Immune checkpoint molecules in canine T-cell lymphomas: flow cytometric assessment of PD-L1 and CD47 expression

Introduction. The role of immunity in the development, progression and prognosis of neoplasms represents a topic of increasing interest in both human and veterinary medicine. Programmed Death-Ligand 1 (PD-L1) and CD47 are surface proteins involved in the inhibition of the host immune response and can be overexpressed in several different neoplasms, including lymphoma. In particular, PD-L1 acts by inactivation of host cytotoxic lymphoid cells, whereas CD47 represents a "don't-eat-me" signal toward monocytes/macrophages. This study aims to describe preliminary results about those proteins’ surface membrane expression in both aggressive and indolent canine T-cell lymphomas (TCL) assessed via flow cytometry (FC). Material and methods. Analyses were performed on surplus material of nodal aspirates obtained for diagnostic purposes after FC confirmation of TCL. Only cases at first presentation were included. PD-L1 expression was calculated as the ratio between median fluorescence intensity (MFI) of neoplastic cells stained with customized anti-PD-L1 antibody and an isotypic control. Neoplastic cells were considered positive for PD-L1 expression if MFI-ratio was>1. CD47 was considered positive if expressed by>20% of neoplastic cells. Results. Specimens obtained from eighteen dogs with TCL (5 T-Zone Lymphomas (TZL) and 13 aggressive TCL) were analyzed: 7(39%) were positive for PD-L1 (2 TZL, 5 aggressive TCL), and 11(61%) were negative. Median MFI-value for positive samples was 1,16 (1,08-1,49). Assessment of CD47 was performed on 11/17 samples and resulted positive in 7(64%) (1 TZL, 6 aggressive TCL) and negative in 4 (36%). Discussion. Both PD-L1 and CD47 were detected via FC on TCL neoplastic cells, showing that the analysis can be performed beside routinary panels. Our results did not show any difference of expression between TZL and other TCL, but the limited number of samples might have been an important bias. Future studies should include more samples and explore the correlation between PD-L1/CD47 expression and clinical outcome.