Superior mouse eosinophil depletion in vivo targeting transgenic Siglec-8 instead of endogenous Siglec-F: mechanisms and pitfalls

Abstract

Eosinophils are important multifunctional granulocytes. When studying eosinophil function and its contribution to diseases, mouse models are often used. Mouse eosinophils selectively express sialic acid-binding immunoglobulin-like lectin (Siglec)-F. Its closest functional paralog on human eosinophils is Siglec-8. These Siglecs are being used to target eosinophils when exploring their mechanistic roles in disease and for potential therapeutic benefit. In order to facilitate preclinical studies of human Siglec-8, we developed transgenic mouse strains expressing human Siglec-8 only on the surface of eosinophils with or without endogenous Siglec-F and have begun characterizing various cellular functions in vitro and in vivo. Eosinophils from Siglec8+ mice, with or without Siglec-F, responded to Siglec-8 antibody engagement in vitro by upregulating surface CD11b, while Siglec-F antibody had no such effect. Engagement of Siglec-F or Siglec-8 with respective antibodies in vitro resulted in only modest increases in cell death. Administration of rat Siglec-F antibodies to mice led to a significant decrease in Siglec-F surface expression on eosinophils due to internalization, and thus appeared to decrease eosinophil numbers based on SiglecF+ cells, but with proper gaiting strategies did not in fact result in significant eosinophil depletion. In marked contrast, administration of mouse Siglec-8 antibodies rapidly and effectively depleted eosinophils from blood and spleens of mice, but an F(ab’)₂ version did not, indicating an Fc-mediated mechanism for eosinophil depletion in vivo. Siglec-8 expressing mice with or without endogenous Siglec-F will be useful to study Siglec-8-based therapeutics, and may be a preferred approach when acute or chronic eosinophil depletion is needed.

Summary:

We uncovered shortcomings when using certain antibodies that target Siglec-F to deplete mouse eosinophils, while administration of anti-Siglec-8 antibody to Siglec-8 transgenic mice works well.

1. Introduction

Eosinophils are innate immune granulocytes that contribute to a range of host defense, homeostatic and disease-related responses.^{1, 2} Much of our knowledge of the biology of this cell comes from preclinical mouse models that employ mice congenitally or conditionally deficient in eosinophils.^3–6 Other strategies, such as those involving mouse, rat or sheep antibody-based depletion by targeting Siglec-F, have also been used, with variable but consistently incomplete blood and tissue depletion.^7–11 In particular, administration of several commercial rat anti-mouse Siglec-F antibodies, or liposomes that selectively engage Siglec-F, also do a suboptimal job of depleting eosinophils.^12–16 Furthermore, Siglec-F is expressed on other cells including alveolar macrophages and intestinal cells,^{17, 18} so effects seen with these targeting strategies may not be eosinophil-specific. Although ligation of its closest human paralog, Siglec-8,⁷ on human eosinophils induces profound cell death in vitro, especially in cytokine primed cells,^19–21 ligation of Siglec-F on mouse eosinophils is a consistently poor inducer of cell death in vitro.^{8, 22, 23} A similar response shared by Siglec-8 and Siglec-F is that both get internalized following ligand binding.^24–26 This makes it potentially problematic, without proper gating strategies and the use of additional eosinophil markers, to use surface expression Siglec-F to track eosinophils after systemic administration of Siglec-F antibody, even though this is frequently done.^27–30

Recently, transgenic mice have been developed in which human Siglec-8 is expressed on the surface of mouse eosinophils and where effective eosinophil depletion is seen following systemic administration of mouse anti-human Siglec-8 monoclonal antibody (mAb).^{14, 31} In humans, the most effective approaches for depleting eosinophils in vivo rely on antibody-dependent cellular cytotoxicity (ADCC) with humanized antibodies such as benralizumab or AK002, also called antolimab.^32–35 We therefore hypothesized that targeting of Siglec-8 with mouse IgG1 antibodies, rather than targeting Siglec-F with rat IgG antibodies, in mice transgenic for Siglec-8, will prove to be a more effective strategy for eliminating mouse eosinophils in vivo. If true, this could either be due to differences in mechanisms of ligand-induced cell death or because of differences in ADCC activity of the targeting antibody that is separate from of any ligation-induced death. Furthermore, we hypothesized that eosinophil tracking, by detecting either Siglec-8 or Siglec-F, whichever is not being targeted, will allow for more accurate assessment of blood and tissue depletion following mAb administration. Indeed, our experiments have identified potential pitfalls when attempting eosinophil depletion by targeting Siglec-F that are overcome by targeting Siglec-8.

2. Materials and Methods

2.1. Mice

Adult male and female mice on a C57/BL6 genetic background were used. SIGLEC8^Eo mice (Siglec8⁺F⁺ mice) were previously characterized by O’Sullivan et al.¹⁴ Siglec8⁺F⁺ mice were bred with SiglecF^−/− mice²² (a generous gift of Dr. Ajit Varki, University of California San Diego, back-crossed here to C57/BL6 (wild type, WT) mice and re-derived to eliminate the Dock8 carrier mutation) generating Siglec8⁺F⁻ mice. For a few experiments, a related strain of C57/BL6 mice in which Siglec-8 was selectively expressed on mouse mast cells, not eosinophils, were also used.³⁶ Mouse genotyping done on tail snips was outsourced to TransnetYX (Cordova, TN). Surface expression of Siglec-F and Siglec-8 on eosinophils was determined by flow cytometric analysis of a blood sample prior to using mice in in vivo assays (see additional methods below). As a control, SIGLEC8^LSL (lacking EoCre) age- and sex-matched littermate mice were used. Mouse studies were performed with the approval of the Institutional Animal Care and Use Committees of Northwestern University (protocol number IS00007627).

2.2. Eosinophil differentiation from bone marrow precursors and cell imaging

Eosinophils were generated from the bone marrow of WT, Siglec8⁺F⁺, Siglec8⁺F⁻ and SiglecF^−/− mice following a protocol previously described by Dyer et al.³⁷ The surface expression of Siglec-F, Siglec-8 and CCR3 on bone marrow cells throughout the differentiation process was measured with flow cytometry as done previously.¹⁴ Cell viability was assessed either with DAPI (ThermoFisher Scientific) or Ghost fixable viability dye (Tonbo Biosciences, San Diego, CA). Mature eosinophils on day 14 of the differentiation protocol were used for functional assays or apoptosis testing. Cytospins of mature eosinophils were stained with a Diff- Kwik™ stain set (Shandon, ThermoFisher Scientific, Waltham, MA) and imaged on an Olympus DSU microscope (Olympus, Tokyo, Japan).

