Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2001 Jun 5;98(12):6824-9.
doi: 10.1073/pnas.121179998. Epub 2001 May 29.

Maintenance of TCR clonality in T cells expressing genes for two TCR heterodimers

D B Sant'Angelo  1 , P Cresswell, C A Janeway Jr, L K Denzin
Affiliations

Maintenance of TCR clonality in T cells expressing genes for two TCR heterodimers

D B Sant'Angelo et al. Proc Natl Acad Sci U S A. .

Abstract

T cell receptor (TCR) allelic exclusion is believed to be primarily mediated by suppression of further recombination at the TCR locus after the expression of a functional TCR protein. Genetic allelic exclusion has been shown to be leaky for the beta chain and, more commonly, for the alpha chain. Here, we demonstrate an additional mechanism by which T cells can maintain monoclonality. T cells from double TCR transgenic mice express only one or the other of the two available TCRs at the cell surface. This "functional allelic exclusion" is apparently due to control of the TCR assembly process because these T cells express RNA and protein for all four transgenic TCR proteins. Lack of cell surface expression of the second TCR may be controlled by a failure to assemble the TCR heterodimer.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Analysis of CD4+ T cells and thymocytes from AND, MBP, or AND/MBP double transgenic mice. FACS of lymphocytes with antibodies against (A) CD4, Vβ3, and Vβ8, (B) CD4, Vβ3, and Vα11, or (C) CD4, Vβ3, and 19G (clonotypic for the MBP TCR). Genotype of T cells is listed at the top (AND, AND transgenic mouse; MBP, MBP transgenic mouse; AND/MBP, double transgenic mouse). Plots are labeled with the specificity of the antibody. Only CD4+ T cells are shown. (D) Thymocytes were stained as in A. Quadrants in D separate TCRhi from TCRlo cells. The numbers are the percentage of cells within each quadrant. (E and F) Proliferation of CD4+ T cells from double TCR transgenic mice. Cells were sorted based on Vβ3 or Vβ8 expression. Sorted cells were incubated with (E) B10.BR (I-Ek) splenocytes + titrated MCC peptide or (F) B10.PL (I-Au) splenocytes + titrated Ac1–16 peptide, pulsed with 1 μCi of [3H]thymidine at 48 h and harvested at 72 h. Controls with splenocytes, but no added peptide, gave a low background cpm that was subtracted from each data point. Assays were done in triplicate and represent four different experiments.
Figure 2
Figure 2
Phenotypic and functional allelic exclusion in double TCR transgenic mice. (A) CD4+ Lymphocytes and (B) thymocytes from mice transgenic for both the D10 and AND TCR stained with antibodies against CD4, Vβ3, and Vβ8 (Left) or CD4, Vα2, and Vα11 (Right). (C) Lymphocytes were sorted based on Vβ expression and then stimulated with B10.BR splenocytes plus either 1 μg/ml MCC peptide recognized by the AND TCR or 1 μg/ml CA-wt peptide recognized by the D10 TCR. (D, Left) CD4+ cells from double transgenic D10/MBP mice stained with antibodies against CD4, Vα2 (to detect the D10 TCR), and 19G (clonotypic for the MBP TCR). T cells express only the MBP TCR (R1), only the D10 TCR (R3), or the D10 TCR plus reduced levels of the MBP TCR (R2). Most thymocytes from D10/MBP mice (D, Right) express both the D10 TCR and lower level of the MBP TCR. (E) T cells were sorted as shown in the left panel of D and then incubated with B10.BR splenocytes plus 1 μg/ml CA-wt peptide or B10.PL splenocytes plus 1 μg/ml Ac1–16 peptide. Assays were done in triplicate and represent three different experiments.
Figure 3
Figure 3
RNA expression in CD4+ T cells sorted based on cell surface TCR expression. PCR of cDNA from single TCR transgenic mice and from sorted lymphocytes from doubly transgenic mice. (A) AND mice express Vα11 and Vβ3 mRNA, and MBP mice express Vα4 and Vβ8.2 mRNA, whereas T cells from AND/MBP mice sorted for cell surface expression of Vβ3 (AND) or Vβ8 (MBP) express mRNA from both transgenic TCR. (B) Both sorted Vβ3+ (AND) and Vβ8.2+ (MBP) T cells from double transgenic AND/D10 mice express mRNA from both TCR transgenes. Only Vα11 and Vβ3 cDNA are amplified from AND mice, and only Vα2 and Vβ8 cDNA from D10 mice are amplified. (C) All four TCR transcripts are amplified from D10/MBP T cells sorted as shown in Fig. 2D. Different Jβ usage in D10 and MBP allowed for each transcript to be separately amplified (as demonstrated in A and B), although both TCRs use the same Vβ8.2 gene segment. RNA was extracted and reverse transcribed, and the cDNA for the TCR α and β chains was amplified by PCR using primers specific for the Vα-Jα and Vβ-Jβ joins. The AND α chain was amplified using primers specific for Vα11 and Cα. Potential contamination of the cDNA samples with genomic DNA was evaluated by the use of primers specific for the second intron of the MHC class II Eb gene (42). Using identical conditions for PCR to those used to amplify the cDNA, no PCR product could be detected on ethidium bromide-stained agarose gels (data not shown).
Figure 4
Figure 4
Intracellular staining of Vβ3 in Vβ8+ T cells from AND/MBP mice. FACS analysis of lymphocytes and thymocytes harvested from either double transgenic AND/MBP mice or single transgenic MBP mice that were stained with antibodies against CD4, Vβ3, or Vβ8. The histograms show the level of cell surface Vβ3 (thin line) and intracellular Vβ3 staining (thick line) on electronically gated CD4+, Vβ8+ T cells or on total thymocytes. Vβ3 staining of Vβ8+ (A) T cells and (C) thymocytes from AND/MBP mice can only be seen if cells are permeabilized before the addition of the Vβ3 antibody. The specificity of the intracellular staining of the AND TCRβ chain is confirmed by carrying out the same staining procedure on (B) T cells and (D) thymocytes from MBP TCR-only transgenic mice. Sorted T cells from AND (E), MBP (F), and Vβ8+ T cells from AND/MBP (G) transgenic mice were pulse labeled for 30 min with [35S]methionine. The TCR α and β proteins were immunoprecipitated from Triton X-100 solubilized lysates with anti-Vβ8 (mAb F23.2), anti-Vβ3 (mAb KJ25), anti-Vα11 (mAb RR8–1), anti-Cβ (mAb H57), or control anti-HLA-DM (mAb MaP.DMB/c) and analyzed by SDS/PAGE (11%). The positions of the individual TCR β and α chains are indicated.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Malissen M, Trucy J, Jouvin-Marche E, Cazenave P A, Scollay R, Malissen B. Immunol Today. 1992;13:315–322. - PubMed
    1. Von Boehmer H. Ann NY Acad Sci. 1995;766:52–61. - PubMed
    1. Mallick C A, Dudley E C, Viney J L, Owen M J, Hayday A C. Cell. 1993;73:513–519. - PubMed
    1. Dudley E C, Girardi M, Owen M J, Hayday A C. Curr Biol. 1995;5:659–669. - PubMed
    1. Petrie H T, Livak F, Burtrum D, Mazel S. J Exp Med. 1995;182:121–127. - PMC - PubMed

MeSH terms

Substances

LinkOut - more resources