{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T11:10:01Z","timestamp":1788952201107,"version":"build-2803163510"},"reference-count":21,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T00:00:00Z","timestamp":1788912000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"\n Introduction<\/jats:title>\n 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\u2019s extensive marginated intravascular leukocyte pool, allowing circulating cells to contaminate analyses of the lung parenchyma.<\/jats:p>\n <\/jats:sec>\n \n Methods<\/jats:title>\n \n Experimental autoimmune encephalomyelitis (EAE) was induced in mice by immunization with MOG\n 35<\/jats:sub>\n -\n 55<\/jats:sub>\n peptide in complete Freund\u2019s 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.\n <\/jats:p>\n <\/jats:sec>\n \n Results<\/jats:title>\n \n We performed\n in vivo<\/jats:italic>\n CD45 labeling by IV injection of a fluorochrome-conjugated anti-CD45 antibody immediately before euthanasia. Despite extensive transcardial perfusion, 73\u201385% of isolated lung leukocytes were IV-labeled, demonstrating substantial persistence of IV cells. In contrast, true lung parenchymal (IV\n -<\/jats:sup>\n ) CD4\n +<\/jats:sup>\n T cells from both na\u00efve 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-\u03b3 than circulating (IV\n +<\/jats:sup>\n ) cells. Notably, IV\n +<\/jats:sup>\n pulmonary vascular CD4\n +<\/jats:sup>\n T cells closely resembled peripheral blood CD4\n +<\/jats:sup>\n T cells but exhibited moderately increased expression of selected cytokines and transcription factors.\n <\/jats:p>\n <\/jats:sec>\n \n Discussion<\/jats:title>\n \n These findings demonstrate that transcardial perfusion alone is insufficient to accurately distinguish lung parenchymal from intravascular immune cells. We propose that\n in vivo<\/jats:italic>\n intravascular labeling should be incorporated as a methodological standard for studies investigating pulmonary immune responses.\n <\/jats:p>\n <\/jats:sec>","DOI":"10.3389\/fimmu.2026.1950269","type":"journal-article","created":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T10:15:44Z","timestamp":1788948944000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Intravascular immune cell labeling as a critical methodological tool for characterizing CD4+ T cells in the lung in EAE"],"prefix":"10.3389","volume":"17","author":[{"given":"Gholamreza","family":"Azizi","sequence":"first","affiliation":[{"name":"Department of Neurology, Thomas Jefferson University","place":["Philadelphia, PA, United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abdolmohamad","family":"Rostami","sequence":"additional","affiliation":[{"name":"Department of Neurology, 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