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Regulate Peripheral B Cell Numbers in Both Mice and Humans Tina W. Wong, Alfred D. Doyle, James J. Lee and Diane F. Jelinek This information is current as of September 26, 2021. J Immunol published online 10 March 2014 http://www.jimmunol.org/content/early/2014/03/07/jimmun ol.1302241 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 10, 2014, doi:10.4049/jimmunol.1302241 The Journal of Immunology

Eosinophils Regulate Peripheral B Cell Numbers in Both Mice and Humans

Tina W. Wong,* Alfred D. Doyle,† James J. Lee,† and Diane F. Jelinek*,‡

The view of eosinophils (Eos) as solely effector cells involved in host parasite defense and in the pathophysiology of allergic diseases has been challenged in recent years. In fact, there is a growing realization that these cells interact with other components of innate and adaptive . For example, mouse Eos were recently demonstrated to promote retention in the marrow. However, it remains unknown whether Eos influence the biology of normal B . In this study, we specifically assessed the effect of Eos on B cell survival, proliferation, and Ig secretion. Our data first revealed that the genetic deletion of Eos from NJ1638 IL-5 transgenic hypereosinophilic mice (previously shown to display profound B cell expansion) resulted in the near abolishment of the B cell lymphocytosis. In vitro studies using human tissues demonstrated Eos’ proximity to B cell follicles and their ability to

promote B cell survival, proliferation, and Ig secretion via a contact-independent mechanism. Additionally, this ability of Eos to Downloaded from enhance B cell responsiveness was observed in both T-independent and T-dependent B cell activation and appears to be inde- pendent of the activation state of Eos. Finally, a retrospective clinical study of hypereosinophilic patients revealed a direct correlation between peripheral levels and B cell numbers. Taken together, our study identifies a novel role for Eos in the regulation of via their impact on B cell homeostasis and proliferation upon activation. The Journal of Immunology, 2014, 192: 000–000. http://www.jimmunol.org/

osinophils (Eos) are innate immune cells that originate Traditionally, Eos have been best known for their anti-helminthic from pluripotent progenitor cells in the effector functions in host defense against infections as well as their E (BM). Developmentally, their survival, expansion, and involvement in the pathophysiology of airway dysfunction and terminal differentiation are driven by the IL-3, IL-5, and tissue remodeling in asthma (10). However, in recent years, these GM-CSF (1). Upon maturation, these cells exit the BM, circulate cells were demonstrated to be much more multifunctional than briefly in the peripheral blood (PB), and then home to and reside originally understood. With respect to immunoregulation, for ex- in tissues that include the gut, uterus, , BM, and mammary ample, Eos mediate aluminum hydroxide–induced B cell ,

gland (2). serve as APCs for T cells, and release cytokines that influence by guest on September 26, 2021 Of the three aforementioned cytokines, IL-5 is the most specific differentiation (i.e., Th1 versus Th2) (11–13). Eos also se- for eosinophilopoiesis (3, 4). To this extent, the IL-5 gene has been crete chemoattractants for the recruitment of T cells, , used as a genetic tool to create mouse models that have altered and dendritic cells to tissue sites (10, 14–17). numbers of Eos for the study of these cells. IL-5 overexpression Recently, a role for Eos in the homeostasis of long-lived plasma from various promoters uniformly results in Eos expansion (5–7). cells (PCs) within mouse BM was described (18, 19). Specifically, Conversely, genetic deletion of IL-5 or its , IL-5Ra, causes PC retention in the marrow was significantly diminished in the reduced Eos numbers (8, 9). absence of Eos. We subsequently demonstrated that in the human PC malignancy, , Eos within the tumor mi- croenvironment can induce proliferation of the malignant cells, *Department of Immunology, Mayo Clinic, Rochester, MN 55905; †Division of thereby contributing to disease pathology (20). Based on these Pulmonary Medicine, Department of Biochemistry and Molecular Biology, Mayo findings, we questioned whether the proliferation-inducing effect Clinic Arizona, Scottsdale, AZ 85259; and ‡Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, MN 55905 of Eos on myeloma cells is a phenomenon that is restricted to this Received for publication August 22, 2013. Accepted for publication February 7, malignancy, or perhaps it is applicable to normal B-lineage cells 2014. as well. Indeed, via both transgenic mouse models and in vitro This work was supported by the Mayo Foundation, National Institutes of Health study of human Eos, we provide strong evidence that eosinophils Grants HL058723 and HL065228, and National Institutes of Health Pre-doctoral do in fact impact the biology of normal B cells. Significantly, this Immunology Training Grant T32 AI07425. This work was also supported by grants from the Mayo Clinic Medical Scientist Training Program, the Mayo Graduate conclusion was supported by our retrospective evaluation of clinical School, and the Mayo Medical School (to T.W.W.), as well as by a grant from the records from patients with idiopathic hypereosinophilic syndrome Mayo Graduate School and by a Sidney Luckman Family Pre-doctoral Fellowship (HES), which demonstrated a direct correlation between Eos levels (to A.D.D.). and circulating B cell numbers. Address correspondence and reprint requests to Dr. Diane F. Jelinek, Department of Immunology, Mayo Clinic, 200 1st Street SW, Rochester, MN 55905. E-mail address: [email protected] Materials and Methods The online version of this article contains supplemental material. Mice Abbreviations used in this article: BM, bone marrow; CBC, complete blood count; Mouse strains employed in these studies include C57BL/6J wild-type (WT) Eos, eosinophil; EPX, eosinophil peroxidase; HES, hypereosinophilic syndrome; IRB, Institutional Review Board; MBP, major basic ; PB, peripheral blood; controls (The Jackson Laboratory, Bar Harbor, ME), eosinophil-deficient PHIL mice (21), NJ1638 IL-5 transgenic mice (6), and NJ1638.PHIL PC, plasma cell; RPMIEos, RPMI1640 1 20% FCS 1 1 ng/ml IL-5; TBNK, T cell/B cell/NK cell; WT, wild-type. mice generated by crossing NJ1638 and PHIL. All mice were analyzed between 3 and 5 mo of age. All mice used in these studies have been Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 backcrossed to C57BL/6J for .20 generations and were maintained in the

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302241 2 REGULATION OF B CELL HOMEOSTASIS BY EOSINOPHILS

Mayo Clinic Arizona Small Animal Facility (a specific pathogen-free fa- mean cell counts of each cohort 6 SEM. A Spearman correlation coeffi- cility). Studies involving animals were performed in accordance with cient between PB Eos and B cell counts was calculated for data pooled National Institutes of Health and Mayo Clinic Institutional Animal Care from all cohorts. and Use Committee guidelines. Cell isolation and culture Flow cytometry analysis of mouse PB, marrow, and B cells were isolated from human PB, , and via B cell en- Single-cell suspensions from PB, BM (flushed from a single femur), and richment kits (StemCell Technologies, Vancouver, BC, Canada) and an homogenized spleen were treated with Pharm Lyse (BD Biosciences, San automated RoboSep cell separator (StemCell Technologies). B cell subsets Jose, CA) to deplete erythrocytes. Cell suspensions were then stained with were isolated using naive and enrichment kits (StemCell various Abs following blockade of FcR with 5 mg/ml Fc blocker (CD16/32; Technologies) or by sorting on a FACSAria cell sorter (BD Biosciences) BD Biosciences). Abs used to identify Eos include: anti–CCR3-allophycocyanin after pan–B cell enrichment and staining with anti-CD27 mAb (BD (83101; R&D Systems, Minneapolis, MN) and anti–Siglec-F-PE (E50- Biosciences). Eos were isolated from human PB and BM as previously 2440; BD Biosciences). Abs for identifying B cells include: anti–B220- described (24). Unless otherwise indicated, all cells were cultured in allophycocyanin (RA3-6B2; eBioscience, San Diego, CA), anti–CD19- RPMIEos (RPMI1640 1 20% FCS 1 1 ng/ml IL-5) at 37˚C with 5% FITC (1D3; BD Biosciences), anti–CD19-PE-Cy7 (eBio1D3; eBioscience), CO2 (24). anti–IgM-FITC (RMM-1; BioLegend, San Diego, CA), anti–CD5-PE (53- 7.3; eBioscience), anti–CD11b-allophycocyanin (M1/70; eBioscience), Histology and immunofluorescence microscopy anti–CD273-PE (TY25; BioLegend), anti–CD80-allophycocyanin (16-10A1; Human and spleen samples were processed for histological analysis as eBioscience), and anti–CD73-V450 (TY/23; BD Horizon). B1a cells previously described (20). Slides were stained either with H&E or with are defined as CD19+IgM+CD11b+CD5+ cells. B1b cells are defined as + + + 2 + 2 immunofluorescence techniques using anti-CD19 Ab (Abcam, Cambridge, CD19 IgM CD11b CD5 cells. B2 B cells are defined as CD19 CD11b MA) to detect B cells and anti-major basic protein (MBP) Ab (EMD cells. Common lymphoid progenitors were identified as described previously

Millipore, Billerica, MA) to detect Eos. Fluorescein-conjugated goat anti- Downloaded from ε (22). Abs used include: anti–CD3 -PE-Cy7 (145-2C11; eBioscience), anti– rabbit Ig (Molecular Probes, Grand Island, NY) and rhodamine-conjugated B220-PE-Cy7 (RA3-6B2; eBioscience), anti–CD19-PE-Cy7, anti-Ter119-PE- goat anti-mouse Ig (Chemicon International, Billerica, MA) were used as Cy7 (TER-119; eBioscience), anti–CD11c-PE-Cy7 (HL3; BD Biosciences), secondary Abs. anti–CD11b-PE-Cy7 (M1/70; eBioscience), anti–Gr1-PE-Cy7 (RB6-8C5; eBio- science), anti–CD8a-PE-Cy7 (53-6.7; eBioscience), anti–TCRb-PE (H57-597; [3H]thymidine incorporation assays

BD Biosciences), anti–Ly6C-PE (HK1.4; eBioscience), anti–NK1.1-PE 3 (PK136; BD Biosciences), anti–TCRgd-FITC (GL3; BD Biosciences), anti– B cell proliferation was assessed using [ H]thymidine (PerkinElmer, Waltham, MA) incorporation assays. B cells isolated from human PB were IL7Ra-eFluor 450 (A7R34; eBioscience), and anti-FLT3-allophycocyanin http://www.jimmunol.org/ (A2F10; eBioscience). Prepro-B and pro-B cells were identified as de- plated in 96-well plates in triplicates at 100,000 cells/well with or without scribed previously (23). Abs used include: anti–B220-allophycocyanin, anti– Eos using RPMIEos in a final volume of 200 ml. A B cell/Eos ratio of 2:1 CD43-PE (1B11; BioLegend), and anti–IgM-FITC. Flow cytometry was was used unless otherwise indicated. Various stimuli/cytokines were used performed on an LSRFortessa cytofluorimeter (BD Biosciences). Data ac- in some experiments, including 2.5 mg/ml CpG oligodeoxynucleotide 2006 quisition and analysis were performed using FACSDiva (version 6.2; BD (provided by the Mayo Clinic core facility); 10 ng/ml IL-5, 10 ng/ml GM- Biosciences) software. Cell types were analyzed to determine the percentage CSF, 100 ng/ml RANTES, 15 ng/ml IL-10, 15 ng/ml IL-4 (each from of PB, BM, or spleen leukocytes and to obtain cells per microliter based on PeproTech); 15 ng/ml IL-21, 75 ng/ml IFN-g, 100 ng/ml IL-33 (each from total leukocyte counts (assessed via hemocytometer). Results are presented R&D Systems); 2 mg/ml anti-human Ig (Jackson ImmunoResearch Lab- as means 6 SEM. Statistical analysis was performed using t tests with oratories, West Grove, PA); and 100 ng/ml CD40L (Alexis Biochemicals, , Farmingdale, NY). Cultures were incubated for 3 d and pulsed with 1 mCi differences between means considered significant when p 0.05. 3 [ H]thymidine during the last 18 h of culture. Radioactivity was measured by guest on September 26, 2021 Ethical statement and human blood and tissue samples using liquid scintillation spectroscopy. For assessing contact dependency, 250,000 B cells/well and 125,000 Eos were plated in 24-well plates either The Mayo Clinic Institutional Review Board (IRB) reviewed and approved in direct coculture or across 0.4-mm pore transwell inserts (Corning, our request for the use of blood, tonsil, and spleen tissue from healthy Corning, NY). Eos supernatants were collected after 24 h at 3 3 106 cells/ml donors. The need for informed consent for these samples was waived due to and used at a 1:4 dilution. RPMIEos served as control media. In ex- their designation as waste products generated during blood donations or periments where Eos and B cells were stimulated with CpG separately surgical waste materials (tonsils and spleens). A separate Mayo Clinic IRB prior to coculture, cells were cultured with or without 2.5 mg/ml CpG for approval was obtained and blood was drawn from healthy individuals for the 24 h and washed three times with PBS prior to plating. Results are rep- specific isolation of Eos. Blood specimens were collected and used only resentative of three independent experiments. Data are represented as the from donors providing written informed consent in accordance with the mean [3H]thymidine incorporation of triplicate samples 6 SEM. Statistical Declaration of Helsinki. analyses were performed by Student t tests. Patient cohort and healthy control subjects ELISA Mayo Clinic IRB approval was obtained for the abstraction of clinical data Mouse serum IL-5 was measured as described previously using an R&D from the Mayo Clinic electronic medical records of patient cohorts and Quantikine ELISA kit (R&D Systems) (6). Results are represented as control subjects in this retrospective study. The Mayo Clinic IRB waived the means 6 SEM. Human B cells were cultured at 0.5 3 106 cells/ml with need for informed consent because the study was restricted to already 2.5 mg/ml CpG stimulation with or without Eos for 10 d. Eos were also existing data. The Mayo Clinic medical database was queried for patients cultured alone to serve as a negative control for Ig secretion. At day 10 of diagnosed at the Mayo Clinic with HES between January 1, 2007 and culture, cell-free supernatants were collected and analyzed for levels of December 31, 2012 and with a complete blood count (CBC) and differential secreted Ig using IgM, IgG, and IgA sandwich ELISAs as previously de- analysis performed at the Mayo Clinic at the time of diagnosis demon- scribed (25). Data are presented as the mean Ig levels of triplicate samples . strating a PB Eos count 1500 cells/ml. A series of exclusion criteria was 6 SEM. Statistical analyses were performed using Student t tests. applied to the query (Table I) to minimize potential confounding factors that could alter cellularity. A cohort of 67 patients was iden- Western blot analysis tified, and gender- and age-matched controls were obtained with a date of PB analysis matched to their corresponding cases and an Eos count Cell lysates were prepared and Western blot was performed as previously of ,500 cells/ml. Statistical analysis was performed to compare PB described (26). Anti-TLR9 Ab (Cell Signaling Technology) was used at a lymphocyte counts between cases and controls using a paired sample 1:1000 dilution and anti–b-actin Ab (Novus Biologicals, Littleton, CO) t test. In 16 of the 67 HES patients, T cell/B cell/NK cell (TBNK) dif- was used at a 1:5000 dilution. HRP-conjugated secondary Abs (GE Health- ferential analysis data were available for further examination of lymphocyte care) were used at a 1:2000 dilution. subsets. Because the TBNK analysis is infrequently performed during Eos activation standard medical evaluation, an additional set of controls as well as patients with mild hypereosinophilia (Eos count of 500–1500 cells/ml) Freshly isolated human PB Eos were cultured at 0.25 3 106 cells/ml with or with TBNK differential data available was obtained without gender and without B cells (0.5 3 106 cells/ml) with or without 2.5 mg/ml CpG or in age matching. Statistical analyses were performed to compare the various the presence of high-dose IL-5 (10 ng/ml), GM-CSF (10 ng/ml), RANTES cohorts for differences between PB B cell, NK cell, CD4 T cell, and CD8 (100 ng/ml), or IL-33 (100 ng/ml) in 24-well plates. Cells were stained on T cell counts using an unpaired Student t tests. Data are represented as days 0, 1, 2, and 3 with anti–Siglec 8 mAb (BioLegend), anti-CD19 mAb The Journal of Immunology 3

