AbstractsBiology & Animal Science

Abstracts 1. Production of soluble peptide-MHC class II-Ig fusion proteins The aim of the study is to construct soluble variants of the murine MHC class II molecule I-Ed in complex with two tumor-specific peptides. Whereas methods for production of soluble MHC class I are well established, production of soluble class II has proven more challenging. The main obstacles are the lack of well-established methods for achieving correct folding, chain pairing, and secretion. As the soluble I-Ed molecules we produce will be used primarily as detection reagents, and not as therapeutic reagents, we were not constrained by structural requirements, such as small size. It was more important to choose a format that allows easy secretion, visualization and multimerization. We have therefore focused on the production of soluble I-Ed on an Ig scaffold, either fused to complete Ig or to an Fc region. Both formats were made with tumor-specific or control peptides fused N-terminally to I-Edb. We were successful in producing soluble I-Ed in both formats after infection of Sf9 insect cells with recombinant baculovirus. Our preliminary data indicate that addition of the complete extracellular part of I-Ed to the complete Ig seems to allow better secretion than addition to the hinge + Fc. Furthermore, secretion levels were higher for molecules without antigenic peptides. The new reagents will be a very important tool in the development and characterization of soluble TCRs. In addition, they can be used to follow specific T-cell proliferation during tumor establishment. 2. Efficient loading of the l2315 epitope onto I-Ed for presentation to specific T-cells In the ER, MHC class II molecules associate with invariant chain (Ii), which blocks the binding of self-peptides to the peptide-binding groove. It also directs the transport of MHC class II to endocytic compartments where it can encounter peptides derived from endocytosed antigens. Ii is sequentially degraded prior to peptide loading. The last degradation fragment of Ii is the class II associated Ii peptide (CLIP). This peptide remains bound in the peptide-binding groove of MHC class II until it is replaced by an antigenic peptide. In this project we wanted to investigate the antigen presenting capabilities of CA36.2.1 cells transfected with Ii with either of two different immunogenic peptides localized in the CLIP region. To be able to compare the two different peptides, we needed to identify transfectants with equal levels of Ii and I-Ed expression. We also wanted to compare the effect of such endogenous loading of peptide onto the murine class II molecule I-Ed with conventional external peptide loading. We found that the transfected cell lines expressed invariant chain at highly variable levels. Furthermore, surface staining of I-Ed revealed a reduction in I-Ed levels on most clones. However, there was no clear correlation between the observed Ii levels and the degree of I-Ed down-regulation. T-cell activation assays with the different transfectants as APCs confirmed that genetic…