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( A ) Schematic depicting left lung from BALB/c (CD45.2) donor initially transplanted into CSB-treated B6 (CD45.2) primary recipient and then at least 30 days later retransplanted into non-immunosuppressed B6 (CD45.1) or B6 Foxp3-DTR (CD45.1) secondary recipient, with diphtheria toxin (DT) administration and analysis 7 days after retransplantation. ( B and C ) Gross image (first panel, n = 4), H&E histology (second panel, n = 4), CCSP immunofluorescence staining (green, third panel, n = 2), and immunohistochemical staining for <t>complement</t> <t>4d</t> <t>(C4d)</t> (brown, fourth panel; arrows point to alveolar endothelial C4d staining; n = 2) in DT-treated B6 ( B ) and B6 Foxp3-DTR ( C ) secondary recipients. ( D – F ) Representative flow cytometric plots and quantification of abundance of CD69 ( D ), Fas ( E ), and IgM/IgD ( F ) expression in graft-infiltrating CD45.2 – CD45.1 + CD19 + B220 + B cells in control (left panels) and recipient Foxp3-depleted (right panels) retransplants ( n ≥ 4). ( G and H ) Flow cytometric analysis of serum donor-specific IgM antibody titers (expressed as mean fluorescence intensity) ( G ) and International Society for Heart and Lung Transplantation (ISHLT) A rejection grades ( H ) in control and recipient Foxp3-depleted retransplants ( n = 4). Results expressed as mean ± SEM. Scale bars: 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001. DT, diphtheria toxin; DTR, diphtheria toxin receptor; TX, transplanted lung.
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( A ) Schematic depicting left lung from BALB/c (CD45.2) donor initially transplanted into CSB-treated B6 (CD45.2) primary recipient and then at least 30 days later retransplanted into non-immunosuppressed B6 (CD45.1) or B6 Foxp3-DTR (CD45.1) secondary recipient, with diphtheria toxin (DT) administration and analysis 7 days after retransplantation. ( B and C ) Gross image (first panel, n = 4), H&E histology (second panel, n = 4), CCSP immunofluorescence staining (green, third panel, n = 2), and immunohistochemical staining for <t>complement</t> <t>4d</t> <t>(C4d)</t> (brown, fourth panel; arrows point to alveolar endothelial C4d staining; n = 2) in DT-treated B6 ( B ) and B6 Foxp3-DTR ( C ) secondary recipients. ( D – F ) Representative flow cytometric plots and quantification of abundance of CD69 ( D ), Fas ( E ), and IgM/IgD ( F ) expression in graft-infiltrating CD45.2 – CD45.1 + CD19 + B220 + B cells in control (left panels) and recipient Foxp3-depleted (right panels) retransplants ( n ≥ 4). ( G and H ) Flow cytometric analysis of serum donor-specific IgM antibody titers (expressed as mean fluorescence intensity) ( G ) and International Society for Heart and Lung Transplantation (ISHLT) A rejection grades ( H ) in control and recipient Foxp3-depleted retransplants ( n = 4). Results expressed as mean ± SEM. Scale bars: 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001. DT, diphtheria toxin; DTR, diphtheria toxin receptor; TX, transplanted lung.
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( A ) Schematic depicting left lung from BALB/c (CD45.2) donor initially transplanted into CSB-treated B6 (CD45.2) primary recipient and then at least 30 days later retransplanted into non-immunosuppressed B6 (CD45.1) or B6 Foxp3-DTR (CD45.1) secondary recipient, with diphtheria toxin (DT) administration and analysis 7 days after retransplantation. ( B and C ) Gross image (first panel, n = 4), H&E histology (second panel, n = 4), CCSP immunofluorescence staining (green, third panel, n = 2), and immunohistochemical staining for <t>complement</t> <t>4d</t> <t>(C4d)</t> (brown, fourth panel; arrows point to alveolar endothelial C4d staining; n = 2) in DT-treated B6 ( B ) and B6 Foxp3-DTR ( C ) secondary recipients. ( D – F ) Representative flow cytometric plots and quantification of abundance of CD69 ( D ), Fas ( E ), and IgM/IgD ( F ) expression in graft-infiltrating CD45.2 – CD45.1 + CD19 + B220 + B cells in control (left panels) and recipient Foxp3-depleted (right panels) retransplants ( n ≥ 4). ( G and H ) Flow cytometric analysis of serum donor-specific IgM antibody titers (expressed as mean fluorescence intensity) ( G ) and International Society for Heart and Lung Transplantation (ISHLT) A rejection grades ( H ) in control and recipient Foxp3-depleted retransplants ( n = 4). Results expressed as mean ± SEM. Scale bars: 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001. DT, diphtheria toxin; DTR, diphtheria toxin receptor; TX, transplanted lung.
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Image Search Results


( A ) Schematic depicting left lung from BALB/c (CD45.2) donor initially transplanted into CSB-treated B6 (CD45.2) primary recipient and then at least 30 days later retransplanted into non-immunosuppressed B6 (CD45.1) or B6 Foxp3-DTR (CD45.1) secondary recipient, with diphtheria toxin (DT) administration and analysis 7 days after retransplantation. ( B and C ) Gross image (first panel, n = 4), H&E histology (second panel, n = 4), CCSP immunofluorescence staining (green, third panel, n = 2), and immunohistochemical staining for complement 4d (C4d) (brown, fourth panel; arrows point to alveolar endothelial C4d staining; n = 2) in DT-treated B6 ( B ) and B6 Foxp3-DTR ( C ) secondary recipients. ( D – F ) Representative flow cytometric plots and quantification of abundance of CD69 ( D ), Fas ( E ), and IgM/IgD ( F ) expression in graft-infiltrating CD45.2 – CD45.1 + CD19 + B220 + B cells in control (left panels) and recipient Foxp3-depleted (right panels) retransplants ( n ≥ 4). ( G and H ) Flow cytometric analysis of serum donor-specific IgM antibody titers (expressed as mean fluorescence intensity) ( G ) and International Society for Heart and Lung Transplantation (ISHLT) A rejection grades ( H ) in control and recipient Foxp3-depleted retransplants ( n = 4). Results expressed as mean ± SEM. Scale bars: 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001. DT, diphtheria toxin; DTR, diphtheria toxin receptor; TX, transplanted lung.

Journal: The Journal of Clinical Investigation

Article Title: Maintenance of graft tissue–resident Foxp3 + cells is necessary for lung transplant tolerance in mice

doi: 10.1172/JCI178975

Figure Lengend Snippet: ( A ) Schematic depicting left lung from BALB/c (CD45.2) donor initially transplanted into CSB-treated B6 (CD45.2) primary recipient and then at least 30 days later retransplanted into non-immunosuppressed B6 (CD45.1) or B6 Foxp3-DTR (CD45.1) secondary recipient, with diphtheria toxin (DT) administration and analysis 7 days after retransplantation. ( B and C ) Gross image (first panel, n = 4), H&E histology (second panel, n = 4), CCSP immunofluorescence staining (green, third panel, n = 2), and immunohistochemical staining for complement 4d (C4d) (brown, fourth panel; arrows point to alveolar endothelial C4d staining; n = 2) in DT-treated B6 ( B ) and B6 Foxp3-DTR ( C ) secondary recipients. ( D – F ) Representative flow cytometric plots and quantification of abundance of CD69 ( D ), Fas ( E ), and IgM/IgD ( F ) expression in graft-infiltrating CD45.2 – CD45.1 + CD19 + B220 + B cells in control (left panels) and recipient Foxp3-depleted (right panels) retransplants ( n ≥ 4). ( G and H ) Flow cytometric analysis of serum donor-specific IgM antibody titers (expressed as mean fluorescence intensity) ( G ) and International Society for Heart and Lung Transplantation (ISHLT) A rejection grades ( H ) in control and recipient Foxp3-depleted retransplants ( n = 4). Results expressed as mean ± SEM. Scale bars: 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001. DT, diphtheria toxin; DTR, diphtheria toxin receptor; TX, transplanted lung.

Article Snippet: Slides were stained with rabbit anti-mouse polyclonal C4d (1:100; Hycult Biotech, catalog HP8033) overnight at 4°C, washed, and then stained with biotinylated goat anti-rabbit IgG (1:200; Vector Laboratories, catalog BA-1000-1.5) for 30 minutes.

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Expressing, Control, Fluorescence, Transplantation Assay