Innate T-αβ lymphocytes as new immunological components of anti-tumoral "off-target" effects of the tyrosine kinase inhibitor dasatinib

Abstract

Kinase inhibitors hold great potential as targeted therapy against malignant cells. Among them, the tyrosine kinase inhibitor dasatinib is known for a number of clinically relevant off-target actions, attributed in part to effects on components of the immune system, especially conventional T-cells and natural killer (NK)-cells. Here, we have hypothesized that dasatinib also influences non-conventional T-αβ cell subsets known for their potential anti-tumoral properties, namely iNKT cells and the distinct new innate CD8 T-cell subset. In mice, where the two subsets were originally characterized, an activated state of iNKT cells associated with a shift toward an iNKT cell Th1-phenotype was observed after dasatinib treatment in vivo. Despite decreased frequency of the total memory CD8 T-cell compartment, the proportion of innate-memory CD8 T-cells and their IFNγ expression in response to an innate-like stimulation increased in response to dasatinib. Lastly, in patients administered with dasatinib for the treatment of BCR-ABL-positive leukemias, we provided the proof of concept that the kinase inhibitor also influences the two innate T-cell subsets in humans, as attested by their increased frequency in the peripheral blood. These data highlight the potential immunostimulatory capacity of dasatinib on innate T-αβ cells, thereby opening new opportunities for chemoimmunotherapy.

Figure 1

Dasatinib promotes type 1 iNKT cells in mice in vivo. (A–C) Flow cytometry analysis of thymic cells from BALB/c WT mice orally gaved with dasatinib (Dasa, n = 12) or its excipient (CTR, n = 8) for 8 weeks. Analysis of iNKT cell frequency (A) and CD69 positive iNKT cell frequency (B). (C) iNKT cell differentiation into NKT1, NKT2 and NKT17 subtypes: frequency (upper panel) and T-bet, PLZF and RORγt MFI in iNKT cells (lower panel) are shown. Representative plots are shown. Statistical analysis: Mann-Whitney two-tailed.

Figure 2

iNKT-cell frequency increases in CML patients under dasatinib treatment PBMCs isolated from patients (n = 47) at CML diagnosis (Dg) or after 3 months of dasatinib treatment (Dasa) were analyzed by flow cytometry for iNKT cells frequency (A) (box plot representation: median, quartiles, bars: 5 and 95 percentiles) and PLZF MFI in iNKT cells (n = 24) (B). Representative plots are shown. Statistical analysis: paired two-tailed Wilcoxon test.

Figure 3

Dasatinib promotes CD8 T_TM cells in mice in vivo. Flow cytometry analysis of thymic cells from BALB/c WT mice orally gaved with dasatinib (Dasa, n = 12) or its excipient (CTR, n = 8) for 8 weeks. Analysis of CD8 T_MEM cells among CD8 T-cells (A) and population distribution between T_VM and T_TM cells among CD8 T_MEM cells (B). Representative plots and histograms are shown. Statistical analysis: two-tailed Mann-Whitney test.

Figure 4

Dasatinib targets CD8 T_TM and CD8 T_VM cells in mice in vitro. (A–C) BALB/c Eomes-GFP derived splenocytes were cultured 7 days in the presence of IL-15 with (Dasa, n = 6) or without (CTR, n = 6) dasatinib, and analyzed by flow cytometry. (A) Analysis of total CD8 T-cells among live lymphocytes and population distribution between T_N, T_CM and T_EM cells among CD8 T-cells. (B) Analysis of CD8 T_MEM cells among CD8 T-cells and population distribution between T_VM and T_TM cells among CD8 T_MEM cells. (C) Splenocytes were further stimulated for 16 h with IL-12 and IL-18 and IFNγ secretion was analyzed in T_MEM, T_VM and T_TM cells (n = 8). Representative plots and histograms are shown. Statistical analysis: paired two-tailed Wilcoxon test.

Figure 5

Innate CD8 T-cell frequency increases in CML patients under dasatinib treatment. PBMCs isolated from patients (n = 15) at CML diagnosis (Dg) or after 3 months of dasatinib (Dasa) treatment were analyzed by flow cytometry for panKIR/NKG2A⁺ Eomes⁺ innate CD8 T-cells (A). Eomes and CD49d MFI were analyzed in innate CD8 T-cells (B). Representative plots are shown. Statistical analysis: paired two-tailed Wilcoxon test.