Abstract

Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies1,2,3. In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells4,5. Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient’s second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient’s CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.

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Acknowledgements

L. Tian, V. Gonzalez, N. Kengle, J. Scholler, Y. Wu, A. Bagg, C. Pletcher, B. Carreno, A. Bigdeli and A. Chew are acknowledged for research support. D. Campana and C. Imai provided the CD19-directed CAR under material transfer agreements. B. Jena and L. Cooper provided the CAR anti-idiotypic antibody. The OSU-CLL cell line was a kind gift from J. C. Byrd. This work was supported by funding from NCI T32CA009140 (J.A.F.), P01CA214278 (C.H.J.), AI104400, AI 082020, AI045008, AI117950 (F.D.B), NIAID K08AI101008 (S.A.C.), NIGMS R01GM118501 (R.M.K.), R01CA165206 (D.L.P. and C.H.J.), a Stand Up to Cancer Phillip A. Sharp Innovation in Collaboration Award (S.L.B. and C.H.J.) and Novartis.

Reviewer information

Nature thanks M. Maus and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

    • Morgan A. Sammons

    Present address: Department of Biology, University at Albany, State University of New York, Albany, NY, USA

    • Shannon A. Carty

    Present address: Department of Internal Medicine and Rogel Cancer Center, University of Michigan, Ann Arbor, MI, USA

Affiliations

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

    • Joseph A. Fraietta
    • , Stefan Lundh
    • , Tyler J. Reich
    • , Alexandria P. Cogdill
    • , Mercy Gohil
    • , Irina Kulikovskaya
    • , Farzana Nazimuddin
    • , Minnal Gupta
    • , Fang Chen
    • , Xiaojun Liu
    • , Regina M. Young
    • , David Ambrose
    • , Yan Wang
    • , Jun Xu
    • , Katherine T. Marcucci
    • , Bruce L. Levine
    • , Yangbing Zhao
    • , Michael Kalos
    • , David L. Porter
    • , Simon F. Lacey
    • , Carl H. June
    •  & J. Joseph Melenhorst
  2. Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    • Joseph A. Fraietta
    • , Stefan Lundh
    • , Shannon A. Carty
    • , Tyler J. Reich
    • , Alexandria P. Cogdill
    • , Mercy Gohil
    • , Irina Kulikovskaya
    • , Farzana Nazimuddin
    • , Minnal Gupta
    • , Fang Chen
    • , Xiaojun Liu
    • , Regina M. Young
    • , David Ambrose
    • , Yan Wang
    • , Jun Xu
    • , Martha S. Jordan
    • , Katherine T. Marcucci
    • , Bruce L. Levine
    • , Yangbing Zhao
    • , Michael Kalos
    • , David L. Porter
    • , Simon F. Lacey
    • , Carl H. June
    •  & J. Joseph Melenhorst
  3. Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    • Joseph A. Fraietta
    • , Jennifer J. D. Morrissette
    • , Martha S. Jordan
    • , Bruce L. Levine
    • , Michael Kalos
    • , Simon F. Lacey
    • , Carl H. June
    •  & J. Joseph Melenhorst
  4. Parker Institute for Cancer Immunotherapy, University of Pennsylvania, Philadelphia, PA, USA

    • Joseph A. Fraietta
    • , Carl H. June
    •  & J. Joseph Melenhorst
  5. Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    • Christopher L. Nobles
    • , Shantan Reddy
    • , Young Hwang
    • , John K. Everett
    • , Rahul M. Kohli
    •  & Frederic D. Bushman
  6. Department of Cell and Developmental Biology, Epigenetics Program, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    • Morgan A. Sammons
    • , Katherine A. Alexander
    • , Enrique Lin-Shiao
    •  & Shelley L. Berger
  7. Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    • Jamie E. DeNizio
    • , David L. Porter
    •  & Rahul M. Kohli
  8. Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    • Jamie E. DeNizio
    •  & Rahul M. Kohli
  9. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA

    • Marvin H. Gee
    •  & K. Christopher Garcia

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Contributions

J.A.F., C.L.N., M.A.S., S.A.C., J.J.D.M., R.M.Y., M.S.J., K.T.M., B.L.L., K.C.G., Y.Z., M.K., D.L.P., R.M.K., S.F.L., S.L.B., F.D.B, C.H.J. and J.J.M. designed experiments. J.A.F., C.L.N., M.A.S., S.L., S.A.C., T.R., A.P.C., J.J.D.M., J.E.D., S.R., Y.H., M.G., I.K., F.N., M.G., F.C., J.K.E., K.A.A., E.L.-S., M.H.G., X.L., D.A., Y.W. and J.X. performed experiments and/or analysed data. J.A.F., F.D.B., C.H.J. and J.J.M. wrote and edited the manuscript, with all authors providing feedback.

