4-1BB stimulation with concomitant inactivation of adenosine A2B receptors enhances CD8+ T cell antitumor response
Jihae Ahn, Ping Xie, Siqi Chen, Guilan Shi, Jie Fan, Minghui Zhang, Hui Tang, Amanda R. Zuckerman, Deyu Fang, Yong Wan, Timothy M. Kuzel, Yi Zhang, Bin Zhang
Jihae Ahn, Ping Xie, Siqi Chen, Guilan Shi, Jie Fan, Minghui Zhang, Hui Tang, Amanda R. Zuckerman, Deyu Fang, Yong Wan, Timothy M. Kuzel, Yi Zhang, Bin Zhang
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Research Article Immunology Oncology

4-1BB stimulation with concomitant inactivation of adenosine A2B receptors enhances CD8+ T cell antitumor response

Abstract

Activating the immune costimulatory receptor 4-1BB (CD137) with agonist antibody binding and crosslinking-inducing agents that elicit 4-1BB intracellular signaling potentiates the antitumor responses of CD8+ T cells. However, the underlying in-depth mechanisms remain to be defined. Here, we show that agonistic 4-1BB treatment of activated CD8+ T cells under continuous antigenic stimulation makes them more metabolically vulnerable to redox perturbation by ablation of intracellular glutathione (GSH) and glutathione peroxidase 4 (GPX4) inhibition. Further, genetic deletion of adenosine A2B receptor (A2BR) induces superior survival and expansion advantage of competent CD8+ T cells with agonistic 4-1BB costimulation, leading to more effective antitumor efficacy of adoptive cell therapy (ACT). Mechanistically, A2BR deletion helps sustain the increased energy and biosynthetic requirements through the GSH/GPX4 axis upon 4-1BB costimulation. A2BR deletion in combination with agonistic 4-1BB costimulation displays a greater ability to promote antitumor CD8+ effector T cell survival and expansion while mitigating T cell exhaustion. Thus, the A2BR pathway plays an important role in metabolic reprogramming with potentiation of the GSH/GPX4 cascade upon agonistic 4-1BB costimulation that allows the fine-tuning of the antitumor responses of CD8+ T cells.

Authors

Jihae Ahn, Ping Xie, Siqi Chen, Guilan Shi, Jie Fan, Minghui Zhang, Hui Tang, Amanda R. Zuckerman, Deyu Fang, Yong Wan, Timothy M. Kuzel, Yi Zhang, Bin Zhang

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Figure 4

NF-κB–dependent transcriptional modulation of gpx4 expression involving GSH biosynthesis and utilization in potentiating CD8+ T cell responses by 4-1BB agonism.

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NF-κB–dependent transcriptional modulation of gpx4 expression involving ...
CD8+ T cells were stimulated by anti-CD3 and anti-CD28 with or without α4-1BB (clone 3H3, 100 ng/ml). Expression levels of gclc (A), gpx4 (B), NFkb1 (C), and Nfkb2 (D) transcripts were determined by quantitative reverse-transcription PCR (qRT-PCR). The NF-κB inhibitors (Bay 11-7082) and/or cell-permeable GSH was added to α4-1BB–treated CD8+ T cell cultures. Intracellular GSH levels (E), viable cell counts (F), Ki-67+ cells (G), IFN-γ+ cells (H), GCLC+ cells (I), GPX4+ cells (J), and BODIPY-PEhimBBrhi cells (K) were analyzed by flow cytometry. NF-κB–binding sites on the GPX4 promoter were predicted by PROMO-ALGGEN (L) and Chip-qPCR analysis confirming the enhanced binding capacity of NF-κb1 and NF-κb2 on gpx4 in α4-1BB–treated CD8+ T cells compared with control cells (M). (N) Flow cytometric analysis of p-IKKα/β (Ser176/180) levels in WT and GPX4–/– CD8+ T cells treated with anti-CD3 and anti-CD28 with or without α4-1BB (clone 3H3, 100 ng/ml) for 24 hours. Data (means ± SEM) are representative of 2 (A–K and N) independent experiments. (A–E) Unpaired Student’s t test. (F–K, M, and N) Two-way ANOVA with Bonferroni’s post test correction was used. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.