Article Figures & Data
Figures
- Figure 1.
Selinexor disrupts normal immune homeostasis. A, C57BL/6 mice were treated with vehicle or KPT-330 (15 mg/kg) by oral gavage 3 times per week (Monday, Wednesday, and Friday) for 2 or 4 weeks. Total thymocyte cell numbers are shown. n = 10, vehicle; n = 5 KPT-330 groups. B, Thymocytes from vehicle or 14-day treated mice in A were stained with antibodies to CD4 and CD8 and analyzed by flow cytometry. Representative image is shown. C, Quantification of thymic populations from vehicle or 14-day treated mice. D, Thymocytes from vehicle or 14-day treated mice in A were stained with antibodies to CD4, CD8, CD25, and CD44 and analyzed by flow cytometry. Representative image is shown, gated on CD4−CD8− cells. E, C57BL/6 mice were treated with vehicle or KPT-330 (15 mg/kg) by oral gavage 3 times per week (Monday, Wednesday, and Friday) for 4 weeks. Bone marrow cells were harvested, stained with antibodies, and analyzed by flow cytometry. CD8 T cells = CD45+CD8+; CD4 T cells = CD45+CD4+; myeloid = CD45+CD11b+Gr1−; pre, proB = CD45+B220+IgD−; immature/mature B = CD45+B220+IgD+; NK cells = CD45+NK1.1+; neutrophils = CD45+CD11b+Gr1+; DN T cells = CD45+CD3+CD4−CD8−; lineage− = CD45+ negative for all other markers. Pie chart shows average frequencies from n = 5 mice per group. F, C57BL/6 mice were treated with vehicle or KPT-330 (15 mg/kg) by oral gavage 3 times per week (Monday, Wednesday, and Friday) for 1, 2, or 4 weeks. Spleen cells were harvested, stained with antibodies, and analyzed by flow cytometry. B cells = CD45+B220+; CD4 T cells = CD45+CD4+Foxp3−; Tregs = CD45+CD4+Foxp3+; macrophages = CD45+CD11b+Gr1−; CD8 T cells = CD45+CD8+; NK cells = CD45+NK1.1+; monocytes = CD45+CD11b+Ly6C+Gr1−; neutrophils = CD45+CD11b+Gr1+. Pie charts show averages from n = 5 mice per group.
- Figure 2.
Antibody production is only modestly affected by selinexor. A, Diagram of KPT-330 dosing and immunization schedule. M, Monday; W, Wednesday; F, Friday. B, C57BL/6 mice were immunized according to diagram shown in A. Serum was collected at the indicated time points, diluted at the indicated ratios, and used in an ELISA with ovalbumin-coated plates. Secondary anti-mouse Igκ coupled to HRP was used for detection. Each line represents one mouse. n = 3 mice per group. C, Serum was analyzed as in B, using secondary anti-mouse IgG1, anti-mouse IgG2b, or anti-mouse IgG2c coupled to HRP for detection.
- Figure 3.
Decreased frequency of selinexor treatment restores immune homeostasis better than decreased dose. A, C57BL/6 mice were dosed for 3 weeks according to the indicated schedules. n = 5 mice per group. M, Monday; W, Wednesday; F, Friday. B, Total thymocyte cell numbers. C, Bone marrow cells were harvested and stained as in Fig. 1E. CD8 T cells and immature B cells were quantified. D, Mesenteric lymph node cells were harvested and stained as in Fig. 1F. Monocytes were quantified. E, Spleen cells were harvested and stained as in Fig. 1F. Monocytes and CD8 T cells were quantified.
- Figure 4.
