Expansion of CD4+CD25+ suppressive regulatory T cells from rhesus macaque peripheral blood by FN18/antihuman CD28-coated Dynal beads

SummaryCD4+CD25+ regulatory T cells (Tregs) play an important role in allograft and self-tolerance and thus have potential therapeutic application in transplantation, autoimmunity, and allergy. Although nonhuman primate (NHP) provide the most accepted preclinical models for translational studies in allograft tolerance and infectious diseases, CD4+CD25+ Tregs have been rarely studied in NHP. The low frequencies of Tregs in peripheral blood will likely necessitate ex vivo expansion to enable Tregs adaptive immune therapy in NHP and humans. Tregs were isolated by magnetic and flow sorting and then stimulated weekly with antirhesus CD3 clone FN18 and antihuman CD28-coated Dynal beads plus 100 U/ml rhIL-2. Under these conditions, the Tregs were expanded 300- to 2000-fold in 4 weeks. Expanded CD4+CD25+ Tregs expressed high to moderate levels of FOXP3 as well as CD95, CD62L, CD69, and CCR7 surface antigens. Expanded rhesus Tregs were anergic and suppressed the proliferation of autologous peripheral blood mononuclear cells (PBMC) in a dose-dependent fashion, and the suppression was partially reversed by anti-transforming growth factor (TGF)-β1 neutralizing antibody (Ab). These results demonstrate that rhesus macaque suppressive regulatory CD4+CD25+FOXP3+ Tregs can be efficiently expanded in vitro under rhesus-specific stimulation, which enables preclinical testing of Treg therapy in the NHP model.