60 results on '"Levings, Megan K."'
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2. Ustekinumab for type 1 diabetes in adolescents: a multicenter, double-blind, randomized phase 2 trial
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Tatovic, Danijela, Marwaha, Ashish, Taylor, Peter, Hanna, Stephanie J., Carter, Kym, Cheung, W. Y., Luzio, Steve, Dunseath, Gareth, Hutchings, Hayley A., Holland, Gail, Hiles, Steve, Fegan, Greg, Williams, Evangelia, Yang, Jennie H. M., Domingo-Vila, Clara, Pollock, Emily, Wadud, Muntaha, Ward-Hartstonge, Kirsten, Marques-Jones, Susie, Bowen-Morris, Jane, Stenson, Rachel, Levings, Megan K., Gregory, John W., Tree, Timothy I. M., and Dayan, Colin
- Abstract
Immunotherapy targeting the autoimmune process in type 1 diabetes (T1D) can delay the loss of β-cells but needs to have minimal adverse effects to be an adjunct to insulin in the management of T1D. Ustekinumab binds to the shared p40 subunit of interleukin (IL)-12 and IL-23, targeting development of T helper 1 cells and T helper 17 cells (TH1 and TH17 cells) implicated in the pathogenesis of T1D. We conducted a double-blind, randomized controlled trial of ustekinumab in 72 adolescents aged 12–18 years with recent-onset T1D. Treatment was well tolerated with no increase in adverse events. At 12 months, β-cell function, measured by stimulated C-peptide, was 49% higher in the intervention group (P= 0.02), meeting the prespecified primary outcome. Preservation of C-peptide correlated with the reduction of T helper cells co-secreting IL-17A and interferon-γ (TH17.1 cells, P= 0.04) and, in particular, with the reduction in a subset of TH17.1 cells co-expressing IL-2 and granulocyte–macrophage colony-stimulating factor (IL-2+GM-CSF+TH17.1 cells, P= 0.04). A significant fall in β-cell-targeted (proinsulin-specific) IL-17A-secreting T cells was also seen (P= 0.0003). Although exploratory, our data suggest a role for an activated subset of TH17.1 cells in T1D that can be targeted with minimal adverse effects to reduce C-peptide loss, which requires confirmation in a larger study. (International Standard Randomised Controlled Trial Number Registry: ISRCTN 14274380).
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- 2024
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3. Regulatory T Cell Biomarkers Identify Patients at Risk of Developing Acute Cellular Rejection in the First Year Following Heart Transplantation
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Kim, Ji-Young V., Assadian, Sara, Hollander, Zsuzsanna, Burns, Paloma, Shannon, Casey P., Lam, Karen, Toma, Mustafa, Ignaszewski, Andrew, Davies, Ross A., Delgado, Diego, Haddad, Haissam, Isaac, Debra, Kim, Daniel, Mui, Alice, Rajda, Miroslaw, West, Lori, White, Michel, Zieroth, Shelley, Keown, Paul A., McMaster, W. Robert, Ng, Raymond T., McManus, Bruce M., Levings, Megan K., and Tebbutt, Scott J.
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- 2023
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4. Preclinical assessment of antigen-specific chimeric antigen receptor regulatory T cells for use in solid organ transplantation
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Proics, Emma, David, Marion, Mojibian, Majid, Speck, Madeline, Lounnas-Mourey, Nadia, Govehovitch, Adeline, Baghdadi, Wissam, Desnouveaux, Justine, Bastian, Hervé, Freschi, Laura, Privat, Geoffrey, Pouzet, Cédric, Grossi, Mauro, Heimendinger, Pierre, Abel, Tobias, Fenard, David, Levings, Megan K., Meyer, François, and Dumont, Céline
- Abstract
A primary goal in transplantation medicine is the induction of a tolerogenic environment for prevention of transplant rejection without the need for long-term pharmacological immunosuppression. Generation of alloantigen-specific regulatory T cells (Tregs) by transduction with chimeric antigen receptors (CARs) is a promising strategy to achieve this goal. This publication reports the preclinical characterization of Tregs (TR101) transduced with a human leukocyte antigen (HLA)-A*02 CAR lentiviral vector (TX200) designated to induce immunosuppression of allograft-specific effector T cells in HLA-A*02-negative recipients of HLA-A*02-positive transplants. In vitro results demonstrated specificity, immunosuppressive function, and safety of TX200-TR101. In NOD scidgamma (NSG) mice, TX200-TR101 prevented graft-versus-host disease (GvHD) in a xenogeneic GvHD model and TX200-TR101 Tregs localized to human HLA-A*02-positive skin transplants in a transplant model. TX200-TR101 persisted over the entire duration of a 3-month study in humanized HLA-A*02 NSG mice and remained stable, without switching to a proinflammatory phenotype. Concomitant tacrolimus did not impair TX200-TR101 Treg survival or their ability to inhibit peripheral blood mononuclear cell (PBMC) engraftment. These data demonstrate that TX200-TR101 is specific, stable, efficacious, and safe in preclinical models, and provide the basis for a first-in-human study.
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- 2023
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5. A Century-long Journey From the Discovery of Insulin to the Implantation of Stem Cell–derived Islets
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Ramzy, Adam, Belmonte, Paul J, Braam, Mitchell J S, Ida, Shogo, Wilts, Emily M, Levings, Megan K, Rezania, Alireza, and Kieffer, Timothy J
- Abstract
For the past century, insulin injections have saved millions of lives, but glycemic instability is still a persistent challenge for people with diabetes, leading to tremendous morbidity and premature mortality. Research in the field of islet transplantation has demonstrated that replacing insulin-producing β cells can restore euglycemia comparable to individuals without diabetes. However, a short supply of cadaveric islet donors, the technically challenging process of isolating islets, and the requirement for chronic immune suppression have impeded widespread clinical adoption. Rather than relying on cadaveric cells, pluripotent stem cells could serve as a virtually unlimited supply of insulin-producing β cells. Protocols have been developed that mimic the normal in vivo development of the human pancreas to generate pancreatic progenitor cells in vitro. Ongoing investigations have yielded progressively more mature β-like cells in vitro that produce insulin but do not yet fully mimic healthy mature β cells. Alongside development of differentiation protocols, other work has provided insight into potential implantation sites for stem cell–derived islet cells including the subcutaneous space, portal vein, and omentum. To optimize implanted cell survival and function, development of immune modulation therapies is ongoing, including selection of immunomodulatory medications and genetic modification of implanted cells to evade immune responses. Further, macroencapsulation or microencapsulation devices could be used to contain and/or immunoprotect implanted cells from the immune response including by using 3-dimensional bioprinting to facilitate the process. Remarkably, ongoing clinical trials have now yielded the first patient relying on differentiated stem cells rather than syringes as their insulin replacement therapy.
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- 2023
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6. Alleviating graft-versus-host disease by directing regulatory T cells to the gut … It is all about location, location, location
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Haque, Manjurul, Boardman, Dominic A., and Levings, Megan K.
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- 2023
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7. Emerging strategies for treating autoimmune disorders with genetically modified Treg cells.
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Boardman, Dominic A. and Levings, Megan K.
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Gene editing of living cells is a cornerstone of present-day medical research that has enabled scientists to address fundamental biologic questions and identify novel strategies to treat diseases. The ability to manipulate adoptive cell therapy products has revolutionized cancer immunotherapy and promises similar results for the treatment of autoimmune diseases, inflammatory disorders, and transplant rejection. Clinical trials have recently deemed polyclonal regulatory T (Treg) cell therapy to be a safe therapeutic option, but questions remain regarding the efficacy of this approach. In this review, we discuss how gene editing technologies are being applied to transform the future of Treg cell therapy, focusing on the preclinical strategies that are currently being investigated to enhance the efficacy, function, and survival of human Treg cells. We explore approaches that may be used to generate immunoregulatory cells ex vivo , detail emerging strategies that are being used to modify these cells (such as using chimeric antigen receptors to confer antigen specificity), and outline concepts that have been explored to repurpose conventional T cells to target and destroy autoreactive and alloreactive lymphocytes. We also describe the key hurdles that currently hinder the clinical adoption of Treg cell therapy and propose potential future avenues of research for this field. [ABSTRACT FROM AUTHOR]
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- 2022
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8. Suppressive and Gut-Reparative Functions of Human Type 1 T Regulatory Cells.
