Exogenous Addition of Minor H Antigen HA-1+ Dendritic Cells to Skin Tissues Ex Vivo Causes Infiltration and Activation of HA-1-Specific Cytotoxic T Cells

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Exogenous Addition of Minor H Antigen HA-1+ Dendritic Cells to Skin Tissues Ex Vivo Causes Infiltration and Activation of HA-1-Specific Cytotoxic T Cells Yeung-Hyen Kim, Florry A. Vyth-Dreese, Ellen Schrama, Stan Pavel, Ingeborg Bajema, Els Goulmy, Eric Spierings Biology of Blood and Marrow Transplantation Volume 17, Issue 1, Pages 69-77 (January 2011) DOI: 10.1016/j.bbmt.2010.08.006 Copyright © 2011 Terms and Conditions

Figure 1 Influx of mature DCs causes skin destruction in skin explant tissues. CFDA-SE-labeled mature DCs were incubated with skin explant tissues as indicated. (A) Schematic time schedule of the DC reconstitution experiment. Mature DCs were added to the skin explant assay at the start of the experiments and cocultured untill fixation. (B) GVHR scoring of the samples treated as depicted in A. (∗P <.05; ∗∗P <.001). (C) H&E staining of the skin explant assays executed as depicted in A. Scale bar represents 10 μm. Black arrows indicate dermal-epidermal cleft formation. (D) Schematic time schedule of the adapted DC infiltration protocol. Mature DCs and skin samples were cocultured. After 24 hours the samples were washed and culturing was extended for another 24 hours (with a total assay time of 48 hours) or 72 hours (with a total assay time of 96 hours) in standard culturing medium. (E) GVHR scoring of the samples following the adapted DC infiltration protocol executed as depicted in D at various time points. (F) Detailed analysis of the GVHR background at various time points using 0.125 × 106 mDC following the protocol depicted in D. In all cases, GVHR scores were grade II or lower. (G) Mature DC infiltration after 96 hours with 0.125 × 106 mDC. The arrows indicate CFDA-SE-labeled DCs. Biology of Blood and Marrow Transplantation 2011 17, 69-77DOI: (10.1016/j.bbmt.2010.08.006) Copyright © 2011 Terms and Conditions

Figure 2 Activation of HA-1-specific CTLs depends upon recognition of their cognate antigen. HA-1 specific CTLs were cocultured with HA-1-positive (A, C) or HA-1-negative (B, D) EBV-BLCL. Activation was monitored by immunofluorescent triple-color staining on cytocentrifuge preparations. EBV-LCL were visualized with anti-CD20 (blue) and T cells with anti-CD3 (green) in combination with either activated cytotoxic cell marker Granzyme B (red; A and B) or nuclear activation marker Ki67 (red; C and D). Activated and unactivated HA-1-specific CTLs have been marked by red and white arrows, respectively. Original magnification is ×400; scale bar represents 10 μm. Biology of Blood and Marrow Transplantation 2011 17, 69-77DOI: (10.1016/j.bbmt.2010.08.006) Copyright © 2011 Terms and Conditions

Figure 3 Infiltration and activation of HA-1-specific CTLs detected by in situ combined tetramer staining. HA-1 CTLs were cocultured with DC-preincubated skin explants according to the adapted skin infiltration protocol described in Materials and Methods. Activated HA-1-specific CTLs (yellow arrows) were identified by triple-color staining for HLA-A2/HA-1 tetramer (blue), CD8 (green), and polarized Granzyme B (red). Scale bar represents 10 μm. Biology of Blood and Marrow Transplantation 2011 17, 69-77DOI: (10.1016/j.bbmt.2010.08.006) Copyright © 2011 Terms and Conditions