Additional file 1 of Ibrutinib protects T cells in patients with CLL from proliferation-induced senescence
Creators: name: Davis, Joanne E.
name: Sharpe, Chia
name: Mason, Kylie
name: Tam, Constantine S.
name: Koldej, Rachel M.
name: Ritchie, David S.
Publisher: name: figshare
Year: 2021
Subjects: subject: Cell Biology
subject: Physiology
subject: FOS: Biological sciences
subject: Pharmacology
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Hematology

description: Additional file 1: Figure S1. Flow cytometry gating strategy. PBMC from HD or CLL patients were gated on single cells, lymphocytes, live cells, and CD19 + B or CD3 + T cells. The memory phenotype of CD8 + or CD4 + T cells was measured using Naïve (N, CD27 + CD45RA +), Central memory (CM, CD27 + CD45RA-), effector memory (EM, CD27-CD45RA-), and terminally differentiated effector memory (TEMRA, CD27-CD45RA +) subsets. CD4 + and CD8 + T cells were also tested for the expression of PD-1, TIM3 and LAG3. Flow gating in the bottom row shows representative CD8 + T cell subsets. Figure S2. Generation 0–2 cell proliferation analysis. Proliferation of CD4 + and CD8 + T cells was measured using FlowJo cell proliferation analysis to identify 9 generations of dividing cells. Generation 0 represents undivided cells, and generation 9 represents cells that have undergone at least 8 divisions. The percentage of cells in generations 0, 1, and 2 was added to give a combined total % of cells in generations 0–2, representing undivided or early generation T cells. Figure S3. Stable memory phenotype CD8 + T cell subset frequency in CLL patients on long-term BTKi therapy. PBMC from CLL patients collected prior to treatment (baseline), or after long-term treatment of ibrutinib (n = 7) or zanubrutinib (n = 8) were compared to Healthy Donor (HD, n = 7). Naïve (N, CD27 + CD45RA +), Central memory (CM, CD27 + CD45RA-), effector memory (EM, CD27-CD45RA-), and terminally differentiated effector memory (TEMRA, CD27-CD45RA +) cells (A-D) were calculated as a percentage of CD8 + T cells. Figure S4. Increased naive CD4 + T cell subset frequency in CLL patients on long-term BTKi therapy. PBMC from CLL patients collected prior to treatment (baseline), or after long-term treatment of ibrutinib (n = 7) or zanubrutinib (n = 8) were compared to Healthy Donor (HD, n = 7). Naïve (N, CD27 + CD45RA +), Central memory (CM, CD27 + CD45RA-), effector memory (EM, CD27-CD45RA-), and terminally differentiated effector memory (TEMRA, CD27-CD45RA +) cells (A-D) were calculated as a percentage of CD4 + T cells. Figure S5. Ibrutinib inhibits cytokine release in CD4 + T cells from CLL patients in vitro. PBMC from untreated CLL patients (n = 5) were incubated with 1 μM ibrutinib, zanubrutinib, or acalabrutinib, or DMSO vehicle control for 18 h prior to activation with T cell stimulation beads for 4 h. CD4 + T cell intracellular IFNγ production is shown, and statistical analysis was performed using mixed effects analysis with Tukey–Kramer test for multiple comparisons. Figure S6. Ibrutinib inhibits T cell cytotoxicity, cytokine release and proliferation of healthy donor T cells in vitro. PBMC from Healthy Donor (HD, n = 6) were incubated with 1 μM ibrutinib, zanubrutinib, or acalabrutinib, or DMSO vehicle control for 18 h prior to activation with T cell stimulation beads for 4 h. CD8 + T cell degranulation (A) intracellular IFNγ production (B) are shown, and statistical analysis was performed using mixed effects analysis with Tukey–Kramer test for multiple comparisons. IL-2, IFNγ, TNFα and IL-4 levels in the supernatant were measured after 24 h of T cell activation bead stimulation of PBMC from HD (n = 4) in the presence of BTKi (C). The sensitivity of the assay was limited to 5000 pg/ml. Data was analysed using RM one-way ANOVA with Fisher’s LSD. Figure S7. Long-term BTKi treatment normalises CD8 + and CD4 + exhaustion marker expression in dividing T cells. Proliferation of PBMC from CLL patients collected at baseline or after long-term treatment of ibrutinib (n = 7) or zanubrutinib (n = 8), and Healthy Donor (HD, n = 7). PD-1, TIM3 and LAG3 cell surface expression (MFI) was measured on total CD8 + and CD4 + T cells (A-F). Data was analysed using Mann–Whitney (HD vs patient sample) or Wilcoxon matched-pairs signed rank (baseline vs treatment sample) tests.