Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

CCR5-targeted allogeneic gamma–delta CD19 chimeric antigen receptor T cells for HIV-associated B cell-malignancy immunotherapy

Nature Biomedical Engineering (2025)Cite this article

Subjects

Abstract

Immune-based cell therapy offers a promising approach to cancer treatment. While autologous chimeric antigen receptor (CAR) T cells have shown success, production is time-consuming, costly and patient specific. Gamma–delta (γδ) T cells are promising for ‘off-the-shelf’ CAR T cell therapy. However, clinical translation of γδ CAR T cells is hampered by low frequency, resistance to genetic manipulation and advanced differentiation after expansion, limiting therapeutic feasibility. Here we demonstrate a method for in vitro activation and expansion of peripheral blood γδ T cells, facilitating high rates of gene editing and efficient CAR integration. Using artificial antigen-presenting cells, we produce minimally differentiated, highly functional γδ CAR T cells. By targeting a US Food and Drug Administration-approved CD19 CAR to the CCR5 locus, we generate CCR5-deficient γδ CD19 CAR T cells (γδ CCR5KI-CAR19), which demonstrated resistance to HIV-mediated depletion and robust antitumour responses against B cell lymphoma and leukaemia. γδ CCR5KI-CAR19 T cells enable the immunotherapy of HIV-associated B cell malignancies. These studies provide preclinical evidence supporting large-scale development of potent allogeneic γδ CAR T cells for diverse immunotherapies.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Generation and expansion of CCR5-deficient γ9δ2 CAR T cells (γδ CCR5KI-CAR19).
Fig. 2: Single-cell transcriptomic profiling of γδ CCR5KI-CAR19 T cells.
Fig. 3: Characterization and killing dynamics of γδ CCR5KI-CAR19 T cells.
Fig. 4: CCR5 deficiency mediates HIV resistance in human γδ CCR5KI-CAR19 T cells.
Fig. 5: In vivo antitumour efficacy of γδ CCR5KI-CAR19 T cells in a chicken embryo model and NSG xenograft model of B cell lymphoma and leukaemia.

Similar content being viewed by others

Data availability

All data from this study are included in the main text, Extended Data and Supplementary Materials. Raw and processed single-cell RNA-sequencing data have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession number GSE294637 and are publicly available65. Raw FASTQ files, processed count matrices and cell-level metadata can be retrieved directly from this repository. For requests for additional data or materials not included in these sources, contact the Penn Center for Innovation at pciinfo@pci.upenn.edu. All such requests will be reviewed to address any potential intellectual property or confidentiality issues, and approved data and materials will be shared following completion of a material transfer agreement. For further assistance, contact the corresponding authors.

References

  1. Maude, S. L. et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N. Engl. J. Med. 371, 1507–1517 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Frigault, M. J. & Maus, M. V. State of the art in CAR T cell therapy for CD19+ B cell malignancies. J. Clin. Invest. 130, 1586–1594 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Park, J. H. et al. Long-term follow-up of CD19 CAR therapy in acute lymphoblastic leukemia. N. Engl. J. Med. 378, 449–459 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Finney, O. C. et al. CD19 CAR T cell product and disease attributes predict leukemia remission durability. J. Clin. Invest. 129, 2123–2132 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Shah, N. N. & Fry, T. J. Mechanisms of resistance to CAR T cell therapy. Nat. Rev. Clin. Oncol. 16, 372–385 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Rafiq, S., Hackett, C. S. & Brentjens, R. J. Engineering strategies to overcome the current roadblocks in CAR T cell therapy. Nat. Rev. Clin. Oncol. 17, 147–167 (2020).

    Article  PubMed  Google Scholar 

  7. Liu, P. et al. Acute graft-versus-host disease after humanized anti-CD19-CAR T therapy in relapsed B-ALL patients after allogeneic hematopoietic stem cell transplant. Front. Oncol. 10, 573822 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Lv, Z., Luo, F. & Chu, Y. Strategies for overcoming bottlenecks in allogeneic CAR-T cell therapy. Front. Immunol. 14, 1199145 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Chiesa, R. et al. Base-edited CAR7 T cells for relapsed T-cell acute lymphoblastic leukemia. N. Engl. J. Med. 389, 899–910 (2023).

    Article  CAS  PubMed  Google Scholar 

  10. Morita, C. T., Jin, C., Sarikonda, G. & Wang, H. Nonpeptide antigens, presentation mechanisms, and immunological memory of human Vgamma2Vdelta2 T cells: discriminating friend from foe through the recognition of prenyl pyrophosphate antigens. Immunol. Rev. 215, 59–76 (2007).

    Article  CAS  PubMed  Google Scholar 

  11. Correia, D. V., Lopes, A. & Silva-Santos, B. Tumor cell recognition by gammadelta T lymphocytes: T-cell receptor vs. NK-cell receptors. Oncoimmunology 2, e22892 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Kabelitz, D., Serrano, R., Kouakanou, L., Peters, C. & Kalyan, S. Cancer immunotherapy with gammadelta T cells: many paths ahead of us. Cell. Mol. Immunol. 17, 925–939 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Gentles, A. J. et al. The prognostic landscape of genes and infiltrating immune cells across human cancers. Nat. Med. 21, 938–945 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Sanz, M., Mann, B. T., Chitrakar, A. & Soriano-Sarabia, N. Human Vdelta2 T cells and their versatility for immunotherapeutic approaches. Cells https://doi.org/10.3390/cells11223572 (2022).

  15. Silva-Santos, B., Mensurado, S. & Coffelt, S. B. gammadelta T cells: pleiotropic immune effectors with therapeutic potential in cancer. Nat. Rev. Cancer 19, 392–404 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hudspeth, K. et al. Engagement of NKp30 on Vdelta1 T cells induces the production of CCL3, CCL4, and CCL5 and suppresses HIV-1 replication. Blood 119, 4013–4016 (2012).

    Article  CAS  PubMed  Google Scholar 

  17. Deniger, D. C., Moyes, J. S. & Cooper, L. J. Clinical applications of gamma delta T cells with multivalent immunity. Front. Immunol. 5, 636 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Deniger, D. C. et al. Bispecific T-cells expressing polyclonal repertoire of endogenous gammadelta T-cell receptors and introduced CD19-specific chimeric antigen receptor. Mol. Ther. 21, 638–647 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Nishimoto, K. P. et al. Allogeneic CD20-targeted gammadelta T cells exhibit innate and adaptive antitumor activities in preclinical B-cell lymphoma models. Clin. Transl. Immunol. 11, e1373 (2022).

    Article  CAS  Google Scholar 

  20. Sanchez Martinez, D. et al. Generation and proof-of-concept for allogeneic CD123 CAR-Delta One T (DOT) cells in acute myeloid leukemia. J. Immunother. Cancer https://doi.org/10.1136/jitc-2022-005400 (2022).

  21. Lee, D. et al. Unlocking the potential of allogeneic Vdelta2 T cells for ovarian cancer therapy through CD16 biomarker selection and CAR/IL-15 engineering. Nat. Commun. 14, 6942 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Rozenbaum, M. et al. Gamma-delta CAR-T cells show CAR-directed and independent activity against leukemia. Front. Immunol. 11, 1347 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Ghilardi, G. et al. T cell lymphoma and secondary primary malignancy risk after commercial CAR T cell therapy. Nat. Med. 30, 984–989 (2024).

    Article  CAS  PubMed  Google Scholar 

  24. Uldrick, T. S. et al. Modernizing clinical trial eligibility criteria: recommendations of the American Society of Clinical Oncology-Friends of Cancer Research HIV Working Group. J. Clin. Oncol. 35, 3774–3780 (2017).

    Article  CAS  PubMed  Google Scholar 

  25. Horner, M. J., Shiels, M. S., Pfeiffer, R. M. & Engels, E. A. Deaths attributable to cancer in the US human immunodeficiency virus population during 2001–2015. Clin. Infect. Dis. 72, e224–e231 (2021).

    Article  PubMed  Google Scholar 

  26. Li, H. et al. Association between Vgamma2Vdelta2 T cells and disease progression after infection with closely related strains of HIV in China. Clin. Infect. Dis. 46, 1466–1472 (2008).

