Date of Award

2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Michael C. Milone

Second Advisor

Steven M. Albelda

Abstract

Chimeric Antigen Receptor (CAR) T cell therapies for hematologic malignancies have been astonishingly successful in driving cancer remissions. However, early loss of CAR T cells and return of normal B cells is a predictor of relapse in pediatric acute lymphoblastic leukemia; the duration of remission is associated with the persistence of CAR T cells for more than three months. These CAR T cells are the product of over 30 years of research and innovation in T cell biology and engineering, which began with the desire to understand how the T cell receptor (TCR) activates T cells in response to antigen. In this thesis, we return to that approach again, but this time to investigate signaling downstream of the costimulatory receptor, 4-1BB, a TNF Receptor Superfamily (TNFRSF) member. 4-1BB-costimulated CAR (BBz) T cells exhibit longer persistence following adoptive transfer than CD28-costimulated CAR (28z) T cells, which contain the first costimulatory domain commonly added to CARs. 4-1BB signaling improves T cell persistence even in the context of 28z CAR activation, which indicates that the 4-1BB cytoplasmic domain contributes unique pro-survival signals. In order to specifically study CAR signaling, we developed a cell-free ligand-based activation and ex vivo culture system for CD19-specific CAR T cells. In this system, we observed greater ex vivo expansion and survival of BBz CAR T cells compared to 28z T cells. We used this system to isolate a pathway activated most by BBz CARs, the non-canonical NF-kB (ncNF-kB) pathway, which is associated with the survival benefit from imparted by other TNFRSFs. We observed that BBz CARs uniquely activate non-canonical NF-kB (ncNF- kB) signaling in T cells basally, and the anti-CD19 BBz CAR further enhances ncNF-kB signaling following ligand engagement. Reducing ncNF-kB signaling specifically diminishes anti-CD19 BBz T cell expansion and survival and is associated with a significant increase in expression of the most pro-apoptotic isoforms of Bim. Although our findings do not exclude the importance of other signaling differences between these CARs, they demonstrate the necessary and non-redundant role of ncNF-kB signaling in promoting BBz CAR T cell survival that likely underlies the engraftment persistence observed with this CAR design.

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