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

PD-1阻断剂作为抗癌治疗剂已经取得了显著的成功。机制如何 PD-1的抗肿瘤功能仍然知之甚少。虽然在PD-1的反式接合中， 通过APC或癌细胞上表达的配体在T细胞中表达，抑制T细胞活化， 提供证据表明，当发生PD-L1：B7-1（CD 80）顺式和PD-1：PD-L1顺式非典型相互作用时， 这些分子在APC上共表达，并破坏PD-1和PD-L1之间的典型相互作用， 反式和T细胞抑制信号传导。因此，PD-L1在肿瘤微环境（TME）中的表达并不 与PD-1介导的T细胞抑制同义。我们产生了识别PD-1 pY 248（pPD-1）的抗体， 其是PD-1抑制信号传导所需的，并在小鼠和人中检测到pPD-1。我们发现pPD-1+ T 在培养物、小鼠肿瘤模型和患者活检中，以及在CD 8 + T中枢记忆（TCM）细胞中， 健康个体的外周血。在荷瘤小鼠中，pPD-1在肿瘤浸润的CD 8 + T细胞中表达。 T淋巴细胞，但主要是Treg。我们产生了条件性靶向Pdcd 1基因（PD-1f/f）的小鼠， 选择性消除Treg中的PD-1（Pdcd 1f/fFoxP 3cre）。在携带肿瘤的Pdcd 1f/fFoxP 3cre小鼠中，Treg显示 富含调节脂质代谢、脂肪酸氧化和 单核细胞/巨噬细胞趋化蛋白-1（MCP-1）和GABA能神经递质， 免疫抑制功能这些特征与B细胞和M2样细胞的显著增加相关。 在某些实施方案中，肿瘤细胞可以通过抑制巨噬细胞的增殖、减少肿瘤浸润性T细胞的活化和增加肿瘤生长来抑制肿瘤细胞的增殖。我们的研究结果揭示 一种以前未被认识到的网络，通过该网络，Treg选择性阻断PD-1信号传导重塑了 免疫景观，并表明在不同细胞类型中消除PD-1信号传导差异性地影响 的TME。我们的研究结果表明，pPD-1是一个强大的标志物，以确定T细胞受到PD-1抑制， 并支持新的假设，即通过pPD-1对PD-1信号传导的细胞特异性检测可以预测 检查点免疫疗法的结果。为了调查这些问题，我们将追求以下具体目标： 1.鉴定癌症背景下pPD-1+ T细胞的免疫学和生物化学特性。 我们将通过单细胞免疫分析来表征pPD-1+ CD 8 + TIL和Treg，并研究T细胞亚群- 通过使用我们的Pdcd 1f/fCD 8 cre和Pdcd 1f/fFoxP 3cre小鼠，特异性PD-1信号转导重塑了TME。 2.鉴定健康个体中pPD-1+细胞的分子和功能特性。我们将 研究PD-1信号如何塑造健康个体和癌症患者中T细胞的特性， 揭示为什么只有癌症介导的PD-1信号传导诱导TEX。 3.目的探讨pPD-1+ TIL在肿瘤患者中的表达、功能及预后意义。我们 将检查患者活检组织中细胞特异性PD-1表达和信号传导，PD-1/pPD-1，PD-1/pPD-1的共表达。 L1和CD 80的表达，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.