Optimization of methods to assess human mucosal T-cell responses to HIV infection

Barbara Shacklett, Otto Yang, Mary Ann Hausner, Julie Elliott, Lance Hultin, Charles Price, Marie Fuerst, Jose Matud, Patricia Hultin, Catherine Cox, Javier Ibarrondo, Johnson T. Wong, Douglas F. Nixon, Peter A. Anton, Beth D. Jamieson

Research output: Contribution to journalArticle

82 Scopus citations

Abstract

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 × 106). Furthermore, 0.5-1 × 106 MMC could be polyclonally expanded to yield 17 × 106 CD8+ T cells allowing mapping of responses to overlapping peptides spanning the HIV-1 genome using IFN-γ enzyme-linked immunospot (ELISpot). Expansion also reduced the spontaneous IFN-γ 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.

Original languageEnglish (US)
Pages (from-to)17-31
Number of pages15
JournalJournal of Immunological Methods
Volume279
Issue number1-2
DOIs
StatePublished - Aug 2003

Keywords

  • ELISpot
  • GALT
  • HIV
  • Mucosal immunity
  • T cells
  • Tetramer

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

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    Shacklett, B., Yang, O., Hausner, M. A., Elliott, J., Hultin, L., Price, C., Fuerst, M., Matud, J., Hultin, P., Cox, C., Ibarrondo, J., Wong, J. T., Nixon, D. F., Anton, P. A., & Jamieson, B. D. (2003). Optimization of methods to assess human mucosal T-cell responses to HIV infection. Journal of Immunological Methods, 279(1-2), 17-31. https://doi.org/10.1016/S0022-1759(03)00255-2