Lipopolysaccharide-dependent down-regulation of CD27 expression on T cells activated with superantigen

Abstract

To investigate the mechanisms underlying T-cell responses during superantigen (SAg) stimulation, we analysed the effects of SAg on CD27 expression with or without lipopolysaccharide (LPS) as a novel regulator of T-cell function. CD27 is expressed on the majority of resting peripheral blood T cells (CD27low). Activation of T cells by SAg induces high levels of CD27 surface expression (CD27high) accompanied with simultaneous CD30 receptor expression. After prolonged activation in vitro, the level of CD27 expression became intermediate. The effects of LPS on down-regulation of CD27high expression on CD30+ T cells were dose-dependent. Separating LPS-stimulated monocytes from T cells by mechanical dispersion abolished its inhibitory effect, indicating the requirement for direct interactions between monocytes and T cells. We also found that SAg up-regulated CD80 expression on CD14+ monocytes and LPS inhibited SAg-induced CD80 expression after 24 hr of stimulation. Up-regulation of CD152 (CTLA-4) was selective, since it was found to be preferentially expressed on the CD30+ population. Competitive experiments using soluble blocking peptides showed that addition of CD28 or CD80 peptide recovered LPS-induced down-regulation of CD27high expression on CD30+ T cells. These observations suggested that the presence of low levels of CD80 on monocytes may partially inhibit CD27 expression due to inefficient delivery of positive signals via CD28/CD80 interaction, and that the increased levels of CD80 enhance the inhibition through interactions with CD152 which is expressed at the highest levels after 48 hr of activation.

Figure 1

Flow cytometric analysis of CD27 expression. PBMC (1×10⁶/ml) were cultured with Con A (10 μg/ml), SEB (1 μg/ml), or SPM-2 (1 μg/ml) for 4 days and stained with FITC-conjugated anti-CD27 and PE-conjugated anti-CD3 mAb (a), or FITC-conjugated anti-CD25 and PE-conjugated anti-CD27 mAb (b). The percentages of respective cell types in the CD3⁺ lymphocytes (a) or in total lymphocytes (b) are shown. Profiles in (a) illustrate results of the highest frequency of CD27^high T cells after stimulation.

Figure 2

Kinetics of enhanced CD27 expression after T-cell activation. PBMC (1×10⁶/ml) were cultured with Con A (10 μg/ml), SPM-2 (1 μg/ml), or medium alone. Cells were harvested at various time-points, and level of CD27 expression of the CD3⁺ lymphocytes was measured.

Figure 3

Flow cytometric analysis of coexpression of CD27^high and CD30. PBMC (1×10⁶/ml) were cultured with Con A (10 μg/ml), SEB (1 μg/ml), SPM-2 (1 μg/ml), or medium alone for 72 hr and stained with mouse anti-CD30 mAb, followed by addition of FITC-conjugated goat anti-mouse IgG, and PE-conjugated anti-CD27 mAb. Numbers represent the percentages of cells expressing both CD27^high and CD30 of the total lymphocyte population.

Figure 4

Effects of blocking of cell interactions on induction of CD27^high CD30⁺ T cells. PBMC (1×10⁶/ml) were stimulated with SPM-2 (1 μg/ml) plus LPS (1 μg/ml) for 48 hr, and the clustered cells were dispersed with a pipette tip (P), followed by culture for a further 24 hr. Cells were also preincubated with anti-CD14 mAb (MY4) or control mouse IgG for 1 hr and then stimulated with SPM-2 plus LPS for 72 hr. Cells were stained with mouse anti-CD30 mAb, followed by addition of FITC-conjugated goat anti-mouse IgG, PE-conjugated anti-CD27 mAb and PerCP-conjugated anti-CD3 mAb. The SPM-2-induced CD27^high CD30⁺ T-cell population is expressed as 100%, and the effects of treatments are shown as percentages of the CD27^high CD30⁺ population. SPM-2-induced CD27^high CD30⁺ T-cell populations ranges from 8·2% to 10·5%. A representative result from three independent experiments is shown.

Figure 5

Expression of costimulatory molecules on responsive cells. PBMC (1×10⁶/ml) were cultured in medium alone or in that containing SPM-2 (1 μg/ml) with or without LPS (1 μg/ml). Each day, cells were stained with FITC-conjugated anti-CD80 and PE-conjugated anti-CD14 mAb (a), FITC-conjugated anti-CD3 and PE-conjugated anti-CD152 mAb (b), or PE-conjugated anti-CD28 and PerCP-conjugated anti-CD3 mAb (c). Cells were gated for CD14⁺ monocytes (a), CD3⁺ blasts (b), or CD3⁺ cells (c).

Figure 6

Expression of CD152 on activated CD30⁺ T cells. PBMC (1×10⁶/ml) were cultured with Con A (10 μg/ml), or SPM-2 (1 μg/ml) for 48 hr and stained with FITC-conjugated anti-CD30 and PE-conjugated anti-CD152 mAb. Data represent the percentages of CD152⁺ T cells in CD30⁺ or CD30⁻ blasts. A representative result from three independent experiments is shown.

Figure 7

Effects of blocking peptides on induction of CD27^high CD30⁺ T cells. PBMC (1×10⁶/ml) were cultured with SPM-2 (1 μg/ml) or SPM-2 plus LPS (1 μg/ml) in the presence or absence of blocking peptides (CD80, CD28; 5 μg/ml) for 72 hr. Cells were stained for the presence of CD27^high CD30⁺ T cells. The SPM-2-induced CD27^high CD30⁺ T-cell population is expressed as 100%, and the effects of treatments with LPS and/or blocking peptides are shown as percentages of the CD27^high CD30⁺ T-cell population. SPM-2-induced CD27^high CD30⁺ T-cell populations ranged from 9·9% to 15·5%. A representative result from three independent experiments is shown.