Multivalent viral epitopes induce rapid, robust and T cell-independent humoral immune responses, but the biochemical basis for such potency remains incompletely understood. We take advantage of a set of liposomes of viral size engineered to display affinity mutants of the model antigen (Ag) hen egg lysozyme. Particulate Ag induces potent ‘all-or-none’ B cell responses that are density dependent but affinity independent. Unlike soluble Ag, particulate Ag induces signal amplification downstream of the B cell receptor by selectively evading LYN-dependent inhibitory pathways and maximally activates NF-κB in a manner that mimics T cell help. Such signaling induces MYC expression and enables even low doses of particulate Ag to trigger robust B cell proliferation in vivo in the absence of adjuvant. We uncover a molecular basis for highly sensitive B cell responses to viral Ag display that is independent of encapsulated nucleic acids and is not merely accounted for by avidity and B cell receptor cross-linking.
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No large data sets were generated for this manuscript. All data generated or analyzed during this study are included in the published article or are available upon request. Source data depicting unprocessed western blots and all statistical analyses are provided with this paper.
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We thank the following funders: R01AI155653 (W.C. and J.Z.), R01AI148487 (J.Z.) and R01AR069520 (J.Z.), an AAI Postdoctoral Fellowship (J.F.B. and J.Z.), NIH Training Grants T32 AI007334 (J.R. and H.V.N.) and T32 AR079068 (H.V.N.) and Rheumatology Research Foundation Scientist Development Award A138750 (H.V.N.). We thank A. Weiss for helpful discussions and critical reading of the manuscript. We thank A. Roque for help with mouse husbandry.
J.Z. serves as a scientific consultant for Walking Fish Therapeutics. W.C. has a patent pending on SVLSs. All other authors have no competing interests.
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Nature Immunology thanks Andrew Getahun and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: L. A. Dempsey, in collaboration with the Nature Immunology team.
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a. Hen egg lysozyme (HEL) engineered with c-terminal cysteine residue and point mutations (zoomed box) described to reduce binding affinity to Hy10 BCR38. b. Schematic of SVLS library constructed with varying densities of HELD or HELT conjugated to liposomes yielding pHELD/T particles (3–500 epitope density/particle, calculated as [HEL]/[lip] in M). Each bar represents a single pHEL preparation to illustrate range. c. Congenically-marked NUR77-GFP reporter MD4 (Hy10) mixed with wild-type splenocytes and stimulated with pHELD or naked control liposome (pLIP02) for 24 hr. GFP was analysed in B220+ cells of each gt as in Main Fig. 1c. d. NUR77-GFP expression was analysed in MD4 B cells at 24 hr post-stimulation with the same batch of pHELD at various times post-synthesis, normalized to the pLIP02 condition in each experiment. e. As in main Fig. 1e but with pHELT at varying ED. Data is from a single experiment and representative of 3 independent experiments. f. As in E, but stimulated with high or ultra-low density pHELD/T. Data is from a single experiment. g. As in E, but depicting CD69 after 24 hr with pHELD/T. Data are from a single experiment and representative of 3 independent experiments. Curves were modelled with three-parameter nonlinear regression (C, E, F, G). h, i. SVLS with HELD conjugated at ED 124 and encapsulated AF594, herein termed pHELD(AF594), mixed at serial dilutions with MD4 cells for 24 hr and analyzed by flow cytometry. H. Quantification of AF594 fluorescence in B220+ lymphocytes. I. Representative histograms (left) depict AF594 fluorescence in B220+ cells. Inset values: AF594 MFI. Average particles/B cell in culture based on concentrations of each. Right-hand histograms depict 0.156pM-stimulated B cells sub-gated according to AF594 fluorescence to represent capture of particles. Data are from a single experiment and representative of 3 independent experiments.
Extended Data Fig. 2 pHEL signaling is sensitive to ED but not affinity and is potent in both follicular and MZ B cells.
a-c. MD4 splenocytes and lymphocytes were stimulated as in Main Fig. 2A,B but assessed for intra-cellular pS6 over a time course. Graphs (left) and histograms (middle) depict pS6 in B220+ cells from a single time course and is representative of 2 independent experiments. Graphs (right) depict B cell pS6 MFI after 20 min. Data are pooled from n = 5 (HELD), n = 6 (HELT) experiments. C. Graph depicts pHELT and pHELD data from A, B graphed together for comparison. A-C were compared by two-tailed paired parametric T-test, with mean depicted. d. As in 2 A, B but comparing pErk in stimulated B220+ splenocytes and lymph node cells separately at 20 mins. Histograms are representative of 2 independent experiments. e-g. As in 2 A, but splenocytes were stained with B220, CD21, and CD23 to identify pErk expression in subsets. Representative histograms show pErk in gated subsets following sHEL-WT 1 μg/ml, 1pM pHELT-HD (ED 256), or PMA stimulation. F. Quantification and mean of %pErk+ cells from three independent experiments (G). Groups were compared by one-way ANOVA. Data in E-G are representative of 8 independent experiments. h, i. Pooled MD4 splenocytes and lymphocytes were loaded with Indo-1 calcium indicator dye, stained with surface markers to detect B220+ subsets (H) as in S2E, and stimulated with anti-IgM (10 μg/ml), sHELD (1 μg/ml), or pHELD-HD (1pM). Calcium entry was assessed by flow cytometry for >= 3 minutes. MFI of bound/unbound Indo-1 fluorescence over time is plotted (I). Data are representative of 4 independent experiments. J. As in Main Fig. 3a,b except CD23+ B cells stimulated with 10, 1, or 0.1pM doses of either pHELT-HD (ED 256) or pHELT (ED 3). Data are representative of 4 independent experiments.
Extended Data Fig. 3 CD19 is dispensable in vitro but promotes B cell expansion in response to particulate Ag in vivo.
a. 20’ Phosflow assay to detect pErk in Cd19+/– or Cd19–/– MD4 splenocytes after stimulation with either control pLIP02 1pM, 10 μg/ml anti-IgM, 1 mg/ml sHELD, or 1pM pHELD-HD. Samples were co-stained with B220, CD23, and CD21 to detect B cell subsets. Histograms depict CD23+ B cells and are representative of 4 independent experiments. b-g. As in Main Fig. 1i, lymphocytes from MD4 mice were loaded with CTV and those from MD4.CD19-/- mice were loaded with CTY. Cells were mixed at a 1:1 ratio prior to adoptive transfer i.v. of 5 × 106 cells into CD45.1 hosts. Time course, immunization, and d3 harvest as in Fig. 1i. MD4 data correspond to those displayed in Main Fig. 1i–k. b. Experimental schematic. c. Representative gating scheme to identify donor IgM[a]+ B cells with either CTV or CTY fluorescence; L/D, LIVE/DEAD stain. d. Representative histograms depict live IgM[a]+ donor lymphocytes in unimmunized (top) or immunized hosts. e. Graph depicts mean + SEM for % distribution of IgM[a]+ donor B cells of each genotype across cell divisions. Statistical analysis by Two-way ANOVA in supplement. f. Graphs depict division and proliferation indices. g. Ratio of CTV/CTY for donor T cells and IgM[a]+ donor B220+ B cells are plotted for each host according to immunization condition. Data in F and G depict mean of 4 biological replicates and were compared by a two-tailed paired parametric T-test.
a, b. As in Main Fig. 6b,c but with broader titration of pHELT-HD (ED 256) dose from 0.1–10 pM. Histograms depict pErk in CD23+ B220+ cells. MD4 histograms in A correspond to those depicted in Extended Data Fig. 2j and are representative of 3 independent experiments. c. As in Main Fig. 6b, but with sHELT and pHELT (HD/LD 256, 64) stimuli to compare varied epitope density. Data are representative of 4 independent experiments. d. As in Main Fig. 6b,c, except pHELD ultra-low ED 6 and pHELT ultra-low ED 3 are used at 10 pM and 1 pM doses. Data from unstimulated and anti-IgM conditions are re-plotted from panel B for ease of comparison. Graphs in B and D each depict data from 3 independent experiments with mean. Pre-specified groups were compared by two-tailed unpaired parametric T-tests. e-h. corresponding to Main Fig. 6e, lymphocytes from MD4 mice were loaded with CTV and those from MD4.Lyn-/- mice were loaded with CTY and mixed at a 1:3 ratio for adoptive transfer of 5 × 106 cells into CD45.1 hosts prior to immunization one day later with pHELT-HD ~ 0.1 μg. e. Representative gating scheme to identify live donor IgM[a]+ B220+ cells with either CTV or CTY fluorescence; L/D, LIVE/DEAD stain. f. Ratio of CTV/CTY in donor T cells and IgM[a]+ donor B220+ cells are plotted for each host according to immunization condition with mean depicted. g. Graph depicts mean + SEM for % distribution of IgM[a]+ donor B cells of each genotype across cell divisions and data was compared by two-way ANOVA with Holm-Sidak correction for multiple comparisons. h. Graphs depict division and proliferation indices. Lines connecting samples indicate donor cells of each genotype from an individual host. Groups in H were compared by a two-tailed parametric paired T-test. Data in E-H represents 5 biological replicates.
a. As in Main Fig. 6f, but with inclusion of anti-IgM 10 μg/ml as second stimulus following pHELD pre-treatment. Data are representative of 4 independent experiments. b. Data correspond to Main Fig. 6g but depict same samples with single cell resolution. c. As in Main Fig. 6g but with sHEL 1 μg/ml added at peak calcium response following pHEL stimulation. Data are representative of 3 independent experiments.
a. As in Main Fig. 7b-d except histograms showing IκBα degradation at 20 minutes post-stimulation for soluble stimuli sHELD/T and pHELT/D with high or low ED (HD vs. LD). Inset triangles represent decreasing concentration of pHEL (10, 1, 0.1 pM). Data are representative of >= 4 independent experiments. b. As in Main Fig. 6b, except splenocytes from Lyn+/+ and Lyn–/– MD4 mice were stained to detect intracellular IκB in CD23+ B cells at 20 minutes. Data are representative of 3 independent experiments. c. Graph corresponds to data in Main Fig. 7g,h except nuclear p65 MFI rather than % p65 positive nuclei are quantified. Graph depicts data with mean from 3 independent experiments. Groups were compared by paired two-tailed parametric T-tests. d. Nuclear p65 translocation is detected as in Fig. 7f except stimuli are applied on ice for 15 min each, first 10pM pHELT-HD +/- superimposed soluble stimuli (anti-IgM 10 μg/ml or sHEL-WT 1 μg/ml), followed by 20 min 37 C incubation as in Main Fig. 6f. e. As in Main Fig. 4c,d except stained to detect intracellular IκB in CD23 + B cells at 20 minutes. Data are representative of 3 independent experiments. In panels A, B and E, line in offset histograms references internal negative control.
a. MD4.NUR77-GFP lymphocytes were cultured for 24 hr with sHELD 1 μg/ml or 1pM pHEL. Ratio of NUR77-GFP and intra-cellular MYC among B220+ cells from a single experiment, representative of 3 independent experiments. b. MD4 lymphocytes incubated at 4 C with 1st stimulus (1pM pHELT-HD) for 15 min, followed by 2nd stimulus (or media) for 15 min. Soluble stimuli: 1 μg/ml anti-IgM or 0.1 μg/ml sHEL-WT. After 48 hr culture at 37 C without BAFF, viable frequencies of B220+ cells are plotted from 4 independent experiments, compared by a two-tailed unpaired parametric T-test. c. As in Main Fig. 8e except histograms depict B220 + FSC-A, representative of 4 independent experiments. d. CTV-loaded NUR77-GFP splenocytes (+/-MD4 Tg) cultured for 72 hr with 20 ng/ml BAFF + either 6.4 μg/ml anti-IgM or 30 ng/ml sHEL, and ibrutinib titration (0–50 nM). Plots depict CTV and GFP expression in B220+ cells, representative of 3 independent experiments. e. NUR77-GFP splenocytes cultured with 20 ng/ml BAFF as in D except with anti-IgM +/-10ng/ml IL-4. Plots depict CTV and GFP expression in B220+ cells, representative of 6 independent experiments. f. As in Main Fig. 8f,g MD4.NUR77-GFP lymphocytes were cultured with stimuli +20 ng/ml BAFF for 72 hrs: 1 μg/ml sHELD/T, 1pM pHELD/T-HD/LD. These data correspond to Fig. 8f,g but include plots and quantification for pHELD/T-LD for comparison. Shaded bars reference NUR77-GFP levels in dividing B cells stimulated with sHELD/sHELT. (Bottom) Quantification of NUR77-GFP levels in the first division. Data are representative of 5 independent experiments. g. As in Main Fig. 8h, but comparing intra-cellular MYC among CTV-loaded B220+ cells after 72 hr culture: pHELT (1, 0.1, 0.01pM) and sHELT (500, 50, 5 ng/ml). Inset values: MFI of division 1 cells. Data are representative of 3 independent experiments. h. As in E, but cultured with anti-IgM(6.4 μg/ml) and titration of ibrutinib, comparing MYC among CTV-loaded B220+ cells after 72 hr culture. Data are representative of 6 independent experiments.
