The HLA class II restriction of Equ c 1 protein-specific TCLs and clones from allergic subjects was assessed by inhibiting the responses with anti-HLA-DQ and -DR antibodies (representative examples shown in Fig. 5b) and by using partially HLA-matched PBMCs for antigen presentation. As shown

in Table 1, restriction by HLA-DQ was seen in three and by HLA-DR in six out of the nine TCLs investigated. In line with the findings with the TCLs, both HLA-DQ and -DR restrictions were detected with the seven Equ c 1 protein-reactive T-cell clones from five different subjects (Fig. 5b and BMN 673 mouse Table 1). More detailed investigations using partially HLA-matched allogeneic PBMCs as APCs revealed that two of the DQ-restricted TCLs were restricted by DQB1*0501 and one by DQB1*0602 and both of the DQ-restricted T-cell clones were restricted by DQB1*0603 (Table 1). Interestingly, we observed that five of the six DR-restricted TCLs and all of the five DR-restricted T-cell clones were restricted by either DRB1*0404 or DRB4*0101 (one TCL was not determined). As the DRB1*0404 and DRB4*0101 restrictions could not be distinguished with partially HLA-matched PBMCs in this experimental setting because of the linkage disequilibrium between these two alleles, we stained one monoclonal

and one oligoclonal TCL from a DRB1*0404/DRB4*0101 positive horse-allergic subject with a DRB4*0101:Equ c 1143–160 Trametinib cost HLA class II tetramer

(Fig. 6). Positive staining with the tetramer confirmed that the DRB4*0101 allele is involved in restricting the CD4+ T-cell response to Equ c 1143–160. Taken together, our findings suggest that a wide array of HLA class II alleles, including DRB4*0101, is able to bind and present the immunodominant epitope region of Equ c 1. In the present study, we have examined allergen-specific peripheral blood CD4+ T-cell responses of subjects sensitized to the major allergen of horse, Equ c 1, and compared them with those of non-allergic horse dust-exposed individuals. As we have previously PAK5 found that Equ c 1 contains one immunodominant epitope region between the amino acids 143 and 160 against which almost all Equ c 1-sensitized individuals mount a strong T-cell response,[11] we chose to analyse the CD4+ T-cell responses to this particular region. Recent studies with lipocalin and non-lipocalin allergens have suggested that there is a difference in the frequency of allergen-specific CD4+ T cells between allergic and non-allergic subjects.[1-7] In line with these findings we observed here that the number of Equ c 1 protein-specific TCLs, but not the number of Equ c 1143–160 peptide-specific TCLs, from allergic subjects tended to be higher than that from non-allergic subjects (Fig. 1).