Background & Aims

Ligand binding to inhibitory receptors on immune cells, such as programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte associated protein 4 (CTLA4), down-regulates the T-cell–mediated immune response (called immune checkpoints). Antibodies that block these receptors increase antitumor immunity in patients with melanoma, non–small-cell lung cancer, and renal cell cancer. Tumor-infiltrating CD4⁺ and CD8⁺ T cells in patients with hepatocellular carcinoma (HCC) have been found to be functionally compromised. We analyzed HCC samples from patients to determine if these inhibitory pathways prevent T-cell responses in HCCs and to find ways to restore their antitumor functions.

Methods

We collected HCC samples from 59 patients who underwent surgical resection from November 2013 through May 2017, along with tumor-free liver tissues (control tissues) and peripheral blood samples. We isolated tumor-infiltrating lymphocytes (TIL) and intra-hepatic lymphocytes. We used flow cytometry to quantify expression of the inhibitory receptors PD-1, hepatitis A virus cellular receptor 2 (TIM3), lymphocyte activating 3 (LAG3), and CTLA4 on CD8⁺ and CD4⁺ T cells from tumor, control tissue, and blood; we studied the effects of antibodies that block these pathways in T-cell activation assays.

Results

Expression of PD-1, TIM3, LAG3, and CTLA4 was significantly higher on CD8⁺ and CD4⁺ T cells isolated from HCC tissue than control tissue or blood. Dendritic cells, monocytes, and B cells in HCC tumors expressed ligands for these receptors. Expression of PD-1, TIM3, and LAG3 was higher on tumor-associated antigen (TAA)-specific CD8⁺ TIL, compared with other CD8⁺ TIL. Compared with TIL that did not express these inhibitory receptors, CD8⁺ and CD4⁺ TIL that did express these receptors had higher levels of markers of activation, but similar or decreased levels of granzyme B and effector cytokines. Antibodies against CD274 (PD-ligand1 [PD-L1]), TIM3, or LAG3 increased proliferation of CD8⁺ and CD4⁺ TIL and cytokine production in response to stimulation with polyclonal antigens or TAA. Importantly, combining antibody against PD-L1 with antibodies against TIM3, LAG3, or CTLA4 further increased TIL functions.

Conclusions

The immune checkpoint inhibitory molecules PD-1, TIM3, and LAG3 are up-regulated on TAA-specific T cells isolated from human HCC tissues, compared with T cells from tumor-free liver tissues or blood. Antibodies against PD-L1, TIM3, or LAG3 restore responses of HCC-derived T cells to tumor antigens, and combinations of the antibodies have additive effects. Strategies to block PD-L1, TIM3, and LAG3 might be developed for treatment of primary liver cancer.

中文翻译：

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抗免疫检查点分子的抗体可恢复肝细胞癌中浸润肿瘤的T细胞的功能

背景与目标

配体与免疫细胞抑制受体的结合，例如程序性细胞死亡1（PD-1）和细胞毒性T淋巴细胞相关蛋白4（CTLA4），下调了T细胞介导的免疫反应（称为免疫检查点）。阻断这些受体的抗体可提高黑色素瘤，非小细胞肺癌和肾细胞癌患者的抗肿瘤免疫力。已发现肝细胞癌（HCC）患者的肿瘤浸润CD4 ⁺和CD8 ⁺ T细胞功能受损。我们分析了来自患者的HCC样品，以确定这些抑制途径是否能阻止HCC中的T细胞反应，并找到恢复其抗肿瘤功能的方法。

方法

我们收集了2013年11月至2017年5月接受手术切除的59例患者的HCC样本，以及无肿瘤的肝组织（对照组织）和外周血样本。我们分离了肿瘤浸润淋巴细胞（TIL）和肝内淋巴细胞。我们使用流式细胞仪来量化肿瘤，对照组织和血液中CD8 ⁺和CD4 ⁺ T细胞上抑制性受体PD-1，甲型肝炎病毒细胞受体2（TIM3），淋巴细胞活化3（LAG3）和CTLA4的表达。 ; 我们研究了在T细胞活化分析中阻断这些途径的抗体的作用。

结果

从HCC组织分离的CD8 ⁺和CD4 ⁺ T细胞上的PD-1，TIM3，LAG3和CTLA4的表达明显高于对照组织或血液。HCC肿瘤中的树突状细胞，单核细胞和B细胞表达这些受体的配体。与其他CD8 ⁺ TIL相比，肿瘤相关抗原（TAA）特异性CD8 ⁺ TIL上PD-1，TIM3和LAG3的表达更高。与不表达这些抑制性受体的TIL相比，CD8 ⁺和CD4 ⁺确实表达了这些受体的TIL具有更高水平的活化标记，但颗粒酶B和效应细胞因子水平相似或降低。针对CD274（PD-配体1 [PD-L1]），TIM3或LAG3的抗体会响应多克隆抗原或TAA的刺激而增加CD8 ⁺和CD4 ⁺ TIL的增殖以及细胞因子的产生。重要的是，将针对PD-L1的抗体与针对TIM3，LAG3或CTLA4的抗体相结合，可以进一步提高TIL功能。

结论

免疫检查点抑制分子PD-1，TIM3和LAG3在从人肝癌组织分离的TAA特异性T细胞上比从无肿瘤肝组织或血液中的T细胞上调。针对PD-L1，TIM3或LAG3的抗体可恢复源自HCC的T细胞对肿瘤抗原的反应，并且抗体的组合具有累加作用。可能会开发出阻断PD-L1，TIM3和LAG3的策略来治疗原发性肝癌。