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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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.
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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.
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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.
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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.
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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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DOI: https://doi.org/10.1038/s41586-023-06704-2
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