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Elfaki, Yassin, Juhao Yang, Julia Boehme, Kristin Schultz, Dunja Bruder, Christine S. Falk, Jochen Huehn, and Stefan Floess. "Tbx21 and Foxp3 Are Epigenetically Stabilized in T-Bet+ Tregs That Transiently Accumulate in Influenza A Virus-Infected Lungs." International Journal of Molecular Sciences 22, no. 14 (July 14, 2021): 7522. http://dx.doi.org/10.3390/ijms22147522.
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During influenza A virus (IAV) infections, CD4+ T cell responses within infected lungs mainly involve T helper 1 (Th1) and regulatory T cells (Tregs). Th1-mediated responses favor the co-expression of T-box transcription factor 21 (T-bet) in Foxp3+ Tregs, enabling the efficient Treg control of Th1 responses in infected tissues. So far, the exact accumulation kinetics of T cell subsets in the lungs and lung-draining lymph nodes (dLN) of IAV-infected mice is incompletely understood, and the epigenetic signature of Tregs accumulating in infected lungs has not been investigated. Here, we report that the total T cell and the two-step Treg accumulation in IAV-infected lungs is transient, whereas the change in the ratio of CD4+ to CD8+ T cells is more durable. Within lungs, the frequency of Tregs co-expressing T-bet is steadily, yet transiently, increasing with a peak at Day 7 post-infection. Interestingly, T-bet+ Tregs accumulating in IAV-infected lungs displayed a strongly demethylated Tbx21 locus, similarly as in T-bet+ conventional T cells, and a fully demethylated Treg-specific demethylated region (TSDR) within the Foxp3 locus. In summary, our data suggest that T-bet+ but not T-bet− Tregs are epigenetically stabilized during IAV-induced infection in the lung.
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Sanz-Rubio, David, Arianne Sanz, Luis Varona, Rosa Bolea, Marta Forner, Ana V. Gil, Pablo Cubero, Marta Marin-Oto, Inmaculada Martin-Burriel, and Jose M. Marin. "Forkhead Box P3 Methylation and Expression in Men with Obstructive Sleep Apnea." International Journal of Molecular Sciences 21, no. 6 (March 23, 2020): 2233. http://dx.doi.org/10.3390/ijms21062233.
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Background: Epigenetic changes in obstructive sleep apnea (OSA) have been proposed as a mechanism for end-organ vulnerability. In children with OSA, Forkhead Box P3 (FOXP3) DNA methylation were associated with inflammatory biomarkers; however, the methylation pattern and its effect in the expression of this gene have not been tested in adults with OSA. Methods: Plasma samples from subjects without comorbid conditions other than OSA were analyzed (the Epigenetics Status and Subclinical Atherosclerosis in Obstructive Sleep Apnea (EPIOSA) Study: NCT02131610). In 16 patients with severe OSA (Apnea-Hypopnea Index—AHI- > 30 events/h) and seven matched controls (AHI < 5), methylation of FOXP3 gen was evaluated by PCR of the promoter and by pyrosequencing of the intron 1 Treg-specific demethylated region (TSDR). In another 74 patients with OSA (AHI > 10) and 31 controls, we quantified FOXP3 protein expression by ELISA and gene expression by quantitative real-time PCR. C-reactive protein (CRP) and plasma Treg cells were also evaluated. Results: Neither the levels of the promoter nor the TSDR demethylated region were different between controls and patients with OSA, whether they were grouped by normal or high CRP. FOXP3 protein and mRNA expression did not differ between groups. Conclusions: FOXP3 methylation or its expression is not altered in adults with OSA, whatever their inflammatory status.
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Schoenbrunn, Anne, Jacqueline Keye, Siegfried Kohler, Marco Frentsch, Beate Moewes, Alexander Scheffold, Chiara Romagnani, and Andreas Thiel. "High Efficiency Isolation of Alloantigen-Reactive Foxp3+ Natural Regulatory T Cells According to Specific Activation Marker Signatures." Blood 118, no. 21 (November 18, 2011): 1915. http://dx.doi.org/10.1182/blood.v118.21.1915.1915.
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Abstract Abstract 1915 Transplant tolerance induction and avoidance of exposure to destructive immunosuppressive drugs are still major objectives in transplantation medicine. Many preclinical animal models have proven the adoptive transfer of polyclonal CD4+CD25+Foxp3+ regulatory T (Treg) cells to be an important and effective tool for the prevention of graft rejection or graft-versus-host-disease. However, polyclonal Treg may also modulate immunity to foreign or tumor antigens. It is unclear yet, whether and how their application might lead to an unwanted general immunosuppression. Therefore the selective transfer of alloantigen-reactive Treg represents a very attractive therapeutic option. However, so far this strategy has been hampered so far by the lack of appropriate marker to assess and isolate antigen-reactive Treg cells with high efficiency. In order to get access to alloantigen-reactive Treg, we established cytometric methodologies allowing a clear dissection between (allo)antigen-specific Treg and Teff based on expression of 4-1BB and the lack of expression of CD40L. After allogeneic stimulation CD4+ T cells expressing 4-1BB but lacking CD40L expression were highly enriched in Foxp3+ T cells. Further molecular analysis of the Foxp3 gene locus revealed that only 4-1BB+CD40L− T cells were characterised by a completely demethylated TSDR (Treg specific demethylated region). 4-1BB+CD40L− T cells highly expressed the transcription factor HELIOS recently demonstrated to specify thymic-derived Treg. Alloantigen-reactive Treg isolated according to 4-1BB and CD40L were superior with respect to their alloantigen-specific in vitro suppression as compared their polyclonal Treg counterparts. Finally 4-1BB+CD40L− alloantigen-reactive Treg could be easily expanded in vitro up to 300 fold during 3 weeks culture, maintaining alloantigen-specific suppression. Our results offer the possibility to improve current approaches for adoptive cell-therapy with alloantigen-specific Treg to achieve transplantation tolerance aiming at a specific inhibition of pathology. Disclosures: Scheffold: Miltenyi Biotec GmbH: Employment.
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Černý, Viktor, Olga Novotná, Petra Petrásková, Kateřina Hudcová, Kristýna Boráková, Ludmila Prokešová, Libuše Kolářová, and Jiří Hrdý. "Lower Functional and Proportional Characteristics of Cord Blood Treg of Male Newborns Compared with Female Newborns." Biomedicines 9, no. 2 (February 9, 2021): 170. http://dx.doi.org/10.3390/biomedicines9020170.
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Understanding the early events involved in the induction of immune tolerance to harmless environmental antigens and microbiota compounds could reveal potential targets for allergic disease therapy or prevention. Regulatory T cells (Treg), particularly induced Treg (iTreg), are crucial for the induction and maintenance of tolerance against environmental antigens including allergens. A decrease in the number and/or function of Treg or iTreg could represent an early predictor of allergy development. We analyzed proportional and functional properties of Treg in the cord blood of children of allergic mothers (neonates at high risk of allergy development) and healthy mothers (neonates with relatively low risk of allergy development). We observed a higher number of induced Treg in the cord blood of females compared to males, suggesting an impaired capacity of male immunity to set up tolerance to allergens, which could contribute to the higher incidence of allergy observed in male infants. The decreased proportion of iTreg in cord blood compared with maternal peripheral blood documents the general immaturity of the neonatal immune system. We observed a positive correlation in the demethylation of the Treg-specific demethylated region (TSDR) and the proportion of Treg in cord blood. Our data suggest that immaturity of the neonatal immune system is more severe in males, predisposing them to increased risk of allergy development.
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Gao, Yu-Lei, Yan-Fen Chai, An-Long Qi, Ying Yao, Yan-Cun Liu, Ning Dong, Li-Jun Wang, and Yong-Ming Yao. "Neuropilin-1highCD4+CD25+ Regulatory T Cells Exhibit Primary Negative Immunoregulation in Sepsis." Mediators of Inflammation 2016 (April 27, 2016): 1–11. http://dx.doi.org/10.1155/2016/7132158.
