CD40 ligand and lipopolysaccharide enhance the in vitro generation of melanoma-reactive T-cells

Cancer vaccine trials require sensitive assays for evaluating T-cell responses in immunized patients. In addition, these methods are used for identifying novel tumor-associated antigens (TAA). Therefore, our aim was to improve the methods for evaluating patients receiving the cancer vaccines by enhancing the in vitro detection of tumor-specific T cells from the peripheral blood. We have developed an efficient and reproducible method for detecting tumor-specific T cells by optimizing the activation of antigen presenting cells (APC) in peripheral blood mononuclear cells (PBMC) of metastatic melanoma patients with soluble trimeric CD40-ligand (CD40L) or lipopolysaccharide (LPS). This method significantly improved the generation of Melan-A/MART-1:27-35 and Melan-A/MART-1:26-35(27L) peptide/tumor-specific cells as well as lower frequency tyrosinase:368-376(370D) specific T cells from the PBMC of melanoma patients. T-cell enhancement from activated PBMC cultures was found to be reproducible within individual patients and was observed after the addition of either CD40L or LPS to PBMC cultures. Additionally, PBMC activation improved the detection of tumor-specific precursors from melanoma patients previously immunized with peptides derived from Melan-A/MART-1, tyrosinase and gp100. Collectively, these findings describe a novel approach for evaluating patients receiving the cancer vaccines and may provide a useful method for the characterization of novel tumor-associated antigens.