2.3. Flow cytometric analyses

Gating and Siglec surface detection

Single cell suspensions of peripheral blood, spleen and bone marrow were depleted of erythrocytes by hypotonic lysis. FcR receptors on cells were blocked on ice using 1 μg/mL of anti-mouse CD16/CD32 (BD Biosciences, San Jose, CA). Unless otherwise stated, eosinophils in blood, spleen and bone marrow were gated as single cells, viable, CD45⁺ (PE-Cy7, mAb clone 30-F11, Biolegend, San Diego, CA), CD11b⁺ (AF700, mAb clone M1/70, ThermoFisher Scientific), CCR3⁺ (FITC, J073E5, Biolegend), high SSC cells and presented as percentage of CD45⁺ cells (see representative gating strategy in Supplementary Figure 1A. Dead cells were excluded by DAPI. Anti-mouse Siglec-F PE (mAb clone E50-2240, rat IgG2a, BD Biosciences) was used to detect Siglec-F surface expression following depletion with anti-mouse Siglec-F (mAb clone 238047, rat IgG2a, R&D Systems, Minneapolis, MN). In addition, to determine whether these two anti-Siglec-F antibodies recognized similar or different epitopes on Siglec-F, eosinophil suspensions were preincubated with rIgG isotype control or unconjugated anti-Siglec-F (mAb clone 238047) at different concentrations for 20 min on ice to prevent internalization. Following a washing step, cells were stained with antibodies used to distinguish eosinophils as listed above including an anti-Siglec-F PE detection mAb clone (E50-2440 or REA798, a recombinant human IgG1 mAb from Miltenyi Biotec, Auburn, CA). Geometric mean of Siglec-F PE on eosinophils was analyzed by flow cytometry. In other experiments, the cell suspension was left unstained or was incubated with 5 μg/mL of one or both unconjugated anti-mouse Siglec-F mAb clones (E50-2440 and 238047) for 20 min. Following a washing step, a secondary polyclonal anti-rIgG conjugated with AF-488 (H+L, goat anti-rat, ThermoFisher Scientific) was added to all samples in order to detect bound anti-Siglec-F antibodies. Cells were washed and resuspended in buffer containing DAPI. Geometric mean of AF-488 (FITC) on viable high SSC cells was acquired with flow cytometry to analyze the additive effect of different mAb clone labeling.

Eosinophil CD11b upregulation assay

Mature bone marrow-derived eosinophils from 4 different genotypes mentioned above were seeded in flat bottom 96 well plates (200,000 cells/well) and primed with 30 ng/mL of rmIL-5 for 4 h. Vehicle, mIgG1 (mAb MOPC-21, Tonbo Biosciences), anti-Siglec-8 (mouse IgG1 mAb clones 2E2 and 2C4,¹⁹ or anti-Siglec-F (mAb clone E50-2440, BD) were added to cells in a final concentration of 2.5 μg/mL for 2 h. Eosinophils were stained with anti-mouse CD11b-AF700 (mAb clone M1/70, 1:20, ThermoFisher Scientific) and CD11b expression levels were analyzed by flow cytometry.

Quantification of Siglec receptors on eosinophils

Quantitative analysis of Siglec-F and Siglec-8 receptors on mouse eosinophils in peripheral blood and spleen from various strains of mice was performed with Quantum™ Simply Cellular® microspheres (Bangs Laboratories, Fishers, IN) according to manufacturer’s protocol using anti-Siglec-F PE (mAb clone E50-2440) and anti-Siglec-8 (mAb clone 2C4-AF647).³⁸ The channel values and geometric means from five distinct populations of beads yielded a calibration curve, which, analyzed with QuickCal® software, enabled the quantitative analysis of Siglec-F and Siglec-8 receptor numbers per cell.

2.4. Chemotaxis and calcium flux assays

The in vitro chemotaxis assays were performed in HTS Transwell plates (Corning) with a 5 μm pore size polycarbonate membrane as previously described.³⁷ Mature bone marrow-derived eosinophils were diluted to a concentration of 10⁶ cells/mL in recombinant mouse IL-5 free differentiation medium. Recombinant mouse eotaxin-2 (R&D Systems) or platelet activating factor (PAF, Sigma, St. Louis, MO) was prepared in 10-fold dilutions using the same differentiation medium. Then, 100 μL of cell suspension was added to the upper well and 100 μL of chemoattractant or media was added to the bottom well. Cell migration was performed for 1 h at 37°C. Migrated eosinophils obtained from the bottom compartment were enumerated by flow cytometric counting and normalized to vehicle (media without chemoattractant) control.

Calcium flux of mature bone marrow-derived eosinophils was measured with a Fluo-4 Direct™ Calcium Assay kit (ThermoFisher Scientific) following stimulation with mouse eotaxin-2. Assays were performed according to the manufacturer’s instructions. Mature eosinophils were loaded with calcium dye at a concentration of 2.5x10⁶ cells/mL for 60 min at 37 °C. Cells were washed and resuspended in PBS containing Ca²⁺. Changes in Ca²⁺ were detected in the FITC channel with an LSR II flow cytometer. Baseline fluorescence was acquired for 30 s, after which cells were stimulated either with vehicle or mouse eotaxin-2 in two different concentrations to induce Ca²⁺ flux.

2.5. In vitro apoptosis assay

Viability of bone marrow-derived eosinophils was assessed with FITC-labelled Annexin V (BD Biosciences) and DAPI. Eosinophils were preincubated with anti-Siglec-F (clone E50-2440 or 238047), anti-Siglec-8 mAb (2C4) or saporin-conjugated antibodies and their respective isotype controls as indicated in the figure legend. Saporin conjugated anti-Siglec-F (clone 9C7, rat IgG2b, a generous gift of Dr. James Paulson, The Scripps Research Institute, La Jolla, CA)¹⁸ or anti-Siglec-8 (clone 2C4) or their isotype controls were custom produced by Advanced Targeting Systems (San Diego, CA).²⁵

