The expression of different immunological markers by cultured human melanocytes (MC) in relation to immune phenomena, were investigated on ten different MC cell lines from early (1st) to late (22nd) passage. Four melanocyte lines (MC-a) which had undergone changes in growth behaviour during prolonged culture were included in the study, together with two melanoma lines. Cytospin preparations of the cells were stained for the presence of a set of different immunological markers and a melanoma-associated antigen (MAA). All MC lines, including the MC-a and the melanoma lines, showed expression of MHC class I, IL-1, IL-2, ICAM-1 and the MAA, NKI-Beteb, during all passages tested. Interestingly, four of the MC lines showed staining for the Fc receptor. A tendency towards a stronger expression of ICAM-1 on a higher percentage of cells was observed on MC with increasing passage number, the MC-a and the melanoma lines. Expression of the MAA was strongly reduced for the MC-a lines in comparison with the MC and the M14 melanoma lines. Positive staining for the HLA class II molecules was obtained on MC of intermediate and late passages, and on the MC-a and the melanoma lines in the decreasing order HLA-DR, DP and DQ. Additionally, we carried out a preliminary study showing that cultured MC also produce IL-1 and IL-6. However, we were not able to show the production of biologically active IL-2 testing several cultured MC lines. Nevertheless, the overall results taken together suggest that MC are immunologically important cells that are susceptible to changes during long-term culture.
Extracorporeal Photochemotherapy (ECP) is a widely used immunotherapy for cutaneous T cell lymphoma, as well as an immunomodulatory treatment for graft versus host disease (GVHD) and rejection of allografts. We hypothesized that ECP's physiologic induction of large-scale monocyte-to-dendritic antigen presenting cell (DC-E) conversion is mechanistically responsible for both its anti-cancer effect (immunizing mature DC-E) and its tolerogenic impact in the transplant setting (8-MOP-maturationally truncated DC-E). To interrogate this possibility, we developed an ECP device that is scalable from mouse to man and tested its capacity to produce DC that, when advantageously tuned and tumor antigen-loaded, can limit the growth of otherwise lethal tumors in the engineered Yale University Mouse Melanoma(YUMM1.7) model (driven by PTEN loss and BRAF V600E activation). Untreated control mouse tumors (N¼217) were 10-fold (p<0.01) larger than ECP treated (N¼50) tumors at the 28-day termination point, as required due to control tumor size. Depletion of DC-E from the cellular vaccine preparation prevented the immunoprotective effect(p<0.01), indicating a primary role for ECP-induced DC. Depletion of platelets from the cellular suspension also prevented the immunizing effect(p<0.01), substantiating prior in vitro evidence that physiologic induction of DC-E is signaled by transient monocyte adherence to platelets. Addition of 8-MOP-UVA-treated PBMCs to the otherwise immunoprotective DC vaccine completely reversed the immunoprotective effect(p<0.01), revealing that maturationally truncated DC are counterproductively tolerogenic to the melanoma tumor antigens which they process. Collectively, these results show that, in ECP, platelet-induced tumor-loaded mature DC-E are immunotherapeutic for established murine melanoma, while immature DC-E are tolerogenic. These findings verify a pivotal role for DC-E and suggest the intriguing possibility that ECP can be modified for immunotherapy of solid tumors.
Using metal ion affinity chromatography and mass spectrometry, we have previously shown that phosphorylated peptides are endogenously processed and presented by many different MHC I and II molecules, and have identified differentially displayed phosphopeptides presented by HLA-A2 on cancer cells. Many of the source proteins for these phosphopeptides are involved in tumorigenesis and metastasis. These peptides are appealing immunotherapeutic targets since mutations in or downregulation of the source proteins as a means of immune escape may compromise malignancy. We focused on one phosphopeptide spanning residues 30-39 of β-catenin (βcatpS33). When used to immunize HLA-A2 transgenic mice, βcatpS33 was very weakly immunogenic. Replacement of a suboptimal Ala anchor residue at P10 with a Val (βcatpS33(V)) enhanced the HLA-A2 binding affinity 10 fold and substantially improved immunogenicity. CD8 T cells from βcatpS33(V)-immunized mice were crossreactive to βcatpS33. The T cells also recognized endogenously processed βcatpS33 on several HLA-A2+ melanoma cells. Thus, crossreactivity to the natural βcatpS33 phosphopeptide can be maintained by making minimal changes in the sequence while preserving the ability of T cells to discriminate between the phosphorylated and unphosphorylated forms of the peptide. Enhancing immunogenicity by fine-tuning such an attractive tumor antigen linked to tumorigenic and metastatic processes should improve the efficiency of tumor control.
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