The American Journal of Pathology, Vol. 184, No. 11, November 2014

ajp.amjpathol.org

VASCULAR BIOLOGY, ATHEROSCLEROSIS, AND ENDOTHELIUM BIOLOGY

B2 Cells Suppress Experimental Abdominal Aortic Aneurysms Akshaya K. Meher,* William F. Johnston,* Guanyi Lu,* Nicolas H. Pope,* Castigliano M. Bhamidipati,y Daniel B. Harmon,z Gang Su,* Yunge Zhao,* Coleen A. McNamara,z Gilbert R. Upchurch, Jr,zx and Gorav Ailawadi*z From the Division of Thoracic and Cardiovascular Surgery,* Department of Surgery, and the Division of Vascular and Endovascular Surgery,x University of Virginia, Charlottesville, Virginia; the Department of Surgery,y State University of New York Upstate Medical University, Syracuse, New York; and the Robert M. Berne Cardiovascular Research Center,z Charlottesville, Virginia Accepted for publication July 10, 2014. Address correspondence to Gorav Ailawadi, M.D., Division of Thoracic and Cardiovascular Surgery, University of Virginia, P.O. Box 800679, Charlottesville, VA 22908. E-mail: gorav@ virginia.edu.

Recent reports of rupture in patients with abdominal aortic aneurysm (AAA) receiving B-cell depletion therapy highlight the importance of understanding the role of B cells (B1 and B2 subsets) in the development of AAA. We hypothesized that B2 cells aggravate experimental aneurysm formation. The IHC staining revealed infiltration of B cells in the aorta of wild-type (C57BL/6) mice at day 7 after elastase perfusion and persisted through day 21. Quantification of immune cell types using flow cytometry at day 14 showed significantly greater infiltration of mononuclear cells, including B cells (B2: 93% of total B cells) and T cells in elastase-perfused aortas compared with saline-perfused or normal aortas. muMT (mature B-cell deficient) mice were prone to AAA formation similar to wild-type mice in two different experimental AAA models. Contradicting our hypothesis, adoptive transfer of B2 cells suppressed AAA formation (102.0%  7.3% versus 75.2%  5.5%; P < 0.05) with concomitant increase in the splenic regulatory T cell (0.24%  0.03% versus 0.92%  0.23%; P < 0.05) and decrease in aortic infiltration of mononuclear cells. Our data suggest that B2 cells constitute the largest population of B cells in experimental AAA. Furthermore, B2 cells, in the absence of other B-cell subsets, increase splenic regulatory T-cell population and suppress AAA formation. (Am J Pathol 2014, 184: 3130e3141; http://dx.doi.org/10.1016/j.ajpath.2014.07.006)

Abdominal aortic aneurysms (AAAs) remain a life-threatening disease. Rupture of AAAs causes acute hemorrhage leading to shock and death. Moreover, recent reports indicate AAA rupture in patients receiving rituximab (a commercially available anti-CD20 antibody) for the treatment of various autoimmune diseases and B-cell lymphomas.1e3 With no available nonsurgical therapies to treat AAAs, there is a pressing need to understand the mechanisms of AAA growth and rupture. During AAA formation, aortic medial and adventitial layers undergo major pathological changes, such as degradation of extracellular matrix and marked infiltration of inflammatory cells, such as T cells, B cells, macrophages, mast cells, neutrophils, natural killer cells, and dendritic cells.4e10 Therefore, understanding the role of the inflammatory cells is crucial to develop a therapy to suppress aneurysm growth. B cells have long been known to be present in human AAA. By immunohistochemistry (IHC), an estimated 60% to 80% of the inflammatory infiltrate in the adventitia is classified as B cells (CD20þ). By using flow cytometry, nearly 20% to 41.1% Copyright ª 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2014.07.006

of total mononuclear hematopoietic cells (CD45þ) are considered B cells (CD19þ).4,8 Flow cytometry analysis of lymphocytes4 in human AAA wall identified activated memory B cells with homing properties and expressing activation markers. The presence of B-cellebound immunoglobulins, such as IgM and IgG, in human AAA tissue has also been well documented.4,7,8,11 B cells are known to form lymphoid follicles called vascular-associated lymphoid tissue12,13 with T cells, which are prominent in AAA. B cells are also detected in AAA of apolipoprotein Eedeficient mice after 14 days of angiotensin II infusion.14 Similar to AAA, atherosclerosis is characterized by a chronic inflammatory cell infiltrate, including B and T cells.15 Genetically B-celledeficient mice, muMT, develop atherosclerosis.16 Doran et al17 have reported adoptive transfer of total B-cell population to muMT mice protected mice against Supported by NIH grant 5K08HL98560 (G.A.). Disclosures: None declared.

Protective Role of B2 Cells in AAA atherosclerosis with significant decrease in macrophage content in aortas. Specific roles for B-cell subsets B1a, B1b, and B2 in the pathogenesis and progression of atherosclerosis have also been identified.16e26 Specifically, B1a cells appear to be protective in atherosclerosis via production of natural antibodies IgM, whereas B2 cells are proatherogenic via activation/proliferation of T cells. As far as role of T cells is concerned, CD4þ T cells are atherogenic,27 whereas T-regulatory cells (Tregs; CD4þCD25þFoxp3þ) are atheroprotective.28,29 Although the role of T cells in AAA formation has been widely investigated,30e35 the role of B cells or B-cell subsets in AAA remains elusive. We hypothesized that B cells were necessary for AAA formation, and the largest constituent of B cells in AAA would be B2 cells. We further hypothesized that adoptive transfer of B2 cells to a B-celledeficient mouse would exacerbate AAA formation. We have previously detected neutrophils, macrophages, and T cells in the murine aorta at day 3 using a well-established model of aortic elastase perfusion.36 Because B cells have never been fully characterized in experimental AAA, we first examined if B cells are present in murine AAA and, subsequently, developed a flow cytometry method to methodically phenotype and quantify B-cell subsets in AAA. To understand the role of B2 cells, we adoptively transferred isolated B2 cells to muMT mice and examined AAA formation. Our study addresses a putative protective role of B2 cells in experimental AAA.

Materials and Methods Tissue Collection from Humans Tissue collection from humans was approved by the University of Virginia (Charlottesville, VA) Institutional Review Board for Health Sciences Research (Institutional Review Board number 13178). AAA tissues from humans were collected from patients undergoing open AAA repair. The tissues collected were immediately rinsed with saline and stored at 10% formalin. The patient and tissue characteristics are given in Supplemental Table S1.