2.6. In vivo treatment of mice for studies of eosinophil depletion

In the acute eosinophil depletion protocol, Siglec8⁺F⁻, Siglec8⁺F⁺ or littermate control mice were given a single i.p. injection of the indicated amount of anti-Siglec mAb (238047, 2C4, 2E2 or 2E2 F(ab’)₂, the latter generously provided by Dr. Bradford Youngblood, Allakos, Inc., Redwood City, CA) anti-Siglec mAb conjugated to saporin (9C7, 2C4) or matched isotype controls. Blood was collected at baseline by cheek bleed into EDTA-containing tubes (or in heparinized capillary tubes) and after 24 h - 48 h yielding paired blood data for each mouse. For the prolonged model, mice (including Siglec-8+ eosinophil or mast cell mice, see text) were treated with anti-Siglec mAb or matched isotype control i.p. every two days. Before each consecutive treatment, a blood sample was obtained to follow eosinophil population percentages and Siglec surface expression over time. At the end of the protocol, mice were sacrificed, spleen and bone marrow were collected, then processed as described previously to generate a single cell suspension for analysis of cell depletion.¹⁴ A total of 50 μL of blood was collected separately into EDTA-coated tubes and absolute eosinophil numbers were determined after staining with Discombés fluid.³⁹ For experiments examining depletion with anti-Siglec-8 (clone 2C4, mouse IgG1 mAb recognizing domain 1 of the extracellular region of Siglec-8), a different anti-Siglec-8-AF647 mAb (clone 1H10, mouse IgG1 recognizing domain 3 of the extracellular portion of Siglec-8³³ and thus not cross-reactive with mAb 2C4, also generously provided by Dr. Bradford Youngblood) was used to detect residual Siglec-8 surface expression. Fluorescence minus one (FMO) controls were used to determine proper population gating. Samples were acquired using a BD LSRII flow cytometer (BD Biosciences). Data analysis was performed using FlowJo v.10 (TreeStar, Inc, Ashland, OR).

2.7. Statistical analysis

Statistical analysis was preformed using the GraphPad Prism™ 6 software (GraphPad Software, Inc, CA, USA). Differences between groups were tested by one-way or two-way ANOVA followed by Dunnett’s or Sidak’s posttest as indicated in the figure legends. When comparing two sets of data paired or unpaired Student’s t-test was used as appropriate. All results are presented as mean ± standard error of the means. P values ≤ 0.05 were considered significant.

3. Results

3.1. Phenotypic and functional characterization of WT and Siglec-8/Siglec-F-altered eosinophils differentiated from bone marrow of mice

In order to validate and compare eosinophils from mice with different SIGLEC8 and SIGLECF phenotypes beyond tail-snip genotyping, mouse eosinophils were differentiated from bone marrow cells ex vivo^{37, 40} as shown in Figure 1A. The development of eosinophils was assessed by measuring the surface expression of Siglec-8, Siglec-F and CCR3 by direct immunofluorescence and flow cytometry. As expected, the expression profile and kinetics of these surface markers on eosinophils differentiated from wild type (WT) and Siglec8⁺F⁺ mice mirrored published data (Figure 1B–C).^{14, 37} Siglec-F and Siglec-8 expression followed a similar kinetic and were detected on the cell surface prior to CCR3. Also as expected, eosinophils differentiated from bone marrow of Siglec8⁺F⁻ animals expressed Siglec-8, but lacked Siglec-F (Figure 1D), while SiglecF^−/− eosinophils did not express Siglec-F or Siglec-8 (Figure 1E). A representative overlay of Siglec-8 and Siglec-F expression from mature eosinophils of all four Siglec phenotypes is shown in Figure 1F. Eosinophils were considered mature by day 14 of the protocol and exhibited typical mouse eosinophil morphology with ring-shaped nuclei and eosin-stained granules (Figure 1G).

Figure 1.

Phenotypic characterization of bone marrow derived eosinophils. Eosinophils from mice of different genetic backgrounds were differentiated from bone marrow hematopoietic stem cells according to protocol presented in (A). Surface expression of Siglec-8, Siglec-F and CCR3 was followed over time by flow cytometry in differentiating cells from WT (B), Siglec8⁺F⁺ (C), Siglec8⁺F⁻ (D), SiglecF^−/− (E) mice. Data from 4-5 mice per genetic model are shown. (F) Mouse Siglec8⁺F⁻ eosinophils stained with Diff-Qwik on day 14 of differentiation. Siglec-F (G-H) and Siglec-8 (I-J) expression on mouse eosinophils was confirmed and quantified in peripheral blood and spleen of animals. Data from 2- 6 mice per genetic model are shown.

The expression of Siglec-8 and Siglec-F was further quantified on eosinophils in peripheral blood and spleen of animals. Similar to bone marrow-derived eosinophils, eosinophils in blood and spleen of Siglec8⁺F⁻ animals did not express Siglec-F (Figure 1 H–I). Expression of Siglec-8 was undetectable in blood and spleen of WT animals (Figure 1 J–K). The quantification of Siglec receptors showed approximately 5-10 fold higher numbers of Siglec-8 receptors per eosinophil (under the control of the CAG promotor)¹⁴ compared to numbers of Siglec-F receptors per eosinophil, and about 5 times more Siglec-8 receptors per cell than what has been reported on human blood eosinophils.³⁴ Finally, transgenic manipulation of Siglec receptors did not result in any significant alterations of eosinophil responses to eotaxin-2 or PAF, such as chemotactic responses and calcium flux, as shown in Supplementary Figure 2.

3.2. Siglec-8 engagement increases CD11b expression on bone marrow-derived eosinophils

We next set out to investigate whether Siglec-8 expressed on the surface of mature mouse bone marrow eosinophils is functional. Previously published data showed that Siglec-8 mAb engagement induced rapid upregulation of CD11b on the surface of human eosinophils.²¹ Indeed, preincubation with two different clones of Siglec-8 mouse IgG1 mAb, 2C4 and 2E2, induced a modest, but significant increase in CD11b expression in mouse eosinophils expressing Siglec-8 (Figure 2C–D), albeit less than that seen with human eosinophils,²¹ but did not increase CD11b expression in eosinophils lacking Siglec-8 (Figure 2A–B). In contrast, Siglec-F engagement (with rat IgG2a mAb E50-2440) under the same conditions had no effect on CD11b expression (Figure 2 A, C). Therefore, the CD11b upregulation mediated by Siglec was functional for Siglec-8 but not for Siglec-F in mouse eosinophils.

Figure 2.

CD11b expression on bone marrow-derived eosinophils following Siglec antibody engagement. Mature bone marrow derived eosinophils were primed with 30 ng/mL of recombinant mouse IL-5 for 4 h and afterwards incubated with vehicle or 2.5 μg/mL of isotype control (mIgG1), anti Siglec-8 (2C4 or 2E2) or anti Siglec-F (E50-2440) for 2 h. CD11b expression was evaluated by flow cytometry and normalized to vehicle control. (A) WT eosinophils, (B) SiglecF^−/− eosinophils, (C) Siglec8⁺F⁺ eosinophils and (D) Siglec8⁺F⁻ eosinophils. Data from 4-5 biological replicates per genetic model are presented and analyzed with one-way ANOVA followed by Dunnett’s posttest (antibody treated versus isotype control).

