ICOSL Signaling in Macrophages Promotes Anti-Tumor Immunity

项目概要/摘要： 胰腺导管腺癌（PDA）是一种侵袭性疾病，很少有长期存活者。 已知炎症性肿瘤微环境（TME）通过以下方式影响胰腺癌进展： 或者产生细胞毒性T细胞应答，或者更常见地，通过诱导肿瘤容许耐受。 许多研究表明，肿瘤相关巨噬细胞（TAM）在培养T细胞方面起着至关重要的作用 在PDA中向免疫原性或耐受原性分化。然而，监管机制 这种疾病中的巨噬细胞表型还不清楚。ICOS的同源配体， T细胞上的刺激受体是在巨噬细胞上表达的B7家族40 kD蛋白。虽然ICOS信令 已经得到了很好的表征，ICOSL在与巨噬细胞接合后向巨噬细胞"反向信号传导"的概念， ICOS没有得到很好的描述。我的初步工作表明IXSL对肿瘤有上调作用 相关巨噬细胞在胰腺癌的背景下。基因缺失或抗体介导 中和巨噬细胞上的ICOSL导致它们向交替活化的M2样细胞分化， 表型和加速癌症进展。相反，我发现ICOSL的参与或激活 在巨噬细胞中通过用ICOS Fc处理导致体外的深刻免疫原性程序。质量 光谱分析表明，ICOSL与巨噬细胞中的STAT1共沉淀，巨噬细胞是已知的 M1样表型。然而，ICOSL作为调节剂的特定分子机制， 巨噬细胞表型及其作为PDA中抗肿瘤免疫的主要调节因子的适用性仍然存在 未知 我的建议将试图通过两个主要的具体目标来弥合这一知识差距：目标1将寻求 确定ICOSL介导的PDA中的肿瘤保护是否是T细胞依赖性现象。具体而言，这一目标 将探索巨噬细胞中ICOSL激活或中和对T细胞表型的影响;采用 流式细胞术、抗原呈递分析和肿瘤内免疫细胞的10 x单细胞RNA测序 来自多种PDA小鼠模型。目标2将寻求解决特定的细胞内信号传导机制 通过它ICOSL调节巨噬细胞极化。通过采用定点诱变来定位 介导ICOSL与STAT 1结合的相互作用表面，这一目标将揭示迄今未知的 信号通路，其驱动免疫原性巨噬细胞编程，涉及ICOSL的胞质尾区。

Project Summary/Abstract: Pancreatic ductal adenocarcinoma (PDA) is an aggressive disease for which there are few long-term survivors. The inflammatory tumor microenvironment (TME) is known to influence pancreatic cancer progression by either generating cytotoxic T cell responses, or, more commonly, by inducing tumor-permissive tolerance. Many studies have shown that tumor-associated macrophages, or TAMs, play a vital role in educating T cells toward immunogenic or tolerogenic differentiation in PDA. However, the mechanisms that regulate macrophage phenotype in this disease are not well understood. ICOSL, the cognate ligand for the ICOS co- stimulatory receptor on T cells, is a B7-family 40 kD protein expressed on macrophages. While ICOS signaling has been well-characterized, the concept of ICOSL ‘back-signaling’ into macrophages upon engagement by ICOS has not been well described. My preliminary work indicates that ICOSL is up-regulated on tumor associated macrophages in the context of pancreatic cancer. Genetic deletion or antibody-mediated neutralization of ICOSL on macrophages results in their differentiation toward an alternatively activated M2-like phenotype and acceleration of cancer progression. Conversely, I found that ICOSL engagement or activation in macrophages by treatment with an ICOS Fc leads to a profound immunogenic program in vitro. Mass spectrometric analysis has shown that ICOSL co-precipitates with STAT1 in macrophages, a known driver of M1-like phenotype. However, the specific molecular mechanisms through which ICOSL acts as a regulator of macrophage phenotype and its applicability as a master regulator of anti-tumor immunity in PDA remains unknown. My proposal will attempt to bridge this gap in knowledge through two main specific aims: Aim 1 will seek to determine if ICOSL-mediated tumor protection in PDA is a T-cell dependent phenomenon. Specifically, this aim will explore the effects of ICOSL-activation or neutralization in macrophages on T-cell phenotype; employing flow cytometry, antigen presentation assays, and 10x single cell RNA sequencing of intra-tumoral immune cells from multiple murine models of PDA. Aim 2 will seek to address the specific intracellular signaling mechanisms through which ICOSL regulates macrophage polarization. By employing site directed mutagenesis to map the interaction surface that mediates ICOSL binding to STAT1, this aim will uncover a heretofore unknown signaling pathway that drives immunogenic macrophage programming involving the cytoplasmic tail of ICOSL.