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Regulatory T cells (Tregs) appear to be involved in sepsis-induced immune dysfunction; neuropilin-1 (Nrp-1) was identified as a surface marker for CD4+CD25+Tregs. In the current study, we investigated the negative immunoregulation of Nrp-1highCD4+CD25+Tregs and the potential therapeutic value of Nrp-1 in sepsis. Splenic CD4+CD25+Tregs from cecal ligation and puncture (CLP) mouse models were further segregated into Nrp-1highTregs and Nrp-1lowTregs; they were cocultured with CD4+CD25− T cells. The expression of forkhead/winged helix transcription factor-3 (Foxp-3), cytotoxic T-lymphocyte associated antigen-4 (CTLA-4), membrane associated transforming growth factor-β (TGF-βm+), apoptotic rate, and secretive ability [including TGF-β and interleukin-10 (IL-10)] for various types of Tregs, as well as the immunosuppressive ability of Tregs on CD4+CD25− T cells, were determined. Meanwhile, the impact of recombinant Nrp-1 polyclonal antibody on the demethylation of Foxp-3-TSDR (Treg-specific demethylated region) was measured in in vitro study. Sepsis per se markedly promoted the expression of Nrp-1 of CD4+CD25+Tregs. Foxp-3/CTLA-4/TGF-βm+ of Nrp-1highTregs were upregulated by septic challenge. Nrp-1highTregs showed strong resilience to apoptosis and secretive ability and the strongest immunosuppressive ability on CD4+CD25− T cells. In the presence of lipopolysaccharide (LPS), the recombinant Nrp-1 polyclonal antibody reduced the demethylation of Foxp-3-TSDR. Nrp-1highTregs might reveal primary negative immunoregulation in sepsis; Nrp-1 could represent a new potential therapeutic target for the study of immune regulation in sepsis.
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Costantini, Benedetta, Shahram Kordasti, Marc Martinez-Llordella, Giovanni Povoleri, Judith C. W. Marsh, Frederic Toulza, and Ghulam J. Mufti. "Towards the Potential Use of In Vitro Expanded Regulatory T-Cells (Tregs) in Aplastic Anemia (AA): Opportunities for Therapy." Blood 128, no. 22 (December 2, 2016): 2673. http://dx.doi.org/10.1182/blood.v128.22.2673.2673.
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Abstract Introduction Severe aplastic anemia (SAA) is an immune mediated bone marrow failure syndrome characterized, among other features, by reduced numbers and dysfunctional regulatory T cells, as shown by our group. Using mass cytometry, we identified an immune signature that predicts clinical response toimmunosuppression (IST)consisting of two Treg subsets (A and B), differentially expressing CD45RA, CD95, CD7, CD28, and CCR4(Kordasti Blood 2016). Treg B are more suppressive and enriched in cell cycle related proteins, suggesting their proliferative potential. The aim of this study was to investigate the in vitro expandability of Treg subpopulations as well as their function, stability, plasticity, clonality and gene signature following expansion with the aim of using autologous in vitro expanded Tregs as a therapeutic option for AA patient. Methods and results Treg expansion, phenotype, and function Total Tregs from AA and HDs are sensitive to low dose IL-2 as shown by STAT5 phosphorylation (p < 0.001, n = 6 AA and 2 HDs). Moreover Tregs from AA and HDs can be expanded at a comparable rate (fold increase 33x vs 21x, p = n.s. n = 6 AA and 8 HDs) in a Treg promoting culture (stimulation with anti CD3/CD28 beads and IL-2 1,000 IU/ml for 4 weeks with all-trans retinoic acid and rapamycin). The expanded total Tregs have the phenotype of Treg B. To investigate the potential differences between Treg A and B in terms of function and expandability, Treg A (CD4+CD25hiCD127loCD45RAhiCD95-CCR4lo) and B (CD4+CD25hiCD127loCD45RAloCD95+CCR4hi) were sorted from 6 HDs and expanded for 4 weeks (as above). On average Treg A and B expanded equally (Treg A 45x, and B 33x fold increase, p = n.s.). After expansion Treg A up-regulate CD95 expression (p = 0.005) similar to Treg B phenotype. Expanded Treg A and B both suppress proliferation of autologous Tconv (n = 3, p < 0.001) as well as the IFN-g and TNF-a secretion. Only Treg B suppressed IL-17A secretion (n = 3, p < 0.05) (figure 1). Treg-specific demethylated region (TSDR) Treg stability was assessed by DNA methylation analysis by deep ampliconbisulphitesequencing of the TSDR. TSDR CpG sites in expanded HDs and AA Tregs are > 98% unmethylated, confirming their stability. Interestingly, TSDR sites were less methylated in Treg B compared to Treg A (5% v 35%), but it decreases to a similar level as Treg B after expansion. Treg plasticity after expansion To investigate the plasticity, total Tregs, Treg A, and Treg B were cultured for 5 days in a Th17 polarizing culture (containing IL-1b,IL-6, IL-2). After 5 days of culture, only total Tregs were secreting IL-17A (p = 0.013), but neither Treg A, nor Treg B. Despite IL-17A secretion, Tregs were still able to suppress autologous Tconv proliferation (n = 3, p= 0.001). T cell receptor diversity To track the clonal origin of Treg A and B, we performed amplification and sequencing of CDR3 using immunoSEQ Platform, as already described. Our results show that both AA and HDs expanded Tregs show a comparable level of TCR VbCDR3 diversity (average clonality score 0.12); moreover Tregs, Treg A, and Treg B are polyclonal, pre and post expansion (clonality score 0.01 for all the subsets). Finally, Treg A and B do not share any TCR sequence and do not have any overlap in their TCR repertoire, suggesting they originate from different clones. Gene expression Expanded Tregs (total, A and B) were compared to established human Treg gene signature (Ferraro PNAS 2014). Treg B aremore enriched in Treg genes compared Treg A (Tregs B vs A false discovery rate (FDR) < 0.0001). IL-7 and CD80 are among the top 5 expressed genes in all 3 subpopulations, which are important for Tregs' motility and homeostasis. Conclusions In summary, AA Tregs can be expanded in vitro and expanded Tregs are more similar to Treg B, which are lacking in SAA as shown before. Although we were not able to sort Treg subpopulations from AA patients due to very low numbers, the expanded Treg A and B from HDs demonstrate a functional Treg profile with minimal plasticity. Interestingly the main difference between Treg A and B is the ability of Treg B to suppress IL-17A secretion. This is important, as IL-17 plays a crucial role in AA pathophysiology (Peffault de Latour Blood 2010). Overall, the current study suggests that in vitro expansion of AA Tregs is feasible. The expanded Tregs show the phenotype of the most functional Treg subset and likely to control the inflammatory response inAA, hence pave the way toward a novel cellular therapy for SAA. Disclosures Marsh: Alexion pharmaceuticals: Honoraria.
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APPEL, HEINER, PEIHUA WU, REBECCA SCHEER, CLAUDIA KEDOR, BIRGIT SAWITZKI, ANDREAS THIEL, ANDREAS RADBRUCH, JOACHIM SIEPER, and UTA SYRBE. "Synovial and Peripheral Blood CD4+FoxP3+ T Cells in Spondyloarthritis." Journal of Rheumatology 38, no. 11 (September 15, 2011): 2445–51. http://dx.doi.org/10.3899/jrheum.110377.
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Objective.Regulatory T cells are characterized by expression of the transcription factor FoxP3 and are thought to be involved in the pathogenesis of autoimmune diseases. We determined the frequency and phenotypic characteristics of CD4+FoxP3+ T cells in the blood and synovial fluid (SF) of patients with inflammatory joint diseases.Methods.SF from 10 patients with ankylosing spondylitis (AS), 20 patients with other spondyloarthritides or with peripheral arthritis (pSpA), and 12 patients with rheumatoid arthritis (RA), and peripheral blood (PB) from 22 patients with AS, 19 with pSpA, 15 with RA, and 12 healthy controls were stained for CD4, FoxP3, CD25, and CD127 and different effector cytokines and then analyzed by flow cytometry. Methylation pattern of the Treg-specific demethylated region (TSDR) was determined after bisulfite treatment by quantitative polymerase chain reaction.Results.In all groups of patients we observed higher frequencies of Foxp3+ cells/CD4+ T cells within SF compared to PB. The frequency of synovial Foxp3+ cells/CD4+ T cells was significantly higher in patients with pSpA (18.79% ± 6.41%) compared to patients with AS (9.69% ± 4.11%) and patients with RA (5.95% ± 2.21%). CD4+FoxP3+ T cells were CD25+ and CD127− and lacked effector cytokine production in any of the different patient groups. The majority of the CD4+CD25+CD127− T cells showed demethylation of the TSDR within the Foxp3 locus, confirming its regulatory phenotype.Conclusion.Our data show accumulation of Foxp3+ T cells within inflamed joints. These Foxp3+ T cells are mainly of stable T regulatory phenotype. The high frequency of Foxp3+ T cells in pSpA might contribute to the spontaneous resolution and remitting course of arthritis in pSpA as compared to the more persistent joint inflammation in RA.
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Azevedo, Rita I., Ekaterina Minskaia, Cláudia Lobato da Silva, Ana Fernandes Platzgummer, Ana I. S. Vieira, Joaquim M. S. Cabral, and Joao F. Lacerda. "Epigenetic Profile of Treg-like Cells Induced By Mesenchymal Stem Cells in Vitro Resembles That of Natural Treg." Blood 132, Supplement 1 (November 29, 2018): 2578. http://dx.doi.org/10.1182/blood-2018-99-112452.
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Abstract 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. Disclosures No relevant conflicts of interest to declare.
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Suh, Koung Jin, Jin Won Kim, Ji Eun Kim, Ji Hea Sung, Jiwon Koh, Kui-Jin Kim, Ji-Won Kim, et al. "Correlation between tumor infiltrating immune cells and peripheral regulatory T cell determined using methylation analyses and its prognostic significance in resected gastric cancer." PLOS ONE 16, no. 6 (June 4, 2021): e0252480. http://dx.doi.org/10.1371/journal.pone.0252480.
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Peripheral regulatory T cells (pTregs) are a highly immunosuppressive fraction of CD4+ T cells. We aimed to evaluate the clinical significance of pTregs in patients with gastric cancer and to determine the correlation between pTregs and immune cell infiltration in tumor microenvironment. pTregs status was determined by assessing the pTreg/total T-cell ratio (ratio of Foxp3 Treg-specific demethylated region (TSDR) to CD3G/CD3D demethylation, so-called Cellular Ratio of Immune Tolerance “ImmunoCRIT”) using methylation analyses in 433 patients with gastric cancer who received curative surgery. Among 422 evaluable patients, 230 (54.5%) had high ImmunoCRIT (> 21.0). Patients with high ImmunoCRIT had significantly shorter disease-free survival (DFS) and overall survival (OS) than those with high ImmunoCRIT (p = 0.030, p = 0.008, respectively). In multivariate analysis, high ImmunoCRIT kept a prognostic role for shorter OS (hazard ratio [HR] 1.9, 95% confidence interval [CI] 1.4–2.9; p = 0.005). CD3+ cell density and CD4+ cell density was significantly higher within the tumor in high ImmunoCRIT group than those in low ImmunoCRIT group (CD3+ cell, 202.12/mm2 vs. 172.2/mm2, p = 0.029; CD4+ cell, 56.5/mm2 vs. 43.5/mm2, p = 0.007). In conclusion, the peripheral ImmunoCRIT determined by epigenetic methylation analysis provides prognostic information in resected gastric tumors.
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Suh, Koung Jin, Jin Won Kim, Ji Eun Kim, Jiwon Koh, Ji-Won Kim, Hye Seung Lee, and Keun-Wook Lee. "Correlation between tumor infiltrating immune cells and peripheral regulatory T cell determined by methylation analyses and its prognostic significance in gastric cancer." Journal of Clinical Oncology 36, no. 4_suppl (February 1, 2018): 66. http://dx.doi.org/10.1200/jco.2018.36.4_suppl.66.
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66 Background: Peripheral regulatory T cells (pTregs) might involve in tumor immune microenvironment. We aim to evaluate the correlation between pTregs and tumor immune microenvironment. Methods: pTregs status was determined from assessment of the pTreg/total T cell ratio (ratio of Foxp3 Treg-specific demethylated region (TSDR) to CD3G/CD3D demethylation) through epigenetic pattern of bisulfite pyrosequencing in long-term stored peripheral blood of 442 gastric cancer patients who received curative surgery. Immunohistochemical staining of multiple immune-related markers including CD3, CD4, CD8, Foxp3, 1-selectin arginase, ADAM metallopeptidase domain 17, CD163 and CD45RO within tumor microenvironment were performed in resected gastric tumor specimen. Results: The median value of FoxP3-TSDR and CD3G/CD3D demethylation was 5.8% and 32.3%, respectively. When pTreg/total T cell ratio was divided into eight equal groups from the lowest to the highest value, the extreme pTreg/total T cell ratio of the upper eighth and the lower eighth was significantly associated with lower CD45RO expressed cell counts within tumor microenvironment. In terms of arginase and CD163, inverse results were observed. Patients with extreme pTreg/total T cell ratio had significantly shorter disease-free survival (DFS) and overall survival (OS) compared to the patients with non-extreme pTreg/total T cell ratio. Multivariate analysis which included age, stage, lymphatic invasion, vascular invasion, and perineural invasion as covariates demonstrated that the extreme pTreg/total T cell ratio was an independent predictor for shorter DFS (HR 1.740, 95% CI 1.128 – 2.682; p = 0.012) and OS (HR 1.900, 95% CI 1.175 – 3.070; p = 0.003). Conclusions: Our results suggest that the pTreg/total T cell ratio determined by epigenetic methylation analysis is correlated with specific immune cell infiltration within tumor microenvironment in resected gastric tumors, and gives prognostic information in gastric cancer patients. pTreg/total T cell ratio could be an easy-obtained potential biomarker for prognosis and future immunotherapeutic treatment strategies.
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Choi, Yoon Seok, Jeewon Lee, Ik-Chan Song, Deog-Yeon Jo, and Eui-Cheol Shin. "Lineage-Plasticity and Inflammatory Change of Human FoxP3+ Regulatory T Cells in Acute Viral Infection: Implication in Immune-Mediated Tissue Injury." Blood 124, no. 21 (December 6, 2014): 4132. http://dx.doi.org/10.1182/blood.v124.21.4132.4132.
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Abstract FoxP3+ CD4+CD25hi regulatory T (Treg) cells play a major role in maintaining the immune homeostasis by preventing the activation of self-reactive T cells as well as in controlling a series of immune responses in viral infections. Recent studies suggest that lineage-commitment of CD4+T cells, including Treg cells, is not a fixed fate, rather a status with a wide range of plasticity. Functional changes and lineage-plasticity of Treg cells during acute viral infection, especially of human, have not been reported so far. Herein, we investigated whether Treg cells show the functional plasticity and whether such changes can affect the regulation of immunopathology in a human acute viral infection. As a model of human acute viral infection, we used a cohort of patients with acute hepatitis A (AHA), since tissue (liver) injury in AHA is mediated exclusively by activated T cells, not by direct cytopathic effect of virus. To assess the plasticity of Treg cell lineage, first, we examined the production of a variety of inflammatory cytokines from Treg cells following T cell receptor (TCR) stimulation of peripheral blood lymphocytes with anti-CD3/CD28 antibody, using intracellular cytokine staining and multicolor flow cytometry. We found that a significant proportion of FoxP3+ CD4+CD25hi Treg cells produced TNF-α following TCR stimulation in patients with AHA, but not in heathy subjects. Analyses at multiple time points during the course of infection showed that TNF-α production from Treg cells decreased in convalescent phase. Likewise, we observed that liver-infiltrating Treg cells also produced TNF-α after TCR stimulation. Moreover, highly-purified CD4+CD25hiCD127lo/-Treg cells could also produce TNF-α following TCR stimulation, indicating that Treg cells of AHA patients can produce TNF-α in direct response to TCR stimulation. Next, to exclude the possibility that TNF-α might be secreted from transiently FoxP3-expressing activated non-Treg CD4+ T cells, we examined the expression level of CD127 on TNF-α-secreting FoxP3+ CD4+ T cells. TNF-α+ Treg cells expressed CD127 in the level similar to conventional TNF-α- counterpart, and CD127 expression levels of both Treg populations were much lower than FoxP3- CD4+ T cells. Furthermore, DNA methylation analysis of Treg cell-specific demethylated region (TSDR) after sorting TNF-α+ Treg cells revealed completely demethylated pattern in highly conserved CpG island of FOXP3 gene. These findings support that TNF-α is produced from bona fide Treg cells, not from FoxP3-expressing activated non-Treg CD4+T cells. In analysis of immunophenotypes, TNF-α+ Treg cells were enriched in CD45RA-FoxP3lo population, implying their reduced in vivo suppressive activity. Along with the lower level of FoxP3, TNF-α+ Treg cells showed lower level of CD39 expression, a surrogate marker of Treg cell suppressive activity, compared to TNF-α- Treg cells. Furthermore, TNF-α+Treg cells showed a robust evidence of lineage-plasticity toward Th17 lineage, expressing a key transcription factor RORγt. Consistently, they expressed CCR6 and co-produced IL-17A following TCR stimulation, which are the hallmark of Th17 effector function. To analyze the clinical implication of attenuate suppressive function and plasticity shown by TNF-α+ Treg cells, we examined correlation between production of proinflammatory cytokines from Treg cells and severity of liver damage in AHA. As a result, proportion of TNF-α-producing Treg cells closely and linearly correlated with severity of liver damage, suggesting the critical role of TNF-α+ Treg cells in the immunopathogenesis of AHA. However, Treg cell suppression assay in the absence or presence of anti-TNF-α antibody showed that Treg cell suppressive function was not affected by TNF-α blockade. This indicates that attenuated function of TNF-α+Treg cells is not attributed simply to production of a kind of inflammatory cytokine, rather to more complicated reprogramming mechanism. Taken together, these data provide a clear evidence of attenuated suppressive activity and Th17-toward lineage plasticity of FoxP3+ Treg cells, represented by TNF-α production, in a human acute viral infection. Also, we suggest one possible mechanism that lineage plasticity and inflammatory changes of Treg cells could be implicated in the immunopathogenesis of human diseases. Disclosures No relevant conflicts of interest to declare.
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Costantini, Benedetta, Shahram Kordasti, Seidl Thomas, Smith Alexander, Mian Syed, Aytug Kizilors, Martinez-Llordella Marc, Judith C. Marsh, and Ghulam J. Mufti. "Autologous Ex-Vivo Regulatory T Cells(Tregs) Expansion in Aplastic Anemia for Potential Clinical Use." Blood 126, no. 23 (December 3, 2015): 3617. http://dx.doi.org/10.1182/blood.v126.23.3617.3617.
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Abstract Introduction Most cases of acquired idiopathic severe aplastic anemia (SAA) are immune mediated. Following an insult which is likely to be viral, there is an increase in auto-reactive oligoclonal T-cells that result in apoptotic death of stem and progenitor cells, due to lack or absence of regulatory T-cells (Tregs) and a pro-inflammatory environment with clonal expansion of Th1 cells and increased Th17 cells. We have previously shown a correlation between low Treg numbers and disease severity, and that Tregs are not only reduced in number but are also unable to effectively suppress T effectors (Teff)1. The only curative treatment option for SAA is hematopoietic stem cell transplantation (HSCT) but this option is not accessible to everyone because of lack of donor or unfitness for the procedure. Moreover, patients who fail standard immunosuppressive treatment (ATG and Cyclosporine A) have limited alternative therapeutic options. The aim of this study was to investigate the expandability of Tregs in AA, their subsequent function and stability following expansion. Such data may provide the rationale for a novel future approach to treatment of refractory SAA. Methods and results IL-2 sensitivity assay One of the hallmarks of response to IL-2 by Tregs is STAT5 phosphorylation. To evaluate AA Tregs response to IL-2, freshly isolated cells from 6 AA patients were cultured in the presence of IL-2 (ranging from 0.1 to 1000 IU/ml). STAT5 phosphorylation was assessed by intracellular staining for pSTAT5 after 15 and 30 minutes. Tregs from AA patients have shown an increase in STAT5 phosphorylation similar to 2 healthy donors (HD) Tregs, confirming their responsiveness to IL-2. Tregs expansion Peripheral blood mononuclear cells (PBMCs) were obtained from six AA patients and eight HDs and enriched for Tregs using a magnetic beads regulatory T cells isolation kit according to the manufacturer instructions. Tregs were then stimulated with anti CD3/CD28 beads (1:1 ratio) and high dose IL-2 (1,000 IU/ml) for four weeks in a "Treg friendly" culture with all-trans retinoic acid 2 mM and rapamycin 100 nM. After four weeks of culture, expanded Tregs were then 'rested' for 5 days with decreasing doses of IL-2. Tregs from AA patients were expanded in a comparable rate to HD Tregs, with a median fold increase of 33 (range 29-149) compared to 21 fold increase (range 8-36) in HD. There was no significant difference between AA and HDs in terms of fold increase after 4 weeks. The expanded Tregs demonstrated more than 90% FoxP3+ expression in both AA and HDs. To assess IL-2 dependency of the expanded Tregs, Tregs were gradually deprived of IL-2 following expansion and STAT5 phosphorylation evaluated in both AA and HDs Tregs. Although expanded AA Tregs showed slightly lower pSTAT5 level following IL-2 deprivation, they responded to low dose IL-2 and pSTAT5 returned to similar level as in HDs. Expanded Tregs function and methylation status of TSDR To assess the suppressive activity of expanded Tregs, Teff were stained with a fluorescent proliferation dye (CellTrace Violet, Life Technologies) and co-cultured with autologous expanded Tregs (1:1 ratio) for 5 days in the presence of anti CD3/CD28 beads (Teff:Treg:beads = 20:20:1). The expanded Tregs from AA patients suppressed the proliferation of both autologous and allogeneic CD4+ T effectors (43% versus 5%, respectively, p = 0.009) and the suppressive function of expanded AA Tregs was not significantly different from expanded HD Tregs. In order to assess the stability of our Tregs we investigated the methylation status of 15 CpG sites within the Foxp3 Treg-specific demethylated region (TSDR) by amplicon sequencing of bisulfite treated DNA on an Illumina MiSeq instrument. The TSDR CpG sites in expanded Tregs from HDs and AA patients were found to be >98% unmethylated (no significant difference between AA and HD), which confirms the stability of expanded Tregs. Conclusions Our data show that Tregs from AA patients can be expanded efficiently ex vivo. Importantly, expanded Tregs are functional and stable with similar phenotypic characteristics to HD Tregs. These results may lead to the development of a novel treatment option for patients who fail/relapse after standard immunosuppression and for those who are not eligible for HSCT. References 1. Kordasti, S., et al. Functional characterization of CD4+ T cells in aplastic anemia. Blood119, 2033-2043 (2012). Disclosures Mufti: Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
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Fan, Huahua, Xiaona Huo, Juan Sun, Yiming Yang, and Xiao Li. "Efficient Induction and Expansion Of CD8+CD28+Foxp3+ Regulatory T Cells By TGF-beta1 and Rapamycin." Blood 122, no. 21 (November 15, 2013): 190. http://dx.doi.org/10.1182/blood.v122.21.190.190.
