Figure 2.
Extramedullary hematopoiesis in the spleen of Calr-deficient mice. (A) Spleen weight of 4- to 6-month-old mice. Mx1-cre;Calrf/− mice (n = 13) exhibited splenomegaly compared with Mx1-cre;Calr+/+ mice (n = 9). On the other hand, no difference was found between Mx1-cre;Calr+/− mice (n = 10) and Mx1-cre;Calr+/+ mice. (B) Histology of the spleen. The spleen was stained with hematoxylin and eosin at 6 months of age. (Ba,c) The margin of white pulp is obscured in Mx1-cre;Calrf/− mice compared with Mx1-cre;Calr+/+ mice (original magnification ×20). (Bb,d) Infiltration of myeloid cells such as megakaryocytes, erythroblasts, and granulocytes is clearly observed in the spleen of Mx1-cre;Calrf/− mice (original magnification ×200). These findings were not apparent in the spleen of Mx1-cre;Calr+/− mice (not shown). (C) Proportions of myeloid cells (Mac1+ or Gr1+), T cells (CD3+), B cells (B220+), erythroid cells (CD71+ and Ter119+), and megakaryocytes (CD41+) in the spleen (top). In spleens of Mx1-cre;Calrf/− mice (n = 13), B cells were decreased, whereas erythroblasts, megakaryocytes, and granulocytes were increased compared with Mx1-cre;Calr+/+ mice (n = 8). No difference was found between Mx1-cre;Calr+/− mice (n = 9) and Mx1-cre;Calr+/+ mice. Proportions of HSCs and progenitors in the spleen (bottom). Compared with Mx1-cre;Calr+/+ mice, Mx1-cre;Calrf/− mice showed increased frequencies of LT-HSC, MPPs, LSK cells, common myeloid progenitors, GMPs, erythromegakaryocyte progenitors, and megakaryocyte progenitors (MKPs). No differences were found in the frequencies of any progenitors between Mx1-cre;Calr+/+ mice and Mx1-cre;Calr+/− mice. (D) Number of hematopoietic colonies per 105 spleen cells. The proportions of CFU-GEMMs, CFU-GMs, and CFU-Es were higher in Mx1-cre;Calrf/− mice (n = 12) than in Mx1-cre;Calr+/+ mice (n = 9). In the spleen of Mx1-cre;Calr+/− mice (n = 10), only CFU-GMs were mildly increased. (E) Heat maps comparing cytokines levels in plasma from Mx1-cre;Calr+/+ (n = 7), Mx1-cre;Calr+/− (n = 6), and Mx1-cre;Calrf/− (n = 7) mice. The levels are shown by the color gradient from green (low levels) to red (high levels). *P < .05; **P < .01 vs Mx1-cre Calr+/+; †P < .05 vs Mx1-cre Calr+/−. (F) Kaplan-Meier plot of Mx1-cre;Calr+/+, Mx1-cre;Calr+/−, and Mx1-cre;Calrf/− mice (n = 30 in each group). All data are presented as the means ± SEM. To assess statistical significance among groups, 1-way ANOVA followed by the Tukey–Kramer test was used. *P < .05; **P < .01 (A,C,D); n.s., not significant.

Extramedullary hematopoiesis in the spleen of Calr-deficient mice. (A) Spleen weight of 4- to 6-month-old mice. Mx1-cre;Calrf/− mice (n = 13) exhibited splenomegaly compared with Mx1-cre;Calr+/+ mice (n = 9). On the other hand, no difference was found between Mx1-cre;Calr+/− mice (n = 10) and Mx1-cre;Calr+/+ mice. (B) Histology of the spleen. The spleen was stained with hematoxylin and eosin at 6 months of age. (Ba,c) The margin of white pulp is obscured in Mx1-cre;Calrf/− mice compared with Mx1-cre;Calr+/+ mice (original magnification ×20). (Bb,d) Infiltration of myeloid cells such as megakaryocytes, erythroblasts, and granulocytes is clearly observed in the spleen of Mx1-cre;Calrf/− mice (original magnification ×200). These findings were not apparent in the spleen of Mx1-cre;Calr+/− mice (not shown). (C) Proportions of myeloid cells (Mac1+ or Gr1+), T cells (CD3+), B cells (B220+), erythroid cells (CD71+ and Ter119+), and megakaryocytes (CD41+) in the spleen (top). In spleens of Mx1-cre;Calrf/− mice (n = 13), B cells were decreased, whereas erythroblasts, megakaryocytes, and granulocytes were increased compared with Mx1-cre;Calr+/+ mice (n = 8). No difference was found between Mx1-cre;Calr+/− mice (n = 9) and Mx1-cre;Calr+/+ mice. Proportions of HSCs and progenitors in the spleen (bottom). Compared with Mx1-cre;Calr+/+ mice, Mx1-cre;Calrf/− mice showed increased frequencies of LT-HSC, MPPs, LSK cells, common myeloid progenitors, GMPs, erythromegakaryocyte progenitors, and megakaryocyte progenitors (MKPs). No differences were found in the frequencies of any progenitors between Mx1-cre;Calr+/+ mice and Mx1-cre;Calr+/− mice. (D) Number of hematopoietic colonies per 105 spleen cells. The proportions of CFU-GEMMs, CFU-GMs, and CFU-Es were higher in Mx1-cre;Calrf/− mice (n = 12) than in Mx1-cre;Calr+/+ mice (n = 9). In the spleen of Mx1-cre;Calr+/− mice (n = 10), only CFU-GMs were mildly increased. (E) Heat maps comparing cytokines levels in plasma from Mx1-cre;Calr+/+ (n = 7), Mx1-cre;Calr+/− (n = 6), and Mx1-cre;Calrf/− (n = 7) mice. The levels are shown by the color gradient from green (low levels) to red (high levels). *P < .05; **P < .01 vs Mx1-cre Calr+/+; †P < .05 vs Mx1-cre Calr+/−. (F) Kaplan-Meier plot of Mx1-cre;Calr+/+, Mx1-cre;Calr+/−, and Mx1-cre;Calrf/− mice (n = 30 in each group). All data are presented as the means ± SEM. To assess statistical significance among groups, 1-way ANOVA followed by the Tukey–Kramer test was used. *P < .05; **P < .01 (A,C,D); n.s., not significant.

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