Romy E. Hoeppli

Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor

Adoptive immunotherapy with regulatory T cells (Tregs) is a promising treatment for allograft rejection and graft-versus-host disease (GVHD). Emerging data indicate that, compared with polyclonal Tregs, disease-relevant antigen-specific Tregs may have numerous advantages, such as a need for fewer cells and reduced risk of nonspecific immune suppression. Current methods to generate alloantigen-specific Tregs rely on expansion with allogeneic antigen-presenting cells, which requires access to donor and recipient cells and multiple MHC mismatches. The successful use of chimeric antigen receptors (CARs) for the generation of antigen-specific effector T cells suggests that a similar approach could be used to generate alloantigen-specific Tregs. Here, we have described the creation of an HLA-A2-specific CAR (A2-CAR) and its application in the generation of alloantigen-specific human Tregs. In vitro, A2-CAR-expressing Tregs maintained their expected phenotype and suppressive function before, during, and after A2-CAR-mediated stimulation. In mouse models, human A2-CAR-expressing Tregs were superior to Tregs expressing an irrelevant CAR at preventing xenogeneic GVHD caused by HLA-A2+ T cells. Together, our results demonstrate that use of CAR technology to generate potent, functional, and stable alloantigen-specific human Tregs markedly enhances their therapeutic potential in transplantation and sets the stage for using this approach for making antigen-specific Tregs for therapy of multiple diseases.

The Environment of Regulatory T Cell Biology: Cytokines, Metabolites, and the Microbiome

Regulatory T cells (Tregs) are suppressive T cells that have an essential role in maintaining the balance between immune activation and tolerance. Their development, either in the thymus, periphery, or experimentally in vitro, and stability and function all depend on the right mix of environmental stimuli. This review focuses on the effects of cytokines, metabolites, and the microbiome on both human and mouse Treg biology. The role of cytokines secreted by innate and adaptive immune cells in directing Treg development and shaping their function is well established. New and emerging data suggest that metabolites, such as retinoic acid, and microbial products, such as short-chain fatty acids, also have a critical role in guiding the functional specialization of Tregs. Overall, the complex interaction between distinct environmental stimuli results in unique, and in some cases tissue-specific, tolerogenic environments. Understanding the conditions that favor Treg induction, accumulation, and function is critical to defining the pathophysiology of many immune-mediated diseases and to developing new therapeutic interventions.

Discarded Human Thymus Is a Novel Source of Stable and Long-Lived Therapeutic Regulatory T Cells.

Regulatory T cell (Treg)–based therapy is a promising approach to treat many immune‐mediated disorders such as autoimmune diseases, organ transplant rejection, and graft‐versus‐host disease (GVHD). Challenges to successful clinical implementation of adoptive Treg therapy include difficulties isolating homogeneous cell populations and developing expansion protocols that result in adequate numbers of cells that remain stable, even under inflammatory conditions. We investigated the potential of discarded human thymuses, routinely removed during pediatric cardiac surgery, to be used as a novel source of therapeutic Tregs. Here, we show that large numbers of FOXP3+ Tregs can be isolated and expanded from a single thymus. Expanded thymic Tregs had stable FOXP3 expression and long telomeres, and suppressed proliferation and cytokine production of activated allogeneic T cells in vitro. Moreover, expanded thymic Tregs delayed development of xenogeneic GVHD in vivo more effectively than expanded Tregs isolated based on CD25 expression from peripheral blood. Importantly, in contrast to expanded blood Tregs, expanded thymic Tregs remained stable under inflammatory conditions. Our results demonstrate that discarded pediatric thymuses are an excellent source of therapeutic Tregs, having the potential to overcome limitations currently hindering the use of Tregs derived from peripheral or cord blood.

How antigen specificity directs regulatory T-cell function: self, foreign and engineered specificity

Regulatory T cells (Tregs) are a suppressive subset of T cells that have important roles in maintaining self‐tolerance and preventing immunopathology. The T‐cell receptor (TCR) and its antigen specificity play a dominant role in the differentiation of cells to a Treg fate, either in the thymus or in the periphery. This review focuses on the effects of the TCR and its antigen specificity on Treg biology. The role of Tregs with specificity for self‐antigen has primarily been studied in the context of autoimmune disease, although recent studies have focused on their role in steady‐state conditions. The role of Tregs that are specific for pathogens, dietary antigens and allergens is much less studied, although recent data suggest a significant and previously underappreciated role for Tregs during memory responses to a wide range of foreign antigens. The development of TCR‐ or chimeric antigen receptor (CAR)‐transduced T cells means we are now able to engineer Tregs with disease‐relevant antigen specificities, paving the way for ensuring specificity with Treg‐based therapies. Understanding the role that antigens play in driving the generation and function of Tregs is critical for defining the pathophysiology of many immune‐mediated diseases, and developing new therapeutic interventions.

Harnessing Advances in T Regulatory Cell Biology for Cellular Therapy in Transplantation

Abstract 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.

