Antigen-presenting cells (APCs) are key players in response to pathogens, bridging innate and adaptive immune responses. This heterogeneous set of populations includes dendritic cells (myeloid, myDC; plasmacytoid, pDC), monocytes (classical, cMo; intermediate, iMo, and non-classical, ncMo), and macrophages (MAC), which perform specialized functions while sharing the potential to recognize antigens. However, there is a lack of studies addressing how distinct stimuli elicit different responses in the same population and the impact of the cell background on the response to antigens. Furthermore, the limitations associated with studying ex vivo MAC populations led to the use of in vitro cell models, but little is known regarding the comparability of these models to their ex vivo counterparts. This study aimed to perform a thorough proteomics analysis of ex vivo human APC populations under viral and bacterial stimulated and unstimulated conditions, alongside distinct in vitro cell models.

cMo, iMo, ncMo, pDC, and myDC were isolated from human peripheral blood (PB) samples (n=3-7) by cell sorting. cMo, pDC, and myDC subsets were stimulated for 18 h with either viral (R848, CpG ODN) or bacterial (LPS) ligands to evaluate the stimulation effects. To assess MAC models, THP1 cells were differentiated into MAC (dTHP1) (200nM PMA, 72h), and CD14+ monocytes were differentiated with M-CSF (M0 MAC) and polarized into M1 and M2 using IFNg and IL-4, respectively. Tissue MAC were isolated from healthy colon (n=5), skin (n=4), and peritoneal dialysate (n=3) samples. Cell samples were lysed, and 4 or 20 μg of protein were enzymatically digested and purified using a bead-based method. Peptide samples were labeled with tandem mass tags (TMTpro16) for quantitative analysis, measured on an Orbitrap Fusion™Lumos™Tribrid™ mass spectrometer, and analyzed using Proteome Discoverer v2.4.

A total of 4,503 different proteins were identified across ex vivo APC populations, with 3,207 (71%) common proteins. Despite the lack of unique proteins due to their close maturational relationship, a modulation of protein patterns was observed. In vitro stimulation of PB populations led to changes in protein abundance, with a role for both the type of stimulus and the cellular background being observed e.g. proteases, like CTSZ, CPQ, and CTSH, potentially leading to different protein cutting patterns and antigen presentation processes. The comparison of in vitro and ex vivo MAC showed that there is not an optimal universal model. Proteome profiles of colon MAC were more similar to M0 and anti-inflammatory M2 MAC, suggesting a more homeostatic/regulatory role in the colon environment. In contrast, skin MAC were more akin to pro-inflammatory M1 MAC, and PD MAC displayed features closer to dTHP1 cells.

Overall, this study shed insight on the processes involved in responses to antigens by APCs and highlighted the importance of selecting the appropriate MAC models when studying these cells.