Intravascular immune cell labeling as a critical methodological tool for characterizing CD4⁺ T cells in the lung in...

Growing evidence highlights the lung -brain axis as a key contributor to CNS autoimmunity. However, accurate characterization of lung immune cells remains technically challenging because standard transcardial perfusion does not eliminate the lung's extensive marginated intravascular leukocyte pool, allowing circulating cells to contaminate analyses of the lung parenchyma.

Experimental autoimmune encephalomyelitis (EAE) was induced in mice by immunization with MOG₃₅-₅₅ peptide in complete Freund's adjuvant, followed by pertussis toxin administration. Lung mononuclear cells were isolated after intravascular (IV) CD45 labeling, transcardial perfusion, and enzymatic digestion. Immune cell populations and intracellular cytokine production were analyzed by multicolor flow cytometry following ex vivo stimulation and intracellular staining.

We performed in vivo CD45 labeling by IV injection of a fluorochrome-conjugated anti-CD45 antibody immediately before euthanasia. Despite extensive transcardial perfusion, 73-85% of isolated lung leukocytes were IV-labeled, demonstrating substantial persistence of IV cells. In contrast, true lung parenchymal (IV^-) CD4⁺ T cells from both naïve and EAE mice exhibited a markedly enriched effector-memory phenotype, elevated expression of tissue residency-associated markers (CD69 and CD11a), and significantly greater production of IL-17, GM-CSF, and IFN-γ than circulating (IV⁺) cells. Notably, IV⁺ pulmonary vascular CD4⁺ T cells closely resembled peripheral blood CD4⁺ T cells but exhibited moderately increased expression of selected cytokines and transcription factors.

These findings demonstrate that transcardial perfusion alone is insufficient to accurately distinguish lung parenchymal from intravascular immune cells. We propose that in vivo intravascular labeling should be incorporated as a methodological standard for studies investigating pulmonary immune responses.