FIGURE 4.

In vivo survival of fresh and thawed nTreg. (A) Experimental schematic, created with biorender.com. Freshly expanded or cryopreserved nTreg were recovered and stained with violet proliferation dye (VPD). Before injection into mice cells were characterized for Treg markers. VPD staining ensured the traceability to Treg-positive cells for later investigations. Immunodeficient BALB/c Rag2^{– /–}cγ^{– /–} mice received 5 × 10⁶ VPD-labeled nTreg from one of two donors which were recovered after 5 days by peritoneal lavage (B). Cell viability was quantified by light microscopy with 0.05% Trypan Blue dead-cell exclusion after transport and bead depletion (fresh nTreg) or transport and thawing (frozen nTreg). The number of nTreg recovered from lavage after 5 day incubation was quantified by flow cytometry. (C) Phenotype and proliferation of recovered human (mCD45^–CD3⁺) lymphocytes were quantified by expression of CD4 and CD25. Division was defined as lymphocytes with VPD staining intensity less than the maximally stained (undivided) peak. Each point represents a separate mouse with n = 2 Treg donors. Data are represented as mean ± SEM and statistical significance determined using unpaired t-tests.