Cytolytic T cell (CTL) responses are predicted to be biased towards membrane proteins. The peptide-binding grooves of most alleles of histocompatibility complex class I (MHC-I) are relatively hydrophobic, therefore peptide fragments derived from human transmembrane helices (TMHs) are predicted to be presented more often as would be expected based on their abundance in the proteome. However, the physiological reason why membrane proteins are presented more often is unclear, and with current research focusing on soluble antigens, little is known about the processing and (cross)-presentation of membrane-derived antigens.

To answer this, we developed a platform to study how known antigen presentation pathways contribute to the presentation of membrane-buried epitopes in human monocyte-derived dendritic cells (moDCs). We engineered a dual-spanning integral TM FRET sensor that contained the NY-ESO-1₁₅₇–₁₆₅ epitope and compared its presentation with soluble full-length NY-ESO1 antigen and peptide controls. To examine endogenous presentation, moDCs were transfected with in vitro–transcribed mRNA encoding the sensor. Using a T cell activation assay, we demonstrated MHC class I presentation of a TMH-derived epitope on the moDC’s cell surface to elicit a CTL response. Antigen presentation was measured with an NY-ESO-1–specific NFAT-luciferase Jurkat T cell reporter system. The relative contributions of the cytosolic and vacuolar pathways to the process of membrane vs. soluble antigens were assessed using chemical perturbations.

Our results demonstrated that moDCs present TMH-derived epitopes on their cell surface to elicit CTL responses. Inhibitor screens revealed that TMH presentation strongly relies on ER-associated degradation (ERAD) and proteasomal activity. Furthermore, these antigens localize to endosomal compartments and require lysosomal maturation and vacuolar (v)-ATPase activity, suggesting that low pH facilitates the extraction of TMHs from the membrane. Together, these findings indicated that membrane-buried antigens use a distinct processing route and differ fundamentally from the processing of soluble antigens.