Accelerated Blood Clearance of Lipid Nanoparticles Entails a Biphasic Humoral Response of B-1 Followed by B-2 Lymphocytes to Distinct Antigenic Moieties

LNPs associate with surfaces of B cells via both PEG-dependent and PEG-independent mechanisms.

(A) Splenocytes from C57BL/6 WT mice were incubated in the presence of medium, nonfluorescent LNP, or rhodamine-labeled LNP (LNP-PE) and analyzed by flow cytometry for fluorescence incorporation in CD19⁺ B cells at 24 h. Representative dotplots (left) and quantitation of percentages of PE⁺CD19⁺ cells (right) are shown. Significance was calculated using one-way ANOVA with Holm–Sidak posttest versus PBS. (B) Splenocytes from WT mice were incubated in the presence of PE-labeled LNP for 24 h and stained with DAPI (blue) to visualize the nuclei, anti–early endocytic vesicle (EEV) (green) to identify endosomal compartments, and anti-CD19 (white) to identify B cells. Cells were imaged by fluorescence microscopy to visualize LNP colocalization with either lysosomes (top) or the plasma membrane (bottom). (C) Splenocytes from WT mice were incubated in the presence of PE-labeled LNP for 24 h. Cells were analyzed by flow cytometry for PE⁺CD19⁺ B cells (left); CD86 expression (histograms) and mean fluorescence intensity (MFI) correlation (right) were calculated using a χ² test. (D) Splenocytes from WT mice were incubated in the presence of medium, PE-labeled, PEG-containing LNP (PEG LNP-PE), or PE-labeled and PEGLess LNP (PEGLess LNP-PE) and analyzed by flow cytometry for fluorescence incorporation in CD19⁺ B cells at 24 h. Representative dot plots (top) and quantitation of percentages of PE⁺CD19⁺ cells (bottom) are shown. Significance was calculated using one-way ANOVA with Holm–Sidak posttest versus medium. (E and F) PEG LNP-PE or PEGLess LNP-PE were injected into WT mice i.v.. Splenocytes were harvested 6 h postinjection and either (E) LNP-PE⁺ fluorescence (red) signals were visualized by confocal microscopy alone (top) or colocalized with CD19⁺ (green) B cells (bottom) or (F) analyzed for PE fluorescence by flow cytometry. Significance was calculated using one-way ANOVA with Tukey posttest. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

LNP PC epitopes lead to direct activation of a subset of B cells (B-1), driving ABC via natural IgM.

(A) C57Bl6/J WT splenocytes were incubated with medium (white bars), PC-containing LNP (gray bars), or PC-containing LNP-PE (red bars) for 24 h. PE fluorescence was analyzed in the indicated B cell populations by flow cytometry. Significance was calculated using two-way ANOVA with Dunnet posttest versus medium. (B) FACS-sorted splenic CD19⁺CD5⁺ B-1 cells from WT mice were evaluated for calcium flux during stimulation with PC-containing LNP, PCLess LNP, PC-containing liposomes, or anti-IgM Ab, followed by ionomycin. (C) C57Bl6/J WT mice (n = 6 for each group) were injected i.v. with human Epo encoding mRNA formulated in PC- or PCLess-containing LNP, PBS, or nothing. Spleens were harvested 24 postinjection and splenocytes were analyzed for CD19⁺CD5⁺ B-1a cell frequency by flow cytometry. Significance was calculated using one-way ANOVA with Dunn posttest versus WT mice injected with PC LNP. (D) WT mice (n = 12) were injected i.v. weekly for 3 wk with PC LNP. Sera were collected before the first injection and 24 h after each injection and analyzed for the presence of anti-PC IgM by flow cytometric bead assay. Significance was calculated using one-way ANOVA with Dunn posttest versus preinjection serum. (E–G) WT (black symbols) or IFNAR^−/− (blue symbols) mice (n = 5 for each group) were injected i.v. with human Epo encoding mRNA formulated with PC-containing LNP weekly for 5 wk (arrows on x-axis indicate the day of injection). (E) Human Epo protein expression by ELISA is shown. (F) Anti-PEG IgM and (G) anti-PC IgM levels were quantified by flow cytometric bead assay in serum 24 h postinjection. Significance was calculated using two-way ANOVA with Sidak posttest versus WT mice at each timepoint. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n.s., not significant.

Removal of both B-1–mediated anti-PC natural IgM and B-2–mediated anti-PEG IgM responses abrogates ABC in mice.

(A–E) C57Bl6/J WT or sIgM^−/− mice were injected i.v. with human Epo encoding mRNA formulated with LNP weekly for 5 wk (arrows on x-axis indicate the day of injection). Human Epo protein expression was assessed in the serum of WT (open symbol) or sIgM^−/− (closed symbol) mice by ELISA 6 and 24 h postinjection. Significance was calculated using two-way ANOVA with Sidak posttest versus C57Bl6/J WT mice at each timepoint. (B) Anti-PEG IgM or (C) anti-PC IgM levels were quantified by flow cytometric bead assay 24 h postinjection. Significance was calculated using one-way ANOVA with Sidak posttest versus WT mice at each timepoint. (D) Anti-PEG IgG1 (triangles) or anti-PEG IgG3 (squares) levels in serum were quantified from C57Bl6/J WT (open symbols) or sIgM^−/− (closed symbols) mice by flow cytometric bead assay 24 h postinjection. Significance was calculated using one-way ANOVA with Sidak posttest versus WT mice at each timepoint. (E) Human Epo mRNA distribution (brown) and nuclei (blue) in the liver 24 h after fifth injection was visualized by in situ hybridization and hematoxylin staining, respectively. Scale bar, 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n.s., not significant.