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Latent human herpesvirus 6 is reactivated in CAR T cells

Nature volume 623, pages 608–615 (2023)Cite this article

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Abstract

Cell therapies have yielded durable clinical benefits for patients with cancer, but the risks associated with the development of therapies from manipulated human cells are understudied. For example, we lack a comprehensive understanding of the mechanisms of toxicities observed in patients receiving T cell therapies, including recent reports of encephalitis caused by reactivation of human herpesvirus 6 (HHV-6)1. Here, through petabase-scale viral genomics mining, we examine the landscape of human latent viral reactivation and demonstrate that HHV-6B can become reactivated in cultures of human CD4+ T cells. Using single-cell sequencing, we identify a rare population of HHV-6 ‘super-expressors’ (about 1 in 300–10,000 cells) that possess high viral transcriptional activity, among research-grade allogeneic chimeric antigen receptor (CAR) T cells. By analysing single-cell sequencing data from patients receiving cell therapy products that are approved by the US Food and Drug Administration2 or are in clinical studies3,4,5, we identify the presence of HHV-6-super-expressor CAR T cells in patients in vivo. Together, the findings of our study demonstrate the utility of comprehensive genomics analyses in implicating cell therapy products as a potential source contributing to the lytic HHV-6 infection that has been reported in clinical trials1,6,7,8 and may influence the design and production of autologous and allogeneic cell therapies.

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Fig. 1: Petabase-scale analysis of viral nucleic acids reveals that HHV-6 is reactivated in human T cells.
Fig. 2: scRNA-seq identifies rare HHV-6-expressing cells in CAR T cell culture.
Fig. 3: HHV-6 reactivation in FDA-approved CAR T cells and clinically used CAR T cells in vitro and in vivo.
Fig. 4: Allogeneic CAR T cells discriminate donor and host reactivation of HHV-6B in vivo.

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Data availability

Raw and processed sequencing data used in this study are available at GEO (accession GSE210063). The HHV-6B reference genome and transcriptome used in this work is available at Genbank (accession AF157706).

Code availability

Code to reproduce all custom analyses in this manuscript is available at https://github.com/caleblareau/hhv6-reactivation and archived at https://doi.org/10.5281/zenodo.8218129.

References

  1. Spanjaart, A. M., van der Valk, F. M., van Rooijen, G., Brouwer, M. C. & Kersten, M. J. Confused about confusion. N. Engl. J. Med. 386, 80–87 (2022).

    Article  PubMed  Google Scholar 

  2. Haradhvala, N. J. et al. Distinct cellular dynamics associated with response to CAR-T therapy for refractory B-cell lymphoma. Nat. Med.28, 1848–1859 (2022).

  3. Wilson, T. L. et al. Common trajectories of highly effective CD19-specific CAR T cells identified by endogenous T cell receptor lineages. Cancer Discov. https://doi.org/10.1158/2159-8290.CD-21-1508 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Talleur, A. et al. Preferential expansion of CD8+ CD19-CAR T cells postinfusion and the role of disease burden on outcome in pediatric B-ALL. Blood Adv. https://doi.org/10.1182/bloodadvances.2021006293 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Hu, Y. et al. Genetically modified CD7-targeting allogeneic CAR-T cell therapy with enhanced efficacy for relapsed/refractory CD7-positive hematological malignancies: a phase I clinical study. Cell Res. 32, 995–1007 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Locke, F. L. et al. Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicentre, phase 1–2 trial. Lancet Oncol. 20, 31–42 (2019).

    Article  CAS  PubMed  Google Scholar 

  7. Baird, J. H. et al. Immune reconstitution and infectious complications following axicabtagene ciloleucel therapy for large B-cell lymphoma. Blood Adv. 5, 143–155 (2021).

  8. Maude, S. L. et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N. Engl. J. Med. 378, 439–448 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Cohen, J. I. Herpesvirus latency. J. Clin. Invest. 130, 3361–3369 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Traylen, C. M. et al. Virus reactivation: a panoramic view in human infections. Future Virol. 6, 451–463 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Düver, F. et al. Viral reactivations following hematopoietic stem cell transplantation in pediatric patients - a single center 11-year analysis. PLoS One 15, e0228451 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Grinde, B. Herpesviruses: latency and reactivation – viral strategies and host response. J. Oral Microbiol. 5, 22766 (2013).

  13. Kampouri, E. et al. CMV and HHV-6 after chimeric antigen receptor (CAR)-T-cell immunotherapy for B-cell malignancies: a prospective study. Transplant. Cell. Ther. 29, S210–S211 (2023).

    Article  Google Scholar 

  14. Labanieh, L. & Mackall, C. L. CAR immune cells: design principles, resistance and the next generation. Nature 614, 635–648 (2023).

    Article  ADS  CAS  PubMed  Google Scholar 

  15. Edgar, R. C. et al. Petabase-scale sequence alignment catalyses viral discovery. Nature 602, 142–147 (2022).

    Article  ADS  CAS  PubMed  Google Scholar 

  16. Leinonen, R., Sugawara, H. & Shumway, M., International Nucleotide Sequence Database Collaboration. The sequence read archive. Nucleic Acids Res. 39, D19–D21 (2011).

    Article  CAS  PubMed  Google Scholar 

  17. Hulo, C. et al. ViralZone: a knowledge resource to understand virus diversity. Nucleic Acids Res. 39, D576–D582 (2011).

    Article  CAS  PubMed  Google Scholar 

  18. LaMere, S. A., Thompson, R. C., Komori, H. K., Mark, A. & Salomon, D. R. Promoter H3K4 methylation dynamically reinforces activation-induced pathways in human CD4 T cells. Genes Immun. 17, 283–297 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Pellett, P. E. et al. Chromosomally integrated human herpesvirus 6: questions and answers. Rev. Med. Virol. 22, 144–155 (2012).

    Article  CAS  PubMed  Google Scholar 

  20. Ablashi, D. et al. Classification of HHV-6A and HHV-6B as distinct viruses. Arch. Virol. 159, 863–870 (2014).

    Article  CAS  PubMed  Google Scholar 

  21. Tang, H. et al. CD134 is a cellular receptor specific for human herpesvirus-6B entry. Proc. Natl Acad. Sci. USA 110, 9096–9099 (2013).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  22. Shytaj, I. L. et al. Alterations of redox and iron metabolism accompany the development of HIV latency. EMBO J. 39, e102209 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Jones, A. C. et al. Personalized transcriptomics reveals heterogeneous immunophenotypes in children with viral bronchiolitis. Am. J. Respir. Crit. Care Med. 199, 1537–1549 (2019).

    Article  CAS  PubMed  Google Scholar 

  24. Holtan, S. G. et al. Stress responses, M2 macrophages, and a distinct microbial signature in fatal intestinal acute graft-versus-host disease. JCI Insight 5, e129762 (2019).

    Article  PubMed  Google Scholar 

  25. Qu, K. et al. Chromatin accessibility landscape of cutaneous T cell lymphoma and dynamic response to HDAC inhibitors. Cancer Cell 32, 27–41 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Reading, J. L. et al. Augmented expansion of Treg cells from healthy and autoimmune subjects via adult progenitor cell co-culture. Front. Immunol. 12, 716606 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Bost, P. et al. Host-viral infection maps reveal signatures of severe COVID-19 patients. Cell 181, 1475–1488 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Gravel, A. et al. Mapping the human herpesvirus 6B transcriptome. J. Virol. https://doi.org/10.1128/JVI.01335-20 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  29. White, E. A. & Spector, D. H. in Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis (eds Arvin, A. et al.) (Cambridge Univ. Press, 2007).

  30. Wyżewski, Z., Gregorczyk, K. P., Szczepanowska, J. & Szulc-Dąbrowska, L. Functional role of Hsp60 as a positive regulator of human viral infection progression. Acta Virol. 62, 33–40 (2018).

    Article  PubMed  Google Scholar 

  31. Nguyen, N. N. T. et al. Hepatitis C virus modulates solute carrier family 3 member 2 for viral propagation. Sci. Rep. 8, 15486 (2018).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  32. Pfaender, S. et al. LY6E impairs coronavirus fusion and confers immune control of viral disease. Nat. Microbiol. 5, 1330–1339 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Kriesel, J. D., Jones, B. B., Dahms, K. M. & Spruance, S. L. STAT1 binds to the herpes simplex virus type 1 latency-associated transcript promoter. J. Neurovirol. 10, 12–20 (2004).

    Article  CAS  PubMed  Google Scholar 

  34. Mesquita, P. M. M. et al. Role of interleukin 32 in human immunodeficiency virus reactivation and its link to human immunodeficiency virus–herpes simplex virus coinfection. J. Infect. Dis. 215, 614–622 (2016).

    PubMed Central  Google Scholar 

  35. Lukhele, S., Boukhaled, G. M. & Brooks, D. G. Type I interferon signaling, regulation and gene stimulation in chronic virus infection. Semin. Immunol. 43, 101277 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Foo, J., Bellot, G., Pervaiz, S. & Alonso, S. Mitochondria-mediated oxidative stress during viral infection. Trends Microbiol. https://doi.org/10.1016/j.tim.2021.12.011 (2022).

    Article  PubMed  Google Scholar 

  37. Zerr, D. M. et al. A population-based study of primary human herpesvirus 6 infection. N. Engl. J. Med. 352, 768–776 (2005).

    Article  CAS  PubMed  Google Scholar 

  38. Zerr, D. M. et al. HHV-6 reactivation and its effect on delirium and cognitive functioning in hematopoietic cell transplantation recipients. Blood 117, 5243–5249 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Stenberg, K., Skog, S. & Tribukait, B. Concentration-dependent effects of foscarnet on the cell cycle. Antimicrob. Agents Chemother. 28, 802–806 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Panowski, S. H. et al. Preclinical development and evaluation of allogeneic CAR T cells targeting CD70 for the treatment of renal cell carcinoma. Cancer Res. 82, 2610–2624 (2022).

    Article  CAS  PubMed  Google Scholar 

  41. Telford, M., Navarro, A. & Santpere, G. Whole genome diversity of inherited chromosomally integrated HHV-6 derived from healthy individuals of diverse geographic origin. Sci. Rep. 8, 3472 (2018).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  42. Deng, Q. et al. Characteristics of anti-CD19 CAR T cell infusion products associated with efficacy and toxicity in patients with large B cell lymphomas. Nat. Med. 26, 1878–1887 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Han, X. et al. Construction of a human cell landscape at single-cell level. Nature 581, 303–309 (2020).

    Article  ADS  CAS  PubMed  Google Scholar 

  44. Yazar, S. et al. Single-cell eQTL mapping identifies cell type–specific genetic control of autoimmune disease. Science 376, eabf3041 (2022).

    Article  CAS  PubMed  Google Scholar 

  45. Ciszewski, C. et al. Identification of a γc receptor antagonist that prevents reprogramming of human tissue-resident cytotoxic T cells by IL15 and IL21. Gastroenterology 158, 625–637 (2020).

    Article  CAS  PubMed  Google Scholar 

  46. Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589–595 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Sommer, C. et al. Allogeneic FLT3 CAR T cells with an off-switch exhibit potent activity against AML and can be depleted to expedite bone marrow recovery. Mol. Ther. 28, 2237–2251 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Sánchez-Ponce, Y. et al. Simultaneous detection of beta and gamma human herpesviruses by multiplex qPCR reveals simple infection and coinfection episodes increasing risk for graft rejection in solid organ transplantation. Viruses 10, 730 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Bray, N., Pimentel, H., Melsted, P. & Pachter, L. Near-optimal RNA-seq quantification with kallisto. Nat. Biotechnol. 34, 525–527 (2016).

    Article  CAS  PubMed  Google Scholar 

  50. Melsted, P., Ntranos, V. & Pachter, L. The barcode, UMI, set format and BUStools. Bioinformatics 35, 4472–4473 (2019).

