Fig 2: High carbohydrate feeding increases peripheral B cells(A) Contribution of macronutrient composition to proportion of splenic B220+ cells (n = 8 per diet, quantified by flow cytometry) was modeled with a mixture model and projected onto an RMT plot. 3-component mixture model represented on the RMT consists of carbohydrate (y-axis), protein (x-axis), and fat (implicit-axis), with splenic B cell proportion (number within isolines) as the response variables. Red and blue areas represent high and low experimental readouts respectively. Dots within the response surface correspond to 10 different diets used in experiments.(B) Representative flow cytometry plot for splenocytes from mice fed on high protein (HP), high carbohydrate (HC) and high fat (HF) diets. Splenic B cells were assessed as B220+ cells based on gating strategy described in Figure S1A by flow cytometry.(C) Linear regression analysis for the absolute energy intake from each macronutrient (protein, carbohydrate and fat) versus the B cell proportion in spleen. Each point represents averaged cage data.(D) Contribution of macronutrient composition to B cell proportion in mesenteric lymph nodes (mLN) (n = 8 per diet, quantified using flow cytometry) was modeled with mixture model and projected onto an RMT plot.(E) Linear regression analysis for the absolute energy intake from each macronutrient (protein, carbohydrate and fat) versus the B cell proportion in mLN. Each point represents averaged cage data.(F) Contribution of macronutrient composition to B cell proportion in Peyer's Patches (PP) (n = 8 per diet, quantified using flow cytometry) was modeled with mixture model and projected onto an RMT plot.(G) Linear regression analysis for the absolute energy intake from each macronutrient (protein, carbohydrate and fat) versus the B cell proportion in PP. Each point represents averaged cage data.See also Figures S1–S4, and Table S1.

Fig 3: 3DNA rapidly binds to target B cells in vivo. AlexaFluor647-labeled anti-CD19 or isotype control 3DNA was injected intravenously into C57BL/6 mice. Cells from the (A, B) peripheral blood and (C, D) spleen were harvested after (A, C) 10 minutes or (B, D) 30 minutes and analyzed by flow cytometry, with cell type defined with the following markers: B cells (B220), T cells (CD4 + CD8), macrophages (MF, CD11b), and neutrophils (GR-1). Graphs show the mean percentage of labeled cells ± SEM for n = 3 mice/group. (E) The proportion of the 2 cell types that primarily bind 3DNA, B cells, and macrophages is shown in the spleen and peripheral blood. Graphs show mean percentage of labeled cells ± SEM for n = 3 to 6 mice/group. (F) 3DNA in peripheral blood and spleen was measured by PCR. Graph shows the mean pg 3DNA ± SEM for n = 3 mice/group. Statistical significance is determined by t test with the Holm-Sidak correction for multiple comparisons. *P < 0.05; ***P < 0.001. n.s. indicates not significant; PCR, polymerase chain reaction; SEM, standard error of mean.

Fig 4: Anti-CD19-targeted 3DNA is internalized in B cells with limited binding of isotype control 3DNA. (A) Anti-CD19-3DNA or Rat Ig-3DNA hybridized with AlexaFluor488 was incubated for 60 minutes at 37°C with spleen cells from C57BL/6 mice and analyzed by imaging cytometry. Anti-CD19 3DNA targets B cells (left panel), while isotype control 3DNA does not (right panel). (B) B220 staining was used to define the cell surface based on Imagestream images. Intensity of internalized anti-CD19 or Rat Ig-3DNA staining defined as the intensity of AlexaFluor488 staining in an area defined as the cell boundary eroded toward the cell center by 4 pixels. (C) Representative images show B220 surface staining (red) and anti-CD19 or isotype control 3DNA (green) on individual cells. A representative experiment of 3 independent trials is shown.

Fig 5: B cells promote primary CD8 T cell responses to subunit vaccines(A) Total splenic B220+CD19+ B cells from WT C57BL/6 mice 0–7 days after i.v. administration of combined-adjuvant vaccine (anti-CD40, poly(I:C), and OVA).(B–D) 200 CD45.1+ OT1 T cells were transferred into WT or µMT-/- mice 1 day before combined-adjuvant vaccination. Spleens were harvested 7 days later. (B) Representative tetramer and CD45.1 staining. (C) Total OT1 cells (left) and tetramer+ endogenous CD8 T cells (right). (D) Tetramer as a percentage of endogenous CD8 T cells.(E) Representative immunofluorescent staining of 2 naive mice per group with DAPI (blue), CD3 (yellow), B220 (magenta), and CD169 (green). Scale bar represents 50 µm for all images.(F) Total splenic B220+CD19+ B cells from WT or MD4 mice 0–7 days after combined-adjuvant vaccine.(G and H) Total splenic tetramer+ CD8 T cells (top) or tetramer+ cells as a percentage of CD8 T cells (bottom) were measured 7 days after subunit vaccination or infection. (G) Ag-specific CD8 T cell responses after i.v. (left) or s.c. (right) administration of combined-adjuvant + OVA vaccine. (H) Responses after i.v. subunit immunization with the single adjuvants poly(I:C) (left) or Pam3Cys (right).(I) Responses after i.p. administration of OVA emulsified in alum.(J) Responses after infection with LM-OVA.Data shown are means ± SEM; n = 3 mice per group, representative of = 2 experiments, except for (F) and (H), which are from one experiment each. Significance was defined by two-tailed, unpaired Student’s t tests, where *p < 0.05, **p < 0.01, and ***p < 0.001.See also Figure S2.