    Article  CAS  PubMed  Google Scholar 

  27. Bordon, J. et al. Association between longer duration of HIV-suppressive therapy and partial recovery of the V gamma 2 T cell receptor repertoire. J. Infect. Dis. 189, 1482–1486 (2004).

    Article  CAS  PubMed  Google Scholar 

  28. Liu, R. et al. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell 86, 367–377 (1996).

    Article  CAS  PubMed  Google Scholar 

  29. Park, R. J. et al. A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors. Nat. Genet. 49, 193–203 (2017).

    Article  CAS  PubMed  Google Scholar 

  30. Hultquist, J. F. et al. A Cas9 ribonucleoprotein platform for functional genetic studies of HIV-host interactions in primary human T cells. Cell Rep. 17, 1438–1452 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Long, A. H. et al. 4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat. Med. 21, 581–590 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Thakur, A., Scholler, J., Schalk, D. L., June, C. H. & Lum, L. G. Enhanced cytotoxicity against solid tumors by bispecific antibody-armed CD19 CAR T cells: a proof-of-concept study. J. Cancer Res. Clin. Oncol. 146, 2007–2016 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Wilhelm, M. et al. Gammadelta T cells for immune therapy of patients with lymphoid malignancies. Blood 102, 200–206 (2003).

    Article  CAS  PubMed  Google Scholar 

  34. Sandoz, P. A. et al. Modulation of lytic molecules restrain serial killing in gammadelta T lymphocytes. Nat. Commun. 14, 6035 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Graydon, C. G., Mohideen, S. & Fowke, K. R. LAG3’s enigmatic mechanism of action. Front. Immunol. 11, 615317 (2020).

    Article  CAS  PubMed  Google Scholar 

  36. Pauza, C. D., Poonia, B., Li, H., Cairo, C. & Chaudhry, S. gammadelta T cells in HIV disease: past, present, and future. Front. Immunol. 5, 687 (2014).

    PubMed  Google Scholar 

  37. Li, H. & Pauza, C. D. HIV envelope-mediated, CCR5/alpha4beta7-dependent killing of CD4-negative gammadelta T cells which are lost during progression to AIDS. Blood 118, 5824–5831 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Li, H. & Pauza, C. D. The alpha4beta7 integrin binds HIV envelope but does not mediate bystander killing of gammadelta T cells. Blood 120, 698–699 (2012).

    Article  CAS  PubMed  Google Scholar 

  39. Poccia, F. et al. Phosphoantigen-reactive Vgamma9Vdelta2 T lymphocytes suppress in vitro human immunodeficiency virus type 1 replication by cell-released antiviral factors including CC chemokines. J. Infect. Dis. 180, 858–861 (1999).

    Article  CAS  PubMed  Google Scholar 

  40. Pontel, L. B. et al. Acute lymphoblastic leukemia necessitates GSH-dependent ferroptosis defenses to overcome FSP1-epigenetic silencing. Redox Biol. 55, 102408 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Nipper, A. et al. 222 The chick embryo chorioallantoic membrane (CAM) as a platform for assessing the in vivo efficacy of chimeric antigen receptor (CAR) T cell therapy in solid tumors. J. Immunother. Cancer 11, A253–A253 (2023).

    Google Scholar 

  42. Pupo, A. et al. AAV vectors: the Rubik’s cube of human gene therapy. Mol. Ther. 30, 3515–3541 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Morris, E. C., Neelapu, S. S., Giavridis, T. & Sadelain, M. Cytokine release syndrome and associated neurotoxicity in cancer immunotherapy. Nat. Rev. Immunol. 22, 85–96 (2022).

    Article  CAS  PubMed  Google Scholar 

  44. Gomes-Silva, D. et al. Tonic 4-1BB costimulation in chimeric antigen receptors impedes T cell survival and is vector-dependent. Cell Rep. 21, 17–26 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Kawalekar, O. U. et al. Distinct signaling of coreceptors regulates specific metabolism pathways and impacts memory development in CAR T cells. Immunity 44, 380–390 (2016).

