Research ArticleCAR-T CELLS

Engineering γδT cells limits tonic signaling associated with chimeric antigen receptors

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Science Signaling  10 Sep 2019:
Vol. 12, Issue 598, eaax1872
DOI: 10.1126/scisignal.aax1872
  • Fig. 1 Expansion augments subsequent activation of CD8+ T cells.

    (A) Example DREMI analysis of correlation between molecules X and Y. A high DREMI score indicates that Y expression is highly dependent on X. (B) Mass cytometry analysis of pSLP-76 and pERK abundance in fresh CD8+ T cells at resting state (US) or after 60 or 360 s of CD3 stimulus. Conventional biaxial scatter plots (upper) and corresponding DREVI plots (lower) are from three independent experiments. (C) Mass cytometry analysis of pSLP-76 correlation with CD3 and pERK correlation with pSLP-76 in fresh or expanded CD8+ T cells before and after 360-s stimulus with CD3 or CD3+CD28. DREVI plots (right) are representative of and DREMI scores (left) are means ± SEM pooled from at least three biological replicates. (D) Mass cytometry analysis of pAKT in fresh or expanded CD8+ T cells stimulated as indicated. Histograms (left) are representative of and EMD scores (left) are means ± SEM pooled from at least three biological replicates. Individual Spearman’s correlation values are indicated (B) and ***P = 0.0002 and ****P < 0.0001 by one-way analysis of variance (ANOVA) test with Sidak’s correction (C and D).

  • Fig. 2 CAR expression promotes tonic activation of signaling networks.

    (A) Mass cytometry analysis of baseline phosphoprotein abundance in expanded CD8+ T cells transduced to express the CD19-28ζ CAR as indicated. Histograms are representative of three independent donors. (B) Mass cytometry analysis of baseline phosphoprotein abundance in CD4+ (green), CD8+ (blue), and Vδ2+ (yellow) T cells transduced to express the CD19-28ζ CAR. Correlation of EMD score with transduction efficiency is from analysis of at least three independent donors. (C and D) Mass cytometry analysis of phosphoprotein abundance in expanded T cells transduced with CD19-28ζ stimulated with antibodies against CD3 (blue), CD28 (green), CD3+CD28 (red), or CAR (violet). Correlation of EMD scores in untransduced and CAR-transduced cells (C) and EMD scores for pSLP-76, pERK, and pMAPKAPK2 (D) are means ± SEM of at least three independent donors. (E) Flow cytometry analysis of TIM-3 and PD-1 abundance on T cells expressing a CD19-28ζ, GD2-28ζ (huk666), or GD2-28ζ (14G2A) CAR. Data are means ± SEM pooled from at least four biological replicates (see also fig. S11). Pearson correlation (B) and analysis of covariance (ANCOVA) (C) P values are displayed; (D) *P < 0.05, **P < 0.01 by paired t test. ns, not significant.

  • Fig. 3 Expansion and CAR expression have distinct effects on CD4+ and CD8+ T cells.

    (A and B) Mass cytometry analysis of the CD3 and pSLP-76 abundance in stimulated fresh (green), expanded (blue), or CD19-28ζ–transduced (red) CD4+ and CD8+ T cells, as indicated. DREMI scores after CD3, CD28, or CD3+CD28 stimulation with means ± SEM (A) are pooled from three independent donors. DREVI plots with DREMI scores after CD3 stimulation alone (B) are representative of all donors. (C to H) Analysis of DREMI mean and variance in the indicated cells across all stimuli (C) identified two distinct types of responses (pink and blue areas). pAKT signaling edges (D) exhibited greater variance than known TCR signaling pathways (E). Differences between CD4+ (F) and CD8+ (G) T cells were most apparent in known TCR signaling pathways. DREMI scores for known PI3K and TCR signaling interactions in fresh, expanded, and transduced CD4+ and CD8+ T cells (H) with means ± SEM are pooled from all experiments. (I) Mass cytometry analysis of pERK and pRelA in expanded and CAR-transduced cells without further stimulus. Data with means ± SEM are pooled from three independent donors. *P = <0.05 and ****P < 0.0001 by one-way ANOVA with Sidak’s correction (A) or two-way ANOVA with Sidak’s multiple comparison correction (I).

  • Fig. 4 Engineering Vδ2+ T cells to express CCRs promotes TCR signaling and effector functions.

    (A) A schematic highlighting that CARs provide all activation signals from one receptor, whereas the CCR requires a CD3ζ signal to be provided from another source. In this case, the engagement of tumor target butyrophilin (BTN3A1) by the Vδ2 γδTCR and the CCR is required for activation. (B) Phospho-flow analysis of pAKT and pERK abundance in CD33-CD28 CCR–transduced Vδ2+ T cells stimulated as indicated up to 600 s. Data are means ± SEM pooled from three independent donors. (C) Flow cytometry analysis of TNFα production by CD33-CD28 CCR–transduced Vδ2+ T cells stimulated as indicated. Data are means ± SEM pooled from three independent donors. (D) 51Cr-release assay of cytotoxicity by untransduced or GD2-CD28 CCR–transduced Vδ2+ T cells cocultured for 4 hours with GD2+ neuroblastoma (LAN1) target cells. Data are means ± SEM pooled from four independent donors. (E) Mass cytometry analysis of the indicated phosphoproteins in expanded Vδ2+ T cells after stimulation with antibodies against CD3 (red), NKG2D (green), or CD3+NKG2D (blue). EMD scores are means ± SEM pooled from four independent donors. (F) Mass cytometry analysis of the indicated phosphoproteins in expanded, CD33-CD28 CCR–transduced Vδ2+ T cells after stimulation with antibodies against CD3 (red), CCR (green), or CD3+CCR (blue). EMD scores are means ± SEM pooled from three independent donors. *P < 0.05, **P < 0.01, ****P < 0.0001 by one-way ANOVA with Sidak’s correction (C and D) or two-way ANOVA with Tukey’s correction (B, E, and F).

  • Fig. 5 DAP10-CCR does not induce tonic signaling and synergizes with CD3 stimulation.

    (A) Mass cytometry analysis of basal phosphoprotein abundance in expanded untransduced, CD33-DAP10 CCR–transduced, or CD33-28ζ CAR–transduced Vδ2+ T cells. Histograms (A, upper) are representative of and EMD scores in expanded CD33-DAP10 (purple) or CD33-28ζ (red) cells (A, lower) are means + SEM pooled from three independent donors. (B to E) Mass cytometry analysis of the indicated phosphoprotein abundance in expanded untransduced or CD33-DAP10 CCR–transduced Vδ2+ T cells after stimulation. DREMI scores with means ± SEM (B) are pooled from three independent donors. DREVI plots of pAKT versus pMAPKAPK2 (C) and pERK versus pSLP-76 (D) are representative of all donors. Network representation of EMD and DREMI scores in DAP10-CCR–transduced Vδ2+ cells (E) stimulated with CCR alone (middle) or CD3+CCR (right). Stimulus inputs (green), outputs colored according to EMD score, and connections indicated by line color and thickness determined by the mean DREMI score are from the analysis of all donors. *P < 0.05 and **P < 0.01 by one-way ANOVA with Sidak’s correction for multiple comparisons (B).

  • Fig. 6 CCR expression in Vδ2+ T cells avoids on-target off-tumor toxicity.

    (A to C) Flow cytometry analysis of TIM-3 and PD-1 abundance on untransduced, CD19-28ζ CAR–transduced, and CD19-DAP10 CCR–transduced Vδ2+ cells (A) or GD2-CD28 CCR–transduced and untransduced Vδ2+ cells (B) or untransduced and CD33-DAP10–transduced Vδ2+ cells 8 days after transduction (see also fig. S15). Data are means + SEM pooled from at least three independent donors. (D) Flow cytometry analysis of cell proliferation by CD33-DAP10 CCR ± or CD33-CD28 CCR ± Vδ2+ T cells cultured with irradiated MV4-11 AML cells. Data are means ± SEM pooled from three independent donors. (E) Flow cytometry analysis of IFN-γ and TNFα production by DAP10-CCR–transduced Vδ2+ T cells cocultured overnight with either MV4-11 or allogeneic monocytes with and without butyrophilin blockade. Data are means + SEM pooled from three independent donors. (F and G) 51Cr-release assay of cytotoxicity by untransduced, CD33-DAP10 CCR–transduced (purple), CD33-28ζ CAR–transduced (yellow), or CD33-CD28 CAR lacking CD3ζ domain–transduced (green) Vδ2+ T cells cocultured with allogeneic monocytes or MV4-11 AML cells and treated with zoledronic acid where indicated. Data are means ± SEM of 3 to 10 independent donors (F) and 3 to 9 independent donors (G). (H) Myeloid colony formation assay by healthy bone marrow cultured overnight with or without untransduced, CD33-DAP10 CCR–transduced, or CD33-CD28ζ CAR–transduced Vδ2+ T cells. Data are means + SEM from three independent donors. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with Sidak’s correction.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/598/eaax1872/DC1

    Fig. S1. Map of signaling networks analyzed in distinct cell populations.

    Fig. S2. Response of αβ T cells to stimulus.

    Fig. S3. Network behavior of activated αβ T or CD19-28ζ CAR-T cells.

    Fig. S4. Representative DREVI plots for expanded CD8+ T cells.

    Fig. S5. DREMI scores for expanded transduced and nontransduced T cells.

    Fig. S6. Representative DREVI plots for expanded transduced and nontransduced T cells.

    Fig. S7. TIM-3 abundance in expanded transduced and nontransduced αβ T cells.

    Fig. S8. Phosphoprotein abundance in CD28-CCRs in Vδ2+ γδT cells.

    Fig. S9. TNFα production in response to CCR stimulus.

    Fig. S10. DREMI scores in expanded or CD33-DAP10 CCR+ γδT cells.

    Fig. S11. SYK, ABL, and ZAP70 activity are increased in Vδ2+ γδT cells cultured with AML cells.

    Fig. S12. CD33-DAP10 CCR+ Vδ2+ γδT cells produce cytokines in response to AML cells.

    Fig. S13. Schematics of CAR and CCR constructs used in this study.

    Fig. S14. Cartoon illustrating stimulation of CAR-transduced αβ T cells by antibody cross-linking.

    Fig. S15. Flow and mass cytometry gating strategies.

    Table S1. Statistical analysis of data displayed in Fig. 3H (CD4+).

    Table S2. Statistical analysis of data displayed in Fig. 3H (CD8+).

    Table S3. Statistical analysis of data displayed in fig. S5.

    Table S4. Antibodies used in mass cytometry.

    Table S5. Antibodies used in flow cytometry.

  • This PDF file includes:

    • Fig. S1. Map of signaling networks analyzed in distinct cell populations.
    • Fig. S2. Response of αβ T cells to stimulus.
    • Fig. S3. Network behavior of activated αβ T or CD19-28 ζ CAR-T cells.
    • Fig. S4. Representative DREVI plots for expanded CD8+ T cells.
    • Fig. S5. DREMI scores for expanded transduced and nontransduced T cells.
    • Fig. S6. Representative DREVI plots for expanded transduced and nontransduced T cells.
    • Fig. S7. TIM-3 abundance in expanded transduced and nontransduced αβ T cells.
    • Fig. S8. Phosphoprotein abundance in CD28-CCRs in Vδ2+ γδT cells.
    • Fig. S9. TNFα production in response to CCR stimulus.
    • Fig. S10. DREMI scores in expanded or CD33-DAP10 CCR+ γδT cells.
    • Fig. S11. SYK, ABL, and ZAP70 activity are increased in Vδ2+ γδT cells cultured with AML cells.
    • Fig. S12. CD33-DAP10 CCR+ Vδ2+ γδT cells produce cytokines in response to AML cells.
    • Fig. S13. Schematics of CAR and CCR constructs used in this study.
    • Fig. S14. Cartoon illustrating stimulation of CAR-transduced αβ T cells by antibody cross-linking.
    • Fig. S15. Flow and mass cytometry gating strategies.
    • Table S1. Statistical analysis of data displayed in Fig. 3H (CD4+).
    • Table S2. Statistical analysis of data displayed in Fig. 3H (CD8+).
    • Table S3. Statistical analysis of data displayed in fig. S5.
    • Table S4. Antibodies used in mass cytometry.
    • Table S5. Antibodies used in flow cytometry.

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