Dissection of T-cell tolerance induction by dendritic cell surface glycoproteins

Abstract

Abstract Tolerogenic dendritic cells (DC) induce T-cell clonal deletion, anergy, generation and activation of regulatory T cells. In vitro production of TolDC has been accomplished, however the full molecular circuitry is not known. We aim to decipher the chemical signals and physical characteristics leading to T-cell tolerance in an artificial system. We have performed surface proteomic analysis of human peripheral blood cDC2 subset without and with in vitro stimulation with polyIC in vitro. The surface proteomics is based on biotinylation of sialic acids and the signal strength was 55-fold higher in the resting compared to the activated condition, presumably due to decreased sialation of glycoproteins in the activated cDC2. We found that label free quantification (LFQ), corrected for differences in sialation, agreed well with quantitative surface expression by flow cytometry. We combined the two datasets and ranked the top 120 glycoproteins with extracellular domains (ECD) > 60 amino acids by LFQ with approximately one third invariant, one third up-regulated/induced and one one-third down-regulated/lost on activation. The 120 ECD were expressed in HEK293 cells with C-terminal 10His tags and purified by Ni2+ affinity chromatography. Supported lipids bilayers (SLB) representing the tolerogenic and activating DC are being designed with different combinations of the 120 ECDs. We will adjust sialation to match resting and activated states. The SLB compositions will be tested for immunological synapse formation (fluorescent self-pMHC/CD80, ICAM-1 and CD58 for the cSMAC, pSMAC and dSMAC) and tolerance/activation spectrum with identification of relevant molecular combinations by machine learning.