Soluble CD25 imposes a low-zone IL-2 signaling environment that favors competitive outgrowth of antigen-experienced CD25high regulatory and memory T cells.

This study focused on soluble (s)CD25-mediated regulation of IL-2 signaling in murine and human CD4+ T cells. Recombinant sCD25 reversibly sequestered IL-2 to limit acute maximal proliferative responses while preserving IL-2 bioavailability to subsequently maintain low-zone IL-2 signaling during prolonged culture. By inhibiting IL-2 signaling during acute activation, sCD25 suppressed T-cell growth and inhibited IL-2-evoked transmembrane CD25 expression, thereby resulting in lower prevalence of CD25high T cells. By inhibiting IL-2 signaling during quiescent IL-2-mediated growth, sCD25 competed with transmembrane CD25, IL2Rßγ, and IL2Rαßγ receptors for limited pools of IL-2 such that sCD25 exhibited strong or weak inhibitory efficacy in IL-2-stimulated cultures of CD25low or CD25high T cells, respectively. Preferential blocking of IL-2 signaling in CD25low but not CD25high T cells caused competitive enrichment of CD25high memory/effector and regulatory FOXP3+ subsets. In conclusion, sCD25 modulates IL-2 bioavailability to limit CD25 expression during acute activation while enhancing CD25highT-cell dominance during low-zone homeostatic IL-2-mediated expansion, thereby 'flattening' the inflammatory curve over time.

Partial CD25 Antagonism Enables Dominance of Antigen-Inducible CD25high 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-ß 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 in vitro propagation. Because Tregs and Tcons differentially express CD25, we hypothesized that anti-CD25 monoclonal antibodies (mAbs) would only partially block IL-2 signaling in CD25high FOXP3+ Tregs while completely blocking IL-2 responses of CD25low-intermediate Tcons to enable preferential outgrowth of Tregs during in vitro propagation. Indeed, murine TGF-ß-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 FOXP3high phenotype during long-term in vitro propagation (>90% FOXP3+ Tregs), whereas parallel cultures lacking PC61 rapidly lost FOXP3. These results pertained to TGF-ß-inducible "iTregs" because Tregs from 2D2-FIG Rag1-/- 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-ß fully stabilized FOXP3+ Tregs during cellular activation in the presence of dendritic cells and antigen/mitogen. Adoptive transfer of blastogenic CD25high 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 CD25high 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.