Staphylococcus aureus convert neonatal conventional CD4(+) T cells into FOXP3(+) CD25(+) CD127(low) T cells via the PD-1/PD-L1 axis.
ABSTRACT: The gut microbiota provides an important stimulus for the induction of regulatory T (Treg) cells in mice, whether this applies to newborn children is unknown. In Swedish children, Staphylococcus aureus has become a common early colonizer of the gut. Here, we sought to study the effects of bacterial stimulation on neonatal CD4(+) T cells for the induction of CD25(+) CD127(low) Treg cells in vitro. The proportion of circulating CD25(+) CD127(low) Treg cells and their expression of FOXP3, Helios and CTLA-4 was examined in newborns and adults. To evaluate if commensal gut bacteria could induce Treg cells, CellTrace violet-stained non-Treg cells from cord or peripheral blood from adults were co-cultured with autologous CD25(+) CD127(low) Treg cells and remaining mononuclear cells and stimulated with S. aureus. Newborns had a significantly lower proportion of CD25(+) CD127(low) Treg cells than adults, but these cells were Helios(+) and CTLA-4(+) to a higher extent than in adults. FOXP3(+) CD25(+) CD127(low) T cells were induced mainly in neonatal CellTrace-stained non-Treg cells after stimulation with S. aureus. In cell cultures from adults, S. aureus induced CD25(+) CD127(low) T cells only if sorted naive CD45RA(+) non-Treg cells were used, but these cells expressed less FOXP3 than those induced from newborns. Sorted neonatal CD25(+) CD127(low) T cells from S. aureus-stimulated cultures were still suppressive. Finally, blocking PD-L1 during stimulation reduced the induction of FOXP3(+) CD25(+) CD127(low) T cells. These results suggest that newborns have a higher proportion of circulating thymically derived Helios(+) Treg cells than adults and that S. aureus possess an ability to convert neonatal conventional CD4(+) T cells into FOXP3(+) CD25(+) CD127(low) Treg cells via the PD-1/PD-L1 axis.
Project description:OBJECTIVE:To assess the use of Helios in combination with FoxP3 as a superior method for identifying non-cytokine-producing human Treg cells in patients with systemic lupus erythematosus (SLE) and to determine if FoxP3+Helios+ Treg cells are maintained at normal levels in patients with clinically active disease. METHODS:Peripheral blood mononuclear cells (PBMCs) were purified from the blood of healthy volunteer donors and from 52 consecutive patients with SLE of varying clinical activity (Systemic Lupus Erythematosus Disease Activity Index scores of 0, 2-4, and ≥ 5). PBMCs (either fresh or after 4 hours of stimulation for cytokine production) were then analyzed by flow cytometry for the expression of cell surface markers (CD4, CD25, CD127, and CD45RA) and transcription factors (FoxP3 and Helios), as well as for the production of cytokines (interleukin-2 and interferon-γ). RESULTS:FoxP3+Helios+ Treg cells were found to be non-cytokine producing in both SLE patients and healthy controls. Patients with clinically active SLE had higher percentages of FoxP3+Helios+ Treg cells than did patients with inactive SLE or healthy controls. When corrected for the total CD4 cell count, the absolute numbers of FoxP3+Helios+ Treg cells in patients with moderately-to-highly active SLE were normal. CONCLUSION:Previous reports of a deficiency in Treg cell number or function in SLE are limited by their use of CD25, either alone or in combination with other markers, to identify human Treg cells. Helios in combination with FoxP3 is a superior method for detecting all non-cytokine-producing Treg cells, irrespective of CD25 or CD45RA expression. Using this method, we showed that FoxP3+Helios+ Treg cell numbers are not reduced in patients with clinically active SLE.
