Articles

B cells promote insulin resistance through modulation of T cells and production of pathogenic IgG antibodies
Daniel A Winer1,2,7, Shawn Winer2,3,7, Lei Shen1,7, Persis P Wadia4, Jason Yantha3, Geoffrey Paltser3, Hubert Tsui3, Ping Wu3, Matthew G Davidson1, Michael N Alonso1, Hwei X Leong1, Alec Glassford5, Maria Caimol1, Justin A Kenkel1, Thomas F Tedder6, Tracey McLaughlin5, David B Miklos4, H-Michael Dosch3 & Edgar G Engleman1
2011 Nature America, Inc. All rights reserved. Chronic inflammation characterized by T cell and macrophage infiltration of visceral adipose tissue (VAT) is a hallmark of obesity-associated insulin resistance and glucose intolerance. Here we show a fundamental pathogenic role for B cells in the development of these metabolic abnormalities. B cells accumulate in VAT in diet-induced obese (DIO) mice, and DIO mice lacking B cells are protected from disease despite weight gain. B cell effects on glucose metabolism are mechanistically linked to the activation of proinflammatory macrophages and T cells and to the production of pathogenic IgG antibodies. Treatment with a B celldepleting CD20 antibody attenuates disease, whereas transfer of IgG from DIO mice rapidly induces insulin resistance and glucose intolerance. Moreover, insulin resistance in obese humans is associated with a unique profile of IgG autoantibodies. These results establish the importance of B cells and adaptive immunity in insulin resistance and suggest new diagnostic and therapeutic modalities for managing the disease. Obesity and its associated metabolic abnormalities, including insulin resistance and type 2 diabetes, have reached epidemic proportions, adversely affecting health and global mortality rates1. Multiple factors contribute to reduced insulin sensitivity, but chronic inflammation in VAT, which results in local and systemic increases in proinflammatory cytokines and adipokines is a major driver2,3. One of these drivers, macrophage infiltration of VAT, is a key event in the establishment of adipose inflammation and insulin resistance4,5. Classically activated, or CD11c+CD206 M1 macrophages, are elevated in VAT of DIO mice and produce proinflammatory cytokines such as tumor necrosis factor- (TNF-), interleukin-1 (IL-1) and IL-6 (refs. 68). T cells are also major participants in VAT inflammation, with proinflammatory CD8+ T cells and interferon- (IFN-)-producing CD4+ T cells contributing to inflammation, glucose intolerance and insulin resistance in DIO mice911. On the contrary, VAT-resident forkhead box P3expressing (Foxp3+) regulatory T cells, which produce IL-10 and TGF-, as well as IL-4 and IL-13secreting type 2 helper T cells (TH2 cells), can have protective roles1113. Notably, the clonal diversity of VAT T cells is highly restricted, which suggests that an active adaptive immune response expanding potentially autoimmune T cells occurs in obese VAT1114. In contrast to macrophages and T cells, little is known about the role of B cells in the development of insulin resistance, despite evidence that such cells are recruited to adipose tissue shortly after the initiation of a high-fat diet (HFD)15 and that their activation is increased in people with type 2 diabetes16. Here we show that B cells
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and IgG are key pathogenic effectors in the development of obesityassociated insulin resistance and glucose intolerance, but not of excess weight gain, in DIO mice. Manipulation of B cells, antibodies or their Fc (fragment crystallizable) receptors may yield promising new therapies for the management of insulin resistance and its associated comorbidities. RESULTS B cells and antibodies in DIO mice We analyzed early immune cell infiltration into epididymal VAT of 6-week-old C57BL/6 mice fed a high-fat diet (60% kcal) for several weeks and compared the immune cell composition to age-matched C57BL/6 mice that were fed a normal chow diet (NCD) (Fig. 1a). HFD induced a significant accumulation of B cells in VAT by 4 weeks that was maintained after 612 weeks on HFD (Fig. 1a). This increase in B cell numbers included total B cells, B1a cells and B2 cells. Total T cell numbers were also increased by 4 weeks, and absolute numbers continued to rise while the mice were on a HFD, consistent with previous reports11,15,17. Despite the increase in absolute B cell numbers in DIO VAT, the relative proportions of B1 and non-B1 subsets were unchanged (Fig. 1a). However, DIO VAT had increased numbers and proportions of class-switched mature B cells, such as IgG+ cells, a pattern suggesting an actively progressing immune process in this fat depot of obese mice (Fig. 1b). To investigate the effects of HFD on systemic B cells, we analyzed spleens from age-matched 12- to 18-week-old HFD-fed and NCD-fed

of Pathology, Stanford University, Palo Alto, California, USA. 2Department of Laboratory Medicine and Pathobiology, University Health Network, University of Toronto, Toronto, Ontario, Canada. 3Neuroscience & Mental Health Program, Research Institute, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada. 4Department of Medicine, Stanford University, Palo Alto, California, USA. 5Division of Endocrinology, Stanford University School of Medicine, Palo Alto, California, USA. 6Department of Immunology, Duke University Medical Center, Durham, North Carolina, USA. 7These authors contributed equally to this work. Correspondence should be addressed to D.A.W. (dan.winer@uhn.on.ca) or E.G.E. (edengleman@stanford.edu). Received 28 January; accepted 4 March; published online 17 April 2011; corrected after print 6 June 2011; doi:10.1038/nm.2353

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Figure 1 B cell and antibody profile in DIO mice. NCD IgM IgM 3,000 (a) Left, time course of T cell (T), B cell (B) and HFD 2,000 macrophage (M) infiltration of VAT after initiation of HFD (two experiments, five mice, *P < 0.05). * Middle and right, B cell subsets in VAT in response 1,000 to 612 weeks of HFD in absolute numbers of 500 B cells (*P = 0.005), B1a cells (*P = 0.04), IgG IgG B1b cells, B2 cells (*P = 0.04) and T cells 0 (*P = 0.03) (middle) and in percentages of CD19+ cells (right). Middle and right, three experiments, nine mice. (b) VAT B cells in absolute numbers (left, *P < 0.05) and as a proportion of CD19+ cells (right, *P < 0.05); three experiments each, nine 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 (right, *P = 0.01) from mouse splenocytes. (e) Serum antibody concentrations in mice (n = 10): IgA (*P = 0.03) and IgG2c (*P = 0.004). (f) Antibody subtypes in VAT lysates from mice (*P = 0.0001) (two experiments, five mice). (g) IgM (top left) and IgG (bottom left) staining in VAT of DIO mice in regions of few and multiple CLSs (IgM top right; IgG bottom right). Arrows indicate antibody-stained cells. Scale bars, 50 m (left images) and 25 m (right images). Error bars in graphs indicate means s.e.m.

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mice. No significant differences were seen in total spleen cell counts or the percentages of naive IgD+ B cells, marginal zone B cells or IgM+IgD follicular B cells (Fig. 1c). However, in contrast to DIO VAT, DIO spleens contained reduced percentages of IgM+IgD cells (Fig. 1c). Consistent with these results, total spleen B cells from DIO mice showed reduced spontaneous production of IgM antibody but elevated IgG secretion (Fig. 1d), suggesting that HFD induces a systemic humoral immune response. We confirmed this systemic response by comparing concentrations of immunoglobulin isotypes in serum and VAT of NCD and DIO mice. DIO mice had reduced concentrations of serum IgA and an increase in serum IgG2c (Fig. 1e), a proinflammatory isotype present in C57BL/6, C57BL/10 and nonobese diabetic mice18. VAT lysates from HFD-fed mice had higher concentrations of IgM compared to IgG and a marked (more than threefold) enrichment in proinflammatory IgG2c (Fig. 1f). Notably, antibody staining in VAT showed a preferred localization of IgG and IgM to regions of crown-like structures (CLSs) (Fig. 1g)19,20. Many of these stained cells were at the interface of large mononucleate and multinucleate giant macrophages and dying adipocytes inside the CLSs. CLSs appeared bathed in tissue fluid enriched for IgM and IgG, whereas the remaining fat tissue showed either very weak or no Ig staining. Enrichment of IgG and IgM in CLSs implicates the involvement of antibodies in the clearance of dying adipocytes19,20. B cells are pathogenic in glucose metabolism in DIO mice To assess the effect of B cells on the regulation of obesity and insulin resistance, we investigated C57BL/6 immunoglobulin heavy-chain
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knockout mice (Bnull) which do not produce mature B cells, for their response to HFD started at 6 weeks of age21. After 8 weeks of HFD, there was no difference in body weight or VAT adipocyte size (Fig. 2a,b), but DIO Bnull mice had a lower ratio of VAT to subcutaneous adipose tissue (SAT) fat pad weight than DIO WT mice (Fig. 2c). Compared with HFD-fed WT mice, 16-week-old HFD-fed Bnull mice had lower fasting glucose (Fig. 2d) and improved glucose tolerance (Fig. 2e). Similarly, 16-week-old HFD-fed Bnull mice had reduced fasting insulin (Fig. 2f) and improved insulin sensitivity upon insulin challenge (Fig. 2g). B cell deficiency did not affect weight, fasting glucose, fasting insulin or glucose and insulin tolerance in NCD mice (Fig. 2a,dg), suggesting that metabolic influences of B cells require a HFD. To confirm that abnormal glucose metabolism in HFD-fed mice is directly attributable to B cells, age-matched 16 to 18 weeks old HFD-fed Bnull mice were reconstituted intraperitoneally (i.p.) with 1 107 total B cells from spleens of DIO WT mice. After 23 weeks, B cells reconstituted primarily in VAT over spleen (Supplementary Fig. 1a) and produced low concentrations of serum antibody (Supplementary Fig. 1b). B cell transfer did not affect total weight (Fig. 2h). However, Bnull mice reconstituted with B cells from DIO mice had worsened glucose tolerance (Fig. 2h) and higher fasting insulin levels (Fig. 2h) compared to control Bnull recipients. B cells from NCD mice did not promote impairment in glucose homeostasis (Fig. 2i), despite similar reconstitution profiles as B cells from DIO mice (Supplementary Fig. 1c), thus indicating that development of pathogenic B cells requires exposure to a HFD. Collectively, these results suggest a pathogenic role for HFD-derived B cells in the promotion of obesityassociated insulin resistance and glucose intolerance.

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3 Figure 2 B cell deficiency modulates glucose metabolism in DIO 40 mice. (a) Body weights of WT (control) and Bnull mice over time 2 20 30 (n = 10 per group). (b) Relative fat cell diameter of 14- to 20 18-week-old HFD mice (n = 3). (c) Ratio of epididymal VAT and SAT 1 10 pad weights in DIO mice (*P = 0.004, n = 10). (d,e) Fasting glucose 10 (*P = 0.04, n = 10) (d) and glucose tolerance test (GTT) (e) of WT 0 0 0 0 2 or Bnull mice on NCD or HFD (*P < 0.05, representative GTT from 0 15 30 45 60 75 90 Time after transfer three experiments, n = 10 per group on HFD and two experiments, Time (min) (weeks) n = 5 per group on NCD). (f) Fasting serum insulin concentrations of 16-week-old WT or Bnull mice on NCD or HFD (*P = 0.04, n = 10). (g) Insulin tolerance test (ITT) in WT or Bnull 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 DIO Bnull mice 2 weeks after reconstitution with DIO WT B cells (representative of three independent experiments). (i) Body weight (left), GTT (middle, n = 5), and fasting insulin (right, n = 5) of DIO Bnull mice 2 weeks after reconstitution with NCD WT B cells (representative of 2 independent experiments). Brackets represent comparison groups for statistics. Error bars on graphs show means s.e.m. Glucose (mmol I1) Insulin (ng ml1) 30 Weight (g)

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DIO Bnull mice show reduced immune cell activation in VAT Inflamed adipose tissue is a key feature of insulin resistance. As B cells reside in VAT and worsen metabolic parameters upon adoptive transfer, we examined the possibility that these cells promote inflammation in VAT. VAT total T cell and macrophage counts were similar in DIO WT and DIO Bnull mice (Fig. 3a), but DIO Bnull mice had fewer proinflammatory M1 macrophages (Fig. 3b). To examine the functional profiles of these immune cells, we measured cytokines known to affect insulin resistance (TNF- and IFN-)9,22,23. The supernatants of total VAT stromal vascular cell (SVC) cultures from DIO Bnull mice had lower concentrations of IFN- compared to DIO WT mice (Fig. 3c). The number of VAT-associated CD8+ T cells producing IFN- from DIO Bnull mice was approximately 30% less compared to DIO WT mice (Fig. 3d). DIO Bnull CD8+ T cells also expressed less of the cytotoxic activation marker CD107a (Fig. 3d). IFN- expression was variable in CD4+ T cells of DIO WT and Bnull mice (Supplementary Fig. 2a). Supernatants of VAT SVC cultures from DIO Bnull mice also contained less TNF- compared to DIO WT mice (Fig. 3e). Macrophages, especially M1 macrophages, are a major source of TNF-, and its decrease in DIO Bnull VAT is partly attributable to reduced numbers of macrophages producing this cytokine (Fig. 3e). In addition, fewer DIO Bnull VAT macrophages expressed co-stimulatory CD86, consistent with an overall decrease in macrophage activation in these mice (Fig. 3f). Analysis of serum revealed that concentrations of resistin and plasminogen activator inhibitor-1 (PAI-1), both previously associated with insulin resistance24,25, were markedly lower in DIO Bnull than DIO WT

mice (Supplementary Fig. 2b). This result suggests that in DIO mice, B cells induce systemic as well as local (VAT) inflammation. B cells modulate VAT associated T cells in vivo B cell functions are influenced by other lymphocyte populations (especially T cells) and vice versa26. To begin to discern a role for T cells in facilitating B cellmediated glucose intolerance, we reconstituted 16-week-old HFD-fed C57BL/6 recombinase activating gene-1null (RAG-1null) mice, which lack lymphocytes, with 1 107 total DIO splenic B cells. After 2 weeks, despite reconstitution (Supplementary Fig. 3), the B cells did not worsen fasting glucose, insulin, or glucose tolerancecontrasting with transfers into DIO Bnull micethereby suggesting that B cells require other lymphocytes to fully promote the impairment of metabolic parameters (Fig. 3g). B cells can activate CD8+ and CD4+ T cells by presenting antigen via major histocompatibility complex (MHC) class I and MHC class II, respectively. To determine whether B celldependent T cell activation is involved in the promotion of glucose intolerance, we reconstituted age-matched 16-week-old HFD-fed Bnull mice with 1 107 purified (> 95%) splenic B cells from either DIO WT mice, or DIO mice lacking MHC class I (C57BL/6 MHC-Inull) or MHC class II (C57BL/6 MHC-IInull). Two weeks after i.p. transfer, CD19+ B cells had successfully reconstituted VAT but not spleen in the DIO Bnull mice (Fig. 3h and Supplementary Fig. 1a). There were no significant differences in weight after adoptive transfer (Fig. 3i). However, in contrast to recipients of B cell grafts from WT mice, recipients of B cells from

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either MHC-Inull or MHC-IInull mice did not develop glucose intolerance or hyperinsulinemia (Fig. 3i). Improved glucose homeostasis in mice receiving MHCnull B cells compared to mice receiving WT B cells,was associated with reduced total VAT SVC production of IFN- (Fig. 3j), attributable to either reduced numbers of VAT CD8+ T cells producing IFN- (MHC-Inull recipients, Fig. 3j) or reduced numbers of VAT CD4+ T cells producing IFN- (MHC-IInull recipients, Fig. 3j). The data show that B cell modulation of VAT T cells occurs in an MHC-dependent manner, probably through B cell antigen presentation to T cells, and that both MHC class I (CD8+ T cells) and class II (CD4+ T cells) are needed for the B cells to maximally affect glucose tolerance in the adoptive transfer model. IgG antibodies are mediators of glucose intolerance In addition to their functions in the modulation of T cell activation, B cells produce antibodies, which are known regulators of immune function27. Because obesity is associated with increased IgG production and IgG+ B cells in VAT, as well as with increased concentrations of systemic and local IgG2c (Fig. 1bf), we next investigated a possible role of IgG in glucose intolerance. We purified IgG (>98% pure) from pooled sera of 16- to 24-week-old HFD-fed (HFD IgG) or NCDfed (NCD IgG) WT mice and injected it i.p. into age-matched DIO Bnull mice. One week after transfer, IgG was present in the serum of all recipient mice (Fig. 4a). Within this time frame, antibody transfer had no effect on body weight (Fig. 4b). However, HFD IgG induced a dramatic worsening of glucose tolerance, which was absent in NCD IgG recipients (Fig. 4c); this suggests that pathogenic IgG specificities are induced during the course of a HFD but not during NCD. This antibody-mediated metabolic effect was associated with worsened fasting insulin, a hallmark of insulin resistance, in HFD IgG recipient mice compared to NCD IgG recipient mice (Fig. 4c). Transferred antibodies also localized to VAT, where they were in close association

2011 Nature America, Inc. All rights reserved.

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PBS DIO MHC-l B 8 Figure 3 B cells influence VAT T cell and macrophage 60 DIO WT B DIO MHC-llnull B * * * * 25 6 function. (a) Numbers of cell subsets in VAT of * 20 14- to 18-week-old mice (four experiments, 4 40 * ten mice). (b) Percentage of VAT macrophages 2 15 20 (CD11b+F4/80+Gr-1) with M1 phenotype 0 10 (*P = 0.049, three experiments, eight mice). 0 5 (c) IFN- production from SVC cultures of VAT 0 2 (three experiments, nine mice, *P = 0.02). 0 Time after transfer (weeks) 0 15 30 45 60 75 90 105120 (d) Intracellular IFN- staining of CD8+ T cells Time (min) isolated from VAT (left, four experiments, ten mice, * *P = 0.04) and percentage of total VAT CD8+ * 300 * 40 100 * * T cells expressing CD107a (right, *P = 0.02, two 80 30 experiments, six mice). (e) TNF- production from 200 60 VAT SVC cultures (left, *P = 0.04, two experiments, 20 40 six mice) and intracellular staining of TNF- in VAT 100 10 20 macrophages (right, two experiments, six mice, 0 0 0 *P = 0.02). (f) CD80 and CD86 expression on VAT macrophages (representative of three experiments, nine mice). (g) GTT (left), fasting glucose (middle) and fasting insulin (right) of recipient DIO RAG-1null (Rag1/) mice 2 weeks after transfer of DIO B cells (n = 10). (h) CD19+ B cells in VAT of Bnull mice 2 weeks after reconstitution with DIO WT, DIO MHC-I null or DIO MHC-IInull B cells (three experiments, nine mice). (i) Weights (left), GTT (middle) and fasting insulin (right) of recipient mice 2 weeks after transfer of DIO WT, DIO MHC-I null or DIO MHC-IInull B cells (*P < 0.05, representative of three experiments, n = 3 per group). (j) IFN- production from VAT SVC cultures (left) and intracellular IFN- in VAT CD8+ T cells (middle) and VAT CD4+ T cells (right) isolated from recipient Bnull mice receiving either PBS or DIO WT, DIO MHC-Inull or DIO MHC-IInull B cells (*P < 0.05, two experiments, six mice). WT, control. Brackets represent comparison groups for statistics. Error bars in graphs are means s.e.m.
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Figure 4 HFD IgG induces abnormal glucose metabolism in recipient Bnull mice. (a) Serum concentration of IgG in Bnull mice 1 week after i.p. IgG injection (n = 3). (b) Body weights of HFD Bnull recipient mice after IgG transfer (representative of three experiments, n = 4). (c) GTT (left, * P < 0.05) and fasting insulin (right, *P < 0.05) 1 week after the transfer of IgG into 16-week-old HFD Bnull mice (representative of three experiments, n = 4). (d) GTT (left) and fasting insulin (right) 4 weeks after the transfer of IgG (representative of two experiments, n = 4). (e) GTT (left, *P < 0.05) and fasting insulin (right, *P = 0.048) 1 week after the 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 two experiments, n = 4). (g) TNF- from VAT SVC cultures (left, *P = 0.04, two experiments, six mice) and M1 macrophages in HFD Bnull VAT 1 week after HFD IgG transfer (right, *P = 0.007, two experiments, six mice). (h) GTT (left, *P < 0.05) and fasting insulin (right, *P = 0.04) 1 week after the transfer of HFD IgG or HFD F(ab)2 (n = 5). (i) TNF- from HFD Bnull 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 Bnull mice 1 week after receiving HFD Ig (n = 5, *P < 0.05). Serum concentration (right) of IgM in HFD Bnull mice 1 week after IgM injection (n = 3). Brackets represent comparison groups for statistics. Error bars show means s.e.m.

with CLSs (Supplementary Fig. 4a). The effects of antibody transfer on glucose metabolism were transient and, as expected on the basis of normal IgG half-life, by 4 weeks after transfer there was no difference in glucose tolerance or fasting insulin between IgG and control recipients (Fig. 4d). To determine whether pathogenic antibodies arise early after the initiation of HFD, we purified IgG from 9- to12-week-old HFD-fed mice (early HFD IgG), as well as from 20- to 24-week-old HFDfed mice (late HFD IgG), and injected each type separately i.p. into 20-week-old HFD-fed Bnull mice. We observed a much stronger effect on glucose tolerance and fasting insulin concentrations with late HFD IgG compared to early HFD IgG; this suggests a possible role for affinity maturation of antibody or late unmasking of HFD antigen in the process (Fig. 4e). To investigate whether HFD IgGinduced disease depends on HFD exposure in recipient mice, we transferred HFD IgG (IgG purified from 16- to 24-week-old HFD-fed C57BL/6 mice) into 6-week-old lean mice. One week after transfer, there was little change in weight, glucose tolerance or fasting insulin levels (Fig. 4f),

thus indicating that the effects of HFD IgG on glucose metabolism are dependent on the recipients exposure to HFD. To investigate the mechanism by which HFD IgG induces glucose intolerance, we first examined its effects on VAT and systemic inflammation. HFD IgG recipient mice had higher concentrations of TNF- in VAT SVC cultures and more pronounced M1 macrophage polarization in VAT when compared to controls (Fig. 4g). In addition, these mice had elevated serum concentrations of proinflammatory mediators including monocyte chemoattractant protein-3, IL-6 and granulocytemacrophage colony-stimulating factor (Supplementary Fig. 4b). IgG antibodies, through their Fc portions, can bind Fc receptors (FcRs) on macrophages and directly induce macrophage oxidative burst, cytotoxicity and proinflammatory cytokine production27. To determine whether the observed effect of HFD IgG antibodies on glucose intolerance is mediated through their Fc components, we generated F(ab)2 fragments from HFD IgG and compared their effect on glucose metabolism to that of intact HFD IgG. One week after i.p. transfer into age-matched DIO Bnull mice, HFD IgG F(ab)2 did not worsen glucose tolerance and
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Table 1 Top ten antibody targets most strongly associated with insulin resistance (IR; top) and with insulin sensitivity (IS; bottom) in human male subjects
Antigen GOSR1 BTK GFAP ASPA NIF3L1 PGD ALDH16A1 KCNAB1 RNA polymerase GSTA3 CTNNA1 CDC37 LGALS14 BM88 NCBP2 P value 8.54E-05 0.001224 0.001224 0.003399 0.003399 0.003399 0.003399 0.003399 0.004574 0.004574 0.001027 0.001224 0.001224 0.002961 0.002961 0.003399 0.003399 0.004574 0.006057 0.007749 IR prevalence 72.22% 50% 50% 44.44% 44.44% 44.44% 44.44% 44.44% 61.11% 61.11% 11.11% 5.56% 5.56% 11.11% 11.11% 5.56% 5.56% 16.67% 27.78% 44.44% IS prevalence 11.11% 5.56% 5.56% 5.56% 5.56% 5.56% 5.56% 5.56% 16.67% 16.67% 61.11% 50% 50% 55.56% 55.56% 44.44% 44.44% 61.11% 72.22% 88.89%

HFD IgM had no effect on metabolic parameters (Fig. 4j). These data point to an unexpected pathogenic role for HFD-induced IgG antibody in promoting glucose intolerance and insulin resistance. Insulin resistance is linked to distinct profiles of IgG As HFD IgG can exert pathologic effects on insulin resistance in obese mice, we next examined whether IgG autoantibodies are present in insulin-resistant humans and, if so, whether they recognize a distinct cluster of antigenic targets. We probed Invitrogen ProtoArray V5.0 chips containing more than 9,000 spotted antigens with serum from 32 age- and weight-matched, overweight to obese, otherwise healthy, male human subjects (Supplementary Table 1). The two groups of subjects differed only in their insulin sensitivities, as determined by a modified insulin-suppression test, and individuals were defined as either insulin resistant or insulin sensitive on the basis of their steadystate plasma glucose concentration. We identified 122 IgG targets that differentially segregated with insulin resistance, whereas 114 targets segregated with insulin sensitivity. The ten antigens that were most highly associated with either insulin resistance or insulin sensitivity in our obese male subjects are shown in Table 1. Antibodies to the top three targets segregating with insulin resistance were validated in these subjects by western blotting (Supplementary Fig. 5). Notably, in both groups the antigens are mostly intracellular proteins, many of which are expressed ubiquitously in tissues including immune cells, pancreas, nervous tissues, muscle or fat. B cell depletion ameliorates metabolic disease To determine whether the manipulation of B cells can be exploited therapeutically in obesity-related insulin resistance, we treated HFD-fed WT mice with a depleting antibody specific to mouse CD20 (MB20-11; ref. 28). We injected antibody 67 weeks after initiation of HFD and maintained the mice on an HFD. B cells, which were depleted by >95% locally in VAT and systemically in spleen 8 d after injection (Fig. 5a), began to repopulate tissues by day 28 after injection; nonetheless, there was still >70% depletion. CD20-specific monoclonal antibody (mAb) treatment had no effect on weight (Fig. 5b) or total serum concentrations of IgG or IgM (Fig. 5c) by 28 d after injection. However, treated mice showed improvements compared to control-treated mice in

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PDDC1 ALS2CR8 PAFAH G subunit XRCC4 Influenza A antigen (H3N2)

