Moreover, previous studies demonstrated the upregulated expression of CD80 and CD86 on murine splenic antigen presenting cells such as dendritic cells and macrophages at the pre-implantation period (day 3.5 pc). B cells bearing upregulated expression of co-stimulatory molecules CD80 and CD86 and activation marker CD27. Our investigations herein demonstrate that during the crucial stages surrounding implantation, uterine B cells are amplified and phenotypically altered to act in a regulatory manner that potentially contributes toward the establishment of maternal immunological tolerance in early pregnancy. experiments that uterine B cells collected from pregnant females at day 5.5 pc significantly suppressed proliferation and activation of syngeneic CD4+ T cells via cell-cell interactions. We thus posit that uterine B cells at peri-implantation exhibit immunosuppressive characteristics and likely take part in fostering the generation of maternal immune tolerance during early pregnancy. Materials and Methods Animals All mice were housed in a specific-pathogen free (SPF) animal facility with optimal lighting and food and water Suppression Assay To examine the suppressive activity of B cells collected from pregnant or virgin mice on CD4+ T cells, B cells from the spleen and PALNs and T cells from a syngeneic spleen were purified by magnetic isolation while B cells from the uterus were purified by FACS sorting. Purified CD4+ splenic T cells were labeled with Cell BR102375 Proliferation Dye e450 (eBioscience) for 10 min at 37C in the dark, then washed with 10% cold RPMI culture medium twice before resuspending in pre-warmed 10% RPMI culture medium supplemented with IL-2 (10 ng/ml, Peprotech, NJ, USA) and DynabeadsTM mouse T-activator CD3/CD28 (eBioscience) for T cell growth and activation. Cells were then dispensed into a 96-well round-bottom plate at 1 105 cells/well, with purified B cells subsequently added to a final ratio of 0.5:1, 1:1, and 2:1 relative to T cell numbers, with unstimulated T cells and T cells with Dynabeads alone as controls. After 3 days in culture, cells were analyzed using flow cytometry to determine T cell proliferation as indicated by sequential dye dilution. T cell proliferation was expressed BR102375 as the proliferative index (PI) (15). The PI denotes the total number of divisions divided by the number of cells that went into division, and is calculated using the following formula: = number of cells in each fluorescent peak, with the peaks identified as follows: pp refers to the parental undivided peak of T cells, G1 is the first T-cell division peak, G2 the second, G3 the third etc. until peak differentiation is not discernible from the background fluorescence. The PI for each sample was calculated using the fitting and modeling processes of the software FCS Express on cell division profiles (DeNovo Software, California, USA). Activation of proliferating CD4+ cells was assessed by staining with CD4-FITC (RM4-5; eBioscience) and CD25-APC (PC61.5; eBioscience) antibodies for 40 min in the dark on ice and assessing expression by flow cytometric methods. Unstained, single-color controls, and FMOs were used as gating controls. Intracellular Staining of IL-10+ B Cells Single cell suspensions were incubated for 5 h in a stimulation cocktail [50 ng/mL phorbol-myristate-acetate (PMA); 500 ng/mL ionomycin; 5 g/mL lipopolysaccharide (LPS 0111:B4, Sigma)] and 1 g/mL Brefeldin A, to induce cytokine production and inhibit Golgi transport enabling accumulation of cytokines within the cell. Cells were then washed twice and incubated with anti-mouse CD16/32 (eBioscience). Cells were washed once and stained with anti-mouse B220/CD45R-BV650 (RA3-6B2; BD Biosciences) for 40 min in the dark on ice. Post-staining, cells were fixed and permeabilized using the BD Fix/Perm Kit (BD Biosciences) as per manufacturer’s instructions. Permeabilized cells were incubated with PE anti-mouse IL-10 (JES5-16E3; BioLegend) or isotype control PE Rat IgG2b (RTK4530; BioLegend) for 40 min at room temperature. Lastly, cells were washed twice with Perm Buffer, resuspended in FACS buffer, and analyzed for B220+IL-10+ cells via flow cytometry. Matched isotype controls were used to ensure correct gating for IL-10+ cells. Generation of Induced IL-10+ B Cells Splenic B cells were magnetically isolated as above and cultured with purified anti-mouse CD40 (HM40-3; BD Biosciences) for 48 h as previously described (16). The cell culture supernatant was also collected and used BR102375 for the measurement of secreted IL-10 levels by ELISA. IL-10 Detection by ELISA IL-10 quantities in supernatants were measured using the ELISA MaxTM Standard Set Mouse IL-10 Kit (Biolegend) following the manufacturer’s LAMA5 instructions. Briefly, high-binding.