Additional file 1 of CAR T cells and T cells phenotype and function are impacted by glucocorticoid exposure with different magnitude
Creators: name: Poiret, Thomas
name: Vikberg, Sara
name: Schoutrop, Esther
name: Mattsson, Jonas
name: Magalhaes, Isabelle
Publisher: name: figshare
Year: 2024
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 1: Figure S1. UT T cells phenotype. A Representative plot of the differentiation subsets defined by CCR7 and CD45RA markers. B Memory phenotype of CAR + and untransduced (UT) fractions of CD3 + T cells in CD19, M28z and MBBz CAR T cell products.GR expression (MFI) in the naïve and TCM memory subsets of (C) UT (from CD19 CAR cell product) CD4 + and CD8 + T cells; (D) of the UT (from M28z and MBBz CAR cell products) CD4 + (left) and CD8 + (right) T cells. n = 5 donors for CD19 CAR T cells and n = 4 donors for M28z and MBBz CAR T cells. Student t test was used to compare GR in different subsets. Medians are represented. *p &lt; 0.05, **p &lt; 0.01. Figure S2. Impact of GC exposure on UT T cells phenotype. Frequency of CD19 CAR T cells (A) and CD4/CD8 ratio in CD19 CAR T cells (B) overtime after Dx (green) or MP (blue) exposure. C. Representative plot of LAG-3, PD-1 and TIM-3 expression without GC exposure or with 10µg/ml Dx. D. Frequency of LAG-3, PD-1, and TIM-3 in CD19 CAR + T cells without or after 1 and 3 exposures with Dx or MP. Relative surface expression of LAG-3, PD-1, andTIM-3 in CAR + and UT CD4 + (E) and CD8 + (F) T cells after 3 exposures with Dx (left) or MP (right). G Frequency of PD-1 in CD4 + and CD8 + CD19 CAR + T cells without or after 3 exposures with Dx or after 3 exposures with Dx and RU-486 (10−5M). H Relative expression of LAG-3, PD-1, and TIM-3 in CD4 + and CD8 + UT T cells after 3 exposures of Dx (left) or MP (right). n = 6 donors. Friedman test with Dunn’s correction was used to compare marker’s expression between 3 conditions. Wilcoxon matched-pairs signed rank test was used to compare marker’s expression in CAR + vs. UT or CD4 + vs. CD8 + T cells. Medians are represented. * p &lt; 0.05, ** p &lt; 0.01, *** p &lt; 0.001. Figure S3. Impact of GCs exposure on CD19 CAR + T cells effector functions. A. Relative frequency of IFNγ + , TNF + or IL2 + CD4 + or CD8 + CD19 CAR and UT after a single exposure with Dx (top) or MP (bottom) after PMA/ionomycin stimulation. B. Frequency of CD107a- IFNγ- TNF- IL2- CD4 + and CD8 + CAR (circle) and UT (square) T cells after 3 exposures with Dx or Dx + RU-486 after PMA/ionomycin stimulation. Frequency of CD107a + , IFNγ + , TNF and IL-2 + CAR CD4 + and CD8 + T cells without GC exposure (0) or 1 exposure with low or high doses of GC (0.1 µg/ml and 10 µg/ml Dx and 0.5µg/ml, 50 µg/ml, and 100 µg/ml MP) following a single (C) or 3 (D) stimulations with CD19 + K562 tumor cells. E. Pie chart showing the number of functions of CD8 + CAR T cells without GC exposure compared to Dx or MP exposure following a single (n = 8 donors) or 3 stimulations (n = 5 donors) with CD19 + K562 tumor cells. Two-way ANOVA with Sidak’s correction was used to compare CD107a + , IFNγ + , TNF and IL-2 + cells frequency at different doses of Dx and MP. Friedman test with Dunn’s correction was used to compare multifunction between the 3 different conditions. Medians are represented. * p &lt; 0.05, ** p &lt; 0.01, *** p &lt; 0.001. Figure S4. Impact of GCs exposure on M28z and MBBz CAR + T cells phenotype and functions. Frequency of M28z and MBBz CAR T cells (A) and CD4/CD8 ratio in CAR T cells (B) without GC exposure (0) or after 3 exposures with low and high concentration of Dx or MP. Frequency of LAG-3 (C), PD-1 (D) and TIM-3 (E) in M28z and MBBz CAR T cells without GC exposure (0) or after 3 exposures with low and high concentration of Dx or MP. Relative surface expression of LAG-3, PD-1 and TIM-3 in CAR + and UT fractions of CD8 + M28z and MBBz T cells following 3 exposures with Dx (F) or MP (G). Frequency of CD107a + , IFNγ + , TNF and IL-2 + CD4 + (H) and CD8 + (I) M28z and MBBz CAR T cells without GC exposure (0) compared to Dx or MP exposure following 3 stimulations with MSLN + K562 tumor cells. A-E n = 6, F&amp;G n = 11 and H&amp;I n = 5 donors. Two-way ANNOVA with Sidak’s correction was used to compare CD107a + , IFNγ + , TNF and IL-2 + cells frequency at different doses of Dx and MP. Mann–Whitney test was used to the surface expressions and functions in M28z vs. MBBz. Medians are represented. * p &lt; 0.05, ** p &lt; 0.01, *** p &lt; 0.001. Figure S5. M28z and MBBz CD4 + and CD8 + CAR T cells phenotype after GC removal. A Representative histograms of GFP and MSLN expression and co-expression on K562 target cells as compared to control K562 (GFP-MSLN-). Relative frequency of LAG-3, PD-1 and TIM-3 in M28z and MBBz CD4 + (B&amp;D) and CD8 + (C&amp;E) CAR T cells after 3 exposures with Dx (B-C) or MP (D-E) and 48h rest in a GC-free medium. n = 5 donors. Student t test was used to compare surface marker expression in GC-exposed vs. GC-rested conditions. * p &lt; 0.05, ** p &lt; 0.01, *** p &lt; 0.001.