Background: TIGIT, as a novel immune checkpoint molecule involved in T cell and NK cell anergy, could induce the immune tolerance and escape through binding with its ligand PVR. Blockade of TIGIT/PVR is considered as a promising strategy in cancer immunotherapy. However, to facilitate the design of inhibitors targeting TIGIT/PVR, the structural characteristics and binding mechanism still need to be further studied.Methods: In this study, molecular dynamics (MD) simulations and in silico mutagenesis were used to analyze the interaction between TIGIT and its ligand PVR. Then, PVR mutants were designed and their activities were determined by using TIGIT overexpressed Jurkat cells.Results: The results suggested that the loops of PVR (CC ' loop, C ' C '' loop, and FG loop) underwent a large intra-molecular rearrangement, and more hydrogen bond crosslinking between PVR and TIGIT were formed during MD simulations. The potential residues for PVR to interact with TIGIT were identified and utilized to predict high affinity PVR mutants. Through the biological activity evaluation, four PVR mutants ((PVR)S72W, (PVR)S72R, (PVR)G131V and (PVR)S132Q) with enhanced affinity to TIGIT were discovered, which could elicit more potent inhibitory effects compared with the wild type PVR.Conclusions: The MD simulations analysis provided new insights into the TIGIT/PVR interaction model, and the identified PVR mutants ((PVR)S72W, (PVR)S72R, (PVR)G131V and (PVR)S132Q) could serve as new candidates for immunotherapy to block TIGIT/PVR.

食管鳞状细胞癌（esophageal squamous cell carcinoma,ESCC）是世界上最常见的恶性肿瘤之一,虽然近几年手术和新辅助放化疗在临床治疗上已经取得很大进展,但ESCC患者的5年生存率仍然很低。基于肿瘤浸润淋巴细胞（tumor infiltrating lymphocytes,TILs）的免疫治疗是治疗肿瘤的有效方法之一,可能因为CD8+TIL含有不同的细胞亚群,从而导致患者的临床疗效差异较大。因此,深入探究CD8+T淋巴细胞有助于我们发现和肿瘤诊断、治疗、预后更相关的T淋巴细胞亚群。组织定居记忆（tissue-resident memory,TRM）T细胞是最近几年发现的新的淋巴细胞亚群。TRM细胞可产生效应细胞因子,对病原体感染的细胞能直接产生细胞毒性,从而参与抗肿瘤免疫反应。CD103+CD8+TRM细胞在人肿瘤组织中已有报道,但这些研究仅限于用免疫组化的方法回顾性分析TRM细胞的浸润比例和患者生存预后的关系,对其在人肿瘤组织中的特性及功能还未做进一步研究。CD103+CD8+TRM细胞在人ESCC肿瘤组织中是否存在及其在抗肿瘤免疫反应中的免疫学特性及功能至今未有研究报道。为了探索CD103+CD8+TRM细胞在ESCC中的存在情况,我们首先对TCGA数据Ⅰ-Ⅳ期ESCC的转录组测序（RNA-sequencing,RNA-Seq）结果进行分析。