ICOSL Signaling in Macrophages Promotes Anti-Tumor Immunity

项目摘要/摘要： 胰腺导管腺癌(PDA)是一种侵袭性疾病，长期存活者很少。 已知炎性肿瘤微环境(TME)通过以下途径影响胰腺癌的进展 要么产生细胞毒性T细胞反应，要么更常见的是通过诱导肿瘤容许性耐受来实现。 许多研究表明，肿瘤相关巨噬细胞或TAMs在培养T细胞方面起着至关重要的作用 向免疫原性或耐受性分化的方向发展。然而，监管的机制 巨噬细胞在本病中的表型尚不清楚。ICOSL，ICOS co-的同源配体 T细胞刺激性受体是一种表达于巨噬细胞的B7家族40kD蛋白。当ICOS信令 已经得到了很好的表征，ICOSL的概念是在与巨噬细胞接触时向巨噬细胞发出回传信号 ICOS还没有得到很好的描述。我的初步工作表明ICOSL在肿瘤上的表达上调 与胰腺癌相关的巨噬细胞。基因缺失或抗体介导 ICOSL在巨噬细胞上的中和导致其分化为交替激活的M2样细胞 癌症进展的表型和加速。相反，我发现ICOSL参与或激活 在巨噬细胞中，用ICOS Fc处理会在体外产生深刻的免疫原性程序。质量 光谱分析表明，ICOSL与STAT1在巨噬细胞中共沉淀，这是已知的驱动因素 M1样表型。然而，ICOSL作为一种调节因子的特定分子机制 PDA残留物中巨噬细胞表型及其作为抗肿瘤免疫主要调节剂的适用性 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肿瘤保护是否是T细胞依赖现象。具体地说，这个目标 将探索ICOSL激活或中和巨噬细胞对T细胞表型的影响； 肿瘤内免疫细胞的流式细胞术、抗原提呈分析和10倍单细胞RNA测序 来自多个PDA小鼠模型。目标2将寻求解决特定的细胞内信号机制 ICOSL通过其调节巨噬细胞极化。通过使用定点突变来定位 介导ICOSL与STAT1结合的相互作用面，这一目的将揭示一个迄今未知的 驱动免疫原性巨噬细胞编程的信号通路，涉及ICOSL的细胞质尾部。

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.