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

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Presentation on theme: "Xin-Zi Tang, PhD, James B. Jung, BS, Christopher D.C. Allen, PhD "— Presentation transcript:

1 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 e6 (April 2019) DOI: /j.jaci Copyright © 2019 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 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  , e6DOI: ( /j.jaci ) Copyright © 2019 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 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  , e6DOI: ( /j.jaci ) Copyright © 2019 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 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  , e6DOI: ( /j.jaci ) Copyright © 2019 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 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  , e6DOI: ( /j.jaci ) Copyright © 2019 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 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  , e6DOI: ( /j.jaci ) Copyright © 2019 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 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  , e6DOI: ( /j.jaci ) Copyright © 2019 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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