GOSR1, golgi SNAP receptor complex member 1, transcript variant 1; BTK, Bruton agammaglobulinemia tyrosine kinase; GFAP, glial fibrillary acidic protein; ASPA, aspartoacylase; NIF3L1, NIF3 NGG1 interacting factor 3-like 1, transcript variant 2; PGD, phosphogluconate dehydrogenase; ALDH16A1, aldehyde dehydrogenase 16 family, member A1; KCNAB1, potassium voltage-gated channel, shaker-related subfamily, beta member 1,transcript variant 1; GSTA3, glutathione S-transferase alpha 3; CTNNA1, catenin, cadherin-associated protein, alpha 1; CDC37, CDC37 cell division cycle 37 homolog; LGALS14, lectin, galactoside-binding, soluble, 14; BM88, BM88 antigen; NCBP2, nuclear cap binding protein subunit 2; PDDC1, Parkinsons disease 7 domain containing 1; ALS2CR8, amyotrophic lateral sclerosis 2 (juvenile) chromosome region, candidate 8; PAFAH G subunit, platelet-activating factor acetylhydrolase, isoform Ib, gamma subunit; XRCC4, X-ray repair complementing defective repair in Chinese hamster cells 4, transcript variant 2.

fasting insulin compared to intact HFD IgG, thereby indicating that the pathogenic properties of HFD IgG are mediated through the Fc region (Fig. 4h). Consistent with these findings, macrophages isolated from VAT of DIO Bnull mice stimulated with HFD IgG show an Fc-dependent increase in TNF- production in vitro (Fig. 4i). In contrast to HFD IgG,

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Figure 5 A CD20-specific B cell-depleting antibody improves obesity-induced glucose abnormalities. (a) Percentage of CD19+ cells depleted in VAT and spleen 8 d after administration of CD20 mAb. (b,c) Weights of mice (b) and percentage depletion of IgG and IgM antibody in serum (c) 28 d after CD20-specific mAb (CD20 mAb) treatment (representative of two experiments, n = 5). (df) Fasting glucose (*P = 0.06) (d), GTT (*P < 0.05) (e) and fasting insulin (*P = 0.04) (f) in HFD WT mice 28 d after receiving either CD20 mAb or control (IgG2c or PBS) (representative of two experiments, n = 5). (g,h) IFN- (*P = 0.003) and TNF- (*P = 0.005) production from SVC cultures of VAT isolated from 17-week-old mice treated with CD20-specific mAb at 13 weeks of age (two experiments, eight mice). (i) Percentage of VAT macrophages expressing TNF- 4 weeks after treatment with CD20 mAb (*P = 0.01, two experiments, eight mice). Error bars show means s.e.m.