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Cook, Laura, Stahl, Martin, Han, Xiao, Nazli, Aisha, MacDonald, Katherine N., Wong, May Q., Tsai, Kevin, Dizzell, Sara, Jacobson, Kevan, Bressler, Brian, Kaushic, Charu, Vallance, Bruce A., Steiner, Theodore S., and Levings, Megan K.
- Abstract
T-regulatory (Treg) cells suppress the immune response to maintain homeostasis. There are 2 main subsets of Treg cells: FOXP3 (forkhead box protein 3)-positive Treg cells, which do not produce high levels of effector cytokines, and type 1 Treg (Tr1) cells, which are FOXP3-negative and secrete interleukin (IL) 10. IL10 is an anti-inflammatory cytokine, so Tr1 cells might be used in the treatment of inflammatory bowel diseases. We aimed to develop methods to isolate and expand human Tr1 cells and define their functions. We obtained blood and colon biopsy samples from patients with Crohn's disease or ulcerative colitis or healthy individuals (controls). CD4
+ T cells were isolated from blood samples and stimulated with anti-CD3 and anti-CD28 beads, and Tr1 cells were purified by using an IL10 cytokine-capture assay and cell sorting. FOXP3-positive Treg cells were sorted as CD4+ CD25high CD127low cells from unstimulated cells. Tr1 and FOXP3-positive Treg cells were expanded, and phenotypes and gene expression profiles were compared. T cells in peripheral blood mononuclear cells from healthy donors were stimulated with anti-CD3 and anti-CD28 beads, and the suppressive abilities of Tr1 and FOXP3-positive Treg cells were measured. Human colon organoid cultures were established, cultured with supernatants from Tr1 or FOXP3-positive cells, and analyzed by immunofluorescence and flow cytometry. T84 cells (human colon adenocarcinoma epithelial cells) were incubated with supernatants from Tr1 or FOXP3-positive cells, and transepithelial electrical resistance was measured to determine epithelial cell barrier function. Phenotypes of Tr1 cells isolated from control individuals vs patients with Crohn's disease or ulcerative colitis did not differ significantly after expansion. Tr1 cells and FOXP3-positive Treg cells suppressed proliferation of effector T cells, but only Tr1 cells suppressed secretion of IL1B and tumor necrosis factor from myeloid cells. Tr1 cells, but not FOXP3-positive Treg cells, isolated from healthy individuals and patients with Crohn's disease or ulcerative colitis secreted IL22, which promoted barrier function of human intestinal epithelial cells. Tr1 cell culture supernatants promoted differentiation of mucin-producing goblet cells in intestinal organoid cultures. Human Tr1 cells suppress proliferation of effector T cells (adaptive immune response) and production of IL1B and TNF by myeloid cells (inmate immune response). They also secrete IL22 to promote barrier function. They might be developed as a cell-based therapy for intestinal inflammatory disorders. [ABSTRACT FROM AUTHOR]- Published
- 2019
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9. Donor‐specific chimeric antigen receptor Tregs limit rejection in naive but not sensitized allograft recipients
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Sicard, Antoine, Lamarche, Caroline, Speck, Madeleine, Wong, May, Rosado‐Sánchez, Isaac, Blois, Mathilde, Glaichenhaus, Nicolas, Mojibian, Majid, and Levings, Megan K.
- Abstract
Cell therapy with autologous donor‐specific regulatory T cells (Tregs) is a promising strategy to minimize immunosuppression in transplant recipients. Chimeric antigen receptor (CAR) technology has recently been used successfully to generate donor‐specific Tregs and overcome the limitations of enrichment protocols based on repetitive stimulations with alloantigens. However, the ability of CAR‐Treg therapy to control alloreactivity in immunocompetent recipients is unknown. We first analyzed the effect of donor‐specific CAR Tregs on alloreactivity in naive, immunocompetent mice receiving skin allografts. Tregs expressing an irrelevant or anti‐HLA‐A2‐specific CAR were administered to Bl/6 mice at the time of transplanting an HLA‐A2+Bl/6 skin graft. Donor‐specific CAR‐Tregs, but not irrelevant‐CAR Tregs, significantly delayed skin rejection and diminished donor‐specific antibodies (DSAs) and frequencies of DSA‐secreting B cells. Donor‐specific CAR‐Treg–treated mice also had a weaker recall DSA response, but normal responses to an irrelevant antigen, demonstrating antigen‐specific suppression. When donor‐specific CAR Tregs were tested in HLA‐A2‐sensitized mice, they were unable to delay allograft rejection or diminish DSAs. The finding that donor‐specific CAR‐Tregs restrain de novo but not memory alloreactivity has important implications for their use as an adoptive cell therapy in transplantation. Donor‐specific, chimeric antigen receptor regulatory T cells can diminish skin graft rejection and humoral alloreactivity in naïve but not sensitized immunocompetent mice.
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- 2020
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10. First-in-human therapy with Treg produced from thymic tissue (thyTreg) in a heart transplant infant
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Bernaldo-de-Quirós, Esther, Camino, Manuela, Martínez-Bonet, Marta, Gil-Jaurena, Juan Miguel, Gil, Nuria, Hernández-Flórez, Diana, Fernández-Santos, Maria Eugenia, Butragueño, Laura, Dijke, I. Esmé, Levings, Megan K., West, Lori J., Pion, Marjorie, and Correa-Rocha, Rafael
- Abstract
Due to their suppressive capacity, regulatory T cells (Tregs) have attracted growing interest as an adoptive cellular therapy for the prevention of allograft rejection, but limited Treg recovery and lower quality of adult-derived Tregs could represent an obstacle to success. To address this challenge, we developed a new approach that provides large quantities of Tregs with high purity and excellent features, sourced from thymic tissue routinely removed during pediatric cardiac surgeries (thyTregs). We report on a 2-year follow-up of the first patient treated worldwide with thyTregs, included in a phase I/II clinical trial evaluating the administration of autologous thyTreg in infants undergoing heart transplantation. In addition to observing no adverse effects that could be attributed to thyTreg administration, we report that the Treg frequency in the periphery was preserved during the 2-year follow-up period. These initial results are consistent with the trial objective, which is to confirm safety of the autologous thyTreg administration and its capacity to restore the Treg pool.
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- 2023
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11. Antigen-specific regulatory T cells: are police CARs the answer?
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Dawson, Nicholas A.J. and Levings, Megan K.
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Cellular therapy with T-regulatory cells (Tregs) is a promising strategy to control immune responses and restore immune tolerance in a variety of immune-mediated diseases, such as transplant rejection and autoimmunity. Multiple clinical trials are currently testing this approach, typically by infusing a single dose of polyclonal Tregs that have been expanded in vitro. However, evidence from animal models of Treg therapy has clearly shown that antigen-specific Tregs are vastly superior to polyclonal cells, meaning that fewer cells are needed for the desired therapeutic effect. Traditional methods to obtain antigen-specific Tregs include antigen-stimulated expansion or T-cell receptor (TCR) overexpression. However, these methods are limited by low cell numbers, complex manufacturing procedures, and knowledge of patient-specific TCRs which recognize disease-relevant MHC-peptide complexes. Recently, several groups have explored the potential to use chimeric antigen receptors (CARs) to generate antigen-specific Tregs. Here, we discuss the progress in this field and highlight the major outstanding questions that remain to be addressed as this approach moves toward clinical applications. [ABSTRACT FROM AUTHOR]
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- 2017
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12. Engineering therapeutic T cells to suppress alloimmune responses using TCRs, CARs, or BARs
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Sicard, Antoine, Levings, Megan K., and Scott, David W.
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Adoptive cell therapy with therapeutic T cells has become one of the most promising strategies to stimulate or suppress immune responses. Using virus‐mediated genetic manipulation, the antigen specificity of T cells can now be precisely redirected. Tailored specificity has not only overcome technical limitations and safety concerns but also considerably broadened the spectrum of therapeutic applications. Different T cell–engineering strategies have now become available to suppress alloimmune responses. We first provide an overview of the allorecognition pathways and effector mechanisms that are responsible for alloimmune injuries in the setting of vascularized organ transplantation. We then discuss the potential to use different T cell–engineering approaches to suppress alloimmune responses. Specifically, expression of allospecific T cell receptors, single‐chain chimeric antigen receptors, or antigen domains recognized by B cell receptors (B cell antibody receptors) in regulatory or cytotoxic T cells are considered. The ability of these strategies to control the direct or indirect pathways of allorecognition and the cellular or humoral alloimmune responses is discussed. An intimate understanding of the complex interplay that occurs between the engineered T cells and the alloimmune players is a necessary prerequisite for the design of safe and successful strategies for precise immunomodulation in transplantation. This review discusses the potential use of different T cell engineering approaches to suppress alloimmune responses.
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- 2018
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13. Engineering therapeutic T cells to suppress alloimmune responses using TCRs, CARs, or BARs
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Sicard, Antoine, Levings, Megan K., and Scott, David W.
- Abstract
Adoptive cell therapy with therapeutic T cells has become one of the most promising strategies to stimulate or suppress immune responses. Using virus-mediated genetic manipulation, the antigen specificity of T cells can now be precisely redirected. Tailored specificity has not only overcome technical limitations and safety concerns but also considerably broadened the spectrum of therapeutic applications. Different T cell–engineering strategies have now become available to suppress alloimmune responses. We first provide an overview of the allorecognition pathways and effector mechanisms that are responsible for alloimmune injuries in the setting of vascularized organ transplantation. We then discuss the potential to use different T cell–engineering approaches to suppress alloimmune responses. Specifically, expression of allospecific T cell receptors, single-chain chimeric antigen receptors, or antigen domains recognized by B cell receptors (B cell antibody receptors) in regulatory or cytotoxic T cells are considered. The ability of these strategies to control the direct or indirect pathways of allorecognition and the cellular or humoral alloimmune responses is discussed. An intimate understanding of the complex interplay that occurs between the engineered T cells and the alloimmune players is a necessary prerequisite for the design of safe and successful strategies for precise immunomodulation in transplantation.
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- 2018
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14. Guiding regulatory T cells to the allograft
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Lamarche, Caroline and Levings, Megan K.
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- 2018
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15. Harnessing Advances in T Regulatory Cell Biology for Cellular Therapy in Transplantation
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Lam, Avery J., Hoeppli, Romy E., and Levings, Megan K.
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Cellular therapy with CD4+FOXP3+T regulatory (Treg) cells is a promising strategy to induce tolerance after solid-organ transplantation or prevent graft-versus-host disease after transfer of hematopoietic stem cells. Treg cells currently used in clinical trials are either polyclonal, donor- or antigen-specific. Aside from variations in isolation and expansion protocols, however, most therapeutic Treg cell-based products are much alike. Ongoing basic science work has provided considerable new insight into multiple facets of Treg cell biology, including their stability, homing, and functional specialization; integrating these basic science discoveries with clinical efforts will support the development of next-generation therapeutic Treg cells with enhanced efficacy. In this review, we summarize recent advances in knowledge of how Treg cells home to lymphoid and peripheral tissues, and control antibody production and tissue repair. We also discuss newly appreciated pathways that modulate context-specific Treg cell function and stability. Strategies to improve and tailor Treg cells for cell therapy to induce transplantation tolerance are highlighted.A timely review of recent developments in regulatory T cell biology and how this information may be used to improve their utility in inducing transplantation tolerance in the clinic.
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- 2017
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16. Thymic progenitors of TCRαβ+ CD8αα intestinal intraepithelial lymphocytes require RasGRP1 for development
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Golec, Dominic P., Hoeppli, Romy E., Henao Caviedes, Laura M., McCann, Jillian, Levings, Megan K., and Baldwin, Troy A.
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Strong T cell receptor (TCR) signaling largely induces cell death during thymocyte development, whereas weak TCR signals induce positive selection. However, some T cell lineages require strong TCR signals for differentiation through a process termed agonist selection. The signaling relationships that underlie these three fates are unknown. RasGRP1 is a Ras activator required to transmit weak TCR signals leading to positive selection. Here, we report that, despite being dispensable for thymocyte clonal deletion, RasGRP1 is critical for agonist selection of TCRαβ+CD8αα intraepithelial lymphocyte (IEL) progenitors (IELps), even though both outcomes require strong TCR signaling. Bim deficiency rescued IELp development in RasGRP1−/− mice, suggesting that RasGRP1 functions to promote survival during IELp generation. Additionally, expression of CD122 and the adhesion molecules α4β7 and CD103 define distinct IELp subsets with differing abilities to generate TCRαβ+CD8αα IEL in vivo. These findings demonstrate that RasGRP1-dependent signaling underpins thymic selection processes induced by both weak and strong TCR signals and is differentially required for fate decisions derived from a strong TCR stimulus.
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- 2017
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17. Effect of Ex Vivo–Expanded Recipient Regulatory T Cells on Hematopoietic Chimerism and Kidney Allograft Tolerance Across MHC Barriers in Cynomolgus Macaques
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Duran-Struuck, Raimon, Sondermeijer, Hugo P., Bühler, Leo, Alonso-Guallart, Paula, Zitsman, Jonah, Kato, Yojiro, Wu, Anette, McMurchy, Alicia N., Woodland, David, Griesemer, Adam, Martinez, Mercedes, Boskovic, Svetlan, Kawai, Tatsuo, Cosimi, A. Benedict, Wuu, Cheng-Shie, Slate, Andrea, Mapara, Markus Y., Baker, Sam, Tokarz, Rafal, D'Agati, Vivette, Hammer, Scott, Pereira, Marcus, Lipkin, W. Ian, Wekerle, Thomas, Levings, Megan K., and Sykes, Megan
- Abstract
Duran-Struuck and colleagues describe the generation of polyclonally expanded CD4+CD25high Treg from Cynomolgus monkeys, and show that these Treg promote multilineage allogeneic chimerism and robust tolerance, if the effects of early cytomegalovirus activation and anti-viral drug toxicities are ameliorated. Supplemental digital content is available in the text.
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- 2017
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18. Isolation, Expansion, and Characterization of Human Natural and Adaptive Regulatory T Cells.
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Walker, John M., Fairchild, Paul J., Gregori, Silvia, Bacchetta, Rosa, Passerini, Laura, Levings, Megan K., and Roncarolo, Maria Grazia
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Regulatory T cells play a central role in controlling homeostasis, and in inducing and maintaining tolerance to both foreign and self-antigens. Several types of T cells with regulatory activity have been described both in mice and humans, and those within the CD4+ subset have been extensively studied. Among them, the best characterized are the naturally occurring CD4+CD25+ regulatory T (Treg) cells, and the adaptive type 1 regulatory T (Tr1) cells. Natural Treg cells can arise directly from the thymus, are characterized by the constitutive expression of the transcription factor Foxp3, and suppress T cell responses in a cell-cell contact mediated mechanism. On the contrary, adaptive Tr1 cells arise in the periphery upon encountering antigen in a tolerogenic environment, produce high levels of interleukin (IL)-10 and mediate suppression via IL-10. During the last decade, much effort has been placed on developing protocols to generate regulatory T-cell lines and clones, to further define the similarities and differences between various regulatory T-cell subsets. In this chapter, we will outline protocols to expand naturally occurring Treg cells, to differentiate homogeneous population of Tr1 cells in vitro, and to generate natural Treg and Tr1 cell clones and cell lines. [ABSTRACT FROM AUTHOR]
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- 2007
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19. CCL22 Prevents Rejection of Mouse Islet Allografts and Induces Donor-Specific Tolerance
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Montane, Joel, Obach, Merce, Alvarez, Sigrid, Bischoff, Loraine, Dai, Derek L., Soukhatcheva, Galina, Priatel, John J., Hardenberg, Gijs, Levings, Megan K., Tan, Rusung, Orban, Paul C., and Verchere, C. Bruce
- Abstract
Manipulation of regulatory T cell (Treg) migration by islet expression of the chemokine CCL22 prevents diabetes in NOD mice and delays recurrent autoimmunity in syngeneic islet transplants. We sought to determine whether attracting Tregs with CCL22 also prevents islet allograft rejection. Isolated Bl/6 mouse islets were transduced overnight with adenovirus expressing CCL22 (Ad-CCL22) downstream of the CMV promoter. Islets were transplanted under the renal capsule of Balb/c recipients made diabetic by streptozotocin. To assess immunologic tolerance, graft-bearing kidneys from recipients of CCL22-expressing islet grafts were removed, and mice received a second transplant of naive islets from the same donor strain or third-party islets into the contralateral kidney. Adenoviral expression of CCL22 conferred prolonged protection of islet allografts in MHC-mismatched, diabetic recipients, maintaining normoglycemia in 75% of recipients for at least 80 days. Increased frequency of Treg cells was observed in islet grafts transduced with Ad-CCL22 compared with untreated grafts. Normoglycemic recipients of CCL22-expressing islet grafts showed complete absence of antidonor antibodies and no lymphocyte proliferation after exposure to donor splenocytes. After removal of the primary graft at day 80, mice that received a second transplant with untreated islets from the same donor strain did not reject the grafts, suggesting the development of tolerance. Expression of CCL22 recruits Treg cells to transplanted islets, prevents activation of alloreactive T-cells and islet allograft failure and induces alloantigen-specific tolerance. Manipulation of Treg cells by CCL22 in transplanted islets may be a novel therapeutic strategy for diabetes.
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- 2015
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20. 213.2: Preliminary Results of the First Clinical Trial to Prevent Graft Rejection in Heart Transplant Children Employing a Cellular Therapy With Autologous Treg Obtained From Thymic Tissue (thyTreg)
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Bernaldo-de-Quirós, Esther, Camino, Manuela, Miguel Gil-Jaurena, Juan, Gil, Nuria, López-Esteban, Rocío, Martínez-Bonet, Marta, Hernández-Flórez, Diana, Fernández-Santos, María Eugenia, Butrageño, Laura, Levings, Megan K, Dijke, Esme I, West, Lori J, Pion, Marjorie, and Correa-Rocha, Rafael
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- 2022
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21. 216.6: Regulatory T Cell Biomarkers Identify Patients at Risk of Developing Acute Cellular Rejection in the First Year Following Heart Transplantation
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Kim, Ji-Young, Levings, Megan K, and Tebbutt, Scott J
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- 2022
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22. Point mutants of forkhead box P3 that cause immune dysregulation, polyendocrinopathy, enteropathy, X-linked have diverse abilities to reprogram T cells into regulatory T cells.
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McMurchy, Alicia N., Gillies, Jana, Allan, Sarah E., Passerini, Laura, Gambineri, Eleonora, Roncarolo, Maria Grazia, Bacchetta, Rosa, and Levings, Megan K.
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GENETIC mutation ,FORKHEAD transcription factors ,INTESTINAL diseases ,T cells ,IMMUNE response ,IMMUNOLOGIC diseases ,IMMUNODEFICIENCY ,TRANSCRIPTION factors - Abstract
Background: Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) is a primary immunodeficiency with autoimmunity caused by mutations in forkhead box P3 (FOXP3), which encodes a transcription factor involved in regulatory T (Treg) cell function. The mechanistic basis for how different mutations in FOXP3 cause distinct manifestations of IPEX remains unclear. Objective: To determine whether 3 different point mutants of FOXP3 that cause severe or mild IPEX differ in their ability to reprogram conventional T cells into Treg cells. Methods: Human CD4
+ T cells were transduced with wild-type or point mutant forms of FOXP3, and changes in cell surface marker expression, cytokine production, proliferation and suppressive capacity were assessed. Ex vivo TH 17 cells were also transduced with different forms of FOXP3 to monitor changes in IL-17 production. Results: The forkhead mutant F373A failed to upregulate CD25 and CCR4, did not suppress cytokine production, and induced suppressive activity less effectively than wild-type FOXP3. In contrast, although the forkhead mutant R347H was also defective in upregulation of CD25, it suppressed the production of cytokines, conferred suppressive capacity on CD4+ T cells, and suppressed IL-17 production. F324L, a mutant outside the forkhead domain associated with mild IPEX, was equivalent to wild-type FOXP3 in all aspects tested. Conclusion: Mutations in FOXP3 that cause IPEX do not uniformly abrogate the ability of FOXP3 to regulate transcription and drive the development of Treg cells. These data support the notion that factors in addition to functional changes in Treg cells, such as alterations in conventional T cells, are involved in the pathogenesis of IPEX. [ABSTRACT FROM AUTHOR]- Published
- 2010
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23. Functional Dynamics of Naturally Occurring Regulatory T Cells in Health and Autoimmunity.
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Levings, Megan K., Allan, Sarah, D'Hennezel, Eva, and Piccirillo, Ciriaco A.
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T cells ,LYMPHOCYTES ,CYTOKINES ,IMMUNOLOGICAL tolerance ,ANIMAL models in research ,AUTOIMMUNE diseases - Abstract
A network of regulatory T (Treg) cells exists to downregulate immune responses in various inflammatory circumstances and ultimately assure peripheral T cell tolerance. Naturally occurring CD4
+ CD25+ Treg cell represents a major lymphocyte population engaged in the dominant control of self-reactive T responses and maintenance of tolerance within this network. CD4+ CD25+ Treg cells differentiate in the normal thymus as a functionally distinct subpopulation of T cells bearing a broad T cell receptor repertoire endowing these cells with the capacity to recognize a wide spectrum of self-Ag and non-self-Ag specificities. The development of CD4+ CD25+ Treg cells is genetically determined, influenced by Ag-specific and nonspecific signals, costimulation, and cytokines that control their activation, expansion, and suppressive activity. Functional abrogation of these cells in vivo, or genetic defects that affect their development or function, unequivocally predisposes animals and humans to the onset of autoimmune and other inflammatory diseases. Studies have shed light in our understanding of the cellular and molecular basis of CD4+ CD25+ Treg cell-mediated immune regulation. In this chapter, we discuss the contribution of naturally occurring CD4+ CD25+ Treg cells in the induction of immunologic self-tolerance in animal models and humans and attempt to provide a comprehensive overview of recent findings regarding the phenotype, functional dynamics, and effector mechanism of these cells in autoimmune diseases. [ABSTRACT FROM AUTHOR]- Published
- 2006
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24. A novel function for FOXP3 in humans: intrinsic regulation of conventional T cells
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McMurchy, Alicia N., Gillies, Jana, Gizzi, Maria Concetta, Riba, Michela, Garcia-Manteiga, Jose Manuel, Cittaro, Davide, Lazarevic, Dejan, Di Nunzio, Sara, Piras, Ignazio S., Bulfone, Alessandro, Roncarolo, Maria Grazia, Stupka, Elia, Bacchetta, Rosa, and Levings, Megan K.
- Abstract
The role of forkhead box P3 (FOXP3) is well-established in T-regulatory cells, but the function of transient FOXP3 expression in activated human conventional T (Tconv) cells is unknown. In the present study, we used 2 approaches to determine the role of FOXP3 in human Tconv cells. First, we obtained Tconv clones from a female subject who is hemizygous for a null mutation in FOXP3, allowing the comparison of autologous T-cell clones that do or do not express FOXP3. Second, we knocked down activation-induced FOXP3 in Tconv cells from healthy donors with small interfering RNA against FOXP3. We found that FOXP3-deficient Tconv cells proliferate more and produce more cytokines than wild-type Tconv cells and have differential expression of 274 genes. We also investigated the role of FOXP3 in Th1 and Th17 cells and found that the expression of activation-induced FOXP3 was higher and more sustained in Th17 cells compared with Th1 cells. Knocking down FOXP3 expression in Th17 cells significantly increased the production of IFN-γ and decreased the expression of CCR4, but had no effect on IL-17 expression. These data reveal a novel function of FOXP3 in Tconv cells and suggest that expression of this protein is important in the function of multiple CD4+ T-cell lineages.
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- 2013
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25. A novel function for FOXP3 in humans: intrinsic regulation of conventional T cells
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McMurchy, Alicia N., Gillies, Jana, Gizzi, Maria Concetta, Riba, Michela, Garcia-Manteiga, Jose Manuel, Cittaro, Davide, Lazarevic, Dejan, Di Nunzio, Sara, Piras, Ignazio S., Bulfone, Alessandro, Roncarolo, Maria Grazia, Stupka, Elia, Bacchetta, Rosa, and Levings, Megan K.
- Abstract
The role of forkhead box P3 (FOXP3) is well-established in T-regulatory cells, but the function of transient FOXP3 expression in activated human conventional T (Tconv) cells is unknown. In the present study, we used 2 approaches to determine the role of FOXP3 in human Tconv cells. First, we obtained Tconv clones from a female subject who is hemizygous for a null mutation in FOXP3, allowing the comparison of autologous T-cell clones that do or do not express FOXP3. Second, we knocked down activation-induced FOXP3 in Tconv cells from healthy donors with small interfering RNA against FOXP3. We found that FOXP3-deficient Tconv cells proliferate more and produce more cytokines than wild-type Tconv cells and have differential expression of 274 genes. We also investigated the role of FOXP3 in Th1 and Th17 cells and found that the expression of activation-induced FOXP3 was higher and more sustained in Th17 cells compared with Th1 cells. Knocking down FOXP3 expression in Th17 cells significantly increased the production of IFN-γ and decreased the expression of CCR4, but had no effect on IL-17 expression. These data reveal a novel function of FOXP3 in Tconv cells and suggest that expression of this protein is important in the function of multiple CD4+T-cell lineages.
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- 2013
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26. Sa449 SERUM ANALYTE PROFILES ASSOCIATED WITH CROHN'S DISEASE AND DISEASE LOCATION.
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Boucher, Gabrielle, Paradis, Alexandre, Chabot-Roy, Geneviève, Coderre, Lise, Hillhouse, Erin E., Bitton, Alain, Rosiers, Christine Des, Levings, Megan K., Schumm, L. Philip, Lazarev, Mark, Brant, Steven R., Duerr, Richard H., Mcgovern, Dermot P.B., Silverberg, Mark S., Cho, Judy H., Lesage, Sylvie, and Rioux, John D.
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- 2021
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27. Fecal Microbiota Transplantation for Recurrent Clostridioides difficile Infection Enhances Adaptive Immunity to C difficile Toxin B.
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Cook, Laura, Rees, William D., Wong, May Q., Peters, Hannah, Levings, Megan K., and Steiner, Theodore S.
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- 2021
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28. T regulatory cell therapy in transplantation
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MacDonald, Katherine G., Orban, Paul C., and Levings, Megan K.
- Abstract
There is great hope that cellular therapy with regulatory T cells (Tregs) will be an effective way to induce alloantigen specific tolerance, ultimately allowing for reduction or elimination of nonspecific immunosuppression. In the past, considerable effort was focused on defining the optimal ways to isolate and expand Tregs from peripheral or cord blood. Now that expansion of therapeutically relevant numbers of Tregs is feasible, we need to consider what is going to happen to the cells when they are transferred in vivo.
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- 2012
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29. Cutaneous GVHD is associated with the expansion of tissue-localized Th1 and not Th17 cells
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Broady, Raewyn, Yu, Jie, Chow, Vickie, Tantiworawit, Adisak, Kang, Christine, Berg, Kyra, Martinka, Magdalena, Ghoreishi, Mehran, Dutz, Jan, and Levings, Megan K.
- Abstract
Studies in mice have shown that proinflammatory Th17 cells can cause acute graft-versus-host disease (aGVHD) related tissue damage; however, whether they play a role in human aGVHD remains unclear. In a prospective study, we measured the proportion of Th17 cells in the blood and skin of patients at the onset of aGVHD. We found no difference in the proportion or amount of IL-17 produced by T cells in the blood of patients with aGVHD (n = 20) compared with time-matched patients without GVHD (n = 14). Moreover, Th17 cells were not increased in the skin of patients with cutaneous aGVHD (n = 7) compared with healthy controls (n = 10). In contrast, we found significantly more interferon-γ–producing T cells in the skin of patients with aGVHD compared with controls. These data support the long-standing paradigm that tissue localized interferon-γ–producing cells are the perpetrators of aGVHD.
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- 2010
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30. Cutaneous GVHD is associated with the expansion of tissue-localized Th1 and not Th17 cells
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Broady, Raewyn, Yu, Jie, Chow, Vickie, Tantiworawit, Adisak, Kang, Christine, Berg, Kyra, Martinka, Magdalena, Ghoreishi, Mehran, Dutz, Jan, and Levings, Megan K.
- Abstract
Studies in mice have shown that proinflammatory Th17 cells can cause acute graft-versus-host disease (aGVHD) related tissue damage; however, whether they play a role in human aGVHD remains unclear. In a prospective study, we measured the proportion of Th17 cells in the blood and skin of patients at the onset of aGVHD. We found no difference in the proportion or amount of IL-17 produced by T cells in the blood of patients with aGVHD (n = 20) compared with time-matched patients without GVHD (n = 14). Moreover, Th17 cells were not increased in the skin of patients with cutaneous aGVHD (n = 7) compared with healthy controls (n = 10). In contrast, we found significantly more interferon-γ–producing T cells in the skin of patients with aGVHD compared with controls. These data support the long-standing paradigm that tissue localized interferon-γ–producing cells are the perpetrators of aGVHD.
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- 2010
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31. ATG-induced expression of FOXP3 in human CD4+ T cells in vitro is associated with T-cell activation and not the induction of FOXP3+ T regulatory cells
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Broady, Raewyn, Yu, Jie, and Levings, Megan K.
- Abstract
Several recent reports have suggested that in vitro exposure of CD4+ T cells to rabbit antithymocyte globulin (rATG), which is commonly used to prevent and treat graft-versus-host disease and allograft rejection, is an effective method to induce CD4+CD25+FOXP3+ T regulatory cells (Tregs). We and others, however, have shown that FOXP3 is also expressed in activated T cells. We therefore investigated whether the induction of FOXP3 expression by rATG resulted in a stable population of suppressive Tregs. We found that exposure of peripheral blood mononuclear cells (PBMCs) or conventional T cells to rATG resulted in induction of transient rather than stable expression of CD25 and FOXP3. Furthermore, rATG-treated T effector cells acquired neither an immunosuppressive profile of cytokine production nor suppressive capacity, even at the time of maximal FOXP3 expression. These findings indicate that the notion that rATG can be used to induce Tregs in vitro for cellular therapy in vivo should be re-evaluated.
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- 2009
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32. ATG-induced expression of FOXP3 in human CD4+T cells in vitro is associated with T-cell activation and not the induction of FOXP3+T regulatory cells
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Broady, Raewyn, Yu, Jie, and Levings, Megan K.
- Abstract
Several recent reports have suggested that in vitro exposure of CD4+T cells to rabbit antithymocyte globulin (rATG), which is commonly used to prevent and treat graft-versus-host disease and allograft rejection, is an effective method to induce CD4+CD25+FOXP3+T regulatory cells (Tregs). We and others, however, have shown that FOXP3 is also expressed in activated T cells. We therefore investigated whether the induction of FOXP3 expression by rATG resulted in a stable population of suppressive Tregs. We found that exposure of peripheral blood mononuclear cells (PBMCs) or conventional T cells to rATG resulted in induction of transient rather than stable expression of CD25 and FOXP3. Furthermore, rATG-treated T effector cells acquired neither an immunosuppressive profile of cytokine production nor suppressive capacity, even at the time of maximal FOXP3 expression. These findings indicate that the notion that rATG can be used to induce Tregs in vitro for cellular therapy in vivo should be re-evaluated.
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- 2009
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33. Wild-type FOXP3 is selectively active in CD4+CD25hi regulatory T cells of healthy female carriers of different FOXP3 mutations
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Di Nunzio, Sara, Cecconi, Massimiliano, Passerini, Laura, McMurchy, Alicia N., Baron, Udo, Turbachova, Ivana, Vignola, Silvia, Valencic, Erica, Tommasini, Alberto, Junker, Anne, Cazzola, Giantonio, Olek, Sven, Levings, Megan K., Perroni, Lucia, Roncarolo, Maria Grazia, and Bacchetta, Rosa
- Abstract
Forkhead box P3 (FOXP3) is constitutively expressed by CD4+CD25hi regulatory T cells (nTregs). Mutations of FOXP3 cause a severe autoimmune syndrome known as immune dysregulation polyendocrinopathy enteropathy X-linked, in which nTregs are absent or dysfunctional. Whether FOXP3 is essential for both differentiation and function of human nTreg cells remains to be demonstrated. Because FOXP3 is an X-linked gene subject to X-chromosome inactivation (XCI), we studied 9 healthy female carriers of FOXP3 mutations to investigate the role of wild-type (WT) versus mutated FOXP3 in different cell subsets. Analysis of active WT versus mutated (mut)–FOXP3 allele distribution revealed a random pattern of XCI in peripheral blood lymphocytes and in naive and memory CD4+T cells, whereas nTregs expressed only the active WT-FOXP3. These data demonstrate that expression of WT-FOXP3 is indispensable for the presence of a normal nTreg compartment and suggest that FOXP3 is not necessary for effector T-cell differentiation in humans.
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- 2009
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34. Wild-type FOXP3is selectively active in CD4+CD25hiregulatory T cells of healthy female carriers of different FOXP3mutations
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Di Nunzio, Sara, Cecconi, Massimiliano, Passerini, Laura, McMurchy, Alicia N., Baron, Udo, Turbachova, Ivana, Vignola, Silvia, Valencic, Erica, Tommasini, Alberto, Junker, Anne, Cazzola, Giantonio, Olek, Sven, Levings, Megan K., Perroni, Lucia, Roncarolo, Maria Grazia, and Bacchetta, Rosa
- Abstract
Forkhead box P3 (FOXP3) is constitutively expressed by CD4+CD25hiregulatory T cells (nTregs). Mutations of FOXP3 cause a severe autoimmune syndrome known as immune dysregulation polyendocrinopathy enteropathy X-linked, in which nTregs are absent or dysfunctional. Whether FOXP3 is essential for both differentiation and function of human nTreg cells remains to be demonstrated. Because FOXP3is an X-linked gene subject to X-chromosome inactivation (XCI), we studied 9 healthy female carriers of FOXP3mutations to investigate the role of wild-type (WT) versus mutated FOXP3 in different cell subsets. Analysis of active WT versus mutated (mut)–FOXP3allele distribution revealed a random pattern of XCI in peripheral blood lymphocytes and in naive and memory CD4+T cells, whereas nTregs expressed only the active WT-FOXP3. These data demonstrate that expression of WT-FOXP3 is indispensable for the presence of a normal nTreg compartment and suggest that FOXP3 is not necessary for effector T-cell differentiation in humans.
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- 2009
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35. Implanted pluripotent stem-cell-derived pancreatic endoderm cells secrete glucose-responsive C-peptide in patients with type 1 diabetes
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Ramzy, Adam, Thompson, David M., Ward-Hartstonge, Kirsten A., Ivison, Sabine, Cook, Laura, Garcia, Rosa V., Loyal, Jackson, Kim, Peter T.W., Warnock, Garth L., Levings, Megan K., and Kieffer, Timothy J.
- Abstract
An open-label, first-in-human phase 1/2 study is being conducted to evaluate the safety and efficacy of pancreatic endoderm cells (PECs) implanted in non-immunoprotective macroencapsulation devices for the treatment of type 1 diabetes. We report an analysis on 1 year of data from the first cohort of 15 patients from a single trial site that received subcutaneous implantation of cell products combined with an immunosuppressive regimen. Implants were well tolerated with no teratoma formation or severe graft-related adverse events. After implantation, patients had increased fasting C-peptide levels and increased glucose-responsive C-peptide levels and developed mixed meal-stimulated C-peptide secretion. There were immunosuppression-related transient increases in circulating regulatory T cells, PD1highT cells, and IL17A+CD4+T cells. Explanted grafts contained cells with a mature β cell phenotype that were immunoreactive for insulin, islet amyloid polypeptide, and MAFA. These data, and associated findings (Shapiro et al., 2021), are the first reported evidence of meal-regulated insulin secretion by differentiated stem cells in patients.
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- 2021
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36. Altered activation of AKT is required for the suppressive function of human CD4+CD25+ T regulatory cells
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Crellin, Natasha K., Garcia, Rosa V., and Levings, Megan K.
- Abstract
Suppression by T regulatory cells (Treg cells) is a major mechanism by which the immune system controls responses to self and nonharmful foreign proteins. Although there are many different types of Treg cells, the best characterized are those that constitutively express cell-surface IL-2Rα (CD25). We investigated whether altered T-cell–receptor (TCR)–mediated signaling in pure populations of ex vivo human CD4+CD25+ Treg cells might underlie their unique phenotype, including hyporesponsiveness to TCR–mediated activation and lack of cytokine production. CD4+CD25+ Treg cells displayed a consistent defect in phosphorylation of AKT at serine 473 and reduced phosphorylation of the AKT substrates FOXO and S6. Restoration of AKT activity via lentiviral-mediated expression of an inducibly active form of the kinase revealed that reduced activity of this pathway was necessary for the suppressive function of CD4+CD25+ Treg cells. These data represent the first demonstration of a causal association between altered signaling and the function of CD4+CD25+ Treg cells. Moreover, we have created the first system allowing inducible abrogation of suppression through manipulation of the suppressor cells. This system will be a powerful tool to further study the mechanism(s) of suppression by CD4+CD25+ Treg cells.
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- 2007
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37. Altered activation of AKT is required for the suppressive function of human CD4+CD25+T regulatory cells
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Crellin, Natasha K., Garcia, Rosa V., and Levings, Megan K.
- Abstract
Suppression by T regulatory cells (Treg cells) is a major mechanism by which the immune system controls responses to self and nonharmful foreign proteins. Although there are many different types of Treg cells, the best characterized are those that constitutively express cell-surface IL-2Rα (CD25). We investigated whether altered T-cell–receptor (TCR)–mediated signaling in pure populations of ex vivo human CD4+CD25+Treg cells might underlie their unique phenotype, including hyporesponsiveness to TCR–mediated activation and lack of cytokine production. CD4+CD25+Treg cells displayed a consistent defect in phosphorylation of AKT at serine 473 and reduced phosphorylation of the AKT substrates FOXO and S6. Restoration of AKT activity via lentiviral-mediated expression of an inducibly active form of the kinase revealed that reduced activity of this pathway was necessary for the suppressive function of CD4+CD25+Treg cells. These data represent the first demonstration of a causal association between altered signaling and the function of CD4+CD25+Treg cells. Moreover, we have created the first system allowing inducible abrogation of suppression through manipulation of the suppressor cells. This system will be a powerful tool to further study the mechanism(s) of suppression by CD4+CD25+Treg cells.
- Published
- 2007
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38. Differentiation of Tr1 cells by immature dendritic cells requires IL-10 but not CD25+CD4+Tr cells
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Levings, Megan K., Gregori, Silvia, Tresoldi, Eleonora, Cazzaniga, Sabrina, Bonini, Chiara, and Roncarolo, Maria Grazia
- Abstract
Dendritic cells (DCs) are specialized antigen-presenting cells that monitor the antigenic environment and activate naive T cells. The role of DCs is not only to sense danger but also to tolerize the immune system to antigens encountered in the absence of maturation/inflammatory stimuli. Indeed, if a naive T cell encounters its antigen on immature DCs (iDCs), it may differentiate into a T-regulatory (Tr) rather than a T-effector cell. However, little is known about the mechanisms by which iDCs differentiate Tr cells. We developed a standardized and highly reproducible protocol to differentiate Tr cells by repetitive exposure of naive peripheral blood CD4+T cells to allogeneic iDCs. The resultant Tr cells are phenotypically and functionally identical to type 1 Tr (Tr1) cells because their generation requires production of IL-10 by iDCs, and they suppress T-cell responses through an interleukin-10 (IL-10)– and a transforming growth factor β (TGF-β)–dependent mechanism. In addition, Tr1 cells induced by iDCs do not require the presence of CD4+CD25+Tr cells for their generation, nor do they express high constitutive levels of CD25 or the transcription factor FoxP3. Thus, iDCs can drive the differentiation of Tr1 cells and can be used to generate large numbers of alloantigen-specific Tr1 cells for clinical use as a cellular therapy to restore peripheral tolerance.
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- 2005
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39. Differentiation of Tr1 cells by immature dendritic cells requires IL-10 but not CD25+CD4+ Tr cells
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Levings, Megan K., Gregori, Silvia, Tresoldi, Eleonora, Cazzaniga, Sabrina, Bonini, Chiara, and Roncarolo, Maria Grazia
- Abstract
Dendritic cells (DCs) are specialized antigen-presenting cells that monitor the antigenic environment and activate naive T cells. The role of DCs is not only to sense danger but also to tolerize the immune system to antigens encountered in the absence of maturation/inflammatory stimuli. Indeed, if a naive T cell encounters its antigen on immature DCs (iDCs), it may differentiate into a T-regulatory (Tr) rather than a T-effector cell. However, little is known about the mechanisms by which iDCs differentiate Tr cells. We developed a standardized and highly reproducible protocol to differentiate Tr cells by repetitive exposure of naive peripheral blood CD4+ T cells to allogeneic iDCs. The resultant Tr cells are phenotypically and functionally identical to type 1 Tr (Tr1) cells because their generation requires production of IL-10 by iDCs, and they suppress T-cell responses through an interleukin-10 (IL-10)– and a transforming growth factor β (TGF-β)–dependent mechanism. In addition, Tr1 cells induced by iDCs do not require the presence of CD4+CD25+ Tr cells for their generation, nor do they express high constitutive levels of CD25 or the transcription factor FoxP3. Thus, iDCs can drive the differentiation of Tr1 cells and can be used to generate large numbers of alloantigen-specific Tr1 cells for clinical use as a cellular therapy to restore peripheral tolerance.
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- 2005
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40. Human CD25+CD4+ T Suppressor Cell Clones Produce Transforming Growth Factor β, but not Interleukin 10, and Are Distinct from Type 1 T Regulatory Cells
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Levings, Megan K., Sangregorio, Romina, Sartirana, Claudia, Moschin, Anna Lisa, Battaglia, Manuela, Orban, Paul C., and Roncarolo, Maria-Grazia
- Abstract
T regulatory (Tr) cells are essential for the induction of peripheral tolerance. Several types of Tr cells exist, including CD4+ T cells which express CD25 constitutively and suppress immune responses via direct cell-to-cell interactions, and type 1 T regulatory (Tr1) cells, which function via secretion of interleukin (IL)-10 and transforming growth factor (TGF)-β. The relationship between CD25+CD4+ T cells and Tr1 cells remains unclear. Here, we demonstrate at the clonal level that Tr1 and CD25+CD4+ T cells are two distinct subsets of regulatory cells with different cytokine production profiles. Furthermore, CD25−CD4+ T cells can be rendered anergic by IL-10 and differentiated into Tr1 cells in the absence of CD25+CD4+ T cells. Cloned human CD25+CD4+ T cell populations are heterogeneous and only a subset of clones continues to express high levels of CD25 and is suppressive. The intensity of CD25, cytotoxic T lymphocyte antigen (CTLA)-4, and glucocorticoid-induced tumor necrosis factor (TNF) receptor expression correlates with the suppressive capacity of the T cell clones. None of the CD25+CD4+ T cell clones with suppressive function produce IL-10, but all produce TGF-β. Suppression mediated by CD25+CD4+ T cell clones is partially dependent on TGF-β, but not on constitutive high expression of CD25. Together these data indicate that naturally occurring human CD25+CD4+ T cells are distinct from IL-10–producing Tr1 cells.
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- 2002
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41. Growth and expansion of human T regulatory type 1 cells are independent from TCR activation but require exogenous cytokines
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Bacchetta, Rosa, Sartirana, Claudia, Levings, Megan K., Bordignon, Claudio, and Narula, Satwant
- Abstract
Cloned T regulatory type 1 (Tr1) cells produce IL-10, TGF-β, IFN-γ, and very low or non-detectable levels of IL-2 and IL-4, following TCR-mediated activation. In addition, upon TCR stimulation, Tr1 cell clones up-regulate activation markers but show low proliferative responses, partially due to the suppressive effect of autocrine IL-10 and TGF-β. Here we show that Tr1 cells have growth requirements different from those of Th1 and Th2 cells. Exogenous IL-15, and to a lesser extent IL-2, induce and support the proliferation of Tr1 cells in the absence of TCR activation. This strong cytokine response correlates with high constitutive levels of the IL-2/15Rβ and common γ chains expressed by Tr1 cell clones. Furthermore, suboptimal doses of IL-15, in combination with IL-2, induce a significant growth (median value: 25-fold increase in cell number) of Tr1 cell clones during a culture period of 11 days, which leads to an in vitro expansion of Tr1 cell clones comparable to that of Th1 and Th2 cell clones. Tr1 cell clones cultured in IL-15 continue to secrete immunosuppressive cytokines and to proliferate poorly upon reactivation via TCR. These findings indicate that Tr1 cells are constitutively capable of responding to cytokines and mainly to IL-15. This growth factor enables a significant in vitro expansion of Tr1 cells facilitating further biological and biochemical characterization of this unique T cell subset.
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- 2002
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42. Human Cd25+Cd4+ T Regulatory Cells Suppress Naive and Memory T Cell Proliferation and Can Be Expanded in Vitro without Loss of Function
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Levings, Megan K., Sangregorio, Romina, and Roncarolo, Maria-Grazia
- Abstract
Active suppression by T regulatory (Tr) cells plays an important role in the downregulation of T cell responses to foreign and self-antigens. Mouse CD4+ Tr cells that express CD25 possess remarkable suppressive activity in vitro and in autoimmune disease models in vivo. Thus far, the existence of a similar subset of CD25+CD4+ Tr cells in humans has not been reported. Here we show that human CD25+CD4+ Tr cells isolated from peripheral blood failed to proliferate and displayed reduced expression of CD40 ligand (CD40L), in response to T cell receptor–mediated polyclonal activation, but strongly upregulated cytotoxic T lymphocyte–associated antigen (CTLA)-4. Human CD25+CD4+ Tr cells also did not proliferate in response to allogeneic antigen-presenting cells, but they produced interleukin (IL)-10, transforming growth factor (TGF)-β, low levels of interferon (IFN)-γ, and no IL-4 or IL-2. Importantly, CD25+CD4+ Tr cells strongly inhibited the proliferative responses of both naive and memory CD4+ T cells to alloantigens, but neither IL-10, TGF-β, nor CTLA-4 seemed to be directly required for their suppressive effects. CD25+CD4+ Tr cells could be expanded in vitro in the presence of IL-2 and allogeneic feeder cells and maintained their suppressive capacities. These findings that CD25+CD4+ Tr cells with immunosuppressive effects can be isolated from peripheral blood and expanded in vitro without loss of function represent a major advance towards the therapeutic use of these cells in T cell–mediated diseases.
