Research ArticleImmunology

4-1BB costimulation promotes CAR T cell survival through noncanonical NF-κB signaling

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Science Signaling  31 Mar 2020:
Vol. 13, Issue 625, eaay8248
DOI: 10.1126/scisignal.aay8248
  • Fig. 1 4-1BB–costimulated CAR activation drives greater ex vivo T cell expansion and survival than does CD28-costimulated CAR activation.

    (A) Representative ex vivo expansion of the indicated CAR T cells stimulated by irradiated Nalm6 target cells at the indicated times at a T cell to target cell ratio of 2:1. (B) Quantification of T cell expansion after 14 days of ex vivo culture from the experiments represented in (A). (C) Quantification of cell death by measurement of the percentage of 7AAD+mCherry cells in all events collected on day 14 of ex vivo culture from the experiments represented in (A). (D) Representative Western blotting analysis of PCNA abundance in the indicated CAR T cells at 0, 7, and 14 days of ex vivo culture with Nalm6 target cells. (E) Quantification of the relative amounts of PCNA in the indicated CAR T cells on day 14 of ex vivo culture with Nalm6 target cells. Data in (B), (C), and (E) are from three donors in two independent experiments and were analyzed by paired Student’s t test. (F) Representative ex vivo expansion of the indicated CAR T cells stimulated by anti-CD19 idiotype–coated target beads (at a T cell to bead ratio of 1:3) added at day 0 (green arrow) and removed at day 14 of culture (black arrow). (G) Quantification of T cell expansion after 14 days of ex vivo culture from the experiments represented in (F). (H) Quantification of cell death as assessed by measurement of the percentage of 7AAD+mCherry cells in all events collected on day 14 of ex vivo culture from the experiments represented in (F). Data in (G) and (H) are from three donors in three independent experiments and were analyzed by paired Student’s t test. (I) Quantification of the relative abundance of PCNA as assessed by Western blotting analysis of the indicated CAR T cells on days 0, 7, and 14 of ex vivo culture with target beads. Data are from donors in three independent experiments and were analyzed by paired Student’s t test.

  • Fig. 2 Anti-CD19 idiotype target beads activate T cell signaling in anti-CD19 CAR T cells.

    (A) Representative Western blotting of total and phosphorylated Zap-70, p65, and ERK1/2 proteins before and at 30 min and 12 hours after the addition of anti-CD19 beads to the indicated CAR T cells at a bead–to–T cell ratio of 5:1. The anti-CD19 CAR T cells used expressed CARs with no signaling domain (Δζ), the CD3ζ chain intracellular domain alone (ζ), the CD28 and CD3ζ chain intracellular domains (28ζ), or the 4-1BB and CD3ζ chain intracellular domains (BBζ). (B to E) Quantification of the relative abundances of phosphorylated Zap-70 (B), p65 (C), ERK1 (D), and ERK2 (E) normalized to their respective total proteins from the experiments represented in (A). P values were determined by two-way ANOVA with Tukey’s multiple comparisons test of data from three donors analyzed in three independent experiments. FC, fold change; TP, time point; NTD, non-transduced.

  • Fig. 3 4-1BB–costimulated anti-CD19 CAR activation stimulates CAR T cell ncNF-κB signaling.

    (A) Representative Western blotting analysis of p100 phosphorylation and the processing of p100 to p52 at the indicated times before and after the addition of target beads to anti-CD19 BBζ CAR T cells. (B and C) Quantitative analyses of the relative amounts of phosphorylated p100 (B) (P = 0.0397 as determined by analyzing the data from two independent experiments using CAR T cells derived from three healthy donors by one-way ANOVA with the factor being time, as is the case for all of the P values in this figure) and the relative amounts of p52 generated by p100 processing (C) (p52/p100 ratio, P = 0.0244) from the experiments represented in (A). (D) Representative Western blotting analysis of NIK protein at the indicated times before and after target bead addition to anti-CD19 BBζ CAR T cells. Actin was used as a loading control. (E) Quantitative analysis of the relative amounts of NIK protein at the indicated times after the addition of beads to the cells in the experiments represented in (D) (P = 0.011). (F) Representative Western blotting analysis of the cytoplasmic and nuclear fractions from anti-CD19 BBζ CAR T cells before and at the indicated times after the addition of target beads. The blot is representative of two separate experiments using CAR T cells generated from two healthy donors (see fig. S6 for the other experiment).

  • Fig. 4 4-1BB, but not CD28, CAR costimulation drives basal and enhances CAR activation–induced ncNF-κB signaling.

    (A) Representative Western blotting analysis of the phosphorylation of p100 and of its processing to p52 in the indicated CAR T cells at the indicated times before and after the addition of target beads at a bead–to–T cell ratio of 5:1. The anti-CD19 CARs used had no signaling domain (Δζ), the CD3ζ chain intracellular domain alone (ζ, green), the CD28 and CD3ζ chain intracellular domains (28ζ, red), or the 4-1BB and CD3ζ chain intracellular domains (BBζ, blue). (B and C) Quantitative analyses of the relative abundances of phosphorylated p100 (B) and of the ratio of p52 to p100 (C) from the experiments represented in (A). (D) Representative Western blotting analysis of NIK protein in the indicated CAR T cells at the indicated times before and after the addition of target beads as described for (A). Actin was used as a loading control. (E) Quantitative analysis of relative amounts of NIK protein in the indicated cells from the experiments represented in (D). (F) Representative Western blotting analysis of RelB, p52, and TBP (loading control) in nuclear fractions from the indicated anti-CD19 CAR T cells before and at the indicated times after the addition of target beads as described for (A). (G and H) Quantitative analyses of the relative amounts of nuclear RelB (G) and nuclear p52 (H) in the indicated cells from the experiments represented in (F). Data in (B), (C), (E), (G), and (H) are from three donors in three independent experiments. All log2(fold change relative to nonsignaling group) quantifications of band fluorescence intensities were normalized to those of the loading control. P values reported were determined by two-way ANOVA with Tukey’s multiple comparisons test.

  • Fig. 5 Reducing 4-1BB costimulation–mediated ncNF-κB signaling diminishes BBζ CAR T cell ex vivo expansion and survival.

    (A) Representative Western blotting analysis of p100 processing to p52 before, 30 min, and 12 hours after the addition of target beads to anti-CD19 BBζ CAR T cells expressing either mCherry (control) or mCherry and dnNIK. (B) Quantitative analysis of the ratio of p52 to p100 ratio from the experiments represented in (A). Data are from CAR T cells generated from three donors in three independent experiments. P value was determined by two-way ANOVA with Holm-Sidak’s multiple comparisons test. (C) Representative Western blotting analysis of RelB, p52, and TBP (loading control) in nuclear fractions of anti-CD19 BBζ CAR T cells treated as described for (A). (D and E) Quantitative analyses of the nuclear localization of RelB (D) and p52 (E) in cells from the experiments represented in (C) using TBP as the loading control. Data are from two independent experiments of CAR T cells generated from three donors. P values were determined by two-way ANOVA with Holm-Sidak’s multiple comparisons test. (F) Representative ex vivo expansion of control or dnNIK-expressing BBζ CAR T cells stimulated by anti-CD19 idiotype–coated target beads (at a T cell–to–bead ratio of 1:3) and added at day 0 (green arrow) and removed at day 14 of culture (black arrow). (G) Quantification of T cell expansion after 14 days of ex vivo culture from the experiments represented in (F). (H) Quantification of cell death by measurement of the percentage of 7AAD+mCherry cells in all events collected on day 14 of the ex vivo culture represented in (F). (I) Representative ex vivo expansion of control or dnNIK-expressing 28ζ CAR T cells stimulated by anti-CD19 idiotype–coated target beads at a T cell–to–bead ratio of 1:3 and added at day 0 (green arrow) and removed at day 14 of culture (black arrow). (J) Quantification of T cell expansion after 14 days of ex vivo culture from the experiments represented in (I). (K) Quantification of cell death by measurement of the percentage of 7AAD+mCherry cells in all events collected on day 14 of the ex vivo culture represented in (I). All P values were determined by paired Student’s t tests of data from four donors in four independent experiments.

  • Fig. 6 4-1BB costimulation–mediated ncNF-κB signaling opposes expression of the pro-apoptotic protein Bim.

    (A) Representative Western blotting analysis of Bim isoforms in control and dnNIK-expressing BBζ CAR T cells before and 14 days after ex vivo expansion in response to the addition of beads. (B to E) Quantitation of the relative basal amounts of BimL (B) and BimS (C), as well as of the amounts of BimL (D) and BimS (E) 14 days after the addition of beads from the experiments represented in (A). Data in (B) to (E) are from four donors in four independent experiments. All P values were derived from paired Student’s t tests. (F) Representative Western blotting analysis of Bim isoforms in control or dnNIK-expressing 28ζ CAR T cells before and 14 days after ex vivo expansion in response to the addition of beads. (G and H) Quantitation of the relative basal amounts of BimL (G) and BimS (H) from the experiments represented in (F). Data are from three independent experiments with three donors. P values were derived from paired Student’s t tests. (I and J) Quantitation of the relative amounts of BimL (I) and BimS (J) 14 days after bead addition from the experiments represented in (F). Data in (I) and (J) are from four donors in four independent experiments. No pre-bead sample was taken for fourth donor shown in (I) and (J). P values were derived from paired Student’s t tests. (K and L) Comparison of the basal amounts of BimL (K) and BimS (L) between 28ζ and BBζ CAR T cells. (M and N) Comparison of the relative amounts of BimL (M) and BimS (N) between 28ζ and BBζ CAR T cells 14 days after expansion in response to beads. Data in (K) to (N) are from three donors in three independent experiments. All P values were derived from paired Student’s t tests.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/13/625/eaay8248/DC1

    Fig. S1. The ncNF-κB pathway.

    Fig. S2. CAR expression on T cells from three donors expanded by irradiated Nalm6 cells.

    Fig. S3. CAR expression on T cells from three donors expanded by anti-CD19 beads.

    Fig. S4. CAR expression on T cells from three donors activated by anti-CD19 beads for signaling assays.

    Fig. S5. Anti-CD19 BBζ CAR activation induces T cell signaling.

    Fig. S6. Additional representative Western blotting analysis of nuclear and cytoplasmic fractions to compare anti-CD19 CAR signaling.

    Fig. S7. Anti-mesothelin BBζ CAR also drives ncNF-κB signaling.

    Fig. S8. Dual-transduced T cell production scheme, day 0 CAR, and mCherry expression.

    Fig. S9. Western blotting analysis of nuclear and cytoplasmic fractions to compare control and dnNIK-expressing anti-CD19 BBζ T cells.

    Fig. S10. dnNIK does not affect PCNA abundance in CAR T cells.

    Fig. S11. BimEL abundance in anti-CD19 CAR T cells.

    Fig. S12. ERK1/2 phosphorylation in control and dnNIK-expressing BBζ CAR T cells.

    Fig. S13. FOXO3a abundance and phosphorylation in control and dnNIK-expressing BBζ CAR T cells.

    Fig. S14. CAR constructs.

    Fig. S15. Representative gating strategy to quantify cell death by flow cytometry and assess the elimination of CD19+ Nalm6 cells.

  • This PDF file includes:

    • Fig. S1. The ncNF-κB pathway.
    • Fig. S2. CAR expression on T cells from three donors expanded by irradiated Nalm6 cells.
    • Fig. S3. CAR expression on T cells from three donors expanded by anti-CD19 beads.
    • Fig. S4. CAR expression on T cells from three donors activated by anti-CD19 beads for signaling assays.
    • Fig. S5. Anti-CD19 BBζ CAR activation induces T cell signaling.
    • Fig. S6. Additional representative Western blotting analysis of nuclear and cytoplasmic fractions to compare anti-CD19 CAR signaling.
    • Fig. S7. Anti-mesothelin BBζ CAR also drives ncNF-κB signaling.
    • Fig. S8. Dual-transduced T cell production scheme, day 0 CAR, and mCherry expression.
    • Fig. S9. Western blotting analysis of nuclear and cytoplasmic fractions to compare control and dnNIK-expressing anti-CD19 BBζ T cells.
    • Fig. S10. dnNIK does not affect PCNA abundance in CAR T cells.
    • Fig. S11. BimEL abundance in anti-CD19 CAR T cells.
    • Fig. S12. ERK1/2 phosphorylation in control and dnNIK-expressing BBζ CAR T cells.
    • Fig. S13. FOXO3a abundance and phosphorylation in control and dnNIK-expressing BBζ CAR T cells.
    • Fig. S14. CAR constructs.
    • Fig. S15. Representative gating strategy to quantify cell death by flow cytometry and assess the elimination of CD19+ Nalm6 cells.

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