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 1

Prf–/– CAR T cells produce increased proinflammatory cytokines.

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Prf–/– CAR T cells produce increased proinflammatory cytokines. (A) Cell...
(A) Cell product characterization: cells were stained for surface CAR expression, CD4/CD8, and CD62L/CD44 and analyzed by flow cytometry 48 hours after the completion of CAR transduction. Gray-dotted histogram overlays represent the untransduced T cell control. (B) CD107a expression on CAR T cells after 4 hours of incubation with CD19+ or CD19– E2aPBX cells. Representative histogram shows CD107a expression on CAR T cell after stimulation with CD19+ E2aPBX cells; gray-dotted histogram overlays represent the isotype control. (C) In vitro cytotoxicity measured by IncuCyte Zoom: GFP-transduced E2aPBX cells were cocultured with CAR T cells (E:T = 2:1). Green objects (GFP+ leukemia cells) were counted at each time point and normalized to untransduced T cell wells (n = 2, biological duplicate). AUC for Prf–/– CAR T cell was 37.8 (95% CI: 37.6–38.1). AUC for WT CAR T cells was 17.8 (95% CI: 17.6–18.0). (D) IFN-γ levels in the 12-hour coculture supernatant of CAR T cells with CD19+ or CD19– E2aPBX cells (E:T = 1:1), as measured by ELISA. (E) Proliferation assay: CAR T cells labeled with CellTrace Violet were cocultured with either CD19+ or CD19– E2aPBX cells (E:T = 1:1) for 3 days and analyzed by flow cytometry. Gray-dotted histogram overlays represent CAR T cells incubated with CD19– E2aPBX cells (unstimulated controls). Representative histograms from 3 biological replicates are shown. (F) CD4+ CAR T cells, CD8+ CAR T cells, or CD4+ and CD8+ CAR T cells (1:1 mixture) were cocultured with E2aPBX cells overnight (E:T = 1:1). Cytokine levels were measured in the coculture supernatant using the Meso Scale Discovery U-PLEX kit. Data are reported as the mean ± SD (B, D, and F). n = 3 (B and D); n = 4–5 (F). Figures are representative of 3 replicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by Kruskal-Wallis test with Dunn’s correction (B and D) and 1-way ANOVA with Šidák’s correction (F).