Unveiling the influence of CAR-negative T-cells: enhancing efficacy and ensuring safety in CAR-T therapies
Abstract Background Despite the promising results of CAR-T cell therapy, still a significant proportion of patients experience treatment failure due to absence of response, progression or treatment-related toxicities. Our research aims to investigate how the CAR-T cell product characteristics and pa...
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BMC
2025-08-01
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| Series: | Journal of Translational Medicine |
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| Online Access: | https://doi.org/10.1186/s12967-025-06899-0 |
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| author | Belén Sierro-Martínez Beatriz Guijarro-Albaladejo Ricardo Fernández-Cisnal Alfonso Rodríguez-Gil Paola Hernández-Díaz María de la Rosa-Garrido Inmaculada Carrasco-Brocal Maribel Lara-Chica Virginia Escamilla-Gómez Raquel Muñoz-García Ángela Bella Francisco Jose García-Cózar Jose Antonio Pérez-Simón Estefanía García-Guerrero |
| author_facet | Belén Sierro-Martínez Beatriz Guijarro-Albaladejo Ricardo Fernández-Cisnal Alfonso Rodríguez-Gil Paola Hernández-Díaz María de la Rosa-Garrido Inmaculada Carrasco-Brocal Maribel Lara-Chica Virginia Escamilla-Gómez Raquel Muñoz-García Ángela Bella Francisco Jose García-Cózar Jose Antonio Pérez-Simón Estefanía García-Guerrero |
| author_sort | Belén Sierro-Martínez |
| collection | DOAJ |
| description | Abstract Background Despite the promising results of CAR-T cell therapy, still a significant proportion of patients experience treatment failure due to absence of response, progression or treatment-related toxicities. Our research aims to investigate how the CAR-T cell product characteristics and particularly the percentage of CAR-negative cells present in the infusion product affects its efficacy and safety. Methods CAR-positive (CAR-pos) and CAR-negative (CAR-neg) cells were analyzed during in vitro expansion with IL-2 or IL-7/IL-15. FACS-sorted populations were assessed for cytotoxic effect and secretion of cytokines. Transcriptomic analyses were conducted for CAR-pos and CAR-neg populations. Results A significant reduction in the percentage of transduced (CAR-pos) T cells was observed throughout the in vitro cell culture, where CAR-neg T cells displayed greater expansion along the procedure compared to CAR-pos T cells, using either IL-2 or IL-7/IL-15. CAR-pos T cells exhibited increased expression of activation and exhaustion markers. Comparative analyses of CAR-pos and CAR-neg showed an increased cytotoxic effect in purified samples with 100% CAR-pos T cells compared to non-purified product. Quantification of cytokine levels revealed a significant decrease in IL-6 in purified compared to non-purified products. RNA sequencing analysis revealed significant differential gene expression profile between CAR-pos and CAR-neg cells, the prior exhibiting an upregulation of genes associated with immune checkpoint regulation, cytokine-cytokine receptor interaction and Th1/Th2 and Th17 cell differentiation pathways. Conclusion CAR-neg T cells exhibit a significantly greater expansion compared to CAR-pos T cells alongside CAR-T cell production. The percentage of CAR-neg cells influences the specific cytotoxicity of the product and on the cytokine pattern, which might influence both efficacy and toxicity and, therefore, the number of CAR-neg T cells within the infusion product must be considered in the clinical setting. Purification of CAR-pos T cells could potentially optimize product characteristics. |
| format | Article |
| id | doaj-art-0c37a21d4dab4d9ea8e61753bbfb8772 |
| institution | Kabale University |
| issn | 1479-5876 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Translational Medicine |
| spelling | doaj-art-0c37a21d4dab4d9ea8e61753bbfb87722025-08-24T11:48:00ZengBMCJournal of Translational Medicine1479-58762025-08-0123111410.1186/s12967-025-06899-0Unveiling the influence of CAR-negative T-cells: enhancing efficacy and ensuring safety in CAR-T therapiesBelén Sierro-Martínez0Beatriz Guijarro-Albaladejo1Ricardo Fernández-Cisnal2Alfonso Rodríguez-Gil3Paola Hernández-Díaz4María de la Rosa-Garrido5Inmaculada Carrasco-Brocal6Maribel Lara-Chica7Virginia Escamilla-Gómez8Raquel Muñoz-García9Ángela Bella10Francisco Jose García-Cózar11Jose Antonio Pérez-Simón12Estefanía García-Guerrero13Instituto de Biomedicina de Sevilla, IBiS/Hospital Universitario Virgen del Rocío/CSIC/Universidad de SevillaInstituto de Biomedicina de Sevilla, IBiS/Hospital Universitario Virgen del Rocío/CSIC/Universidad de SevillaInstituto de Investigación E Innovación Biomédica (INiBICA)Instituto de Biomedicina de Sevilla, IBiS/Hospital