Xin-Zi Tang, PhD, James B. Jung, BS, Christopher D.C. Allen, PhD

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Presentation transcript:

A case of mistaken identity: The MAR-1 antibody to mouse FcεRIα cross-reacts with FcγRI and FcγRIV Xin-Zi Tang, PhD, James B. Jung, BS, Christopher D.C. Allen, PhD Journal of Allergy and Clinical Immunology Volume 143, Issue 4, Pages 1643-1646.e6 (April 2019) DOI: 10.1016/j.jaci.2018.11.045 Copyright © 2019 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 The MAR-1 antibody stains cells from FcεRIα KO mice but not from FcRγc KO mice. Results of flow cytometric analysis of MAR-1 staining is indicated by representative flow cytometric plots and summary graphs. A and B, MAR-1 staining on moDCs from the lungs and mLNs 3 days after intranasal HDM (Fig 1, A) or poly I:C (Fig 1, B) exposure in wild-type (WT; black lines) and FcεRIα KO (red lines) mice. C, MAR-1 staining on blood basophils to confirm the FcεRIα KO genotype. D, MAR-1 staining on moDCs in the lungs and mLNs 3 days after HDM exposure in FcεRIα KO (red lines) and FcRγc KO mice (blue lines) compared with WT mice (black lines). E, MAR-1 staining on splenic red pulp macrophages, alveolar macrophages, and peritoneal macrophages from untreated mice of the indicated genotypes. gMFI, Geometric mean fluorescence intensity. F, MAR-1 staining on blood monocytes from untreated mice. In histograms dashed lines in Fig 1, A-C, and gray lines in Fig 1, D-F, denote isotype control staining. Data shown are representative of at least 2 independent experiments. In summary graphs each data point represents 1 mouse, and bars indicate means. Gating strategies to identify the represented populations of moDCs, basophils, macrophages, and monocytes are shown in Fig E1. Journal of Allergy and Clinical Immunology 2019 143, 1643-1646.e6DOI: (10.1016/j.jaci.2018.11.045) Copyright © 2019 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 MAR-1 cross-reacts with FcγRI and FcγRIV. A, Representative plots showing flow cytometric analysis of MAR-1 and FcγRI (CD64) costaining on moDCs in lungs and mLNs 3 days after HDM exposure. B, Representative plots showing flow cytometric analysis of MAR-1 costaining with FcγRI or FcγRIV on blood monocytes. For Fig 2, A and B, black dots denote isotype control staining, and red dots denote MAR-1 staining. The gating strategy to identify moDCs and monocytes is shown in Fig E1. C, Phoenix cells were transfected individually with the indicated Fc receptors along with a yellow fluorescent protein reporter gene. Transfected cells were gated by using yellow fluorescent protein expression and stained with MAR-1 or antibodies to FcγRI, FcγRII/III (CD16/32 clone 2.4G2), or FcγRIV (CD16-2), as indicated. Data shown are representative of at least 3 independent experiments. Journal of Allergy and Clinical Immunology 2019 143, 1643-1646.e6DOI: (10.1016/j.jaci.2018.11.045) Copyright © 2019 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Gating strategy. A, Gating strategy for splenic red pulp macrophages and peritoneal macrophages. B, Gating strategy for alveolar macrophages and moDCs from the lung. A similar gating scheme was used for moDCs in mLNs. C, Gating strategy for blood basophils and monocytes. Siglec-F, Sialic acid–binding immunoglobulin-like lectin F. Journal of Allergy and Clinical Immunology 2019 143, 1643-1646.e6DOI: (10.1016/j.jaci.2018.11.045) Copyright © 2019 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 The majority of MAR-1 staining is accounted for by FcγR expression. Flow cytometric analysis of Fc receptor expression and MAR-1 staining in splenic cells from wild-type (WT; black lines), FcRγc KO mice (blue lines), and mice lacking α chains from all 4 FcγR (FcγRα 4 KO) mice (brown lines). Histograms are representative of 3 mice of each genotype. Journal of Allergy and Clinical Immunology 2019 143, 1643-1646.e6DOI: (10.1016/j.jaci.2018.11.045) Copyright © 2019 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Myeloid cells in BALB/c and B6 mice show similar staining with MAR-1. Flow cytometric analysis of Fc receptor expression and MAR-1 staining in splenic cells from B6 (black lines), BALB/c (brown lines), and B6 FcRγc KO mice (blue lines). Histograms are representative of 3 mice of each genotype. Journal of Allergy and Clinical Immunology 2019 143, 1643-1646.e6DOI: (10.1016/j.jaci.2018.11.045) Copyright © 2019 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 MAR-1 does not deplete known macrophages, monocytes, or moDCs. Cell counts of blood basophils (A), Ly6C+ (B) and Ly6C− (C) monocytes, moDCs in the lung (D) and mLNs (E), and tissue macrophages (F) after MAR-1 or isotype control antibody treatment. In Fig E4, A-E, mice underwent 2 protocols for MAR-1 treatment. One set of mice were injected intraperitoneally with 5 μg of MAR-1 or isotype control antibody every 12 hours on days −2, −3, and −4 for a total of 6 times, and another set of mice were injected intravenously with 100 μg of MAR-1 or isotype control antibody once on day −3. All mice were immunized with HDM intranasally on day 0. Myeloid cell populations were assessed on day 3. In Fig E4, F, mice received 100 μg of MAR-1 or isotype control antibody 1 day before macrophage populations were assessed. Each data point represents 1 mouse, and horizontal bars indicate means. *P < .05 and ***P < .001. n.s., Not significant. Journal of Allergy and Clinical Immunology 2019 143, 1643-1646.e6DOI: (10.1016/j.jaci.2018.11.045) Copyright © 2019 American Academy of Allergy, Asthma & Immunology Terms and Conditions