Mice C57BL/6 (stock number 000664) and muMT mice on C57BL/6 background (stock number 002288) were obtained from Jackson Laboratory (Bar Harbor, ME). Only male mice were used for induction of AAA or cell isolation. All protocols were approved by the University of Virginia Animal Care and Use Committee. All mice were given water and standard chow diet (catalog number 7012; Teklad Diets, Madison, WI) ad libitum.

elastase model of mouse AAA was performed as described by Bhamidipati et al,39 and aortas were harvested at day 14. Video micrometry measurements were performed before perfusion, after perfusion, and at the time of harvest. Changes in aortic diameter were calculated as percentage dilation over baseline at day 14.

Phenotyping and Quantification of Immune Cells in AAA Samples Dilated aortas were carefully collected from mice and digested with an enzymatic cocktail of 1000 U/mL collagenase type I, 400 U/mL collagenase type XI, 125 U/mL hyaluronidase type I-s, and 60 U/mL DNase.17 Subsequently, the samples were stained with fluorescent dyeeconjugated antibodies and run on the 16-color flow cytometer machine LSRFortessa (equipped with laser lines 488, 405, 561, and 640 nm; Becton Dickinson, Franklin Lakes, NJ) located in the Flow Cytometry Core Facility at University of Virginia. The flow antibodies used were obtained from BD Pharmingen (San Jose, CA) [phosphatidylethanolamine (PE) rat anti-mouse Foxp3, MF23] and Biolegend (San Diego, CA) (purified anti-mouse CD16/32, clone 93; PE/Cy7 anti-mouse CD3ε, clone 145-2C11; allophycocyanin anti-mouse CD4, GK1.5; Alexa Fluor 647 antimouse CD5, 53-7.3; PerCP/Cy5.5 anti-mouse CD19, 6D5; PE anti-mouse CD23, B3B4; allophycocyanin/Cy7 antimouse CD45, 30-F11; Alexa Fluor 488 anti-mouse/human CD45R/B220, RA3-6B2; and PE anti-mouse IgM, RMM1). Dead cells were eliminated from analysis by staining with DAPI (Sigma-Aldrich, St. Louis, MO) or LIVE/DEAD Fixable Dead Cell Stain Kit from Molecular Probes, Invitrogen (Eugene, OR). For Foxp3 staining, surface-stained cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% saponin. The flow data were analyzed using FlowJo software. To count the number of cells, CountBright Absolute Counting Beads (Molecular Probes, Grand Island, NY) were added to the cell suspension before flow cytometry. Fluorescent minus one controls were used for each antibody in the experiment. To quantify intracellular cytokines, isolated B cells were first stained with live/dead cell stain, then surface stained with PE/Cy7 anti-mouse CD19 (6D5; Biolegend), fixed, and permeabilized with saponin. Intracellular cytokines were stained with antibodies from eBiosciences (San Diego, CA) (antimouse IL-6 PE, MP5-20F3) and Biolegend [Alexa Fluor 700 anti-mouse IL-2, JES6-5H4; Alexa Flour 647 anti-mouse IL-4, 1B11; PerCP/Cy5.5 anti-mouse IL-10, JES5-16E3; and Alexa Fluor 488 anti-mouse interferon (IFN)-g, XMG1.2], and counting beads were added before running on LSRFortessa.

IHC Data Elastase Perfusion and Topical Model of Mouse AAA Elastase perfusion model of mouse AAA was performed using saline (control) or elastase, as previously described,37,38 and aortas were harvested at day 0, 3, 7, 14, or 21. A topical

The American Journal of Pathology

-

ajp.amjpathol.org

Standard immunoperoxidase or immunofluorescence staining methods were performed on paraffin sections (5 mm thick) of human or mouse AAA. In human sections, B cells were detected using anti-CD79a antibody (HM47/A9; Abcam,

3131

Meher et al

Figure 1

Presence of B and T cells in the aortic wall of human and mouse AAA. A: Left: Human AAA section stained with B cells (CD79a, brown). Right: A consecutive section stained for both B cells (CD79a, green) and T cells (CD3ε, red); image was acquired on a confocal microscope. B: Mouse aortic sections from day 0, 3, 7, 14, and 21 after elastase perfusion stained for B cells (B220, brown) (n Z 3). C: Mouse aortic section from 14 days after elastase perfusion stained for B cells (B220, green) and T cells (CD3ε, red); image was acquired on an epifluorescent microscope. The asterisk indicates lumen. Scale bars: 500 mm (A, left); 10 mm (A, right); 50 mm (B and C).

Cambridge, MA) and T cells using anti-CD3 antibody (SP7; Abcam). In mouse aortic sections, B cells were detected using rat anti-mouse CD45R (RA3-6B2; BD Pharmingen, San Jose, CA), T cells using anti-CD3 antibody (sc-1127; Santa Cruz Biotechnology, Inc., Dallas, TX), macrophages using antie Mac-2 antibody (Cedarlane, Burlington, NC), and neutrophils using rat anti-mouse Ly-6B.2 antibody (AbD Serotec, Kidlington, UK). Immunoperoxidase and epifluorescent images were acquired on a Nikon Eclipse TieU inverted microscope (Melville, NY) and NIS-Elements Br microscope imaging software. Confocal images were acquired on Zeiss LSM 700 (Peabody, MA) using 405-, 488-, and 633-nm lasers and ZEN 2012 lite software (Zeiss).

3132

Isolation of B2 Cell B2 cells were prepared from spleen of 8- to 10-week-old wild-type (WT) mice. Briefly, mice were euthanized by CO2 inhalation, followed by cervical dislocation. Spleens were collected, and a single-cell suspension was prepared by passing through a 70-mm mesh. Red blood cells were lysed and B2 cells were isolated using CD43 (ly-48) microbeads (130-049-801) from Miltenyi Biotec (Auburn, CA) using negative selection strategy as the recommended protocol. Soon after isolation, B2 cells were suspended in phosphate-buffered saline (PBS) and transferred to muMT mice.

ajp.amjpathol.org

-

The American Journal of Pathology

Protective Role of B2 Cells in AAA

Isolation and Culture of B Cells from Aorta AAA was induced in WT mice using an elastase perfusion method. For controls, aortas were perfused with saline. Day 14 after perfusion, aortas were harvested as described before. Two elastase- or saline-perfused aortas were pulled together and digested with the aorta-digesting enzymes. Thereafter, the cell suspension was washed with PBS and untouched pan B cells were isolated using pan B cell isolation kit (130-095-813; Miltenyi Biotec, San Diego, CA) using the recommended protocol. Immediately after purification, the cells were washed and suspended in B-cell medium [Iscove’s modified Dulbecco’s medium supplemented with 1 mmol/L sodium pyruvate, 2 mmol/L L-glutamine, 55 mmol/L b-mercaptoethanol, 0.1 mg/mL bovine serum albumin, and 1% antibiotic-antimycotic (Gibco, Grand Island, NY)] and incubated at 37 C. Two hours after incubation, the aortic B cells were cultured for a further 18 hours with 5 ng/ mL phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich), calcium ionophore A23187, or 1.25 mmol/L ionomycin (Sigma-Aldrich) and 10 mg/mL brefeldin A (Sigma-Aldrich) and intracellular cytokines, which were quantified on a flow cytometer, as described earlier in Materials and Methods.

was induced using elastase perfusion model after 7 days. Fourteen days after elastase perfusion, mice were harvested.

