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Utility of the housekeeping genes 18S rRNA, β-actin, and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) for normalisation in real-time quantitative RT-PCR analysis of gene expression in human T lymphocytes
Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
2004 (English)In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 59, no 6, 566-573 p.Article in journal (Refereed) Published
Abstract [en]

The accuracy of 18S rRNA, β-actin mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as indicators of cell number when used for normalization in gene expression analysis of T lymphocytes at different activation stages was investigated. Quantitative real-time reverse transcriptase-polymerase chain reaction was used to determine the expression level of 18S rRNA, β-actin mRNA, GAPDH mRNA and mRNA for six cytokines in carefully counted samples of resting human peripheral blood mononuclear cells (PBMCs), intestinal lymphocytes and PBMCs subjected to polyclonal T-cell activation. The 18S rRNA level in activated and resting PBMCs and intestinal lymphocytes was essentially the same, while the levels of β-actin and GAPDH mRNAs fluctuated markedly upon activation. When isolated γδTCR+, CD4+ and CD8+ subpopulations were studied, 18S rRNA levels remained unchanged after 21 h of activation but increased slightly after 96 h. In contrast, there was a 30–70-fold increase of GAPDH mRNA/cell in these cell populations upon activation. Cytokine analysis revealed that only normalization to 18S rRNA gave a result that satisfactorily reflected their mRNA expression levels per cell. In conclusion, 18S rRNA was the most stable housekeeping gene and hence superior for normalization in comparative analyses of mRNA expression levels in human T lymphocytes.

Place, publisher, year, edition, pages
John Wiley & Sons, 2004. Vol. 59, no 6, 566-573 p.
National Category
Immunology
Identifiers
URN: urn:nbn:se:umu:diva-3295DOI: 10.1111/j.0300-9475.2004.01440.xOAI: oai:DiVA.org:umu-3295DiVA: diva2:141838
Available from: 2003-12-18 Created: 2003-12-18 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Extrathymic T cell receptor gene rearrangement in human alimentary tract
Open this publication in new window or tab >>Extrathymic T cell receptor gene rearrangement in human alimentary tract
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

T lymphocytes regulate the initiation, duration, and magnitude of adaptive immune responses and function as effector cells in cell mediated immunity. To become immunologically competent they must generate functional antigen receptors. This process takes place in the thymus and requires somatic recombination of T cell receptor (TCR) genes. It is mediated by the endonucleases recombination activating gene-1 (RAG1) and RAG2. Although the thymus regresses at puberty, T cells are present throughout life implying that other tissues must provide the proper milieu for T cell development. This thesis describes extrathymic T cell maturation in man. RAG1, RAG2, and the preTα-chain (pTα), which is exclusively utilized in developing T cells, were used as markers for TCR gene rearrangement. Two new exons (1A and 1B) encoding sequences in the 5’ untranslated region (5’UTR) of mRNA were discovered in the human RAG1 gene. The previously described 5’UTR exon (renamed 1C) was located between the new exons and exon 2, the latter containing the entire coding sequence. We found that small intestinal lymphocytes of the T cell lineage expressed the new exons in three different splice forms. RAG1 mRNA containing the 1C exon was not expressed in small intestinal lymphocytes. In contrast, splice forms containing the 1A exon were not expressed in thymocytes. RAG1 and pTα mRNA expressing lymphocytes were seen both within the epithelium and in lamina propria. Thymocyte-like CD2+CD7+CD3-, CD4+CD8+, CD1a+, and IL7-R+ lymphocytes were identified in the small intestinal mucosa. CD2+CD7+CD3- cells had the highest expression levels of mRNA for RAG1 and pTα, suggesting that the small intestinal mucosa is indeed a site for T cell maturation. Small intestinal T lymphocytes were also shown to kill via the Fas/FasL pathway in a TCR/CD3 independent manner and via the perforin/granzyme pathway in a TCR/CD3 dependent manner. The Fas/FasL-mediated cytotoxicity may reflect an ongoing selection process of extrathymically maturated T cells.

The nasopharyngeal tonsil is the major inductive site for immune reactions against inhaled antigens. Previous demonstration of RAG1 expression in tonsillar B cells was interpreted as antigen driven receptor revision. The present study confirms the expression of RAG1 in B cells. We also found that RAG1, RAG2, and pTa mRNAs were expressed in lymphocytes of the T cell lineage. A small population of cells with the immature phenotype CD2+CD7+CD3- was demonstrated. This population had the highest expression levels of mRNA for RAG1, RAG2, pTα and terminal deoxynucleotidyl transferase. All four splice-forms of RAG1 mRNA were expressed. RAG1 and pTα mRNA expressing cells were mainly located in the proximity of the surface epithelium and in the outer rim of the follicles. These results suggest that the nasopharyngeal tonsil is a site where extrathymic T cell development and antigen driven TCR revision are occurring in parallel.

Celiac disease (CD) is a small intestinal enteropathy characterized by permanent intolerance to gluten. Gluten reactive intestinal T cells are central in the pathogenesis and CD can be regarded as a failure to maintain tolerance to this food antigen. Expression of the RAG1 1A/2 splice form was significantly decreased in small intestinal T cell subsets of CD patients suggesting that impaired TCR gene rearrangement could contribute to failure of maintain tolerance in CD.

Together, these findings show that both small intestinal and nasopharyngeal tonsillar lymphocytes of T cell lineage have the molecular machinery for antigen receptor rearrangement and that thymocyte-like lymphocytes are present in both tissues. Thus these organs are likely sites of T lymphocyte ontogeny as well as for secondary T cell receptor rearrangement in man.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2003. 69 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 856
Keyword
Immunology, T cell maturation, recombination activating gene, preTα, human intestinal mucosa, intraepithelial lymphocytes, lamina propria lymphocytes, nasopharyngeal tonsil, Immunologi
National Category
Immunology in the medical area
Research subject
Immunology
Identifiers
urn:nbn:se:umu:diva-169 (URN)91-7305-524-7 (ISBN)
Public defence
2004-01-16, E04, 6E, Umeå Universitet, Umeå, 09:00
Opponent
Supervisors
Available from: 2003-12-18 Created: 2003-12-18 Last updated: 2013-03-25Bibliographically approved

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Bas, AnnaForsberg, GöteHammarström, StenHammarström, Marie-Louise
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