Studies on binding of HIV-1 p24gag peptide to HLA-Cw3+ cells.

Human major histocompatibility complex class I antigens, HLA-C, are expressed on the cell surface at approximately a tenfold lower level than HLA-A and -B. We hypothesized that the expression of HLA-C is limited by the quantity of high affinity peptides which bind to these molecules, thus allowing only a small fraction of HLA-C molecules to be transported and/or to remain stable on the cell surface. If this assumption is correct, then the addition of exogenous peptide should increase cell surface HLA-C expression. To verify the hypothesis, we pulsed lymphoblastoid cell line PAJ (HLA-Cw3+) with synthetic HIV-1 p24gag 145-152 peptide, known to be presented to T-lymphocytes by HLA-Cw3 molecule. PAJ (HLA-Cw3+) cells bound approximately two times more of the peptide than HAJ (HLA-Cw3-), and four times more than 500/C9 (HLA-Cw3-) cells. Accordingly, overnight pulsing of PAJ cells with the p24gag 145-152 peptide caused an increase in class I HLA expression detected on the cell surface by flow cytofluorimetric analysis with anti-HLA-B,C monoclonal antibodies but not by anti-HLA-A antibody. In contrast, HLA-Cw3- cells treated in the same manner did not show any increase of HLA class I expression. Our data suggest that low concentration of high affinity peptides within the cell may be one of the factors limiting cell surface expression of HLA-C molecules.

Human in vitro cell lines verification by minisatellite DNA restriction fragment length polymorphism.

Four families of human in vitro cell lines were tested for minisatellite restriction fragment length polymorphism (RFLP) using multilocus probes MZ1.3 and/or 33.15 after digestion of DNA with restriction enzymes HinfI or HaeIII. These results confirmed that (i) the RFLP pattern is relatively stable in established cell lines and, therefore, could be used as a specific marker of a cell line identity, (ii) the use of MZ1.3 and 33.15 probes permits the identification of hybridomas and (iii) one of the cell lines tested, a lymphoblastoid cell line HAJ, may possess a hot spot of mutation.

A new lymphoblastoid cell line defective in class II HLA expression.

A lymphoblastoid cell line, HAJ, was derived by in vitro transformation with Epstein-Barr virus of peripheral blood lymphocytes (PBL) from a patient with renal insufficiency awaiting kidney graft. Cell surface expression of class I and class II HLA molecules was determined by flow cytofluorimetry using monoclonal antibodies and compared with that of cell line PAJ similarly derived from a healthy donor. HAJ cells expressed class I antigens at levels comparable with PAJ cells. In contrast, class II antigens were absent from the cell surface of HAJ cells while they were abundant on PAJ cells. Permeabilization and fixation of cells with acetone/formaldehyde solution revealed intracellular Ki-67 antigen but not class II HLA molecules. The genes for HLA-DR beta, DQ alpha and DP alpha were present in the HAJ genome as detected with polymerase chain reaction (PCR) using locus-specific primers amplifying a second exon. In RT (reverse transcriptase)-PCR, transcripts of DQA1 and DPA1 genes were easily detectable in PAJ (positive control) but not in HAJ cells. These results suggest a defect in HAJ cells of transcription of genes for all class II antigens. The cell line HAJ may prove to be an interesting model for in vitro studies of molecular mechanisms of the regulation of class II expression.