PO-396 Soluble mediators released by human melanoma-associated fibroblasts interfere with cytotoxic T cell response

IntroductionMelanoma associated fibroblasts (MAFs) represent a highly heterogeneous population of fibroblast-like cells residing in the tumour stroma, the exact origin of which is a matter of ongoing debate. In many aspects, MAFs recapitulate key features of both mesenchymal stem cells (MSCs) having well-known immunosuppressive properties, and activated fibroblasts actively participating in matrix reorganisation, a prerequisite of tumour invasion. Compared to normal fibroblasts, MAFs display high proliferation rate, show increased motility, release a skewed spectrum of extracellular matrix components and growth factors, and actively secrete both inflammatory and immunosuppressive cytokines, such as TGFβ, VEGF, and IL-6 into their environment.Material and methodsMAFs and normal fibroblasts (NFs) were isolated from enzymatically disintegrated melanomas and healthy skin, respectively. MAFs were identified as Melan-A-/gp100-/α-SMA+cells by flow cytometry. Untouched CD8 +cytotoxic T cells were isolated from healthy donors by magnetic sorting, and activated by anti-CD3/CD28 in the presence of MAF- and NF-conditioned media (CM). Expression of activation markers, markers of degranulation and cytokine production were analysed and redirected killing assays were performed to define the impact of MAF-released soluble mediators on CD8 +T cell activity.Results and discussionsWe found that upon cognate activation of MAF-CM-treated CD8 +T cells, expression of the early activation molecule CD69 and granzyme B production were suppressed as compared to NF-treated cells. On the other hand, intracellular IFNg staining remained unaffected by MAF-CM and there was no difference in activation-induced LAMP-1 staining, i.e. in cytotoxic degranulation either. Killing capacity of CD8 +T cells was markedly reduced by MAF-CM, too, as judged by redirected killing assays utilising anti-CD3-/CD28-loaded P815 cells as targets. To reveal key mechanisms responsible for this phenomenon, several soluble factors and immunomodulatory pathways were measured in MAFs and their CMs by ELISA and flow cytometry.ConclusionTaken together, these data indicate that MAF cells are involved in the regulation of the antitumor response within the tumour microenvironment.