Competing interests

J.A.F., C.L.N., R.M.Y., B.L.L., M.K., D.L.P., S.F.L., F.D.B., C.H.J. and J.J.M. hold patents related to CTL019 cell therapy. These authors declare no additional competing interests. The remaining authors declare no competing interests.

Corresponding authors

Correspondence to Carl H. June or J. Joseph Melenhorst.

Extended data figures and tables

  1. Extended Data Fig. 1 Timeline of disease clearance by CAR T cells in Patient-10.

    An outline of clinical findings in Patient-10, including the results of bone marrow assessments, tumour cytogenetics and CAR T cell persistence. CTL019 cell infusion time points are indicated by red arrows.

  2. Extended Data Fig. 2 CAR T cell detection and profiling of immune cell populations in Patient-10 and other responders.

    a, Pre- and post-infusion kinetics of CAR T cell expansion (CD3+, CD8+ and CD8) are shown in Patient-10 compared to other responders. The number of circulating CTL019 cells was calculated based on frequencies of CD3+, CD8+ and CD8 CAR T cell populations and absolute cell counts. b, Flow cytometry gating strategy to identify peripheral blood CAR T cells in Patient-10. c, Relative percentages of CTL019 cells in the CD4 and CD8 compartments of this patient. T cells from a healthy subject served as a negative control. d, The persistence of CAR T cells in the peripheral blood of Patient-10 was determined by qPCR. The average threshold cycle (Ct) value obtained from three replicates and a standard deviation (SD) is listed. Calculations of CAR T cell abundance are reported as average marking per cell as well as transgene copies per microgram of genomic DNA. e, Frequencies of circulating B cells in Patient-10 compared to a healthy subject. Pre-gating was performed to exclude dead cells as well as doublets, and all gating thresholds were based on fluorescence minus one (FMO) controls (representative of two independent experiments). f, Enumeration of various immune cell populations in the blood of Patient-10. The frequency of each population is listed in a separate column that corresponds to its phenotypic marker.

  3. Extended Data Fig. 3 Clonal composition of CAR T cells from Patient-10 and other subjects treated with CTL019.

    a, TCRVβ distribution in CD8 (left) and CD8+ (right) CAR T cells in the cellular infusion product of Patient-10. b, Relative frequencies of CAR T cell clones in three patients who had complete responses to CTL019 therapy, summarized as stacked bar graphs. Each colour (horizontal bar) denotes a major cell clone, as marked by lentiviral integration sites. c, Integration site analysis in CAR T cells from two partially responding patients. d, In vivo expansion of CAR T cells in the above patients as determined by quantifying the average CAR transgene copies per microgram DNA at each time point.

  4. Extended Data Fig. 4 Detection of TET2 chimaeric transcripts in Patient-10 CAR T cells and DNA sequencing for mutation detection.

    a, The strategy for detection of polyadenylated RNA corresponding to truncated TET2 transcripts is depicted. Boxes represent the genomic regions between TET2 exons 9 and 10 with the integrated vector present. Blue and red arrows indicate general locations of the forward and reverse primers, which are listed below the diagram. LTR, long terminal repeat; cPPT, polypurine tract; EF1α, elongation factor 1-α promoter. Sequences corresponding to the splice junctions for the three chimaeric messages (five total junctions) are listed in the bottom chart. Underlines indicate consensus splice donors and acceptors. b, Visualization of chimaeric TET2 RT–PCR products. PCR products were separated on a native agarose gel and stained with ethidium bromide. Expected sizes of amplicons are listed above the gel. Truncated transcripts are highlighted by blue boxes. A key to the RT–PCR reactions is shown below the diagram. *Band size not determined (two independent experiments). c, Genes interrogated by the next generation sequencing panel used to analyse DNA isolated from CD8+CAR+ T cells and CAR T cells in Patient-10 at the peak of his response. d, Sanger sequencing of specific amplifications corresponding to the allele that was disrupted by integration of the CAR lentivirus is shown. The mutation that was detected by next generation sequencing of total genomic DNA from CAR+ T cells (Fig. 3c) is not present in the TET2 allele hosting the lentiviral integration site.

