The interaction between staphylococcal superantigen-like proteins and human dendritic cells

Abstract

Staphylococcus aureus produce a family of exotoxins (staphylococcal superantigen like proteins, SSLs) with structural, but not functional, homology to superantigens. These proteins have previously been shown to interact selectively with antigen presenting cells, including dendritic cells. The functional consequences of this interaction are now explored. SSL7 and 9 had no effect on viability or morphology of dendritic cells. The proteins did not induce dendritic cell maturation, as measured by cell surface phenotype. Exposure to SSL did not alter the ability of dendritic cells to take up FITC-dextran. Finally, exposure to SSLs did not impair the ability of the dendritic cells to stimulate allogeneic or antigen specific T cell responses. However, dendritic cells loaded with SSL7 or 9 were able to stimulate a T cell proliferative response in 3/8 healthy individuals tested. Sera from nine out of 10 individuals tested contained antibodies against both SSL7 and SSL9, and the response to each SSL was specific and not cross-reactive. The results demonstrate that SSLs are immunogenic in humans at both the B and T cell level, but it remains unclear whether this response is to the benefit of the bacterium or the host.

Fig. 1

SSL7 and SSL9 interact selectively with dendritic cells. DCs (unpurified) were incubated in the presence of SSL7-FITC or SSL9-FITC for one hour at 37°C, and the cells were then stained for CD1a. The cells were fixed and analysed by flow cytometry, measuring SSL as FL1 (horizontal) and CD1a by FL2 (vertical). The numbers show the percentage of SSL positive cells that were also CD1a positive.

Fig. 2

SSLs do not alter DC morphology or cell surface phenotype. (a) Morphology of DC treated with SSL proteins. DC were cultured in the presence of 4·16 µM SSL7 or SSL9 for 18 h at 37°C. Lipopolysaccharide (LPS, 100 ng/ml) and peptidoglycan (PG, 5 µg/ml) were used as positive control. Representative of 3 experiments. (b) Phenotypic analysis of DC treated with SSL proteins. Data are shown for expression of cell surface molecules on DC that had been treated for 18 h with 4·16 µM SSL7 or SSL9. Lipopolysaccharide (LPS, 100 ng/ml) and peptidoglycan (PG, 5 µg/ml) were used as positive control. Expression of the indicated markers is shown by the solid histograms, whereas cells stained with relevant control mAb are indicated by the open line histograms. The numbers on each histogram correspond to the median fluorescence intensity (MFI) of mAb staining. Results shown are from one donor and are representative of similar data obtained from experiments carried out with DC from four different donors.

Fig. 3

Endocytosis of FITC-Dx by DCs exposed to SSL proteins. DCs were incubated for (a) 1 h or (b) 18 h in presence or absence of SSL7 (▪) or SSL9 (▴) (4·16 µM). Medium control (♦). Excess proteins were removed by washing, and the cells were incubated with different concentrations of FITC-dextran (1, 3, 10 and 30 µg/ml). Excess dextran was removed by washing, and the cells were fixed. The total cell associated dextran was measured by flow cytometry, and expressed as mean fluorescent intensity for a minimum of 5000 DC. The results of one of three separate experiments are shown.

Fig. 4

Effect of SSL proteins on the T cell stimulatory capacity of DCs. (a) DCs (10⁴) were treated with different concentrations of SSL7 (▪) or SSL9 (□)(0·42, 1·25 and 4·16 µM) or medium alone () and cultured for 18 h before the addition of autologous T cells (2 × 10⁵) in the presence or absence (M) of purified protein derivative (PPD; 500 U/ml). Data are mean ± SEM of 5 experiments. (b) DCs (10⁴) were treated with different concentrations of SSL7 (▪) or SSL9 (□)(0·42, 1·25 and 4·16 µM) or medium alone () and cultured for 18 h before the addition of allogeneic T cells (2 × 10⁵). DC – dendritic cells cultured without allogeneic T cells; TC-allogeneic T cells cultured without dendritic cells. Data are mean ± SEM of 3 experiments. Proliferation was assessed by incorporation of ³H-thymidine.

Fig. 5

T cell responses to DC loaded with SSL protein. DCs (10⁴) were incubated for 6 days with autologous T cells (2 × 10⁵) in presence of different concentrations of SSL7 (▪) or SSL9 (▴) (0·42, 1·25 and 4·16 µM). Data are mean ± SD of triplicate cultures from individual experiments. Proliferation was assessed by incorporation of ³H-thymidine.

Fig. 6

Effects of SSL protein on cytokine production. DCs (10⁴) prepared from an individual showing a proliferative response to SSLs (volunteer 1 in Fig. 5) were incubated for 4 days with autologous T cells (2 × 10⁵) in presence of different concentrations of SSL7 (□) or SSL9 (▪) (0·42, 1·25 and 4·16 µM) and purified protein derivative was used as control (PPD; 500 U/ml). Supernatants were tested using ELISA as described in the methods. Similar experiments were carried out using cells from individuals 2 and 8 (Fig. 5), as described in text.

Fig. 7

Antibody responses to SSLs in human sera. (a) Serum from 10 individuals (1–8 are the same individuals as in Fig. 5) was diluted by 1 : 2000 and tested for binding to SSL7 (□) or SSL9 (▪) by ELISA as described in the methods. A polyclonal rabbit antibody raised against purified His-tagged SSL7 was included as a positive control (M-SSL7). Binding of an alkaline-phosphatase coupled anti-human IgG was detected by adding the chromogen OPD (o-phenylenediamine dihydrochloride) and measuring the optical density at 405 nanometers. Data are representative of 3 experiments. (b,c) Serum from sample 1 above was diluted 1 : 2000 (final dilution) and mixed with varying concentrations of SSL7 (♦), SSL9 (▪) or Emblp32 (▴) as shown. The sera were then added to plates coated with either SSL7 (b) or SSL9 (c) as shown. One experiment of two.