Date of Award
Doctor of Philosophy (PhD)
Cancer is the leading cause of premature death in the United States and other developed countries. Current decreases in cancer mortality rates are largely being driven by immunotherapies, which leverage an understanding of T cell activation and inhibition to produce an anti-tumor response. T-cell-based therapies may help decrease mortality rates even further. CAR T cell therapies and T cell activators like bispecific T-cell engagers and dendritic cell vaccines have increased survival in patients otherwise out of treatment options. While these therapies have been successful for niche patient populations, applying these therapies to wider patient populations has been difficult. In a specific example, no CAR T cell therapies have made it to Phase III clinical trials for cancers with solid tumors. A fundamental understanding of T cell activation and inhibition mechanisms may expand the use of targeted and adoptive cell therapies to more indications and increase the efficacy of them for current indications.First, we identified CXCR5 as an exploitable target for angioimmunoblastic T-cell lymphoma (AITL). Unlike other T cell markers, CXCR5 can be targeted without risking severe immunodeficiencies. In addition, CXCR5 signaling is implicated as being pro-tumorigenic in AITL and other cancer types. We present the utility of a small-molecule antagonist of CXCR5, YU241279. This compound inhibits CXCR5 signaling at submicromolar concentrations and has favorable pharmacokinetics. YU241279 is capable of inhibiting physiological responses to CXCR5 signaling like migration towards CXCL13, but the compound demonstrated no direct cytostatic/cytotoxic activity on Raji cells. In a patient-derived xenograft mouse model of AITL the compound induced reductions of AITL cells through inhibitions of growth and/or migration, and YU241279 specifically increased the expression of GPR15 mRNA in AITL cells. The activity of YU241279 on AITL cells seems specific to AITL cells as peripheral T cells from healthy donors did not increase GPR15 expression after drug treatment. Overall, the compound demonstrated activity specific to AITL cells and may have a potential role as part of a combination therapy for AITL patients. Next, we identified IRE1α as a potential bifunctional protein to be used in T cell engineering. The cytoplasmic splicing properties of IRE1α could generate a logic gate in CAR T cells, increasing the antigen specificity of cells. The ability of IRE1α to support protein synthesis under ER stress in other cell types and to induce signaling through mediators such as TRAF2 may also promote T cell survival/effector activity in the tumor microenvironment (TME). Pilot experiments with a single-chain variable fragment (scFv)-IRE1α fusion demonstrated no specific splicing activity in response to antigen stimulation and Xbp1s-expressing CD8+ T cells demonstrated no phenotype under stress conditions. We then shifted focus to a CXCL13 ligand CAR because CXCR5 has potential as an exploitable antigen in T-cell lymphomas. Early versions of the CARs demonstrated tonic signaling and were unable to cause CXCR5-specific cytotoxicity. This led to the development of a first-ever high-throughput CAR screen. The screen indicated that CXCL13 ligand CARs experience tonic signaling as a whole, possibly as a result of the CXCL13 domain. We then generated two different CXCL13 non-signaling chimeric antigen receptors (NSCARs) for use in NK cells, but optimization of the experimental assays is required before conclusions can be reached about their efficacy. Third, we performed single-cell RNA sequencing (scRNAseq) and single-cell TCR sequencing (scTCRseq) on paired pre- and post-REP TIL specimens to ascertain how this decades-old and relatively unmodified clinical production process affects T cells, and how to improve the process to increase the efficacy of TIL therapies. We discovered that several “atypical” T cell markers, such as CD40, are relatively prominent in TILs and that the APC-associated molecules like OX40L are expressed on TILs with variable prevalence. There is some indication that these markers are functional and inducing signaling in cis, but further experiments are warranted. TILs also demonstrated a degree of multipotency during REP and a cell-specific predisposition to responding external stimuli, like IFNγ. There are several limitations of the technologies used (e.g., empirically excluding γδ cells), but we have proposed several methods of overcoming these limitations and controlling for confounding variables. We hope to further our understanding of the REP process to enhance the efficacy of TIL therapies. In conclusion, cancer is a prominent public health threat and fundamental understandings of T cell activation and inhibition have led to the revolutionary new therapies that are currently driving the decreases in cancer mortality rates. Furthering our understanding and exploitation of these pathways may promote a healthier future.
Kerr, Samuel Robert, "Modulation of T Cells to Promote an Anti-Tumor Response: Activation And Inhibition Of T Cells For Efficacy In T-Cell Lymphomas And Melanoma" (2022). Yale Graduate School of Arts and Sciences Dissertations. 613.