Background
Specific allergen immunotherapy (SIT) is recommended for persistent allergic rhinoconjunctivitis and asthma resistant to pharmacotherapy, and is the only disease-modifying treatment in that benefit persists after stopping treatment [
1,
2]. However, subcutaneous injection of immunotherapy (SCIT) extracts carries a risk of anaphylaxis, whilst sublingual therapy (SLIT) requires daily treatment over three years. One approach to reducing the risk of anaphylaxis with SCIT is to modify allergen extracts to reduce IgE binding [
3]. Such allergoids include extracts treated with acid (“depigmented”), prior to polymerisation with glutaraldehyde [
4]. These extracts showed efficacy in clinical trials for both birch and grass pollen-related allergic rhinitis, and house dust mite-related asthma [
5‐
8]. In these studies and large observational series, side effects were minimal [
5‐
9]. The resulting depigmented-polymerised (Depig-pol) allergen extracts are of very large molecular mass (0.5-3 MDa as opposed to less than 100 KDa for native allergen extracts), and have at least 50 times reduced IgE binding in vitro in competition assays and in vivo in skin prick testing [
4,
10,
11]. Analysis of sequences obtained by enzymatic digestion of Depig-pol and native extracts suggests conservation of major allergen sequences for both dust mite and pollen extracts [
10,
11], and in vivo immunogenicity studies show induction of IgG directed against the majority of major allergens upon treatment of patients with these extracts and in studies involving immunization of experimental animals [
5‐
7,
11].
Whilst allergoids have reduced IgE binding (allergenicity) which may improve safety, it has also been suggested that they have reduced T cell stimulating activity (immunogenicity) which could compromise efficacy [
12]. Studies of T cell activity of allergoids in terms of proliferation of primary T cells, lines or clones, have suggested variable degrees of loss of ability to stimulate T cells, either generally or restricted to some epitopes [
12‐
16]. However, the immunological efficacy of SIT most likely relates to induction of tolerance to allergens through interactions with regulatory T cells (Treg) [
17]. Studies with unmodified allergen extracts suggest increased numbers and activity of both CD4
+CD25
hiFoxp3
hi T cells and IL-10 producing T cells after SIT treatment, together with induction of the IL-10-dependent antibody IgG4 [
17]. Thus expansion of Tregs may be a more relevant activity to assess for allergen extracts for SIT.
In order to improve efficacy, reduce dosing and improve safety of allergen immunotherapy a number of adjuvants have been examined. In particular, there is much current interest in 1α, 25-dihydroxyvitamin D3, which can expand both induced IL-10-producing and Foxp3+ Treg in vitro, and improved effects of immunotherapy in animal models [
18‐
21]. Vitamin D status and/or treatment of human volunteers with vitamin D derivatives correlated with or increased Foxp3+ T cell frequency in the periphery [
19] and the airway in asthmatics [
20] and IL-10 producing Treg in the periphery [
18].
Here we have examined the relative stimulation of effector and regulatory T cells in vitro by unmodified and depigmented-polymerised allergen extracts, together with the effects of 1α, 25-dihydroxyvitamin D3.
Discussion
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 [
22]. 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 [
10]. 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 [
23].
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 [
20] suggest that depigmented-polymerised extracts and 1α, 25-dihydroxyvitamin D3 both favour Foxp3
hi 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 [
18]. However, our recent data shows that 1α, 25-dihydroxyvitamin D3 can also expand Foxp3
hi Tregs [
20]. 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 Foxp3
hi 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 Foxp3
hi Treg population [
20].
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.
Conclusion
Depigmented-polymerised allergen extracts appear to favour Treg expansion over activation of effector T cells to a greater extent than unmodified allergen extracts. 1α, 25-dihydroxyvitamin D3 further reduces effector T cell activation by both allergen extracts, and may be a useful adjuvant for SIT.
Acknowledgements
This study was funded by Laboratorios Leti, Spain, including salary support for ZLU. MEM and JC are paid employees of Laboratorios Leti, Spain, and DSR is a paid consultant to Laboratorios Leti. CB was supported by the National Institute for Health Research (NIHR) Clinical Research Facility at Guy’s & St Thomas’ NHS Foundation Trust and NIHR Biomedical Research Centre based at Guy’s & St Thomas’ NHS Foundation Trust and King’s College London. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department for Health. We gratefully acknowledge the support of the divisional research nurses, Mrs Kheem Jones and Mrs Cherylin Reinholtz.
Competing interests
Authors declare no competing financial interests.
Authors’ contributions
CMH and DS conceived and secured funding for the project. ZLU, DFR, CB, MEM, JC, CMH and DSR contributed to the design of, performed and analyzed data from all experiments. ZLU, DSR and CMH designed the study. ZLU, DR and CB performed all experiments. MEM measured serum allergen specific IgE and with JC supervised preparation of extracts. ZLU, DFR, DSR and CMH analysed data and ZLU, DSR and CMH wrote the manuscript. All authors read and approved the final manuscript.