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eji2586-sup-0001-FigureS1.pdf469K

Figure S1. Splenocyte distribution is normal in both naïve and immunized CD37−/−mice. FACS analyses of the major splenocyte and T lymphocyte populations in WT and CD37−/− mice that were (A) naïve, (B) 10 days post-immunization and (C) 14 days post-immunization with B16-OVA. The frequencies of NK cells (CD3NK1.1+), T-cells (CD3+), B cells (CD19+), DC (CD11c+MHC-II+), Granulocytes (F4/80Gr1+) and Macrophages (F4/80+CD11c) are expressed as a percentage relative to the total number of viable cells (left axis). The frequencies of T-cell subpopulations including NKT (CD3+NK1.1+), Th (CD3+CD4+), Tc (CD3+CD8+) and T regulatory (CD3+Foxp3+) cells are expressed as a percentage relative to the total number of viable CD3+ T-cells (right axis). Histogram bars represent the mean frequency of the given population +/-SD and significance tested via ANOVA (n = 3–4 mice/group).

Figure S2. The Th1-polarizing cytokine IL-12p70 is secreted at normal levels by CD37−/-DC. Purified naïve splenic DC were pooled from four mice/group and cultured with either media alone, 10 ng/mL CpG peptide or 1 ng/mL LPS. All conditions were supplemented with GM-CSF, IL-4 and IFNy. After 24 hours, IL-6, TNFa, IL-10 and IL-12p70 secretion was quantified in supernatants via flow cytometric bead array. Histogram bars represent the average cytokine concentration from three independent experiments + SEM and significance tested via ANOVA (n = 3 experiments/group). (B) BMDC maturation was assessed by flow cytometric analysis of surface CD80, CD86 and MHC Class II upregulation post LPS activation (1 ng/mL).

eji2586-sup-0002-S1.avi3123KSupporting Information Video 1. Wild-type DC migration. Video shows a region of skin in a CD37+/+ CD11c-YFP mouse, showing migrating dermal DCs 18–20 h after application of oxazolone to the skin. DCs migrate extensively but randomly throughout the skin, extending pseudopods at the front end of the cell. Dermal collagen (blue) is visible via second harmonic generation. Real-time duration = 29 min.
eji2586-sup-0002-S2.avi2693KSupporting Information Video 2. CD37−/− DC migration. Video of a region of skin in a CD37−/− CD11c-YFP mouse 18–20 h after application of oxazolone to the skin. CD37−/− DCs are very active in extending pseudopods, in some cases extending pseudopods in more than one direction simultaneously. However, their migration is largely unproductive as the cells migrate only minimally away from their point of origin. Dermal collagen (blue) is visible via second harmonic generation. Real-time duration = 23 min.

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