Exposure to Melan-A/MART-126-35 tumor epitope specific CD8+T cells reveals immune escape by affecting the ubiquitin-proteasome system (UPS)

The generation of antitumor cytotoxic T cell (CTL) response involves the processing and presentation of tumor antigens onto MHC class I molecules1,2. These specialized T cells can detect target cells that endogenously express protein molecules (i.e. mutated, over-expressed and/or tissue differentiation antigens) and subsequently remove these cells from the body3,4. The vast majority of peptides presented by MHC class I molecules at the cell surface for recognition by specific cytotoxic T-cells (CTL) is generated by the ubiquitin-proteasome system (UPS) with its central multicatalytic proteinase complex, the proteasome5,6. Peptides generated by the proteasome system are transported by TAP proteins (transporter associated with antigen presentation) into the ER where peptides of appropriate length and affinity will bind to MHC class I proteins to be presented at the cell surface for immune recognition by CTL7,8,9. The standard 20S proteasome (s-20S proteasome) with its active site β-subunits β1, β2 and β5 represents the central catalytic unit of the UPS and the catalytic core of the 30S proteasome which is built by the association of two 19S regulator complexes with the 20S core complex. IFN-γ induces the synthesis of alternative catalytic immunosubunits (i-subunits), i.e. β1i/LMP2, β2i/MECL1 and β5i/LMP7 and the concomitant formation of immunoproteasome (i-proteasome) subtypes8,9,10. The 30S proteasome complexes are responsible for the degradation of proteins in the nucleus and the cytosol, which are marked for degradation by a poly-ubiquitin chain and consequently recognized by specific subunits of the 19S regulator complex. A special problem arises for the degradation and processing of membrane proteins, which are co-translationally transported into the endoplasmic reticulum (ER). These proteins, if misfolded or mutated, are re-translocated to the cytosolic side of the ER to be degraded by the 30S proteasome complex in an ubiquitin-dependent manner11,12,13. This process is called ER associated degradation pathway (ERAD) and essentially requires the so-called ERAD-complex within the ER-membrane. This complex is composed of a number of different proteins including Derlin, VIMP, Herp and the E3-ligase HRD114,15. Functionally associated with the ERAD pathway on the cytosolic site of the ER is the p97/VCP ATPase complex. The p97/VCP complex binds and extracts poly-ubiquitinated proteins from the membrane making them available for proteasomal degradation at the cytosolic site of the ER16,17. Efficient processing and generation of the target antigenic peptides by the UPS is essential for treatment of cancers by T-cell therapy. However, immune escape due to inefficient processing of HLA dependent tumor epitopes can be one important reason for failure of such therapies. It is known that tumors can down-regulate or completely lose expression of tumor antigens and HLA class I molecules, thereby escaping from T cell recognition18,19. Modulation of the UPS has also been observed and, in particular, the expression of the IFN-γ inducible components of the UPS such as PA28α/β and the i-subunits β1i/LMP2 and β5i/LMP7 were found to be altered in tumor cells, affecting both the quantity and in certain cases also the quality of the generated epitopes20,21,22. In some cases, a deficient expression of proteasome components could be reverted in the presence of IFN-γ, thereby also reconstituting MHC class I surface expression23. However, due to the complexity of the UPS and its associated pathways, only a few immune escape mechanisms have been characterized so far, although knowledge of these mechanisms is a prerequisite for the improvement of cancer immunotherapy. To identify novel mechanisms by which tumors can become refractory to immune elimination, human melanoma cells expressing the transmembrane Melan-A/MART-1 tumor antigen were exposed to two rounds of brief co-culture with Melan-A/MART-126-35-specific CTL. Immune selected melanoma cell clones, being resistant to lysis by Melan-A/MART-126-35 CTL were investigated for the underlying mechanism focusing on the role of the proteasomal antigen processing machinery. We detected a deregulated ERAD pathway as a so far unknown mechanism for immune escape of melanoma cells. In particular, the essential non-inducible ERAD component p97/VCP has been found to be down regulated in CTL-resistant melanoma cells. Rescue experiments reconstituting p97/VCP expression in melanoma cells resulted in enhanced Melan-A/MART-126-35 epitope recognition, underlining the in vivo functional relevance of our results.