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. 2011 Jul;8(4):333-40.
doi: 10.1038/cmi.2011.14. Epub 2011 May 30.

The development and functions of CD4(+) T cells expressing a transgenic TCR specific for an MHC-I-restricted tumor antigenic epitope

Xue Han  1 , Peiying Ye, Liqun Luo, Linghua Zheng, Yang Liu, Lieping Chen, Shengdian Wang
Affiliations

The development and functions of CD4(+) T cells expressing a transgenic TCR specific for an MHC-I-restricted tumor antigenic epitope

Xue Han et al. Cell Mol Immunol. 2011 Jul.

Abstract

It has been reported that the ratio of CD4(+) to CD8(+) T cells has no bias in a few class I major histocompatibility complex (MHC-I)-restricted T-cell receptor (TCR)-transgenic mice specific for alloantigens or autoantigens, in which most CD4(+) T cells express an MHC-I-restricted TCR. In this study, we further showed that more than 50% of CD4(+) T cells in MHC-I-restricted P1A tumor antigen-specific TCR (P1ATCR)-transgenic mice could specifically bind to MHC-I/P1A peptide complex. P1A peptide could stimulate the transgenic CD4(+) T cells to proliferate and secrete both type 1 helper T cell and type 2 helper T cell cytokines. The activated CD4(+) T cells also showed cytotoxicity against P1A-expressing tumor cells. The analysis of TCR α-chains showed that these CD4(+) T cells were selected by co-expressing endogenous TCRs. Our results show that CD4(+) T cells from P1ATCR transgenic mice co-expressed an MHC-I-restricted transgenic TCR and another rearranged endogenous TCRs, both of which were functional.

