B Cell−Mediated Presentation through MHC Class II Is Dispensable for Atherosclerosis Progression

Jesse W. Williams, Andrew Elvington, Skyler Kessler, Mary Wohltmann, Gregory F. Wu and

Gwendalyn J. Randolph Downloaded from

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Adaptive Immunity

B Cell–Mediated through MHC Class II Is Dispensable for Atherosclerosis Progression

Jesse W. Williams,* Andrew Elvington,* Skyler Kessler,* Mary Wohltmann,* Gregory F. Wu,*,† and Gwendalyn J. Randolph* *Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110; and †Department of Neurology,

Washington University School of Medicine, St. Louis, MO 63110 Downloaded from

ABSTRACT

Depletion of B cells attenuates plaque development and modulates responses in mouse models of atherosclerosis, suggesting http://www.immunohorizons.org/ that Ag presentation by B cells may promote disease progression. Thus, we set out to determine the role of B cell–mediated MHC class II (MHC II) Ag presentation during atherosclerotic plaque development. We developed murine conditional MHC II deletion and expression systems under control of the B cell–restricted CD19 promoter in an experimental model of atherosclerosis. Mice lacking MHC II expression only on B cells exhibited systemic shifts in germinal center and marginal zone B cell populations, leading to a reduced Ab response compared with littermate control animals. However, all populations were present and normal cholesterol uptake was detected in the plasma following high-fat diet treatment. In a second model, in which conditional expression of MHC II is limited only to B cells, showed similar overall cellularity characteristics compared with mice with complete MHC II deficiency. High- fat diet feeding showed no major changes in atherosclerotic plaque size or plaque cellular content in either conditional deletion or conditional expression approaches, compared with control animals. By testing the necessity and sufficiency of MHC II on B cells in the progression of atherosclerosis, we determine that MHC II on B cells does not directly regulate lesion development in murine by guest on October 2, 2021 models. ImmunoHorizons, 2019, 3: 37–44.

INTRODUCTION lesion, the role of the adaptive in disease progression has become increasingly recognized (3). B cell Atherosclerotic plaques are driven by a subintimal influx of deficiency was tested in bone marrow (BM) transplant models of (potentially modified) cholesterol particles that are subsequently atherosclerosis, showing accelerated lesion development (4). taken up by local , leading to the formation of foamy However, B cell depletion using anti-CD20 Ab showed the cells that constitute the majority of total plaque cells. Of opposite results, suggesting a protective function for B cells in hematopoietic cells present, plaques predominantly consist of atherosclerosis progression (5). These seemingly opposing results innate immune cells, which are the first cells to be detected and are reconciled when anti-CD20 Ab depletion was suggested to most numerous, particularly the and macrophage- selectively remove atherosclerosis-promoting B2 B cells and have lineage cells (1, 2). However, despite modest cell numbers in the incomplete depletion of protective IgM-producing B cell subsets.

Received for publication February 20, 2018. Accepted for publication January 7, 2019. Address correspondence and reprint requests to: Prof. Gwendalyn J. Randolph, Department of Pathology and Immunology, Washington University School of Medicine, 425 S. Euclid Avenue, Box 8118, St. Louis, MO 63110. E-mail address: [email protected] ORCIDs: 0000-0003-3992-4908 (S.K.); 0000-0003-3588-0637 (G.F.W.). J.W.W. and G.J.R. wrote and edited the manuscript. J.W.W., G.F.W., and G.J.R. conceived and supervised the project. J.W.W., A.E., S.K., and M.W. performed and analyzed experiments. This work was supported by National Institutes of Health (NIH) R01 HL118206, R37 AI049653, and DP1DK109668 (to G.J.R.). J.W.W. was supported by NIH Training Grant 2T32DK007120-41, American Heart Association Grant 17POST33410473, and NIH K99 HL138163. A.E. was supported by NIH Training Grant T32-HL07081-38. G.F.W. was supported by Grant R01NS083678 from the National Institute of Neurological Disorders and Stroke. Abbreviations used in this article: ATLO, artery-associated tertiary lymphoid organ; BM, bone marrow; DC, ; GC, germinal center; HFD, high-fat diet; MHC II, MHC class II; MZ, marginal zone; ORO, Oil Red O; oxLDL, oxidized low-density lipoprotein; Tfh, T follicular helper. The online version of this article contains supplemental material. This article is distributed under the terms of the CC BY 4.0 Unported license. Copyright © 2019 The Authors https://doi.org/10.4049/immunohorizons.1800015 37

ImmunoHorizons is published by The American Association of Immunologists, Inc. 38 B CELL MHC II ROLE IN ATHEROSCLEROSIS ImmunoHorizons

The observed B cell–dependent shift in disease kinetics has to promote plaque growth compared with control MHC II– 2 2 typically been attributed to the Ab-producing function of B cells, deficientmicewhenontheLdlr / background. Together, where IgG-producing cells are generally considered proinflam- these data argue that B cells are not instrumental in promot- matory and IgM-producing cells regarded as atherosclerosis- ing atherosclerotic pathology through MHC II–mediated protective (3, 6, 7). Recent reviews have highlighted the differential presentation. roles of Ab production and have expanded on the potential contributions of individual B cell subsets to atherosclerosis (8–11). Importantly, specific depletion of the B2 subset of B cells, using MATERIALS AND METHODS the BAFF-R–deficient mouse, in atherosclerotic models results in a systemic reduction in CD4+ T cell activation (12). This phenotype Mice was observed even in the presence of normal dendritic cell (DC) Mice were housed in specific pathogen-free animal facilities, activation and profiling, suggesting that changes in T cell activa- maintained by Washington University School of Medicine. Six- tion during atherosclerosis are a direct effect of B2 cell deficiency. week-old animals were used for all experiments and either given These results may suggest that one potential mechanism of B2 HFDfor8or20wkorreceivedadonorthymuswith8wkrecovery cre

cells is to promote T cell activation through Ag presentation. with subsequent HFD treatment for 8 wk. Strains CD19 Downloaded from Therefore, we sought to address whether B cells mediate MHC [no. 006785, B6.129P2(C)-Cd19tm1(cre)Cgn/J; Jackson Laboratory], fl/fl b f/f 2/2 class II (MHC II) might regulate plaque development in models MHC II , IAb stop ,andLdlr (no. 002207, B6.129S7- of atherosclerosis. Ldlrtm1Her/J; Jackson Laboratory) mice were housed and main- Identifying the cells responsible for Ag presentation to CD4+ tainedat Washington University Schoolof Medicine. HFD (Envigo T cells, and determining the locations where cognate interactions Teklad TD.88137) containing 21% milk fat and 0.15% cholesterol occur, has been a major interest to the cardiovascular field. Yet was used for atherosclerosis studies. All experimental procedures http://www.immunohorizons.org/ few studies have successfully assessed individual cell subset con- were approved by the Washington University School of Medicine tributions in models of atherosclerosis. Sage et al. (13) found Institutional Animal Care and Use Committee. plasmacytoid DCs, a rare lymphoid-derived DC subset, are nec- essary for the promotion of atherosclerotic plaques following Flow cytometry an 8-wk high-fat diet (HFD) regiment. Subsequent studies have Blood samples were collected from the superficial temporal vein. suggested plasmacytoid DCs may have specificprotectivefunc- Spleens were mechanically separated and digested in 1 mg/ml tions in eliciting the T regulatory cell response (14). However, Collagenase II for 45 min at 37°C. For both blood and spleen further analysis will be needed to reconcile these conflicting samples, RBCs were lysed (Pharm Lyse, BD Biosciences) prior to results. Using a mixed BM chimera approach into Ldlr2/2 counting (Cellometer X4, Nexcelom Bioscience) and pro- recipient mice, a recent study suggested that MHC II expression cessed for flow cytometeric staining. Samples were FcR-blocked by guest on October 2, 2021 by B cells was necessary for development of atherosclerosis (15). (2.4G2; BioLegend), then stained on ice with specific Abs; CD45 Surprisingly, other cell types typically considered professional Pacific Blue conjugated (30-Fl1; BioLegend), CD19 PE conjugated APC subsets, such as conventional DC and macrophage have not (eBio1D3; eBioscience), B220 PE conjugated (RA3-6B2; eBio- been individually assessed for their contributions to Ag pre- science), MHC II allophycocyanin/Cy7 conjugated (I-A/I-E) sentation in atherosclerosis. (M5/114; eBioscience), GL7 allophycocyanin conjugated (GL7; It is established that B cells can use the BCR to recognize native BioLegend), CD4 allophycocyanin conjugated (CK1.5; eBio- or modified Ags, allowing for processing and presentation to science), CD8a PerCP conjugated (53-6.7; BD Pharmingen), T cells (16). Ags like oxidized low-density lipoprotein (oxLDL) CD23 Fitc conjugated (B3B4; BioLegend), CD21 PEcy7 conjugated have been demonstrated as targets of Abs produced by B cells. (7E9; BioLegend), TCRb Fitc conjugated (H57-597; eBioscience), Additionally, B cells were found to even endocytose and present PD1 PE conjugated (29F.1A12; BioLegend), CXCR5 PerCPcy5.5 transferred Ag on MHC II in regions that are relevant to disease conjugated (L138D7; BioLegend), CD44 allophycocyanin/Cy7 progression (17). B cell–mediated Ag presentation is functionally conjugated (IM7; BioLegend), and CD62L PEcy7 conjugated relevant in models of in mice (18–20). Specifically, (MEL-14; BD Biosciences). Samples were run on BD LSRFortessa marginal zone (MZ) B cells, but not the more common follicular instrument and analyzed using FlowJo 10 (Tree Star). B cell B cell, has been found to be potentially important players in Ag subsets were identified as follicular (CD19+ CD212 CD23+), presentation (21). Thus, it is quite plausible that B cells may indeed MZ (CD19+ CD21+ CD232), or germinal center (GC) (CD19+ play pivotal roles in promoting the adaptive T cell response in the GL7+). pathogenesis of atherosclerosis. Using conditional deletion or expression systems in vivo, we address in this study whether Ag Plaque analysis presentation by B cells on MHC II is a primary mechanism in the Hearts were collected and immediately fixed in 4% paraformal- promotion of atherosclerosis. Interestingly, Ldlr2/2 mice in which dehyde containing 30% sucrose. Hearts were then embedded in MHCIIisspecifically deleted on B cells showed no significant OCT (no. 4583; Tissue-Tek), and cryosections (10 mm) were changes in plaque development following HFD feeding. Further- prepared through the aortic sinus. Samples were stained with Oil more, the expression of MHC II on B cells alone was not sufficient Red O (ORO; no. 01516; Sigma-Aldrich) to determine lipid

https://doi.org/10.4049/immunohorizons.1800015 ImmunoHorizons B CELL MHC II ROLE IN ATHEROSCLEROSIS 39 deposition. For sinus immunostaining for macrophage and smooth conditional deletion system by crossing the CD19cre/+MHC IIf/f muscle area, sections were blocked with 5% donkey serum and mouse (18) to an atherosclerotic mouse model on the Ldlr2/2 stained with rat anti-MOMA2 (MOMA-2; Bio-Rad) and mouse background. Efficient and constitutive MHC II deletion was anti-SMA (1A4; Thermo Fisher Scientific). Sections were washed detected by flow cytometry of splenic B cells (Fig. 1A). Importantly, and incubated with secondary Abs conjugated to Cy3 (catalog no. other CD19-negative APCs, including DCs and macrophages, 712-165-153; Jackson ImmunoResearch) or Cy2 (catalog no. maintained normal levels of MHC II expression (Fig. 1A, 715-545-150; Jackson ImmunoResearch) raised in donkey against Supplemental Fig. 1A, 1B) (18). The spleens of CD19cre/+ MHC IIf/f the appropriate species. Sections were images on a Leica SPE Ldlr2/2 or control (MHC IIf/f Ldlr2/2) mice on an HFD were microscope. Plaque area and content were measured using examined for their ability to generate B cell populations. Although Image J analysis software. For en face analysis, aortae were fixed thetotalBcellpoolandfollicularBcellswerenormalinnumber, with 4% paraformaldehyde, adipose tissue was carefully removed, GC B cells (CD19+ GL7+)andMZBcells(CD19+ CD21+ CD23+) and the arteries were pinned onto wax dishes. Aortae were were reduced when MHC II was deleted on B cells (Fig. 1B). CD19 pretreated with 100% propylene glycol (no. P4347; Sigma-Aldrich) has been previously established as a regulator of MZ B cell for 15 min, then incubated with ORO (no. 01516; Sigma-Aldrich) development (22), consistent with our finding that partial deletion

for 3 h at room temperature. Aortae were washed with 85% pro- of CD19 led to a 50% reduction in MZ cells numbers (Fig. 1B). Total Downloaded from pylene glycol and then washed multiple times in PBS. Aortae were ablation of MZ B cells was previously associated with loss of T imaged on a Zeiss dissecting microscope, and plaque area follicular helper (Tfh) cells (23). However, we observed no shift in measurements were made using Image J analysis software. Tfh cells when MZ cells were only partially depleted (Supplemental Fig. 1C). Although MZ and GC B cells were reduced, those remaining ELISA in the spleen were localized in typical, anticipated architecture.

For total Ig titers, plasma levels were measured using Southern To test the role of MHC II on B cells in atherosclerosis http://www.immunohorizons.org/ Biotech Mouse Ig Panel (catalog no. 5300-01B), following progression, CD19cre/+MHC IIf/f Ldlr2/2 or control (MHC IIf/f manufacturers instruction. For Ag-specificoxLDLELISA,plates Ldlr2/2) mice were fed a cholesterol-rich HFD for 8 or 20 wk. (Nunc MaxiSorp) were coated with anti-oxLDL Ab (no. AB3230; The aortic root and aortic arch are the primary and best studied MilliporeSigma) 1:250 in carbonate buffer at room temp for 3 h, sites where plaques form in mice; therefore, we assessed plaque then washed with TBST. Plates were then incubated with copper- development in these locations by ORO staining to identify areas oxidized human low-density lipoprotein (no. L34357; Invitrogen) of neutral lipid deposition (Fig. 1C, 1D). Conditional deletion of diluted 1:100 in PBS for 2 h at room temp, then blocked with MHCIIonBcellsshowednosignificant changes in plaque size at 0.5% BSA and washed with TBST. Plasma samples were loaded either time point analyzed when compared with their littermate at 1:10 and 1:100 dilutions into wells and incubated overnight at controls (Fig. 1E), suggesting that B cells are notessential players in

4°C, then washed three times with TBST. Detection Ab (Alkaline MHC II–dependent Ag presentation during atherosclerosis. This by guest on October 2, 2021 phosphatase–conjugated anti-IgG1 or anti-IgM; SouthernBiotech) conclusion was further supported by the observation that there was incubated at room temp for 1 h, then washed with TBST and were no changes in CD4 T cell activation markers observed in finally rinsed in TBS. Substrate was added (catalog no. 5300-04B; the aorta-draining mediastinal lymph nodes (Fig. 2D). Cellular SouthernBiotech) and incubated at 37°C for ;30 min and read at compositions of plaques at 8 wk of HFD feeding, including foamy 405 nm on a Cytation 5 plate reader. macrophages and smooth muscle cells, were also similar between experimental groups (Fig. 1F, 1G). Cholesterol analysis Importantly, and consistent with plaque measurements, serum Mouse plasma was collected from whole blood with 5 mmol/L cholesterol levels were unaffected at 4 or 8 wk of HFD treatment EDTA to reduce blood clotting, then stored at 280°C. Total plasma between CD19cre/+MHC IIf/f Ldlr2/2 or control (MHC IIf/f Ldlr2/2) cholesterol levels were measured using Dako Cholesterol-E kit mice(Fig.2A).BymodulatingMHCIIexpressiononBcells,itwas (439-17501) following the manufacturer’sinstructions. expected there would be defects in the development of IgG and potentially reduced Ag-specificresponses(20).Weob- Statistical analysis served reduced levels of total IgG1 and IgG2c in the serum of The statistical significance of differences in mean values was CD19cre/+MHC IIf/f Ldlr2/2 mice compared with controls following analyzed by unpaired two-tailed Student t test, using Prism HFD feeding (Fig. 2B), whereas these mice showed similar titers for 2 2 software. The p values ,0.05 were considered statistically IgM and IgG2b. CD19cre/+MHC IIf/f Ldlr / mice were also found significant. Error bars show the SEM. to have reductions in atherosclerosis-relevant anti-oxLDL–specific Ag responses in IgM and IgG1 isotypes in the serum (Fig. 2C). These data confirm that deletion of MHC II on B cells leads to attenuation RESULTS of Ab responses in atherosclerosis. However, they also support a conclusion in which class-switch associated with the development Bcell–mediated Ag uptake and presentation is a potential of oxLDL-specific IgG1 responses is not a regulator of lesion mechanism for the initiation of adaptive T cell responses development when IgM affinity maturation to relevant Ags is in atherosclerosis. To test this hypothesis, we developed a likewise reduced.

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FIGURE 1. Conditional deletion of MHC II on B cells does not regulate atherosclerotic plaque development in Ldlr2/2 mice. (A) Spleens from naive CD19cre/+MHC IIf/f Ldlr2/2 or littermate control mice were assayed for B cell expression of MHC II by flow cytometry. (B) Spleen B cell subsets were identified in the spleens of chow fed CD19cre/+MHC IIf/f Ldlr2/2 mice to assess cellularity changes. Following 8 or 20 wk of HFD feeding, CD19cre/+MHC IIf/f Ldlr2/2 mice were assessed for plaque development in the aortic sinus (scale bar, 250 mm) (C) and aortic arch (original magnification 310) (D) by ORO staining. (E) Total plaque area and percentage of plaque staining area was quantified. (F) Representative image of an aortic sinus from 8 wk HFD-fed wild-type mouse stained for macrophage (MOMA2) and smooth muscle cell content (smooth muscle ; SMA). Scale bar, 100 mm. (G) Percentage of macrophage and SMA content was quantified. (A) Representative or (B) combined data from two independent experiments containing three to five mice per group. (C–E and G) Data are representative of two or three independent experiments containing five to nine animals per group each. *p # 0.05, **p # 0.01, by nonparametric Student t test.

Because other APCs (such as DC or macrophage) or B cells not background. Effective conditional expression of MHC II was efficiently deleting MHC II in our conditional deletion model may observed only on CD19+ cells when spleens were assessed by flow be able to compensate for the loss of MHC II expression on the cytometry (Fig. 3A). Expression levels of MHC II on the surface of majority of B cells, we next sought to determine whether B cell B cells were at levels comparable to those of control B cells subsets were able to drive atherosclerosis as independently as (Fig.3B).Asexpected,becauseofalackofMHCII–mediated se- +/+ b f/f APCs when all other cells were deficient for expression. To lection in the , both control cre-negative CD19 IAb stop conditionally express MHC II on only B cells in atherosclerosis, Ldlr2/2mice (completely MHC II deficient) and conditional expres- cre/+ b f/f 2/2 cre/+ b f/f 2/2 we bred the CD19 IAb stop mouse (19) to the Ldlr sing mice CD19 IAb stop Ldlr (only B cells expressing MHC

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FIGURE 2. MHC II expression on B cells is required for oxLDL-specific Ab responses in atherosclerosis. Conditional deletion of B Cell MHC II in a model of atherosclerosis. (A) Total cholesterol levels in plasma of CD19cre/+ MHC IIf/f Ldlr2/2 or control mice, following 0, 4, or 8 wk HFD feeding. (B) Total serum Ab titers were measured by specific ELISA (isotype as indicated) in control (MHC IIf/f Ldlr2/2) or CD19cre/+ MHC IIf/f Ldlr2/2 mice following 8 wk of HFD feeding. (C) oxLDL-specific IgM and IgG1 levels in the serum of CD19cre/+ MHC IIf/f Ldlr2/2 mice following 8 wk of HFD feeding. (D) Mediastinal lymph node CD4 T cells were analyzed for expression of activation markers CD44 and CD62L following 8 wk HFD feeding. (A–D) Data shown are n $ 5, repeated in two independent experiments. *p # 0.05, **p # 0.01, by nonparametric Student t test.

+ II) were largely devoid of circulating CD4 T cells (Fig. 3C, 3D). To differences in plaque development as measured by ORO staining by guest on October 2, 2021 study animals with a competent CD4+ T cell population, we (Fig. 3G–I) or cellularity (Supplemental Fig. 2D), indicating that performed thymic transplants. In this approach, young Ldlr2/2 exclusive expression of MHC II by B cells alone does not enhance pup thymi were transplanted into the axillary region of recipient atherosclerosis. Mice were also observed to have similar levels of mice and given 8 wk to reconstitute a T cell compartment in the serum Ab responses following HFD feeding (Supplemental transplant host (24). Following 8 wk, CD4+ Tcellpopulationswere Fig. 2E, 2F). Together, these data show that MHC II–mediated expanded 10-fold in the peripheral blood (Fig. 3C, 3E) and spleen Ag presentation by B cells is neither necessary nor sufficient for (Supplemental Fig. 2A) compared with nontransplanted controls, atherosclerotic disease progression. albeit at levels below that of MHC II–sufficient Ldlr2/2 mice. B cell populations in these animals were largely found to be in normal numbers and proportions. However, GC and MZ B cells were DISCUSSION partially reduced in CD19cre/+ mice similar to our conditional deletion model (Supplemental Fig. 2B), likely because of hetero- Despite adaptive immunity being implicated in murine models of zygosity of CD19 (22). atherosclerotic disease since the early 1990s, little is understood To determine whether expression of MHC II on B cells is in terms of where Ag presentation occurs or which cells are sufficient to promote atherosclerosis progression we directly responsible for cognate interactions with T cells. Targeting the b f/f 2/2 cre/+ b f/f 2/2 compared IAb stop Ldlr with CD19 IAb stop Ldlr for therapeutic intervention of high-risk mice following thymic transplantation and an additional patients has been gaining momentum and may be more feasible 8 wk HFD feeding regimen. Both strains of mice showed thanbroadlytargetingtheinnateimmunesystem(25). similarly elevated cholesterol levels following HFD treatment The role of MHC II on B cells was previously investigated using (Supplemental Fig. 2C) and no significant changes in CD4 T cell a mixed BM chimera approach. Specifically, (80%) B cell–deficient activation in the aorta-draining mediastinal lymph nodes (Fig. 3F). BM (mMT) was mixed with (20%) MHC II2/2 BM andtransferred Subsequent plaque analysis in the aortic sinus and aortic arch into irradiated Ldlr2/2 mice, creating a system in which all donor found that complete MHC II–deficient (control) or conditional BcellswereMHC II2/2, and the majority of other cells would cre/+ b f/f 2/2 expressing CD19 IAb stop Ldlr mice showed no significant presumably derive from 80% MHC II–sufficient precursors. In

