Figure 4. Specificity of gp41 MPER interactions with total B cells.

A) Representative histograms showing cold inhibition of gp41 MPER binding to BALB/c B cells (100 ng/10⁶ splenocytes). Homologous competitor (unlabeled gp41 MPER tetramer) or heterologous competitor (scrambled gp41 MPER tetramer) were pre-incubated at 10-fold molar excess. Numbers represent the percentage of gp41 MPER-reactive B cells within singlet, live, CD19⁺ populations. B) Graphical representation of inhibition with increasing amounts of unlabeled gp41 MPER or heterologous (scrambled gp41 or dsDNA mimic) tetramer competitors. Data were calculated as in A) and represent the average of three experiments. C) Interactions of total BALB/c splenic B cells with various HIV-1 epitope-bearing B cell tetramers. Splenocytes were stained (100 ng tetramers/10⁶ cells) with gp120 V3, gp41 immunodominant, and gp41 MPER-specific B cell tetramers or their scrambled counterparts. Plotted are average percentages of tetramer-reactive B cells within singlet, live, CD19⁺ populations from three independent experiments. D) B cell tetramer reactivity in BALB/c splenocytes treated with BCR and MCLR ligands. 10⁶ splenocytes from naïve BALB/c mice were stained with 100 ng gp41 MPER tetramers, either alone, or pre-treated for 45 min at 37°C with anti-Ig, mannan, or α-methyl-mannopyranoside. Plotted are the percentages of tetramer-reactive B cells within singlet, live, CD19⁺ populations from an average of three experiments. E) Effect of BCR internalization on gp41 MPER surface staining of BALB/c splenic B cells. Anti-Ig stimulation of purified BALB/c splenic B cells for various times (in min.) and calculation of remaining surface-bound APC-labeled gp41 MPER was done as described in materials and methods; shown are results of a representative experiment. F) Cap formation of anti-Ig/gp41 MPER B cell tetramer complexes in BALB/c splenic B cells after BCR cross-linking, as described in materials and methods. An example of a capped B cell after 15 min of anti-Ig stimulation is shown, either as individual immunofluorescent stains of Alexa488 Wheat Germ Agglutinin, APC gp41 MPER tetramers, R-PE-anti IgM+IgG, or merged.