Enriching leukapheresis improves T cell activation and transduction efficiency during CAR T processing
Open accessAbstract
The majority of CD19-directed CAR T cell products are manufactured using an autologous process. Although using a patient's leukapheresis reduces the risks of rejection, it introduces variability in starting material composition and the presence of cell populations that might negatively affect production of chimeric antigen receptor (CAR) T cells, such as myeloid cells. In this work, the effect of monocytes (CD14) on the level of activation, growth, and transduction efficiency was monitored across well plate and culture bag platforms using healthy donor leukapheresis. Removal of monocytes from leukapheresis improved the level of activation 2-fold, achieving the same level of activation as when initiating the process with a purified T cell starting material. Two activation reagents were tested in well plate cultures, revealing differing sensitivities to starting material composition. Monocyte depletion in culture bag systems had a significant effect on transduction efficiency, improving consistency and increasing the level of CAR expression by up to 64% compared to unsorted leukapheresis. Cytotoxicity assays revealed that CAR T cell products produced from donor material depleted of monocytes and isolated T cells consistently outperformed those made from unsorted leukapheresis. Analysis of memory phenotypes and gene expression indicated that CAR T cells produced using depleted starting material displayed a more rested and naive state.
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The presence of MDSCs and monocytes can hamper the ex vivo activation and expansion of T cells, and other contaminants such as granulocytes and red blood cells suppress T cell proliferation.
“The presence of MDSCs and monocytes can hamper the ex vivo activation and expansion of T cells (40,42-44). Other cellular contaminants such as granulocytes and red blood cells suppress T cell proliferation (45,46).”
From Scalable Manufacturing of CAR T Cells for Cancer Immunotherapy (2021) · cites this paper as [44]
The Discussion (page unavailable) says monocytes can inhibit T-cell activation and expansion, and the Results report improved activation after monocyte removal in Dynabead-stimulated cultures. The source does not establish the claim about MDSCs. The separate sentence about granulocytes and red blood cells carries different citations, so it is not attributed to this source. (LLM, conf=0.86)
Experimental — accuracy not yet measuredSeveral ranges/measures exist to determine total T cell number via CD3 expression, characterize CD4+ and CD8+ subsets, detect transduced and not transduced cells, and identify contaminating cell subsets such as monocytes/macrophages (CD14).
“Several ranges/measures exist to determine the total T cell number via CD3 expression, to characterize the CD4+ and CD8+ subsets within this population (54), to detect transduced and not transduced cells, and to identify any contaminating cell subsets, such as monocytes/macrophages (CD14) (44).”
From Scalable Manufacturing of CAR T Cells for Cancer Immunotherapy (2021) · cites this paper as [44]
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- Authors
- Noaks, E., Peticone, C., Kotsopoulou, E., & Bracewell, D. G.
- Affiliations
- University College London (GB)
- Autolus (United Kingdom) (GB)
- Corresponding author
- Bracewell, Daniel G.
- Venue
- Molecular Therapy - Methods & Clinical Development
- Publisher
- Elsevier BV
- Published
- 2021-03
- Volume
- 20
- Pages
- 675-687
- ISSN
- 2329-0501 (ISSN-L)
- PMC
- PMC7917474
- OpenAlex
- W3128412377
- Cited by
- 62
- References
- 42
- Citations by year
- 2026: 9 · 2025: 12 · 2024: 16 · 2023: 14 · 2022: 6 · 2021: 4 · 2020: 1
- FWCI
- 3.64
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Molecular Therapy — Methods & Clinical Development (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- UCL Discovery (University College London) (submittedVersion)
- Funders
- EPSRC
- Topics
- Health Sciences › Medicine › Oncology › CAR-T cell therapy research
- Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
- Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
- Keywords
- CAR T cell manufacturing, leukapheresis, monocyte depletion, CD19 CAR, T cell activation, transduction efficiency, cytotoxicity, T cell memory phenotype, naive T cells
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