Foxp3 Treg Cells Research Articles

Circulating regulatory T cells are reduced in obesity and may identify subjects at increased metabolic and cardiovascular risk

Reduced numbers of regulatory T (Treg ) cells have been observed in visceral adipose tissue of obese mice and humans. However, it is unknown whether human obesity affects circulating Treg cells and whether their number is associated with markers of systemic inflammation or glucose intolerance. Peripheral blood mononuclear cells were isolated from venous blood of obese (BMI ≥ 27 kg/m(2) ; n = 30) and nonobese (BMI ≥ 27 kg/m(2) ; n = 13) individuals and analyzed using flow cytometry for the expression of CD4, CD25, and Foxp3. Reduced circulating Treg -cell numbers were detected in obese compared with nonobese study participants (P = 0.038). Circulating CD4(+) CD25(+) CD127(-) Foxp3 Treg cells inversely correlated with body weight (P = 0.009), BMI (P = 0.004) and plasma leptin levels (P = 0.004) and were reduced in subjects with hsCRP ≥ 3.0 mg/L (P = 0.034) or HbA1c ≥ 5.5% (P < 0.005). Receiver operating characteristic curve analysis revealed a cutoff of circulating Treg cells < 1.06% to be predictive for hsCRP levels ≥ 3.0 mg/L, and logistic regression showed that the risk of having hsCRP levels ≥ 3.0 mg/L was increased 9.6-fold (P = 0.008), if Treg cells were below this threshold. The Treg cutoff for HbA1c levels ≥ 5.5% was 0.73%, and this cutoff also predicted an increased risk of having elevated levels of both hsCRP and HbA1c, if only obese subjects were examined. Our findings thus reveal an association between circulating Treg cells and measures of adiposity, inflammation, and glucose intolerance. Although further prospective studies are needed, we present data suggesting that the determination of Treg cells might be useful to identify obese subjects at increased risk of developing cardiovascular and/or metabolic complications.

Overcoming regulatory T‐cell suppression by a lyophilized preparation of Streptococcus pyogenes

Cancer vaccines have yet to yield clinical benefit, despite the measurable induction of humoral and cellular immune responses. As immunosuppression by CD4(+) CD25(+) regulatory T (Treg) cells has been linked to the failure of cancer immunotherapy, blocking suppression is therefore critical for successful clinical strategies. Here, we addressed whether a lyophilized preparation of Streptococcus pyogenes (OK-432), which stimulates Toll-like receptors, could overcome Treg-cell suppression of CD4(+) T-cell responses in vitro and in vivo. OK-432 significantly enhanced in vitro proliferation of CD4(+) effector T cells by blocking Treg-cell suppression and this blocking effect depended on IL-12 derived from antigen-presenting cells. Direct administration of OK-432 into tumor-associated exudate fluids resulted in a reduction of the frequency and suppressive function of CD4(+) CD25(+) Foxp3(+) Treg cells. Furthermore, when OK-432 was used as an adjuvant of vaccination with HER2 and NY-ESO-1 for esophageal cancer patients, NY-ESO-1-specific CD4(+) T-cell precursors were activated, and NY-ESO-1-specific CD4(+) T cells were detected within the effector/memory T-cell population. CD4(+) T-cell clones from these patients had high-affinity TCRs and recognized naturally processed NY-ESO-1 protein presented by dendritic cells. OK-432 therefore inhibits Treg-cell function and contributes to the activation of high-avidity tumor antigen-specific naive T-cell precursors.

Foxp3+ regulatory T cells are activated in spite of B7‐CD28 and CD40‐CD40L blockade

Costimulatory signals are required for priming and activation of naive T cells, while it is less clear how they contribute to induction of regulatory T (Treg)-cell activity. We previously reported that the blockade of the B7-CD28 and CD40L-CD40 interaction efficiently suppresses allogeneic T-cell activation in vivo. This was characterized by an initial rise in Foxp3(+) cells, followed by depletion of host-reactive T cells. To further investigate effects of costimulatory blockade on Treg cells, we used an in vitro model of allogeneic CD4(+) cell activation. When CTLA-4Ig and anti-CD40L mAb (MR1) were added to the cultures, T-cell proliferation and IL-2 production were strongly reduced. However, Foxp3(+) cells proliferated and acquired suppressive activity. They suppressed activation of syngeneic CD4(+) cells much more efficiently than did freshly isolated Treg cells. CD4(+) cells activated by allogeneic cells in the presence of MR1 and CTLA-4Ig were hyporesponsive on restimulation, but their response was restored to that of naive CD4(+) cells when Foxp3(+) Treg cells were removed. We conclude that natural Treg cells are less dependent on B7-CD28 or CD40-CD40L costimulation compared with Foxp3(-) T cells. Reduced costimulation therefore alters the balance between Teff and Treg-cell activation in favor of Treg-cell activity.

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

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

Vitamin D Levels and FoxP3 Treg Cells Expression in North Indian Children with Atopic Diseases

Vitamin D Levels and FoxP3 Treg Cells Expression in North Indian Children with Atopic Diseases

IL‐2 promotes the function of memory‐like autoregulatory CD8+T cells but suppresses their development via FoxP3+Treg cells

IL-2 plays a critical role in both effector T-cell development and FoxP3(+) CD4(+) Treg-cell homeostasis. A reduction in Il2 transcription results in impaired FoxP3(+) CD4(+) Treg-cell recruitment and function, and accounts for the association between murine Il2 and type 1 diabetes (T1D). The progression of T1D elicits a disease-countering negative feedback regulatory loop that involves the differentiation of low-avidity autoreactive CD8(+) T cells into memory-like autoregulatory T cells in a CD4(+) Th-dependent manner. Since these auto-regulatory T cells express IL-2Rβ (CD122), we hypothesized that their development might also be regulated by IL-2. Here, we investigate the effects of differences in IL-2 expression on this autoregulatory subset. We show that decreased IL-2 production impairs the regulatory capacity of memory-like autoregulatory CD8(+) CD122(+) T cells. Surprisingly, we also find that a reduction in IL-2 production capacity increases memory autoregulatory CD8(+) T-cell formation indirectly, by decreasing the development and function of FoxP3(+) Treg cells in nonobese diabetic mice. These results illustrate a complex homeostatic interplay between IL-2, CD4(+) Th cells, FoxP3(+) CD4(+) Treg cells and autoregulatory CD8(+) T-cell memory whereby IL-2 controls the function of both Treg-cell subsets, but IL-2-potentiation of FoxP3(+) CD4(+) Treg-cell function results in the suppression of CD4(+) Th-cell activation and autoregulatory memory CD8(+) T-cell formation.

Protection against allergic airway inflammation during the chronic and acute phases of Trichinella spiralis infection

Modulation of the host immune response by helminths has been reported to be essential for parasite survival and also to benefit the host by suppressing inflammatory diseases such as allergies. We have previously shown that excretory-secretory products of Trichinella spiralis muscle larvae have immunomodulatory properties and induce in vitro the expansion of CD4(+) CD25(+) FOXP3(+) Treg cells in a TGF-β-dependent manner. We aimed at determining the effect of the acute (intestinal) and the chronic (muscle) phase of T. spiralis infection on experimental allergic airway inflammation (EAAI) to Ovalbumin (OVA) and the involvement of Treg cells. The chronic phase was established before OVA-sensitization/challenge and the acute phase at two-time points, before and after OVA-sensitization. Mice were infected with 400 T. spiralis larvae and after euthanasia different pathological features of EAAI were measured. Adoptive transfer of CD4(+) T cells from Trichinella infected mice to OVA sensitized/challenged recipients was also performed. We found that the chronic as well as the acute phase of Trichinella infection suppress EAAI as indicated by reduction in airway inflammation, OVA-specific IgE levels in sera, Th2-cytokine production and eosinophils in bronchoalveolar lavage. This protective effect was found to be stronger during the chronic phase and to be associated with increased numbers of splenic CD4(+) CD25(+) FOXP3(+) Treg cells with suppressive activity. Adoptive transfer of splenic CD4(+) T cells from chronically infected mice with elevated numbers of Treg cells resulted in partial protection against EAAI. These results demonstrate that the protective effect of T. spiralis on EAAI increases as infection progresses from the acute to the chronic phase. Here, Treg cells may play an essential role in the suppression of EAAI. Elucidating the mechanisms and molecular helminth structures responsible for this regulatory process is relevant to develop alternative tools for preventing or treating allergic asthma.

HCV-infected hepatocytes drive CD4+ CD25+ Foxp3+ regulatory T-cell development through the Tim-3/Gal-9 pathway.

HCV is remarkable at disrupting human immunity to establish chronic infection. The accumulation of Treg cells at the site of infection and upregulation of inhibitory signaling pathways (such as T-cell Ig and mucin domain protein-3 (Tim-3) and galectin-9 (Gal-9)) play pivotal roles in suppressing antiviral effector T (Teff) cells that are essential for viral clearance. While Tim-3/Gal-9 interactions have been shown to negatively regulate Teff cells, their role in regulating Treg cells is poorly understood. To explore how Tim-3/Gal-9 interactions regulate HCV-mediated Treg-cell development, here we provide pilot data showing that HCV-infected human hepatocytes express higher levels of Gal-9 and TGF-β, and upregulate Tim-3 expression and regulatory cytokines TGF-β/IL-10 in co-cultured human CD4(+) T cells, driving conventional CD4(+) T cells into CD25(+) Foxp3(+) Treg cells. Additionally, recombinant Gal-9 protein can transform TCR-activated CD4(+) T cells into Foxp3(+) Treg cells in a dose-dependent manner. Importantly, blocking Tim-3/Gal-9 ligations abrogates HCV-mediated Treg-cell induction by HCV-infected hepatocytes, suggesting that Tim-3/Gal-9 interactions may regulate human Foxp3(+) Treg-cell development and function during HCV infection.

Functional changes in regulatory T cells during an experimental infection with sparganum (plerocercofid of Spirometra mansoni)

Regulatory T (Treg) cells are important in the regulation of immune response, but the exact regulation of Treg-cell function in vivo is still not well known. In the present study, we investigated the functional activity of CD4(+) CD25(+) Treg cells as well as the frequency and number of CD4(+) CD25(+) FoxP3(+) Treg cells in the spleens of experimentally infected mice with a tissue-migrating parasite, sparganum (plerocercoid of Spirometra mansoni) for 3 weeks. The results demonstrated fluctuations in the Treg-cell function during the parasite infection, being up-regulated at day 3, down-regulated until day 14, and thereafter up-regulated again at day 21. We also investigated the cytokine-producing capability of the splenocytes to study the pattern of immune response of the mice to the parasite. The results showed decreased capabilities of interleukin-2 (IL-2), interferon-γ (IFN-γ) and IL-17α production, whereas IL-4-producing and IL-10-producing capabilities were increased along with the parasitic infection. Meanwhile, IL-6-producing capability was increased to reach a peak at week 2, and thereafter was decreased to the baseline level. As a regulatory mechanism, we found that Treg-cell function was attenuated in the presence of the crude extracts of sparganum, but was enhanced in the presence of the excretory-secretory products, suggesting that sparganum products were involved in the triggering and regulation of immune response in the acute and chronic phases, respectively. Results show that Treg cells are central in the immune homeostasis in vivo that is maintained by host-parasite interactions during the parasitic infection.

The Ratio of Treg/Th17 Cells Correlates with the Disease Activity of Primary Immune Thrombocytopenia

BackgroundPrimary immune thrombocytopenia (ITP) is an autoimmune heterogeneous disorder that is characterized by decreased platelet count. Regulatory T (Treg) cells and T helper type 17 (Th17) cells are two subtypes of CD4+ T helper (Th) cells. They play opposite roles in immune tolerance and autoimmune diseases, while they share a common differentiation pathway. The imbalance of Treg/Th17 has been demonstrated in several autoimmune diseases. In this study, we aimed to investigate the ratio of the number of Treg cells to the number of Th17 cells in ITP patients and evaluate the clinical implications of the alterations in this ratio.MethodsThirty adult patients with newly diagnosed ITP enrolled in this study. Twelve patients had been clinically followed up for 12 months. The percentages of CD4+CD25hiFoxp3+ Treg cells and CD3+CD4+IL-17-producing Th17 cells in these patients and healthy controls (n = 17) were longitudinally analyzed by flow cytometry.ResultsThe percentage of Treg cells in ITP patients was significantly lower than that of healthy controls, and the percentage of Th17 cells increased significantly at disease onset. The ratio of Treg/Th17 correlated with the disease activity.ConclusionThe ratio of Treg/Th17 might be relevant to the clinical diversity of ITP patients, and this Treg/Th17 ratio might have prognostic role in ITP patients.

IL-36g Promotes Intestinal Inflammation Via Intestinal Macrophage/DC Activation

The two most common forms of inflammatory bowel disease (IBD), Crohn's disease and ulcerative colitis, affect approximately 1.4 million people in the United States. Uncontrolled APCs reactivity toward commensal bacteria and the consequent pro-inflammatory cytokine production is implicated in disease pathogenesis. Neutralization of TNF is currently one of the most effective biological therapies for Crohn's patients, however, there remains a pressing need for the development of novel therapeutics targeting pro-inflammatory cytokines. Several members of the IL-1 family of cytokines, including IL-1a, IL-1b, IL-18 and IL-33 are associated with the pathophysiology of IBD. Since we have identified a member of the IL-1 family of cytokines, termed IL-36g, in a genetic screen for factors selectively expressed by inflammatory colonic lamina propria (cLP) macrophages, in this work we focus on defining the pathogenic role for IL-36g during intestinal inflammation. C57BL/6 mice were fed for 5 days with 3% DSS dissolved in drinking water, and IL-36Ra was injected intraperitoneally on days -1, 1 and 3. Animals were assessed daily for stool consistency, fecal blood, and weight loss. cLP cells were isolated by magnetic positive selection or cell sorting. Cell surface staining and intracellular cytokine detection was performed using labeled antibodies for CD45, CD103, CD11c, MHC-II, CD11b, F4/80, Ly6C, Cx3cr1-FITC, live/dead staining, CD4, TCRb, CD45, IL-17A, IFNg, and FoxP3. qPCR on total RNA from macrophages was performed using SYBR Green. Treg induction was performed by co-culture of macrophages, OT-II CD4+ T cells and OVA for 3 days. Our results demonstrated that IL-36g is highly induced in the inflamed colonic tissue of mice during acute and chronic intestinal inflammation with one of the major cell types expressing IL-36g in the inflamed colonic mucosa being inflammatory Ly6C+CX3CR1(lo) macrophages. This specific expression prompted us to investigate the modulation of the function of resident inflammatory macrophages, and we observed that inflammatory cLP macrophages isolated from DSStreated mice failed to promote Foxp3 induction in responding CD4+ T cells when compared to control macrophages. The addition of IL-36g to co-cultures of T cells and cLP macrophages isolated from healthy mouse colon confirmed this specific inhibition of FoxP3 cell differentiation in the presence of IL-36g. Together, these data suggest that one important biological function of IL-36g is to inhibit Foxp3 Treg cell differentiation. We next examined the role of IL-36g in vivo during DSSinduced colitis by administration of the IL-36 receptor antagonist (IL-36Ra) on days -1, 1, and 3 of the DSS regimen, which resulted in a significant reduction in the overall disease when compared to mice that did not receive IL-36Ra. Since IL-36Ra blocks the function of IL-36R, which can bind IL-36a, IL-36b, and IL-36g, we conclude from these studies that at least one of these IL-36R ligands is important in the pathogenesis of intestinal inflammation. Collectively, our data demonstrate an important role for the macrophage-derived inflammatory cytokine IL-36g and the IL-36g/IL-36R axis during the pathogenesis of intestinal inflammation. The present work will aid in understanding appropriate targets for immunotherapy. This work was supported by CCFA (RFA, CDA) and NIH (AA017870 and AI083554) awards.

