Endosomal Targeting Sequences from Non-Classical Antigen Presenting Molecules Can Direct Antigens into the MIIC and Other Antigen Processing Compartments.

The ultimate goal of these studies is to enhance the development of MHC class II-restricted helper T cell responses that are required for effective and durable immunotherapy. In order to optimize the processing and presentation of MHC class II-restricted epitopes, we are using a differential targeting strategy that relies upon the unique endosomal targeting sequences from the individual isoforms of the CD1 family of non-classical antigen presenting molecules. Distinct endosomal compartments differ with respect to pH and resident proteases. MHC class II molecules can load antigen in each of the different endosomal compartments. This is in addition to the traditional antigen loading compartment, MIIC. Therefore, by forcing the expression of a tumor antigen across different endosomal compartments, we hope to maximize the number of successfully processed epitopes. To achieve this, we have developed a PCR-based strategy to link the leader, transmembrane, and endosomal targeting sequences from the four different human CD1 isoforms, CD1a-d, to DNA encoding for a surrogate antigen, green fluorescent protein (GFP). Using this strategy, we have demonstrated that fusion of individual CD1 tails to the sequence for GFP resulted in four distinct expression patterns in HELA cells transfected with these constructs. Using immunofluorescent staining and confocal laser microscopy, we determined the pattern of antigen expression in various endosomal compartments. GFP variably colocalized with recycling endosomes (ARF-6), early endosomes (CD71, transferrin receptor), late endosomes (CD63), and late endosomes/lysosmes/MIIC (CD107a, LAMP-1). Three of the four CD1 tails also resulted in co-expression of GFP with the MHC class II molecule, HLA-DR. The expression pattern for each CD1 tail is shown in Table 1. These experiments validate the feasibility of the CD1 targeting approach. Current studies are underway to apply this strategy to the leukemia antigen, WT1, using recently completed and sequenced WT1/CD1 fusion constructs.

Table 1. Pattern of GFP/CD1 fusion expression in different endosomal compartments. Fusion of the endosomal targeting sequences from human CD1 molecules to the model antigen, GFP, results in different patterns of antigen expression within distinct endosomal compartments. The degree of colocalization between GFP and the indicated markers of endosomal compartments is represented as follows: (−), no colocalization; (−/+), infrequent colocalization; (+), frequent colocalization; (++), extensive colocalization.