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- 2001
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43. Differentiation of T Regulatory Cells by Immature Dendritic Cells
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Roncarolo, Maria-Grazia, Levings, Megan K., and Traversari, Catia
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- 2001
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44. Recurrent Clostridioides difficile Infection Is Associated With Impaired T Helper Type 17 Immunity to C difficile Toxin B.
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Cook, Laura, Rees, William D., Wong, May Q., Kwok, William W., Levings, Megan K., and Steiner, Theodore S.
- Published
- 2021
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45. 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.
- Abstract
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 vitromanifest 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.
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- 2021
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46. Heterodimerization of the ? and ß Chains of the Interleukin-3 (IL-3) Receptor Is Necessary and Sufficient for IL-3–Induced Mitogenesis
- Author
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Orban, Paul C., Levings, Megan K., and Schrader, John W.
- Abstract
The high-affinity receptor for interleukin-3 (IL-3) is a complex of the IL-3–binding subunit (?IL-3) and a larger ß chain—ßc, or, in the mouse, ßc or its close relative ßIL-3. There is evidence that the critical event that initiates signaling is not the approximation of the cytoplasmic domains of ?IL-3 and ßIL-3, but is, rather, the formation of a ß-ß homodimer. Many of these studies involved the analyses of receptor chimeras where the cytoplasmic domains were derived from ?IL-3, ßc or ßIL-3, and the extracellular domains were derived from other cytokine receptors, such as the erythropoietin receptor (EpoR). However, evidence that the EpoR may also associate with other receptors clouds the interpretation of these experiments. Therefore, we reevaluated the structure of the functional IL-3R using chimeric receptors with extracellular domains derived not from members of the cytokine-receptor family, but from CD8 or CD16. We show, by expression of these chimeras in Ba/F3 or CTLL-2 cells, that mitogenic signals were only generated by heterodimerization of the cytoplasmic domains of ?IL-3 and ßIL-3. Homodimers of either ?IL-3 or ßIL-3, alone or in combination, were nonfunctional. Furthermore, the ability of heterodimers to stimulate mitogenesis correlated with their ability to induce tyrosine phosphorylation of JAK-2. These data suggest that the physiological activation of the IL-3R involves the generation of simple heterodimers of ?IL-3 and ßIL-3.
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- 1999
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47. Interleukin-4 Synergizes With Raf-1 to Promote Long-Term Proliferation and Activation of c-jun N-terminal Kinase
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Levings, Megan K., Bessette, Darrell C., and Schrader, John W.
- Abstract
This report shows that interleukin-4 (IL-4), which plays a key role in regulating immune responses, fails to support cellular growth. We investigated whether this failure of IL-4 to promote growth was because of its unique inability to activate the Ras/Raf/Erk pathway. Consistent with other reports, expression in Ba/F3, a factor-dependent hematopoietic cell line, of either activated Q61KN-Ras or a hormone-inducible activated Raf-1, resulted in suppression of apoptosis but not in long-term growth. However, in the presence of IL-4, Ba/F3 cells that expressed either Q61KN-Ras or activated Raf-1 grew continuously at a rate comparable with that stimulated by IL-3. Investigation of the biochemical events associated with the stimulation of long-term growth showed that, as expected, the presence of activated Raf-1 resulted in an increased activity of extracellular signal regulated kinase (ERK) mitogen-activated protein kinase (MAPK) but not of c-jun N-terminal kinase/stress-activated protein kinase (JNK). However, surprisingly, if IL-4 was present, cells expressing active Raf-1 exhibited increases in JNK activity. These observations point to a novel mechanism for JNK activation involving synergy between Raf-1 and pathways activated by IL-4 and suggest that in hematopoietic cells proliferation is correlated not only with “mitogen activated” ERK activity, but also with JNK activity.
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- 1999
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48. Interleukin-4 Synergizes With Raf-1 to Promote Long-Term Proliferation and Activation of c-jun N-terminal Kinase
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Levings, Megan K., Bessette, Darrell C., and Schrader, John W.
- Abstract
This report shows that interleukin-4 (IL-4), which plays a key role in regulating immune responses, fails to support cellular growth. We investigated whether this failure of IL-4 to promote growth was because of its unique inability to activate the Ras/Raf/Erk pathway. Consistent with other reports, expression in Ba/F3, a factor-dependent hematopoietic cell line, of either activated Q61KN-Ras or a hormone-inducible activated Raf-1, resulted in suppression of apoptosis but not in long-term growth. However, in the presence of IL-4, Ba/F3 cells that expressed either Q61KN-Ras or activated Raf-1 grew continuously at a rate comparable with that stimulated by IL-3. Investigation of the biochemical events associated with the stimulation of long-term growth showed that, as expected, the presence of activated Raf-1 resulted in an increased activity of extracellular signal regulated kinase (ERK) mitogen-activated protein kinase (MAPK) but not of c-jun N-terminal kinase/stress-activated protein kinase (JNK). However, surprisingly, if IL-4 was present, cells expressing active Raf-1 exhibited increases in JNK activity. These observations point to a novel mechanism for JNK activation involving synergy between Raf-1 and pathways activated by IL-4 and suggest that in hematopoietic cells proliferation is correlated not only with “mitogen activated” ERK activity, but also with JNK activity.
- Published
- 1999
- Full Text
- View/download PDF
49. Heterodimerization of the α and β Chains of the Interleukin-3 (IL-3) Receptor Is Necessary and Sufficient for IL-3–Induced Mitogenesis
- Author
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Orban, Paul C., Levings, Megan K., and Schrader, John W.
- Abstract
The high-affinity receptor for interleukin-3 (IL-3) is a complex of the IL-3–binding subunit (αIL-3) and a larger β chain—βc, or, in the mouse, βcor its close relative βIL-3. There is evidence that the critical event that initiates signaling is not the approximation of the cytoplasmic domains of αIL-3and βIL-3, but is, rather, the formation of a β-β homodimer. Many of these studies involved the analyses of receptor chimeras where the cytoplasmic domains were derived from αIL-3, βcor βIL-3, and the extracellular domains were derived from other cytokine receptors, such as the erythropoietin receptor (EpoR). However, evidence that the EpoR may also associate with other receptors clouds the interpretation of these experiments. Therefore, we reevaluated the structure of the functional IL-3R using chimeric receptors with extracellular domains derived not from members of the cytokine-receptor family, but from CD8 or CD16. We show, by expression of these chimeras in Ba/F3 or CTLL-2 cells, that mitogenic signals were only generated by heterodimerization of the cytoplasmic domains of αIL-3and βIL-3. Homodimers of either αIL-3or βIL-3, alone or in combination, were nonfunctional. Furthermore, the ability of heterodimers to stimulate mitogenesis correlated with their ability to induce tyrosine phosphorylation of JAK-2. These data suggest that the physiological activation of the IL-3R involves the generation of simple heterodimers of αIL-3and βIL-3.
- Published
- 1999
- Full Text
- View/download PDF
50. Functional effects of chimeric antigen receptor co-receptor signaling domains in human regulatory T cells
- Author
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Dawson, Nicholas A. J., Rosado-Sánchez, Isaac, Novakovsky, German E., Fung, Vivian C. W., Huang, Qing, McIver, Emma, Sun, Grace, Gillies, Jana, Speck, Madeleine, Orban, Paul C., Mojibian, Majid, and Levings, Megan K.
- Abstract
Comparison of 10 co-receptor variant CARs revealed a requirement for CD28wt for optimal suppression by human regulatory T cells.
- Published
- 2020
- Full Text
- View/download PDF
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