3.3. Incubation with Siglec antibodies in vitro results in weak cell death responses in bone marrow-derived eosinophils

Engagement of Siglec-8 on human eosinophils in vitro can induce robust cell death responses, while this is much less remarkable for engagement of Siglec-F on mouse eosinophils.^{8, 19–23} Therefore, we examined whether Siglec mAb ligation leads to cell death of bone marrow-derived eosinophils. In accordance to previously published data,²³ Siglec-F mAb (clone E50-2440) induced a modest, but significant, WT eosinophil cell death in vitro after 48 h (Figures 3A and B). As expected, there were no changes in cell viability following Siglec-F mAb incubation of Siglec-F^−/− eosinophils (data not shown). Incubation of Siglec8⁺F⁺ eosinophils with either Siglec-8 (2C4) or Siglec-F (E50-2440) mAb lead to an equally modest decrease in cell viability (Figure 3C) highlighting distinct differences between Siglec-8 engagement on mouse compared to human eosinophils.^{8, 19–23} A similarly small effect on cell death was observed following 24 h incubation of Siglec8⁺F⁻ mouse eosinophils with Siglec-8 mAb (Figure 3D). Moreover, even the addition of antibodies conjugated to saporin for 24 h did not enhance the cell death of mouse eosinophils (Figure 3E), unlike what has been observed with human eosinophils,²⁵ suggesting that, at least for bone marrow-derived mouse eosinophils, Siglec-8 engagement results in much less cell death than with human eosinophils, even when involving internalization of toxic payloads.

Figure 3.

Effect of Siglec engagement in vitro on eosinophil viability. Cell viability of mature bone marrow eosinophils was assessed with Annexin V / DAPI staining. (A) WT eosinophils were pretreated with isotype control or anti Siglec-F (E50–2440, 1.25 – 10 μg/mL) for 48 h. (B) Representative histograms of Annexin V / DAPI staining of eosinophils in (A) treated with vehicle or isotype control and anti Siglec-F at 10 μg/mL. (C) Siglec8⁺F⁺ eosinophils were pretreated with 1.25 – 10 μg/mL isotype control, anti Siglec-F (E50-2440) or anti Siglec-8 (2C4) for 48 h. Data from 4 independent experiments performed in duplicates are shown and analyzed with two-way ANOVA followed by Sidak’s posttest. ** p<0.01 anti Siglec-F versus isotype control; * p<0.05 anti Siglec-F versus isotype control, # p<0.05 anti Siglec-8 versus isotype control. (D) Siglec8⁺F⁻ mouse eosinophils were pretreated with 10 μg/mL isotype controls (mouse IgG1 or rat IgG2), anti Siglec-8 (2C4) or anti Siglec-F (E50-2440 or 238047) for 24 h. (E) Siglec8⁺F⁻ mouse eosinophils were pretreated with 10 μg/mL saporin-conjugated isotype controls (mouse IgG1 or rat IgG2), anti Siglec-8 (2C4) or anti Siglec-F (9C7) antibodies for 24 h

3.4. Systemic administration of Siglec-F antibody does not deplete eosinophils after a single dose, but does alter Siglec-F receptor surface expression on eosinophils

After observing very modest Siglec mAb-mediated cell death in vitro (Figure 3), the effect of Siglec-F administration on eosinophil depletion in vivo was explored. The effect of a single i.p. injection of a Siglec-F mAb (rat IgG2a clone 238047) on eosinophil percentages in blood and spleen (see representative gating strategy in Supplementary Figure 1A). After a single injection of Siglec-F mAb (15 μg) no significant decrease in eosinophils from baseline was observed when compared to isotype control (Figures 4A and B). Furthermore, there was no reduction in manual eosinophil cell counts performed on blood samples as measured following staining with Discombe’s fluid (Supplementary Figure 1D). This was unexpected, because an identical commercially available rat IgG2a Siglec-F mAb was previously used in studies to acutely deplete eosinophils.^{28, 41} In contrast to our study design, these investigators used Siglec-F mAb (clone E50-2440) after depletion with Siglec-F mAb (clone 238047) in order to detect eosinophils. However, following administration of the Siglec-F mAb, the expression of surface Siglec-F on circulating eosinophils was drastically decreased (Figure 4C), consistent with either epitope overlap between these two antibodies and/or mAb-mediated receptor internalization as seen in vitro.²⁴ Siglec-F surface expression was also significantly reduced on eosinophils from the spleen of Siglec-F mAb-treated animals (Supplementary Figure 1C). As shown with representative flow cytometry data in Supplementary Figure 1B, eosinophil percentages using Siglec-F as a marker are artifactually only about half of that detected when an additional eosinophil cell surface marker, CCR3, is included in the gating strategy (following pre-gating for single/viable/CD45⁺/CD11b⁺ granulocytic cells).

Figure 4.

Single administration of Siglec-F mAb in vivo decreases the surface expression of Siglec-F on eosinophils, but does not effectively deplete eosinophils. Siglec8⁺F⁺ mice received a single i.p. injection of 15 μg of isotype control or anti-Siglec F (238047). (A) Percentage of eosinophils (analyzed as % of CD45⁺ cells, see representative gating strategy in Supplementary Figure 1) at baseline and 48 h after the administration of anti Siglec-F. (B) Eosinophil percentage in blood of isotype treated compared to anti Siglec-F treated animals. Data from 2 independent experiments are shown. n=5-9, ns (non-significant), unpaired Student’s t-test. (C) Geometric mean of Siglec-F PE (clone E50-2440 used for detection) on blood eosinophils before and 48 h after anti Siglec-F (clone 238047) administration. (D) Additivity trial of two Siglec-F mAb clones. Following red blood cell lysis, mouse blood leukocytes were incubated with vehicle or anti Siglec-F (clone 238047 or clone E50-2440, 5 μg/mL) or both (5 μg/mL each). A secondary FITC-labelled polyclonal anti-rat IgG was then added to detect bound primary antibodies. 2^nd Ab=vehicle treated cells incubated only with secondary antibody. (E-F) Following red blood cell lysis, mouse blood leukocytes were incubated with isotype control or anti Siglec-F (clone 238047) at the indicated concentrations for 20 min, washed, and then incubated with a different anti Siglec-F-PE clone. (E) Geometric mean of Siglec-F PE (staining clone E50-2440) is quantified with representative histograms of anti Siglec-F pretreated cells shown in (F). Mean ± SEM from 2-3 independent experiments is shown. Data were analyzed with two-way ANOVA followed by Sidak’s posttest.*** p<0.001 anti Siglec-F (238047) versus isotype treated cells.

