Immigration Of CD49b~+CD25~+NK Cells Into Pregnant Uterus

ObjectivesTo investigate the potential roles of regulatory natural killer cells in pregnancy tolerance by comparing NOD (non-obese diabetic) mice and BALB/c mice.Materials and MethodsNOD mice are characterized by NK-cell deficiency and widely used in immuno logical tolerance research. Both BALB/c and NOD mice are inbred strains. Allogeneic BALB/c×C57BL/6 and NOD×C57BL/6 mating combinations were established. Pregnant mice were randomized and either injected intraperitoneally with rabbit anti-mouse asialo ganglio-N-tetraosylceramide (ASGM1) stock solution to deplete NK cells or injected intravenously with either magnetic-affinity cell sorting (MACS)-purified CD49b+CD25+cells or CD49b+CD25- cells. Solvent control groups were injected with RPMI1640 medium via the same route. All injections were made at gestational day 2.5, and the females were killed on gestational day 12.5 to remove the uterus and calculate the percentage of embryo resorption.Mononuclear cells were isolated from placenta and decidua. CD49b+cells were separated using microbead-conjugated anti-mouse CD49b and magnetic affinity cell sorting separation columns. The expression of cytokines within CD49b+cells was detected by flow cytometry.Using similar methods, the expression of Foxp3 in CD45+, CD3+and CD49b+ lymphocytes and the expression of CXCR4 in splenic NK cells and uterine NK cells on gestational day 12.5 were detected by flow cytometry.MACS-purified splenic CD49b+cells obtained from virgin BALB/c (wild-type, WT) or NOD mice were added to the upper chamber of a 24-well transwell plate with an 8μm pore size. A total of 600μl RPMI1640 culture medium supplemented with 10%fetal bovine serum in the presence or absence of l00ng/mL recombinant mouse CXCL12 was added into the lower chamber to observe the migration of CXCR4+NK cells under the regulation of CXCL12.Splenic CD49b+CD25+and CD49b+CD25- cells from virgin C57BL/6 mice were purified by MACS and labeled with CFSE (5,6-carboxyfluorescein diacetate succinimidyl ester). The pregnant mice were killed on gestational day 12.5 for the collection of placental mononuclear cells. To determine whether the CFSE-labeled cells migrated into the pregnant uterus, the CFSE signal in these cells was analyzed by flow cytometry.ResultsFollowing the depletion of NK cells with anti-ASGM1, the resorption rate of embryos was increased both in allogeneic BALB/c breedings (P< 0.01) and in allogeneic NOD breedings (P< 0.05). Adoptive transfer of CD49b+CD25+cells into allogeneic pregnant NOD mice decreased the percentage of embryo loss (P< 0.05). Conversely, no change in embryo loss was observed when CD49b+CD25-cells were transferred. The percentage of NK1 cells present in the uterine CD49b+cell population of allogeneic pregnant NOD mice was significantly higher than in BALB/c mice (P< 0.01). In contrast, the percentages of NK3 and NKr1 cells were significantly lower in NOD mice than in BALB/c mice (P< 0.01). No significant difference was observed in the percentage of the NK2 subset between allogeneic pregnant BALB/c mice and NOD mice (P> 0.05). The percentage of Foxp3+cells in both the CD3+population (P< 0.01) and the CD49b+population (P< 0.05) of BALB/c mice was higher than that of NOD mice. Additionally, the percentage of Foxp3+cells in the CD49b+population was higher than that found in the CD3+ population of both mouse strains (BALB/c, P< 0.01; NOD, P< 0.05). The percentage of CXCR4+cells was significantly higher among uterine CD49b+CD25+population than in CD49b+CD25-population isolated from both allogeneic pregnant BALB/c mice and NOD mice. However, the percentage of CXCR4+splenic CD49b+CD25+ cells isolated from virgin NOD mice was lower than virgin BALB/c mice (P< 0.01). Additionally, splenic CD49b+CD25+cells from virgin BALB/c expressed a higher percentage of CXCR4 than CD49b+CD25- cells from the same mice (P< 0.01). No such trends were detected in virgin NOD mice. In contrast, only a low level of CXCR4 expression was detected among the splenic CD49b+CD25+and CD49b+CD25- cells from NOD mice. As detected in a transwell system, CXCL12 added to the lower chamber was more likely to preferentially attract splenic CXCR4+ cells from both strains of mice, whereas the CXCR4- cells remained in the upper chamber. The percentage of CD49b+CXCR4+cells that migrated to the lower chamber was higher than that found in the upper chamber (P< 0.01). More CFSE-labeled cells were found in the CD49b+CD25+cell transfer group than in the CD49b+CD25-cell transfer group (P< 0.01).ConclusionsThe recruitment of peripheral CXCR4 expressing CD49b+CD25+NK cells into pregnant uteri may be important to pregnancy success.