Professional Education


  • PhD, University of British Columbia, Experimental Medicine (Immunology) (2021)
  • BArts Sc, McMaster University, Arts & Science and Biochemistry (2015)

Stanford Advisors


All Publications


  • PTEN is required for human Treg suppression of costimulation in vitro. European journal of immunology Lam, A. J., Haque, M., Ward-Hartstonge, K. A., Uday, P., Wardell, C. M., Gillies, J. K., Speck, M., Mojibian, M., Klein Geltink, R. I., Levings, M. K. 2022

    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 signalling, 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 discernable 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 signalling 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. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1002/eji.202249888

    View details for PubMedID 35746855

  • Suppression of Human Dendritic Cells by Regulatory T Cells BIO-PROTOCOL Huang, Q., Lam, A. J., Boardman, D. A., Dawson, N. J., Levings, M. K. 2021; 11 (21)
  • Optimized CRISPR-mediated gene knockin reveals FOXP3-independent maintenance of human Treg identity. Cell reports Lam, A. J., Lin, D. T., Gillies, J. K., Uday, P., Pesenacker, A. M., Kobor, M. S., Levings, M. K. 2021; 36 (5): 109494

    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 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.

    View details for DOI 10.1016/j.celrep.2021.109494

    View details for PubMedID 34348163

  • Helios is a marker, not a driver, of human Treg stability. European journal of immunology Lam, A. J., Uday, P., Gillies, J. K., Levings, M. K. 2021

    Abstract

    Regulatory T cell (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 Tregs 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. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1002/eji.202149318

    View details for PubMedID 34561855

  • Innate Control of Tissue-Reparative Human Regulatory T Cells. Journal of immunology (Baltimore, Md. : 1950) Lam, A. J., MacDonald, K. N., Pesenacker, A. M., Juvet, S. C., Morishita, K. A., Bressler, B., Pan, J. G., Sidhu, S. S., Rioux, J. D., Levings, M. K. 2019; 202 (8): 2195-2209

    Abstract

    Regulatory T cell (Treg) therapy is a potential curative approach for a variety of immune-mediated conditions, including autoimmunity and transplantation, in which there is pathological tissue damage. In mice, IL-33R (ST2)-expressing Tregs mediate tissue repair by producing the growth factor amphiregulin, but whether similar tissue-reparative Tregs exist in humans remains unclear. We show that human Tregs in blood and multiple tissue types produced amphiregulin, but this was neither a unique feature of Tregs nor selectively upregulated in tissues. Human Tregs in blood, tonsil, synovial fluid, colon, and lung tissues did not express ST2, so ST2+ Tregs were engineered via lentiviral-mediated overexpression, and their therapeutic potential for cell therapy was examined. Engineered ST2+ Tregs exhibited TCR-independent, IL-33-stimulated amphiregulin expression and a heightened ability to induce M2-like macrophages. The finding that amphiregulin-producing Tregs have a noneffector phenotype and are progressively lost upon TCR-induced proliferation and differentiation suggests that the tissue repair capacity of human Tregs may be an innate function that operates independently from their classical suppressive function.

    View details for DOI 10.4049/jimmunol.1801330

    View details for PubMedID 30850479

  • Characterization of regulatory T cells in obese omental adipose tissue in humans. European journal of immunology Wu, D., Han, J. M., Yu, X., Lam, A. J., Hoeppli, R. E., Pesenacker, A. M., Huang, Q., Chen, V., Speake, C., Yorke, E., Nguyen, N., Sampath, S., Harris, D., Levings, M. K. 2019; 49 (2): 336-347

    Abstract

    Obesity-associated visceral adipose tissue (AT) inflammation promotes insulin resistance and type 2 diabetes (T2D). In mice, lean visceral AT is populated with anti-inflammatory cells, notably regulatory T cells (Tregs) expressing the IL-33 receptor ST2. Conversely, obese AT contains fewer Tregs and more proinflammatory cells. In humans, however, there is limited evidence for a similar pattern of obesity-associated immunomodulation. We used flow cytometry and mRNA quantification to characterize human omental AT in 29 obese subjects, 18 of whom had T2D. Patients with T2D had increased proportions of inflammatory cells, including M1 macrophages, with positive correlations to body mass index. In contrast, Treg frequencies negatively correlated to body mass index but were comparable between T2D and non-T2D individuals. Compared to human thymic Tregs, omental AT Tregs expressed similar levels of FOXP3, CD25, IKZF2, and CTLA4, but higher levels of PPARG, CCR4, PRDM1, and CXCL2. ST2, however, was not detectable on omental AT Tregs from lean or obese subjects. This is the first comprehensive investigation into how omental AT immunity changes with obesity and T2D in humans, revealing important similarities and differences to paradigms in mice. These data increase our understanding of how pathways of immune regulation could be targeted to ameliorate AT inflammation in humans.

    View details for DOI 10.1002/eji.201847570

    View details for PubMedID 30566246

  • An optimized method to measure human FOXP3+ regulatory T cells from multiple tissue types using mass cytometry. European journal of immunology Dawson, N. A., Lam, A. J., Cook, L., Hoeppli, R. E., Broady, R., Pesenacker, A. M., Levings, M. K. 2018; 48 (8): 1415-1419

    Abstract

    We optimized a method to detect FOXP3 by mass cytometry and compared the resulting data to conventional flow cytometry. We also demonstrated the utility of the protocol to profile antigen-specific Tregs from whole blood, or Tregs from tissues such as cord blood, thymus and synovial fluid.

    View details for DOI 10.1002/eji.201747407

    View details for PubMedID 29676458

  • Human Regulatory T Cell Potential for Tissue Repair Via IL-33/ST2 and Amphiregulin Lam, A. J., Huang, H., Pan, J. G., Sidhu, S. S., Charron, G., Ivison, S. M., Rioux, J. D., Levings, M. K., IGenoMed Consortium LIPPINCOTT WILLIAMS & WILKINS. 2018: S331
  • Harnessing Advances in T Regulatory Cell Biology for Cellular Therapy in Transplantation. Transplantation Lam, A. J., Hoeppli, R. E., Levings, M. K. 2017; 101 (10): 2277-2287

    Abstract

    Cellular therapy with CD4FOXP3 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.

    View details for DOI 10.1097/TP.0000000000001757

    View details for PubMedID 28376037

  • Janus and PI3-kinases mediate glucocorticoid resistance in activated chronic leukemia cells. Oncotarget Oppermann, S., Lam, A. J., Tung, S., Shi, Y., McCaw, L., Wang, G., Ylanko, J., Leber, B., Andrews, D., Spaner, D. E. 2016; 7 (45): 72608-72621

    Abstract

    Glucorticoids (GCs) such as dexamethasone (DEX) remain important treatments for Chronic Lymphocytic Leukemia (CLL) but the mechanisms are poorly understood and resistance is inevitable. Proliferation centers (PC) in lymph nodes and bone marrow offer protection against many cytotoxic drugs and circulating CLL cells were found to acquire resistance to DEX-mediated killing in conditions encountered in PCs including stimulation by toll-like receptor agonists and interactions with stromal cells. The resistant state was associated with impaired glucocorticoid receptor-mediated gene expression, autocrine activation of STAT3 through Janus Kinases (JAKs), and increased glycolysis. The JAK1/2 inhibitor ruxolitinib blocked STAT3-phosphorylation and partially improved DEX-mediated killing of stimulated CLL cells in vitro but not in CLL patients in vivo. An automated microscopy-based screen of a kinase inhibitor library implicated an additional protective role for the PI3K/AKT/FOXO pathway. Blocking this pathway with the glycolysis inhibitor 2-deoxyglucose (2-DG) or the PI3K-inhibitors idelalisib and buparlisib increased DEX-mediated killing but did not block STAT3-phosphorylation. Combining idelalisib or buparlisib with ruxolitinib greatly increased killing by DEX. These observations suggest that glucocorticoid resistance in CLL cells may be overcome by combining JAK and PI3K inhibitors.

    View details for DOI 10.18632/oncotarget.11618

    View details for PubMedID 27579615

    View details for PubMedCentralID PMC5341931