Journal: The Journal of investigative dermatology

Article Title: CCR6 is required for epidermal trafficking of γδ T cells in an IL-23-induced model of psoriasiform dermatitis

doi: 10.1038/jid.2012.260

Figure Lengend Snippet: IL-23 was injected into ears of WT mice at day 0, 2, and 4. Mice were euthanized on the indicated days and ears were stained with anti-CCL20 antibody (red) and DAPI (nuclear counterstain in blue). Isotype control staining showed no epidermal staining with either IL-23 or PBS-injected ears (data not shown). Scale bar = 50 μm.

Article Snippet: In the same way, we also performed intradermal injection of 20 μl PBS containing 10 μg monoclonal anti-mouse CCL20/ MIP3α antibody (R&D Systems, Minneapolis, MN, USA) mixed with 500 ng recombinant mouse IL-23, 20 μl PBS containing 10 μg rat IgG 1 isotype control (R&D Systems) mixed with 500 ng recombinant mouse IL-23, or 20 μl PBS alone.

Techniques: Injection, Staining

Journal: The Journal of investigative dermatology

Article Title: CCR6 is required for epidermal trafficking of γδ T cells in an IL-23-induced model of psoriasiform dermatitis

doi: 10.1038/jid.2012.260

Figure Lengend Snippet: Following IL-23 ( a ) or PBS ( b ) injection for 6 days, mice were euthanized and ears were stained with anti-CCR6 mAbs (green) and anti-Laminin 332 mAbs (basement membrane in red), and the 3D images were acquired using a confocal microscope. The figure shows the perspective from the dermis toward the epidermis. The width of basement membrane in red shows the depth of the sections. Larger numbers of CCR6-expressing cells in the epidermis (above the basement membrane) were revealed in IL-23-, but not PBS-injected, mice. CCR6-expressing cells ( a ) at (arrow head), immediately adjacent to (double arrowheads), or above (arrow) the basement membrane in the dashed rectangular area marked in the upper figure are shown at higher magnification below. Isotype control staining showed no staining with either IL-23 or PBS-injected ears (data not shown). Scale bar = 10 μm. In ( c ), IL-23-treated skin sections were stained with labeled mAbs against CCR6 (green) and the γδ-TCR (red). Images were acquired by confocal microscopy at the level of the epidermis (x63 objective, scale bar as indicated). Similar results were obtained in 3 independent experiments.

Article Snippet: In the same way, we also performed intradermal injection of 20 μl PBS containing 10 μg monoclonal anti-mouse CCL20/ MIP3α antibody (R&D Systems, Minneapolis, MN, USA) mixed with 500 ng recombinant mouse IL-23, 20 μl PBS containing 10 μg rat IgG 1 isotype control (R&D Systems) mixed with 500 ng recombinant mouse IL-23, or 20 μl PBS alone.

Techniques: Injection, Staining, Microscopy, Expressing, Labeling, Confocal Microscopy

Journal: The Journal of investigative dermatology

Article Title: CCR6 is required for epidermal trafficking of γδ T cells in an IL-23-induced model of psoriasiform dermatitis

doi: 10.1038/jid.2012.260

Figure Lengend Snippet: Following IL-23 injection for 6 days, both WT and CCR6 KO mice were euthanized. Both epidermal and dermal cell suspensions were analyzed by flow cytometry with γδ TCR, CD3, and IL-22 mAbs. ( a ) Total numbers of GDL T cells per PBS- or IL-23-injected ear of WT and CCR6 KO mice were counted. The percentages of CD3 + GDL T cells ( b ) and IL-22 + GDL T cells ( c ) were calculated. *P<0.01 vs. all other groups. **P<0.05 vs. all other groups. Each treatment group consisted of 4 ears. All P values were calculated using Student’s t-test, 2-sided. In ( c , epidermis), p values are provided for comparisons between PBS and IL-23-treatment in WT and CCR6 KO mice. Similar results were obtained in 2 independent experiments.

Article Snippet: In the same way, we also performed intradermal injection of 20 μl PBS containing 10 μg monoclonal anti-mouse CCL20/ MIP3α antibody (R&D Systems, Minneapolis, MN, USA) mixed with 500 ng recombinant mouse IL-23, 20 μl PBS containing 10 μg rat IgG 1 isotype control (R&D Systems) mixed with 500 ng recombinant mouse IL-23, or 20 μl PBS alone.

