Research ArticleIMMUNOLOGY

Single-cell transcriptomics identifies multiple pathways underlying antitumor function of TCR- and CD8αβ-engineered human CD4+ T cells

See allHide authors and affiliations

Science Advances  03 Jul 2020:
Vol. 6, no. 27, eaaz7809
DOI: 10.1126/sciadv.aaz7809
  • Fig. 1 Coexpression of CD8αβ with TCR redirects CD4+ T cells to the targeted class I epitope.

    (A) Scheme of retroviral vectors containing the survivin-specific TCR (top, TCR) or the combination of TCR and CD8αβ and the selectable marker gene ΔCD271 (bottom, TCR8). (B and C) Determination of monomeric dissociation kinetics with survivin-specific reversible NTAmers. (B) Representative analysis of temperature-controlled (4°C) pMHC-TCR or pMHC-TCR8 monomeric dissociation rates. No NTAmer staining in TCR+CD4+ T cells; ND, not detected. (C) Summary of monomeric dissociation constants [koff (s−1)], n = 3 donors, three independent experiments with technical replicates. Mean ± SD, P = NS, one-way analysis of variance (ANOVA) test. (D and E) Analysis of early TCR signaling events. (D) Representative FACS histograms of pLCK-Y394 phosphorylation NT (gray), TCR+ (blue), and TCR8+ (green) CD4+ or CD8+ T cells. (E) Summary of pLCK MFI normalized to MFI in NT control cells. n = 4 donors, mean ± SD, CD4: TCR+ versus TCR8+: 104 ± 11% versus 173 ± 35%, CD8: TCR+ versus TCR8+: 106 ± 7% versus 126 ± 13%, TCR8+ CD8 versus CD4: 126 ± 13% versus 173 ± 35%. NS, not significant, *P < 0.05. (F) Antigen sensitivity measured by IFN-γ ELISpot against peptide-pulsed T2 cells. SFC, spot-forming cells, n = 3 donors, three technical replicates each, mean ± SD, nonlinear regression (curve fit). (G) Coculture of NT, TCR+, or TCR8+ CD4+ (red bars) or CD8+ (black bars) T cells with BV173 leukemia cells (HLA-A2*02:01+survivin+); E:T ratio 1:5, residual BV173 cells quantified on day 3, n = 7. (H) Coculture of NT, TCR+, or TCR8+CD4+ (left) or CD8+ (right) T cells with wild-type (WT) BV173 (solid bars) or β2-microglobulin knockout (B2M-KO) BV173 cells (open bars); E:T ratio 1:5, residual BV173 cells quantified on day 3, n = 3. Mean ± SD, ***P < 0.001 and ****P < 0.0001. t test on log-transformed data.

  • Fig. 2 Single-cell transcriptomics reveals distinct T cell subpopulations.

    (A) Schematic overview of experimental setup and corresponding groups. (B) t-SNE plot of all 25,474 cells analyzed, color code indicates sample origin, and each dot represents one cell. (C) t-SNE plot of all cells separated into 19 distinct clusters by k-nearest neighbor clustering analysis; each cluster is color coded. (D) Heat map showing the top 20 DE genes of each cluster for all cocultured samples with selected genes identified on the right. Cluster 18 contains cocultured TCR+CD8+ and cocultured TCR8+CD8+ cells, depicted as clusters 18a and 18b on the heat map. (E) Venn diagram of DE genes (0.25 log fold change) up-regulated from expanded to cocultured state. Distinct or shared representative genes from each cocultured sample are indicated and color coded by biological function.

  • Fig. 3 Sustained proliferative capacity and preservation of a less differentiated phenotype in cocultured TCR8+CD4+ T cells.

    (A) t-SNE plot of cell cycle–associated genes (GO_0007049) revealing location of proliferating cells in each sample. (B) Mean expression pseudo time course of cell cycle–related genes for each sample. Pseudo time points represent fresh (F), expanded (E), and cocultured (C) conditions. (C) Bar plot representing proportion of replicating T cells per sample. The replicating cells are defined by their association to clusters 10, 13, 14, and 18. (D) t-SNE plot of TN, TCM, TEM, and TEFF cells identified by hierarchical clustering. Each dot represents one cell color coded by differentiation status. (E) Combined plot overlaying % cells for each subset by t-SNE (bars) and two-marker–based FACS validation (line with dots, CD62L and CD45RO, n = 9 independent donors, mean ± SD and individual values, Mann-Whitney U test). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (F) t-SNE plot (top) and corresponding bar plot (bottom) showing mean gene expression of representative TN/TCM markers TCF7, IL7R, CCR7, and CXCR4.

  • Fig. 4 TCR8 promotes cytotoxicity and costimulation in TCR8+CD4+ T cells in the absence of exceeding activation/exhaustion.

    (A) t-SNE plot of cytotoxicity-related genes (list of genes provided in table S3). (B) t-SNE plot of GZMB and GZMA (top) and intracellular FACS validation of T cells stimulated with BV173 cells (bottom, n = 6 independent donors, mean ± SD and individual values, Mann-Whitney U test). **P < 0.01. (C) Single-cell quantification of interaction kinetics between T cells and target cells by TIMING assay. tSeek, time to first encounter of effector and target cell; tContact, time of conjugation between effector and target cell; tDeath, time from first contact to target cell apoptosis. (D) Cumulative incidence of a single T cell in finding (tSeek) one (left, E:T 1:1) or two (right, E:T 1:2) target cells (top row), in forming a stable synapse with the target (tContact, middle row), or in killing the target (tDeath). TCR+CD4+ T cells (red dotted lines), TCR8+CD4+ T cells (red solid lines), TCR+CD8+ T cells (black dotted lines), and TCR8+CD8+ T cells (black solid lines). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, log-rank test. (E) t-SNE plot of costimulation-related genes (list of genes provided in table S3). (F) t-SNE plot of CD28 and SLAMF1 (top) and FACS validation (bottom, n = 6 to 9 independent donors, mean ± SD and individual values, Mann-Whitney U test). *P < 0.05, ***P < 0.001, and ****P < 0.0001. (G) t-SNE plot of activation/exhaustion related-genes (list of genes provided in table S3). (H) t-SNE plot of PD-1 and CTLA-4 (top) and FACS validation (bottom, n = 5 independent donors, mean ± SD and individual values, Mann-Whitney U test).

  • Fig. 5 TCR8+CD4+ T cells are characterized by metabolic programs that support enhanced T cell performance.

    (A) t-SNE plot of OXPHOS-related genes [HUGO Gene Nomenclature Committee (HGNC) database group 639, four to five representative genes per complex]. (B) Mean expression pseudo time course of representative OXPHOS genes. (C) Absolute MFI values for mitochondrial membrane potential (left) and mass (right) determined by FACS (n = 7 to 8 independent donors, mean ± SD and individual values, Mann-Whitney U test). *P < 0.05 and ***P < 0.001. (D) t-SNE plot of glycolysis-related genes (KEGG database). (E) Mean expression pseudo time course of indicated genes. (F) t-SNE plot of FAO-related genes (KEGG database). (G) Mean expression pseudo time course of indicated genes.

  • Fig. 6 TCR8+CD4+ T cells efficiently kill and expand under stress conditions in vitro and in vivo.

    (A) Experimental setup of bulk sequential coculture stress test. (B) Sequential cocultures of CD4+ (left) or CD8+ (right) T cells. Quantification of tumor (top) and T cells (bottom) over time, with tumor cell rechallenge (+), n = 7. Number of tumor cell killings by condition: CD4+ T cells: NT 0 ± 0, TCR+ 0 ± 0, TCR8+ 3.2 ± 0.5; CD8+ T cells: NT 0 ± 0, TCR+ 1.3 ± 1.1, TCR8+ 2 ± 1.4. TCR+CD8+ versus TCR8+CD8+ T cells: P = 0.04; TCR8+CD4+ versus TCR+CD8+ T cells: P = 0.01; TCR8+CD4+ versus TCR8+CD8+ T cells: P = NS, t test. T cell expansion: TCR+CD8+ versus TCR8+CD8+: P = NS; TCR8+CD4+ versus TCR+CD8+: P = 0.002; TCR8+CD4+ versus TCR8+CD8+, P = 0.015, t test on log AUC. (C) Cytokine quantification in coculture supernatants 24 hours after first tumor challenge (D1) and 24 hours after third tumor challenge (D10), n = 6. Mean ± SD, *P < 0.05 and **P < 0.01. (D) Experimental setup mouse xenograft experiment. XRT, radiation; BLI, bioluminescence imaging; iv, intravenously. (E and F) BLI results from mice treated with (E) CD8+ T cells or (F) CD4+ T cells. BLI pictures show three representative mice per group, color scale 5 × 103 to 5 ×104 p/sec/cm2/sr (left) and summary of total flux (right). Nontransduced (NT) control T cells (n = 5, gray), TCR+ T cells (n = 5, blue), TCR8+ T cells (n = 5, green). Mean ± SD. **P < 0.01, ***P < 0.001, and ****P < 0.0001. t test on log AUC, day 28 for CD8+, day 35 for CD4+ T cells.

  • Fig. 7 TCR8+CD4+ T cell performance is characterized by sustained expression of diverse transcriptional programs.

    (A) Summary of the main findings identified by scRNAseq in cocultured cytotoxic engineered CD4+ and CD8+ T cells. (B) Summary of main findings from in vitro and in vivo functional assays and overall engineered T cell performance. Low to high intensity of red indicates low to high T cell function.

Supplementary Materials

  • Supplementary Materials

    Single-cell transcriptomics identifies multiple pathways underlying antitumor function of TCR- and CD8αβ-engineered human CD4+ T cells

    Jan A. Rath, Gagan Bajwa, Benoit Carreres, Elisabeth Hoyer, Isabelle Gruber, Melisa A. Martínez-Paniagua, Yi-Ru Yu, Nazila Nouraee, Fatemeh Sadeghi, Mengfen Wu, Tao Wang, Michael Hebeisen, Nathalie Rufer, Navin Varadarajan, Ping-Chih Ho, Malcolm K. Brenner, David Gfeller, Caroline Arber

    Download Supplement

    The PDF file includes:

    • Figs. S1 to S7
    • Tables S1
    • Legends for tables S2 to S4

    Other Supplementary Material for this manuscript includes the following:

    Files in this Data Supplement:

Stay Connected to Science Advances

Navigate This Article