CD26 Is Down-Regulated In Inducible Regulatory T Cells

CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg) play an important role in allograft- and self-tolerance and thus have the potential for therapeutic application in immunological and allergic disorders. And several attempts of ex vivo expansion of Treg to enable the adaptive immunoregulatory therapy in humans. However the quality of the final products is hardly assessed because of no unique surface markers of the Treg to be distinguished from activated T cells. CD127 is useful surface markers for naïve Treg, but after induction culture with anti-CD28 and anti-CD3-MAbs, CD127 becomes negative and no more helpful to distinguish the iTreg in cultured T cells. Here we studied gene expressions and the influence factors in inducible Treg (iTreg) compared to those in activated T cells and report.

CD4⁺ T cells from cord blood cells (CB) were isolated by anti-CD4 monoclonal antibody (MAb)-conjugated magnetic beads, and cultured using a plastic plate coated with anti-CD28 and anti-CD3-MAbs in the medium containing recombinant human (rh) IL-2 and rhTGF-β. After 2-weeks of culture, Treg-rich populations were obtained in the culture with rhTGF-β. The gene expression array was performed between the cells cultured with and without TGF-β. To study the influence of mTOR inhibitor on Treg expansion, we added rapamycin to the culture system with TGF-β. CFSE-labeled responder T cells and autologous or allogeneic dendritic cells (DC) with or without expanded Treg-rich populations.

Mean fold induction of CD4⁺CD25⁺Foxp3⁺Treg/Treg % in the cultured cells derived from CB naïve CD4+ T cells was 7,403/7% of the cultured cells without TGF-β, 31,795/32% with TGF-β and 6,128/49% with TGF-β and rapamycin, respectively. Dipeptidyl peptidase IV (DPP-IV:CD26) is significantly down-regulated from high to intermediate (inter)/negative in the cultured cells with TGF-β compared to those without TGF-β, while CD127 indicated all negatice in the cultured cells. Mean of median intensity of CD26 indicated 64.2 in the cultured cells without TGF-β, 0.44 with TGF-β and 0.23 with TGF-β and rapamycin, respectively, while CD45RO became positive for all cultured cells from CD45RA+naive phenotype. Rapamycin augmented Foxp3 expression of the cultured cells with TGF-β, although expansion rate itself of iTreg was reduced. The resulting Treg-rich populations inhibited the proliferative response of CFSE-labeled CD4⁺ and CD8⁺ T cells to allogeneic DC.

CD45RO⁺CD45RA^-CD26^high T cells is defined as memory phenotype, while iTreg cultured under the presence of TGF-β with or without Rapamycin indicated the unique population, CD45RO⁺CD26^{inter to negative}. The mechanism of the down-regulation of CD26 remained to be solved. Our results indicated that CD26 may be useful marker to assess the final products of iTreg to distinguish from the activated T cells and mTOR inhibitor such as rapamycin will be helpful reagent to induce Treg and applicable to clinical use.

No relevant conflicts of interest to declare.