Glycosylation‐dependent interaction between CD69 and S100A8/S100A9 complex is required for regulatory T‐cell differentiation

Abstract

Cluster of differentiation (CD)69 is a leukocyte activation receptor involved in the maintenance of immune homeostasis and is positively selected in activated regulatory T (Treg) cells, implicating its role during Treg-cell differentiation. By RNA interference, we show that CD69 is not sufficient to support the conversion of CD4(+) naive T cells into Treg cells, whereas it does that of human peripheral blood mononuclear cells (hPBMCs) (P < 0.01), suggesting that a ligand-receptor interaction is required for CD69 function. Using immunoprecipitation and mass spectrometry, we identified the S100A8/S100A9 complex as the natural ligand of CD69 in hPBMCs. CD69 specifically associates with S100A8/S100A9 complex as confirmed by in vitro binding and competition assay, and the treatment of CD69 with peptide-N-glycosidase significantly abolishes such association. In agreement, the glycomics analysis determines the glycosylation site and the N-glycan composition of CD69, and terminal removal of sialic acid from that N-linked glycans reverses the generation of forkhead box P3-positive Treg cells (23.21%; P < 0.05). More specifically, we showed that CD69-S100A8/S100A9 association is required for the up-regulation of suppressor of cytokine signaling 3 resulting in inhibited signaling of signal transducer and activator of transcription 3 (36.54% increase upon CD69 silencing; P < 0.01). This might in turn support the secretion of key regulator TGF-β (∼ 3.28-fold decrease upon CD69 silencing; P < 0.05), leading to reduced production of IL-4 in hPBMCs. Our results demonstrate the functional and mechanistic interplays between CD69 and S100A8/S100A9 in supporting Treg-cell differentiation.

Thymus-Derived Regulatory T Cell Development Is Regulated by C-Type Lectin-Mediated BIC/MicroRNA 155 Expression

“…The role of CD69 as a negative regulator of the immune system has remained a controversial issue during the last years (23). However, very recent studies by independent groups show that CD69 plays a crucial role in the suppressor function of mouse and human Treg cells as well as in the generation of in vitro-induced Treg cells (12,16,(24)(25)(26). Nevertheless, the specific role of the C-type lectin in the development of Treg cells in the thymus remains elusive.…”

Aurora A and NF-κB Survival Pathway Drive Chemoresistance in Acute Myeloid Leukemia via the TRAF-Interacting Protein TIFA

“…All resulting STR profiles were matched with known ATCC fingerprints (www.ATCC.org). Maintenance of respective cell lines followed our previous protocols (21)(22)(23). 293T and HeLa cells were maintained in DMEM(Gibco).…”

“…All cells were incubated in a humidified atmosphere at 37 C with 5% CO 2 . To isolate human PBMCs, freshly acquired whole-blood samples were layered over Ficoll (GE Healthcare) as described previously (23). Isolated primary cells were maintained according to a previously described protocol (24) and subjected to experimental analyses within two weeks.…”

“…Immunoprecipitation experiment was conducted as described previously (23). Subconfluent 293T cells in a p100 petri dish were transfected with 5 mg of the vectors or HA-TIFA using Jet-PEI (Polyplus Transfection).…”

Crucial role for CD69 in allergic inflammatory responses: CD69‐Myl9 system in the pathogenesis of airway inflammation

“…Recently, various CD69 interacting molecules were reported . Human CD69 was reported to interact with galectin‐1 expressed on DCs, which was identified by pull‐down assay using human DCs and recombinant human CD69 extracellular domain protein.…”