Fig 2: Effect of B cell depletion on natural IgM mediated immunity against S. pneumoniae. Flow cytometry analysis of splenocytes recovered from C57B/6 mice 7 days after IP administration of 100ug of aCD20 antibody (six mice/group). (A) Percentage of persisting natural IgM secreting cells (B1a, CD5+CD23-) and total B cells (CD19+CD3-) compared to undepleted controls. (B) A representative flow cytometry plot of the relative level of IgM expression in splenic B cells from aCD20 treated (solid line) and untreated controls (dotted line). Unstained sample data are also shown (gray filling). (C) IgM expression level (Mean fluorescence intensity - MFI) on B1a cells (CD5+CD23-) in the depleted (white bar) versus undepleted mice (black bar). (D) A representative flow cytometry plot of IgM binding to S. pneumoniae 6B strain incubated in serum from undepleted (dotted line) and B cell depleted (solid line) mice; unstained samples (gray filling). (E) Mean MFI of IgM binding to S. pneumoniae 6B strain in serum from B cell depleted (white column) or undepleted (black columns) C57B/6 mice. (F, G) Effects of B cell depletion of immune naïve C57B/6 mice on susceptibility to S. pneumoniae pneumonia. Mice were inoculated intranasally with 2 x 106 CFU S. pneumoniae 6B (F) and D39 strain (G) CFU in 50 ul PBS under deep anaesthesia and CFU were measured at 24 h by plating serial dilutions of blood and lungs. For all graphs, bars represent the mean values for each group, error bars indicate standard deviations and Mann Whitney U test was used for statistical analysis (*P < 0.05, ns, not significant).

Fig 3: Reconstitution of B cell population after depletion. (A) Flow cytometry analysis of total number of B (CD19+) and T cells (CD3+) in single cell spleen preparations from C57Bl/6 mice colonized with S. pneumoniae 3 days after B cell depletion treatment. B cell reconstitution has been assessed 26 days after aCD20 antibody IP administration (3 mice/group). Undepleted mice are shown in black, depleted in white. (B, C) Reconstituted B cell subset populations in splenocytes after one (B) or two (C) depletion treatments were analyzed by flow cytometry 31 and 26 days after depletion, respectively. Marginal zone (CD5-CD21+CD23-IgM+), follicular (CD5-CD23+IgM-), and regulatory (CD5+CD21+IgM+) B cells populations are shown. Depleted/vaccinated (white bars), undepleted/vaccinated (black bars) mice were analyzed (n=6/group). Bars represent mean values for each group, error bars indicate SDs and Mann Whitney U test was used for statistical analysis (*P < 0.05, ns, not significant).

Fig 4: Interchain disulfide bonds are absent and intrachain disulfide bonds are partially formed in cytosolic IgGs. (a) Schematic representation of 3D8 IgG1 variants. The absence of interchain and intrachain disulfide bonds is denoted by a superscript asterisk (*) or sharp (#) symbol, respectively, in front of the chain. The locations of asterisk and sharp symbols indicate Cys-to-Ser substitution sites. (b) Purified WT 3D8 IgG1 protein was subjected to 15% SDS-PAGE in reducing conditions in the presence of different concentrations of DTT. (c,d) Immunoblotting. Lysates of HEK293 transfectants were pretreated with 100 mM DTT for 30 min at room temperature (or not), then subjected to non-reducing SDS-PAGE. H (c) and L (d) chains were detected by immunoblotting with the indicated antibodies. (e,f) Co-IP of H and L chains. Lysates of transfectants were immunoprecipitated using Protein A/G-agarose. Input and IP samples were resolved by non-reducing SDS-PAGE (e) and reducing SDS-PAGE. (f) Input represents 10% of the total amount of lysate used for IP. (g) Immunoblotting. At 24 hours after transfection, cells were incubated in the presence or absence of 10 µM MG132 for 12 hours. Lysates of transfectants were then resolved by reducing SDS-PAGE. Membranes were probed with antibodies recognizing human IgG/Fc or human C?. (h,i) Evaluation of non-covalent H:L chain association by ELISA. Lysates of transfectants were heated at 100 °C for 10 min or mixed with DTT (final concentration, 2 mM), and then placed in wells coated with Protein L. Captured IgGs were detected with AP-conjugated anti-human IgG/Fc antibody. (j) Evaluation of formation of the correct antigen-binding site by ELISA. Lysates of transfectants were placed in wells coated with O2F3 IgM, and bound IgGs were detected with AP-conjugated anti-human IgG/Fc. In (h–j), data are presented as mean ± SEM, n = 3.

Fig 5: Effects of IP or IV administration of aCD20 on splenocyte B cell populations in C57Bl/6 mice. Splenocyte B cell (anti-CD19 antibody) and T cell (anti-CD3 antibody) populations were quantified using flow cytometry 7 days after the administration of the aCD20 antibody (SA271G2 BioLegend). (A–C) IV administration of either 50µg or 100µg aCD20, showing representative flow cytometry plots (A), total cell numbers (B), and cell population proportions (C). (D, E) IP administration of either 25 µg or 100 µg aCD20, showing representative flow cytometry plots (D), total cell numbers (E), and cell population proportions (F). (G) Effects of B cell depletion on B cell subsets for IP treated mice; marginal zone (CD5-CD21+CD23-IgM+), follicular (CD5-CD23+IgM-) and regulatory (CD5+CD21+IgM+) B cell populations are shown. For all histograms columns represent means, error bars SDs: black bars, undepleted; gray bars, low-dose depleted; white, high-dose depleted (n=3). Mann Whitney U test was used for statistical analysis (***P < 0.001, ****P < 0.0001).