Immunoproteomic analysis of house dust mite antigens reveals distinct classes of dominant T cell antigens according to function and serological reactivity

Abstract

BACKGROUND House dust mite (HDM) allergens are a common cause of allergy and allergic asthma. A comprehensive analysis of proteins targeted by T cells, which are implicated in the development and regulation of allergic disease independent of their antibody reactivity, is still lacking. OBJECTIVE To comprehensively analyze the HDM-derived protein targets of T cell responses in HDM-allergic individuals, and investigate their correlation with IgE/IgG responses and protein function. METHODS Proteomic analysis (liquid chromatography tandem mass spectrometry) of HDM extracts identified 90 distinct protein clusters, corresponding to 29 known allergens and 61 novel proteins. Peripheral blood mononuclear cells (PBMC) from 20 HDM-allergic individuals were stimulated with HDM extracts and assayed with a set of ~2500 peptides derived from these 90 protein clusters and predicted to bind the most common HLA class II types. 2D immunoblots were made in parallel to elucidate IgE and IgG reactivity and putative function analyses were performed in silico according to gene ontology (GO) annotations. RESULTS Analysis of T cell reactivity revealed a large number of T cell epitopes. Overall response magnitude and frequency was comparable for known and novel proteins, with 15 antigens (nine of which were novel) dominating the total T cell response. Most of the known allergens that were dominant at the T cell level were also IgE-reactive, as expected, while few novel dominant T cell antigens were IgE reactive. Among known allergens, hydrolase activity and detectable IgE/IgG reactivity are strongly correlated, while no protein function correlates with immunogenicity of novel proteins. A total of 106 epitopes accounted for half of the total T-cell response, underlining the heterogeneity of T cell responses to HDM allergens. CONCLUSIONS AND CLINICAL RELEVANCE Herein, we define the T cell targets for both known allergens and novel proteins, which may inform future diagnostics and immunotherapeutics for allergy to HDM.

Strategies to Study T Cells and T Cell Targets in Allergic Disease

“…Using predicted peptide binding as a preselection criterion to decrease the number of peptides to screen for T cell epitope identification is less thorough than using overlapping peptides and may therefore increase the risk of missing T cell-reactive peptides. However, it has been reported that it is a reliable approach to identify the vast majority of T cell epitopes [28,29], and it has been successfully used in several allergen systems, including Timothy grass [11], German cockroach [30], house dust mite [31], and others [32], to perform large-scale epitope identification studies. Therefore, the decision between using overlapping and predicted peptides is likely dictated by the size and number of allergens studied as well as the amount of cells available from the clinical cohort.…”

Blueprint for the House Dust Mite

“…The ability to make recombinant D. pteronyssinus homologues of these components from the information in the genome not only opens up these investigations to studies in the many more regions where people are substantively or almost exclusively sensitized by D. pteronyssinus, but also to the possibility that investigators in these regions are more interested in making authenticable assessments. The T-cell responses of house dust mite-allergic subjects from Europe and America made to peptides representing predicted epitopes of many of these proteins showed that they did not induce substantive T-cell responses compared to the responses induced by the high IgE-binding components authenticated earlier with quantitative analyses [6], Why would you want to sequence the house dust mite genome? Randall et al [1] help explain this in an analysis of their assembly of the genomic sequence of Derma tophagoides pteronyssinus.…”

“…IgE binding to Der p 14 and a small group of other apparently minor IgE-binding proteins has been associated with an additional propensity of house dust mite-allergic children to develop asthma [21], and it has also been found to be one of the most stimulatory allergens as assessed by the T-cell epitope study by Oseroff et al [6]. Antibodies produced to recombinant Der p 14 have not only revealed the presence of a Der p 14-like molecule in house dust mite eggs, which would be expected to be a vitellogenin known to belong to this type of protein, but also a protein in the bodies of male mites that correspond to the related lipid transporter apolipophorin [22].…”

“…Currently, there is almost no information on proteins that are in inhalable air besides that gleaned for Der p 1 by monoclonal antibody assays, but this has been highly instructive in showing that nasal filters and personal monitors can collect enough protein to measure and to provide baseline information on Der p 1 for a reference [29]. From measuring T-cell responses to peptides representing the epitopes of allergens and other proteins represented in the mite transcriptome, there is evidence that quite a few nonallergenic proteins induce T-cell re- 124 sponses in humans [6]. Therefore, these can now be studied comprehensively and be extended by studying responses to proteins known to be in the inhalable air at known doses.…”

“…Randall et al [1] now provide the first analysis of the genomic organization of the house dust mite as it affects our knowledge of allergens. The advances have come in tandem with other big data on the transcriptome [3,6,30] and proteome [30], noting that both these depend on growth conditions and can now be referenced to the genome data. Although not so up-scalable, the implementation and refinement of megapool peptide techniques for comparing T-cell responses to large assemblies of house dust mite proteins provides a powerful tool with which to interrogate the interactions of mite proteins with the human immune system [6], as does the systematic production of recombinant proteins for serology.…”