(BD Biosciences), and anti-CD69 mAb (BD Biosciences) and analyzed on Human Eos are found near B cell follicles in secondary a FACSCalibur flow cytometer (BD Biosciences). Mouse IgG1 Abs (BD lymphoid organs Biosciences) were used as control. Data analysis was performed using FlowJo software (TreeStar, Ashland, OR). Eos were gated based on The observations described above, along with our recent prior work 2 side scatter profile and as Siglec 8+/CD19 cells. Data shown are repre- (20), prompted us to investigate the ability of Eos to impact normal sentative of three independent experiments. human B cell biology. We initially evaluated Eos localization Cell survival analysis within secondary lymphoid tissues.Inmouselymphnodes,Eos have been reported to localize to the paracortex near the B cell Eos were prestained with PKH26 red fluorescent dye (Sigma-Aldrich, St. Louis, MO) following the manufacturer’s protocol. B cells were cultured at follicles (i.e., T cell/B cell border) (32). Immunofluorescence 0.5 3 106 cells/ml in 24-well plates with or without 2.5 mg/ml CpG with or analysis of human spleens and tonsils revealed that similar to mice, without PKH26-stained Eos (0.25 3 106 cells/ml). Cells were stained on day Eos are found on the border of, or in close proximity to, B cell 0 and every 24 h for 6 consecutive days thereafter with annexin V-FITC and follicles (Fig. 3). It is noteworthy that although we did not observe 7-aminoactinomycin D (BD Biosciences) and analyzed on a FACSCalibur flow cytometer (BD Biosciences). Data analysis was performed using FlowJo Eos deep within germinal centers, we did find their occasional software. Eos in coculture were excluded from the live/dead analysis based on localization within the perimeter of the follicle (Fig. 3D, 3H). PKH26 and side scatter profile. Fold changes in percentage live B cells cocultured with Eos were calculated relative to B cells cultured alone. Data Human Eos promote B cell proliferation in vitro are presented as mean of the fold changes across three independent experi- We stimulated PB B cells with the TLR9 agonist CpG in culture with ments 6 SEM. Statistical analyses were performed using Student t tests. increasing concentrations of Eos to evaluate whether human Eos can directly impact B cell proliferation. These data revealed that Eos Results Downloaded from B cell lymphocytosis in IL-5 transgenic hypereosinophilic mice indeed enhanced B cell proliferation in a dose-dependent manner is dependent on the presence of Eos (Fig. 4A) and that maximal enhancement was achieved at a B cell/ Eos ratio of 2:1. Because exogenous IL-5 was added to our cultures NJ1638 IL-5 transgenic hypereosinophilic mice exhibit age- to maintain Eos survival, we confirmed that IL-5 does not affect dependent B cell lymphocytosis with absolute B cell counts 4.5- human B cell proliferation (Supplemental Fig. 2). We also con- . fold more than that of WT mice at 1 mo of age and 30-fold more firmed that mature Eos, as nonproliferating terminal effector cells, than that of WT mice beyond 12 mo of age (6). We crossed NJ1638 do not incorporate [3H]thymidine (Fig. 4A, right bar). Thus, the http://www.jimmunol.org/ mice with PHIL (designated NJ1638.PHIL), a mouse whereby [3H]thymidine values observed in our cocultures result from B cell committed Eos-lineage cells are deleted via the expression of DNA synthesis. Based on these findings, we performed all of our diphtheria toxin under eosinophil peroxidase (EPX), an Eos- subsequent in vitro studies at a 2:1 B cell/Eos ratio. specific promoter (21), to assess the etiology of the B cell ex- To address whether B cells from different compartments are pansion in these mice (i.e., IL-5 overexpression versus hyper- differentially influenced by Eos, PB, tonsillar, and splenic B cells eosinophilia). Examination of serum demonstrated equal or higher were assessed for proliferation with or without CpG stimulation levels of IL-5 in the NJ1638.PHIL compared with NJ1638 with and without Eos. Our data demonstrated that the proliferation (Fig. 1A). Furthermore, we confirmed that NJ1638.PHIL exhibited of B cells from all tissues could be promoted by Eos (Fig. 4B–D). by guest on September 26, 2021 a dramatic reduction in the total number of PB leukocytes (Fig. 1B) Furthermore, Eos modestly augmented B cell DNA synthesis in and the number of PB Eos (Fig. 1C) as compared with NJ1638. the absence of CpG. Lastly, consistent with our findings in the When the PB of WT, PHIL, NJ1638, and NJ1638.PHIL mice was mouse where all of the examined PB B cell subsets were expanded analyzed for B cell numbers, our data revealed that whereas NJ1638 with hypereosinophilia, we observed that the proliferation of hu- mice had significantly elevated numbers of B cells relative to WT man PB CD19+CD272 naive and CD19+CD27+ memory B cells mice, this elevation was largely abolished in NJ1638.PHIL mice was similarly enhanced by Eos (Fig. 4E, 4F). (Fig. 1D). These findings were confirmed (data not shown) in a separate cross between NJ1638 and another Eos-deficient mouse, Ig secretion by CpG-activated B cells is enhanced by Eos 2/2 2/2 MBP-1 /EPX (27). Notably, the splenomegaly observed in We next determined whether the presence of Eos during CpG- NJ1638 (6) was also reduced in the NJ1638.PHIL mice to a spleen mediated activation of B cells leads to an increase in Ig secretion. size comparable to that of WT mice (Supplemental Fig. 1). Fur- Significantly higher concentrations of IgM, IgG, and IgA were ther characterization of the PB B cell expansion in NJ1638 mice detected in cell-free supernatants from Eos/B cell cocultures as demonstrated increased numbers of all B cell subsets examined, compared with those from B cell cultures alone (Fig. 4G), sug- including the spectrum of naive-like to memory-like cells (28, 29) gesting that Eos can promote B cell proliferation in a manner that as well as B1 B cells (30) (Fig. 2A, 2B). Additionally, analysis of similarly supports enhanced Ab production. the BM and spleens of WT and NJ1638 mice was performed and revealed a trend of reduced B cell progenitors in the marrow but Eos can enhance B cell proliferation via a contact-independent elevated populations in the spleen (Fig. 2C, left and middle). This mechanism apparent shift in B cell from marrow to spleen in Eos express numerous cell surface molecules as well as soluble NJ1638 mice most likely reflects the result of crowding out due to factors that are stored within their granules (1, 33). We cultured the dramatic Eos expansion within the marrow of these mice (27). B cells either alone or in coculture with Eos in direct contact or However, when the total number of B cell progenitors was quan- across transwells to determine whether surface-bound molecules tified for each mouse, no difference was found between the number or soluble factors or both mediate augmented B cell proliferation of progenitors in WT and in NJ1638 mice (Fig. 2C, right). Taken by Eos. Fig. 5A demonstrated that B cell proliferation was sig- together, our data demonstrate that the ablation of Eos in the IL-5 nificantly enhanced when cultured in direct contact with Eos as transgenic mice leads to a near abolishment of the B cell lympho- well as when the Eos and B cells were physically separated by cytosis despite sustained elevation in IL-5 levels, suggesting that the a transwell, thus indicating that the promotion of B cell prolifer- observed B cell expansion in IL-5 transgenic mice, which occurs ation by Eos can be achieved via a contact-independent mecha- predominantly after B cell development, is the direct consequence nism. Additionally, Eos culture supernatants also promoted B cell of hypereosinophilia. proliferation, thereby providing further evidence that an Eos- 4 REGULATION OF B CELL HOMEOSTASIS BY EOSINOPHILS Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. B cell lymphocytosis in NJ1638 IL-5 transgenic mice is driven by the eosinophilia. (A and B) Serum IL-5 levels (A) and total PB leukocyte (WBC) counts (B) were assessed in WT, PHIL, NJ1638, and NJ1638.PHIL mice. (C and D) PB leukocytes of WT, PHIL, NJ1638, and NJ1638.PHIL mice were analyzed for percentage (left; pregated on Siglec-F+/SSChi cells) and absolute numbers (right) of Eos (C) and B cells (D). Data are shown as means 6 SEM of three animals for each genotype. *p , 0.05, **p , 0.01. n.d., not detected. derived soluble factor may contribute to the observed augmenta- Western blot analysis (Fig. 6A). Furthermore, we directly tested tion in B cell responsiveness (Fig. 5B). However, note that the whether CpG might affect the ability of Eos to promote B cell Eos-mediated augmentation of B cell proliferation was greater proliferation by first culturing B cells and Eos separately with or when the two cell types were in direct contact (Fig. 5A), and thus without CpG followed by the coculturing of the unstimulated and it is possible that a contact-dependent mechanism also exists. the prestimulated cells. Our data demonstrated that the unstimu- lated and the prestimulated Eos enhanced B cell proliferation to an CpG-mediated direct activation of Eos is not required for the equivalent degree (Fig. 6B), further suggesting that CpG has no support of B cell proliferation direct impact on Eos with respect to their ability to enhance B cell In the described Eos/B cell cocultures, both Eos and B cells have proliferation. been exposed to CpG stimulation, raising the possibility that CpG- We next questioned whether the activation state of Eos would 1) induced Eos activation caused enhanced B cell proliferation. Be- be influenced by coculture with B cells, and 2) impact the ability of cause a consensus view of Eos TLR9 expression has not been Eos to promote B cell proliferation. We first examined for changes reached (34–40), we began by assessing human Eos TLR9 ex- in Eos morphology as an indicator of cell activation. We observed pression. Although trace levels of TLR9 mRNA were found in Eos that whereas Eos cultured in vitro for 3 d with or without CpG purified from human PB and BM by PCR (data not shown), the displayed an unactivated spherical morphology, sporadic appear- expression of TLR9 protein by these cells was undetectable via ance of Eos with elongated morphology was detected in those The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. PB, BM, and spleen analyses of WT, PHIL, NJ1638, and NJ1638.PHIL mice. (A) The spectrum of naive-like to memory-like phenotype of B cells is characterized by surface expression of CD80, CD273, and CD73 (28, 29). (B) PB from WT, PHIL, NJ1638, and NJ1638.PHIL were analyzed for naive- like/memory-like phenotype as described in (A) or for B1 versus B2 phenotypes. (C) Spleen and BM isolated from a single femur of WT and NJ1638 mice were analyzed for common lymphoid progenitors (CLP), prepro-B cells, and pro-B cells. Total numbers of B cell progenitors in each mouse were calculated by combining the numbers of spleen and marrow cells using the approximation that the total BM hematopoietic volume is 15.8 times the hematopoietic volume in a single femur; that is, estimated total hematopoiesis = cells in spleen + (cells in one femur 3 15.8) (31). *p , 0.05, ** p , 0.01, *** p , 0.001. cultured together with B cells (data not shown). We then assessed evidence of Eos degranulation upon activation. Flow cytometric for surface expression of the activation marker, CD69, in these analysis revealed a mild increase in CD69 expression in Eos cultures as well as levels of EPX in the culture supernatants as cultured with B cells compared with those cultured alone, and this 6 REGULATION OF B CELL HOMEOSTASIS BY EOSINOPHILS

