Perforin-deficient CAR T cells recapitulate late-onset inflammatory toxicities observed in patients
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
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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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 2

Prf–/– CAR T cells exhibit inferior cytotoxicity compared with WT CAR T cells.

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Prf–/– CAR T cells exhibit inferior cytotoxicity compared with WT CAR T ...
(A) In vivo treatment scheme: B6-CD45.1 mice were injected with 1 × 106 E2aPBX (CD45.2+) cells via tail vein injection (i.v.) on day –6, lymphodepleted with cyclophosphamide i.p. injection (200 mg/kg) on day –1, and administered CAR T cells (CD45.2+) i.v. on day 0. (B–F) Leukemia-bearing B6-CD45.1 mice were treated with either WT or Prf–/– CAR T cells at a cell dosage of 5 × 104, 1 × 105, or 5 × 106. (B) BM leukemia burden (CD45.2+CD19+) on day 7 was assessed by flow cytometry. (C) Kaplan-Meier survival curve. (D and E) Total splenic B cells (CD19+B220+) on day 14 were assessed by flow cytometry. (D) Representative dot plots and (E) statistical comparisons are shown. (F) Adoptively transferred T cells (CD45.2+ and either CD8+ or CD4+) in spleens on day 7 were assessed by flow cytometry. (G and H) CAR T cells were manufactured from CD4+ or CD8+ purified splenic T lymphocytes. Leukemia-bearing B6-CD45.1 mice were treated with either CD4+, CD8+, or a 1:1 mixture of CD4+ and CD8+ CAR T cells manufactured from WT or Prf–/– donors (total CAR T cells: 1 × 105 cells/mouse), according to the experimental scheme depicted in A. (G) Kaplan-Meier survival curve and (H) BM leukemia burden (CD45.2+CD19+) on day 14, as assessed by flow cytometry. Data are reported as the mean ± SD (B, E, F, and H). n = 5 (B, C, F, G, and H); n = 10 (E, pooled data from 2 independent experiments). Figures are representative of 2 replicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by Kruskal-Wallis test with Dunn’s correction (B, E, and H), 1-way ANOVA with Šidák’s correction (F), or log-rank (Mantel-Cox) test (C and G).