Cryopreservation is a widely used method for long-term storage of biological materials, but it may significantly reduce cell viability and alter immune cell function. We have previously demonstrated that virus-specific tissue-resident T cells can be directly studied from swabs of the human nasal cavity, enabling minimally invasive assessment of local immune responses1-3. To date, these analyses have relied exclusively on freshly processed samples. However, for large multicenter clinical studies, logistical constraints may necessitate sample cryopreservation and centralized analysis. It is therefore critical to understand how freezing and thawing affect the recovery and functionality of nasal mucosal T cells.
In this study, nasal swab samples were collected, processed into single-cell suspensions, and analyzed to compare T cell quantity, composition, and antigen-specific function before and after cryopreservation. Flow cytometry was used to quantify total lymphocytes and CD4⁺ and CD8⁺ T cells, while ELISpot and cytokine release assays (CRA) were employed to assess antigen-specific responses.
We found that nasal mucosal T cells are predominantly CD8⁺, accounting for approximately 73.7% of lymphocytes, whereas CD4⁺ T cells represent 16.0%. Cryopreservation markedly reduced cell recovery, with only 21.8% of CD8⁺ and 24.2% of CD4⁺ T cells retained after two weeks at −80 °C. Although the relative frequency of T cells appeared increased after thawing, this likely reflects preferential loss of non-T cell populations rather than true enrichment. Importantly, antigen-specific T cell responses were substantially diminished following freezing. Functional analyses after thawing were only feasible in donors with strong baseline responses, as responses in low responders fell below the limit of detection. Consistently, CRA measurements showed reduced secretion of IFN-γ and CXCL10 in frozen samples compared to fresh controls.
In summary, cryopreservation severely compromises both the yield and functional capacity of nasal mucosal T cells. Given the limited number of cells obtainable from nasal swabs, these findings support the use of fresh samples for immunological analyses. For multicenter studies, implementing simple functional assays such as CRA at the point of care may represent a more effective strategy to avoid the detrimental effects of freezing.