Supplementary Material for: PD-1/CTLA-4 blockade leads to expansion of CD8+PD-1int TILs and results in tumor remission in experimental liver cancer

Background: Checkpoint inhibitors act on exhausted CD8+ T cells and restore their effector function in chronic infections and cancer. The underlying mechanisms of action appear to differ between different types of cancer and are not yet fully understood.
Methods: Here, we established a new orthotopic HCC model to study the effects of checkpoint blockade on exhausted CD8+ tumor-infiltrating lymphocytes (TILs). The tumors expressed endogenous levels of HA, which allowed the study of tumor-specific T cells
Results: The induced tumors developed an immune-resistant TME in which few T cells were found. The few recovered CD8+ TILs were mostly terminally exhausted and expressed high levels of PD-1. PD-1/CTLA-4 blockade resulted in a strong increase in the number of CD8+ TILs expressing intermediate amounts of PD-1, also called progenitor-exhausted CD8+ TILs, while terminally exhausted CD8+ TILs were almost absent in the tumors of treated mice. Although transferred naïve tumor-specific T cells did not expand in the tumors of untreated mice, they expanded strongly after treatment and generated progenitor-exhausted but not terminally exhausted CD8+ TILs. Unexpectedly, although progenitor-exhausted CD8+ TILs mediated the anti-tumor response after treatment with minimal changes in their transcriptional profile.
Conclusion: In our model, few doses of checkpoint inhibitors during the priming of transferred CD8+ tumor-specific T cells were sufficient to induce tumor remission. PD-1/CTLA-4 blockade neither reinvigorated the effector function of CD8+PD-1high TILs nor changed the functionality of CD8+PD-1int TILs. Therefore, PD-1/CTLA-4 blockade has an ameliorative effect the expansion of recently primed CD8+ T cells while preventing their development into CD8+PD-1high TILs in the TME. This finding could have important implications for future T cell therapies.

研究背景：免疫检查点抑制剂（checkpoint inhibitors）可作用于耗竭性CD8+ T细胞（exhausted CD8+ T cells），在慢性感染及癌症中恢复其效应子功能。但其作用的潜在机制在不同癌症类型中存在差异，目前尚未完全阐明。
研究方法：本研究构建了一种新型原位肝细胞癌（hepatocellular carcinoma, HCC）模型，用于探究免疫检查点阻断（checkpoint blockade）对耗竭性CD8+肿瘤浸润淋巴细胞（tumor-infiltrating lymphocytes, TILs）的作用效果。该肿瘤可表达内源性水平的HA，从而支持肿瘤特异性T细胞的相关研究。
研究结果：诱导形成的肿瘤构建出免疫耐受型肿瘤微环境（tumor microenvironment, TME），该微环境中仅存在极少量T细胞。所回收的少量CD8+ TILs大多为终末耗竭状态，且高表达PD-1。PD-1/细胞毒性T淋巴细胞相关抗原4（CTLA-4）阻断治疗可使表达中等水平PD-1的CD8+ TILs数量显著增加，这类细胞也被称为祖细胞样耗竭CD8+ TILs；而经治疗的小鼠肿瘤中几乎无法检测到终末耗竭CD8+ TILs。尽管输注的初始肿瘤特异性T细胞在未治疗小鼠的肿瘤中无法扩增，但经治疗后其扩增能力显著增强，并分化为祖细胞样耗竭而非终末耗竭的CD8+ TILs。出乎意料的是，祖细胞样耗竭CD8+ TILs在治疗后介导抗肿瘤应答，但其转录谱仅发生极小幅度的改变。
研究结论：在本模型中，在输注的CD8+肿瘤特异性T细胞致敏阶段给予少量免疫检查点抑制剂即可诱导肿瘤缓解。PD-1/CTLA-4阻断治疗既无法恢复CD8+PD-1high TILs的效应子功能，也未改变CD8+PD-1int TILs的功能特性。因此，PD-1/CTLA-4阻断治疗可促进新近致敏CD8+ T细胞的扩增，同时阻止其在肿瘤微环境中分化为CD8+PD-1high TILs。这一发现可为未来的T细胞疗法提供重要的理论参考。