CD25 High Foxp3 Research Articles

Title: Repeated implantation failure is associated with increased Th17/Treg cell ratio, during the secretory phase of the human endometrium

Repeated implantation failure (RIF) is a significant limiting factor in assisted reproduction. Chronic endometrial inflammation has been noted in RIF women, therefore we sought to investigate the potential association of endometrial Th17/Treg ratio and endometrial inflammation in these cases. Endometrial pipelle biopsies were obtained from volunteers, 29 women with RIF (failure to achieve pregnancy following at least 3 transfers of high-grade embryos in IVF-cycles) and 27 fertile women (at least one child) in total, at the secretory phase of the menstrual cycle. Using tissues from 17 fertile and 18 RIF endometrial samples, stromal and immune cells were isolated and flow cytometry analysis was performed to determine Th17 and CD4+CD25high FOXP3+ cell populations in endometrial stromal cell suspensions. Another group of tissues from 10 fertile and 11 RIF samples were used for mRNA expression levels of Treg and Th17-cell transcription factors, FOXP3 and RORγt respectively. Endometrial inflammatory mediators’ mRNA expression was also analyzed. A statistically significant increase in protein flow cytometry analysis of Th17/Treg ratio (p≤0.05) as well as a reduction in absolute Treg cells in the endometrium (p≤0.05) was noted in women with RIF. Additionally, RNA analysis on the same set of women indicated RORγt/FOXP3 significantly increased in women with RIF compared to fertile ones (p≤0.05). Finally, women with RIF exhibited significantly (p≤0.05) elevated mRNA levels of pro-inflammatory mediators (ΤΝF-a, ΙL-6, IL-8 and CCl2). Women with RIF exhibit elevated Th17/Treg ratio, mostly due to endometrial Treg depletion, as well as a pro-inflammatory state in the endometrium.

Investigating the T regulatory cells and Sirtuin-I serum level in immunotherapy treated house dust mite allergic asthma patients

Objectives House dust mite aeroallergens are predominant triggers of frequent asthma attacks among adults and children. The intensity of asthma and immune reaction necessitates treatment alternatives based on adjusting chosen immunity biomarkers to control the exacerbation of symptoms and establish long-term immune tolerance. In this study, we selected CD4+CD25+Foxp3+ regulatory T cells (Tregs), FOXP3, and Sirtuin-1 as they are known to have a potential role in the immune reaction in different allergic diseases. We investigated their interplay during HDM allergic asthma and its respective immunotherapy. Methods Eighty-four subjects were divided into 3 groups; healthy controls (CT), HDM asthma patients without immunotherapy (WOIT), and HDM asthma patients treated with subcutaneous immunotherapy for 6 months before recruitment (WIT). They were enrolled according to the pulmonary function, skin prick tests, and HDM-specific IgE. CD4+ CD25+ and CD4+ CD25+ FOXP3+hi T cells Cell percentages, FOXP3 gene expression, and Sirtuin-1 (Sirt1) serum level were analyzed. Results We found that there is a significant difference between WOIT and WIT groups in the CD4+ CD25+ and CD4+ CD25+ FOXP3+hi T cell percentages. While there is no statistically significant difference between WOIT and WIT groups in FOXP3 level. On the controversy, the SIRT1 level in the CT group (4.53 ± 3.880) significantly decreased in the WOIT and WIT groups. Conclusion This study revealed that both CD4 CD25 and CD4 CD25 high FOXP3 cell percentages increased in the WIT group and declined in the WOIT group. While, FOXP3 gene expression increased in both groups. In addition, the Sirt1 serum level showed some improvement in WIT group after a serious drop in the WOIT group comparing with the CT group. The modulation of these biomarkers for the remission and control of allergic asthma can be a prognostic outcome of immunotherapy which needs to be confirmed by larger scale studies.

Immunomodulatory Functions of Adipose Mesenchymal Stromal/Stem Cell Derived From Donors With Type 2 Diabetes and Obesity on CD4 T Cells.

For adipose stromal/stem cell (ASCs)-based immunomodulatory therapies, it is important to study how donor characteristics, such as obesity and type 2 diabetes (T2D), influence ASCs efficacy. Here, ASCs were obtained from 2 groups, donors with T2D and obesity (dASCs) or nondiabetic donors with normal-weight (ndASCs), and then cultured with anti-CD3/CD28-stimulated allogeneic CD4 T cells. ASCs were studied for the expression of the immunomodulators CD54, CD274, and indoleamine 2, 3 dioxygenase 1 (IDO) in inflammatory conditions. CD4 T cells cultured alone or in cocultures were assessed to evaluate proliferation, activation marker surface expression, apoptosis, the regulatory T cells (Tregs; CD4+ CD25high FOXP3+) frequency, and intracellular cytokine expression using flow cytometry. Modulation of T-cell subset cytokines was explored via ELISA. In inflammatory conditions, the expression of CD54, CD274, and IDO was significantly upregulated in ASCs, with no significant differences between ndASCs and dASCs. dASCs retained the potential to significantly suppress CD4 T-cell proliferation, with a slightly weaker inhibitory effect than ndASCs, which was associated with significantly reduced abilities to decrease IL-2 production and increase IL-8 levels in cocultures. Such attenuated potentials were significantly correlated with increasing body mass index. dASCs and ndASCs comparably reduced CD4 T-cell viability, HLA-DR expression, and interferon-gamma production and conversely increased CD69 expression, the Tregs percentage, and IL-17A production. Considerable amounts of the immunomodulators prostaglandin E2 (PGE2) and IL-6 were detected in the conditioned medium of cocultures. These findings suggest that ASCs obtained from donors with T2D and obesity are receptive to the inflammatory environment and able to modulate CD4 T cells accordingly.

The Relationship Between FoxP3 and SOCs3 Gene Expressions and Disease Activity in Rheumatoid Arthritis.

Immune dysregulation plays an important role in the pathogenesis of rheumatoid arthritis (RA). The CD4+CD25 high FoxP3+ subset of regulatory T cells plays an essential role in preventing autoimmunity and maintaining immune homeostasis. Negative regulation of JAK/STAT signaling is controlled by Suppressor of Cytokine Signaling (SOCs3) proteins. SOCs is produced at lower levels in RA. Our aim was to evaluate the expressional dysregulation of SOCs3 and FoxP3 genes in RA patients in relation to disease activity. We have recruited 90 patients with RA and 60 healthy controls in case control study. Whole blood samples were collected from RA patients and healthy subjects. The measurement of FoxP3 and SOCs3 gene expression was performed by real-time PCR (qPCR). Patients with RA had significantly decreased expression levels of FoxP3 and SOCs3 genes in comparison with controls (P<0.001), in addition to the insignificance correlation of both genes with disease activity in RA patients. FoxP3 and SOCs3 genes showed significant defects in rheumatoid arthritis patients with no significant difference in disease activity.

Interleukin-18 binding protein regulates the apoptosis and necroptosis of fibroblast-like synoviocytes and chondrocytes in rheumatoid arthritis.

Interleukin (IL)-18 plays a pro-inflammatory role in rheumatoid arthritis (RA), and its soluble inhibitor IL-18 binding protein (IL-18BP) has a potential therapeutic role. We investigated the role of IL-18BP on the joint destruction process of RA by accessing the effects of IL-18BP on fibroblast-like synoviocytes (FLSs) and chondrocytes. Peripheral blood mononuclear cells (PBMCs) from patients with RA and healthy controls were cultured under T cell proliferative conditions with 10, 50, or 100 ng/mL of IL-18BP. After three days of culture, flow cytometry for CD4+ T cells was performed using various IL-18BP concentrations. The apoptosis and necroptosis of FLSs and chondrocytes were measured by flow cytometry using annexin V and propidium iodide (PI) and western blot under TNF-α stimulation with IL-18BP (10, 50, and 100 ng/mL). Differentiation of CD4+ IL-17A+ and CD4+ IL-4+ cells decreased and that of CD4+ CD25high Foxp3+ and CD4+ interferon (IFN)-γ+ cells increased on addition of IL-18BP to cultured RA patient-driven PBMCs. RA-FLS migration ability was not suppressed by IL-18BP after 12 or 24 h. IL-18BP increased annexin V+ FLS level and reduced annexin V+ chondrocyte level in a dose-dependent manner, whereas PI+ annexin V- FLS and chondrocyte levels were suppressed by 50, 100 ng/mL IL-18BP in culture. The administration of IL-18BP regulated the type 17 helper T cell/ regulatory T cell imbalance and attenuated the production of pro-inflammatory cytokines. IL-18BP further increased FLS apoptosis and decreased the necroptosis of FLS/chondrocytes and apoptosis of chondrocytes suggesting the joint preservative potential of IL-18BP.

Identification of a CD4+ T cell line with Treg-like activity

Regulatory T cells (Tregs) suppress adaptive immunity and inflammation. Although they play a role in suppressing anti-tumor responses, development of therapeutics that target Tregs is limited by their low abundance, heterogeneity, and lack of specific cell surface markers. We isolated human PBMC-derived CD4+ CD25high Foxp3+ Tregs and demonstrate they suppress stimulated CD4+ PBMCs in a cell contact-dependent manner. Because it is not possible to functionally characterize cells after intracellular Foxp3 staining, we identified a human T cell line, MoT, as a model of human Foxp3+ Tregs. Unlike Jurkat T cells, MoT cells share common surface markers consistent with human PBMC-derived Tregs such as: CD4, CD25, GITR, LAG-3, PD-L1, CCR4. PBMC-derived Tregs and MoT cells, but not Jurkat cells, inhibited proliferation of human CD4+PBMCs in a ratio-dependent manner. Transwell membrane separation prevented suppression of stimulated CD4+PBMC proliferation by MoT cells and Tregs, suggesting cell–cell contact is required for suppressive activity. Blocking antibodies against PD-L1, LAG-3, GITR, CCR4, HLA-DR, or CTLA-4 did not reverse the suppressive activity.We show that human PBMC-derived Tregs and MoT cells suppress stimulated CD4+PBMCs in a cell contact-dependent manner, suggesting that a Foxp3+Treg population suppresses immune responses by an uncharacterized cell contact-dependent mechanism.

Pim Kinases as Therapeutic Targets in Early RheumatoidArthritis.

