Hagan Natalie B, Inaku Charles, Kunder Nikesh, White Tayleur, Iraguha Thierry, Meyer Anna, Pauken Kristen E, Schenkel Jason M
Department of Immunology, University of Texas MD Anderson Cancer Center, Houston, TX, United States.
Department of Translational Molecular Pathology, University of Texas MD Anderson Cancer Center, Houston, TX, United States.
J Immunol. 2025 Apr 1;214(4):828-838. doi: 10.1093/jimmun/vkae051.
Leukocytes migrate through the blood and extravasate into organs to surveil the host for infection or cancer. Recently, we demonstrated that intravenous (IV) anti-CD45.2 antibody labeling allowed for precise tracking of leukocyte migration. However, the narrow labeling window can make this approach challenging for tracking rare migration events. Here, we show that altering antibody administration route and fluorophore can significantly extend the antibody active labeling time. We found that while both IV and intraperitoneal (IP) anti-CD45.2 antibody labeled circulating leukocytes after injection, they had different kinetic properties that impacted labeling time and intensity. Quantification of circulating antibody revealed that while unbound IV anti-CD45.2 antibody rapidly decreased, unbound IP anti-CD45.2 antibody increased over 1 h. Using in vitro and in vivo serial dilution assays, we found that Alexa Fluor 647 and Brilliant Blue 700 (BB700) dyes had the greatest labeling sensitivity compared with other fluorophores. However, IP antibody injection with anti-CD45.2 BB700, but not Alexa Fluor 647, resulted in continuous blood leukocyte labeling for over 6 h. Finally, we leveraged IP anti-CD45.2 BB700 antibody to track slower migrating leukocytes into tumors. We found that IP anti-CD45.2 antibody injection allowed for the identification of ∼7 times as many tumor-specific CD8+ T cells that had recently migrated from blood into tumors. Our results demonstrate how different injection routes and fluorophores affect anti-CD45.2 antibody leukocyte labeling and highlight the utility of this approach for defining leukocyte migration in the context of homeostasis and cancer.
白细胞通过血液迁移并渗出到器官中，以监测宿主是否受到感染或患癌症。最近，我们证明静脉注射（IV）抗CD45.2抗体标记能够精确追踪白细胞迁移。然而，标记窗口狭窄会使这种方法在追踪罕见迁移事件时具有挑战性。在这里，我们表明改变抗体给药途径和荧光团可以显著延长抗体活性标记时间。我们发现，虽然静脉注射和腹腔注射（IP）抗CD45.2抗体在注射后都能标记循环白细胞，但它们具有不同的动力学特性，影响标记时间和强度。循环抗体的定量分析表明，未结合的静脉注射抗CD45.2抗体迅速减少，而未结合的腹腔注射抗CD45.2抗体在1小时以上的时间里有所增加。通过体外和体内系列稀释试验，我们发现与其他荧光团相比，Alexa Fluor 647和亮蓝700（BB700）染料具有最大的标记灵敏度。然而，腹腔注射抗CD45.2 BB700抗体而非Alexa Fluor 647抗体，可使血液白细胞持续标记超过6小时。最后，我们利用腹腔注射抗CD45.2 BB700抗体追踪迁移较慢的白细胞进入肿瘤。我们发现，腹腔注射抗CD45.2抗体能够识别出约7倍数量的最近从血液迁移到肿瘤中的肿瘤特异性CD8 + T细胞。我们的结果表明不同的注射途径和荧光团如何影响抗CD45.2抗体对白细胞的标记，并突出了这种方法在稳态和癌症背景下定义白细胞迁移的实用性。
