Treg-specific Demethylated Region Research Articles

Targeting CXCR4 impaired T regulatory function through PTEN in renal cancer patients

BackgroundTregs trafficking is controlled by CXCR4. In Renal Cell Carcinoma (RCC), the effect of the new CXCR4 antagonist, R54, was explored in peripheral blood (PB)-Tregs isolated from primary RCC patients.MethodsPB-Tregs were isolated from 77 RCC patients and 38 healthy donors (HDs). CFSE-T effector-Tregs suppression assay, IL-35, IFN-γ, IL-10, TGF-β1 secretion, and Nrp-1+Tregs frequency were evaluated. Tregs were characterised for CTLA-4, PD-1, CD40L, PTEN, CD25, TGF-β1, FOXP3, DNMT1 transcriptional profile. PTEN-pAKT signalling was evaluated in the presence of R54 and/or triciribine (TCB), an AKT inhibitor. Methylation of TSDR (Treg-Specific-Demethylated-Region) was conducted.ResultsR54 impaired PB-RCC-Tregs function, reduced Nrp-1+Tregs frequency, the release of IL-35, IL-10, and TGF-β1, while increased IFN-γ Teff-secretion. The CXCR4 ligand, CXCL12, recruited CD25+PTEN+Tregs in RCC while R54 significantly reduced it. IL-2/PMA activates Tregs reducing pAKT+Tregs while R54 increases it. The AKT inhibitor, TCB, prevented the increase in pAKT+Tregs R54-mediated. Moreover, R54 significantly reduced FOXP3-TSDR demethylation with DNMT1 and FOXP3 downregulation.ConclusionR54 impairs Tregs function in primary RCC patients targeting PTEN/PI3K/AKT pathway, reducing TSDR demethylation and FOXP3 and DNMT1 expression. Thus, CXCR4 targeting is a strategy to inhibit Tregs activity in the RCC tumour microenvironment.

Targeted Demethylation of FOXP3-TSDR Enhances the Suppressive Capacity of STAT6-deficient Inducible T Regulatory Cells.

In vitro induced T regulatory cells (iTregs) are promising for addressing inflammation-driven diseases. However, current protocols for the generation and expansion of iTregs fail to induce extensive demethylation of the Treg-specific demethylated region (TSDR) within the FOXP3 gene, recognized as the master regulator for regulatory T cells (Tregs). This deficiency results in the rapid loss of Foxp3 expression and an unstable regulatory phenotype. Nevertheless, inhibition of STAT6 signaling effectively stabilizes Foxp3 expression in iTregs. Thus, this study aimed to develop a protocol combining epigenetic editing with STAT6 deficiency to improve iTregs' ability to maintain stable suppressive function and a functional phenotype. Our findings demonstrate that the combination of STAT6 deficiency (STAT6-/-) with targeted demethylation of the TSDR using a CRISPR-TET1 tool leads to extensive demethylation of FOXP3-TSDR. Demethylation in STAT6-/- iTregs was associated with enhanced expression of Foxp3 and suppressive markers such as CTLA-4, PD-1, IL-10, and TGF-β. Furthermore, the edited STAT6-/- iTregs exhibited an increased capacity to suppress CD8+ and CD4+ lymphocytes and could more efficiently impair Th1-signature gene expression compared to conventional iTregs. In conclusion, the deactivation of STAT6 and TSDR-targeted demethylation via CRISPR-TET1 is sufficient to induce iTregs with heightened stability and increased suppressive capacity, offering potential applications against inflammatory and autoimmune diseases.

Abstract 2463: Targeting CXCR4 impaired T regulatory function through PTEN in renal cancer patients

