Table_1_Targeting immunosuppressive Ly6C+ classical monocytes reverses anti-PD-1/CTLA-4 immunotherapy resistance.xlsx

IntroductionDespite significant clinical advancement with the use of immune checkpoint blockade (ICB) in non-small cell lung cancer (NSCLC) there are still a major subset of patients that develop adaptive/acquired resistance. Understanding resistance mechanisms to ICB is critical to developing new therapeutic strategies and improving patient survival. The dynamic nature of the tumor microenvironment and the mutational load driving tumor immunogenicity limit the efficacy to ICB. Recent studies indicate that myeloid cells are drivers of ICB resistance. In this study we sought to understand which immune cells were contributing to resistance and if we could modify them in a way to improve response to ICB therapy.ResultsOur results show that combination anti-PD-1/CTLA-4 produces an initial antitumor effect with evidence of an activated immune response. Upon extended treatment with anti-PD-1/CTLA-4 acquired resistance developed with an increase of the immunosuppressive populations, including T-regulatory cells, neutrophils and monocytes. Addition of anti-Ly6C blocking antibody to anti-PD-1/CTLA-4 was capable of completely reversing treatment resistance and restoring CD8 T cell activity in multiple KP lung cancer models and in the autochthonous lung cancer KrasLSL-G12D/p53fl/fl model. We found that there were higher classical Ly6C+ monocytes in anti-PD-1/CTLA-4 combination resistant tumors. B7 blockade illustrated the importance of dendritic cells for treatment efficacy of anti-Ly6C/PD-1/CTLA-4. We further determined that classical Ly6C+ monocytes in anti-PD-1/CTLA-4 resistant tumors are trafficked into the tumor via IFN-γ and the CCL2-CCR2 axis. Mechanistically we found that classical monocytes from ICB resistant tumors were unable to differentiate into antigen presenting cells and instead differentiated into immunosuppressive M2 macrophages or myeloid-derived suppressor cells (MDSC). Classical Ly6C+ monocytes from ICB resistant tumors had a decrease in both Flt3 and PU.1 expression that prevented differentiation into dendritic cells/macrophages.ConclusionsTherapeutically we found that addition of anti-Ly6C to the combination of anti-PD-1/CTLA-4 was capable of complete tumor eradication. Classical Ly6C+ monocytes differentiate into immunosuppressive cells, while blockade of classical monocytes drives dendritic cell differentiation/maturation to reinvigorate the anti-tumor T cell response. These findings support that immunotherapy resistance is associated with infiltrating monocytes and that controlling the differentiation process of monocytes can enhance the therapeutic potential of ICB.

尽管在非小细胞肺癌（NSCLC）治疗中应用免疫检查点阻断（ICB）技术取得了显著的临床进展，但仍有一部分患者表现出适应性/获得性耐药。深入理解ICB的耐药机制对于开发新型治疗方案和提升患者生存率至关重要。肿瘤微环境的动态特性以及驱动肿瘤免疫原性的突变负荷限制了ICB的治疗效果。近期研究指出，髓系细胞是ICB耐药的驱动因素。在本研究中，我们旨在探究哪些免疫细胞参与了耐药过程，以及是否可以通过某种方式对其进行调节以增强对ICB疗法的反应。研究结果揭示，抗PD-1/CTLA-4联合疗法能够产生初始的抗癌效果，并显示出免疫反应被激活的证据。然而，在抗PD-1/CTLA-4的长期治疗过程中，耐药性逐渐产生，表现为免疫抑制细胞群体的增加，包括调节性T细胞、中性粒细胞和单核细胞。将抗Ly6C阻断抗体添加至抗PD-1/CTLA-4联合疗法中，能够完全逆转治疗耐药性，并在多个KP肺癌模型以及自发性肺癌KrasLSL-G12D/p53fl/fl模型中恢复CD8 T细胞的活性。我们发现，在抗PD-1/CTLA-4联合耐药肿瘤中存在更高比例的经典Ly6C+单核细胞。B7阻断实验表明，树突状细胞对于抗Ly6C/PD-1/CTLA-4治疗的有效性至关重要。我们进一步确定，抗PD-1/CTLA-4耐药肿瘤中的经典Ly6C+单核细胞通过IFN-γ和CCL2-CCR2轴被运送到肿瘤内部。从机制上分析，我们发现来自ICB耐药肿瘤的经典单核细胞无法分化为抗原呈递细胞，而是分化为免疫抑制性M2巨噬细胞或髓源性抑制细胞（MDSC）。来自ICB耐药肿瘤的经典Ly6C+单核细胞的Flt3和PU.1表达水平降低，这阻碍了其向树突状细胞/巨噬细胞的分化。治疗学上，我们发现将抗Ly6C添加至抗PD-1/CTLA-4联合疗法中，能够实现肿瘤的完全清除。经典Ly6C+单核细胞分化为免疫抑制细胞，而经典单核细胞的阻断则驱动树突状细胞的分化/成熟，从而重新激活抗肿瘤T细胞反应。这些发现支持了免疫治疗耐药性与浸润性单核细胞相关，并且控制单核细胞的分化过程能够增强ICB的潜在治疗效能。