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. 2022 Feb;602(7897):503-509.
doi: 10.1038/s41586-021-04390-6. Epub 2022 Feb 2.

Decade-long leukaemia remissions with persistence of CD4+ CAR T cells

J Joseph Melenhorst #  1   2   3   4   5 , Gregory M Chen #  6 , Meng Wang #  7   8   9 , David L Porter #  9   10 , Changya Chen  11   12 , McKensie A Collins  7   8   9   13 , Peng Gao  11   12 , Shovik Bandyopadhyay  13 , Hongxing Sun  7   8   9 , Ziran Zhao  7   8   9 , Stefan Lundh  7   8   9 , Iulian Pruteanu-Malinici  14 , Christopher L Nobles  15 , Sayantan Maji  7   8   9 , Noelle V Frey  9 , Saar I Gill  9 , Alison W Loren  16 , Lifeng Tian  7   9 , Irina Kulikovskaya  7   8   9 , Minnal Gupta  7   8   9 , David E Ambrose  7   8   9 , Megan M Davis  7   8   9 , Joseph A Fraietta  7   8   9   15 , Jennifer L Brogdon  14 , Regina M Young  7   8   9 , Anne Chew  7   8   9 , Bruce L Levine  7   8   9 , Donald L Siegel  7   8   17 , Cécile Alanio  18   19   20 , E John Wherry  18   19   20 , Frederic D Bushman  15 , Simon F Lacey  7   8   9 , Kai Tan  21   22   23   24   25 , Carl H June  26   27   28   29   30
Affiliations

Decade-long leukaemia remissions with persistence of CD4+ CAR T cells

J Joseph Melenhorst et al. Nature. 2022 Feb.

Erratum in

  • Author Correction: Decade-long leukaemia remissions with persistence of CD4+ CAR T cells.
    Melenhorst JJ, Chen GM, Wang M, Porter DL, Chen C, Collins MA, Gao P, Bandyopadhyay S, Sun H, Zhao Z, Lundh S, Pruteanu-Malinici I, Nobles CL, Maji S, Frey NV, Gill SI, Loren AW, Tian L, Kulikovskaya I, Gupta M, Ambrose DE, Davis MM, Fraietta JA, Brogdon JL, Young RM, Chew A, Levine BL, Siegel DL, Alanio C, Wherry EJ, Bushman FD, Lacey SF, Tan K, June CH. Melenhorst JJ, et al. Nature. 2022 Dec;612(7941):E22. doi: 10.1038/s41586-022-05376-8. Nature. 2022. PMID: 36477542 No abstract available.

Abstract

The adoptive transfer of T lymphocytes reprogrammed to target tumour cells has demonstrated potential for treatment of various cancers1-7. 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 leukaemia1-4 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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Conflict of interest statement

Competing interests 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.

