CXCL12 enhances exogenous CD4+CD25+ T cell migration and prevents embryo loss in non-obese diabetic mice  Yi Lin, M.D., Liang Xu, M.D., Haiyan Jin, M.D., Yanmin Zhong, M.Sc., Jingfang Di, M.Sc., Qi-de Lin, M.D.  Fertility and Sterility  Volume 91, Issue 6, Pages 2687-2696 (June 2009) DOI: 10.1016/j.fertnstert.2008.01.109 Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Treg cells and CXCR4 + T cells in NOD × C57BL/6 and BALB/c × C57BL/6 mice. CD3+, CD4+, CD3+CD25+, and CD3+CD25- cells were purified with MACS. (A) Isotype control stained with FITC- and PE-conjugated isotype antibodies. (B) Single CD3+ control derived from costaining of FITC-conjugated isotype antibody and anti-CD45-PE. (C, E, G, I, K, L) Cells derived from WT mice. (D, F, H, J) Cells derived from NOD mice. (K) Purified CD3+CD25+ cells. (L) Purified CD3+CD25- cells. Data are representative of one of three independently conducted experiments. (M) Summary of flow cytometric data derived from groups C–L. P values are shown in M. WT = wild-type or non-immunodeficient female BALB/c mice impregnated by C57BL/6 male mice; NOD = female NOD mice impregnated by C57BL/6 male mice; PL 9.5d = placenta harvested on gestational day 9.5; SP 9.5d = spleen harvested on gestational day 9.5. Fertility and Sterility 2009 91, 2687-2696DOI: (10.1016/j.fertnstert.2008.01.109) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Intracellular CXCL12 production in placental CK7+ cells. Summary of flow cytometric data regarding placentas harvested on gestational days 9.5, 13.5, and 17.5. n = 3 for each group. P values are indicated. Fertility and Sterility 2009 91, 2687-2696DOI: (10.1016/j.fertnstert.2008.01.109) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 3 In vitro migratory assay of Treg cells. MACS-purified splenic CD3+ cells from wild-type BALB/c × C57BL/6 mice (WT) and NOD × C57BL/6 mice (NOD) mice were seeded into the upper chamber of an 8.0-μm pore size transwell plate, with or without CXCL12 in the lower chamber. After cultivation for 48 hours the number of cells that migrated into the lower chamber was counted with a hemocytometer, and the percentage of Foxp3+ cells in the migrated CD3+ cells was detected by flow cytometry with FITC-conjugated anti-mouse Foxp3 and PE-conjugated anti-mouse CD3. (A) Cell count of migrated CD3+ cells. (B) Summary of flow cytometric data. 104 cells were analyzed in each case. n = 3 for each group. P values are indicated. 0 h = at the beginning of cultivation. 48 h = after a 48 hours' cultivation. Fertility and Sterility 2009 91, 2687-2696DOI: (10.1016/j.fertnstert.2008.01.109) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 4 In vivo Treg cell migration induced by CXCL12. (A) Unstained splenic Treg cells from BALB/c (negative control). (B) CFSE-labeled splenic Treg cells from BALB/c at the beginning of cell transfer. (C–E) Flow cytometric analysis of MACS-purified CD3+CD4+CD25+ Treg cells based on CFSE signal; the cells were harvested from placentas on gestational day 9.5. (C) CFSE-labeled Treg cell transfer with CXCL12 injection. (D) CFSE-labeled Treg cell transfer without CXCL12 injection. (E) CXCL12 injection without Treg cell transfer. (F–H) MACS-purified placental CD3+ cells. Data are representative of one of three independently conducted experiments. No significant increase in the Foxp3+ T cell percentage was detected when exogenous Treg cells were not transferred but CXCL12 was injected beforehand. Fertility and Sterility 2009 91, 2687-2696DOI: (10.1016/j.fertnstert.2008.01.109) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions