![Figure 1. MPO deficiency enhances T-cell responses leading to increased skin DTH and AIA. (A-F) WT (n = 8) and Mpo−/− mice (n = 8) were immunized subcutaneously with OVA/CFA and CD4+ T-cell activation (A) and proliferation (B) assessed in LNs and spleen 6 days later (flow cytometry). (C) Concentrations of cytokines in 72-hour supernatants from OVA-stimulated splenocytes (ELISA). (D-E) Intracellular staining for IFN-γ and IL-17A in OVA-stimulated CD4-labeled splenocytes. Spleen cells were cultured (48 hours) in the presence of OVA (10 μg/mL) with brefeldin-A (5 μg/mL) added for the last 8 hours. CD4-labeled cells were then stained intracellularly for IFN-γ and IL-17A. The percentage of CD4+ T cells producing IFN-γ and IL-17A (Di), and IFN-γ and IL-17A expression by CD4+ T-cell (expressed as mean fluorescence intensity; Dii) are shown. (E) Representative flow cytometry plots showing IFN-γ (Ei) and IL-17A (Eii) production by CD4+ T cells from WT and Mpo−/− mice. (F) Skin DTH, expressed as the difference in thickness of footpads injected with OVA (right footpad) or BSA (left footpad). (G) CD4-enriched CFSE-labeled naïve OT-II T cells were transferred intravenously to WT (n = 4) and Mpo−/− recipient mice (n = 4) on d0. Recipient mice were injected subcutaneously with OVA/CFA on d2. (Gi) Proliferation of OT-II T cells in draining LNs of recipient mice 72 hours after OVA/CFA immunization (based on CFSE dilution; flow cytometry). (Gii-iii) IFN-γ production by OT-II T cells in recipient mice (intracellular staining of CD4/Vβ5-labeled splenocytes cultured with OVA323-339 [1 μM]). (Hi) The development of mBSA-induced AIA in WT (n = 8) and Mpo−/− (n = 9) mice. (Hii) Representative photomicrographs of formalin-fixed, paraffin-embedded 3-μm-thick Safranin-O-stained (fast green/hematoxylin counterstain) joint sections of WT and Mpo−/− mice with mBSA-AIA showing more severe disease in MPO-deficient animals. Sections were examined on a Leica BMLB Laboratory microscope with a ×5/0.70 NA objective lens. Images were captured with a Leica DC 300F digital camera using Photoshop CS software (Adobe Systems). Original magnification, ×50. Data are representative of 2-3 independent experiments. *P < .05, **P < .01, ***P < .001, •P = .07, #P = .1.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/121/20/10.1182_blood-2012-09-456483/2/4195f1.jpeg?Expires=1735841792&Signature=c8GC6R89MHMLgu91HKnODByy5hQpLpyqMvOfEk~PSi2MdMNkQgbQvQ3a55Evoj-f1n3lbZ9vPTbjKl5Dj~NJk~PvDAU9M0gapEckoxYTGNk8UCjRJonier26vZiZFnWFdckBVcvabSVOMXP9iv4FSWGnT5q2l6JAQvJs64~zMUDyEH4YEXHYrKN8pIBMDlO-YS7Mowku4ZxHWq5T4wWcpsPyiYz9m-6sQUsNaHhvWdP8v3RAqoO8XtmCX5AC6eZAQTlWNIQNHD3zFS02iGLKletF-JPQEV1cRDD9MQMtr0bIGwTPG~h-m6JP4g612yudsL7Jsy86pjBDOO26Abwf-Q__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
MPO deficiency enhances T-cell responses leading to increased skin DTH and AIA. (A-F) WT (n = 8) and Mpo−/− mice (n = 8) were immunized subcutaneously with OVA/CFA and CD4+ T-cell activation (A) and proliferation (B) assessed in LNs and spleen 6 days later (flow cytometry). (C) Concentrations of cytokines in 72-hour supernatants from OVA-stimulated splenocytes (ELISA). (D-E) Intracellular staining for IFN-γ and IL-17A in OVA-stimulated CD4-labeled splenocytes. Spleen cells were cultured (48 hours) in the presence of OVA (10 μg/mL) with brefeldin-A (5 μg/mL) added for the last 8 hours. CD4-labeled cells were then stained intracellularly for IFN-γ and IL-17A. The percentage of CD4+ T cells producing IFN-γ and IL-17A (Di), and IFN-γ and IL-17A expression by CD4+ T-cell (expressed as mean fluorescence intensity; Dii) are shown. (E) Representative flow cytometry plots showing IFN-γ (Ei) and IL-17A (Eii) production by CD4+ T cells from WT and Mpo−/− mice. (F) Skin DTH, expressed as the difference in thickness of footpads injected with OVA (right footpad) or BSA (left footpad). (G) CD4-enriched CFSE-labeled naïve OT-II T cells were transferred intravenously to WT (n = 4) and Mpo−/− recipient mice (n = 4) on d0. Recipient mice were injected subcutaneously with OVA/CFA on d2. (Gi) Proliferation of OT-II T cells in draining LNs of recipient mice 72 hours after OVA/CFA immunization (based on CFSE dilution; flow cytometry). (Gii-iii) IFN-γ production by OT-II T cells in recipient mice (intracellular staining of CD4/Vβ5-labeled splenocytes cultured with OVA323-339 [1 μM]). (Hi) The development of mBSA-induced AIA in WT (n = 8) and Mpo−/− (n = 9) mice. (Hii) Representative photomicrographs of formalin-fixed, paraffin-embedded 3-μm-thick Safranin-O-stained (fast green/hematoxylin counterstain) joint sections of WT and Mpo−/− mice with mBSA-AIA showing more severe disease in MPO-deficient animals. Sections were examined on a Leica BMLB Laboratory microscope with a ×5/0.70 NA objective lens. Images were captured with a Leica DC 300F digital camera using Photoshop CS software (Adobe Systems). Original magnification, ×50. Data are representative of 2-3 independent experiments. *P < .05, **P < .01, ***P < .001, •P = .07, #P = .1.