As well as being an established oncoprotein and therapeutic target in cancer, proviral integration site for Moloney murine leukemia virus 1 (Pim-1) is implicated in human autoimmunity. This study was undertaken to investigate Pim-1 and its family members as potential therapeutic targets in early rheumatoid arthritis (RA). A flow cytometry assay for PIM1 transcript measurement in peripheral blood mononuclear cells from patients with early arthritis was validated and applied as a biomarker of Pim-1 activity at the cellular level. Synovial protein expression was similarly determined by multiplex immunofluorescence in tissue samples from untreated RA patients and non-RA disease controls. Functional consequences of Pim kinase family manipulation in freshly isolated CD4+ T cells from these individuals were ascertained, along with the impact of Pim inhibition on mice with collagen-induced arthritis (CIA). The percentage of circulating CD4+ T cells positive for PIM1 transcript by flow cytometry proved a faithful surrogate for gene expression and was significantly higher in patients with early RA than in those with other diseases. Pim-1 protein levels were similarly up-regulated in synovial CD4+ T cells from patients with early RA. Ex vivo, exposure of T cell receptor-stimulated early RA CD4+ T cells to Pim kinase inhibitors restrained their activation and proliferative capacity. Diminished production of proinflammatory cytokines (interferon-γ and interleukin-17) and an expanded CD25high FoxP3+ Treg cell fraction were also observed in exposed versus unexposed cells. Finally, administration of Pim inhibitors robustly limited arthritis progression and cartilage destruction in CIA. Our findings indicate that Pim kinases are plausible therapeutic targets in a readily identifiable subgroup of patients with early RA. Repurposing of Pim inhibitors for this disease should be considered.

Mechanisms of Regulatory T Cell Recruitment By Mesenchymal Stem Cells in Vitro

Regulatory T cells (Treg) play a critical role in the maintenance of immune tolerance. The scarcity of clinical trials using adoptive Treg therapy in humans has been largely due to the difficulty in obtaining sufficient Treg numbers under GMP conditions. Due to the paucity of Treg in the peripheral blood, several groups are investigating the possibility of expanding these cells in vitro .We sought to investigate the potential of mesenchymal stem cells (MSC) to recruit Treg in vitro . Previous studies suggest that MSC-mediated immunomodulation may be partly driven by Treg induction and/or expansion. However, the mechanisms of Treg recruitment by MSC are so far poorly understood.We performed co-cultures of human peripheral blood mononuclear cells (PBMC) from healthy donors with allogeneic bone marrow (BM)-derived MSC expanded in xeno-free conditions. Our data show an increase in the absolute counts and frequency within the T cell pool of CD4+ CD25high Foxp3+ CD127low Treg cells (4 and 6fold, respectively) after a 14day coculture, both compared to PBMC cultured alone and to Day 0. This increase appears to be specific for Treg, as the levels of conventional CD4 T cells (Tcon) and CD8 T cells are not increased in the presence of MSC. Co-culture with MSC does not induce Treg proliferation, as assessed by Ki67 expression. In terms of susceptibility to apoptosis, MSC increase Bcl-2 levels within Treg but not within Tcon, further suggesting a preferential impact on Treg.The observation of increased Treg numbers after MSC co-culture in the absence of Treg proliferation suggests that MSC recruit Treg by inducing Tcon cells to acquire a Treg-like phenotype. In order to determine the mechanism of MSC-mediated Treg recruitment, we co-cultured either purified CFSE-labelled Treg or Tcon with MSC. In Treg:MSC co-cultures, cell numbers are maintained but Treg do not proliferate. In Tcon:MSC co-cultures, only the cells that acquire a Treg-like phenotype proliferate, whereas the cells that maintain a Tcon phenotype do not. These results indicate that the increase in Treg numbers after co-culture of PBMC with MSC is mainly due to the induction of Tcon-derived Treg-like cells, rather than to Treg expansion.In order to determine if these Treg-like cells induced by MSC also resemble Treg in terms of functional ability, we co-cultured PBMC or purified Tcon with MSC and FACSorted the resulting Treg-like populations after 10 days. The suppressive ability of these purified cells, as well as of unsorted cells from the bulk co-cultures, was assessed in suppression assays using autologous fresh Tcon labelled with CFSE as responder cells. We further compared their suppressive ability to that of Treg co-cultured with MSC, as well as of fresh Treg. Our data suggest that bulk co-cultures of PBMC:MSC, Tcon:MSC and Treg:MSC significantly suppress Tcon proliferation (% divided cells at 1:1 Tcon:Treg ratio compared to Tcon alone, Mann Whitney test: P=0.0022, P=0.0043 and P=0.0303, respectively). As for the functional ability of Treg purified after MSC co-culture, both induced Treg-like cells isolated from Tcon:MSC co-cultures and natural Treg isolated from Treg:MSC co-cultures significantly suppress Tcon proliferation (Mann Whitney test: P=0.0095 for both).We also assessed the expression of functional markers that are associated with Treg induction and/or expansion. After Tcon:MSC co-culture, Treg-like cells express higher levels of PD-1 and CD15s than the cells that maintain a Tcon phenotype, whereas the expression of GITR was similar within Treg-like and Tcon cells. Interestingly, the frequency of MSC expressing PD-1 ligand (PDL-1) increases after co-culture, both compared to Day 0 and to MSC cultured alone.In order to investigate which cytokines may potentially be driving the induction of Treg-like cells by MSC, we measured cytokine levels in co-culture supernatants. The anti-inflammatory cytokines IL-10 and TGF-β are increased, whilst the pro-inflammatory cytokines IFN-γ and TNF-α are reduced in PBMS:MSC co-cultures when compared to PBMC cultured alone.Overall, our data suggest that Treg recruitment by MSC is due to Tcon conversion into Treg-like cells, rather than Treg expansion. Our results demonstrate that MSC induce the generation of a Treg-like population, both in terms of the phenotypic hallmarks of Treg cells and of the functional ability to suppress autologous Tcon proliferation. DisclosuresNo relevant conflicts of interest to declare.