    Article  CAS  PubMed  Google Scholar 

  46. Blanco, B. et al. Overcoming CAR-mediated CD19 downmodulation and leukemia relapse with T lymphocytes secreting anti-CD19 T-cell engagers. Cancer Immunol. Res. 10, 498–511 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Ramirez-Fernandez, A. et al. Synapse topology and downmodulation events determine the functional outcome of anti-CD19 T cell-redirecting strategies. Oncoimmunology 11, 2054106 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Gober, H. J. et al. Human T cell receptor gammadelta cells recognize endogenous mevalonate metabolites in tumor cells. J. Exp. Med. 197, 163–168 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Di Lorenzo, B. et al. Broad cytotoxic targeting of acute myeloid leukemia by polyclonal Delta One T cells. Cancer Immunol. Res. 7, 552–558 (2019).

    Article  PubMed  Google Scholar 

  50. Sandstrom, A. et al. The intracellular B30.2 domain of butyrophilin 3A1 binds phosphoantigens to mediate activation of human Vgamma9Vdelta2 T cells. Immunity 40, 490–500 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Seagar, M. et al. LGI1 tunes intrinsic excitability by regulating the density of axonal Kv1 channels. Proc. Natl Acad. Sci. USA 114, 7719–7724 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Liu, Y. et al. Biosynthesis of 4-thiouridine in tRNA in the methanogenic archaeon Methanococcus maripaludis. J. Biol. Chem. 287, 36683–36692 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Hayday, A. C. & Vantourout, P. The innate biologies of adaptive antigen receptors. Annu. Rev. Immunol. 38, 487–510 (2020).

    Article  CAS  PubMed  Google Scholar 

  54. Hoeres, T. et al. Improving immunotherapy against B-cell malignancies using gammadelta T-cell-specific stimulation and therapeutic monoclonal antibodies. J. Immunother. 42, 331–344 (2019).

    Article  CAS  PubMed  Google Scholar 

  55. Capietto, A. H., Martinet, L. & Fournie, J. J. Stimulated gammadelta T cells increase the in vivo efficacy of trastuzumab in HER-2+ breast cancer. J. Immunol. 187, 1031–1038 (2011).

    Article  CAS  PubMed  Google Scholar 

  56. Alnaggar, M. et al. Allogenic Vgamma9Vdelta2 T cell as new potential immunotherapy drug for solid tumor: a case study for cholangiocarcinoma. J. Immunother. Cancer 7, 36 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  57. Xu, Y. et al. Allogeneic Vgamma9Vdelta2 T-cell immunotherapy exhibits promising clinical safety and prolongs the survival of patients with late-stage lung or liver cancer. Cell. Mol. Immunol. 18, 427–439 (2021).

    Article  CAS  PubMed  Google Scholar 

  58. Hu, Y. et al. gammadelta T cells: origin and fate, subsets, diseases and immunotherapy. Signal Transduct. Target. Ther. 8, 434 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Polito, V. A. et al. Universal ready-to-use immunotherapeutic approach for the treatment of cancer: expanded and activated polyclonal gammadelta memory T cells. Front. Immunol. 10, 2717 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Fraietta, J. A. et al. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat. Med. 24, 563–571 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Kloss, C. C. et al. Dominant-negative TGF-beta receptor enhances PSMA-targeted human CAR T cell proliferation and augments prostate cancer eradication. Mol. Ther. 26, 1855–1866 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Ciminale, V., Felber, B. K., Campbell, M. & Pavlakis, G. N. A bioassay for HIV-1 based on Env-CD4 interaction. AIDS Res. Hum. Retroviruses 6, 1281–1287 (1990).

    Article  CAS  PubMed  Google Scholar 

  63. Bak, R. O., Dever, D. P. & Porteus, M. H. CRISPR/Cas9 genome editing in human hematopoietic stem cells. Nat. Protoc. 13, 358–376 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Jung, I. Y. et al. Tissue-resident memory CAR T cells with stem-like characteristics display enhanced efficacy against solid and liquid tumors. Cell Rep. Med. 4, 101053 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Ramírez-Fernández, Á., Dimitri, A. J., Chen, G. M. & Fraietta, J. A. CCR5-targeted allogeneic gamma-delta CD19 CAR T cells for HIV-associated B cell malignancy immunotherapy. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE294637 (Gene Expression Omnibus, 2025).

Download references

Acknowledgements

This study was supported by the following grants: AMC Scholar Award from the AIDS Malignancy Consortium (UM1CA121947) (to A.R.-F.), T32 AI007632 (to A.J.D.) and the FUNDAME Fellowship from Alfonso Martin Escudero (to A.R.-F.). F.C. and J.A.F. received support through a Penn Center for AIDS Research (CFAR) Pilot Grant Program (Core Grant: NIAID P30 AI045008). The study additionally received funding from NIH grant NCI P01 CA214278 (to M.R., D.L.S., C.H.J. and J.A.F.) and the Bob Levis Funding Group (to B.L.L., C.H.J. and J.A.F.). Further support was provided by the National Science Foundation Engineering Research Center for Cell Manufacturing Technologies (to B.L.L. and J.A.F.), the Alliance for Cancer Gene Therapy Investigator Award in Cell and Gene Therapy for Cancer (to J.A.F.) and funding from the Parker Institute for Cancer Immunotherapy (to C.H.J.). J.A.F. also receives funding for correlative data science from a Parker Institute for Cancer Immunotherapy Innovation Challenge Award and a Swim Across American Gene Editing Innovation Award. In addition, funding was received from a U01 AG066100 via the Samuel Waxman Cancer Research Foundation and a Chambers Centurion gift (to C.H.J. and J.A.F.) and an Abramson Cancer Center P30 Core Grant P30 CA016520 (to J.A.F.). Further support was provided by the Spanish Ministry of Economy and Competitiveness (PID2021-123039OB-C21) and the Catalan Agency for Management of University and Research Grants (AGAUR 2021SGR01535) (to G.R.). Also, this work was supported by Programa Investigo (AGAUR) funded by Next Generation EU—PRTR (2023 INV-2 00011 (200011TC5)) (to M.P.). We acknowledge the Human Immunology Core at the University of Pennsylvania (RRID SCR_022380) and the Apheresis Unit at the Hospital of the University of Pennsylvania for providing peripheral blood mononuclear cells. We are grateful to the Stem Cell and Xenograft Core at the University of Pennsylvania (RRID SCR_010035) for their husbandry services and assistance with in vivo mouse studies, as well as the Cell and Animal Radiation Core at the University of Pennsylvania (RRID SCR_022377) for access to the xRad irradiator. We appreciate the support from the Translational and Correlative Sciences Laboratory (TCSL) and the Product Development Laboratory (PDL) at the University of Pennsylvania Center for Cellular Immunotherapies. We also thank the Lymphoma Translational Group and Laura Perich from the Josep Carreras Leukaemia Research Institute. We are especially grateful to S. F. Lacey (University of Pennsylvania) for his helpful suggestions. Schematic illustrations were created with BioRender.

Author information

Author notes

  1. These authors contributed equally: Ángel Ramírez-Fernández, Alexander J. Dimitri.

Authors and Affiliations

  1. Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Ángel Ramírez-Fernández, Alexander J. Dimitri, Robert Bartoszek, Gregory M. Chen, Yuqi Zhou, Reyes Acosta, James L. Riley & Joseph A. Fraietta

  2. Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Ángel Ramírez-Fernández, Alexander J. Dimitri, Fang Chen, Robert Bartoszek, Gregory M. Chen, Laura Córdoba-Espejo, Yuqi Zhou, Yun-hsin Tang, Chien-Ting Lin, Reyes Acosta, John Scholler, Guido Ghilardi, Patrizia Porazzi, Anne Chew, Julie K. Jadlowsky, Vanessa E. Gonzalez, Donald L. Siegel, Bruce L. Levine, Marco Ruella, Carl H. June, James L. Riley & Joseph A. Fraietta

  3. Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Ángel Ramírez-Fernández, Alexander J. Dimitri, Robert Bartoszek, Gregory M. Chen, Laura Córdoba-Espejo, Guido Ghilardi, Patrizia Porazzi, Vanessa E. Gonzalez, Donald L. Siegel, Bruce L. Levine, Marco Ruella, Carl H. June, James L. Riley & Joseph A. Fraietta

