Chemokine Receptor Expression on Neoplastic and Reactive T Cells in the Skin at Different Stages of Mycosis Fungoides Tilmann Kallinich, J. Marcus Muche,

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Chemokine Receptor Expression on Neoplastic and Reactive T Cells in the Skin at Different Stages of Mycosis Fungoides Tilmann Kallinich, J. Marcus Muche, Shixin Qin, Wolfram Sterry, Heike Audring, Richard A. Kroczek Journal of Investigative Dermatology Volume 121, Issue 5, Pages 1045-1052 (November 2003) DOI: 10.1046/j.1523-1747.2003.12555.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Chemokine receptor expression at the plaque stage of MF (patient 2). (A) Perivascular and epidermotropic MF infiltrate stained for CD3 (original magnification 100×). (B)-(G) High-power view of the region demarcated by arrows in (A) after staining for CD3, and for the chemokine receptors CCR4, CCR6, CXCR3, and CXCR4; also included is the staining of the same area with isotype control monoclonal antibody MOPC-21 Scale bar: (A) 100 μm; (B)-(G) 50 μm. Journal of Investigative Dermatology 2003 121, 1045-1052DOI: (10.1046/j.1523-1747.2003.12555.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Chemokine receptor expression at the tumor stage of MF in a case of loss of CD3 on the neoplastic cells (patient 9). (A) Staining of perivascular CD3+ reactive T cells (arrows), surrounded by tumor cells. (B)-(D) Staining for chemokine receptors CXCR3, CXCR4, and CXCR5. (E) Control staining of the same area with isotype monoclonal antibody MOPC-21. Scale bar: (A)-(E) 200 μm. Journal of Investigative Dermatology 2003 121, 1045-1052DOI: (10.1046/j.1523-1747.2003.12555.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Chemokine receptor expression on folliculotropic and epidermotropic tumor cells in MF (patient 7). (A) Staining for CD3 (white arrows indicate the follicular epithelium). (B) Staining for CCR4 (the inset (400×) shows an intradermal Pautrier's microabscess with all cells positive for CCR4); gray arrows indicate the position of the basement membrane. (C) Staining for CCR6 (inset (630×) shows an intraepithelial Pautrier's microabscess with all cells positive for CCR6). (D) Staining for CXCR3. (E) Staining for CXCR4, with tumor cell growth resulting in a superficial erosion (white arrows point to the position of the basement membrane). (F) Control staining with isotype monoclonal antibody MOPC-21. Scale bar: (A)-(D), (F) 100 μm; (E) 200 μm; (B) insert, (C) insert, 50 μm. Journal of Investigative Dermatology 2003 121, 1045-1052DOI: (10.1046/j.1523-1747.2003.12555.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Chemokine receptor expression in a diffuse dermal tumor cell infiltrate (patient 8). (A) Staining of the infiltrate for CD3 (50×). (B) Higher magnification of CD3+ T cells with large atypical (black arrows) or small (white arrows) nuclei. (C) Staining for CCR4, with many large cells positive. (D) Staining for CCR5, with large cells negative. (E) Staining for CXCR3, with large cells negative. (F) Staining for CXCR4, with large cells partially positive. (G) Staining for CXCR5, with large cells negative. (H) Control staining with isotype monoclonal antibody MOPC-21. Scale bar: (A) 200 μm; (B)–(H) 50 μm. Journal of Investigative Dermatology 2003 121, 1045-1052DOI: (10.1046/j.1523-1747.2003.12555.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Flow cytometric analysis of activation cell surface markers on T cells isolated from skin at the tumor stage of MF (patients 8 and 11). Cells were isolated from lesional skin as described in Materials and Methods and examined by flow cytometry. For analysis, the cells were gated on the CD3 marker. In both patients, approximately half of the CD3+ cells expressed the clonal TCR Vβ chains Vβ22 or Vβ2, respectively, and could thus be identified as MF tumor cells, as only 2%–5% of all cells in the normal T cell repertoire express these Vβ chains. Shown is the staining of tumor cells and reactive T cells for CD45RO, CD69, CD25, and ICOS using two-color flow cytometry. The percentage given in the upper quadrants represents the proportion of reactive T cells and tumor cells positive for the respective cell surface activation marker. (A) Patient 8; (B) patient 11. Journal of Investigative Dermatology 2003 121, 1045-1052DOI: (10.1046/j.1523-1747.2003.12555.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Flow cytometric analysis of chemokine receptors on T cells isolated from skin at the tumor stage of MF (patients 8 and 11). Isolation of cells and staining for the clonal TCR Vβ chains Vβ22 and Vβ2 were as described in the legend to Figure 5. Shown is the flow cytometric analysis of reactive T cells and tumor cells for expression of the indicated chemokine receptors after gating on CD3. (A) Patient 8; (B) patient 11. Journal of Investigative Dermatology 2003 121, 1045-1052DOI: (10.1046/j.1523-1747.2003.12555.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions