MCB Undergraduate Program, University of California, Berkeley, CA, United States of America.
Department of Pathology, University of California, San Francisco, CA, United States of America.
PLoS One. 2022 Aug 17;17(8):e0272857. doi: 10.1371/journal.pone.0272857. eCollection 2022.
Effective immune responses depend on efficient antigen uptake in the periphery, transport of those antigens to, and presentation in draining lymph nodes (LNs). These processes have been studied intensively using stable fluorescent proteins (FPs) as model antigens. To date, ZsGreen is the only FP that can be tracked efficiently towards LNs, hence, it is difficult to compare studies using alternated tracking proteins. Here, we systematically compared six different FPs. We included ZsGreen, ZsYellow, DsRed, AsRed, mCherry, and mRFP based on sequence homology and/or origin species, and generated FP-expressing tumor cell lines. Stability of fluorescent signal was assessed in vitro over time, across different pH environments, and in vivo through FP antigen uptake and transfer to immune cells isolated from tumors and tumor-draining LNs. ZsGreen could be detected in high percentages of all analyzed tumor-infiltrating immune cells, with highest amounts in tumor-associated macrophages (TAMs) and type 2 conventional dendritic cells (cDC2s). ZsYellow, AsRed, and DsRed followed a similar pattern, but percentages of FP-containing immune cells in the tumor were lower than for ZsGreen. Strikingly, mRFP and mCherry demonstrated a 'non-canonical' antigen uptake pattern where percentages of FP-positive tumor-infiltrating immune cells were highest for cDC1s not TAMs and cDC2s despite comparable stabilities and localization of all FPs. Analysis of antigen-containing cells in the LN was hindered by intracellular degradation of FPs. Only ZsGreen could be efficiently tracked to the LN, though some signal was measurable for ZsYellow and DsRed. In summary, we find that detection of antigen uptake and distribution is subject to variabilities related to fluorophore nature. Future experiments need to consider that these processes might be impacted by protein expression, stability, or other unknown factors. Thus, our data sheds light on potential under-appreciated mechanisms regulating antigen transfer and highlights potential uses and necessary caveats to interpretation based on FP use.
有效的免疫反应取决于在外周有效地摄取抗原，将这些抗原运输到引流淋巴结 (LNs) 并进行呈递。这些过程已经使用稳定的荧光蛋白 (FPs) 作为模型抗原进行了深入研究。迄今为止，ZsGreen 是唯一一种可以有效地追踪到 LNs 的 FP，因此，很难比较使用交替追踪蛋白的研究。在这里，我们系统地比较了六种不同的 FP。我们包括 ZsGreen、ZsYellow、DsRed、AsRed、mCherry 和 mRFP，基于序列同源性和/或起源物种，并生成了 FP 表达的肿瘤细胞系。我们评估了荧光信号在体外随时间的稳定性、不同 pH 环境下的稳定性，以及通过 FP 抗原摄取和转移到从肿瘤和肿瘤引流 LNs 中分离的免疫细胞在体内的稳定性。ZsGreen 可以在所有分析的肿瘤浸润免疫细胞中以高比例检测到，在肿瘤相关巨噬细胞 (TAMs) 和 2 型常规树突状细胞 (cDC2s) 中含量最高。ZsYellow、AsRed 和 DsRed 遵循类似的模式，但 ZsGreen 中 FP 阳性免疫细胞的比例较低。引人注目的是，mRFP 和 mCherry 表现出“非典型”的抗原摄取模式，尽管所有 FP 的稳定性和定位都相似，但 FP 阳性肿瘤浸润免疫细胞的比例在 cDC1s 中最高，而不是 TAMs 和 cDC2s。在 LN 中分析含抗原的细胞受到 FP 细胞内降解的阻碍。只有 ZsGreen 可以有效地追踪到 LN，尽管可以测量到一些 ZsYellow 和 DsRed 的信号。总之，我们发现抗原摄取和分布的检测受到与荧光团性质相关的可变性的影响。未来的实验需要考虑到这些过程可能受到蛋白质表达、稳定性或其他未知因素的影响。因此，我们的数据揭示了调节抗原转移的潜在被低估的机制，并强调了基于 FP 使用的潜在用途和必要的解释注意事项。
