Partial CD25 Antagonism Enables Dominance of Antigen-Inducible CD25 FOXP3 Regulatory T Cells As a Basis for a Regulatory T Cell-Based Adoptive Immunotherapy.

FOXP3 regulatory T cells (Tregs) represent a promising platform for effective adoptive immunotherapy of chronic inflammatory disease， including autoimmune diseases such as multiple sclerosis. Successful Treg immunotherapy however requires new technologies to enable long-term expansion of stable， antigen-specific FOXP3 Tregs in cell culture. Antigen-specific activation of naïve T cells in the presence of TGF-β elicits the initial differentiation of the FOXP3 lineage， but these Treg lines lack phenotypic stability and rapidly transition to a conventional T cell (Tcon) phenotype during propagation. Because Tregs and Tcons differentially express CD25， we hypothesized that anti-CD25 monoclonal antibodies (mAbs) would only partially block IL-2 signaling in CD25 FOXP3 Tregs while completely blocking IL-2 responses of CD25 Tcons to enable preferential outgrowth of Tregs during propagation. Indeed， murine TGF-β-induced MOG-specific Treg lines from 2D2 transgenic mice that were maintained in IL-2 with the anti-CD25 PC61 mAb rapidly acquired and indefinitely maintained a FOXP3 phenotype during long-term propagation (>90% FOXP3 Tregs)， whereas parallel cultures lacking PC61 rapidly lost FOXP3. These results pertained to TGF-β-inducible "iTregs" because Tregs from 2D2-FIG mice， which lack thymic or natural Tregs， were stabilized by continuous culture in IL-2 and PC61. MOG-specific and polyclonal Tregs upregulated the Treg-associated markers Neuropilin-1 (NRP1) and Helios (IKZF2). Just as PC61 stabilized FOXP3 Tregs during expansion in IL-2， TGF-β fully stabilized FOXP3 Tregs during cellular activation in the presence of dendritic cells and antigen/mitogen. Adoptive transfer of blastogenic CD25 FOXP3 Tregs from MOG35-55-specific 2D2 TCR transgenic mice suppressed experimental autoimmune encephalomyelitis in pretreatment and therapeutic protocols. In conclusion， low IL-2 concentrations coupled with high PC61 concentrations constrained IL-2 signaling to a low-intensity range that enabled dominant stable outgrowth of suppressive CD25 FOXP3 Tregs. The ability to indefinitely expand stable Treg lines will provide insight into FOXP3 Treg physiology and will be foundational for Treg-based immunotherapy.