Shacklett Barbara L, Yang Otto, Hausner Mary Ann, Elliott Julie, Hultin Lance, Price Charles, Fuerst Marie, Matud Jose, Hultin Patricia, Cox Catherine, Ibarrondo Javier, Wong Johnson T, Nixon Douglas F, Anton Peter A, Jamieson Beth D
Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA, USA.
J Immunol Methods. 2003 Aug;279(1-2):17-31. doi: 10.1016/s0022-1759(03)00255-2.
The majority of HIV-1 infections occur via sexual transmission at mucosal epithelia lining the vagina, cervix or rectum. Mucosal tissues also serve as viral reservoirs. However, our knowledge of human mucosal T-cell responses is limited. There is a need for reliable, sensitive, and reproducible methods for assessing mucosal immunity. Here we report on the collaborative efforts of two laboratories to optimize methods for processing, culturing, and analyzing mucosal lymphocytes. Rectal biopsy tissue was obtained by flexible sigmoidoscopy, which is rapid, minimally invasive, and well tolerated. Of the four methods compared for isolating mucosal mononuclear cells (MMC), collagenase digestion reproducibly yielded the most lymphocytes (4-7 x 10(6)). Furthermore, 0.5-1 x 10(6) MMC could be polyclonally expanded to yield 17 x 10(6) CD8+ T cells allowing mapping of responses to overlapping peptides spanning the HIV-1 genome using IFN-gamma enzyme-linked immunospot (ELISpot). Expansion also reduced the spontaneous IFN-gamma production normally detected in fresh MMC. Piperacillin-tazobactam and amphotericin B reduced contamination of MMC cultures to 4%. Taken together, these methods will be useful for studies of mucosal immunity to HIV-1 and other pathogens during natural infection and following vaccination.
大多数HIV-1感染是通过性传播在阴道、宫颈或直肠的黏膜上皮发生的。黏膜组织也是病毒储存库。然而，我们对人类黏膜T细胞反应的了解有限。需要有可靠、灵敏且可重复的方法来评估黏膜免疫。在此，我们报告两个实验室为优化处理、培养和分析黏膜淋巴细胞的方法所做的合作努力。通过乙状结肠镜检查获取直肠活检组织，该方法快速、微创且耐受性良好。在比较的四种分离黏膜单核细胞（MMC）的方法中，胶原酶消化法可重复性地产生最多的淋巴细胞（4 - 7×10⁶）。此外，0.5 - 1×10⁶个MMC可多克隆扩增产生17×10⁶个CD8⁺T细胞，从而能够使用γ干扰素酶联免疫斑点法（ELISpot）绘制针对跨越HIV-1基因组的重叠肽段的反应图谱。扩增还减少了新鲜MMC中通常检测到的自发γ干扰素产生。哌拉西林-他唑巴坦和两性霉素B将MMC培养物的污染率降低到4%。综上所述，这些方法将有助于研究自然感染期间及接种疫苗后对HIV-1和其他病原体的黏膜免疫。
