Journal: The Journal of Clinical Investigation

Article Title: Perforin-deficient CAR T cells recapitulate late-onset inflammatory toxicities observed in patients

doi: 10.1172/JCI130059

Figure Lengend Snippet: (A) Cell product characterization: cells were stained for surface CAR expression, CD4/CD8, and CD62L/CD44 and analyzed by flow cytometry 48 hours after the completion of CAR transduction. Gray-dotted histogram overlays represent the untransduced T cell control. (B) CD107a expression on CAR T cells after 4 hours of incubation with CD19+ or CD19– E2aPBX cells. Representative histogram shows CD107a expression on CAR T cell after stimulation with CD19+ E2aPBX cells; gray-dotted histogram overlays represent the isotype control. (C) In vitro cytotoxicity measured by IncuCyte Zoom: GFP-transduced E2aPBX cells were cocultured with CAR T cells (E:T = 2:1). Green objects (GFP+ leukemia cells) were counted at each time point and normalized to untransduced T cell wells (n = 2, biological duplicate). AUC for Prf–/– CAR T cell was 37.8 (95% CI: 37.6–38.1). AUC for WT CAR T cells was 17.8 (95% CI: 17.6–18.0). (D) IFN-γ levels in the 12-hour coculture supernatant of CAR T cells with CD19+ or CD19– E2aPBX cells (E:T = 1:1), as measured by ELISA. (E) Proliferation assay: CAR T cells labeled with CellTrace Violet were cocultured with either CD19+ or CD19– E2aPBX cells (E:T = 1:1) for 3 days and analyzed by flow cytometry. Gray-dotted histogram overlays represent CAR T cells incubated with CD19– E2aPBX cells (unstimulated controls). Representative histograms from 3 biological replicates are shown. (F) CD4+ CAR T cells, CD8+ CAR T cells, or CD4+ and CD8+ CAR T cells (1:1 mixture) were cocultured with E2aPBX cells overnight (E:T = 1:1). Cytokine levels were measured in the coculture supernatant using the Meso Scale Discovery U-PLEX kit. Data are reported as the mean ± SD (B, D, and F). n = 3 (B and D); n = 4–5 (F). Figures are representative of 3 replicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by Kruskal-Wallis test with Dunn’s correction (B and D) and 1-way ANOVA with Šidák’s correction (F).

Article Snippet: Some mice were euthanized on day 13, which was 24 hours after the last administration of antibodies, while the rest were kept for survival analyses. ( B ) Kaplan-Meier survival curve. ( C ) The percentages of CD8 + CAR T cells in spleens on day 13 were assessed by flow cytometry. ( D ) Surface CAR expression, ( E ) the percentages of CD44 + CD62L – (Tem or Teff) cells, and ( F ) PD-1 expression among adoptively transferred CD8 + CAR T cell (CD45.2 + /CD8 + ) subsets in spleens on day 13 were analyzed by flow cytometry. ( G – J ) Leukemia-bearing mice were treated with CAR T cells (5 × 10 6 ) derived from WT, Prf –/– , or ARE-Del (homozygous- or heterozygous-KO) mice according to the treatment scheme in . ( G ) Serum IFN-γ was measured on day 0 (before adoptive T cell transfer) and on days 3, 14, and 29 after adoptive T cell transfer using the Meso Scale Discovery U-PLEX kit. ( H ) Surface CAR expression, ( I ) the percentages of CD44 + CD62L – (Tem or Teff) cells, and ( J ) PD-1 expression within the CD8 + CAR T cell subset (CD45.2 + CD8 + ) in spleens on day 13 were analyzed by flow cytometry.

Techniques: Staining, Expressing, Flow Cytometry, Transduction, Incubation, In Vitro, Enzyme-linked Immunosorbent Assay, Proliferation Assay, Labeling

Journal: The Journal of Clinical Investigation

Article Title: Perforin-deficient CAR T cells recapitulate late-onset inflammatory toxicities observed in patients

doi: 10.1172/JCI130059

Figure Lengend Snippet: (A) Treatment scheme of neutralizing IFN-γ in CAR T cell recipients. Mice were treated with i.p. injection of an IFN-γ–neutralizing antibody (XMG1.2, 200 μg) or an isotype control (rat IgG1, 200 μg) on days 0, 2, 4, 6, 8, 10, and 12. Some mice were euthanized on day 13, which was 24 hours after the last administration of antibodies, while the rest were kept for survival analyses. (B) Kaplan-Meier survival curve. (C) The percentages of CD8+ CAR T cells in spleens on day 13 were assessed by flow cytometry. (D) Surface CAR expression, (E) the percentages of CD44+CD62L– (Tem or Teff) cells, and (F) PD-1 expression among adoptively transferred CD8+ CAR T cell (CD45.2+/CD8+) subsets in spleens on day 13 were analyzed by flow cytometry. (G–J) Leukemia-bearing mice were treated with CAR T cells (5 × 106) derived from WT, Prf–/–, or ARE-Del (homozygous- or heterozygous-KO) mice according to the treatment scheme in Figure 2A. (G) Serum IFN-γ was measured on day 0 (before adoptive T cell transfer) and on days 3, 14, and 29 after adoptive T cell transfer using the Meso Scale Discovery U-PLEX kit. (H) Surface CAR expression, (I) the percentages of CD44+CD62L– (Tem or Teff) cells, and (J) PD-1 expression within the CD8+ CAR T cell subset (CD45.2+CD8+) in spleens on day 13 were analyzed by flow cytometry. Data are reported as the mean ± SD (C–J). n = 4–5 (B, C, E, F, and H–J); n = 9–10 (D and G). Figures are representative of 3 replicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by log-rank (Mantel-Cox) test (B), 1-way ANOVA with Šidák’s correction (C–F and H–J), or Kruskal-Wallis test with Dunn’s correction (G).

