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
View: Text | PDF
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

×

Figure 3

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

Options: View larger image (or click on image) Download as PowerPoint
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).