IFN-γ–driven skewing towards Th1 over Th17 differentiation underlies CRS and neutropenia in CAR-T therapy
Payal Goala, Yongliang Zhang, Nolan Beatty, Allan Pavy, Shannon McSain, Cooper Sailer, Muhammad Junaid Tariq, Showkat Hamid, Eduardo Cortes Gomez, Jianmin Wang, Duna Massillon, Maxwell Ilecki, Justin C. Boucher, Constanza Savid-Frontera, Sae Bom Lee, Hiroshi Kotani, Meredith L. Stone, Michael D. Jain, Marco L. Davila
Payal Goala, Yongliang Zhang, Nolan Beatty, Allan Pavy, Shannon McSain, Cooper Sailer, Muhammad Junaid Tariq, Showkat Hamid, Eduardo Cortes Gomez, Jianmin Wang, Duna Massillon, Maxwell Ilecki, Justin C. Boucher, Constanza Savid-Frontera, Sae Bom Lee, Hiroshi Kotani, Meredith L. Stone, Michael D. Jain, Marco L. Davila
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Research Article Hematology Immunology Oncology

IFN-γ–driven skewing towards Th1 over Th17 differentiation underlies CRS and neutropenia in CAR-T therapy

Abstract

Chimeric antigen receptor T cell (CAR-T) therapy has led to significant improvements in patient survival. However, a subset of patients experience high-grade toxicities, including cytokine release syndrome (CRS) and immune cell–associated hematological toxicity (ICAHT). We utilized IL-2Ra knockout mice to model toxicities with elevated levels of IL-6, IFN-γ, and TNF-α and increased M1-like macrophages. Onset of CRS was accompanied by a reduction in peripheral blood neutrophils due to disruption of bone marrow neutrophil homeostasis characterized by an increase in apoptotic neutrophils and a decrease in proliferative and mature neutrophils. Both nontumor-bearing and Em-ALL tumor-bearing mice recapitulated the cooccurrence of CRS and neutropenia. IFN-γ–blockade alleviated CRS and neutropenia without affecting CAR-T efficacy. Mechanistically, a Th1-Th17 imbalance was observed to drive cooccurrence of CRS and neutropenia in an IFN-γ–dependent manner leading to decreased IL-17A and G-CSF, neutrophil production, and neutrophil survival. In patients, we observed an increase in the IFN-γ–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 IFN-γ blockade to reduce both CRS and neutropenia.

Authors

Payal Goala, Yongliang Zhang, Nolan Beatty, Allan Pavy, Shannon McSain, Cooper Sailer, Muhammad Junaid Tariq, Showkat Hamid, Eduardo Cortes Gomez, Jianmin Wang, Duna Massillon, Maxwell Ilecki, Justin C. Boucher, Constanza Savid-Frontera, Sae Bom Lee, Hiroshi Kotani, Meredith L. Stone, Michael D. Jain, Marco L. Davila

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Figure 3

Evaluating cooccurrence of CRS and Neutropenia in IL-2Ra KO mice.

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Evaluating cooccurrence of CRS and Neutropenia in IL-2Ra KO mice. (A) Sc...
(A) Schematic representation of IL-2Ra KO and WT mice treated with 2M WT CAR-T (i.v.) cells. Data are pooled from 2 independently performed experiments. (B–D) Time course analysis of cytokines IL-6 (B), TNF-α (C) and IFN-γ (D) in post WT CAR-T treated IL-2Ra KO and WT mice. IL-2Ra KO pre and postcytokines are paired values taken from each mouse alive until week 4 (n = 10). WT paired cytokine values represent a subset of live mice until week 4 (n = 7). (E–G) Time course of neutrophil concentration (K/mL) (E), red blood cell (RBC) concentration (M/mL) (F) and platelet concentration (K/mL) (G) in the peripheral blood from IL-2Ra KO and C57BL/6J mice using complete blood profiling (CBC). Pre and post levels from CBC are paired values taken from each mouse alive until week 4 per cohort (n = 8 for IL-2Ra KO and n = 8 for C57BL/6J). (H) Schematic of IL-2Ra KO and C57BL/6J mice treated with WT CAR-T cells sacrificed periodically followed by kit-based isolation of neutrophils from bone marrow (BM). Cell turnover of the purified neutrophils was analyzed for apoptosis using Annexin V, and proliferation using BrDU. Neutrophil maturation rate was analyzed using BMMC. Data are pooled from 2 independently performed experiments. I–J. Time point comparison in IL-2Ra KO versus C57BL/6J mice of % Annexin V+ apoptotic cells as a frequency of neutrophils (kit-based purification) (I) and % Ly6G+CXCR2+ cells as a frequency of mature neutrophils (gated on BMMC as Live+Lineage-CD11b+Gr1+ckit-CXCR4- cells) (J). (K) IL-2Ra KO and C57BL/6J mice were injected with 2mg BrDU i.p. 48 hours prior to harvest, followed by staining for BrDU-labelled neutrophils. Line plot represents % BrDU+ cells as a frequency of live neutrophils (kit-based purification) at weeks 1 through 4. Error bars represent SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. P values for line plots (B–G) were generated using paired t test. P values for line plots (I–K) were generated using unpaired t test.