Shared and restricted T-cell receptor use is crucial for carbamazepine-induced Stevens- Johnson syndrome Tai-Ming Ko, MS, Wen-Hung Chung, MD, PhD, Chun-Yu.

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Shared and restricted T-cell receptor use is crucial for carbamazepine-induced Stevens- Johnson syndrome Tai-Ming Ko, MS, Wen-Hung Chung, MD, PhD, Chun-Yu Wei, MS, Han-Yu Shih, MS, Jung-Kuei Chen, MS, Chia-Hsien Lin, MS, Yuan- Tsong Chen, MD, PhD, Shuen-Iu Hung, PhD Journal of Allergy and Clinical Immunology Volume 128, Issue 6, Pages 1266-1276.e11 (December 2011) DOI: 10.1016/j.jaci.2011.08.013 Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 CBZ-specific T-cell responses of patients with CBZ-SJS and CBZ-tolerant control subjects. A and B, Levels of IFN-γ (Fig 1, A) and granulysin (Fig 1, B) were determined in the culture supernatants of CBZ-stimulated T cells (after 2 cycles of culture with CBZ) from patients (n = 4) and tolerant control subjects (n = 2). The dashed line indicates a mean level of IFN-γ or granulysin detected by means of ELISA in the T-cell culture supernatants in the absence of drug stimulation (n = 8). C and D, CBZ-specific T-cell (after 2 cycles of culture with CBZ) cytotoxicity was determined by using 51Cr release assays. E, Flow cytometric immunophenotypic analysis of the CD8+ subsets in CBZ-stimulated T cells obtained from the fifth cycle for 2 representative patients with CBZ-SJS/TEN. Numbers indicate the percentage of cells in the CD3+/CD8+ quadrant. ∗∗P < .01 and ∗∗∗P < .001, paired t test. Journal of Allergy and Clinical Immunology 2011 128, 1266-1276.e11DOI: (10.1016/j.jaci.2011.08.013) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Common skewed TCR VB and VA repertoires of CBZ-specific CTLs. A and B, CDR3 spectratype patterns of VB (Fig 2, A) and VA (Fig 2, B) of CBZ-stimulated CD8+ T cells obtained from the fifth cycle of in vitro cultures from 8 patients with CBZ-SJS/TEN. C, The x-axis displays CDR3 length, and the y-axis indicates fluorescence intensity of the runoff products of each TCR subfamily. Black squares indicate a normal CDR3 size profile (quasi-Gaussian distribution, >5 peaks), gray squares indicate a restricted CDR3 size profile (skewed TCR repertoire, 1-4 peaks), and open squares indicate an undetectable CDR3 size profile (undetectable TCR, 0 peak). Journal of Allergy and Clinical Immunology 2011 128, 1266-1276.e11DOI: (10.1016/j.jaci.2011.08.013) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Upregulation of skewed VB and VA subfamilies in patients with CBZ-SJS/TEN. A and B, cDNA expression levels of TCR VB (Fig 3, A) and VA (Fig 3, B) subfamilies of 8 patients with SJS/TEN or 8 tolerant control subjects (2 CBZ-tolerant and 6 OXC-tolerant subjects) were measured by using quantitative real-time PCR and normalized by the levels of the TCR constant region (CB or CA). Bars indicate the expansion fold of TCR VB (Fig 3, A) and VA (Fig 3, B) in CBZ-stimulated T cells (after 2 cycles of culture with CBZ), with levels compared with those of T cells in the absence of CBZ stimulation. ∗P < .05 and ∗∗P < .01, unpaired t test. Journal of Allergy and Clinical Immunology 2011 128, 1266-1276.e11DOI: (10.1016/j.jaci.2011.08.013) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Correlation of CBZ-specific CDR3 clonotypes in patients with CBZ-SJS/TEN and disease onset. A, Expression levels of 2 TCR clonotypes (VB-11-ISGSY and VB-11-GLAGVDN) in PBMCs or blister fluid cells of patients (n = 3) in the indicated disease stage of CBZ-SJS/TEN was determined by using real-time PCR. B and C, Levels of CBZ-specific TCR clonotypes increased in PBMCs from patients with CBZ-SJS/TEN (n = 14) yet were absent in CBZ-tolerant control subjects (n = 11) and showed different degrees of increase in healthy subjects (n = 29). Each point of data (Fig 4, B and C) represents the expression levels of the clonotype of 1 subject. N.D., Not detectable. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001, unpaired t test. Journal of Allergy and Clinical Immunology 2011 128, 1266-1276.e11DOI: (10.1016/j.jaci.2011.08.013) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 In vitro priming of disease-specific clonotypes and blockade of CBZ-specific cytotoxicity with anti-TCR antibodies. A, Increased release of granulysin by the CBZ-priming T cells (after 2 cycles of culture with CBZ) from a patient with CBZ-SJS (S2) and 3 healthy donors (H1-H3) but absent in 4 healthy donors (H4-H7) and a tolerant control subject (T2) who did not have VB-11-ISGSY clonotypes. B and C, Cytotoxicities of the CBZ-priming T cells isolated from different subjects after 2 cycles of culture with CBZ were determined by using 51Cr release assays. D, The CBZ-priming T cells were incubated with anti–TCR-VB-11 mAb or isotype control antibodies before the cytotoxicity assay. Ab, Antibody. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001; data in Fig 5, A and D, by unpaired t test and data in Fig 5, B and C, by paired t test. Journal of Allergy and Clinical Immunology 2011 128, 1266-1276.e11DOI: (10.1016/j.jaci.2011.08.013) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 T-cell clones validate the functional role of TCRs in patients with CBZ-SJS/TEN. A, Amino acid sequences of VA and VB CDR3 clonotypes of 7 T-cell clones (CS-02, 04, 08, and 10-13) from patients with CBZ-SJS/TEN. B, Cytotoxicity was determined by incubating T-cell clones with CBZ (25 μg/mL) and 51Cr-loaded C1R-B∗1502 or C1R-B∗5801 cells. ∗P < .05 and ∗∗P < .01, unpaired t test. See the Methods section in this article’s Online Repository. Journal of Allergy and Clinical Immunology 2011 128, 1266-1276.e11DOI: (10.1016/j.jaci.2011.08.013) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 Patterns of cross-reactivity with CBZ and OXC in the TCR transfectants expressing disease-associated clonotypes. We determined the levels of IL-2 secretion in the culture supernatants of the TF-10 TCR (from CS-10 single clone) transfectants obtained from a 24-hour incubation with indicated concentrations (in micromoles) of CBZ or OXC by means of ELISA. The assay sensitivity for IL-2 was 4 pg/mL. Data are expressed as means ± SDs of triplicate measurements per condition and representative of 3 independent experiments. Journal of Allergy and Clinical Immunology 2011 128, 1266-1276.e11DOI: (10.1016/j.jaci.2011.08.013) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 TCR VB repertoires in CBZ-stimulated T cells obtained from drug-tolerant subjects. CDR3 spectratype patterns of CD8+ cells were obtained from CBZ-stimulated T cells of CBZ/OXC-tolerant subjects (n = 8) at the second cycle of CBZ incubation. Black squares indicate a normal CDR3 size profile (quasi-Gaussian distribution, >5 peaks), gray squares indicate a restricted CDR3 size profile (skewed TCR repertoire, 1-4 peaks), and open squares indicate an undetectable CDR3 size profile (undetectable TCR, 0 peak). Data were obtained by using duplicate measurements of the PCR products with the cDNA of T-cell expansions of 8 donors and are representative of 2 independent experiments. Journal of Allergy and Clinical Immunology 2011 128, 1266-1276.e11DOI: (10.1016/j.jaci.2011.08.013) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Spectratype profiles of skewed TCR VB-11 with identical CDR3 length. CDR3 spectratype patterns of the VB-11 subfamily of CBZ-stimulated CD8+ T cells were obtained from the fifth cycle of in vitro cultures from patients with CBZ-SJS (S1, S2, and S7). The x-axis displays CDR3 length (amino acid), and the y-axis indicates the fluorescence intensity of the runoff products of VB-11. Data are representative of at least 2 independent experiments that measured the PCR products of T-cell expansions of 3 donors. Journal of Allergy and Clinical Immunology 2011 128, 1266-1276.e11DOI: (10.1016/j.jaci.2011.08.013) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Expression of CBZ-specific T-cell receptor in Jurkat T cells. The full-length cDNA fragments encoding TCR VA-22 and VB-11 of the T-cell clone (CS-10) were amplified by means of PCR with specific primers (Tables E4 and E5). The expression vectors containing TCR VA-22, VB-11, and CD8 cDNA fragments, respectively, were cotransfected into the Jurkat T-cell line. The transfected Jurkat cells were maintained in the culture medium containing 10 μg/mL blasticidin. The TCR transfectant (TF-10, VA-22-FISGTY, and VB-11-ISGSY; black histogram) and control parental cells (gray histogram) were stained with anti–VB-11 mAb and quantified by means of flow cytometry. The number above the bracketed line indicates the percentage of TCR VB-11–expressing cells. Data are representative of at least 2 independent experiments. Journal of Allergy and Clinical Immunology 2011 128, 1266-1276.e11DOI: (10.1016/j.jaci.2011.08.013) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 IL-2 secretion of TCR transfectants on stimulation with CBZ. The TF-10 (VA-22-FISGTY and VB-11-ISGSY) TCR transfectants with CD8 expression were incubated with parental C1R cells or C1R cells expressing HLA-B∗1502 (C1R-B∗1502 cells) or HLA-B∗5801 (C1R-B∗5801 cells) in the presence of CBZ (50 μg/mL) or GBP (50 μg/mL). We determined the levels of IL-2 secretion in Jurkat cell-culture supernatants obtained from a 24-hour incubation with drug stimulation (CBZ of 50 μg/mL or GBP of 50 μg/mL) by using an ELISA kit. The assay sensitivity for IL-2 was 4 pg/mL. Data are expressed as means ± SDs of triplicate measurements per condition and are representative of at least 2 independent experiments. ∗∗∗P < .001, unpaired t test. Journal of Allergy and Clinical Immunology 2011 128, 1266-1276.e11DOI: (10.1016/j.jaci.2011.08.013) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 The reactivity of TCR transfectants to CBZ and OXC. We determined levels of IL-2 secretion in the culture supernatants of the TCR transfectants TF-2 (A), TF-4 (B), TF-8 (C), and TF-11 (D) after a 24-hour incubation with the indicated concentration (in micromoles) of CBZ or OXC. The assay sensitivity of ELISA for IL-2 was 4 pg/mL. Data are expressed as means ± SDs of triplicate measurements per condition and are representative of at least 2 independent experiments. Journal of Allergy and Clinical Immunology 2011 128, 1266-1276.e11DOI: (10.1016/j.jaci.2011.08.013) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions