Differential impact of cryopreservation on CD4+ and CD8+ T cell repertoires and clonotypic expansion

PBMC cryopreservation is a fundamental technique in immunology research and clinical applications, which is used to assess T cell responses to antigens across disease settings. However, its differential impact on CD4+ and CD8+ T cell repertoire dynamics has not been studied and is of high relevance owing to the increasing number of T cell assays using clonotypic expansions as a functional readout. In this study, we systematically compared the effects of cryopreservation on CD4+ and CD8+ T cell clonotypic expansions using a functional T cell receptor (TCR) sequencing assay. We found that cryopreservation significantly reduces the reproducibility of antigen-specific CD4+ T cell clonotypic expansions compared to CD8+ T cells. The clonality of uncultured CD4+ T cells at baseline was lower than CD8+ T cells, but this alone did not account for the observed differences post-cryopreservation. Our results suggest that freeze-thaw cycles stochastically affect CD4+ T cell clone survival and expansion capacity, leading to inconsistent repertoire changes after antigenic stimulation. These findings have important implications for experimental design and data interpretation in T cell studies, particularly those relying on cryopreserved samples for CD4+ T cell functional assays. We recommend caution when interpreting TCR repertoire data from cryopreserved CD4+ T cell data and suggest using fresh samples where possible for CD4+ T cell studies. we tested the impact of varying cryopreservation times on the reproducibility of CD8+ and CD4+ T cell expansions within the same donors and against the same classes of antigens in a more controlled and rigorous manner. To adopt a conservative approach, we selected the CEF immunodominant antigenic peptide pool for testing. This decision was based on the rationale that if we could not reproducibly detect responses to these dominant antigens, such effects would likely be amplified with less dominant antigens (i.e. those derived from cancer). After 10 days of culture, we sequenced the TCR Vb CDR3 region to profile the resulting TCR repertoire. *************************************************************** Raw files for human/patient samples were not submitted to GEO due to concerns about submitting personally identifiable sequence data for open access. ***************************************************************