CD8+ T细胞是一种高度多样的细胞群体，具有明显的表型功能，可以影响免疫治疗的结果。对天然生成的肿瘤特异性T细胞的CD8+特异性和TCR亲和力的作用进行进一步研究非常重要，其中在同一肿瘤中既存在高亲和力又存在低亲和力的T细胞，它们识别相同的肽-MHC（pMHC）。这有助于理解T细胞衰竭和对PD-1免疫治疗的耐受性。在皮下肿瘤植入后的第3、6和9天，用PD-1抗体对CT26模型进行处理，导致早期肿瘤发展期间产生不同的反应。利用四聚体染色确定靶向肿瘤特异表位GSW11的CD8+ T细胞的频率和亲和力，并通过四聚体竞争实验进行确认。采用流式细胞术和批量RNA测序对高和低亲和力的GSW11特异性CD8+ T细胞进行功能特征化。通过体外细胞毒性试验和体内移植试验来确定高和低亲和力群体的细胞毒性。成功的PD-1免疫治疗与低亲和力（Tetlo）的GSW11特异性CD8+ T细胞的优势扩增以及表达克隆VβTCR相关。高亲和力T细胞（Tethi）只在进展的PD-1耐药性肿瘤中找到，如果存在的话。与Tethi相比，Tetlo表现出前体衰竭或祖细胞T细胞表型，其Tcf-1和T-bet的表达更高，CD39、PD-1和Eomes的衰竭标记的表达较低，而Tethi细胞则处于终末衰竭状态。转录组学分析显示，与Tethi相比，在逆行和进展中的肿瘤中都存在丰富的Tetlo，这些细胞中显著富集了与TCR信号传导、细胞毒性和氧化磷酸化相关的通路，而与DNA损伤、凋亡和自嗜作用相关的基因则下调。体外研究显示，Tetlo表现出比Tethi更高的细胞毒性。Tetlo的移植在肿瘤控制方面的效果比Tethi更为有效，并且在联合应用两剂PD-1抗体时可以达到治愈效果。针对亲和力较低的次优T细胞反应以靶向pMHC亲和力新表位，显示了改善PD-1免疫治疗的潜力。未来的干预可能会考虑通过疫苗接种或移植来扩增低亲和力群体。© 作者（或其雇主）2023年。在CC BY许可下允许重复使用。由BMJ出版。

CD8+ T cells are a highly diverse population of cells with distinct phenotypic functions that can influence immunotherapy outcomes. Further insights on the roles of CD8+ specificities and TCR avidity of naturally arising tumor-specific T cells, where both high and low avidity T cells recognizing the same peptide-major histocompatibility complex (pMHC) coexist in the same tumor, are crucial for understanding T cell exhaustion and resistance to PD-1 immunotherapy.CT26 models were treated with anti-PD-1 on days 3, 6 and 9 following subcutaneous tumor implantation generating variable responses during early tumor development. Tetramer staining was performed to determine the frequency and avidity of CD8+ T cells targeting the tumor-specific epitope GSW11 and confirmed with tetramer competition assays. Functional characterization of high and low avidity GSW11-specific CD8+ T cells was conducted using flow cytometry and bulk RNA-seq. In vitro cytotoxicity assays and in vivo adoptive transfer experiments were performed to determine the cytotoxicity of high and low avidity populations.Treatment success with anti-PD-1 was associated with the preferential expansion of low avidity (Tetlo) GSW11-specific CD8+ T cells with Vβ TCR expressing clonotypes. High avidity T cells (Tethi), if present, were only found in progressing PD-1 refractory tumors. Tetlo demonstrated precursor exhausted or progenitor T cell phenotypes marked by higher expression of Tcf-1 and T-bet, and lower expression of the exhaustion markers CD39, PD-1 and Eomes compared with Tethi, whereas Tethi cells were terminally exhausted. Transcriptomics analyses showed pathways related to TCR signaling, cytotoxicity and oxidative phosphorylation were significantly enriched in Tetlo found in both regressing and progressing tumors compared with Tethi, whereas genes related to DNA damage, apoptosis and autophagy were downregulated. In vitro studies showed that Tetlo exhibits higher cytotoxicity than Tethi. Adoptive transfer of Tetlo showed more effective tumor control than Tethi, and curative responses were achieved when Tetlo was combined with two doses of anti-PD-1.Targeting subdominant T cell responses with lower avidity against pMHC affinity neoepitopes showed potential for improving PD-1 immunotherapy. Future interventions may consider expanding low avidity populations via vaccination or adoptive transfer.© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.