  5. Extended Data Fig. 5 Analysis of DNA methylation variants from HEK293T cells overexpressing TET2.

    a, Depiction of sequential oxidations of 5-mC to 5-hmC, 5-fC and 5-caC catalysed by TET2 (top). Dot blots for 5-mC, 5-hmC, 5-fC and 5-caC in genomic DNA (gDNA) isolated from HEK293T cells transfected with the E1879Q TET2 mutant are shown. Assay controls include an empty vector, wild-type TET2 and a catalytically inactive (HxD) TET2 mutant (bottom left). A western blot using anti-FLAG antibody to detect hTET2 in the above cells is also shown. Hsp90α/β was used as a loading control (bottom right). Brightness and contrast were adjusted evenly across blots. b, Original, uncropped DNA (left) and western (right) blots. A dilution series was used for semiquantitative analysis of DNA methylation variants. Representative results of three independent experiments are shown. c, Validation of anti-FLAG and anti-Hsp90α/β antibodies. Serial dilutions of lysates (0.008, 0.04, 0.2, 0.8, 4.0 µg µl−1) obtained from HEK293T cells transfected with wild-type TET2. Gels were probed by western blot. Both chemiluminescence (left) and digital (right) images were captured, demonstrating that these antibodies exhibit specificity for the expected FLAG tag and Hsp90 based on molecular weight markers. d, 5-mC, 5-hmC, 5-fC and 5-caC nucleosides were analysed at fixed concentrations using LC–MS/MS to generate standard curves. The area under the curve (AUC) was calculated for each MS/MS fragment. In the case of 5-hmC, the slope was further adjusted because of quality control analysis of an equimolar mixture of oligonucleotides, each containing a single modification. e, Analysis of gDNA from individual biological replicates within each HEK293T cell group. The top chart lists the raw AUCs that were converted to relative amounts of modified cytosine (middle) according to their signal intensities from the respective standard curves. The percentage of each modified cytosine calculated for each sample is shown in the bottom chart. Results are from three independent experiments.

  6. Extended Data Fig. 6 Global chromatin profiling of CAR+ and CAR T cells from Patient-10.

    a, Venn diagrams of high-confidence differential ATAC–seq regions (left) and enrichment of those peaks in regions of the diagrams (right) in CAR+ and CARCD8+ T cells expanded from Patient-10 (two biological replicates analysed in two independent experiments). Boxes extend from the 25th to 75th percentiles, the middle line denotes the median and whiskers show minimum and maximum. b, Gene Ontology terms associated with chromatin regions that are significantly more open in CD8+CAR+ T cells from Patient-10 compared to their matched CD8+CAR T cell counterparts. c, Ontology analysis for chromatin regions that are less accessible in CD8+CAR+ T cells than in CD8+CAR T cells. d, Enrichment of transcription factor (TF) binding motifs in chromatin regions gained or lost in CAR+ compared to CAR T cells from Patient-10. Transcription factor motifs that were potentially more accessible in increased ATAC–seq peaks of CAR+ T cells included E26 transformation-specific (ETS) (GABPα, ELF1, Elk4) and zinc finger (ZF) transcription factor (Sp1) binding sites that are known to be enriched in human CD8+ T cells before differentiation occurs47. Transcription factor motifs that were potentially less accessible owing to reduced ATAC–seq peaks in CAR+ T cells from Patient 10 (NF-κB, IRF1, NFAT–AP1 and CTCF) are enriched in terminally differentiated effector and exhausted T cells and have known key roles in forming the epigenetic landscape that programs their biology38.

  7. Extended Data Fig. 7 The differentiation state of CAR T cells in Patient-10 compared to other responders over time.

    a, Representative contour plots of flow cytometric data depicting the frequency of CAR+ and CARCD8+ T cells in Patient-10 that express HLA-DR. The proportions of these cells that express CD45RO and CCR7 as determinants of differentiation status are shown. Contour plot insets indicate the frequencies of the gated cell populations. b, Example gating strategy used to determine the differentiation phenotype of CD8+CAR+ and CAR T cells from a complete responder (top left). Line graphs depict the differentiation state of these cell populations in other responding patients over time and are plotted with corresponding CAR T cell levels in the blood, as determined by qPCR.

  8. Extended Data Fig. 8 Effect of TET2 expression on T cell differentiation and viability.

    a, Representative flow cytometry plots showing the differentiation state of healthy donor CD8+CAR+ T cells after transduction with a scrambled shRNA (control) or shRNA targeting TET2. Insets define frequencies of gated populations. b, Frequencies of healthy subject CAR+CD8+ (top) and CAR+CD4+ (bottom) T cells according to differentiation phenotype following control or TET2 shRNA transduction (n = 10; pooled results from four independent experiments). P values were determined using a two-tailed, paired Student’s t-test. c, Comparison of the expression levels of TET2 in naive and memory CD8+ T cell subsets from three healthy donors. Two variants encoding different isoforms have been identified for this gene in humans. Expression levels of each TET2 variant were estimated by measuring the probe intensity from microarray analysis. d, Viability of CAR+ T cells transduced with a TET2 shRNA or scrambled control and restimulated with K562 cells expressing CD19 (n = 12; pooled results from three independent experiments). Each arrow indicates the time point at which CAR T cells were exposed to antigen. Error bars depict s.e.m.

  9. Extended Data Fig. 9 CAR T cell cytokine profiles following TET2 inhibition.

    a, Representative flow cytometry of acute intracellular cytokine production by healthy donor (n = 6; three independent experiments) CAR T cells transduced with a TET2 shRNA or a scrambled control shRNA (left). Production of IFNγ, TNFα and IL-2 by total CD3+, CD4+ and CD8+ CAR T cells is shown. These cells were stimulated with beads coated with anti-CD3 and anti-CD28 antibodies (top right) or CAR anti-idiotypic antibodies (bottom right). Boxes represent the 25th to 75th percentiles, the middle line denotes the median and whiskers depict minimum and maximum. b, Heat map and cluster analysis of cytokine profiles for CAR T cells transduced with a TET2 shRNA or scrambled control and serially restimulated with irradiated K562 cells expressing CD19 are shown. Colours represent scaled cytokine data corresponding to each stimulation time point. Hierarchal clustering was used to generate the cluster dendrogram and cytokine response groups. c, Production of IFNγ (top), TNFα (middle) and IL-2 (bottom) by TET2 knockdown or control CAR T cells (n = 6; three independent experiments) following restimulation with CD19 antigen. Black arrows indicate when CAR T cells were exposed to CD19-expressing K562 cells. Error bars denote s.e.m. All P values were determined using a two-tailed, paired Student’s t-test.

  10. Extended Data Fig. 10 Effect of TET2 knockdown on the cytotoxic machinery of CAR T cells.

    a, Flow cytometry plots showing the frequency of TET2 knockdown or control CAR T cells expressing CD107a (a marker of cytolysis) following CD3 and CD28 or CAR-specific stimulation (left). Summarized data from analysis of CAR T cells manufactured from n = 6 different healthy donors is shown (right). b, Representative histograms illustrating expression levels of granzyme B and perforin in CAR T cells in the setting of TET2 inhibition as compared to its counterpart control (left). Pooled data from CAR T cells of n = 5 healthy donors are summarized on the right. c, Cytotoxic capacity of CTL019 cells (transduced with a TET2 or scrambled control shRNA) after overnight co-culture with luciferase-expressing OSU-CLL (left) or NALM-6 (right) cells. Untransduced T cells were included as an additional group to control for non-specific lysis. P values were determined using a two-tailed, paired Student’s t-test. All data were pooled from three independent experiments.

  11. Extended Data Fig. 11 Effector and memory molecule expression by CAR T cells from Patient-10 compared to those from other responding subjects.

    a, Expression of granzyme B (left) and the frequency of CAR and CAR+ T cells co-expressing granzyme B/Ki-67 (right panel) at the peak of in vivo CTL019 expansion in Patient-10 compared to three other complete responders. b, Representative histograms of intracellular Eomes expression (left), and contour plots depicting frequencies of CD27- (middle) and KLRG1-expressing (right) lymphocytes in the same cell populations of these patients. These results are representative of three experiments repeated independently with comparable findings.

Supplementary information

  1. Supplementary Tables

    This file contains Supplementary Tables 1-3.

  2. Reporting Summary

  3. Supplementary Table 4

    Genes associated with more open or closed ATAC peak regions in CD8+ CAR+ compared to CD8+ CAR− Tcells from Patient-10.

  4. Supplementary Table 5

    Gene set enrichment analysis reports showing GO and KEGG pathways and hallmark gene sets associated with ATAC-seq peaks gained or lost in CD8+ CAR+ compared to CD8+ CAR− Patient 10 Tcells.

  5. Supplementary Table 6

    List of uniquely enriched transcription factor binding motifs present in peaks gained or lost in CD8+ CAR+ compared to CD8+ CAR− Tcells from Patient-10.

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https://doi.org/10.1038/s41586-018-0178-z

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