24 results on '"Levings, Megan K."'
Search Results
2. Interactions between islets and regulatory immune cells in health and type 1 diabetes
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Budd, Matthew A., Monajemi, Mahdis, Colpitts, Sarah J., Crome, Sarah Q., Verchere, C. Bruce, and Levings, Megan K.
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- 2021
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3. Eras of designer Tregs: Harnessing synthetic biology for immune suppression.
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Tuomela, Karoliina, Salim, Kevin, and Levings, Megan K.
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IMMUNOSUPPRESSION ,REGULATORY T cells ,SYNTHETIC biology ,ANTIGEN receptors ,T cells - Abstract
Summary: Since their discovery, CD4+CD25hiFOXP3hi regulatory T cells (Tregs) have been firmly established as a critical cell type for regulating immune homeostasis through a plethora of mechanisms. Due to their immunoregulatory power, delivery of polyclonal Tregs has been explored as a therapy to dampen inflammation in the settings of transplantation and autoimmunity. Evidence shows that Treg therapy is safe and well‐tolerated, but efficacy remains undefined and could be limited by poor persistence in vivo and lack of antigen specificity. With the advent of new genetic engineering tools, it is now possible to create bespoke "designer" Tregs that not only overcome possible limitations of polyclonal Tregs but also introduce new features. Here, we review the development of designer Tregs through the perspective of three 'eras': (1) the era of FOXP3 engineering, in which breakthroughs in the biological understanding of this transcription factor enabled the conversion of conventional T cells to Tregs; (2) the antigen‐specificity era, in which transgenic T‐cell receptors and chimeric antigen receptors were introduced to create more potent and directed Treg therapies; and (3) the current era, which is harnessing advanced genome‐editing techniques to introduce and refine existing and new engineering approaches. The year 2022 marked the entry of "designer" Tregs into the clinic, with exciting potential for application and efficacy in a wide variety of immune‐mediated diseases. [ABSTRACT FROM AUTHOR]
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- 2023
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4. Targeting regulatory T cell metabolism in disease: Novel therapeutic opportunities.
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Halvorson, Torin, Tuomela, Karoliina, and Levings, Megan K.
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REGULATORY T cells ,CELL metabolism ,AUTOIMMUNE diseases ,IMMUNOSUPPRESSION ,GENOME editing ,DRUG therapy - Abstract
Regulatory T cells (Tregs) are essential for immune homeostasis and suppression of pathological autoimmunity but can also play a detrimental role in cancer progression via inhibition of anti‐tumor immunity. Thus, there is broad applicability for therapeutic Treg targeting, either to enhance function, for example, through adoptive cell therapy (ACT), or to inhibit function with small molecules or antibody‐mediated blockade. For both of these strategies, the metabolic state of Tregs is an important consideration since cellular metabolism is intricately linked to function. Mounting evidence has shown that targeting metabolic pathways can selectively promote or inhibit Treg function. This review aims to synthesize the current understanding of Treg metabolism and discuss emerging metabolic targeting strategies in the contexts of transplantation, autoimmunity, and cancer. We discuss approaches to gene editing and cell culture to manipulate Treg metabolism during ex vivo expansion for ACT, as well as in vivo nutritional and pharmacological interventions to modulate Treg metabolism in disease states. Overall, the intricate connection between metabolism and phenotype presents a powerful opportunity to therapeutically tune Treg function. [ABSTRACT FROM AUTHOR]
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- 2023
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5. Acidity promotes the differentiation of immunosuppressive regulatory T cells.
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Tuomela, Karoliina and Levings, Megan K
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REGULATORY T cells ,LACTIC acid ,T cells ,CELL populations ,ACIDITY - Abstract
The metabolic milieu is emerging as a major contributing factor in the maintenance of the immunosuppressive microenvironment within tumors. In particular, the presence of lactic acid produced by highly glycolytic cancer cells is known to suppress antitumor immune cell subsets while promoting immunosuppressive cell populations, such as regulatory T cells (Tregs). Unlike conventional T cells, Tregs have a unique, potent ability to take up lactic acid to fuel both mitochondrial metabolism and gluconeogenesis, thus supporting suppressive function and proliferation. In this issue of the European Journal of Immunology [Eur. J. Immunol. 2023.53:2250258], Rao et al. uncover a novel mechanism by which lactic acid can support Treg accumulation within tumors in mice. This study shows that lactic acid, through a pH‐dependent mechanism rather than lactate itself, promotes TGFβ‐induced differentiation of Tregs from conventional CD4+ T cells. These findings build on the already multifaceted role of lactic acid in maintaining an immunosuppressive tumor microenvironment. [ABSTRACT FROM AUTHOR]
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- 2023
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6. PTEN is required for human Treg suppression of costimulation in vitro.
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Lam, Avery J., Haque, Manjurul, Ward‐Hartstonge, Kirsten A., Uday, Prakruti, Wardell, Christine M., Gillies, Jana K., Speck, Madeleine, Mojibian, Majid, Klein Geltink, Ramon I., and Levings, Megan K.
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GRAFT versus host disease ,GRAFT rejection ,GENOME editing ,REGULATORY T cells ,T cells ,AUTOIMMUNE diseases - Abstract
Regulatory T‐cell (Treg) therapy is under clinical investigation for the treatment of transplant rejection, autoimmune disease, and graft‐versus‐host disease. With the advent of genome editing, attention has turned to reinforcing Treg function for therapeutic benefit. A hallmark of Tregs is dampened activation of PI3K‐AKT signaling, of which PTEN is a major negative regulator. Loss‐of‐function studies of PTEN, however, have not conclusively shown a requirement for PTEN in upholding Treg function and stability. Using CRISPR‐based genome editing in human Tregs, we show that PTEN ablation does not cause a global defect in Treg function and stability; rather, it selectively blocks their ability to suppress antigen‐presenting cells. PTEN‐KO Tregs exhibit elevated glycolytic activity, upregulate FOXP3, maintain a Treg phenotype, and have no discernible defects in lineage stability. Functionally, PTEN is dispensable for human Treg‐mediated inhibition of T‐cell activity in vitro and in vivo but is required for suppression of costimulatory molecule expression by antigen‐presenting cells. These data are the first to define a role for a signaling pathway in controlling a subset of human Treg activity. Moreover, they point to the functional necessity of PTEN‐regulated PI3K‐AKT activity for optimal human Treg function. [ABSTRACT FROM AUTHOR]
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- 2022
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7. Helios is a marker, not a driver, of human Treg stability.
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Lam, Avery J., Uday, Prakruti, Gillies, Jana K., and Levings, Megan K.
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REGULATORY T cells ,IMMUNOLOGICAL tolerance ,T cells ,TRANSCRIPTION factors ,IMMUNE response - Abstract
Treg therapy holds promise as a potentially curative approach to establish immune tolerance in transplantation and autoimmune disease. An outstanding question is whether therapeutic Tregs have the potential to transdifferentiate into effector T‐cells and, thus, exacerbate rather than suppress immune responses. In mice, the transcription factor Helios is thought to promote Treg lineage stability in a range of inflammatory contexts. In humans, the role of Helios in Tregs is less clear, in part, due to the inability to enrich and study subsets of Helios‐positive versus Helios‐negative Tregs. Using an in vitro expansion system, we found that loss of high Helios expression and emergence of an intermediate Helios (Heliosmid)‐expressing population correlated with Treg destabilization. We used CRISPR/Cas9 to genetically ablate Helios expression in human naive or memory Tregs and found that Helios‐KO and unedited Tregs were equivalent in their suppressive function and stability in inflammation. Thus, high Helios expression is a marker, but not a driver, of human Treg stability in vitro. These data highlight the importance of monitoring Helios expression in therapeutic Treg manufacturing and provide new insight into the biological function of this transcription factor in human T‐cells. [ABSTRACT FROM AUTHOR]
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- 2022
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8. The parasite cytokine mimic Hp‐TGM potently replicates the regulatory effects of TGF‐β on murine CD4+ T cells.