研究发现在ESCC肿瘤组织中,CD8A基因表达与TRM细胞标记基因-整合素αE亚单位（ITGAE,又名CD103）基因表达呈正相关性（r=0.4786）,CD8A高表达的肿瘤组织样本富集较高的ITGAE基因（P＜0.001）,并且在CD8A高达的肿瘤组织样本中,TRM细胞相关基因RUNX3、CD69、CXCR6明显高于CD8A低表达的肿瘤组织样本（P＜0.01、P＜0.0001、P＜0.0001）;进一步分析 CD9、RUNX3、CXC6与ITGAE基因的关系,发现CD69、RUNX3、CXCR6与ITGAE基因的表达也具有显著正相关性（r=0.3189、r=0.2478、r=0.5142）,并且 CD69、RUNX3、CXCR6在ITGAE基因高表达的肿瘤组织样本中表达明显高于ITGAE基因低表达的肿瘤组织样本（P＜0.01、P＜0.05、P＜0.0001）;此外,ITGAE与PD-1、TIM-3、CTLA-4、LAG-3、TIGIT等免疫检查点分子及颗粒酶B、干扰素-γ杀伤性分子对应的基因之间显著正相关（r=0.4631、r=0.4108、r=0.3869、r=0.4394、r=0.4788、r=0.5054、r=0.3682）;与ITGAE低表达的肿瘤组织相比,ITGAE高表达的肿瘤组织样本也高表达PD-1、TIM-3、CTLA-4、LAG-3和TIGIT等免疫检查点基因（P＜0.0001、P＜0.0001、P＜0.001、P＜0.001、P＜0.001）,并高表达颗粒酶B和干扰素-γ（Interferon-γ,IFN-γ）（P＜0.0001、P＜0.001）等杀伤性分子基因。以上结果表明食管鳞癌在基因水平可能存在CD103+CD8+TRM细胞,并且可能是肿瘤免疫反应中的主要效应细胞。ESCC中表达较高的CD103+CD8+TRM细胞相关基因,接下来通过流式细胞术（flow cytometry,FCM）验证新鲜初治ESCC手术标本中CD103+CD8+TRM细胞的存在情况。结果显示ESCC肿瘤组织中存在CD103+CD8+TRM细胞,为（60.47±3.99）%;并且高表达PD-1、TIM-3免疫检查点,PD-1+、TIM-3+、PD-1+TIM-3+表达依次为（48.51±4.74）%、（29.09 ± 4.61）%、（39.73±5.03）%;同时也高表达CD69及低表达CD62L、CCR7,依次为（57.66±7.65）%、（2.42±0.89）%、（4.42±1.20）%,表明 CD103+CD8+T细胞有可能是一群肿瘤特异性T细胞,并具有组织定居T细胞的表型特征。为了了解化疗药物对肿瘤组织样本中CD103+CD8+TRM细胞的影响,我们进一步用FCM方法分析新辅助化疗后的ESCC患者中CD103+CD8+TRM细胞的浸润情况,结果发现新辅助化疗后ESCC肿瘤组织中CD103+CD8+T细胞的浸润比例为（65.11±6.20）%,并且高表达PD-1、TIM-3免疫检查点,PD-1+、TIM-3+、PD-1+TIM-3+表达依次为（52.40±6.31）%、（42.15±6.09）%、（52.43±5.80）%;并且,新辅助化疗后肿瘤组织中CD103+CD8+TRM细胞的比例,及 CD103+CD8+TRM 细胞上 PD-1+、TIM-3+、PD-1+TIM-3+的表达比例与初治患者相比无统计学差异（P＞0.05）,推测CD103+CD8+TRM细胞可能不受化疗药物的影响。回顾性分析2012年9月到2013年5月初治的76例ESCC癌旁组织和癌组织蜡块标本中CD103和CD8 T细胞的浸润情况,并收集患者的临床病理信息。结果显示,癌组织和癌旁组织均有CD8+和CD103+细胞浸润,但组织多色免疫荧光显示癌组织中大多数CD103+细胞共表达CD8+,显示CD103+CD8+T细胞表型,而在癌旁组织中大多数CD103+细胞不共表达CD8+,表明癌组织中CD103+细胞是T细胞来源。另外,Kaplan-Meier生存分析和Log-Rank检验结果发现CD103+和CD8+T细胞的高表达与ESCC患者较好的临床预后有关。以上结果表明CD103+CD8+TRM细胞和ESCC好的临床预后关系密切。我们进一步研究观察到,在ESCC新鲜组织标本中,与CD103-CD8+T细胞相比,CD103+CD8+T细胞Ki67增殖能力强,为（8.29±1.10）%,可分泌IFN-γ、IL-2 并且表达 CD107a,为（6.55±2.08）%、（4.24 ± 1.52）%、（11.71±1.94）%。另外,由于CD103+CD8+T细胞高表达PD-1,为了探究PD-1阻断对CD103+CD8+T细胞的影响,用anti-PD-1抗体和CD103+CD8+T细胞共孵育,发现CD103+T细胞在anti-PD-1抗体阻断后IFN-γ和 IL-2分泌能力增强,依次为（11.00±3.15）%、（5.71±1.64）%。为了更充分研究 anti-PD-1 药物对 CD103+CD8+T细胞的影响,我们运用化学致癌剂4-硝基喹啉-1-氧化物（4-NQO）饮水法建立小鼠原发食管癌模型,检测anti-PD-1抗体治疗原位食管癌小鼠后,小鼠体内CD103+CD8+T细胞的变化情况。结果显示食管癌小鼠经anti-PD-1抗体治疗后,食管组织肿瘤灶数目明显较少,对照组和anti-PD-1抗体治疗组肿瘤数目分别为4.14±0.59,2.28±0.35,有统计学差异（P＜0.05）;免疫组化结果显示食管肿瘤组织浸润的CD103+淋巴细胞（H-Score评分）在对照组和治疗组分别为0.08±0.01,0.14±0.01,两组之间有统计学差异（P＜0.05）,由此推测anti-PD-1抗体对小鼠的治疗作用有可能和CD103+T细胞的浸润增加有关。以上几部分研究对CD103+CD8+TRM细胞的存在及特性有了初步认识,最后以4-NQO诱导的食管癌小鼠为研究对象,FCM分选脾脏CD103-CD8+T细胞和CD103+CD8+T细胞,利用新一代RNA-seq技术,初步探索CD103-CD8+T细胞和CD103+CD8+T细胞的转录组差异表达基因。结果显示CD103+CD8+T细胞相对于CD103-CD8+T细胞差异表达基因的功能富集主要集中在细胞毒功能、细胞免疫反应、细胞粘附功能及相关的信号通路。这与以往的研究相吻合,表明CD103+CD8+T细胞可能参与了食管癌肿瘤组织中的免疫反应。综上所述,ESCC中存在CD103+CD8+TRM细胞,并且与患者的预后密切相关,是一群可能不受化疗药物影响,在肿瘤环境中容易被活化,容易产生应答的肿瘤反应性T细胞。并且,靶向PD-1后,CD103+CD8+TRM细胞功能有所增强,为食管癌免疫治疗的联合治疗提供有效的理论基础。