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Figures

Figure 1
Figure 1
Detection of MHC-I-restricted TgTCR on CD4+ T cells in P1ATCR-transgenic mice. (a, b) Lymph node cells (1×106) from P1ATCR-transgenic or non-transgenic DBA/2 mice were stained with the indicated amounts of Ld/P1A (open) or control Ld/HBV (filled) dimer, anti-CD3, anti-CD4 and anti-CD8 mAb simultaneously. (a) Binding of Ld/P1A dimer to CD4+ and CD8+ T cells was analyzed; (b) 1 µg of Ld/P1A (open) or control Ld/HBV (filled) dimer was incubated with anti-H-2Ld before staining. The values are the percentages of Ld/P1A dimer-positive cells. (c) Expression of TCR Vα8 was detected by RT-PCR in Ld/P1A+, Ld/P1A− P1ATCR-transgenic CD4+ T cells and non-transgenic CD4+ T cells. GAPDH were used as an internal reference. (d) Expression levels of TCR Vα8 transgene on Ld/P1A dimer-positive (open) and dimer-negative T cells (filled, grey) were detected by anti-TCR Vα8 antibody. Rat IgG was used for the isotype control (filled, black). The results are representative of at least three independent experiments. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IgG, immunoglobulin G; mAb, monoclonal antibody; MHC, major histocompatibility complex; P1ATCR, P1A tumor antigen-specific T-cell receptor; TgTCR, transgenic T-cell receptor; TCR, T-cell receptor.
Figure 2
Figure 2
P1A peptide stimulates the proliferation of P1ATCR-transgenic CD4+ T cells. Lymph node and spleen cells from P1ATCR-transgenic DBA/2 mice were labeled with CFSE and stimulated with the indicated concentrations of P1A peptide for 3 days. (a) Proliferation, evaluated using CFSE, was analyzed for gated CD8+ (left) and CD4+ (right) T cells. (b) CD8-depleted lymph node and spleen cells were stimulated with the indicated concentrations of P1A peptide, and the proliferation of CD4+ T cells was analyzed. The numbers above the gating bars depict the percentages of proliferated cells. The results are representative of at least three independent experiments. CFSE, carboxyfluorescein succinimidyl ester; P1ATCR, P1A tumor antigen-specific T-cell receptor.
Figure 3
Figure 3
Cytokine production and P1A-specific cytotoxicity of P1ATCR-transgenic CD4+ and CD8+ T cells stimulated with P1A peptide. CD8- or CD4-depleted splenocytes from P1ATCR-transgenic DBA/2 mice were stimulated with the indicated concentrations of P1A peptide for 3 days. (a) Cytokines in the supernatants were detected using Cytometric Bead Array Kits. The values in the graphs indicate the mean±s.e.m. values of triplicates. (b) The expression of IFN-γ by CD8+ or CD4+ T cells was analyzed by intracellular staining after stimulation with the indicated concentrations of P1A peptide for 2 days. The percentages of IFN-γ+ cells among the gated CD4+ and CD8+ T cells are shown. (c) Activated CD8+ or CD4+ splenocytes were incubated at the indicated E/T ratios with CFSE-labeled specific target P815 cells (P1A+ and H-2Ld+) and nonspecific target CT26 cells (P1A− and H-2Ld+). After 16 h, the CFSE profiles were analyzed in 7-AAD−CFSE+ cells to determine the level of cytotoxicity. The numbers above the gating bars indicate the percentages of CT26 cells (left) and P815 cells (right) among the total 7-AAD−CFSE+ cells in each panel. The results are representative of at least three independent experiments.
Figure 4
Figure 4
Analysis of endogenous TCRs in P1ATCR-transgenic CD4+ T cells. (a) RT-PCR was performed with 1×106 sorted Ld/P1A+ and Ld/P1A−CD4+ P1ATCR-transgenic T cells and non-transgenic CD4+ T cells using a conserved Cα-specific primer paired with Vα1-, 2-, 3-, 4-, 5- and 8-specific primers. PCR products were electrophoresed on a 1.2% agarose gel. (b) CD4+ T cells were stained with anti-TCR Vα2 and Vα8.3 mAbs (left) or anti-TCR Vα11.1/11.2 and Vα8.3 mAbs (right). (c) The frequencies of Vα2+CD4+ T cells among the total CD4+ T cell population of PBMCs from transgenic and non-transgenic DBA/2 mice. The statistical results show the mean±s.e.m. values of five mice for each group. The results are representative of at least three independent experiments. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mAb, monoclonal antibody; P1ATCR, P1A tumor antigen-specific T-cell receptor; PBMC, peripheral blood mononuclear cell; RT-PCR, reverse transcription polymerase chain reaction; TCR, T-cell receptor.
Figure 5
Figure 5
Endogenous TCRs on P1ATCR-transgenic CD4+ T cells are functional. Lymph node cells from P1ATCR-transgenic and non-transgenic DBA/2 mice were labeled with CFSE and cocultured with irradiated CD11c+ splenocytes isolated from C57BL/6 mice at indicated ratios for 5 days. The cells were harvested and stained with anti-CD4 and Ld/P1A dimer. The CFSE profiles were analyzed for gated transgenic Ld/P1A+CD4+ cells, Ld/P1A−CD4+ cells and non-transgenic CD4+ cells. The numbers beside the CFSE histograms indicate the percentages of proliferating cells. The results are representative of at least two independent experiments. CFSE, carboxyfluorescein succinimidyl ester; P1ATCR, P1A tumor antigen-specific T-cell receptor; TCR, T-cell receptor.
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References

    1. Kisielow P, Bluthmann H, Staerz UD, Steinmetz M, von Boehmer H. Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes. Nature. 1988;333:742–746. - PubMed
    1. Pircher H, Burki K, Lang R, Hengartner H, Zinkernagel RM. Tolerance induction in double specific T-cell receptor transgenic mice varies with antigen. Nature. 1989;342:559–561. - PubMed
    1. Morgan DJ, Liblau R, Scott B, Fleck S, McDevitt HO, Sarvetnick N, et al. CD8+ T cell-mediated spontaneous diabetes in neonatal mice. J Immunol. 1996;157:978–983. - PubMed
    1. McGargill MA, Mayerova D, Stefanski HE, Koehn B, Parke EA, Jameson SC, et al. A spontaneous CD8 T cell-dependent autoimmune disease to an antigen expressed under the human keratin 14 promoter. J Immunol. 2002;169:2141–2147. - PubMed
    1. Asnagli H, Schmitt-Verhulst AM, Guimezanes A. Class I- and class II-reactive TCRs coexpressed on CD4+ T cells both trigger CD4/CD8-shared and CD4-unique functions. J Immunol. 1997;158:4533–4542. - PubMed

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