The study of T cell repertoire in patients with Carbamazepine-induced Stevens-Johnson syndrome (CBZ-SJS)

Abstract

Stevens-Johnson Syndrome (SJS) is a severe, life-threatening cutaneous adverse reaction, most often caused by medication. Growing evidence indicates that CD8+ cytotoxic T lymphocytes, which can be activated by specific antigen-MHC complex expressed on the surface of antigen-presenting cells (APC), are involved in the pathogenesis of SJS. In our previous studies, we found that SJS induced by carbamazepine (CBZ), a commonly prescribed anticonvulsant, is strongly associated with HLA-B*1502 allele. Because specific T cell receptor (TCR) recognition of drug-bound peptide-MHC complex is likely to be driven by HLA-B*1502 with CBZ-bound endogenous peptides, we hypothesized that T cell populations having restricted TCR usage that recognized the CBZ modified antigens may be selected during the course of the disease in the SJS patients. My thesis is therefore to aim at analyzing TCR usage of CBZ-specific T cells in PBMC or blister cells from CBZ-SJS patients. To examine if CBZ-SJS patients share common T-cell receptor usage, I first enriched drug-specific T cells from PBMC and blister cells of patients by co-culturing with irradiated autologous EBV- transformed B cells and CBZ major metabolite, 10,11-epoxide CBZ. There was a biased reactivity of the T cells after co-culturing for 3 to 5 weeks, with CD8+ T cells increasing 1.8 to 7.5 times (from average 30% to average 90%) in three CBZ-SJS PBMC and to 85.43% in one CBZ-SJS blister, which were detected by FLOW cytometry. The mRNAs of these T cells were extracted by Trizol reagent and reverse-transcripted into cDNA. These cDNAs were used to detect both α-chain and β-chain TCR phenotype by PCR with 47 sequence specific primers for different TCRV gene types. The PCR result showed a selected pattern of these biased T cells that were composed of restricted T cell gene types comparing to normal control or patient before co-culturing. After cloning and sequencing the transcripts of each TCRV gene type, the gene rearrangement and the amino acid sequences of each TCR were analyzed IMGT website. The result showed that there was preferential TCRAV and TCRBV gene usage in the SJS patients. Among four CBZ-SJS patients, TRAV14 and TRBV3-1*01 are shared by three patients, while TRAV19*01 and TRBV7-8*03/D1*01/J2-7*01 and TRAV17*01 and TRBV28*01/D1*01/ J1-1*01 are shared by two patients. This finding suggests selective activation of T cells in the pathogenesis of CBZ-SJS. More samples and further functional studies are needed to demonstrate that these TCRs are indeed involved in the induction of T cell activation.