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a common gastrointestinal malignancy with poor patient prognosis. Current treatment for ESCC, including immunotherapy, is only beneficial for a small subset of patients. Better characterization of the tumor microenvironment (TME) and the development of novel therapeutic targets are urgently needed.; METHODS: In the present study, we hypothesized that integration of single-cell transcriptomic sequencing and large microarray sequencing of ESCC biopsies would reveal the key cell subtypes and therapeutic targets that determine the prognostic and tumorigenesis of ESCC. We characterized the gene expression profiles, gene sets enrichment, and the TME landscape of a microarray cohort including 84 ESCC tumors and their paired peritumor samples. We integrated single-cell transcriptomic sequencing and bulk microarray sequencing of ESCC to reveal key cell subtypes and druggable targets that determine the prognostic and tumorigenesis of ESCC. We then designed and screened a blocking peptide targeting Chemokine C-C motif ligand 18 (CCL18) derived from tumor associated macrophages and validated its potency by MTT assay. The antitumor activity of CCL18 blocking peptide was validated in vivo by using 4-nitroquinoline-1-oxide (4-NQO) induced spontaneous ESCC mouse model.; RESULTS: Comparative gene expression and cell-cell interaction analyses revealed dysregulated chemokine and cytokine pathways during ESCC carcinogenesis. TME deconvolution and cell interaction analyses allow us to identify the chemokine CCL18 secreted by tumor associated macrophages could promote tumor cell proliferation via JAK2/STAT3 signaling pathway and lead to poor prognosis of ESCC. The peptide Pep3 could inhibit the proliferation of EC-109 cells promoted by CCL18 and significantly restrain the tumor progression in 4-NQO-induced spontaneous ESCC mouse model.; CONCLUSIONS: For the first time, we discovered and validated that CCL18 blockade could significantly prevent ESCC progression. Our study revealed the comprehensive cell-cell interaction network in the TME of ESCC and provided novel therapeutic targets and strategies to ESCC treatment. © 2023. The Author(s).