a, b. As in Main Figs. 2e,f and 3c except cells from MD4 or MD4.ML5 mice were stimulated with indicated reagents: anti-IgM 10 μg/ml, sHELD 1 μg/ml, or pHELD-HD (ED 286) 1 pM. Indo-1 ratio is shown on a linear scale. Gated in B are corresponding time gates to capture fraction of responding or non-responding B cells. Data are representative of 3 independent experiments. c, d. As in 8 A, B except pooled splenocytes and lymphocytes from MD4 (naïve) or MD4.ML5 (anergic) mice were incubated with indicated stimuli for 24 hours (sHEL-WT 1 μg/ml or pHELT-HD 1pM). Histograms and graphs depict MFI of CD86, CD69, or intra-cellular MYC in live B220 B cells. Graphs depict data and mean from and histograms are representative of 3 (MD4/ML5) or 4 (MD4) independent experiments. MD4 data in graphs corresponds to a subset of data plotted in Fig. 8b. Data were compared by two-tailed unpaired parametric t-test with Holm-Sidak correction. e-g. Viable frequencies of CTV-loaded MD4 (naïve) or MD4.ML5 (anergic) lymph node B cells in culture at 72-hours post-stimulation with sHELD/T and pHELD/T in the presence or absence of 20 ng/ml BAFF assessed via live/dead indicator dye. E. Representative plots depict gating to identify live B cells from total B220+ cells at 72 hr with corresponding histograms below depicting vital dye dilution of live B220+ cells at the same time point. Data are representative of 4 independent experiments. F. Absolute live B cell counts at sequential time points assessed using counting beads from a single experiment. G. Graphs depict %live B220 + B cells from total lymphocyte gate. Each datapoint is a sample from 4 independent experiments (except N = 3 for MD4 with no BAFF) independent experiments; lines connect samples from the same experiment. Groups were compared by a two-tailed paired parametric T-test.
a. Signalosome assembly in naïve B cells upon BCR stimulation by soluble Ag. BCR signal transduction requires sequential action of Src family kinases (SFKs) and SYK kinase. CD19 engagement amplifies PI3K activation and production of PI(3,4,5)P3. While multiple SFKs can mediate ITAM signaling downstream of the BCR, the SFK LYN plays a non-redundant role in phosphorylating ITIM-containing inhibitory coreceptors which in turn recruit PTPases SHP1 and SHIP1 that suppress PIP3. Dynamic regulation of PIP3 at the plasma membrane controls amplitude of signaling by recruiting downstream mediators including AKT, BTK, and PLCγ2 to orchestrate transcriptional programs mediated by NFAT, NF-κB and other factors. b. SVLS with appropriately spaced epitopes robustly engage pre-existing BCR nanoclusters but evade co-inhibitory receptors and results in downstream signal amplification. SVLS do not rely upon CD19 engagement for signal amplification in vitro. In the absence of inhibitory PTPase engagement via ITIM -containing inhibitory receptors, PIP3 accumulates at the plasma membrane leading to enhanced and prolonged signalosome assembly and activity downstream of SVLS stimulation. Robust NFAT and NF-κB accumulation in the nucleus and AKT-dependent signals mimic co-stimulation and promote MYC expression, resulting in T-independent cell growth, survival, and proliferation.
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Brooks, J.F., Riggs, J., Mueller, J.L. et al. Molecular basis for potent B cell responses to antigen displayed on particles of viral size. Nat Immunol 24, 1762–1777 (2023). https://doi.org/10.1038/s41590-023-01597-9