Glioblastoma-targeted CD4+ CAR T cells mediate superior antitumor activity
Dongrui Wang, … , Stephen J. Forman, Christine E. Brown
Dongrui Wang, … , Stephen J. Forman, Christine E. Brown
Published May 17, 2018
Citation Information: JCI Insight. 2018;3(10):e99048. https://doi.org/10.1172/jci.insight.99048.
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Research Article Immunology Oncology

Glioblastoma-targeted CD4+ CAR T cells mediate superior antitumor activity

Abstract

Chimeric antigen receptor–modified (CAR-modified) T cells have shown promising therapeutic effects for hematological malignancies, yet limited and inconsistent efficacy against solid tumors. The refinement of CAR therapy requires an understanding of the optimal characteristics of the cellular products, including the appropriate composition of CD4+ and CD8+ subsets. Here, we investigated the differential antitumor effect of CD4+ and CD8+ CAR T cells targeting glioblastoma-associated (GBM-associated) antigen IL-13 receptor α2 (IL13Rα2). Upon stimulation with IL13Rα2+ GBM cells, the CD8+ CAR T cells exhibited robust short-term effector function but became rapidly exhausted. By comparison, the CD4+ CAR T cells persisted after tumor challenge and sustained their effector potency. Mixing with CD4+ CAR T cells failed to ameliorate the effector dysfunction of CD8+ CAR T cells, while surprisingly, CD4+ CAR T cell effector potency was impaired when coapplied with CD8+ T cells. In orthotopic GBM models, CD4+ outperformed CD8+ CAR T cells, especially for long-term antitumor response. Further, maintenance of the CD4+ subset was positively correlated with the recursive killing ability of CAR T cell products derived from GBM patients. These findings identify CD4+ CAR T cells as a highly potent and clinically important T cell subset for effective CAR therapy.

Authors

Dongrui Wang, Brenda Aguilar, Renate Starr, Darya Alizadeh, Alfonso Brito, Aniee Sarkissian, Julie R. Ostberg, Stephen J. Forman, Christine E. Brown

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Figure 2

CD4+ CAR T cells retain effector potency after repetitive tumor challenge.

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CD4+ CAR T cells retain effector potency after repetitive tumor challeng...
(A) PBT030-2 GBM cells were cocultured with CD4+ or CD8+ CAR T cells at E:T ratios of 1:4, 1:6, 1:10, and 1:20, and the numbers of viable tumor cells were enumerated at the denoted time points. n = 3 replicates per time point. **P < 0.01 using an unpaired Student’s t test. (B) Schema of repetitive tumor challenge assay. CAR T cells were cocultured with PBT030-2 GBM cells (4,000 T cells; 16,000 GBM cells; E:T = 1:4) and rechallenged with 32,000 GBM cells every other day. (C) Remaining viable tumor cell numbers were quantified at the indicated time points during the rechallenge assay (arrows indicate tumor rechallenge). n = 3 replicates per data point. ***P < 0.001, unpaired Student’s t test comparing the CD4+ and CD8+ groups at the indicated time points. (D) CFSE-labeled, repetitively tumor challenged CD4+ and CD8+ CAR T cell were analyzed for CFSE intensity (gated on CD19+ T cells) to determine their proliferation status at D3 and D7. (E–H) Activation potential after in vivo tumor stimulation. (E) Isolated CD4+ or CD8+ CAR T cells were administered to tumors via intratumoral injection and harvested after 5 days for in vitro restimulation with PBT030-2 cells (E:T = 1:1, equivalent CAR [CD19+] T cell number across different groups) to evaluate their activation potential. (F–H) In vivo tumor stimulated CD4+ and CD8+ CAR T cells were evaluated for their activation potential through degranulation (F), intracellular IFN-γ (G), and surface expression of activation markers CD69 and 4-1BB (H) following in vitro restimulation. (F and G) n = 3 replicates.*P < 0.05, **P < 0.01 unpaired Student’s t test compared with the non–in vivo–stimulated cells (None). All data are representative of 3 different donors; data represents ± SEM.