The Role of Checkpoint inhibitors in CIA

摘要 在10%-43%的患者中发现了与严重炎症性关节炎有关的免疫相关不良事件 接受免疫检查点抑制剂，αCTLA4，αPD-1或αPD-L1，表明免疫的重要性 维持关节内动态平衡的抑制途径。我们提取了人体滑液，发现高密度 滑膜巨噬细胞表达PD-L1的频率。我们建议研究PD-L1表达的作用 在小鼠胶原性关节炎(CIA)过程中滑膜巨噬细胞和T细胞的表达。我们发现这种免疫力 与其他免疫检查点相比，检查点抑制因子PD-L1主要在关节中表达。 此外，我们还发现滑膜巨噬细胞和CD3+T细胞是关节中表达PD-L1的主要细胞， 在CIA过程中，PD-L1的表达增加。我们对滑膜巨噬细胞进行了广泛的分析，发现 三种PD-L1表达细胞：F4/80intCD115+CCR2-MHCII-CD73-Tim4-PD-L1hi(CD115+ 巨噬细胞)、F4/80hiCD115-CCR2intMHCIIhiCD73intTim4intPD-L1int(巨噬细胞)和F4/80intCD115- CCR2lowMHCII-CD73loTim4intPD-L1int(F4/80+巨噬细胞)。我们还发现PD-L1具有最高的 在CD4+和CD8+T细胞中的表达频率。此外，我们还鉴定了一个新的关节组织亚群。 幼稚关节内的常驻记忆T细胞，表型为CD8+CD69+和CD8+CD69+CD103+。我们 假设巨噬细胞和/或T细胞表达PD-L1对滑膜是必需的 关节炎症的动态平衡和调节。在目标1中，我们将检查PD-L1的重要性 滑膜巨噬细胞通过使用cre/flx系统选择性删除PD-L1表达。我们会 建立巨噬细胞特异性PD-L1KO小鼠LyzcrePD-L1fl/fl和CD115crePD-L1fl/fl。我们会诱使中情局 LyzcrePD-L1fl/fl、CD115crePD-L1fl/fl、Lyzcre和CD115cre小鼠，观察其对关节的保护作用 CIA滑膜巨噬细胞PD-L1的表达(Subaim 1.1)。我们还将通过以下方式进行异种繁殖实验 CD45.1 C57BL6和CD45.2 CD115crePD-L1fl/fl联合检测PD-L1+滑膜巨噬细胞 是中情局期间的常驻或骨髓来源(Subaim 1.2)。我们发现PD-L1高表达 在滑膜T细胞中。在目标2中，我们将研究CIA后滑膜T细胞的保护功能。我们将使用 Lockcrep D-L1fl/fl小鼠，在CD3+T细胞中缺乏PD-L1表达，以检测PD-L1特异性的影响 CIA期间T细胞的缺失(Subaim 2.1)。为了测试组织驻留记忆T细胞是否在 CIA，我们将利用组织驻留嵌合体小鼠模型选择性地删除LockcrePD- L1fl/fl小鼠。来自LockcrePD-L1fl/fl或Lockcre小鼠的供体T细胞将取代外周T细胞群以证实 关节中组织驻留记忆T细胞的保护功能(Subaim 2.2)。我们的项目有很高的 由于关节特异性PD-L1的表达可以根据治疗目的进行调节，因此具有翻译潜力 人类用于类风湿性关节炎或检查点抑制剂引起的关节炎。

Abstract Immune related adverse events linked to severe inflammatory arthritis have been identified in 10-43% of patients receiving immune checkpoint inhibitors, αCTLA4, αPD-1 or αPD-L1, indicating the importance of immune inhibitory pathways in maintaining homeostasis in the joints. We obtained human synovial fluid and found a high frequency of PD-L1 expression by synovial macrophages. We propose to examine the role of PD-L1 expression in synovial macrophages and T cells during murine collagen induced arthritis (CIA). We found that immune checkpoint inhibitor, PD-L1, is primarily expressed in the joint compared to other immune checkpoints. Additionally, we found synovial macrophages and CD3+ T cells are the main PD-L1 expressing cells in the joint, and PD-L1 expression is increased during CIA. We extensively profiled the synovial macrophages and found three populations of PD-L1 expressing cells: F4/80intCD115+CCR2-MHCII-CD73-Tim4-PD-L1hi (CD115+ macrophages), F4/80hiCD115-CCR2intMHCIIhiCD73intTim4intPD-L1int (MHCII+ macrophages), and F4/80intCD115- CCR2lowMHCII-CD73loTim4intPD-L1int (F4/80+ macrophages). We have also found that PD-L1 has the highest frequency of expression in CD4+ and CD8+ T cells. Furthermore, we identified a novel subset of joint tissue resident memory T cells in the naïve joint that are phenotypically CD8+CD69+ and CD8+CD69+CD103+. We hypothesize that the expression of PD-L1 by macrophages and/or T cells are essential for synovial homeostasis and regulation of joint inflammation. In Aim 1, we will examine the importance of PD-L1 expression by synovial macrophages through the selective deletion of PD-L1 using the cre/flox system. We will generate two macrophages specific PD-L1 KO mice, LyzcrePD-L1fl/fl and CD115crePD-L1fl/fl. We will induce CIA in LyzcrePD-L1fl/fl, CD115crePD-L1fl/fl, Lyzcre and CD115cre mice and harvest the joint to determine the protective role of synovial macrophage PD-L1 expression in CIA (Subaim 1.1). We will also perform parabiosis experiments by surgically joining CD45.1 C57BL6 and CD45.2 CD115crePD-L1fl/fl to determine if PD-L1+ synovial macrophages are resident or bone marrow derived during CIA (Subaim 1.2). We have found that PD-L1 is highly expressed in synovial T cells. In Aim 2, we will examine the protective function of synovial T cells following CIA. We will use LckcreP D-L1fl/fl mice, which lack PD-L1 expression in CD3+ T cells to examine the effects of PD-L1 specific deletion in T cells during CIA (Subaim 2.1). To test if tissue resident memory T cells play a protective role during CIA, we will utilize a tissue resident chimera mouse model to selectively delete peripheral T cells in LckcrePD- L1fl/fl mice. Donor T cells from LckcrePD-L1fl/fl or Lckcre mice will replace peripheral T cell populations to confirm the protective function of tissue resident memory T cells in the joint (Subaim 2.2). Our project has high translational potential since joint specific PD-L1 expression could be modulated with therapeutic purposes in humans for rheumatoid arthritis or check point inhibitors induced arthritis.