In order to further study the interaction of these two mAb clones, an antibody additivity binding assay was performed, where whole blood leukocytes were incubated with either of the two clones alone, or both, and bound antibody was detected with a fluorochrome-conjugated secondary anti-rat polyclonal antibody. As shown in Figure 4D, the amount of secondary antibody binding was the same with preincubation of either mAb alone, or both Siglec-F antibodies, suggesting that the two mAb clones bind to a cross-reactive epitope. In fact, a progressive, dose-dependent decrease in anti-rat antibody binding was seen, suggesting that the binding of the first Siglec-F mAb interfered with the ability of the second mAb to bind (Figures 4E and F). This was not due to receptor internalization by the first mAb, because the mAb incubations were performed on ice to block internalization.²⁴ Finally, this effect was also observed using a third clone of Siglec-F mAb (REA798, human IgG) for detection (Supplementary Figure 1E).

3.5. Transgenic Siglec- 8 as an alternative to endogenous Siglec-F as a surface marker for tracking mouse eosinophil numbers in blood and spleen following Siglec-F antibody administration in vivo

To further test the extent of Siglec-F mAb-mediated eosinophil depletion and the kinetics of its surface expression on remaining eosinophils, a prolonged model of Siglec-F mAb (clone 238047, rat IgG2a isotype) administration was performed as shown in Figure 5A. During the course of the protocol, the surface expression of Siglec-F and Siglec-8 on eosinophils was followed (identified by the previously mentioned gating strategy including CCR3 positivity) in Siglec8⁺F⁺ animals. In Siglec-F mAb-treated animals, the percentage of eosinophils as assessed by detectable Siglec-F⁺ cells decreased significantly after a single dose, and remained low with subsequent doses. This was remarkably lower than, and discordant with, the percentage of blood eosinophils as assessed by either CCR3⁺ or Siglec-8⁺ cell quantification, which were minimally altered by Siglec-F mAb administration (Figure 5B). This discordance was further reflected in the levels of each of these surface markers, as assessed by flow cytometry. The geometric means for Siglec-F decreased in Siglec-F mAb treated animals when compared to control rat IgG treated mice (Figure 5C), while the geometric means for CCR3 (data not shown) and Siglec-8 were comparable between the two mAb treatment groups (Figure 5D). Furthermore, repeated administration of Siglec-F mAb significantly decreased the geometric mean of Siglec-F-PE mAb labeling of eosinophils in the spleen, but did not alter the geometric mean of Siglec-8 (Figures 5 E and F). These data strongly suggest that following systemic administration of Siglec-F mAb, the use of Siglec-F alone to track eosinophils significantly underestimates the number of eosinophils remaining in blood and spleen. By day 7 of the prolonged depletion protocol (4 repeated injections of Siglec-F mAb), eosinophils were partially and significantly depleted from spleen and blood of mice compared to control rat IgG treated animals (Supplemental Figure 3A and B). This was confirmed by performing manual microscopy counts from cytospins of the same blood samples (Supplemental Figure 3C).

Figure 5.

Siglec-8 is a more reliable marker for detecting eosinophils than Siglec-F following systemic administration of Siglec-F antibody. (A) protocol used for the prolonged model of anti Siglec-F treatment. (B) Eosinophil percentage in blood samples as determined by Siglec-F, Siglec-8 or CCR3 positivity over time. Mean ± SEM of 3 biologic replicates from 1-2 independent experiments are shown. **p<0.01 Siglec-F versus CCR3 as a marker, two-way ANOVA with Dunnett’s posttest. (C-D) Geometric mean of Siglec-F-PE or Siglec-8-AF647 labeling of eosinophils in isotype versus anti Siglec-F treated animals over time. ns=non-significant, *p<0.05, **p<0.01, ***p<0.001 analyzed with two-way ANOVA followed with Sidak’s posttest. (E-F) Geometric mean of Siglec-F-PE or Siglec-8-AF647 labeling of eosinophils from spleens of isotype and anti Siglec-F treated animals. Data from 1-2 independent experiments are shown and analyzed with unpaired Student’s t-test. **p<0.01, ns (non-significant, p>0.05).

3.6. Siglec-8 antibody administration leads to rapid and profound eosinophil depletion after a single injection

Having only obtained partial depletion of eosinophils when targeting Siglec-F in vivo, even after multiple injections of Siglec-F mAb, similar experiments were performed using Siglec-8 mAb (mAb clone 2C4, mouse IgG1). Remarkably, a single 10 μg i.p. injection was sufficient to cause near complete depletion of eosinophils from blood of Siglec-8⁺ mice as assessed by gating on CCR3⁺ or Siglec-F⁺ cells (Figure 6A). Administration of saporin-conjugated 2C4 (2C4-SAP) showed a similar, perhaps even more consistent and profound depletion effect (Figure 6B). A single dose of 2C4 with or without saporin conjugation also partially depleted eosinophils from spleens and bone marrow of Siglec-8⁺ mice (Figure 6 C–E). In order to elucidate the minimal dose needed, a range of mAb doses given as a single injection of 2C4-Saporin per mouse were tested. Paired data in blood (0 h-24 h) and percentages in spleen indicated that a 2.5 μg dose was the minimum dose needed for optimal depletion of eosinophils, because higher doses up to 10 μg/mouse were just as effective (S4).

Figure 6.

A single dose of 2C4 or 2C4-saporin effectively depletes eosinophils in vivo. A single application of anti Siglec-8 (with or without saporin) effectively depletes eosinophils in vivo. (A-B) Eosinophil percentages at baseline and 24 h following a single i.p. injection of 10 μg anti Siglec-8 (2C4 or 2C4-saporin) or isotype control (mIgG1 or mIgG1-saporin). Data from 5-7 mice per treatment are shown and analyzed with two-way ANOVA followed by Sidak’s posttest (0 h versus 24 h). Eosinophil percentages in spleen (C-D) and bone marrow (E) 24 h after treatment are shown and compared to isotype control with unpaired Student’s t-test. (F-G) Duration of anti Siglec-8-mediated eosinophil depletion was followed with repeated blood sampling and is represented as eosinophil percentages in blood over time. Means ± SEM of 3 mice per treatment group are shown and analyzed with two-way ANOVA followed by Dunnett’s posttest (anti Siglec-8 versus isotype control treated mice).

To explore the duration of depletion, and to facilitate using 2C4 in Siglec8⁺ mice as a method of transient eosinophil depletion, mice received a single injection of 2C4 (Figure 6F) or 2C4-saporin (Figure 6G) and circulating eosinophil percentages were determined over time. Efficient depletion, especially with 2C4-saporin, was observed for up to 2 days, with recovery, albeit <50%, beginning by day 3 after injection.

3.7. Repeated injections of 2C4 result in profound and selective eosinophil depletion from blood, spleen and bone marrow

To further examine the extent of Siglec-8 mAb-mediated eosinophil depletion, 2C4 mAb was tested in a repeated-dose prolonged model, as shown in Figure 7A. Mice were injected with 2C4 mAb or isotype control and eosinophil percentages were followed by tracking CCR3, Siglec-F or Siglec-8. In contrast to Siglec-F targeting results, the eosinophil population was markedly depleted regardless of the gating strategy used (Figure 7B). Multiple injections of 2C4 were more effective at reducing eosinophil numbers in bone marrow and spleen (Figure 7C–D) compared to a single injection (Figure 6D–E). No other cell types were depleted, and repeated injections were well-tolerated (data not shown). In order to confirm that the observed effect was due to specific engagement of Siglec-8 on eosinophils, additional control experiments were performed in EoCre⁻ animals (lacking Siglec-8) and in Siglec8+MCPT5+ animals where Siglec-8 is expressed on mast cells instead of eosinophils.³⁶ As shown in Supplemental Figure 5, 2C4 mAb did not alter eosinophil numbers in these control experiments.

Figure 7.

Repeated dosing of 2C4 mAb effectively depletes eosinophils in vivo from multiple body compartments. (A) protocol used for anti Siglec-8 (2C4) treatment. (B) Eosinophil percentage in blood samples as determined by Siglec-F, Siglec-8 or CCR3 positivity over time. Mean ± SEM of 3 biologic replicates. Two-way ANOVA with Sidak’s posttest. ***p<0.001 CCR3⁺ population versus time zero baseline, §§§ p<0.001 Siglec-F⁺ population versus time zero baseline, ### p<0.001 Siglec-8⁺ population versus time zero baseline. (C) Percentage of eosinophils in bone marrow on day 6 of the protocol. (D) Percentage of eosinophils in spleen on day 6 of the protocol. Differences between isotype treated animals (n=2) and 2C4 treated animals (n=3) were evaluated with unpaired Student’s t-test.

3.8. Mechanism of 2C4-mediated eosinophil depletion in vivo: role of the Fc region of the mIgG1

Finally, pursuing the cause for discordance between the in vitro and in vivo effects seen with mouse IgG1 anti-Siglec-8 versus rat IgG2a anti-Siglec-F antibodies, the contribution of Fc-mediated depletion, such as ADCC, was investigated. Mice were injected with F(ab’)₂ fragments of 2E2 mouse IgG1 anti-human Siglec-8 mAb, previously shown to induce human eosinophil death as effectively as the intact antibody.²¹ Using the same gating strategies as for Figure 7, the intact 2E2 mAb was found to deplete mouse eosinophils from Siglec-8⁺ mice in a manner comparable to 2C4. In contrast, the administration of an identical dose of F(ab’)₂ fragments of 2E2 had no depleting effect (Figure 8A–C). The F(ab’)₂ fragments of 2E2 were able to bind to mouse eosinophils in vivo because they internalized Siglec-8 (Figure 8D, as detected by flow cytometry using a non-cross-reactive clone 1H10 Siglec-8 mAb). Taken together, these data demonstrate the distinctive mIgG1 Fc dependency of Siglec-8 mAb-mediated eosinophil depletion in vivo.

Figure 8.

The effect of 2C4-mediated eosinophil depletion in vivo is dependent on the Fc region of the mIgG1. Siglec8⁺F⁺ mice were given a single i.p. injection of 10 μg isotype control, anti Siglec-8 (2C4 or 2E2) or the F(ab’)₂ fragment of 2E2. (A) Eosinophil percentages (CCR3⁺Siglec-F⁺) were determined from blood samples at baseline and 24 h after treatment. Data from 5-7 biological replicates per treatment are shown and analyzed with two-way ANOVA followed with Sidak’s posttest (baseline vs 24 h). (B) Eosinophil percentages in spleen 24 h after treatment are presented. Data are analyzed by one-way ANOVA followed by Tukey’s posttest. (C) Eosinophil percentages in blood of 2E2 and F(ab’)₂ 2E2 treated animals are shown and compared with unpaired Student’s t-test. (D) Geometric mean of Siglec-8-AF647 (1H10 staining clone) labeling of eosinophils at baseline and 24 h following treatment with non-cross-reactive 2E2 F(ab’)₂. Data from 5 biologic replicates are shown and analyzed with paired Student’s t-test.

4. Discussion

In the present study we describe a novel strain of Siglec8⁺F⁻ mice generated from crossing Siglec8⁺F⁺ and Siglec-F null strains of mice. We demonstrate that eosinophils can be differentiated from bone marrow of these mice and that these eosinophils exhibit comparable functional responses to external migratory stimuli (Figure 1 and Supplementary Figure 2). Moreover, eosinophils differentiated from Siglec8+ mice increase their expression of CD11b on their surface following Siglec-8 (but interestingly not Siglec-F) antibody engagement (Figure 2), an indication that the Siglec-8 present on the cell surface is functional and capable of downstream signaling.

It has previously been reported that antibody engagement of Siglec-8 on human eosinophils and its closest functional paralog Siglec-F on murine eosinophils, results in cell death in vitro. Cell death of human cytokine-primed eosinophils was quite marked and shown to be dependent on ROS production,^19–21 while targeting Siglec-F on mouse eosinophils resulted in very modest levels of caspase-dependent apoptosis.^{8, 22, 23} Our data show that antibodies targeting Siglec-8 on mouse eosinophils from transgenic mice have a modest effect on cell viability in vitro. These modest in vitro murine eosinophil responses (cell death and CD11b upregulation, Figures 2 and 3) might be explained by the distinct mechanisms of mouse and human eosinophil activation and ROS production.⁴² Importantly, modest in vitro death responses bring into question the mechanisms of eosinophil reductions sometimes seen following Siglec-F mAb administration in vivo.^7–16

Previously published work has demonstrated that antibody binding to Siglec-F and Siglec-8 induces their rapid internalization in vitro and in vivo.^{14, 24, 25} Additionally, we now demonstrate that there is complete cross-reactivity among several frequently used commercially available Siglec-F antibodies. For instance, the binding of one Siglec-F mAb clone can completely prevent the binding of another mAb clone. Taken together, these two phenomena can result in artifactual lowering of detectable Siglec-F levels on eosinophils, making the tracking of Siglec-F a fallible strategy for flow cytometric tracking of eosinophils following administration of Siglec-F antibodies. Instead, proper additional labeling and gating strategies that rely on detection of an alternative, non-targeted, specific eosinophil cell surface marker (e.g. CCR3 in WT mice) would be needed. Furthermore, one of the benefits of our available Sig8+F+ mouse strain is that it enables tracking of eosinophil numbers by means of detecting Siglec-8, even when Siglec-F antibodies have been administered. Since Siglec-8 in SIGLEC8^Eo mice is specifically expressed on the surface of eosinophils¹⁴ and its surface expression is not affected by Siglec-F targeting (Figure 4) it serves as an alternative flow cytometric marker to CCR3 for detecting eosinophils in murine blood and tissues. Furthermore, in the case of Siglec-8 targeting, a non-cross-reactive clone (1H10) is available that recognizes a separate receptor domain, enabling accurate tracking of Siglec-8 expression and internalization when Sigec-8 mAb is administered.

