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Background HLA-E in humans and Qa1 in mice primarily present canonical leader peptides derived from the signal sequences of classical MHC class I molecules—specifically HLA-A, HLA-C, and HLA-G in humans, and H2-D and H2-L in mice. These peptides serve as inhibitory ligands for the NKG2A/CD94 receptor complex on NK cells and CD8 + T cells, helping to maintain immune homeostasis. Disruptions in this presentation axis—such as during viral infection—can lead to the display of novel peptides that override this inhibition and activate immune responses. However, the full set of mechanisms governing canonical peptide presentation remains unclear.Methods To investigate the regulation of Qa1-restricted inhibitory peptide presentation, we knocked out Signal Peptide Peptidase (SPP), an ER-resident protease responsible for signal peptide cleavage, in two tumor models: YUMMER melanoma and KPC pancreatic adenocarcinoma. We analyzed changes in Qa1-bound peptides using mass spectrometry and evaluated tumor growth in vivo. To precisely quantify the presentation of canonical peptides, we employed novel tools including the EXXI antibody and an NKG2A CAR cell-based reporter assay. We are also now targeting other SPP family members that are mechanistically and structurally related to SPP to explore potential alternative peptide-processing pathways.Results SPP knockout led to a substantial reduction in canonical peptide loading in both tumor models. While SPP-deficient melanoma tumors underwent robust rejection in vivo, pancreatic tumors lacking SPP did not, despite comparable decreases in canonical inhibitory peptide levels. Surprisingly, the dominant peptide bound to Qa1 in both models remained the canonical peptide, indicating the presence of alternative, SPP-independent mechanisms for its production. In the KPC model, we also identified novel leader peptides derived from other MHC class I molecules (H2-K) that may exert similar inhibitory functions. Ongoing studies are evaluating whether these peptides act as alternative inhibitory ligands, potentially compensating for the loss of the canonical peptide and preserving immune suppression.Conclusions Our findings reveal that redundant or alternative mechanisms sustain inhibitory peptide presentation in tumors even after SPP deletion. Ongoing studies targeting related SPP-like proteases and evaluating the function of newly identified leader peptides aim to elucidate these compensatory pathways. These insights have significant implications for fully disrupting the Qa1/NKG2A/CD94 inhibitory axis to unleash anti-tumor immunity.