INSIGHTS ON THE CROSSTALK BETWEEN THE IMMUNE SYSTEMAND CANCER: IMMUNE CHECKPOINT MOLECULES AND LYMPHOIDPOPULATIONS ASSESSMENT IN CANINE ROUND CELL TUMORS

The crosstalk between the immune system and cancer represents a fundamental aspect of oncology, since a better understanding of disease mechanisms could lead to the development of novel therapeutic strategies. This PhD project focused on three main topics: (1) the expression and prognostic role of programmed death-ligand 1 (PD-L1) in canine lymphoma, (2) the expression and prognostic role of CD47 in canine leukemias, and (3) the characterization of lymphocyte populations in peripheral blood and sentinel lymph nodes (SLNs) of dogs with mast cell tumors (MCTs). Regarding PD-L1, the project evaluated the expression of the surface membrane protein (mPD-L1) by means of flow cytometry (FC), mRNA by real-time qPCR, and soluble protein (sPD-L1) plasmatic concentrations by competitive ELISA in dogs with nodal lymphoma. Their potential prognostic role was also investigated. B-cell lymphomas showed the highest frequency and level of mPD-L1 expression, aggressive T-cell lymphomas exhibited the lowest one, and T-zone lymphomas displayed frequent but overall low expression. No correlation was identified among mPD-L1, mRNA levels, and sPD-L1, although the latter was increased in FC-negative dogs. No statistically significant influence on prognosis was found. However, trends suggested longer survival in mPD-L1-negative cases in both B-cell and T-cell lymphomas, and all progressing T-zone lymphomas were mPD-L1-positive. These findings indicate a potential, although not definitive, prognostic role of mPD-L1, warranting further investigation in larger cohorts. In the second part of the project, CD47 was assessed in peripheral blood cells of healthy dogs to establish baseline values and support interpretation in pathological conditions. It resulted consistently expressed across all leukocyte populations and red blood cells, with low intra-assay variability but notable inter-individual differences, particularly in lymphocytes and erythrocytes. Subsequently, CD47 expression was evaluated in canine leukemias. No differences were observed between acute and chronic leukemias, but myeloid acute leukemias exhibited significantly higher expression compared to lymphoid forms. Although not statistically significant, survival was lower in dogs with acute leukemia and CD47 overexpression, suggesting a possible prognostic implication that requires confirmation in larger studies. In the third part of the project, we investigated lymphocyte populations in SLNs and peripheral blood of dogs with mast cell tumors. Variations in nodal T/B and CD4/CD8 ratios were influenced by tumor grade, metastatic status, and glucocorticoid pre-treatment, highlighting a complex interplay between tumor biology and immune response, whereas no significant changes were observed in circulating lymphocytes. As part of this project, the impact of SLN mapping dyes on FC fluorescence measurements was assessed. Results demonstrated that commonly used dyes can significantly alter baseline fluorescence across channels, potentially introducing bias in quantitative FC analyses. In conclusion, this PhD project provides further insights into tumor-immune system interactions while also addressing critical technical aspects of FC. PD-L1 appears to be overall low expressed in canine lymphoma, while CD47 emerges as a more promising marker. As expected, different tumor characteristics influenced the composition of lymphoid population in SLNs.