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Background Advanced glycation end products (AGEs) can modulate immune responses through binding to the receptor for AGEs (RAGE). 1 RAGE activation promotes pro-inflammatory signaling and may influence immune checkpoint pathways like PD-1, which regulates T cell exhaustion in cancer.2 Dysregulation of RAGE and PD-1 signaling can impair anti-tumor immunity.3 The effect of a novel glycation product, MAGE (melibiose-derived AGE)4 remains to be described, especially in context of anti-cancer immune response. Here we assessed MAGE effect on PD-1 and RAGE expression in peripheral blood mononuclear cells (PBMCs) to explore how MAGE might contribute to cancer-associated immune suppression.Methods PBMC were isolated from fresh blood of healthy volunteers by density gradient centrifugation of LymphoSep solution. The obtained cells were stimulated with 250 µg/mL MAGE 4 or myoglobin as a carrier protein for 24 hours in 5% CO2, at 37°C, and processed for FACS analysis. PBMCs were gated on FSC-A/SSC-A and single cells (FSC-A/FSC-H). Next, purity of monocytes was assessed by determining the CD14/SSC-A positive cell population. RAGE expression and PD-1 was determined with an appropriate antibody. In addition, M1/M2 polarization of macrophages (CD14+CD68+) based on CD14+CD68+CD80-CD206+ (M2) and CD14+CD68+CD80+/-CD206- (M1) staining was performed. Other cell populations were also analyzed by differential staining and gaiting for lymphocytes B (CD3-CD56-), TC (CD3+CD8+CD56-CD16+/-), TH (CD3+CD56-), NK (CD3-CD56+CD16+/-) or NKT (CD3+CD56+). Samples were analysed in duplicates and data were averaged from at least 4 independent donors. Statistical analysis by t-Test was run to identify significant changes.Results RAGE expression was decresed on monocytes (CD14+) treated with MAGE, below the MB effect, but had no effect on the receptor status in all other cell types.There was small non-significant decrese in PD-1+ cells in most populations (NK, NKT, Tc, TH, monocytes), except B lymphocytes that did not change in presence of MAGE. However, cells incubation with MAGE activated the population of T lymphocytes (CD3+), as assessed by expression of a degranulation marker CD107a. Monocyte population was the most responding cells with significant PD-1 decrease when treated with MAGE. Interestingly, the carrier protein (MB) also showed significant effect on decrease of PD-1+ cell number showing additional immunosuppression effect.In addition, MAGE induced polarisation of macrophages towards pro-inflammatory M1 phenotype.Conclusions We observed significant immunomodulatory effect of the melibiose-derived AGEs on monocytes, macrophages, and T lymphocytes isolated from PBMC. MAGE-related decrease in cells expressing RAGE and PD-1 might have a positive impact for anti-cancer strategies thus should be further explored.Acknowledgements Project was supported by funds from the Polish National Science Centre, OPUS13 No. 2017/25/B/NZ4/01198 and OPUS27 No. 2024/53/B/NZ4/0911 for Magdalena Staniszewska, Principle Investigator.References Bierhaus A, et al. Understanding RAGE, the receptor for advanced glycation end products. J Clin Invest. 2005;115(5):1115–1125.Pauken KE, Wherry EJ. Overcoming T cell exhaustion in infection and cancer. Trends Immunol. 2015;36(4):265–276.Chen DS, Mellman I. Elements of cancer immunity and the cancer-immune set point. Nature. 2017;541(7637):321–330.Staniszewska M, et al. The melibiose derived glycation product mimics a unique epitope present in human and animal tissues. Sci Rep. 2021;11:2940.