(a) Time course of T cell (T), B cell (B) and monocyte (M) infiltration of VAT after initiation of HFD (left, 2 experiments, 5 mice, *P < 0.05). B cell subsets in VAT in response to 6–12 weeks of HFD in absolute numbers (middle) of B cells (*P = 0.005), B1a cells (*P = 0.04), B1b cells, non-B1 cells/B2 (*P = 0.04) and T cells (*P = 0.03), and in percentages of CD19⁺ cells (right); middle and right, 3 experiments, 9 mice. (b) VAT B cells in absolute numbers (left,*P < 0.05) and proportion of CD19⁺ cells (right, *P < 0.05); left and right, 3 experiments each, 9 mice. (c) Spleen B cell subsets in response to HFD (MZ, marginal zone; FC, follicular cells, *P = 0.01, n = 5). (d) Spontaneous production of IgM (left, *P = 0.0006) and IgG (*P = 0.01) from mouse splenocytes (e) Serum antibody concentrations in mice: IgA (*P = 0.03) and IgG2c (*P = 0.004) (n = 10). (f) Antibody subtypes in VAT lysates from mice (*P = 0.0001) (2 experiments, 5 mice). (g) IgM (top left) and IgG (bottom left) staining in VAT of HFD mice in regions of few and multiple CLSs (IgM top right; IgG bottom right) (arrows indicate antibody stained cells, bar 50 μm left and 25 μm right). Graphs are means ± s.e.m.

(a) Numbers of cell subsets in VAT of 14–18 week old mice (4 experiments, 10 mice). (b) Percentage of VAT macrophages (CD11b⁺F4/80⁺Gr-1^-) with M1 phenotype (*P = 0.049, 3 experiments, 8 mice). (c) IFN-γ production from SVC cultures of VAT (3 experiments, 9 mice, *P = 0.02). (d) Intracellular IFN-γ staining of CD8⁺ T cells isolated from VAT (left, 4 experiments,10 mice, *P = 0.04) and percentage of total VAT CD8⁺ T cells expressing CD107a (right, *P = 0.02, 2 experiments, 6 mice). (e) TNF-α production from VAT SVC cultures (left, *P = 0.04, 2 experiments, 6 mice) and intracellular staining of TNF-α in VAT macrophages (right, 2 experiments, 6 mice, *P = 0.02). (f) CD80 and CD86 expression on VAT macrophages (representative of 3 experiments, 9 mice). (g) GTT (left), fasting glucose (middle) and fasting insulin (right) of recipient HFD RAG^null mice 2 weeks after transfer of HFD B cells (n = 10). (h) CD19⁺ B cells in VAT of B^null mice 2 weeks after reconstitution with various B cells (3 experiments, 9 mice). (i) Weights (left), GTT (middle) and fasting insulin (right) of recipient mice 2 weeks after transfer of various B cells (*P < 0.05, representative of 3 experiments, n = 3 per group). (j) IFN-γ production from VAT SVC cultures (left), and intracellular IFN-γ in VAT CD8⁺ T cells (middle) and VAT CD4⁺ T cells (right) isolated from recipient B^null mice receiving either PBS orvarious B cells (*P < 0.05, 2 experiments, 6 mice). Graphs are means ± s.e.m.

Age matched 12–13 week old (6–7 weeks on HFD) HFD WT mice were treated with MB20-11 CD20 mAb (100 μg i.v.). (a) Percentage of CD19⁺ cells depleted in VAT and spleen ≥8 days following administration of CD20 mAb. (b) Weights of mice and (c) percentage depletion of IgG and IgM antibody in serum 28 days post CD20 mAb treatment (representative of two experiments, n = 5). (d) Fasting glucose (*P = 0.06), (e) GTT (*P < 0.05) and (f) fasting insulin (*P = 0.04) in HFD WT mice 28 days after receiving either CD20 mAb or control (IgG2c or PBS) (representative of two experiments, n = 5). (g, h) IFN-γ (*P = 0.003) and TNF-α (*P = 0.005) production from SVC cultures of VAT isolated from 17 week old mice treated with CD20 mAb at 13 weeks of age (2 experiments, 8 mice). i) Percentage of VAT macrophages expressing TNF-α 4 weeks after treatment with CD20 mAb (*P = 0.01, 2 experiments, 8 mice). Graphs are means ± s.e.m.

(a) Body weights of WT and B^null mice over time (n = 10 per group). (b) Relative fat cell diameter of 14–18 week old HFD mice (n = 3). (c) Ratio of epididymal VAT and SAT pad weights in HFD mice (*P = 0.004, n = 10) (d) Fasting glucose (*P = 0.04, n = 10) and (e) glucose tolerance test (GTT) of WT or B^null mice on NCD or HFD (*P < 0.05, representative GTT from 3 experiments, n=10 per group on HFD and 2 experiments, n=5 per group on NCD). (f) Fasting serum insulin concentrations of 16 week old WT or B^null mice on NCD or HFD (*P = 0.04, n = 10). (g) Insulin tolerance test (ITT) in WT or B^null mice on NCD or HFD (*P < 0.05, n = 5 per group). (h) Body weight (left), GTT (middle, *P < 0.05, n = 6), and fasting insulin (right, *P = 0.02, n = 6) of HFD B^null mice 2 weeks following reconstitution with HFD WT B cells (representative of 3 independent experiments). (i) Body weight (left), GTT (middle, n = 5), and fasting insulin (right, n = 5) of HFD B^null mice 2 weeks following reconstitution with NCD WT B cells (representative of 2 independent experiments). Graphs are means ± s.e.m.

(a) Serum concentration of IgG in B^null mice one week following i.p. IgG injection (n = 3). (b) Body weights of HFD B^null recipient mice after IgG transfer (representative of 3 experiments, n = 4). (c) GTT (left, * P < 0.05) and fasting insulin (right, *P < 0.05) one week following transfer of IgG into 16 week old HFD B^null mice (representative of 3 experiments, n = 4). (d) GTT (left) and fasting insulin (right) four weeks following transfer of IgG (representative of 2 experiments, n = 4). (e) GTT (left, *P < 0.05) and fasting insulin (right, *P = 0.048) one week following transfer of late or early IgG (n = 5). (f) Weights (left), GTT (center), and fasting insulin (right) of 6 week old NCD B^null mice 1 week after IgG transfer (representative of 2 experiments, n = 4). (g) TNF-α from VAT SVC cultures (left, *P = 0.04, 2 experiments, 6 mice) and M1 macrophages in HFD B^null VAT one week after IgG transfer (right, *P = 0.007, 2 experiments, 6 mice). (h) GTT (left, *P < 0.05) and fasting insulin (right, *P = 0.04) one week after transfer of HFD Ig (n =5). (i) TNF-α from HFD B^null VAT macrophages stimulated in vitro with HFD IgG (*P= 0.007), or HFD F(ab′)₂ (n = 3). j) GTT (left) and fasting insulin (middle) of HFD B^null mice 1 week after receiving HFD Ig (n = 5, *P < 0.05). Serum concentration (right) of IgM in HFD B^null mice 1 week following IgM injection (n = 3). Graphs are means ± s.e.m.