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. 2020 Dec 2;28(12):2577-2592.
doi: 10.1016/j.ymthe.2020.07.023. Epub 2020 Jul 25.

A Distinct Transcriptional Program in Human CAR T Cells Bearing the 4-1BB Signaling Domain Revealed by scRNA-Seq

Angela C Boroughs  1 , Rebecca C Larson  1 , Nemanja D Marjanovic  2 , Kirk Gosik  2 , Ana P Castano  3 , Caroline B M Porter  2 , Selena J Lorrey  3 , Orr Ashenberg  2 , Livnat Jerby  2 , Matan Hofree  2 , Gabriela Smith-Rosario  2 , Robert Morris  4 , Joshua Gould  2 , Lauren S Riley  3 , Trisha R Berger  3 , Samantha J Riesenfeld  2 , Orit Rozenblatt-Rosen  2 , Bryan D Choi  3 , Aviv Regev  5 , Marcela V Maus  6
Affiliations

A Distinct Transcriptional Program in Human CAR T Cells Bearing the 4-1BB Signaling Domain Revealed by scRNA-Seq

Angela C Boroughs et al. Mol Ther. .

Abstract

T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have produced impressive outcomes for the treatment of B cell malignancies, but different products vary in kinetics, persistence, and toxicity profiles based on the co-stimulatory domains included in the CAR. In this study, we performed transcriptional profiling of bulk CAR T cell populations and single cells to characterize the transcriptional states of human T cells transduced with CD3ζ, 4-1BB-CD3ζ (BBζ), or CD28-CD3ζ (28ζ) co-stimulatory domains at rest and after activation by triggering their CAR or their endogenous T cell receptor (TCR). We identified a transcriptional signature common across CARs with the CD3ζ signaling domain, as well as a distinct program associated with the 4-1BB co-stimulatory domain at rest and after activation. CAR T cells bearing BBζ had increased expression of human leukocyte antigen (HLA) class II genes, ENPP2, and interleukin (IL)-21 axis genes, and decreased PD1 compared to 28ζ CAR T cells. Similar to previous studies, we also found BBζ CAR CD8 T cells to be enriched in a central memory cell phenotype and fatty acid metabolism genes. Our data uncovered transcriptional signatures related to costimulatory domains and demonstrated that signaling domains included in CARs uniquely shape the transcriptional programs of T cells VSports手机版. .

Keywords: CAR T cells; T cell activation; adoptive cellular therapy; single-cell RNA sequencing; transcriptional profiling. V体育安卓版.

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Figures

None
Graphical abstract
Figure 1
Figure 1
Antigen Stimulation of CAR T Cells through Their CAR Yields a Weaker but Similar T Cell Activation Signal Compared to Stimulation via Anti-CD3-TCR (A) Vector maps of CD19 CAR constructs (L, leader sequence; TM, hinge and transmembrane domain). (B) Experimental design of bulk RNA sequencing (RNA-seq). T cells were isolated from a leukopak from three normal donors, sorted on CD3+ and CD8+ or CD4+, mixed at a 1:1 CD4-to-CD8 ratio, activated with anti-CD3/CD28 T cell expansion beads, and transduced with one of the four CAR constructs or left untransduced (UT). Cells were expanded for 7 days before the beads were removed. T cells were then rested for an additional 7 days prior to stimulation with anti-CD3/CD28 beads or irradiated Nalm6 cells at a 1:1 effector-to-target cell ratio for 0, 4 or 24 h (see Figures S2A and S2B for transduction efficiencies). Samples were then sorted on mCherry+ CD4+ or CD8+ T cells and sequenced separately as bulk populations. Data were collected for T cells from three human donors with technical duplicates. (C) Experimental design of single-cell RNA-seq. T cells from two additional normal donors were isolated, activated, and transduced and expanded as in (B). UT T cells, BBζ, 28ζ, and ζ CAR T cells were either left unstimulated or stimulated with irradiated Nalm6 cells at a 1:1 effector-to-target cell ratio for 24 h. In addition, the UT T cells were stimulated through the TCR with irradiated K562 cells expressing an scFv against CD3 at a 1:1 effector-to-target cell ratio. Stimulated and unstimulated samples were then sorted as live mCherry+ cells and then sequenced using 10x Technology. More than 3,500 cells were sequenced per sample from two human donors. (D–F) Principal component analysis of the expression profiles from the bulk RNA-seq samples corrected for donor variation. Colored from left to right by (D) stimulation condition, (E) CD4+ versus CD8+, and (F) CAR construct. (G) t-Distributed Stochastic Neighbor Embedding (tSNE) of 83,123 single-cell expression profiles (dots) of all single cells sequenced from two donors after donor-specific batch correction (see Figures S2B and S2C). Cells are labeled by their CAR (color hue) and the type of stimulation (light tones for unstimulated). (H) tSNE expression profiles as in (G), colored by the degree of CD8A and CD4 expression. (I) Violin plots showing the distribution of T cell activation signature scores across each condition in CD4+ or CD8+ T cells. T cell activation gene signature were defined as the 15 most upregulated genes shared between CAR-activated CAR T cells and anti-CD3-activated UT cells compared to resting T cells (∗p < 2.2 × 10−16, Wilcoxon test; gene signatures are shown in Tables S1–S3).
Figure 2
Figure 2
Transcriptional Signatures of Resting CAR T Cells Indicate Tonic Signaling through Both CD3ζ and the Co-stimulatory Domain (A) Heatmap of normalized expression from bulk RNA-seq of both the upregulated and downregulated genes that were differentially expressed (DE). FDR < 0.1 in all three functional CD19 CARs: 28ζ, BBζ, and ζ at rest in each case compared to Δζ at rest in either CD8 or CD4 T cells. Each column represents a different donor, and columns of heatmaps are always ordered as donors 1 to 3. (B) tSNE of scRNA-seq from all CAR T cells at rest, colored by the cluster assigned using a graph-based clustering approach. Assigned clusters were then defined based on the gene expression of several key genes (see also Figures S3H and S3I). (C) The same tSNE plot as in (B), depicting degree of CD8A and CD4 expression. (D) Violin plots of cells showing the degree of expression of SELL (CD62L) and CCR7 used to describe the clusters shown in (B). (E) Violin plots of cells as clustered in (B), showing degree of S phase and G2M phase signatures. (F) Of all the cells in a defined cluster, the percent contribution of each CAR to these cells is shown. (G) Graphical display showing the correlation matrix of the chi-square test residual values (difference between observed and expected values; see also Table S4).
Figure 3
Figure 3
CAR T Cells with the 4-1BB Co-stimulation Domain Have Persistent Upregulation of Markers Associated with Activation, Particularly MHC Class II Genes but Not PD1 (A) Volcano plots of DE genes between BBζ (positive x axis) and 28ζ CARs at 24 h post-CAR activation in CD4+ (left) and CD8+ (right) T cells. Genes with FDR <0.05 are colored red (see also Figure S4 for 0- and 4-h time points; see Table S5 for gene list). (B) Heatmap showing normalized HLA class II gene expression in CD4+ T cells from all three donors of 28ζ and BBζ CAR T cells 24 h post-CAR stimulation. (C–E) Mean fluorescence intensity (MFI) of HLA-DR surface expression measured by flow cytometry on CD19-CAR T cells (C) at rest or (D) 24 h after activation of CD19-specific CARs with Nalm6 cells at a 1:1 ratio, or (E) U87-mediated activation of EGFR-specific CAR T cells at a 2:1 effector-to-target cell ratio. (F) IL21R expression in bulk RNA-seq BBζ and 28ζ CAR T samples with CAR stimulation. Individual TPM values shown with mean and SEM; adj-p values were calculated by DESeq2 using a Holm-Bonferroni correction. (G) IL-21 cytokine levels measured in the supernatants of bulk CD4+/CD8+ CD19-CAR T cells stimulated for 24 h with Nalm6 cells at a 1:1 ratio or (H) EGFR-CAR T cells stimulated with U87 cells at a 2:1 effector-to-target cell ratio. (I) MFI of PD1 expression in CD19-CAR T cells with CAR stimulation (via Nalm6 cells, 1:1 effector-to-target cell ratio). (C–I) N = 3 normal donors that were not used for RNA-seq studies; mean and SEM plotted. (C–F) When required, p values were adjusted for multiple comparisons using a Holm-Bonferroni method adjustment (∗adj-p < 0.05, ∗∗adj-p < 0.01) or (G–I) p values were determined using a paired Student’s t test between BBζ and 28ζ (∗p < 0.05, ∗∗p < 0.01). See also Table S6.
Figure 4
Figure 4
Antigen Stimulation of CARs Bearing the 4-1BB Co-stimulation Domain Results in Marked Th1 Polarization (A) Gene set enrichment analysis of an early polarizing Th1 signature. Position of signature genes depicted in a rank fold-change list of the DE genes between RNA-seq BBζ and 28ζ profiles 24 h post-CAR activation with Nalm6 cells (see also Figure S7E). (B) Heatmap of known Th1 cell polarizing genes in CD4+ T cells from three donors in 28ζ versus BBζ CARs 24 h post-Nalm6 stimulation (see also Figure S7B). (C) IL-4 soluble cytokine detected in the supernatants of CD19-CAR T cells after 24 h of Nalm6 stimulation at a 1:1 ratio and in (D) EGFR-CAR T cells stimulated for 24 h with U87 cells at a 2:1 effector-to-target ratio measured by Luminex (see Figure S7F for IL-5). (C and D) N = 3 normal donors that were not used for RNA-seq studies. Mean and SEM plotted. p values were determined using a paired Student’s t test between BBζ and 28ζ. ∗p < 0.05.
Figure 5
Figure 5
Antigen-Specific Activation of 4-1BB CAR T Cells Induces a Distanced Program with Additional Genes Networks Than 4-1BB Ligand-Mediated Triggering of 4-1BB Single-cell expression profiles of CAR T cells after 24 h of stimulation with Nalm6 cells were normalized and aligned across two donors. (A) tSNE plot of single-cell expression profiles (dots) colored by CAR T cell construct. (B) Results of latent Dirichlet allocation on T cells with 16 topics and a tolerance parameter of 0.1 (see Materials and Methods). For each topic shown, there is a bar plot of top-scoring genes (y axis), ranked by a uniqueness score. The genes in topic 11, which were also found as DE and upregulated (adj-p < 0.05) in BBζ versus 28ζ CAR T cells at any time point from bulk RNA-seq data, are in red. Each cell (dot) of the tSNE is colored by the weight of the topic (see also Figure S8 and Table S7). (C) Degree of CD4 and CD8A expression in cells plotted in the tSNE as in (A). (D) Gene regulators discovered using network analysis of the top 100 genes in each topic. Transcription factors with a statistical association (FDR < 0.1) were plotted by −log(pval) for each topic of interest (see also Table S8). (E) T cell activation signature expression level in cells plotted in the tSNE as in (A) (see Table S3 for activation signature). (F) Box-and-whisker plot and cumulative distribution plot of the topic 11 weights per cell across the different CAR T cell groups. (G) Network of predicted transcription factors identified and the genes they regulate in the 4-1BB program (topic 11). (H) UT T cells were stimulated for 24 h with irradiated K562 expressing αCD3 with or without 4-1BBL at a 1:1 effector-to-target cell ratio. Heatmap of 4-1BB CAR signature genes in UT T cells stimulated with and without 4-1BBL (normalized by row) gene signatures. N = 3 normal donors.
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References (VSports在线直播)