    Article  CAS  PubMed  Google Scholar 

  51. Zhang, H. et al. Fast alignment and preprocessing of chromatin profiles with Chromap. Nat. Commun. 12, 6566 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  52. Yang, K. et al. T cell-derived lymphotoxin limits Th1 response during HSV-1 infection. Sci. Rep. 8, 17727 (2018).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  53. Sharon, E., Volchek, L. & Frenkel, N. Human herpesvirus 6 (HHV-6) alters E2F1/Rb pathways and utilizes the E2F1 transcription factor to express viral genes. Proc. Natl Acad. Sci. USA 111, 451–456 (2014).

    Article  ADS  CAS  PubMed  Google Scholar 

  54. Hines, M. R. et al. Hemophagocytic lymphohistiocytosis-like toxicity (carHLH) after CD19-specific CAR T-cell therapy. Br. J. Haematol. 194, 701–707 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Heaton, H. et al. Souporcell: robust clustering of single-cell RNA-seq data by genotype without reference genotypes. Nat. Methods 17, 615–620 (2020).

    Article  CAS  PubMed  Google Scholar 

  56. Eraslan, G. et al. Single-nucleus cross-tissue molecular reference maps toward understanding disease gene function. Science 376, eabl4290 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Cao, J. et al. A human cell atlas of fetal gene expression. Science 370, eaba7721 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Calderon, D. et al. Landscape of stimulation-responsive chromatin across diverse human immune cells. Nat. Genet. 51, 1494–1505 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Richards, R. M. et al. NOT-gated CD93 CAR T cells effectively target AML with minimized endothelial cross-reactivity. Blood Cancer Discov. 2, 648–665 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank members of the laboratory of A.T.S. for helpful discussions; M. Green and G. Syal for assistance with public sequencing data; and S. Schell for assistance with clinical samples at St. Jude. C.A.L. is supported by a Stanford Science Fellowship, a Parker Institute for Cancer Immunotherapy Scholarship, a seed award from the Center for Human Systems Immunology and NIH K99 HG012076. A.T.S. is supported by the Burroughs Wellcome Fund Career Award for Medical Scientists, the Parker Institute for Cancer Immunotherapy, a Pew-Stewart Scholars for Cancer Research Award, a Cancer Research Institute Lloyd J. Old STAR Award and a Baxter Foundation Faculty Scholar Award. S.L. is supported by the NCI Research Specialist Award (R50CA251956). This work was supported by a sponsored research agreement with Allogene Therapeutics. Part of the analysis was carried out by the Center for Translational Immunology and Immunotherapy (CeTI2), which is supported by St. Jude Children’s Research Hospital.

Author information

Author notes

  1. Caleb A. Lareau

    Present address: Computational and Systems Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

Authors and Affiliations

  1. Department of Pathology, Stanford University, Stanford, CA, USA

    Caleb A. Lareau, Yajie Yin, Katalin D. Sandor, Bence Daniel, Jacob C. Gutierrez, Tsion Abay, Robert R. Stickels, Jeffrey M. Verboon, Vincent Liu, Frank A. Buquicchio, Fangyi Wang, Theodore L. Roth & Ansuman T. Satpathy

  2. Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA

    Caleb A. Lareau, Yajie Yin, Katalin D. Sandor, Bence Daniel, Jacob C. Gutierrez, Tsion Abay, Robert R. Stickels, Vincent Liu, Frank A. Buquicchio, Fangyi Wang & Ansuman T. Satpathy

  3. Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA

    Caleb A. Lareau, Robbie G. Majzner & Ansuman T. Satpathy

  4. Department of Genetics, Stanford University, Stanford, CA, USA

    Caleb A. Lareau, Vincent Liu & Anshul Kundaje

  5. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    Katie Maurer & Catherine J. Wu

  6. Harvard Medical School, Boston, MA, USA

    Katie Maurer, Gad Getz, Marcela V. Maus & Catherine J. Wu

  7. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Katie Maurer, Nicholas J. Haradhvala, Jackson Southard, Gad Getz, Marcela V. Maus, Shuqiang Li & Catherine J. Wu

  8. Allogene Therapeutics, South San Francisco, CA, USA

    Garima Yagnik, José Peña, Ren Song, Wenjing Li, Aastha Shrestha, Alison Moore, Zachary J. Roberts, Houman Dehghani & Thomas Pertel

  9. Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Jeremy Chase Crawford & Paul G. Thomas

  10. Department of Hematology, University of Amsterdam, Amsterdam, the Netherlands

    Anne M. Spanjaart & Marie J. Kersten

  11. Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Janice M. Riberdy & Stephen Gottschalk

  12. Translational Immunogenomics Laboratory, Dana-Farber Cancer Institute, Boston, MA, USA

    Jackson Southard & Shuqiang Li

  13. IBM Research, New York, NY, USA

    Laxmi Parida

  14. Cancer Center, Massachusetts General Hospital, Boston, MA, USA

    Gad Getz & Marcela V. Maus

  15. Berlin Institute of Health at Charité – Universitätsmedizin Berlin, Berlin, Germany

    Leif S. Ludwig

  16. Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin Institute for Medical Systems Biology (BIMSB), Berlin, Germany

    Leif S. Ludwig & Aimee C. Talleur

  17. Department of Computer Science, Stanford University, Stanford, CA, USA

    Anshul Kundaje

  18. Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA

    Robbie G. Majzner

  19. Division of Pediatric Hematology, Oncology, Stem Cell Transplantation & Regenerative Medicine, Department of Pediatrics, Stanford University, Stanford, CA, USA

    Robbie G. Majzner

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  1. Caleb A. Lareau
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  2. Yajie Yin
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Contributions

C.A.L. and A.T.S. conceived and designed the study with input from T.P., H.D. and R.G.M. C.A.L. led all analyses. Y.Y., K.M., K.D.S., B.D., T.A., R.R.S., J.M.R., F.A.B. and F.W. carried out experiments and interpreted data. J.C.C., J.C.G., N.J.H., J.M.V., V.L. and A.K. supported the informatics analyses. K.M., J.C.C., L.P., G.G., M.V.M., A.C.T., P.G.T., S.G. and C.J.W. provided data and/or insights related to the in vivo HHV-6 expression. G.Y., J.P., R.S., W.L. and A.S. designed, executed and analysed data related to HHV-6 in vitro CAR T data. A.M., Z.J.R., T.P. and H.D. provided technical review, strategy and guidance for in vitro CAR experiments. A.M.S., L.S.L., T.L.R., M.J.K. and R.G.M. interpreted experiments and impact. C.A.L. and A.T.S. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Caleb A. Lareau or Ansuman T. Satpathy.

Ethics declarations

Competing interests

A.T.S. is a founder of Immunai and Cartography Biosciences and receives research funding from Allogene Therapeutics and Merck Research Laboratories. C.A.L. and L.S.L. are consultants to Cartography Biosciences. N.J.H. is a consultant for Constellation Pharmaceuticals. C.J.W. holds equity in BioNTech Inc and receives research funding from Pharmacyclics. G.G. receives research funds from IBM and Pharmacyclics, and is a founder of, consultant for and holder of private equity in Scorpion Therapeutics. M.V.M. is an inventor on patents related to CAR T cell therapies held by the University of Pennsylvania (some licensed to Novartis), holds equity in TCR2, Century Therapeutics, Genocea, Oncternal and Neximmune, has served as a consultant for multiple companies involved in cell therapies, receives research support from KitePharma and Moderna, and serves on the Board of Directors of 2Seventy Bio. M.J.K. has received research funding from Kite, a Gilead Company, as well as honoraria from Kite, a Gilead Company, Novartis, Takeda, Miltenyi Biotec, BMS/Celgene, Beigene and Adicet Bio. S.G. is a co-inventor on patent applications in the fields of cell or gene therapy for cancer, a consultant for TESSA Therapeutics and a member of the Data and Safety Monitoring Board of Immatics, and has received honoraria from Tidal, Catamaran Bio, Sanofi and Novartis within the past 2 years. J.C.C. and P.G.T. are named on patent applications in the fields of T cell or gene therapy for cancer. G.Y., J.P., R.S., W.L., A.S., A.M., Z.J.R., T.P. and H.D. are employees and shareholders of Allogene Therapeutics. A.K. is a scientific cofounder of Ravel Biotechnology Inc., is on the scientific advisory board of PatchBio Inc., SerImmune Inc., AINovo Inc., TensorBio Inc. and OpenTargets, is a consultant with Illumina Inc. and owns shares in DeepGenomics Inc., Immunai Inc. and Freenome Inc. R.G.M. is a cofounder of and holds equity in Link Cell Therapies and CARGO Therapeutics, and is a consultant for Lyell Immunopharma, NKarta, Arovella Pharmaceuticals, Innervate Radiopharmaceuticals, Aptorum Group, Gadeta, FATE Therapeutics (Data and Safety Monitoring Board) and Waypoint Bio. All other authors declare no competing interests.

Peer review

Peer review information

Nature thanks Artem Babaian, Joshua Hill and Richard O'Neil for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Characterization of OX40 and CD21 gene expression in bulk sequencing experiments.

(a) Expression of TNFRSF4 (OX40), the canonical receptor of HHV-6B, in unstimulated and stimulated immune cell populations. OX40 is not expressed in unstimulated immune cells but highly expressed in CD4 and CD8 T cells after activation/stimulation via anti-CD3/anti-CD28 and IL-2. (b) Same as in (a) but for CD21, the canonical receptor for EBV. Gamma-delta T cells are highlighted for their high baseline expression of this receptor. (c) Broad expression of OX40 across healthy tissues from the GTEx bulk atlas. Data is summarized over 948 donors across the tissues shown from the GTEx v8 release. Boxplots: center line, median; box limits, first and third quartiles; whiskers, 1.5× interquartile range.

Extended Data Fig. 2 Characterization of OX40 gene expression in resting and stimulated endothelial cells.

(a) Refinement of cell-type specific HHV-6B expression patterns using the single-cell GTEx atlas. Data is summarized over 25 donors across the tissues shown from the GTEx v8 release Boxplots: center line, median; box limits, first and third quartiles; whiskers, 1.5× interquartile range. (b) Pseudobulk expression patterns of OX40 from the Human Fetal Atlas.

Extended Data Fig. 3 Supporting analyses for HHV-6 reactivation using Serratus.

(a) Comparison of read number matching to any of the annotated 16 T-cell libraries from Serratus for HHV-6A or HHV-6B. A distinct library is compared for HHV-6A and HHV-6B, connected by a line, quantification from Serratus. Libraries are sorted by HHV-6B expression, which was higher for all libraries than HHV-6A. (b) Heatmap of HHV-6B transcripts across the four samples with highest RNA expression in libraries from Serratus. Shown are the first 40 genes (based on genomic coordinate order) from the HHV-6B transcriptome and the number of reads that pseudoalign to each transcript. (c) Summary of % RNA molecules aligning to the HHV-6B reference in the naive CD4+ culture from the LaMere et al. dataset; compare to Fig. 1e. (d) Summary of HHV-6-SNV analysis and overlap across the two RNA-seq samples with highest HHV-6 reactivation (from Fig. 1d,e) (e) Summary of HHV-6B expression in a previously reported ATAC-seq atlas25, showing sorted T cells from Patient 59, an individual with CTCL, who had detectable levels of HHV-6B DNA within cells. (f) Smoothed coverage (rolling mean of 500 base pairs) over the four libraries from Patient 59, showing the coverage across the HHV-6B reference genome. (g) Schematic and results of previously described26 adoptive Treg therapy culture showing HHV-6 reactivation after reanalysis of primary data.

Extended Data Fig. 4 Supporting analyses for HHV-6 expression during in vitro CAR T cell culture.

(a) Summary of observed (red) and permuted (gray) HHV-6B expression for four donors at day 19 in culture. The dotted line is at 10 UMIs, the threshold for a super-expressor. (b) Heatmap of HHV-6B expression for selected cells across 3 donors with detectable super-expressors. Columns are grouped based on HHV-6B gene programs (immediate early; early; late). (c) UMAP for 3 samples, noting marker genes and HHV-6B UMI expression (log transformed). The WPRE feature indicates the presence of the CAR transgene.

Extended Data Fig. 5 Association of viral DNA and RNA in CAR T cell cultures.

(a) Schematic of the experiment where CAR T cells from D98 were profiled using the 10x Genomics Multiome workflow to detect both viral DNA and RNA. (b) Scatter plot of the abundance of viral DNA and RNA at single-cell resolution. Pearson correlation between the log10 abundances is shown. (c) Per-cell viral gene expression signatures. The proportion of viral gene expression belonging to each class (late, early, immediate early) per cell is shown. (d) Same plot as in (c) but colored by the log2 number of viral DNA fragments. The population of cells highly expressing early HHV-6 transcripts show a corresponding high HHV-6 DNA copy number is highlighted by the arrow. (e) Pearson correlation of HHV-6 transcript signatures with their log abundance of DNA fragments per cell. The two-sided p-value for the Pearson correlation test is noted by each bar. (f) Bulk-level RNA and DNA correspondence in the four donors studied in the day 19 allogeneic CAR products.

Extended Data Fig. 6 Supporting analyses of HHV-6 expression dynamics during in vitro CAR T cell culture.

(a) Immunofluorescence staining of two viral proteins (p41 and gB) and nuclei (DAPI) over the course of the cell therapy product culture. The donor and day of culture are noted for each panel. Images are representative of independent experiments that confirmed results of qPCR assay. (b) Summary of HHV-6B transcript expression in re-cultured samples for donors D61 and D34. (c) Summary of HHV-6B +/− cells from differential expression testing for host factors. Arrows indicate lymphotoxin α (LTα/LTA) and downregulation of lymphotoxin β (LTβ/LTB). (d) Correlation statistics of HHV-6B transcript signatures with OX40 expression across 3 recultured samples, including p-values from Pearson’s product-moment correlation coefficient. The consistently positive, significant correlation statistic represents an association uniquely between OX40 expression and the immediate early HHV-6B gene signature.

Extended Data Fig. 7 Quantification of HVV6 expression during the manufacturing of allogeneic CAR T cells from a donor with ciHHV-6 under different conditions.

(a) Schematic of experimental design. PBMCs from a ciHHV-6 donor were activated and subjected to four different conditions to assess the impact of individual steps of the CAR T cell manufacturing process. (b) Quantification of conditions in (a) over the 19 day culture process. HHV-6 copy number is shown and stable over the culture. (c) Per-gene HHV-6 viral RNA abundance at Day 1 and Day 19 of the full CAR T manufacturing process for the ciHHV-6 donor (left) and a donor previously analyzed with scRNA-seq (D98, right).

Extended Data Fig. 8 Mitigation of HHV-6 reactivation and spreading via foscarnet treatment in vitro.

(a) Schematic of CAR T product reculture experiment. Donor D97, which at day 19 showed a low but detectable level of HHV-6, was selected for reculture for five days. (b) Summary of RT-qPCR at the control and two treatment levels of Foscarnet. Each dot represents a technical replicate over one biological replicate per condition (validated in panel d). HHV-6 was not detected (n.d.) at the 1 mM concentration. Error bars represent the standard error of the mean. Comparison of foscarnet treated to untreated resulted in significantly lower abundance of HHV-6 RNA (p = 0.00026; two-sided ordinary least squares linear model). (c) Schematic of D34 reculture +/− foscarnet at 1 mM. (d) Difference between untreated and treated in the abundance of HHV-6+ cells. Comparing the two 10x Genomics scRNA-seq data channels, foscarnet-treated cells had a lower incidence of HHV-6 positive cells (OR = 6.25; p = 8.3e-122; Fisher’s exact test, two-sided). (e) Reduced dimensionality analysis of treated and untreated D34 cells profiled with scRNA-seq. Host gene expression was used for the analysis, showing overlapping clustering of populations irrespective of treatment status. (f) Differential gene expression analysis comparing foscarnet treated and control CAR T cells. The three most significant differential genes are noted. 0 genes were differentially expressed with a minimum log2 fold-change exceeding 1 (noted by the red).

Extended Data Fig. 9 Gene expression annotation for the Day 19 PBMC allogeneic CD7 dataset.

Expression of select marker genes supporting the annotations contained in Fig. 4c.

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Lareau, C.A., Yin, Y., Maurer, K. et al. Latent human herpesvirus 6 is reactivated in CAR T cells. Nature 623, 608–615 (2023). https://doi.org/10.1038/s41586-023-06704-2

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