Bacterial superantigen facilitates epithelial presentation of allergen to T helper 2 cells

Abstract

Although clinical and laboratory evidence support roles for both staphylococcal infection and environmental allergens in the pathogenesis of atopic dermatitis, human studies have largely considered these variables independently. We sought to test the hypothesis that staphylococcal superantigen influences the allergen-specific T cell response. We first mapped a Der p 1 epitope and used HLA DRB1*1501 class II tetramer-based cell sorted populations to show that specific CD4(+) T cells were able to recognize the peptide presented by HLA DR-matched keratinocytes. We observed that staphylococcal enterotoxin B (SEB) enhanced the IL-4 Der p 1-specific T cell response. This response was mediated by two synergistic mechanisms: first, SEB-induced IFN-gamma promoted class II and intercellular adhesion molecule-1 expression by presenting keratinocytes; and second, SEB-induced IL-4 directly amplified allergen-specific CD4(+) T cell production of many cytokines. We propose that handling of staphylococcal infection is a critical step in the amplification of the allergen-specific T cell response, linking two common disease associations and with implications for the prevention and treatment of atopic disease.

Fig. 1.

Cultured IFN-γ and IL-4 ELISpot responses to Der p 1 peptides by nonatopics (a and b) and individuals with AD (c and d). Cultured ELISpot data from nonatopics (NA) (n = 10) and individuals with AD individuals (n = 13). Responses to overlapping peptides of Der p 1 as defined by ELISpot IFN-γ (a and c) and IL-4 (b and d) cytokine secretion measured as spot- forming units (sfu) per million cells are shown. The horizontal bar represents the cutoff for a positive response (mean + 3 × SD of control wells of the population). Data are presented as mean ± SD of sfu.

Fig. 2.

HLA DRB1*1501 tetramer staining of T cells. (a) Flow cytometric analysis of a short-term T cell line grown with peptide 18.10 (Upper) or recombinant Der p 1 (Lower). (b) Tetramer-sorted clonal population. (c) The histogram demonstrates a control Vβ 22-negative cell population (filled) vs. CD4⁺ tetramer-positive cells used in b (open).

Fig. 3.

SEB enhances the IL-4 Der p 1-specific T cell response. (a) HLA DRB1*1501 keratinocytes (HaCaT cells) were incubated overnight with SEB alone or with unstimulated PBMCs or SEB-stimulated PBMCs. The keratinocytes were subsequently washed, pulsed with peptide, and then incubated with a peptide-specific T cell line or clone 14 in an IL-4 ELISpot assay. Only when keratinocytes were incubated with the supernatant from SEB-stimulated PBMCs were they able to present antigen to the peptide-specific CD4⁺ T cell line and clone. Responses are demonstrated as sfu by using IL-4 ELISpot with mean ± SD. (b) Flow cytometric analysis of HLA class II and ICAM-1 expression of keratinocytes incubated overnight with supernatant from PBMCs alone (gray area) or PBMCs and SEB (black line). (c) As b but with IFN-γ-depleted supernatant. (d) IL-4 ELISpot assay using the undepleted (Nil) or IFN-γ-depleted (IFNg) supernatant-incubated keratinocytes to present peptide 18.10 to specific T cell line. Data are presented as mean ± SD.

Fig. 4.

SEB enhances IL-4 responses of antigen-specific T cells by IL-4 mediated activation. (a) IL-4 ELISpot assay. Polyclonal peptide-specific T cell lines from six separate donors show significantly (P < 0.01) enhanced IL-4 production when stimulated with combined peptide + SEB compared with the sum of the isolated peptide and SEB responses. Three different lines were tested from donor 1. (b) Cytokine bead assay. Cytokine production by peptide-stimulated (filled) antigen-specific T cell line from donor 1 compared with irrelevant stimulus (open). The line produces both Th1 and Th2 cytokines in response to peptide. (c) Percentage reduction of cytokine production in a low IL-4 environment. Of the cytokines showing responses in b, the majority were significantly (P < 0.01) reduced in conditions that sequestered IL-4. (d) IL-4 ELISpot assay. Blocking IL-4R signaling results in significantly (P < 0.01) reduced IL-4 production after combined stimulation of peptide-specific T cell line with SEB and peptide. Data are presented as the mean ± SD of sfu.