Foxp3 Expression In CD4 Research Articles

Cyclosporine A May Affect the Expression of Foxp3 by Regulation of Akt and Stat3 Phosphorylation in CD4+CD25+ Regulatory T Cells from Mouse Model of Severe Aplastic Anemia

Although Cyclosporine A(CsA) is an effective immunosuppressant in the treatment of aplastic anemia(AA), its affects on CD4+CD25+ regulatory T cells(Tregs) from patients with AA still remains controversial. We have first developed a novel mouse model of severe aplastic anemia(SAA) induced by administration with IFN-γ and busulphan. In this study, the SAA model female mice were intragastrically administered with CsA at daily dose of 5 mg/kg for 10 days(the CsA-treated group, n=60), the SAA group(n=60) and the un-treated group (n=60) were as control. All mice selected to set up the SAA model developed the typical clinical and pathological patterns of SAA from day 10 post dosing. Most of them presented bleedings in association with anemia and infections. At day 15 after treatment, a mortality of 35% was found in the SAA group, while a mortality of 20% was found in the CsA-treated group because of impaired liver function and infections. In order to explore the effect of CsA on CD4+CD25+ Tregs from the novel mouse SAA model, the mononuclear cells of the peripheral blood and spleen were subjected to assess the percentage of CD4+CD25+ Tregs and the intracellular Foxp3 expression in CD4+CD25+ Tregs by flow cytometry(FCM). In the SAA group, the FCM analysis showed decreased ratios of CD4+CD25+ Tregs in CD4+ T cells and down-expression of Foxp3 in CD4+CD25+ Tregs compared with the un-treated group(P<0.05, respectively). These results were in accordance with our previous observations. However, after treatment with CsA, the expression of Foxp3 in CD4+CD25+ Tregs was increased(P<0.05) but not the ratios of CD4+CD25+ Tregs in CD4+ T cells(P>0.05). In addition, after being pured by immunomagnetic beads, the splenic CD4+CD25+ Tregs was subjected to assess the Akt and Stat3 phosphorylation using Western blot. Compared with the un-treated group, CD4+CD25+ Tregs with increased Akt phosphorylation accompanied by increased Stat3 phosphorylation was found in SAA group(P<0.05, respectively). On the contrary, CsA-treated group exhibited CD4+CD25+ Tregs with a reduction in both Akt phosphorylation and Stat3 phosphorylation compared with the SAA group(P<0.05, respectively). These results indicated that CsA may increase the expression of Foxp3 by up-regulation the levels of Akt and Stat3 phosphorylation in CD4+CD25+ Tregs from the novel mice model of severe aplastic anemia. Disclosures:No relevant conflicts of interest to declare.

Expression of Ets-1 and FOXP3 mRNA in CD4+CD25+ T regulatory cells from patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease with complex genetic predisposing factors involved. Ets-1 transcription factor plays an important role in the suppressive activity of CD4(+)CD25(+) Treg cells and stable expression of FOXP3. To find its potential role in the pathogenesis of SLE, we investigate the mRNA expression of Ets-1 and FOXP3 mRNA in CD4(+)CD25(+) Treg cells from patients with SLE. Real-time transcription-polymerase chain reaction analysis was used to determine the expression of Ets-1 and FOXP3 mRNA in CD4(+)CD25(+) Treg cells from 36 patients with SLE and 18 sex-and-age-matched healthy controls. The Ets-1 mRNA expression level was decreased in patients with SLE [0.225 (0.135, 0.337)] than healthy controls [0.528 (0.303, 0.681)] (P < 0.001). The expression levels of FOXP3 mRNA were lower in SLE patients [0.608 (0.272, 1.164)] than healthy controls [0.919 (0.690, 1.223)], but the difference was not significant (P = 0.106). Significant reduction in Ets-1 and FOXP3 expression was also found in new-onset SLE subgroup when compared with healthy controls (P < 0.001). The level of Ets-1 and FOXP3 mRNA was not significantly different in hyperactive and lower active SLE group when compared with inactive SLE group, respectively (P > 0.05). There were no significant differences between SLE with lupus nephritis (LN) and SLE without LN either (P > 0.05). Associations of Ets-1 and FOXP3 mRNA expression levels with major clinical and laboratory parameters of SLE patients were also analyzed. However, no significant association was found. Significant positive correlation was found between Ets-1 and FOXP3 mRNA expression in CD4(+)CD25(+) Treg cells from SLE patients (r = 0.698, P < 0.001). Our results found that the expression levels of Ets-1 mRNA were decreased in SLE patients and Ets-1 expression was positively correlated with the expression of FOXP3. It indicated that Ets-1 may play an important role in the stable expression of FOXP3 in CD4(+)CD25(+) Treg cells.

Mechanistic target of rapamycin activation triggers IL-4 production and necrotic death of double-negative T cells in patients with systemic lupus erythematosus.

The mechanistic target of rapamycin (mTOR) is recognized as a sensor of mitochondrial dysfunction and effector of T cell lineage development; however, its role in autoimmunity, including systemic lupus erythematosus, remains unclear. In this study, we prospectively evaluated mitochondrial dysfunction and mTOR activation in PBLs relative to the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) during 274 visits of 59 patients and 54 matched healthy subjects. Partial least square-discriminant analysis identified 15 of 212 parameters that accounted for 70.2% of the total variance and discriminated lupus and control samples (p < 0.0005); increased mitochondrial mass of CD3(+)/CD4(-)/CD8(-) double-negative (DN) T cells (p = 1.1 × 10(-22)) and FOXP3 depletion in CD4(+)/CD25(+) T cells were top contributors (p = 6.7 × 10(-7)). Prominent necrosis and mTOR activation were noted in DN T cells during 15 visits characterized by flares (SLEDAI increase ≥ 4) relative to 61 visits of remission (SLEDAI decrease ≥ 4). mTOR activation in DN T cells was also noted at preflare visits of SLE patients relative to those with stable disease or healthy controls. DN lupus T cells showed increased production of IL-4, which correlated with depletion of CD25(+)/CD19(+) B cells. Rapamycin treatment in vivo blocked the IL-4 production and necrosis of DN T cells, increased the expression of FOXP3 in CD25(+)/CD4(+) T cells, and expanded CD25(+)/CD19(+) B cells. These results identify mTOR activation to be a trigger of IL-4 production and necrotic death of DN T cells in patients with SLE.

Expression of CD4&lt;sup&gt;+&lt;/sup&gt;CD25&lt;sup&gt;high+&lt;/sup&gt;&lt;sup&gt; &lt;/sup&gt;Regulatory T Cells in Miniature Swine

In mice, there has been increasing evidence that FOXP3-expressing CD4+CD25+ regulatory T cells (Tregs) inhibit potentially reactive T cells to allo-antigens and play an important role in transplant (Tx) tolerance. Human CD4+CD25 high+ T cells represent Tregs, characterized by anergic and antigen-specific suppressive properties. Despite the significance of miniature swine as a preclinical model, only limited data are available regarding the existence of a porcine counterpart to the murine or human Tregs. Methods: From peripheral blood of five adult miniature swine, CD4+ cells (CD4+), CD4+CD25 high+ cells (CD25high+), CD4+CD25low+ cells (CD25low+), and CD4+CD25- cells (CD25-) were isolated with a cell sorter. Expression of the homologous gene to murine FOXP3 was quantified by RT-PCR in isolated fractions, and normalized to GAPDH. Proliferation of CD25high+ and suppressive property of CD25high+ to CD4+ responder cells upon allogeneic stimulation was evaluated by MLR and dilution assay, respectively, after isolation and 9-day culture with allogeneic cells in the presence of IL-2. Results: FOXP3 was expressed ten times higher in CD25 high+, compared to that in CD25low+ (p&lt;0.05). CD25- expressed almost no FOXP3. CD25high+ was anergic and could suppress the proliferation of responder CD4+ upon allogeneic stimulation after isolation. After culture, CD25high+ remained anergic and showed a more suppressive effect on cells used for culture than on third party cells. Conclusions: 1) CD25high+ in miniature swine peripheral blood expressed an extremely high level of FOXP3. 2) This fraction was anergic and has suppressive properties, showing antigen-specificity after exposure to alloantigens. 3) We found for the first time that miniature swine CD25high+ represents the homologous population to the murine and human Tregs. Miniature swine Tx models, therefore, may provide opportunities for pre-clinical evaluation of innovative Tregs-based tolerance strategy.