Project description:Regulatory T (Treg) cells are characterized by the expression of CD4, CD25 and the intracellular Foxp3. However, these markers do not indicate whether Treg cells are thymic derived Treg (tTreg) cells or peripherally induced Treg (pTreg) cells. Recently, Helios and Neuropilin-1 (Nrp1) has been reported as potential markers for tTreg cells. Herein, we used flow cytometry to examine the proportion of CD4(+)CD8(-)CD25(+) Treg cells expressing Helios, Nrp1 and Foxp3 in thymus, pancreatic draining lymph nodes (PDLNs) and spleen of CD-1 mice, and thymus of NOD and C57BL/6 mice. The frequency of Helios(+) cells was higher than that of Nrp1(+) cells in CD4(+)CD8(-)CD25(+) and CD4(+)CD8(-)CD25(+)Foxp3(+) Treg cells in thymus. Interestingly, the proportion of IL-10(+), Ebi3(+)and CTLA-4(+) cells was higher in Helios(+) than Nrp1(+) tTreg cells. The anti-apoptotic activity of Helios(+) tTreg cells was higher in thymus compared to Nrp1(+) tTreg cells. Nrp1 seems to be expressed at a later developmental stage compared to Helios and Foxp3. Furthermore, the expression of Nrp1 in CD4(+)CD25(+) T cells of younger mice did not increase after stimulating them in vitro with anti-CD3 and -CD28. Thus, under these conditions, Helios could be considered a more reliable marker for distinguishing tTreg cells from pTreg cells than Nrp1.
Project description:Regulatory T cells (Tregs) constitute an attractive therapeutic target given their essential role in controlling autoimmunity. However, recent animal studies provide evidence for functional heterogeneity and lineage plasticity within the Treg compartment. To understand better the plasticity of human Tregs in the context of type 1 diabetes, we characterized an IFN-?-competent subset of human CD4(+)CD127(lo/-)CD25(+) Tregs. We measured the frequency of Tregs in the peripheral blood of patients with type 1 diabetes by epigenetic analysis of the Treg-specific demethylated region (TSDR) and the frequency of the IFN-?(+) subset by flow cytometry. Purified IFN-?(+) Tregs were assessed for suppressive function, degree of TSDR demethylation, and expression of Treg lineage markers FOXP3 and Helios. The frequency of Tregs in peripheral blood was comparable but the FOXP3(+)IFN-?(+) fraction was significantly increased in patients with type 1 diabetes compared to healthy controls. Purified IFN-?(+) Tregs expressed FOXP3 and possessed suppressive activity but lacked Helios expression and were predominately methylated at the TSDR, characteristics of an adaptive Treg. Naive Tregs were capable of upregulating expression of Th1-associated T-bet, CXCR3, and IFN-? in response to IL-12. Notably, naive, thymic-derived natural Tregs also demonstrated the capacity for Th1 differentiation without concomitant loss of Helios expression or TSDR demethylation.
Project description:Regulatory T cells (Tregs) play a central role in counteracting inflammation and autoimmunity. A more complete understanding of cellular heterogeneity and the potential for lineage plasticity in human Treg subsets may identify markers of disease pathogenesis and facilitate the development of optimized cellular therapeutics. To better elucidate human Treg subsets, we conducted direct transcriptional profiling of CD4(+)FOXP3(+)Helios(+) thymic-derived Tregs and CD4(+)FOXP3(+)Helios(-) T cells, followed by comparison with CD4(+)FOXP3(-)Helios(-) T conventional cells. These analyses revealed that the coinhibitory receptor T cell Ig and ITIM domain (TIGIT) was highly expressed on thymic-derived Tregs. TIGIT and the costimulatory factor CD226 bind the common ligand CD155. Thus, we analyzed the cellular distribution and suppressive activity of isolated subsets of CD4(+)CD25(+)CD127(lo/-) T cells expressing CD226 and/or TIGIT. We observed TIGIT is highly expressed and upregulated on Tregs after activation and in vitro expansion, and is associated with lineage stability and suppressive capacity. Conversely, the CD226(+)TIGIT(-) population was associated with reduced Treg purity and suppressive capacity after expansion, along with a marked increase in IL-10 and effector cytokine production. These studies provide additional markers to delineate functionally distinct Treg subsets that may help direct cellular therapies and provide important phenotypic markers for assessing the role of Tregs in health and disease.