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FIGURE 3. Conditional expression of MHC II on B cells does not regulation atherosclerotic plaque development in Ldlr2/2 mice. 2/2 b f/f 2/2 cre/+ b f/f 2/2 (A) Spleens from Ldlr , IAb stop Ldlr , and CD19 IAb stop Ldlr mice were assayed for B cell expression of MHC II. (B) Histogram of + + b f/f 2/2 cre/+ MHC II expression levels in CD19 spleen cells from indicated strains. (C) CD4 T cell counts for the blood of IAb stop Ldlr and CD19 b f/f 2/2 2/2 IAb stop Ldlr mice before or after thymic transplantation, compared with control Ldlr mouse. Representative flow plots showing T cell b f/f 2/2 cre/+ b f/f 2/2 populations (D) before or (E) following thymic transplantation and 8 wk recovery of IAb stop Ldlr and CD19 IAb stop Ldlr , and compared against control Ldlr2/2 mice. Following thymic transplantation, 8 wk reconstitution, and 8 wk HFD feeding, mice were assayed for CD4 T cell activation in the mediastinal lymph node (F), as well as staining for plaque deposition in the aortic sinus (scale bar, 250 mm) (G) and aortic arch (original magnification 310) (H), quantified in (I). Scale bar, 250 mm. (A)–(F) are representative or combined data from two independent experiments containing three to five mice per group. (F)–(H) contain five to six animals per group and are representative of three independent experiments. **p # 0.01, ***p # 0.001, by nonparametric Student t test. contrast to our findings, this approach reported a remarkable may be a confounding factor when interpreting the results. That is, reduction in overall plaque size and no change in the plaque it remains controversial whether loss of MHC II on B cells with content (15). In agreement, IgG against oxLDL (our study) or total DCs fully sufficient in MHC II would markedly alter T cell IgG was dramatically suppressed (15). However, they also activation systemically, even if effects on immunity to particular observed a reduction in activated CD4 T cells systemically, which AgsdoesrequireMHCIIonBcells(15,19,20,26–29). Both of our

https://doi.org/10.4049/immunohorizons.1800015 ImmunoHorizons B CELL MHC II ROLE IN ATHEROSCLEROSIS 43 loss- and gain-of-function approaches in our study showed control DISCLOSURES levels of CD4 T cell activation in draining lymph node and spleen (data not shown). Possible explanations for the divergent results The authors have no financial conflicts of interest. may relate to the impact of radiation on atherosclerosis (30) or that the BM chimera left the DC compartment with a substantial ACKNOWLEDGMENTS proportion of DCs also lacking MHC II. Indeed, data provided in + thestudyseemstosuggestthepresenceofmoreCD11c cells We thank the Washington University Flow Cytometry Core Facility for lacking MHC II than observed in control mice (15), although no technical assistance in experiments and members of the Randolph formal analysis of this issue was reported. However, we cannot Laboratory for help in preparation of this manuscript. eliminate the possibility that our study failed to delete MHC II as fully as Tay et al. (15). Nonetheless, we underscore our additional REFERENCES finding that MHC II expressed only in B cells did not drive plaque progression. 1. Randolph, G. J. 2014. Mechanisms that regulate macrophage burden – Our findings may rule out a role for B cells in Ag presentation in in atherosclerosis. Circ. Res. 114: 1757 1771.

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https://doi.org/10.4049/immunohorizons.1800015 44 B CELL MHC II ROLE IN ATHEROSCLEROSIS ImmunoHorizons

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