Figures

Extended Data Fig. 1 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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.
Fig. 1 |
Fig. 1 |. Molecular tracking of effectors and targets in long-term responders to anti-CD19 CAR T cell therapy for CLL.
a, Representative flow cytometry data showing persistence of mostly CD4+ CTL019 cells in patient 1, eight years after infusion. b, c, Kinetics of CAR T cell expansion and persistence (red triangles, by quantitative PCR with vector-specific primers; blue circles, by flow cytometry using an anti-CAR antibody) and response of B cells (blue squares, by anti-CD19 flow cytometry) to anti-CD19 CAR T cell therapy in patient 1 (b) and patient 2 (c). d, e, Clonal evolution of CAR T cells based on lentiviral vector integration site analysis. Pairwise Morisita’s overlap index (shown as two decimal points in each circle) was computed between all timepoints (days (D), months (M) or years (Y) after infusion) for patient 1 (d) and patient 2 (e). f, g, Integration sites with abundance above 10% at at least one time point were tracked over time for patient 1 (f) and patient 2 (g). The integration sites were labelled using the nearest genes of integration.
Fig. 2 |
Fig. 2 |. Analysis of CD3+ CAR+ T cells using CyTOF across multiple time points.
a, Uniform manifold approximation and projection (UMAP) of CD3+CAR+ gated cells from CyTOF data generated from samples at five time points for each of patient 1 (PT1) and patient 2 (PT2). Each colour represents cells from one patient time point after infusion. b, Protein expression of selected CyTOF markers, revealing a prominent Ki67hi population of CD4+ CAR T cells, as well as a CD4−CD8− population expressing Helios, GZMB and 2B4. c, UMAP grouped by five major clusters of CAR T cells: a CD4+Ki67hi population, a CD4+ population without this Ki67hi phenotype, a CD8+ population highly expressing GZMK, a CD8+ population highly expressing GZMB, and a CD4−CD8− population expressing Helios. The adjacent stacked bar plots indicate the proportion of each CAR T cell population at different time points, revealing an initial response phase involving CD8+ and CD4−CD8− CAR T cells, followed by a long-term remission stage dominated by this CD4+Ki67hi population. This transition to CD4+Ki67hi CAR T cells was more delayed in patient 2 compared with patient 1. d, Ki67 expression in CD4+CAR+ and CD4+CAR− cells; and CD8+CAR+ and CAR− cells across time points. Statistical testing was performed using the Wilcoxon rank-sum test. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; NS, not significant (P > 0.05). e, Heat map indicating z-score normalized expression of CyTOF markers across five major CAR T cell clusters.
Fig. 3 |
Fig. 3 |. Multi-omic single-cell analysis reveals clonal expansion, proliferation and activation in CAR T cells from patient 1 at year 9.3.
a, UMAP of 1,437 T cells sorted from peripheral blood from patient 1 at year 9.3. UMAP coordinates were computed using the single-cell RNA-seq component of the 5’ TCR-seq and CITE-seq protocol. b, Normalized expression of the CAR construct detected from 5’ single-cell RNA-seq reads. c, UMAP coloured by the eight detected TCR clonotypes with at least ten cells each. Minor clusters with fewer than ten detected cells were coloured light grey. d, e, Normalized CITE-seq antibody expression for the CD4 (d) and CD8a (e) proteins. f, g, Cell cycle scores for cells in the S phase (f) or G2/M (g) phases. h, i, Normalized CITE-seq antibody expression for the activation markers CD38 (h) and HLA-DR (i). j, Gene set enrichment analysis for genes significantly upregulated in CD4+CAR+ T cells compared with CD4+CAR− T cells. Gene Ontology (GO) biological pathways and cellular components and Reactome pathways were considered. TCA, tricarboxylic acid.
Fig. 4 |
Fig. 4 |. Evidence of functional activation, metabolic reprogramming and antigen-driven signalling in CAR T cells from patient 1 at year 9.3.
a, UMAP indicating upregulated RNA expression of cytotoxic genes PRF1, GZMA and GZMK in CD4+ CAR T cells, with GZMB and GZMM being expressed only in the normal CD8+ T cells. b, Evidence of cytotoxic function in CD4+ CAR T cells from patient 1 at year 9.3 in response to CAR-specific stimulation. T cells from patient 1 at year 9.3 were subjected to CAR-specific stimulation by co-culture with CD19-expressing K562 cells. Shown are gated CD3+CAR+CD4+ T cells, and the bars represent the frequency of cells double-positive for CD107a and MIP-1β, perforin and GZMA. c, UMAP indicating upregulated RNA expression of cytokine genes IL10 and IL32 among CAR T cells compared to normal T cells. d, AUCell scores for Reactome TCR signalling and Gene Ontology antigen receptor-mediated signalling pathways. e, RNA expression of key glycolytic gene GAPDH and fermentative glycolysis gene LDHA; AUCell scores for oxidative phosphorylation, respiratory electron transport and mitochondrial protein complex upregulated among CAR T cells, particularly in the active cell cycling phases. f, Proposed model of mechanistic basis of sustained remission with B cell aplasia mediated by few, metabolically active but immune checkpoint inhibitor-restrained CAR T cell clones.
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