Fig. 7: In vivo IL-9 blockade in Eμ-TCL1/p66^−/− mice normalizes PD-1 expression in CD8⁺ cells.

A Schematic representation of the experimental design of intraperitoneal injection of wild-type C57BL/6 mice (n = 3) or Eμ-TCL1/p66Shc^−/− mice (n = 6) with overt leukemia (~40% CD5⁺CD19⁺ leukemic cells in PB) with 100 μg of either isotype control (n = 3) or anti-IL-9 (n = 3) antibodies dissolved in 100 μl PBS. B Flow cytometric analysis of PD-1⁺CD8⁺ cells in spleens isolated from either wild type mice injected with PBS (n = 3) or Eμ-TCL1/p66Shc^−/− mice (n = 6) injected with anti-IL-9 (IL-9 Ab, n = 3) or isotype (iso Ab, n = 3) mAb. Data are expressed as MFI of PD-1 in CD8⁺-gated cells (left) and as % PD-1⁺CD8⁺ cells (right) (RM one-way ANOVA). C Flow cytometric analysis of PD-1⁺CD8⁺ cells in 220 μm-thick slices obtained from spleens of wild-type mice (n = 3) and cultured for 48 h in the presence of conditioned media, with the addition of either control (iso Ab), anti-IL-9 (IL-9 Ab) or recombinant IL-9 (rIL-9) (n independent experiments ≥3; one-way ANOVA). D Quantification by ELISA of IL-9 released in media conditioned by splenic B cells from either wild-type (n = 6) or Eμ-TCL1/p66^−/− (n = 6) mice. Mann Whitney Rank Sum test. E. Quantification by ELISA of IL-9 released in the culture supernatants of leukemic cells isolated from peripheral blood of CLL patients (n = 13) and treated in vitro for 48 h with either DMSO or 10 μM Ibrutinib. Paired t test. F qRT-PCR analysis of mRNA expression of IL-9 in leukemic cells isolated from peripheral blood of CLL patients (n = 4) who received 420 mg Ibrutinib once a day. Blood samples were collected on the starting day of Ibrutinib treatment (before Ibrutinib) and at follow-up of Ibrutinib treatment (follow-up Ibrutinib; range follow-up 26.2 ± 7.3 months). Paired t test. Data are expressed as mean ± SD. ****p ≤ 0.0001; **p ≤ 0.01; *p ≤ 0.05.