generation-of-human-chimeric-antigen-receptor-regulatory-t-cells

Generation of Human Chimeric Antigen Receptor Regulatory T Cells

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable ...

Medical University of South Carolina

The requisites for protein translation in T cells are poorly understood and how translation shapes the antitumor efficacy of T cells is unknown. Here we demonstrated that IL15-conditioned T cells were primed by the metabolic energy sensor AMP-activated protein kinase to undergo diminished translation relative to effector T cells. However, we showed that IL15-conditioned T cells exhibited a remarkable capacity to enhance their protein translation in tumors, which effector T cells were unable to duplicate. Studying the modulation of translation for applications in cancer immunotherapy revealed that direct  pharmacologic inhibition of translation elongation primed robust T-cell antitumor immunity. Our work elucidates that altering protein translation in CD8 T cells can shape their antitumor capability.

Remodeling Translation Primes CD8 T-cell Antitumor Immunity.

Autoimmune disease, caused by unwanted immune responses to self-antigens, affects millions of people each year and poses a great social and economic burden to individuals and communities. In the course of autoimmune disorders, including rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes mellitus, and multiple sclerosis, disturbances in the balance between the immune response against harmful agents and tolerance towards self-antigens lead to an immune response against self-tissues. In recent years, various regulatory immune cells have been identified. Disruptions in the quality, quantity, and function of these cells have been implicated in autoimmune disease development. Therefore, targeting or engineering these cells is a promising therapeutic for different autoimmune diseases. Regulatory T cells, regulatory B cells, regulatory dendritic cells, myeloid suppressor cells, and some subsets of innate lymphoid cells are arising as important players among this class of cells. Here, we review the roles of each suppressive cell type in the immune system during homeostasis and in the development of autoimmunity. Moreover, we discuss the current and future therapeutic potential of each one of these cell types for autoimmune diseases.

The emerging role of regulatory cell-based therapy in autoimmune disease.

The adaptive immune system exhibits exquisite specificity and memory and is involved in virtually every process in the human body. Redirecting adaptive immune cells, in particular T cells, to desired targets has the potential to lead to the creation of powerful cell-based therapies for a wide range of maladies. While conventional effector T cells (Teff) would be targeted towards cells to be eliminated, such as cancer cells, immunosuppressive regulatory T cells (Treg) would be directed towards tissues to be protected, such as transplanted organs. Chimeric antigen receptors (CARs) are designer molecules comprising an extracellular recognition domain and an intracellular signaling domain that drives full T cell activation directly downstream of target binding. Here, we describe procedures to generate and evaluate human CAR CD4 helper T cells, CD8 cytotoxic T cells, and CD4FOXP3 regulatory T cells.

Redirecting Human Conventional and Regulatory T Cells Using Chimeric Antigen Receptors.

Chimeric antigen receptor (CAR) T cell therapy has proven to be a successful treatment option for leukemias and lymphomas. These encouraging outcomes underscore the potential of adoptive cell therapy for other oncology applications, namely, solid tumors. However, CAR T cells are yet to succeed in treating solid tumors. Unlike liquid tumors, solid tumors create a hostile tumor microenvironment (TME). CAR T cells must traffic to the TME, survive, and retain their function to eradicate the tumor. Nevertheless, there is no universal preclinical model to systematically test candidate CARs and CAR targets for their capacity to infiltrate and eliminate human solid tumors in vivo. Here, we provide a detailed protocol to evaluate human CAR CD4 helper T cells and CD8 cytotoxic T cells in immunodeficient (NSG) mice bearing antigen-expressing human solid tumors.

How to Test Human CAR T Cells in Solid Tumors, the Next Frontier of CAR T Cell Therapy.

Regulatory T cells (Tregs) are promising cellular therapies to induce immune tolerance in organ transplantation and autoimmune disease. The success of chimeric antigen receptor (CAR) T-cell therapy for cancer has sparked interest in using CARs to generate antigen-specific Tregs. Here, we compared CAR with endogenous T cell receptor (TCR)/CD28 activation in human Tregs. Strikingly, CAR Tregs displayed increased cytotoxicity and diminished suppression of antigen-presenting cells and effector T (Teff) cells compared with TCR/CD28 activated Tregs. RNA sequencing revealed that CAR Tregs activate Teff cell gene programs. Indeed, CAR Tregs secreted high levels of inflammatory cytokines, with a subset of FOXP3 CAR Tregs uniquely acquiring CD40L surface expression and producing IFNγ. Interestingly, decreasing CAR antigen affinity reduced Teff cell gene expression and inflammatory cytokine production by CAR Tregs. Our findings showcase the impact of engineered receptor activation on Treg biology and support tailoring CAR constructs to Tregs for maximal therapeutic efficacy.

High affinity chimeric antigen receptor signaling induces an inflammatory program in human regulatory T cells.

Regenerative medicine is a rapidly expanding field harnessing human pluripotent stem cell (hPSC)-derived cells and tissues to treat many diseases, including type 1 diabetes. However, graft immune protection remains a key challenge. Chimeric antigen receptor (CAR) technology confers new specificities to effector T cells and immunosuppressive regulatory T cells (Tregs). One challenge in CAR design is identifying target molecules unique to the cells of interest. Here, we employ combinatorial genetic engineering to confer CAR-Treg-mediated localized immune protection to stem cell-derived cells. We engineered hPSCs to express truncated epidermal growth factor receptor (EGFRt), a biologically inert and generalizable target for CAR-Treg homing and activation, and generated CAR-Tregs recognizing EGFRt. Strikingly, CAR-Tregs suppressed innate and adaptive immune responses in vitro and prevented EGFRt-hPSC-derived pancreatic beta-like cell (sBC [stem cell-derived beta cell]) graft immune destruction in vivo. Collectively, we provide proof of concept that hPSCs and Tregs can be co-engineered to protect hPSC-derived cells from immune rejection upon transplantation.

Combinatorial genetic engineering strategy for immune protection of stem cell-derived beta cells by chimeric antigen receptor regulatory T cells.

Regulatory T cells (Tregs) are promising cellular therapies to induce immune tolerance in organ transplantation and autoimmune disease. The success of chimeric antigen receptor (CAR) T cell therapy for cancer has sparked interest in using CARs to generate antigen-specific Tregs. Here, we compared CAR with endogenous T cell receptor (TCR)/CD28 activation in human Tregs. Strikingly, CAR Tregs displayed increased cytotoxicity and diminished suppression of antigen-presenting cells and effector T (Teff) cells compared with TCR/CD28-activated Tregs. RNA sequencing revealed that CAR Tregs activate Teff cell gene programs. Indeed, CAR Tregs secreted high levels of inflammatory cytokines, with a subset of FOXP3 CAR Tregs uniquely acquiring CD40L surface expression and producing IFN-γ. Interestingly, decreasing CAR antigen affinity reduced Teff cell gene expression and inflammatory cytokine production by CAR Tregs. Our findings showcase the impact of engineered receptor activation on Treg biology and support tailoring CAR constructs to Tregs for maximal therapeutic efficacy.

Article	Year
Remodeling Translation Primes CD8 T-cell Antitumor Immunity. Cancer immunology research\| PubMed ID: 32075802	2020
The emerging role of regulatory cell-based therapy in autoimmune disease. Frontiers in immunology\| PubMed ID: 36591309	2022
Redirecting Human Conventional and Regulatory T Cells Using Chimeric Antigen Receptors. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 38070117	2024
How to Test Human CAR T Cells in Solid Tumors, the Next Frontier of CAR T Cell Therapy. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 38070118	2024
High affinity chimeric antigen receptor signaling induces an inflammatory program in human regulatory T cells. bioRxiv : the preprint server for biology\| PubMed ID: 38617240	2024
Combinatorial genetic engineering strategy for immune protection of stem cell-derived beta cells by chimeric antigen receptor regulatory T cells. Cell reports\| PubMed ID: 39561045	2024
High-affinity chimeric antigen receptor signaling induces an inflammatory program in human regulatory T cells. Molecular therapy. Methods & clinical development\| PubMed ID: 39687729	2024

Rob A. Robino

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