SummaryThe limited access to the nuclear compartment may constitute one of the major barriers after bacteriamediated expression plasmid DNA delivery to eukaryotic cells. Alternatively, a self-destructing Listeria monocytogenes strain was used to release translation-competent mRNA directly into the cytosol of epithelial cells, macrophages and human dendritic cells. Enhanced green fluorescent protein (EGFP)-encoding mRNA, adapted for translation in mammalian cells by linking an IRES element to the 5 ¢ ¢ ¢ ¢ -end of the egfp coding sequence, was produced by T7 RNA polymerase in the carrier bacteria upon entry into the cytosol where the mRNA is efficiently released from the lysed bacteria and immediately translated in eukaryotic host cells. Besides the much earlier expression of EGFP being detectable already 4 h after infection, the number of EGFP expressing mammalian cells obtained with this novel RNA delivery technique is comparable to or -especially in phagocytic cellseven higher than that obtained with the expression plasmid DNA delivery strategy. Accordingly, bacteriamediated delivery of ovalbumin-encoding mRNA to macrophages resulted in efficient antigen processing and presentation in vitro indicating that this approach may also be adapted for the in vivo delivery of antigen-encoding mRNA leading to a more efficient immune response when applied to vaccine development.
MHC molecules present peptides in their binding groove to T-cell receptors inducing proliferation or cytotoxicity of alloreactive T cells. A previously generated human monoclonal antibody (mAb) UL-5 A1, recognizing a conformational epitope formed by HLA DR1/DRB1*0101 molecules and HLA-A2 derived peptides, demonstrates T-cell-like recognition of the peptide/MHC complex (PMC). To study the genes of the antigen binding region, the nucleotide sequences of the rearranged genes in the variable regions of UL-5 A1 were determined and the V-gene usage (VH3, V lambda 2) was identified by comparison with published germlines. The genes encoding heavy (Fd) and light (L) chains of UL-5 A1 were linked and expressed in a bacterial system. Specificity of the recombinant Fab-5 A1 was determined with HLA-typed LCLs by flow cytometric analysis. As demonstrated in competitive inhibition assays, UL-5 A1 and Fab-5 A1 recognize the same PMC epitope on HLA-A2+, -DR1/DRB1*0101+ typed LCLs. Additionally, mAb UL-5 A1 and Fab-5 A1 both recognize HLA-A2-, -DR1/DRB1*0101+ LCLs exogenously loaded with HLA-A2 peptides (105-117, 103-117). UL-5 A1-like antibodies against peptide/MHC complexes could prove valuable tools for research on T-cell recognition and MHC function.
MHC molecules present peptides in their binding groove to T-cell receptors inducing proliferation or cytotoxicity of alloreactive T cells. A previously generated human monoclonal antibody (mAb) UL-5 Al, recognizing a conformationa l epitope formed by HLA DRl/DRBl *0101 molecules and HLA-A2 derived peptides, demonstrates Tcell-like recognition of the peptide/MHC complex (PMC). To stud y the genes of the antigen binding region, the nucleotide sequences of the rearranged genes in the variable regions of UL-5 Al were determined and the Yge.ne usage (VH3, YA2) was identified by comparison With publi hed germlines. The genes encodi ng heavy (Fd) and light (L) chains of UL-5 A 1 were linked and expressed in a bacterial system. Specificity of the reco mbinant Fab-5 A 1 was determined with HLA -typed LCLs by ~ow cytometric analysis. As demonstrated in competitive 1111 'b' . 1 • 1t1on as ays, UL-5 Al and Fa b-5 A 1 recogni ze the sa me PM epitope on HLA-A2+, -DR 1/DRBl *0101 + typed L Ls. Additionally, mAb UL-5 Al and Fab-5 Al both recognize HLA-Ar, -DRl/DRBl *OlOr LCLs exogenously loaded with HLA-A2 peptides (105-11 7, 103-11 7). UL-5 Al -like antibodies again t peptide/MH complexes could prove va luable tools for research on Tcell recognition and MH fun ction.
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