Granulocyte Colony-Stimulating Factor Induces Osteoblast Inhibition by B Lymphocytes and Osteoclast Activation by T Lymphocytes during Hematopoietic Stem/Progenitor Cell Mobilization  Sidan Li, Tianshou Li, Yongbing Chen, Yinchao Nie, Changhong Li, Lanting Liu, Qiaochuan Li, Lugui Qiu  Biology of Blood and Marrow Transplantation  Volume 21, Issue 8, Pages 1384-1391 (August 2015) DOI: 10.1016/j.bbmt.2015.05.005 Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Reduced mobilization in mice with defective B lymphocytes but not with defective T lymphocytes. (A) Total WBC counts in PB of C57BL/6 (white), B6.129-Cd3etm1Lov/J (black), and B6.129P2(C)-Cd19tm1(cre)Cgn/J mice (gray) treated with G-CSF (n = 5 to 7 each time point). (B) Representative scatter plots showing LSK stem cells in PB of C57BL/6 (filled circle), B6.129-Cd3etm1Lov/J (filled square), and B6.129P2(C)-Cd19tm1(cre)Cgn/J mice (filled triangle) during G-CSF mobilization (n = 5 to 7 each time point). Data are mean ± SEM. (C) Representative scatter plots showing LSK stem cells in BM of C57BL/6 (filled circle), B6.129-Cd3etm1Lov/J (filled square), and B6.129P2(C)-Cd19tm1(cre)Cgn/J mice (filled triangle) during G-CSF mobilization (n = 5 to 7 each time point). Data are mean ± SEM. (D) Colony-forming cells in mouse PB, C57BL/6 (white), B6.129-Cd3etm1Lov/J (black), and B6.129P2(C)-Cd19tm1(cre)Cgn/J mice (gray), treated with G-CSF (n = 5 to 7 each time point). (E) Colony-forming cells in mouse BM, C57BL/6 (white), B6.129-Cd3etm1Lov/J (black), and B6.129P2(C)-Cd19tm1(cre)Cgn/J mice (gray), treated with G-CSF (n = 5 to 7 each time point). Biology of Blood and Marrow Transplantation 2015 21, 1384-1391DOI: (10.1016/j.bbmt.2015.05.005) Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Decrease of mature osteoblast was not seen in B lymphocyte defective mice. Representative photomicrographs showing endosteal osteoblasts (red arrows) in untreated (day 0) or G-CSF treated on day 3 and day 5 of C57BL/6 mice (A), B6.129-Cd3etm1Lov/J (B), and B6.129P2(C)-Cd19tm1(cre)Cgn/J mice (C). Original magnification, 400×. (D) Bone marrow osteocalcin mRNA expression of C57BL/6, B6.129-Cd3etm1Lov/J, and B6.129P2(C)-Cd19tm1(cre)Cgn/J mice. The expression of osteocalcin mRNA relative to b-actin mRNA is shown. (n = 5 to 7 each time point). (E) Serum osteocalcin concentrations of C57BL/6, B6.129-Cd3etm1Lov/J, and B6.129P2(C)-Cd19tm1(cre)Cgn/J mice (n = 5 to 7 each time point; data are mean ± SEM). (This Figure is available in color online at www.bbmt.org). Biology of Blood and Marrow Transplantation 2015 21, 1384-1391DOI: (10.1016/j.bbmt.2015.05.005) Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 SDF-1 and osteopontin expression were unchanged in B lymphocyte defective mice. (A) BM SDF-1 and osteopontin mRNA expression of C57BL/6 mice during G-CSF mobilization (n = 5 to 7 each time point). (B) BM SDF-1 and osteopontin mRNA expression of B6.129-Cd3etm1Lov/J mice during G-CSF mobilization (n = 5 to 7 each time point). (C) BM SDF-1 and osteopontin mRNA expression of B6.129P2(C)-Cd19tm1(cre)Cgn/J mice during G-CSF mobilization (n = 5 to 7 each time point). (D) Representative photomicrographs showing immunoreactivity of SDF-1 and osteopontin in femoral metaphysial trabeculae of untreated (day 0) or G-CSF–treated (days 3 and 5) C57BL/6 mice. Arrowheads indicate endosteal bone-lining