Development of a mouse model for studying in vivo T-cell receptor mutations

Umeki, Shigeko; Suzuki, Takehiko; Kusunoki, Yoichiro; Seyama, Toshio; Fujita, Shoichiro; Kyoizumi, Seishi

doi:10.1016/s1383-5718(97)00084-3

Cited by 18 publications

(22 citation statements)

References 34 publications

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“…Even for the measurement of the frequency of mutants in a population of human cells-i.e., to measure f, as distinct from lonly a few model genes have been used: hprt (25, 26), glycophorin A (GPA; refs 27, 28), HLA (29-31), CD3 (32), and h globin (33). This list of genes is extremely limited because for any potential ''sentinel gene'', a wide range of mutants must be viable, with a readily detectable phenotype, which can result from a single inactivating mutation.…”

Section: Discussionmentioning

confidence: 99%

A Quantitative Measurement of the Human Somatic Mutation Rate

Araten

Golde²,

Zhang³

et al. 2005

Cancer Research

144

133

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The mutation rate (m) is a key biological feature of somatic cells that determines risk for malignant transformation, and it has been exceedingly difficult to measure in human cells. For this purpose, a potential sentinel is the X-linked PIG-A gene, because its inactivation causes lack of glycosylphosphatidylinositol-linked membrane proteins. We previously found that the frequency ( f ) of PIG-A mutant cells can be measured accurately by flow cytometry, even when f is very low. Here we measure both f and m by culturing B-lymphoblastoid cell lines and first eliminating preexisting PIG-A mutants by flow sorting. After expansion in culture, the frequency of new mutants is determined by flow cytometry using antibodies specific for glycosylphosphatidylinositol-linked proteins (e.g., CD48, CD55, and CD59). The mutation rate is then calculated by the formula m = f/d, where d is the number of cell divisions occurring in culture. The mean m in cells from normal donors was 10.6 Â 10 À7 mutations per cell division (range 2.4 to 29.6 Â 10 À7 ). The mean m was elevated >30-fold in cells from patients with Fanconi anemia (P < 0.0001), and m varied widely in ataxia-telangiectasia with a mean 4-fold elevation (P = 0.002). In contrast, m was not significantly different from normal in cells from patients with Nijmegen breakage syndrome. Differences in m could not be attributed to variations in plating efficiency. The mutation rate in man can now be measured routinely in B-lymphoblastoid cell lines, and it is elevated in cancer predisposition syndromes. This system should be useful in evaluating cancer risk and in the design of preventive strategies. (Cancer Res 2005; 65(18): 8111-7)

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Section: Discussionmentioning

confidence: 99%

A Quantitative Measurement of the Human Somatic Mutation Rate

Araten

Golde²,

Zhang³

et al. 2005

Cancer Research

144

133

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“…Alternatively, B6NZB mice may be more resistant to radiation-induced cell damage and mutations. Such strain differences have been suggested in rejection of allogeneic marrow transplants (43) and in radiation-induced mutations (19).…”

Section: Discussionmentioning

confidence: 93%

“…The frequencies of TCR-negative CD4 cells gradually decreased over time, possibly as a result of defective proliferation in vivo (19,21,22). The rapid decrease in frequencies of H-2K-deficient mutants suggested a mechanism involving active elimination in vivo.…”

Section: ϫ5mentioning

confidence: 99%

“…Nylon-wool-nonadherent spleen cells (2 ϫ 10 6 ) were stained with 10 l PE-labeled anti-CD3 Ab and 10 l FITC-labeled anti-CD4 Ab and then analyzed by flow cytometry as described previously (19). In brief, fluorescence data from a minimum of 2 ϫ 10 5 lymphocyte-gated events were acquired, and a window for normal T cells was set as a rectangle with the width extending from one-half of the modal FL-1 value through twice the modal value.…”

Section: Enumeration Of Tcr-negative Cd4 Cellsmentioning

confidence: 99%

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NK-Mediated Elimination of Mutant Lymphocytes that Have Lost Expression of MHC Class I Molecules

Kusunoki

Kyoizumi

Honma

et al. 2000

The Journal of Immunology

Self Cite

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Mutant cells generated in vivo can be eliminated when mutated gene products are presented as altered MHC/peptide complexes and recognized by T cells. Diminished expression of MHC/peptide complexes enables mutant cells to escape recognition by T cells. In the present study, we tested the hypothesis that mutant lymphocytes lacking expression of MHC class I molecules are eliminated by autologous NK cells. In H-2b/k F1 mice, the frequency of H-2Kb-negative T cells was higher than that of H-2Kk-negative T cells. The frequency of H-2K-deficient T cells increased transiently after total body irradiation. During recovery from irradiation, H-2Kk-negative T cells disappeared more rapidly than H-2Kb-negative T cells. The disappearance of H-2K-deficient T cells was inhibited by administration of Ab against asialo-GM1. H-2Kk-negative T cells showed higher sensitivity to autologous NK cells in vitro than H-2Kb/k heterozygous or H-2Kb-negative T cells. Adding syngeneic NK cells to in vitro cultures prevented emergence of mutant cells lacking H-2Kk expression but had little effect on the emergence of mutant cells lacking H-2Kb expression. Results in the H-2b/k F1 strain correspond with the sensitivity of parental H-2-homozygous cells in models of marrow graft rejection. In H-2b/d F1 mice, there was no significant difference between the frequencies of H-2Kb-negative and H-2Kd-negative T cells, although the frequencies of mutant cells were different after radiation exposure among the strains examined. H-2b/d F1 mice also showed rapid disappearance of the mutant T cells after irradiation, and administration of Ab against asialo-GM1 inhibited the disappearance of H-2K-deficient T cells in H-2b/d F1 mice. Our results provide direct evidence that autologous NK cells eliminate mutant cell populations that have lost expression of self-MHC class I molecules.

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“…Using mouse models, the in vivo kinetics and dose-response of radiation-induced TCR mutants have been observed Umeki et al, 1997). In this system, expression of TCR mutant could be observed 3 days after whole-body irradiation, and it reached the maximum level between 2-3 weeks and then gradually decreased.…”

Section: Introductionmentioning

confidence: 99%

Measurement of Mutant Frequency in T-Cell Receptor (Tcr) Gene by Flow Cytometry After X-Irradiation on El-4 Mice Lymphoma Cells

et al. 2007

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-It is well known that somatic mutations are induced by ionizing irradiation. We have previously reported the measurement of mutant frequency (MF) on the T-cell receptor (TCR) gene in mouse T-lymphocytes after irradiation by flow cytomery. In this study, we developed an in vitro system using murine EL-4 lymphoma cells and observed frequency of cells defective in TCR gene expression after exposure to ionizing irradiation. EL-4 cells were stained with fluorescein-labeled anti-CD4 and phycoerythrin-labeled anti-CD3 antibodies. They were analyzed with a flow cytometer to detect mutant EL-4 cells lacking surface expression of TCR/CD3 complexes which showed CD3− , CD4 + due to a somatic mutation at the TCR genes. Mutant cells could be observed at 2 days after 3 Gy irradiation. MF of EL-4 cells was 6.7 × 10 −4 for 0 Gy and the value increased to the maximum level of 39 × 10 −4 between 4 and 8 days after 3 Gy irradiation and these data were found to be best fitted by a linear-quadratic doseresponse model. After the peak value the TCR MF gradually decreased with a half-life of approximately 3.2 days. We also examined the hprt mutant frequencies at seven days after irradiation and the cytokinesisblocked micronucleus frequency at 20 hrs after irradiation. The frequencies of hprt mutation and micronuclei were found to be best fitted by a linear-quadratic dose-response model and a linear dose-response model, respectively. The method to detect mutation on TCR gene is quick and easy in comparison with other methods and is considered useful for the mutagenicity test.

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Development of a mouse model for studying in vivo T-cell receptor mutations

Cited by 18 publications

References 34 publications

A Quantitative Measurement of the Human Somatic Mutation Rate

A Quantitative Measurement of the Human Somatic Mutation Rate

NK-Mediated Elimination of Mutant Lymphocytes that Have Lost Expression of MHC Class I Molecules

Measurement of Mutant Frequency in T-Cell Receptor (Tcr) Gene by Flow Cytometry After X-Irradiation on El-4 Mice Lymphoma Cells

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