Abstract Purpose: In Renal Cell Carcinoma (RCC) a crucial role is played by tumor microenvironment (TME). The high presence of T regulatory cells (Tregs) in TME cause a suppressive environment that promotes the tumor growth and its treatments failure. Tregs trafficking is controlled by CXCR4. In RCC, the new CXCR4 antagonist R54 was explored in peripheral blood Tregs isolated from primary RCC patients. Experimental Design: To investigate the mechanism by which the new CXCR4 antagonist R54 impairs Tregs activity, peripheral blood (PB) Tregs were explored on primary RCC patients. PB-Tregs were isolated from 77 RCC patients and 38 healthy donors-(HD) and CFSE-Tregs suppression assay, IL-35 secretion and Nrp-1+Tregs frequency was conducted. PB-Tregs were analyzed for PTEN, CD25, TGF-β1, FOXP3, DNMT1 expression. PTEN-pAKT signaling was evaluated in the presence of R54 and/or triciribine (TCB), Akt inhibitor. TSDR (Treg-specific Demethylated Region) methylation analysis was conducted. Result: Ex vivo R54 impaired PB-RCC-Tregs function, reduced IL-35 release and Nrp-1+Tregs. As PTEN and CD25 expression significantly decreased in R54-PB-RCC-Tregs, PTEN signaling was evaluated. While CXCL12 recruited PTEN+CD25+Tregs cells, R54 significantly reduced it in PB-RCC-Tregs. Tregs activation through IL-2/PMA impaired pAKT+Tregs while R54 increased it. The Akt inhibitor, triciribine, prevented the pAKT+Tregs R54 induction. As active Tregs present demethylated Treg-specific region (TSDR), a significant decrease in demethylation rate (DMR) of Foxp3-TSDR was observed in R54 treated PB-RCC-Tregs. As consequence of TSDR modulation, the expression of Tregs regulating genes was evaluated with significant reduction in DNMT1 and FOXP3 expression. Taken together, our findings demonstrated that R54 causes an impairment of peripheral Tregs functionality in primary RCC patients through regulation of PTEN/PI3K/AKT pathway, resulting in impaired Tregs activity. Conclusion: The CXCR4 antagonist R54 affects Tregs function in primary RCC patients through regulation of PTEN/PI3K/AKT pathway, reduction in TSDR demethylation and Foxp3 and DNMT1 expression. CXCR4 targeting is thus a strategy to inhibit Tregs activity contributing to the CXCR4 function in RCC tumor microenvironment. Citation Format: Giuseppina Rea, Sara Santagata, Anna Maria Bello, Anna Capiluongo, Maria Napolitano, Sonia Desicato, Alessandra Fragale, Crescenzo D'Alterio, Anna Maria Trotta, Caterina Ieranò, Luigi Portella, Marilena Di Napoli, Salvatore Di Maro, Florinda Feroce, Rosa Azzaro, Lucia Gabriele, Nicola Longo, Sandro Pignata, Sisto Perdonà, Stefania Scala. Targeting CXCR4 impaired T regulatory function through PTEN in renal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2463.

Abstract 6325: Lineage tracing defines responding CAR T cells in patients with B cell malignancies

Abstract Autologous T cells engineered to express a chimeric antigen receptor (CAR) have transformed the standard of care for patients with B cell malignancies, but >50% of patients progress following therapy. Here, we sought to understand key T cell intrinsic factors impacting efficacy: CAR T cell expansion, persistence, and homing to the tumor. Using an endogenous T cell receptor (TCR) sequence as a ‘barcode’, we followed individual T cell clonotypes at the single-cell level from pre-manufacture apheresis and infusion products to tumor-involved lymph nodes and blood at peak and late expansion in 22 adult patients with relapsed or refractory large B cell lymphoma (LBCL) or acute lymphoblastic leukemia (ALL) treated with axicabtagene ciloleucel, an FDA-approved CD19-CAR T cell therapy, or bispecific CD19/CD22-CAR T cells on an investigator-initiated trial (NCT03233854). The resulting CAR T cell atlas comprises matched transcriptome (scRNA-seq) and surface protein expression (CITE-seq) for 846,344 cells from 97 samples, with 251,175 unique TCR clonotypes identified, including 17,479 clonotypes that could be traced across ≥2 timepoints in CAR+ cells, enabling lineage tracing at scale for the first time. We found that T cells present in apheresis that preferentially expand during manufacturing primarily comprise T central memory (Tcm) cells and are enriched for predicted specificity to common viruses, including influenza, but also include a subset of T regulatory (Treg) cells. In infusion products, CAR T conventional (non-Treg) cells with superior abilities to home to the tumor, expand, and persist post-infusion share features of activated T effector memory (Tem) phenotype enriched for expression of BATF3 and high TCF4 transcription factor activity. Baseline apheresis and infusion products were enriched for cells classified as Tregs in patients with poor response. This observation aligned with our recent work linking post-infusion CAR Tregs to progression following CD19-CAR T cell therapy. We therefore performed lineage tracing and methylation analysis of the Treg-specific demethylated region (TSDR) in the FOXP3 gene and found that post-infusion CAR Tregs are primarily derived from pre-existing Tregs. Of interest, flow cytometry analysis of baseline blood from a cohort of 53 patients treated with CD19-CAR for LBCL showed that Tregs are elevated in baseline blood relative to healthy donors. Removing Tregs from healthy donor T cells prior to manufacturing enhanced CAR T cell expansion and anti-tumor activity against JeKo-1 human lymphoma cell line, whereas adding 5% Tregs to mimic patient T cells rendered the resulting CAR T cells less active. These analyses pinpoint the identities of apheresis T cells and infusion CAR T cells with properties impacting efficacy, and also identify pre-existing Tregs as limiting efficacy of CAR T cell immunotherapies. Citation Format: Zinaida Good, Mark P. Hamilton, Jay Y. Spiegel, Moksha H. Desai, Zachary J. Ehlinger, Patrick J. Quinn, Yiyun Chen, Snehit Prabhu, Shin-Heng Chiou, Sreevidya Kurra, Eric Yang, Michael G. Ozawa, Matthew J. Frank, Lori Muffly, Gursharan K. Claire, Sushma Bharadwaj, Saurabh Dahiya, Katherine A. Kong, Mark M. Davis, Sylvia K. Plevritis, Elena Sotillo, Bita Sahaf, David B. Miklos, Crystal L. Mackall. Lineage tracing defines responding CAR T cells in patients with B cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6325.

Clinical grade multiparametric cell sorting and gene-marking of regulatory T cells

Background aimsRegulatory T cells (Tregs) are the main mediators of peripheral tolerance. Treg-directed therapy has shown promising results in preclinical studies of diverse immunopathologies. At present, the clinical applicability of adoptive Treg transfer is limited by difficulties in generating Tregs at sufficient cell dose and purity. MethodsWe developed a Good Manufacturing Practice (GMP) compliant method based on closed-system multiparametric Fluorescence-Activated Cell Sorting (FACS) to purify Tregs, which are then expanded in vitro and gene-marked with a clinical grade retroviral vector to enable in vivo fate tracking. Following small-scale optimization, we conducted four clinical-scale processing runs. ResultsWe showed that Tregs could be enriched to 87– 92% purity following FACS-sorting, and expanded and transduced to yield clinically relevant cell dose of 136–732×106 gene-marked cells, sufficient for a cell dose of at least 2 × 106 cells/kg. The expanded Tregs were highly demethylated in the FOXP3 Treg-specific demethylated region (TSDR), consistent with bona fide natural Tregs. They were suppressive in vitro, but a small percentage could secrete proinflammatory cytokines, including interferon-γ and interleukin-17A. ConclusionsThis study demonstrated the feasibility of isolating, expanding and gene-marking Tregs in clinical scale, thus paving the way for future phase I trials that will advance knowledge about the in vivo fate of transferred Tregs and its relationship with concomitant Treg-directed pharmacotherapy and clinical response.

Suppressor T helper type 17 cell responses in intestinal transplant recipients with allograft rejection

BackgroundIntestinal transplant (ITx) rejection is associated with memory T helper type 17 cell (Th17) infiltration of grafted tissues. Modulation of Th17 effector cell response is facilitated by T regulatory (Treg) cells, but a phenotypic characterization of this process is lacking in the context of allograft rejection. MethodsFlow cytometry was performed to examine the expression of surface receptors, cytokines, and transcription factors in Th17 and Treg cells in ITx control (n = 34) and rejection patients (n = 23). To elucidate key pathways guiding the rejection biology, we utilized RNA sequencing (RNAseq) and assessed epigenetic stability through pyrosequencing of the Treg-specific demethylated region (TSDR). ResultsWe found that intestinal allograft rejection is characterized by Treg cellular infiltrates, which are polarized toward Th17-type chemokine receptor, ROR-γt transcription factor expression, and cytokine production. These Treg cell subsets have maintained epigenetic stability, as defined by FoxP3-TSDR methylation status, but displayed upregulation of functional Treg and purinergic signaling genes by RNAseq analysis such as CD39, in keeping with suppressor Th17 properties. ConclusionWe show that ITx rejection is associated with increased polarized cells that express a Th17-like phenotype concurrent with regulatory purinergic markers.

OP02 IL23R-CAR-Tregs: creating a therapeutic breakthrough for Crohn’s Disease

Abstract Background Regulatory T cells (Tregs) are key immunomodulators to maintain immune homeostasis and self-tolerance, including in the intestine. Disrupted immune homeostasis is associated with Crohn’s Disease (CD) pathogenesis. As a living drug, Treg therapy is a promising treatment for restoring immune balance and achieving durable tissue tolerance in the diseased intestine. By targeting a disease-relevant protein, Interleukin-23 receptor (IL23R), we engineered Tregs expressing IL23R-chimeric antigen receptor (CAR) to create IL23R-CAR-Tregs for treating CD. Methods IL23R protein expression in CD intestine was verified by immunohistochemical analysis. Tonic signaling and CAR-specific activation were quantified during the IL23R-CAR screening. The IL23R-CAR-Treg phenotype was assessed by monitoring the expression of FoxP3 and Helios transcription factors, and the demethylation status of FoxP3 Treg-specific demethylated region (TSDR). The suppressive function of IL23R-CAR-Tregs was evaluated using in vitro assays and a colitis mouse model. The functionality of IL23R-CAR-Tregs were further assessed using intestinal biopsy-derived cells from CD patients in an in vitro activation assay. Transcriptomic and proteomic analyses were performed to associate molecular profiles with IL23R-CAR-Treg activation. Results Our highly potent IL23R-CAR lead candidate displays negligible tonic signalling and a strong signal-to-noise ratio. The resulting IL23R-CAR-Tregs maintain their regulatory phenotype with high FoxP3 and Helios expression and TSDR demethylation during in vitro expansion, CAR-specific stimulation, and in the presence of proinflammatory cytokines. In accordance with their regulatory phenotype, IL23R-CAR-Tregs exhibit CAR-dependent suppressive activity, promote a tolerogenic environment, and protect mice from colitis. The expression of IL23R was also confirmed in the intestinal mucosa from CD patients. Importantly, IL23R-CAR-Tregs elicit activation following exposure to intestinal biopsy-derived cells from CD, demonstrating the engagement and potency of IL23R-CAR towards their target in CD. We also identified molecular profiles that correlated with IL23R-CAR activation and, more broadly, showed their potential to monitor IL23R-CAR-Treg activity in CD patients. Conclusion Our results demonstrate that an IL23R-CAR-Treg product could be a promising therapy for CD patients, with the potential long-term benefits of immune tolerance.

Identification of unstable regulatory and autoreactive effector T cells that are expanded in patients with FOXP3 mutations.

Studies of the monogenic autoimmune disease immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX) have elucidated the essential function of the transcription factor FOXP3 and thymic-derived regulatory T cells (Tregs) in controlling peripheral tolerance. However, the presence and the source of autoreactive T cells in IPEX remain undetermined. Here, we investigated how FOXP3 deficiency affects the T cell receptor (TCR) repertoire and Treg stability in vivo and compared T cell abnormalities in patients with IPEX with those in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED). To study Tregs independently of their phenotype and to analyze T cell autoreactivity, we combined Treg-specific demethylation region analyses, single-cell multiomic profiling, and bulk TCR sequencing. We found that patients with IPEX, unlike patients with APECED, have expanded autoreactive T cells originating from both autoreactive effector T cells (Teffs) and Tregs. In addition, a fraction of the expanded Tregs from patients with IPEX lost their phenotypic and functional markers, including CD25 and FOXP3. Functional experiments with CRISPR-Cas9-mediated FOXP3 knockout Tregs and Tregs from patients with IPEX indicated that the patients' Tregs gain a TH2-skewed Teff-like function, which is consistent with immune dysregulation observed in these patients. Analyses of FOXP3 mutation-carrier mothers and a patient with IPEX after hematopoietic stem cell transplantation indicated that Tregs expressing nonmutated FOXP3 prevent the accumulation of autoreactive Teffs and unstable Tregs. These findings could be directly used for diagnostic and prognostic purposes and for monitoring the effects of immunomodulatory treatments.

Human HLA-DR+CD27+ regulatory T cells show enhanced antigen-specific suppressive function.

Regulatory T cells (Tregs) have potential for the treatment of autoimmune diseases and graft rejection. Antigen specificity and functional stability are considered critical for their therapeutic efficacy. In this study, expansion of human Tregs in the presence of porcine PBMCs (xenoantigen-expanded Tregs, Xn-Treg) allowed the selection of a distinct Treg subset, coexpressing the activation/memory surface markers HLA-DR and CD27 with enhanced proportion of FOXP3+Helios+ Tregs. Compared with their unsorted and HLA-DR+CD27+ double-positive (DP) cell-depleted Xn-Treg counterparts, HLA-DR+CD27+ DP-enriched Xn-Tregs expressed upregulated Treg function markers CD95 and ICOS with enhanced suppression of xenogeneic but not polyclonal mixed lymphocyte reaction. They also had less Treg-specific demethylation in the region of FOXP3 and were more resistant to conversion to effector cells under inflammatory conditions. Adoptive transfer of porcine islet recipient NOD/SCID IL2 receptor γ-/- mice with HLA-DR+CD27+ DP-enriched Xn-Tregs in a humanized mouse model inhibited porcine islet graft rejection mediated by 25-fold more human effector cells. The prolonged graft survival was associated with enhanced accumulation of FOXP3+ Tregs and upregulated expression of Treg functional genes, IL10 and cytotoxic T lymphocyte antigen 4, but downregulated expression of effector Th1, Th2, and Th17 cytokine genes, within surviving grafts. Collectively, human HLA-DR+CD27+ DP-enriched Xn-Tregs expressed a specific regulatory signature that enabled identification and isolation of antigen-specific and functionally stable Tregs with potential as a Treg-based therapy.

Unlocking the potential of Tregs: innovations in CAR technology.

