Targeting KLF2 in macrophages to improve immune checkpoint therapy for hepatocellular cancer

摘要 抗程序性死亡-1抗体（αPD-1 Ab）作为单一药物治疗人肝细胞癌（HCC） 于2021年7月26日退出美国市场，因为一项多中心III期研究未证明 其在改善患者存活率方面优于对照组的功效。因此，迫切需要确定和针对关键的 细胞和分子调节剂，以设计针对HCC的新免疫检查点治疗（ICT）策略。一 建立了独特的、原位的和临床相关的鼠HCC模型，其模拟HCC起始， 在人类中的进展，并反映了肿瘤生物学，免疫学和人类疾病的典型组织学。在 在该模型中，SV 40 T抗原（TAg）仅在肿瘤中作为可追踪的肿瘤特异性抗原（TSA）表达， 使得能够在肿瘤起始、进展和对治疗的反应期间进行TSA免疫研究。利用这个模型， 开发了几种基于免疫的HCC治疗策略， 最近建立的，显著提高了αPD-1 Ab对HCC的治疗效果。具体地说， 多形拟杆菌（Bacteroides thetaiotaomicron，B.th）是拟杆菌属的一个成员，具有富含CpG的核酸， 作为TLR 9激动剂，被鉴定为微生物调节剂，可显著增强αPD-1 Ab治疗 HCC的疗效。这一令人兴奋的发现导致了对潜在细胞和分子机制的研究。单- 细胞RNA测序（scRNA-seq）显示，HCC生长上调肿瘤中的Kruppel样因子-2（KLF 2）， 相关巨噬细胞（TAM）和B.th添加活化效应CD 8 + T细胞和改善αPD-1 对HCC的治疗效果与TAM中KLF 2表达降低相关。此外，委员会认为， KLF 2敲低（KD）的巨噬细胞（MΦ）的过继细胞转移（ACT）使TSA免疫能够 显著抑制HCC生长。相反，在MΦ中KLF 2过表达（OE）会损害B.th/αPD-1-β细胞。 诱导HCC的治疗性抑制。这些令人信服的结果突出了KLF 2作为介导 微生物通过调节MΦs对肝癌发生和B.th/αPD-1免疫治疗的影响。进一步研究 表明KLF 2通过调节MΦ TLR 9和信号调节蛋白α（SIRPα）表达来调节MΦ 肿瘤吞噬和免疫调节功能。这些发现产生了以下假设：B.th， 富含CpG的核酸通过表型和功能编程MΦ在HCC中重振αPD-1 Ab ICT 通过KLF 2控制TLR 9和SIRPα的表达。在目的1中，KLF 2介导的MΦ作为细胞基础介导了 肝癌的发病机制和免疫耐受将被研究，以发展新的治疗方法 通过将KLF 2-KO MΦ与 αPD-1抗体在目标2中，分子机制和调节剂 B.th/αPD-1通过调控TLR 9和SIRPα表达抑制KLF 2重编程MΦ。的 从这项研究中产生的知识不仅可以识别出未被识别的内源性调节因子， 在HCC中编程MΦ，但也说明这些因素是触发MΦ的有效目标，并导致 改善总部合同委员会的信息和通信技术。

ABSTRACT Anti-programmed death-1 antibody (αPD-1 Ab) as a single agent for treating human hepatocellular cancer (HCC) was withdrawn from the US market on July 26, 2021, because a multi-center phase III study did not demonstrate its efficacy in improving patient survival over controls. Thus, there is an urgent need to identify and target critical cellular and molecular regulators to design new immune checkpoint therapy (ICT) strategies against HCC. A unique, orthotopic, and clinically relevant murine HCC model was established that mimics HCC initiation and progression in humans and reflects the tumor biology, immunology, and histology typical of human disease. In this model, SV40 T antigen (TAg) is expressed solely in tumors as a trackable tumor-specific antigen (TSA), enabling TSA immunity study during tumor initiation, progression, and response to treatments. Using this model, several immune-based therapeutic strategies for HCC were developed and a novel microbe-based strategy was recently established that significantly improves the therapeutic efficacy of αPD-1 Ab for HCC. Specifically, Bacteroides thetaiotaomicron (B.th), one member of genus Bacteroides, with CpG-rich nucleic acid which functions as TLR9 agonist, was identified as a microbial regulator to significantly boost αPD-1 Ab therapeutic efficacy for HCC. This exciting finding led to studies of the underlying cellular and molecular mechanisms. Single- cell RNA sequencing (scRNA-seq) revealed that HCC growth upregulated Kruppel like factor-2 (KLF2) in tumor- associated macrophages (TAMs), and B.th addition activated effector CD8+ T cells and improved αPD-1 therapeutic effect against HCC, which was associated with reduced KLF2 expression in TAMs. Moreover, adoptive cell transfer (ACT) of macrophages (MΦs) with KLF2 knockdown (KD) enabled TSA immunization to significantly suppress HCC growth. Conversely, KLF2 overexpression (OE) in MΦs compromised B.th/αPD-1- induced therapeutic suppression of HCC. These compelling results highlight KLF2 as a key regulator mediating microbes’ impact on hepatocarcinogenesis and B.th/αPD-1 immunotherapy by modulating MΦs. Further studies indicate that KLF2 controls MΦ expression of TLR9 and signal-regulatory protein α (SIRPα) to regulate MΦ tumor phagocytosis and immune regulatory function. These findings generate the following hypothesis: B.th, with CpG-rich nucleic acid, reinvigorates αPD-1 Ab ICT in HCC by phenotypically and functionally programming MΦ via KLF2-controlled expression of TLR9 and SIRPα. In Aim 1, KLF2-directed MΦs as the cellular basis mediating HCC pathogenesis and immune tolerance will be studied toward the development of a new therapeutic approach by integrating KLF2-KO MΦs with αPD-1 Ab. In Aim 2, the molecular mechanism and regulators by which B.th/αPD-1 suppress KLF2 to reprogram MΦ by controlling TLR9 and SIRPα expression will be studied. The knowledge generated from this study will not only identify unrecognized endogenous regulators with a role in programming MΦ in HCC, but also make a case that these factors are effective targets to trigger MΦs and lead to improved ICT for HCC.