osteoblasts. Original magnification, 400×. (E) Representative photomicrographs showing immunoreactivity of SDF-1 and osteopontin in femoral metaphysial trabeculae of B6.129-Cd3etm1Lov/J mice. Original magnification, 400×. (F) Representative photomicrographs showing immunoreactivity of SDF-1 and osteopontin in femoral metaphysial trabeculae of B6.129P2(C)-Cd19tm1(cre)Cgn/J mice. Original magnification, 400×. Biology of Blood and Marrow Transplantation 2015 21, 1384-1391DOI: (10.1016/j.bbmt.2015.05.005) Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 T cells involved in osteoclast activation after G-CSF administration. (A-C) TRAP staining of femoral metaphysic of untreated (day 0) and treated with G-CSF for 5 days (day 5) in C57BL/6 (A), B6.129P2(C)-Cd19tm1(cre)Cgn/J (B), and B6.129-Cd3etm1Lov/J (C) mice. Arrowheads indicate active TRAP+ osteoclasts stained in red (original magnification, 400×). (D) TRACP-5b concentrations were analyzed by ELISA, C57BL/6 (white), B6.129P2(C)-Cd19tm1(cre)Cgn/J (black), and B6.129-Cd3etm1Lov/J (gray) (n = 5 to 7 each time point; data are mean ± SEM). (This Figure is available in color online at www.bbmt.org). Biology of Blood and Marrow Transplantation 2015 21, 1384-1391DOI: (10.1016/j.bbmt.2015.05.005) Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 Wnt antagonist Dkk1 expressed by BM B lymphocytes partially involved in osteoblast inhibition. (A) mRNA levels of Dkk1 in steady and mobilized state B lymphocytes were detected by real-time quantitative PCR. (B) Protein levels of Dkk1 and β-catenin in steady and mobilized state B lymphocytes were detected by Western blotting. Biology of Blood and Marrow Transplantation 2015 21, 1384-1391DOI: (10.1016/j.bbmt.2015.05.005) Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 6 Increased T lymphocyte–induced RANKL/OPG ratio potently stimulates osteoclast activity during G-CSF mobilization. (A) mRNA levels of RANKL in steady and mobilized state T lymphocytes were detected by real-time quantitative PCR. (B) mRNA levels of OPG in steady and mobilized state T lymphocytes were detected by real-time quantitative PCR. (C) The protein levels of RANKL/OPG and β-catenin in steady and mobilized state T lymphocytes were detected by Western blotting. Biology of Blood and Marrow Transplantation 2015 21, 1384-1391DOI: (10.1016/j.bbmt.2015.05.005) Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions

Supplemental Figure 1 Measurement of the HSPC cell content of the PB by immunophenotypic analysis. Representative flow cytometry plot of 5 to 7 independent experiments of SCA-1 and c-Kit expression of C57BL/6 (A), B6.129-Cd3etm1Lov/J (B), and B6.129P2(C)-Cd19tm1(cre)Cgn/J (C) mice is shown with the mean frequency indicated. Biology of Blood and Marrow Transplantation 2015 21, 1384-1391DOI: (10.1016/j.bbmt.2015.05.005) Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions

Supplemental Figure 2 Measurement of the HSPC cell content of the BM by immunophenotypic analysis. Representative flow cytometry plot of 5 to 7 independent experiments of SCA-1 and c-Kit expression of C57BL/6 (A), B6.129-Cd3etm1Lov/J (B), and B6.129P2(C)-Cd19tm1(cre)Cgn/J (C) mice is shown with the mean frequency indicated. Biology of Blood and Marrow Transplantation 2015 21, 1384-1391DOI: (10.1016/j.bbmt.2015.05.005) Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions