Research ArticleIMMUNOTHERAPY

Gasdermin E–mediated target cell pyroptosis by CAR T cells triggers cytokine release syndrome

See allHide authors and affiliations

Science Immunology  17 Jan 2020:
Vol. 5, Issue 43, eaax7969
DOI: 10.1126/sciimmunol.aax7969
  • Fig. 1 CAR T cells induced tumor cell pyroptosis.

    (A to D) Primary B leukemic cells were cocultured with CD19 or HER2-CAR T cells at an effector/target ratio (E/T) of 2:1 for 6 hours. Tumor cell viability was measured by using the CellTiter-Glo Luminescent Cell Viability Assay Kit [(A), n = 10]. The representative images were shown (B). Cell death was determined by flow cytometry [(C), n = 6], and LDH level in the supernatants was measured [(D), n = 10]. Scale bar, 20 μm. White arrows indicate pyroptotic cells. (E to H) Luc-Raji or NALM-6 cells were cocultured with CD19- or HER2-CAR T cells at different ratios as indicated for 4 hours or at the ratio of 1:1 for the indicated time. Cell viability was measured (E), and the representative images were shown (F). The percentage of annexin V+/PI+ tumor cells was determined by flow cytometry (G). LDH levels in the supernatants were measured (H). Scale bar, 10 μm. (I) The same as (A), except that primary B leukemic cells were imaged by AFM. Pore diameter and depth were calculated within a cellular membrane area of 5 μm by 5 μm from 10 cells. Pore number was quantified from three areas of 5 μm by 5 μm per cell. A pore was defined as a cavity deeper than 10 nm in the plasma membrane. White arrows indicate pores. (J) Raji cells were cocultured with CD19-CAR T cells for 1 hour. Pore size and number were measured and counted. **P < 0.01, ***P < 0.001, by Student’s t test (C, D, I, and J) or one-way ANOVA (A, E, G, and H). Data are means ± SD of three independent experiments.

  • Fig. 2 Tumor cell pyroptosis by CAR T cells is mediated by GSDME.

    (A) GSDME and GSDMD in primary B leukemic cells isolated from B-ALL patients (n = 5) or cell lines were determined by Western blot. (B) CD19+ B leukemic cells from B-ALL patients (n = 8) were cocultured with or without CD19-CAR T cells for 6 hours. GSDME, caspase 3 (Casp3), and cleaved caspase 3 (C-Casp3) were analyzed by Western blot. (C and D) SGGFP or GSDME-SGs Raji or NALM-6 cells were cocultured with HER2 or CD19-CAR T cells for 4 hours. The percentage of annexin V+/PI+ or annexin V+ cells (C) and the LDH level from the supernatant (D) were measured. (E to G) GSDME-deficient Raji or NALM-6 cells with overexpressed vector, WT-GSDME, or D270-GSDME were cocultured with CD19-CAR T cells at an E/T ratio of 2:1 for 4 hours. Cell morphology was observed under a microscope (E). The LDH level from the supernatant was detected (F). The expression of GSDME was determined by Western blot (G). Scale bar, 20 μm. White arrows indicate the pyroptotic cell. **P < 0.01, by one-way ANOVA (C, D, and F). Data are means ± SD of three independent experiments.

  • Fig. 3 CAR T cell–released granzyme B triggers the activation of GSDME.

    (A) CD19+ Raji or NALM-6 cells were cocultured with or without CD19-CAR T cells for 4 hours. GSDME, Casp1, C-Casp1, Casp3, C-Casp3, Casp4, and C-Casp4 were analyzed by Western blot. (B to D) Scramble (Scr) or GZMB-siRNAs CD19-CAR T cells were cocultured with Luc-Raji or NALM-6 cells for 4 hours. Casp3, C-Casp3, Casp7, and C-Casp7 were analyzed by Western blot (B). Cell viability (C) and LDH levels in the supernatants (D) were measured. (E to G) The same as (B) to (D), except that PRF1-siRNAs CD19-CAR T cells were used. **P < 0.01, by one-way ANOVA (C, D, F, and G). Data are means ± SD of three independent experiments.

  • Fig. 4 Superior affinity is critical for CAR T cell–mediated tumor cell pyroptosis.

    (A to D) hCD19-mCAR T cells were cocultured with CD19-B16 (E/T = 2:1), HER2-B16 (E/T = 2:1), or vector-B16 pulsed with OVA peptide (E/T = 20:1) for 6 hours. Representative cell morphology was shown (A). LDH levels in supernatants, cell viability (B), and the annexin V+ or annexin V+/PI+ cells (C) were measured. The CD107a+ or GrB+ cells were determined by flow cytometry (D). White arrows indicate pyroptotic cells. (E) CD19-recognizing CAR OT-I T cells were cocultured with CD19 or HER2-B16 cells for 6 hours at a 2:1 E/T ratio. The expression of GSDME was determined by Western blot. (F to H) The same as (A), except that SGGFP or GSDME-SGs CD19-B16 cells were used. Cell morphology was observed by microscopy (F). The LDH level from the supernatant (G) was measured, and cell viability (H) was detected by a microplate luminometer. Scale bar, 20 μm. (I to K) Luc-OVA-B16 or B16 cells were cocultured with tumor-specific CD8+ T cells in the presence or absence of 10 nM perforin and granzyme B. A representative image of cell death is shown (I). Six hours later, cell viability (J) and LDH levels (K) were measured. White arrows indicate the pyroptotic cells. **P < 0.01, by one-way ANOVA (B to D, G, J, and K). Data are means ± SD of three independent experiments.

  • Fig. 5 Co-signaling domain(s) is important for pyroptosis by CAR T cells.

    (A to D) CD19-recognizing CD3ζ-CAR T cells were cocultured with CD19-expressing B16 cells in the presence of a CD28 antibody. Meanwhile, OT-I T cells were cocultured with OT-I peptide-pulsed, CD19-expressing B16 cells. CD3ζ-CD28-CAR T cells were used as a positive control. Cell viability was measured by a microplate luminometer (A), and the LDH level from the supernatant was detected (B). The percentage of annexin V+/PI+ or annexin V+ cells was determined by flow cytometry (C). Cell morphology was observed by microscopy (D). Scale bar, 20 μm. (E to G) CD19-B16 cells were cocultured with HER2-CAR T cells or CD3ζ, CD3ζ-CD28, CD3ζ–4-1BB, or CD3ζ–CD28–4-1BB–CD19–CAR T cells for 4 hours. Cell morphology was observed under a microscope (E). The LDH level (F) and cell viability (G) were also measured. Scale bar, 20 μm. **P < 0.01, by one-way ANOVA (A to C, F, and G). Data are means ± SD of three independent experiments.

  • Fig. 6 Tumor cell pyroptosis triggered macrophages to release proinflammatory cytokines.

    (A) Macrophages isolated from healthy volunteers were treated with control or pyroptotic Raji supernatants. IL-1β and IL-6 in the culture medium were determined by enzyme-linked immunosorbent assay (ELISA). (B) The same as (A), except that pyroptotic primary B leukemic cell supernatants were used. (C and D) Human macrophages were treated with supernatants from coculturing SGGFP or GSDME-SGs Raji or NALM-6 cells with or without CD19-CAR T cells. IL-1β and IL-6 were determined by ELISA (C), and GSDMD, Casp1, C-Casp1, and NLRP3 were analyzed by Western blot (D). (E) SGGFP, CASP1-SGs, or GSDMD-SGs THP-1 cells were treated with supernatants from coculturing Raji cells and CAR T cells. IL-1β and IL-6 were determined by ELISA. **P < 0.01, by one-way ANOVA (A to C and E). Data are means ± SD of three independent experiments.

  • Fig. 7 Tumor cell pyroptosis triggers CRS in CAR T cell–treated mice.

    (A to E) CD19-CAR T cells were transferred to SCID-beige mice with a high Raji tumor burden. The change of weight [(A), n = 6] and body temperature [(B), n = 6] was calculated. The serum levels of SAA (C), IL-6 (D), and IL-1β (E) were measured by ELISA (n = 5). (F to I) SGGFP or GSDME-SGs–Luc-Raji cells were intraperitoneally injected into mice for 3 weeks, followed by the intravenous injection of CD19-CAR T cells. Weight [(F), n = 6] and temperature [(G), n = 6] changes were calculated. Serum levels of SAA, IL-6, and IL-1β were measured by ELISA [(H), n = 5]. Mice survival was analyzed [(I), n = 10]. (J) The same as (A), except that intraperitoneal macrophages were isolated to perform Western blot with anti-GSDMD, Casp1, and C-Casp1 (n = 4). (K to M) Mice with high Raji tumor burden were treated with a control liposome, clophosome-A (intravenous, 200 μl), or belnacasan (100 mg/kg) once daily for 3 days, followed by CD19-CAR T cell injection. Thirty-six hours later, serum levels of SAA, IL-6, and IL-1β were measured by ELISA [(K), n = 5]. Weight and temperature changes were calculated [(L), n = 6]. Mouse survival was recorded [(M), n = 10]. (N) Primary B leukemic cells isolated from B-ALL patients (n = 11) were lysed for Western blot against GSDME. (O) Correlation between GSDME expression and grade of CRS (n = 11). (P) Correlation between LDH level and CRS grade (n = 11). **P < 0.01, ***P < 0.001, by Student’s t test (A and B), one-way ANOVA (C to H, K, and L) or by log-rank survival analysis (I and M). Data are means ± SD.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/5/43/eaax7969/DC1

    Materials and Methods

    Fig. S1. CAR T cells induced tumor cells to enter pyroptosis.

    Fig. S2. GSDME-mediated CAR T cell induced tumor cell pyroptosis.

    Fig. S3. Perforin/granzyme B–caspase 3–GSDME pathway–mediated CAR T cell induced tumor cell pyroptosis.

    Fig. S4. Tumor-specific CD8+ T cells induced tumor cells to enter apoptosis, not pyroptosis.

    Fig. S5. High levels of perforin and granzyme B induce tumor cell pyroptosis.

    Fig. S6. Tumor cell pyroptosis activated macrophages to release inflammatory cytokines.

    Fig. S7. Pyroptotic tumor cells released ATP to activate macrophages.

    Fig. S8. Tumor cell pyroptosis initiated CRS induced by CAR T cell therapy in vivo.

    Fig. S9. ATP-Casp1-GSDMD pathway regulated the CRS induced by CAR T cell therapy in vivo.

    Table S1. The general information from patients enrolled.

    Table S2. Body temperature (ºC) from B-ALL patients after CD19 CAR T cell therapy.

    Table S3. Serum level of IL-6 (pg/ml) from patients after CAR T cell therapy.

    Table S4. Serum level of IFN-γ (pg/ml) from patients after CAR T cell therapy.

    Table S5. Serum level of IL-10 (pg/ml) from patients after CAR T cell therapy.

    Movie S1. HER2+ m-Cherry–MCF-7 cells were cocultured with HER2-CAR T cells.

    Movie S2. HER2+ m-Cherry–SGC-7901 cells were cocultured with HER2-CAR T cells.

  • Supplementary Materials

    The PDF file includes:

    • Materials and Methods
    • Fig. S1. CAR T cells induced tumor cells to enter pyroptosis.
    • Fig. S2. GSDME-mediated CAR T cell induced tumor cell pyroptosis.
    • Fig. S3. Perforin/granzyme B–caspase 3–GSDME pathway–mediated CAR T cell induced tumor cell pyroptosis.
    • Fig. S4. Tumor-specific CD8+ T cells induced tumor cells to enter apoptosis, not pyroptosis.
    • Fig. S5. High levels of perforin and granzyme B induce tumor cell pyroptosis.
    • Fig. S6. Tumor cell pyroptosis activated macrophages to release inflammatory cytokines.
    • Fig. S7. Pyroptotic tumor cells released ATP to activate macrophages.
    • Fig. S8. Tumor cell pyroptosis initiated CRS induced by CAR T cell therapy in vivo.
    • Fig. S9. ATP-Casp1-GSDMD pathway regulated the CRS induced by CAR T cell therapy in vivo.
    • Table S1. The general information from patients enrolled.
    • Table S2. Body temperature (ºC) from B-ALL patients after CD19 CAR T cell therapy.
    • Table S3. Serum level of IL-6 (pg/ml) from patients after CAR T cell therapy.
    • Table S4. Serum level of IFN-γ (pg/ml) from patients after CAR T cell therapy.
    • Table S5. Serum level of IL-10 (pg/ml) from patients after CAR T cell therapy.
    • Legends for movies S1 and S2

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). HER2+ m-Cherry–MCF-7 cells were cocultured with HER2-CAR T cells.
    • Movie S2 (.avi format). HER2+ m-Cherry–SGC-7901 cells were cocultured with HER2-CAR T cells.

    Files in this Data Supplement:

Stay Connected to Science Immunology

Navigate This Article