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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-1? 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.

SUBMITTER: Ishii K

PROVIDER: S-EPMC7524496 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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

Ishii Kazusa K Pouzolles Marie M Chien Christopher D CD Erwin-Cohen Rebecca A RA Kohler M Eric ME Qin Haiying H Lei Haiyan H Kuhn Skyler S Ombrello Amanda K AK Dulau-Florea Alina A Eckhaus Michael A MA Shalabi Haneen H Yates Bonnie B Lichtenstein Daniel A DA Zimmermann Valérie S VS Kondo Taisuke T Shern Jack F JF Young Howard A HA Taylor Naomi N Shah Nirali N NN Fry Terry J TJ

The Journal of clinical investigation 20201001 10

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 w ...[more]

PMID: 32925169

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Project description:The impact of neuroinflammation and microglial activation to Parkinson's disease (PD) progression is still debated. Post-mortem analysis of PD brains has shown that neuroinflammation and microgliosis are key features of end-stage disease. However, microglia neuroimaging studies and evaluation of cerebrospinal fluid (CSF) cytokines in PD patients at earlier stages do not support the occurrence of a pronounced neuroinflammatory process. PD animal models recapitulating the motor and non-motor features of the disease, and the slow and progressive neuropathology, can be of great advantage in understanding whether and how neuroinflammation associates with the onset of symptoms and neuronal loss. We recently described that 18-month-old NF-κB/c-Rel deficient mice (c-rel-/-) develop a spontaneous late-onset PD-like phenotype encompassing L-DOPA-responsive motor impairment, nigrostriatal neuron degeneration, α-synuclein and iron accumulation. To assess whether inflammation and microglial activation accompany the onset and the progression of PD-like pathology, we investigated the expression of cytokines (interleukin 1 beta (Il1b), interleukin 6 (Il6)) and microglial/macrophage activation markers (Fc gamma receptor III (Fcgr3), mannose receptor 1 (Mrc1), chitinase-like 3 (Ym1), arginase 1 (Arg 1), triggering receptor expressed on myeloid cells 2 (Trem2)), together with microglial ionized calcium binding adapter molecule 1 (Iba1) and astrocyte glial fibrillary acidic protein (GFAP) immunolabeling, in the substantia nigra (SN) of c-rel-/- mice, at premotor (4- and 13-month-old) and motor phases (18-month-old). By quantitative real-time RT-PCR we found increased M2c microglial/macrophage markers expression (Mrc1 and Arg1) in 4-month-old c-rel-/- mice. M2-type transcription dropped down in 13-month-old c-rel-/- mice. At this age, the pro-inflammatory Il1b, but not Il6 or the microglia-macrophage M1-polarization marker Fcgr3/CD16, increased when compared to wild-type (wt). Furthermore, no significant variation in the transcription of inflammatory and microglial/macrophage activation genes was present in 18-month-old c-rel-/- mice, that display motor dysfunctions and dopaminergic neuronal loss. Immunofluorescence analysis of Iba1-positive cells in the SN revealed no sign of overt microglial activation in c-rel-/- mice at all the time-points. MRC1-Iba1-positive cells were identified as non-parenchymal macrophages in 4-month-old c-rel-/- mice. Finally, no sign of astrogliosis was detected in the SN of the diverse animal groups. In conclusion, this study supports the presence of a mild inflammatory profile without evident signs of gliosis in c-rel-/- mice up to 18 months of age. It suggests that symptomatic PD-like phenotype can develop in the absence of concomitant severe inflammatory process.

Project description:BACKGROUNDChimeric antigen receptor (CAR) T cell immunotherapy has resulted in complete remission (CR) and durable response in highly refractory patients. However, logistical complexity and high costs of manufacturing autologous viral products limit CAR T cell availability.METHODSWe report the early results of a phase I/II trial in B cell acute lymphoblastic leukemia (B-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT) using donor-derived CD19 CAR T cells generated with the Sleeping Beauty (SB) transposon and differentiated into cytokine-induced killer (CIK) cells.RESULTSThe cellular product was produced successfully for all patients from the donor peripheral blood (PB) and consisted mostly of CD3+ lymphocytes with 43% CAR expression. Four pediatric and 9 adult patients were infused with a single dose of CAR T cells. Toxicities reported were 2 grade I and 1 grade II cytokine-release syndrome (CRS) cases at the highest dose in the absence of graft-versus-host disease (GVHD), neurotoxicity, or dose-limiting toxicities. Six out of 7 patients receiving the highest doses achieved CR and CR with incomplete blood count recovery (CRi) at day 28. Five out of 6 patients in CR were also minimal residual disease negative (MRD-). Robust expansion was achieved in the majority of the patients. CAR T cells were measurable by transgene copy PCR up to 10 months. Integration site analysis showed a positive safety profile and highly polyclonal repertoire in vitro and at early time points after infusion.CONCLUSIONSB-engineered CAR T cells expand and persist in pediatric and adult B-ALL patients relapsed after HSCT. Antileukemic activity was achieved without severe toxicities.TRIAL REGISTRATIONClinicalTrials.gov NCT03389035.FUNDINGThis study was supported by grants from the Fondazione AIRC per la Ricerca sul Cancro (AIRC); Cancer Research UK (CRUK); the Fundación Científica de la Asociación Española Contra el Cáncer (FC AECC); Ministero Della Salute; Fondazione Regionale per la Ricerca Biomedica (FRRB).

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

Publications

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

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