Human immune compartment comparisons: Optimization of proliferative assays for blood and gut T lymphocytes.
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.
Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, CA 90095, United States.
Jeffrey
J
Dock
Division of Hematology and Oncology, Department of Medicine, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, CA 90095, United States; UCLA AIDS Institute, David Geffen School of Medicine at UCLA, United States.
Lance
L
Hultin
Department of Epidemiology, Fielding School of Public Health, University of California-Los Angeles, Los Angeles, CA 90095, United States; UCLA AIDS Institute, David Geffen School of Medicine at UCLA, United States.
Patricia
P
Hultin
Division of Digestive Diseases, Department of Medicine, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, CA 90095, United States; UCLA AIDS Institute, David Geffen School of Medicine at UCLA, United States.
Julie
J
Elliot
Division of Infectious Diseases, Department of Medicine, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, CA 90095, United States; Department of Microbiology Immunology & Molecular Genetics, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, CA 90095, United States; UCLA AIDS Institute, David Geffen School of Medicine at UCLA, United States; AIDS Healthcare Foundation, Los Angeles, CA 90028, United States.
Otto O
OO
Yang
Division of Digestive Diseases, Department of Medicine, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, CA 90095, United States; UCLA AIDS Institute, David Geffen School of Medicine at UCLA, United States.
Peter A
PA
Anton
Division of Hematology and Oncology, Department of Medicine, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, CA 90095, United States; UCLA AIDS Institute, David Geffen School of Medicine at UCLA, United States.
Beth D
BD
Jamieson
Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, CA 90095, United States; UCLA AIDS Institute, David Geffen School of Medicine at UCLA, United States. Electronic address: reffros@mednet.ucla.edu.
Rita B
RB
Effros
D000328:Adult; D049109:Cell Proliferation; D002478:Cells, Cultured; D005260:Female; D005753:Gastric Mucosa / Q000166:cytology / Q000276:immunology*; D006801:Humans; D008297:Male; D008875:Middle Aged; D013601:T-Lymphocytes / Q000276:immunology*
CD3 T cell; CD8 T cell; GALT; Mucosa; Proliferation assay; Replicative senescence