Functionality of stored cryopreserved lymphocytes and preservation of antigen-specific responses

Donor lymphocyte infusions (DLI) are routinely used as second line treatment to protect against relapse of haematological malignancies following haematopoietic stem cell transplant (HSCT). Lymphocyte collections are increasingly being used as starting materials for chimaeric antigen receptor-T (CAR-T) manufacture. Current protocols for storage and cryopreservation of lymphocytes are based on those developed for CD34+ HSC grafts and may not be appropriate for lymphocytes. As clinical use of lymphocyte products increases, there is a pressing need to identify cryopreservation and storage protocols that will optimise lymphocyte recovery and function when cells are stored or shipped prior to use. This study investigated the effects of storage time at 4°C prior to cryopreservation on post-thaw recovery and functionality of T lymphocytes from nonmobilised and granulocyte-colony stimulating factor (G-CSF) mobilised peripheral blood. Non-mobilised lymphocytes were extracted from sixteen apheresis cones from platelet donations and purified by density gradient separation. Lymphocytes exposed to G-CSF were obtained from the residual quality control samples from nine G-CSF mobilised apheresis harvests from volunteer donors. Lymphocyte preparations were phenotyped by flow cytometry and the total number of viable T cells present determined. Cells were stored for either 24, 24-48 or 48-72 hours at 4°C prior to rate-controlled cryopreservation. Following a minimum of 7 days storage, cells were thawed and stimulated with CD3/CD28 activation beads for 4, 24 and 72 hours followed by flow cytometric assessment of CD25 and CD69 expression on CD4+ and CD8+ cells. Post-thaw recovery of viable T cells from each cryopreserved sample was calculated. Expression of CD25/CD69 and CD3+ viability and recovery were compared for cells cryopreserved after the different times in storage. The results demonstrated a significant loss of viable T cells after cryopreservation that became greater over time from collection to cryopreservation. If cryopreserved within 24 hours of collection, means of 77% of non-mobilised and 57% of mobilised CD3+ cells were recovered post thaw. After >48 hours, the mean recovery dropped to 28% and 15% respectively. Expression of activation markers after stimulation appeared unaffected by length of time to cryopreservation in both sample groups indicating that T cells that survived the freeze thaw process were functionally unharmed. Exposure to G-CSF did not affect T cell responses to stimulation. These findings demonstrate that current protocols for storing and handling lymphocytes require modification to optimise recovery of T cells post thaw. Further study on a larger sample group, is required to determine optimum protocols. As functionality post thaw was unaffected by time to cryopreservation or G-CSF stimulation, this positive finding indicates current freezing protocols do not influence lymphocyte functionality which will be advantageous for future therapies using T cells.