CD25 High Foxp3 Research Articles

Comparison of the percentages of CD4+ CD25high FOXP3+ , CD4+ CD25low FOXP3+ , and CD4+ FOXP3+ Tregs, in the umbilical cord blood of babies born to mothers with and without preeclampsia.

Little is known about how preeclampsia affects regulatory T-cell count and functions in umbilical cord blood of babies born to preeclamptic mothers. Here, we analyze the percentage of CD4+ CD25high FOXP3+ , CD4+ CD25low FOXP3+ , and CD4+ FOXP3+ Tregs, in the umbilical cord blood of babies born to mothers with and without preeclampsia. The percentage of umbilical cord blood CD4+ CD25high FOXP3+ , CD4+ CD25low FOXP3+ , and CD4+ FOXP3+ Tregs were analyzed by flow cytometry. CD4+ CD25high FOXP3+ Treg (%) and CD4+ FOXP3+ Treg (%) were significantly lower, while CD4+ CD25low (%) was significantly higher in umbilical cord blood of babies born to preeclamptic mothers. Preeclampsia is associated with immune dysregulation which leads to a deficiency in Treg (CD4+ CD25high FOXP3+ ) in the umbilical cord blood of babies born to preeclamptic mothers.

Ascaris suum infection modulates inflammation: Implication of CD4+ CD25high Foxp3+ T cells and IL-10.

Helminth infections have the ability to modulate host's immune response through mechanisms that allow the chronic persistence of the worms in the host. Here, we investigated the mechanisms involved on the suppressive effect of Ascaris suum infection using a murine experimental model of LPS-induced inflammation. We found that infection with A.suum markedly inhibited leucocyte influx induced by LPS into air pouches, suppressed secretion of pro-inflammatory cytokines (IL-1β, TNF-α and IL-6) and induced high levels of IL-10 and TGF-β. Augmented frequency of CD4+ CD25high Foxp3+ T cells was observed in the mesenteric lymph nodes of infected mice. Adoptive transfer of purified CD4+ CD25+ T cells to recipient uninfected mice demonstrated that these cells were able to induce a suppressive effect in the LPS-induced inflammation in air pouch model. In addition, adoptive transfer of CD4+ CD25+ T cells derived from IL-10 knockout mice suggests that this suppressive effect of A.suum infection involves IL-10 cytokine. In conclusion, our results demonstrated that A.suum experimental infection was capable of suppressing LPS-induced inflammation by mechanisms, which seem to be dependent on responses of CD4+ CD25+ T cells and secretion of IL-10 cytokine.

FICZ generates human tDCs that induce CD4+ CD25high Foxp3+ Treg-like cell differentiation

Dendritic cells (DCs) play a central role in the maintenance of immune homeostasis, their participation as professional antigen presenting cells is essential to the initiation of the adaptive immune response as well as to the induction of tolerance. The recently described role of the aryl hydrocarbon receptor (AhR) in the immune system, particularly in the modulation of the adaptive immune response has attracted the attention as a potential player in the induction of immune tolerance. However, the effects of AhR activation through endogenous ligands on human DCs have been poorly evaluated. In this study, we investigated the effect of FICZ, a natural AhR ligand, on monocyte-derived dendritic cells (Mo-DCs) from healthy subjects. We found that the activation of AhR through FICZ during DCs differentiation and maturation processes resulted in a decreased expression of CD83, an increased expression of the enzyme IDO and a reduced production of the pro-inflammatory cytokines IL-6 and TNF-α. More importantly, FICZ-treated DCs were able to induce the differentiation of naive T lymphocytes into CD4+ CD25high Foxp3+ T reg-like cells. Our results show that the activation of the AhR on human DCs induces a tolerogenic phenotype with potential implications in immunotherapy.