Anti-CD25 Treatment Depletes Treg Cells and Decreases Disease Severity in Susceptible and Resistant Mice Infected with Paracoccidioides brasiliensis

Regulatory T (Treg) cells are fundamental in the control of immunity and excessive tissue pathology. In paracoccidioidomycosis, an endemic mycosis of Latin America, the immunoregulatory mechanisms that control the progressive and regressive forms of this infection are poorly known. Due to its modulatory activity on Treg cells, we investigated the effects of anti-CD25 treatment over the course of pulmonary infection in resistant (A/J) and susceptible (B10.A) mice infected with Paracoccidioides brasiliensis. We verified that the resistant A/J mice developed higher numbers and more potent Treg cells than susceptible B10.A mice. Compared to B10.A cells, the CD4+CD25+Foxp3+ Treg cells of A/J mice expressed higher levels of CD25, CTLA4, GITR, Foxp3, LAP and intracellular IL-10 and TGF-β. In both resistant and susceptible mice, anti-CD25 treatment decreased the CD4+CD25+Foxp3+ Treg cell number, impaired indoleamine 2,3-dioxygenase expression and resulted in decreased fungal loads in the lungs, liver and spleen. In A/J mice, anti-CD25 treatment led to an early increase in T cell immunity, demonstrated by the augmented influx of activated CD4+ and CD8+ T cells, macrophages and dendritic cells to the lungs. At a later phase, the mild infection was associated with decreased inflammatory reactions and increased Th1/Th2/Th17 cytokine production. In B10.A mice, anti-CD25 treatment did not alter the inflammatory reactions but increased the fungicidal mechanisms and late secretion of Th1/Th2/Th17 cytokines. Importantly, in both mouse strains, the early depletion of CD25+ cells resulted in less severe tissue pathology and abolished the enhanced mortality observed in susceptible mice. In conclusion, this study is the first to demonstrate that anti-CD25 treatment is beneficial to the progressive and regressive forms of paracoccidioidomycosis, potentially due to the anti-CD25-mediated reduction of Treg cells, as these cells have suppressive effects on the early T cell response in resistant mice and the clearance mechanisms of fungal cells in susceptible mice.

IκBNS Protein Mediates Regulatory T Cell Development via Induction of the Foxp3 Transcription Factor

Forkhead box P3 positive (Foxp3(+)) regulatory T (Treg) cells suppress immune responses and regulate peripheral tolerance. Here we show that the atypical inhibitor of NFκB (IκB) IκB(NS) drives Foxp3 expression via association with the promoter and the conserved noncoding sequence 3 (CNS3) of the Foxp3 locus. Consequently, IκB(NS) deficiency leads to a substantial reduction of Foxp3(+) Treg cells invivo and impaired Foxp3 induction upon transforming growth factor-β (TGF-β) treatment invitro. Moreover, fewer Foxp3(+) Treg cells developed from IκB(NS)-deficient CD25(-)CD4(+) Tcells adoptively transferred into immunodeficient recipients. Importantly, IκB(NS) was required for the transition of immature GITR(+)CD25(+)Foxp3(-) thymic Treg cell precursors into Foxp3(+) cells. In contrast to mice lacking c-Rel or Carma1, IκB(NS)-deficient mice do not show reduced Treg precursor cells. Our results demonstrate that IκB(NS) critically regulates Treg cell development in the thymus and during gut inflammation, indicating that strategies targeting IκB(NS) could modulate the Treg cell compartment.