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White, Madeleine P J, Smyth, Danielle J, Cook, Laura, Ziegler, Steven F, Levings, Megan K, and Maizels, Rick M
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REGULATORY T cells ,CYTOKINES ,T cells ,HOMEOSTASIS ,GENE expression profiling ,IMMUNE system ,INTERLEUKIN-7 - Abstract
Transforming growth factor‐beta (TGF‐β) family proteins mediate many vital biological functions in growth, development and regulation of the immune system. TGF‐β itself controls immune homeostasis and inflammation, including conversion of naïve CD4+ T cells into Foxp3+ regulatory T cells (Tregs) in the presence of interleukin‐2 and T‐cell receptor ligands. The helminth parasite Heligmosomoides polygyrus exploits this pathway through a structurally novel TGF‐β mimic (Hp‐TGM), which binds to mammalian TGF‐β receptors and induces Tregs. Here, we performed detailed comparisons of Hp‐TGM with mammalian TGF‐β. Compared with TGF‐β, Hp‐TGM induced greater numbers of Foxp3+ Tregs (iTregs), with more intense Foxp3 expression. Both ligands upregulated Treg functional markers CD73, CD103 and programmed death‐ligand 1, but Hp‐TGM induced significantly higher CD39 expression than did TGF‐β. Interestingly, in contrast to canonical TGF‐β signaling through Smad2/3, Hp‐TGM stimulation was slower and more sustained. Gene expression profiles induced by TGF‐β and Hp‐TGM were remarkably similar, and both types of iTregs suppressed T‐cell responses in vitro and experimental autoimmune encephalomyelitis‐driven inflammation in vivo. In vitro, both types of iTregs were equally stable under inflammatory conditions, but Hp‐TGM‐induced iTregs were more stable in vivo during dextran sodium sulfate‐induced colitis, with greater retention of Foxp3 expression and lower conversion to a ROR‐γt+ phenotype. Altogether, results from this study suggest that the parasite cytokine mimic, Hp‐TGM, may deliver a qualitatively different signal to CD4+ T cells with downstream consequences for the long‐term stability of iTregs. These data highlight the potential of Hp‐TGM as a new modulator of T‐cell responses in vitro and in vivo. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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9. Induction of stable human FOXP3+ Tregs by a parasite‐derived TGF‐β mimic.
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Cook, Laura, Reid, Kyle T, Häkkinen, Elmeri, de Bie, Brett, Tanaka, Shigeru, Smyth, Danielle J, White, Madeleine PJ, Wong, May Q, Huang, Qing, Gillies, Jana K, Ziegler, Steven F., Maizels, Rick M, and Levings, Megan K
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INFLAMMATORY bowel diseases ,REGULATORY T cells ,T helper cells ,THERAPEUTICS ,T cells ,TH1 cells - Abstract
Immune homeostasis in the intestine is tightly controlled by FOXP3+ regulatory T cells (Tregs), defects of which are linked to the development of chronic conditions, such as inflammatory bowel disease (IBD). As a mechanism of immune evasion, several species of intestinal parasites boost Treg activity. The parasite Heligmosomoides polygyrus is known to secrete a molecule (Hp‐TGM) that mimics the ability of TGF‐β to induce FOXP3 expression in CD4+ T cells. The study aimed to investigate whether Hp‐TGM could induce human FOXP3+ Tregs as a potential therapeutic approach for inflammatory diseases. CD4+ T cells from healthy volunteers were expanded in the presence of Hp‐TGM or TGF‐β. Treg induction was measured by flow cytometric detection of FOXP3 and other Treg markers, such as CD25 and CTLA‐4. Epigenetic changes were detected using ChIP‐Seq and pyrosequencing of FOXP3. Treg phenotype stability was assessed following inflammatory cytokine challenge and Treg function was evaluated by cellular co‐culture suppression assays and cytometric bead arrays for secreted cytokines. Hp‐TGM efficiently induced FOXP3 expression (> 60%), in addition to CD25 and CTLA‐4, and caused epigenetic modification of the FOXP3 locus to a greater extent than TGF‐β. Hp‐TGM‐induced Tregs had superior suppressive function compared with TGF‐β‐induced Tregs, and retained their phenotype following exposure to inflammatory cytokines. Furthermore, Hp‐TGM induced a Treg‐like phenotype in in vivo differentiated Th1 and Th17 cells, indicating its potential to re‐program memory cells to enhance immune tolerance. These data indicate Hp‐TGM has potential to be used to generate stable human FOXP3+ Tregs to treat IBD and other inflammatory diseases. [ABSTRACT FROM AUTHOR]
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- 2021
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10. Cross talk between human regulatory T cells and antigen‐presenting cells: Lessons for clinical applications.
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Wardell, Christine M, MacDonald, Katherine N, Levings, Megan K, and Cook, Laura
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SUPPRESSOR cells ,CROSSTALK ,INFLAMMATORY bowel diseases ,HUMAN biology ,TYPE 1 diabetes - Abstract
Regulatory T cells (Tregs) have a critical role in maintaining self‐tolerance and immune homeostasis. There is much interest in using Tregs as a cell therapy to re‐establish tolerance in conditions such as inflammatory bowel disease and type 1 diabetes, with many ongoing clinical studies testing the safety and efficacy of this approach. Manufacturing of Tregs for therapy typically involves ex vivo expansion to obtain sufficient cell numbers for infusion and comes with the risk of altering the activity of key biological processes. However, this process also offers an opportunity to tailor Treg function to maximize in vivo activity. In this review, we focus on the roles of antigen‐presenting cells (APCs) in the generation and function of Tregs in humans. In addition to stimulating the development of Tregs, APCs activate Tregs and provide signals that induce specialized functional and homing marker expression. Cross talk between Tregs and APCs is a critical, often under‐appreciated, aspect of Treg biology, with APCs mediating the key properties of infectious tolerance and bystander suppression. Understanding the biology of human Treg‐APC interactions will reveal new ways to optimize Treg‐based therapeutic approaches. [ABSTRACT FROM AUTHOR]
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- 2021
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11. Ectopic germline recombination activity of the widely used Foxp3‐YFP‐Cre mouse: a case report.
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Wu, Dan, Huang, Qing, Orban, Paul C., and Levings, Megan K.
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DELETION mutation ,INSULIN receptors ,MICE ,TRANSGENIC mice ,SUPPRESSOR cells - Abstract
Summary: Regulatory T (Treg) cell‐specific deletion of a gene of interest is a procedure widely used to study mechanisms controlling Treg development, homeostasis and function. Accordingly, several transgenic mouse lines have been generated that bear the Cre recombinase under control of the Foxp3 promoter either as a random transgene insertion or knocked into the endogenous Foxp3 locus, with the Foxp3YFP‐Cre strain of mice being one of the most widely used. In an attempt to generate Treg cells that lacked expression of the insulin receptor (Insr), we crossed Foxp3YFP‐Cre mice with Insrfl/fl mice. Using a conventional two‐band PCR genotyping method we found that offspring genotypes did not correspond to the expected Mendelian ratios. We therefore developed a quantitative PCR‐based genotyping method to investigate possible ectopic recombination outside the Treg lineage. With this method we found that ~50% of the F1‐generation mice showed evidence of ectopic recombination and that ~10% of the F2‐generation mice had germline Cre recombination activity leading to a high frequency of offspring with global Insr deletion. Use of the quantitative PCR genotyping method enabled accurate selection of mice without ectopic recombination and only the desired Treg cell‐specific Insr deletion. Our data highlight the need to use genotyping methods that allow for assessment of possible ectopic recombination driven by the Foxp3YFP‐Cre allele, particularly when studying genes that are systemically expressed. [ABSTRACT FROM AUTHOR]
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- 2020
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12. An optimized method to measure human FOXP3+ regulatory T cells from multiple tissue types using mass cytometry.
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Dawson, Nicholas A. J., Lam, Avery J., Cook, Laura, Hoeppli, Romy E., Broady, Raewyn, Pesenacker, Anne M., and Levings, Megan K.
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- 2018
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13. Engineered Tolerance: Tailoring Development, Function, and Antigen-Specificity of Regulatory T Cells.
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Dawson, Nicholas A. J., Vent-Schmidt, Jens, and Levings, Megan K.
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TISSUE engineering ,T cells ,IMMUNE response - Abstract
Regulatory T cells (Tregs) are potent suppressors of immune responses and are currently being clinically tested for their potential to stop or control undesired immune responses in autoimmunity, hematopoietic stem cell transplantation, and solid organ transplantation. Current clinical approaches aim to boost Tregs in vivo either by using Treg-promoting small molecules/proteins and/or by adoptive transfer of expanded Tregs. However, the applicability of Treg-based immunotherapies continues to be hindered by technical limitations related to cell isolation and expansion of a pure, well-characterized, and targeted Treg product. Efforts to overcome these limitations and improve Treg-directed therapies are now under intense investigation in animal models and pre-clinical studies. Here, we review cell and protein engineering-based approaches that aim to target different aspects of Treg biology including modulation of IL-2 signaling or FOXP3 expression, and targeted antigen-specificity using transgenic T cell receptors or chimeric antigen receptors. With the world-wide interest in engineered T cell therapy, these exciting new approaches have the potential to be rapidly implemented and developed into therapies that can effectively fine-tune immune tolerance. [ABSTRACT FROM AUTHOR]
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- 2017
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14. The environment of regulatory T cell biology: cytokines, metabolites, and the microbiome.
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Hoeppli, Romy E., Dan Wu, Cook, Laura, and Levings, Megan K.
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HUMAN T cells ,FORKHEAD transcription factors ,PHYSIOLOGICAL effects of cytokines ,METABOLITES ,PHYSIOLOGICAL effects of tretinoin ,MICROORGANISMS - Abstract
Regulatory T cells (Tregs) are suppressive T cells that have an essential role in maintaining the balance between immune activation and tolerance. Their development, either in the thymus, periphery, or experimentally in vitro, and stability and function all depend on the right mix of environmental stimuli.This reviewfocuses on the effects of cytokines, metabolites, and the microbiome on both human and mouse Treg biology. The role of cytokines secreted by innate and adaptive immune cells in directing Treg development and shaping their function iswell established. Newand emerging data suggest that metabolites, such as retinoic acid, and microbial products, such as short-chain fatty acids, also have a critical role in guiding the functional specialization of Tregs. Overall, the complex interaction between distinct environmental stimuli results in unique, and in some cases tissue-specific, tolerogenic environments. Understanding the conditions that favorTreg induction, accumulation, and function is critical to defining the pathophysiology of many immune-mediated diseases and to developing new therapeutic interventions. [ABSTRACT FROM AUTHOR]
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- 2015
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15. Control of tissue-localized immune responses by human regulatory T cells.
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Pesenacker, Anne M., Broady, Raewyn, and Levings, Megan K.
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Treg cells control immune responses to self and nonharmful foreign antigens. Emerging data from animal models indicate that Treg cells function in both secondary lymphoid organs and tissues, and that these different microenvironments may contain specialized subsets of Treg cells with distinct mechanisms of action. The design of therapies for the restoration of tissue-localized immune homeostasis is dependent upon understanding how local immune responses are influenced by Treg cells in health versus disease. Here we review the current state of knowledge about human Treg cells in four locations: the skin, lung, intestine, and joint. Despite the distinct biology of these tissues, there are commonalities in the biology of their resident Treg cells, including phenotypic and functional differences from circulating Treg cells, and the presence of cytokine-producing (e.g. IL-17
+ ) FOXP3+ cells. We also highlight the challenges to studying tissue Treg cells in humans, and opportunities to use new technologies for the detailed analysis of Treg cells at the single-cell level. As emerging biological therapies are increasingly targeted toward tissue-specific effects, it is critical to understand their potential impact on local immune regulation. [ABSTRACT FROM AUTHOR]- Published
- 2015
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16. Interleukin-10-secreting type 1 regulatory T cells in rodents and humans.
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Roncarolo, Maria Grazia, Gregori, Silvia, Battaglia, Manuela, Bacchetta, Rosa, Fleischhauer, Katharina, and Levings, Megan K.
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INTERLEUKIN-10 ,INTERLEUKINS ,T cells ,LYMPHOCYTES ,IMMUNE response - Abstract
Interleukin-10 (IL-10)-secreting T regulatory type 1 (Tr1) cells are defined by their specific cytokine production profile, which includes the secretion of high levels of IL-10 and transforming growth factor-β(TGF-β), and by their ability to suppress antigen-specific effector T-cell responses via a cytokine-dependent mechanism. In contrast to the naturally occurring CD4
+ CD25+ T regulatory cells (Tregs) that emerge directly from the thymus, Tr1 cells are induced by antigen stimulation via an IL-10-dependent process in vitro and in vivo. Specialized IL-10-producing dendritic cells, such as those in an immature state or those modulated by tolerogenic stimuli, play a key role in this process. We propose to use the term Tr1 cells for all IL-10-producing T-cell populations that are induced by IL-10 and have regulatory activity. The full biological characterization of Tr1 cells has been hampered by the difficulty in generating these cells in vitro and by the lack of specific marker molecules. However, it is clear that Tr1 cells play a key role in regulating adaptive immune responses both in mice and in humans. Further work to delineate the specific molecular signature of Tr1 cells, to determine their relationship with CD4+ CD25+ Tregs, and to elucidate their respective role in maintaining peripheral tolerance is crucial to advance our knowledge on this Treg subset. Furthermore, results from clinical protocols using Tr1 cells to modulate immune responses in vivo in autoimmunity, transplantation, and chronic inflammatory diseases will undoubtedly prove the biological relevance of these cells in immunotolerance. [ABSTRACT FROM AUTHOR]- Published
- 2006
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17. Optimized CRISPR-mediated gene knockin reveals FOXP3-independent maintenance of human Treg identity.
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Lam, Avery J., Lin, David T.S., Gillies, Jana K., Uday, Prakruti, Pesenacker, Anne M., Kobor, Michael S., and Levings, Megan K.
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Regulatory T cell (Treg) therapy is a promising curative approach for a variety of immune-mediated conditions. CRISPR-based genome editing allows precise insertion of transgenes through homology-directed repair, but its use in human Tregs has been limited. We report an optimized protocol for CRISPR-mediated gene knockin in human Tregs with high-yield expansion. To establish a benchmark of human Treg dysfunction, we target the master transcription factor FOXP3 in naive and memory Tregs. Although FOXP3-ablated Tregs upregulate cytokine expression, effects on suppressive capacity in vitro manifest slowly and primarily in memory Tregs. Moreover, FOXP3-ablated Tregs retain their characteristic protein, transcriptional, and DNA methylation profile. Instead, FOXP3 maintains DNA methylation at regions enriched for AP-1 binding sites. Thus, although FOXP3 is important for human Treg development, it has a limited role in maintaining mature Treg identity. Optimized gene knockin with human Tregs will enable mechanistic studies and the development of tailored, next-generation Treg cell therapies. [Display omitted] • Optimized method for human Treg expansion with CRISPR-mediated gene knockin • FOXP3 ablation causes more pronounced defects in memory Tregs than naive Tregs • FOXP3-KO Tregs retain a Treg-type protein, transcript, and DNA methylation profile • FOXP3 maintains DNA methylation at regions enriched for AP-1 binding sites Lam et al. optimize efficient CRISPR-based targeted gene integration in naive and memory human Tregs. Using this method, the authors find FOXP3 has subset-specific roles in regulating mature Treg function and a limited role in maintaining lineage identity. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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18. Building a CAR-Treg: Going from the basic to the luxury model.
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Rosado-Sánchez, Isaac and Levings, Megan K.
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HEMATOPOIETIC stem cell transplantation , *STEM cell transplantation , *SUPPRESSOR cells , *CHIMERIC antigen receptors , *CELLULAR therapy - Abstract
• Advances in use of chimeric antigen receptors (CARs) in Tregs are reviewed. • Factors to consider when creating CAR-Tregs are discussed. • How progress in CAR-T cell design can be applied to Tregs is discussed. Regulatory T cells (Tregs) control immune homeostasis and prevent exacerbated immune responses, and can be used as cell therapy to dampen a variety of autoimmune or autoinflammatory responses. Treg therapy is significantly more effective if the cells are antigen-specific. One way to re-direct the specificity of Tregs is to engineer them to express a Chimeric Antigen Receptor (CAR). Proof-of-concept studies have shown the potential for "basic" models of CAR-Tregs to be used as cellular therapy in autoimmunity, organ transplantation and hematopoietic stem cell transplantation. In parallel, work in the context of cancer has significantly advanced knowledge of how to optimise CAR-T cell structure and function for more precise and potent function. In this review, we summarize the current state of knowledge about important considerations when generating CAR-Tregs. We also extrapolate from emerging findings with CAR-T cells about strategies to further improve CAR-Treg function, creating "luxury" models with refined activity. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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19. Tregs with an MHC class II peptide-specific chimeric antigen receptor prevent autoimmune diabetes in mice.
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Spanier, Justin A., Fung, Vivian, Wardell, Christine M., Alkhatib, Mohannad H., Yixin Chen, Swanson, Linnea A., Dwyer, Alexander J., Weno, Matthew E., Silva, Nubia, Mitchell, Jason S., Orban, Paul C., Mojibian, Majid, Verchere, C. Bruce, Fife, Brian T., and Levings, Megan K.
- Subjects
- *
CHIMERIC antigen receptors , *T cell receptors , *REGULATORY T cells , *TYPE 1 diabetes , *T cells , *PEPTIDES - Abstract
Adoptive immunotherapy with Tregs is a promising approach for preventing or treating type 1 diabetes. Islet antigen-specific Tregs have more potent therapeutic effects than polyclonal cells, but their low frequency is a barrier for clinical application. To generate Tregs that recognize islet antigens, we engineered a chimeric antigen receptor (CAR) derived from a monoclonal antibody with specificity for the insulin B chain 10-23 peptide presented in the context of the IAg7 MHC class II allele present in NOD mice. Peptide specificity of the resulting InsB-g7 CAR was confirmed by tetramer staining and T cell proliferation in response to recombinant or islet-derived peptide. The InsB-g7 CAR redirected NOD Treg specificity such that insulin B 10-23-peptide stimulation enhanced suppressive function, measured via reduction of proliferation and IL-2 production by BDC2.5 T cells and CD80 and CD86 expression on dendritic cells. Cotransfer of InsB-g7 CAR Tregs prevented adoptive transfer diabetes by BDC2.5 T cells in immunodeficient NOD mice. In WT NOD mice, InsB-g7 CAR Tregs prevented spontaneous diabetes. These results show that engineering Treg specificity for islet antigens using a T cell receptor-like CAR is a promising therapeutic approach for the prevention of autoimmune diabetes. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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20. Regulatory T cells produce profibrotic cytokines in the skin of patients with systemic sclerosis.
