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TET2 guards against unchecked BATF3-induced CAR T cell expansion

Nature volume 615, pages 315–322 (2023)Cite this article

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Abstract

Further advances in cell engineering are needed to increase the efficacy of chimeric antigen receptor (CAR) and other T cell-based therapies1,2,3,4,5. As T cell differentiation and functional states are associated with distinct epigenetic profiles6,7, we hypothesized that epigenetic programming may provide a means to improve CAR T cell performance. Targeting the gene that encodes the epigenetic regulator ten–eleven translocation 2 (TET2)8 presents an interesting opportunity as its loss may enhance T cell memory9,10, albeit not cause malignancy9,11,12. Here we show that disruption of TET2 enhances T cell-mediated tumour rejection in leukaemia and prostate cancer models. However, loss of TET2 also enables antigen-independent CAR T cell clonal expansions that may eventually result in prominent systemic tissue infiltration. These clonal proliferations require biallelic TET2 disruption and sustained expression of the AP-1 factor BATF3 to drive a MYC-dependent proliferative program. This proliferative state is associated with reduced effector function that differs from both canonical T cell memory13,14 and exhaustion15,16 states, and is prone to the acquisition of secondary somatic mutations, establishing TET2 as a guardian against BATF3-induced CAR T cell proliferation and ensuing genomic instability. Our findings illustrate the potential of epigenetic programming to enhance T cell immunity but highlight the risk of unleashing unchecked proliferative responses.

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Fig. 1: Effect of TET2 disruption on CAR T cell therapeutic efficacy is dependent on CAR design.
Fig. 2: Effect of CAR design on long-term T cell accumulation upon CRISPR–Cas9 editing of the TET2 locus.
Fig. 3: Hyperproliferative TET2-edited CAR T populations are oligoclonal and biallelically edited for TET2.
Fig. 4: Loss of effector function in hyperproliferative TET2bed CAR T cells.
Fig. 5: The BATF3–MYC axis drives hyperproliferation of TET2bed CAR T cells.

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Data availability

Data generated from RNA-seq and ATAC-seq have been deposited in the Gene Expression Omnibus with the accession number GSE220259. The publicly available datasets used in this study are GSE23321 for central memory and effector memory phenotype comparison, AKL_HTLV1_UP (M7705), AKL_HTLV1_DN (M9815), the angioimmunoblastic T cell lymphoma dataset (GSE6338) and HALLMARK_MYC_V1 (M5926). Source data are provided with this paper.

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Acknowledgements

We thank members of the Sadelain laboratory for helpful discussion and feedback; C. Zebley, B. Youngblood, K. Helin and the Sloan Kettering Institute Centre of Epigenetics Research for advice on epigenetic analysis; J. Boyer for western blot support; N. Socci for advice on exome analysis; M. Schmidt for retroviral integration site analysis; S. Monette and A. Michel from the SKI/CUMC laboratory of Comparative Pathology for conducting pathology analysis; and the following SKI core facilities for their support: Flow Cytometry, Centre of Comparative Medicine and Pathology, Anti-tumour Assessment, Molecular Cytology, Bioinformatics, Integrated Genomics Operation and Cell Therapy and Cell Engineering. Illustrations in Figs. 1a, 2c and 5h and Extended Data Figs. 4a, 7a and 10a were generated using Servier Medical Art. This work was supported by the Pasteur–Weizmann/Servier award, the Leopold Griffuel award, the Leukemia and Lymphoma society (LLS ID: 7014-17) and the MSKCC core grant (P30 CA008748).

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Author notes

  1. Judith Feucht

    Present address: University Children’s Hospital, Tübingen, Germany

  2. These authors contributed equally: Nayan Jain, Zeguo Zhao

Authors and Affiliations

  1. Louis V. Gerstner Jr Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Centre, New York, NY, USA

    Nayan Jain & Anton Dobrin

  2. Centre for Cell Engineering and Immunology Program, Memorial Sloan Kettering Cancer Centre, New York, NY, USA

    Nayan Jain, Zeguo Zhao, Judith Feucht, Archana Iyer, Anton Dobrin, Jorge Mansilla-Soto, Michael Lopez, Gertrude Gunset & Michel Sadelain

  3. Centre for Epigenetics Research, Memorial Sloan Kettering Cancer Centre, New York, NY, USA

    Richard Koche, Julie Yang & Yingqian Zhan

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Contributions

N.J. and Z.Z. designed the study, performed the experiments, analysed and interpreted data and wrote the manuscript. A.I. and M.L. contributed to RNA-seq and exome analysis. R.K., J.Y. and Y.Z. contributed to ATAC-seq analysis. J.F. and A.D. contributed to animal studies. J.M.-S. contributed to gene targeting. G.G. contributed to vector construction, T cell transduction and animal studies. M.S. designed the study, analysed and interpreted data and wrote the manuscript.

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Correspondence to Michel Sadelain.

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Extended data figures and tables

Extended Data Fig. 1 Rv-1928z and Rv-19BBz pre-infusion and in vivo CAR T cell phenotyping.

a,b, Pre-infusion transduction efficiency and phenotyping by flow cytometry of Rv-1928z (a) and Rv-19BBz (b) CAR T cells. c,d, Tumour monitoring of NALM6 bearing mice treated with Rv-1928z (c) and Rv-19BBz (d) CAR T cells. e,f, Bone marrow (e) and Splenic (f) CAR T cell quantification at 3 weeks post infusion. Data is represented as mean±SE [n = 5 (Rv-1928z), n = 6 (Rv-19BBz)]. g,h Differentiation phenotyping of pooled bone marrow CAR T cells at week 3 post infusion. Data from another experiment included in supplementary information. i, CAR T cell inhibitory receptor expression at week 3 post infusion from mouse bone marrow (n = 3). p values were determined by two-sided Mann–Whitney test (e,f) and two-sided χ2 test (h). p < 0.05 was considered statistically significant. p values are denoted: p > 0.05, not significant, NS; *, p < 0.05. Replicate information for g,i are available in Supplementary Table 3. Exact p values are available in Supplementary Table 4.

Source data

Extended Data Fig. 2 Rv-1928z+41BBL and TRAC-1928z pre-infusion and in vivo CAR T cell phenotyping.

a,b, Pre-infusion transduction efficiency and phenotyping by flow cytometry of Rv-1928z+ 41BBL (a) and TRAC-1928z (b) CAR T cells. c,d, Tumour monitoring of NALM6 bearing mice treated with Rv-1928z+41BBL (c) and TRAC-1928z (d) CAR T cells. e,f, Differentiation phenotyping of pooled bone marrow CAR T cells at week 3 post infusion. Data from another experiment included in supplementary information. g, CAR T cell inhibitory receptor expression at week 3 post infusion from mouse bone marrow (n = 3). p values were determined by two-sided χ2 test (f). p < 0.05 was considered statistically significant. p values are denoted: p > 0.05, not significant, NS; *, p < 0.05. Replicate information for e,g are available in Supplementary Table 3.

Source data

Extended Data Fig. 3 Long-term CAR T cell phenotypes upon CRISPR/Cas9 editing of TET2 locus.

a,b, Differentiation phenotyping of retrovirally encoded CAR T cells (day 90) and TRAC-1928z CAR T cells (day 75) isolated from the bone marrow. c, Inhibitory receptor expression of bone marrow Rv-1928z, Rv-19BBz, Rv-1928z+41BBL (day 90) and TRAC-1928z (day 75) CAR T cells. p values were determined by two-sided χ2 test (b). p < 0.05 was considered statistically significant. p values are denoted: p > 0.05, not significant, NS; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Replicate information for a,c are available in Supplementary Table 3.

Extended Data Fig. 4 Effect of TET2 editing on CAR T cell accumulation in a prostate cancer model.

a, Schematics of the prostate cancer experimental design. TET2 was edited with the previously discussed gRNA (g1) and an alternative gRNA (g2). PSMA28z+41BBL (PSMA targeted, CD28 costimulated CAR that expresses 41BBL ligand) was used in this study (Dose: 2e5). b, CAR T cell counts in the peripheral blood 30 days post infusion of T cells. Bars show median values. c, Mice with the top 4 CAR T cell peripheral counts at day 30 across both TET2 targeting gRNA (g1, n = 2. g2, n = 2) were euthanized at day 45 along with 5 scrambled gRNA treated PSMA28z+41BBL mice and their splenic CAR T cell numbers were quantified. p values were determined by two-sided Mann-Whitney (b, c) [n = 5 (WT PSMA28z+41BBL), n = 8 (TET2etdg1 PSMA29z+41BBL), n = 11 (TET2etdg2 PSMA29z+41BBL)]. p < 0.05 was considered statistically significant. p values are denoted: *, p < 0.05; **, p < 0.01. Exact p values are available in Supplementary Table 4. The mouse illustration in part a was generated using Servier Medical Art, CC BY 3.0.

Source data

Extended Data Fig. 5 Clonal expansion in all 4 hyper-proliferative CAR T cell populations.

a, Gel image of PCR product for WT CAR T cells and hyper-proliferative TET2-edited CAR T cells. The PCR is designed to amplify the site of gRNA editing. b, Enrichment of TET2-editing from pre-infusion (day 0) in mice to day 21 in Rv-1928z and Rv-1928z+41BBL CAR T cells. p values were determined by two-sided χ2 test. c,d, TCRvβ sequencing reveals hyper-proliferative populations that are dominant for a single clone in TET2bed Rv-1928z (c) and Rv-1928z+41BBL (d). Part of the retroviral vector that was inserted in the TET2 alleles of these clones is highlighted in the figures. e–g, Examples of hyper-proliferative Rv-1928z+41BBL CAR T cell populations that are oligoclonal (left panel) with biallelic TET2 editing (right panel). h, Western blot showing total loss of TET2 at protein level in different hyper-proliferative populations. i,j, Examples of oligoclonality in TET2bed TRAC-1928z (i) and Rv-19BBz (j). p < 0.05 was considered statistically significant. p values are denoted: p > 0.05, not significant, NS; *, p < 0.05; **, p < 0.01.

Source data

Extended Data Fig. 6 TCR is dispensable for emergence of hyper-proliferative phenotype in TET2-edited Rv-1928z+41BBL CAR T cells.

a,b, Differentiation phenotyping of TCR+TET2etd RV-1928z+41BBL (a) and TCR−TET2etd RV-1928z+41BBL (b) CAR T cells. c, Summary of emergence of hyper-proliferative phenotype post CAR T cell infusion in mice for different donors. Mice were monitored for 90 days. 2e5 CAR T cells were used for both the groups.

Extended Data Fig. 7 Properties of the chimeric antigen receptor design determine composition of TET2bed hyper-proliferative populations.

a, Rv-1928z or Rv-1928z+41BBL CAR T cells were generated from the same donor to assess the effect of CAR design on clonal persistence. 5 Mice were euthanized at day 21 to assess clonal diversity post tumour clearance. 15 mice were followed for emergence of a hyper-proliferative phenotype. b,c, Pair-wise analysis of Rv-1928z (b) and Rv-1928z+41BBL (c) at day 0 and day 21. d, Top 100 Rv-1928z clones at infusion were mapped in the Rv-1928z+41BBL infusion product. These clones were then assessed at day 21 for both the CAR receptors. p values were determined by two-tailed Mann-Whitney test. e,f, Pair-wise analysis (day 0 vs day 90) of the lone hyper-proliferative population found at day 90 for Rv-1928z CAR receptor (e). Representative pair-wise analysis (day 0 vs day 90) of a Rv-1928z+41BBL hyper-proliferative population (f). g, Changes in clonality index over time in Rv-1928z and Rv-1928z+41BBL CAR T cells. h,i, Tracking the fate of the 100 most abundant pre-infusion clones in the hyper-proliferative populations of Rv-1928z (h) and Rv-1928z+41BBL (i). (j) Retro-tracking late-stage dominant clones in the infusion product (Day 0). All dominant clones were isolated at day 90 except for 2-00 which was isolated at day 200. p < 0.05 was considered statistically significant. p values are denoted: p > 0.05, not significant, NS; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. The human, mouse and lipid bilayer illustrations in part a were generated using Servier Medical Art, CC BY 3.0.

Source data

Extended Data Fig. 8 In vitro and in vivo effector function assessment of TET2-edited and hyper-proliferative TET2bed CAR T cells.

a,b, In vitro cytolytic activity assessment upon co-culture with NALM6 for 16-h as determined by luciferase activity for pre-infusion TET2-edited Rv-1928z (n = 3) and hyper-proliferative TET2bed Rv-1928z (2-2) (n = 3) (a) and pre-infusion TET2-edited Rv-1928z+41BBL (n = 3) and hyper-proliferative TET2bed Rv-1928z+41BBL (17-1) (n = 3) (b). Data is represented as mean±SD. c, NALM6 bearing NSG mice were treated with 2e6 hyper-proliferative TET2bed Rv-1928z (n = 7) or TET2bed Rv-1928z+41BBL (n = 7) CAR T cells to assess their in vivo anti-tumour efficacy. d, Normalized transcript counts of WT Rv-1928z+41BBL and TET2bed Rv-1928z+41BBLCAR T cells isolated from mice at day 90. R=Rest (Transcript counts at isolation). S= Stimulated (Transcript counts 24 h post CD3/28 stimulation). Data is represented as mean±SD (n = 3). e, Schematic of in vitro repeated rechallenge assay for effector function analysis. f,g, Day 1 in vitro cytolytic activity assessment (f) and effector cytokine assessment (g). h,i, Day 8 in vitro cytolytic activity assessment (h) and effector cytokine assessment (i). j,k, Day 15 in vitro cytolytic activity assessment (j) and effector cytokine assessment (k). Data in f–k is represented as mean±SD (n = 3). l, TCF1 staining of WT Rv-1928z+41BBL and TET2bed Rv-1928z+41BBL CAR T cells isolated from mice at day 90. WT samples were a pool of 5 mice. TCF1 staining of other hyper-proliferative TET2bed CAR T cells in Supplementary Table 3. p values in a,b,f,h,j were determined by two-sided Student’s unpaired t-test corrected by BKY method. p values in c were determined by two-sided Mann-Whitney test. p values in d,g,i,k were determined by two-sided unpaired t-test. p < 0.05 was considered statistically significant. p values are denoted: p > 0.1, not significant, ns. p < 0.1 are indicated. *, p < 0.05. **, p < 0.01. ***, p < 0.001. ****, p < 0.0001. Exact p values are available in Supplementary Table 4.

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Extended Data Fig. 9 No conserved secondary genetic mutation between different hyper-proliferative TET2bed CAR T populations dominant for a single clone.

a, (Right panel) Copy number changes in TET2bed Rv-1928z+41BBL (17-1). The top panel displays log (ratio) denoted by “(logR)” with chromosomes alternating in the blue and gray. The middle panel displays log (odds-ratio) denoted by “(logOR)”. Segment means are plotted in red lines. In the bottom panel total (black) and minor (red) copy number are plotted for each segment. The bottom bar shows the associated cellular fraction (cf). Dark blue indicates high cf. Light blue indicates low cf. Beige indicates a normal segment (total=2, minor=1). The table shows genetic events occurring at >0.1 cf. (Left panel) CAR T cell clonality as determined by vβ sequencing in TET2bed Rv-1928z+41BBL (17-1). b, Nonsynonymous acquired point mutations in TET2bed Rv-1928z+41BBL (17-1). Mutations that occur at a frequency > ((dominant TCRvβ frequency/2) -0.1) or >0.3 whichever is lower is annotated. c, (Right panel) Copy number changes in TET2bed Rv-1928z (2-2). (Left panel) CAR T cell clonality as determined by vβ sequencing in TET2bed Rv-1928z (2-2). d, Nonsynonymous acquired point mutations in TET2bed Rv-1928z (2-2). e, (Right panel) Copy number changes in TET2bed TRAC-1928z (4-1). (Left panel) CAR T cell clonality as determined by vβ sequencing in TET2bed TRAC-1928z (4-1). f, Nonsynonymous acquired point mutations in TET2bed TRAC-1928z (4-1).

Extended Data Fig. 10 Hyper-proliferative TET2bed Rv-1928z+41BBL do not achieve uncontrolled proliferative state upon secondary transplant.

a, Schematics of secondary transplant of hyper-proliferative TET2bed Rv-1928z+41BBL cells. The exogenous cytokine supplement had to be stopped at day 60 due to deteriorating mice condition in response to frequent injections. b, CAR T cell quantification in peripheral blood under different exogenous supplementation at day 30, day 60 and day 75. Each dot represents a mouse. UD: undetected. Data is represented as mean±SD (n = 5). c, CAR T cell quantification in bone marrow and spleen at day 150 post CAR T cell infusion. Data is represented as mean±SD (n = 5 for no supplement, and IL2. n = 4 for IL7/15). p values were determined by two-sided Mann–Whitney test (b). p < 0.05 was considered statistically significant. p values are denoted: p > 0.05, not significant, NS; *, p < 0.05; **, p < 0.01. (b). Exact p values are available in Supplementary Table 4. The mouse illustration in part a was generated using Servier Medical Art, CC BY 3.0.

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Supplementary information

Supplementary Figures

This file contains Supplementary Figs. 1–4.

Reporting Summary

Supplementary Table 1

Exome analysis of hyper-proliferative TET2bed CAR T cells. 1a, Translocation analysis in TET2bed hyper-proliferative CAR T cells. 1b, Mutation analysis in TET2bed hyper-proliferative CAR T cells. 1c, Copy number analysis in TET2bed hyper-proliferative CAR T cells.

Supplementary Table 2

Retroviral integration site analysis in hyper-proliferative TET2bed CAR T cells. 2a, Retrovirus integration site analysis for TET2bed Rv-1928z+41BBL (17-1). 2b, Retrovirus integration site analysis for TET2bed Rv-1928z (2-2).

Supplementary Table 3

Replicate information on representative figures. 3a-d, Replicate information for selected panels in Extended Data Fig. 1 (a), Extended Data Fig. 2 (b), Extended Data Fig. 3 (c), and Extended Data Fig. 8 (d).

Supplementary Table 4

Exact p values for figures. 4a-i, Exact p values for selected panels in Fig. 2 (a), Fig. 4 (b), Fig. 5 (c), Extended Data Fig. 1 (d), Extended Data Fig. 4 (e), Extended Data Fig. 8 (f), Extended Data Fig. 10 (g), Supplementary Fig. 1 (h), Supplementary Fig. 2 (i).

Supplementary Table 5

List of antibodies used in the flow cytometry.

Supplementary Data

Source Data for Supplementary Fig. 2.

Source data

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Jain, N., Zhao, Z., Feucht, J. et al. TET2 guards against unchecked BATF3-induced CAR T cell expansion. Nature 615, 315–322 (2023). https://doi.org/10.1038/s41586-022-05692-z

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