Regulatory T cells (Tregs) adoptive immunotherapy is emerging as a viable treatment option for both autoimmune and alloimmune diseases. However, numerous challenges remain, including limitations related to cell number, availability of target-specific cells, stability, purity, homing ability, and safety concerns. To address these challenges, cell engineering strategies have emerged as promising solutions. Indeed, it has become feasible to increase Treg numbers or enhance their stability through Foxp3 overexpression, post-translational modifications, or demethylation of the Treg-specific demethylated region (TSDR). Specificity can be engineered by the addition of chimeric antigen receptors (CARs), with new techniques designed to fine-tune specificity (tandem chimeric antigen receptors, universal chimeric antigen receptors, synNotch chimeric antigen receptors). The introduction of B-cell targeting antibody receptor (BAR) Tregs has paved the way for effective regulation of B cells and plasma cells. In addition, other constructs have emerged to enhance Tregs activation and function, such as optimized chimeric antigen receptors constructs and the use of armour proteins. Chimeric antigen receptor expression can also be better regulated to limit tonic signaling. Furthermore, various opportunities exist for enhancing the homing capabilities of CAR-Tregs to improve therapy outcomes. Many of these genetic modifications have already been explored for conventional CAR-T therapy but need to be further considered for CAR-Tregs therapies. This review highlights innovative CAR-engineering strategies that have the potential to precisely and efficiently manage immune responses in autoimmune diseases and improve transplant outcomes. As these strategies are further explored and optimized, CAR-Treg therapies may emerge as powerful tools for immune intervention.

Disabled C3ar1/C5ar1 Signaling in Foxp3+ T Regulatory Cells Leads to TSDR Demethylation and Long-Term Stability.

Demethylation of the T regulatory cell (Treg)-specific demethylation region (TSDR) of the Foxp3 gene is the hallmark of Foxp3+ Treg stability, but the cellular signaling that programs this epigenetic state remains undefined. In this article, we show that suppressed C3a and C5a receptor (C3ar1/C5ar1) signaling in murine Tregs plays an obligate role. Murine C3ar1-/-C5ar1-/- Foxp3+ cells showed increased suppressor of cytokine signaling 1/2/3 expression, vitamin C stabilization, and ten-eleven translocation (TET) 1, TET2, and TET3 expression, all of which are linked to Treg stability. C3ar1-/-C5ar1-/- Foxp3+ cells additionally were devoid of BRD4 signaling that primes Th17 cell lineage commitment. Orally induced OVA-specific C3ar1-/-C5ar1-/- Foxp3+ OT-II Tregs transferred to OVA-immunized wild-type recipients remained >90% Foxp3+ out to 4 mo, whereas identically generated CD55-/- (DAF-/-) Foxp3+ OT-II Tregs (in which C3ar1/C5ar1 signaling is potentiated) lost >75% of Foxp3 expression by 14 d. After 4 mo invivo, the C3ar1-/-C5ar1-/- Foxp3+ OT-II Tregs fully retained Foxp3 expression even with OVA challenge and produced copious TGF-β and IL-10. Their TSDR was demethylated comparably with that of thymic Tregs. They exhibited nuclear translocation of NFAT and NF-κB reported to stabilize thymic Tregs by inducing hairpin looping of the TSDR to the Foxp3 promoter. Thus, disabled CD4+ cell C3ar1/C5ar1 signaling triggers the sequential cellular events that lead to demethylation of the Foxp3 TSDR.

Responders to low-dose ATG induce CD4+ T cell exhaustion in type 1 diabetes.

BACKGROUNDLow-dose anti-thymocyte globulin (ATG) transiently preserves C-peptide and lowers HbA1c in individuals with recent-onset type 1 diabetes (T1D); however, the mechanisms of action and features of the response remain unclear. Here, we characterized the post hoc immunological outcomes of ATG administration and their potential use as biomarkers of metabolic response to therapy (i.e., improved preservation of endogenous insulin production).METHODSWe assessed gene and protein expression, targeted gene methylation, and cytokine concentrations in peripheral blood following treatment with ATG (n = 29), ATG plus granulocyte colony-stimulating factor (ATG/G-CSF, n = 28), or placebo (n = 31).RESULTSTreatment with low-dose ATG preserved regulatory T cells (Tregs), as measured by stable methylation of FOXP3 Treg-specific demethylation region (TSDR) and increased proportions of CD4+FOXP3+ Tregs (P < 0.001) identified by flow cytometry. While treatment effects were consistent across participants, not all maintained C-peptide. Responders exhibited a transient rise in IL-6, IP-10, and TNF-α (P < 0.05 for all) 2 weeks after treatment and a durable CD4+ exhaustion phenotype (increased PD-1+KLRG1+CD57- on CD4+ T cells [P = 0.011] and PD1+CD4+ Temra MFI [P < 0.001] at 12 weeks, following ATG and ATG/G-CSF, respectively). ATG nonresponders displayed higher proportions of senescent T cells (at baseline and after treatment) and increased methylation of EOMES (i.e., less expression of this exhaustion marker).CONCLUSIONAltogether in these exploratory analyses, Th1 inflammation-associated serum and CD4+ exhaustion transcript and cellular phenotyping profiles may be useful for identifying signatures of clinical response to ATG in T1D.TRIAL REGISTRATIONClinicalTrials.gov NCT02215200.FUNDINGThe Leona M. and Harry B. Helmsley Charitable Trust (2019PG-T1D011), the NIH (R01 DK106191 Supplement, K08 DK128628), NIH TrialNet (U01 DK085461), and the NIH NIAID (P01 AI042288).