  4. Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Ángel Ramírez-Fernández, Alexander J. Dimitri, Robert Bartoszek, Gregory M. Chen, Laura Córdoba-Espejo, Anne Chew, Julie K. Jadlowsky, Donald L. Siegel, Bruce L. Levine, Carl H. June, James L. Riley & Joseph A. Fraietta

  5. Parker Institute for Cancer Immunotherapy, University of Pennsylvania, Philadelphia, PA, USA

    Ángel Ramírez-Fernández, Alexander J. Dimitri, Robert Bartoszek, Gregory M. Chen, Carl H. June & Joseph A. Fraietta

  6. Lymphoma Program, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA

    Guido Ghilardi, Patrizia Porazzi, Stefan K. Barta & Marco Ruella

  7. Division of Hematology-Oncology, Department of Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA, USA

    Guido Ghilardi, Patrizia Porazzi, Stefan K. Barta & Marco Ruella

  8. Lymphoma Translational Group, Josep Carreras Leukaemia Research Institute (IJC), Badalona, Barcelona, Spain

    Mireia Pellicer, Núria Profitós-Pelejà & Gaël Roué

  9. Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA, USA

    Michael T. Lotze

Authors
  1. Ángel Ramírez-Fernández
    View author publications

    Search author on:PubMed Google Scholar

  2. Alexander J. Dimitri
    View author publications

    Search author on:PubMed Google Scholar

  3. Fang Chen
    View author publications

    Search author on:PubMed Google Scholar

  4. Robert Bartoszek
    View author publications

    Search author on:PubMed Google Scholar

  5. Gregory M. Chen
    View author publications

    Search author on:PubMed Google Scholar

  6. Laura Córdoba-Espejo
    View author publications

    Search author on:PubMed Google Scholar

  7. Yuqi Zhou
    View author publications

    Search author on:PubMed Google Scholar

  8. Yun-hsin Tang
    View author publications

    Search author on:PubMed Google Scholar

  9. Chien-Ting Lin
    View author publications

    Search author on:PubMed Google Scholar

  10. Reyes Acosta
    View author publications

    Search author on:PubMed Google Scholar

  11. John Scholler
    View author publications

    Search author on:PubMed Google Scholar

  12. Guido Ghilardi
    View author publications

    Search author on:PubMed Google Scholar

  13. Patrizia Porazzi
    View author publications

    Search author on:PubMed Google Scholar

  14. Mireia Pellicer
    View author publications

    Search author on:PubMed Google Scholar

  15. Núria Profitós-Pelejà
    View author publications

    Search author on:PubMed Google Scholar

  16. Stefan K. Barta
    View author publications

    Search author on:PubMed Google Scholar

  17. Anne Chew
    View author publications

    Search author on:PubMed Google Scholar

  18. Julie K. Jadlowsky
    View author publications

    Search author on:PubMed Google Scholar

  19. Vanessa E. Gonzalez
    View author publications

    Search author on:PubMed Google Scholar

  20. Donald L. Siegel
    View author publications

    Search author on:PubMed Google Scholar

  21. Bruce L. Levine
    View author publications

    Search author on:PubMed Google Scholar

  22. Gaël Roué
    View author publications

    Search author on:PubMed Google Scholar

  23. Marco Ruella
    View author publications

    Search author on:PubMed Google Scholar

  24. Michael T. Lotze
    View author publications

    Search author on:PubMed Google Scholar

  25. Carl H. June
    View author publications

    Search author on:PubMed Google Scholar

  26. James L. Riley
    View author publications

    Search author on:PubMed Google Scholar

  27. Joseph A. Fraietta
    View author publications

    Search author on:PubMed Google Scholar

Contributions

