Activation of human CD4+ T cells by targeting MHC class II epitopes to endosomal compartments using human CD1 tail sequences.

Distinct CD4(+) T-cell epitopes within the same protein can be optimally processed and loaded into major histocompatibility complex (MHC) class II molecules in disparate endosomal compartments. The CD1 protein isoforms traffic to these same endosomal compartments as directed by unique cytoplasmic tail sequences, therefore we reasoned that antigen/CD1 chimeras containing the different CD1 cytoplasmic tail sequences could optimally target antigens to the MHC class II antigen presentation pathway. Evaluation of trafficking patterns revealed that all four human CD1-derived targeting sequences delivered antigen to the MHC class II antigen presentation pathway, to early/recycling, early/sorting and late endosomes/lysosomes. There was a preferential requirement for different CD1 targeting sequences for the optimal presentation of an MHC class II epitope in the following hierarchy: CD1b > CD1d = CD1c > > > CD1a or untargeted antigen. Therefore, the substitution of the CD1 ectodomain with heterologous proteins results in their traffic to distinct intracellular locations that intersect with MHC class II and this differential distribution leads to specific functional outcomes with respect to MHC class II antigen presentation. These findings may have implications in designing DNA vaccines, providing a greater variety of tools to generate T-cell responses against microbial pathogens or tumours.

Roles for SR proteins and hnRNP A1 in the regulation of c-src exon N1.

The splicing of the c-src exon N1 is controlled by an intricate combination of positive and negative RNA elements. Most previous work on these sequences focused on intronic elements found upstream and downstream of exon N1. However, it was demonstrated that the 5' half of the N1 exon itself acts as a splicing enhancer in vivo. Here we examine the function of this regulatory element in vitro. We show that a mutation in this sequence decreases splicing of the N1 exon in vitro. Proteins binding to this element were identified as hnRNP A1, hnRNP H, hnRNP F, and SF2/ASF by site-specific cross-linking and immunoprecipitation. The binding of these proteins to the RNA was eliminated by a mutation in the exonic element. The activities of hnRNP A1 and SF2/ASF on N1 splicing were examined by adding purified protein to in vitro splicing reactions. SF2/ASF and another SR protein, SC35, are both able to stimulate splicing of c-src pre-mRNA. However, splicing activation by SF2/ASF is dependent on the N1 exon enhancer element whereas activation by SC35 is not. In contrast to SF2/ASF and in agreement with other systems, hnRNP A1 repressed c-src splicing in vitro. The negative activity of hnRNP A1 on splicing was compared with that of PTB, a protein previously demonstrated to repress splicing in this system. Both proteins repress exon N1 splicing, and both counteract the enhancing activity of the SR proteins. Removal of the PTB binding sites upstream of N1 prevents PTB-mediated repression but does not affect A1-mediated repression. Thus, hnRNP A1 and PTB use different mechanisms to repress c-src splicing. Our results link the activity of these well-known exonic splicing regulators, SF2/ASF and hnRNP A1, to the splicing of an exon primarily controlled by intronic factors.