Megan Levings

Professor

Relevant Degree Programs

 

Biography

Research in my laboratory is focused on a novel subset of CD4+ T cells, termed T regulatory (Treg) cells, which control immune homeostasis. Current work is focused on determining how Treg cells differ from normal CD4+ T cells at both the biochemical and molecular levels, and elucidating their role in transplantation tolerance, diabetes and inflammatory bowel disease. We are also developing methods to use Tregs as a cellular therapy to replace standard immunosuppression in the context of organ transplantation or to restore tolerance in the context of autoimmunity.

Postdoctoral Fellows

Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2019)
Tailoring the migration capacity of regulatory T cells to enhance their efficacy as a cellular therapy (2018)

No abstract available.

Function and control of adipose tissue regulatory T cells : implications for obesity (2015)

Obesity is associated with chronic low-grade inflammation in visceral adipose tissue (VAT), which promotes the development of insulin resistance. The role of adaptive immunity in VAT inflammation has only recently been investigated. Initial studies suggest that VAT-resident regulatory T cells (Tregs) have a prominent role in suppressing VAT inflammation and correcting metabolic dysfunction in obese mice. I sought to investigate how Tregs in the VAT are regulated.Obesity is accompanied by a rise in insulin levels, and whether this hyperinsulinemia affects the progression of inflammation is not known. I first found that Tregs express the insulin receptor, and high levels of insulin inhibited IL-10 production and the ability of Tregs to suppress macrophages. In parallel, Tregs from the VAT of obese mice showed a similar decrease in IL-10 production, suggesting that hyperinsulinemia may contribute to the development of obesity-associated inflammation via an effect of insulin on Treg function. I then found that the majority of IL-10-expressing Tregs in the VAT expressed the ST2 chain of the IL-33 receptor. The proportion of ST2+ Tregs in VAT was severely diminished in obese mice, and this effect could be completely reversed by treatment with IL-33. IL-33 treatment also reversed VAT inflammation in obese mice, and resulted in a reduction of hyperinsulinemia and insulin resistance. These data suggested that IL-33 is critical for the maintenance of ST2+ Tregs in the VAT, and that delivery of IL-33 may be a new therapeutic approach to reverse obesity-associated Treg deficiency, inflammation and insulin resistance. It is not known whether Tregs and adipocytes can directly interact in the VAT. I found that soluble factors produced by adipocytes significantly increased survival and IL-10 production from Tregs, and caused a shift towards oxidative metabolism in vitro. Similarly, Tregs resident in mouse VAT have substantially increased expression of IL-10 compared to those found in the periphery, indicating that the interaction between Tregs and adipocytes may contribute to the functional specialization of Tregs in the VAT. Taken together, these data suggest that insulin, IL-33 and adipocyte-produced factors regulate IL-10-expressing Tregs and their ability to control inflammation in the VAT.

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Immune regulation of autoinflammation caused by cellular stress (2015)

During chronic inflammation and tissue injuries, various danger-associated molecules can be released and are able to potentiate inflammation and T cell responses. Among the many possible danger signals, I focused on studying high concentrations of extracellular ATP since it has been implicated in a variety of autoinflammatory diseases. ATP activates the inflammasome in macrophages, stimulates dendritic cell (DC) maturation, and inhibits regulatory T cell (Treg) function. However, how ATP regulates Toll-like receptor (TLR) responses in intestinal epithelial cells (IECs), which represent the front line of enteric defense, remains unclear. Therefore, I examined how ATP modulates TLR responses in IECs and found that it enhanced the response of IECs to a TLR1/2 ligand Pam₃CSK₄ primarily through the P2X7 purinergic receptor, leading to increased DC maturation and antigen-specific T cell proliferation. Furthermore, intra-rectal delivery of ATP lowered the activation threshold of epithelial cells to endogenous TLR ligands, making IECs more prone to immune activation. Since ATP is an important molecule that can potentiate inflammatory responses, the second aim of the study was to investigate if Tregs, including Foxp3+ Tregs and IL-10 producing Tr1 cells, can regulate ATP induced inflammasome activation and IL-1β production. I found that Tr1 cells inhibited the production of Il1b mRNA, inflammasome-mediated activation of caspase-1, and secretion of mature IL-1β, in an IL-10 dependent manner. Surprisingly, Foxp3+ Tregs, despite the production of IL-10, failed to inhibit IL-1β production. The important role of IL-10 in regulating inflammasome activation was further illustrated in the monosodium urate induced peritonitis model, where IL-10R-deficient mice had an increased influx of peritoneal neutrophils compared to wild type mice. Moreover, IL-1β production from macrophages derived from Nlrp3A350V knock-in mice, which carry a mutation found in cryopyrin associated periodic syndrome patients, was suppressed by Tr1 cells, but not Foxp3+ Tregs. Using an adoptive transfer model, I found Tr1 cells can protect against weight loss in mice expressing a gain-of-function mutation in NLRP3. Collectively, these data demonstrated the complex regulation of host response to cellular stress signal ATP, and that IL-10 producing Tr1 cells may have unique therapeutic effects in controlling ATP-mediated inflammasome activation via IL-10-mediated suppression.

