BACKGROUND With immunotherapy gaining increasing approval for treatment of different tumor types, scientists rely on cutting edge methods for the monitoring of immune responses and biomarker development in patients. Due to the lack of tools to efficiently detect rare circulating human tumor-specific CD4 T cells, their characterization in patients still remains very limited. METHODS We have used combinatorial staining strategies with peptide major histocompatibility complex class II (pMHCII) multimer constructs of different alleles to establish an optimized staining procedure for in vitro and direct ex-vivo visualization of tumor-specific CD4 T cells, in patient samples. Furthermore, we have generated reversible multimers to achieve optimal cell staining and yet disassemble prior to in vitro cell expansion, thus preventing activation induced cell death. RESULTS We observed a vastly improved detection of tumor-specific, viral-specific and bacterial-specific cells with our optimization methods compared with the non-optimized staining procedure. By increasing the variety of fluorochromes used to label the pMHCII multimers, we were also able to increase the parallel detection of different specificities within one sample, including antigen-specific CD8 T cells. A decrease in cell viability was observed when using the full optimization method, but this was mitigated by the removal of neuraminidase and the use of reversible multimers. CONCLUSION This new optimized staining procedure represents an advance toward better detection and analysis of antigen-specific CD4 T cells. It should facilitate state-of-the art precision monitoring of tumor-specific CD4 T cells and contribute to accelerate the use and the targeting of these cells in cancer immunotherapy.

中文翻译：

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优化的组合pMHC II类多聚体标记，可对患者的肿瘤特异性CD4 T细胞进行精确的免疫监测。

背景技术随着免疫疗法在治疗不同肿瘤类型方面获得越来越多的认可，科学家们依靠先进的方法来监测患者的免疫应答和生物标记物的发育。由于缺乏有效检测稀有循环人类肿瘤特异性CD4 T细胞的工具，其在患者中的表征仍然非常有限。方法我们将组合染色策略与不同等位基因的主要II型肽主要组织相容性复合体（pMHCII）多聚体构建体结合使用，以建立优化的染色程序，用于在体外和直接离体可视化患者样品中的肿瘤特异性CD4 T细胞。此外，我们已经产生了可逆的多聚体，以实现最佳的细胞染色，并在体外细胞扩增之前进行拆卸，从而防止激活诱导的细胞死亡。结果与非优化的染色程序相比，我们的优化方法观察到了肿瘤特异性，病毒特异性和细菌特异性细胞的极大改善。通过增加用于标记pMHCII多聚体的荧光染料的种类，我们还能够提高在一个样品中，包括抗原特异性CD8 T细胞，对不同特异性的并行检测。当使用完全优化方法时，观察到细胞活力降低，但是通过去除神经氨酸酶和使用可逆多聚体可以缓解这种情况。结论这种新的优化的染色程序代表了更好地检测和分析抗原特异性CD4 T细胞的进步。