Functional characterization of double-negative T cells isolated from leukoreduction filter residues

ObjectiveTo characterize the biological properties of double-negative T (DNT) cells isolated from leukoreduction filter residues.MethodsLeukoreduction filters containing residues from 400 mL whole blood units (n=6) were collected from a blood center. Filters were back-flushed with normal saline, and the eluate was concentrated to obtain leukoreduction filter residues. Leukocytes in the residues were counted by dual-fluorescence staining. DNT cells were then isolated from the residues using antibody-mediated adsorption and density gradient centrifugation. Both cryopreserved-thawed and non-cryopreserved DNT cells derived from the residues were subjected to in vitro culture. Cells were assessed for expansion fold, viability, immunophenotype, differentiation status, and cytotoxicity against target cells using dual-fluorescence staining and flow cytometry, with comparisons made to DNT cells derived from whole blood.ResultsThe leukocyte recovery rate achieved through reverse flushing of the leukocyte reduction filter was (41.9±14.7)%. Compared to whole blood, the DNT cells starting material obtained from filter residues showed no significant difference in total T-cells content (P>0.05). However, the viability and purity of the resulting DNT cells starting materials were significantly lower (both PP>0.05). However, the viability of DNT cells from residues was significantly lower than that of whole blood-derived DNT cells [(86.0±4.2)% vs. (92.2±1.2)%, PP>0.05). However, the viability of the 3-day cryopreservation recovery group [92.4% (91.8%, 92.8%) vs. 87.8% (82.0%, 89.0%)] and the cytotoxicity against target cells of the 15-day cryopreservation recovery group [91.3% (89.4%, 95.1%) vs. 70.9% (67.3%, 80.2%)] were both significantly higher than those of non-cryopreserved DNT cells (all PConclusionsDNT cells derived from leukoreduction filter residues exhibited highly comparable characteristics to those from whole blood in terms of expansion, purity, differentiation, and biological potency. Furthermore, their biological activity post-cryopreservation and revival remained largely similar to non-cryopreserved cells. These findings suggest that leukoreduction filter residues represent a promising alternative source of starting material for manufacturing off-the-shelf, allogeneic DNT cell therapeutics.