Project description:OBJECTIVE: The gut environment modulates the pathogenesis of type 1 diabetes (T1D), but how it affects autoimmunity toward pancreatic ?-cells, a self-tissue located outside the intestine, is still unclear. In the small intestine, lamina propria dendritic cells (LPDCs) induce peripheral differentiation of FoxP3(+) regulatory T (Treg) cells. We tested the hypothesis that the intestinal milieu impinges on human T1D by affecting differentiation of FoxP3(+) Treg cells. RESEARCH DESIGN AND METHODS: We collected duodenal biopsies of 10 T1D patients, 16 healthy subjects, and 20 celiac individuals and performed a fluorescent-activated cell sorter analysis to measure percentages of various immune cell subsets, including CD4(+) and CD8(+) T cells, NK cells, ?? T cells, CD103(+)CD11c(+) LPDCs, and CD4(+)CD25(+)FoxP3(+)CD127(-) Treg cells. In parallel, we assessed the tolerogenic function (i.e., capacity to induce differentiation of FoxP3(+) Treg cells) by LPDCs of T1D patients and control subjects. RESULTS: Our analysis revealed a significant reduction in the percentage of intestinal CD4(+)CD25(+)FoxP3(+)CD127(-) Treg cells in T1D patients compared with healthy subjects (P = 0.03) and celiac individuals (P = 0.003). In addition, we found that LPDCs from T1D patients completely lacked their tolerogenic function; they were unable to convert CD4(+)CD25(-) T cells into CD4(+)CD25(+)FoxP3(+)CD127(-) Treg cells. CONCLUSIONS: Our data indicate that T1D patients have a reduced number of intestinal FoxP3(+) Treg cells as a result of their defective differentiation in the gut. These findings suggest that intestinal immune regulation is not only calibrated to tolerate commensal bacteria and food components but also is instrumental in maintaining immune tolerance toward pancreatic ?-cells and preventing T1D.
Project description:Regulatory T cells (Tregs) are functionally suppressive CD4 T cells, critical for establishing peripheral tolerance and controlling inflammatory responses. Previous reports of Tregs during chronic HIV disease have conflicting results with higher or lower levels compared with controls. Identifying true Tregs with suppressive activity proves challenging during HIV infection, as traditional Treg markers, CD25 and FOXP3, may transiently upregulate expression as a result of immune activation (IA). Helios is an Ikaros family transcription factor that marks natural Tregs with suppressive activity and does not upregulate expression after activation. Coexpression of FOXP3 and Helios has been suggested as a highly specific marker of "bona fide" Tregs. We evaluated Treg subsets by FOXP3 coexpressed with either CD25 or Helios and their association with HIV disease progression in perinatally infected HIV-positive children. Identifying Tregs by FOXP3 coexpression with Helios rather than CD25 revealed markedly higher Treg frequencies, particularly in HIV+ children. Regardless of antiretroviral therapy, HIV-infected children had a selective expansion of memory FOXP3+Helios+ Tregs. The rise in memory Tregs correlated with declining HIV clinical status, indicated by falling CD4 percentages and CD4:CD8 ratios and increasing HIV plasma viremia and IA. In addition, untreated HIV+ children exhibited an imbalance between the levels of Tregs and activated T cells. Finally, memory Tregs expressed IA markers CD38 and Ki67 and exhaustion marker, PD-1, that tightly correlated with a similar phenotype in memory CD4 T cells. Overall, HIV-infected children had significant disruptions of memory Tregs that associated with advancing HIV disease.
