Cryopreserved T-cells and Anti-tumor Activity
Cancer immunotherapies based on antigen-specific T-cell treatment are gaining ground as a means of treating cancer, due to their encouraging success in clinical trials. Extended culturing is known to reduce or alter the function of T-cells, however, so it would be highly advantageous if isolated T-cells could be rapidly expanded, then cryopreserved to preserve their functional value. The problem with this idea is that very few studies have been specifically designed to confirm the extent to which cryopreserved T-cells retain functionality. A short report recently published in Nature/Immunology and Cell Biology  will help fill that gap by reporting their results on anti-tumor activity in cryopreserved cytotoxic T-cells.
To carry out their studies, the authors compared tumor reactive mouse CD8+T-cells which had been cryopreserved during their initial cell expansion, to identical mouse cells that were freshly isolated. The primary goal of the research, which took place at the University of Sydney Medical School in Australia, was to test whether cryopreserved cytotoxic T-lymphocytes (CTLs) should be considered a viable source of T-cells for cellular therapy in cancer patients. This means that not only should the cells have high viability and recovery rates, but also that they retain full functionality in all aspects of their anti-tumor activity.
Choosing the right freezing and thawing method is crucial to optimizing cell survival and functionality, particularly when cells will be used for translational research and clinical studies. The Sydney-based research team cryopreserved the CTLs for their studies using a slow cooling method that guaranteed cells would cool at the recommended rate of 1°C/minute. Cells were then placed in a -80°C freezer for 48 hours, at which time they were transferred to liquid nitrogen and stored for up to 12 months before recovery. Following cryopreservation, CTLs were thawed in a 37°C water bath, and cultured, like their freshly isolated cohorts, in media containing IL-2. Cryopreserved CTLs spent 3-4 days in culture prior to being analyzed in various functional assays. Freshly isolated cells were continuously cultured for the same time period as the cryopreserved cells prior to functional analysis.
The authors performed a comprehensive array of CTL characterization and activity assays. Flow cytometry was used to examine viability and expression of cell surface molecules, resulting in the finding that both cell populations displayed high viability rates and expressed high levels of the same activation markers. Other assays measured tumor and lymph node infiltration capacity, cytotoxic capacity, and 3D migration characteristics. Differentiation capacity, cytokine production, and tumor rejection potential were all measured, each time with the same result—there was no significant difference between cryopreserved cells and freshly isolated cells for any of the measured parameters.
The authors came to the clear conclusion that cryopreservation was “a viable method of preserving fully functional T-cells for immunotherapy”, and that cryopreservation enables scientists to store large numbers of T-cells with their functionality unimpaired for extended periods of time. This is truly good news for the future of cellular immunotherapy.
 Galeano Niño, G., et al. Antigen-specific T cells fully conserve antitumour function following cryopreservation. Immunology and Cell Biology 1–8, Jan 2016.