Effect of red blood suspension on CD4~+CD25~+Treg differentiation

Objective To explore the effect of red blood suspension on differentiation of CD4+CD25+Treg derived from allogene T lymphocytes in vitro and its underlying mechanism.Methods CD3+T lymphocytes from human peripheral blood were isolated and purified by Ficoll density gradient centrifugation combined with adherence MACS selection.Purified human T cells were stimulated with anti-CD3/anti-CD28 and then exposed to allogene leukocyte-containing red blood suspension or leukocyte-depleted red blood suspension for 72 h.The ratios of CD4+CD25+Tregs from different co-culture systems were detected by flow cytometry.The expression of Foxp3 mRNA was determined by real-time qPCR.The levels of pro-inflammatory cytokines such as interleukin-2(IL-2)and interferon-γ(IFN-γ)and anti-inflammatory cytokines like,interleukin-10(IL-10)and transforming growth factor beta(TGF-β)in the culture supernatant were determined by enzyme-linked immunoabsorbent assay(ELISA).Results Compared with those of negative group(culture media),CD4+CD25+Tregs cells rate and Foxp3 mRNT expression in leukocyte-containing group and leukocyte-depleted group increased.At the same time,the cytokine of pro-inflammatory cytokines,IL-2 and IFN-γ,significantly decreased,and the antiinflammatory cytokines,TGF-βand IL-10 increased significantly(P0.01).The results of leukocyte-containing group were more prominent(P0.05).Conclusion Red blood suspension could promote differentiation of T lymphocytes to CD4+CD25+Treg by regulating balance of proinflammatory/anti-inflammatory cytokines.The regulation,to a large extent,depends on the remaining white blood cells in red blood suspension.