Using commercially available rat anti-mouse antibodies, whose IgG2a and IgG2b subclasses are not particularly effective at binding mouse FcɣR, systemic administration of anti Siglec-F did not result in robust reductions of eosinophil numbers after a single dose and only showed modest depletion effects after four repeated injections in a more prolonged model that resembled those used in prior publications, including those that may have relied on detection of Siglec-F+ cells without additional gating.^{16, 27, 41} This approach can be problematic as shown in Supplemental Figure 1B. A more accurate assessment of Siglec-F antibody-mediated eosinophil reductions was accomplished with gating strategies using CCR3 and/or Siglec-8 as markers of eosinophils and by performing manual eosinophil counts by microscopy (Figure 4 and 5).

Perhaps most remarkable in the present study was the finding that targeting Siglec-8 on eosinophils from Siglec8⁺F⁻ and Siglec8⁺F⁺ mice with a mouse IgG1 mAb, either alone or conjugated with the ribosomal toxin saporin, resulted in profound and sustained depletion in blood, spleen and bone marrow of mice (Figure 6–7). Siglec-8 mAb mediated-depletion was fully dependent on Siglec-8 expression on the eosinophil (see Supplemental Figure 5) without any detectable off-target effects against any other cells and was well-tolerated by the mice. The saporin-conjugated mAb was perhaps slightly more efficacious in reducing eosinophil numbers, suggesting that mAb internalization and toxin delivery was at least partially responsible for the depleting effect. While not compared directly, levels of Siglec-8 antibody-mediated eosinophil depletion were faster and showed similar or stronger effect as the use of iPHIL mice⁴³ or anti IL-5 treatment (approx. 50% reduction with 20x the amount of antibody).^{44, 45} More importantly from a mechanistic perspective, by using F(ab’)₂ fragments of mouse IgG1 Siglec-8 mAb, we demonstrated that the Fc portion of the mAb was necessary for eosinophil depletion (Figure 8). This strongly suggests that the greater eosinophil-depleting efficacy seen in vivo achieved via Siglec-8 targeting (with mouse IgG1) compared to anti-Siglec-F targeting (rat IgG2) is primarily, if not exclusively, due to Fc biology occurring in vivo via ADCC or ADCP. It remains possible that some of the differing effects seen with various Siglec-F mAb clones are related to their subclass (e.g., rat IgG2a, rat IgG2b) and their engagement, or lack thereof, of FcɣR on NK cells and others. Another possibility is that the 10-fold higher number of Siglec-8 receptors (≈100,000/cell) compared to Siglec-F receptors (≈10,000/cell) on the Siglec8⁺ mouse eosinophils (Figure 1) contributed to differences in mAb mediated depletion, although we did not study eosinophils under inflammatory conditions, where higher Siglec-F levels can be observed. Regardless, the enhanced efficacy and consistency of eosinophil depletion in blood and tissues with Siglec-8 mAb targeting is striking.

One shortcoming of the present work is that we have not yet clarified whether NK cells or some other cell type is responsible for the Fc-mediated effects of anti Siglec-8 eosinophil depletion. This could be tested with NK cell or FcɣR deficient mice, but they would need to be bred with Siglec8⁺F⁺ or Siglec8⁺F⁻ mice. In order to directly compare the Fc receptor-mediated effects of anti Siglec-F and anti Siglec-8 mAb in vivo, we would need to create and test a mouse IgG anti-Siglec-F mAb and a rat IgG2 anti-Siglec-8 mAb for their relative depletion activity in vivo, neither of which are currently available. One prior publication used a mouse anti-mouse Siglec-F mAb to deplete eosinophils in WT mice, but even then the depletion was incomplete.⁸ It is also unclear why repeated dosing is needed to maintain aggressive eosinophil depletion (Figure 6), as we did not actually measure the half-life of any of the mouse or rat mAb used in our experiments. Finally, we have yet to explore the Siglec-8 mAb targeting on mast cell numbers in mast cell specific Siglec-8 knock in mice.

Our present study may have important implications regarding past published work, where anti Siglec-F mAb was used to “deplete” eosinophils as a stand-alone method or in various disease models. For example, following the administration of anti Siglec-F mAb (be it mouse anti-mouse, rat anti-mouse or sheep polyclonal) the reductions of eosinophil numbers were significant, but transient or incomplete.⁸ Moreover, a number of studies used Siglec-F as a flow cytometric marker to track eosinophil depletion after administration of anti Siglec-F.^{27, 41} As we have shown in Figure 5 and Supplemental Figures 1 and 2, this strategy may overestimate the extent of eosinophil depletion, whether due to receptor internalization or simply antibody cross-reactivity between the antibody administered for depletion and a second anti Siglec-F antibody used for detection ex vivo. In order to comprehensively track eosinophil depletion, especially in tissues, methods other than flow cytometry can be used, such as tissue MBP staining.^9–12 Finally, given the potential for less eosinophil depletion than anticipated, one wonders whether the biology observed in some of these prior studies might either be due to effects on other Siglec-F expressing cells (e.g., macrophages) or if Siglec-F and its ligand(s) play a direct role in the observed effects. If the latter is the case, the use of Siglec-F null mice that have normal numbers of eosinophils²² might mirror the functional phenotype observed in anti Siglec-F treated WT mice. Finally, the lack of effects of “eosinophil depletion” by administration of anti-Siglec-F mAb seen in some studies might simply be due to incomplete eosinophil depletion.

In summary, our study is the first to describe a novel mouse strain of Siglec8⁺F⁻ eosinophils- a useful tool for studying human Siglec biology in preclinical models. We demonstrate that targeting Siglec-8 on the surface of these eosinophils in vivo results in rapid, selective, and extensive eosinophil depletion that was not simply due to Siglec-8 engagement and antibody-mediated Siglec-8 cross-linking, but instead due to mouse IgG1 Fc-mediated effects, likely ADCC or a similar Fc-dependent process. We incidentally identified potential shortcomings of using Siglec-F antibodies to deplete eosinophils in vivo, particularly with regards to solely using Siglec-F as a surface marker to detect eosinophils after anti Siglec-F antibody administration. In contrast, by using non-targeted surface receptors such as endogenous CCR3 or transgenic Siglec-8 as specific eosinophil markers, we were able to accurately track eosinophil numbers in blood and tissues over time using flow cytometry, highlighting some important advantages of these novel Siglec-8 knock-in strains of mice.

Supplementary Material

Supp Figure 2

Supplementary Figure 2. Eosinophils differentiated ex vivo from the bone marrow of various Siglec genotype mice respond comparatively to different migratory stimuli. Mature eosinophils (day 14 of the differentiation process) were collected, washed and used in assays. (A-B) Chemotactic index (number of eosinophils migrated towards chemoattractant / number of eosinophils migrated towards vehicle control) of eosinophils with different Siglec-F and Siglec-8 surface expression patterns migrating towards the indicated concentrations of mouse eotaxin-2 (A) or platelet activating factor (PAF) (B). Data from 3-5 biological replicates (each condition performed in duplicates) per genotype are shown. Data was analyzed with two-way ANOVA , followed by Dunnett’s posttest and was found not to be significantly altered between genotype groups. (C) Calcium flux measurements in eosinophils differentiated from Siglec8⁺F⁻ mice. Eosinophils were loaded with calcium indicator dye and baseline signals were acquired for 30 s. Cells were then stimulated with either 10 or 30 nM mouse eotaxin-2 or with vehicle. Calcium responses were recorded with flow cytometry and normalized to baseline set at 1. Mean ±SEM two independent experiments is shown.

Supp Figure 1

Supplementary Figure 1. Representative and comparative eosinophil gating strategies showing potential pitfalls after Siglec-F antibody administration in vivo. (A) Representative gating strategy used to determine eosinophil percentages. Unless otherwise stated, eosinophil percentages were presented as % of CD45⁺ cells and gated as single cells / viable (DAPI⁻)/ CD45⁺ / CD11b⁺ / CCR3⁺ high side scatter (hiSSC) cells. Cellular debris was gated out as shown by avoiding low FSC/SSC events. (B) Representative flow cytometric data of pregated CD11b⁺ population depicting Siglec-F positive cells at time point 0h and 48h after a single injection of 10 μg anti Siglec-F (clone 238047). CCR3 labeling and eosinophil percentage of the same sample at 48 h time point is shown on the bottom right. (C-D) WT mice were given a single i.p. injection (10 μg) of isotype control or anti Siglec-F (clone 238047) and eosinophils in blood samples were analyzed 48 h later. (C) Geometric mean fluorescence of a different Siglec-F-PE mAb labeling of eosinophils in the spleen. Data from one of two independent experiments are shown. (D) Eosinophil cell number as determined by manual microscopic counting of blood stained with Discombe’s fluid. (E) Following red blood cell lysis, blood leukocytes were incubated with isotype control or anti Siglec-F (clone 238047) at the indicated concentrations for 20 min, washed, and then a different anti Siglec-F-PE mAb (clone REA 798) was added and geometric mean fluorescence was determined. Mean ± SEM from 2-3 independent experiments is shown. Data were analyzed with two-way ANOVA followed by Sidak’s posttest.*** p<0.001 anti Siglec-F (238047) versus isotype treated cells.

Suppl Figure 3

Supplementary Figure 3. Repeated injections of anti Siglec-F into Siglec8⁺F⁻ mice partially deplete eosinophils. A prolonged model of anti Siglec-F or isotype control treatment was used (as shown in Figure 5A, 10 μg per mouse, every second day) and eosinophil percentages in blood (A) and spleen (B) on day 7 of the protocol were determined with flow cytometry. Data from 5-6 biologic replicates per group from 2 independent experiments are shown and analyzed using unpaired Student’s t test ( *p<0.05 ). (C) Eosinophil numbers in blood on day 7 of the protocol as determined by manual microscopic counting of blood stained with Discombe’s fluid. Data from 3 biologic replicates per group is shown. Data was analyzed with unpaired Studentś t-test and found to be non-significant (ns, p>0.05).

Suppl Figure 4

Supplementary Figure 4. Dose dependent depletion of eosinophils in vivo using 2C4-saporin. Siglec8⁺F⁻ mice received a single i.p. injection of 2C4-saporin (0.1 – 10 μg). Blood eosinophil percentages were evaluated at baseline and 24 h after injection (A-E). Change in eosinophil percentage was evaluated using paired Student’s t-test. *p<0.05, **p<0.01, ***p<0.001, ns=non-significant. (F) Eosinophil percentages in spleen 24 h after i.p. injection of 2C4-saporin analyzed with one-way ANOVA with Dunnett’s posttest. Data from 4-5 biological replicates per dose are shown.

Suppl Figure 5

Supplementary Figure 5. Siglec-8 expression on eosinophils is necessary for 2C4-saporin-mediated eosinophil depletion. Mice were given a single 5 μg i.p. injection of 2C4-saporin. (A-B) Eosinophil percentages at baseline and 24 h after injection were analyzed in Siglec8⁺EoCre⁺ mice (Siglec8⁺F⁺) and Siglec8⁺EoCre⁻ (Siglec8⁻F⁺ littermate controls). (A) Paired blood sample data from 5 biological replicates are shown and analyzed with two-way ANOVA followed with Sidak’s posttest, *p<0.05, ns (non-significant, p>0.05). (B) Eosinophil percentages in spleens harvested 24 h after injection from the same 5 animals per genotype as in (A) are shown and compared with unpaired Student’s t-test, *p<0.05. (C-D) Mice with Siglec-8 expressed on mast cells (Siglec8⁺MCPT5⁺ mice) were given a 10 μg i.p. injection of 2C4-saporin or mIgG1-saporin (isotype control). Eosinophil percentages in blood (C) and spleen (D) 24 h after treatment were analyzed with unpaired Student’s t-test, ns (non-significant, p>0.05).

Abbreviations

ADCC

antibody-dependent cellular cytotoxicity

ADCP

antibody-dependent cellular phagocytosis

FcR

receptors for the Fc portion of immunoglobulins

mAb

monoclonal antibody

Siglec

sialic acid-binding immunoglobulin-like lectin

WT

wild-type