    1. Kalos M., Levine B.L., Porter D.L., Katz S., Grupp S.A., Bagg A., June C.H. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci. Transl. Med. 2011;3:95ra73. - "V体育平台登录" PMC - PubMed
    1. Brentjens R.J., Davila M.L., Riviere I., Park J., Wang X., Cowell L.G., Bartido S., Stefanski J., Taylor C., Olszewska M. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci. Transl. Med. 2013;5:177ra38. - PMC (VSports在线直播) - PubMed
    1. Grupp S.A., Kalos M., Barrett D., Aplenc R., Porter D.L., Rheingold S.R., Teachey D.T., Chew A., Hauck B., Wright J.F. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N. Engl. J. Med. 2013;368:1509–1518. - PMC - PubMed
    1. Kochenderfer J.N., Dudley M.E., Kassim S.H., Somerville R.P., Carpenter R.O., Stetler-Stevenson M., Yang J.C., Phan G.Q., Hughes M.S., Sherry R.M. Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor. J. Clin. Oncol. 2015;33:540–549. - PMC - PubMed
    1. Hamza T., Barnett J.B., Li B. Interleukin 12 a key immunoregulatory cytokine in infection applications. Int. J. Mol. Sci. 2010;11:789–806. - PMC (VSports注册入口) - PubMed

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