Identification of overrepresented T cell receptor genes in blood and tissue biopsies by PCR-ELISA.
The analysis of T cell receptor variable (TCR V) gene repertoires in blood or tissues may provide important information when studying immunopathological mechanisms. The overexpression of a TCR gene may indicate the expansion of the corresponding T cell subset. In autoimmune diseases, clonally expanded T cell subsets in the affected organs may represent pathogenic lymphocytes. We describe a simple, rapid and sensitive method to determine the TCR AV and BV gene repertoire using a PCR-ELISA method. RNA is extracted from lymphocytes, transcribed to cDNA, which is then used as a template for PCR with 19 different TCR AV gene and 20 BV gene specific primers as the forward primer, and a digoxigenin (DIG) labeled AC/BC primer as the reverse primer. The DIG labeled PCR amplicons are hybridized with a fluorescein isothiocyanate (FITC) labeled TCR C region specific probe. Finally, the amplicons are quantified by ELISA using anti-FITC coated microtiter plates, and an anti-DIG conjugated peroxidase. Although PCR-ELISA cannot accurately quantify the expression level of a given TCR gene, overrepresented TCR V genes are easily identified by comparing the relative expression levels of each individual V gene in the total V gene repertoire. We demonstrate that this technique can be used to determine TCR profiles in blood and tissue samples containing as few as 50,000 T cells. In combination with CDR3 fragment size analysis, this method is an efficient tool to identify clonally expanded T cell subsets in the synovial biopsies of rheumatoid arthritis patients.
VanderBorght
A
A
Department of Autoimmune Disease, Dr. L. Willems-Instituut, Diepenbeek, Belgium.
van der Aa
A
A
Geusens
P
P
Vandevyver
C
C
Raus
J
J
Stinissen
P
P
D001172:Arthritis, Rheumatoid / Q000097:blood / Q000276:immunology / Q000473:pathology*; D001427:Bacterial Toxins; D002999:Clone Cells; D017931:DNA Primers; D004768:Enterotoxins / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D015329:Gene Rearrangement, T-Lymphocyte; D019674:Genes, T-Cell Receptor beta; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008213:Lymphocyte Activation; D018655:Lymphocyte Count; D010835:Phytohemagglutinins / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011948:Receptors, Antigen, T-Cell / Q000097:blood* / Q000235:genetics*; D015203:Reproducibility of Results; D013211:Staphylococcus aureus / Q000276:immunology; D018089:Superantigens; D013582:Synovial Fluid / Q000276:immunology; D013583:Synovial Membrane / Q000276:immunology / Q000473:pathology; D016176:T-Lymphocyte Subsets / Q000378:metabolism / Q000473:pathology
D001427:Bacterial Toxins; D017931:DNA Primers; D004768:Enterotoxins; D010835:Phytohemagglutinins; D011948:Receptors, Antigen, T-Cell; D018089:Superantigens; C030107:enterotoxin F, Staphylococcal