title: Optimization of methods to assess human mucosal T-cell responses to HIV infection.
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 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.
authors.affiliation: Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA, USA.
authors.forename: Barbara L
authors.initials: BL
authors.lastname: Shacklett
authors.forename: Otto
authors.initials: O
authors.lastname: Yang
authors.forename: Mary Ann
authors.initials: MA
authors.lastname: Hausner
authors.forename: Julie
authors.initials: J
authors.lastname: Elliott
authors.forename: Lance
authors.initials: L
authors.lastname: Hultin
authors.forename: Charles
authors.initials: C
authors.lastname: Price
authors.forename: Marie
authors.initials: M
authors.lastname: Fuerst
authors.forename: Jose
authors.initials: J
authors.lastname: Matud
authors.forename: Patricia
authors.initials: P
authors.lastname: Hultin
authors.forename: Catherine
authors.initials: C
authors.lastname: Cox
authors.forename: Javier
authors.initials: J
authors.lastname: Ibarrondo
authors.forename: Johnson T
authors.initials: JT
authors.lastname: Wong
authors.forename: Douglas F
authors.initials: DF
authors.lastname: Nixon
authors.forename: Peter A
authors.initials: PA
authors.lastname: Anton
authors.forename: Beth D
authors.initials: BD
authors.lastname: Jamieson
mesh_terms: D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D016207:Cytokines / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods; D015658:HIV Infections / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology*; D015395:Histocompatibility Antigens Class I / Q000276:immunology; D006801:Humans; D007158:Immunologic Techniques; D009092:Mucous Membrane / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D016207:Cytokines; D018984:Epitopes, T-Lymphocyte; D015395:Histocompatibility Antigens Class I