Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays

Abstract

T cell monitoring is increasingly performed using cryopreserved PBMC. It has been suggested that resting of PBMC after thawing, that is, culturing them overnight in test medium, produces higher antigen-induced spot counts in ELISPOT assays. To evaluate the importance of overnight resting, we systematically tested cryopreserved PBMC from 25 healthy donors. CEF peptides (comprising CMV, EBV and flu antigens) were used to stimulate CD8 cells and mumps antigen to stimulate CD4 cells. The data show that resting significantly increased antigen-elicited T cell responses only for CEF high responder PBMC. The maximal gain observed was doubling of spot counts. For CEF low responders, and for mumps responders of either low- or high reactivity levels, resting had no statistically significant effect on the observed spot counts. Therefore, resting is not a generally applicable approach to improve ELISPOT assay performance, but can be recommended only for clinical subject cohorts and antigens for which it has a proven benefit. Because resting invariably leads to losing about half of the PBMC available for testing, and because doubling the PBMC numbers plated into the assay reliably doubles the antigen-induced spot counts, we suggest the latter approach as a simple and reliable alternative to resting for enhancing the performance of ELISPOT assays. Our data imply that resting is not required if PBMC were cryopreserved and thawed under conditions that minimize apoptosis of the cells. Therefore, this study should draw attention to the need to optimize freezing and thawing conditions for successful T cell work.

Serial Measurements of Apoptotic Cell Numbers Provide Better Acceptance Criterion for PBMC Quality than a Single Measurement Prior to the T Cell Assay

“…To further investigate the potential paracrine effects of apoptotic cells, PBMC were kept in a 37 °C incubator overnight. Such “overnight resting” leads to ~ 50% loss in PBMC [ 34 ], which is also visible in Figure 2 for the B cell-depleted PBMC. This loss of cells, due to apoptosis, will result in the engulfment of apoptotic cells by macrophages leading to the release of anti-inflammatory cytokines.…”

Circulating mycobacterial-reactive CD4+ T cells with an immunosuppressive phenotype are higher in active tuberculosis than latent tuberculosis infection

“…Resting PBMC before use enhances the sensitivity of T-cell assays through depletion of dead cells but PBMC may be lost [50]. Another difference between the study of Harari et al [8] and ours was that we included 31 active TB patients compared to 8 active TB patients in their study.…”

How much of Virus-Specific CD8 T Cell Reactivity is Detected with a Peptide Pool when Compared to Individual Peptides?

“…Following the thaw and wash procedures, PBMC were resuspended at a final concentration of 4 X 10 6 PBMC/ml in CTL-Test Medium (CTLT-005) of which 100 µl (400,000 cells) was plated per well into the ELISPOT assay. The thawed PBMC were not “rested” before testing, but were plated directly into the assay as resting does not significantly improve the performance of these PBMC [32]. …”