Drug Specific Cytotoxic T-Cells in the Skin Lesions of a Patient with Toxic Epidermal Necrolysis Amal Nassif, Armand Bensussan, Nicolas Bachot, Martine.

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Drug Specific Cytotoxic T-Cells in the Skin Lesions of a Patient with Toxic Epidermal Necrolysis Amal Nassif, Armand Bensussan, Nicolas Bachot, Martine Bagot, Laurence Boumsell, Jean-Claude Roujeau Journal of Investigative Dermatology Volume 118, Issue 4, Pages 728-733 (April 2002) DOI: 10.1046/j.1523-1747.2002.01622.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Phenotype of lymphocytes present in the blister fluid of a patient with cotrimoxazole-related TEN.(a) FACS analysis of cells present in the blister fluid and gated as lymphocytes. Most lymphocytes were CD3+, CD8+ (99%) and coexpressed CD56 (72%), CLA (83%), HLA-DR (85%). Most cells had an αβ T cell receptor (89%). (b) Cytometry analysis of the TCR Vβ repertoire. Two major Vβ chains were expressed on the blister lymphocytes (gray bars), Vβ 13.1 (56%) and Vβ14 (23%), suggesting an oligoclonal response, in contrast with normal control blood lymphocytes (black bars); however, minor populations of blister cells expressed other Vβ (not shown in the figure). Journal of Investigative Dermatology 2002 118, 728-733DOI: (10.1046/j.1523-1747.2002.01622.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Drug-dependent cytotoxicity of blister fluid cells on autologous lymphocytes. (a) Black bars: unstimulated blister fluid cells were lytic for autologous EVB-tranformed lymphocytes preincubated with cotrimoxazole (SMX 80 mg per liter, TMP 16 mg per liter). Blister fluid cells induced no cytotoxicity against autologous EVB-transformed lymphocytes in the absence of drug (gray bars) and against unrelated allogeneic cells preincubated with cotrimoxazole (white bars). (b) Preincubation of target cells with sulfamethoxazole-SMX alone, 80 mg per liter (white bar) or with the nitroso metabolite 20 mg per liter (dotted bar) resulted in a lysis by blister fluid cells similar to that observed with cotrimoxazole, i.e., SMX 80 mg per liter plus TMP 16 mg per liter (gray bar). The hydroxylamine metabolite of sulfamethoxazole (black bar) had no significant effect as compared with the medium alone (dark gray bar) Journal of Investigative Dermatology 2002 118, 728-733DOI: (10.1046/j.1523-1747.2002.01622.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 MHC class I restriction of drug-dependent cytotoxicity.(a) Anti-class I monoclonal antibody W6-32, but not an anti-class II, abolished drug-dependent lysis of autologous lymphocytes by blister fluid cells. Grey bars, lysis of target cells without drug; black bars, lysis of target cells in the presence of cotrimoxazole (SMX 80 mg per liter plus TMP 16 mg per liter); white bars, lysis of target cells in the presence of both cotrimoxazole and anti-class I monoclonal antibody. Dark gray bars, lysis of target cells in the presence of both cotrimoxazole and anti-class II monoclonal antibody. (b) Blister fluid cells were tested, at an effector/target ratio of 40/1, in the presence of cotrimoxazole on EBV-transformed lymphocytes from two allogeneic controls sharing with the patient a variety of serologically determined HLA class I molecules and on a mutant cell line C1R, expressing only CW4 by serologic determination. Lysis segregated with HLA-Cw4. Journal of Investigative Dermatology 2002 118, 728-733DOI: (10.1046/j.1523-1747.2002.01622.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Mediators of cytotoxicity.(a) Blister cells preincubated with IL-2 (100 UI per ml for 24 h) exhibited an increased specific drug-dependent cytotoxicity (black bar), but no LAK cytotoxicity on target cells in the absence of drug (gray bar). This drug-dependent lysis was totally abolished by preincubation of target cells with EGTA (dark gray bar) but not by preincubation with monoclonal antibodies anti-CD95-Fas (white bar) or anti-TRAIL R1 and R2 (TRAIL) (dotted bar).(b) When effector cells were preincubated with Brefeldin A (white bar) the drug-dependent lysis (black bar) was unaffected. In contrast it was abolished by CMA (dark gray bar). (c) Although not implicated in the lysis, CD95-Fas and TRAIL receptor 2 were expressed on target lymphocytes as shown by FACS analysis. Journal of Investigative Dermatology 2002 118, 728-733DOI: (10.1046/j.1523-1747.2002.01622.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions