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HLA Restriction of Carbamazepine-Specific T‑Cell Clones from an HLA-A*31:01-Positive Hypersensitive Patient Maike Lichtenfels,† John Farrell,† Monday O. Ogese,† Catherine C. Bell,† Sidonia Eckle,‡ James McCluskey,‡ B. Kevin Park,† Ana Alfirevic,† Dean J. Naisbitt,*,† and Munir Pirmohamed† †
Department of Molecular and Clinical Pharmacology, University of Liverpool, Sherrington Building, Ashton Street, Liverpool L69 3GE, England ‡ Department of Microbiology and Immunology, University of Melbourne, Gate 11 Royal Parade, Parkville, Victoria 3010, Australia S Supporting Information *
ABSTRACT: HLA-A*31:01 is associated with carbamazepine (CBZ) hypersensitivity in Caucasian and Japanese populations. Herein, we show that HLA-A*31:01+ restricted the activation of carbamazepine-specific CD8+ T-cells, which provides an immunological basis for the genetic association. Furthermore, CD4+ T-cells were activated with carbamazepine in a HLA-DRB1*04:04restricted manner, indicating that a common HLA haplotype may contribute to the multiclonal T-cell response seen in European patients with CBZ hypersensitivity.
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arbamazepine (CBZ) is known to cause hypersensitivity reactions of varying severity in a small proportion of patients treated with the drug. The reactions are thought to have an underlying immune-mediated etiology, and drugreactive T-lymphocytes of CD4 and CD8 phenotype have been isolated from patients’ blood and skin. Genetic factors predisposing individuals to CBZ hypersensitivity have long been postulated to be important, and recent genetic studies have revealed strong associations with specific human leukocyte antigen (HLA) alleles. HLA-B*15:02 was detected in almost all cases of CBZ-induced Stevens-Johnson syndrome (SJS) in patients of Han Chinese or South-East Asian ancestry.1 Its functional role in the development of CBZ-induced SJS has been well characterized, showing CD8 T-cell responses in these patients to be HLA-B*15:02 restricted and mediated through restricted T-cell receptor (TCR) usage.2,3 Genome wide association studies have found HLA-A*31:01 to be associated with all clinical phenotypes of CBZ hypersensitivity in Caucasian and Japanese populations.4,5 A study by Niihara et al. aimed to determine whether lymphocyte activation was correlated with HLA-A31 status by comparing lymphocyte responses in HLA-A31 positive to A31 negative CBZ hypersensitive patients, but no significant difference could be detected.6 However, functional studies investigating whether Tcell responses to CBZ are dependent on the drug interacting specifically with HLA-A*31:01 have not been performed. The importance of the CD4+ T-cell response in CBZ hypersensitivity is currently not known. © 2014 American Chemical Society
Figure 1. CBZ-induced release of cytolytic molecules from a representative CD8+ (A) and CD4+ (B) T-cell clone, measured by ELISpot.
In the present study, we investigated HLA class I and II restriction of CBZ-reactive T-cell clones generated from a patient carrying HLA-A*31:01. The patient presented with a generalized maculopapular exanthema with eosinophilia and lymphocytosis 6 days after start of CBZ treatment. Blood samples were collected after approval by the local ethics committee, and informed consent was obtained from the patient. T-cell reactivity was confirmed in vitro by the lymphocyte transformation test with a maximal stimulation index of 15.9 recorded using 50 μg/mL CBZ (data not shown). Drug-reactive T-cells were enriched by repeated stimulation with CBZ over two weeks and T-cell clones (TCC) generated by the serial dilution method (see Supporting Information).7 Epstein−Barr-virus-transformed B-cell lines were used as Received: December 9, 2013 Published: January 29, 2014 175
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Figure 2. HLA restriction of CD8+ T-cell clones. (A) Proliferative responses to CBZ after blocking of APC with anti-HLA antibodies. (B) Response of clones to HLA-A30 A31 block. (C) Reactivity to CBZ in the presence of allogeneic HLA-mismatched APC. TCC were incubated with APC ± CBZ for 48 h and proliferation determined by [3H]-thymidine incorporation. Data represent the mean cpm ± SE of 3 clones. Wilcoxon test was used to compare responses between paired samples (A,B) and Mann−Whitney U test for independent samples (C) (*p < 0.05, **p < 0.01).
