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

To characterize the biological properties of double-negative T (DNT) cells isolated from leukoreduction filter residues.

Leukoreduction 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.

The 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 P<0.05). After 17 days of culture, DNT cells from filter residues and whole blood showed no significant differences in expansion fold, immunophenotype, differentiation status, or cytotoxicity toward target cells (all P>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)%, P<0.05]. After thawing (post 3 or 15 days of cryopreservation) and 17 days of culture, DNT cell starting materials from residues showed comparable immunophenotype, expansion fold, and differentiation status to their non-cryopreserved counterparts from the same source (all P>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 P<0.05).

DNT 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.