Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT

Using CMV pp65 peptide pool stimulation or pp65 HLA-A2 tetramer staining, there was significant correlation between shipped and cryopreserved samples for each assay (p ≤ 0.001). The differences in response magnitude between cryopreserved and shipped PBMC specimens were not significant for most antigens and assays. There was significant correlation between CFC and ELISPOT assay using pp65 peptide pool stimulation, in both shipped and cryopreserved samples (p ≤ 0.001). Strong correlation was observed between CFC (using HLA-A2-restricted pp65 peptide stimulation) and tetramer staining (p < 0.001). Roughly similar sensitivity and specificity were observed between the three assays and between shipped and cryopreserved samples for each assay.We conclude that all three assays show concordant results on shipped versus cryopreserved specimens, when using a peptide-based readout. The assays are also concordant with each other in pair wise comparisons using equivalent antigen systems.An increasing number of experimental vaccines are being developed for diseases in which cellular immunity is likely to be required for protection [1]. These include HIV [2], hepatitis C [3], malaria [4], and cancer [5]. In these settings, immunological monitoring of antigen-specific T cell responses is likely to be an important part of vaccine assessment [6-10]. Unfortunately, surrogate markers of protection have not been established for vaccines that induce cellular immunity, although correlations between T cell IFNγ production and clinical responses have been reported in small numbers of patients [11,12] and in mouse models [13-17].Even in the absence of surrogate markers of protection, the degree to which vaccines can induce T cell responses can be taken as a measure of immunogenicity, or potency, and as such can be used to compare different vaccine candidates. It is likely that inducing a particular level of antigen-specific T cell response to a vaccine will be necessary, though perhaps not su