Data Sheet 2_Detection of spontaneous anti-neoepitope T-cell responses in non-metastatic bladder cancer patients.docx

BackgroundBladder carcinomas are immunogenic, and patients with bladder cancer benefit from immune checkpoint therapy. This is correlated to a high tumor mutation burden, which provides a higher number of neoepitopes that can be recognized by tumor-specific CD8+ T cells. Intravesical Bacillus Calmette-Guérin (BCG) is used to treat non-muscle invasive bladder cancer (NMIBC), but its mechanism of action remains elusive. Most lymphocytes appearing in the urine of BCG-treated patients are CD4+ T cells though preclinical studies showed that CD8+ T cells are also necessary for BCG treatment efficacy. It is currently unknown which proportion of patients with non-metastatic bladder cancer develop a spontaneous antitumor CD8+ response, and if BCG treatment influences this response.
MethodsIn a first cohort of 15 NMIBC and 9 muscle invasive bladder cancer patients, we used IFN-y ELISPOT assays to screen for the presence of anti-neoepitope CD8+ T cells in the blood, tumor and urine. In a second cohort of 4 NMIBC patients, we analyzed the features and specificity of CD8+ T cells infiltrating the tumoral or bladder tissues before and after BCG using single cell transcriptomic analyses. A total of 31 tumor-infiltrating CD8+ clonotypes were screened against neoepitopes and tumor cDNA libraries.
Results9 out of 24 patients from the first cohort mounted a spontaneous and functional anti-neoepitope T-cell response in blood and/or tumor. In 5 patients from this cohort who were treated with BCG, no neoantigen-specific T cells were detected in urine during treatment. In the second cohort, 6 out of 6 TCRs from exhausted CD8+ TILs from one patient recognized 5 different neoepitopes. T-cell receptor (TCR) repertoire analyses indicated that the frequencies of these tumor-specific T cells did not increase after BCG instillations, neither in the bladder nor in the blood. None of the 25 other TCRs of CD8+ T cells recognized tumor-specific antigens.
ConclusionsWe show that one third of patients with non-metastatic bladder cancer mount a spontaneous and functional anti-neoepitope CD8+ T-cell response detectable in blood or tumor. In 4 patients with NMIBC, BCG treatment did not boost or induce the anti-neoepitope response, suggesting alternative mechanisms of action for its efficacy.

背景：膀胱癌具有免疫原性，膀胱癌患者可从免疫检查点治疗（immune checkpoint therapy）中获益。这一现象与高肿瘤突变负荷（tumor mutation burden）相关，该负荷可产生更多可被肿瘤特异性CD8+ T细胞（CD8+ T cell）识别的新表位（neoepitopes）。膀胱内灌注卡介苗（Bacillus Calmette-Guérin, BCG）是目前治疗非肌层浸润性膀胱癌（non-muscle invasive bladder cancer, NMIBC）的常用手段，但其具体作用机制仍未明确。经BCG治疗的患者尿液中出现的淋巴细胞多为CD4+ T细胞，但临床前研究表明CD8+ T细胞同样对BCG的治疗疗效至关重要。目前尚不明确，在非转移性膀胱癌患者中，有多大比例会自发产生抗肿瘤CD8+ T细胞应答，以及BCG治疗是否会对该应答产生影响。
方法：本研究第一队列纳入15例非肌层浸润性膀胱癌患者与9例肌层浸润性膀胱癌（muscle invasive bladder cancer）患者，通过干扰素γ酶联免疫斑点试验（IFN-γ ELISPOT assay）筛查血液、肿瘤组织及尿液中抗新表位CD8+ T细胞的存在情况。第二队列纳入4例非肌层浸润性膀胱癌患者，利用单细胞转录组分析（single cell transcriptomic analyses），对比BCG治疗前后浸润于肿瘤组织或膀胱组织的CD8+ T细胞的特征与特异性。我们共针对31种肿瘤浸润性CD8+ T细胞克隆型，开展新表位与肿瘤cDNA文库的筛查实验。
结果：第一队列的24例患者中，有9例在血液或/和肿瘤组织中出现了自发的、具有功能活性的抗新表位T细胞应答。该队列中接受BCG治疗的5例患者，在治疗期间的尿液样本中均未检测到新抗原特异性T细胞。第二队列中，来自同一名患者的耗竭性肿瘤浸润性CD8+ T细胞的6种T细胞受体（T-cell receptor, TCR），可识别5种不同的新表位。T细胞受体库分析显示，在BCG膀胱灌注后，这些肿瘤特异性T细胞的频率在膀胱组织及血液中均未出现升高。其余25种CD8+ T细胞受体均未识别肿瘤特异性抗原。
结论：本研究证实，三分之一的非转移性膀胱癌患者，可在血液或肿瘤组织中检测到自发产生的、具有功能活性的抗新表位CD8+ T细胞应答。在4例非肌层浸润性膀胱癌患者中，BCG治疗并未增强或诱导抗新表位T细胞应答，这提示BCG的治疗疗效可能存在其他作用机制。