Analyses of thymocyte commitment to regulatory T cell lineage in thymus of healthy subjects and patients with 22q11.2 deletion syndrome.

The Chromosome 22q11.2 deletion syndrome (22q11.2DS) results in an inborn error of immunity due to defective thymic organogenesis. Immunological abnormalities in 22q11.2DS patients are thymic hypoplasia, reduced output of T lymphocytes by the thymus, immunodeficiency and increased incidence of autoimmunity. While the precise mechanism responsible for increased incidence of autoimmunity is not completely understood, a previous study suggested a defect in regulatory T cells (Treg) cell lineage commitment during T cell development in thymus. Here, we aimed to analyze this defect in more detail. Since Treg development in human is still ill-defined, we first analyzed where Treg lineage commitment occurs. We performed systematic epigenetic analyses of the Treg specific demethylation region (TSDR) of the FOXP3 gene in sorted thymocytes at different developmental stages. We defined CD3+CD4+CD8+ FOXP3+CD25+ as the T cell developmental stage in human where TSDR demethylation first occurs. Using this knowledge, we analyzed the intrathymic defect in Treg development in 22q11.2DS patients by combination of TSDR, CD3, CD4, CD8 locus epigenetics and multicolor flow cytometry. Our data showed no significant differences in Treg cell frequencies nor in their basic phenotype. Collectively, these data suggest that although 22q11.2DS patients present with reduced thymic size and T cell output, the frequencies and the phenotype of Treg cell at each developmental stage are surprisingly well preserved.

STAT6 controls the stability and suppressive function of regulatory T cells.

Signal transducer and activator of transcription 6 (STAT6) promotes tumorigenesis by decreasing the Forkhead box P3+ (Foxp3+) cell frequency allowing for the infiltration of inflammatory cells during the early stages of colitis-associated cancer (CAC). In this study, we dissected the role of STAT6 in the generation of inducible in vitro regulatory T cells (iTregs) and peripheral in vivo Tregs (pTregs) under inflammatory conditions. In in vitro assays, when STAT6 was lacking, iTregs preserved a stable phenotype and expressed high levels of Foxp3 and CD25 during long expansion periods, even in the presence of IL-6. This effect was associated with increased in vitro suppressive ability, over-expression of programmed death-1 (PD-1), CTLA-4, and Foxp3, and decreased IFN-γ expression. Furthermore, iTregs developed during STAT6 deficiency showed a higher demethylation status for the FOXP3 Treg-specific demethylated region (TSDR), coupled with lower DNA methyltransferase 1 (DNMT1) mRNA expression, suggesting that STAT6 may lead to Foxp3 silencing. Using a mouse model of CAC, the STAT6-/- pTregs expressed a more activated phenotype at the intestine, had higher suppressive capacity, and expressed more significant levels of PD-1 and latency-associated peptide of TGF-β (LAP) associated with their ability to attenuate tumor development. These data suggest that STAT6 signaling impairs the induction, stability, and suppressive capacity of Tregs developed in vitro or in vivo during gut inflammation.

P53 promotes the expansion of regulatory T cells via DNMT3a- and TET2- mediated Foxp3 expression in sepsis.

