4-1BB enhancement of CAR T function requires NF-κB and TRAFs
Gongbo Li, Justin C. Boucher, Hiroshi Kotani, Kyungho Park, Yongliang Zhang, Bishwas Shrestha, Xuefeng Wang, Lawrence Guan, Nolan Beatty, Daniel Abate-Daga, Marco L. Davila
Gongbo Li, Justin C. Boucher, Hiroshi Kotani, Kyungho Park, Yongliang Zhang, Bishwas Shrestha, Xuefeng Wang, Lawrence Guan, Nolan Beatty, Daniel Abate-Daga, Marco L. Davila
View: Text | PDF
Research Article Immunology Oncology

4-1BB enhancement of CAR T function requires NF-κB and TRAFs

Abstract

Chimeric antigen receptors (CARs) have an antigen-binding domain fused to transmembrane, costimulatory, and CD3ζ domains. Two CARs with regulatory approval include a CD28 or 4-1BB costimulatory domain. While both CARs achieve similar clinical outcomes, biologic differences have become apparent but not completely understood. Therefore, in this study we aimed to identify mechanistic differences between 4-1BB and CD28 costimulation that contribute to the biologic differences between the 2 CARs and could be exploited to enhance CAR T cell function. Using CD19-targeted CAR T cells with 4-1BB we determined that enhancement of T cell function is driven by NF-κB. Comparison to CAR T cells with CD28 also revealed that 4-1BB is associated with more antiapoptotic proteins and dependence on persistence for B cell killing. While TNF receptor–associated factor 2 (TRAF2) has been presupposed to be required for 4-1BB costimulation in CAR T cells, we determined that TRAF1 and TRAF3 are also critical. We observed that TRAFs impacted CAR T viability and proliferation, as well as cytotoxicity and/or cytokines, in part by regulating NF-κB. Our study demonstrates how 4-1BB costimulation in CAR T cells impacts antitumor eradication and clinical outcomes and has implications for enhanced CAR design.

Authors

Gongbo Li, Justin C. Boucher, Hiroshi Kotani, Kyungho Park, Yongliang Zhang, Bishwas Shrestha, Xuefeng Wang, Lawrence Guan, Nolan Beatty, Daniel Abate-Daga, Marco L. Davila

×

Figure 5

NF-κB signaling regulates the viability and proliferation of 4-1BB–based CAR T cells.

Options: View larger image (or click on image) Download as PowerPoint
NF-κB signaling regulates the viability and proliferation of 4-1BB–based...
(A) CAR expression (mCherry) after transduction (left) and NF-κB upregulation (right) in NF-κB/293/GFP-Luc reporter cells. Reporter cells were transduced with mouse CD19-targeted CARs and NF-κB signaling was measured by flow cytometry for GFP. Data are representative of 2 independent experiments. (B) m19-humBBz CAR T cells have greater NF-κB signaling than m1928z CAR T cells after antigen stimulation. Three million CAR T cells derived from NF-κB-RE-luc transgenic mice were cocultured with 3T3-mCD19 cells at a 10:1 ratio for 4 hours. Cell lysates were evaluated using a luciferase assay. Data are representative of 3 independent experiments in triplicate. (C) Amino acid sequences of human wild-type and mutated 4-1BB endodomains evaluated in hCD19-targeted CARs. Putative TRAF binding domains or mutations are in red. Amino acid numbers of the 4-1BB endodomain are shown. (D) NF-κB signaling of hCD19 CAR–transduced reporter cells. NF-κB/293/GFP-Luc reporter cells were retrovirally transduced with hCD19-targeted CARs. Percentages of GFP+ cells, which reflect NF-κB signaling, were measured by flow cytometry. Data are from 1 experiment and done in triplicate. (E) Viability on day 16 and (F) proliferation of hCD19-targeted CAR T cells cultured in vitro. Human T cells were isolated from healthy donor peripheral blood mononuclear cells at day 0. CAR T cells were harvested, beads removed and cocultured with 3T3-hCD19 cells at a 5:1 ratio for 2 weeks. Cell numbers were measured at indicated time points. (G) Cytotoxicity of hCD19-targeted CAR T cells. CAR T cells were cocultured with 3T3-hCD19 cells at a 10:1 ratio. Target cell killing was monitored by xCELLigence RTCA (real-time cell analysis). For E, F, and G, data are from a single experiment in triplicate. Data in B–F represent the mean ± SD. Cytotoxicity curves show the mean only. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by unpaired t test (B, D, and E) or 2-way ANOVA (F). ns, not significant.