Effects of Engineered Costimulation on the Function of T Cell Subsets

Abstract

Progress in clinical adoptive immunotherapy was initially hindered by the T cells’ lack of antigen specificity, poor engraftment, and limited persistence in the host. The introduction of chimeric antigen receptors (CARs) into T cells has overcome these obstacles by re-directing polyclonal T cells to a specific antigen with an extracellular binding domain and by including costimulation domains that enhance engraftment and persistence. From this observation, we hypothesized that the overexpression of CARs in different T cell subsets fundamentally changes their biology. CAR T cell products bearing either 4-1BB or CD28 costimulatory domains have been approved as therapies for leukemia and lymphoma. However, the engraftment kinetics, persistence, and toxicity profiles of CD28 versus 4‑1BB CAR T cells are distinct. To obtain an in‑depth understanding of the functional state of different types of CAR-modified T cells, we performed RNA sequencing on first- and second-generation CAR T cells both at rest and following CAR or endogenous T cell receptor (TCR) stimulation. We describe a high-resolution view of the transcriptional differences between 4‑1BB and CD28 containing CAR T cells, including variances in cytokine profiles, cytokine receptors, and metabolic pathways. These transcriptional profiles define CAR signaling pathways that ultimately determine CAR T cell fate. Adoptive immunotherapy with regulatory T cells (Tregs) also holds promise in transplantation, graft-versus-host disease, and autoimmune diseases. Tregs are key modulators of inflammation and are important for peripheral tolerance. The challenges of using Tregs as adoptive immunotherapy mirror those for cancer: namely antigen specificity, engraftment, and persistence. We modified primary human Tregs with CARs bearing different costimulatory domains and performed rigorous analyses in vitro of the functional potential of Tregs. We showed that the presence of a CAR and the type of costimulation domain does not affect the Treg’s expression of Foxp3+. However, the costimulation domain does affect CAR-Treg cytokine production and surface marker expression. Furthermore 4‑1BB costimulation decreases the suppressive function of CAR-Tregs. In vivo experiments, demonstrated that CAR-Tregs traffic to antigen expressing sites and can suppress antigen specific effector T cell responses. Our findings support the use of CD28-based CARs for tissue-specific suppression in the clinic.