Graduation Year

2025

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Marco L. Davila,MD, Ph.D.

Committee Member

Michael D. Jain,MD, Ph.D.

Committee Member

Paulo Rodriguez, Ph.D.

Committee Member

Jose Alejandro Guevara,MD, Ph.D.

Keywords

Anti-CD19, CAR-T Therapy, B Cell Acute lymphoblastic Leukemia, Cytokine Release Syndrome, Neutropenia, IFNγ-blockade, IL-2Rα, Regulatory T cells

Abstract

CAR-T therapy has improved overall and complete response rates, andprolonged progression free survival of patients with relapsed/refractory hematological malignancies. The short-term and long-term impacts of anti-CD19 CAR-T products on the immune landscape of B cell malignancies has garnered considerable attention in recent years. Most notably, a subset of patients experience high-grade cytokine toxicities, including cytokine release syndrome and immune cell-associated hematologic toxicity that manifest as prolonged cytopenia. Whereas etiology of CRS is more well established, the mechanisms leading to CAR-T associated cytopenia remains elusive. Rejeski et. al. showed that severe cytopenia in patients post CAR-T treatment led to frequent infections and increased hospitalizations. Onset of cytopenia was associated with worse survival owing to non-relapse mortality in patients post CAR-T treatment, thereby emphasizing the need to address these hematological toxicities. An observation drawn from two independent clinical trials was the co-occurrence of cytopenia with CRS. Among the cytopenias neutropenia was most predominant, where the decline in neutrophils intensified with the grade of CRS. We utilized an IL-2Rα knockout mouse model to recapitulate cytokine toxicities with elevated levels of IL6, IFNγ, and TNFα and increased M1-like-like macrophages. Onset of CRS was accompanied by a reduction in peripheral blood neutrophils due to a disruption of bone marrow

neutrophil homeostasis characterized by an increase in apoptotic neutrophils, and adecrease in proliferative and mature neutrophils. Both non-tumor-bearing and Eμ- ALL tumor-bearing mice recapitulated the co-occurrence of CRS and neutropenia. IFNγ-blockade alleviated both CRS and neutropenia without affecting CAR-T efficacy. Mechanistically, a Th1-Th17 imbalance was observed to drive cooccurrence of CRS and neutropenia in an IFNγ-dependent manner, leading to decreased IL-17A and G-CSF, neutrophil production, and neutrophil survival. In patients, we observed an increase in the IFNγ-to-IL-17A ratio in the peripheral blood during high-grade CRS and neutropenia. We have uncovered a biological basis for ICAHT and provide support for the use of IFNγ-blockade to reduce CRS and neutropenia.

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