A.R.-F.: conceptualization, resources, funding acquisition, investigation, methodology, writing (original draft) and writing (review and editing). A.J.D.: investigation, methodology and writing (review and editing). F.C.: investigation and methodology. R.B.: investigation and methodology. G.M.C.: investigation and methodology. L.C.-E.: investigation and methodology. Y.Z.: investigation and methodology. Y.-h.T.: investigation and methodology. C.-T.L.: investigation and methodology. R.A.: investigation and methodology. J.S.: investigation and methodology. G.G.: investigation and methodology. P.P.: investigation and methodology. M.P.: investigation and methodology. N.P.-P.: investigation and methodology. S.K.B.: investigation and methodology. A.C.: investigation and methodology. J.K.J.: investigation and methodology. V.E.G.: investigation and methodology. D.L.S.: investigation and methodology. B.L.L.: investigation, methodology and writing (review and editing). G.R.: resources, supervision, validation, investigation, methodology and writing (review and editing). M.R.: resources, investigation, methodology and writing (review and editing). M.T.L.: investigation, methodology and writing (review and editing). C.H.J.: investigation, methodology and writing (review and editing). J.L.R.: supervision, validation, investigation, methodology and writing (review and editing). J.A.F.: conceptualization, resources, formal analysis, supervision, funding acquisition, validation, investigation, writing (original draft) and writing (review and editing).

Corresponding authors

Correspondence to Ángel Ramírez-Fernández or Joseph A. Fraietta.

Ethics declarations

Competing interests

A.C.: co-founder and equity holder in Tmunity Therapeutics. D.L.S.: founder’s equity and licensed IP with Verismo Therapeutics, Vetigenics and Chimeric Therapeutics. B.L.L.: consultancy and advisory roles with Terumo, GSK and Kite; co-founder and equity holder in Tmunity Therapeutics (acquired by Kite) and Capstan Therapeutics; advisory board memberships with Avectas, Capstan (Chair), Immuneel, Immusoft, In8bio, Ori Biotech, Oxford Biomedica, Thermo Fisher Pharma Services and UTC Therapeutics; member of the Alliance for Cancer Gene Therapy Board of Directors. M.R.: holds patents related to CD19 CAR T cells; consultant for NanoString, Bristol Myers Squibb, GlaxoSmithKline, Scailyte, Bayer and AbClon; research funding from AbClon, NanoString, Oxford NanoImaging, viTToria Biotherapeutics, CURIOX and Beckman Coulter; scientific founder of viTToria Biotherapeutics. C.H.J.: receives royalties from Novartis and Kite paid to the University of Pennsylvania; scientific co-founder and equity holder in Capstan Therapeutics, Dispatch Biotherapeutics and BlueWhale Bio; board member of AC Immune; scientific advisory roles with various companies, including BluesphereBio, Cabaletta, Carisma, Cartography, Cellares, Cellcarta, Celldex, Danaher, Decheng, ImmuneSensor, Kite, Poseida, Verismo, Viracta and WIRB-Copernicus Group. J.L.R.: received grants from Tmunity/Kite outside of the submitted work; co-founder of Tmunity Therapeutics and BlueWhale Bio with monetary compensation and equity. J.A.F.: holds patents and intellectual property in T cell-based cancer immunotherapy with royalties; receives funding from Tmunity Therapeutics and Danaher Corporation; consultancy with Retro Biosciences; scientific advisory board memberships with Cartography Bio, Shennon Biotechnologies Inc., CellFe Biotech, OverT Bio, Inc., and Tceleron Therapeutics, Inc. All other authors declare no competing interests. G.R.: received grants from Onconova Therapeutics outside of the submitted work.

Peer review

Peer review information

Nature Biomedical Engineering thanks Jurgen Kuball and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Efficiency of CCR5KI-CAR19 KO/KI gene-edited strategy.

a, Wild-type expression of the CCR5 receptor in γδ T cells before (gray) and after (blue) gene editing. b, CCR5-KO efficiency in αβ CCR5KI-CAR19 (red) and γδ CCR5KI-CAR19 (blue) T cells. c, CCR5 expression in CAR− and CAR+ γδ T CCR5KI-CAR19 cells and d, Correlation analysis between donor-matched control CCR5 expression and CAR expression (two-tailed Pearson simple linear regression test). e, Percentage of CAR+ cells using different multiplicity of infection (MOI) ratios. a-d, In vitro experiments were conducted using CAR T cells manufactured from different healthy subjects. Results are shown as mean ± SD from 9 different donors.

