Longitudinal Intravascular Antibody Labeling Identified Regulatory T Cell Recruitment as a Therapeutic Target in a Mouse Model of Lung Cancer.

Anticancer immunity is predicated on leukocyte migration into tumors. Once recruited, leukocytes undergo substantial reprogramming to adapt to the tumor microenvironment. A major challenge in the field is distinguishing recently recruited from resident leukocytes in tumors. In this study, we developed an intravascular Ab technique to label circulating mouse leukocytes before they migrate to tissues, providing unprecedented insight into the kinetics of recruitment. This approach unveiled the substantial role of leukocyte migration in tumor progression using a preclinical mouse model of lung adenocarcinoma. Regulatory T cells (Tregs), critical mediators of immunosuppression, were continuously and rapidly recruited into tumors throughout cancer progression. Moreover, leukocyte trafficking depended on the integrins CD11a/CD49d, and CD11a/CD49d blockade led to significant tumor burden reduction in mice. Importantly, preventing circulating Treg recruitment through depletion or sequestration in lymph nodes was sufficient to decrease tumor burden, indicating that Treg migration was crucial for suppressing antitumor immunity. These findings underscore the dynamic nature of the immune compartment within mouse lung tumors and demonstrate the relevance of a temporal map of leukocyte recruitment into tumors, thereby advancing our understanding of leukocyte migration in the context of tumor development.

In vivo antibody labeling route and fluorophore dictate labeling efficiency, sensitivity, and longevity.

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 one hour. Using in vitro and in vivo serial dilution assays, we found that Alexa Fluor 647 (AF647) and Brilliant Blue 700 (BB700) dyes had the greatest labeling sensitivity compared to other fluorophores. However, IP antibody injection with anti-CD45.2 BB700, but not AF647, resulted in continuous blood leukocyte labeling for over 6 hours. 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 ~seven 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.