MiRNA Landscape in Pathogenesis and Treatment of Vogt-Koyanagi-Harada Disease.

miRNAs, one of the members of the noncoding RNA family, are regulators of gene expression in inflammatory and autoimmune diseases. Changes in miRNA pool expression have been associated with differentiation of CD4+ T cells toward an inflammatory phenotype and with loss of self-tolerance in autoimmune diseases. Vogt–Koyanagi–Harada (VKH) disease is a chronic multisystemic pathology, affecting the uvea, inner ear, central nervous system, and skin. Several lines of evidence support an autoimmune etiology for VKH, with loss of tolerance against retinal pigmented epithelium-related self-antigens. This deleterious reaction is characterized by exacerbated inflammation, due to an aberrant TH1 and TH17 polarization and secretion of their proinflammatory hallmark cytokines interleukin 6 (IL-6), IL-17, interferon γ, and tumor necrosis factor α, and an impaired CD4+ CD25high FoxP3+ regulatory T cell function. To restrain inflammation, VKH is pharmacologically treated with corticosteroids and immunosuppressive drugs as first and second line of therapy, respectively. Changes in the expression of miRNAs related to immunoregulatory pathways have been associated with VKH development, whereas some genetic variants of miRNAs have been found to be risk modifiers of VKH. Furthermore, the drugs commonly used in VKH treatment have great influence on miRNA expression, including those miRNAs associated to VKH disease. This relationship between response to therapy and miRNA regulation suggests that these small noncoding molecules might be therapeutic targets for the development of more effective and specific pharmacological therapy for VKH. In this review, we discuss the latest evidence regarding regulation and alteration of miRNA associated with VKH disease and its treatment.

Low-Zone IL-2 Signaling: Fusion Proteins Containing Linked CD25 and IL-2 Domains Sustain Tolerogenic Vaccination in vivo and Promote Dominance of FOXP3+ Tregs in vitro.

Low-zone IL-2 signaling is key to understanding how CD4+ CD25high FOXP3+ regulatory T cells (Tregs) exhibit dominance and overgrow conventional effector T cells (Tcons) that typically express lower levels of the IL-2 receptor alpha chain (i.e., CD25). Thus, modalities such as low-dose IL-2 or IL-2/anti-IL-2 antibody complexes have been advanced in the clinic to selectively expand Treg populations as a treatment for chronic inflammatory autoimmune diseases. However, more effective reagents that efficiently lock IL-2 signaling into a low signaling mode are needed to validate and exploit the low-zone IL-2 signaling niche of Tregs. This study focuses on CD25-IL2 and IL2-CD25 fusion proteins (FPs) that were approximately 32 and 320-fold less potent than IL-2. These FPs exhibited transient binding to transmembrane CD25 on human embryonic kidney (HEK) cells, had partially occluded IL-2 binding sites, and formed higher order multimeric conformers that limited the availability of bioactive IL-2. These FPs exhibited broad bell-shaped concentration ranges that favored dominant Treg outgrowth during continuous culture and were used to derive essentially pure long-term Treg monocultures (∼98% Treg purity). FP-induced Tregs had canonical Treg suppressive activity in that these Tregs suppressed antigen-specific proliferative responses of naïve CD4+ T cells. The in vivo administration of CD25-IL2/Alum elicited robust increases in circulating Tregs and selectively augmented CD25 expression on Tregs but not on Tcons. A single injection of a Myelin Oligodendrocyte Glycoprotein (MOG35-55)-specific tolerogenic vaccine elicited high levels of circulating MOG-specific Tregs in vivo that waned after 2–3 weeks, whereas boosting with CD25-IL2/Alum maintained MOG-specific CD25high Tregs throughout the 30-day observation period. However, these FPs did not antagonize free monomeric IL-2 and lacked therapeutic efficacy in experimental autoimmune encephalomyelitis (EAE). In conclusion, these data reveal that CD25-IL2 FPs can be used to select essentially pure long-term lines of FOXP3+ CD25high Tregs. This study also shows that CD25-IL2 FPs can be administered in vivo in synergy with tolerogenic vaccination to maintain high circulating levels of antigen-specific Tregs. Because tolerogenic vaccination and Treg-based adoptive immunotherapy are limited by gradual waning of Tregs, these FPs have potential utility in sustaining tolerogenic Treg responses in vivo.

