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. 1999 Apr;96(4):642-8.
doi: 10.1046/j.1365-2567.1999.00720.x.

Enhancement of B7-1 (CD80) expression on B-lymphoma cells by irradiation

Affiliations

Enhancement of B7-1 (CD80) expression on B-lymphoma cells by irradiation

A Seo et al. Immunology. 1999 Apr.

Abstract

Irradiation of A20.2J mouse B-lymphoma cells enhanced their antigen-presenting ability to induce interleukin-2 (IL-2) production by 42-6A T cells specific for ovalbumin (OVA)323-339/I-Ad. Irradiated and fixed A20.2J cells were more efficient antigen-presenting cells (APC) to present OVA323-339 peptide than the unirradiated and fixed cells. Irradiation selectively increased the expression of B7-1 molecules, but not of the major histocompatibility complex class II molecules, B7-2, lymphocyte function-associated antigen-1, or intracellular adhesion molecule-1. Irradiation of A20.2J cells with 100 Gy followed by overnight incubation was optimal for the enhancement of B7-1 expression. Anti-B7-1 monoclonal antibody inhibited the irradiation-induced enhancement of APC function. Irradiation of A20.2J cells induced the accumulation of B7-1 mRNA. Thus, it was concluded that the enhancement of APC function by irradiation was due to the up-regulation of B7-1 molecules through the accumulation of its mRNA. Although partial inhibition of protein synthesis has been shown to enhance the accumulation of B7-1 mRNA and its expression, irradiation did not decrease the protein synthesis in A20.2J cells. The incubation with irradiated A20.2J cells stimulated unirradiated A20.2J cells to increase B7-1 expression, suggesting that irradiation of A20.2J cells induced expression or secretion of some molecule(s) to enhance B7-1 expression.

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Figures

Figure 1
Figure 1
Irradiation of A20.2J cells enhances their APC function. (a) A20.2J cells were irradiated with 0, 40, or 100 Gy, and 1×105 irradiated cells were incubated overnight with 1×104 42-6A cells in the presence of OVA at the indicated concentrations. Their culture supernatants were assayed for IL-2 activity using CTLL-2 cells. The proliferation of CTLL-2 cells was determined by the incorporation of [3H]thymidine, using a Matrix 96 direct beta-counter. The results were expressed as the average counts/3 min of triplicate assay. Standard deviation was shown by small bar, which was hidden by the symbol and thus invisible in some cases. (b) A20.2J cells were irradiated, incubated overnight and fixed with 0·5% paraformaldehyde. The treated cells (2·5×105) were incubated overnight with 4×104 42-6A cells and OVA323–339 peptide at indicated concentrations. The culture supernatants were assayed for IL-2 activity.
Figure 2
Figure 2
Enhancement of B7-1 expression by irradiation. A20.2J cells unirradiated or 100 Gy-irradiated and incubated overnight were treated with anti-B7-1 mAb (16-10A.1, hamster IgG), anti-B7-2 (GL-1, rat IgG2a), anti-LFA-1 (FD441.8, rat IgG2b), anti-ICAM-1 (YN1/L7.4), or anti-MHC class II (M5/114, rat IgG2b), followed by the staining with FITC-conjugated anti-hamster IgG second antibody, or with biotinylated anti-rat κ second antibody and FITC-conjugated avidine. Hamster IgG, R35-95 mAb (rat IgG2a), or anti-GK1.5 mAb (rat IgG2b) was used as a control. The stained cells were analyzed on a flow cytometer. Solid lines represent staining with experimental antibodies (bold line, 100 Gy-irradiated A20.2J cells; fine line, unirradiated A20.2J cells), and shaded areas represent staining with negative control antibodies. Each staining profile of irradiated A20.2J cells with the control antibody was almost superimposable on that of the non-irradiated cells.
Figure 3
Figure 3
Time kinetics and dose–response curve of the enhancement of B7-1 expression by irradiation. (a) A20.2J cells were irradiated with 100 Gy and incubated for the number of days indicated, then stained by the sequential treatment with anti-B7-1 mAb, 16-10A.1 (hamster IgG), biotinylated anti-hamster IgG antibody, and FITC-conjugated avidine. As a control, hamster IgG was used. They were analyzed on a flow cytometer with log scale of fluorescent intensity and the fluorescent intensity was expressed as the mean channel. From these data, Δ mean channel was calculated as follows; Δ mean channel = (mean channel with anti-B7-1 mAb) – (mean channel with control IgG). (b) A20.2J cells were irradiated with indicated doses and incubated for 1 day. Then, they were stained for B7-1 molecules on the surface and analysed as described above.
Figure 4
Figure 4
Inhibition of the enhanced APC function of irradiated A20.2J cells. The 100 Gy-irradiated and overnight incubated A20.2J cells (a) or unirradiated A20.2J cells (b) were fixed with 0·5% paraformaldehyde. They (2·5×105) were incubated with 4×104 42-6A cells and 1·6 μg/ml OVA323–339 peptide in the presence of one of the following mAbs. Anti-B7-1 mAb (1G10, rat IgG2a), anti-B7-2 mAb (RMMP-1, rat IgG2a), anti-H-2Kd mAb (SF1-1.1, rat IgG2a), or control mAb with unknown specificity (R35-95, rat IgG2a) was added at indicated doses. After overnight incubation, their supernatants were assayed for IL-2 activity. Standard deviation was shown by a small bar, but was too small and invisible in some cases.
Figure 5
Figure 5
Northern blot analysis of B7-1 mRNA expression. Cellular RNA was obtained from A20.2J cells unirradiated or 100 Gy-irradiated, electrophoresed, and hybridized with 32P-labelled B7-1 cDNA probe. As a control, the membrane was re-hybridized with β-actin cDNA. The membrane was subjected autoradiographic analysis (a), and densitometric analysis (b). The ratio B7-1:actin was calculated and the ratio obtained from unirradiated A20.2J cells was referred to as 100%. The treatment with 0·313 μm emetine was used as a control for the increase in the B7-1 mRNA accumulation.
Figure 6
Figure 6
B7-1 expression on unirradiated A20.2J cells incubated with irradiated A20.2J cells. A20.2J cells were irradiated and labelled with BCECF-AM. The treated cells and unirradiated A20.2J cells were incubated separately or together overnight, and examined for the expression of B7-1 molecules by staining with anti-B7-1 mAb (16-10A.1, hamster IgG) and PE-conjugated goat antihamster IgG antibody. (a) Unirradiated A20.2J cells alone, (b) the 100 Gy-irradiated, A20.2J cells labelled with BCECF-AM and overnight incubated alone, (c) A20.2J cells, 100 Gy-irradiated, labelled with BCECF, and overnight incubated were mixed with unirradiated A20.2J cells and the expression of B7-1 was examined. (d) A20.2J cells 100 Gy-irradiated and labelled with BCECF-AM and unirradiated A20.2J cells were incubated overnight, and examined for the expression of B7-1.

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