Tumor necrosis factor α-induced proteins: Natural brakes on inflammation

Peggy Jacques and Dirk ElewautOne of the principal tasks of our immune systemis to protect our body against pathogens. Of equalimportance, however, is the capacity to preserve theintegrity of the host by protecting against autoimmunity.To accomplish this, a number of regulatory pathwayshave been revealed. They include certain regulatoryT cell subsets, such as FoxP3 Treg cells, natural killerT cells, and Tr1 cells, as well as other cell types, such ascertain macrophage subsets. While some of these occurnaturally as the immune system develops, interestingly,certain subsets may be actively induced in adults. Thishas sparked interest in their potential use in therapeuticapproaches. Also at the cellular level, a number ofproteins with antiinflammatory capacity have been dis-covered, some of which are induced in response to theproinflammatory cytokine tumor necrosis factor (TNF).TNF operates both upstream and downstream ofdiverse signaling cascades, and numerous proteins areinduced in response to TNF. In recent years, some ofthese so-called TNF -induced proteins (TNFAIPs) havegained a lot of interest. One of these proteins is A20, orTNFAIP-3, a deubiquitinating protein that negativelyregulates NF- B–dependent gene expression in re-sponse to different immune-activating stimuli, includingTNF, interleukin-1 (IL-1), and in response to triggeringof Toll-like receptors (TLRs) and nucleotide-bindingoligomerization domain–containing protein 2 (NOD-2)receptor 1. A20 is considered to inhibit NF- B func-tion by deubiquitinating specific NF- B signaling mole-cules, such as receptor-interacting protein 1 (RIP-1),RIP-2, and TNF receptor–associated factor 6 (TRAF-6).Several single-nucleotide polymorphisms in the humanTNFAIP-3 locus have been shown to be associated withincreased susceptibility to rheumatoid arthritis, type 1diabetes mellitus, systemic lupus erythematosus, celiacdisease, Crohn’s disease, psoriasis, and multiple sclerosis(for review, see ref. 1).Another TNF-induced protein, TNFAIP-5, orpentraxin 3, is expressed in rheumatoid arthritis synovialtissue and in astrocytes from patients with Alzheimer’sdisease (2,3), while the apoptosis regulator TNFAIP-8contains a death-effector domain and is capable ofinhibiting caspase-mediated apoptosis (4). Knock-downof TNFAIP-8 expression in tumor cells decreases theironcogenicity, which suggests that it may be involved incarcinogenesis (5). TNFAIP-6 (TNF -stimulated gene 6[TSG-6]), a protein involved in cell–cell and cell–matrixinteractions, is expressed within the synovium and car-tilage of arthritic joints and is thought to play a protec-tive role in articular inflammation (6). It further inhibitsRANKL-induced osteoclast differentiation and activa-tion (7).The important roles of these proteins in themammalian immune system have been demonstrated inmice, where a deficiency in the proteins generally leadsto systemic inflammation. Mice fully deficient in A20spontaneously develop multiple organ inflammation andcachexia and die within 2 weeks of birth (8). Micedeficient in TNFAIP-8–like 2 (TIPE-2; a member of theTNFAIP-8 family) develop multiple organ inflammationand splenomegaly, are hypersensitive to septic shock,and die prematurely (9).TNF -induced adipose-related protein (TIARP),also called 6-transmembrane protein of prostate 2(STAMP-2) or the human homolog 6-transmembraneepithelial antigen of prostate 4 (STEAP-4), belongs to afamily of 4 mammalian proteins that were all originallycharacterized in the prostate, including STAMP-1(STEAP-2), STEAP, and STEAP-3 (pHyde) (10,11).The expression of TIARP/STAMP-2 dramatically in-creases during TNF exposure, and yet also during thecourse of adipose differentiation, as demonstrated in the3T3-L1 preadipose cell line (11). Tissue distribution ofTIARP messenger RNA is not restricted to white andbrown adipose tissues, but is also detectable in liver,kidney, heart, and skeletal muscle.

Astrocytic Fas ligand expression is required to induce T‐cell apoptosis and recovery from experimental autoimmune encephalomyelitis

In T-cell-mediated autoimmune diseases of the CNS, apoptosis of Fas(+) T cells by FasL contributes to resolution of disease. However, the apoptosis-inducing cell population still remains to be identified. To address the role of astrocytic FasL in the regulation of T-cell apoptosis in experimental autoimmune encephalomyelitis, we immunized C57BL/6 glial fibrillary acid protein (GFAP)-Cre FasL(fl/fl) mice selectively lacking FasL in astrocytes with MOG(35-55) peptide. GFAP-Cre FasL(fl/fl) mice were unable to resolve EAE and suffered from persisting demyelination and paralysis, while FasL(fl/fl) control mice recovered. In contrast to FasL(fl/fl) mice, GFAP-Cre FasL(fl/fl) mice failed to induce apoptosis of Fas(+) activated CD4(+) T cells and to increase numbers of Foxp3(+) Treg cells beyond day 15 post immunization, the time point of maximal clinical disease in control mice. The persistence of activated and GM-CSF-producing CD4(+) T cells in GFAP-Cre FasL(fl/fl) mice also resulted in an increased IL-17, IFN-γ, TNF, and GM-CSF mRNA expression in the CNS. In vitro, FasL(+) but not FasL(-) astrocytes induced caspase-3 expression and apoptosis of activated T cells. In conclusion, FasL expression of astrocytes plays an important role in the control and elimination of autoimmune T cells from the CNS, thereby determining recovery from EAE.

