Apoptosis of Hematopoietic Stem Cells Contributes to Bone Marrow Suppression Following CAR-T Therapy

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 in which patients develop fever, hypotension, hypoxia and capillary leak syndrome due to the production of multiple pro-inflammatory cytokines upon T cell activation. In addition to these acute effects, patients who develop CRS frequently suffer from prolonged cytopenias and delayed bone marrow recovery. Moreover, those who experience the most severe symptoms of CRS were found to have the greatest delay in full marrow recovery, placing patients at risk for life-threatening infections. Although an association between severe CRS and prolonged neutropenia has been clearly established, the precise mechanism underlying this phenomenon remains undefined.

To address the impact of the hyperinflammatory state of CRS on the bone marrow in patients treated with CAR-T cells, we adapted an existing model of CRS in which human CD19.28z CAR-T cells are injected intraperitoneally to mice bearing tumors derived from human CD19+ Raji cell lines. Mice treated with CAR-T cells were found to have elevation of several pro-inflammatory cytokines, including profound elevation of human IFNgamma several orders of magnitude higher than seen in common chronic infection models. HSPCs were isolated for RNA sequencing and gene set enrichment analysis of the RNA sequencing data revealed that genes associated with apoptosis were significantly upregulated in HSPCs from mice that developed CRS. Additionally, quantification of HSPCs by flow cytometry revealed that an EPCR negative population of HSPCs, which represents an inflammation responsive subset of HSPCs intermediate in its commitment toward terminal differentiation, was found to be decreased in mice that were treated with CAR-T cells.

Though mice bearing Raji cell tumors that were treated with CAR-T cells exhibited signs of CRS and elevated pro-inflammatory cytokines, they did not develop cytopenias as typically seen in patients who develop CRS secondary to CAR-T therapy. We reasoned that this may be partly due to the fact that several of the pro-inflammatory cytokines elevated in this model are derived from the human CAR-T cells, and thus have a diminished effect on mouse HSPCs. To address this question, we injected mice with mIFNgamma and mIL-6 on three consecutive days to mimic the effects of CRS in a fully murine model. Bone marrow was harvested and flow cytometric assays were conducted to evaluate the degree of apoptosis and proliferation on specific HSPC populations. HSPCs were found to have increased levels of apoptosis upon treatment with mIFNgamma and mIL-6, while ST-HSC and multipotent progenitors exhibited increases in proliferation with mIFNgamma treatment alone.

This study provides evidence that pro-inflammatory cytokines are elevated far more significantly in CAR-T therapy compared to chronic infection models and impact the bone marrow through a combined mechanism. Pluripotent HSCs that are exposed to elevated levels of IFNgamma and IL-6 undergo increased cell death, while more committed progenitor cells become more proliferative in response to elevated IFNgamma. These bystander effects contribute to depleted stores of repopulating HSCs and, ultimately, cytopenias. We speculate that administration of administration of IFNgamma antibodies or JAK inhibitors may help preserve bone marrow function in the setting of CAR-T therapy.

Mamonkin:Fate Therapeutics: Patents & Royalties; Allogene Therapeutics: Patents & Royalties. Rouce:Tessa Therapeutics: Research Funding; Kite Pharmaceuticals: Research Funding; Novartis: Honoraria; Pfizer: Consultancy.