tivation in these cocultures and result in further augmentation of B cell proliferation. However, our results demonstrated that none of these cytokines led to augmented Eos-mediated stimulation of B cell DNA synthesis (Fig. 6D). Eos promote B cell proliferation in both T-independent and T-dependent B cell activation Activation of B cells via CpG stimulation mimics a T-independent B cell activation process. We questioned whether Eos might influ- ence the proliferation of B cells activated in a T-dependent manner. Thus, we cultured B cells with or without Eos and subjected them to either CpG or anti-Ig/CD40L. These data revealed that Eos enhanced proliferation of both CpG- and anti-Ig/CD40L–activated B cells (Fig. 6E). Furthermore, the addition of various cytokines, includ- ing IL-10/IL-21, IL-4, and IFN-g, differentially affected B cell proliferation but did not abolish the effect of Eos, suggesting that the Eos-derived soluble factor that promotes B cell proliferation is independent of the effect of these known modulators of B cell ac-

tivation, proliferation, and differentiation (44–49). Downloaded from Eos support B cell survival in vitro Eos have been described to play a role in BM PC homeostasis by providing PC survival factors (18, 19). To determine the effect of Eos on B cell survival, we cultured B cells with or without Eos in

the absence or presence of CpG and analyzed the cultures for http://www.jimmunol.org/ apoptosis. We observed that whereas the presence of Eos in- creased the relative percentage of live cells in the unstimulated B cell cultures over the course of 6 d, the relative percentage of live cells in the CpG-stimulated cultures was largely unaffected by Eos during this period (Fig. 7). Human PB Eos counts correlate with B cell counts To assess the clinical relevance of our findings in the mouse and in our in vitro studies, we performed a clinical retrospective study by guest on September 26, 2021 comparing the PB counts of patients with HES to healthy con- trols. We obtained CBCs and differential analyses from hyper- eosinophilic patients (Eos . 1500 cells/ml) and gender- and age- matched controls (Eos , 500 cells/ml). Because a number of conditions may intrinsically cause alterations in B cell counts and serve as confounding factors (Table I), we excluded patients with such conditions from our analysis. In a total of 67 pairs of sub- jects, we observed a significantly elevated total lymphocyte count in HES patients compared with healthy controls (2.8 6 0.2 versus 2.2 6 0.2 3 109/l, p , 0.001; Fig. 8A). Further analysis of FIGURE 3. Eos can be found in close proximity to B cell follicles in the lymphocyte subsets in 16 of the 67 HES patients where TBNK human tonsils and spleens. Human tonsils (A–D)andspleens(E–H)were differential data were available revealed that PB B cell numbers stainedwithH&E(A, B, E, F) or using immunofluorescence (C, D, G, H)for were significantly elevated in HES patients and patients with mild specific visualization of Eos and B cells. In H&E-stained images, B cell hypereosinophilia (Eos 500–1500 cells/ml) compared with control A E follicles are outlined in the dotted regions ( , ) and Eos are highlighted by subjects (Fig. 8B). Additionally, despite the small sample size, black arrows (B, F). In immunofluorescence-stained slides, anti-MBP–labeled analysis of pooled cohorts showed a weak, but statistically sig- Eos are in red and anti-CD19–labeled B cells are in green. Autofluorescent RBCs are white in these overlaid images. (B), (D), (F), and (H) show higher nificant, positive correlation between PB Eos and B cell numbers magnifications of areas enclosed in white box in (A), (D), (E), and (F), re- (correlation coefficient, 0.25; 95% CI, [0.05, 0.44]; p = 0.01; Fig. 8C). spectively. Scale bars, 100 mm(A, E); 50 mm(B, C, F, G); 20 mm(D, H). In contrast, NK cell, CD4 T cell, and CD8 T cell numbers were comparable between control subjects, patients with mild hyper- eosinophilia, and HES patients (Fig. 8D–F). increase was noted to be generally less than that observed when stimulated with various known Eos activators (Fig. 6C) (41–43). In Discussion contrast, ELISA analysis of the culture supernatants demonstrated IL-5 transgenic hypereosinophilic mice exhibit B cell lymphocy- undetectable levels of EPX in Eos cultured with or without B cells tosis (6). It was presumed that IL-5, being a known rodent B cell whereas varying levels of EPX were detected in culture super- growth factor (50, 51), was the driver of this B cell expansion. In natants from Eos stimulated with the various activating cytokines light of the recent work describing Eos’ ability to support malig- (IL-33 . high-dose IL-5 . RANTES . GM-CSF; data not nant PC growth (20), the possibility exists that Eos may similarly shown). We thus tested whether addition of these Eos activators to influence other proliferating B-lineage cells, that is, those found Eos and B cell cocultures would enhance the degree of Eos ac- during normal immune activation, such that the expanded B cell The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. Human Eos promote human B cell proliferation and Ig secretion. (A) CpG-stimulated B cells were cultured in the absence or presence of in- creasing numbers of Eos and proliferation was assessed by [3H]thymidine incorporation. (B–D) B cells isolated from PB (B), tonsils (C), and spleens (D)were assessed for proliferation in culture with or without CpG stimulation and in the absence or presence of Eos. (E and F) PB B cells were separated into naive (E)and memory (F) cells and proliferation was assessed with or without CpG stimulation in the absence or presence of Eos. (G) CpG-stimulated B cells were cultured with or without Eos for 10 d and Ig levels were measured in the cell-free supernatants by ELISA. *p , 0.05, **p , 0.01, ***p , 0.001. n.d., not detected. pool in IL-5 transgenic mice is the direct consequence of having B cells occurs predominantly after, and not during, development. too many Eos. The findings from the present work strongly support The in vitro studies using human cells further demonstrate an this hypothesis, demonstrating a B cell lymphocytosis in the IL-5 Eos-induced enhancement of B cell proliferation upon peripheral transgenic mouse that is dramatically reduced when Eos are ge- activation. Additionally, a prolongation in lifespan of unac- netically deleted. The observation that a small elevation in PB tivated human B cells by Eos was observed, and this may also in B cell counts persists in NJ1638.PHIL mice and is primarily at- part contribute to the expanded B cell pool. Taken together, the tributable to elevated B1 B cell levels compared with WT mice is effect that Eos have on B cells appears to be multifaceted, in- consistent with the previous characterization of IL-5 as a B1 B cell volving B cell peripheral activation and homeostasis but not growth factor (52), but the degree to which IL-5 drives B cell development. proliferation appears minor in comparison with the effect of Eos. In the mouse node, Eos have been described to localize Analysis of the combined hematopoietic compartments (i.e., spleen to the paracortex just outside of B cell follicles (31). An accumula- plus BM) revealed comparable numbers of progenitor cells in WT tion of Eos in regions surrounding the follicles was also noted in and in NJ1638 mice, suggesting that the influence Eos exert on NJ1638 hypereosinophilic mice (A. Doyle, unpublished observa- 8 REGULATION OF B CELL HOMEOSTASIS BY EOSINOPHILS

FIGURE 5. Eos can augment B cell pro- liferation via a contact-independent mecha- nism. (A) Proliferation of B cells either alone, in direct coculture with B cells, or in the presence of Eos across a 0.4-mm transwell was assessed. (B) Culture supernatants (supe) werecollectedfromEosandusedtotreatB cells in a proliferation assay with or without CpG stimulation. **p , 0.01, ***p , 0.001.

tions). Examination of human secondary lymphoid organs in this presence of a contact-independent mechanism in the Eos/B cell study revealed similar localization of human Eos near or within interaction, the positioning of Eos deep within germinal centers the perimeter of B cell follicles. Because in vitro data point to the would not be required for Eos to exert their effect on B cells. Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 6. Activation of Eos is not required for the support of B cell proliferation. (A) Eos isolated from human BM and PB and B cells isolated from PB, tonsils, and spleens were analyzed for TLR9 protein expression. b-actin was used as loading control. (B) B cells and Eos were stimulated independently with CpG. Proliferation of unstimulated and CpG-stimulated B cells was assessed either alone or in the presence of unstimulated or CpG-stimulated Eos. (C) Eos were cultured alone or with B cells with or without CpG or in the presence of various Eos-activating cytokines. Eos were analyzed for surface expression of CD69 on days 0, 1, 2, and 3 by flow cytometry (solid gray, isotype; open black, anti-CD69 mAb). Numbers in FACS plots represent change in mean fluorescence intensity. Data are representative of three independent experiments. (D and E) B cells or B plus Eos cultures were stimulated with various cytokines for Eos activation (D) or B cell activation (E) and proliferation was evaluated. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 9

FIGURE 7. Eos promote B cell survival in vitro but not upon activation by CpG. (A)Flowcyto- metric analysis of B cell survival in culture with or without CpG stimulation in the absence or presence of Eos over 6 d. (B) Fold change in percentage of live B cells was calculated between B cells cultured with Eos and those cultured alone. Statistical tests were performed to compare fold changes at each time point to that at day 0. *p , 0.05, **p , 0.01. Downloaded from http://www.jimmunol.org/

Alternatively, their localization to the perimeter of the follicles respect to promotion of B cell growth, 1) the activation state of may reflect their potential involvement in the initial B cell acti- Eos is irrelevant, 2) partial activation of Eos is sufficient to provide vation event that occurs at the T/B border. Whereas the numbers maximal benefit for B cells, or 3) the nature of Eos activation of Eos present in secondary lymphoid organs appear quite limited differs between that achieved via coculture with B cells compared and thus call into question the relevance of the in vitro findings, with the exogenous cytokines used. It is possible that stimuli other we suggest that the paucity of these cells under normal states may than the ones used in this study could in fact enhance Eos’ ability in fact be advantageous such that nonspecific or aberrant B cell to promote B cell proliferation. This would be consistent with proliferation is not observed. Of note, eosinophilic infiltrates are a previous study showing that activation of Eos resulted in the by guest on September 26, 2021 not uncommonly found in classical Hodgkin and may enhanced expression of PC survival factors in the mouse (18). contribute to the disease pathogenesis (53). Additionally, Eos are Furthermore, results from this study suggest the possible existence recruited to secondary lymphoid organs or other nonlymphoid tis- of bidirectional crosstalk between Eos and B cells such that Eos sues upon active infection, or Ag challenge, potentially supporting are rendered more poised to interact with and influence B cells. the humoral response in a more pathogen-specific manner (32, 54). Studies are currently underway to further characterize this Eos/ A small number of Eos were observed to acquire a partially B cell interaction. activated appearance when cultured with B cells, and yet attempts Of note, although primary eosinopenia has yet to be described in to further activate Eos via exogenous cytokines did not improve humans, in IL-5 knockout or IL-5Ra knockout mice, a concomi- Eos’ ability to support B cell growth. This may suggest that, with tant reduction in PB Eos and peritoneal B1, but not PB B2, B cells was observed (8, 9). However, previous reports and current analysis show that the specific genetic deletion of Eos in both PHIL and Table I. Inclusion and exclusion criteria used for retrospective study MBP-12/2/EPX2/2 mice did not result in alterations in the PB (predominantly B2) B cell pool (Fig. 1 and data not shown) (21, 27). Inclusion Exclusion Together, these observations suggest that, at least in mice, Eos can Diagnosis of Use of cytoreductive therapy promote B cell survival and/or proliferation but do not play a req- hypereosinophilia at time of CBC evaluation uisite role in these activities. The presence of other support cells CBC + Differential at Mayo Diagnosis of parasitic infection during B cell development in the BM and activation in the periphery at time of diagnosis within 6 mo Blood Eos .1500 cells/ml Diagnosis of Hodgkin’s or may explain the nonessential nature of Eos. As such, we speculate non-Hodgkin’s lymphoma that Eos might be a critical amplifier of humoral immunity in a Diagnosis of allergic disorders regulated manner in response to specific pathogens. (i.e., asthma, allergic Eos have been shown to produce a vast array of mediators, a rhinitis, drug ) Diagnosis of eosinophilic number of which are known B cell proliferation factors. Although granulomatosis the precise mechanism of the Eos-induced augmentation of B cell with polyangiitis proliferation remains elusive, the data presented in the present (Churg–Strauss syndrome) study point toward a soluble factor not found among the usual Diagnosis of pulmonary suspects. Notably, APRIL and IL-6 were demonstrated to drive the eosinophilia/Loffler’s syndrome Eos/PC interaction in the mouse BM (19); however, the interaction Diagnosis of eosinophilic between human Eos and B cells appears to be independent of fasciitis/gastroenteritis/ the effects of these two cytokines because in vitro stimulation of colitis/esophagitis human B cells with IL-6 has no impact on their proliferation and 10 REGULATION OF B CELL HOMEOSTASIS BY EOSINOPHILS Downloaded from

FIGURE 8. Human PB B cell numbers uniquely correlate with Eos counts. (A) PB lymphocyte counts were obtained from CBC analysis of HES patients http://www.jimmunol.org/ and age- and gender-matched healthy controls; n = 67. (B–F) Peripheral blood B cell (B, C), NK cell (D), CD4 T cell (E), and CD8 T cell (F) counts were obtained from TBNK differential analysis of control subjects (n = 21), patients with mild hypereosinophilia (n = 58), and HES patients (n = 16). Data were pooled from control subjects, patients with mild hypereosinophilia, and HES patients and plotted for PB Eos versus B cell counts (C). *p , 0.05, **p , 0.01. soluble TACI does not abolish the augmentation of B cell pro- differences in B cell biology between these two species (e.g., liferation by Eos (data not shown). The potential for lipid medi- responses toward IL-5), the results from this study implicate a ators and granule to be the driver of this Eos/B cell fundamental, evolutionarily conserved mechanism used specifi- by guest on September 26, 2021 interaction is currently under investigation. cally by Eos to regulate the humoral . Additional The present study, although providing evidence toward a role for studies are warranted to gain further understanding of the mediator Eos in the promotion of B cell proliferation, raises the question of (s) and molecular pathway(s) involved in the Eos/B cell interaction. whether Eos are unique among all in their ability to support B cell growth. A number of studies have demonstrated that Acknowledgments other granulocytes (including , , and mast We thank Drs. Hirohito Kita and Joseph Butterfield for mentorship and cells) can interact with B cells to aid in B cell activation and Ig intellectual contribution to the work. We thank the Mayo Clinic Division of class-switching and secretion (55–59), yet with the exception of Biomedical Statistics and Informatics and especially Pauline Funk for mast cells, the influence of these granulocytes on B cell homeo- assistance in abstracting clinical data. Lastly, we thank Diane Squillace, Jasmina Suko, Jake Kloeber, Kelly Shim, Joseph Neely, and David Nguyen stasis and proliferation remains unknown. Thus, in consideration for technical assistance. of the newly discovered B cell growth-promoting role of Eos described in this report, future studies are warranted to assess Disclosures whether similar roles exist in other granulocytes as well. The authors have no financial conflicts of interest. Finally, PB analysis in patients with idiopathic HES, albeit small in number of subjects examined, revealed a significant increase in B cell counts compared with healthy controls. Importantly, other lymphocyte References subsets in these patients, including CD4 T cells, CD8 T cells, and NK 1. Lee, J. J., E. A. Jacobsen, S. I. Ochkur, M. P. McGarry, R. M. Condjella, A. D. Doyle, H. Luo, K. R. Zellner, C. A. Protheroe, L. Willetts, et al. 2012. cells, were not found to be elevated in numbers, suggesting that the Human versus mouse eosinophils: “that which we call an eosinophil, by any observed B cell expansion is unlikely to be a mere reflection of a other name would stain as red”. J. Clin. Immunol. 130: 572–584. generalized immune hyperactivation. Given the findings from the 2. Kita, H. 2011. Eosinophils: multifaceted biological properties and roles in health and disease. Immunol. Rev. 242: 161–177. transgenic mouse work, it is of interest whether blood B cell counts 3. Sanderson, C. J. 1992. -5, eosinophils, and disease. Blood 79: 3101– could be normalized in hypereosinophilic patients simply by the re- 3109. 4. Mori, Y., H. Iwasaki, and K. Akashi. 2013. Eosinophil lineage-committed pro- moval of excess Eos. However, because most therapies used for the genitors. In Eosinophils in Health and Disease, 1st Ed. J. J. Lee, and treatment of HES use anti-neoplastic, chemotherapeutic, and immu- H. F. Rosenberg, eds. Academic, San Diego, p. 89–96. nosuppressive agents, which can cause cytolysis in lymphocytes as 5. Dent, L. A., M. Strath, A. L. Mellor, and C. J. Sanderson. 1990. Eosinophilia in transgenic mice expressing interleukin 5. J. Exp. Med. 172: 1425–1431. well (60, 61), the direct impact of Eos reduction on B cell numbers in 6. Lee, N. A., M. P. McGarry, K. A. Larson, M. A. Horton, A. B. Kristensen, and these patients is difficult to assess. J. J. Lee. 1997. Expression of IL-5 in /T cells leads to the develop- The fact that Eos were found to modulate peripheral B cell ment of a massive eosinophilia, extramedullary eosinophilopoiesis, and unique histopathologies. J. Immunol. 158: 1332–1344. numbers across two species, that is, mice and humans, highlights 7. Tominaga, A., S. Takaki, N. Koyama, S. Katoh, R. Matsumoto, M. Migita, the importance of these findings. Especially in light of the known Y. Hitoshi, Y. Hosoya, S. Yamauchi, Y. Kanai, et al. 1991. Transgenic mice The Journal of Immunology 11

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