Limited heterogeneity of T-cell receptor Vβ gene expression in the early stage of insulitis in NOD mice

ABSTRACTRationale: evaluation of the T-cell receptor (TCR) Vβ-chain repertoire by PCR-based CDR3 length analysis allows fine resolution of the usage of the TCR Vβ repertoire and is a sensitive tool to monitor changes in the T-cell compartment. A multiplex PCR method employing 24 labeled upstream Vβ primers instead of the conventionally labeled downstream Cβ primer is described. Method: RNA was isolated from purified CD4 and CD8 T-cell subsets from umbilical cord blood and clinical samples using TRI reagent followed by reverse transcription using a Cβ primer and an Omniscript RT kit. The 24 Vβ primers were multiplexed based on compatibility and product sizes into seven reactions. cDNA was amplified using 24 Vβ primers (labeled with tetrachloro-6-cardoxyfluorescein, 6-carboxyfluorescein, and hexachloro-6-carboxyfluorescein), an unlabeled Cβ primer, and Taqgold polymerase. The fluorescent PCR products were resolved on an automated DNA sequencer and analyzed using the Genotyper 2.1 software. Results: Vβ spectratypes of excellent resolution were obtained with RNA amounts of 250 ng using the labeled Vβ primers. The resolution was superior to that obtained with the labeled Cβ primer assay. Also the numbers of PCRs were reduced to 7 from the 12 required in the Cβ labeling method, and the sample processing time was reduced by half. Conclusion: The method described for T-cell receptor Vβ-chain repertoire analysis eliminates tedious dilutions and results in superior resolution with small amounts of RNA. The fast throughput makes this method suitable for automation and offers the feasibility to perform TCR Vβ repertoire analyses in clinical trials.

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The ht beta gene encodes a novel CACCC box-binding protein that regulates T-cell receptor gene expression

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5691-5701.1993 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5691-5701

Author(s):

Y Wang ◽

J A Kobori ◽

L Hood

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Binding Protein ◽

Cell Receptor ◽

Regulatory Function ◽

Human T Cell ◽

Alpha Gene ◽

Beta Gene

A gene encoding a novel CACCC box-binding protein that binds to the promoter region of the human T-cell receptor (TCR) V beta 8.1 gene and the mouse TCR alpha gene silencer has been cloned. This gene, termed ht beta, contains four zinc fingers of the class Cys2-X12-His2 that may be responsible for DNA binding and a highly negatively charged region that defines a putative transcriptional activation domain. Analysis of the expression of ht beta mRNA revealed similar expression levels and patterns in various cell lines. The bacterially expressed ht beta protein can bind to the CACCC box in both the human TCR V beta 8.1 gene promoter and the mouse TCR alpha gene silencer. The CACCC box is essential for efficient transcription of the V beta 8.1 promoter. Cotransfection with an ht beta expression plasmid and a reporter vector indicated that ht beta can activate human TCR V beta 8.1 gene transcription. ht beta also is able to counteract the silencing effect of the mouse TCR alpha gene silencer. The CACCC box has been found in almost all V beta 8.1 gene subfamily members and in both TCR alpha and beta gene enhancers in humans and mice. These results suggest that the CACCC box-binding protein may have an important regulatory function for TCR gene expression in alpha beta T cells versus gamma delta T cells.

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