Abstract
The adoptive transfer of T lymphocytes reprogrammed to target tumour cells has demonstrated potential for treatment of various cancers1,2,3,4,5,6,7. However, little is known about the long-term potential and clonal stability of the infused cells. Here we studied long-lasting CD19-redirected chimeric antigen receptor (CAR) T cells in two patients with chronic lymphocytic leukaemia1,2,3,4 who achieved a complete remission in 2010. CAR T cells remained detectable more than ten years after infusion, with sustained remission in both patients. Notably, a highly activated CD4+ population emerged in both patients, dominating the CAR T cell population at the later time points. This transition was reflected in the stabilization of the clonal make-up of CAR T cells with a repertoire dominated by a small number of clones. Single-cell profiling demonstrated that these long-persisting CD4+ CAR T cells exhibited cytotoxic characteristics along with ongoing functional activation and proliferation. In addition, longitudinal profiling revealed a population of gamma delta CAR T cells that prominently expanded in one patient concomitant with CD8+ CAR T cells during the initial response phase. Our identification and characterization of these unexpected CAR T cell populations provide novel insight into the CAR T cell characteristics associated with anti-cancer response and long-term remission in leukaemia.
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Data availability
Raw sequencing data for this study are available at dgGaP under accession no. phs002931.v1.p1.
Code availability
Analysis code are available on request.
Change history
07 December 2022
A Correction to this paper has been published: https://doi.org/10.1038/s41586-022-05376-8
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Acknowledgements
We acknowledge the contributions of the following research cores at the University of Pennsylvania and Children’s Hospital of Philadelphia: the Translational and Correlative Studies Laboratory for providing standardized flow cytometric, quantitative PCR and cytokine multiplexing analyses, and biobanking of patient specimens on CAR T cell trials; the Clinical Cell and Vaccine Production Facility for cell processing and biobanking; the Human Immunology Core for providing healthy donor cells and analytical support; the Flow Cytometry Core for flow cytometry equipment maintenance and access to electronic sorters; the Stem Cell and Xenograft Core for providing de-identified patient specimens; and the High-Performance Computing Facility. This work was supported by Novartis Institute for Biomedical Research (to J.J.M. and C.H.J.), NIH grant R01-CA-241762-01 (to J.J.M. and F.D.B.); NIH grant CA233285 (to K.T.); CIHR Doctoral Foreign Study Award no. 433117 (to G.M.C.); and NIH Medical Scientist Training Program T32 GM07170 (to S.B.).
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J.J.M., G.M.C., M.W., D.L.P., C.C., M.A.C., P.G., S.B., H.S., Z.Z., S.L., I.P.-M., C.L.N., S.M., L.T., I.K., M.G., D.E.A., F.D.B., S.F.L., K.T. and C.H.J. performed experiments or analysed the data. All authors helped design the experiments and contribute to data interpretation. J.J.M., G.M.C., M.W., D.L.P., K.T. and C.H.J. wrote the manuscript.
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J.J.M., D.L.P., J.A.F., S.F.L. and C.H.J. hold patents related to CAR T cell manufacturing and biomarker discovery. I.P.-M. and J.B. are employees of Novartis. The remaining authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Clonal evolution for patient 1 and 2 based on TCR sequencing data.
Pairwise Morisita’s overlap index was computed between all timepoints (row and column labels) for patient 1 (a) and 2 (b). TCR clones (rows) with maximum abundance > 1% across time points were retained and tracked over time for patient 1 (c) and 2 (d).
Extended Data Fig. 2 Genomic annotation of integration sites for infusion product, post infusion timepoints < 60d and > 60d.
a, graphic of the annotation scheme. The integration sites from patient 1 (b) and 2 (c) were annotated based on its position relative to known genes (UCSC hg38) and permissive enhancers (FANTOM 5). The counts of integration sites that fall into each annotation category in infusion product were summarized (left). The mean and standard deviation of the number of sites for each category were also computed for 1–60 days post infusion (middle) and > 60 days post infusion (right).
Extended Data Fig. 3 Gating strategy and CyTOF marker expression profiles.
a, Gating strategy performed computationally on CyTOF data to filter to CD3+CAR+ T cells for downstream analysis. b, Protein expression of our CyTOF panel depicted on a single-cell basis on our UMAP.
Extended Data Fig. 4 CITE-Seq with 5’ TCR profiling reveals the presence of double-negative gamma-delta CAR T-cells in patient 2 at month 3 and year 3.
a, UMAP showing expression of key marker genes and TCRαβ clonotype for patient 2 at month 3, and (b) patient 2 at year 3. Aliquots of peripheral blood were sorted for CD3+CD14−CAR+ cells, and 5’ CITE-Seq with TCRαβ clonotyping was performed. High-quality cells were computationally identified by retaining cells with 200–5000 genes detected and less than 5% mitochondrial RNA, and shown are the 552 (month 3) and 242 (year 3) cells that were verified as CAR T-cells with at least one read aligned to the 5’ CAR construct. UMAP plots showing normalized RNA expression are colored in shades of blue; plots showing protein expression via CITE-Seq antibody-derived tags are colored in shades of green; TCRαβ clonotype plots (bottom-right of panels a and b) are colored using a spectral color scheme, with cells with no detected TCRαβ clonotype colored light grey. Red arrows indicate the double-negative CAR T-cell population in both time points, with gamma-delta identity demonstrated by protein expression of the γδ TCR, lack of protein expression of the αβ TCR, specific RNA expression of TRDV1 and TRGV4, and non-detection of the TCRαβ clonotype.
Extended Data Fig. 5 Re-analysis of previous published flow cytometry data performed on patient 2 demonstrates the functional capacity of CD4+, CD8+, and double-negative CAR T-cells.
a, Gating strategy for the identification of CD4+, CD8+, and double-negative CAR T-cells. Flow cytometry data are from the functional experiment described by Porter et al. Sci Transl Med (2015), in which the authors stimulated cells from patient 2 with CD19 expressing K562 cells. Re-examination of the flow cytometry data identified prominent CD4+, CD8+, and double-negative CAR T-cell populations. Shown is representative gating at day 259, in which 31.2% of CAR T-cells were double-negative CAR T-cells. b, Line plots and box plots showing CAR-specific activation of CD4+, CD8+, and double-negative CAR T-cells supported by greater proportions of CAR+ T-cells expression MIP-1B and CD107a compared to CAR− cells in response to CAR specific stimulation. Pairwise statistical testing was performed using the two-sided Welch’s t-test.
Extended Data Fig. 6 Analysis of CAR T-cell clonotype, cell cycle, and differential gene expression from patient 1 at year 9.3.
a, Heatmap showing the relative frequencies of TCR clonotypes at the 2-month, 3-month, 15-month, 18-month, 21-month, and 9-year time points. Note that the first five columns were estimated from bulk TCR sequencing, whereas the rightmost column was estimated from the single-cell TCR/CITE-Seq data from year 9. b, UMAPs indicating strong up-regulation of RNA expression of cell cycle genes. c, UMAP colored by cell cycle phase using Seurat. d, Proportions of cells in each cell cycle phase, compared between CAR− T and CAR+ T cells. Chi-squared p-value = 8.97e-15. e, Proportions of cells in each cell cycle phase, compared between the top six CAR T-cell clonotypes. Pairwise statistical significance was assessed with the Chi-Squared test, and multiple-testing correction was performed using the Benjamini-Hochberg method. Numbers within the bars indicate the number of cells observed. f, Volcano plot indicating genes up-regulated in CAR T-cells compared to normal CD4+ T cells (rightward direction) and genes down-regulated in CAR T-cells compared to normal CD4+ T cells (leftward direction). Differentially expressed genes were determined using the Wilcoxon rank-sum test with a Bonferroni-adjusted p-value cutoff of 0.001 (dark red) and 0.05 (red). g, Gene Set Enrichment Analysis plot for the effector CD4+ gene signature. h, Heatmap indicating normalized gene expression values for the 32 differentially expressed genes with a Bonferroni-adjusted p-value cutoff of 0.001.
Extended Data Fig. 7 CITE-Seq protein expression and correlation for patient 1 at year 9.3.
a, UMAP colored by normalized expression of CITE-Seq protein expression determined by antibody-derived tags. b, Pairwise Spearman correlations between CITE-Seq protein expression values across cells.
Extended Data Fig. 8 Flow cytometry analysis of functional experiment on CAR T-cells from patient 1 year 9.3.
a. Representative gating strategy to identify CD4+ CAR T-cells from the functional assay. b. Identification of populations expressing functional markers CD107a, MIP-1β, Perforin, and Granzyme A. Gates were defined based on FMO controls.
Extended Data Fig. 9 Transcriptional regulation of CAR T-cells in patient 1 at year 9.
a, Volcano plot indicating transcription factors (TFs) up-regulated in CAR T-cells compared to normal CD4+ T cells (rightward direction) and TFs down-regulated in CAR T-cells compared to normal CD4+ T cells (leftward direction). Differentially expressed TFs were determined using the Wilcoxon rank-sum test with a Bonferroni-adjusted p-value cutoff of 0.001 (dark red) and 0.05 (red). b, Pairwise correlation of TF regulon scores determined by GENIE3 and AUCell in the comparison between CAR T-cells and CD4+ CAR− T cells. c, UMAP indicating RNA expression of selected differentially expressed TFs TCF7, TOX, IKZF3, and PRDM1. d, UMAP indicating RNA expression of differentially expressed AP-1 TFs, FOS, JUNB, JUN, and BATF.
Extended Data Fig. 10 CITE-Seq with 5’ TCR profiling reveals the presence of CD4+ CAR T-cells with characteristic expression of GZMA and GZMK in patients 1 and 2.
a, UMAP embeddings showing expression of key marker genes for patient 2 at year 6.5 post-infusion, as well as (b) patient 1 at month 12 and (c) patient 1 at month 15. Sample processing and data analysis was the same as in Extended Data Fig. 4. Shown are high-quality cells that were verified as CAR T-cells with at least one read aligned to the 5’ CAR construct. UMAP plots showing normalized RNA expression are colored in shades of blue; plots showing protein expression via CITE-Seq antibody-derived tags are colored in shades of green. Red arrows in panel a indicate a CD4+ CAR T-cell population with high expression of GZMA and GZMK, similar to the the long-persisting CAR T-cell population in patient 1 at year 9.3 described in Figs. 3–4 and Extended Data Figs. 6, 7, and 9. The CAR T-cells from patient 1 at months 12 and 15 characteristically expressed GZMA and GZMK at high levels, with the observation of cells expressing CD4 at a protein and RNA level, and cells that expressed CD8B at the RNA level.
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Melenhorst, J.J., Chen, G.M., Wang, M. et al. Decade-long leukaemia remissions with persistence of CD4+ CAR T cells. Nature 602, 503–509 (2022). https://doi.org/10.1038/s41586-021-04390-6
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DOI: https://doi.org/10.1038/s41586-021-04390-6
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