The role of the CD58:CD2 axis in cancer immune evasion and resistance to immunotherapy

项目总结 免疫检查点抑制物(ICI)，如抗PD-1抗体(APD1)，使CD8+T细胞介导的抗 在部分转移性黑色素瘤患者中，该药物可增强肿瘤免疫力，并产生持久反应。然而， 大多数患者没有从ICI中受益，耐药的潜在机制仍然难以捉摸。整体而言 本工作的目的是阐明癌细胞介导的ICI耐药机制，并开发新的 以分子或免疫为基础的疗法来克服耐药性。先前的工作表明，co-1的表达 CD8+T细胞上的刺激蛋白CD28是aPD1疗效所必需的，然而，我们发现 CD28通常在肿瘤浸润性CD8+T淋巴细胞(TIL)中缺失，包括那些保存的 祖细胞功能。相反，所有TIL都保持CD2的表达，这是最有效的替代共刺激 蛋白。事实上，我们发现患者来源的CD28-TIL具有保持增殖、克隆性的能力 自体癌细胞的扩增、细胞因子的产生和裂解。CD2的配体是CD58，我们 发现CD58在黑色素瘤细胞上表达。CD58：CD2轴在抗肿瘤免疫中的作用 对ICI的反应和抵抗还没有被探索。在初步研究中，我们建立了患者来源的配对 黑色素瘤细胞和自体TIL。我们开发了perturb-cite-seq，这是一种支持池的新方法 CRISPR-CAS9通过单细胞RNA序列和蛋白质读数进行筛选。我们进行了一次扰动引用序列筛选 在患者模型中，干扰数百个在ICI抵抗中可能起作用的基因，然后与 TIL和特写的幸存细胞逃脱了T细胞的杀伤。我们恢复了已知的癌症免疫机制 规避和新奇的行为，包括CD58的丢失/下调。在人源化小鼠模型中，CD58在 肿瘤细胞导致T细胞浸润受损，对过继细胞转移(ACT)产生抵抗。此外，我们 CD58缺失时PD-L1代偿性上调。使用基因组规模的CRISPR-Cas9筛查和联合 免疫沉淀质谱学筛选，我们确定了这种相互作用的一个可能的调节因子，CMTM6。 挽救CD58重新致敏的癌细胞对T细胞的杀伤，增加T细胞的激活，增强T细胞的渗透 体内，并取消PD-L1的代偿性表达。基于这些发现，我们将剖析 CD58：CD2轴在抗肿瘤免疫、肿瘤免疫逃避和抵抗ICI中的三个目的：i) 为了确定CD58缺失导致癌症免疫逃避的机制，ii)确定CD58缺失的作用 CMTM6在调节CD58丢失中代偿性PD-L1上调中的作用，以及iii)确定aPD1是否 耐药与患者CD58：CD2轴受损有关。我们建议系统地解决 这些利用遗传、生化、患者和活体模型，以及多模式单细胞基因组学进行分析 使用新的机器学习工具。我们进一步测试了两种克服由于缺陷引起的ICI耐药性的疗法。 在CD58：CD2轴上。这项工作完成后，将阐明CD58：CD2通路在癌症中的作用 和癌症免疫疗法，并可能为治疗发展提供新的途径。

PROJECT SUMMARY Immune checkpoint inhibitors (ICI), such as anti-PD-1 antibodies (aPD1), enable CD8+ T cell-mediated anti- tumor immunity and produce durable responses in a portion of patients with metastatic melanoma. However, most patients do not benefit from ICI and the underlying mechanisms of resistance remain elusive. The overall goal of this work is to elucidate mechanisms of resistance to ICI mediated by cancer cells, and develop novel molecular or immune-based therapies to overcome resistance. Prior work demonstrated that expression of co- stimulatory protein CD28 on CD8+ T cells is required for efficacy of aPD1, however, we show that expression of CD28 is frequently absent in tumor-infiltrating CD8+ T lymphocytes (TILs), including those with preserved progenitor function. In contrast, all TILs maintain expression of CD2, the most potent alternative co-stimulatory protein. Indeed, we find that patient-derived CD28- TILs had preserved capacity for proliferation, clonal expansion, cytokine production and lysis of autologous cancer cells. The ligand of CD2 is CD58, and we discovered that CD58 was expressed on melanoma cells. The role of the CD58:CD2 axis in anti-tumor immunity, and response and resistance to ICI is unexplored. In preliminary studies, we established patient-derived pairs of melanoma cells and autologous TILs. We developed Perturb-CITE-seq, a novel method enabling pooled CRISPR-Cas9 screens with single-cell RNA-seq and protein readouts. We performed a Perturb-CITE-seq screen in patient models, perturbing hundreds of genes with putative roles in ICI resistance, followed by co-culture with TILs and profiled survivor cells that escaped T cell killing. We recovered known mechanisms of cancer immune evasion and novel ones, including loss/downregulation of CD58. In humanized mouse models, loss of CD58 in tumor cells resulted in impaired T cell infiltration and resistance to adoptive cell transfer (ACT). Additionally, we observed compensatory PD-L1 upregulation in CD58 loss. Using genome-scale CRISPR-Cas9 screens and co- immunoprecipitation mass spectrometry screens, we identified a putative regulator of this interaction, CMTM6. Rescue of CD58 re-sensitized cancer cells to T cell killing, increased T cell activation, enhanced T cell infiltration in vivo, and abolished compensatory PD-L1 expression. Based on these findings, we will dissect the role of the CD58:CD2 axis in anti-tumor immunity, cancer immune evasion and resistance ICI resistance in three aims: i) to determine the mechanisms by which CD58 loss confers cancer immune evasion, ii) to determine the role of CMTM6 in regulating compensatory PD-L1 upregulation in CD58 loss, and iii) to determine whether aPD1 resistance is associated with an impaired CD58:CD2 axis in patients. We propose to systematically address these using genetic, biochemical, patient and in vivo models, and multi-modal single-cell genomics analyzed with novel machine-learning tools. We further test two therapies that overcome resistance to ICI due to defects in the CD58:CD2 axis. Upon completion, this work will shed light on the role of the CD58:CD2 pathway in cancer and cancer immunotherapy, and may provide novel avenues for therapeutic development.