T-bet+ B cells accumulate in adipose tissue and exacerbate metabolic disorder during obesity

Abstract

Obesity is accompanied by inflammation in adipose tissue, impaired glucose tolerance, and changes in adipose leukocyte populations. These studies of adipose tissue from humans and mice revealed that increased frequencies of T-bet⁺ B cells in adipose tissue depend on invariant NKT cells and correlate with weight gain during obesity. Transfer of B cells enriched for T-bet⁺ cells exacerbates metabolic disorder in obesity, while ablation of Tbx21 specifically in B cells reduces serum IgG2c levels, inflammatory cytokines, and inflammatory macrophages in adipose tissue, ameliorating metabolic symptoms. Furthermore, transfer of serum or purified IgG from HFD mice restores metabolic disease in T-bet⁺ B cell-deficient mice, confirming T-bet⁺ B cell-derived IgG as a key mediator of inflammation during obesity. Together, these findings reveal an important pathological role for T-bet⁺ B cells that should inform future immunotherapy design in type 2 diabetes and other inflammatory conditions.

Keywords: B cells; CD11c(+) T-bet(+) B cells; IgG2c; adipose tissue; glucose intolerance; iNKT cells; inflammation; metabolic disorder; obesity; type 2 diabetes.

Figure 1.. Increased expression of T-bet, CD11c, and CD69 in adipose B cells correlates with increasing BMI in humans with obesity.

(A) Flow cytometry plots display gating and frequency of B cells (gate 5) and iNKT cells (gate 3) in human subcutaneous adipose tissue. Dot plot quantifies B cell and iNKT cell frequency in adipose tissue from patients that are lean (white), overweight (gray), or obese (black); each dot represents one patient. (See also Table S1 and Figure S1). (B-E) Representative histograms, dot plot quantification, and correlation between body mass index (BMI; kg/m²) of forward scatter-area (FSC-A) (B), intracellular T-bet protein expression (C), surface CD11c (D) and CD69 protein expression (E) for human adipose tissue B cells. Data are from >30 experiments. (F) Flow cytometry plot of CD19⁺ cells and gating of T-bet⁺ B cells from human adipose tissue. (G) Frequency of adipose tissue B cells that are T-bet⁺. Dots represent individual patients that are lean (white), overweight (gray), or obese (black). (H-I) Frequency of T-bet⁺ B cell correlation with patient age (H) or BMI (I). Quantification of CD11c (J) and CD1d protein expression (K) for T-bet⁻ (white) and T-bet⁺ (black) human adipose tissue B cells. (See also Figure S2) Dots represent one patient, line shows mean, bars indicate SEM; 1-way ANOVA. (A-E) Lines indicate linear regression trend with coefficient of determination (r²); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Figure 2.. Murine adipose and splenic T-bet⁺ B cell frequency and surface phenotype correlate with increasing body weight.

(A) Body weight and epididymal white adipose fat pad weight (week 15) of WT mice on the normal chow diet (NCD; white) or the high fat diet (HFD; black). (B) Glucose tolerance and resting serum glucose (week 15) measured by GTT. (C) Flow cytometry captures frequency and number of splenic and WAT CD19⁺ B cells in WT. (D-E) Flow cytometry of B cells from adipose tissue (D) or spleen (E) of WT mice on the NCD or HFD. Zebra plots show gating of T-bet⁺ CD11c⁺ B cells, dot plots show frequency and number of T-bet⁺ CD11c⁺ cells from tissues indicated; correlation between frequency of tissue T-bet⁺ CD11c⁺ B cells and body weight. See also Figure S3. Representative histograms and quantification of FSC-A (F), CD69 (G), intracellular Nur77 protein (H), surface CD21 (i), CD23 (j), and CD1d protein expression (K) for splenic CD11c⁻ T-bet⁻ B cells (circles; white, dark grey) and T-bet⁺ CD11c⁺ B cells (squares; light grey, black). Symbols represent one mouse on NCD (white, dark grey) or HFD (light grey, black). Data pooled from 4 (A-B), 2 (C), 7 (D-E), or 3 (F-K) experiments with 2–5 mice/grp and represented as mean ± SEM; 2way ANOVA, (D-E) Student’s t-test; (F-K) Wilcoxon matched pairs signed-rank. (D-E) Lines indicate linear regression trend with coefficient of determination (r²); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3.. Expansion of T-bet⁺ CD11c⁺ B cells in diet-induced obesity depends on iNKT cells.

