T‐bet regulates differentiation of forkhead box protein 3+ regulatory T cells in programmed cell death‐1‐deficient mice

Programmed cell death‐1 (PD‐1) plays an important role in peripheral T cell tolerance, but whether or not it affects the differentiation of helper T cell subsets remains elusive. Here we describe the importance of PD‐1 in the control of T helper type 1 (Th1) cell activation and development of forkhead box protein 3 (FoxP3⁺) regulatory T cells (T_regs). PD‐1‐deficient T cell‐specific T‐bet transgenic (P/T) mice showed growth retardation, and the majority died within 10 weeks. P/T mice showed T‐bet over‐expression, increased interferon (IFN)‐γ production by CD4⁺ T cells and significantly low FoxP3⁺ T_reg cell percentage. P/T mice developed systemic inflammation, which was probably induced by augmented Th1 response and low FoxP3⁺ T_reg count. The study identified a unique, previously undescribed role for PD‐1 in Th1 and T_reg differentiation, with potential implication in the development of Th1 cell‐targeted therapy.     

Regulatory T cells and T helper 17 cells in viral infection

CD4⁺ T cells are the central element of the adaptive immune responses and protect the body from a variety of pathogens. Starting from naive cells, CD4⁺ T cells can differentiate into various effector cell subsets with specialized functions including T helper (Th) 1, Th2, Th17, regulatory T (Treg) and T follicular helper (Tfh) cells. Among them, Tregs and Th17 cells show a strong plasticity allowing the functional adaptation to various physiological and pathological environments during immune responses. Although they are derived from the same precursor cells and their differentiation pathways are interrelated, the terminally differentiated cells have totally opposite functions. Studies have shown that Tregs and Th17 cells have rather complex interplays in viral infection: Th17 cells may contribute to immune activation and disease progression while Tregs may inhibit this process and play a key role in the maintenance of immune homoeostasis, possibly at the cost of compromised viral control. In this review, we take respiratory syncytial virus (RSV), hepatitis B virus (HBV)/hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections as examples to discuss these interplays and their impacts on disease progression in viral infection.     

Critical stoichiometric ratio of CD4+ CD25+ FoxP3+ regulatory T cells and CD4+ CD25− responder T cells influence immunosuppression in patients with B‐cell acute lymphoblastic leukaemia

Regulatory T (Treg) cells act to suppress activation of the immune system and thereby maintain immunological homeostasis and tolerance to self-antigens. The frequency and suppressing activity of Treg cells in general are high in different malignancies. We wanted to identify the role and regulation of CD4⁺ CD25⁺ FoxP3⁺ Treg cells in B-cell acute lymphoblastic leukaemia (B-ALL). We have included patients at diagnosis (n = 54), patients in clinical remission (n = 32) and normal healthy individuals (n = 35). These diagnosed patients demonstrated a lower number of CD4⁺ CD25⁺ cells co-expressing a higher level of FoxP3, interleukin-10, transforming growth factor-β and CD152/CTLA-4 than the normal population. Treg cells from patients showed a higher suppressive capability on CD4⁺ CD25⁻ responder T (Tresp) cells than normal. The frequency and immunosuppressive potential of CD4⁺ CD25⁺ FoxP3⁺ Treg cells became high with the progression of malignancy in B-ALL. Relative distribution of Tresp and Treg cells was only ˜5 : 1 in B-ALL but ˜35 : 1 in normal healthy individuals, further confirming the elevated immunosuppression in patients. A co-culture study at these definite ex vivo ratios, indicated that Treg cells from B-ALL patients exhibited higher immunosuppression than Treg cells from normal healthy individuals. After chemotherapy using the MCP841 protocol, the frequency of CD4⁺ CD25⁺ cells was gradually enhanced with the reduction of FoxP3, interleukin-10 positivity corresponded with disease presentation, indicating reduced immunosuppression. Taken together, our study indicated that the CD4⁺ CD25⁺ FoxP3⁺ Treg cells played an important role in immunosuppression, resulting in a positive disease-correlation in these patients. To the best of our knowledge, this is the first detailed report on the frequency, regulation and functionality of Treg cells in B-ALL.     

