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

Stefanie Kuerten,Edith Karacsony,Paul V Lehmann,Helena Batoulis,Mascha S Recks,Wenji Zhang,Ramu A Subbramanian

doi:10.3390/cells1030409

Stefanie Kuerten, Edith Karacsony + Show 5 more

Open Access

https://doi.org/10.3390/cells1030409

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Journal: Cells	Publication Date: Jul 30, 2012
Citations: 59	License type: CC BY 4.0

Affiliation: Shaker Heights Public Library, University of Cologne

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.

Highlights

Because T cells are key mediators of immunity, monitoring antigen-specific T cells has become central to progress in many fields of medical research, including infectious diseases and vaccinology [1±3], transplantation [4,5], allergies [6], autoimmunity [7±9], and tumor immunology [10±12].Among the techniques available to measure T cell immunity, ELISPOT has found wide use because it is simple, reliable, sensitive, quantitative, and efficient in cell utilization [10,12,13].The ELISPOT technology permits enumeration of individual antigen-specific T cells through the detection of their secretory products such as the antigen-triggered release of interferon gamma
When it comes to the detection of polyfunctional T cells, dual color ELISPOT assays permit the measurement of cytokine coexpression in individual T cells with the same sensitivity as intracytoplasmic cytokine staining (ICS), low frequency responses that are difficult to detect by ICS can be readily detected by dual color ELISPOT [22]
Test results obtained with freshly isolated PBMC, can be considered as the baseline against which variations of PBMC handling, such as cryopreservation or resting, can be compared

Summary

Introduction

Because T cells are key mediators of immunity, monitoring antigen-specific T cells has become central to progress in many fields of medical research, including infectious diseases and vaccinology [1±3], transplantation [4,5], allergies [6], autoimmunity [7±9], and tumor immunology [10±12]. While initially IFN-Ȗ(/,6327DVVD\V found wide use for the detection of Th1/Tc1 cells, continued advancement in the ELISPOT field has enabled the detection of Th2/Tc2, Th17, cytolytic, and regulatory T cells via measurements of antigen-induced release of IL-2, IL-4, IL-5 [5,15,16], IL-17 [17], Granzyme B and perforin [18±20], and IL-10 [21], respectively When it comes to the detection of polyfunctional T cells, dual color ELISPOT assays permit the measurement of cytokine coexpression in individual T cells with the same sensitivity as ICS, low frequency responses that are difficult to detect by ICS can be readily detected by dual color ELISPOT [22]. One of the major goals of immune monitoring is to lower the detection limit of ELISPOT assays for antigen-specific T cells Towards this goal, it has been reported WKDW3overnight restingof cryopreserved PBMC after thawing before their utilization in ELISPOT assays would increase the sensitivity of the assay, that is, provide higher antigen-induced spot counts [24,25]. The study reported here was designed to fill this gap

Working Hypothesis

Resting of PBMC Is Associated with Significant Cell Loss

Frequencies of CD4 and CD8 Cells Are Unchanged between Fresh and Rested PBMC

Thawing and Resting of Cryopreserved PBMC

Flow Cytometry

Statistics

Conclusions