Extended Data Fig. 2 Differential expression analysis of all genes and transcription factors of γδ CCR5KI-CAR19 cultured with or without APCs.

a, Scatterplot showing differentially expressed genes shown by mean normalized expression between Feeder and No Feeder groups (x axis) compared to the log2 fold-change between Feeder and No Feeder groups. Red points indicate genes with FDR < 0.05 and log2 fold-change greater than 1; blue points indicate genes with FDR < 0.05 and log2 fold-change less than -1. b, Scatterplot showing differentially expressed transcription factors shown by mean normalized expression between Feeder and No Feeder groups (x axis) compared to the log2 fold-change between Feeder and No Feeder groups. Red points indicate genes with FDR < 0.05 and log2 fold-change greater than 1; blue points indicate genes with FDR < 0.05 and log2 fold-change less than -1.

Extended Data Fig. 3 Efficiency of CCR5KI-CAR19 KO/KI expanded with APCs and killing dynamics.

a, Summary of CCR5KI-CAR19 and CCR5-KO in αβ mock (light red) and CCR5KI-CAR19 (red, n = 9) T cells, γδ mock (light blue, n = 9) and CCR5KI-CAR19 (blue, n = 12) T cells and γδ mock (light purple, n = 8) and CCR5KI-CAR19 (purple, n = 8) T cells culture with antigen-presenting cells (APCs, two-tailed Kruskal–Wallis test, n indicates different donors). b Summary of CCR5-KO in αβ mock (light red, n = 9) and CCR5KI-CAR19 (red, n = 9) T cells, γδ mock (light blue, n = 9) and CCR5KI-CAR19 (blue, n = 12) T cells, and γδ mock (light purple, n = 5) and CCR5KI-CAR19 (purple, n = 8) T cells cultured with APCs (two-tailed Kruskal–Wallis test; n indicates independent donors). c, Cytolytic activity of αβ mock (light red) and CCR5KI-CAR19 (red) T cells, and γδ mock* (light purple) and CCR5KI-CAR19* (purple) T cells, measured 24 h after co-culture (two-tailed Mann–Whitney U test; n = 4 per group; n indicates independent donors). Data are presented as mean ± SD from different donors. Asterisk (*) indicates expansion with APCs.

Extended Data Fig. 4 Caspase‑3 immunohistochemical staining in tumor tissues from γδ CCR5KI CAR19 and γδ mock T cell treated mice.

Immunohistochemical (IHC) staining for caspase-3 (indicated by red arrows) in two different representative tissue sections of tumors treated with γδ CCR5KI-CAR19 (Tumor samples; CAM-19 and CAM-24) and γδ mock (samples; CAM-74 and CAM-35) T cells are shown. Tumors samples were taken from the experiment shown in Fig. 5a-e at termination. Scale bars are indicated.

Supplementary information

Supplementary Information (download PDF )

Supplementary Figs. 1–9.

Reporting Summary (download PDF )

Supplementary Table 1 (download XLSX )

This table lists all genes and pathways showing significant expression differences between γδ CCR5KI-CAR19 T cells expanded with irradiated K562-derived artificial antigen-presenting cells (aAPCs) and those expanded without APCs, specifically in cells assigned to G1 phase by single-cell RNA-seq analysis. Gene.name: HGNC symbol for each gene; p_val: uncorrected P value from the Wilcoxon rank sum test comparing expression levels between conditions; avg_log2FC: average log2 fold change in expression (APC versus no-APC); p_val_adj: false discovery rate-adjusted P value (Benjamini–Hochberg correction); mean.expr: mean normalized expression level across all cells; Transcription.factor: Boolean indicator (‘True’/‘False’) of whether the gene encodes a known transcription factor.

Supplementary Table 2 (download XLSX )

This table summarizes the exact pairwise tests and corresponding P values corresponding to main Figs. 1–5 and Extended Data Fig. 3.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ramírez-Fernández, Á., Dimitri, A.J., Chen, F. et al. CCR5-targeted allogeneic gamma–delta CD19 chimeric antigen receptor T cells for HIV-associated B cell-malignancy immunotherapy. Nat. Biomed. Eng (2025). https://doi.org/10.1038/s41551-025-01527-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Version of record:

  • DOI: https://doi.org/10.1038/s41551-025-01527-0

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer