Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea.
Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea.
Gastroenterology. 2018 Dec;155(6):1936-1950.e17. doi: 10.1053/j.gastro.2018.08.030. Epub 2018 Aug 24.
BACKGROUND & AIMS: T-cell exhaustion, or an impaired capacity to secrete cytokines and proliferate with overexpression of immune checkpoint receptors, occurs during chronic viral infections but has also been observed in tumors, including hepatocellular carcinomas (HCCs). We investigated features of exhaustion in CD8 T cells isolated from HCC specimens.
We obtained HCC specimens, along with adjacent nontumor tissues and blood samples, from 90 patients who underwent surgical resection at Asan Medical Center (Seoul, Korea) from April 2016 through April 2018. Intrahepatic lymphocytes and tumor-infiltrating T cells were analyzed by flow cytometry. Tumor-infiltrating CD8 T cells were sorted by flow cytometry into populations based on expression level of programmed cell death 1 (PDCD1 or PD1): PD1-high, PD1-intermediate, and PD1-negative. Sorted cells were analyzed by RNA sequencing. Proliferation and production of interferon gamma (IFNG) and tumor necrosis factor (TNF) by CD8 T cells were measured in response to anti-CD3 and antibodies against immune checkpoint receptors including PD1, hepatitis A virus cellular receptor 2 (HAVCR2 or TIM3), lymphocyte activating 3 (LAG3), or isotype control. Tumor-associated antigen-specific CD8 T cells were identified using HLA-A*0201 dextramers. PDL1 expression on tumor tissue was assessed by immunohistochemistry.
PD1-high, PD1-intermediate, and PD1-negative CD8 T cells from HCCs had distinct gene expression profiles. PD1-high cells expressed higher levels of genes that regulate T-cell exhaustion than PD1-intermediate cells. PD1-high cells expressed TIM3 and LAG3, and low proportions of TCF1, TBET/eomesodermin, and CD127. PD1-high cells produced the lowest amounts of IFNG and TNF upon anti-CD3 stimulation. Differences in the PD1 expression patterns of CD8 T cells led to the identification of 2 subgroups of HCCs: HCCs with a discrete population of PD1-high cells were more aggressive than HCCs without a discrete population of PD1-high cells. HCCs with a discrete population of PD1-high cells had higher levels of predictive biomarkers of response to anti-PD1 therapy. Incubation of CD8 T cells from HCCs with a discrete population of PD1-high cells with antibodies against PD1 and TIM3 or LAG3 further restored proliferation and production of IFNG and TNF in response to anti-CD3.
We found HCC specimens to contain CD8 T cells that express different levels of PD1. HCCs with a discrete population of PD1-high CD8 T cells express TIM3 and/or LAG3 and produce low levels of IFNG and TNF in response to anti-CD3. Incubation of these cells with antibodies against PD1 and TIM3 or LAG3 further restore proliferation and production of cytokines; HCCs with a discrete population of PD1-high CD8 T cells might be more susceptible to combined immune checkpoint blockade-based therapies.
T 细胞耗竭，即细胞因子分泌和增殖能力受损，同时过度表达免疫检查点受体，这种现象发生在慢性病毒感染期间，但也存在于肿瘤中，包括肝细胞癌（HCC）。本研究旨在探究 HCC 标本中 CD8 T 细胞耗竭的特征。
我们从 2016 年 4 月至 2018 年 4 月期间在韩国首尔 Asan 医疗中心接受手术切除的 90 名 HCC 患者的 HCC 标本、相邻非肿瘤组织和血液样本中获得了 HCC 标本。通过流式细胞术分析了肝内淋巴细胞和肿瘤浸润性 T 细胞。通过流式细胞术将肿瘤浸润性 CD8 T 细胞按程序性死亡受体 1（PDCD1 或 PD1）的表达水平分为 PD1-高、PD1-中、PD1-低三组。对分选的细胞进行 RNA 测序分析。用抗 CD3 和针对免疫检查点受体（包括 PD1、肝炎 A 病毒细胞受体 2（HAVCR2 或 TIM3）、淋巴细胞激活 3（LAG3）或同型对照）的抗体检测 CD8 T 细胞的增殖和干扰素 γ（IFNG）和肿瘤坏死因子（TNF）的产生。使用 HLA-A*0201 右旋体鉴定肿瘤相关抗原特异性 CD8 T 细胞。用免疫组织化学法评估肿瘤组织上的 PDL1 表达。
HCC 中的 PD1-高、PD1-中、PD1-低 CD8 T 细胞具有不同的基因表达谱。与 PD1-中细胞相比，PD1-高细胞表达调节 T 细胞耗竭的基因水平更高。PD1-高细胞表达 TIM3 和 LAG3，而 TCF1、TBET/eomesodermin 和 CD127 的比例较低。PD1-高细胞在抗 CD3 刺激下产生的 IFNG 和 TNF 最少。CD8 T 细胞 PD1 表达模式的差异导致鉴定出 2 组 HCC：具有离散 PD1-高细胞群的 HCC 比没有离散 PD1-高细胞群的 HCC 更具侵袭性。具有离散 PD1-高细胞群的 HCC 具有更高水平的抗 PD1 治疗反应预测生物标志物。用抗 PD1 和 TIM3 或 LAG3 孵育来自具有离散 PD1-高细胞群的 HCC 的 CD8 T 细胞，进一步恢复了对抗 CD3 的增殖和 IFNG 和 TNF 的产生。
我们发现 HCC 标本中含有表达不同 PD1 水平的 CD8 T 细胞。具有离散 PD1-高 CD8 T 细胞群的 HCC 表达 TIM3 和/或 LAG3，并在抗 CD3 时产生低水平的 IFNG 和 TNF。用抗 PD1 和 TIM3 或 LAG3 孵育这些细胞可进一步恢复细胞因子的增殖和产生；具有离散 PD1-高 CD8 T 细胞群的 HCC 可能更容易受到基于免疫检查点阻断的联合治疗的影响。