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fasting glucose (Fig. 5d), glucose tolerance (Fig. 5e) and fasting insulin (Fig. 5f). Improved glucose tolerance persisted for more than 40 d and diminished with the return of B cells (Supplementary Fig. 6a). Consistent with a role for B cells in altering the local VAT cytokine milieu, VAT SVCs of treated mice showed reduced concentrations of the key inflammatory mediators IFN- (Fig. 5g) and TNF- (Fig. 5h); the latter change was attributable, at least partly, to reduced production by macrophages (Fig. 5i). Notably, IgG from CD20 mAbtreated mice was unable to transfer metabolic disease (Supplementary Fig. 6b), suggesting that CD20-specific mAb treatment may lead to alterations in IgG function in addition to its other effects on B cells29. DISCUSSION We discovered a fundamental role for B cells in the pathogenesis of obesity-associated insulin resistance. Previous studies have identified other immune cells as metabolic controllers with pathogenic potential in obesity911. In healthy nonobese individuals, VAT-resident regulatory T cells and TH2 cells have a beneficial effect by reducing VAT inflammation. During DIO, these cells are overwhelmed by proinflammatory CD8+ and TH1 cells, which promote insulin resistance and glucose intolerance11. B lymphocytes can now be added to the list of immune cells participating in this process, in which they activate CD8+ and TH1 cells and release pathogenic antibodies. Consistent with a previous report15, we show that B cell accumulation in VAT occurs early (by 4 weeks) after initiation of HFD. B cells worsen glucose tolerance, in part, by inducing MHC-dependent pro-inflammatory cytokine production by both CD4+ and CD8+ T cells. Similar mechanisms occur in models of infection, cancer30 and autoimmunity28. As B and T cells are recruited early to VAT in response to HFD10,15, the data support a role for B cells in modulating T cell function in DIO VAT. Alternatively, T cells may function by inducing IgG class switching in B cells. Indeed, we observed elevated concentrations of proinflammatory IgG2c in serum and VAT of DIO mice. Class switching could also be influenced by lipids in the HFD VAT environment acting directly through TLRs on B cells. It is also possible that cytokines or antibodies produced by B cells can directly interact with and affect insulin sensitivity in adipocytes. B cells also exacerbate metabolic disease through production of IgG. Although autoantibodies have not been previously recognized as having a crucial role in type 2 diabetes, an estimated 10% of people with type 2 diabetes have antibodies to islet cell antigens, and these antibodies are correlated with the need for insulin therapy31. Almost one-third of people with advanced type 2 diabetes have autoantibodies that inhibit endothelial cell function32. The presence of antibodies to glial fibrillary acidic protein (GFAP), which predicts insulin resistance as shown by our array (Table 1), is also reported at higher rates in type 2 diabetes33. We show that transfer of IgG from DIO WT mice to DIO Bnull mice induces rapid local and systemic changes in inflammatory cytokine production, and that it changes VAT macrophages to a proinflammatory M1 phenotype. These effects required exposure to HFD, suggesting that factors related to diet, possibly including diet-induced conditioning or induction of target autoantigens, are required for antibodies to exert their effects on glucose metabolism. Furthermore, we show that HFD IgG antibodies induce insulin resistance through an Fc-mediated process. As DIO VAT is a site of increased apoptotic and necrotic load, and because antibodies concentrate in regions of VAT CLSs, it is conceivable that interactions between antibodies and FcRs on macrophages occur in VAT and promote clearance of apoptotic and necrotic debris and inflammation34,35. Identification of the precise FcR responsible for IgG effects on glucose metabolism warrants further investigation. Antibodies also fix complement; recently, the complement protein C3a and its receptor C3aR on macrophages were identified as key mediators of insulin resistance36. We further show that insulin resistance in obese humans is linked to autoantibodies directed against a specific profile of self antigens. Antibodies to one of the top three antigens linked to insulin resistance, GFAP, occur in approximately 30% of people with type 2 diabetes33. Notably, we detected antibodies to Golgi SNAP receptor complex member-1 (GOSR1) in more than 70% of obese insulin resistant males. GOSR1 is an essential component of the Golgi SNAP receptor complex, where it functions in trafficking proteins between the endoplasmic reticulum and the Golgi37. It is unknown whether expression of this protein changes in response to endoplasmic reticulum stress, which is thought to be a prominent initiator of insulin resistance38. Our array data also show distinct antigenic targets associated with insulin sensitivity, thereby raising the possibility that some IgG antibodies may be protective. It will be crucial to validate antigenic targets in larger cohorts. Finally, we show that depletion of B cells with a CD20-targeting mAb early in disease has therapeutic benefit in abnormal glucose metabolism. These results are consistent with a role for B cells early in disease pathogenesis, similar to observations in several autoimmune diseases39. Recently, CD20-specific mAb was used in the treatment of atherosclerotic lesions in apoliprotein Edeficient (Apoe/) and low-density lipoprotein receptordeficient (Ldlr/) mice40. In CD20specific mAb experiments, beneficial anti-inflammatory effects are linked to reduced T cell activation. Accordingly, we observed reduced concentrations of proinflammatory IFN- and TNF- in VAT after CD20-specific mAb treatment. Consistent with other reports, total serum levels of IgM and IgG after CD20-specific mAb treatment were not drastically changed despite a prominent therapeutic benefit41. One possible explanation for this finding is that in our studies, CD20-specific mAb was administered by 67 weeks after HFD, just a few weeks after B cells substantially infiltrate VAT. Because HFD IgG became more pathogenic with longer exposure to HFD, we hypothesized that the antibodies present after early treatment were not fully pathogenic. This was verified by the inability of antibodies from mice treated with CD20specific mAb to transfer metabolic disease. The lack of pathogenicity of HFD IgG from CD20-specific mAbtreated mice at this time point could be a result of reduced affinity maturation, reduced class switching or both41. Rituximab, a mAb specific for human CD20 and used in the treatment of rheumatoid arthritis as well as in B cell malignancies, can cause both hyperglycemia and severe hypoglycemia42. Other B cell and antibody-modulating agents are either approved for human use or in clinical trials, including intravenous immunoglobulin (IvIg), transmembrane activator and calcium modulator and cyclophilin ligand (TACI) fusion proteins, and antibodies or small-molecule inhibitors to CD19, CD22, CD79a and b, B lymphocyte stimulator (BLyS), spleen tyrosine kinase (Syk) and a proliferation-inducing ligand (APRIL). Our findings suggest new possible uses for such agents, and agents that modulate FcR function and signaling, in the management of obesity-related abnormalities in glucose metabolism. Collectively, our data support a model wherein early recruitment of B cells promotes VAT T cell activation and proinflammatory cytokine production, which potentiate M1 macrophage polarization and insulin resistance. B cells can also exert their detrimental effects systemically through the production of pathogenic IgG antibodies, which target distinct clusters of self proteins. Comparative mass sequencing of T and B cell antigen receptors in obesity-related insulin resistance

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is under way and promises to yield additional insights into the fundamental cause of this pervasive disease. METHODS Methods and any associated references are available in the online version of the paper at http://www.nature.com/naturemedicine/.
Note: Supplementary information is available on the Nature Medicine website. ACKNoWLEDGMENTS We thank D. Jones for secretarial assistance; C. Benike for critical review of the manuscript; A. Chawla for critical review of the figures; C. Wang, L. Tolentino and K. Heydari for assistance with flow cytometry; and Y. Yang and L. Herzenberg for help in developing macrophage and B cell subset gates. These studies were supported by US National Institutes of Health grants CA141468 and DK082537 (E.G.E) and Canadian Institutes of Health Research Grant 111156 (H.M.D.). AUTHoR CoNTRIBUTIoNS D.A.W. and S.W. conceived the study, did experimental work and wrote the manuscript. L.S. was involved in experimental work, project planning and manuscript preparation. P.P.W., A.G., T.M. and D.B.M. contributed the human array data. J.Y., G.P., M.G.D., M.N.A., H.T., P.W., H.X.L., J.A.K. and M.C. did experimental work; T.F.T. contributed the CD20-specific mAb and was involved in manuscript preparation. H.M.D. supervised parts of the project and was involved in manuscript preparation; E.G.E. was involved in project planning, financing, supervision, data analysis and manuscript preparation. E.G.E. and H.M.D. are both senior authors. CoMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.
Published online at http://www.nature.com/naturemedicine/. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html.
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Mice. We purchased C57BL/6 (B6), Bnull B6 (2288, B6.129S2-Igh-6tm1Cgn/J), MHC-Inull (2087, B6.129P2-B2mtm1Unc), MHC-IInull (3239, B6.129S2C2tatm1Ccum/J), and RAG-1null (2216, B6.129S7-Rag1tm1Mom/J) mice from Jackson Laboratory and maintained them in a pathogen-free, temperaturecontrolled environment on a 12-h light and dark cycle. The mice received either NCD (LabDiet, 15 kcal% fat) or HFD (Research Diets, 60 kcal% fat) beginning at 6 weeks of age. Mice fed an HFD for at least 8 weeks were obese and considered DIO mice. All mice used in comparative studies were males and were age-matched between groups within individual experiments. Studies used protocols approved by the Institutional Animal Care and Use Committee of Stanford University and the Hospital for Sick Children. B cell purification. We mechanically dissociated spleens on 70-m nylon cell strainers; this was followed by negative selection with a mouse B cell enrichment kit (Stemcell Technologies) (see Supplementary Methods). Metabolic studies. We measured GTTs, ITTs, serum insulin and fat cell diameter as previously described11.

F(ab)2 fragments with a Pierce F(ab)2 Preparation Kit (Thermo Scientific) according to the manufacturers instructions (Supplementary Methods). Purification and transfer of IgM. We purified IgM from mouse serum with an IgM Purification Kit from Pierce Biotechnology (see Supplementary Methods). In vitro effects of HFD IgG. We positively selected monocytes and/or macrophages by CD11b microbeads (Miltenyi) from total VAT cells and then plated them at 1 105 cells per well in 96-well ELISA plates coated with HFD IgG (50 g ml1). We collected the supernatant after 24 h. B cell depletion with CD20 mAb. Mouse CD20-specific mAb (MB20-11, IgG2c isotype) was provided by T.F. Tedder (Duke University Medical Center). We suspended sterile CD20-specific mAb and isotype control (Southern Biotech) at 100 g in 100 l PBS and administered it to HFD-fed mice i.v. via retro-orbital injection. No marked differences were identified between isotype-treated and PBS-treated mice, and these mice were pooled in the control population. Histology and immunohistochemistry. We fixed and stained VAT as previously described11. IgG (Vector) and IgM (Sigma) stains were done according to the manufacturers protocol. Western blotting. We ran 1 g of recombinant purified human GOSR1, BTK (Novus) and human purified GFAP (US Biological) on an SDS-PAGE gel and transferred it onto a polyvinylidene fluoride membrane. We probed the blots with human subject serum samples (1 in 10 for GOSR1 and BTK, 1 in 100 for GFAP) or commercial GOSR1-specific (1 in 160), BTK-specific (1 in 500), (Abcam) and GFAP-specific (1 in 500, BioLegend) antibodies as positive controls. Proteins were detected using a chemiluminescence kit (Invitrogen). Human subjects. We obtained sera from 32 age- and BMI-matched overweight to obese male subjects (Supplementary Methods). Serum samples were obtained with the approval of the Stanford Internal Review Board for Human Subjects and with informed consent. Human antibody array. We used ProtoArrays Version 5.0 (Invitrogen) with 1:500 diluted human sera run according to the manufacturers instructions (see Supplementary Methods). Statistical analyses. Statistical significance between two means was determined by unpaired t tests. Statistics comparing proportions of race in human subjects were determined by Fishers exact test. Statistics for the human array are described separately (Supplementary Methods). In figure legends involving multiple experiments from pooled animal tissue, the number of experiments is listed, followed by the total number of pooled mouse samples. All data are presented as means s.e.m. Statistical significance was two-tailed and set at <0.05. Additional methods. Detailed methodology is described in the Supplementary Methods.

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Isolation of VAT-associated immune cells and VAT lysates. We isolated VATassociated immune cells as previously described11. We cultured 2.5 105 to 3.5 105 VAT-derived SVCs for 1216 h in a 96-well, round-bottom plate in RPMI medium (Lonza) supplemented with 10% (vol/vol) FCS (Omega Scientific) for cytokine measurements. We prepared VAT lysates by homogenizing VAT tissue in radioimmunoprecipitation lysis buffer (Santa Cruz Technologies) and then incubating the homogenized tissue on ice for 20 min. We centrifuged the lysates at 15,000g for 10 min at 4 C before protein quantification with a BSA protein quantification kit (Thermo Scientific). Antibody ELISA and ELISpot. We measured IgA; IgE; IgM; and IgG subclasses IgG1, IgG2b, IgG2c and IgG3 in serum or VAT lysate by ELISA, using kits from Bethyl Laboratories. The frequency of spontaneous IgM- and IgG-producing B cells in spleen was determined with mouse IgG or IgM ELISpotPLUS Kits (Mabtech). See Supplementary Methods. Cytokine measurement. We measured serum cytokines by Luminex bead assay and cytokines in the supernatants of SVC VAT cultures by ELISA according to the manufacturers instructions (eBioscience). We did intracellular cytokine staining as described11 with antibodies listed in the Supplementary Methods. We acquired data on an LSR II flow cytometer (BD Biosciences) and analyzed it with FlowJo software (Tree Star). Flow cytometry. We stained splenocytes or fat-associated cells for 30 min with commercial antibodies (see Supplementary Methods). We gated B cell subsets as B1a: CD19+IgM+IgDB220/lo CD5+; B1b: CD19+IgM+IgDB220/lo CD5; B2/non-B1: all other CD19+ cells. Splenic MZ B cells were gated as CD19+IgM+IgDCD21+CD23, whereas IgM+IgDFC cells were defined as CD19+IgM+IgDCD23+CD21. Purification and transfer of IgG. We purified IgG from mouse serum with a Melon Gel IgG Spin Purification Kit (Pierce Biotechnology) and generated

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doi:10.1038/nm.2353