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Understanding & optimizing human T regulatory cell function in patients with autoimmunity and/or undergoing transplantation (2015)

CD4⁺FOXP3⁺ T regulatory cells (Tregs) are potent suppressors of inflammatory immune activity. Cellular therapy with Tregs is a promising way to induce antigen specific tolerance in transplantation and autoimmunity, as it would allow the reduction of nonspecific immunosuppression. Currently, Tregs are being tested in clinical trials; however, outstanding questions regarding stability, specificity, and longevity of transferred cells remain. The aim of this research was to better understand the potential plasticity of Tregs, develop novel methods of creating antigen specific Tregs, and determine the optimal signals for Tregs to persist after transfer. To better understand the pathological conversion of Tregs to inflammatory cells, I examined the phenotype of Tregs in systemic sclerosis, a Th2-biased disease. I found that Tregs in patient skin and blood had acquired Th2-cytokine and homing marker expression, respectively, and that both tissue-localized and homing cells express the receptor for IL-33, which was expressed in patient skin. This work suggests that sub-populations of Tregs have the capacity to become pathogenic upon encountering tissue-specific inflammatory signals. Next, in order to create antigen specific Tregs, I developed a novel chimeric antigen receptor (CAR) against HLA-A2 and tested its function. A2CAR-Tregs were highly activated and proliferative in response to HLA-A2, but they retained their suppressive capacity and expression of the transcription factors FOXP3 and Helios, and the effector molecules CD25 and CTLA-4. A2CAR-Tregs were also superior to polyclonal Tregs at preventing xenoGVHD in mice, even at low doses. Thus, A2CAR-Treg cell therapy is a promising new technology to create potent Tregs for antigen-specific transplant tolerance. Finally, to optimize Treg activity and persistence in patients, I developed a series of CARs containing different co-stimulatory domains. Four TNFR superfamily domains were cloned, from 4-1BB, OX40, GITR and TNFR2, and three signalling domains were cloned from the B7/CD28 superfamily, from ICOS, PD-1, and CTLA-4. 4-1BB, OX40, ICOS, and PD-1 containing CARs were expressed on the surface of cells. These tools will provide valuable information in future research on Treg survival in vivo. Collectively, these studies have provided insights to improve both the safety and efficacy of Treg cell therapy.

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Phenotypic and functional characterization of T cells and Foxp3⁺ T regulatory cells in inflammatory bowel disease : steps towards T regulatory cell therapy in mucosal disease (2012)

Because of their potent suppressive capacity and critical role in the normal function of the human immune system, T regulatory cells (Tregs) have long been considered candidates for the therapeutic treatment of autoimmune and chronic inflammatory diseases. However, the clinical implementation of these cells has proven challenging in practice, in part due to a lack of knowledge surrounding this T cell subset. Specifically, an evaluation of the unique functions of individual Treg cell lineages, along with a comprehensive investigation of the non-suppressive capacities of these cells, including chemokine production, is necessary. Furthermore, in the application of Treg cellular therapy in mucosal diseases such as inflammatory bowel disease, the identification of putative antigens that can be targeted by Tregs is warranted. To these aims, I evaluated the phenotypic and functional characteristics of Helios⁺ and Helios⁻ Treg subsets, with the knowledge that expression of Helios, an Ikaros family transcription factor, may differentiate natural, thymic derived Tregs from their in vivo peripherally induced counterparts. I found that Helios positive and negative Treg subsets expressed similar Treg markers and displayed a similar capacity for suppression and plasticity. However, these Tregs did differ in terms of cytokine/chemokine production as well as methylation state of the FOXP3 Treg-specific demethylated region. Futhermore, total populations of FOXP3⁺ Tregs were evaluated for chemokine expression; I found that Tregs produce significant quantities of CXCL8 and other acute phase chemokines, and are able to attract inflammatory cells of the innate immune system. In addition, FOXP3 expression enhances CXCL8 production, likely because of its ability to bind the CXCL8 gene promoter. To evaluate putative antigens that can be targeted by Treg therapy in inflammatory bowel disease, I assessed the role of flagellin in disease. Flagellin exacerbates colitic disease in mice in a TLR5 independent manner and flagellin-specific T cells can be identified in patients with CD. Collectively, these findings bring us closer to the effective application of Treg cellular therapy in the setting of mucosal disease.

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Phenotypic and functional characterization of T regulatory cells (2012)

FOXP3⁺ T regulatory cells (Tregs) normally function to restrain immune responses, but when their activities go awry diseases such as autoimmunity and cancer can result. Animal models have proven that enhancing or inhibiting the function of Tregs is an effective way to prevent, and in some cases cure, many immune-mediated diseases. Approaches to specifically modulate the activity of Tregs are already being translated to humans, yet we know remarkably little about how Tregs achieve their potent immunosuppressive effects. The aim of this research was to further understand the factors that regulate the molecular phenotype and functionality of Tregs in order to better use them for therapeutic purposes. To achieve this goal, the interaction between Tregs and adenoviral-transduced human monocyte-derived dendritic cells in the context of cancer immunotherapy was explored. I found that these genetically-engineered DCs designed to boost the immune response were still susceptible to Treg suppressive influence. Next, I investigated the biological relevance of chemokine secretion by Tregs and determined that chemokine-mediated active recruitment of their targets of inhibition may be a novel mechanism of action. Finally, I established a human Treg-specific gene signature using Affymetrix microarray technology in order to define better ways to isolate and track these cells. Taken together, these studies have contributed significantly to understanding how Tregs exert their homeostatic control of immunity and revealed potential tactics to manipulate their activity in clinical aspects.

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The role of human forkhead box P3 in CD4⁺ T cell subsets (2012)

The immune system eliminates threats to the body, but it must also prevent immune-mediated damage caused by inflammation and autoimmune disease. One way that immune responses are limited is by specialized T cells known as T regulatory (Treg) cells. The transcription factor forkhead box P3 (FOXP3) is highly expressed in Treg cells and is critical for their suppressive function. The importance of FOXP3 is demonstrated in humans with a severe autoimmune disease called immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) caused by mutations in FOXP3. Furthermore, conventional T (Tconv) cells can be re-programmed into suppressive cells upon stable over-expression of FOXP3. Therefore, there is tremendous interest in manipulating FOXP3 function and/or using Treg cells as a cellular therapy to modify immune responses of cancer patients, patients suffering from autoimmune disease, and transplantation patients. To better understand the function of FOXP3, the first goal was to investigate how mutant forms of FOXP3 found in IPEX patients were defective at programming Treg characteristics. Surprisingly, mutant forms of FOXP3 were not completely deficient at converting Tconv cells into Treg cells, suggesting that factors besides a defect in Treg cells may contribute to IPEX pathogenesis. FOXP3 is transiently up-regulated in human Tconv cells upon activation, but its role in these cells is unknown. Hence, the second goal was to examine the function of FOXP3 in Tconv cells by comparing FOXP3-deficient with wild type Tconv cells. FOXP3-deficient Tconv cells proliferated more and produced more cytokines than wild type Tconv cells. This finding suggests that FOXP3 has a role in the regulation of Tconv cell activation, especially in Th17 cells which were found to highly express activation-induced FOXP3. Lastly, the possibility of using FOXP3⁺ cells as a cellular therapy was investigated. A method to expand large, pure populations of human and cynomolgus Treg cells was developed, and ex vivo expanded Treg cells were able to promote mixed chimerism and tolerance to a kidney transplant in cynomolgus macaques. Together, this work sheds light on the role of FOXP3 in CD4⁺ T cell subsets and helps pave the way for use of Treg cell therapy in the clinic.

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Defining the biological role of FOXP3 in human CD4+ T cells (2008)

The involvement of regulatory T cells (Tregs) in immune homeostasis is now recognized as one of the fundamental mechanisms of immune tolerance. While several different types of Tregs cooperate to establish and maintain immune homeostasis, much current research is focused on defining the characteristics of the CD4⁺CD25⁺ Treg subset, as these cells can mediate dominant, long-lasting and transferable tolerance in many experimental models. The aim of this research was to characterize the biological role of a protein known as forkhead box P3 (FOXP3) that was initially identified as an essential transcription factor for the development of mouse CD4⁺CD25⁺ Tregs, in human CD4⁺ T cells. Following confirmation that, like mouse Tregs, human Tregs also expressed high levels of FOXP3, several approaches were used to investigate the role of this protein in human CD4⁺ T cells. 1) Characterization of endogenous FOXP3 expression in CD4⁺ T cell subsets revealed that this protein is not a Treg-specific marker as was previously thought. Instead, low-level and transient expression was found to be typical of highly activated non-regulatory effector T cells. 2) To generate large numbers of Tregs suitable for cellular therapy, the capacity of ectopic FOXP3 expression to drive Treg generation in vitro was explored. It was found that high and constitutive expression mediated by a lentiviral vector, but not fluctuating expression driven by a retroviral vector, was sufficient to generate suppressive cells. Over-expression strategies were also used to characterize a novel splice isoform unique to human cells, FOXP3Δ2 (FOXP3b). 3) To further probe the requirements of FOXP3 to induce suppressor function, a system for conditionally-active FOXP3 ectopic expression was developed. These studies established that FOXP3 acts a quantitative regulator rather than a “master switch” for Tregs, and that there is a temporal component to its capacity to direct Treg phenotype and function. In summary, this research has significantly expanded the understanding of the biological function of FOXP3 in human CD4⁺ T cells. Based on the potential of these cells to be manipulated for therapy, this work contributes to the field of immunology on both academic and clinical research fronts.

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