Measurement of leukocyte trafficking kinetics in macaques by serial intravascular staining

Abstract

Leukocyte trafficking enables detection of pathogens, immune responses, and immune memory. Dysregulation of leukocyte trafficking is often found in disease, highlighting its important role in homeostasis and the immune response. Whereas some of the molecular mechanisms mediating leukocyte trafficking are understood, little is known about the regulation of trafficking, including trafficking kinetics and its impact on immune homeostasis. We developed a method of serial intravascular staining (SIVS) to measure trafficking kinetics in nonhuman primates using infusions of fluorescently labeled antibodies to label circulating leukocytes. Because antibody infusions labeled only leukocytes in the blood, cells were “barcoded” according to their location at the time of each infusion, providing positional histories that could be used to infer trafficking kinetics. We used SIVS and multiparameter flow cytometry to quantitate cellular trafficking into lymphoid tissues of healthy animals at homeostasis and to identify perivascular cells that could be unique to nonlymphoid organs. To investigate how these parameters could be influenced during disease, SIVS was used to quantify lymphocyte trafficking in macaques infected with the bacterial pathogen Mycobacterium tuberculosis and to enumerate intravascular leukocytes in lung granulomas. We showed that whereas most cells in lung granulomas were localized there for more than 24 hours, granulomas were dynamic with a slow continual cellular influx, the rate of which predicted clearance of M. tuberculosis from the granulomas. SIVS, in combination with intracellular staining and multiparametric flow cytometry, is a powerful method to quantify the kinetics of leukocyte trafficking in nonhuman primates in vivo.

Mild SARS-CoV-2 infection in rhesus macaques is associated with viral control prior to antigen-specific T cell responses in tissues

“…Using intravenous (i.v.) antibody staining to distinguish between tissue parenchymal and intravascular cells [73][74][75] , we confirmed that most cells in the BAL, nasal turbinates, salivary gland, tonsils, and lymph nodes were from the tissue parenchyma (Fig S5). As expected for such a highly vascularized tissue, most cells from the lung tissue were intravascular stain positive, but a small population of CD69 + ivcells were detectable in the lungs confirming that tissue resident cells were also detected in pulmonary tissue.…”

Tissue Trafficking Kinetics of Rhesus Macaque Natural Killer Cells Measured by Serial Intravascular Staining

“…In our liver sample, most NK cells were IVas+, which was expected given that the liver contains primarily sinusoidal capillaries. The liver IVas+ cells had a distinct NK phenotype from blood cells in the same animal (Figure S4A), indicating that a large fraction of NK cells in liver may occupy a perivascular niche, as previously reported for T cells in the liver (38). Within our lung sample, the majority of DN and CD16 NK cells were IVas+, while the majority of CD56 NK cells were IVas-, indicating a possible difference in localization within lung tissue (Figure S4B).…”

“…The National Institutes of Health (NIH) and Bioqual Inc. (where some animals were housed) are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care and are in full compliance with the Animal Welfare Act and Public Health Service Policy on Humane Care and Use of Laboratory Animals. Sedation and euthanization followed previously described protocols ( 38 ).…”

“…Potter et al advanced this protocol by developing a method for serial intravascular staining (SIVS) in which non-human primates are given repeated injections of differently colored anti-CD45 antibodies ( 38 ). Antibodies are given at a sub-saturating dose with an in vivo half-life of 8.5 minutes; thus the labeling can be considered a pulse which instantly tags leukocytes in circulation at the time of infusion.…”

A virtual host model of Mycobacterium tuberculosis infection identifies early immune events as predictive of infection outcomes

“…Using our virtual population of 500 hosts, we are able to show that early events at both granuloma-and host-scales can be predictive of clinical-scale outcomes ~150 days later (at 200 days p.i.). These predictions are potentially useful for experimentalists, who can use analogous experimental techniques (such as serial intravascular staining (95), or IHC cytokine staining of granulomas ( 96)) to make educated predictions about downstream clinical-scale outcomes. Further, these HostSim predictions contribute to a growing body of evidence that suggests early immune events matter in TB (15,82,97).…”