CCL2 is critical for immunosuppression to promote cancer metastasis

We previously found that cancer metastasis is accelerated by immunosuppression during Snail-induced epithelial-to-mesenchymal transition (EMT). However, the molecular mechanism still remained unclear. Here, we demonstrate that CCL2 is a critical determinant for both tumor metastasis and immunosuppression induced by Snail(+) tumor cells. CCL2 is significantly upregulated in various human tumor cells accompanied by Snail expression induced by snail transduction or TGFβ treatment. The Snail(+) tumor-derived CCL2 amplifies EMT events in other cells including Snail(-) tumor cells and epithelial cells within tumor microenvironment. CCL2 secondarily induces Lipocalin 2 (LCN2) in the Snail(+) tumor cells in an autocrine manner. CCL2 and LCN2 cooperatively generate immunoregulatory dendritic cells (DCreg) having suppressive activity accompanied by lowered expression of costimulatory molecules such as HLA-DR but increased expression of immunosuppressive molecules such as PD-L1 in human PBMCs. The CCL2/LCN2-induced DCreg cells subsequently induce immunosuppressive CD4(+)FOXP3(+) Treg cells, and finally impair tumor-specific CTL induction. In murine established tumor model, however, CCL2 blockade utilizing the specific siRNA or neutralizing mAb significantly inhibits Snail(+) tumor growth and metastasis following systemic induction of anti-tumor immune responses in host. These results suggest that CCL2 is more than a chemoattractant factor that is the significant effector molecule responsible for immune evasion of Snail(+) tumor cells. CCL2 would be an attractive target for treatment to eliminate cancer cells via amelioration of tumor metastasis and immunosuppression.

Immunological homeostasis of the eye

Uveitis is a sight-threatening disease caused by autoimmune or infection-related immune responses. Studies in experimental autoimmune uveitis and in human diseases imply that activated CD4(+) T cells, Th1 and Th17 cells, play an effector role in ocular inflammation. The eye has a unique regional immune system to protect vision-related cells and tissues from these effector T cells. The immunological balance between the pathogenic CD4(+) T cells and regional immune system in the eye contributes to the maintenance of ocular homeostasis and good vision. Current studies have demonstrated that ocular parenchymal cells at the inner surface of the blood-ocular barrier, i.e. corneal endothelial (CE) cells, iris pigment epithelial (PE) cells, ciliary body PE cells, and retinal PE cells, contribute to the regional immune system of the eye. Murine ocular resident cells directly suppress activation of bystander T cells and production of inflammatory cytokines. The ocular resident cells possess distinct properties of immunoregulation that are related to disparate anatomical location. CE cells and iris PE cells, which are located at the anterior segment of the eye and face the aqueous humor, suppress activation of T cells via cell-to-cell contact mechanisms, whereas retinal PE cells suppress the activation of T cells via soluble factors. In addition to direct immune suppression, the ocular resident cells have another unique immunosuppressive property, the induction of CD25(+)Foxp3(+) Treg cells that also suppress the activation of bystander T cells. Iris PE cells convert CD8(+) T cells into Treg cells, while retinal PE cells convert CD4(+) T cells greatly and CD8(+) T cells moderately into Treg cells. CE cells also convert both CD4(+) T cells and CD8(+) T cells into Treg cells. The immunomodulation by ocular resident cells is mediated by various soluble or membrane-bound molecules that include TGF-β TSP-1, B7-2 (CD86), CTLA-2α, PD-L1 (B7-H1), galectin 1, pigment epithelial-derived factor PEDF), GIRTL, and retinoic acid. Human retinal PE cells also possess similar immune properties to induce Treg cells. Although there are many issues to be answered, human Treg cells induced by ocular resident cells such as retinal PE cells and related immunosuppressive molecules can be applied as immune therapy for refractive autoimmune uveitis in humans in the future.