Techniques: Injection, Flow Cytometry

Journal: JCI Insight

Article Title: Allograft dendritic cell p40 homodimers activate donor-reactive memory CD8 + T cells

doi: 10.1172/jci.insight.96940

Figure Lengend Snippet: (A) Groups of A/J mice (n = 4–8/group) received cardiac allografts subjected to 0.5 or 8 hours of CIS from either WT C57BL/6 (WTB6) or B6.IL12p40–/– donors. The indicated recipients of WT allografts were treated with 200 μg anti–IL-12 p40 mAb i.p. on days 0 and 1. All recipients were injected with 100 μg BrdU i.p. on days 0 and 1 after transplant. The following day, allografts were harvested and digested, and cell aliquots were stained with antibody and analyzed by flow cytometry to assess total numbers and proliferation of infiltrating memory CD4+ and CD8+ T cells. *P < 0.05; **P < 0.01, as determined by the Mann-Whitney nonparametric test. (B) Groups of A/J mice (n = 4/group) received cardiac allografts subjected to 0.5 or 8 hours of CIS from WT C57BL/6 or B6.IL12p35–/– donors. All recipients were injected with 100 μg BrdU i.p. on days 0 and 1. On day 2 after transplant, allografts were harvested and digested, and cell aliquots were stained with antibody and analyzed by flow cytometry to assess total numbers and proliferation of infiltrating memory CD4+ and CD8+ T cells. *P < 0.05 as determined by the Mann-Whitney nonparametric test. (C) Groups of C57BL/6 mice (n = 5–6/group) received C57BL/6 cardiac isografts or A/J cardiac allografts subjected to 0.5 or 8 hours of CIS. Grafts were harvested on day 2 after transplant, total RNA was isolated, and expression of the indicated cytokine mRNA was tested by qPCR. Results shown indicate relative expression vs. expression in naive hearts of A/J mice. **P < 0.01, as determined by the Mann-Whitney nonparametric test.

Article Snippet: Quantification of IL-12 heterodimer and p40 homodimer protein was performed on heart iso- and allograft lysates, and serum was collected at 48 hours after transplant using Mouse IL-12 p70 and Mouse p40 Quantikine ELISA Kits, respectively, from R&D Systems.

Techniques: Injection, Staining, Flow Cytometry, MANN-WHITNEY, Isolation, Expressing

Journal: JCI Insight

Article Title: Allograft dendritic cell p40 homodimers activate donor-reactive memory CD8 + T cells

doi: 10.1172/jci.insight.96940

Figure Lengend Snippet: (A) Groups of C57BL/6 mice (n = 5/group) received A/J cardiac allografts or C57BL/6 isografts subjected to 0.5 or 8 hours of CIS. The indicated recipients of 8 hours of CIS allografts were treated with 200 μg control rat IgG or anti-CD4 mAb on days –3, –2, and –1 prior to transplant. On day 2 after transplant, serum and graft lysates were prepared and quantities of p40 homodimers and IL-12 p70 heterodimers were tested by ELISA. *P < 0.05, as determined by the Mann-Whitney nonparametric test. (B) Groups of C57BL/6 mice (n = 5/group) received A/J cardiac allografts subjected to 0.5 or 8 hours of CIS and the indicated recipients were injected with 200 μg recombinant p40 homodimers i.p. on days 0 and 1. All recipients were injected with 100 μg BrdU i.p. on days 0 and 1 after transplant; the next day, allografts were harvested and digested, and cell aliquots were stained with antibody and analyzed by flow cytometry to assess total numbers and numbers of proliferating memory CD8+ T cells infiltrating the grafts. *P < 0.05; **P < 0.01, as determined by the Mann-Whitney nonparametric test. (C) Groups of C57BL/6 mice (n = 4–7/group) received A/J cardiac allografts subjected to 0.5 or 8 hours of CIS, and on days 0 and 1, were treated with 200 μg control rat IgG or 250 μg CTLA-4Ig i.p. with or without 200 μg recombinant p40 homodimers i.p. Allograft survival was monitored by daily abdominal palpation and rejection confirmed by laparotomy. ***P < 0.001 vs. all other groups, as determined using the Log-rank/Mantel-Cox test.

Article Snippet: Quantification of IL-12 heterodimer and p40 homodimer protein was performed on heart iso- and allograft lysates, and serum was collected at 48 hours after transplant using Mouse IL-12 p70 and Mouse p40 Quantikine ELISA Kits, respectively, from R&D Systems.

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Injection, Recombinant, Staining, Flow Cytometry