The effect of IFN-α on Jak2V617F;Tyk2−/− progenitors and HSCs in a chimeric BM transplant model. (A) Schematic representation of the generation of mixed WT/Jak2V617F chimeric mice (competitive transplantation, donor; 2 × 106Jak2V617F BM cells with competitor 2 × 105 WT BM cells, recipient; WT mice) or mixed WT/Jak2V617F;Tyk2−/− chimeric mice (competitive transplantation, donor; 2 × 106Jak2V617F;Tyk2−/− BM cells with competitor 2 × 105 WT BM cells, recipient; WT mice). After 4 weeks, recipient mice were treated with PBS or 600 ng of ropeg-IFN-α for 8 weeks. PBS-treated mixed WT/Jak2V617F chimeric mice (n = 10), ropeg-IFN-α–treated mixed WT/Jak2V617F chimeric mice (n = 10), PBS-treated mixed WT/Jak2V617F;Tyk2−/− chimeric mice (n = 17), and ropeg-IFN-α–treated mixed WT/Jak2V617F;Tyk2−/− chimeric mice (n = 17); shown in panels B-G, respectively. (B) Complete blood cell count after 8 weeks of PBS or ropeg-IFN-α treatment in mixed WT/Jak2V617F chimeric mice and mixed WT/Jak2V617F;Tyk2−/− chimeric mice. The absence of inhibitory effects of IFN-α on leukocyte count and Jak2V617F chimerism in leukocytes in mixed WT/Jak2V617F;Tyk2−/− chimeric mice. (C) The change in Jak2V617F chimerism in PB leukocytes. The decrement of Jak2V617F chimerism in PB leukocytes from mixed WT/Jak2V617F chimeric mice after ropeg-IFN-α treatment was absent in mixed WT/Jak2V617F;Tyk2−/− chimeric mice. (D) The change in hematopoietic compartments of the PB after 8 weeks of PBS or ropeg-IFN-α treatment. Purple bar, WT cells; blue bar, Jak2V617F cells; green bar, Jak2V617F;Tyk2−/− cells. (E) The proportion of MKPs after treatment with ropeg-IFN-α in the BM from mixed WT/Jak2V617F chimeric mice and mixed WT/Jak2V617F;Tyk2−/− chimeric mice. At 48 hours after the last treatment, the proportion of MEPs, MKPs, and CD41+ cells in the BM was analyzed. Data are presented as mean ± SD. (F) Upper: the proportion of LT-HSCs and other progenitors after treatment with ropeg-IFN-α in the BM from mixed WT/Jak2V617F chimeric mice and mixed WT/Jak2V617F;Tyk2−/− chimeric mice. At 48 hours after the last treatment, the proportions of LT-HSCs, CMPs, and GMPs in the BM were analyzed. Data are presented as mean ± SD. Lower: the change in Jak2V617F chimerism in LT-HSCs and progenitors after 8 weeks of PBS or ropeg-IFN-α treatment. The decrement of Jak2V617F chimerism in LT-HSCs, CMPs, and GMPs from mixed WT/Jak2V617F chimeric mice after ropeg-IFN-α treatment was absent in mixed WT/Jak2V617F;Tyk2−/− chimeric mice. (G) Analysis of the number of Jak2V617F HSCs and their subpopulations after 8 weeks of treatment with ropeg-IFN-α. Left: the total number of LT-HSCs in 2 femurs and 1 tibia from mixed WT/Jak2V617F chimeric mice and mixed WT/Jak2V617F;Tyk2−/− chimeric mice. Purple bar, WT LT-HSCs; blue bar, Jak2V617F LT-HSCs; green bar, Jak2V617F;Tyk2−/− LT-HSCs. Right: percentage of CD41hi and CD41lo LT-HSCs. Orange bar, CD41lo LT-HSCs; dark orange bar, CD41hi LT-HSCs. (H) Effects of IFN-α treatment on HSC cell cycles in Jak2V617F and Jak2V617F;Tyk2−/− mice. BM cells from 2 mice of each type were mixed, and cell cycle analysis was performed. The sample size for each treatment group: PBS-treated mixed WT/Jak2V617F chimeric mice (n = 5), ropeg-IFN-α–treated mixed WT/Jak2V617F chimeric mice (n = 5), PBS-treated mixed WT/Jak2V617F;Tyk2−/− chimeric mice (n = 8), and ropeg-IFN-α–treated mixed WT/Jak2V617F;Tyk2−/− chimeric mice (n = 8). Left: G0 phase (Ki67lowH333422n). Middle: G1 phase (Ki67+H333422n). Right: SG2M phase (Ki67+H333424n). Reduction in quiescent (Ki67lowH333422n) Jak2V617F HSCs but not in Jak2V617F;Tyk2−/− HSCs after IFN-α treatment. Increase in actively cycling HSCs (Ki67+H333424n) in Jak2V617F HSCs but not in Jak2V617F;Tyk2−/− HSCs after IFN-α treatment. Data are presented as mean ± SD. Statistical significance was determined by the Tukey test after 1-way ANOVA; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. (I) Schematic representation of generation of Tyk2−/−/Jak2V617F;Tyk2−/− chimeric mice (noncompetitive transplantation, donor; 2 × 106Jak2V617F;Tyk2−/− BM cells, recipient; Tyk2−/− mice). After 4 weeks, recipient mice were treated with PBS or 600 ng of ropeg-IFN-α for 8 weeks. PBS-treated Tyk2−/−/Jak2V617F;Tyk2−/− chimeric mice (n = 8) and ropeg-IFN-α–treated Tyk2−/−/Jak2V617F;Tyk2−/− chimeric mice (n = 8; panels J and K, respectively). (J) The proportion of HSCs and progenitors in BM from Tyk2−/−/Jak2V617F;Tyk2−/− chimeric mice after treatment with ropeg-IFN-α. At 48 hours after the last treatment, the proportion of LT-HSCs, CMPs, GMPs, MEPs, MKPs, and CD41+ cells in the BM was analyzed. Data are presented as mean ± SD. The absence of inhibitory effects of IFN-α on the proportion of MKPs from Tyk2−/−/Jak2V617F;Tyk2−/− chimeric mice. (K) PB complete cell count in leukocytes after 8 weeks of PBS or ropeg-IFN-α treatment in Tyk2−/−/Jak2V617F;Tyk2−/− chimeric mice. The absence of inhibitory effects of IFN-α on leukocyte count but not on platelet count in Tyk2−/−/Jak2V617F;Tyk2−/− chimeric mice. WBC, white blood cell; Hb, hemoglobin; Plt, platelet; n.s., not significant.

The effect of IFN-α on Jak2V617F;Tyk2−/− progenitors and HSCs in a chimeric BM transplant model. (A) Schematic representation of the generation of mixed WT/Jak2V617F chimeric mice (competitive transplantation, donor; 2 × 106Jak2V617F BM cells with competitor 2 × 105 WT BM cells, recipient; WT mice) or mixed WT/Jak2V617F;Tyk2−/− chimeric mice (competitive transplantation, donor; 2 × 106Jak2V617F;Tyk2−/− BM cells with competitor 2 × 105 WT BM cells, recipient; WT mice). After 4 weeks, recipient mice were treated with PBS or 600 ng of ropeg-IFN-α for 8 weeks. PBS-treated mixed WT/Jak2V617F chimeric mice (n = 10), ropeg-IFN-α–treated mixed WT/Jak2V617F chimeric mice (n = 10), PBS-treated mixed WT/Jak2V617F;Tyk2−/− chimeric mice (n = 17), and ropeg-IFN-α–treated mixed WT/Jak2V617F;Tyk2−/− chimeric mice (n = 17); shown in panels B-G, respectively. (B) Complete blood cell count after 8 weeks of PBS or ropeg-IFN-α treatment in mixed WT/Jak2V617F chimeric mice and mixed WT/Jak2V617F;Tyk2−/− chimeric mice. The absence of inhibitory effects of IFN-α on leukocyte count and Jak2V617F chimerism in leukocytes in mixed WT/Jak2V617F;Tyk2−/− chimeric mice. (C) The change in Jak2V617F chimerism in PB leukocytes. The decrement of Jak2V617F chimerism in PB leukocytes from mixed WT/Jak2V617F chimeric mice after ropeg-IFN-α treatment was absent in mixed WT/Jak2V617F;Tyk2−/− chimeric mice. (D) The change in hematopoietic compartments of the PB after 8 weeks of PBS or ropeg-IFN-α treatment. Purple bar, WT cells; blue bar, Jak2V617F cells; green bar, Jak2V617F;Tyk2−/− cells. (E) The proportion of MKPs after treatment with ropeg-IFN-α in the BM from mixed WT/Jak2V617F chimeric mice and mixed WT/Jak2V617F;Tyk2−/− chimeric mice. At 48 hours