Bone marrow-derived dendritic cells rather than spleen-derived dendritic cells can generate regulatory T cells

There are many evidences that dendritic cells (DC) can establish and maintain immunological tolerance through inducing the differentiation of regulatory T cells Treg. This study was purposed to explore the possibility to gene-rate Treg from bone marrow-derived DC (BM-DC) or spleen-derived DC (spDC) generated CD4(+) CD25(+) FOXP3(+) Treg by induction. Bone marrow immature DC (imDC) induced from bone marrow precursor cells of C57BL/6 mice by GM-CSF and IL-4; after culture for 6 day, imDC were stimulated by LPS for additional 16 hours and the mature DC (mDC) have been got; the spDC were collected from spleen of C57BL/6 mice by MACS. Co-culturing fresh BALB/c mouse CD4(+) T cells with these three sorts of DC above mentioned respectively was performed to generate CD4(+) CD25(+) FOXP3(+) Treg. The expression of FOXP3 in CD4(+) T cells was detected by flow cytometry, and the capacity of different DC generated CD4(+) CD25(+) FOXP3(+) Treg was evaluated. The results showed that stimulated by C57BL/6 immature or mature DC, the positive rate of FOXP3 in BALB/c CD4(+) T cells increased from (8.57 ± 1.14)% to (15.80 ± 1.35)%, (17.93 ± 1.45)% respectively (P < 0.01); while stimulated by spDC, the positive rate of FOXP3 in BALB/c CD4(+) T cells decreased from (8.57 ± 1.14)% to (3.95 ± 0.79)% (P < 0.05). It is concluded that the BM-DC but not spDC can generate Treg from CD4(+) T cells, BM-DC may mediate immune tolerance rather than the immune response.

FRI0009 Il4-10 synerkine: a novel anti-inflammatory drug to inhibit immunopathology in rheumatic diseases

BackgroundA considerable percentage of patients shows a limited response to biologics targeting one specific inflammatory mediator, largely because of redundancy of these mediators. An attractive alternative is inhibition of multiple...

Exposure to Factor VIII Protein in the Presence of Phosphatidylserine Induces Hypo-responsiveness toward Factor VIII Challenge in Hemophilia A Mice

Administration of recombinant factor VIII (FVIII), an important co-factor in blood clotting cascade, elicits unwanted anti-FVIII antibodies in hemophilia A (HA) patients. Previously, FVIII associated with phosphatidylserine (PS) showed significant reduction in the anti-FVIII antibody response in HA mice. The reduction in the immune response to FVIII-PS could be due either to a failure of the immune system to recognize the antigen (i.e. immunological ignorance) or to an active induction of an antigen-specific nonresponsiveness (i.e. immunological tolerance). If it were a result of tolerance, one would predict that pre-exposure to FVIII-PS would render the mice hypo-responsive to a subsequent FVIII challenge. Here, we have demonstrated that naive HA mice that were pretreated with FVIII-PS showed a significantly reduced FVIII immune response to further challenge with native FVIII and that this decreased responsiveness could be adoptively transferred to other mice. An increase in number of FoxP3-expressing CD4(+) regulatory T-cells (Treg) was observed for the FVIII-PS-immunized group as compared with animals that received FVIII alone, suggesting the involvement of Treg in PS-mediated hypo-responsiveness. The PS-mediated reduction in antibody response was reversed by the co-administration of function-blocking anti-TGF-β antibody with FVIII-PS. The decreased response to FVIII induced by FVIII-PS was determined to be antigen-specific because the immune response to another non-cross-reactive antigen (ovalbumin) was not altered. These results are consistent with the notion that FVIII-PS is tolerogenic and suggest that immunization with this tolerogenic form of the protein could be a useful treatment option to minimize immunogenicity of FVIII and other protein-based therapeutics.

DNA hypermethylation leads to lower FOXP3 expression in CD4+ T cells of patients with primary Sjögren's syndrome

DNA hypermethylation leads to lower FOXP3 expression in CD4+ T cells of patients with primary Sjögren's syndrome

T-regulatory cell-mediated immune tolerance as a potential immunotherapeutic strategy to facilitate graft survival.

T-regulatory cell-mediated immune tolerance as a potential immunotherapeutic strategy to facilitate graft survival.

Peroxisome proliferator-activated receptor-gamma in CD11c+ lung dendritic cells programs peripheral CD4+ T cells for Foxp3 induction during airway tolerance. (P6001)

Abstract Peroxisome proliferator-activated receptor-gamma (PPARγ) is a widely expressed molecular regulator with anti-inflammatory functions. However, whether it has an anti-inflammatory role in dendritic cells (DCs) that influence the Treg/Teffector balance has not been experimentally addressed. In this study, we investigated the role of PPARγ in DCs regulating the delicate balance between tolerance versus inflammation in the periphery using a model of airway induced tolerance by inhaled allergen. Using mice with selective deletion of PPARγ in CD11c+ cells, we observed that PPARγ expression in CD11chi lung DCs is essential for de novo Foxp3 expression in CD4+ T cells. We demonstrate that PPARγ plays a crucial role in enhancing aldh1a2 expression in lung CD103+ DCs under tolerogenic conditions with significantly less expression of the enzyme in these cells when mice are immunized for airway inflammation. When tolerized CD11ccre/+ PPARγfl/fl mice were antigen-challenged, a complete loss of immune tolerance with increased neutrophil-dominated airway inflammation was observed. Taken together, our study identifies PPARγ as a central regulator in DC-mediated programming of Foxp3 expression in naive CD4+ T cells dictating tolerance versus inflammation upon antigen provocation.

CD4+FoxP3+ regulatory T cell-mediated suppression of dendritic cells (P1049)

Abstract CD4+FoxP3+ regulatory T cells are a subset of T cells that play central role in regulating inflammatory responses during many different immunological diseases. However the mechanisms of CD4+FoxP3+ regulatory T cells-mediated suppression remain controversial and poorly understood. Several studies have shown that CD4+FoxP3+ regulatory T cells can interact with and suppress a large number of cell types including CD4+ T cells and antigen presenting cells. For instance in vitro studies have shown that CD4+FoxP3+ regulatory T cells can inhibit cytokine production especially IL-2 production on CD4+ T cells hence the inhibition of CD4+ T cell proliferation; CD4+FoxP3+ regulatory T cells can also confer infectious tolerance by converting conventional T cells into functional FoxP3+ T cells in a cell contact TGF-β-dependent manner.In addition, CD4+FoxP3+ regulatory T cells suppress dendritic cell maturation and function by down-regulating the expression of co-stimulatory molecules CD80/CD86 via the interaction with CTLA-4.In our study, we have observed that CD4+FoxP3+ regulatory T cells may also modulate dendritic cell function via the PD-1:PD-L pathway. Dendritic cells that have been previously cultured with CD4+FoxP3+ regulatory T cells have decreased ability to support CD4+CD25- T cell proliferation and they can also induce higher level of FoxP3 expression in CD4+CD25- T cells.

Transplant tolerance depends on regulatory T cells induced by TGF-β-producing regulatory B cells (P2168)

Abstract Regulatory B cells (Bregs) have been shown to play a critical role in immune homeostasis and in autoimmunity models. We have recently demonstrated that combined anti-TIM-1 and anti-CD45RB antibody treatment results in tolerance to full MHC mismatch islet allografts in mice, generating Bregs that are necessary for tolerance, and capable of transferring tolerance to untreated animals. Herein, we demonstrate that adoptively transferred Bregs require the presence of Tregs to establish tolerance, and that adoptive transfer of Bregs increases the number of Tregs. Bregs induce significantly more Foxp3 expression in CD4+CD25- T cells, in vivo, compared to naive B cells. We also show that Bregs express the TGF-β associated latency-associated peptide (LAP). Furthermore, Breg-mediated graft prolongation post-adoptive transfer is abrogated by neutralization of TGF-β activity. Collectively, these findings suggest that in this model of antibody-induced transplantation tolerance, Bregs promote graft survival by promoting Treg development, possibly via TGF-β production.

The microbiota-derived signal indole regulates T-cell fate by reciprocal control of Th17 and Treg development (P3178)

Abstract The presence of a normal microbiota and immune regulation in the gut limits the onset of several inflammatory disorders. However, the specific microbiota mechanisms or chemical effectors that promote homeostasis are largely unknown. Previously, we reported that indole, a microbiota-derived (not host derived) tryptophan-metabolite, attenuates indicators of inflammation in intestinal epithelial cells. In addition, we show that indole treatment rescues mice from colitis in vivo. Based on our results, we hypothesized that indole influences mucosal CD4 T-cell function. Because Foxp3+ regulatory T cells (Tregs) and pro-inflammatory Th17 cells are key players in the balance of gut homeostasis, we tested the impact of indole on Treg and Th17 development. Here, we reveal that indole regulates Treg/Th17 lineage fate by dramatically augmenting TGF-β-induced Treg expansion, function, and stability. Interestingly, indole also promoted Treg Foxp3 expression independent of TGF-β or IL-2 suggesting a novel mechanism for the induction of Foxp3 expression in CD4 T cells. In a reciprocal fashion, indole inhibited Th17 development via STAT3 and RORgt pathways and decreased IL-17 production. These data reveal a novel mechanistic paradigm on how the microbiota influence mucosal immunobiology and establish the metabolite indole as a potent regulator of Treg and Th17 cell balance. Moreover, our results suggest microbiota-derived metabolites offer a rich pool of potent immunomodulatory compounds.

Expression of interferon-inducible genes is influenced by sex hormones and gender in SLE (P1380)

Abstract Women may be more susceptible than men to SLE and other autoimmune diseases in part because many healthy women have higher interferon regulated genes and fewer regulatory T cells. Similarly, female BWF1 lupus mice have increased expression of interferon genes compared to age and sex- matched males. Estradiol regulates interferon-inducible genes differentially in healthy controls and in SLE, with higher induction of IFN-inducible genes in the SLE population. Plasma 17b-estradiol levels are significantly increased in female SLE patients compared to healthy females (p&amp;lt;0.01). Testosterone (100 pg/ml) significantly increased FoxP3 expression in CD4+CD25+hi cells from women with SLE (p&amp;lt; 0.03). Estradiol increases proinflammatory cytokines and positively correlated with plasma levels of IL-6/ IL-21 and miR21, miR25 and miR186.

Soluble and Membrane-Bound TGF-β-Mediated Regulation of Intratumoral T Cell Differentiation and Function in B-Cell Non-Hodgkin Lymphoma

While the effect of TGF-β on malignant B cells in non-Hodgkin lymphoma (NHL) has been previously evaluated, studies to specifically define the role of TGF-β in tumor immunity in B-cell NHL are limited. We found that soluble TGF-β, secreted by both lymphoma cells and intratumoral T cells, is present in the serum of patients with B-cell NHL. Soluble TGF-β promoted regulatory T (Treg) cells by enhancing expression of Foxp3 in CD4+ T cells and suppressed effector helper T (TH) cells by inhibiting expression of IFN-γ and IL-17. Blockade of the IL-2 signaling pathway diminished the effect of soluble TGF-β on T cell differentiation. Furthermore, we found that membrane-bound TGF-β is expressed specifically on the surface of malignant B cells in B-cell NHL. TGF-β was able to bind to the surface of lymphoma B cells through an interaction with heparan sulfate (HS) but not through the TGF-β receptor. We showed that pretreatment of lymphoma B cells with TGF-β significantly inhibits the proliferation and cytokine production of intratumoral T cells. Taken together, these results suggest that tumor-associated soluble and membrane-bound TGF-β are involved in the regulation of intratumoral T cell differentiation and function in B-cell NHL.

Myeloid-derived suppressor cells in non-malignant and non-infectious liver disease

BackgroundMyeloid-derived suppressor cells (MDSC) have been described as potent immunosuppressive cells in malignant and infectious liver disease. However, little is known about their role in non-infectious or non-malignant disease. We sought to characterise MDSC in patients with chronic non-infectious or non-malignant liver disease. MethodsExplants obtained from 12 patients undergoing liver transplantation and blood from 30 patients treated for haemochromatosis at the Queen Elizabeth Hospital, Birmingham, were analysed for the frequency of functional CD14+ HLA-DR– monocytic MDSC. Functional capacity was defined as the capability to suppress proliferation of maximally stimulated, CFSE-labelled CD4 T cells using CD3/CD28-beads (Dynabeads, LifeTechnologies, UK) at a ratio of 1:1. Additionally, MDSC were analysed for their capacity to induce CD4 regulatory T cells (assessed by FoxP3 expression) in cells activated with CD2/CD3/CD28-beads (Miltenyi, Germany). Both MDSC and CD4 cells were isolated by magnet-activated cell-sorting using a combination of depletion steps and positive-selection-steps. Analysis of frequency and immunotyping of MDSC was performed with flow-cytometry. FindingsCD14+ HLA-DR– MDSC obtained both from liver tissue and peripheral blood were able to suppress proliferation of CD4 T cells and to induce FOXP3-expression in CD4 T cells, typical of regulatory T cells. No such findings were observed when using CD14+ HLA-DR+ monocytes. Moreover, MDSC depleted of CD16+ monocytes showed weaker immunosuppressive capacity. In patients with haemochromatosis, the frequency of CD14+ HLA-DR– MDSC in peripheral blood ranged from 0·5% to 79% and in the liver of cirrhotic patients from 9·1% to 75·5%. InterpretationCD14+ HLA-DR– MDSC are fully functional in patients who have non-infectious or non-malignant liver disease. Similar to HLA-DR+ monocytes, CD16 expression may identify subtypes of monocytic MDSC with distinct immunoregulatory properties. Given the varying frequency of MDSC in the patients analysed, the clinical relevance of MDSC in non-malignant and non-infectious liver-disease has to be further analysed since they may influence the course of disease in these patients. FundingDeutsche Forschungsgemeinschaft and Liver Foundation Trust Fund.

Abstract IA14: Investigating the ICOS/ICOSL pathway as a target for combination therapy with anti-CTLA-4.

Abstract Biomarker studies used with immunotherapeutic strategies in the clinic have typically involved monitoring immunologic changes within the systemic circulation; however, recent data indicate that immunological changes within tumor tissues will be more likely to predict clinical responses. To obtain such data we conducted the first pre-surgical clinical trial with anti-CTLA-4 (ipilimumab) in a cohort of patients with localized bladder cancer. We are also conducting the first combination therapy pre-surgical trial with ipilimumab plus leuprolide acetate in patients with localized prostate cancer. Immunological data from these trials are obtained from both tumor tissues and blood samples. We found an increased frequency of CD4 and CD8 T cells expressing high levels of inducible costimulator (ICOS) and decreased frequency of FOXP3-expressing CD4 T cells within tumor tissues of treated patients. The CD4+ICOShi population contained effector T cells that produced IFN-γ and recognized the cancer-testis antigen NY-ESO-1 expressed on tumor cells. We therefore identified ICOS as marker of a subset of effector T cells that is increased in frequency after anti-CTLA-4 therapy. ICOS+ T cells are being explored as both a pharmacodynamic marker for treatment with anti-CTLA-4 as well as a novel target to improve the efficacy of anti-CTLA-4 therapy. These observations led us to test the possibility that engagement of ICOS could enhance the efficacy of anti-CTLA-4 therapy. To this end we transduced mouse B16F10 melanoma cells with a cDNA encoding ICOSL or a control construct. B16-ICOSL+ cells (IVAX) and control B16 cells were irradiated and used alone or in combination with anti-CTLA-4 to treat mice bearing established B16F10 tumors. We found that combination of the IVAX with anti-CTLA-4 was markedly more effective than the control vaccine plus anti-CTLA-4 or that of any single treatment alone. The increase in therapeutic efficacy was accompanied by a marked in increase in the density and functionality of CD4 and CD8 T cells within the tumor. These results suggest a novel strategy for manipulating the immune system to enhance anti-tumor responses: checkpoint blockade coupled with provision of agonist signals to enhance costimulation mediated by ICOS. Citation Format: Padmanee Sharma. Investigating the ICOS/ICOSL pathway as a target for combination therapy with anti-CTLA-4. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr IA14.

Regulatory T Cells in Clinical Liver Transplantation

Background: Regulatory T-cells (Tregs) (CD4+CD25+Foxp3+) have a key role in tolerance and may influence allograft outcome. Tregs have been extensively studied in animals however their role in clinical transplantation remains unknown. Aim: Identify and characterize Tregs in the peripheral blood of liver transplant recipients. Methods: We performed a cross-sectional study phenotyping peripheral blood lymphocytes by flow cytometry and correlate these with clinical parameters. Groups examined included: (a) Normal Healthy Controls (n=10); (b) Patients with liver failure on the waiting list (n=15) and (c) Liver Transplant Reicpients (LTR) (n=101). Results: The proportion of CD25+ cells in CD4+ population was shown to be lower in LTR compared with the two controls. The level of CD4+CD25+Foxp3+ T cells (within CD4+ population) was higher in patients with chronic liver disease awaiting transplantation and LTR compared to the normal controls. LTR with a history of pre-transplant malignancy (hepatocellular carcinoma) had significantly higher levels of CD4+CD25+Foxp3+ cells in the CD4+ population compared to patients without pre-transplant malignancy. Treg phenotype correlated with age. An inverse correlation was observed between CD127 and Foxp3 expression in CD4+CD25+ cells. Conclusion: (1) Lower CD4+CD25+ cells in LTR may relate to immunosuppression; (2) Higher levels of CD4+CD25+Foxp3+ cells in LTR and patients with liver disease may contribute to a suppressed immune state; (3) CD4+CD25+Foxp3+ may be a marker of tendency to malignancy. Future studies are required to understand the biological significance of these cells and confirm these findings.

Foxp3 Expression in Bovine CD8&lt;sup&gt;+&lt;/sup&gt; T Cells Is Associated with the Intensity of CD25 Expression

The aim of the undertaken research was to determine whether Foxp3(+)CD25(+)CD8(+) cells exist in cattle and whether Foxp3 expression in CD8(+) T cells is correlated with the intensity of CD25 expression. It has been found that 0.66 and 2.36% of CD8(+) cells on average showed high and low expression of the CD25 molecule, respectively. On average, 11.61% of CD25(high)CD8(+) cells expressed Foxp3, while the mean percentage of Foxp3(+) cells within CD25(low)CD8(+) cells was 3.66%. The absolute count of Foxp3(+)CD25(high)CD8(+) cells was not significantly different from the absolute count of Foxp3(+)CD25(low)CD8(+) cells. The obtained results indicate that CD8(+) cells with the regulatory phenotype, i.e. Foxp3(+)CD25(high)CD8(+) and Foxp3(+)CD25(low)CD8(+) cells, occur naturally in bovine peripheral blood, although both of these subpopulations are relatively small. There is a positive correlation between the intensity of CD25 expression and the expression of the transcription Foxp3 factor in bovine CD8(+) cells.

N‐acetylcysteine reduces disease activity by blocking mammalian target of rapamycin in T cells from systemic lupus erythematosus patients: A randomized, double‐blind, placebo‐controlled trial

Systemic lupus erythematosus (SLE) patients exhibit T cell dysfunction, which can be regulated through mitochondrial transmembrane potential (Δψm) and mammalian target of rapamycin (mTOR) by glutathione (GSH). This randomized, double-blind, placebo-controlled study was undertaken to examine the safety, tolerance, and efficacy of the GSH precursor N-acetylcysteine (NAC). A total of 36 SLE patients received either daily placebo or 1.2 gm, 2.4 gm, or 4.8 gm of NAC. Disease activity was evaluated monthly by the British Isles Lupus Assessment Group (BILAG) index, the SLE Disease Activity Index (SLEDAI), and the Fatigue Assessment Scale (FAS) before, during, and after a 3-month treatment period. Mitochondrial transmembrane potential and mTOR were assessed by flow cytometry. Forty-two healthy subjects matched to patients for age, sex, and ethnicity were studied as controls. NAC up to 2.4 gm/day was tolerated by all patients, while 33% of those receiving 4.8 gm/day had reversible nausea. Placebo or NAC 1.2 gm/day did not influence disease activity. Considered together, 2.4 gm and 4.8 gm NAC reduced the SLEDAI score after 1 month (P = 0.0007), 2 months (P = 0.0009), 3 months (P = 0.0030), and 4 months (P = 0.0046); the BILAG score after 1 month (P = 0.029) and 3 months (P = 0.009); and the FAS score after 2 months (P = 0.0006) and 3 months (P = 0.005). NAC increased Δψm (P = 0.0001) in all T cells, profoundly reduced mTOR activity (P = 0.0009), enhanced apoptosis (P = 0.0004), reversed expansion of CD4-CD8- T cells (mean ± SEM 1.35 ± 0.12-fold change; P = 0.008), stimulated FoxP3 expression in CD4+CD25+ T cells (P = 0.045), and reduced anti-DNA production (P = 0.049). This pilot study suggests that NAC safely improves lupus disease activity by blocking mTOR in T lymphocytes.