BATF and IRF4 cooperate to counter exhaustion in tumour-infiltrating CAR T cells (ATAC-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. For transcriptional profiling using ATAC-seq, 50,000 cells were sorted from the Live/Dead dye-negative CD8+Thy1.1+GFP+ population of the isolated tumor infiltrating lymphocytes or cultured CD8+ T cells. The cells were resuspended in FACS buffer and filtered with a 70 M filter before sorting.

转录因子之间的协同相互作用对于基因转录过程至关重要。我们此前的研究表明，NFAT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细胞低应答（耗竭）的负反馈程序。本研究证实，BATF与IRF4可协同拮抗T细胞耗竭。在表达嵌合抗原受体（chimeric antigen receptor, CAR）的CD8+ 42 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，提升其效应细胞因子的分泌量，降低其抑制性受体及耗竭相关转录因子TOX的表达水平，并诱导产生可抑制肿瘤复发的长寿记忆性T细胞。上述效应依赖于BATF-IRF的相互作用：表达无法与Irf4结合的Batf突变体的细胞，既无法在肿瘤内存活，也不能有效延缓肿瘤生长。我们提出，过表达BATF是一种具备临床转化潜力的抗肿瘤治疗策略，可通过重编程CAR肿瘤浸润淋巴细胞（CAR tumor-infiltrating lymphocytes, CAR-TILs）的表型与转录谱，使其脱离耗竭状态并增强效应功能。对于采用转座酶可及性测序（Assay for Transposase-Accessible Chromatin sequencing, ATAC-seq）的转录谱分析实验，我们从分离得到的肿瘤浸润淋巴细胞或体外培养的CD8+ T细胞的活细胞染料阴性的CD8+Thy1.1+GFP+群体中分选了50,000个细胞。具体操作如下：将细胞重悬于流式分选缓冲液（FACS buffer）中，经70 μm滤膜过滤后再进行分选。