- Author
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MacDonald, Katherine G., Dawson, Nicholas A.J., Huang, Qing, Dunne, James V., Levings, Megan K., and Broady, Raewyn
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Background Systemic sclerosis (SSc) is an autoimmune disorder characterized by fibrosis of the skin and internal organs. Pathologic conversion of regulatory T (Treg) cells into inflammatory cytokine–producing cells is thought to be an important step in the progression of autoimmunity, but whether loss of normal Treg cell function contributes to SSc is unknown. Objective We sought to determine whether Treg cells in the blood and skin of patients with SSc acquired abnormal production of effector cytokines. Methods Peripheral blood and skin biopsy specimens were collected from control subjects and patients with limited or diffuse SSc. Flow cytometry was used to evaluate expression of cell-surface proteins and the cytokine production profile of forkhead box protein 3–positive Treg cells compared with forkhead box protein 3–negative conventional T cells. Results Treg cells in the blood of patients with SSc had a normal phenotype and did not produce T-effector cytokines. In contrast, Treg cells from skin affected by SSc produced significant amounts of IL-4 and IL-13. Although Treg cells in the blood of patients with SSc did not make T H 2 cytokines, they contained a significantly higher proportion of skin-homing cells expressing T H 2 cell–associated chemokine receptors. Evidence that IL-33 caused the differentiation of skin Treg cells into T H 2-like cells, combined with high tissue-localized expression of this cytokine in patients with SSc and expression of the ST2 chain of the IL-33 receptor on skin-localized Treg cells, suggests that IL-33 might be an important stimulator of tissue-localized loss of normal Treg cell function. Conclusion These data are the first evidence for the presence of T H 2-like Treg cells in human autoimmunity and show that Treg cell plasticity can be tissue specific. Localized dysfunction of Treg cells is a previously unknown factor that might contribute to fibrosis in patients with SSc. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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21. Manufacturing next-generation regulatory T-cell therapies.
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MacDonald, Katherine N, Salim, Kevin, and Levings, Megan K
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REGULATORY T cells , *HEMATOPOIETIC stem cells , *PLURIPOTENT stem cells , *GENETIC overexpression , *GRAFT versus host disease , *GENETIC engineering - Abstract
Regulatory T-cell (Treg) therapy has shown promise in treating autoimmune diseases, transplant rejection, or graft-versus-host disease in early clinical trials. These trials have demonstrated that cell therapy using polyclonal Tregs is feasible and safe, however, the field has been limited by the lack of polyclonal cell specificity and consequent large cell numbers required, and the difficulty in generating autologous products for some patients. Thus, the field is moving toward 'next generation' Treg cell therapies that include genetic modification strategies to engineer specificity and/or modify function, as well as methods to generate Tregs in vitro. In this review, we describe how genetic modification of Tregs using viral transduction or gene editing may be incorporated into Treg manufacturing protocols. We also describe how Tregs may be generated via FOXP3 gene editing or overexpression, or by differentiation from pluripotent stem cells. The application of these various types of engineered Tregs is discussed. • 'Next generation' Treg cell therapy products will use genetic engineering to enhance function. • Methods to scale up Treg cell therapy products depend on cell source and type of genetic engineering. • Genetic engineering of Tregs can be achieved through viral or non-viral means. • Therapeutic Tregs can be made from conventional T cells or hematopoietic stem cells. • Future therapeutic Tregs could be made from pluripotent stem cells. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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22. Consequences of adjusting cell density and feed frequency on serum-free expansion of thymic regulatory T cells.
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MacDonald, Katherine N., Hall, Michael G., Ivison, Sabine, Gandhi, Sanjiv, Klein Geltink, Ramon I., Piret, James M., and Levings, Megan K.
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REGULATORY T cells , *NUTRIENT density , *SERUM-free culture media , *T cells , *IMMUNOGLOBULINS , *DENSITY - Abstract
Given the promising results from phase 1/2 clinical trials of therapy involving regulatory T cells (Tregs), it is critical to develop Treg manufacturing methods that use well-defined reagents. Seeking to maximize expansion of human thymic Tregs activated with anti-CD3/CD28 antibody-coated beads and cultured in serum-free medium, the authors investigated the effect of adjusting process parameters including cell density and cell concentration, and feeding strategy on Treg yield and quality. The authors found that levels of expansion and viability varied with cell density on the day of restimulation. Tregs restimulated at low cell densities (1 × 105 cells/cm2) initially had high growth rates, viability and FOXP3 expression, but these parameters decreased with time and were less stable than those observed in cultures of Tregs restimulated at high cell densities (5 × 105 cells/cm2), which had slower growth rates. High-density expansion was associated with expression of inhibitory molecules and lower intracellular oxygen and extracellular nutrient concentrations as well as extracellular lactate accumulation. Experiments to test the effect of low oxygen revealed that transient exposure to low oxygen levels had little impact on expansion, viability or phenotype. Similarly, blockade of inhibitory molecules had little effect. By contrast, replenishing nutrients by increasing the feeding frequency between 2 days and 4 days after restimulation increased FOXP3, viability and expansion in high-density cultures. These data show the previously undescribed consequences of adjusting cell density on Treg expansion and establish a Good Manufacturing Practice-relevant protocol using non-cell-based activation reagents and serum-free media that supports sustained expansion without loss of viability or phenotype. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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23. Flagellin-specific human CAR Tregs for immune regulation in IBD.
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Boardman, Dominic A., Wong, May Q., Rees, William D., Wu, Dan, Himmel, Megan E., Orban, Paul C., Vent-Schmidt, Jens, Zachos, Nicholas C., Steiner, Theodore S., and Levings, Megan K.
- Subjects
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REGULATORY T cells , *INFLAMMATORY bowel diseases , *BACTERIAL flagella , *CHIMERIC antigen receptors , *CYTOSKELETAL proteins - Abstract
Regulatory T cell (Treg) therapy is a promising strategy to treat inflammatory bowel disease (IBD). Data from animal models has shown that Tregs specific for intestinal antigens are more potent than polyclonal Tregs at inhibiting colitis. Flagellins, the major structural proteins of bacterial flagella, are immunogenic antigens frequently targeted in IBD subjects, leading to the hypothesis that flagellin-specific Tregs could be an effective cell therapy for IBD. We developed a novel chimeric antigen receptor (CAR) specific for flagellin derived from Escherichia coli H18 (FliC). We used this CAR to confer FliC-specificity to human Tregs and investigated their therapeutic potential. FliC-CAR Tregs were activated by recombinant FliC protein but not a control flagellin protein, demonstrating CAR specificity and functionality. In a humanized mouse model, expression of the FliC-CAR drove preferential migration to the colon and expression of the activation marker PD1. In the presence of recombinant FliC protein in vitro , FliC-CAR Tregs were significantly more suppressive than control Tregs and promoted the establishment of colon-derived epithelial cell monolayers. These results demonstrate the potential of FliC-CAR Tregs to treat IBD and more broadly show the therapeutic potential of CARs targeting microbial-derived antigens. • Chimeric antigen receptors (CAR) can be used to confer microbe specificity to Tregs. • Flagellin-specific CAR Tregs preferentially home to damaged intestinal tissues. • CAR Tregs elicit antigen-dependent immunosuppression. • Activated CAR Tregs secrete factors that promote intestinal barrier integrity. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
24. A method for expansion and retroviral transduction of mouse regulatory T cells.
- Author
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Wu, Dan, Wong, May Q., Vent-Schmidt, Jens, Boardman, Dominic A., Steiner, Theodore S., and Levings, Megan K.
- Subjects
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SUPPRESSOR cells , *GENETIC transduction , *TRANSGENE expression , *CHIMERIC antigen receptors , *GRAFT rejection - Abstract
Adoptive cell therapy with genetically modified regulatory T cells (Tregs) is under clinical investigation for the treatment of transplant rejection and various autoimmune conditions. A limitation of modelling this approach in mice is the lack of optimized protocols for expanding and transducing mouse Tregs. Here we describe a protocol for purifying, expanding and retrovirally transducing mouse Tregs with a vector encoding a chimeric antigen receptor as a model transgene. We found that isolation of Tregs from C57Bl/6J Foxp3E GFP mice solely based on eGFP expression resulted in sufficiently pure cells; co-sorting of CD25hi cells was not essential. Although expansion with rapamycin reduced Treg expansion, it promoted maximal in vitro suppressive activity. Retroviral transduction of Tregs following 2 days of stimulation with anti-CD3/CD28 beads achieved a transduction efficiency of ~40% and did not impair their suppressive capacity. When injected into a conventional T cell (Tconv)-transfer-induced colitis model, transduced Tregs inhibited colitis progression at ratios as low as 1 Treg to 100 Tconvs, and maintained Foxp3 and transgene expression throughout an 8-week period. This method facilitates the study of transduced Tregs in animal models and will enable the study of genetically engineered Treg therapy for a variety of inflammatory diseases. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
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