Project description:BACKGROUND: Upon antigen exposure, cord blood derived T cells respond to ubiquitous environmental antigens by high proliferation. To date it remains unclear whether these "excessive" responses relate to different regulatory properties of the putative T regulatory cell (Treg) compartment or even expansion of the Treg compartment itself. METHODS: Cord blood (>37 week of gestation) and peripheral blood (healthy controls) were obtained and different Treg cell subsets were isolated. The suppressive potential of Treg populations after antigen exposure was evaluated via functional inhibition assays ([(3)H]thymidine incorporation assay and CFSE staining) with or without allergen stimulation. The frequency and markers of CD4(+)CD25(high)FoxP3(+) T cells were characterized by mRNA analysis and flow cytometry. RESULTS: Cord blood derived CD4(+)CD25(high) cells did not show substantial suppressor capacity upon TCR activation, in contrast to CD4(+)CD25(high) cells freshly purified from adult blood. This could not be explained by a lower frequency of FoxP3(+)CD4(+)CD25(high)cells or FOXP3 mRNA expression. However, after antigen-specific stimulation in vitro, these cells showed strong proliferation and expansion and gained potent suppressive properties. The efficiency of their suppressive capacity can be enhanced in the presence of endotoxins. If T-cells were sorted according to their CD127 expression, a tiny subset of Treg cells (CD4(+)CD25(+)CD127(low)) is highly suppressive even without prior antigen exposure. CONCLUSION: Cord blood harbors a very small subset of CD4(+)CD25(high) Treg cells that requires antigen-stimulation to show expansion and become functional suppressive Tregs.
Project description:OBJECTIVE:Despite the high frequency of CD4+ T cells with a regulatory phenotype (CD25+CD127(low) FoxP3+) in the joints of patients with rheumatoid arthritis (RA), inflammation persists. One possible explanation is that human Treg cells are converted into proinflammatory interleukin-17 (IL-17)-producing cells by inflammatory mediators and thereby lose their suppressive function. The aim of this study was to investigate whether activated monocytes, which are potent producers of inflammatory cytokines and are abundantly present in the rheumatic joint, induce proinflammatory cytokine expression in human Treg cells and impair their regulatory function. METHODS:The presence and phenotype of CD4+CD45RO+CD25+CD127(low) T cells (memory Treg cells) and CD14+ monocytes in the peripheral blood (PB) and synovial fluid (SF) of patients with RA were investigated by flow cytometry. Memory Treg cells obtained from healthy control subjects underwent fluorescence-activated cell sorting and then were cocultured with autologous activated monocytes and stimulated with anti-CD3 monoclonal antibodies. Intracellular cytokine expression, phenotype, and function of cells were determined by flow cytometry, enzyme-linked immunosorbent assay, and proliferation assays. RESULTS:In patients with RA, the frequencies of CD4+CD45RO+CD25+CD127(low) Treg cells and activated CD14+ monocytes were higher in SF compared with PB. In vitro-activated monocytes induced an increase in the percentage of IL-17-positive, interferon-? (IFN?)-positive, and tumor necrosis factor ? (TNF?)-positive Treg cells as well as IL-10-positive Treg cells. The observed increase in IL-17-positive and IFN?-positive Treg cells was driven by monocyte-derived IL-1?, IL-6, and TNF? and was mediated by both CD14+CD16- and CD14+CD16+ monocyte subsets. Despite enhanced cytokine expression, cells maintained their CD25+FoxP3+CD39+ Treg cell phenotype and showed an enhanced capacity to suppress T cell proliferation and IL-17 production. CONCLUSION:Treg cells exposed to a proinflammatory environment show increased cytokine expression as well as enhanced suppressive activity.
Project description:Regulatory T cells (Treg) are important for immune homeostasis and are considered of great interest for immunotherapy. The paucity of Treg numbers requires the need for ex vivo expansion. Although therapeutic Treg flow-sorting is feasible, most centers aiming at Treg-based therapy focus on magnetic bead isolation of CD4+CD25+ Treg using a good manufacturing practice compliant closed system that achieves lower levels of cell purity. Polyclonal Treg expansion protocols commonly use anti-CD3 plus anti-CD28 monoclonal antibody (mAb) stimulation in the presence of rhIL-2, with or without rapamycin. However, the resultant Treg population is often heterogeneous and pro-inflammatory cytokines like IFN? and IL-17A can be produced. Hence, it is crucial to search for expansion protocols that not only maximize ex vivo Treg proliferative rates, but also maintain Treg stability and preserve their suppressive function. Here, we show that ex vivo expansion of low purity magnetic bead isolated Treg in the presence of a TNFR2 agonist mAb (TNFR2-agonist) together with rapamycin, results in a homogenous stable suppressive Treg population that expresses FOXP3 and Helios, shows low expression of CD127 and hypo-methylation of the FOXP3 gene. These cells reveal a low IL-17A and IFN? producing potential and hardly express the chemokine receptors CCR6, CCR7 and CXCR3. Restimulation of cells in a pro-inflammatory environment did not break the stability of this Treg population. In a preclinical humanized mouse model, the TNFR2-agonist plus rapamycin expanded Treg suppressed inflammation in vivo. Importantly, this Treg expansion protocol enables the use of less pure, but more easily obtainable cell fractions, as similar outcomes were observed using either FACS-sorted or MACS-isolated Treg. Therefore, this protocol is of great interest for the ex vivo expansion of Treg for clinical immunotherapy.
Project description:The control of peripheral immune responses by FOXP3+ T regulatory (Treg) cells is essential for immune tolerance. However, at any given time, Treg frequencies in whole blood can vary more than fivefold between individuals. An understanding of factors that influence Treg numbers and migration within and between individuals would be a powerful tool for cellular therapies that utilize the immunomodulatory properties of Tregs to control pathology associated with inflammation. We sought to understand how season could influence Treg numbers and phenotype by monitoring the proportion of natural thymus-derived Tregs (nTregs) defined as (CD3+CD4+CD25+FOXP3+CD127-/low ) cells as a proportion of CD4+ T cells and compared these to all FOXP3+ Tregs (allTregs, CD3+CD25+FOXP3+CD127-/low ). We were able to determine changes within individuals during 1 year suggesting an influence of season on nTreg frequencies. We found that, between individuals at any given time, nTreg/CD4+ T cells ranged from 1.8% in February to 8.8% in the summer where median nTreg/CD4 in January and February was 2.4% (range 3.75-1.76) and in July and August was 4.5% (range 8.81-3.17) p = 0.025. Importantly we were able to monitor individual nTreg frequencies throughout the year in donors that started the year with high or low nTregs. Some nTreg variation could be attributed to vitamin D status where normal linear regression estimated that an absolute increase in nTreg/CD4+ by 0.11% could be expected with 10 nmol increase in serum 25 (OH) vitamin D3 (p = 0.005, 95% CI: 0.03-0.19). We assessed migration markers on Tregs for the skin and/or gut. Here cutaneous lymphocyte associated antigen (CLA+) expression on CD25+FOXP3+CD4+/CD4+ was compared with the same population expressing the gut associated integrin, ?7. Gut tropic CD25+FOXP3+?7+Tregs/CD4+ had similar dynamics to nTreg/CD4+. Conversely, CD25+FOXP3+CLA+Tregs/CD4+ showed no association with vitamin D status. Important for cellular therapies requiring isolation of Tregs, the absolute number of ?7+CD4+CD25+FOXP3+Tregs was positively associated with 25(OH)vitamin D3 (R 2 = 0.0208, r = 0.184, p = 0.021) whereas the absolute numbers of CLA+CD4+CD25+FOXP3+Tregs in the periphery were not influenced by vitamin D status. These baseline observations provide new opportunities to utilize seasonal variables that influence Treg numbers and their migratory potential in patients or donors.