Aggregation of Antigen-Specific T Cells at the Inoculation Site of Mature Dendritic Cells David Schrama, Lars Østergaard Pedersen, Petra Keikavoussi,

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Aggregation of Antigen-Specific T Cells at the Inoculation Site of Mature Dendritic Cells David Schrama, Lars Østergaard Pedersen, Petra Keikavoussi, Mads Hald Andersen, Per thor Straten, Eva-Bettina Bröcker, Eckhart Kämpgen, Jürgen C. Becker Journal of Investigative Dermatology Volume 119, Issue 6, Pages 1443-1448 (December 2002) DOI: 10.1046/j.1523-1747.2002.19604.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Immunohistologic characterization of vaccination sites. Skin biopsies were obtained from injection sites of either nonpulsed (A, C, D) or oncolysate-pulsed (B) DC. Sections of these were subjected to staining with antibodies directed against CD83 (A, B), PNAd (C), or TCA-4 (D). Magnification: 50× (C) and 200× (all others). Journal of Investigative Dermatology 2002 119, 1443-1448DOI: (10.1046/j.1523-1747.2002.19604.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 T cell infiltrate at antigen-pulsed and nonpulsed DC vaccination sites. Skin biopsies were obtained from vaccination sites of nonpulsed (A, C, E) or oncolysate-pulsed (B, D, F) DC. Antibodies directed against CD62L (A, B; open arrowhead) or CD3 (C-F) were visualized by a red color, those directed against CD45RA (A, B) or CD83 (E, F) by a blue color. Double-positive cells appear purple (black arrows). Magnifications: 50× (A, B) and 400× (all others). Journal of Investigative Dermatology 2002 119, 1443-1448DOI: (10.1046/j.1523-1747.2002.19604.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Relative overexpression of DC-CK1 and SDF-1 at vaccination sites compared to normal skin. Messenger RNA encoding DC-CK1, SDF-1, and the housekeeping gene GAPDH expressed within vaccination sites 48 h after injection of DC and normal skin was measured by real-time PCR. The relative expression of DC-CK1 (gray) and SDF-1 (dark gray) was calculated by the ΔΔCT method with normal skin as calibrator. Samples 1 and 2 were obtained from patient 3 receiving either gp100209–217 or tyrosinase192–200 peptide pulsed DC; sample 3 was derived from patient 4 receiving tyrosinase214–222 peptide pulsed DC. The insert depicts original real-time PCR graphs for DC-CK1 (black) and GAPDH (gray) amplification of normal skin (circled lines) and sample 1 (line). Rn represents the level of fluorescence detected, the dotted black line the detection threshold. Journal of Investigative Dermatology 2002 119, 1443-1448DOI: (10.1046/j.1523-1747.2002.19604.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Clonotype maps of vaccination sites. Skin biopsies from injection sites of nonpulsed (A) or oncolysate-pulsed (B) DC were subjected to clonotype mapping. Within the clonotype maps covering the BV regions 1–24 each distinct band represents a clonally expanded T cell. To ensure the feasibility of the applied method, the extent of respective T cell BV families was evaluated by immunohistochemistry as exemplified for Vβ12 and Vβ14. Magnification of immunohistochemistry figures: 100× (A), 200× (B). Journal of Investigative Dermatology 2002 119, 1443-1448DOI: (10.1046/j.1523-1747.2002.19604.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Specificity of infiltrating T cells. Sections of skin biopsies, obtained 24 h after cutaneous injection of oncolysate-pulsed DC, were subjected to double staining with anti-TCR Vβ12 antibodies (red) and MAA/HLA-A*0201 multimers (green). The MAA-derived peptides used for generation of multimers are MART26–35 (upper part), gp100209–217 (middle part), or MAGE-3271–279 peptides (lower part). The images at the far right represent the digital overlay of the first two rows. Double positive cells appear yellow. Journal of Investigative Dermatology 2002 119, 1443-1448DOI: (10.1046/j.1523-1747.2002.19604.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 TCR repertoire usage at vaccination sites differs from that of antigen-reactive cells circulating in peripheral blood. Comparative clonotype mapping for selected TCR BV families of (1) bulk peripheral blood lymphocytes and (2) gp100209–217/HLA-A*0201 reactive T cells, both obtained 24 h prior to vaccination with gp100209–217 peptide pulsed DC, or (3) the respective vaccination site after 24 h. Identical clones are retained at the same position in the gel. Journal of Investigative Dermatology 2002 119, 1443-1448DOI: (10.1046/j.1523-1747.2002.19604.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions