Surface receptor proteins are crucial for understanding and targeting immune responses. They are central to (i) the cluster of differentiation (CD) concept for classifying immune cells and phenotypes, (ii) coordinating immune signaling and effector functions, and (iii) serving as prime diagnostic and therapeutic targets in human diseases. Virtually all surface proteins are N- and/or O-glycosylated, with these glycosylated domains significantly influencing intra- and intermolecular interactions. Untargeted glycoproteomics using LC-MS is the gold standard for providing comprehensive and unbiased information on immune-regulatory surface proteins.

Here, we present the first surfaceome study of primary human mucosal-associated invariant T cells (MAITs) and their glycosylation response under infection-like conditions. MAITs are the most abundant anti-infective T cell population in the liver, lung, and gut. These innate-like effector cells can directly recognize, via their T cell receptor, bacterial-derived metabolites presented by MR1 on antigen-presenting cells. Activated MAIT cells are multifunctional, providing regulatory, inflammatory, or cytotoxic responses enabling direct killing of infected cells as well as maintenance of organ homeostasis. Recently, we provided the first proteome study of MAITs indicating their unique effector phenotype (Bulitta et al., 2018), and discovered their antiviral properties by translatomics (Jakob et al., 2023). In order to elucidate the possibilities for selective regulation of their different immune responses, we established a robust glycoproteome pipeline to study surfaceomes of primary human immune cells. Bacterial-activated MAITs from six individuals were characterized by flow cytometry and total glycoproteomes were examined using an adapted hydrazide chemistry approach (Zhang et al. 2003). This allowed the enrichment of N- and O-glycosylated proteins which were characterized donor-dependently by LC-MS/MS (Dionex 3000 RSLC-Orbitrap Exploris 480 MS). In total, we identified 2300 glycoproteins containing over 200 surface proteins including known (e.g. CD25, CD26, CD45) and novel surface receptors, which we started to confirm by flow cytometry. Additionally, PNGase treatment of glycosylated proteins at hydrazide beads allowed the selective release and investigation of N-glycosylated asparagines. Interestingly, infection-like conditions caused the regulation of only 6 N-glycosylation sites suggesting a functional role in anti-infective MAIT cell responses. In conclusion, we established robust surfaceome analytics which (i) unravels the human MAIT cell surfaceome and receptors as novel targets for anti-infective strategies and (ii) is now used for characterizing additional immune cell types such as monocytes/macrophages.