(A) Flow cytometry of lymphocytes from WT or Cd1d1^−/− mice fed the NCD or HFD, presented with the t-SNE algorithm. See also Figure S4. Color in top row cell maps indicate expression of CD19: blue (low) to red (high). Second row cell maps indicate subgating of B cell cluster 1 (blue) and 2 (red). Histograms show expression of T-bet (third row) and CD11c (bottom row) by clusters identified in t-SNE maps; B cell cluster 1 (blue) and 2 (red). (B-D) Representative flow cytometry of splenic (B) and quantification of WAT and splenic (C,D) T-bet⁺ CD11c⁺ cell frequency of total B cells; WT NCD (white) and HFD (black), or Cd1d1^−/− NCD (light blue) and HFD (dark blue). (E) Flow cytometric quantification of numbers of NK1.1+ iNKT cells in WAT from WT mice fed a NCD (grey) or HFD (red). (F-G) ELISA measure of serum IgG1 (F), IgG2c (G), and IgM (H) antibodies from WT or Cd1d1^−/− mice fed the NCD or HFD; correlation between frequency of splenic T-bet⁺ CD11c⁺ B cells and IgG1 (F), IgG2c (G), and IgM (H), assessed by flow cytometry and ELISA, respectively. WT NCD (white) and HFD (black), or Cd1d1^−/− NCD (light blue) and HFD (dark blue). Dots represent individual mice. Data pooled from 7 (A-D), 2 (E), or 3 (F-G) experiments with at least 3 mice/grp. Mean ± SEM; (B-C) Two-way ANOVA; (D-F) Student’s t-test and Student’s paired t-test. Lines indicate linear regression trend with coefficient of determination (r²); *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.. Innate signal-induced expansion of T-bet⁺ CD11c⁺ B cells is iNKT cell-dependent.

(A) Flow cytometry of FSC-A, intracellular T-bet protein, surface CD69, CD86, CD21, CD23, and CD1d protein expression for splenic CD19⁺ B cells from WT mice administered 50μg R848 on day 0, 2 and harvested day 4 (black) or control (white). (B-G) Flow cytometry plots display gating strategy and frequency of B cell subsets in spleen of WT mice with (right) or without (left) R848 injection, as in (A). (H) Splenic B cell subset frequency from (B-G). Transitional type 1 (T1) gate 1, Transitional type 2 (T2) gate 2, Transitional type 2- Marginal zone precursor (T2-MZP) gate 3, Follicular B (FOB) gate 4, marginal zone B gate 5, CD21⁻ CD23⁻ gate 6, germinal center B (GC B) gate 7, plasmablast gate 8, CD11c⁺ gate 9. (I) Flow cytometry quantification of Tbet⁺ CD11c⁺ B cell frequency, isolated from spleens harvested 4 days after injection of wild-type or Cd1d1^−/− mice with (black) or without (white) R848. (J) Flow cytometry quantification of IFN-γ⁺ cells among iNKT cells and conventional (c)T cells, isolated from spleens of GREAT C.129S4(B6)-Ifng^tm3.1Lky/J mice with (black) or without (white) R848 injection, harvested on day 4. (K) Flow cytometry of splenic T-bet⁺ CD11c⁺ B cells from wild-type and Jα18^−/− mice with (black) or without (white) i.p. injection of 0.5 μg αGalCer. Spleens were harvested 4 days after injection. Data are from at 3 (A, I-J, N), 2 (B-H), or 5 (K) experiments with at least 2 mice/grp. Dots represent one mouse and/or show mean ± SEM; bar height is mean ± SEM; (A-H) Student’s t-test; (I-K) 1-way ANOVA. Line indicates linear regression trend with coefficient of determination (r²); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5.. T-bet-expressing B cells secrete CXCL10 and contribute to metabolic disorder in obesity.