Effects of p300/CBP on histone acetylation of Foxp3 gene in children with Kawasaki disease

Objective To investigate the effects of p300/CBP on histone acetylation of Foxp3 gene and its roles in the immunological pathogenesis of Kawasaki disease (KD). Methods Forty-six children with KD and twenty-eight age-matched health children were consented to participate in this study. Co-immunoprecipitation and real-time PCR were performed to detect Foxp3-associated acetylation levels of histone H4 and binding abilities of p300, CBP, pSmad3 (phosphorylated mothers against decapentaplegic homolog 3) and NF-AT (nuclear factor of activated T cells) with Foxp3 gene in CD4+ T cells. The percentages of CD4+ CD25high Foxp3+ cells (Treg) and the expression of Foxp3, CTLA4 (cytotoxic T-lymphocyte-associated protein 4), p300, CBP, TGF-βRⅡ (transforming growth factor β receptor Ⅱ) and pLAT1 at protein level were analyzed by flow cytometry. Quantitative real-time PCR was used to measure the expression of Foxp3, IL-10, TGF-β, TGF-βRⅠ, Egr-1 (early growth response protein 1), RARα (retinoic acid receptor α) and PLCγ1 (phospholipase C-γ1) in Treg cells at mRNA level. Plasma concentrations of TGF-β and retinol acid (RA) were measured by enzyme-linked immunosorbent assay. Results (1) The percentages of Treg cells, levels of Foxp3 and molecules associated with suppressive function of Treg cells (TGF-β, IL-10 and CTLA4), acetylation levels of histone H4 associated with promoter, conserved non-coding DNA sequence 1 (CNS1) and CNS2 of Foxp3 gene decreased remarkably during acute KD (P 0.05). (2) The levels of p300 and CBP in Treg cells and their binding abilities with Foxp3 gene were down-regulated significantly during acute KD (P 0.05). Conclusion Hypoacetylation of histone H4 associated with Foxp3 gene caused by insufficient expression of p300/CBP and their impaired binding abilities might be involved with immune dysfunction in KD. IVIG therapy regulates the expression of p300/CBP and their binding abilities with Foxp3 gene through up-regulating TGF-β signal. Key words: Kawasaki disease; Foxp3; Histone acetylation; Regulatory T cell; Immunity

Phenotypical analysis of ectoenzymes CD39/CD73 and adenosine receptor 2A in CD4+ CD25high Foxp3+ regulatory T-cells in psoriasis.

Background CD39 and CD73 are two novel cell surface markers of CD25highFoxp3+ regulatory T‐cells (Tregs). Concordant expression of these two ectoenzymes not only discriminate Tregs from other cell populations, but also generates pericellular adenosine, which has been reported to suppress proliferation of activated T effector (Teff) cells. Because it is currently unclear whether human ectoenzymes (CD39/CD73) are involved in the impaired suppressive activity of Tregs in psoriasis, we examined the frequencies and phenotypes of CD39/CD73‐expressing Tregs and related receptor adenosine receptor 2A (A2A R) in peripheral blood of patients with different types of psoriasis.MethodsPeripheral blood mononuclear cells (PMBC) were prepared from patients with three different types of psoriasis (psoriasis vulgaris, pustular psoriasis and erythrodermic psoriasis). CD4+ cells were separated from PBMC by negative selection on midiMACS columns, and the frequencies and phenotypes of CD39 and CD73 expressing Tregs, and A2A R expressing Teff were all determined by flow cytometry analysis. Blood from healthy volunteers served as controls.ResultsThe expression of single CD73+ Tregs was markedly reduced (approximately 50%) in psoriasis vulgaris, compared to normal controls. In pustular psoriasis, the mean numbers of CD39+ Tregs and A2AR+ Teff was significantly lower than in normal controls. Among three different types of psoriasis, CD39 expression was strikingly reduced in the blood Treg population of pustular psoriasis patients. Decreased CD73+ Tregs levels were observed in psoriasis vulgaris compared to pustular psoriasis and erythrodermic psoriasis.ConclusionsThe differences in the expression of CD39− and CD73− Tregs may be a factor in the pathogenesis of psoriasis.

Prevention of Allograft Rejection by Use of Regulatory T Cells With an MHC-Specific Chimeric Antigen Receptor.

CD4+ CD25high FOXP3+ regulatory T cells (Tregs) are involved in graft-specific tolerance after solid organ transplantation. However, adoptive transfer of polyspecific Tregs alone is insufficient to prevent graft rejection even in rodent models, indicating that graft-specific Tregs are required. We developed a highly specific chimeric antigen receptor that recognizes the HLA molecule A*02 (referred to as A2-CAR). Transduction into natural regulatory T cells (nTregs) changes the specificity of the nTregs without alteration of their regulatory phenotype and epigenetic stability. Activation of nTregs via the A2-CAR induced proliferation and enhanced the suppressor function of modified nTregs. Compared with nTregs, A2-CAR Tregs exhibited superior control of strong allospecific immune responses in vitro and in humanized mouse models. A2-CAR Tregs completely prevented rejection of allogeneic target cells and tissues in immune reconstituted humanized mice in the absence of any immunosuppression. Therefore, these modified cells have great potential for incorporation into clinical trials of Treg-supported weaning after allogeneic transplantation.

Association of Higher CD4+ CD25high CD127low , FoxP3+ , and IL-2+ T Cell Frequencies Early After Lung Transplantation With Less Chronic Lung Allograft Dysfunction at Two Years.

Regulatory T cells (Treg) can regulate alloantigens and may counteract chronic lung allograft dysfunction (CLAD) in lung transplantation. We analyzed Treg in peripheral blood prospectively and correlated percentages of subpopulations with the incidence of CLAD at 2 years. Among lung-transplanted patients between January 2009 and July 2011, only patients with sufficient Treg measurements were included into the study. Tregs were measured immediately before lung transplantation, at 3 weeks and 3, 6, 12, and 24 months after transplantation and were defined as CD4+ CD25high T cells and further analyzed for CTLA4, CD127, FoxP3, and IL-2 expressions. Between January 2009 and July 2011, 264 patients were transplanted at our institution. Among the 138 (52%) patients included into the study, 31 (22%) developed CLAD within 2 years after transplantation. As soon as 3 weeks after lung transplantation, a statistically significant positive association was detected between Treg frequencies and later absence of CLAD. At the multivariate analysis, increasing frequencies of CD4+ CD25high CD127low , CD4+ CD25high FoxP3+ and CD4+ CD25high IL-2+ T cells at 3 weeks after lung transplantation emerged as protective factors against development of CLAD at 2 years. In conclusion, higher frequencies of specific Treg subpopulations early after lung transplantation are protective against CLAD development.

Functional defects in CD4+ CD25high FoxP3+ regulatory cells in ankylosing spondylitis.

Forkhead box P3 (FoxP3)-positive regulatory T cells (Tregs) play a pivotal role in the preservation of self-tolerance, and Treg dysfunction has been implicated in many autoimmune diseases. Whether and how Tregs participate in the pathogenesis of ankylosing spondylitis (AS) has not been fully elucidated. Here, we investigated Treg function and found that Tregs in peripheral blood (PB) from patients with active AS had lower FoxP3 mean fluorescence intensity (MFI) than those from healthy controls and could not fully suppress naïve T cell (Tn) proliferation. We also studied the mechanisms underlying PB Treg dysfunction in this context and found that PB Tregs failed to effectively utilize IL-2 and had relatively little STAT5 phosphorylation in active AS. Moreover, PB Tregs from patients with active AS exhibited greater CpG island methylation in the CNS2 region of the FOXP3 gene. Therefore, our findings indicate that functional defects in Tregs are present in AS. Abnormal IL-2 signalling and aberrant CNS2 epigenetic control induced functional defects in PB Tregs and represents a potential new mechanism for AS pathogenesis. These findings may aid the design of new treatment approaches for AS.

Regulatory Cell Populations in Relapsing-Remitting Multiple Sclerosis (RRMS) Patients: Effect of Disease Activity and Treatment Regimens.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) of autoimmune etiology that results from an imbalance between CNS-specific T effector cells and peripheral suppressive mechanisms mediated by regulatory cells (RC). In this research, we collected blood samples from 83 relapsing remitting MS (RRMS) patients and 45 healthy persons (HC), to assess the sizes of their RC populations, including CD4+CD25highFoxp3+ (nTregs), CD3+CD4+HLA−G+, CD3+CD8+CD28−, CD3+CD56+, and CD56bright cells, and how RC are affected by disease activity (acute phase or remission) and types of treatment (methylprednisolone, interferon, or natalizumab). In addition, we isolated peripheral blood mononuclear cells (PBMC) and cultured them with peptides mapping to myelin antigens, to determine RC responsiveness to autoantigens. The results showed decreased levels of nTregs in patients in the acute phase ± methylprednisolone and in remission + natalizumab, but HC levels in patients in remission or receiving interferon. Patients + interferon had the highest levels of CD3+CD4+HLA−G+ and CD3+CD8+CD28− RC, and patients in the acute phase + methylprednisolone the lowest. Patients in remission had the highest levels of CD3+CD56+, and patients in remission + natalizumab the highest levels of CD56bright cells. Only nTregs responded to autoantigens in culture, regardless of disease activity or treatment. The highest suppressive activity was exhibited by nTregs from patients in remission. In conclusion, in RRMS disease activity and type of treatment affect different RC populations. nTregs respond to myelin antigens, indicating that it is possible to restore immunological tolerance through nTreg induction.

Divergence of helper, cytotoxic, and regulatory T cells in the decidua from miscarriage

The aim of this prospective study was to evaluate phenotypic differences of helper T (Th), cytotoxic T (Tc), and regulatory T (Treg) cells in the deciduae of missed miscarriage with a normal chromosome karyotype of a fetus (MN) and missed miscarriage with an abnormal chromosome karyotype of a fetus (MA). The decidua of 19 MN and 28 MA was obtained. Additionally, the decidua of 15 induced abortion (IA) and the endometrium of 19 non-pregnant women (EM) were obtained. IFN-γ(+) , IL-17(+) , CD25(high) Foxp3(+) cells in CD4(+) (Th) cells, and IFN-γ(+) cells in CD8(+) (Tc) cells were evaluated by flow cytometry. The percentages of IFN-γ(+) Tc and CD4(+) CD25(high) Foxp3(+) (Treg) cells in MN were significantly increased as compared with MA and IA. The percentage of IFN-γ(+) Th in MN was increased as compared with IA. Activation of IFN-γ(+) Tc and Treg cells in the decidua might be associated with the pathophysiology underlying MN.

IL-33 polymorphisms are associated with increased risk of hay fever and reduced regulatory T cells in a birth cohort.

IL-33 polymorphisms influence the susceptibility to asthma. IL-33 indirectly induces Th2-immune responses via dendritic cell activation, being important for development of atopic diseases. Furthermore, IL-33 upregulates regulatory T cells (Tregs), which are critical for healthy immune homeostasis. This study investigates associations between IL-33 polymorphisms during the development of childhood atopic diseases and underlying mechanisms including immune regulation of Tregs. Genotyping of IL-33-polymorphisms (rs928413, rs1342326) was performed by MALDI-TOF-MS in 880 of 1133 PASTURE/EFRAIM children. In 4.5-year-old German PASTURE/EFRAIM children (n = 99), CD4+ CD25high FOXP3+ Tregs were assessed by flow cytometry following 24-h incubation of PBMCs with PMA/ionomycin, LPS or without stimuli (U). SOCS3, IL1RL1, TLR4 mRNA expression and sST2 protein levels ex vivo were measured in PASTURE/EFRAIM children by real-time PCR or ELISA, respectively. Health outcomes (hay fever, asthma) were assessed by questionnaires at the age of 6 years. rs928413 and rs1342326 were positively associated with hay fever (OR = 1.77, 95%CI = 1.02-3.08; OR = 1.79, 95%CI = 1.04-3.11) and CD4+ CD25high FOXP3+ Tregs (%) decreased in minor allele homozygotes/heterozygotes compared to major allele homozygotes (p(U) = 0.004; p(LPS) = 0.005; p(U) = 0.001; p(LPS) = 0.012). SOCS3 mRNA expression increased in minor allele homozygotes and heterozygotes compared with major allele homozygotes for both IL-33-polymorphisms (p(rs928413) = 0.032, p(rs1342326) = 0.019) and negatively correlated to Tregs. IL-33-polymorphisms rs928413 and rs1342326 may account for an increased risk of hay fever with the age of 6 years. Lower Tregs and increased SOCS3 in combined heterozygotes and minor allele homozygotes may be relevant for hay fever development, pointing towards dysbalanced immune regulation and insufficient control of allergic inflammation.