Selinexor blocks activation of naïve T cells. A, CD8 T cells from an OT-I mouse were labeled with CellTrace violet and cocultured with CD40-activated B cells pulsed with ovalbumin peptide (SIINFEKL) at the indicated concentrations in the presence or absence of 1 μmol/L KPT-330. T-cell proliferation was measured by flow cytometry 3 days later. B, CD8 T cells from C57BL/6 mice were labeled with CellTrace violet and cocultured with anti-CD3/CD28–coated beads or PMA/Ion in the presence or absence of 1 μmol/L KPT-330. Three days later, cells were stained with anti-CD25 and anti-CD8 and analyzed by flow cytometry. C, Total lymph node cells from C57BL/6 mice were cultured with anti-CD3/CD28 beads and the indicated concentrations of KPT-330 added at the indicated time points relative to CD3/CD28 stimulation. Twenty-four hours later, cells were stained with antibodies to CD4, CD8, and CD25 and analyzed by flow cytometry.
- Figure 5.
Selinexor blocks the activation of effector CD8 T cells. A, CD8 T cells from an OT-I mouse were cultured with anti-CD3/CD28 beads for 7 days to generate effector cells. Effector cells were washed, counted, and plated onto B16OVA tumor cells that had been pretreated with IFNγ to induce MHC class I expression. KPT-330 was added to the cocultures at the indicated concentrations. Twenty-four hours later, OT-I cells were collected, fixed and permeabilized, and stained with antibodies to CD25, CD69, IFNγ, and granzyme B, and analyzed by flow cytometry. No response was seen from OT-I effector cells cultured with B16 cells not transduced with ovalbumin. Representative of three independent experiments. B, C57BL/6 mice were dosed with KPT-330 10 mg/kg by oral gavage. Serum was collected at various time points and analyzed for KPT-330 concentration by mass spectrometry. Dashed line, 100 nmol/L concentration.
- Figure 6.
Optimized selinexor dosing does not inhibit antitumor immunity in vivo. A, C57BL/6 mice were inoculated subcutaneously with 1 × 105 B16 cells on day 0. On days 7, 9, 14, and 16, mice were dosed with vehicle or 10 mg/kg KPT-330 by oral gavage. Tumors were harvested on day 21, digested and infiltrates analyzed by flow cytometry. Total CD45+ cells were quantified. n = 5 mice per group. Representative of two experiments. B, Tumor infiltrates from A were stained with antibodies to CD45, CD8, CD4, NK1.1, B220, MHC class II, Gr1, Foxp3, CD11b, and CD11c and analyzed by flow cytometry. n = 5 mice per group. Representative of two experiments. M, Monday; W, Wednesday. C, C57BL/6 mice were inoculated with B16 tumors and dosed with 10 mg/kg KPT-330 on days 6, 8, 13, and 15. On day 12 after tumor inoculation, all mice received adoptive transfer of 5 × 105 activated TRP1high;CD45.1+ CD8 T cells. On day 17, tumor infiltrates were harvested, stained with the indicated antibodies, and analyzed by flow cytometry after gating on CD45+CD8+CD45.1+ TRP1-specific cells. n = 5 mice per group. D, Tumor infiltrates from C were gated on endogenous CD8 T cells (CD45+CD8+CD45.1−). E, Tumor infiltrates from C were stained with antibodies to PD-L1 and other cell surface markers. Mean fluorescence intensity (MFI) of PD-L1 expression was quantified. Tumor = CD45−; Mac = CD45+CD11b+Gr1−CD11c−; MDSCs = CD45+CD11b+Gr1+; dendritic cells = CD45+MHCII+CD11c+. n = 5 mice per group. F, Tumor growth of mice analyzed in C and D.
Additional Files
Supplementary Data
- Supplemental Figure 1 - Supplemental Figure 1: Response to immunization is only modestly affected by selinexor.
- Supplemental Figure 2 - Supplemental Figure 2: IFNγ secretion is impaired by selinexor.
- Supplemental Figure 3 - Supplemental Figure 3: Degranulation of effector CD8 T cells is affected by selinexor.
- Supplemental Figure legends - Legends for supplemental figures
Volume 16, Issue 3
