Figure 3.

Complex tri/tetra-antennary N-glycan surface expression correlates with expression levels of Treg markers and suppressive potency. (A,B) The expression of CD39, CD73, ICOS, GITR, Helios, PD-1, PDL-1, CTLA-4, and CD103 was evaluated in Treg subpopulations within freshly isolated spleen and lymph node cells from C57BL/6 FoxP3.EGFP mice defined according to Phaseolus vulgaris leucoagglutinin (PHA-L) binding intensities [low (−/+), mid (+) or high (++)]. Dashed tinted histograms represent the median fluorescence intensities for the eF710 and PE fluorescence minus one (FMO) controls for lectin and antibody labeling, respectively (n = 3 individual animals). (C) Gating strategy for FACS purification of cell populations used in the functional assay (D,E). (D) CD4⁺ and (E) CD8⁺ responder T-cells were labeled with CellTrace™ Violet and cocultured in the presence of CD4⁻CD8⁻ antigen-presenting cells and purified PHA-L^high or PHA-L^low Treg at Treg: responder T-cell ratios of 0:1, 1:4, 1:6, 1:8, and 1:16 for 4 days with anti-CD3 stimulation. Suppressive function was quantified for PHA-L^high and PHA-L^low Treg based on responder T-cell division index (DI) and presented as the calculated percent suppression [%Suppression (DI) = 100 − [(DI of the 1:X)/(DI of 0:1)] × 100 in which 1:X represent cocultures with Treg and 0:1 refers to cocultures with responder T-cell alone]. Data represent mean ± SD (n = 3 technical replicates). Results for proliferative assays were confirmed in a repeat experiment. Statistical analysis was performed by permutation test with an unpaired design (*p value ≤ 0.1).