A Study on T Regulatory Cells in Patients with Respiratory Allergies in Zagazig University Allergy and Immunology Unit

Background: A positive autologous serum skin test (ASST) reflects the presence of anti-FceRI and/or anti-IgE autoantibodies and used to show autoimmunity in cases with respiratory allergies. Impaired CD4+CD25 high FoxP3+ T regulatory cell activity can cause autoimmune diseases and allergy. Objective: To investigate the association between respiratory allergies and CD4+CD25 high FoxP3+ T regulatory T cell count and re-evaluate the significance of ASST reactivity in respiratory allergy patients and healthy controls. Methodology: 16 patients with persistent asthma, 14 seasonal allergic rhinitis patients and 30 control subjects were included. ASST and measurement of FoxP3+ regulatory T cell count was performed in all subjects. Results: ASST was positive in 18/30 (60%) respiratory allergic patients and none of control subjects. FoxP3+Treg cell count was lower in respiratory allergic patients than control subjects with negative correlation with disease severity. Conclusion: Respiratory allergic patients have lower level of FoxP3+ regulatory T cell than normal individuals. A positive ASST response found in respiratory diseases patients suggests that it may occur as a result of some inflammatory events during the diseases process and could be related to disease activity .

Clinical Significance of Monitoring T Lymphocyte Subsets after Allogeneic Hematopoietic Stem Cell Transplantation

To evaluate the relationship between T lymphocyte subsets and the incidence of graft-versus-host disease (GVHD) and its clinical significance of monitoring the changes of T lymphocyte subsets dynamicly on 1, 3, 6, 12 month after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Twenty cases received allo-HSCT in Department of Hematology of General Hospital of Beijing Military Command from January 2013 to January 2014, including 10 males and 10 females with average age of 20.3 years (3-46 years old), among them 4 cases rectived HLA matched transplantation and 16 cases rectived HLA mismatched transplantation. The levels of T lymphocyte subsets including CD3(+), CD4(+), CD8(+), CD4(+)/CD8(+), CD4(+)CD25(high) FOXP3(+) in the peripheral blood were manitored with flow cytometry (FCM) on +1, +3, +6, +12 month after transplantation dynamicly. (1) Follow up to March 2015, the levers of CD3(+), CD4(+), CD8(+), CD4(+)/CD8(+), CD4(+) CD25(high) FOXP3(+) showed a different degree of recovery after transplantation for all cases and returned to the lever of pre-transplantation on 12 month basically, and CD8(+) T cells recovered earlier than CD4(+) T cells, while the decrease of CD4(+) T cells lasted more than 1 year; The proportion inversion of CD4(+)/CD8(+) also lasted for more than 1 year;(2) The level of CD4(+) CD25(high) FOXP3(+) in patients with acute GVHD was lower than that in patients without acute GVHD. The dynamic monitoring of the T lymphocyte subsets, especially CD4(+) CD25(high) FOXP3(+) after transplantation has importent clinical significance, it can forecast the incidence of acute GVHD before symptoms appeared; the dynamic monitoring of the T-lymphocyte subsets also can be used as reference indicator for prediction of GVHD, theraby it can reduce mortality of patients after transplantation.

Relationship between regulatory T cells subsets and lipid profile in dyslipidemic patients: a longitudinal study during atorvastatin treatment.

BackgroundThe CD4+ T-lymphocytes and their subtype CD4 + CD25highFoxP3+ regulatory T cells are receiving growing interest as major regulators of atherogenesis. We sought to investigate 1) whether the CD4 + cell subsets were expressed differently in dyslipidemic patients (Pts) and healthy subjects (HS) and 2) whether atorvastatin treatment could be associated in-vivo and in-vitro with cell changes in expression and functional response.MethodsCD4+ subsets frequency (CD4 + CD25highFoxP3+, CD4 + CD25-FoxP3+) and mRNA expression for FoxP3, IL-10 and TGF-β were evaluated in 30 consecutive Pts at baseline and after a 3-month atorvastatin therapy, and in 17 HS.ResultsThe % of CD4 + cells did not differ between HS and Pts. The % of CD4 + CD25highFoxP3+ was higher in Pts than HS and did not change during treatment. The CD4 + CD25-FoxP3+ cells were similar between the two groups and were lower in Pts at visit 2. Cytokine expression and FoxP3 did not differ in HS and Pts and no substantial change was observed during treatment. At visit 1, CD4 + CD25highFoxP3+ cells were significantly correlated with both total-cholesterol (r = 0.570, P = 0.0002), LDL-cholesterol (r = 0.715, P = 0.0001), Apolipoprotein B (r = 0.590, P = 0.0001). In-vitro atorvastatin (up to 5 μM) failed to induce any significant modulation of cell functions.ConclusionCD4 + CD25highFoxP3+ regulatory cells seem to be over-stimulated in the early pre-clinical phase of atherosclerosis and a relationship exists between their frequency and circulating lipids. A potential immuno-modulation by statin treatment is not achieved through a normalization in peripheral CD4 + cell subsets.

Bovine leukemia virus reduces anti-viral cytokine activities and NK cytotoxicity by inducing TGF-β secretion from regulatory T cells.

CD4(+)CD25(high)Foxp3(+) T cells suppress excess immune responses that lead to autoimmune and/or inflammatory diseases, and maintain host immune homeostasis. However, CD4(+)CD25(high)Foxp3(+) T cells reportedly contribute to disease progression by over suppressing immune responses in some chronic infections. In this study, kinetic and functional analyses of CD4(+)CD25(high)Foxp3(+) T cells were performed in cattle with bovine leukemia virus (BLV) infections, which have reported immunosuppressive characteristics. In initial experiments, production of the Th1 cytokines IFN-γ and TNF-α was reduced in BLV-infected cattle compared with uninfected cattle, and numbers of IFN-γ or TNF-α producing CD4(+) T cells decreased with disease progression. In contrast, IFN-γ production by NK cells was inversely correlated with BLV proviral loads in infected cattle. Additionally, during persistent lymphocytosis disease stages, NK cytotoxicity was depressed as indicated by low expression of the cytolytic protein perforin. Concomitantly, total CD4(+)CD25(high)Foxp3(+) T cell numbers and percentages of TGF-β(+) cells were increased, suggesting that TGF-β plays a role in the functional declines of CD4(+) T cells and NK cells. In further experiments, recombinant bovine TGF-β suppressed IFN-γ and TNF-α production by CD4(+) T cells and NK cytotoxicity in cultured cells. These data suggest that TGF-β from CD4(+)CD25(high)Foxp3(+) T cells is immunosuppressive and contributes to disease progression and the development of opportunistic infections during BLV infection.

Frequency of FOXP3+ Regulatory T-cells in the Blood of Chronic Hepatitis C Patients with Immune Mediated Skin Manifestations; Relationship to Hepatic Condition and Viral Load.

Chronic hepatitis C (CHC) infection causes a wide range of immune mediated hepatic and extrahepatic manifestations. Cutaneous manifestations constitute a major portion of the latter group. Host immune response to the virus - particularly regulatory T lymphocytes - is important in determining the outcome of the infection and the possibility of extrahepatic manifestations. The present study aims to investigate the relationship between immune-mediated cutaneous manifestations of CHC infection and the frequency of CD4+CD25 high FOXP3+ T regulatory lymphocytes. We also investigated the relationships to viral load and hepatic conditions. A total of 58 CHC patients (30 had cutaneous manifestations and 28 did not) and 30 healthy HCV free subjects were enrolled in this study. The frequency of CD4+CD25 high FOXP3+ Treg cells was measured in the peripheral blood of all participants. Additionally, serum ALT, RNA viral load, and hepatic ultrasonographic studies were performed for all patients. Skin manifestations were dominated by small vessel vasculitis and to a lesser extent lichen planus. Treg cell frequency was significantly lower in CHC patients with skin manifestations compared to those without. Chronic hepatic insult was significantly more common among patients with skin manifestations, although serum ALT levels were significantly lower in this group. Treg frequencies did not correlate to either ALT level or viral load. HCV-associated skin lesions are associated with low frequency of Treg cells. They are not related to liver enzymes or viral load; however, they are associated with a more advanced gross liver insult.

Altered Expression of Functional Proteins in CD4 Regulatory T Cells during Therapy with Idelalisib

Introduction: Idelalisib, a specific small molecule inhibitor of the delta isoform of PI3 kinase (PI3Kd), has recently been FDA approved for the treatment of relapsed CLL. Expression of PI3Kd is predominantly restricted to leukocytes, and inhibition of PI3Kd promotes apoptosis of neoplastic CLL cells. Patients who receive idelalisib often experience toxicities that appear to be immune mediated. In an ongoing phase II study of idelalisib-ofatumumab at DFCI, 16 of 21 patients experienced grade 3 or greater autoimmune toxicities (14 transaminitis, 2 enteritis/colitis and 3 presumed drug-induced pneumonitis).Methods: To identify potential causes of autoimmunity in patients receiving idelalisib, we used single cell mass cytometry (CyTOF) with a panel of 26 surface membrane and 9 intracellular markers to provide a comprehensive phenotypic and functional analysis of all peripheral blood lymphocytes. Blood samples were obtained at baseline prior to therapy and at onset of autoimmune symptoms, which occurred at a median of 28 days from start of therapy. Results in 5 severely affected patients were compared with 3 patients who did not develop autoimmune toxicities and 15 healthy donors. The second sample from unaffected patients was matched in time to the affected patient samples.Results: Within CD3 T cells, idelalisib therapy led to an increased percentage of CD4 T cells and decreased percentage of CD8 T cells. Within CD4 T cells, CD25high Foxp3+ regulatory T cells (Treg) were reduced, resulting in a marked increase of the ratio of conventional CD4 T (Tcon)/Treg and increased CD8/Treg ratio. These findings were similar in patients who developed autoimmune toxicities and those who did not. Analysis of effector memory (EM; 45RA- CCR7-), central memory (CM; 45RA- CCR7+), terminal effector (TEMRA; 45RA+ CCR7-) and naive (45RA+ CCR7+) subsets within Treg, Tcon and CD8 T cells was comparable before and after idelalisib therapy and with healthy individual controls. To evaluate the heterogeneity of each population we utilized viSNE, which allows visualization of complex high-dimensional cytometry data on a two-dimensional plot. Treg in patients who developed autoimmune phenomena were compared with Treg from patients who did not. In patients who developed autoimmunity, baseline Treg had higher expression of PD-1 and lower expression of functional markers, such as GITR, Tbet, CXCR3, PDL-1, granzyme-β and TIM-3. In some cases, expression of these functional markers declined further with idelalisib therapy (Figure 1). Cytotoxic T lymphocyte antigen 4 (CTLA4) is constitutively expressed by Treg, and its deficiency has been shown to limit suppressive function of Treg. Inducible co-stimulator (ICOS) also mediates the suppressive functions of CD4 Treg by regulating cell surface expression of CTLA-4. Interestingly, expression of CTLA-4 and ICOS did not change during idelalisib therapy (Figure 1). Susceptibility to apoptosis was monitored by expression of pro-survival Bcl-2 and pro-apoptotic CD95 (FAS). Treg from patients developing autoimmunity expressed lower levels of Bcl-2 and higher levels of CD95 compared to those who did not. Conversely, Treg of non-affected patients retained higher expression of functional markers and became more active, assessed by increased expression of HLA-DR. The use of CyTOF combined with our comprehensive panel allowed us to delineate up to 13 distinct Treg sub-populations among healthy controls. In contrast, CLL patients often lacked such broad Treg heterogeneity. Expression of functional markers by Tcon and CD8 T cells from patients who would develop side effects, were also slightly lower, suggesting that autoimmune toxicities were not due to expansion or activation of CD8 or Tcon effector cells but due to the defect in both number and function of Tregs, which became insufficient to maintain the immune tolerance.Conclusion: These studies allowed us to identify defects in both Treg number and function, which may be responsible for the autoimmune toxicities in patients receiving idelalisib therapy. Further studies may allow the development of a reliable predictor of these toxicities by evaluation of the Treg markers prior to drug exposure which could be used to guide therapy. Moreover, the identification of idelalisib as a Treg inhibitor suggests that it could be used as an immunomodulatory agent to target the Treg pathway, in patients with solid tumors. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Ex Vivo Signaling Protein Mapping in T Lymphocytes in the Psoriatic Arthritis Joints.

We assessed signaling protein mapping in total T cells, to analyze the proportions of T regulatory (Treg) and TCD4+ effector (Teff) cell phenotypes, and the respective interleukin 6Rα (IL-6Rα) expression in the inflammatory microenvironment of synovial fluid (SF) of patients with sustained psoriatic arthritis (PsA). Our approach was to measure the IL-6 level in SF using a multiplex bead immunoassay. Reverse-phase protein array was used to assess Janus kinase (JAK) 1 and JAK2, extra-cellular regulated kinase (ERK) 1 and 2, protein kinase Cδ (PKCδ), signal transducer and activator and transcription (STAT) 1, STAT3, and STAT5 phosphoproteins in total T cell lysates from SF of patients with PsA. Frequencies of CD4+IL-17A-F+IL-23+ CD4+ Th cells producing IL-17A and IL-17F (Th17) and CD4+CD25high intracellular forkhead box transcription factor+ (FOXP3+) phenotypes, and the percentage of Treg- and Teff- cells were quantified in SF and matched peripheral blood (PB) of patients with PsA and PB of healthy controls (HC) by flow cytometry. Our results were the following: In PsA SF samples, a coordinate increase of JAK1, ERK1/2, STAT1, STAT3, and STAT5 phosphoproteins was found in total T cells in SF of PsA; where IL-6 levels were higher than in PB from HC. Expanded CD4+IL-17A-F+IL-23+ Th17, CD4+ CD25- Teff- and CD4+CD25(high) FoxP3+Treg subsets, showing similar levels of enhanced IL-6Rδ expression, were confined to PsA joints. In our studies, the transcriptional network profile identified by ex vivo signaling protein mapping in T lymphocytes in PsA joints revealed the complex interplay between IL-1, IL-6, and IL-23 signaling and differentiation of Th17 cells and CD4+Tregs in sustained joint inflammation in PsA.

T regulatory cell phenotypes in peripheral blood and bronchoalveolar lavage from non-asthmatic and asthmatic subjects.

An unresolved issue in T regulatory cells' cell biology is the lack of consensus on phenotypic markers that accurately define the natural Treg (nTreg) population. To examine nTreg frequency and functional capacity in healthy controls and their frequency in asthmatic subjects using three different phenotypic strategies. We hypothesized that phenotypically different nTreg are quantitatively and functionally different. Thirty-four healthy, non-asthmatic and 17 asthmatic subjects were studied. Three nTreg phenotypes were defined as follows: nTreg1 (CD4(+) CD25(+) Foxp3(+) ), nTreg2 (CD4(+) CD25(+) CD127(low) Foxp3(+) ), and nTreg3 (CD4(+) CD25(high) Foxp3(+) ). The flow cytometric determination of nTreg frequency in peripheral blood (PB) and bronchoalveolar lavage (BAL) was performed using fluorescently labelled antibodies. Peripheral blood nTreg functional capacity was assessed using a CFSE-based suppression assay. There was a significantly lower frequency of PB nTreg3 compared to nTreg2 and nTreg1 (P<0.05). Both nTreg2 and nTreg3 had a significantly greater suppressive capacity than nTreg1 at T responder (Tresp) to nTreg ratios of 16:1 up to 1:1 (P<0.01). Asthmatics exhibited a significantly lower PB nTreg3 and nTreg1 frequency than healthy controls (P<0.05). There were no differences between healthy controls and asthmatic subjects when comparing BAL nTreg frequency. Phenotypically different nTreg subsets are quantitatively and functionally different and are variably observed in asthma. The CD4(+) CD25(high) Foxp3(+) phenotype was the least frequent, but demonstrated the greatest suppression, and was significantly lower in PB of asthmatic subjects. Consequently, it is imperative that nTreg phenotypes be clearly defined and that the interpretation of their frequency and function be phenotype specific.

Overexpression of CD45RA isoforms in carriers of the C77G mutation leads to hyporeactivity of CD4+CD25highFoxp3+ regulatory T cells.

Disorders in regulatory T-cell (T(reg)) function can result in the breakdown of immunological self-tolerance. Thus, the identification of mechanisms controlling the activity of T(reg) is of great relevance. We used T(reg) from individuals carrying the C77G polymorphism as models to study the role of CD45 molecules in humans. C77G prevents splicing of CD45 exon A thereby leading to an aberrant expression pattern of CD45 isoforms in affected individuals. Resting and in vitro expanded/activated CD4(+)CD25(high)Foxp3(+) T(reg) from carriers of C77G strongly expressed CD45RA isoforms whereas these isoforms were almost absent in cells from individuals with wild-type CD45. C77G T(reg) showed diminished upregulation of activation markers, lower phosphorylation of p56(lck)(Y505) and a reduced proliferative potential when stimulated with anti-TcR or anti-TcR plus CD28 mAb suggesting decreased responsiveness to activating stimuli. In addition, the capacity to suppress proliferation of conventional CD4(+) T cells was impaired in C77G T(reg). Furthermore, microarray studies revealed distinct gene expression patterns in T(reg) from C77G carriers. These data suggest that the changes in CD45 isoform combination resulting from the C77G mutation alter the responsiveness of T(reg) to TcR-mediated signaling. Targeting CD45 isoform expression might be a useful approach to modulate T(reg) function.