after the last treatment, the proportion of MEPs, MKPs, and CD41+ cells in the BM was analyzed. Data are presented as mean ± SD. (F) Upper: the proportion of LT-HSCs and other progenitors after treatment with ropeg-IFN-α in the BM from mixed WT/Jak2V617F chimeric mice and mixed WT/Jak2V617F;Tyk2−/− chimeric mice. At 48 hours after the last treatment, the proportions of LT-HSCs, CMPs, and GMPs in the BM were analyzed. Data are presented as mean ± SD. Lower: the change in Jak2V617F chimerism in LT-HSCs and progenitors after 8 weeks of PBS or ropeg-IFN-α treatment. The decrement of Jak2V617F chimerism in LT-HSCs, CMPs, and GMPs from mixed WT/Jak2V617F chimeric mice after ropeg-IFN-α treatment was absent in mixed WT/Jak2V617F;Tyk2−/− chimeric mice. (G) Analysis of the number of Jak2V617F HSCs and their subpopulations after 8 weeks of treatment with ropeg-IFN-α. Left: the total number of LT-HSCs in 2 femurs and 1 tibia from mixed WT/Jak2V617F chimeric mice and mixed WT/Jak2V617F;Tyk2−/− chimeric mice. Purple bar, WT LT-HSCs; blue bar, Jak2V617F LT-HSCs; green bar, Jak2V617F;Tyk2−/− LT-HSCs. Right: percentage of CD41hi and CD41lo LT-HSCs. Orange bar, CD41lo LT-HSCs; dark orange bar, CD41hi LT-HSCs. (H) Effects of IFN-α treatment on HSC cell cycles in Jak2V617F and Jak2V617F;Tyk2−/− mice. BM cells from 2 mice of each type were mixed, and cell cycle analysis was performed. The sample size for each treatment group: PBS-treated mixed WT/Jak2V617F chimeric mice (n = 5), ropeg-IFN-α–treated mixed WT/Jak2V617F chimeric mice (n = 5), PBS-treated mixed WT/Jak2V617F;Tyk2−/− chimeric mice (n = 8), and ropeg-IFN-α–treated mixed WT/Jak2V617F;Tyk2−/− chimeric mice (n = 8). Left: G0 phase (Ki67lowH333422n). Middle: G1 phase (Ki67+H333422n). Right: SG2M phase (Ki67+H333424n). Reduction in quiescent (Ki67lowH333422n) Jak2V617F HSCs but not in Jak2V617F;Tyk2−/− HSCs after IFN-α treatment. Increase in actively cycling HSCs (Ki67+H333424n) in Jak2V617F HSCs but not in Jak2V617F;Tyk2−/− HSCs after IFN-α treatment. Data are presented as mean ± SD. Statistical significance was determined by the Tukey test after 1-way ANOVA; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. (I) Schematic representation of generation of Tyk2−/−/Jak2V617F;Tyk2−/− chimeric mice (noncompetitive transplantation, donor; 2 × 106Jak2V617F;Tyk2−/− BM cells, recipient; Tyk2−/− mice). After 4 weeks, recipient mice were treated with PBS or 600 ng of ropeg-IFN-α for 8 weeks. PBS-treated Tyk2−/−/Jak2V617F;Tyk2−/− chimeric mice (n = 8) and ropeg-IFN-α–treated Tyk2−/−/Jak2V617F;Tyk2−/− chimeric mice (n = 8; panels J and K, respectively). (J) The proportion of HSCs and progenitors in BM from Tyk2−/−/Jak2V617F;Tyk2−/− chimeric mice after treatment with ropeg-IFN-α. At 48 hours after the last treatment, the proportion of LT-HSCs, CMPs, GMPs, MEPs, MKPs, and CD41+ cells in the BM was analyzed. Data are presented as mean ± SD. The absence of inhibitory effects of IFN-α on the proportion of MKPs from Tyk2−/−/Jak2V617F;Tyk2−/− chimeric mice. (K) PB complete cell count in leukocytes after 8 weeks of PBS or ropeg-IFN-α treatment in Tyk2−/−/Jak2V617F;Tyk2−/− chimeric mice. The absence of inhibitory effects of IFN-α on leukocyte count but not on platelet count in Tyk2−/−/Jak2V617F;Tyk2−/− chimeric mice. WBC, white blood cell; Hb, hemoglobin; Plt, platelet; n.s., not significant.

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