4-1BB+ Tregs and inhibitory progenitor exhausted T cells confer resistance to anti-PD-L1 and anti-CTLA-4 combination therapy

Predictors of immune checkpoint inhibitor response in cancer remain elusive. From a previous phase 2 neoadjuvant immunotherapy window-of-opportunity study, we present the single-cell RNA and T cell receptor (TCR) sequencing analysis of 57 pre- and post-treatment tumor biopsies from head and neck cancer patients treated with durvalumab (anti-PD-L1) alone or with tremelimumab (anti-CTLA-4), identifying key cellular and molecular predictors of immune checkpoint inhibitor (ICI) response. Malignant cells and neutrophil senescence promote ICI response. While CXCL13+ exhausted T (Tex) cells enhance response through 4-1BB signaling, anti-CTLA-4 induces 4-1BB+ regulatory T cells (Tregs) restricting ICI efficacy. These opposing roles of 4-1BB in different cellular contexts may explain the limited benefit of combinatorial immunotherapy observed in clinical trials. We identify two subsets of tumor-reactive progenitor Tex (Tpex): ICI-responsive Tpex1 and ICI-resistant Tpex2, a subset characterized by KLRB1 and IL17R. The balance of Tpex1 and Tpex2 associates with ICI response across multiple cancers, offering insights into sustaining response. This study was registered at ClinicalTrials.gov (NCT03737968). Overall design: 57 HNSCC tumor biopsies, extracted from various tumor primary sites, were sequenced for TCR or GEX at single cell resolution. Samples are either treated with immune checkpoint inhibitor (Durvalumab or Durvalumab and Tremelimumab) or at extracted at baseline (pre-treatment).

癌症免疫检查点抑制剂（immune checkpoint inhibitor, ICI）应答的预测标志物仍不明确。本研究基于一项既往II期新辅助免疫治疗机会窗口期研究，对57例头颈部癌症患者治疗前后的肿瘤活检组织开展单细胞RNA测序与T细胞受体（T cell receptor, TCR）测序分析，这些患者接受度伐利尤单抗（durvalumab，抗PD-L1）单药或联合替西利姆单抗（tremelimumab，抗CTLA-4）治疗，最终鉴定出ICI应答的关键细胞与分子预测标志物。
恶性细胞与中性粒细胞衰老可促进ICI应答。CXCL13阳性耗竭性T（Tex）细胞可通过4-1BB信号通路增强应答，而抗CTLA-4治疗可诱导4-1BB阳性调节性T细胞（Tregs），进而限制ICI的疗效。4-1BB在不同细胞微环境中的这种双向作用，或可解释临床试验中联合免疫治疗获益有限的现象。
我们还鉴定出两类肿瘤反应性祖耗竭性T（Tpex）细胞亚群：ICI应答型Tpex1与ICI耐药型Tpex2，其中Tpex2亚群以KLRB1和IL17R为特征。Tpex1与Tpex2的平衡状态与多种癌症中的ICI应答相关，为维持免疫治疗应答提供了新的研究视角。
本研究已在ClinicalTrials.gov注册（注册号：NCT03737968）。整体实验设计：57例头颈部鳞状细胞癌（HNSCC）肿瘤活检组织取材于不同肿瘤原发部位，以单细胞分辨率进行TCR或基因表达（GEX）测序；样本分为免疫检查点抑制剂治疗组（度伐利尤单抗单药或度伐利尤单抗联合替西利姆单抗）及基线（治疗前）取材的对照组。