{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T20:52:12Z","timestamp":1790628732450,"version":"4.1.0"},"reference-count":35,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,5,22]],"date-time":"2026-05-22T00:00:00Z","timestamp":1779408000000},"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":"<jats:p>\n                    Gamma delta (\u03b3\u03b4) T cells are promising candidates for adoptive immunotherapy due to their unique biological and safety profiles. In particular, ex vivo expanded V\u03b39V\u03b42 T cells engineered with chimeric antigen receptors (CARs) have gained considerable interest, as they combine potent innate-like antitumor activity with a low risk of graft-versus-host disease and cytokine release syndrome, making them ideal for off-the-shelf cancer immunotherapies. However, as they represent only 1\u20135% of peripheral T cells, effective activation and expansion protocols are essential to generate sufficient numbers for clinical use. In this study, we describe an automated and highly efficient process for the expansion of V\u03b39V\u03b42 T cells and the manufacturing of CAR \u03b3\u03b4 T cells using the CliniMACS Prodigy closed system. This platform integrates cell separation, activation, genetic modification and expansion ensuring reproducibility and compliance with GMP standards. Starting with an alpha-beta (\u03b1\u03b2) T cell and B cell magnetic depletion, we achieved a 4.59-log \u03b1\u03b2T cell depletion and a 3.97-log B cell depletion. By carefully optimizing activation, transduction and expansion, we were able to achieve up to 374-fold increase in V\u03b39V\u03b42 T cells, resulting in an average number of 6.64 x 10\n                    <jats:sup>9<\/jats:sup>\n                    (4.76\u00d710\n                    <jats:sup>9<\/jats:sup>\n                    \u2013 8.98\u00d710\n                    <jats:sup>9<\/jats:sup>\n                    ) cells after 14 days of culture using the TCT-LS process. Using a GMP-grade lentiviral vector pseudotyped with the envelope protein from baboon endogenous virus (BaEV) encoding a CD123 chimeric antigen receptor (CAR) at a multiplicity of infection (MOI) of 0.12, we achieved a mean \u03b3\u03b4 T cell transduction efficiency of 57.4%. Flow cytometry analysis of the final cell product showed an average composition of 89.83% \u03b3\u03b4 T cells, 9.82% NK cells, and 0.012% residual \u03b1\u03b2 T cells. The low average vector copy number (VCN) of 1.4 underscores the safety and efficiency of the transduction protocol. Functional assays against a leukemic cell line confirmed robust anti-tumor activity of CAR-\u03b3\u03b4 T cells produced using this platform, demonstrating the feasibility of large-scale manufacturing and highlighting their potential for future clinical immunotherapy applications.\n                  <\/jats:p>","DOI":"10.3389\/fimmu.2026.1779035","type":"journal-article","created":{"date-parts":[[2026,5,22]],"date-time":"2026-05-22T06:08:31Z","timestamp":1779430111000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["A clinical-grade automated platform for the manufacturing of CAR-\u03b3\u03b4 T cells for immunotherapy"],"prefix":"10.3389","volume":"17","author":[{"given":"Jan","family":"Kuska","sequence":"first","affiliation":[{"name":"Research and Development, Miltenyi Biotec B.V. & Co. KG","place":["Bergisch Gladbach, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julia","family":"Kostyra","sequence":"additional","affiliation":[{"name":"Research and Development, Miltenyi Biotec B.V. & Co. KG","place":["Bergisch Gladbach, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lorraine","family":"Pinot","sequence":"additional","affiliation":[{"name":"Research and Development, Miltenyi Biotec B.V. & Co. KG","place":["Bergisch Gladbach, Germany"]},{"name":"Department of Immunology, Institute for Cell Biology, University of T\u00fcbingen","place":["T\u00fcbingen, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Evgeny","family":"Egorov","sequence":"additional","affiliation":[{"name":"Research and Development, Miltenyi Biotec B.V. & Co. 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KG","place":["Bergisch Gladbach, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mario","family":"Assenmacher","sequence":"additional","affiliation":[{"name":"Research and Development, Miltenyi Biotec B.V. & Co. KG","place":["Bergisch Gladbach, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rimas","family":"Orentas","sequence":"additional","affiliation":[{"name":"Research and Development, Miltenyi Biotec B.V. & Co. KG","place":["Bergisch Gladbach, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sabine","family":"Mueller","sequence":"additional","affiliation":[{"name":"Research and Development, Miltenyi Biotec B.V. & Co. KG","place":["Bergisch Gladbach, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jos\u00e9 Villacorta","family":"Hidalgo","sequence":"additional","affiliation":[{"name":"Research and Development, Miltenyi Biotec B.V. & Co. 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