To study the role of T regulatory cells in developing asthma in a birth cohort of babies with family history of allergies

Asthma is known to be correlated with TH2 immune response. There is evidence that T-regulatory cells are important to prevent allergic diseases like asthma. There are few studies based on the role of Treg cell in birth cohort babies with family history of allergy. Fifty children with asthma and respiratory allergy (study group), 20 children born with a positive family history and being followed up as a birth cohort (cohort group) and a group of 10 healthy children with no history of allergy in the family were considered to be satisfactory (Control group) were recruited in this study. Total IgE level, pulmonary function test (PFT) was assessed. The levels of TH1, TH2, TH17 and CD4+ CD25high FoxP3+ Treg cells expression were determined by Flow cytometry [FACSCanto (Becton Dickinson, Mountain View, CA, USA) with FACS DivaTM software]. The Ethics Committee of PGIMER approved this study (Micro / 2006 / 754 / 8th May 2006). The average level of total IgE in the studied group was 316.8±189.8 IU/mL and significantly higher than cohort (124±28.5 IU/mL, p<0.0001) and control (50±17.5 IU/mL, p<0.0001). The frequency of TH17 cells and culture supernatant level of IL-17 were 12.09±8.67 pg/ml and significantly higher in study group than corresponding level in cohort [5.91±3.56 pg/mL, p<0.001] and control group [2.01±1.27 pg/mL, p<0.001], whereas the frequency of Foxp3 level were significantly lower in study group [(49.00±13.47) %] and cohort group [(68.22±8.55) %] compare to the control group [(95.91±2.63) %]. The total serum IgE level is negatively correlated with FoxP3 level (r= -0.5273 p<0.0001). The FoxP3 expression is negatively correlated with the IL-17 levels (r= -0.5631, p< 0.0001) and IL-4 levels (r= -0.2836 p=0.0460). Imbalance in TH17/Treg was found in patients with allergic asthma. Elevated IL-17 and IL-4 cytokine response and down regulation of FoxP3 were associated with asthma. We also found that T regulatory cells expression decreases as asthma progression.

Mesenchymal stromal cells induce regulatory T cells via epigenetic conversion of human conventional CD4 T cells in vitro.

Regulatory T cells (Treg) play a critical role in immune tolerance. The scarcity of Treg therapy clinical trials in humans has been largely due to the difficulty in obtaining sufficient Treg numbers. We performed a preclinical investigation on the potential of mesenchymal stromal cells (MSCs) to expand Treg in vitro to support future clinical trials. Human peripheral blood mononuclear cells from healthy donors were cocultured with allogeneic bone marrow‐derived MSCs expanded under xenogeneic‐free conditions. Our data show an increase in the counts and frequency of CD4+ CD25high Foxp3+ CD127low Treg cells (4‐ and 6‐fold, respectively) after a 14‐day coculture. However, natural Treg do not proliferate in coculture with MSCs. When purified conventional CD4 T cells (Tcon) are cocultured with MSCs, only cells that acquire a Treg‐like phenotype proliferate. These MSC‐induced Treg‐like cells also resemble Treg functionally, since they suppress autologous Tcon proliferation. Importantly, the DNA methylation profile of MSC‐induced Treg‐like cells more closely resembles that of natural Treg than of Tcon, indicating that this population is stable. The expression of PD‐1 is higher in Treg‐like cells than in Tcon, whereas the frequency of PDL‐1 increases in MSCs after coculture. TGF‐β levels are also significantly increased MSC cocultures. Overall, our data suggest that Treg enrichment by MSCs results from Tcon conversion into Treg‐like cells, rather than to expansion of natural Treg, possibly through mechanisms involving TGF‐β and/or PD‐1/PDL‐1 expression. This MSC‐induced Treg population closely resembles natural Treg in terms of phenotype, suppressive ability, and methylation profile.

A modified flow cytometry method for objective estimation of human CD4+ regulatory T cells (CD4+ Tregs) in peripheral blood, via CD4/CD25/CD45RO/FoxP3 labeling.

Several methods exist for flow-cytometric estimation of human peripheral blood CD4+ T regulatory cells (CD4+ Tregs). We report our experience with the estimation of human CD4+ Tregs via three different characterizations using flow cytometry (CD25high FoxP3+ , CD25high CD127low/- FoxP3+ , and CD4+ CD25high/int CD45ROFoxP3+ ) in normal subjects. We have used these methods on the control populations from two studies (32 and 36 subjects, respectively), the latter two methods retrospectively on the subjects of the first study. The six CD4+ T cell fractions obtained by the third method were differentially colored to ascertain the distribution of these cell fractions in the CD25/FoxP3, CD45RO/FoxP3, and CD25/CD127 dot plots from CD4/CD25/CD45RO/FoxP3 and CD4/CD25/CD45RO/CD127 panels. Each approach gives significantly different estimates of Tregs (expressed as percentage of CD4+ T cells), with the second almost invariably yielding higher percentages than the other two. Only the third approach can distinguish among effector and naïve Tregs and FoxP3+ non-Tregs. Analysis of CD25/CD127 dot plots reveals that Treg delineation via the widely used definition of CD4+ CD25high CD127low/- cells unavoidably yields a mixture of nearly all effector and most of naïve Tregs, as well as FoxP3+ non-Tregs plus other cells. Delineation of effector/naïve Tregs and FoxP3+ non-Tregs is possible via CD45RO/CD25 dot plots but not by CD45RO/FoxP3 counterparts (as done previously) because of overlapping FoxP3 intensities among Tregs and non-Tregs. Our comparison shows that CD4/CD25/CD45RO/FoxP3 panels are an objective means of estimating effector and naïve Tregs via colored dot plots, aiding thus in Treg delineation in health and detecting aberrations in disease.

