Characterization of natural killer cells in nonobese diabetic/severely compromised immunodeficient mice during pregnancy Yi Lin, M.D., Yanmin Zhong,

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Characterization of natural killer cells in nonobese diabetic/severely compromised immunodeficient mice during pregnancy Yi Lin, M.D., Yanmin Zhong, M.Sc., Shigeru Saito, M.D., Yijing Chen, M.D., Weizai Shen, M.D., Jingfang Di, M.Sc., Shan Zeng, M.Sc. Fertility and Sterility Volume 91, Issue 6, Pages 2676-2686 (June 2009) DOI: 10.1016/j.fertnstert.2007.08.087 Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Intracellular IFN-γ production in DX5+ cells after indicated stimulation or inhibition in vitro. Cultured DX5+ cells were from PB and purified by magnetic affinity cell sorting, based on syngeneic pregnant BALB/c and NOD/SCID mice. The indicated inhibitor was added 4 hours before the stimulator was added into the culture medium. The secretion of IFN-γ was blocked by brefeldin A 6 hours before the cultured cells were harvested. The IFN-γ+ cell percentage was indicated in the histogram. (A) Data are representative of one of three independently conducted flow cytometries. (B) Data summary of flow cytometry. ∗P<.05 vs. NOD/SCID groups, BALB/c no stimulus group, and LY294002-blocked groups. P+L = poly I:C + LPS. Fertility and Sterility 2009 91, 2676-2686DOI: (10.1016/j.fertnstert.2007.08.087) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 2 DX5+ NK cell cytotoxicity detected by 51Cr release assay. Filled squares = BALB/c treated with poly (I:C); empty squares = BALB/c treated with PBS; filled circles = NOD/SCID treated with poly (I:C); empty circles = NOD/SCID treated with PBS. (A) Spleen. There was lower but detectable cytotoxicity in NOD/SCID mice, which could be boosted slightly upon poly (I:C) stimulation. In comparison, the cytotoxicity could be significantly increased in response to poly (I:C) stimulation in BALB/c. (B) Peripheral blood. NK cell cytotoxicity could be detected in BALB/c, which was boosted slightly upon poly (I:C) stimulation. In comparison, no apparent increase of cytotoxicity could be detected in NOD/SCID mice in response to poly (I:C) stimulation. (C) Placenta. A low level of cytotoxicity could be detected in both BALB/c and NOD/SCID mice at day 13.5 of gestation. However, the cytotoxicity was increased significantly in BALB/c in response to poly (I:C) stimulation, whereas no apparent increase was detected in the NOD/SCID mice. Fertility and Sterility 2009 91, 2676-2686DOI: (10.1016/j.fertnstert.2007.08.087) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Expression of iNOS in placental NK cells. Placental NK cells were purified by magnetic affinity cell sorting and cultured. (A–D): From BALB/c. (E–H): From NOD/SCID mice. (A, E) Double-negative control derived from co-staining of AF- and PE-isotype. (B, F) Single DX5+ control derived from co-staining of AF-isotype and PE-conjugated anti-mouse DX5. (C, G) Without LPS induction. (D, H) Induced with LPS for 48 hours. Data are representative of one of three independently conducted experiments. (I) Summary of flow cytometry data. AF-isotype = Alexa Fluor 488-conjugated isotype control antibody; PE-isotype = PE-conjugated isotype control antibody; AF-iNOS = Alexa Fluor 488-conjugated anti-mouse iNOS; %iNOS+ = the percentage of iNOS+ cells in DX5+ cell population. Fertility and Sterility 2009 91, 2676-2686DOI: (10.1016/j.fertnstert.2007.08.087) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions