Gene editing to induce FOXP3 expression in human CD4+ T cells leads to a stable regulatory phenotype and function

2020 ◽  
Vol 12 (546) ◽  
pp. eaay6422 ◽  
Author(s):  
Yuchi Honaker ◽  
Nicholas Hubbard ◽  
Yufei Xiang ◽  
Logan Fisher ◽  
David Hagin ◽  
...  
Keyword(s):  
T Cells ◽  
Gene Editing ◽  
Foxp3 Expression ◽  
Cell Therapies ◽  
Homology Directed Repair ◽  
Engineering Strategy ◽  
And Function ◽  
Regulatory Phenotype

Thymic regulatory T cells (tTregs) are potent inhibitors of autoreactive immune responses, and loss of tTreg function results in fatal autoimmune disease. Defects in tTreg number or function are also implicated in multiple autoimmune diseases, leading to growing interest in use of Treg as cell therapies to establish immune tolerance. Because tTregs are present at low numbers in circulating blood and may be challenging to purify and expand and also inherently defective in some subjects, we designed an alternative strategy to create autologous Treg-like cells from bulk CD4+ T cells. We used homology-directed repair (HDR)–based gene editing to enforce expression of FOXP3, the master transcription factor for tTreg. Targeted insertion of a robust enhancer/promoter proximal to the first coding exon bypassed epigenetic silencing, permitting stable and robust expression of endogenous FOXP3. HDR-edited T cells, edTregs, manifested a transcriptional program leading to sustained expression of canonical markers and suppressive activity of tTreg. Both human and murine edTregs mediated immunosuppression in vivo in models of inflammatory disease. Further, this engineering strategy permitted generation of antigen-specific edTreg with robust in vitro and in vivo functional activity. Last, edTreg could be enriched and expanded at scale using clinically relevant methods. Together, these findings suggest that edTreg production may permit broad future clinical application.

scholarly journals Androgen receptor modulates Foxp3 expression in CD4+CD25+Foxp3+ regulatory T-cells

2015 ◽  
Vol 26 (15) ◽  
pp. 2845-2857 ◽  
Author(s):  
Magdalena Walecki ◽  
Florian Eisel ◽  
Jörg Klug ◽  
Nelli Baal ◽  
Agnieszka Paradowska-Dogan ◽  
...  
Keyword(s):  
T Cells ◽  
Androgen Receptor ◽  
Treg Cell ◽  
Foxp3 Expression ◽  
Treg Cells ◽  
Strong Increase ◽  
Histone H4 ◽  
And Function

CD4+CD25+Foxp3+ regulatory T (Treg) cells are able to inhibit proliferation and cytokine production in effector T-cells and play a major role in immune responses and prevention of autoimmune disease. A master regulator of Treg cell development and function is the transcription factor Foxp3. Several cytokines, such as TGF-β and IL-2, are known to regulate Foxp3 expression as well as methylation of the Foxp3 locus. We demonstrated previously that testosterone treatment induces a strong increase in the Treg cell population both in vivo and in vitro. Therefore we sought to investigate the direct effect of androgens on expression and regulation of Foxp3. We show a significant androgen-dependent increase of Foxp3 expression in human T-cells from women in the ovulatory phase of the menstrual cycle but not from men and identify a functional androgen response element within the Foxp3 locus. Binding of androgen receptor leads to changes in the acetylation status of histone H4, whereas methylation of defined CpG regions in the Foxp3 gene is unaffected. Our results provide novel evidence for a modulatory role of androgens in the differentiation of Treg cells.

scholarly journals PD-L1 regulates the development, maintenance, and function of induced regulatory T cells

2009 ◽  
Vol 206 (13) ◽  
pp. 3015-3029 ◽  
Author(s):  
Loise M. Francisco ◽  
Victor H. Salinas ◽  
Keturah E. Brown ◽  
Vijay K. Vanguri ◽  
Gordon J. Freeman ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Cell Function ◽  
Foxp3 Expression ◽  
Cell Development ◽  
Peripheral Tolerance ◽  
Cell Conversion ◽  
And Function

Both the programmed death (PD) 1–PD-ligand (PD-L) pathway and regulatory T (T reg) cells are instrumental to the maintenance of peripheral tolerance. We demonstrate that PD-L1 has a pivotal role in regulating induced T reg (iT reg) cell development and sustaining iT reg cell function. PD-L1−/− antigen-presenting cells minimally convert naive CD4 T cells to iT reg cells, showing the essential role of PD-L1 for iT reg cell induction. PD-L1–coated beads induce iT reg cells in vitro, indicating that PD-L1 itself regulates iT reg cell development. Furthermore, PD-L1 enhances and sustains Foxp3 expression and the suppressive function of iT reg cells. The obligatory role for PD-L1 in controlling iT reg cell development and function in vivo is illustrated by a marked reduction in iT reg cell conversion and rapid onset of a fatal inflammatory phenotype in PD-L1−/−PD-L2−/− Rag−/− recipients of naive CD4 T cells. PD-L1 iT reg cell development is mediated through the down-regulation of phospho-Akt, mTOR, S6, and ERK2 and concomitant with the up-regulation of PTEN, all key signaling molecules which are critical for iT reg cell development. Thus, PD-L1 can inhibit T cell responses by promoting both the induction and maintenance of iT reg cells. These studies define a novel mechanism for iT reg cell development and function, as well as a new strategy for controlling T reg cell plasticity.

Decreased Generation and Function of CD4+CD25hi T Regulatory Cells in Human STAT5b Deficiency.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 768-768
Author(s):  
Aileen Cleary ◽  
Kari Nadeau ◽  
Wenwei Tu ◽  
Vivian Hwa ◽  
Kira Y. Dionis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Gene Dosage ◽  
Foxp3 Expression ◽  
Intermediate Phenotype ◽  
Gamma Chain ◽  
Gene Dosage Effect ◽  
And Function

Abstract CD4+ CD25+ regulatory T cells (Tregs) are a well characterized population of cells that play an important role in limiting inflammation and in the maintenance of tolerance to self. Here we describe a patient with a homozygous missense mutation (A630P) in the STAT5b gene who clinically displays immune dysregulation in association with decreased numbers and function of Tregs. Freshly isolated or in vitro-derived CD4+CD25high Treg cells from this patient had low Foxp3 expression, did not suppress non Treg T-cell proliferation, and were unable to kill autologous CD4+CD25neg T cells compared to controls. CD25 expression in response to IL-2 did not increase on freshly isolated CD4 T cells and was decreased on T-cell blasts derived from the patient. The patients mother who was heterozygous for this mutation had an intermediate phenotype for all of these immune abnormalities, indicating a gene dosage effect. In contrast, IL-2 upregulated expression of the common gamma chain (γc) cytokine receptor and perforin by T cells normally. Activation-induced T-cell expression of CD40-ligand (CD154) and interferon-gamma (IFN-γ) were also normal in the patient. These results suggest that the STAT5 pathway propagates an important IL-2 mediated signal that is necessary for Treg generation and function in humans in vivo.

Epigenetic Regulation of Regulatory T-Cells: Impact on Autoimmunity and Graft Rejection

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. SCI-23-SCI-23
Author(s):  
Wayne W. Hancock
Keyword(s):  
T Cells ◽  
Catalytic Activity ◽  
Hdac Inhibitors ◽  
Foxp3 Expression ◽  
Epigenetic Mechanisms ◽  
Treg Function ◽  
Class Iib ◽  
And Function

Abstract Abstract SCI-23 Mutations of Foxp3, a transcription factor characteristic of T regulatory (Treg) cells, often cause lethal autoimmunity, leading to much research into how Foxp3+ Tregs control inflammatory and immune responses. This presentation will emphasize therapeutic aspects of our ongoing studies showing that the functions of Foxp3 are regulated by histone/protein deacetylases (HDAC), histone acetyltransferases (HAT), and DNA methyltransferases (DNMT), and for the sake of time will focus on the effects of HDAC inhibitors (HDACi). We have found that Foxp3 acetylation promotes DNA binding and can induce or suppress expression of multiple Foxp3-dependent genes in Tregs. Acetylation is catalyzed by specific HATs whose neutralization can diminish Treg function, with relevance to cancer and HIV, whereas use of selected HDAC inhibitors (HDACi) can increase acetylation and Treg suppression, with relevance to control of autoimmunity and transplant rejection. Many HDAC inhibitors (HDACi) were evaluated for their effects on Treg function. As class I-selective HDACi agents (e.g. benzamides) had little or no effect on Treg function, but pan-HDACi (e.g. hydroxymates) enhanced Treg function, we focused on class II HDACs. There are 2 class II subfamilies; class IIa members are thought to largely function in a tissue-specific manner through recruitment of other proteins since they display only weak catalytic activity, whereas class IIb family members display bona fide catalytic activity. We therefore analyzed class IIb members, of which HDAC6 is the best established and for which selective HDACi are available. HDAC6 exists in the cytoplasm and regulates acetylation of alpha-tubulin and other proteins, including HSP90. Blocking HDAC6 through the use of an HDAC6-specific inhibitor promotes HSP90 acetylation and release of HSP90 client proteins. Use of HDAC6 or HSP90 inhibitors increased Foxp3 expression and enhanced Treg function in vitro and in vivo, and could prevent, or treat pre-existing, autoimmunity in a Treg-dependent manner. We have also investigated the various class IIa family members, of which HDAC9 is of particular interest since its expression is increased 30-fold in Treg vs. regular T cells. HDAC9 decreases Foxp3 expression and function, and its neutralization promotes Treg survival by regulating expression of HSP70 and related HSPs. Our ongoing studies show that HDAC6 neutralization leads to acetylation of HSP90, release of HSF-1 and induction of HSP70, and also suggest that HDAC9 may regulate the acetylation and stabilization of HSF-1. Once produced, HSP70 can chaperone and promote Foxp3 nuclear translocation and function, such that the HDAC6 and HDAC9 pathways are closely intertwined with regard to control of Treg biology. In summary, acetylation, methylation and other epigenetic mechanisms in Tregs are being probed using genetic and pharmacologic approaches. Various currently approved drugs influence Foxp3-dependent Treg functions by affecting epigenetic mechanisms, and while additional HDAC-specific regulators are needed, a rationale is now in place for use of HDAC inhibitors as powerful tools to promote the development and functions of Foxp3+ Tregs in vitro and in vivo. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The post sepsis-induced expansion and enhanced function of regulatory T cells create an environment to potentiate tumor growth

Blood ◽  
2010 ◽  
Vol 115 (22) ◽  
pp. 4403-4411 ◽  
Author(s):  
Karen A. Cavassani ◽  
William F. Carson ◽  
Ana Paula Moreira ◽  
Haitao Wen ◽  
Matthew A. Schaller ◽  
...  
Keyword(s):  
T Cells ◽  
Severe Sepsis ◽  
Regulatory T Cells ◽  
Tumor Growth ◽  
Tumor Model ◽  
Foxp3 Expression ◽  
And Function ◽  
Immune Defects

Abstract One of the more insidious outcomes of patients who survive severe sepsis is profound immunosuppression. In this study, we addressed the hypothesis that post septic immune defects were due, in part, to the presence and/or expansion of regulatory T cells (Tregs). After recovery from severe sepsis, mice exhibited significantly higher numbers of Tregs, which exerted greater in vitro suppressive activity compared with controls. The expansion of Tregs was not limited to CD25+ cells, because Foxp3 expression was also detected in CD25− cells from post septic mice. This latter group exhibited a significant increase of chromatin remodeling at the Foxp3 promoter, because a marked increase in acetylation at H3K9 was associated with an increase in Foxp3 transcription. Post septic splenic dendritic cells promoted Treg conversion in vitro. Using a solid tumor model to explore the function of Tregs in an in vivo setting, we found post septic mice showed an increase in tumor growth compared with sham-treated mice with a syngeneic tumor model. This observation could mechanistically be related to the ability of post septic Tregs to impair the antitumor response mediated by CD8+ T cells. Together, these data show that the post septic immune system obstructs tumor immunosurveillance, in part, by augmented Treg expansion and function.

98 ATA3271: an armored, next-generation off-the-shelf, allogeneic, mesothelin-CAR T cell therapy for solid tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A109-A109
Author(s):  
Jiangyue Liu ◽  
Xianhui Chen ◽  
Jason Karlen ◽  
Alfonso Brito ◽  
Tiffany Jheng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Next Generation ◽  
Phase 3 ◽  
T Cell Therapy ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T

BackgroundMesothelin (MSLN) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein with high expression levels in an array of malignancies including mesothelioma, ovaria, non-small cell lung cancer, and pancreatic cancers and is an attractive target antigen for immune-based therapies. Early clinical evaluation of autologous MSLN-targeted chimeric antigen receptor (CAR)-T cell therapies for malignant pleural mesothelioma has shown promising acceptable safety1 and have recently evolved with incorporation of next-generation CAR co-stimulatory domains and armoring with intrinsic checkpoint inhibition via expression of a PD-1 dominant negative receptor (PD1DNR).2 Despite the promise that MSLN CAR-T therapies hold, manufacturing and commercial challenges using an autologous approach may prove difficult for widespread application. EBV T cells represent a unique, non-gene edited approach toward an off-the-shelf, allogeneic T cell platform. EBV-specific T cells are currently being evaluated in phase 3 trials [NCT03394365] and, to-date, have demonstrated a favorable safety profile including limited risks for GvHD and cytokine release syndrome.3 4 Clinical proof-of-principle studies for CAR transduced allogeneic EBV T cell therapies have also been associated with acceptable safety and durable response in association with CD19 targeting.5 Here we describe the first preclinical evaluation of ATA3271, a next-generation allogeneic CAR EBV T cell therapy targeting MSLN and incorporating PD1DNR, designed for the treatment of solid tumor indications.MethodsWe generated allogeneic MSLN CAR+ EBV T cells (ATA3271) using retroviral transduction of EBV T cells. ATA3271 includes a novel 1XX CAR signaling domain, previously associated with improved signaling and decreased CAR-mediated exhaustion. It is also armored with PD1DNR to provide intrinsic checkpoint blockade and is designed to retain functional persistence.ResultsIn this study, we characterized ATA3271 both in vitro and in vivo. ATA3271 show stable and proportional CAR and PD1DNR expression. Functional studies show potent antitumor activity of ATA3271 against MSLN-expressing cell lines, including PD-L1-high expressors. In an orthotopic mouse model of pleural mesothelioma, ATA3271 demonstrates potent antitumor activity and significant survival benefit (100% survival exceeding 50 days vs. 25 day median for control), without evident toxicities. ATA3271 maintains persistence and retains central memory phenotype in vivo through end-of-study. Additionally, ATA3271 retains endogenous EBV TCR function and reduced allotoxicity in the context of HLA mismatched targets. ConclusionsOverall, ATA3271 shows potent anti-tumor activity without evidence of allotoxicity, both in vitro and in vivo, suggesting that allogeneic MSLN-CAR-engineered EBV T cells are a promising approach for the treatment of MSLN-positive cancers and warrant further clinical investigation.ReferencesAdusumilli PS, Zauderer MG, Rusch VW, et al. Abstract CT036: A phase I clinical trial of malignant pleural disease treated with regionally delivered autologous mesothelin-targeted CAR T cells: Safety and efficacy. Cancer Research 2019;79:CT036-CT036.Kiesgen S, Linot C, Quach HT, et al. Abstract LB-378: Regional delivery of clinical-grade mesothelin-targeted CAR T cells with cell-intrinsic PD-1 checkpoint blockade: Translation to a phase I trial. Cancer Research 2020;80:LB-378-LB-378.Prockop S, Doubrovina E, Suser S, et al. Off-the-shelf EBV-specific T cell immunotherapy for rituximab-refractory EBV-associated lymphoma following transplantation. J Clin Invest 2020;130:733–747.Prockop S, Hiremath M, Ye W, et al. A Multicenter, Open Label, Phase 3 Study of Tabelecleucel for Solid Organ Transplant Subjects with Epstein-Barr Virus-Driven Post-Transplant Lymphoproliferative Disease (EBV+PTLD) after Failure of Rituximab or Rituximab and Chemotherapy. Blood 2019; 134: 5326–5326.Curran KJ, Sauter CS, Kernan NA, et al. Durable remission following ‘Off-the-Shelf’ chimeric antigen receptor (CAR) T-Cells in patients with relapse/refractory (R/R) B-Cell malignancies. Biology of Blood and Marrow Transplantation 2020;26:S89.

scholarly journals 173 Expression of a membrane-tethered IL-15/IL-15 receptor fusion protein enhances the persistence of MSLN-targeted TRuC-T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A185-A185
Author(s):  
Michelle Fleury ◽  
Derrick McCarthy ◽  
Holly Horton ◽  
Courtney Anderson ◽  
Amy Watt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
T Cell Receptor ◽  
Cytokine Production ◽  
Cell Receptor ◽  
Great Promise ◽  
And Function

BackgroundAdoptive cell therapies have shown great promise in hematological malignancies but have yielded little progress in the context of solid tumors. We have developed T cell receptor fusion construct (TRuC®) T cells, which are equipped with an engineered T cell receptor that utilizes the full complement of TCR signaling subunits and recognizes tumor-associated antigens independent of HLA. In clinical trials, mesothelin (MSLN)-targeting TRuC-T cells (TC-210 or gavo-cel) have shown unprecedented results in patients suffering from advanced mesothelioma and ovarian cancer. To potentially increase the depth of response, we evaluated strategies that can promote intra-tumoral T cell persistence and function. Among the common ??-chain cytokines, IL-15 uniquely supports the differentiation and maintenance of memory T cell subsets by limiting terminal differentiation and conferring resistance to IL-2 mediated activation-induced cell death (AICD). In the studies described here, we evaluated the potential of IL-15 as an enhancement to TRuC-T cell phenotype, persistence and function against MSLN+ targets.MethodsPrimary human T cells were activated and transduced with a lentiviral vector encoding an anti-MSLN binder fused to CD3ε alone or co-expressed with a membrane-tethered IL-15rα/IL-15 fusion protein (IL-15fu). Transduced T cells were expanded for 9 days and characterized for expression of the TRuC, IL-15rα and memory phenotype before subjecting them to in vitro functional assays to evaluate cytotoxicity, cytokine production, and persistence. In vivo efficacy was evaluated in MHC class I/II deficient NSG mice bearing human mesothelioma xenografts.ResultsIn vitro, co-expression of the IL-15fu led to similar cytotoxicity and cytokine production as TC-210, but notably enhanced T-cell expansion and persistence upon repeated stimulation with MSLN+ cell lines. Furthermore, the IL-15fu-enhanced TRuC-T cells sustained a significantly higher TCF-1+ population and retained a stem-like phenotype following activation. Moreover, the IL-15fu-enhanced TRuCs demonstrated robust in vivo expansion and intra-tumoral accumulation as measured by ex vivo analysis of TRuC+ cells in the tumor and blood, with a preferential expansion of CD8+ T cells. Finally, IL-15fu-enhanced TRuC-T cells could be observed in the blood long after the tumors were cleared.ConclusionsThese pre-clinical studies suggest that the IL-15fu can synergize with TC-210 to increase the potency and durability of response in patients with MSLN+ tumors.Ethics ApprovalAll animal studies were approved by the respective Institutional Animal Care and Use Committees.

scholarly journals Vγ9Vδ2 T cells as a promising innovative tool for immunotherapy of hematologic malignancies

2011 ◽  
Vol 4 (4) ◽  
pp. 211
Author(s):  
Serena Meraviglia ◽  
Carmela La Mendola ◽  
Valentina Orlando ◽  
Francesco Scarpa ◽  
Giuseppe Cicero ◽  
...  
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Ex Vivo ◽  
Γδ T Cells ◽  
Hematologic Malignancies ◽  
Cytolytic Activity ◽  
Cell Therapies ◽  
Stressed Cells

The potent anti-tumor activities of γδ T cells, their ability to produce pro-inflammatory cytokines, and their strong cytolytic activity have prompted the development of protocols in which γδ agonists or ex vivo-expanded γδ cells are administered to tumor patients. γδ T cells can be selectively activated by either synthetic phosphoantigens or by drugs that enhance their accumulation into stressed cells as aminobisphosphonates, thus offering new avenues for the development of γδ T cell-based immunotherapies. The recent development of small drugs selectively activating Vγ9Vδ2 T lymphocytes, which upregulate the endogenous phosphoantigens, has enabled the investigators to design the experimental approaches of cancer immunotherapies; several ongoing phase I and II clinical trials are focused on the role of the direct bioactivity of drugs and of adoptive cell therapies involving phosphoantigen- or aminobisphosphonate-activated Vγ9Vδ2 T lymphocytes in humans. In this review, we focus on the recent advances in the activation/expansion of γδ T cells in vitro and in vivo that may represent a promising target for the design of novel and highly innovative immunotherapy in patients with hematologic malignancies.<br />

scholarly journals Nuclear factor of activated T cells 2 is required for osteoclast differentiation and function in vitro but not in vivo

2018 ◽  
Vol 119 (11) ◽  
pp. 9334-9345 ◽  
Author(s):  
Jungeun Yu ◽  
Stefano Zanotti ◽  
Lauren Schilling ◽  
Ernesto Canalis
Keyword(s):  
T Cells ◽  
Osteoclast Differentiation ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
And Function

scholarly journals IL-2 administration increases CD4+CD25hi Foxp3+ regulatory T cells in cancer patients

Blood ◽  
2006 ◽  
Vol 107 (6) ◽  
pp. 2409-2414 ◽  
Author(s):  
Mojgan Ahmadzadeh ◽  
Steven A. Rosenberg
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Interleukin 2 ◽  
Selective Inhibition ◽  
High Dose ◽  
Protein Levels ◽  
And Function ◽  
The Impact

Abstract Interleukin-2 (IL-2) is historically known as a T-cell growth factor. Accumulating evidence from knockout mice suggests that IL-2 is crucial for the homeostasis and function of CD4+CD25+ regulatory T cells in vivo. However, the impact of administered IL-2 in an immune intact host has not been studied in rodents or humans. Here, we studied the impact of IL-2 administration on the frequency and function of human CD4+CD25hi T cells in immune intact patients with melanoma or renal cancer. We found that the frequency of CD4+CD25hi T cells was significantly increased after IL-2 treatment, and these cells expressed phenotypic markers associated with regulatory T cells. In addition, both transcript and protein levels of Foxp3, a transcription factor exclusively expressed on regulatory T cells, were consistently increased in CD4 T cells following IL-2 treatment. Functional analysis of the increased number of CD4+CD25hi T cells revealed that this population exhibited potent suppressive activity in vitro. Collectively, our results demonstrate that administration of high-dose IL-2 increased the frequency of circulating CD4+CD25hi Foxp3+ regulatory T cells. Our findings suggest that selective inhibition of IL-2-mediated enhancement of regulatory T cells may improve the therapeutic effectiveness of IL-2 administration. (Blood. 2006;107:2409-2414)

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