{"doi":"10.3389/fimmu.2020.541619","title":"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","abstract":"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.","journal":"Frontiers in Immunology","year":2020,"id":88476,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":10,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9535,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":439933,"name":"Cody D. Moorman","orcid":"0000-0001-8967-5917","position":1,"is_corresponding":false},{"id":282573,"name":"Brandon L. Garcia","orcid":"0000-0002-6724-8700","position":2,"is_corresponding":false},{"id":439931,"name":"Mark D. Mannie","orcid":"0000-0003-0475-8516","position":3,"is_corresponding":false},{"id":439932,"name":"Kayla B. DeOca","orcid":"0000-0001-6952-0934","position":0,"is_corresponding":true}],"reference_count":51,"raw_metadata":null,"created_at":"2026-07-18T22:01:22.387926Z","pmid":"33072087","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}