Epigenetic Profile of Treg-like Cells Induced By Mesenchymal Stem Cells in Vitro Resembles That of Natural Treg

Regulatory T cells (Treg) are a key population in immune tolerance and their potential use in the treatment of chronic inflammatory diseases has been increasingly investigated. A major hurdle to adoptive Treg therapy in humans is the difficulty in obtaining sufficient numbers of clinical grade Treg.We and others have investigated the potential of mesenchymal stem cells (MSC) to induce and/or expand Treg in vitro. Our goal is to ascertain whether MSC co-culture supports the recruitment and expansion of Treg from a non-Treg starting population, which would allow for clinically relevant numbers to be reached, whilst bypassing the costly isolation of Treg under GMP conditions prior to expansion.We found that co-cultures of human peripheral blood mononuclear cells (PBMC) from healthy donors with allogeneic bone marrow-derived MSC specifically increase the absolute counts and frequency (4 and 6-fold, respectively) of Treg-like cells with a CD4+ CD25high Foxp3+ CD127low phenotype. We further observed that this increase in Treg numbers after MSC co-culture is mainly due to the induction of conventional CD4 T cells (Tcon) to acquire a Treg-like phenotype, rather than to Treg expansion.These data led us to investigate if these Treg-like cells induced by MSC also resemble Treg in terms of suppressive ability and epigenetic profile, which would inform on their functional potential and stability, two key features for their applicability in a clinical setting.We performed suppression assays using FACSorted Treg-like cells after co-culture of purified Tcon with MSC as suppressor cells and autologous fresh Tcon labelled with CFSE as responder cells. We compared the suppressive ability of Treg-like cells to that of Treg co-cultured with MSC, as well as of fresh Treg. We found that induced Treg-like cells isolated from Tcon:MSC co-cultures (P=0.0121), natural Treg isolated from Treg:MSC co-cultures (P=0.0025) and fresh Treg (P=0.0006) all significantly suppress Tcon proliferation (% divided cells at 1:1 Tcon:Treg ratio compared to Tcon alone, Mann Whitney test).We observed significantly higher levels of TGF-β in PBMS:MSC co-culture supernatants compared to that of PBMC cultured alone (P<0.05 on Day 10, P<0.01 on Day 14, two-way ANOVA), indicating a role for TGF-β in the induction of these Treg-like cells. TGF-β-induced Treg have been suggested to be less stable than natural Treg, likely due to epigenetic regulation. In particular, DNA demethylation has been shown to be a key factor in Treg stability. The potential therapeutic application of MSC-induced Treg-like cells would hinge on these cells being epigenetically stable. Hence, we investigated whether MSC-induced Treg-like cells are more similar to the original Tcon that they arose from or to natural Treg in terms of their DNA methylation profile.Most Treg epigenetic studies focus on the Treg-specific demethylated region (TSDR) of the FOXP3 gene and, given that FOXP3 is encoded on the X chromosome, opt to use male donors to bypass artefacts of X-chromosome inactivation. In order to avoid a gender biased analysis, we sought an alternative to TSDR. CAMTA1 (calmodulin-binding transciption activator 1) is a Ca2+-dependent calmodulin-binding transcription factor encoded on chromosome 1q3.6, which has been proposed as a potential molecular marker that distinguishes Treg from Tcon in both male and female donors. We assessed the methylation status of 13 CpG sites within the 470bp CAMTA1 intronic region 3 based on the coding DNA strand in Treg-like cells purified after Tcon:MSC co-cultures, as well as in fresh Treg and Tcon from the same donor. Fresh Tcon cells from one of the donors showed 100% methylation of CpGs 1-13, whereas fresh Treg and Treg-like cells from the same donor showed much lower levels of methylation (33.8% and 0%, respectively), with the highest differences in methylation observed in CpGs 2 and 11 (15% in fresh Treg; 0% in Treg-like cells; 100% in fresh Tcon). These two CpG positions, which have been previously demonstrated to be the ones distinguishing Treg from Tcon, were consistently more demethylated in fresh Treg and Treg-like cells than in fresh Tcon, with an average methylation levels of 5.8% for fresh Treg, 17% for Treg-like cells and 73% for fresh Tcon.Overall, our data demonstrate that MSC induce a subset of Tcon to acquire a Treg-like phenotype, which is accompanied by a suppressive ability and an epigenetic profile that resemble that of natural Treg. DisclosuresNo relevant conflicts of interest to declare.

Characterization of CD45RO+ memory T lymphocytes in keloid disease

Memory T cells, a highly effective subset of T lymphocytes, have been reported to be involved in many inflammatory skin disorders. However, the potentialrole of memory T cells in keloid disease (KD) remains unclear. Due to their important role in regulating inflammation, we investigated the characteristics of CD45RO+ memory T cells in KD. Primary cutaneous cells were isolated from keloid scars and normal skin by enzymic digestion. Peripheral blood mononuclear cells were isolated from a related blood sample, and flow cytometry was applied to identify the phenotypic and functional abnormalities of memory T cells in KD. We observed that the majority of T lymphocytes in keloid scars had the memory phenotype, and a greater number of the CD8+ memory T cells in keloid scars produced lower levels of tumour necrosis factor (TNF)-α. This abnormal cytokine production was even more distinct in Forkhead box (FOX)P3- CD8- memory T cells, with lower TNF-α production and enhanced interferon-γ production. Furthermore, FOXP3+ CD8- memory T cells in keloid scars were abnormal, including showing reduced CD25 and cytotoxic T-lymphocyte-associated antigen 4 expression and interleukin-10 production. In addition, a significant decrease in the number of CD4+ CD25high FOXP3+ regulatory T cells was identified in patients with multiple keloid scars. We also found that there was significantly increased infiltration of CD103+ CD8+ memory T cells in keloid scars. Our findings preliminarily elucidate the abnormalities of CD45RO+ memory T cells in keloid scars and provide early evidence that a disrupted T-cell response contributes to the progression of KD.

Alternative Th17 and CD4+ CD25+ FoxP3+ cell frequencies increase and correlate with worse cardiac function in Chagas cardiomyopathy.

Immune homeostasis has been suggested to play an important role in the clinical evolution of chronic Chagas disease; however, the immunopathologic factors involved have not been fully elucidated. Therefore, our study aimed to analyse the frequency of CD4+ CD25+ FoxP3+ cells, classic Th17 cells, alternative Th17 cells and IL-17+ B cells from peripheral blood of chronic cardiac patients after invitro stimulation with Trypanosoma cruzi soluble EPI antigen. Patients were selected and classified according to clinical evaluation of cardiac involvement: mild, B1 (CARD1) (n=20) and severe, C (CARD2) (n=11). Patients with the indeterminate form of CD were included as the control group A (IND) (n=17). Blood samples were collected and cultured in the presence of EPI antigen. Cells frequency and median fluorescence intensity (MFI) were obtained by flow cytometry. Our results showed that only CD4+ CD25+ FoxP3+ , CD4+ CD25high FoxP3+ , CD4+ IL-17+ IFN-γ- and CD4+ IL-17+ IFN-γ+ cells are more frequent in patients with severe cardiac disease and correlate with worse global cardiac function. However, while indeterminate patients demonstrated a positive correlation between CD4+ CD25+ FoxP3+ and CD4+ IL-17+ IFN-γ- Th17 cells, this relationship was not observed in cardiac patients. IL-17 expression by Th17 cells and B cells correlated with disease progression. Altogether our results suggest that the clinical progression of Chagas cardiomyopathy involves worsening of inflammation and impairment of immunoregulatory mechanisms.

Oral myeloid cells uptake allergoids coupled to mannan driving Th1/Treg responses upon sublingual delivery in mice.

BackgroundPolymerized allergoids coupled to nonoxidized mannan (PM‐allergoids) may represent novel vaccines targeting dendritic cells (DCs). PM‐allergoids are better captured by DCs than native allergens and favor Th1/Treg cell responses upon subcutaneous injection. Herein we have studied in mice the in vivo immunogenicity of PM‐allergoids administered sublingually in comparison with native allergens.MethodsThree immunization protocols (4‐8 weeks long) were used in Balb/c mice. Serum antibody levels were tested by ELISA. Cell responses (proliferation, cytokines, and Tregs) were assayed by flow cytometry in spleen and lymph nodes (LNs). Allergen uptake was measured by flow cytometry in myeloid sublingual cells.ResultsA quick antibody response and higher IgG2a/IgE ratio were observed with PM‐allergoids. Moreover, stronger specific proliferative responses were seen in both submandibular LNs and spleen cells assayed in vitro. This was accompanied by a higher IFNγ/IL‐4 ratio with a quick IL‐10 production by submandibular LN cells. An increase in CD4+ CD25high FOXP3+ Treg cells was detected in LNs and spleen of mice treated with PM‐allergoids. These allergoids were better captured than native allergens by antigen‐presenting (CD45+ MHC‐II +) cells obtained from the sublingual mucosa, including DCs (CD11b+) and macrophages (CD64+). Importantly, all the differential effects induced by PM‐allergoids were abolished when using oxidized instead of nonoxidized PM‐allergoids.ConclusionOur results demonstrate for the first time that PM‐allergoids administered through the sublingual route promote the generation of Th1 and FOXP3+ Treg cells in a greater extent than native allergens by mechanisms that might well involve their better uptake by oral antigen‐presenting cells.

DNA Methyltransferase Inhibition Promotes Th1 Polarization in Human CD4+CD25high FOXP3+ Regulatory T Cells but Does Not Affect Their Suppressive Capacity.

Regulatory T cells (Treg) can show plasticity whereby FOXP3 expression, the master transcription factor for Treg suppressor function, is lost and proinflammatory cytokines are produced. Optimal FOXP3 expression strongly depends on hypomethylation of the FOXP3 gene. 5-Azacytidine (Aza) and its derivative 5-aza-2′-deoxycytidine (DAC) are DNA methyltransferase inhibitors (DNMTi) that are therapeutically used in hematological malignancies, which might be an attractive strategy to promote Treg stability. Previous in vitro research primarily focused on Treg induction by DAC from naïve conventional CD4+ T cells (Tconv). Here, we examined the in vitro effect of DAC on the stability and function of FACS-sorted human naturally occurring CD4+CD25high FOXP3+ Treg. We found that in vitro activation of Treg in the presence of DAC led to a significant inhibition of Treg proliferation, but not of Tconv. Although Treg activation in the presence of DAC led to increased IFNγ expression and induction of a Thelper-1 phenotype, the Treg maintained their suppressive capacity. DAC also induced a trend towards increased IL-10 expression. In vivo studies in patients with hematological malignancies that were treated with 5-azacytidine (Vidaza) supported the in vitro findings. In conclusion, despite its potential to increase IFNγ expression, DAC does preserve the suppressor phenotype of naturally occurring Treg.

Partial CD25 Antagonism Enables Dominance of Antigen-Inducible CD25high FOXP3+ Regulatory T Cells As a Basis for a Regulatory T Cell-Based Adoptive Immunotherapy.

FOXP3+ regulatory T cells (Tregs) represent a promising platform for effective adoptive immunotherapy of chronic inflammatory disease, including autoimmune diseases such as multiple sclerosis. Successful Treg immunotherapy however requires new technologies to enable long-term expansion of stable, antigen-specific FOXP3+ Tregs in cell culture. Antigen-specific activation of naïve T cells in the presence of TGF-β elicits the initial differentiation of the FOXP3+ lineage, but these Treg lines lack phenotypic stability and rapidly transition to a conventional T cell (Tcon) phenotype during in vitro propagation. Because Tregs and Tcons differentially express CD25, we hypothesized that anti-CD25 monoclonal antibodies (mAbs) would only partially block IL-2 signaling in CD25high FOXP3+ Tregs while completely blocking IL-2 responses of CD25low-intermediate Tcons to enable preferential outgrowth of Tregs during in vitro propagation. Indeed, murine TGF-β-induced MOG-specific Treg lines from 2D2 transgenic mice that were maintained in IL-2 with the anti-CD25 PC61 mAb rapidly acquired and indefinitely maintained a FOXP3high phenotype during long-term in vitro propagation (>90% FOXP3+ Tregs), whereas parallel cultures lacking PC61 rapidly lost FOXP3. These results pertained to TGF-β-inducible “iTregs” because Tregs from 2D2-FIG Rag1−/− mice, which lack thymic or natural Tregs, were stabilized by continuous culture in IL-2 and PC61. MOG-specific and polyclonal Tregs upregulated the Treg-associated markers Neuropilin-1 (NRP1) and Helios (IKZF2). Just as PC61 stabilized FOXP3+ Tregs during expansion in IL-2, TGF-β fully stabilized FOXP3+ Tregs during cellular activation in the presence of dendritic cells and antigen/mitogen. Adoptive transfer of blastogenic CD25high FOXP3+ Tregs from MOG35-55-specific 2D2 TCR transgenic mice suppressed experimental autoimmune encephalomyelitis in pretreatment and therapeutic protocols. In conclusion, low IL-2 concentrations coupled with high PC61 concentrations constrained IL-2 signaling to a low-intensity range that enabled dominant stable outgrowth of suppressive CD25high FOXP3+ Tregs. The ability to indefinitely expand stable Treg lines will provide insight into FOXP3+ Treg physiology and will be foundational for Treg-based immunotherapy.

Broad immunomodulating effect of first-line Pazopanib in metastatic renal cell carcinoma patients

Background: The impact of tyrosine kinase inhibitors (TKIs) on tumor immunity of patients (pts) with metastatic renal cell carcinoma (mRCC) is largely unknown. We investigated the activity of pazopanib in counteracting tumor-induced immunosuppression and boosting adaptive immune response. Methods: Sixteen mRCC pts receiving first-line Pazopanib were prospectively analyzed at baseline, 3 and 6 months for blood Immune profiling by multicolor cytofluorimetry. Gene expression analysis was performed by Illumina HT12v4 BeadChip Arrays. Data were evaluated by t-test, enrichment analysis and deconvolution algorithms. Results: Pazopanib administration (800 mg per os/daily) was associated with a significant decrease of cell subsets involved in immunosuppression, including CD14+ monocytes, monocytic CD14+ HLA-DRneg myeloid derived suppressor cells (MDSC) and CD14+PDL-1+ cells. Similarly, low density CD15+ granulocytic MDSC and CD4+ CD25high Foxp3+ regulatory T cells were reduced by treatment. Concomitantly, a boost of antitumor effectors, such as activated T lymphocytes (identified as CD3+PD-1dim T cells) and cytotoxic CD3-CD16+CD56dim NK cells, was observed. Changes were more evident at 3 months and in pts achieving clinical benefit (69%), defined as the sum of partial response and stable disease at first restaging. Interestingly, a statistically significant increase of lymphocyte/monocyte ratio, as determined by routine blood test was also detected. Gene expression analysis confirmed the immunoregulatory effects of pazopanib. By comparing with those collected after 3 months after treatment start and pre-treatment samples, pathway-enrichment analysis revealed a coherent modulation of NK Granzyme A, IL8 signaling and other immune-related pathways. Similarly, using deconvolution algorithms, we observed an enrichment of NK and CD8+ T cell transcripts. Conclusions: Pazopanib reshapes tumor immunity by reducing immunosuppressive cells (MDSC and Treg) and triggering T cells and NK effectors. These data provide a strong rationale for using Pazopanib both before an immunocheckpoints inhibitors and also in combination strategies based on the synergism between TKIs and immunotherapy.