Article Snippet: Some mice were euthanized on day 13, which was 24 hours after the last administration of antibodies, while the rest were kept for survival analyses. ( B ) Kaplan-Meier survival curve. ( C ) The percentages of CD8 + CAR T cells in spleens on day 13 were assessed by flow cytometry. ( D ) Surface CAR expression, ( E ) the percentages of CD44 + CD62L – (Tem or Teff) cells, and ( F ) PD-1 expression among adoptively transferred CD8 + CAR T cell (CD45.2 + /CD8 + ) subsets in spleens on day 13 were analyzed by flow cytometry. ( G – J ) Leukemia-bearing mice were treated with CAR T cells (5 × 10 6 ) derived from WT, Prf –/– , or ARE-Del (homozygous- or heterozygous-KO) mice according to the treatment scheme in . ( G ) Serum IFN-γ was measured on day 0 (before adoptive T cell transfer) and on days 3, 14, and 29 after adoptive T cell transfer using the Meso Scale Discovery U-PLEX kit. ( H ) Surface CAR expression, ( I ) the percentages of CD44 + CD62L – (Tem or Teff) cells, and ( J ) PD-1 expression within the CD8 + CAR T cell subset (CD45.2 + CD8 + ) in spleens on day 13 were analyzed by flow cytometry.

Techniques: Injection, Flow Cytometry, Expressing, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: Perforin-deficient CAR T cells recapitulate late-onset inflammatory toxicities observed in patients

doi: 10.1172/JCI130059

Figure Lengend Snippet: (A) Treatment scheme for IL-1 blockade in CAR T cell recipients. Mice were treated with a daily i.p. injection of anakinra (10 mg/kg/day in 200 μL) or PBS (200 μL) from days 0 to 13. Some cohorts of mice were euthanized on day 14, which was 24 hours after the last administration of anakinra or PBS, while the rest were kept for survival analyses. (B) Kaplan-Meier survival curve. (C) Leukemia (CD45.2+CD19+) in BM on day 14 was assessed by flow cytometry. (D) Percentages of CD8+ CAR T cell in spleens on day 14. (E) Surface CAR expression and (F) percentages of CD44+CD62L– (Tem or Teff) cells within the CD8+ CAR T cell subset (CD45.2+CD8+) in spleens were evaluated on day 14. (G) Serum levels of IFN-γ, IL-27, and TNF-α were measured on day 0 (before CAR T cell infusion) and on day 14 using the Meso Scale Discovery U-PLEX kit. Healthy controls were age- and sex-matched untreated littermates. Data are reported as the mean ± SD (C–G). n = 5 (B and C); n = 13–15 (D, data were pooled from independent 2 experiments); n = 9–10 (E and F); n = 10 (G, except for n = 6 in the IFN-γ pre-CAR group). Figures are representative of 2 replicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by log-rank (Mantel-Cox) test (B) or 1-way ANOVA with Šidák’s correction (D–G).

Article Snippet: Some mice were euthanized on day 13, which was 24 hours after the last administration of antibodies, while the rest were kept for survival analyses. ( B ) Kaplan-Meier survival curve. ( C ) The percentages of CD8 + CAR T cells in spleens on day 13 were assessed by flow cytometry. ( D ) Surface CAR expression, ( E ) the percentages of CD44 + CD62L – (Tem or Teff) cells, and ( F ) PD-1 expression among adoptively transferred CD8 + CAR T cell (CD45.2 + /CD8 + ) subsets in spleens on day 13 were analyzed by flow cytometry. ( G – J ) Leukemia-bearing mice were treated with CAR T cells (5 × 10 6 ) derived from WT, Prf –/– , or ARE-Del (homozygous- or heterozygous-KO) mice according to the treatment scheme in . ( G ) Serum IFN-γ was measured on day 0 (before adoptive T cell transfer) and on days 3, 14, and 29 after adoptive T cell transfer using the Meso Scale Discovery U-PLEX kit. ( H ) Surface CAR expression, ( I ) the percentages of CD44 + CD62L – (Tem or Teff) cells, and ( J ) PD-1 expression within the CD8 + CAR T cell subset (CD45.2 + CD8 + ) in spleens on day 13 were analyzed by flow cytometry.

Techniques: Injection, Flow Cytometry, Expressing