BATF and IRF4 cooperate to counter exhaustion in tumour-infiltrating CAR T cells (ChIP-seq)

Cooperative interactions among transcription factors are essential for gene transcription. We previously showed that NFAT and AP-1 (Fos-Jun) transcription factors cooperate to promote the effector functions of T cells, but that under conditions where it is unable to cooperate with AP-1, NFAT imposes a negative feedback programme of T cell hyporesponsiveness (“exhaustion”). Here we show that BATF and IRF4 cooperate to counter T cell exhaustion. Overexpression of Batf in CD8+ 42 T cells expressing a chimeric antigen receptor (CAR) promoted the survival and expansion of tumour-infiltrating CAR T cells, increased their production of effector cytokines, decreased their expression of inhibitory receptors and the exhaustion-associated transcription factor TOX, and led to the generation of long-lived memory T cells that controlled tumour recurrence. These responses were dependent on the BATF-IRF interaction, since cells expressing a Batf mutant unable to interact with Irf4 did not survive in tumours and did not effectively delay tumour growth. We suggest that BATF overexpression is a therapeutically viable option for improving the anti-tumour responses of CAR TILs, by skewing their phenotypes and transcriptional profiles away from exhaustion and towards increased effector function. Overall design: ChiP-seq: Determination of BATF or IRF4 binding sites in CD8 T cells in vitro and in Re-Stimulated CD8 T cells in vitro Chromatin Immunoprecipitation followed by Sequencing for CD8 T cells after in vitro culture and Re-Stimulation 6h

转录因子间的协同相互作用对基因转录至关重要。本课题组此前研究表明，活化T细胞核因子（NFAT，Nuclear Factor of Activated T cells）与AP-1（Fos-Jun）转录因子可协同促进T细胞的效应功能；而当NFAT无法与AP-1发生协同作用时，其会诱导T细胞低反应性的负反馈程序，即所谓的“耗竭”。本研究证实，BATF与IRF4协同可拮抗T细胞耗竭。在表达嵌合抗原受体（CAR，Chimeric Antigen Receptor）的CD8+ T细胞中过表达Batf，可促进肿瘤浸润性CAR-T细胞的存活与扩增，增强其效应性细胞因子的分泌，降低其抑制性受体及耗竭相关转录因子TOX的表达，并诱导可抑制肿瘤复发的长寿记忆性T细胞生成。上述效应依赖于BATF-IRF相互作用，因为表达无法与Irf4结合的Batf突变体的细胞无法在肿瘤微环境中存活，也不能有效延缓肿瘤生长。我们提出，过表达BATF可通过将CAR-TILs的表型与转录谱从耗竭状态转向增强的效应功能状态，成为改善CAR-TILs抗肿瘤应答的治疗可行方案。整体实验设计：染色质免疫共沉淀测序（ChIP-seq）：分别检测体外培养的CD8 T细胞，以及经体外再刺激6小时的CD8 T细胞中BATF或IRF4的结合位点。