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Abstract Background Although extensive studies have focused on the CD4+CD25+Foxp3+Tregs in recent years, CD8+Tregs have also been reported to play important roles in maintenance of immune tolerance. Adoptive transfer of CD8+Tregs in rodents can prevent or treat autoimmune diseases, allograft rejection or graft-versus-host disease. Objective Several approaches for induction of Ag-specific CD8+Tregs have been reported, but there is currently no reliable protocol for the ex vivo induction and large-scale expansion of human polyclonal CD8+CD28+Foxp3+Tregs. Our research was designed to investigate an effective method to induce and expand polyclonal CD8+CD28+Foxp3+Tregs in vitro on a large scale to meet the clinical demand. Methods CD8+ T lymphocytes were isolated from adult peripheral blood with immunomagnetic beads and cultured for a week using anti-CD3/CD28 antibody-coated beads and IL-2 in combination with TGF-beta1 and rapamycin. Expanded CD8+Tregs were re-stimulated with fresh anti-CD3/CD28 antibody-coated beads and cytokines for other cycles. The phenotype of CD8+Tregs was analyzed by flow cytometry. In the suppression assay, CFSE progressive dilution was used as readout of responder cells proliferation. For in vivo experiments, the mouse models of collagen-induced arthritis (CIA) were established. 2x10E6 ex vivo induced and expanded human CD8+CD28+Foxp3+Tregs were transferred into CIA recipient mouse with the onset of RA. Clinical and histopathologic scores, cytokine and the secretion of anti-type-two collagen antidody in serum were analyzed. Results A large number of CD8+CD28+Foxp3+Tregs could be efficiently induced and expanded from CD8+ T lymphocytes in polyclonal stimulation culture system added TGF-beta1 and rapamycin. By repeated stimulation of CD8+ T cells for 4 weeks, we could generate larger than 1x10E10 CD8+CD28+Foxp3+Tregs without loss of their suppressive function from every 1x10E6 CD8+ T cells, which can typically be isolated from 5-10ml of peripheral blood. The expanded CD8+Tregs expressed high level of Foxp3, CD28, CD25, PD-1, CD103, CD62L, CCR7, CTLA4, CD39 and CD73, secreted small amount of IL-2, IFN-gamma, IL-10 and TGF-beta, did not secret IL-17A, displayed a partially anergic phenotype, and could inhibit the proliferation of autologous or allogeneic CD4 effective T lymphocytes activated by anti-CD3/CD28 antibody. We found that the suppression of CD8+CD28+Foxp3+Tregs in proliferation of effective T lymphocytes was mainly dependent on cell contact but not dependent on their cytotoxicity. CD8+CD28+Foxp3+Tregs did not secret IL17A or secret small amount of IFN-gamma in the presence of inflammatory cytokines such as IL-1beta and IL-6 or IL-21 and IL-23. Furthermore, CD8+CD28+Foxp3+Tregs are a kind of induced regulatory T cells, because their Treg-cell-specific demethylated region (TSDR) has a methylation status. CD8+CD28+Foxp3+Tregs treatment could significantly alleviate the severity of arthritis, and reduce the cartilage destruction of the joints of CIA mice and the level of total anti-type-two collagen IgG antibody in serum as well. Conclusion we have developed a simple method using TGF-beta1 and rapamycin to induce and expand highly efficient human polyclonal CD8+CD28+Foxp3+Tregs with suppressive capacities from CD8+ T cells on a large scale. This procedure of CD8+Tregs expansion we used fits with the currently available clinical grade isolation tools with the objective of facilitating easy translation into clinical practice. This approach may facilitate the clinical application of CD8+Treg-based immunotherapy in transplantation and autoimmune diseases. Disclosures: No relevant conflicts of interest to declare.
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Roncarolo, Maria Grazia, Manuela Battaglia, Rosa Bacchetta, Megan Levings, and Silvia Gregori. "T-Cell-Mediated Suppression: From Bench to Bedside." Blood 122, no. 21 (November 15, 2013): SCI—37—SCI—37. http://dx.doi.org/10.1182/blood.v122.21.sci-37.sci-37.
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Abstract T regulatory cells (Tregs) play a pivotal role in promoting and maintaining tolerance. Several subsets of Tregs have been identified but, to date, the best characterized are the CD4+FOXP3+ Tregs (FOXP3+Tregs), thymic-derived or induced in the periphery, and the CD4+ IL-10-producing T regulatory type 1 (Tr1) cells. In the past decade much effort has been dedicated to develop methods for the in vitro induction and expansion of FOXP3+Tregs and of Tr1 cells for Treg-based cell therapy to promote and restore tolerance in T-cell mediated diseases, and for expanding antigen (Ag)-specific Tregs in vivo. FOXP3+Tregs constitutively express high levels of CD25 and of the transcription factor FOXP3. FOXP3+Tregs are distinguished from activated CD4+ T cells by the low expression of CD127, and by the DNA demethylation of a specific region of the FOXP3 gene called Treg-specific demethylated region (TSDR). FOXP3+Tregs suppress effector T-cell responses through cell-to-cell contact-dependent mechanisms and suppression requires activation via TCR and is IL-2 dependent. In vitro protocols to expand FOXP3+Tregs for adoptive transfer in vivo have been established. We demonstrated that rapamycin permits the in vitro expansion of FOXP3+Tregs while impairing the proliferation of non-Tregs. Moreover, rapamycin-expanded FOXP3+Tregs maintain their regulatory phenotype in a proinflammatory environment and Th17 cells do not expand in the presence of rapamycin. Despite the progress in FOXP3+Tregs expansion protocols, adoptive transfer of FOXP3+Tregs in humans remains a difficult experimental procedure due to the ability to expand a sufficient number of Ag-specific FOXP3+Tregs in vitro. To propagate a homogenous population of FOXP3+Tregs we developed a lentiviral vector (LV)-based strategy to ectopically express FOXP3 in CD4+ T cells. This method results in the development of suppressive cells that are super-imposable to FOXP3+Tregs. Conversion of effector T cells into FOXP3+Tregs upon LV-mediated gene transfer of wild-type FOXP3 was also obtained in CD4+T cells from immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) patients. We also developed a LV platform, which selectively targets expression of the transgene in hepatocytes, to induce tolerance to self or exogenous Ags. Using this approach we showed that systemic administration of LV encoding for the gene of interest leads to the induction of Ag-specific FOXP3+ Tregs, which mediate tolerance even in pre-immunized hosts. Tr1 cells are identified by their cytokine profile (IL-10+TGF-b+IL-4-IL-17-). Tr1 cells express transiently FOXP3 upon activation; but FOXP3 expression never reaches the high levels characteristic of FOXP3+Tregs. Tr1 cell differentiation and function is independent of FOXP3 since suppressive Tr1 cells can be isolated or generated from peripheral blood of IPEX patients. Tr1 cells were first discovered in peripheral blood of patients who developed tolerance after HLA-mismatched fetal liver hematopoietic stem cell transplant (HSCT). Since their discovery, Tr1 cells have proven to be important in mediating tolerance in several immune-mediated diseases. The immuno-regulatory mechanisms of Tr1 cells have been studied over the years thanks to the possibility to generate these cells in vitro. Tr1 cells suppress T-cell responses via the secretion of IL-10 and TGF-β and by the specific killing of myeloid APC via Granzyme B and perforin. Tr1 cells can be induced in vitro in an Ag-specific manner in the presence of IL-10 or of DC-10. Proof-of-principle clinical trials in allogeneic HSCT demonstrated the safety of Treg-based cell therapy with these polarized Tr1 cells. We are currently planning a phase I/II trial using in vitro polarized Tr1 cells with DC-10 in patients after kidney transplantation. An alternative strategy for the induction of high numbers of human Tr1 cells is the LV-mediated gene transfer of human IL-10 into conventional CD4+ T cells. Stable ectopic expression of IL-10 leads to the differentiation of homogeneous populations of Tr1-like cells displaying potent suppressive functions both in vitro and in vivo. A major hurdle, which limited the studies and the clinical use of Tr1 cells, was the lack of specific biomarkers. By gene expression profiling of human Tr1 cell clones we identified two surface markers (CD49b and LAG-3), which are stably and selectively co-expressed on murine and human Tr1 cells induced in vitro or in vivo. The co-expression of CD49b and LAG-3 enables the isolation of highly suppressive Tr1 cells from in vitro IL-10-polarized Tr1 cells and allows tracking of Tr1 cells in peripheral blood of patients who developed tolerance after allogeneic HSCT. The identification of CD49b and LAG-3 as Tr1-specific biomarkers will facilitate the study of Tr1 cells in vivo in healthy and pathological conditions and the use of Tr1 cells for forthcoming therapeutic interventions. In conclusion, Tregs play a key role in maintaining immunological homeostasis in the periphery. Several open questions regarding FOXP3+ Tregs or Tr1 cell-based therapy in humans remain: how long do Tregs survive after transfer? Is their phenotype stable in pathological conditions and inflammatory environments? Is their mechanism of suppression in vivo Ag-specific? Carefully designed and standardized future clinical protocols reflecting a concerted action among different investigators will help to address these questions and to advance the field. Disclosures: No relevant conflicts of interest to declare.
