Characterization and Reprogramming of T-cell Metabolic Dysfunction in Chronic Lymphocytic Leukemia for Improved Adoptive Cell Therapy

Graduation Year

2024

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Javier Pinilla-Ibarz, M.D., Ph.D.

Committee Member

George Blanck, Ph.D.

Committee Member

Robert Deschenes, Ph.D.

Committee Member

Bala Chandran, Ph.D.

Committee Member

Paulo Rodriguez, Ph.D.

Committee Member

Eva Sahakian, Ph.D.

Keywords

CAR T cell, CLL, Exhaustion, Metabolism, Mitochondria, T cell

Abstract

Chronic lymphocytic leukemia (CLL) is a predominant B-cell malignancy impacting elderly populations in Western societies. Targeted therapies have revolutionized the CLL treatment landscape. Still, patients often require long-term therapy and are subject to drug-induced resistance and severe toxicities, for which CLL is still considered an incurable disease. T-cell-based immunotherapy, particularly chimeric antigen receptor (CAR) T cells, has shown remarkable success, leading to various FDA approvals in hematological malignancies. Nonetheless, CAR T-cell therapy success rate is limited in CLL because of the dysfunctional nature of T cells. CLL T cells exhibit skewing toward immunosuppressive and terminally differentiated phenotypes, leading to hindrances in manufacturing persistent CAR T-cell infusion products for patients. Metabolism plays a significant role in controlling T-cell fate following antigen exposure. Once activated, the T cell engages in a complex differentiation process, each phase of which relies on specific metabolic pathways to support the defined function of this phase. Research has recently shown that metabolic defects of T cells infiltrating the tumor microenvironment (TME) or in chronic viral infection settings pointedly determine the dysfunctional or exhausted state of these cells.

Eμ-TCL1 murine model is the most established preclinical model for studying T-cell dysfunction in CLL. However, this model is not yet validated for investigating the metabolic defects potentially acquired by CLL T cells in the leukemic microenvironment. Using the Eμ-TCL1 model to study T-cell metabolism, this dissertation project demonstrated significant metabolic and mitochondrial abnormalities acquired by CLL T cells along disease progression. This work shows a considerable accumulation of defective depolarized mitochondria associated with significant glycolytic and stress-sensing signaling disturbances in CLL T cells. These metabolic defects were remarkably associated with exhaustion-like transcriptional and epigenetic signatures. Additionally, this project provides preliminary evidence of upregulated endoplasmic reticulum (ER) stress response in CLL T cells, which is potentially involved in the metabolic dysregulation of these cells.

The Phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway plays a central role in T-cell differentiation and metabolic programming following antigen recognition. The work described herein provides a translational approach for reprogramming dysfunctional CLL T cells via ex vivo treatment with PI3K inhibitors (PI3Ki) to improve the manufacturing of adoptive cell therapy (ACT) products. Ex vivo priming with the PI3Ki idelalisib significantly improved the mitochondrial activity and metabolic fitness of CLL T cells. Moreover, idelalisib treatment remarkably shifted T-cell fate into central memory and less-differentiated subsets with reduced exhaustion signature. The established ex vivo treatment protocol enabled us to successfully manufacture an in vivo persistent CAR T-cell therapy product with superior antileukemic activity in the immunocompetent Eμ-TCL1 mice.

Th17 cells are a specific subtype of CD4+ helper T cells previously described as a phenotype with a high degree of plasticity and stem-cell-like properties. Previous studies have shown significant enhancement of in vivo anti-tumor activity and persistence of experimental ACT products using ex vivo polarized murine or human Th17 cells. The work in this dissertation project described, for the first time, the optimization of an in vitro Th17 polarization protocol using CLL T cells for improving ACT. Preliminary findings showed successful engraftment and persistence of in vitro polarized Th17 cells in leukemia-bearing Eμ-TCL1 mice. Additionally, using Eμ-TCL1-derived CD4+ T cells, the optimized protocol was used effectively to generate Th17 CAR T cells that exhibited significant persistence following in vitro challenge with target cells.

In sum, this work contributes to advancing the knowledge and understanding of the metabolic aspects of T-cell dysfunction in CLL. Furthermore, we establish novel approaches for reprogramming T-cell metabolic fitness and ex vivo polarization, generating effective ACT products for CLL. Ultimately, discovering novel techniques for ex vivo manipulating the T-cell dynamic nature is a must to enhance the success rate of T-cell-based immunotherapy in CLL patients.

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