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

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.

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.

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.