Pathway-Based Analysis of a Melanoma Genome-Wide Association Study: Analysis of Genes Related to Tumour-Immunosuppression

Systemic immunosuppression is a risk factor for melanoma, and sunburn-induced immunosuppression is thought to be causal. Genes in immunosuppression pathways are therefore candidate melanoma-susceptibility genes. If variants within these genes individually have a small effect on disease risk, the association may be undetected in genome-wide association (GWA) studies due to low power to reach a high significance level. Pathway-based approaches have been suggested as a method of incorporating a priori knowledge into the analysis of GWA studies. In this study, the association of 1113 single nucleotide polymorphisms (SNPs) in 43 genes (39 genomic regions) related to immunosuppression have been analysed using a gene-set approach in 1539 melanoma cases and 3917 controls from the GenoMEL consortium GWA study. The association between melanoma susceptibility and the whole set of tumour-immunosuppression genes, and also predefined functional subgroups of genes, was considered. The analysis was based on a measure formed by summing the evidence from the most significant SNP in each gene, and significance was evaluated empirically by case-control label permutation. An association was found between melanoma and the complete set of genes (pemp = 0.002), as well as the subgroups related to the generation of tolerogenic dendritic cells (pemp = 0.006) and secretion of suppressive factors (pemp = 0.0004), thus providing preliminary evidence of involvement of tumour-immunosuppression gene polymorphisms in melanoma susceptibility. The analysis was repeated on a second phase of the GenoMEL study, which showed no evidence of an association. As one of the first attempts to replicate a pathway-level association, our results suggest that low power and heterogeneity may present challenges

Formation and kinetics of MHC class I-ovalbumin peptide complexes on immature and mature murine dendritic cells.

Dendritic cells (DC) are professional antigen-presenting cells that are able to induce primary T cell responses. Therefore, several strategies employ peptide-pulsed DC in tumor immunotherapy. For efficient antigen presentation and induction of an immune response by DC the number and stability of MHC I-peptide complexes is crucial. We studied this issue by using the antibody 25-D1.16 that specifically detects OVA peptide SIINFEKL in conjunction with H-2 Kb molecules, and determined its kinetics on mature and immature bone marrow-derived murine DC. Optimal peptide loading was reached after 8-16 h at 50 microM peptide pulse, and was comparable in serum-free versus serum-containing medium. Stimulation of DC with LPS or Poly I:C, and to a lesser extent TNF-alpha, upregulated the total number of surface MHC I molecules and thus improved peptide loading. Pulse-chase experiments revealed a constant half-life of peptide/Kb complexes independent of preceding DC stimulation or their maturation stage. The duration of peptide/Kb complex expression on mature DC, however, could be extended from 24 h to 72 h when the cultures were pretreated with LPS or Poly I:C, but not TNF-alpha. These data might have important implications for the clinical application of peptide-pulsed DC in tumor immunotherapy