This may prove beneficial to mount immune responses against cancer in the future. and and and and 0.01, *** 0.001, **** 0.0001 using unpaired two-tailed test. using 1 nMC1 M peptide was dependent on MHC I, given that no exogenous peptide association was observed on HeLaM-HLA-ABCKO cells at these concentrations. (and and and and and and and ?and2and and shows the MFI of fluorescent peptide binding SD from three independent experiments. ( 0.001, **** 0.0001, n/s not significant, using unpaired two-tailed test. Next, we explored the capability of soluble TAPBPR to promote peptide exchange on surface MHC I molecules by testing its ability to replace naturally presented peptides, with an exogenously added fluorescent peptide. Molidustat Cells were pretreated soluble TAPBPR for 15 min, followed by incubation fluorescent peptide with varying affinity for HLA-A*68:02 for an additional 15 min (Fig. 3and and and and and and and and and and IFN-Ctreated cells Mouse monoclonal to FOXA2 were used. Equivalent experiments of were performed using HeLaM-HLA-ABCKO expressing HLA-A*02:01 and can be found in 0.05, *** 0.001 using unpaired two-tailed test. We subsequently determined whether the peptides loaded via TAPBPR were available for T cell receptor (TCR) detection. Encouragingly, soluble TAPBPR dissociates from cells upon high-affinity peptide binding onto surface MHC I molecules (and 0.0001 using unpaired two-tailed test. Discussion Although TAPBPR usually functions as an intracellular peptide editor on MHC I molecules, we demonstrate that when given access to the surface pool of MHC I molecules, either through targeting full-length TAPBPR to the PM or by adding soluble TAPBPR to cells, TAPBPR retains its function as a peptide-exchange catalyst. Thus, we have developed two cell-based peptide-exchange systems for MHC I, Molidustat which complement those already established (11, 12). Here, we have shown that TAPBPR can mediate peptide editing on three distinct MHC I molecules (HLA-A*68:02, HLA-A*02:01, and H-2Kb) expressed on the surface of cells. As expected, the efficiency of TAPBPR-mediated peptide exchange is dependent on affinity of the incoming peptide for a particular Molidustat MHC I. Intriguingly, our work, particularly when using soluble TAPBPR, demonstrates that TAPBPR can dissociate peptides that apparently have relatively high affinity for MHC I, given that it works on MHC complexes expressed on the surface of cells with an intact antigen-presentation pathway and thus on molecules that have already undergone the process of chaperone-mediated quality control. This raises interesting questions regarding the precise Molidustat criteria by which TAPBPR selects peptides. This ability of TAPBPR to outcompete apparently good peptides from MHC I relatively quickly may explain why TAPBPR levels in cells are quite low. Our cell-based assays for determining the ability of TAPBPR to catalyze peptide exchange on MHC I molecules offer a number of advantages over the already-established cell-free assays, representing a more physiological system for exploring this concept. First, in contrast to the cell-free systems (6, 7, 11, 12), our assays here assess the interaction between TAPBPR and MHC I molecules in their naturally occurring membrane-bound conformations, taking into account the restrictions imposed by a cellular membrane, either on both the MHC I molecules and on TAPBPR, or on MHC I alone. Second, as opposed to the bacterial refolds used in the Chen and Bouvier assay (11), the MHC I molecules present in our system are subjected to the naturally occurring posttranslational modifications within the cell, as is also the case in Wearsch and Cresswells (12) assay; moreover, the MHC I molecules here are loaded with a broad spectrum of peptides instead of being refolded around single individual ones, creating a less-biased and broader range of ligands for TAPBPR. In addition, the cellular assays offer the possibility to screen the ability of TAPBPR to function as a peptide-exchange catalyst on a broad range of MHC molecules in a highly efficient manner, simply by using the MHC I molecules expressed on cells, and without the need to make bacterial refolds of individual MHC I. In Molidustat contrast to TAPBPR, we found that tapasin was not able to perform its peptide-editing function on surface-expressed MHC I molecules. There are a number of potential reasons.