The CD64/CD28/CD3ζ chimeric receptor reprograms T-cell metabolism and promotes T-cell persistence and immune functions while triggering antibody-independent and antibody-dependent cytotoxicity
Abstract Background Recent studies have shown that CD32/CD8a/CD28/CD3ζ chimeric receptor cells directly kill breast cancer cells, suggesting the existence of cell surface myeloid FcγR alternative ligands (ALs). Here, we investigated the metabolism, ALs, cytotoxicity, and immunoregulatory functions o...
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2025-02-01
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| Series: | Experimental Hematology & Oncology |
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| Online Access: | https://doi.org/10.1186/s40164-025-00601-2 |
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| author | Sara Caratelli Francesca De Paolis Domenico Alessandro Silvestris Silvia Baldari Illari Salvatori Apollonia Tullo Giulia Lanzilli Aymone Gurtner Alberto Ferri Cristiana Valle Simona Padovani Valeriana Cesarini Tommaso Sconocchia Loredana Cifaldi Roberto Arriga Giulio Cesare Spagnoli Soldano Ferrone Adriano Venditti Piero Rossi Graziano Pesole Gabriele Toietta Giuseppe Sconocchia |
| author_facet | Sara Caratelli Francesca De Paolis Domenico Alessandro Silvestris Silvia Baldari Illari Salvatori Apollonia Tullo Giulia Lanzilli Aymone Gurtner Alberto Ferri Cristiana Valle Simona Padovani Valeriana Cesarini Tommaso Sconocchia Loredana Cifaldi Roberto Arriga Giulio Cesare Spagnoli Soldano Ferrone Adriano Venditti Piero Rossi Graziano Pesole Gabriele Toietta Giuseppe Sconocchia |
| author_sort | Sara Caratelli |
| collection | DOAJ |
| description | Abstract Background Recent studies have shown that CD32/CD8a/CD28/CD3ζ chimeric receptor cells directly kill breast cancer cells, suggesting the existence of cell surface myeloid FcγR alternative ligands (ALs). Here, we investigated the metabolism, ALs, cytotoxicity, and immunoregulatory functions of CD64/CD28/CD3ζ in colorectal cancer (CRC) and squamous cell carcinoma of the head and neck. Methods The CD64/CD28/CD3ζ -SFG retroviral vector was used to produce viruses for T-cell transduction. T-cell expansion and differentiation were monitored via flow cytometry. Gene expression was assessed by RNA-seq. Bioenergetics were documented on a Seahorse extracellular flux analyzer. CD64/CD28/CD3ζ polarization was identified via confocal microscopy. Cytotoxicity was determined by MTT assay and bioluminescent imaging, and flow cytometry. Tridimensional antitumor activity of CD64/CD28/CD3ζ T cells was achieved by utilizing HCT116-GFP 3D spheroids via the IncuCyte S3 Live-Cell Analysis system. The intraperitoneal distribution and antitumor activity of NIR-CD64/CD28/CD3ζ and NIR-nontransduced T cells were investigated in CB17-SCID mice bearing subcutaneous FaDu Luc + cells by bioluminescent and fluorescent imaging. IFNγ was assessed by ELISA. Results Compared to CD16/CD8a/CD28/CD3ζ T cells, CD32/CD8a/CD28/CD3ζ T cells, and non-transduced T cells, CD64/CD28/CD3ζ T cells exhibited the highest levels of cell expansion and persistence capacity. A total of 235 genes linked to cell division and 52 genes related to glycolysis were overexpressed. The glycolytic phenotype was confirmed by functional in vitro studies accompanied by preferential T-cell effector memory differentiation. Interestingly, oxamic acid was found to inhibit CD64-CR T cell proliferation, indicating the involvement of lactate. Upon CD64/CD28/CD3ζ T-cell conjugation with CRC cells, CD64/CD28/CD3ζ cells polarize at immunological synapses, leading to CRC cell death. CD64/CD28/CD3ζ T cells kill SCCHN cells, and in combination with the anti-B7-H3 mAb (376.96) or anti-EGFR mAb, these cells trigger antibody-dependent cellular cytotoxicity (ADCC) in vitro under 2D and 3D conditions. The 376.96 mAb combined with CD64/CD28/CD3ζ T cells had anti-SCCHN activity in vivo. In addition, they induce the upregulation of PD-L1 and HLA-DR expression in cancer cells via IFNγ. PD-L1 positive SCCHN cells in combination with anti-PD-L1 mAb and CD64-CR T cells were killed by ADCC, which enhanced direct cytotoxicity. These findings indicate that the glycolytic phenotype is involved in CD64-CR T cell proliferation/expansion. These cells mediate long-lasting HLA-independent cytotoxicity and ADCC in CRC and SCCHN cells. Conclusions CD64/CD28/CD3ζ T cells could significantly impact the rational design of personalized studies to treat CRC and SCCHN and the identification of novel FcγR ALs in cancer and healthy cells. |
| format | Article |
| id | doaj-art-0d855708871d4e91a497fc9199679d4a |
| institution | OA Journals |
| issn | 2162-3619 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | BMC |
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| series | Experimental Hematology & Oncology |
| spelling | doaj-art-0d855708871d4e91a497fc9199679d4a2025-08-20T02:15:07ZengBMCExperimental Hematology & Oncology2162-36192025-02-0114112110.1186/s40164-025-00601-2The CD64/CD28/CD3ζ chimeric receptor reprograms T-cell metabolism and promotes T-cell persistence and immune functions while triggering antibody-independent and antibody-dependent cytotoxicitySara Caratelli0Francesca De Paolis1Domenico Alessandro Silvestris2Silvia Baldari3Illari Salvatori4Apollonia Tullo5Giulia Lanzilli6Aymone Gurtner7Alberto Ferri8Cristiana Valle9Simona Padovani10Valeriana Cesarini11Tommaso Sconocchia12Loredana Cifaldi13Roberto Arriga14Giulio Cesare Spagnoli15Soldano Ferrone16Adriano Venditti17Piero Rossi18Graziano Pesole19Gabriele Toietta20Giuseppe Sconocchia21Department of Biomedicine, Institute of Translational Pharmacology, Italian National Research Council (CNR)Department of Biomedicine, Institute of Translational Pharmacology, Italian National Research Council (CNR)Dipartimento di Bioscienze, Biotecnologie e Ambiente, University of BariTumor Immunology and Immunotherapy Unit, IRCCS Regina Elena National Cancer InstituteIRCCS Fondazione Santa LuciaDepartment of Biomedicine, Institute of Biomembranes Bioenergetics and Molecular Biotechnologies, National Research Council (CNR)Department of Biomedicine, Institute of Translational Pharmacology, Italian National Research Council (CNR)Department of Biomedicine, Institute of Translational Pharmacology, Italian National Research Council (CNR)Department of Biomedicine, Institute of Translational Pharmacology, Italian National Research Council (CNR)Department of Biomedicine, Institute of Translational Pharmacology, Italian National Research Council (CNR)Department of Biomedicine, Institute of Translational Pharmacology, Italian National Research Council (CNR)Department of Biomedicine, Institute of Translational Pharmacology, Italian National Research Council (CNR)San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCCS San Raffaele Scientific InstituteDepartment of Clinical Sciences and Translational Medicine, University of Rome “Tor Vergata”Department of Systems Medicine, University of Rome “Tor Vergata”Department of Biomedicine, Institute of Translational Pharmacology, Italian National Research Council (CNR)Department of Surgery, Massachusetts General Hospital, Harvard Medical SchoolDepartment of Biomedicine and Prevention, University of Rome “Tor Vergata”Department of Experimental Medicine and Surgery, University of Rome “Tor Vergata”Dipartimento di Bioscienze, Biotecnologie e Ambiente, University of BariTumor Immunology and Immunotherapy Unit, IRCCS Regina Elena National Cancer InstituteDepartment of Biomedicine, Institute of Translational Pharmacology, Italian National Research Council (CNR)Abstract Background Recent studies have shown that CD32/CD8a/CD28/CD3ζ chimeric receptor cells directly kill breast cancer cells, suggesting the existence of cell surface myeloid FcγR alternative ligands (ALs). Here, we investigated the metabolism, ALs, cytotoxicity, and immunoregulatory functions of CD64/CD28/CD3ζ in colorectal cancer (CRC) and squamous cell carcinoma of the head and neck. Methods The CD64/CD28/CD3ζ -SFG retroviral vector was used to produce viruses for T-cell transduction. T-cell expansion and differentiation were monitored via flow cytometry. Gene expression was assessed by RNA-seq. Bioenergetics were documented on a Seahorse extracellular flux analyzer. CD64/CD28/CD3ζ polarization was identified via confocal microscopy. Cytotoxicity was determined by MTT assay and bioluminescent imaging, and flow cytometry. Tridimensional antitumor activity of CD64/CD28/CD3ζ T cells was achieved by utilizing HCT116-GFP 3D spheroids via the IncuCyte S3 Live-Cell Analysis system. The intraperitoneal distribution and antitumor activity of NIR-CD64/CD28/CD3ζ and NIR-nontransduced T cells were investigated in CB17-SCID mice bearing subcutaneous FaDu Luc + cells by bioluminescent and fluorescent imaging. IFNγ was assessed by ELISA. Results Compared to CD16/CD8a/CD28/CD3ζ T cells, CD32/CD8a/CD28/CD3ζ T cells, and non-transduced T cells, CD64/CD28/CD3ζ T cells exhibited the highest levels of cell expansion and persistence capacity. A total of 235 genes linked to cell division and 52 genes related to glycolysis were overexpressed. The glycolytic phenotype was confirmed by functional in vitro studies accompanied by preferential T-cell effector memory differentiation. Interestingly, oxamic acid was found to inhibit CD64-CR T cell proliferation, indicating the involvement of lactate. Upon CD64/CD28/CD3ζ T-cell conjugation with CRC cells, CD64/CD28/CD3ζ cells polarize at immunological synapses, leading to CRC cell death. CD64/CD28/CD3ζ T cells kill SCCHN cells, and in combination with the anti-B7-H3 mAb (376.96) or anti-EGFR mAb, these cells trigger antibody-dependent cellular cytotoxicity (ADCC) in vitro under 2D and 3D conditions. The 376.96 mAb combined with CD64/CD28/CD3ζ T cells had anti-SCCHN activity in vivo. In addition, they induce the upregulation of PD-L1 and HLA-DR expression in cancer cells via IFNγ. PD-L1 positive SCCHN cells in combination with anti-PD-L1 mAb and CD64-CR T cells were killed by ADCC, which enhanced direct cytotoxicity. These findings indicate that the glycolytic phenotype is involved in CD64-CR T cell proliferation/expansion. These cells mediate long-lasting HLA-independent cytotoxicity and ADCC in CRC and SCCHN cells. Conclusions CD64/CD28/CD3ζ T cells could significantly impact the rational design of personalized studies to treat CRC and SCCHN and the identification of novel FcγR ALs in cancer and healthy cells.https://doi.org/10.1186/s40164-025-00601-2CD64CAR-T cellLigandBiosensorCancerCytotoxicity |
| spellingShingle | Sara Caratelli Francesca De Paolis Domenico Alessandro Silvestris Silvia Baldari Illari Salvatori Apollonia Tullo Giulia Lanzilli Aymone Gurtner Alberto Ferri Cristiana Valle Simona Padovani Valeriana Cesarini Tommaso Sconocchia Loredana Cifaldi Roberto Arriga Giulio Cesare Spagnoli Soldano Ferrone Adriano Venditti Piero Rossi Graziano Pesole Gabriele Toietta Giuseppe Sconocchia The CD64/CD28/CD3ζ chimeric receptor reprograms T-cell metabolism and promotes T-cell persistence and immune functions while triggering antibody-independent and antibody-dependent cytotoxicity Experimental Hematology & Oncology CD64 CAR-T cell Ligand Biosensor Cancer Cytotoxicity |
| title | The CD64/CD28/CD3ζ chimeric receptor reprograms T-cell metabolism and promotes T-cell persistence and immune functions while triggering antibody-independent and antibody-dependent cytotoxicity |
| title_full | The CD64/CD28/CD3ζ chimeric receptor reprograms T-cell metabolism and promotes T-cell persistence and immune functions while triggering antibody-independent and antibody-dependent cytotoxicity |
| title_fullStr | The CD64/CD28/CD3ζ chimeric receptor reprograms T-cell metabolism and promotes T-cell persistence and immune functions while triggering antibody-independent and antibody-dependent cytotoxicity |
| title_full_unstemmed | The CD64/CD28/CD3ζ chimeric receptor reprograms T-cell metabolism and promotes T-cell persistence and immune functions while triggering antibody-independent and antibody-dependent cytotoxicity |
| title_short | The CD64/CD28/CD3ζ chimeric receptor reprograms T-cell metabolism and promotes T-cell persistence and immune functions while triggering antibody-independent and antibody-dependent cytotoxicity |
| title_sort | cd64 cd28 cd3ζ chimeric receptor reprograms t cell metabolism and promotes t cell persistence and immune functions while triggering antibody independent and antibody dependent cytotoxicity |
| topic | CD64 CAR-T cell Ligand Biosensor Cancer Cytotoxicity |
| url | https://doi.org/10.1186/s40164-025-00601-2 |
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