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B.S. Hanna et al.
we utilized the TCL1 AT model of CLL.9,25 We transplanted C57BL/6 WT mice with TCL1 splenocytes, and after 2 weeks mice were assigned to treatment with ibrutinib or vehicle according to tumor load in PB. In line with published data,29 ibrutinib treatment resulted in a significant decrease in CLL development in comparison to vehicle-treated mice, as evident by a decrease in CD5+CD19+ cell counts in PB and decreased splenomegaly and hepatomegaly (Figure 1A and B). Moreover, CLL cell proliferation was significantly reduced, as measured by Ki-67 staining (Figure 1C), confirm- ing efficient cytotoxic activity of the drug.
We then evaluated the effect of ibrutinib on anti- tumoral CD8+ T cells. We have recently shown that CLL development in TCL1 mice induces the differentiation of an anti-tumoral, oligoclonal effector CD8+ T-cell popula- tion that controls tumor development in an IFNγ-depen- dent manner.8 Accordingly, we analyzed the splenic CD8+ T-cell composition using CD44 and CD127, which divide CD8+ T cells into CD127hiCD44low naïve, CD127hiCD44hi memory, and CD127lowCD44int-hi effector subsets (Figure 1D). As expected, CLL development induced a sizable expansion of CD127lowCD44+ effector T cells in vehicle-
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Figure 1. Ibrutinib modulates effector CD8+ T-cell differentiation and proliferation in the TCL1 adoptive transfer model. C57BL/6 mice were transplanted with splenocytes from leukemic TCL1 mice, and after 2 weeks assigned to treatment with ibrutinib (0.16 mg/mL) or vehicle control in drinking water. Mice were sacrificed after 2 weeks of treatment. Untransplanted C57BL/6 mice (n=3) were used as controls. (A) Absolute numbers of CD5+CD19+ chronic lymphocytic leukemia (CLL) cells in peripheral blood analyzed by flow cytometry, and (B) spleen and liver weight of vehicle- or ibrutinib-treated mice (n=4). (C) Flow cytometric analysis of Ki-67 expression in CD5+CD19+ CLL cells in spleen from vehicle- or ibrutinib-treated mice (n=4). (D) Flow cytometric analysis of splenic CD3+CD8+ T cells from vehicle- or ibrutinib-treated mice (n=4). Cell subsets were defined as naïve (CD127hiCD44low), memory (Mem: CD127hiCD44hi), and effector (Eff: CD127lowCD44int-hi) cells. (E) Absolute numbers of splenic CD8+ Eff cells from control, vehicle- or ibrutinib-treated mice (n=4). (F) Flow cytometric analysis of CD69 and CD137, (G) T-bet and Eomes (FMO: fluorescence minus 1), and (H) Ki-67 expression on CD8+ T cells from control, vehicle- or ibrutinib-treated mice (n=4). Results are representative of at least two independent experiments. Graphs show means±standard error of mean. **P<0.01, ***P<0.001.
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