Yuan J, Gallardo H F, Rasalan T, Ranganathan R, Wang J, Zhang Y, Panageas K, Stan R, Young J W, Houghton A N, Wolchok J D
Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Cytotherapy. 2006;8(5):498-508. doi: 10.1080/14653240600868262.
Development of a practical and sensitive assay for evaluating immune responses against cancer Ag has been a challenge for immune monitoring of patients. We have established a reproducible method using peptide-pulsed K562-A*0201 cells as APC to expand Ag-specific T cells in vitro. This method may be applied for monitoring T-cell responses in cancer immunotherapy clinical trials.
Autologous PBMC from HLA-A0201+ healthy donors and patients with melanoma were stimulated with peptide-pulsed K562-A0201 cells under varying conditions. We investigated (1) different culture conditions, including the requirements for serum and cytokines for expansion of CD8+ T lymphocytes; (2) a range of peptide concentrations for Ag loading; (3) phenotypic characterization of responding T cells; and (4) APC:responder ratios and their effects on T-cell expansion. We validated these conditions by ELISPOT and intracellular cytokine staining (ICS) assays using peptides from influenza, Epslein-Barr Virus (EBV) and tyrosinase.
Conditions for optimal T-cell expansion using K562-A0201 APC included input of 2 x 10(6) PBMC, a 10 microg/mL peptide concentration to pulse K562-A0201 cells, a 1:30 APC:responder T-cell ratio and culture in 10% autologous plasma supplemented with IL-2 and IL-15. In these conditions, Ag-specific T cells expanded >100-fold over a 10-day culture period (peak at day 12).
This bulk culture method is simple and reliable for expanding human Ag-specific T cells using peptide-pulsed K562-A*0201 cells. This HLA-matched APC line can be adapted to other HLA haplotypes, and has advantages for monitoring clinical trials of immunotherapy with limited availability of autologous APC and PBMC from patients.
开发一种实用且灵敏的检测方法来评估针对癌症抗原的免疫反应，一直是患者免疫监测面临的一项挑战。我们建立了一种可重复的方法，使用肽脉冲处理的K562 - A*0201细胞作为抗原呈递细胞（APC）在体外扩增抗原特异性T细胞。该方法可应用于癌症免疫治疗临床试验中的T细胞反应监测。
在不同条件下，用肽脉冲处理的K562 - A0201细胞刺激来自HLA - A0201 +健康供体和黑色素瘤患者的自体外周血单核细胞（PBMC）。我们研究了：（1）不同的培养条件，包括CD8 + T淋巴细胞扩增所需的血清和细胞因子；（2）一系列用于抗原负载的肽浓度；（3）反应性T细胞的表型特征；以及（4）APC与反应细胞的比例及其对T细胞扩增的影响。我们通过使用来自流感病毒、爱泼斯坦 - 巴尔病毒（EBV）和酪氨酸酶的肽进行酶联免疫斑点分析（ELISPOT）和细胞内细胞因子染色（ICS）试验来验证这些条件。
使用K562 - A0201 APC进行最佳T细胞扩增的条件包括输入2×10⁶个PBMC、10μg/mL的肽浓度用于脉冲K562 - A0201细胞、1:30的APC与反应性T细胞比例以及在补充有白细胞介素 - 2（IL - 2）和白细胞介素 - 15（IL - 15）的10%自体血浆中培养。在这些条件下，抗原特异性T细胞在10天的培养期内扩增超过100倍（在第12天达到峰值）。
这种批量培养方法对于使用肽脉冲处理的K562 - A*0201细胞扩增人抗原特异性T细胞而言简单且可靠。这种HLA匹配的APC系可适用于其他HLA单倍型，并且对于在患者自体APC和PBMC可用性有限的情况下监测免疫治疗临床试验具有优势。
