Language

English

Publication Date

3-1-2023

Journal

Transplantation and Cellular Therapy

DOI

10.1016/j.jtct.2022.12.020

PMID

36592718

PMCID

PMC9991966

PubMedCentral® Posted Date

3-1-2024

PubMedCentral® Full Text Version

Author MSS

Abstract

Background: CAR-T therapy represents a revolutionary treatment for patients with relapsed/refractory hematologic malignancies. However, its use can result in significant toxicities, including cytokine release syndrome (CRS), a potentially life-threatening clinical syndrome resulting from release of pro-inflammatory cytokines upon T cell activation. In addition, patients who develop CRS often experience prolonged cytopenias and those with the most severe CRS also have the greatest delay in full marrow recovery. While an association between CRS and delayed bone marrow recovery has been established, the precise mechanism underlying this phenomenon remains unknown.

Objective: To test our hypothesis that delayed bone marrow recovery following CAR-T cell therapy is caused by elevation of pro-inflammatory cytokines leading to apoptosis and depletion of hematopoietic stem and progenitor cells (HSPCs).

Study Design: SCID-beige mice bearing intraperitoneal CD19+ Raji cell tumors were treated with injection of human CD19.28z CAR-T cells. Bone marrow was then harvested for analysis by flow cytometry and HSPCs were isolated for whole transcriptome analysis by RNA sequencing. Additionally, complete blood counts and serum cytokine levels were measured. A second model was developed in which SCID-beige mice were treated with murine IFNγ (mIFNγ) or murine IL-6 (mIL-6) or both. Bone marrow was harvested and flow cytometric assays were conducted to evaluate the degree of apoptosis and proliferation on specific HSPC populations.

Results: SCID-beige mice bearing intraperitoneal Raji cell tumors that were treated with CAR-T cells developed CRS, with elevation of several pro-inflammatory cytokines, including profound elevation of human IFNg. Gene set enrichment analysis of RNA sequencing data revealed that genes associated with apoptosis were significantly upregulated in HSPCs from mice that developed CRS. EPCR negative HSCs, a subset of HSCs that is poised for terminal differentiation, was found to be specifically decreased in mice that were treated with CAR-T cells. Furthermore, HSPCs were found to have increased levels of apoptosis upon treatment with mIFNγ and mIL-6, while ST-HSC and multipotent progenitors exhibited increases in proliferation with mIFNγ treatment alone.

Conclusion: The results from this study provide evidence that the elevation of pro-inflammatory cytokines following CAR-T therapy impacts the bone marrow through a combined mechanism: pluripotent HSCs that are exposed to elevated levels of IFNγ and IL-6 undergo increased cell death, while more committed progenitor cells become more proliferative in response to elevated IFNγ. These combined effects lead to depleted stores of repopulating HSCs and ultimately cytopenias.

Keywords

Animals, Humans, Mice, Apoptosis, Bone Marrow, Bone Marrow Diseases, Cytokines, Hematopoietic Stem Cells, Interleukin-6, Mice, SCID, Neoplasms, Receptors, Chimeric Antigen, Immunotherapy, Adoptive

Published Open-Access

yes

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