Regulatory T cell frequencies are increased in preterm infants with clinical early‐onset sepsis

The predisposition of preterm neonates to invasive infection is, as yet, incompletely understood. Regulatory T cells (T_regs) are potential candidates for the ontogenetic control of immune activation and tissue damage in preterm infants. It was the aim of our study to characterize lymphocyte subsets and in particular CD4⁺CD25⁺forkhead box protein 3 (FoxP3)⁺ T_regs in peripheral blood of well‐phenotyped preterm infants (n = 117; 23 + 0 – 36 + 6 weeks of gestational age) in the first 3 days of life in comparison to term infants and adults. We demonstrated a negative correlation of T_reg frequencies and gestational age. T_regs were increased in blood samples of preterm infants compared to term infants and adults. Notably, we found an increased T_reg frequency in preterm infants with clinical early‐onset sepsis while cause of preterm delivery, e.g. chorioamnionitis, did not affect T_reg frequencies. Our data suggest that T_regs apparently play an important role in maintaining maternal‐fetal tolerance, which turns into an increased sepsis risk after preterm delivery. Functional analyses are needed in order to elucidate whether T_regs have potential as future target for diagnostics and therapeutics.     

Regulatory T cells in autoimmune neuroinflammation

Regulatory T cells are the central element for the maintenance of peripheral tolerance. Several subtypes of regulatory T (Treg) cells have been described, and most of them belong to the CD4⁺ T-helper (Th) cell lineage. These specific subtypes can be discriminated according to phenotype and function. Forkhead box protein 3 (FoxP3)-expressing natural Treg cells (Tregs) and IL-10-producing, T-regulatory type 1 cells (Tr1) are the best-studied types of CD4⁺ regulatory T cells in humans and experimental animal models. It was shown that they play a crucial role during autoimmune neuroinflammation. Both cells types seem to be particularly important for multiple sclerosis (MS). Here, we discuss the role of CD4⁺ regulatory T cells in autoimmune neuroinflammation with an emphasis on Tregs and Tr1 cells in MS.     

Membrane‐bound Dickkopf‐1 in Foxp3+ regulatory T cells suppresses T‐cell‐mediated autoimmune colitis

Induction of tolerance is a key mechanism to maintain or to restore immunological homeostasis. Here we show that Foxp3⁺ regulatory T (Treg) cells use Dickkopf‐1 (DKK‐1) to regulate T‐cell‐mediated tolerance in the T‐cell‐mediated autoimmune colitis model. Treg cells from DKK‐1 hypomorphic doubleridge mice failed to control CD4⁺ T‐cell proliferation, resulting in CD4 T‐cell‐mediated autoimmune colitis. Thymus‐derived Treg cells showed a robust expression of DKK‐1 but not in naive or effector CD4 T cells. DKK‐1 expression in Foxp3⁺ Treg cells was further increased upon T‐cell receptor stimulation in vitro and in vivo. Interestingly, Foxp3⁺ Treg cells expressed DKK‐1 in the cell membrane and the functional inhibition of DKK‐1 using DKK‐1 monoclonal antibody abrogated the suppressor function of Foxp3⁺ Treg cells. DKK‐1 expression was dependent on de novo protein synthesis and regulated by the mitogen‐activated protein kinase pathway but not by the canonical Wnt pathway. Taken together, our results highlight membrane‐bound DKK‐1 as a novel Treg‐derived mediator to maintain immunological tolerance in T‐cell‐mediated autoimmune colitis.     

