研究異嘌呤醇所引起嚴重皮膚過敏反應病人 的T淋巴球受器

Abstract

異嘌呤醇 (Allopurinol)被用來治療痛風已經持續了近半個世紀，且到現今還是最常用的藥物之ㄧ，但是在某些服用這個藥物的病人身上會產生嚴重性皮膚不良反應 (SCAR)，包括了過敏症候群 (Hypersensitivity Syndrome)、史蒂文生氏-強生症候群 (Steven-Johnson Syndrome)與毒性表皮壞死溶解症 (Toxic Epidermal Necrolysis)。其中後者造成的死亡率高達30%。在之前的研究中，我們的研究團隊在這些病人的體內發現到HLA-B*5801這一個共同的基因標記，這個標記在所有的病人(51/51)體內都有發現，但是20% (20/135)也帶有這一個基因的病人在吃了異嘌呤醇之後並不會發病，這個發現說明了HLA-B*5801對於造成異嘌呤醇引起的嚴重性皮膚不良反應是必需的，但是單單只帶有這一個基因吃了藥後並不一定會發病。因為嚴重性皮膚不良反應的致病機轉可能包括了由HLA-藥物複合體與T 淋巴受器之間的交互作用並進一步活化藥物專一性的T細胞。因此我們假設這些病人的T細胞可能擁有同樣的T淋巴受器以辨識HLA-藥物所形成的複合體並進一步造成疾病。在這篇研究當中，我們使用一個常被用來培養藥物專一性T細胞的系統，我們使用這個系統將8個異嘌呤醇過敏病人血液中藥物專一性的細胞活化並分離出來。在經過5到6次的藥物與同源B細胞株的刺激之後，我們發現到CD4+ T細胞在過敏症候群與毒性表皮壞死溶解症病人血液活化出來T細胞中活化且成為主要的族群；而CD8+ T細胞則在史蒂文生氏-強生症候群病人血液活化出來的T細胞中成了主要族群。此外7個不帶有HLA-B*5801和2個帶有此基因的正常人的血液也在這個活化系統中依照和病人一樣的條件來刺激，但是這些正常人血液中的T細胞無法被活化並最後凋亡。在那些分離出來的藥物專一性細胞中，經過T淋巴受器的分析與定序之後，我們發現在Vβ2、Vβ13A、Vβ15 和Vβ18中，某些病人的CD4+ T細胞有使用類似的T淋巴受器；而在Vβ12和Vβ13A中，某些病人的CD8+ T細胞也有使用類似的T淋巴受器。更甚於此的是，我們發現在一個HSS、一個TEN的CD4+ T細胞還有一個HSS病人的CD8+ T細胞上發現到一模一樣的Vβ13A T淋巴球受器與互補決定區3序列：CASSYSPGRNEGFF。總的來說，這一篇研究支持了這些上述的相似T淋巴球受器，特別是Vβ13A-CASSYSPGRNEGFF可能參與在異嘌呤醇所引起嚴重皮膚過敏反應的致病機轉當中。

Allopurinol is the most frequently used drug to treat hyperuricemia-related diseases, such as gout. However, some patients who are sensitive to allopurinol may develop severe cutaneous adverse reactions (SCAR), which include hypersensitivity syndrome, Steven Johnson syndrome and toxic epidermal necrolysis. The latter still carries up to 30 % mortality. In the previous study, we found that HLA-B* 5801 allele was a genetic marker for SCAR caused by allopurinol. HLA-B* 5801 was present in 100% (51/51) of patients with allopurinol-SCAR, but 20% (20/135) of tolerant patients also carried this allele. The finding suggests that HLA-B*5801 is necessary but not sufficient for allopurinol-induced SCAR. Since SCAR may involve activation of T cells through recognition of drug-MHC complex by the specific T cell receptors, we hypothesized that T cells with restricted TCR usage could recognize allopurinol bound MHC antigens, and these antigens may be selected during the course of SCAR development. A well established in vitro stimulation system was used to generate the drug specific T cells from 8 allopurinol-induced SCAR patients (4 HSS, 2 SJS, 1 SJS/TEN and 1 TEN). Peripheral blood mononuclear cells from these patients were co-cultured with allopurinol and autologous B cells and after 5~6 times of the in vitro stimulation, CD4+ T cells were found to be dominant in the HSS, SJS/TEN and TEN patients while CD8+ T cells dominant in the SJS patients. In contrast, T cells could not be enriched from 7 subjects who were negative for the HLA-B*5801 allele and from 2 healthy subjects positive for the HLA-B*5801 allele. Examining the TCR usage in the drug-enriched T cells showed restricted TCR usage among Vβ2, 13A, 15 and 18 of the CD4+ T cells and Vβ12, 13A of the CD8+ T cells. Furthermore, three patients showed an identical CASSYSPGRNEGFF CDR3 sequence in the Vβ13A TCR (CD4+ T cells of 1 HSS and 1 TEN and CD8+ T cells of 1 HSS). Taken together, these results suggested the presence of the restricted TCRs usage and that these restricted TCRs, especially Vβ13A-CASSYSPGRNEGFF may be involved in the pathogenesis of allopurinol-induced SCAR.