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 4

Prf–/– CAR T cells induce inflammation in recipients.

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Prf–/– CAR T cells induce inflammation in recipients. Leukemia-bearing m...
Leukemia-bearing mice were treated with CAR T cells (5 × 106 cells) as depicted in Figure 2A. (A) The weight of the recipients’ spleens and (B) absolute total splenocyte counts were monitored. (C) H&E-stained images of recipients’ spleens harvested on day 15. In Prf–/– tissue in far right bottom panel, phagocytes (yellow arrows) and mitotic figures (red arrows) are highlighted. Scale bars: 200 μm, 100 μm, and 20 μm (original magnification, 100× for the enlarged inset showing a representative phagocyte). (D) Giemsa stain of spleen section touch preparation from Prf–/– CAR T cell recipients on day 14. Two representative hemophagocytes are shown (original magnification, 100×). (E) The percentages and (F) absolute numbers of recipient-derived (CD45.1+CD45.2–) CD8+, CD11b+, and CD19+ cells on day 15 in spleens, as assessed by flow cytometry. (G) Representative dot plots and (H) statistical comparison of CD44–CD62L+ naive T cell, CD44+CD62L+ Tcm cell, and CD44+CD62L– Tem or Teff cell composition within recipient-derived CD8+ T cells. (I) Gene expression in whole BM from recipients (NanoString nCounter Inflammation panel). Volcano plot shows gene expression data comparing recipients of Prf–/– CAR T cells with those of WT CAR T cells [–log10(P value) vs. log2(fold change)]. Genes with a differential expression of P < 0.01 are annotated in the legend. Genes involved in inflammatory pathways are further highlighted in red. (J and K) Expression levels of (J) pro–IL-1β and (K) IL-18 were assessed by RT-qPCR. (L–N) Serum levels of (L) IFN-γ, (M) TNF-α, and (N) IL-6 in CAR T cell recipients were measured at the indicated time points using the Cytokine Beads Array kit. Data are reported as the mean ± SD (A, B, E, F, H, and J–N). n = 5 (B and I); n = 4 (A, E, F, H, and L–N); n = 4–5 (J); n = 9–10 (K). Figures are representative of 3 replicate experiments (A–H and J–N). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by Kruskal-Wallis test with Dunn’s correction (B and M) or 1-way ANOVA with Šidák’s correction (E, F, H, J, K, and L). Ctrl, control.