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Schreiber, Lisa, Beate Pietzsch, Stefan Floess, Carla Farah, Lothar Jänsch, Ingo Schmitz, and Jochen Huehn. "The Treg-Specific Demethylated Region Stabilizes Foxp3 Expression Independently of NF-κB Signaling." PLoS ONE 9, no. 2 (February 5, 2014): e88318. http://dx.doi.org/10.1371/journal.pone.0088318.
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Aurich, Christine, Jürgen Weber, Christina Nagel, Maximiliane Merkl, Rony Jude, Sascha Wostmann, Dirk Ollech, Udo Baron, Sven Olek, and Thomas Jansen. "Low levels of naturally occurring regulatory T lymphocytes in blood of mares with early pregnancy loss." Reproduction, Fertility and Development 26, no. 6 (2014): 827. http://dx.doi.org/10.1071/rd13012.
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Early pregnancy loss is a major reason for low reproductive efficiency in the horse. In humans and mice, low numbers of regulatory T cells (Treg cells) are linked to miscarriage. The percentage of Treg cells in oestrous mares at the start of the breeding season was evaluated in relation to the outcome of subsequent pregnancy. For identification and quantification of Treg cells, a highly sensitive and specific qPCR assay targeting the Treg-specific demethylated region in the equine forkhead box transcription factor (FOXP3) gene was established. In a total of 108 mares, pregnancy was followed until detection of early pregnancy loss (n = 17), abortion without identification of an infectious or apparent cause (n = 9) or birth of a viable foal (n = 82). Measured Treg-cell levels did not significantly differ between mares that conceived (82%; 1.50 ± 0.04%) or did not get pregnant (18%; 1.45 ± 0.10%). The Treg-cell percentage at oestrus before breeding was significantly different (P < 0.05) between mares that either underwent early pregnancy loss up to Day 40 of pregnancy (1.29 ± 0.07%) and mares that aborted (1.61 ± 0.15%) or gave birth to a live foal (1.52 ± 0.05%). These results suggest that low levels of Treg cells in mares can contribute to pregnancy loss up to Day 40 after ovulation.
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Liu, Hao-Run, and Wei-Min Li. "Treg-specific demethylated region activity in isolated regulatory t lymphocytes is a surrogate for disease severity in hepatocellular carcinoma." IUBMB Life 67, no. 5 (April 23, 2015): 355–60. http://dx.doi.org/10.1002/iub.1378.
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Rossetti, Maura, Roberto Spreafico, Alessandro Consolaro, Jing Yao Leong, Camillus Chua, Margherita Massa, Suzan Saidin, et al. "TCR repertoire sequencing identifies synovial Treg cell clonotypes in the bloodstream during active inflammation in human arthritis." Annals of the Rheumatic Diseases 76, no. 2 (June 16, 2016): 435–41. http://dx.doi.org/10.1136/annrheumdis-2015-208992.
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ObjectivesThe imbalance between effector and regulatory T (Treg) cells is crucial in the pathogenesis of autoimmune arthritis. Immune responses are often investigated in the blood because of its accessibility, but circulating lymphocytes are not representative of those found in inflamed tissues. This disconnect hinders our understanding of the mechanisms underlying disease. Our goal was to identify Treg cells implicated in autoimmunity at the inflamed joints, and also readily detectable in the blood upon recirculation.MethodsWe compared Treg cells of patients with juvenile idiopathic arthritis responding or not to therapy by using: (i) T cell receptor (TCR) sequencing, to identify clonotypes shared between blood and synovial fluid; (ii) FOXP3 Treg cell-specific demethylated region DNA methylation assays, to investigate their stability and (iii) flow cytometry and suppression assays to probe their tolerogenic functions.ResultsWe found a subset of synovial Treg cells that recirculated into the bloodstream of patients with juvenile idiopathic and adult rheumatoid arthritis. These inflammation-associated (ia)Treg cells, but not other blood Treg cells, expanded during active disease and proliferated in response to their cognate antigens. Despite the typical inflammatory-skewed balance of immune mechanisms in arthritis, iaTreg cells were stably committed to the regulatory lineage and fully suppressive. A fraction of iaTreg clonotypes were in common with pathogenic effector T cells.ConclusionsUsing an innovative antigen-agnostic approach, we uncovered a population of bona fide synovial Treg cells readily accessible from the blood and selectively expanding during active disease, paving the way to non-invasive diagnostics and better understanding of the pathogenesis of autoimmunity.
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Zhu, Ling, Lei Jia, Zhongwei Liu, Yong Zhang, Junkui Wang, Zuyi Yuan, and Rutai Hui. "Elevated Methylation of FOXP3 (Forkhead Box P3)-TSDR (Regulatory T-Cell–Specific Demethylated Region) Is Associated With Increased Risk for Adverse Outcomes in Patients With Acute Coronary Syndrome." Hypertension 74, no. 3 (September 2019): 581–89. http://dx.doi.org/10.1161/hypertensionaha.119.12852.
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Shimazu, Yayoi, Yutaka Shimazu, Masakatsu Hishizawa, Masahide Hamaguchi, Yuya Nagai, Noriko Sugino, Sumie Fujii, et al. "Hypomethylation of the Treg-Specific Demethylated Region in FOXP3 Is a Hallmark of the Regulatory T-cell Subtype in Adult T-cell Leukemia." Cancer Immunology Research 4, no. 2 (December 17, 2015): 136–45. http://dx.doi.org/10.1158/2326-6066.cir-15-0148.
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Sorathia, Nilofer, Hussein Al-Rubaye, and Benham Zal. "The Effect of Statins on the Functionality of CD4+CD25+FOXP3+ Regulatory T-cells in Acute Coronary Syndrome: A Systematic Review and Meta-analysis of Randomised Controlled Trials in Asian Populations." European Cardiology Review 14, no. 2 (July 11, 2019): 123–29. http://dx.doi.org/10.15420/ecr.2019.9.2.
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Acute coronary syndrome (ACS) is characterised by increased effector cells and decreased regulatory T-cells (Tregs). Statins have been shown to be clinically beneficial in ACS patients. This effect could be mediated via the induction of Tregs in ACS patients. The aim of this systemic review and meta-analysis was to evaluate whether statin therapy enhances the frequency of Tregs determined by CD4+CD25+FOXP3+ in this subset of patients. A comprehensive search of PubMed and Embase was performed. Studies were restricted to randomised controlled trials that quantified CD4+CD25+FOXP3+ cell frequency by flow cytometric analysis before and after statin treatment in adults diagnosed with ACS. A minimum of at least two of the conventional markers to identify Tregs was compulsory. Four randomised controlled trials studies (439 participants) were included, all with low-to-moderate risk of bias. Pooled data showed a significant increase in Treg frequency after statin therapy in ACS patients. A further meta-regression and subgroup analysis also showed a negative dose-related effect, and a statin type-related effect (rosuvastatin versus atorvastatin), respectively. The results confirmed that statins positively alter the frequency of Tregs, which may indicate a potential mechanism of their therapeutic effect. However, there was a risk of information bias due to the markers used to identify Tregs, which was not fully explored, therefore, further randomised controlled trials should utilise markers of Tregs, such as the FOXP3 locus (Treg-specific demethylated region), for identification.
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Takata, Katsuyoshi, Lauren C. Chong, Avinash Thakur, Tomohiro Aoki, Anja Mottok, Elizabeth Chavez, Pedro Farinha, et al. "PRAME Expression Is Correlated with Treatment Outcome and Specific Features of the Tumor Microenvironment in Classical Hodgkin Lymphoma." Blood 134, Supplement_1 (November 13, 2019): 1509. http://dx.doi.org/10.1182/blood-2019-129730.
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Background: The tumor-associated antigen PRAME is over-expressed in several types of cancer and is currently investigated as a therapeutic target for T-cell immunotherapy. Our previous integrative genomic study in diffuse large B-cell lymphoma (DLBCL) identified PRAME deletion to be correlated with patient outcome and an immunologically "cold" tumor microenvironment. However, it remains an open question whether PRAME expression significantly contributes to differential treatment outcomes and tumor microenvironment crosstalk across various B-cell lymphoma subtypes. Material and Methods: We performed an immunohistochemical (IHC) screen in a large cohort of B-cell lymphomas (de novo DLBCL; N=347, follicular lymphoma (FL); N= 166, mantle cell lymphoma (MCL); N= 180), and classical Hodgkin lymphoma (HL); N= 166) to assess PRAME expression as a prognostic biomarker. Moreover, to investigate PRAME-expression associated tumor microenvironment composition and function, we correlated PRAME IHC results with single cell RNA sequencing data of more than 127,000 cells from 22 HL tissue specimens. Results: PRAME IHC analysis revealed frequent PRAME over-expression in HL (115/166, 69%), followed by DLBCL (104/319, 33%), FL (13/166, 8%), and MCL (14/180, 8%). Interestingly, only HL showed a significant treatment outcome correlation, whereas other B-cell lymphoma subtypes did not. Specifically, using a previously published HL cohort (Steidl et al, NEJM 2010) PRAME-negative Hodgkin Reed Sternberg (HRS) cells indicated significantly shorter overall survival (P = 0.008) and disease-specific survival (P = 0.042 ). To characterize PRAME-specific microenvironment composition and function in HL, we analyzed T-, B-, NK-cell, and macrophage subsets in PRAME-positive (17 of 22 cases) vs -negative (5 of 22 cases) tumor samples using single cell RNA sequencing data. From 22 expression-based microenvironment cell clusters that were annotated and assigned to a cell type based on gene expression, all three CD4 helper T-cell clusters were de-enriched in PRAME-negative samples, and the CD4 non-Treg proportion was significantly lower in PRAME-negative samples (P = 0.049). Strikingly, when focusing on phenotypic features of cells within the CD4 non-Treg T-cell cluster, CXCL13 was identified as the most up-regulated gene in PRAME-negative samples. When interrogating published HRS cell transcriptome data (Steidl et al, Blood 2012), immune response pathways including chemokine receptors and chemokine ligands were up-regulated in PRAME-negative HRS cell samples. Of specific interest, CXCR5, the cognate receptor for CXCL13, was significantly upregulated as a member of the chemokine pathway (P = 0.0086) in PRAME-negative HRS cell samples. These results suggest that crosstalk between CXCL13 (produced in the microenvironment) and CXCR5 (expressed on HRS cells) contributes to tumor maintenance in PRAME-negative HL. Finally, to explore potential therapeutic approaches for PRAME-negative HL cells, we focused on 3 HL-derived cell lines (L540, L591, DEV) with low PRAME expression and exposed these lines to DNMT or HDAC inhibitors. DNMT inhibitor treatment showed clear restoration of PRAME expression in a dose dependent manner, but no restoration was found by HDAC inhibitor treatment. To investigate the effect of DNA methylation in transcriptional regulation of PRAME in HL cells, we performed bisulfite sequencing in the PRAME CpG promoter region in PRAME down-regulated (L540, L591, DEV) and up-regulated (HD-LM2, KMH-2, L1236) cell lines and found hypermethylation in PRAME low vs high cell lines. Moreover, the CpG promoter region was significantly demethylated by DNMT inhibitor treatment in cell lines with low PRAME expression. Conclusion: We discovered that PRAME protein expression was correlated with outcome in HL and identified specific T-cell subsets in PRAME-negative patients. PRAME restoration by DNMT inhibitors might represent a new therapeutic avenue in combination with modern immunotherapies, such as PRAME-specific T-cell therapy or PD1 inhibition. Disclosures Scott: Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding; Celgene: Consultancy. Steidl:Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Bristol-Myers Squibb: Research Funding; Roche: Consultancy; Seattle Genetics: Consultancy; Bayer: Consultancy; Juno Therapeutics: Consultancy; Tioma: Research Funding.
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Taghadosi, Mahdi, Soraya Bilvayeh, Sasan Ghaffari, Nasrin Iranshahi, Amirreza Esfandiari, and Parisa Zafari. "The Correlation Between Plasma Levels of Vitamin D and Epigenetic Alterations of Treg-Specific Demethylated Region (TSDR) in Rheumatoid Arthritis Patients." ACTA MEDICA IRANICA, November 30, 2019. http://dx.doi.org/10.18502/acta.v57i6.1876.
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Abstract- Rheumatoid arthritis (RA) is the most common autoimmune inflammatory disease of joints among adults. Regulatory T cells (Treg) control immune responses in this illness. Through the expression of FoxP3, a Treg transcription factor, Vitamin D keeps autoimmune diseases in check. Yet, the molecular mechanism of FoxP3 expression by vitamin D is not well-inspected. It may influence FoxP3 expression via epigenetic changes. This study aimed to investigate the correlation between plasma levels of vitamin D and the demethylation of the TSDR region in Foxp3 promoter in patients with RA. Twenty untreated RA patients and 41 healthy controls participated in this study. Plasma levels of 25-OH vitamin D were measured by competitive ELISA method. The demethylation of TSDR regions in Foxp3 gene was also assessed using the quantitative Methylation Specific PCR (qMSP) method. The demethylation of TSDR region was significantly lower in RA patients compared with healthy controls (P=0.006). Vitamin D plasma levels were significantly higher in RA patients rather than healthy people (P=0.034). There was no statistically significant correlation between vitamin D plasma levels and demethylation of TSDR region. As expected, epigenetic alternation showed considerable difference between RA patients and healthy controls, but about vitamin D correlation with methylation modification, more studies are needed
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Reading, James L., Valerie D. Roobrouck, Caroline M. Hull, Pablo Daniel Becker, Jelle Beyens, Alice Valentin-Torres, Dominic Boardman, et al. "Augmented Expansion of Treg Cells From Healthy and Autoimmune Subjects via Adult Progenitor Cell Co-Culture." Frontiers in Immunology 12 (September 1, 2021). http://dx.doi.org/10.3389/fimmu.2021.716606.
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Recent clinical experience has demonstrated that adoptive regulatory T (Treg) cell therapy is a safe and feasible strategy to suppress immunopathology via induction of host tolerance to allo- and autoantigens. However, clinical trials continue to be compromised due to an inability to manufacture a sufficient Treg cell dose. Multipotent adult progenitor cells (MAPCⓇ) promote Treg cell differentiation in vitro, suggesting they may be repurposed to enhance ex vivo expansion of Tregs for adoptive cellular therapy. Here, we use a Good Manufacturing Practice (GMP) compatible Treg expansion platform to demonstrate that MAPC cell-co-cultured Tregs (MulTreg) exhibit a log-fold increase in yield across two independent cohorts, reducing time to target dose by an average of 30%. Enhanced expansion is coupled to a distinct Treg cell-intrinsic transcriptional program characterized by elevated expression of replication-related genes (CDK1, PLK1, CDC20), downregulation of progenitor and lymph node-homing molecules (LEF1 CCR7, SELL) and induction of intestinal and inflammatory tissue migratory markers (ITGA4, CXCR1) consistent with expression of a gut homing (CCR7lo β7hi) phenotype. Importantly, we find that MulTreg are more readily expanded from patients with autoimmune disease compared to matched Treg lines, suggesting clinical utility in gut and/or T helper type1 (Th1)-driven pathology associated with autoimmunity or transplantation. Relative to expanded Tregs, MulTreg retain equivalent and robust purity, FoxP3 Treg-Specific Demethylated Region (TSDR) demethylation, nominal effector cytokine production and potent suppression of Th1-driven antigen specific and polyclonal responses in vitro and xeno Graft vs Host Disease (xGvHD) in vivo. These data support the use of MAPC cell co-culture in adoptive Treg therapy platforms as a means to rescue expansion failure and reduce the time required to manufacture a stable, potently suppressive product.