Altered Decidual DC-SIGN+ Antigen-Presenting Cells and Impaired Regulatory T-Cell Induction in Preeclampsia

Regulatory T (Treg) cell expansion is required for tolerance of the semi-allogeneic fetus in healthy pregnancy and impaired in preeclampsia in humans. However, the reasons remain unknown. Herein, we show that expansion of CD4(+)Helios(-)Foxp3(+) adaptive Treg (iTreg) cells, rather than CD4(+)Helios(+)Foxp3(+) natural Treg cells, accounts for this expansion in healthy pregnancy. This expansion is even more pronounced in the decidua, where there is an overrepresentation of iTreg cells. In preeclampsia, however, there is impaired systemic iTreg cell expansion, associated with a lack of iTreg cell overrepresentation in the decidua. Because decidual antigen-presenting cells (APCs) may be important for iTreg cell induction, we studied decidual CD14(+) APCs using immunohistochemistry and flow cytometry. We show that decidual CD14(+)DC-SIGN(+) APCs are closely associated with Foxp3(+) Treg cells. Furthermore, CD14(+)DC-SIGN(+) cells display a distinct phenotype compared with their CD14(+)DC-SIGN(-) counterparts. In particular, they have increased expression of tolerogenic molecules, HLA-G, and immunoglobulin-like transcript 4. In vitro, CD14(+)DC-SIGN(+) APCs from healthy pregnant women induced iTreg cells significantly more efficiently than CD14(+)DC-SIGN(-) APCs. Conversely, in preeclampsia, both CD14(+)DC-SIGN(+) and CD14(+)DC-SIGN(-) APCs induced iTreg cells poorly. These results suggest that decidual CD14(+)DC-SIGN(+) APCs may play important roles in iTreg cell induction, a process that is defective in preeclampsia and likely contributes to its pathogenesis.

Contribution of regulatory T cells to alleviation of experimental allergic asthma after specific immunotherapy

Allergen-specific immunotherapy (SIT) has been used since 1911, yet its mechanism of action remains to be elucidated. There is evidence indicating that CD4(+)FOXP3(+) regulatory T cells (Treg cells) are induced during SIT in allergic patients. However, the contribution of these cells to SIT has not been evaluated in vivo. To evaluate the in vivo contribution of (i) CD4(+) CD25(+) T cells during SIT and of (ii) SIT-generated inducible FOXP3(+) Treg cells during allergen exposure to SIT-mediated suppression of asthmatic manifestations. We used a mouse model of SIT based on the classical OVA-driven experimental asthma. Treg cells were quantified by flow cytometry 24 and 96 h post SIT treatment. We depleted CD4(+) CD25(+) T cells prior to SIT, and CD4(+)FOXP3(+) T cells prior to allergen challenges to study their contribution to the suppression of allergic manifestations by SIT treatment. Our data show that depletion of CD4(+)CD25(+) T cells at the time of SIT treatment reverses the suppression of airway hyperresponsiveness (AHR), but not of airway eosinophilia and specific IgE levels in serum. Interestingly, the number of CD4(+)CD25(+)FOXP3(+) T cells is transiently increased after SIT in the spleen and blood, suggesting the generation of inducible and presumably allergen-specific Treg cells during treatment. Depletion of CD4(+)FOXP3(+) Treg cells after SIT treatment partially reverses the SIT-induced suppression of airway eosinophilia, but not of AHR and serum levels of specific IgE. We conclude that SIT-mediated tolerance induction towards AHR requires CD4(+)CD25(+) T cells at the time of allergen injections. In addition, SIT generates CD4(+)CD25(+)FOXP3(+) T cells that contribute to the suppression of airway eosinophilia upon allergen challenges. Therefore, enhancing Treg cell number or their activity during and after SIT could be of clinical relevance to improve the therapeutic effects of SIT.

Treg Cells Acquire New Directions, Cytokines Navigate

Foxp3(+) Treg cells express transcription factors normally expressed by specific T helper cells at sites of inflammation. In this issue of Immunity, Koch etal. (2012) and Hall etal. (2012) demonstrate that IFN-γ and IL-27 distinctly induces T-bet(+) Treg cells via STAT-1 transcription factor activation.