Trp-1-/- mice vaccinated with Trp-1 generate high avidity T cells that are protective against melanoma

C264 Cancer immunotherapy involves manipulation of the immune system to promote recognition and elimination of malignant cells by activating immune responses against tumor antigens. Prominent melanoma differentiation antigens under investigation for immunotherapy include MART-1, gp100 and TRP-1. These proteins are specific to both melanocytes and melanomas and can be recognized as antigens by T-cells. Efforts to utilize these antigens for vaccination or expansion of tumor-specific T-cells, however, have failed to achieve dependable therapeutic efficacy in clinical trials. Current theories suggest that immune tolerance is a key barrier to the development of tumor immunity. We elected to focus our experimental work on TRP-1, which has been previously shown to be the most abundant glycoprotein in melanocytic cells. Immunization against this protein has been found to induce autoimmune vitiligo. Additionally, a TRP-1 knockout mouse (KO), which lacks expression of the antigen in peripheral melanocytes, has been described. Based on these findings and our previous identification of novel MHC class II restricted epitope from TRP-1, we sought to utilize TRP-1 as a model antigen for studying self-tolerance and autoimmunity. Our first experiments focused on TRP-1 expression within medullary thymic epithelial cells (mTECs) of WT and KO mice. mTECs are known to systematically eliminate self-reactive T-cells by expressing and presenting self antigens on MHC:peptide complexes. We now report quantitative RT-PCR results which reveal ample ectopic expression of TRP-1 in WT mTECs, suggesting that central tolerance could affect the host T-cell repertoire which recognizes this antigen. Also, mTECS from KO mice demonstrably lack TRP-1 expression, making the KO a suitable control for our tolerance studies. To gauge the effect of host antigen expression on tolerizing the anti-TRP-1 T-cell repertoire, we compare ELISPOT assays performed on splenocytes harvested from WT and KO mice following vaccination with rVV-TRP-1. The potency of T-cell responses is measured by IFN-γ release following stimulation with our class II TRP-1 epitope. Interestingly, splenocytes from the KO demonstrate significantly higher numbers of IFN-γ secreting T-cells compared to the WT (with log-fold differences at micromolar concentrations of TRP-1 peptide). We also utilize functional studies to assess anti-tumor activity of T-cells from the WT and KO backgrounds. WT and KO mice are immunized twice with RVV-TRP-1, after which splenocytes are harvested and T-cells expanded ex-vivo by pulsing with TRP-1. These T-cells are infused into WT mice previously challenged with B16 melanoma; lungs from the treated mice are subsequently extracted and metastatic tumor nodules are counted. We found that T-cells from the KO mice provided statistically superior tumor protection compared to those from the WT. Mice receiving KO T-cells ended up with an average of only 5 metastatic nodules compared to an average 58 nodules in those receiving WT T-cells. Our results suggest that tolerance induced by antigen expression significantly dampens the anti-tumor immune response. They also show that antigen knockout mice can be sources of potent anti-tumor T-cells for future adoptive transfer and gene therapy studies.