Figure 3. HLA-restricted response of CD4+ clones. (A) CBZ-specific proliferation of TCC after HLA blocking of APC. (B) Fine-mapping of HLA class II restriction. (C) CBZ-specific response of clones in the presence of HLA-mismatched APC. (D) Proliferative responses of TCC to CBZ using HLA-transfected T2 cell lines. Data represent the mean cpm ± SE of 3 clones. Data were analyzed using the Mann−Whitney U test (*p < 0.05; **p < 0.01).
antigen-presenting cells (APC). Reactivity of TCC (5 × 104 TCC, 1 × 104 APC per well; 200 μL total volume, 96 well cell culture plates) to CBZ (1−100 μg/mL) was tested by measurement of proliferation using [3H]-thymidine after a 48 h culture period. A total of 947 clones were tested, and 67 were found to respond to CBZ (control, 5,525.8 ± 18,928.0 cpm; 25 μg/mL CBZ, 34,418.8 ± 43,632.5 cpm; see Table S1). TCC proliferated to CBZ in a dose-dependent manner, and when phenotyped for CD expression by flow cytometry, CD4+, CD8+, and CD4+CD8+ clones were detected (35%, 39%, and 26%, respectively). Ten CD4+ and five CD8+ well growing
clones were selected for more detailed functional analysis. Secretion of cytolytic molecules was assessed using enzymelinked immunospot (ELISpot), and both CD4+ and CD8+ clones released granzyme B, perforin, and FasLigand when stimulated with CBZ revealing that both types of lymphocytes display cytotoxic effector functions (Figure 1). The activation of CD8+ clones could be blocked by an antiHLA class I antibody but not anti-HLA class II (Figure 2A). Furthermore, when APCs were pretreated with an anti-HLAA30 A31 antibody, proliferative responses of CD8+ clones were strongly reduced (Figure 2B). In the next step, HLA-mismatch 176
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experiments were conducted to characterize further the role of HLA-A*31:01 on the activation of T-cells with CBZ. Using HLA-A*31:01+ APC from healthy donors and allogeneic APC expressing common HLA-A alleles (HLA-A*01:01, -A*02:01, and -A*03:01; see Table S2), we were able to show a drugspecific HLA- A*31:01-dependent activation of the CD8+ clones (Figure 2C). As described previously, CBZ-specific CD4+ T-cell responses are also readily detectable in Caucasian hypersensitive patients.7 This is in stark contrast to the dominant CD8 T-cell response seen in Han Chinese CBZ-SJS patients, where only minor expansion of CD4+ T-cells could be observed.3 For CD4+ clones, CBZ reactivity was restricted by HLA class II (Figure 3A), and more detailed analysis using HLA-DP-, -DQ-, and -DR-blocking antibodies showed the response to be driven primarily by HLA-DR and -DP (Figure 3B). HLA haplotype analysis revealed the patient to be a carrier of HLA-DRB1*04:04; and this HLA-DRB1 allele is known to form a common haplotype with HLA-A*31:01 in Caucasians. We therefore investigated a possible role of HLA-DRB1*04:04 in CBZ-specific CD4+ T-cell responses using partially HLAmatched APC (Figure 3C). CD4+ clones proliferated upon CBZ stimulation in the presence of allogeneic APC expressing HLA-DRB1*04:04 and HLA-A*31:01 but also HLADRB1*04:04+ APC lacking HLA-A*31:01. APCs expressing other HLA-DRB1 alleles could not stimulate a response in these clones. Furthermore, HLA-DRB1*04:04-transfected T2 cell lines were able to trigger specific proliferation to CBZ in CD4+ clones confirming that this HLA class II allele is functionally relevant for the activation of CBZ-specific Tlymphocytes from our hypersensitive patient (Figure 3D). Interestingly, T-cell activation was only observed if HLA-DM, which stabilizes empty HLA class II molecules and assists the removal of CLIP (class II-associated invariant chain peptide) from the HLA binding groove prior to antigen loading, was expressed at the same time. This suggests that binding of CBZ to HLA-DRB1*04:04 may be hindered in the presence of CLIP, which might be due to the insufficient binding affinity between CBZ and CLIP preventing the formation of a stable drug−peptide−HLA complex. Alternatively, CBZ may only bind to an empty HLA binding groove, thereby inducing a change to the peptide repertoire, which in turn causes T-cell activation.8 The implication of this observation warrants further investigation. In conclusion, we were able to generate drug-specific clones of CD4+ and CD8+ phenotype from our HLA-A*31:01+ patient and characterize the patient’s individual HLA restriction profile. We discovered an HLA class II allele, i.e., HLADRB1*04:04, to be functionally important for the activation of CD4+ T-cells. The strong linkage disequilibrium between these two alleles suggests that a common haplotype may contribute to the multiclonal response seen in all Caucasian carbamazepine hypersensitive patients. Further studies including a larger patient cohort that are carriers of HLA-A*31:01 are needed in order to confirm our initial findings described here.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +44 151 7945346. Fax: +44 151 7945540. E-mail: dnes@ liv.ac.uk. Funding
This work was funded from the European Union’s Seventh Framework Programme FP7/2007-2013 under grant agreement number 238132 and the Centre for Drug Safety Science supported by the Medical Research Council (G0700654). M.P. is a NIHR Senior Investigator and is funded by the NHS Chair of Pharmacogenetics from the UK Department of Health. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the patient for participating in this project and the CDSS nurse who helped collect the sample. ABBREVIATIONS CBZ, carbamazepine; HLA, human leukocyte antigen; TCC, Tcell clones; APC, antigen-presenting cells REFERENCES
(1) Hung, S. I., Chung, W. H., Jee, S. H., Chen, W. C., Chang, Y. T., Lee, W. R., Hu, S. L., Wu, M. T., Chen, G. S., Wong, T. W., Hsiao, P. F., Chen, W. H., Shih, H. Y., Fang, W. H., Wei, C. Y., Lou, Y. H., Huang, Y. L., Lin, J. J., and Chen, Y. T. (2006) Genetic susceptibility to carbamazepine-induced cutaneous adverse drug reactions. Pharmacogenet. Genomics 16, 297−306. (2) Wei, C. Y., Chung, W. H., Huang, H. W., Chen, Y. T., and Hung, S. I. (2012) Direct interaction between HLA-B and carbamazepine activates T cells in patients with Stevens-Johnson syndrome. J. Allergy Clin. Immunol. 129, 1562−1569.e5. (3) Ko, T. M., Chung, W. H., Wei, C. Y., Shih, H. Y., Chen, J. K., Lin, C. H., Chen, Y. T., and Hung, S. I. (2011) Shared and restricted T-cell receptor use is crucial for carbamazepine-induced Stevens-Johnson syndrome. J. Allergy Clin. Immunol. 128, 1266−1276.e11. (4) McCormack, M., Alfirevic, A., Bourgeois, S., Farrell, J. J., Kasperaviciute, D., Carrington, M., Sills, G. J., Marson, T., Jia, X., de Bakker, P. I., Chinthapalli, K., Molokhia, M., Johnson, M. R., O’Connor, G. D., Chaila, E., Alhusaini, S., Shianna, K. V., Radtke, R. A., Heinzen, E. L., Walley, N., Pandolfo, M., Pichler, W., Park, B. K., Depondt, C., Sisodiya, S. M., Goldstein, D. B., Deloukas, P., Delanty, N., Cavalleri, G. L., and Pirmohamed, M. (2011) HLA-A*3101 and carbamazepine-induced hypersensitivity reactions in Europeans. N. Engl. J. Med. 364, 1134−1143. (5) Ozeki, T., Mushiroda, T., Yowang, A., Takahashi, A., Kubo, M., Shirakata, Y., Ikezawa, Z., Iijima, M., Shiohara, T., Hashimoto, K., Kamatani, N., and Nakamura, Y. (2011) Genome-wide association study identifies HLA-A*3101 allele as a genetic risk factor for carbamazepine-induced cutaneous adverse drug reactions in Japanese population. Hum. Mol. Genet. 20, 1034−1041. (6) Niihara, H., Kakamu, T., Fujita, Y., Kaneko, S., and Morita, E. (2012) HLA-A31 strongly associates with carbamazepine-induced adverse drug reactions but not with carbamazepine-induced lymphocyte proliferation in a Japanese population. J. Dermatol. 39, 594−601. (7) Wu, Y., Farrell, J., Pirmohamed, M., Park, B. K., and Naisbitt, D. J. (2007) Generation and characterization of antigen-specific CD4+, CD8+, and CD4+CD8+ T-cell clones from patients with carbamazepine hypersensitivity. J. Allergy Clin. Immunol. 119, 973−981. (8) Illing, P. T., Vivian, J. P., Dudek, N. L., Kostenko, L., Chen, Z., Bharadwaj, M., Miles, J. J., Kjer-Nielsen, L., Gras, S., Williamson, N. A., Burrows, S. R., Purcell, A. W., Rossjohn, J., and McCluskey, J. (2012) Immune self-reactivity triggered by drug-modified HLA-peptide repertoire. Nature 486, 554−558.
ASSOCIATED CONTENT
S Supporting Information *
Experimental procedures, specificity, and phenotype of CBZspecific TCC and HLA haplotypes of APC. This material is available free of charge via the Internet at http://pubs.acs.org. 177
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