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Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines
Published on: November 8, 2011
A new lymphoblastoid cell line defective in class II HLA expression
1Laboratory of Immunogenetics, Polish Academy of Sciences, Wroclaw, Poland.
Insights
A new cell line, HAJ, derived from a patient with kidney disease, shows no surface expression of human leukocyte antigen (HLA) class II molecules. This defect appears to be at the gene transcription level, offering a model for studying HLA class II regulation.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Human Leukocyte Antigen (HLA) class II molecules are crucial for immune responses.
- Defects in HLA class II expression can lead to immune dysregulation.
- Understanding the regulation of HLA class II expression is vital for immunology research.
Purpose of the Study:
- To characterize the expression of HLA class I and class II molecules in a novel lymphoblastoid cell line (HAJ).
- To investigate the molecular basis for any observed differences in HLA expression compared to a control cell line (PAJ).
- To establish the HAJ cell line as a model for studying HLA class II gene regulation.
Main Methods:
- Generation of lymphoblastoid cell lines (HAJ and PAJ) via Epstein-Barr virus transformation of peripheral blood lymphocytes.
- Flow cytofluorimetry to assess cell surface expression of HLA class I and class II antigens.
- Polymerase Chain Reaction (PCR) and Reverse Transcriptase PCR (RT-PCR) to detect HLA gene presence and transcripts.
Main Results:
- HAJ cells exhibited comparable levels of HLA class I antigens to PAJ cells.
- Class II HLA antigens were absent on the surface of HAJ cells, unlike abundant expression on PAJ cells.
- PCR confirmed the presence of HLA-DR beta, DQ alpha, and DP alpha genes in HAJ, but RT-PCR showed no detectable transcripts for DQA1 and DPA1.
Conclusions:
- The HAJ cell line demonstrates a defect in the transcription of genes encoding all class II HLA antigens.
- This deficiency in HLA class II expression is likely due to a transcriptional regulatory issue.
- The HAJ cell line serves as a valuable in vitro model for investigating the molecular mechanisms governing HLA class II gene expression.
Abstract:
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.
