Department of Oncology UNIL CHUV, University of Lausanne, Epalinges, Switzerland.
Ludwig Institute for Cancer Research, Lausanne Branch of Immunology, Epalinges, Switzerland.
J Immunother Cancer. 2020 May;8(1). doi: 10.1136/jitc-2019-000435.
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.
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.
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.
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.
随着免疫疗法在治疗不同肿瘤类型方面越来越受到认可，科学家们依赖于最先进的方法来监测患者的免疫反应和生物标志物的发展。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，因此对患者中这些细胞的特征描述仍然非常有限。
我们使用肽主要组织相容性复合物 II（pMHCII）多聚体构建体的组合染色策略，针对不同等位基因，建立了优化的体外和直接离体可视化患者肿瘤特异性 CD4 T 细胞的染色程序。此外，我们还生成了可逆多聚体，以实现最佳细胞染色，并且在体外细胞扩增之前进行解组装，从而防止激活诱导的细胞死亡。
与非优化染色程序相比，我们的优化方法大大提高了对肿瘤特异性、病毒特异性和细菌特异性细胞的检测。通过增加用于标记 pMHCII 多聚体的荧光染料的种类，我们还能够在一个样本中同时平行检测不同的特异性，包括抗原特异性 CD8 T 细胞。当使用完整的优化方法时，观察到细胞活力下降，但通过去除神经氨酸酶和使用可逆多聚体可以减轻这种影响。
这种新的优化染色程序代表了在更好地检测和分析抗原特异性 CD4 T 细胞方面的进展。它应该有助于对肿瘤特异性 CD4 T 细胞进行最先进的精确监测，并有助于加速这些细胞在癌症免疫治疗中的应用和靶向。
