Detection of PD-1 inhibitory signaling and its molecular relays in T cells: Implications for cancer immunotherapy

PD-1阻断剂在抗癌治疗方面取得了显著的成功。机制(S) PD-1对抗肿瘤功能的影响仍知之甚少。尽管在PD-1的反式结合中 在T细胞中通过其配体表达在APC或癌细胞上表达抑制T细胞激活，不断发展的发现 提供证据表明顺式中的PD-L1：B7-1(CD80)和顺式中的PD-1：PD-L1发生非正则相互作用 这些分子在APC上共表达，并破坏PD-1和PD-L1之间的正则相互作用 反式和T细胞抑制信号转导。因此，PD-L1在肿瘤微环境(TME)中的表达不 与PD-1介导的T细胞抑制同义词。我们产生了一种识别PD-1pY248(PPD-1)的抗体， 它是PD-1抑制信号转导所必需的，并在小鼠和人中检测到PPD-1。我们发现了PPD-1+T 在培养物、小鼠肿瘤模型和患者活检组织中的细胞，也在CD8+T中央记忆(TCM)细胞中 健康人的外周血液。在荷瘤小鼠中，PPD-1在肿瘤浸润性CD8+中表达 T淋巴细胞，但主要分布在Treg。我们建立了条件靶向Pdcd1基因(PD-1f/f)的小鼠，并 选择性清除Treg(Pdcd1f/fFoxP3cre)中的PD-1。在荷瘤的Pdcd1f/fFoxP3cre小鼠中，Treg显示 在调节脂肪代谢、脂肪酸氧化和产生 单核/巨噬细胞趋化蛋白-1与GABA能神经递质的关系 免疫抑制功能。这些特征与B细胞和M2样细胞的显著增加有关 巨噬细胞，减少肿瘤浸润性T细胞的激活，促进肿瘤生长。我们的结果显示 一种以前不为人知的网络，通过它对PD-1信号的Treg选择性封锁重塑了 免疫学格局，并表明在不同类型的细胞中取消PD-1信号会产生不同的影响 TME。我们的发现表明，PPD-1是识别受PD-1抑制的T细胞的一个强有力的标志物 并支持新的假设，即PPD-1对PD-1信号的细胞特异性检测可以预测 检查点免疫治疗的结果。为了调查这些问题，我们将追求以下具体目标： 1.鉴定PPD-1+T细胞在癌症中的免疫学和生化特性。 我们将通过单细胞免疫图谱来鉴定PPD-1+CD8+TIL和Treg，并研究T细胞亚群如何- 特定的PD-1信号通过使用我们的Pdcd1f/fCD8cre和Pdcd1f/fFoxP3cre小鼠重塑TME。 2.鉴定健康人PPD-1+细胞的分子和功能特性。我们会 研究PD-1信号如何塑造健康人和癌症患者的T细胞特性 揭示为什么只有癌症介导的PD-1信号才能诱导TeX。 3.检测肿瘤患者外周血中PPD-1+TIL的表达、功能及预后意义。我们 将检测患者活检组织中细胞特异性PD-1的表达和信号，PD-1/PPD-1，PD-1，PD-1的共同表达 L1和CD80，并确定其预后意义。

PD-1 blocking agents have achieved significant success as anti-cancer therapeutics. The mechanism(s) of how PD-1 compromises anti-tumor function remain poorly understood. Although in trans engagement of PD-1 expressed in T cells by its ligands expressed on APC or cancer cells inhibits T cell activation, evolving discoveries provide evidence that PD-L1:B7-1(CD80) in cis and PD-1:PD-L1 in cis non-canonical interactions occur when these molecules are co-expressed on APC, and disrupt the canonical interaction between PD-1 and PD-L1 in trans and T cell inhibitory signaling. Thus, expression of PD-L1 in the tumor microenvironment (TME) is not synonymous with PD-1-mediated T cell inhibition. We generated an antibody that recognizes PD-1pY248 (pPD-1), which is required for PD-1 inhibitory signaling, and detected pPD-1 in mouse and human. We found pPD-1+ T cells in cultures, in mouse tumor models and patient biopsies, but also in CD8+ T central memory (TCM) cells in the peripheral blood of healthy individuals. In tumor bearing mice, pPD-1 was expressed in tumor infiltrating CD8+ T lymphocytes but mostly in Treg. We generated mice with conditional targeting of Pdcd1 gene (PD-1f/f) and selectively eliminated PD-1 in Treg (Pdcd1f/fFoxP3cre). In tumor-bearing Pdcd1f/fFoxP3cre mice, Treg displayed enrichment in pathways regulating lipid metabolism, fatty acid oxidation, and production of monocyte/macrophage chemotactic protein-1 (MCP-1) and GABAergic neurotransmitter with known immunosuppressive function. These features correlated with a significant increase of B cells and M2-like macrophages, diminished activation of tumor infiltrating T cells, and increased tumor growth. Our results reveal a previously unappreciated network by which Treg-selective blockade of PD-1 signaling reshapes the immunological landscape and suggest that abrogation of PD-1 signaling in distinct cell types differentially impacts the TME. Our findings indicate that pPD-1 is a powerful marker to identify T cells subjected to PD-1 inhibitory signaling and support the novel hypothesis that cell-specific detection of PD-1 signaling by pPD-1 might predict the outcome of checkpoint immunotherapy. To investigate these, we will pursue the following specific aims: 1. To identify the immunological and biochemical properties of pPD-1+ T cells in the context of cancer. We will characterize pPD-1+ CD8+ TIL and Treg by single cell immunoprofiling, and investigate how T cell subset- specific PD-1 signaling reshapes the TME by using our Pdcd1f/fCD8cre and Pdcd1f/fFoxP3cre mice. 2. To identify the molecular and functional properties of pPD-1+ cells in healthy individuals. We will examine how PD-1 signaling shapes the properties of T cells in healthy individuals and in cancer patients and uncover why only cancer-mediated PD-1 signaling induces TEX. 3. To determine expression, function and prognostic significance of pPD-1+ TIL in cancer patients. We will examine cell-specific PD-1 expression and signaling in patient biopsies, co-expression of PD-1/pPD-1, PD- L1 and CD80, and determine their prognostic significance.