(A) Representative flow cytometry histograms and dot plot summaries of cell size (FSC-A), intracellular T-bet protein expression, surface CD69, CD86, CD21, and CD1d protein expression, of isolated WT splenic B cells cultured in vitro with (red dots/histograms) or without (gray dots/histograms) 2.5 μg/ml R848 for 48 hours. (B) ELISA quantification of CXCL10 in supernatants from WT splenic B cells stimulated with (black) or without (white) R848 as in (A). (C) ELISA measured CXCL10 in day 4 serum of WT mice with (black) or without (white) i.p. injection of 50μg R848 on day 0 and day 3. (D-E) GTT and glucose area under curve (AUC) for WT mice fed the NCD (D, circles) or HFD (E, squares), with (black) or without (white) transfer of B cells enriched for T-bet⁺ CD11c⁺ B cells. Donor B cells were from WT mice treated with R848 and NP-KLH in vivo prior to isolation/transfer. See also Figure S5. Bar graphs show GTT AUC, 4 days after transfer. Data are from one experiment with at least 4 mice/grp (A), pooled from three experiments (B-C), or from 2 experiments with 2–4 mice/grp (D-E). Symbol centers or bar heights indicate individual values or group means, respectively and data show mean ± SEM; (A-E) Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 6.. Ablation of Tbx21 in B cells improves metabolic symptoms, limits adipose weight gain, and reduces inflammatory macrophage infiltrate in adipose tissue during obesity.

(A) Representative flow cytometry histograms and scatter plot summary of intracellular CXCL10 protein expression by Tbet+ CD11c+ B or conventional B cells (cB) as compared to fluorescent minus one (FMO) control staining. (B) Flow cytometry plots display gating strategy and frequency of splenic CXCL10⁺ CD11c⁺ B cells isolated from indicated mice fed NCD (top panels) or HFD (bottom panels); frequency summarized in bar graph. (C-D) GTT and glucose area under curve (AUC) for indicated mice fed HFD (C, squares; E, black squares or red triangles) or NCD (D, circles; E, white or grey squares). Bar graphs show AUC for GTT (C,D). Mice lacking Tbet+ B cells (red) compared to littermate controls with intact Tbet+ B cells (black) when fed a HFD vs NCD were compared for total and visceral white adipose tissue weight (F) and frequency of CD45+ leukocytes (G), adipose tissue macrophages (ATM) (H), and M1 macrophages (I). Dots represent individual mice. Data pooled from two experiments with 3–4 mice/grp; lines and bar heights represent mean ± SEM; Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 7.. Mice lacking Tbet+ B cells have reduced serum IgG2c and reduced glucose intolerance, but adoptive transfer of HFD serum or purified IgG from HFD serum restores metabolic disease and expands WAT macrophages.

(A-D) ELISA on serum from Tbet+ B cell deficient mice (red) or intact littermate controls (black) fed NCD or HFD for 20+ weeks revealed concentrations (ug/ml) of total IgM (A), IgG (B), IgG1 (C), and IgG2c (D). GTT and glucose (AUC) of HFD fed Tbet+ B cell deficient mice (red square) are exacerbated by serum transfer from WT HFD fed mice (grey triangle) or purified IgG from WT HFD mice (black circle) (E, F). Transfer of IgG from HFD fed WT mice to obese mice lacking Tbet+ B cells restores frequency, cells/gram, and number of F4/80⁺CD11b⁺ adipose macrophages to levels comparable to HFD fed littermate controls (H).