Anti cd19

Phenotyping of mononuclear cells was performed in 1% BSA and 3% human serum-PBS according to standard methods using a panel of antibodies directed against monocytes, T- and B-lymphocytes, natural killer cells and red cells. The following conjugated antibodies were used: anti-CD19 (Beckman Coulter Cat# IM1284U, RRID:AB_131011), anti-CD56 (Beckman Coulter Cat# IM2073U, RRID:AB_131195), anti-CD3 (Beckman Coulter Cat# IM1282U, RRID:AB_10640418), anti-CD14 (Beckman Coulter Cat# IM0645U, RRID:AB_130992), anti-CD16 (Beckman Coulter Cat# IM0814U, RRID:AB_10640417) were from Beckman Coulter (FL). FACS analysis was performed on a LSRII cytometer (BD Biosciences, CA). Mø phenotype was confirmed by flow cytometry targeting CD68 (R and D Systems Cat# IC20401P, RRID:AB_2074835) and CD80 (R and D Systems Cat# FAB140F, RRID:AB_357027), CD163 (R and D Systems Cat# FAB1607P, RRID:AB_2074536) and CD206 (R and D Systems Cat# FAB25342P, RRID:AB_10889015) antibodies all from R&D Systems (IN). Data were analyzed with Flowing Software (University of Turku, Finland) or FACSDiVa software (BD Biosciences) and represented, when required, with the logical display.

To sort purified macaque CD19⁺CD25^high B_reg cells and CD3⁺CD4⁺ T cells, peripheral blood mononuclear cells (PBMCs) obtained from naïve macaques were surface stained with anti-CD25 (clone BC96, eBioscience), anti-CD3, anti-CD4 and anti-CD19 (Beckman Coulter). The dead cells were gated out using aqua blue dead cell dye (Invitrogen). Sorting was performed using a MoFlo Astrios EQ. Three populations were sorted: CD19⁺CD25^high, CD19⁺CD25⁻ and CD3⁺ CD4⁺. The CD3⁺CD4⁺ T cells were labeled with CFSE (Invitrogen) according to the manufacturer’s instructions. These labeled T cells were co-cultured either with CD19⁺CD25^high or CD19⁺CD25⁻ cells at a 1:1 ratio in an anti-CD3 (FN18, from the Nonhuman Primate Reagent Resource, 10 μg/ml) coated U-bottom 96-well plate in the presence of anti-CD28 (CD28.2, NIH Nonhuman Primate Reagent Resource, 1 μg/ml) and IL-2 (100 units/ml; NIH Resource for Nonhuman Primate Immune Reagents). The cells were cultured at 37°C for 72 hours. As controls, unlabeled and CFSE-labeled CD4⁺ T cells were cultured with or without any of the stimuli. Analysis of the CFSE pattern of CD4⁺ T cells in the different conditions was performed using FlowJo analysis software (Tree Star, Ashland, OR, USA). The proliferating cells were expressed as the percentage of CFSE⁺ CD4⁺ T cells excluding generation 0.