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 3

Prf–/– CAR T cell undergo a late reexpansion in the absence of detectable antigens.

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Prf–/– CAR T cell undergo a late reexpansion in the absence of detectabl...
Leukemia-bearing B6-CD45.1 mice were treated as depicted in Figure 2A and received either WT or Prf–/– CAR T cells (CD45.2+) on day 0 at the indicated doses (A and B) or 5 × 106 cells (C–N). (A) The percentages of CD8+ CAR T cells (CD45.2+CD8+) within total splenocytes were evaluated on day 14. (B) Surface CAR expression on CD8+ CAR T cells was assessed by protein L/streptavidin-PE staining on day 14. (C) Leukemia (CD45.2+CD19+) in BM and (D) B cells (CD19+B220+) in spleens were measured at baseline and at the indicated time points following adoptive T cell transfer. (E) The percentages of adoptively transferred CAR T cell (CD45.2+CD3+) in spleens and (F) surface CAR expression on CD8+ CAR T cell in spleens were monitored at indicated time points. (G) The absolute (Abs.) number of CD8+ CAR T cells expressing surface CAR in spleens was evaluated on day 14. (H–J) The composition of CD44–CD62L+ naive, CD44+CD62L+ Tcm, and CD44+CD62L– Tem or Teff cells within the CD8+ CAR T cell subset in spleens was assessed. (H) Representative dot plots (day 14) and the percentages of (I) Teff and (J) Tcm cells in CD8+ CAR T cells are shown. (K–N) Expression of surface CAR, PD-1, TIM3, and LAG3 on CD8+ CAR T cells in spleens were assessed by flow cytometry on day 14. (K) Representative dot plots and the percentages of WT and Prf–/– CD8+ CAR T cells expressing (L) PD-1, (M) TIM3, and (N) LAG3 are shown. Data are reported as the mean ± SD (A–G, I, J, and L–N). n = 5 (A, B, G, and L–N); n = 4 (C–F, I, and J). Figures are representative of 3 replicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA with Šidák’s correction (A, B, I, and J) or Mann-Whitney U test (G and L–N).