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Human immune compartment comparisons: Optimization of proliferative assays for blood and gut T lymphocytes
- Source :
- Journal of immunological methods. 445
- Publication Year :
- 2016
-
Abstract
- The accumulation of peripheral blood late-differentiated memory CD8 T cells with features of replicative (cellular) senescence, including inability to proliferate in vitro, has been extensively studied. Importantly, the abundance of these cells is directly correlated with increased morbidity and mortality in older persons. Of note, peripheral blood contains only 2% of the total body lymphocyte population. By contrast, the gut-associated lymphoid tissue (GALT) is the most extensive lymphoid organ, housing up to 60% of total body lymphocytes, but has never been assessed with respect to senescence profiles. We report here the development of a method for measuring and comparing proliferative capacity of peripheral blood and gut colorectal mucosa-derived CD8 T cells. The protocol involves a 5-day culture of mononuclear leukocyte populations, from blood and gut colorectal mucosa respectively, labeled with 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) and 5-bromo-2'-deoxyuridine (BrdU) and stimulated with anti-CD2/3/28-linked microbeads. Variables tested and optimized as part of the protocol development include: mode of T cell stimulation, CFSE concentration, inclusion of a second proliferation marker, BrdU, culture duration, initial culture concentration, and inclusion of autologous irradiated feeder cells. Moving forward, this protocol demonstrates a significant advance in the ability of researchers to study compartment-specific differences of in vitro proliferative dynamics of CD8 T cells, as an indicator of replicative senescence and immunological aging. The study's two main novel contributions are (1) Optimization and adaptation of standard proliferative dynamics blood T cell protocols for T cells within the mucosal immune system. (2) Introduction of the novel technique of combining CFSE and BrdU staining to do so.
- Subjects :
- 0301 basic medicine
Senescence
Adult
Male
Cells
1.1 Normal biological development and functioning
Lymphocyte
T cell
T-Lymphocytes
Immunology
Population
Biology
Article
Mucosa
03 medical and health sciences
0302 clinical medicine
Immune system
Proliferation assay
Underpinning research
Opthalmology and Optometry
medicine
GALT
Immunology and Allergy
Cytotoxic T cell
Humans
Proliferation Marker
education
Cells, Cultured
Cancer
Cell Proliferation
education.field_of_study
Cultured
Inflammatory and immune system
CD3 T cell
Middle Aged
Colo-Rectal Cancer
030104 developmental biology
medicine.anatomical_structure
Lymphatic system
CD8 T cell
Medical Microbiology
Gastric Mucosa
HIV/AIDS
Replicative senescence
Female
Digestive Diseases
030217 neurology & neurosurgery
Subjects
Details
- ISSN :
- 18727905
- Volume :
- 445
- Database :
- OpenAIRE
- Journal :
- Journal of immunological methods
- Accession number :
- edsair.doi.dedup.....63c3c1fff379fcaeb02fecf6a4dc2481