{
  "abstract": "CAR-T cell therapy has transformed the treatment of hematologic malignancies, but many patients relapse or fail to respond, and efficacy against solid tumors remains limited. A contributing factor may be the conventional manufacturing process, which depends on strong T-cell activation via anti-CD3/anti-CD28 stimulation prior to gene transfer. While this boosts proliferation and lentiviral transduction efficiency, it also drives differentiation and metabolic remodeling, potentially compromising long-term persistence in vivo. We hypothesized that culturing T cells with IL-7 and IL-15, in the absence of TCR-mediated activation, would sustain a less-differentiated phenotype and greater metabolic fitness while still enabling transduction. Preliminary experiments identified 5 days of IL-7/IL-15 culture as the optimal window for transduction, guiding subsequent cell-cycle, phenotypic, and metabolic characterization. Peripheral Blood Mononuclear Cells (PBMCs) were collected from four healthy platelet donors on day 0 (D0). Non-activated T cells were isolated by negative selection and cultured with IL-7/IL-15 without TCR stimulation. In parallel, activated PBMCs underwent overnight monocyte depletion followed by anti-CD3/anti-CD28 bead stimulation on D1. Both groups were maintained until D6, with sampling at D1, D4, and D6 for flow cytometry (cell cycle: Hoechst/Pyronin Y; proliferation: CFSE; differentiation: CD45RA/CD62L/CD95; activation: CD69/CD25; mitochondrial potential: MitoTracker) and metabolic profiling (Seahorse XF T Cell Metabolic Profiling Kit) at D6. The two groups progressed through the cell cycle from D1 to D6, but activated cells exited quiescence (G0) more markedly and proliferated more extensively, as shown by lower CFSE retention. TCR activation increased effector memory T cells (TEM) among CD3+/CD8+ populations and drove progressive CD69/CD25 upregulation, whereas non-activated cells remained phenotypically stable and predominantly CD69⁻CD25⁻ throughout culture. Non-activated cells retained a higher proportion of functionally intact mitochondria across the culture period, while activated cells showed a transient decline at D4. Seahorse analysis revealed a shift toward a more energetic, glycolysis-driven metabolic profile in activated cells (increased ECAR), whereas non-activated T cells maintained mitochondrial respiratory capacity comparable to activated cells, as reflected by similar spare respiratory capacity between groups. These findings show that IL-7/IL-15 culture alone is sufficient to release T cells from quiescence (G0), a state required for lentiviral transduction, while preserving a less-differentiated phenotype and mitochondrial respiratory capacity on par with activated cells. Although TCR-mediated activation promoted greater proliferation, it was also linked to transient mitochondrial alterations. The IL-7/IL-15-based strategy therefore represents a promising alternative for CAR-T cell manufacturing, enabling efficient gene transfer while preserving the functional fitness, persistence potential, and therapeutic efficacy of the final product. This work was supported by CAPES, CNPq, and FAPESP (2025/11080-2).",
  "abstract_shown": true,
  "affiliations": [
    "Hemocentro de Ribeirão Preto, Faculdade de Medicina de Ribeirao Preto (FMRP), Universidade de São Paulo (USP), São Paulo, SP, Brazil"
  ],
  "article_type": null,
  "arxiv": null,
  "author_total": 7,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "Millena BRANDÃO"
    },
    {
      "affs": [
        0
      ],
      "name": "Izadora Peter FURTADO"
    },
    {
      "affs": [
        0
      ],
      "name": "Matheus Henrique DOS SANTOS"
    },
    {
      "affs": [
        0
      ],
      "name": "Virgínia PICANÇO-CASTRO"
    },
    {
      "affs": [
        0
      ],
      "name": "Rodrigo Tocantins Calado Saloma RODRIGUES"
    },
    {
      "affs": [
        0
      ],
      "name": "Lucas Eduardo Botelho SOUZA"
    },
    {
      "affs": [
        0
      ],
      "name": "Diego Villa CLÉ"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/b7ey501vbd1ad.bib",
  "cited_by": 0,
  "doi": "10.1016/j.htct.2026.106876",
  "fulltext_url": "http://www.sciencedirect.com/science/article/pii/S2531137926006231",
  "id": "b7ey501vbd1ad",
  "issns": [
    "2531-1379",
    "2531-1387"
  ],
  "landing_url": "https://doi.org/10.1016/j.htct.2026.106876",
  "language": "en",
  "licence": "CC-BY",
  "licence_label": "CC BY",
  "licence_source": "journal",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "lcc",
      "RC633-647.5"
    ],
    [
      "openalex",
      "2730|CAR-T cell therapy research"
    ]
  ],
  "openalex": "W7223096344",
  "pdf_url": null,
  "pmcid": null,
  "pmid": null,
  "published": "2026-10-23",
  "publisher": "Elsevier",
  "ris": "https://research.draconitic.com/paper/b7ey501vbd1ad.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T13:43:29Z",
      "native_id": "fbb472a4b65746af9b17a44f17e0f3e6",
      "source": "doaj",
      "url": "https://doi.org/10.1016/j.htct.2026.106876"
    }
  ],
  "subjects": [
    {
      "field": "medicine",
      "origin": "openalex:2730|CAR-T cell therapy research",
      "subfield": "oncology"
    }
  ],
  "title": "IL-7/IL-15 CULTURE RELEASES T CELLS FROM QUIESCENCE WITHOUT TCR ACTIVATION WHILE PRESERVING A LESS-DIFFERENTIATED, METABOLICALLY FIT PHENOTYPE FOR CAR-T MANUFACTURING",
  "title_text": "IL-7/IL-15 CULTURE RELEASES T CELLS FROM QUIESCENCE WITHOUT TCR ACTIVATION WHILE PRESERVING A LESS-DIFFERENTIATED, METABOLICALLY FIT PHENOTYPE FOR CAR-T MANUFACTURING",
  "type": "peer-reviewed",
  "updated_at": "2026-10-30T13:43:29Z",
  "url": "https://research.draconitic.com/paper/b7ey501vbd1ad",
  "venue": "Hematology, Transfusion and Cell Therapy",
  "versions": []
}