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 7

IFN-γ neutralization negatively impacts CAR T cell leukemia clearance.

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IFN-γ neutralization negatively impacts CAR T cell leukemia clearance. (...
(A) Treatment scheme of neutralizing IFN-γ in CAR T cell recipients. Mice were treated with i.p. injection of an IFN-γ–neutralizing antibody (XMG1.2, 200 μg) or an isotype control (rat IgG1, 200 μg) on days 0, 2, 4, 6, 8, 10, and 12. Some mice were euthanized on day 13, which was 24 hours after the last administration of antibodies, while the rest were kept for survival analyses. (B) Kaplan-Meier survival curve. (C) The percentages of CD8+ CAR T cells in spleens on day 13 were assessed by flow cytometry. (D) Surface CAR expression, (E) the percentages of CD44+CD62L– (Tem or Teff) cells, and (F) PD-1 expression among adoptively transferred CD8+ CAR T cell (CD45.2+/CD8+) subsets in spleens on day 13 were analyzed by flow cytometry. (G–J) Leukemia-bearing mice were treated with CAR T cells (5 × 106) derived from WT, Prf–/–, or ARE-Del (homozygous- or heterozygous-KO) mice according to the treatment scheme in Figure 2A. (G) Serum IFN-γ was measured on day 0 (before adoptive T cell transfer) and on days 3, 14, and 29 after adoptive T cell transfer using the Meso Scale Discovery U-PLEX kit. (H) Surface CAR expression, (I) the percentages of CD44+CD62L– (Tem or Teff) cells, and (J) PD-1 expression within the CD8+ CAR T cell subset (CD45.2+CD8+) in spleens on day 13 were analyzed by flow cytometry. Data are reported as the mean ± SD (C–J). n = 4–5 (B, C, E, F, and H–J); n = 9–10 (D and G). Figures are representative of 3 replicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by log-rank (Mantel-Cox) test (B), 1-way ANOVA with Šidák’s correction (C–F and H–J), or Kruskal-Wallis test with Dunn’s correction (G).