Immunosuppression is an important characteristic of sepsis and is closely related to poor outcomes. Regulatory T cells (Tregs) contribute to immune suppression by inhibiting effector T cell (Teff) proliferation and differentiation. We aimed to investigate the role of p53 in Treg expansion after sepsis. We constructed a sepsis model in wild-type (WT) and p53f/f/CD4-Cre+ mice by cecal ligation and puncture (CLP) and evaluated the proportions of CD4+CD25+ Foxp3+ Tregs by flow cytometry. The expression levels of forkhead/winged helix transcription factor p3 (Foxp3), DNA methyltransferase enzyme (DMNT)3a and ten-eleven translocation (TET)2 were examined using quantitative real-time PCR and Western blot analysis. Treg-specific demethylation region (TSDR) methylation sites in cells were analyzed by bisulfite-sequencing PCR. Furthermore, the direct binding of p53 to the Dnmt3a and TET2 promoters was illustrated using a luciferase assay. The suppressive ability of Tregs was indicated by enzyme-linked immunosorbent assay analysis of cytokine levels and the proliferation of cocultured Teffs. Finally, mortality rates after CLP were compared among WT and p53f/f/CD4-Cre+ mice. The proportion of CD4+CD25+ Foxp3+ Tregs was significantly reduced in p53f/f/CD4-Cre+ mice compared to WT mice after CLP. The enhanced expression of Foxp3 in WT mice was downregulated in the p53f/f/CD4-Cre+ group. We found decreased DMNT3a and increased TET2 levels after CLP. However, the dysregulation of DNMT3a and TET2 was significantly reversed in p53f/f/CD4-Cre+ mice. TSDR underwent increased demethylation in p53f/f/CD4-Cre+ mice. Luciferase activity indicated direct binding of p53 to the promoter regions of DNMT3a and TET2 to regulate their transcription. Consequently, Tregs from p53f/f/CD4-Cre+ CLP mice exhibited limited suppressive ability, as indicated by the reduced production of transforming growth factor-β and interleukin 10 (IL-10). In the coculture system, Teffs showed preserved production of IL-2, differentiation into Th1 cells and proliferation in the presence of Tregs isolated from p53f/f/CD4-Cre+ CLP mice. Finally, the mortality rate of the p53f/f/CD4-Cre+ group after CLP was significantly reduced in comparison to that of the WT group. p53 appears to be critical for Foxp3 expression and consequent Treg expansion by regulating the induction of DNMT3a and TET2, thereby resulting in Foxp3-TSDR demethylation in the context of sepsis.

Assessment of regulatory T cells (Tregs) and Foxp3 methylation level in chronic myeloid leukemia patients on tyrosine kinase inhibitor therapy.

The key cell population permits cancer cells to avoid immune-surveillance is regulatory T cells (Tregs). This study evaluates the level of Tregs in chronic myeloid leukemia (CML) patients and the effect of Tyrosine kinase inhibitor (TKI) on Treg levels, as a pathway to understand the immune response and behavior among advance stage and optimal response CML patients using imatinib therapy. Blood samples were collected from 30 CML patients (optimal response to TKI), 30 CML patients (failure response to TKI), and 30 age- and gender-matched controls. Analysis involved measuring percentages of Tregs (CD4 + CD25 + FOXP3 +) by flow cytometer and demethylation levels of FOXP3 Treg-specific demethylated region (TSDR) by PCR. The data revealed that Tregs and the FOXP3-TSDR demethylation percentages significantly increased in failure response group in comparison to the optimal response and control groups, while no significant difference between optimal response and control groups. Tregs and FOXP3 TSDR demethylation percentages showed high sensitivity and specificity, suggesting powerful discriminatory biomarkers between failure and optimal groups. An assessment of the Tregs and demethylation percentage among different BCR-ABL levels of CML patients on TKI revealed no significant differences in parameter percentage in the optimal response to TKI patients with different molecular responses (log 3 reduction or other deeper log 4.5 and 5 reduction levels). Our findings demonstrate an effective role of functional Tregs among different CML stages. Also, the study suggests that the major molecular response to therapy at level 0.1% of BCR-ABL transcript could be enough to induce immune system restoration in patients.

IPSC-Derived HLA-A2 CAR Tregs Showed Suppression of GvHD in a Xenograft Model

Overcoming pathologies caused by excessive immunity, such as graft-versus-host disease (GvHD) after hematopoietic stem cell transplantation and autoimmune diseases such as type1 diabetes and inflammatory bowel disease, is a challenge for medical research. It has been reported that CD4 single positive helper T cells can be induced from iPS cells (iPS-CD4Ts) by organoid culture using Notch ligand-expressing cells. However, these cells are conventional helper T cells (Tconvs) that secrete inflammatory cytokines such as IFN-γ or IL-4 if they are not treated in any way. Now, we report that we have succeeded in inducing FOXP3, a marker of immunosuppressive regulatory T cells (Tregs), in these iPS-CD4Ts at a high rate by combining compounds and cytokines. We used CDK8/19 inhibitor(A), TNFR2 antibody(M), Rapamycin(R), and TGF-β(T). Therefore, we named this process the AMRT. AMRT-treated iPS-CD4Ts didn't secrete inflammatory cytokines even under strong stimulus conditions. And, these cells were accompanied by demethylation of the Treg-specific demethylation region (TSDR), which suggested stable conversion to immunosuppressive cells. Furthermore, the combination of compounds and cytokines that induced FOXP3 expression in iPSC-derived Tconvs and converted them to FOXP3+ cells can also be used to amplify primary natural Tregs (nTregs) while retaining FOXP3. Comprehensive gene expression analysis (Ampliseq) showed close profiling of primary nTregs and AMRT-treated iPS-CD4Ts. These AMRT-treated iPS-CD4Ts were assessed functionally by CAR-mediated immunosuppressive ability. We evaluated the ability of these iPSC-derived HLA-A2 CAR-transduced FOXP3+ T cells to inhibit CTL division in mixed lymphocyte reaction (MLR) assays with HLA-A2+ allo CTLs. AMRT-treated iPS-CD4Ts showed CTL division suppression ability beyond primary nTregs. Surprisingly, this suppressive capacity was independent of the induction of CAR. If proximity is guaranteed in advance, the bystander suppression effect may be sufficient. We then assessed the immunosuppressive capacity of these cells through the extent to which they could suppress the xenograft GvHD that human HLA-A2+ PBMCs cause in NSG mice. AMRT-treated HLA-A2 CAR-transduced iPS-CD4Ts showed a survival benefit comparable to that of primary nTregs. In contrast to the invitro results, the invivo immunosuppressive capacity was CAR-specific. In summary, we have successfully generated large numbers of immune-suppressing regulatory T cells from iPS cells to suppress GvHD. This is a groundbreaking achievement that paves the way for curative therapies for GvHD and autoimmune diseases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PD-L2 controls peripherally induced regulatory T cells by maintaining metabolic activity and Foxp3 stability

Regulatory T (Treg) cells are central to limit immune responses to allergens. Here we show that PD-L2 deficiency prevents the induction of tolerance to ovalbumin and control of airway hyperreactivity, in particular by limiting pTreg numbers and function. In vitro, PD-1/PD-L2 interactions increase iTreg numbers and stability. In mice lacking PD-L2 we find lower numbers of splenic pTregs at steady state, producing less IL-10 upon activation and with reduced suppressive activity. Remarkably, the numbers of splenic pTregs are restored by adoptively transferring PD-L2high dendritic cells to PD-L2KO mice. Functionally, activated pTregs lacking PD-L2 show lower Foxp3 expression, higher methylation of the Treg-Specific Demethylation Region (TSDR) and a decreased Tricarboxylic Acid (TCA) cycle associated with a defect in mitochondrial function and ATP production. Consequently, pyruvate treatment of PD-L2KO mice partially restores IL-10 production and airway tolerance. Together, our study highlights the importance of the PD-1/PD-L2 axis in the control of metabolic pathways regulating pTreg Foxp3 stability and suppressive functions, opening up avenues to further improve mucosal immunotherapy.

Analysis of T Cell Populations in Clinically Stable ISHLT Grade A1 Lesions

<h3>Purpose</h3> CD4+FOXP3+ Regulatory T cells (Tregs) may modulate acute cellular rejection (ACR)-associated immune responses and the progression of ACR to Chronic Lung Allograft Dysfunction (CLAD) following lung transplantation (LTx). Treg stability is enhanced by demethylation of the Treg-specific demethylated region (TSDR) in the Foxp3 locus. Grade A1 ACR is not treated in clinically stable patients at our centre, affording the opportunity to study its natural history in relation to its cellular composition. Our hypothesis was that a greater Treg content in first, clinically stable, grade A1 biopsies is associated with a lower risk of CLAD. <h3>Methods</h3> In this retrospective study, we selected clinically stable patients who received a bilateral LTx between 1 Jan 2010 and 18 Dec 2016 with a first episode of A1B0 ACR and who remained CLAD free for at least 5 years (n=16) or who developed CLAD within 2 years (n=9) following LTx. Immunofluorescence was used to quantify CD4+, CD8+ and FOXP3+ cells in transbronchial biopsies (Figure 1A). Separately, CD3+ cells were fluorescently labelled, micro-dissected (Figure 1B) and, using bisulfite conversion and pyrosequencing, the degree of TSDR methylation was determined. <h3>Results</h3> TSDR methylation was slightly lower in the CLAD group as compared to the CLAD-free group (Figure 1C), indicating increased Treg infiltration. Accordingly, the ratio of CD4+FOXP3+ cells to total CD4+ cells was slightly higher in the CLAD group (Figure 1D). These measures were weakly negatively correlated (r² = 0.250, p=0.0152) (Figure 1E). The ratio of CD4+:CD8+ cells was significantly lower in the CLAD group (p=0.0065) (Figure 1F). <h3>Conclusion</h3> Patients who developed CLAD within 2 years of LTx showed no significant difference in Treg, though greater CD8+ T cell, infiltration compared to stable patients. These results suggest that in asymptomatic patients with a first episode of A1 rejection, greater CD8+ T cell content may be indicative of a poorer long-term outlook. Validation in additional patients is now underway.

Augmented Expansion of Treg Cells From Healthy and Autoimmune Subjects via Adult Progenitor Cell Co-Culture.

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.