A novel oncolytic neutrophil phenotype is induced by IL-10R inhibition [CITE-seq]

While the impact of neutrophils on antitumor T cells has been an active area of study, fewer studies have focused on the potential of neutrophils to directly impact tumor cells. Here we asked whether neutrophils have the capacity to directly kill tumor cells independently of T cells. We find that anti-CD40-based therapy, when combined with interleukin 10 (IL-10) receptor blockade, initiates a Batf3-dependent pathway in which IL-12 and interferon gamma (IFN?) secretion results in oncolytic neutrophil activity. Using a combination of microscopy, single-cell, and functional assays, we observe that killing of tumor cells by neutrophils is dependent on physical contact and degranulation. This degranulation-mediated killing is associated with an atypical dynamic invasive neutrophil phenotype. In line with our preclinical findings, our phase 1 trial of anti-CD40 shows that circulating IL-12, IFN?, and IL-10 increase in response to anti-CD40, while our phase 1b/2 PRINCE study shows that lower circulating IL-10 is associated with favorable overall survival specifically among anti-CD40-treated patients. Finally, we find that neutrophil expansion with granulocyte colony stimulating factor (G-CSF) is associated with improved overall survival specifically in patients treated with anti-CD40, suggesting that this pathway may be amenable to therapeutic intervention in patients with advanced cancer. Overall design: C57BL/6 wild type and Batf3 knockout mice were intradermally inoculated with B16F10 murine melanoma cells on their flanks, and eight days later, they received Isotype control, or a combination of anti-CD40/MPL/anti-IL-10R (CMI). Twelve hours post-treatment, the B16F10 tumors were harvested and processed into single-cell suspensions. Single-cell RNA sequencing (scRNA-Seq) was subsequently performed on these FACS-sorted cell suspensions.

尽管中性粒细胞对抗肿瘤T细胞的影响已成为活跃的研究热点，但聚焦于中性粒细胞直接调控肿瘤细胞潜力的相关研究仍较为匮乏。本研究旨在探究中性粒细胞是否具备不依赖T细胞直接杀伤肿瘤细胞的能力。我们发现，基于抗CD40的治疗方案联合白细胞介素10（IL-10）受体阻断剂时，可激活一条依赖Batf3的信号通路：该通路中IL-12与干扰素γ（IFN-γ）的分泌可介导溶瘤性中性粒细胞的抗肿瘤活性。通过联合运用显微镜成像、单细胞实验与功能实验，我们观察到中性粒细胞对肿瘤细胞的杀伤作用依赖于细胞间直接接触与脱颗粒过程。这种脱颗粒介导的杀伤效应与一种非典型的动态侵袭性中性粒细胞表型密切相关。与临床前研究结果一致，我们开展的抗CD40疗法I期临床试验显示，外周血IL-12、IFN-γ与IL-10水平会在抗CD40治疗后升高；而我们的Ib/II期PRINCE研究则表明，在接受抗CD40治疗的患者中，循环IL-10水平较低者的总生存期更优。此外我们发现，粒细胞集落刺激因子（G-CSF）诱导的中性粒细胞扩增，仅在接受抗CD40治疗的患者中与总生存期改善显著相关，提示该通路可作为晚期癌症患者的潜在治疗干预靶点。整体实验设计：将C57BL/6野生型及Batf3基因敲除小鼠于胁腹部皮内接种B16F10小鼠黑色素瘤细胞，接种8天后分别给予同型对照或抗CD40/MPL/抗IL-10R联合治疗（简称CMI）。治疗12小时后，收获B16F10肿瘤组织并制备为单细胞悬液，随后对经荧光激活细胞分选（FACS）富集的细胞悬液进行单细胞RNA测序（scRNA-Seq）。