Statistical Analysis Means and SEMs were calculated using GraphPad Prism version 5 (La Jolla, CA). An unpaired nonparametric t-test was used to determine differences between two groups, whereas multiple groups were compared using a simple one-way analysis of variance. P < 0.05 was considered significant.

Results B and T Cells Colocalize in Human AAA We performed double-immunofluorescence staining to examine the abundance and localization of both B (antiCD79a) and T (anti-CD3ε) cells in human AAA wall (Figure 1A). Both B and T cells were abundant and contacted each other, forming lymphoid-like structures in the aortic media of AAA. VerhoeffeVan Gieson (VVG) and a-smooth muscle actin staining revealed absence of thrombus in AAA wall (Supplemental Figure S1, A and B).

B-Cell Transfer and Induction of AAA

B Cells in Experimental AAA

Seven-week-old muMT male littermates were transferred with 25 million of isolated B2 cells or PBS via tail vein, and AAA

To determine whether B cells are present in experimental models of mouse AAA, we induced AAA by elastase perfusion

Figure 2 A: Schematic shows a segment of dilated aorta harvested for phenotypic analysis of B cells. B: Representative flow plots and gating strategy to phenotype B cells from day 14 elastase-perfused aorta. C: Representative flow plots (already gated for lymphocytes, live and singlets) from day 14 salineperfused aorta. FSC, forward scatter; IVC, inferior vena cava; SSC, side scatter.

The American Journal of Pathology

-

ajp.amjpathol.org

3133

Meher et al in WT mice and harvested the aortas at days 0, 3, 7, 14, and 21. Staining for CD45R/B220, a marker for B cells, demonstrated appearance of B cells at day 7, which persisted at day 21 in the adventitial layer (Figure 1B and Supplemental Figure S1C). Similar to the human AAA samples, we observed B and T cells are present together at day 14 (Figure 1C). To avoid modelspecific effects, we used an alternate AAA model, which we recently described (using full-strength elastase placed adventitially on WT mice39), and observed similar accumulation of B cells at day 14 (data not shown). Altogether, our results demonstrate prevalence of B cells in experimental models of mouse AAA.

Characterization of B-Cell Subsets in Mouse AAA Next, we developed a unique method to perform flow cytometry on individual mouse AAAs to quantify B-cell subsets. Our optimized protocol for digestion allowed us to prepare a cell suspension from an approximately 5-mm segment of abdominal aorta (Figure 2A) from mouse. Unexpectedly, we observed that surface expression of CD23, a well-studied marker for B-cell phenotyping, was abolished (Supplemental Figure S2A) in our optimized protocol and in the protocol described by Butcher et al.40 Therefore, we followed the gating strategy, as described by Thomas et al,41 which uses the markers CD19 and B220 to determine the B1 and B2 cell populations. In our gating strategy (Figure 2B), lymphocytes were gated first, followed by live cells, singlets, CD45þCD3 mononuclear hematopoietic cells, and B cells (CD19þB220þ). CD19hiB220lo cells were considered B1 cells, whereas CD19loB220hi cells were considered B2 cells. Furthermore, B1-gated cells were phenotyped as B1a (CD19hiCD5hi) and B1b (CD19hiCD5lo) cells. All B cells were found to express IgM (data not shown). We further observed that our surgical procedure, which involved laparotomy of mouse to perform saline or elastase perfusion of abdominal aorta, led to a decrease in B1 cell population (from 46% to 13%) and an increase in B2 cell population (from 21% to 49%) of total hematopoietic cells in peritoneal fluid (Supplemental Figure S2B). However, the populations of B-cell subsets in spleen and blood were unaffected after surgery (data not shown).

B2 Cells Are the Largest Constituent of B Cells in Mouse AAA We used counting beads in flow cytometry to determine the number of immune cells in unperfused normal abdominal aorta, and saline- and elastase-perfused aortas of WT mice. Elastase perfusion led to AAA formation in WT mice (Figure 3). Few immune cells were detected in the normal aorta, whereas the numbers of mononuclear hematopoietic cells, T cells, B cells, and B-cell subsets were significantly higher in elastase-perfused aortas compared with the salineperfused aortas (Figure 3). In elastase-perfused aortas, 23% of mononuclear infiltrates were T cells, whereas 4.5% were

3134

Figure 3 Increased infiltration of immune cells after saline or elastase perfusion of abdominal aorta of WT mice. Increase in aorta diameter and quantification of the number of immune cells in day 14 saline (n Z 6) and elastase (n Z 11) perfused aortas, and equivalent length of unperfused abdominal aorta (n Z 6) from WT mice. Values are expressed as means  SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, respectively.

B cells. B-cell numbers were 3631  578 and 1476  524 in elastase- and saline-perfused aortas, respectively. B2 cells were the largest constituent of B cells, both in elastase (B1 versus B2, 7.2% versus 92.8% of total B cell) and saline (B1 versus B2, 3.7% versus 96.3% of total B cell) perfused aortas (Figures 2, B and C, and 3). Among the B1 cell subsets, B1b cell number was higher than B1a cell. Correlation analysis between the immune cell number aorta diameters (irrespective of elastase or saline perfusion) revealed that total hematopoietic cell, T cell, B1, B1a, and B1b cell counts strongly correlated with increasing aorta diameter (Supplemental Figure S3). However, total B and B2 cell counts poorly correlated with aortic diameter. Because B2 cells were the largest constituent of B cells, B2 cell number affected correlation of total B cell with aorta diameter. Altogether, our data showed that elastase

ajp.amjpathol.org

-

The American Journal of Pathology

Protective Role of B2 Cells in AAA perfusion in WT mice led to increased aortic infiltration of B cells compared with saline-perfused or normal aortas, and many B cells are B2 cells; however, it is undefined whether B2 cells are protective or harmful.

B Cells from Saline- and Elastase-Perfused Aortas Have a Similar Cytokine Secretion Profile Cytokine-producing effector B cells (Be) have been shown to regulate many inflammatory diseases, which are independent of antibodies produced by B cells.42 Harris et al43 divided Be cells into two subsets: Be1 cells that synthesize tumor necrosis factor-a, IFN-g, and IL-12, and Be2 cells that synthesize IL-2, IL-4, IL-6, and IL-10. To determine the Be cell types present in saline- and elastase-perfused aortas, we purified total B cells using magnetic-activated cell sorting (MACS) columns (Figure 4A) stimulated with a mixture of PMA and ionomycin, blocked cytokine secretion using brefeldin A, and examined intracellular synthesis of IFN-g, IL2, IL-4, IL-6, and IL-10 using flow cytometry. We found a large population of B cells synthesized IL-6 and IL-10, indicating predominance of Be2 cell type; however, no difference was observed between these populations of B cells isolated from saline- and elastase-perfused mice (Figure 4B). This suggests that the B cells infiltrated to the aorta have similar Be populations irrespective of saline or elastase perfusion. However, more B cells in elastase-perfused aorta compared with saline-perfused aorta suggests a higher level of Be cytokines in the aneurysms.

B-Cell Deficiency Cannot Protect Mice from Experimental AAA muMT mice are deficient in mature B1 and B2 cells. To understand the role of B cell in AAA formation, we induced AAA in muMT mice using the elastase perfusion model. To our surprise, we observed muMT mice developed AAA similar to the WT mice at day 14 (Figure 5A). Histochemical staining on aortic cross sections revealed increased collagen degradation (VVG) and abundance of T cells,

macrophages, and neutrophils (Figure 5A), which were also found in day 14 WT mice (Supplemental Figure S4). Our findings contradict a study by Zhou et al,44 who reported that muMT mice were protected from AAA by elastase perfusion. Therefore, to further confirm our findings, we studied the effect of B-cell deficiency using a second experimental AAA model. Specifically, we observed that both WT and muMT mice developed AAA at day 14 using a novel topical elastase model that we reported previously39 (Figure 5B). The IHC staining of aortic sections from muMT mice further demonstrated abundance of T cells, macrophages, and neutrophils (Figure 5B), similar to day 14 WT mice (Supplemental Figure S4). These results suggest that in the absence of B cells, other immune cells, such as neutrophils, macrophages, and T cells, potentially regulate aneurysm formation in muMT mice.

Adoptive Transfer of B2 Cells Suppresses AAA Growth and Increases Peripheral Treg Population in muMT Mice Because the muMT mice developed an aneurysm similar to WT mice, we speculated that B1 and B2 cells have opposing roles in AAA formation, as they do in atherosclerosis. Adoptive transfer of total spleen cells or splenic B cells has been shown to protect mice from development of atherosclerosis.26 Because B2 cells worsen experimental atherosclerosis, we hypothesized that adoptive transfer of B2 cells would worsen aneurysm formation in muMT mice. Therefore, we adoptively transferred 25 million MACS column purified (purity >98%) (Supplemental Figure S5) splenic B2 cells in PBS to muMT mice. As described in experimental atherosclerosis studies,19 control muMT mice were injected with an equal volume of PBS. Transfer of isolated B2 cells reconstituted 8.8  1.6  105 B2 cells in the spleen of muMT mice (Figure 6, A and B). This led to a 2.2-fold increase in T cell and a twofold increase in the number of mononuclear hematopoietic cells in spleen (Figure 6, C and D). Contrary to our hypothesis, the aneurysm size was significantly smaller in B2 celletransferred mice compared with PBS controls (75.2%  5.5% in B2 celletransferred

Figure 4

Cytokines synthesized by B cells. A: Elastase- or saline-perfused aortas were collected at day 14 and digested, and untouched total B cells were isolated using Miltenyi column. B: The isolated aortic B cells were cultured for 18 hours with 5 ng/mL PMA, 1.25 mmol/L ionomycin, and 10 mg/mL brefeldin A and then stained with live/dead dye and B cell surface marker (CD19). Subsequently, the cells were fixed, permeabilized, and stained for intracellular cytokines, which were quantified on a flow cytometer. B cells isolated from two aortas were pooled to make n Z 4. Values are expressed as means  SEM.

The American Journal of Pathology

-

ajp.amjpathol.org

3135

Meher et al

Figure 5

muMT mice develop AAA with increased elastin degradation and increased infiltration of immune cells to aorta. muMT mice develop AAA similar to WT mice in elastase perfusion (A; n Z 8 to 10) and topical elastase (B; n Z 6 to 8) model. AAA sections from muMT mice were stained for VVG (elastic fibers, black), T cells (CD3ε, brown), macrophages (Mac2, brown) and neutrophils (Gr1, brown). Values are expressed as means  SEM. There is no significant difference in AAA growth between WT/elastase and muMT/elastase animals in elastase perfusion or topical elastase model. ***P < 0.001. Scale bar Z 50 mm (A and B).

muMT versus 102.0%  7.3% in control muMT, P Z 0.01) (Figure 6E). In accordance with decreased AAA size, the number of mononuclear hematopoietic cells and T cells trended lower in the elastase-perfused aortas of B2 celle transferred mice (Supplemental Figure S6), although it did not reach to a level of statistical significance. This suggests adoptive transfer of B2 celleinduced generation or differentiation of a protective cell type. B2 cells have been shown to promote differentiation of naïve CD4 T cells to Tregs. Therefore, we determined Treg population (CD45þCD3εþCD4þFoxp3þ) in the

3136

spleen, draining lymph nodes, peritoneal fluid, and aneurysm tissues of these mice using flow cytometry. The B2 celletransferred muMT mice showed an increased percentage (% of total CD3εþ T cells), 0.24%  0.03% versus 0.92%  0.23% (P Z 0.02), as well as total number (P Z 0.01) of Tregs in spleen (Figure 6, F and G). The percentage of Tregs in infiltrated T cells trended to be increased in B2 celletransferred aorta (Supplemental Figure S6); however, we did not observe significant differences in Treg population in lymph nodes or peritoneal fluids. The observed increase in Treg population in the

ajp.amjpathol.org

-

The American Journal of Pathology

Protective Role of B2 Cells in AAA

Figure 6 Adoptive transfer of B2 cells suppressed AAA formation and increased peripheral Treg population in muMT mice. A: Representative flow plots show reconstitution of B2 cells in spleen of muMT mice transferred with 25 million WT B2 cells. Increase in absolute numbers of B2 (B), T (C), and mononuclear hematopoietic (D) cells in spleen of muMT mice (n Z 5) transferred with B2 cells. E: Increase in aorta diameter in PBS or B2 celletransferred mice (n Z 7 to 9). F: Representative flow plots show increased Treg (CD4þFoxp3þ) population in B2 celletransferred mice. G: Increase in Treg population in spleen of muMT mice (n Z 5) transferred with B2 cells. Values are expressed as means  SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, respectively.

muMT mice might be originated from the contaminating Treg in B2 cell preparation. Therefore, we examined our B2 cell preparation for the presence of Tregs. We did not find contaminating Tregs in isolated B2 cell preparations

The American Journal of Pathology

-

ajp.amjpathol.org

that were being transferred to the muMT mice (Supplemental Figure S5). These results altogether showed that adoptive transfer of isolated B2 cells to muMT mice suppressed aneurysm formation with a

3137

Meher et al concomitant increase in peripheral Tregs and a decrease in aortic infiltration of immune cells.

Discussion The role of B cells in AAA formation has been poorly understood. By using a mouse elastase perfusion-induced AAA, we first determined the location and phenotype of the B cells. We observed that B and T cells colocalized in human and mouse AAA and that the B2 cell population was significantly higher than B1 cells. By using two models of experimental AAA, we observed that the muMT mice are not protected from AAA and appear similar to the WT mice. Unexpectedly, adoptive transfer of B2 cells into muMT mice suppressed AAA and was associated with an increase in peripheral Treg cells. B cells derived from spleen regulate experimental atherosclerosis.19 In atherosclerosis, B2 cells are considered harmful via activation/proliferation of T cells. However, in vitro, B2 cells promote Treg generation from naïve CD4þ T cells in the presence of transforming growth factor (TGF)-b and IL-2.45 Moreover, resting B cells expand the Treg population via producing TGF-b3.46 Others have also shown that splenic B cells proliferate splenic Tregs in the presence of soluble antiCD3 and irradiated splenic antigen-presenting cells.47 Furthermore, B-cell depletion by anti-CD20 antibody led to encephalomyelitis with concomitant significant reduction in peripheral Tregs.47 In these studies, the B-celledepleted mice have a normal thymic Treg content,45e47 indicating a dominant role of B-cellemediated generation of peripheral Tregs in immunosuppression. Consistent with a decreased Treg population in B-cell depletion studies, muMT mice were found to have significantly fewer peripheral Tregs compared with WT mice. A B-cellemediated increase in Treg population was found to be independent of B-cell production of IL-10. Altogether, there are several lines of evidence that splenic B cells can increase Treg population either by differentiating naïve CD4 T cells to Tregs, expanding the existing population of Tregs, or recruiting Tregs to lymphoid organs. Moreover, these findings support our observation that adoptive transfer of B2 cells increases the splenic Treg population in muMT mice. Furthermore, TGF-b activity, which is required for Treg homeostasis, has been shown to protect WT mice from AAA, and neutralization of TGF-b using antieTGF-b antibody led to development of aortic aneurysm.48 Both atherosclerosis and AAA are aortic diseases, and may share common risk factors, such as smoking, hypertension, and obesity.49,50 It is not clear whether atherosclerosis leads to AAA formation or vice versa. In the Tromsø (Norway) study, lack of consistent dose-response relationship between atherosclerosis and AAA suggests atherosclerosis may develop concomitantly with AAA or secondary to AAA growth.50 Moreover, diabetes is positively associated with atherosclerosis, but negatively associated with AAA, which contradicts the consensus that AAA is a manifestation of atherosclerosis.51

3138

Tregs have been shown to play an important role in suppression of atherogenesis in mice.29 It has also been shown that depletion of Foxp3þ Tregs promotes atherosclerosis in mice.28 As far as human AAA is concerned, Yin et al52 found that the population of CD4þCD25þFoxp3þ Tregs was significantly lower in the peripheral blood of patients with AAA compared with patients with abdominal aortic atherosclerotic occlusive disease or healthy controls. Ait-Oufella et al53 reported that both genetic and anti-CD25 antibody-mediated depletion of Tregs augmented angiotensin IIeinduced aneurysm formation, and supplementing genetically Treg-deficient mice with WT Tregs suppressed aneurysm formation. Although anti-CD25 antibody treatment causes a small decrease in peripheral Treg population (not the natural Treg content of thymus), it significantly affects host immune response.54 Because B2 cells differentiate naïve CD4þ T cells to Tregs to a greater frequency than dendritic cells,45 a potential mechanism for B2-induced suppression of AAA in muMT mice would be Tregs differentiated or expanded in lymphoid organs, such as spleen, suppress inflammation systemically, leading to inhibition of AAA growth. Alternatively, B2 celleinduced newly differentiated Tregs are recruited to the site of AAA and prevent aneurysm growth. In fact, our studies demonstrate increased population of Tregs in the spleen of muMT mice after B2 cell transfer. We found the total B cell isolated from saline- or elastaseperfused aortas produced both proinflammatory (IFN-g and IL-6) and anti-inflammatory (IL-2, IL-4, and IL-10) cytokines. B1 cells are well known to produce natural IgM and protect mice from atherogenesis. In vitro, in contrast to the effect of B2 cells, B1 cells promote the differentiation of proinflammatory type 1 helper T cell (Th1) and Th17 from naïve CD4þ T cells with a greater frequency than dendritic cells.45 Although B1 cells are fewer than B2 cells, they have greater potency of differentiating Th17 than B2 cells differentiating Tregs. As far as regulation of T-cell phenotype is concerned, the effects of B1 and B2 may counteract each other via Th17 and Treg in WT mice. There are several limitations to our study. In most of the studies involving AAA patients, B cells constitute a large percentage of mononuclear cell infiltrate. However, in the elastase perfusion model, we found the percentage of hematopoietic cells that were B cells (CD45þCD3CD19þB220þ) was only 4.5%. A plausible explanation for this is most of the human AAA samples were in late stages of aneurysm growth, which might have taken years to develop. On the other hand, elastase perfusion is a rapid model and it takes approximately 14 days for aneurysm development. Nevertheless, this model has been documented to recapitulate human AAA development55,56 and widely used for experimental AAA studies. Furthermore, B-cell populations are identified by different markers and differ greatly between mice and humans.57,58 To understand the role of B cells in aneurysm formation, we performed elastase perfusioneinduced AAA in muMT mice and found that the muMT mice developed AAA similar to the WT mice. In accordance with this finding, a 62-year-old

ajp.amjpathol.org

-

The American Journal of Pathology

Protective Role of B2 Cells in AAA patient with a history of non-Hodgkin lymphoma receiving rituximab had ruptured AAA,2 whereas patients with immune cytopenia or spondyloarthropathies died after receiving rituximab treatment.1,3 In contrast to our findings, Zhau et al44 have reported that muMT mice were protected from elastase perfusioneinduced AAA and the absence of humoral immune response (IgG or IgM antibody) was attributed to the protection. Supplementing the muMT mice with total IgG antibodies or natural IgM antibodies induced AAA formation. To confirm our observation that muMT mice are susceptible to experimental AAA, we used a second model of AAA (topical elastase model developed in our laboratory)39 and still found no protection in AAA in muMT mice. Overall, formation of AAA in muMT mice emphasizes dominance of other immune cells, such as neutrophils, macrophages, and T cells in AAA in absence of B cells. Increasing B2 cells in the spleen of muMT mice via adoptive transfer can increase Treg population by differentiating CD4þ T cell, expanding the population of existing Tregs, or both. Further experimentation needs to be done to delineate the mechanism of B2 cellemediated increase in Treg population. We observed that adoptive transfer of B2 cells not only increased splenic B2 and Treg populations, but it also increased total hematopoietic and T-cell population. Among the T-cell population, Tregs comprised only 0.9%. Therefore, the presence of other splenic T-cell subpopulations, such as Th1, Th2, and Th17, in spleen of B2 celletransferred muMT mice needs to be determined. Furthermore, it is unknown how splenic Treg content regulates AAA formation. It is possible that adoptively transferred B2 cells may be synthesizing protective antibodies. Although the potent immunosuppressive effects of Tregs can be used in the treatment of multiple inflammatory diseases, the scarce availability and unspecific immune response generate major obstacles in using autologous Treg cells as a means of treatment of AAA. Thus, various methods of Treg generation have been tested, including dendritic celleinduced Treg differentiation.59 Because B2 cells modulate Treg differentiation from naïve CD4þ T cells at a greater frequency than dendritic cells, increasing B2 cell number may represent an alternative and effective approach to increase Treg number and prevent AAA growth. Altogether, we have shown that both B1 and B2 cells are present in mouse experimental AAA, with significantly more B2 than B1 cells. Absence of total B cells does not affect aneurysm formation; however, adoptive transfer of isolated B2 cells can suppress AAA progression, with an increase in peripheral Treg population. Our study has highlighted a possible protective role of B2 cells in vascular diseases.

Acknowledgments We thank Saeko Okutsu and Melissa H. Bevard for providing technical assistance in mouse surgery, and tissue

The American Journal of Pathology

-

ajp.amjpathol.org

processing and IHC, respectively; Tony Herring and Cindy Dodson for maintaining the mouse colonies; and Dr. Rahul Sharma for giving consultations on Treg.

Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2014.07.006.

References 1. Shanafelt TD, Madueme HL, Wolf RC, Tefferi A: Rituximab for immune cytopenia in adults: idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, and Evans syndrome. Mayo Clin Proc 2003, 78:1340e1346 2. Moulin-Romsee G, Mortelmans L: PET versus PET-CT in patient with suspicion of non-Hodgkin lymphoma recurrence. Clin Nucl Med 2007, 32:954e955 3. Nocturne G, Dougados M, Constantin A, Richez C, Sellam J, Simon A, Wendling D, Mariette X, Gottenberg JE: Rituximab in the spondyloarthropathies: data of eight patients followed up in the French Autoimmunity and Rituximab (AIR) registry. Ann Rheum Dis 2010, 69:471e472 4. Ocana E, Bohorquez JC, Perez-Requena J, Brieva JA, Rodriguez C: Characterisation of T and B lymphocytes infiltrating abdominal aortic aneurysms. Atherosclerosis 2003, 170:39e48 5. Rizas KD, Ippagunta N, Tilson MD 3rd: Immune cells and molecular mediators in the pathogenesis of the abdominal aortic aneurysm. Cardiol Rev 2009, 17:201e210 6. Forester ND, Cruickshank SM, Scott DJ, Carding SR: Increased natural killer cell activity in patients with an abdominal aortic aneurysm. Br J Surg 2006, 93:46e54 7. Bobryshev YV, Lord RS, Parsson H: Immunophenotypic analysis of the aortic aneurysm wall suggests that vascular dendritic cells are involved in immune responses. Cardiovasc Surg 1998, 6:240e249 8. Forester ND, Cruickshank SM, Scott DJ, Carding SR: Functional characterization of T cells in abdominal aortic aneurysms. Immunology 2005, 115:262e270 9. Eliason JL, Hannawa KK, Ailawadi G, Sinha I, Ford JW, Deogracias MP, Roelofs KJ, Woodrum DT, Ennis TL, Henke PK, Stanley JC, Thompson RW, Upchurch GR Jr: Neutrophil depletion inhibits experimental abdominal aortic aneurysm formation. Circulation 2005, 112:232e240 10. Moehle CW, Bhamidipati CM, Alexander MR, Mehta GS, Irvine JN, Salmon M, Upchurch GR Jr, Kron IL, Owens GK, Ailawadi G: Bone marrow-derived MCP1 required for experimental aortic aneurysm formation and smooth muscle phenotypic modulation. J Thorac Cardiovasc Surg 2011, 142:1567e1574 11. Pasquinelli G, Preda P, Gargiulo M, Vici M, Cenacchi G, Stella A, D’Addato M, Martinelli GN, Pileri S: An immunohistochemical study of inflammatory abdominal aortic aneurysms. J Submicrosc Cytol Pathol 1993, 25:103e112 12. Millonig G, Schwentner C, Mueller P, Mayerl C, Wick G: The vascular-associated lymphoid tissue: a new site of local immunity. Curr Opin Lipidol 2001, 12:547e553 13. Bobryshev YV, Lord RS: Vascular-associated lymphoid tissue (VALT) involvement in aortic aneurysm. Atherosclerosis 2001, 154: 15e21 14. Saraff K, Babamusta F, Cassis LA, Daugherty A: Aortic dissection precedes formation of aneurysms and atherosclerosis in angiotensin II-infused, apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol 2003, 23:1621e1626 15. Hansson GK, Hermansson A: The immune system in atherosclerosis. Nat Immunol 2011, 12:204e212

3139

Meher et al 16. Major AS, Fazio S, Linton MF: B-lymphocyte deficiency increases atherosclerosis in LDL receptor-null mice. Arterioscler Thromb Vasc Biol 2002, 22:1892e1898 17. Doran AC, Lipinski MJ, Oldham SN, Garmey JC, Campbell KA, Skaflen MD, Cutchins A, Lee DJ, Glover DK, Kelly KA, Galkina EV, Ley K, Witztum JL, Tsimikas S, Bender TP, McNamara CA: B-cell aortic homing and atheroprotection depend on Id3. Circ Res 2012, 110:e1ee12 18. Ait-Oufella H, Herbin O, Bouaziz JD, Binder CJ, Uyttenhove C, Laurans L, Taleb S, Van Vre E, Esposito B, Vilar J, Sirvent J, Van Snick J, Tedgui A, Tedder TF, Mallat Z: B cell depletion reduces the development of atherosclerosis in mice. J Exp Med 2010, 207: 1579e1587 19. Kyaw T, Tay C, Krishnamurthi S, Kanellakis P, Agrotis A, Tipping P, Bobik A, Toh BH: B1a B lymphocytes are atheroprotective by secreting natural IgM that increases IgM deposits and reduces necrotic cores in atherosclerotic lesions. Circ Res 2011, 109: 830e840 20. Sage AP, Tsiantoulas D, Baker L, Harrison J, Masters L, Murphy D, Loinard C, Binder CJ, Mallat Z: BAFF receptor deficiency reduces the development of atherosclerosis in miceebrief report. Arterioscler Thromb Vasc Biol 2012, 32:1573e1576 21. Lipinski MJ, Perry HM, Doran AC, Oldham SN, McNamara CA: Comment on “Conventional B2 B cell depletion ameliorates whereas its adoptive transfer aggravates atherosclerosis”. J Immunol 2011, 186:4. author reply 6 22. Kyaw T, Tay C, Khan A, Dumouchel V, Cao A, To K, Kehry M, Dunn R, Agrotis A, Tipping P, Bobik A, Toh BH: Conventional B2 B cell depletion ameliorates whereas its adoptive transfer aggravates atherosclerosis. J Immunol 2010, 185:4410e4419 23. Kyaw T, Tay C, Hosseini H, Kanellakis P, Gadowski T, MacKay F, Tipping P, Bobik A, Toh BH: Depletion of B2 but not B1a B cells in BAFF receptor-deficient ApoE mice attenuates atherosclerosis by potently ameliorating arterial inflammation. PLoS One 2012, 7: e29371 24. Zhou X, Hansson GK: Detection of B cells and proinflammatory cytokines in atherosclerotic plaques of hypercholesterolaemic apolipoprotein E knockout mice. Scand J Immunol 1999, 50:25e30 25. Lewis MJ, Malik TH, Ehrenstein MR, Boyle JJ, Botto M, Haskard DO: Immunoglobulin M is required for protection against atherosclerosis in low-density lipoprotein receptor-deficient mice. Circulation 2009, 120:417e426 26. Caligiuri G, Nicoletti A, Poirier B, Hansson GK: Protective immunity against atherosclerosis carried by B cells of hypercholesterolemic mice. J Clin Invest 2002, 109:745e753 27. Zhou X, Nicoletti A, Elhage R, Hansson GK: Transfer of CD4(þ) T cells aggravates atherosclerosis in immunodeficient apolipoprotein E knockout mice. Circulation 2000, 102:2919e2922 28. Klingenberg R, Gerdes N, Badeau RM, Gistera A, Strodthoff D, Ketelhuth DF, Lundberg AM, Rudling M, Nilsson SK, Olivecrona G, Zoller S, Lohmann C, Luscher TF, Jauhiainen M, Sparwasser T, Hansson GK: Depletion of FOXP3þ regulatory T cells promotes hypercholesterolemia and atherosclerosis. J Clin Invest 2013, 123: 1323e1334 29. Ait-Oufella H, Salomon BL, Potteaux S, Robertson AK, Gourdy P, Zoll J, Merval R, Esposito B, Cohen JL, Fisson S, Flavell RA, Hansson GK, Klatzmann D, Tedgui A, Mallat Z: Natural regulatory T cells control the development of atherosclerosis in mice. Nat Med 2006, 12:178e180 30. Xiong W, Zhao Y, Prall A, Greiner TC, Baxter BT: Key roles of CD4þ T cells and IFN-gamma in the development of abdominal aortic aneurysms in a murine model. J Immunol 2004, 172: 2607e2612 31. Zhou HF, Yan H, Cannon JL, Springer LE, Green JM, Pham CT: CD43-mediated IFN-gamma production by CD8þ T cells promotes abdominal aortic aneurysm in mice. J Immunol 2013, 190: 5078e5085

3140

32. Romain M, Taleb S, Dalloz M, Ponnuswamy P, Esposito B, Perez N, Wang Y, Yoshimura A, Tedgui A, Mallat Z: Overexpression of SOCS3 in T lymphocytes leads to impaired interleukin-17 production and severe aortic aneurysm formation in miceebrief report. Arterioscler Thromb Vasc Biol 2013, 33:581e584 33. Taleb S, Romain M, Ramkhelawon B, Uyttenhove C, Pasterkamp G, Herbin O, Esposito B, Perez N, Yasukawa H, Van Snick J, Yoshimura A, Tedgui A, Mallat Z: Loss of SOCS3 expression in T cells reveals a regulatory role for interleukin-17 in atherosclerosis. J Exp Med 2009, 206:2067e2077 34. Galle C, Schandene L, Stordeur P, Peignois Y, Ferreira J, Wautrecht JC, Dereume JP, Goldman M: Predominance of type 1 CD4þ T cells in human abdominal aortic aneurysm. Clin Exp Immunol 2005, 142:519e527 35. Chan WL, Pejnovic N, Liew TV, Hamilton H: Predominance of Th2 response in human abdominal aortic aneurysm: mistaken identity for IL-4-producing NK and NKT cells? Cell Immunol 2005, 233: 109e114 36. Sharma AK, Lu G, Jester A, Johnston WF, Zhao Y, Hajzus VA, Saadatzadeh MR, Su G, Bhamidipati CM, Mehta GS, Kron IL, Laubach VE, Murphy MP, Ailawadi G, Upchurch GR Jr: Experimental abdominal aortic aneurysm formation is mediated by IL-17 and attenuated by mesenchymal stem cell treatment. Circulation 2012, 126:S38eS45 37. Ailawadi G, Moehle CW, Pei H, Walton SP, Yang Z, Kron IL, Lau CL, Owens GK: Smooth muscle phenotypic modulation is an early event in aortic aneurysms. J Thorac Cardiovasc Surg 2009, 138: 1392e1399 38. Johnston WF, Salmon M, Su G, Lu G, Stone ML, Zhao Y, Owens GK, Upchurch GR Jr, Ailawadi G: Genetic and pharmacologic disruption of interleukin-1beta signaling inhibits experimental aortic aneurysm formation. Arterioscler Thromb Vasc Biol 2013, 33: 294e304 39. Bhamidipati CM, Mehta GS, Lu G, Moehle CW, Barbery C, DiMusto PD, Laser A, Kron IL, Upchurch GR Jr, Ailawadi G: Development of a novel murine model of aortic aneurysms using peri-adventitial elastase. Surgery 2012, 152:238e246 40. Butcher MJ, Herre M, Ley K, Galkina E: Flow cytometry analysis of immune cells within murine aortas. J Vis Exp 2011, (53). pii:2848 41. Thomas MD, Kremer CS, Ravichandran KS, Rajewsky K, Bender TP: c-Myb is critical for B cell development and maintenance of follicular B cells. Immunity 2005, 23:275e286 42. Lund FE: Cytokine-producing B lymphocytes: key regulators of immunity. Curr Opin Immunol 2008, 20:332e338 43. Harris DP, Haynes L, Sayles PC, Duso DK, Eaton SM, Lepak NM, Johnson LL, Swain SL, Lund FE: Reciprocal regulation of polarized cytokine production by effector B and T cells. Nat Immunol 2000, 1: 475e482 44. Zhou HF, Yan H, Stover CM, Fernandez TM, Rodriguez de Cordoba S, Song WC, Wu X, Thompson RW, Schwaeble WJ, Atkinson JP, Hourcade DE, Pham CT: Antibody directs properdindependent activation of the complement alternative pathway in a mouse model of abdominal aortic aneurysm. Proc Natl Acad Sci U S A 2012, 109:E415eE422 45. Zhong X, Gao W, Degauque N, Bai C, Lu Y, Kenny J, Oukka M, Strom TB, Rothstein TL: Reciprocal generation of Th1/Th17 and T(reg) cells by B1 and B2 B cells. Eur J Immunol 2007, 37: 2400e2404 46. Shah S, Qiao L: Resting B cells expand a CD4þCD25þFoxp3þ Treg population via TGF-beta3. Eur J Immunol 2008, 38:2488e2498 47. Ray A, Basu S, Williams CB, Salzman NH, Dittel BN: A novel IL10-independent regulatory role for B cells in suppressing autoimmunity by maintenance of regulatory T cells via GITR ligand. J Immunol 2012, 188:3188e3198 48. Wang Y, Ait-Oufella H, Herbin O, Bonnin P, Ramkhelawon B, Taleb S, Huang J, Offenstadt G, Combadiere C, Renia L, Johnson JL, Tharaux PL, Tedgui A, Mallat Z: TGF-beta activity

ajp.amjpathol.org

-

The American Journal of Pathology

Protective Role of B2 Cells in AAA

49.

50.

51. 52.

53.

protects against inflammatory aortic aneurysm progression and complications in angiotensin II-infused mice. J Clin Invest 2010, 120:422e432 Golledge J, Norman PE: Atherosclerosis and abdominal aortic aneurysm: cause, response, or common risk factors? Arterioscler Thromb Vasc Biol 2010, 30:1075e1077 Johnsen SH, Forsdahl SH, Singh K, Jacobsen BK: Atherosclerosis in abdominal aortic aneurysms: a causal event or a process running in parallel? the Tromso study. Arterioscler Thromb Vasc Biol 2010, 30: 1263e1268 Lederle FA: The strange relationship between diabetes and abdominal aortic aneurysm. Eur J Vasc Endovasc Surg 2012, 43:254e256 Yin M, Zhang J, Wang Y, Wang S, Bockler D, Duan Z, Xin S: Deficient CD4þCD25þ T regulatory cell function in patients with abdominal aortic aneurysms. Arterioscler Thromb Vasc Biol 2010, 30:1825e1831 Ait-Oufella H, Wang Y, Herbin O, Bourcier S, Potteaux S, Joffre J, Loyer X, Ponnuswamy P, Esposito B, Dalloz M, Laurans L, Tedgui A, Mallat Z: Natural regulatory T cells limit angiotensin IIinduced aneurysm formation and rupture in mice. Arterioscler Thromb Vasc Biol 2013, 33:2374e2379

The American Journal of Pathology

-

ajp.amjpathol.org

54. Kinsey GR, Sharma R, Huang L, Li L, Vergis AL, Ye H, Ju ST, Okusa MD: Regulatory T cells suppress innate immunity in kidney ischemia-reperfusion injury. J Am Soc Nephrol 2009, 20:1744e1753 55. Ailawadi G, Eliason JL, Roelofs KJ, Sinha I, Hannawa KK, Kaldjian EP, Lu G, Henke PK, Stanley JC, Weiss SJ, Thompson RW, Upchurch GR Jr: Gender differences in experimental aortic aneurysm formation. Arterioscler Thromb Vasc Biol 2004, 24:2116e2122 56. Thompson RW, Curci JA, Ennis TL, Mao D, Pagano MB, Pham CT: Pathophysiology of abdominal aortic aneurysms: insights from the elastase-induced model in mice with different genetic backgrounds. Ann N Y Acad Sci 2006, 1085:59e73 57. Garraud O, Borhis G, Badr G, Degrelle S, Pozzetto B, Cognasse F, Richard Y: Revisiting the B-cell compartment in mouse and humans: more than one B-cell subset exists in the marginal zone and beyond. BMC Immunol 2012, 13:63 58. Griffin DO, Holodick NE, Rothstein TL: Human B1 cells in umbilical cord and adult peripheral blood express the novel phenotype CD20þ CD27þ CD43þ CD70. J Exp Med 2011, 208:67e80 59. Yamazaki S, Inaba K, Tarbell KV, Steinman RM: Dendritic cells expand antigen-specific Foxp3þ CD25þ CD4þ regulatory T cells including suppressors of alloreactivity. Immunol Rev 2006, 212:314e329

3141