regulatory T cells (aTreg) in the presence of TGF-β. In particular, when compared with splenic CD8α⁻ DCs, the CD8α⁺ DC subset were superior in inducing higher frequencies of conversion. This was not attributable to the difference in basal level of costimulation, because deficiency of CD40 or CD80/86 signaling did not diminish the differential induction of Foxp3. Conversion was regulated by DC maturation status. Further insights into the molecular mechanisms of conversion were gained by analyzing the contribution of several costimulatory and coinhibitory receptors. Costimulatory signals through GITR suppressed conversion, whereas coinhibitory signaling via programmed death 1 ligand (PD-L1) but not PD-L2 was required for conversion. Ex vivo PD-L1^−/− DCs failed to support Foxp3 induction in the presence of TGF-β. In vivo blocking PD-L1 signaling abolished conversion in a tumor-induced aTreg conversion model. Collectively, this study highlights the cellular and molecular parameters that might be exploited to control the de novo generation of aTregs and peripheral tolerance.     

Tumor-Infiltrating Immune Cells Are Associated With Prognosis of Gastric Cancer

Immune cells contribute to determining the prognosis of gastric cancer. However, their exact role is less clear.We determined the prognostic significance of different immune cells in intratumoral tissue (_T), stromal tissue (_S), and adjacent normal tissue (_N) of 166 gastric cancer cases and their interactions, including CD3⁺, CD4⁺, CD8⁺, CD57⁺, CD68⁺, CD66b⁺, and Foxp3⁺ cells, and established an effective prognostic nomogram based on the immune reactions.We found high densities of _TCD3⁺, _TCD4⁺, _TCD8⁺, _SCD3⁺, _SCD4⁺, _SCD57⁺, _SCD66b⁺, and _NFoxp3⁺ cells, as well as high _TCD8⁺/_SCD8⁺ ratio, _TCD68⁺/_SCD68⁺ ratio, _TCD3⁺/_TFoxp3⁺ ratio, _TCD4⁺/_TFoxp3⁺ ratio, _TCD8⁺/_TFoxp3⁺ ratio, _SCD3⁺/_SFoxp3⁺ ratio, and _SCD4⁺/_SCD8⁺ ratio were associated with better survival, whereas high densities of _TCD66b⁺, _TFoxp3⁺, _SFoxp3⁺ and _NCD66b⁺ cells as well as high _TCD57⁺/_SCD57⁺ ratio, _TCD66b⁺/_SCD66b⁺ ratio, _SCD8⁺/_SFoxp3⁺ ratio, and _TFoxp3⁺/_NFoxp3⁺ ratio were associated with significantly worse outcome. Multivariate analysis indicated that tumor size, longitudinal tumor location, N stage, _TCD68⁺/_SCD68⁺ ratio, _TCD8⁺/_TFoxp3⁺ ratio, density of _TFoxp3⁺ cells, and _TCD66b⁺/_SCD66b⁺ ratio were independent prognostic factors, which were all selected into the nomogram. The calibration curve for likelihood of survival demonstrated favorable consistency between predictive value of the nomogram and actual observation. The C-index (0.83, 95% CI: 0.78 to 0.87) of our nomogram for predicting prognosis was significantly higher than that of TNM staging system (0.70).Collectively, high _TCD68⁺/_SCD68⁺ ratio and _TCD8⁺/_TFoxp3⁺ ratio were associated with improved overall survival, whereas high density of _TFoxp3⁺ cells and _TCD66b⁺/_SCD66b⁺ ratio demonstrated poor overall survival, which are promising independent predictors for overall survival in gastric cancer.     

B7-H1 expression is associated with expansion of regulatory T cells in colorectal carcinoma

AIM: To investigate the expression of B7-H1 in human colorectal carcinoma (CRC) to define its regulating effects on T cells in tumor microenvironment.METHODS: One hundred and two paraffin blocks and 33 fresh samples of CRC tissues were subject to this study. Immunohistochemistry was performed for B7-H1 and CD3 staining in CRC tissues. Ficoll-Hypaque density gradient centrifugation was used to isolate peripheral blood mononuclear cells of fresh CRC tissues; flow cytometry and immunofluorescence staining were used for detection of regulatory T cells. Data was analyzed with statistical software.RESULTS: Costimulatory molecule B7-H1 was found strongly expressed in CRC tissues, localized in tumor cell membrane and cytoplasm, while weak or none expression of B7-H1 was detected in pared normal colorectal tissues. Meanwhile, CD3 positive T cells were found congregated in CRC tumor nest and stroma. Statistic analysis showed that B7-H1 expression level was negatively correlated to the total T cell density in tumor nest (P < 0.0001) and tumor stroma (P = 0.0200) of 102 cases of CRC tissues. Among the total T cells, a variable amount of regulatory T cells with a clear Foxp3⁺ (forkhead box P3) staining could be detected in CRC tissues and patients’ blood. Interestingly, in the 33 samples (15 cases of B7-H1^high CRC tissues and 18 cases of B7-H1^low CRC tissues) of freshly isolated mononuclear cells from CRC tissues, the percentages of CD4⁺Foxp3⁺ and CD8⁺Foxp3⁺ regulatory T cells were found remarkably higher in B7-H1^high CRC tissues than in B7-H1^low CRC tissues (P = 0.0024, P = 0.0182), indicating that B7-H1 expression was involved in proliferation of regulatory T cell. No significant difference was found in CRC peripheral blood (P = 0.0863, P = 0.0678). PD-1 is the specific ligand for B7-H1 pathway transferring inhibitory signal to T cell, which is expressed by activated T cell. Our further analysis of PD-1 expression on T cells in CRC tissues showed that conventional T cells (CD4⁺Foxp3^-/CD8⁺Foxp3^-), which was thought to contribute to the anti-tumor immune response, highly expressed PD-1; while regulatory T cells (CD4⁺Foxp3⁺/CD8⁺Foxp3^-) almost failed to express PD-1. The average percentage of PD-1 expression on regulatory T cells was significantly higher than the percentage of PD-1 on conventional T cells (CD4⁺Foxp3^- T cell, P < 0.0001; CD8⁺Foxp3^- T cell, P < 0.0001). The diverse expression of PD-1 might lead to different fate of T cell subsets in B7-H1 over-expression CRC tumor microenvironment.CONCLUSION: B7-H1 expression in tumor cells can inhibit the conventional T cell proliferation in tumor microenvironment through the PD-1 expression on conventional T cells.