Perforin-deficient CAR T cells recapitulate late-onset inflammatory toxicities observed in patients
Kazusa Ishii, … , Nirali N. Shah, Terry J. Fry
Kazusa Ishii, … , Nirali N. Shah, Terry J. Fry
Published September 14, 2020
Citation Information: J Clin Invest. 2020;130(10):5425-5443. https://doi.org/10.1172/JCI130059.
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Research Article Immunology Inflammation

Perforin-deficient CAR T cells recapitulate late-onset inflammatory toxicities observed in patients

Abstract

Late-onset inflammatory toxicities resembling hemophagocytic lymphohistiocytosis (HLH) or macrophage activation syndrome (MAS) occur after chimeric antigen receptor T cell (CAR T cell) infusion and represent a therapeutic challenge. Given the established link between perforin deficiency and primary HLH, we investigated the role of perforin in anti-CD19 CAR T cell efficacy and HLH-like toxicities in a syngeneic murine model. Perforin contributed to both CD8+ and CD4+ CAR T cell cytotoxicity but was not required for in vitro or in vivo leukemia clearance. Upon CAR-mediated in vitro activation, perforin-deficient CAR T cells produced higher amounts of proinflammatory cytokines compared with WT CAR T cells. Following in vivo clearance of leukemia, perforin-deficient CAR T cells reexpanded, resulting in splenomegaly with disruption of normal splenic architecture and the presence of hemophagocytes, which are findings reminiscent of HLH. Notably, a substantial fraction of patients who received anti-CD22 CAR T cells also experienced biphasic inflammation, with the second phase occurring after the resolution of cytokine release syndrome, resembling clinical manifestations of HLH. Elevated inflammatory cytokines such as IL-1β and IL-18 and concurrent late CAR T cell expansion characterized the HLH-like syndromes occurring in the murine model and in humans. Thus, a murine model of perforin-deficient CAR T cells recapitulated late-onset inflammatory toxicities occurring in human CAR T cell recipients, providing therapeutically relevant mechanistic insights.

Authors

Kazusa Ishii, Marie Pouzolles, Christopher D. Chien, Rebecca A. Erwin-Cohen, M. Eric Kohler, Haiying Qin, Haiyan Lei, Skyler Kuhn, Amanda K. Ombrello, Alina Dulau-Florea, Michael A. Eckhaus, Haneen Shalabi, Bonnie Yates, Daniel A. Lichtenstein, Valérie S. Zimmermann, Taisuke Kondo, Jack F. Shern, Howard A. Young, Naomi Taylor, Nirali N. Shah, Terry J. Fry

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Figure 8

IL-1 blockade improves circulating proinflammatory cytokine levels without inhibiting CAR T cell leukemia clearance.

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IL-1 blockade improves circulating proinflammatory cytokine levels witho...
(A) Treatment scheme for IL-1 blockade in CAR T cell recipients. Mice were treated with a daily i.p. injection of anakinra (10 mg/kg/day in 200 μL) or PBS (200 μL) from days 0 to 13. Some cohorts of mice were euthanized on day 14, which was 24 hours after the last administration of anakinra or PBS, while the rest were kept for survival analyses. (B) Kaplan-Meier survival curve. (C) Leukemia (CD45.2+CD19+) in BM on day 14 was assessed by flow cytometry. (D) Percentages of CD8+ CAR T cell in spleens on day 14. (E) Surface CAR expression and (F) percentages of CD44+CD62L– (Tem or Teff) cells within the CD8+ CAR T cell subset (CD45.2+CD8+) in spleens were evaluated on day 14. (G) Serum levels of IFN-γ, IL-27, and TNF-α were measured on day 0 (before CAR T cell infusion) and on day 14 using the Meso Scale Discovery U-PLEX kit. Healthy controls were age- and sex-matched untreated littermates. Data are reported as the mean ± SD (C–G). n = 5 (B and C); n = 13–15 (D, data were pooled from independent 2 experiments); n = 9–10 (E and F); n = 10 (G, except for n = 6 in the IFN-γ pre-CAR group). Figures are representative of 2 replicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by log-rank (Mantel-Cox) test (B) or 1-way ANOVA with Šidák’s correction (D–G).