Detection and quantification of blood-derived CD8(+) T lymphocytes secreting tumor necrosis factor alpha in response to HLA-A2.1-binding melanoma and viral peptide antigens
Detection and quantification of blood-derived CD8(+) T lymphocytes secreting tumor necrosis factor alpha in response to HLA-A2.1-binding melanoma and viral peptide antigens
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DOI：
10.1016/0022-1759(96)00007-5
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发表时间：
1996-05-27
影响因子：
2.2
通讯作者：
Wolfel, T
中科院分区：
文献类型：
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作者：
Herr, W;Schneider, J;Wolfel, T
我们采用酶联免疫斑点（ELISPOT）法检测和定量识别HLA-A2.1呈递肽抗原的血源性CD8(+) T细胞。从外周血中分离CD8(+) T淋巴细胞，用装载肽的a2.1阳性0.174 x CEM刺激40 h。T2细胞。肿瘤坏死因子α (tnf - α)由单个T细胞对抗原接触反应分泌，被捕获在预涂有抗tnf - α抗体的硝化纤维素膜上，然后免疫化学显示为斑点。通过该试验，在测试期间检测到高达25%的克隆细胞溶解T淋巴细胞（CTL）识别出与HLA-A2.1相关的定义黑色素瘤抗原。CD8（+）淋巴细胞对已知的人类免疫缺陷病毒（HIV）逆转录酶的免疫原性hla - a2.1结合肽有反应，仅在HIV感染患者中检测到，而在抗HIV阴性供者中没有检测到。T细胞与源自突变的周期蛋白依赖性激酶4 （CDK4-R24C）的肽反应，仅在从患者血液中分离的CD8（+）淋巴细胞中检测到，该患者的黑色素瘤先前被发现携带CDK4-R24C等位基因。T细胞对正常黑色素细胞分化抗原酪氨酸酶和黑色素-a /MART-1的hla - a2.1相关肽反应的频率很低，这可能反映了对这些抗原的天然自身免疫。然而，在黑色素瘤患者中，我们发现黑色素瘤转移手术后几天，T细胞对黑色素-a /MART-1和酪氨酸酶肽的频率很高(高达38 / 10（5)CD8(+) T细胞）。在接下来的几个月里逐渐减少。在一名进展性疾病的HIV感染患者中，我们观察到与HIV逆转录酶肽反应的T细胞的损失。这些观察结果提供了证据，证明肽依赖性tnf - α斑点的形成是由于先前体内抗原暴露所致。因此，在疾病的自然过程中，以及在旨在诱导保护性T细胞免疫的治疗干预期间，tnf - α ELISPOT检测可能有助于监测抗原特异性T淋巴细胞反应。此外，它可能有助于识别免疫显性T细胞表位。
We applied an enzyme-linked immunospot (ELISPOT) assay for the detection and quantification of blood-derived CD8(+) T cells recognizing peptide antigens presented by HLA-A2.1. CD8(+) T lymphocytes were isolated from peripheral blood and were stimulated for 40 h with peptide-loaded A2.1-positive 0.174 x CEM.T2 cells. Tumor necrosis factor alpha (TNF-alpha) secreted by single T cells in response to antigen contact was trapped on nitrocellulose membranes precoated with anti-TNF-alpha antibodies and was then immunochemically visualized as spots. With this assay, up to 25% of cloned cytolytic T lymphocytes (CTL) were detected during the test period that recognized defined melanoma antigens in association with HLA-A2.1.CD8(+) lymphocytes responsive to a known immunogenic HLA-A2.1-binding peptide from reverse transcriptase of the human immunodeficiency virus (HIV) were only detectable in HIV-infected patients, but not in anti-HIV-negative donors. T cells reacting with a peptide derived from a mutated cyclin-dependent kinase 4 (CDK4-R24C) were exclusively detected among CD8(+) lymphocytes isolated from blood of the patient, whose melanoma had previously been found to carry the CDK4-R24C allele. T cells responding to HLA-A2.1-associated peptides of normal melanocyte differentiation antigens tyrosinase and Melan-A/MART-1 were found at low frequencies in almost all donors tested, which might reflect a natural autoimmunity to these antigens, However, in a melanoma patient we found a few days after surgery of melanoma metastases high frequencies of T cells against Melan-A/MART-1 and tyrosinase peptides (up to 38 per 10(5) CD8(+) T cells), which gradually decreased during the following months. In an HIV-infected patient with progressive disease we observed a loss of T cells reactive with the HIV reverse transcriptase peptide.These observations provide evidence that peptide-dependent TNF-alpha spot formation in vitro resulted from previous antigen exposure in vivo. Therefore, the TNF-alpha ELISPOT assay might be useful in monitoring antigen-specific T lymphocyte responses during the natural course of diseases as well as during therapeutic interventions aiming at the induction of protective T cell immunity. In addition, it might help to identify immunodominant T cell epitopes.