Development of a unified mass cytometry assay integrating activation-induced markers (AIMs) and cytokine profiling for high-throughput assessment of antigen-specific T cell responses

Accurate profiling of antigen-specific T cells by measuring T cell activation markers and cytokine production has been limited by inconsistent marker usage across studies and substantial methodological variability. To overcome these challenges, we established a unified mass cytometry (CyTOF) platform that integrates optimized activation-induced marker (AIM) panels, intracellular cytokine staining (ICS), and a cadmium-based barcoding system for high-throughput, multiplexed analysis of T cell responses. We optimized stimulation conditions (18–24 h) and validated highly sensitive dual AIM combinations, including CD25+CD134+ and CD25+CD69+ in CD4+ T cells, as well as CD25+CD137+ and CD25+CD69+ in CD8+ T cells. These combinations were stable under protein transport inhibition and closely paralleled cytokine-producing populations. In addition, we developed a cadmium-tagged β2 M/CD298 barcoding strategy that supports 21-plex sample processing without signal distortion. Validation in two independent cohorts confirmed the platform’s high sensitivity, reproducibility, and its ability to simultaneously detect AIM+ T cells and cytokine-producing subsets. Together, this integrated workflow provides a robust and scalable framework for comprehensive immune monitoring in vaccine development and infectious disease research.

通过检测T细胞活化标志物与细胞因子产生来精准分析抗原特异性T细胞（antigen-specific T cells），长期以来受制于不同研究间标志物使用标准不统一，以及方法学层面存在的显著异质性。为解决上述难题，本研究建立了一套统一化的质谱流式细胞术（mass cytometry, CyTOF）平台，该平台整合了优化后的活化诱导标志物（activation-induced marker, AIM）组合、胞内细胞因子染色（intracellular cytokine staining, ICS）技术，以及基于镉的条形码系统（cadmium-based barcoding system），可实现T细胞应答的高通量、多参数分析。
我们优化了刺激培养条件（18~24小时），并验证了高灵敏度的双活化诱导标志物组合：在CD4阳性T细胞中为CD25+CD134+与CD25+CD69+，在CD8阳性T细胞中则为CD25+CD137+与CD25+CD69+。上述组合在蛋白转运抑制（protein transport inhibition）条件下稳定性良好，且与产细胞因子的细胞群体高度匹配。
此外，本研究开发了镉标记的β2微球蛋白/CD298条形码策略，可支持21重样本处理且无信号畸变。在两个独立队列中开展的验证实验证实，该平台兼具高灵敏度与良好重现性，可同时检测活化诱导标志物阳性T细胞与产细胞因子亚群。
综上，这套整合式工作流程为疫苗研发与传染病研究领域的全面免疫监测提供了一套稳健且可扩展的研究框架。