Successful granulocyte‐colony stimulating factor treatment of Crohn's disease is associated with the appearance of circulating interleukin‐10‐producing T cells and increased lamina propria plasmacytoid dendritic cells

Granulocyte‐colony stimulating factor (G‐CSF) has proved to be a successful therapy for some patients with Crohn's disease. Given the known ability of G‐CSF to exert anti‐T helper 1 effects and to induce interleukin (IL)‐10‐secreting regulatory T cells, we studied whether clinical benefit from G‐CSF therapy in active Crohn's disease was associated with decreased inflammatory cytokine production and/or increased regulatory responses. Crohn's patients were treated with G‐CSF (5 µg/kg/day subcutaneously) for 4 weeks and changes in cell phenotype, cytokine production and dendritic cell subsets were measured in the peripheral blood and colonic mucosal biopsies using flow cytometry, enzyme‐linked immunosorbent assay and immunocytochemistry. Crohn's patients who achieved a clinical response or remission based on the decrease in the Crohn's disease activity index differed from non‐responding patients in several important ways: at the end of treatment, responding patients had significantly more CD4⁺ memory T cells producing IL‐10 in the peripheral blood; they also had a greatly enhanced CD123⁺ plasmacytoid dendritic cell infiltration of the lamina propria. Interferon‐γ production capacity was not changed significantly except in non‐responders, where it increased. These data show that clinical benefit from G‐CSF treatment in Crohn's disease is accompanied by significant induction of IL‐10 secreting T cells as well as increases in plasmacytoid dendritic cells in the lamina propria of the inflamed gut mucosa.     

Imbalance of different types of CD4+forkhead box protein 3 (FoxP3)+ T cells in patients with new‐onset systemic lupus erythematosus

The aim of this study was to examine the numbers of CD4⁺CD25⁻forkhead box protein 3 (FoxP3)⁺, CD4⁺CD25⁺FoxP3⁺ and CD4⁺CXCR5⁺FoxP3⁺ T cells in patients with new-onset systemic lupus erythematosus (SLE). The numbers of CD4⁺CD25⁻FoxP3⁺, CD4⁺CD25⁺FoxP3⁺ and CD4⁺CXCR5⁺FoxP3⁺ T cells and the concentrations of serum interleukin (IL)-10 in 23 patients and 20 healthy controls (HC) were measured. The potential correlations between CD4⁺FoxP3⁺ T cells, serum IL-10 and clinical measures in SLE patients were analysed. In comparison with that in the HC, significantly reduced numbers of CD4⁺CD25⁺FoxP3⁺ and CD4⁺CXCR5⁺FoxP3⁺ T cells, but increased numbers of CD4⁺CD25⁻FoxP3⁺ T cells, were detected, accompanied by significantly lower levels of serum IL-10 in the patients. Stratification analysis indicated the numbers of CD4⁺CD25⁺FoxP3⁺ and CD4⁺CXCR5⁺FoxP3⁺ T cells and serum IL-10 levels in the patients with seropositive anti-dsDNA were significantly less than that in those with seronegative anti-dsDNA. Treatment with the anti-SLE therapy, particularly with prednisone, leflunomide and methotrexate, significantly improved the imbalance of these types of FoxP3⁺ T cells and increased the concentrations of serum IL-10 in the drug-responding patients. The numbers of CD4⁺CD25⁺FoxP3⁺ T cells were correlated negatively with the values of SLE disease activity index (SLEDAI), whereas the numbers of CD4⁺CD25⁻FoxP3⁺ T cells were correlated positively with the values of SLEDAI, erythrocyte sedimentation rate (ESR) and serum C3. In addition, the concentrations of serum IL-10 were correlated positively with the numbers of CD4⁺CD25⁺FoxP3⁺ T cells, but negatively with the values of SLEDAI, serum C3, CRP and ESR in these patients. Our data indicate that the imbalance of different types of FoxP3⁺CD4⁺ T cells may contribute to the development of SLE in Chinese patients.     

Interleukin‐21 (IL‐21) synergizes with IL‐2 to enhance T‐cell receptor‐induced human T‐cell proliferation and counteracts IL‐2/transforming growth factor‐β‐induced regulatory T‐cell development

Interleukin‐2 (IL‐2) is a mainstay for current immunotherapeutic protocols but its usefulness in patients is reduced by severe toxicities and because IL‐2 facilitates regulatory T (Treg) cell development. IL‐21 is a type I cytokine acting as a potent T‐cell co‐mitogen but less efficient than IL‐2 in sustaining T‐cell proliferation. Using various in vitro models for T‐cell receptor (TCR)‐dependent human T‐cell proliferation, we found that IL‐21 synergized with IL‐2 to make CD4⁺ and CD8⁺ T cells attain a level of expansion that was impossible to obtain with IL‐2 alone. Synergy was mostly evident in naive CD4⁺ cells. IL‐2 and tumour‐released transforming growth factor‐β (TGF‐β) are the main environmental cues that cooperate in Treg cell induction in tumour patients. Interleukin‐21 hampered Treg cell expansion induced by IL‐2/TGF‐β combination in naive CD4⁺ cells by facilitating non‐Treg over Treg cell proliferation from the early phases of cell activation. Conversely, IL‐21 did not modulate the conversion of naive activated CD4⁺ cells into Treg cells in the absence of cell division. Treg cell reduction was related to persistent activation of Stat3, a negative regulator of Treg cells associated with down‐modulation of IL‐2/TGF‐β‐induced phosphorylation of Smad2/3, a positive regulator of Treg cells. In contrast to previous studies, IL‐21 was completely ineffective in counteracting the suppressive activity of Treg cells on naive and memory, CD4⁺ and CD8⁺ T cells. Present data provide proof‐of‐concept for evaluating a combinatorial approach that would reduce the IL‐2 needed to sustain T‐cell proliferation efficiently, thereby reducing toxicity and controlling a tolerizing mechanism responsible for the contraction of the T‐cell response.     

Peripheral CD4 loss of regulatory T cells is associated with persistent viraemia in chronic HIV infection

Chronic HIV infection is associated with T cell abnormalities and altered effector function. Regulatory T cells (Treg) are CD4+ T cells that play a critical role in regulating the immune system. The impact of regulatory T cells on HIV infection and disease progression may be highly significant. We hypothesize that chronic antigenic stimulation from a persistent, high viraemic state may promote a population of Treg that contributes to HIV-associated immune dysfunction. We evaluated the pattern of Treg in chronically infected, HIV-positive individuals over a course of 6 months. Treg are depleted at a distinct rate from that of absolute CD4 cells and loss of Treg is slower in the presence of viral suppression. In vitro depletion of CD25+ CD4+ cells resulted in increased Gag-specific CD4 and CD8 responses. A significant correlation between ex vivo measurement of Treg and Gag-specific CD4 T cell responses was observed (r=-0 x 41, P=0 x 018) with a trend observed with Gag-specific CD8 T cell responses (P=0 x 07). The impact of HIV infection on the Treg population directly complicates the measured effect of Treg on the immune dysfunction although our data support the important role of Treg on modulating the effector T cell response in chronic infection.     

T‐cell modulation by cyclophosphamide for tumour therapy

The power of T cells for cancer treatment has been demonstrated by the success of co‐inhibitory receptor blockade and adoptive T‐cell immunotherapies. These treatments are highly successful for certain cancers, but are often personalized, expensive and associated with harmful side effects. Other T‐cell‐modulating drugs may provide additional means of improving immune responses to tumours without these disadvantages. Conventional chemotherapeutic drugs are traditionally used to target cancers directly; however, it is clear that some also have significant immune‐modulating effects that can be harnessed to target tumours. Cyclophosphamide is one such drug; used at lower doses than in mainstream chemotherapy, it can perturb immune homeostasis, tipping the balance towards generation of anti‐tumour T‐cell responses and control of cancer growth. This review discusses its growing reputation as an immune‐modulator whose multiple effects synergize with the microbiota to tip the balance towards tumour immunity offering widespread benefits as a safe, and relatively inexpensive component of cancer immunotherapy.     

Glucocorticoid treatment restores the impaired suppressive function of regulatory T cells in patients with relapsing–remitting multiple sclerosis

Patients relapsing from multiple sclerosis (MS) are treated with high‐dose, short‐term intravenous injection of glucocorticoid (GC), although its mechanism of action remains only partly understood. We evaluated the ex vivo and in vitro effects of GC on regulatory T cell (T_reg) function in 14 relapsing–remitting MS (RR‐MS) patients in acute phase and 20 healthy controls (HC). T_reg function was enhanced significantly after 5 days of GC treatment. Furthermore, there was a trend towards increasing proportions of CD4⁺CD25⁺forkhead box P3⁺ T cells and interleukin‐10 secretion with GC treatment when compared with HC. In conclusion, GC treatment restores the impaired T_reg function in patients with RR‐MS in its acute phase.     

Primary human endothelial cells support direct but not antibody‐dependent enhancement of dengue viral infection

Microvascular plasma leakage is the hallmark of dengue hemorrhagic fever and dengue shock syndrome. The precise molecular mechanisms leading to microvascular leakage are yet to be determined, but dengue virus (DENV) infection and consequent endothelial cell death has been suggested as its major cause. However, the extent of endothelial cell permissiveness to DENV infection and the magnitude of cell death following DENV infection are controversial. To clarify this issue, we analyzed the kinetics and consequences of DENV infection of human umbilical vein endothelial cells (HUVEC) using a novel molecularly cloned DENV2‐16681 virus. Viral replication was detected as early as 24 hr post‐infection by RT‐PCR and plaque assays. However, merely 2% of HUVEC were DENV antigen‐positive even after 96 hr of infection as measured by the FACS indirect immunofluorescence assays. Unlike monocytes/macrophages, HUVEC did not support antibody dependent enhancement of dengue viral infection due to a lack of FcγRI and FcγRII. Furthermore, DENV infection did not increase HUVEC apoptosis as compared to mock‐infected cells. Because in vitro only a small percentage of endothelial cells were productively infected in vitro with no significant apoptosis occurring in either infected or bystander cells, it would be important to re‐examine whether direct dengue viral infection of endothelium is the major cause of the extensive vascular leakage observed in patients with dengue hemorrhagic fever and dengue shock syndrome. J. Med. Virol. 81:519–528, 2009. © 2009 Wiley‐Liss, Inc.     

T regulatory cells lacking CD25 are increased in MS during relapse

Dysregulation of inflammatory responses is considered to be a key element in autoreactive immune responses. T regulatory cells (Tregs) are important to maintain self-tolerance and the role of CD4⁺CD25⁺FoxP3⁺ Tregs in autoimmunity has been extensively investigated. Recently, it was shown that Tregs in systemic lupus erythematosus lacked CD25 but were biologically functional. These data warrants for further investigation of CD25^? Tregs in human autoimmunity. We analyzed relapsing–remitting multiple sclerosis (MS) patients by multicolor flow cytometry for the expression of CD3, CD4, IL2R (CD25), FoxP3, and the IL7R (CD127). Further, the level of Tregs was compared in remitting and relapsing patients and correlated with disease duration. Patients in relapse exhibited higher levels of FoxP3-positive Tregs lacking CD25 compared to healthy controls (p < 0.05), indicating that Tregs attempt to restrain immune activity during relapse. The proportion of Tregs tended to be decreased with disease duration, while CD25⁺CD4⁺ and CD25⁺CD8⁺ effector T-cell proportions were elevated and positively correlated with overall disease duration (p < 0.05). In conclusion, while MS patients in remission have normal levels of Tregs of different phenotype, relapsing patients show an increased proportion of systemic CD25^? FoxP3⁺ Tregs. With time, the proportion of Tregs decrease while effector T cells expand.