Fig. 1.

MPER-specific discrimination and engagement by the BCR mediates co-delivery of LACK for MHCII presentation. (A) Schematic of pLACK formulated MPER/liposome engagement by BCR of 4E10 BNAb-expressing WEHI-231B cells and the subsequent detection of MHCII(I-A^d)/LACK complex by antibody 2C44. (B) Ca²⁺ mobilization analysis of 4E10 bNAb-expressing WEHI-231B cells upon stimulation with the various MPER/liposomes. MPER peptides arrayed on the surface of liposome at different molar ratios of peptide to lipid (1:50–1:1000) were tested. Colored lines represent Fluo4-AM fluorescence in the presence of Ca²⁺ ions following stimulation with different MPER ratios and for positive anti-IgM F(ab)₂ (dark brown) and for negative bare liposome (blue) and PBS (red) control treatments. (C and D) Quantification of LACK:I-A^d complexes on the surface of WEHI-231B cells expressing 4E10 BCR (human kappa chain⁺; hC_k⁺) (C) or lacking 4E10 BCR (hC_k⁻) (D) as determined by flow cytometry using antibody 2C44. MPER/liposomes (white bars) or bare liposomes (gray bars) formulated with pLACK at different molar ratios of LACK peptide to lipid were incubated with WEHI cells for 1 h, followed by a 6 h incubation for processing/presentation, and detection with 2C44. (E) Synergistic effect of MPLA on the I-A^d/LACK presentation by 4E10 expressing WEHI cells. pLACK/liposomes containing MPER or MPLA or MPER and MPLA as denoted by (+) signs in table were incubated with 4E10-expressing cells (white bars) and 4E10-negative cells (gray bar in-lays) for 1 h with various liposomes packaged with 1:100 pLACK to lipid followed by a period of time for processing as described in Materials and Methods. Results are representative of three independent experiments. Bars represent the mean with plus/minus SEM shown. (*) p < 0.05, (**) p < 0.01, or (***) p < 0.001 by unpaired student’s T-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)