Human Langerhans cells electroporated with Wilms’ tumor 1 mRNA induce antigen-specific cytolytic T lymphocytes Free

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The Wilms’ tumor 1 (WT1) gene encodes a transcription factor essential for cell growth and development. Overexpression of wild type WT1 occurs in many leukemias and solid tumors, where it correlates with poor prognosis. The WT1 protein may therefore provide a therapeutic target as a tumor-associated antigen (TAA). Vaccination protocols using peptide-pulsed dendritic cells (DCs) to induce anti-tumor responses, however, can only present defined epitopes restricted to certain human leukocyte antigens (HLA). In contrast, vaccination with mRNA-electroporated DCs could support processing and presentation of multiple TAA epitopes tailored to a patient’s own HLA. We have previously shown that human Langerhans-type dendritic cells (LCs) are the most potent DCs for stimulating CTL. We therefore electroporated immature LCs derived from CD34⁺ progenitors with human WT1 mRNA. These LCs were then matured by 48 hrs’ exposure to a standard inflammatory cytokine combination. 80% (±18, SD) of the electroporated LCs expressed the WT1 transgene up to 96 hours later, based on concomitant expression of a GFP reporter gene. After gating on HLA-DR^bright, CD11b^neg LCs, all of the WT1 mRNA and mock electroporated LCs exhibited a mature, activated phenotype characterized by CD86, CD80, and CD40 expression. More than 95% of these cells also displayed the prototypic DC activation marker, CD83. In an allogeneic mixed lymphocyte reaction, a standard assay of DC function, both WT1 mRNA and mock-electroporated LCs stimulated similar allogeneic T cell proliferation, comparable to positive control stimulation by PHA mitogen (n=3 expts). Moreover, after only seven days’ stimulation in vitro, WT1-electroporated LCs induced at least a 2-fold greater increase in Ag-specific T cell secretion of IFN-gamma by ELISpot, compared with mock-electroporated LCs. An additional seven days’ stimulation resulted in a further 3-to-4-fold increase in these Ag-specific effector T cells (n=2 expts to date). Intracellular cytokine staining further revealed that 35% of CD8⁺ and 18% of CD4⁺ T cells secreted IFN-gamma in response to WT1 electroporated-LCs, confirming presentation of both class I and II MHC-restricted epitopes. In a standard ⁵¹Cr release assay, T cells exhibited 80% specific lysis of a WT-1⁺ tumor cell line, compared with <20% specific lysis of a WT1^neg cell line at an effector:target ratio of only 20:1, after only two 7d rounds of stimulation by WT1 mRNA electroporated LCs. These data demonstrate for the first time that WT1 mRNA-electroporated human LCs can elicit potent CD8⁺ cytolytic and CD4⁺ Th responses against a self or differentiation antigen that is strongly conserved in the mammalian genome and to which individuals should normally be tolerant. This approach merits further testing in vivo and would allow any individual with a WT1 expressing tumor to undergo vaccination by WT-1 mRNA electroporated LCs regardless of HLA type.