HLA Restriction of Carbamazepine-Specific T‑Cell Clones from anHLA-A*31:01-Positive Hypersensitive PatientMaike 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 L693GE, 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 Caucasianand Japanese populations. Herein, we show that HLA-A*31:01+ restricted the activation ofcarbamazepine-specific CD8+ T-cells, which provides an immunological basis for the geneticassociation. Furthermore, CD4+ T-cells were activated with carbamazepine in a HLA-DRB1*04:04-restricted manner, indicating that a common HLA haplotype may contribute to the multiclonal T-cellresponse seen in European patients with CBZ hypersensitivity.

Carbamazepine (CBZ) is known to cause hypersensitivityreactions of varying severity in a small proportion of

patients treated with the drug. The reactions are thought tohave an underlying immune-mediated etiology, and drug-reactive T-lymphocytes of CD4 and CD8 phenotype have beenisolated from patients’ blood and skin. Genetic factorspredisposing individuals to CBZ hypersensitivity have longbeen postulated to be important, and recent genetic studieshave revealed strong associations with specific human leukocyteantigen (HLA) alleles. HLA-B*15:02 was detected in almost allcases of CBZ-induced Stevens-Johnson syndrome (SJS) inpatients of Han Chinese or South-East Asian ancestry.1 Itsfunctional role in the development of CBZ-induced SJS hasbeen well characterized, showing CD8 T-cell responses in thesepatients to be HLA-B*15:02 restricted and mediated throughrestricted T-cell receptor (TCR) usage.2,3 Genome wideassociation studies have found HLA-A*31:01 to be associatedwith all clinical phenotypes of CBZ hypersensitivity inCaucasian and Japanese populations.4,5 A study by Niihara etal. aimed to determine whether lymphocyte activation wascorrelated with HLA-A31 status by comparing lymphocyteresponses in HLA-A31 positive to A31 negative CBZhypersensitive patients, but no significant difference could bedetected.6 However, functional studies investigating whether T-cell responses to CBZ are dependent on the drug interactingspecifically with HLA-A*31:01 have not been performed. Theimportance of the CD4+ T-cell response in CBZ hyper-sensitivity is currently not known.

Figure 1. CBZ-induced release of cytolytic molecules from arepresentative CD8+ (A) and CD4+ (B) T-cell clone, measured byELISpot.

In the present study, we investigated HLA class I and IIrestriction of CBZ-reactive T-cell clones generated from a

patient carrying HLA-A*31:01. The patient presented with ageneralized maculopapular exanthema with eosinophilia andlymphocytosis 6 days after start of CBZ treatment. Bloodsamples were collected after approval by the local ethicscommittee, and informed consent was obtained from thepatient. T-cell reactivity was confirmed in vitro by thelymphocyte transformation test with a maximal stimulationindex of 15.9 recorded using 50 μg/mL CBZ (data not shown).Drug-reactive T-cells were enriched by repeated stimulationwith CBZ over two weeks and T-cell clones (TCC) generatedby the serial dilution method (see Supporting Information).7

Epstein−Barr-virus-transformed B-cell lines were used as

Received: December 9, 2013Published: January 29, 2014

Rapid Report

pubs.acs.org/crt

© 2014 American Chemical Society 175 dx.doi.org/10.1021/tx400460w | Chem. Res. Toxicol. 2014, 27, 175−177

antigen-presenting cells (APC). Reactivity of TCC (5 × 104

TCC, 1 × 104 APC per well; 200 μL total volume, 96 well cellculture plates) to CBZ (1−100 μg/mL) was tested bymeasurement of proliferation using [3H]-thymidine after a 48h culture period. A total of 947 clones were tested, and 67 werefound to respond to CBZ (control, 5,525.8 ± 18,928.0 cpm; 25μg/mL CBZ, 34,418.8 ± 43,632.5 cpm; see Table S1). TCCproliferated to CBZ in a dose-dependent manner, and whenphenotyped for CD expression by flow cytometry, CD4+,CD8+, and CD4+CD8+ clones were detected (35%, 39%, and26%, respectively). Ten CD4+ and five CD8+ well growing

clones were selected for more detailed functional analysis.Secretion of cytolytic molecules was assessed using enzyme-linked immunospot (ELISpot), and both CD4+ and CD8+

clones released granzyme B, perforin, and FasLigand whenstimulated with CBZ revealing that both types of lymphocytesdisplay cytotoxic effector functions (Figure 1).The activation of CD8+ clones could be blocked by an anti-

HLA class I antibody but not anti-HLA class II (Figure 2A).Furthermore, when APCs were pretreated with an anti-HLA-A30 A31 antibody, proliferative responses of CD8+ clones werestrongly reduced (Figure 2B). In the next step, HLA-mismatch

Figure 2. HLA restriction of CD8+ T-cell clones. (A) Proliferative responses to CBZ after blocking of APC with anti-HLA antibodies. (B) Responseof 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 usedto 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 HLAclass II restriction. (C) CBZ-specific response of clones in the presence of HLA-mismatched APC. (D) Proliferative responses of TCC to CBZ usingHLA-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).

Chemical Research in Toxicology Rapid Report

dx.doi.org/10.1021/tx400460w | Chem. Res. Toxicol. 2014, 27, 175−177176

experiments were conducted to characterize further the role ofHLA-A*31:01 on the activation of T-cells with CBZ. UsingHLA-A*31:01+ APC from healthy donors and allogeneic APCexpressing common HLA-A alleles (HLA-A*01:01, -A*02:01,and -A*03:01; see Table S2), we were able to show a drug-specific 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 responseseen in Han Chinese CBZ-SJS patients, where only minorexpansion of CD4+ T-cells could be observed.3

For CD4+ clones, CBZ reactivity was restricted by HLA classII (Figure 3A), and more detailed analysis using HLA-DP-,-DQ-, and -DR-blocking antibodies showed the response to bedriven 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 toform a common haplotype with HLA-A*31:01 in Caucasians.We therefore investigated a possible role of HLA-DRB1*04:04in CBZ-specific CD4+ T-cell responses using partially HLA-matched APC (Figure 3C). CD4+ clones proliferated uponCBZ stimulation in the presence of allogeneic APC expressingHLA-DRB1*04:04 and HLA-A*31:01 but also HLA-DRB1*04:04+ APC lacking HLA-A*31:01. APCs expressingother HLA-DRB1 alleles could not stimulate a response inthese clones. Furthermore, HLA-DRB1*04:04-transfected T2cell lines were able to trigger specific proliferation to CBZ inCD4+ clones confirming that this HLA class II allele isfunctionally relevant for the activation of CBZ-specific T-lymphocytes 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 theremoval of CLIP (class II-associated invariant chain peptide)from the HLA binding groove prior to antigen loading, wasexpressed at the same time. This suggests that binding of CBZto HLA-DRB1*04:04 may be hindered in the presence ofCLIP, which might be due to the insufficient binding affinitybetween CBZ and CLIP preventing the formation of a stabledrug−peptide−HLA complex. Alternatively, CBZ may onlybind to an empty HLA binding groove, thereby inducing achange to the peptide repertoire, which in turn causes T-cellactivation.8 The implication of this observation warrants furtherinvestigation.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 restrictionprofile. We discovered an HLA class II allele, i.e., HLA-DRB1*04:04, to be functionally important for the activation ofCD4+ T-cells. The strong linkage disequilibrium between thesetwo alleles suggests that a common haplotype may contributeto the multiclonal response seen in all Caucasian carbamaze-pine hypersensitive patients. Further studies including a largerpatient cohort that are carriers of HLA-A*31:01 are needed inorder to confirm our initial findings described here.

■ ASSOCIATED CONTENT

*S Supporting InformationExperimental procedures, specificity, and phenotype of CBZ-specific TCC and HLA haplotypes of APC. This material isavailable free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Tel: +44 151 7945346. Fax: +44 151 7945540. E-mail: dnes@liv.ac.uk.FundingThis work was funded from the European Union’s SeventhFramework Programme FP7/2007-2013 under grant agree-ment number 238132 and the Centre for Drug Safety Sciencesupported by the Medical Research Council (G0700654). M.P.is a NIHR Senior Investigator and is funded by the NHS Chairof Pharmacogenetics from the UK Department of Health.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank the patient for participating in this project and theCDSS nurse who helped collect the sample.

■ ABBREVIATIONSCBZ, carbamazepine; HLA, human leukocyte antigen; TCC, T-cell 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 susceptibilityto carbamazepine-induced cutaneous adverse drug reactions. Pharma-cogenet. 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 carbamazepineactivates T cells in patients with Stevens-Johnson syndrome. J. AllergyClin. 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-cellreceptor use is crucial for carbamazepine-induced Stevens-Johnsonsyndrome. 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., deBakker, 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 andcarbamazepine-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 associationstudy identifies HLA-A*3101 allele as a genetic risk factor forcarbamazepine-induced cutaneous adverse drug reactions in Japanesepopulation. 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-inducedadverse drug reactions but not with carbamazepine-inducedlymphocyte 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 carbamaze-pine 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-peptiderepertoire. Nature 486, 554−558.

Chemical Research in Toxicology Rapid Report

dx.doi.org/10.1021/tx400460w | Chem. Res. Toxicol. 2014, 27, 175−177177