Universitario Virgen del Rocío/CSIC/Universidad de SevillaInstituto de Biomedicina de Sevilla, IBiS/Hospital Universitario Virgen del Rocío/CSIC/Universidad de SevillaInstituto de Biomedicina de Sevilla, IBiS/Hospital Universitario Virgen del Rocío/CSIC/Universidad de SevillaInstituto de Biomedicina de Sevilla, IBiS/Hospital Universitario Virgen del Rocío/CSIC/Universidad de SevillaInstituto de Biomedicina de Sevilla, IBiS/Hospital Universitario Virgen del Rocío/CSIC/Universidad de SevillaInstituto de Biomedicina de Sevilla, IBiS/Hospital Universitario Virgen del Rocío/CSIC/Universidad de SevillaInstituto de Biomedicina de Sevilla, IBiS/Hospital Universitario Virgen del Rocío/CSIC/Universidad de SevillaInstituto de Biomedicina de Sevilla, IBiS/Hospital Universitario Virgen del Rocío/CSIC/Universidad de SevillaInstituto de Investigación E Innovación Biomédica (INiBICA)Instituto de Biomedicina de Sevilla, IBiS/Hospital Universitario Virgen del Rocío/CSIC/Universidad de SevillaInstituto de Biomedicina de Sevilla, IBiS/Hospital Universitario Virgen del Rocío/CSIC/Universidad de SevillaAbstract Background Despite the promising results of CAR-T cell therapy, still a significant proportion of patients experience treatment failure due to absence of response, progression or treatment-related toxicities. Our research aims to investigate how the CAR-T cell product characteristics and particularly the percentage of CAR-negative cells present in the infusion product affects its efficacy and safety. Methods CAR-positive (CAR-pos) and CAR-negative (CAR-neg) cells were analyzed during in vitro expansion with IL-2 or IL-7/IL-15. FACS-sorted populations were assessed for cytotoxic effect and secretion of cytokines. Transcriptomic analyses were conducted for CAR-pos and CAR-neg populations. Results A significant reduction in the percentage of transduced (CAR-pos) T cells was observed throughout the in vitro cell culture, where CAR-neg T cells displayed greater expansion along the procedure compared to CAR-pos T cells, using either IL-2 or IL-7/IL-15. CAR-pos T cells exhibited increased expression of activation and exhaustion markers. Comparative analyses of CAR-pos and CAR-neg showed an increased cytotoxic effect in purified samples with 100% CAR-pos T cells compared to non-purified product. Quantification of cytokine levels revealed a significant decrease in IL-6 in purified compared to non-purified products. RNA sequencing analysis revealed significant differential gene expression profile between CAR-pos and CAR-neg cells, the prior exhibiting an upregulation of genes associated with immune checkpoint regulation, cytokine-cytokine receptor interaction and Th1/Th2 and Th17 cell differentiation pathways. Conclusion CAR-neg T cells exhibit a significantly greater expansion compared to CAR-pos T cells alongside CAR-T cell production. The percentage of CAR-neg cells influences the specific cytotoxicity of the product and on the cytokine pattern, which might influence both efficacy and toxicity and, therefore, the number of CAR-neg T cells within the infusion product must be considered in the clinical setting. Purification of CAR-pos T cells could potentially optimize product characteristics.https://doi.org/10.1186/s12967-025-06899-0CAR-TFACS-sortingCAR-negativeRNA-seqICANSCRS |
| spellingShingle | Belén Sierro-Martínez Beatriz Guijarro-Albaladejo Ricardo Fernández-Cisnal Alfonso Rodríguez-Gil Paola Hernández-Díaz María de la Rosa-Garrido Inmaculada Carrasco-Brocal Maribel Lara-Chica Virginia Escamilla-Gómez Raquel Muñoz-García Ángela Bella Francisco Jose García-Cózar Jose Antonio Pérez-Simón Estefanía García-Guerrero Unveiling the influence of CAR-negative T-cells: enhancing efficacy and ensuring safety in CAR-T therapies Journal of Translational Medicine CAR-T FACS-sorting CAR-negative RNA-seq ICANS CRS |
| title | Unveiling the influence of CAR-negative T-cells: enhancing efficacy and ensuring safety in CAR-T therapies |
| title_full | Unveiling the influence of CAR-negative T-cells: enhancing efficacy and ensuring safety in CAR-T therapies |
| title_fullStr | Unveiling the influence of CAR-negative T-cells: enhancing efficacy and ensuring safety in CAR-T therapies |
| title_full_unstemmed | Unveiling the influence of CAR-negative T-cells: enhancing efficacy and ensuring safety in CAR-T therapies |
| title_short | Unveiling the influence of CAR-negative T-cells: enhancing efficacy and ensuring safety in CAR-T therapies |
| title_sort | unveiling the influence of car negative t cells enhancing efficacy and ensuring safety in car t therapies |
| topic | CAR-T FACS-sorting CAR-negative RNA-seq ICANS CRS |
| url | https://doi.org/10.1186/s12967-025-06899-0 |
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