Human immune compartment comparisons: Optimization of proliferative assays for blood and gut T lymphocytes
| Autor: | Lance E. Hultin, Beth D. Jamieson, Jeffrey Dock, Rita B. Effros, Otto O. Yang, Julie Elliot, Peter A. Anton, Patricia M. Hultin |
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| Rok vydání: | 2016 |
| Předmět: |
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 |
| Zdroj: | Journal of immunological methods. 445 |
| ISSN: | 1872-7905 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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