Characterization and inhibition of a cholecystokinin-inactivating serine peptidase. targets, thereby eradicating the tumor. A large number of tumor-expressed T cell epitopes have been identified (5), some of which are currently used as vaccines in immunotherapeutic trials. A better understanding of the expression profiles, processing and presentation pathways of the respective antigens will be crucial to select the most promising targets for the next generation of immunotherapies. To this end, it is important to determine which antigenic peptides are generated in tumors and which vaccines would most efficiently limit the development of immune escape variants. Peptides recognized by CTLs typically originate from the degradation of intracellular proteins by the proteasome, which comprises three catalytic subunits: 1, 2, and 5 (2). In immune cells or cells stimulated with interferon- (IFN-), three other catalytic subunits are induced C LMP2 , MECL-1, and LMP7 C which replace the subunits 1, 2, and 5, respectively, to form immunoproteasomes. Intermediate proteasomes containing only one Ligustilide or two immunosubunits (1, 2, LMP7 and LMP2, 2, LMP7) also exist. Standard, intermediate, and immunoproteasomes all have unique cleavage specificities, generating different sets of peptides from the same protein precursors (6, 7). Other cytosolic proteases such as tripeptidyl peptidase 2 (TPP2), insulysin, or nardilysin acting independently or in concert with the proteasome are also involved in the production PITPNM1 of some antigenic peptides (8). Peptides produced in the cytosol are subsequently transported into the lumen of the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). TAP is a heterodimer composed of two subunits, TAP1 and TAP2, both of which are essential for peptide translocation (8). Inside the ER, the chaperone tapasin binds the N-terminal domains (N-domains) of the Ligustilide TAP chains (9) and bridges TAP to MHC I via its lumenal domain. Moreover, tapasin recruits two additional proteins: the thiol oxidoreductase ERp57, captured via a disulfide bond, and the chaperone calreticulin, which Ligustilide binds simultaneous to ERp57 and MHC I (8). The resulting complex consisting of one TAP heterodimer, two tapasin-ERp57 conjugates, and one or two MHC I-calreticulin units is known as the peptide-loading complex (PLC) (10, 11). Within the PLC, antigenic peptides are inserted into the binding groove of MHC I with tapasin acting as a crucial editor skewing the peptide repertoire towards high affinity ligands (12). Some peptides require N-terminal trimming via ER-associated aminopeptidases ERAP1 Ligustilide and/or ERAP2 (13-15). Once a ligand with sufficient affinity is captured, MHC I dissociates from the PLC and migrates to the plasma membrane. Various quality control pathways act along the secretory route (16-18), further ensuring that only optimally loaded MHC I molecules reach the cell surface. At the plasma membrane, peptide antigens are presented to CTLs. Immunotherapeutic treatment can lead to the development of tumor escape variants no longer recognized by the immune system. This frequently occurs through loss of IFN signaling in the tumor (19, 20) leading to a reduction in antigen presentation by impairing the coordinated upregulation Ligustilide of the antigen processing machinery (21). Alternatively, components of the MHC I antigen processing and presentation machinery, such as 2-microglobulin (2m), are often found directly mutated in non-responder or relapsing patients treated by immunotherapy (20, 22, 23). The central role of the classical MHC I antigen presentation pathway in tumor clearance is also highlighted by the large number of cancers in which TAP (24-32) and/or tapasin (33-35) are downregulated or not expressed at all. Both molecules are key players in the process and their loss or downmodulation in tumors causes immune evasion and is frequently associated with a poor prognosis. While.