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.

Low-Zone IL-2 Signaling: Fusion Proteins Containing Linked CD25 and IL-2 Domains Sustain Tolerogenic Vaccination in vivo and Promote Dominance of FOXP3+ Tregs in vitro.

Low-zone IL-2 signaling is key to understanding how CD4+ CD25high FOXP3+ regulatory T cells (Tregs) exhibit dominance and overgrow conventional effector T cells (Tcons) that typically express lower levels of the IL-2 receptor alpha chain (i.e., CD25). Thus, modalities such as low-dose IL-2 or IL-2/anti-IL-2 antibody complexes have been advanced in the clinic to selectively expand Treg populations as a treatment for chronic inflammatory autoimmune diseases. However, more effective reagents that efficiently lock IL-2 signaling into a low signaling mode are needed to validate and exploit the low-zone IL-2 signaling niche of Tregs. This study focuses on CD25-IL2 and IL2-CD25 fusion proteins (FPs) that were approximately 32 and 320-fold less potent than IL-2. These FPs exhibited transient binding to transmembrane CD25 on human embryonic kidney (HEK) cells, had partially occluded IL-2 binding sites, and formed higher order multimeric conformers that limited the availability of bioactive IL-2. These FPs exhibited broad bell-shaped concentration ranges that favored dominant Treg outgrowth during continuous culture and were used to derive essentially pure long-term Treg monocultures (∼98% Treg purity). FP-induced Tregs had canonical Treg suppressive activity in that these Tregs suppressed antigen-specific proliferative responses of naïve CD4+ T cells. The in vivo administration of CD25-IL2/Alum elicited robust increases in circulating Tregs and selectively augmented CD25 expression on Tregs but not on Tcons. A single injection of a Myelin Oligodendrocyte Glycoprotein (MOG35-55)-specific tolerogenic vaccine elicited high levels of circulating MOG-specific Tregs in vivo that waned after 2-3 weeks, whereas boosting with CD25-IL2/Alum maintained MOG-specific CD25high Tregs throughout the 30-day observation period. However, these FPs did not antagonize free monomeric IL-2 and lacked therapeutic efficacy in experimental autoimmune encephalomyelitis (EAE). In conclusion, these data reveal that CD25-IL2 FPs can be used to select essentially pure long-term lines of FOXP3+ CD25high Tregs. This study also shows that CD25-IL2 FPs can be administered in vivo in synergy with tolerogenic vaccination to maintain high circulating levels of antigen-specific Tregs. Because tolerogenic vaccination and Treg-based adoptive immunotherapy are limited by gradual waning of Tregs, these FPs have potential utility in sustaining tolerogenic Treg responses in vivo.

Tolerogenic vaccines: Targeting the antigenic and cytokine niches of FOXP3+ regulatory T cells.

FOXP3+ regulatory T cells (Tregs) constitute a critical barrier that enforces tolerance to both the self-peptidome and the extended-self peptidome to ensure tissue-specific resistance to autoimmune, allergic, and other inflammatory disorders. Here, we review intuitive models regarding how T cell antigen receptor (TCR) specificity and antigen recognition efficiency shape the Treg and conventional T cell (Tcon) repertoires to adaptively regulate T cell maintenance, tissue-residency, phenotypic stability, and immune function in peripheral tissues. Three zones of TCR recognition efficiency are considered, including Tcon recognition of specific low-efficiency self MHC-ligands, Treg recognition of intermediate-efficiency agonistic self MHC-ligands, and Tcon recognition of cross-reactive high-efficiency agonistic foreign MHC-ligands. These respective zones of TCR recognition efficiency are key to understanding how tissue-resident immune networks integrate the antigenic complexity of local environments to provide adaptive decisions setting the balance of suppressive and immunogenic responses. Importantly, deficiencies in the Treg repertoire appear to be an important cause of chronic inflammatory disease. Deficiencies may include global deficiencies in Treg numbers or function, subtle 'holes in the Treg repertoire' in tissue-resident Treg populations, or simply Treg insufficiencies that are unable to counter an overwhelming molecular mimicry stimulus. Tolerogenic vaccination and Treg-based immunotherapy are two therapeutic modalities meant to restore dominance of Treg networks to reverse chronic inflammatory disease. Studies of these therapeutic modalities in a preclinical setting have provided insight into the Treg niche, including the concept that intermediate-efficiency TCR signaling, high IFN-ß concentrations, and low IL-2 concentrations favor Treg responses and active dominant mechanisms of immune tolerance. Overall, the purpose here is to assimilate new and established concepts regarding how cognate TCR specificity of the Treg repertoire and the contingent cytokine networks provide a foundation for understanding Treg suppressive strategy.

A GM-CSF-neuroantigen tolerogenic vaccine elicits inefficient antigen recognition events below the CD40L triggering threshold to expand CD4+ CD25+ FOXP3+ Tregs that inhibit experimental autoimmune encephalomyelitis (EAE).

BACKGROUND: Tolerogenic vaccines represent antigen-specific interventions designed to re-establish self-tolerance and thereby alleviate autoimmune diseases, which collectively comprise over 100 chronic inflammatory diseases afflicting more than 20 million Americans. Tolerogenic vaccines comprised of single-chain GM-CSF-neuroantigen (GMCSF-NAg) fusion proteins were shown in previous studies to prevent and reverse disease in multiple rodent models of experimental autoimmune encephalomyelitis (EAE) by a mechanism contingent upon the function of CD4+ CD25+ FOXP3+ regulatory T cells (Tregs). GMCSF-NAg vaccines inhibited EAE in both quiescent and inflammatory environments in association with low-efficiency T cell receptor (TCR) signaling events that elicited clonal expansion of immunosuppressive Tregs. METHODS: This study focused on two vaccines, including GMCSF-MOG (myelin oligodendrocyte glycoprotein 35-55/MOG35-55) and GMCSF-NFM (neurofilament medium peptide 13-37/NFM13-37), that engaged the transgenic 2D2 TCR with either low or high efficiencies, respectively. 2D2 mice were crossed with FOXP3 IRES eGFP (FIG) mice to track Tregs and further crossed with Rag-/- mice to reduce pre-existing Treg populations. RESULTS: This study provided evidence that low and high efficiency TCR interactions were integrated via CD40L expression levels to control the Treg/Tcon balance. The high-efficiency GMCSF-NFM vaccine elicited memory Tcon responses in association with activation of the CD40L costimulatory system. Conversely, the low-efficiency GMCSF-MOG vaccine lacked adequate TCR signal strength to elicit CD40L expression and instead elicited Tregs by a mechanism that was impaired by a CD40 agonist. When combined, the low- and high-efficiency GMCSF-NAg vaccines resulted in a balanced outcome and elicited both Tregs and Tcon responses without the predominance of a dominant immunogenic Tcon response. Aside from Treg expansion in 2D2-FIG mice, GMCSF-MOG caused a sustained decrease in TCR-ß, CD3, and CD62L expression and a sustained increase in CD44 expression in Tcon subsets. Subcutaneous administration of GMCSF-MOG without adjuvants inhibited EAE in wildtype mice, which had a replete Treg repertoire, but was pathogenic rather than tolerogenic in 2D2-FIG-Rag1-/- mice, which lacked pre-existing Tregs. CONCLUSIONS: This study provided evidence that the GMCSF-MOG vaccine elicited antigenic responses beneath the CD40L triggering threshold, which defined an antigenic niche that drove dominant expansion of tolerogenic myelin-specific Tregs that inhibited EAE.