Use of overlapping peptide mixtures as antigens for cytokine flow cytometry
Use of overlapping peptide mixtures as antigens for cytokine flow cytometry
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DOI：
10.1016/s0022-1759(01)00416-1
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发表时间：
2001-09-01
影响因子：
2.2
通讯作者：
Picker, LJ
中科院分区：
文献类型：
--
作者：
Maecker, HT;Dunn, HS;Picker, LJ
细胞内细胞因子染色和流式细胞术可用于测量t细胞对特定抗原的反应。虽然CD8(+) t细胞对可溶性蛋白的反应通过这种方法检测效率不高，但肽可以用作抗原。利用跨越整个蛋白序列的重叠肽，可以检测到CD8(+) t细胞对多个表位的反应，而不管HLA类型如何。在这项研究中，比较了不同长度的重叠肽混合物，发现有15个氨基酸肽与I个氨基酸重叠，可以刺激CD4（+）和CD8(+) t细胞反应。这种多肽混合物对CD4(+) t细胞的刺激作用与重组蛋白完全相同，同时对CD8(+) t细胞的刺激作用远高于重组蛋白完全。虽然8-12个氨基酸肽产生最高水平的CD8(+) t细胞反应，但15个氨基酸肽仍然非常有效。然而，长度为20个氨基酸的肽在相同的肽剂量下不会刺激强烈的CD8(+) t细胞反应。细胞因子对单个表位的反应加起来大约等于对整个混合物的反应，表明大混合物可以以定量方式检测反应。与全蛋白抗原不同，肽混合物在冷冻保存的PBMC和室温保存24小时的血液中都能有效刺激反应。因此，重叠的15个氨基酸肽混合物可能有助于通过细胞因子流式细胞术分析抗原特异性CD4（+）和CD8(+) t细胞反应，使用临床标本，包括运送的血液或冷冻保存的PBMC。(C) 2001 Elsevier Science B.V.版权所有
Intracellular cytokine staining and flow cytometry can be used to measure T-cell responses to defined antigens. Although CD8(+) T-cell responses to soluble proteins are inefficiently detected by this approach, peptides can be used as antigens. Using overlapping peptides spanning an entire protein sequence, CD8(+) T-cell responses can be detected to multiple epitopes, regardless of HLA type. In this study, overlapping peptide mixes of various lengths were compared and 15 amino acid peptides with I I amino acid overlaps were found to stimulate both CD4(+) and CD8(+) T-cell responses. Such peptide mixes stimulated CD4(+) T-cell responses equivalent to those observed with whole recombinant protein, while simultaneously stimulating CD8(+) T-cell responses much higher than those observed with whole protein. Although 8-12 amino acid peptides produced the highest level of CD8(+) T-cell responses, 15 amino acid peptides were still very effective. Peptides that were 20 amino acids in length, however, did not stimulate strong CD8(+) T-cell responses at the same peptide dose. The cytokine responses to individual epitopes added up approximately to the response to the entire mix, demonstrating that large mixes can detect responses in a quantitative fashion. Unlike whole protein antigens, peptide mixes were effective at stimulating responses in both cryopreserved PBMC and blood stored for 24 h at room temperature. Thus, overlapping 15 amino acid peptide mixes may facilitate the analysis of antigen-specific CD4(+) and CD8(+) T-cell responses by cytokine flow cytometry, using clinical specimens that include shipped blood or cryopreserved PBMC. (C) 2001 Elsevier Science B.V. All rights reserved.