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Iwaszkiewicz-Grzes, Dorota, Magdalena Piotrowska, Mateusz Gliwinski, Zuzanna Urban-Wójciuk, and Piotr Trzonkowski. "Antigenic Challenge Influences Epigenetic Changes in Antigen-Specific T Regulatory Cells." Frontiers in Immunology 12 (March 23, 2021). http://dx.doi.org/10.3389/fimmu.2021.642678.
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BackgroundHuman regulatory T cells (Tregs) are the fundamental component of the immune system imposing immune tolerance via control of effector T cells (Teffs). Ongoing attempts to improve Tregs function have led to the creation of a protocol that produces antigen-specific Tregs, when polyclonal Tregs are stimulated with monocytes loaded with antigens specific for type 1 diabetes. Nevertheless, the efficiency of the suppression exerted by the produced Tregs depended on the antigen with the best results when insulin β chain peptide 9-23 was used. Here, we examined epigenetic modifications, which could influence these functional differences.MethodsThe analysis was pefromed in the sorted specific (SPEC, proliferating) and unspecific (UNSPEC, non-proliferating) subsets of Tregs and Teffs generated by the stimulation with monocytes loaded with either whole insulin (INS) or insulin β chain peptide 9-23 (B:9-23) or polyclonal cells stimulated with anti-CD3/anti-CD28 beads (POLY). A relative expression of crucial Tregs genes was determined by qRT-PCR. The Treg-specific demethylated region (TSDR) in FoxP3 gene methylation levels were assessed by Quantitative Methylation Specific PCR (qMSP). ELISA was used to measure genomic DNA methylation and histone H3 post-translational modifications (PTMs).ResultsTregs SPECB:9-23 was the only subset expressing all assessed genes necessary for regulatory function with the highest level of expression among all analyzed conditions. The methylation of global DNA as well as TSDR were significantly lower in Tregs SPECB:9-23 than in Tregs SPECINS. When compared to Teffs, Tregs were characterized by a relatively lower level of PTMs but it varied in respective Tregs/Teffs pairs. Importantly, whenever the difference in PTM within Tregs/Teffs pair was significant, it was always low in one subset from the pair and high in the other. It was always low in Tregs SPECINS and high in Teffs SPECINS, while it was high in Tregs UNSPECINS and low in Teffs UNSPECINS. There were no differences in Tregs/Teffs SPECB:9-23 pair and the level of modifications was low in Tregs UNSPECB:9-23 and high in Teffs UNSPECB:9-23. The regions of PTMs in which differences were significant overlapped only partially between particular Tregs/Teffs pairs.ConclusionsWhole insulin and insulin β chain peptide 9-23 affected epigenetic changes in CD4+ T cells differently, when presented by monocytes. The peptide preferably favored specific Tregs, while whole insulin activated both Tregs and Teffs.
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Varanasi, Siva Karthik, Pradeep B. J. Reddy, Siddheshvar Bhela, Ujjaldeep Jaggi, Fernanda Gimenez, and Barry T. Rouse. "Azacytidine Treatment Inhibits the Progression of Herpes Stromal Keratitis by Enhancing Regulatory T Cell Function." Journal of Virology 91, no. 7 (January 18, 2017). http://dx.doi.org/10.1128/jvi.02367-16.
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ABSTRACT Ocular infection with herpes simplex virus 1 (HSV-1) sets off an inflammatory reaction in the cornea which leads to both virus clearance and chronic lesions that are orchestrated by CD4 T cells. Approaches that enhance the function of regulatory T cells (Treg) and dampen effector T cells can be effective to limit stromal keratitis (SK) lesion severity. In this report, we explore the novel approach of inhibiting DNA methyltransferase activity using 5-azacytidine (Aza; a cytosine analog) to limit HSV-1-induced ocular lesions. We show that therapy begun after infection when virus was no longer actively replicating resulted in a pronounced reduction in lesion severity, with markedly diminished numbers of T cells and nonlymphoid inflammatory cells, along with reduced cytokine mediators. The remaining inflammatory reactions had a change in the ratio of CD4 Foxp3+ Treg to effector Th1 CD4 T cells in ocular lesions and lymphoid tissues, with Treg becoming predominant over the effectors. In addition, compared to those from control mice, Treg from Aza-treated mice showed more suppressor activity in vitro and expressed higher levels of activation molecules. Additionally, cells induced in vitro in the presence of Aza showed epigenetic differences in the Treg-specific demethylated region (TSDR) of Foxp3 and were more stable when exposed to inflammatory cytokines. Our results show that therapy with Aza is an effective means of controlling a virus-induced inflammatory reaction and may act mainly by the effects on Treg. IMPORTANCE HSV-1 infection has been shown to initiate an inflammatory reaction in the cornea that leads to tissue damage and loss of vision. The inflammatory reaction is orchestrated by gamma interferon (IFN-γ)-secreting Th1 cells, and regulatory T cells play a protective role. Hence, novel therapeutics that can rebalance the ratio of regulatory T cells to effectors are a relevant issue. This study opens up a new avenue in treating HSV-induced SK lesions by increasing the stability and function of regulatory T cells using the DNA methyltransferase inhibitor 5-azacytidine (Aza). Aza increased the function of regulatory T cells, leading to enhanced suppressive activity and diminished lesions. Hence, therapy with Aza, which acts mainly by its effects on Treg, can be an effective means to control virus-induced inflammatory lesions.
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Keefe, Ryan C., Hiroyuki Takahashi, Lisa Tran, Kacie Nelson, Nathan Ng, Willem M. Kühtreiber, and Denise L. Faustman. "BCG therapy is associated with long-term, durable induction of Treg signature genes by epigenetic modulation." Scientific Reports 11, no. 1 (July 22, 2021). http://dx.doi.org/10.1038/s41598-021-94529-2.
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AbstractInduction of immunosuppressive T-regulatory cells (Tregs) is a desirable goal in autoimmunity, and perhaps other immune diseases of activation. One promising avenue is with the bacille-calmette-guérin (BCG) vaccine in autoimmune type 1 diabetes (T1D). Its administration is associated with gradual clinical improvements in human autoimmunity over a 2–3 year post-vaccination period. We hypothesize that those improvements, and their unusually long time course to fully materialize, are partially attributable to BCG’s induction of Tregs. Here we report on a 3 year-long longitudinal cohort of T1Ds and examine the mechanism by which Treg induction occurs. Using the Human Infinium Methylation EPIC Bead Chip, we show that BCG vaccination is associated with gradual demethylation of most of 11 signature genes expressed in highly potent Tregs: Foxp3, TNFRSF18, CD25, IKZF2, IKZF4, CTLA4, TNFR2, CD62L, Fas, CD45 and IL2; nine of these 11 genes, by year 3, became demethylated at the majority of CpG sites. The Foxp3 gene was studied in depth. At baseline Foxp3 was over-methylated compared to non-diabetic controls; 3 years after introduction of BCG, 17 of the Foxp3 gene’s 22 CpG sites became significantly demethylated including the critical TSDR region. Corresponding mRNA, Treg expansion and clinical improvement supported the significance of the epigenetic DNA changes. Taken together, the findings suggest that BCG has systemic impact on the T cells of the adaptive immune system, and restores immune balance through Treg induction.
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Wu, Q., D. Bending, and LR Wedderburn. "PReS-FINAL-1011: Can repeated T cell receptor stimulation lead to epigenetic reprogramming of the treg-specific demethylated region in human conventional T cells?" Pediatric Rheumatology 11, S2 (December 2013). http://dx.doi.org/10.1186/1546-0096-11-s2-p9.
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