Direct Evidence to Support the Role of Antigen-Specific CD8+ T Cells in Melanoma- Associated Vitiligo Frédérique-Anne Le Gal, Philippe Lefebvre, Jean-Christophe.

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Direct Evidence to Support the Role of Antigen-Specific CD8+ T Cells in Melanoma- Associated Vitiligo Frédérique-Anne Le Gal, Philippe Lefebvre, Jean-Christophe Deschemin, Muriel Andrieu, Jean-Gérard Guillet Journal of Investigative Dermatology Volume 117, Issue 6, Pages 1464-1470 (December 2001) DOI: 10.1046/j.0022-202x.2001.01605.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Immunohistochemical staining for CD4+ and CD8+ T lymphocytes in an alcohol-formalin-acetic acid fixed paraffin-embedded vitiligo biopsy specimen from patient 1. (A) A small proportion of CD4+ T cells in the lymphocytic infiltrate are solely located in the superficial dermis. Original magnification 200×. (B) The lymphocytic infiltrate is predominantly CD8+. Interestingly, lymphocytes are exclusively CD8+ in the basal layer of the epidermis, where melanocytes are usually located (in a normally pigmented skin). Scale bar: 5 μm. Journal of Investigative Dermatology 2001 117, 1464-1470DOI: (10.1046/j.0022-202x.2001.01605.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Expanded VIL are T cells expressing the CLA. A biopsy sample was taken from the active margin of a vitiligo patch in patient 3. The specimen was minced and placed in culture medium containing IL-2. VIL were harvested after 3 wk of culture and tested for their expression of CD3, CD4, CD8, CD16+CD56, and CLA (A) and CD8, CD56, and CD45RA (B) by flow cytometry. The distribution of CD56 and CD45RA expression is shown among the viable (R1) CD8+ (R2) T cells. Journal of Investigative Dermatology 2001 117, 1464-1470DOI: (10.1046/j.0022-202x.2001.01605.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Analysis of the TCR repertoire of VIL and PBMC from patient 3. Total RNA recovered from thawed PBMC, and from PBMC and VIL after 3 wk of culture, was extracted and reverse transcribed. cDNAs were PCR-amplified using the 24 Vβ-specific probes and a common fluorescent Cβ-specific probe. Each Vβ-Cβ PCR product was analyzed by electrophoresis in agarose gel. X signifies that the Vβ-Cβ PCR product is detectable using this method; O means that no product could be detected. CDR3 length diversity was evaluated by electrophoresis of PCR products using a DNA sequencer and data were analyzed with Immunoscope software (open squares, no PCR product identified by agarose gel electrophoresis; hatched squares, Gaussian, i.e. polyclonal, distribution of CDR3 sizes; gray squares, oligoclonal distribution; black squares: clonal distribution). Journal of Investigative Dermatology 2001 117, 1464-1470DOI: (10.1046/j.0022-202x.2001.01605.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Specific recognition of melanocyte-antigen-derived peptides by VIL and PBMC determined in an IFN-γ Elispot assay. VIL from patients 1, 2, 3, 4, and 5 were harvested between 2 and 4 wk of culture (A). PBMC from the same patients were cultured for 12 d with IL-2 and melanocyte-antigen-derived peptides able to associate with the patients' HLA molecules (B). As control, cells from other skin conditions (lichen planus and secondary syphilis) were extracted and cultured in the same conditions as VIL and tested in an IFN-γ Elispot assay for their reactivity toward melanoma antigens. The horizontal line crossing the histograms represents the cut-off value defining specific IFN-γ secretion (mean background + 3 SEM). Cells were incubated in nitrocellulose plates coated with an antihuman IFN-γ monoclonal antibody (5 × 104 cells per well), together with the test peptides (1 µg per ml) for 20 h. The number of IFN-γ-secreting cells was determined by revealing the spots formed on the nitrocellulose membrane after incubation of the plates with a biotinylated antihuman IFN-γ monoclonal antibody followed by alkaline-phosphatase-labeled Extravidin and then alkaline phosphatase conjugate substrate. Each spot corresponds to one IFN-γ-secreting cell. Results are the means of triplicates ± SEM. Journal of Investigative Dermatology 2001 117, 1464-1470DOI: (10.1046/j.0022-202x.2001.01605.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Specific recognition of melanoma cells by VIL from patients 1, 3, and 4, determined by an IFN-γ Elispot assay. VIL from patients 1, 3, and 4 were harvested between 3 and 4 wk of culture and tested in an IFN-γ Elispot assay as described in Figure 4. The following melanoma cell lines (5 × 103 cells per well) were used as targets: BOU (A2/66, B50/41), DEL (A1/3, B35/39), CHO (A1/11, B42/44), and IGR1/54 (A2/3 B58/-). +signifies that the target cell line shares one HLA class I locus with the patient's VIL; – signifies no matches. Results are the means of triplicates ± SEM. Journal of Investigative Dermatology 2001 117, 1464-1470DOI: (10.1046/j.0022-202x.2001.01605.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions