Peptide epitope recognized by Toxoplasma gondii-infected cell-specific cytotoxic T lymphocytes

本文从一名慢性弓形虫病患者外周血淋巴细胞诱导的细胞毒性T淋巴细胞(C)中提取刚地弓形虫感染细胞的天然加工肽，并对其进行检测。CTL系是通过每周用弓形虫感染的人B淋巴瘤系（ARH）体外刺激获得的，该淋巴瘤系与患者共享HLA-A2和Cw4决定因子。抗hla - a，B，C单克隆抗体（mAb）和抗hla - a2单克隆抗体抑制了CTL对弓形虫感染的ARH和用反相高效液相色谱（RP-HPLC）提取物29段脉冲的ARH的裂解活性。Anti-HLA-DR mAb未能阻断裂解活性。因此，弓形虫感染的细胞向CTL呈递肽是由HLA-A2分子介导的。从弓形虫感染的ARH细胞裂解液中，用抗HLA-A2单抗偶联免疫吸附柱纯化与HLA-A2分子结合的肽。从HLA-A2分子络合物中解离的肽段，经反相高效液相色谱（RP-HPLC）分离为第29段，用于nh_2末端的顺序Edman降解。29分氨基酸序列分析表明，2号位置为优势亮氨酸，3号位置为异亮氨酸，8号位置为苯丙氨酸，9号位置为优势蛋氨酸和苯丙氨酸。因此，片段29中hla - a2结合肽的氨基酸序列部分符合hla - a2结合肽基序的预测算法。

Naturally processed peptides derived from Toxoplasma gondii (T.gondii) -infected cells were acid extracted and detected by cytotoxic T lymphocytes (C) induced from peripheral blood lymphocytes of a patient with chronic toxoplasmosis. The CTL lines were obtained by weekly in vitro stimulation with a T.gondii-infected human B lymphoma line, ARH,which shares HLA-A2 and Cw4 determinants with the patient. The lytic activity of these CTL lines against T.gondii-infected ARH and ARH pulsed with fraction 29 of a reversed-phase high-performance liquid chromatography (RP-HPLC) extract from T.gondii-infected ARH was inhibited by an anti-HLA-A,B,C monoclonal antibody (mAb) and an anti-HLA-A2 mAb. Anti-HLA-DR mAb failed to block the lytic activity. Thus, the presentation of peptides by T.gondii-infected cells for CTL is mediated by HLA-A2 molecules. The peptides bound to HLA-A2 molecules were purified with an anti-HLA-A2 mAb-coupled immunoadsorbent column from T.gondii-infected ARH cell lysate. The peptides dissociated from the complex of HLA-A2 molecules and fractionated by RP-HPLC in fraction 29 were submitted for sequential NH_2-terminal Edman degradation. The amino acid sequence analysis of fraction 29 revealedthat a dominant leucine was found at position 2, isoleucine was found at position 3, phenylalanine was found at position 8, and methionine and phenylalanine were dominant at position 9. Thus, the amino acid sequence of the HLA-A2-bound peptide in fraction 29 was in part consistent with the predictive algorithm of HLA-A2-binding peptide motifs.