Back to Search
Start Over
Optimization of methods to assess human mucosal T-cell responses to HIV infection
- Source :
- Journal of Immunological Methods. 279:17-31
- Publication Year :
- 2003
- Publisher :
- Elsevier BV, 2003.
-
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.
- Subjects :
- Cellular immunity
Sexual transmission
T-Lymphocytes
T cell
Immunology
Epitopes, T-Lymphocyte
Enzyme-Linked Immunosorbent Assay
HIV Infections
CD8-Positive T-Lymphocytes
Biology
Peripheral blood mononuclear cell
Epitope
medicine
Humans
Immunology and Allergy
Mucous Membrane
ELISPOT
Histocompatibility Antigens Class I
Flow Cytometry
Vaccination
medicine.anatomical_structure
HIV-1
Immunologic Techniques
Cytokines
CD8
Subjects
Details
- ISSN :
- 00221759
- Volume :
- 279
- Database :
- OpenAIRE
- Journal :
- Journal of Immunological Methods
- Accession number :
- edsair.doi.dedup.....a43b3a03d62c17ce9a941b3144049ac1