{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T04:16:12Z","timestamp":1787026572782,"version":"build-2736575974"},"reference-count":30,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T00:00:00Z","timestamp":1759881600000},"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 \n Chronic lymphocytic leukemia (CLL) has proven difficult to treat with chimeric antigen receptor (CAR) T cell therapy. CLL cells can negatively alter T cell fitness and induce a pseudohypoxic state. We hypothesized that production of CAR T cells under restricted oxygen conditions resembling physiological oxygen levels that can be encountered in tissues (\n i.e.<\/jats:italic>\n 2% O\n 2<\/jats:sub>\n ) could promote outgrowth of hypoxia-tolerant CAR T cells.\n <\/jats:p>\n <\/jats:sec>\n \n Methods<\/jats:title>\n \n We performed\n in vitro<\/jats:italic>\n phenotypic and functional assessments of CD19-directed CAR T cells produced in either 21% (\n Nor<\/jats:sup>\n CAR) or 2% (\n Hyp<\/jats:sup>\n CAR) O\n 2<\/jats:sub>\n derived from healthy donors (HDs) or patients with CLL.\n <\/jats:p>\n <\/jats:sec>\n \n Results<\/jats:title>\n \n Production of HD-derived CAR T cells in 2% O\n 2<\/jats:sub>\n promoted the enrichment of a na\u00efve-like subset.\n Hyp<\/jats:sup>\n CAR and\n Nor<\/jats:sup>\n CAR cells were functionally distinct; CD4+\n Hyp<\/jats:sup>\n CAR cells produced more IL-2 and tumor necrosis factor than CD4+\n Nor<\/jats:sup>\n CAR cells. Production in 2% O\n 2<\/jats:sub>\n was not detrimental to viability or proliferation upon cognate antigen-stimulation and led to increased activation. After chronic stimulation in hypoxia,\n Hyp<\/jats:sup>\n CAR-product remained enriched in na\u00efve-like cells, and demonstrated cytotoxic and cytokine production capacity. In CAR T cells derived from patients with CLL,\n Nor<\/jats:sup>\n CAR and\n Hyp<\/jats:sup>\n CAR subsets were functionally and phenotypically comparable, but displayed different mitochondrial metabolism.\n <\/jats:p>\n <\/jats:sec>\n \n Discussion<\/jats:title>\n \n We demonstrated that production in 2% O\n 2<\/jats:sub>\n is not detrimental, confers subtle but lasting functional and phenotypic changes in CAR T cells warranting further research on the impact of hypoxic production on CAR T cell functionality in hypoxic tumor microenvironments.\n <\/jats:p>\n <\/jats:sec>","DOI":"10.3389\/fimmu.2025.1675786","type":"journal-article","created":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T05:40:45Z","timestamp":1759902045000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":2,"title":["Production of functional CD19 CAR T cells under hypoxic manufacturing 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