Regulatory T-cells drive immune dysfunction in CLL

Deepesh Lad, Romy Hoeppli, Qing Huang, Rosa Garcia, Lixin Xu, Cynthia Toze, Raewyn Broady and Megan Levings Department of Internal Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India; British Columbia Children’s Hospital Research Institute, Vancouver, Canada; Leukemia/BMT Program of BC, British Columbia Cancer Agency, University of British Columbia, Vancouver, Canada

Thymic progenitors of TCRαβ+ CD8αα intestinal intraepithelial lymphocytes require RasGRP1 for development

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 TCR&agr;&bgr;+CD8&agr;&agr; 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 &agr;4&bgr;7 and CD103 define distinct IELp subsets with differing abilities to generate TCR&agr;&bgr;+CD8&agr;&agr; 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.

Humanization and Pre-Clinical Validation of an Anti-HLA-A*02: 01 Chimeric Antigen Receptor for use with Regulatory T cells to Enhance Transplant Tolerance.

Introduction Achieving transplant tolerance with regulatory T cell (Treg) adoptive immunotherapy is currently under investigation as a therapy to reduce graft rejection and improve long-term outcomes. Traditional approaches involve the of polyclonal Tregs, which are known to be less potent than antigen-specific cells, or antigen-expanded Tregs, which have several technical limitations. We and others have developed an alternate approach to generate antigen-specific Tregs by expressing a chimeric antigen receptor specific for HLA-A*02:01 (A2-CAR). In our initial studies the antigen-binding region (scFV) of the A2-CAR was derived from the mouse BB7.2 hybridoma, which, due to the high degree of homology between HLA molecules, has been reported to bind to HLA-A alleles in addition to *02:01. Here we sought to systematically define the antigen-specificity of the A2-CAR as well as humanize the sequence to minimize the risk of immunogenicity. Methods We designed 20 humanized versions of the A2-CAR and systematically tested them to determine which were highly expressed on the surface of human Tregs and capable of mediating A2-stimulated activation, expansion, and suppression. We also developed a novel method to systematically, and comprehensively test HLA-allele specificity. Results Of the 20 humanized A2-CARs, 10 were expressed on Tregs and retained A*02:01 binding capacity. We used a series of functional screens to define which of these 10 A2-CARs most effectively stimulated Treg activation, proliferation and proliferation. We then took advantage of the Panel Reactive Antibody (PRA) assay (One Lambda) and created a new method to test CAR-expressing Tregs to bind to specific HLA-alleles. We found that the majority of the humanized A2-CARs had a significantly reduced reactivity to binding to alleles other than A*02:01. We also tested the biological relevance of HLA cross reactivity by stimulating A2-CAR expressing Tregs with cell lines expressing HLA alleles that were or were not found to be cross reactive using the PRA assay. Ultimately, six humanized anti-A2 CARs showed the desired properties, with an ability to activate Tregs, bind to HLA-A2 but not to a comprehensive panel of other common A or B alleles. The potent ability of one of these variants to suppress rejection was confirmed in a humanized model of xenogeneic graft-versus-host disease. Conclusion We successfully developed a series of humanized A2-CARs which were comprehensively screened for desirable properties to generate antigen-specific Tregs. This body of pre-clinical data will support the development of a first-in-human clinical trial of A2-CAR-engineered Tregs to prevent organ allograft rejection.

An optimized method to measure human FOXP3+ regulatory T cells from multiple tissue types using mass cytometry

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.

Tailoring the homing capacity of human Tregs for directed migration to sites of Th1-inflammation or intestinal regions

Cell‐based therapy with CD4+FOXP3+ regulatory T cells (Tregs) is a promising strategy to limit organ rejection and graft‐vs‐host disease. Ongoing clinical applications have yet to consider how human Tregs could be modified to direct their migration to specific inflammation sites and/or tissues for more targeted immunosuppression. We show here that stable, homing‐receptor‐tailored human Tregs can be generated from thymic Tregs isolated from pediatric thymus or adult blood. To direct migration to Th1‐inflammatory sites, addition of interferon‐γ and IL‐12 during Treg expansion produced suppressive, epigenetically stable CXCR3+TBET+FOXP3+ T helper (Th)1‐Tregs. CXCR3 remained expressed after injection in vivo and Th1‐Tregs migrated efficiently towards CXCL10 in vitro. To induce tissue‐specific migration, addition of retinoic acid (RA) during Treg expansion induced expression of the gut‐homing receptors α4β7‐integrin and CCR9. FOXP3+ RA‐Tregs had elevated expression of the functional markers latency‐associated peptide and glycoprotein A repetitions predominant, increased suppressive capacity in vitro and migrated efficiently to healthy and inflamed intestine after injection into mice. Homing‐receptor‐tailored Tregs were epigenetically stable even after long‐term exposure to inflammatory conditions, suppressive in vivo and characterized by Th1‐ or gut‐homing‐specific transcriptomes. Tailoring human thymic Treg homing during in vitro expansion offers a new and clinically applicable approach to improving the potency and specificity of Treg therapy.