Blomia tropicalis allergen 5 (Blo t 5) T-cell epitopes and their ability to suppress the allergic immune response

Abstract

Blomia tropicalis is the major asthma allergen in the tropics comparable to Dermatophagoides pteronyssinus. However, little is known about the B. tropicalis epitopes recognized by T cells. Our aim was to identify the T-cell epitopes in the major B. tropicalis allergen, Blo t 5, and investigate the potential of the corresponding peptides to inhibit the allergic inflammatory lung response. C57BL/6 mice were immunized with plasmid DNA encoding Blo t 5 and T-cell epitopes identified using the interferon-γ ELISPOT assay with 15-mer overlapping peptides. C57BL/6 mice were sensitized with bone-marrow-derived dendritic cells (BMDC) pulsed with Blo t 5 allergen followed by intranasal Blo t 5 challenge. Two H-2^b restricted epitopes (Bt5_76-90 and Bt5_106-115 ) were recognized by CD4 T cells specific for Blo t 5, but no CD8 epitopes were identified. In mice sensitized with Blo t 5-pulsed BMDC and challenged with intranasal Blo t 5 Bt5_76-90 and Bt5_106-115 , peptide-specific CD4 T cells were found to secrete the T helper type 2 cytokines interleukin-5 and interleukin-13. Intradermal administration of synthetic peptides encoding the identified T-cell epitopes suppressed allergic airway inflammation to further allergen challenges. Hence, we have identified novel CD4 T-cell epitopes specific for Blo t 5 and demonstrated that these peptides could be employed therapeutically to suppress the T-cell response in a murine model of allergic airway inflammation.

Keywords: Allergy; Blomia tropicalis; DNA vaccine; mouse model.

Figure 1

Mapping of epitopes recognized by Blo t 5‐specific T cells from C57BL/6 and BALB/c mice. Splenocytes from C57BL/6 mice immunized with pVAX‐Blo t 5 were screened using interferon‐γ (IFN‐γ) ELISPOT. (a) The T‐cell response to Blo t 5 peptides was measured by culture of Blo t 5‐specific T cells with 15‐mer peptides (10‐overlap) mean ± SEM, n = 6. (b) Splenocytes from immunized mice were depleted of CD4 T cells (open columns) or CD8 T cells (closed columns) before the IFN‐γ ELISPOT with the epitope containing peptides previously identified. Each column depicts the mean ± SEM of the pooled results of each group (n = 4). Identified peptides were truncated from both N‐ and C‐termini and the minimal epitope capable of eliciting a T‐cell response was measured (n = 8). Screening of peptides #16 (c), #21 and #22 (d), only amino acids present in the overlap region between the two peptides were tested. (e, f) Blo t 5 epitopes peptides identified in BALB/c mice immunized as above using the same approach. *P < 0·05, **P < 0·01, ***P = 0·001.

Figure 2

Blo t 5‐specific CD4 T cells are recruited to the lung following allergen challenge and participate in the allergic inflammatory response. (a) DNA immunization with Blo t 5 pVAX on days 0, 3 and 6 and intranasal challenge with Blo t 5 protein on days 14, 16 and 18. (b) Immune cell infiltration into the bronchoalveolar lavage (BAL) following allergen challenge analysed by flow cytometry (n = 6). (c) Cytokine production from cells in the draining lymph nodes after 3 days of culture with Blo t 5 protein. Two replicates with pooled lymph node cells from three mice per experiment). (d) Representative intracellular staining for interleukin‐4 (IL‐4), IL‐5 and IL‐13 of lymph node cells after 3 days of culture with different antigens. Cells were gated on live CD3⁺ CD4⁺ cells. (e) Tabulated intracellular cytokine staining results from two different experiments (three mice per experiment). Blo t 5 sensitization and challenge model. Mice were sensitized by Blo t 5‐pulsed bone‐marrow‐derived dendritic cells (BMDC) and challenged with Blo t 5 intranasally. **P < 0·01.

Figure 3

T cells recognizing Blo t 5 epitopes were induced by allergen sensitization and challenge through intranasal exposure. (a) Mice were sensitized through the intranasal administration of bone‐marrow‐derived dendritic cells (BMDC) pulsed with recombinant Blo t 5 allergen and subsequently challenged with the recombinant protein. (b) Mice showed increased bronchoalveolar lavage (BAL) eosinophilia compared with control mice treated with PBS. (c) Percentage of cells secreting interleukin‐5 (IL‐5) and IL‐13 in lymph node cultures from mice sensitized and challenged with Blo t 5 (n = 3). Cells were cultured with medium only (control), Blo t 5 protein or 15‐mer peptides containing Blo t 5 epitopes. Cells were gated on live CD3⁺ CD4⁺ cells for analysis. (d) Cytokine productions from cells isolated from the draining lymph nodes following 3 days of culture with Blo t 5 protein (n = 3). **P < 0·01.

Figure 4

Application of Blo t 5 peptides for peptide immunotherapy of allergic asthma. (a) Experimental protocol for peptide immunotherapy studies. Eosinophilia in the bronchoalveolar lavage (BAL) (b) and lungs (c) were assessed by flow cytometry. Eosinophils were identified by flow cytometry as Siglec‐F⁺ CD11c^– Ly6G^– cells (n = 38 and n = 37 for BAL and n = 15 for lung). Interleukin‐5 (IL‐5) and IL‐13 levels in the BAL (d) and lungs (e) were assessed by ELISA. (f) Draining lymph node cells were cultured for 3 days and the cytokine production was assayed by ELISA (n = 18). (g) Total serum IgE levels were not affected by peptide immunotherapy (n = 24). **P < 0·05, **P < 0·01.

Figure 5

Role of regulatory T cells and interleukin‐10 (IL‐10) in Blo t 5 peptide immunotherapy. (a) Infiltration of CD3⁺ CD4⁺ CD25⁺ FoxP3⁺ cells in lungs of mice that had received either Blo t 5 peptides immunotherapy or ovalbumin (OVA) peptide (mock) were assessed by flow cytometry (n = 14). (b) FoxP3 expression as measured by the MFI of FoxP3 staining in CD3⁺ CD4⁺ CD25⁺ FoxP3⁺ cells (n = 14). ELISA measurement of IL‐10 levels in (c) lymph node culture (n = 18) and (d) lung homogenate (n = 8) of mice that received peptide immunotherapy compared with control mice. (e) Levels of the T helper type 1 cytokine interferon‐γ as measured by ELISA (n = 18).

Figure 6

Response of T helper type 2 (Th2) cells suppressed by peptide immunotherapy. (a) Percentage of CD3⁺ CD4⁺ cells secreting interleukin‐5 (IL‐5) or IL‐13 measured by intracellular cytokine staining (n = 5). (b) The number of IL‐33 receptor (T1/ST2) expressing cells gated on CD3⁺ cells. (c) Programmed death 1 (PD‐1) expression in CD3⁺ cells in lungs of peptide‐treated mice compared with control mice (n = 10). **P < 0·05, **P < 0·01.