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

从弓形虫感染细胞中提取天然处理的多肽，并用从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)进行检测。CTL株是用弓形虫感染的人类B淋巴瘤株ARH通过每周体外刺激获得的，ARH与患者共享HLA-A2和CW4决定簇。抗人类白细胞抗原-A、B、C单抗和抗人类白细胞抗原A2单抗可抑制这些CTL株对弓形虫感染的ARH的杀伤活性，并与弓形虫感染的ARH的反相高效液相色谱(RP-HPLC)提取物的组分29致敏。抗人类白细胞抗原-DR单抗不能阻断其裂解活性。因此，弓形虫感染细胞的CTL呈递多肽是由人类白细胞抗原A2分子介导的。用抗HLA-A2单抗偶联免疫吸附树脂从弓形虫感染的ARH细胞裂解物中纯化与人类白细胞抗原A2分子结合的多肽。从人类白细胞抗原-A2分子复合体中解离出来的多肽，经反相高效液相色谱分离得到29个组分，然后提交给连续的NH_2末端Edman降解。组分29的氨基酸序列分析表明，第2位以亮氨酸为主，第3位以异亮氨酸为主，第8位以苯丙氨酸为主，第9位以蛋氨酸和苯丙氨酸为主。因此，29号组分的氨基酸序列与人类白细胞抗原-A2结合肽基序的预测算法部分一致。

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.