These data suggest reduced proliferation and cytokine production (particularly IL-17 and IFNγ) by PBMC from atopic donors stimulated with Depig-pol extracts compared to native, unmodified allergen extracts for grass pollen and house dust mite. However, Depig-pol allergen extracts for grass pollen and HDM favoured expansion of regulatory T cells over effector T cells in vitro, and this balance was further tilted towards regulation by 1α, 25-dihydroxyvitamin D3. Thus Depig-pol allergen extracts may favour regulatory T cells over effector T cell activation, which might be expected to augment tolerance induction in SIT.
Several reports have previously examined T cell responses to allergoids [12–16]. All showed reduced proliferation of PBMCs stimulated with allergoid when compared to native allergen, and variable reductions in cytokine production. This difference in T cell proliferation between allergoid and native extract was suggested to vary with antigen presenting cell type, although this was not confirmed in a subsequent study [15, 16]. Analysis of responses of T cell lines and clones suggested variable loss of T cell epitopes in allergoid extracts compared to native extracts [13–15]. Our data also suggests reduced proliferation and effector cytokine production by PBMC stimulated by Depig-pol extracts compared with native extract, and these findings were confirmed by reduced expansion of cells with effector phenotype in Depig-pol stimulated cultures. We would argue that reduced activation and expansion of effector T cells by Depig-pol extracts will actually be beneficial in the context of SIT, where effector T cell activation may contribute to side effects. This is demonstrated most graphically for peptide therapy where small allergen peptides which did not cross link IgE nonetheless activated effector T cells and lead to isolated late asthmatic reactions in patients . We did not examine loss of T cell epitopes comparing Depig-pol extracts with native extracts, although previous mass spectroscopic analysis has suggested preservation of most major allergen sequences . Even if some T cell epitopes were lost during the depigmentation and polymerisation process, we would argue that the critical activity for tolerance induction is expansion of regulatory T cells that work in both antigen-specific and non-antigen specific fashion, as shown by linked suppression to cat major allergen Fel d 1 peptides not included in the experimental peptide immunotherapy treatment in an animal model .
Although Depig-pol extracts stimulated reduced effector T cell expansion and activation compared to native extracts, numbers of regulatory T cells were similar, so the ratio of regulatory to effector T cells in cultures stimulated with Depig-pol extracts was significantly higher than for native extract. Activation requirements for effector versus regulatory T cells are incompletely understood, but we would suggest that antigen-presenting cell processing of and activation by these molecules of vastly different mass may be relevant in the case of Depig-pol versus native extracts [10, 11].
Interestingly, our data both here and in previous studies  suggest that depigmented-polymerised extracts and 1α, 25-dihydroxyvitamin D3 both favour Foxp3hi T cells (by reducing expansion of effector T cells). Initial reports suggested that 1α, 25-dihydroxyvitamin D3 acted to expand IL-10 producing T cells . However, our recent data shows that 1α, 25-dihydroxyvitamin D3 can also expand Foxp3hi Tregs . The type of Treg emerging depended on the concentration of 1α, 25-dihydroxyvitamin D3 in cultures, and the cytokine milieu and essentially no co-expression of Foxp3 and IL-10 was observed, although both populations exhibited comparable suppressive activity [19, 20]. The present findings are in keeping with that data in that a relatively higher concentration favoured Foxp3hi Tregs (albeit 10-7 M here rather than 10-6 M in an anti-CD3 driven culture system), and in these conditions IL-10 producing T cells were not detected (data not shown). Our previous report suggested that the major effect of 1α, 25-dihydroxyvitamin D3 was to suppress proliferation of effector T cells and maintain Foxp3 expression by regulatory T cells: our current data agrees with this, as we show reduced effector T cells and cytokines, and a preserved Foxp3hi Treg population .
SIT with depigmented-polymerised extracts, which have at least 95% reduced IgE binding compared to native allergen extract, has been shown to be clinically effective in reducing symptom scores for rhinoconjunctivitis and asthma with minimal side effects and rapid up-dosing [5–8]. Further studies are required to examine the effects of Depig-pol extracts on Tregs in vivo, and to determine whether 1α, 25-dihydroxyvitamin D3 can enhance tolerance in a clinical setting of SIT.