Human immune compartment comparisons: Optimization of proliferative assays for blood and gut T lymphocytes

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.

Functional Studies of T Regulatory Lymphocytes in Human Schistosomiasis in Western Kenya

“…Using the BrdU proliferation assay, 25 we confirmed that optimal responses were obtained at day 3 when PBMC cultures were exposed to the mitogen PHA and by 5 days when cultured with SEA or SWAP. Cultures from those optimum response days are presented in Figure 5 .…”

Distinct aging profiles of CD8+ T cells in blood versus gastrointestinal mucosal compartments

“…We tested the proliferative capacity of CD3/CD28/CD2-stimulated blood and gut derived mononuclear cells in 5-day cultures. For each donor, both stimulated and unstimulated (control) cultures were evaluated, using our recently developed double-label CSFE/BrdU system [ 28 ]. Briefly, proliferating cells registered as CFSE lo BrdU + and non-proliferating cells CFSE hi BrdU - ( Fig 1A ).…”

“…Proliferation status of CD8 + T cells was determined using a recently developed double-label CFSE/BrdU system on 5-day cultures of blood and gut derived mononuclear cells stimulated with CD3/CD28/CD2 antibodies, as previously described in extensive detail [ 28 ]. On day 5, live CD8 + T cells from blood and gut cultures that had undergone proliferation following stimulation were identified as CFSE low BrdU + .…”

B‐cell Therapy for Multiple Sclerosis: Entering an era

“…The generally favorable safety profile of anti-CD20 therapies may be due to several factors. First, although B cell depletion from the circulation is nearly complete, only 2% of the body’s total lymphocyte pool exists in peripheral blood, 91 and depletion of the body’s major stores of B cells in lymphoid organs is only partial. Thus, a large reservoir of B lymphocytes likely remain, even after multiple doses.…”