CD69 Modulates Sphingosine-1-Phosphate-Induced Migration of Skin Dendritic Cells  Amalia Lamana, Pilar Martin, Hortensia de la Fuente, Laura Martinez-Muñoz, Aranzazu Cruz-Adalia, Marta Ramirez-Huesca, Cristina Escribano, Kathrin Gollmer, Mario Mellado, Jens V. Stein, Jose Luis Rodriguez-Fernandez, Francisco Sanchez-Madrid, Gloria Martinez del Hoyo  Journal of Investigative Dermatology  Volume 131, Issue 7, Pages 1503-1512 (July 2011) DOI: 10.1038/jid.2011.54 Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Epidermal Langerhans cells (LCs) and dermal dendritic cells (DCs) express CD69 after maturation. (a) Histograms show the expression of CD69 by epidermal LCs and dermal DCs, after ex vivo isolation (uncultured) and overnight culture in the absence or in the presence of tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS) maturation stimuli, in wild-type (WT) and CD69-/- mice. Percentages of cells expressing CD69 are indicated. Expression profiles are shown on a logarithmic scale along the x-axis. The results shown are from a representative experiment of three performed. (b) Skin sections from WT and CD69-/- mice were stained with anti-CD69 (green), anti- major histocompatibility complex-II (MHC-II; red), and Hoechst (blue) for nuclear localization (scale bar=20μm). Journal of Investigative Dermatology 2011 131, 1503-1512DOI: (10.1038/jid.2011.54) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Toll-like receptor (TLR)-induced maturation of plasmacytoid and conventional dendritic cell (pDC and cDC) subsets is associated with the induction of CD69 surface expression. (a) Histograms show flow cytometry analysis of the surface expression of CD69 and CD86 on cDCs and pDCs derived from fms-related tyrosine kinase 3-ligand (Flt3L)-driven bone marrow cultures, both before and after maturation with lipopolysaccharide (LPS) or CpG. Expression profiles are shown on a logarithmic scale along the x-axis. Dotted lines correspond to the isotype control. The results shown are from a representative experiment of five with similar results. (b) Effect of in vivo TLR-induced DC maturation on CD69 expression. Bar charts show expression of CD69 by splenic pDCs and CD8α+ and CD8α− cDCs at 15hours after intravenous injection of LPS or CpG. The results shown are from a representative experiment of three performed (five mice per group). Journal of Investigative Dermatology 2011 131, 1503-1512DOI: (10.1038/jid.2011.54) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 The absence of CD69 does not affect dendritic cell (DC) differentiation or maturation. (a, top) Dot-plots represent plasmacytoid and conventional DCs (pDCs and cDCs) generated from fms-related tyrosine kinase 3-ligand (Flt3L)-driven cultures from wild-type (WT) and CD69–/– mice, defined according to their expression of B220 and CD11b after 7 days of differentiation. The percentage of each DC population is indicated. (a, bottom) Histograms show major histocompatibility complex-II (MHC-II) and costimulatory molecule expressions by Flt3L-derived DCs from WT and CD69–/– mice before maturation (dotted lines) and after lipopolysaccharide (LPS; thin lines) or CpG (bold lines)-induced maturation. The results shown are from a representative experiment of five performed. (b) MHC-II and CD86 expression on immature (control) and mature splenic DCs 15hours after intravenous injection of LPS or CpG into WT mice (white bars) and CD69–/– mice (black bars). The results shown are from a representative experiment of three performed. Journal of Investigative Dermatology 2011 131, 1503-1512DOI: (10.1038/jid.2011.54) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 In vivo migration of skin dendritic cells (DCs) to draining lymph nodes (LNs) after FITC painting contact sensitization assay in CD69–/– mice. (a) Dot-plots show FITC+ cells correlated with CD11c expression at 24hours; CD207/Langerin and CD69 (b), CD103 (c), and caspase-3 (d) expression in FITC+ cells is shown. (e) DC migration (mean±SD), as (left) the ratio of percentage of CD69–/– FITC+CD11c+ cells with respect to wild-type or (right) the absolute number of FITC+CD11c+ cells at 24hours (*P<0.05, Student's t-test); and (bottom) at 18, 24, and 48hours (representative experiment shown of five performed). (f) Confocal images of epidermal sheets stained with anti-major histocompatibility complex-II (MHC-II). The maximal projections and a detail of Langerhans cell (LC) morphology are shown (scale bar=20μm). Mean±SD of the percentage of MHC-II+ cells from epidermal and dermal skin explants is shown. NS, not significant. Journal of Investigative Dermatology 2011 131, 1503-1512DOI: (10.1038/jid.2011.54) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Interstitial motility and distribution of CD69–/– dendritic cells (DCs) within the lymph node (LN). (a) fms-related tyrosine kinase 3-ligand (Flt3L)-derived conventional DCs (cDCs) were labeled and injected subcutaneously into C57BL/6 recipients and 18hours later, popliteal LNs were analyzed by flow cytometry. Bar chart shows the fold difference in migration, determined as the ratio of the percentage of immigrated CD69–/– cDCs to the corresponding percentage of wild-type DCs. Eight popliteal LNs were analyzed (*P<0.05, Student's t-test). (b) Fluorescently labeled Flt3L-derived cDCs from wild-type and CD69–/– mice were transferred into wild-type recipients, and popliteal LNs were analyzed by two-photon microscopy after 15–22hours. Bar chart shows the mean values of track velocity of wild-type (empty dots) and CD69–/– (filled dots) cDCs (Student's t-test statistical analysis). NS, not significant. (c) The turning angles of wild-type (WT) and CD69–/– cDCs are represented. Journal of Investigative Dermatology 2011 131, 1503-1512DOI: (10.1038/jid.2011.54) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 CD69 deficiency alters the response to sphingosine 1-phosphate (S1P) of fms-related tyrosine kinase 3-ligand (Flt3L)-derived conventional dendritic cells (cDCs). Matured Flt3L-cDC migration toward (a) S1P or (b) CXCL12 (chemokine (C-X-C motif) ligand 12). (Left) Data show the percentage (means±SD) of migrated cells (representative experiment, four performed); (right) fold difference determined as the ratio of the percentage (mean±SD) of migrated CD69–/– cDCs with respect to wild-type DCs (*P<0.05, Student's t-test). (c) Data show the percentage (mean±SD) of FITC+CD11c+ DCs after FTY720 and SEW2871 treatment. A representative experiment is shown of three performed (*P<0.05, Student's t-test). (d) S1P1 and S1P3 expression by western blot. Results show the fold difference (means±SD) relative to expression in wild-type DCs (*P<0.05, Student's t-test) after quantification (β-actin normalization). (e) S1P1 and S1P3 mRNA expression levels in wild-type and CD69–/– Flt3L-cDCs. NS, not significant. Journal of Investigative Dermatology 2011 131, 1503-1512DOI: (10.1038/jid.2011.54) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions