Abstract
As soon as Peripheral Blood Mononuclear Cells (PBMC) are isolated from whole blood, some cells begin dying. The rate of apoptotic cell death is increased when PBMC are shipped, cryopreserved, or stored under suboptimal conditions. Apoptotic cells secrete cytokines that suppress inflammation while promoting phagocytosis. Increased numbers of apoptotic cells in PBMC may modulate T cell functions in antigen-triggered T cell assays. We assessed the effect of apoptotic bystander cells on a T cell ELISPOT assay by selectively inducing B cell apoptosis using α-CD20 mAbs. The presence of large numbers of apoptotic B cells did not affect T cell functionality. In contrast, when PBMC were stored under unfavorable conditions, leading to damage and apoptosis in the T cells as well as bystander cells, T cell functionality was greatly impaired. We observed that measuring the number of apoptotic cells before plating the PBMC into an ELISPOT assay did not reflect the extent of PBMC injury, but measuring apoptotic cell frequencies at the end of the assay did. Our data suggest that measuring the numbers of apoptotic cells prior to and post T cell assays may provide more stringent PBMC quality acceptance criteria than measurements done only prior to the start of the assay.
Highlights
T cell monitoring studies, e.g., assessing whether a vaccination has induced immunity, require the use Peripheral Blood Mononuclear Cells (PBMC)
Based on the data presented here, we suggest that serial measurements of live, dead, and apoptotic cell numbers in PBMC samples provide more accurate acceptance criteria regarding the quality PBMC
As the numbers of PBMC available for testing is frequently limiting in clinical trials, an attractive possibility for the second live/dead apoptotic cell count is to test the PBMC that were plated in an ELISPOT assay following the cell incubation period
Summary
T cell monitoring studies, e.g., assessing whether a vaccination has induced immunity, require the use Peripheral Blood Mononuclear Cells (PBMC). We show that mere measurements of live/dead ratios and apoptotic cell frequencies prior to seeding the PBMC into a T cell assay are not necessarily reliable markers for PBMC functionality. Measuring the apoptotic cell fraction at the beginning and at the end of the assay, was found to be a more reliable marker to detect damage to PBMC and their functional impairment. On the other hand macrophages, upon apoptotic cell engulfment, secrete anti-inflammatory cytokines such as TGF-β and IL-10 [26,27] Since all these processes could potentially affect T cell activation and function, we tested whether the presence of apoptotic bystander cells present PBMC would affect the results of T cell ELISPOT assays
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