BACKGROUND
specific allergen immunotherapy  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  <cit> .
however, subcutaneous injection of immunotherapy  extracts carries a risk of anaphylaxis, whilst sublingual therapy  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  <cit> .
such allergoids include extracts treated with acid , prior to polymerisation with glutaraldehyde  <cit> .
these extracts showed efficacy in clinical trials for both birch and grass pollen-related allergic rhinitis, and house dust mite-related asthma  <cit> .
in these studies and large observational series, side effects were minimal  <cit> .
the resulting depigmented-polymerised  allergen extracts are of very large molecular mass , and have at least  <dig> times reduced ige binding in vitro in competition assays and in vivo in skin prick testing  <cit> .
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  <cit> , 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  <cit> .
whilst allergoids have reduced ige binding  which may improve safety, it has also been suggested that they have reduced t cell stimulating activity  which could compromise efficacy  <cit> .
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  <cit> .
however, the immunological efficacy of sit most likely relates to induction of tolerance to allergens through interactions with regulatory t cells   <cit> .
studies with unmodified allergen extracts suggest increased numbers and activity of both cd4+cd25hifoxp3hi t cells and il- <dig> producing t cells after sit treatment, together with induction of the il-10-dependent antibody igg <dig>  <cit> .
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 d <dig>  which can expand both induced il-10-producing and foxp3+ treg in vitro, and improved effects of immunotherapy in animal models  <cit> .
vitamin d status and/or treatment of human volunteers with vitamin d derivatives correlated with or increased foxp3+ t cell frequency in the periphery  <cit>  and the airway in asthmatics  <cit>  and il- <dig> producing treg in the periphery  <cit> .
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 d <dig>
RESULTS
depigmented-polymerised allergen extracts stimulate less t cell proliferation than native extracts, but similar cytokine production
production of depigmented-polymerised extracts involves two steps which might alter t cell reactivity .
therefore initial timecourse and dose-response experiments were performed to assess proliferation of pbmc from atopic donors  in cultures stimulated with native, depigmented and depigmented-polymerised  house dust mite  and grass pollen  extracts.
maximal proliferation was seen at day  <dig> for all extracts except for depig-pol hdm extract which was highest at day  <dig> .
note that pbmc viability was comparable between hdm native and depig-pol stimulated cultures,  <dig> %  and  <dig> % , respectively.
data for some of the experiments with grass pollen extracts has been presented elsewhere  <cit> .
dermatophagoides pteronyssinus  phleum pratense
donor clinical characteristics are shown in brackets , specific ige concentration and cap class for each allergen, and size of skin prick test weal in mm.
note equal proportions of male and female subjects were studied.
mean  of allergic patients was 38 years old ; mean age of nonatopic individuals was 40 years old .
proliferation for depigmented hdm and grass extracts was very similar to that seen for native extracts, but proliferation was signficantly less for depig-pol extracts of both hdm and grass when compared to native unmodified extracts .
for this reason, further comparisons were between native and depig-pol extracts.
cytokine production was also maximal at day seven for all cytokines except il- <dig> which peaked at day  <dig> .
seven days of culture was therefore used for further comparisons .
production of il- <dig>  il- <dig> and il- <dig> did not differ significantly between cultures stimulated with native or depigmented-polymerised extracts of either hdm or grass , whilst there was a non-significant trend for reduced ifnγ and significantly reduced il- <dig> in depig-pol-stimulated cultures compared to native extract.
depigmented-polymerised allergen extracts expand regulatory t cells more than effector t cells in culture
to examine effects of native and depigmented-polymerised extracts in stimulation of regulatory or effector cells we examined expression of cd <dig>  foxp <dig> and cd <dig> after 7 days of culture of pbmc.
gating strategy was as described, to identify cells that we have previously shown to be regulatory in cultures  <cit> .
for both hdm and grass, depig-pol extracts expanded similar numbers of cd4+cd25+foxp3hicd127lo  compared to native extracts, but reduced numbers of cd4+cd25+foxp3locd127+ .
when expressed as a ratio of cd25+foxp3hi cd127lo to cd25+foxp3locd127+ cells this was significantly higher for depig-pol extracts of both hdm and grass when compared to cultures stimulated with native extracts .
addition of 1α, 25-dihydroxyvitamin d <dig> to cultures further favours cd25hifoxp3hicd127lo t cells
addition of 1α, 25-dihydroxyvitamin d <dig> at 10-7 m further reduced expansion of effector cells in cultures stimulated with both native and depig-pol extracts of hdm and grass , whilst numbers of cd4+cd25+foxp3hicd127lo regulatory cells were not reduced.
thus the ratio of regulatory to effector cells after cultures was further increased by addition of 1α, 25-dihydroxyvitamin d <dig> .
in addition 1α, 25-dihydroxyvitamin d <dig> reduced effector cytokines, particularly il- <dig> and ifnγ, but did not significantly affect il- <dig> production .
discussion
these data suggest reduced proliferation and cytokine production  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 d <dig>  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  <cit> .
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  <cit> .
analysis of responses of t cell lines and clones suggested variable loss of t cell epitopes in allergoid extracts compared to native extracts  <cit> .
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  <cit> .
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  <cit> .
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  <dig> peptides not included in the experimental peptide immunotherapy treatment in an animal model  <cit> .
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  <cit> .
interestingly, our data both here and in previous studies  <cit>  suggest that depigmented-polymerised extracts and 1α, 25-dihydroxyvitamin d <dig> both favour foxp3hi t cells .
initial reports suggested that 1α, 25-dihydroxyvitamin d <dig> acted to expand il- <dig> producing t cells  <cit> .
however, our recent data shows that 1α, 25-dihydroxyvitamin d <dig> can also expand foxp3hi tregs  <cit> .
the type of treg emerging depended on the concentration of 1α, 25-dihydroxyvitamin d <dig> in cultures, and the cytokine milieu and essentially no co-expression of foxp <dig> and il- <dig> was observed, although both populations exhibited comparable suppressive activity  <cit> .
the present findings are in keeping with that data in that a relatively higher concentration favoured foxp3hi tregs , and in these conditions il- <dig> producing t cells were not detected .
our previous report suggested that the major effect of 1α, 25-dihydroxyvitamin d <dig> was to suppress proliferation of effector t cells and maintain foxp <dig> 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  <cit> .
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  <cit> .
further studies are required to examine the effects of depig-pol extracts on tregs in vivo, and to determine whether 1α, 25-dihydroxyvitamin d <dig> can enhance tolerance in a clinical setting of sit.
CONCLUSIONS
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 d <dig> further reduces effector t cell activation by both allergen extracts, and may be a useful adjuvant for sit.

 specific allergen immunotherapy 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. however, subcutaneous injection of immunotherapy extracts carries a risk of anaphylaxis, whilst sublingual therapy 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. such allergoids include extracts treated with acid, prior to polymerisation with glutaraldehyde. these extracts showed efficacy in clinical trials for both birch and grass pollen-related allergic rhinitis, and house dust mite-related asthma. in these studies and large observational series, side effects were minimal. the resulting depigmented-polymerised allergen extracts are of very large molecular mass, and have at least times reduced ige binding in vitro in competition assays and in vivo in skin prick testing. 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, 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. whilst allergoids have reduced ige binding which may improve safety, it has also been suggested that they have reduced t cell stimulating activity which could compromise efficacy. 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. however, the immunological efficacy of sit most likely relates to induction of tolerance to allergens through interactions with regulatory t cells. studies with unmodified allergen extracts suggest increased numbers and activity of both cd4+cd25hifoxp3hi t cells and il- producing t cells after sit treatment, together with induction of the il-10-dependent antibody igg. 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 d which can expand both induced il-10-producing and foxp3+ treg in vitro, and improved effects of immunotherapy in animal models. vitamin d status and/or treatment of human volunteers with vitamin d derivatives correlated with or increased foxp3+ t cell frequency in the periphery and the airway in asthmatics and il- producing treg in the periphery. 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 d  depigmented-polymerised allergen extracts stimulate less t cell proliferation than native extracts, but similar cytokine production production of depigmented-polymerised extracts involves two steps which might alter t cell reactivity. therefore initial timecourse and dose-response experiments were performed to assess proliferation of pbmc from atopic donors in cultures stimulated with native, depigmented and depigmented-polymerised house dust mite and grass pollen extracts. maximal proliferation was seen at day for all extracts except for depig-pol hdm extract which was highest at day. note that pbmc viability was comparable between hdm native and depig-pol stimulated cultures, % and %, respectively. data for some of the experiments with grass pollen extracts has been presented elsewhere. dermatophagoides pteronyssinus phleum pratense donor clinical characteristics are shown in brackets, specific ige concentration and cap class for each allergen, and size of skin prick test weal in mm. note equal proportions of male and female subjects were studied. mean of allergic patients was 38 years old; mean age of nonatopic individuals was 40 years old. proliferation for depigmented hdm and grass extracts was very similar to that seen for native extracts, but proliferation was signficantly less for depig-pol extracts of both hdm and grass when compared to native unmodified extracts. for this reason, further comparisons were between native and depig-pol extracts. cytokine production was also maximal at day seven for all cytokines except il- which peaked at day. seven days of culture was therefore used for further comparisons. production of il- il- and il- did not differ significantly between cultures stimulated with native or depigmented-polymerised extracts of either hdm or grass, whilst there was a non-significant trend for reduced ifnγ and significantly reduced il- in depig-pol-stimulated cultures compared to native extract. depigmented-polymerised allergen extracts expand regulatory t cells more than effector t cells in culture to examine effects of native and depigmented-polymerised extracts in stimulation of regulatory or effector cells we examined expression of cd foxp and cd after 7 days of culture of pbmc. gating strategy was as described, to identify cells that we have previously shown to be regulatory in cultures. for both hdm and grass, depig-pol extracts expanded similar numbers of cd4+cd25+foxp3hicd127lo compared to native extracts, but reduced numbers of cd4+cd25+foxp3locd127+. when expressed as a ratio of cd25+foxp3hi cd127lo to cd25+foxp3locd127+ cells this was significantly higher for depig-pol extracts of both hdm and grass when compared to cultures stimulated with native extracts. addition of 1α, 25-dihydroxyvitamin d to cultures further favours cd25hifoxp3hicd127lo t cells addition of 1α, 25-dihydroxyvitamin d at 10-7 m further reduced expansion of effector cells in cultures stimulated with both native and depig-pol extracts of hdm and grass, whilst numbers of cd4+cd25+foxp3hicd127lo regulatory cells were not reduced. thus the ratio of regulatory to effector cells after cultures was further increased by addition of 1α, 25-dihydroxyvitamin d. in addition 1α, 25-dihydroxyvitamin d reduced effector cytokines, particularly il- and ifnγ, but did not significantly affect il- production. discussion these data suggest reduced proliferation and cytokine production 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 d 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. 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. analysis of responses of t cell lines and clones suggested variable loss of t cell epitopes in allergoid extracts compared to native extracts. 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 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. interestingly, our data both here and in previous studies suggest that depigmented-polymerised extracts and 1α, 25-dihydroxyvitamin d both favour foxp3hi t cells. initial reports suggested that 1α, 25-dihydroxyvitamin d acted to expand il- producing t cells. however, our recent data shows that 1α, 25-dihydroxyvitamin d can also expand foxp3hi tregs. the type of treg emerging depended on the concentration of 1α, 25-dihydroxyvitamin d in cultures, and the cytokine milieu and essentially no co-expression of foxp and il- was observed, although both populations exhibited comparable suppressive activity. the present findings are in keeping with that data in that a relatively higher concentration favoured foxp3hi tregs, and in these conditions il- producing t cells were not detected. our previous report suggested that the major effect of 1α, 25-dihydroxyvitamin d was to suppress proliferation of effector t cells and maintain foxp 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. further studies are required to examine the effects of depig-pol extracts on tregs in vivo, and to determine whether 1α, 25-dihydroxyvitamin d can enhance tolerance in a clinical setting of sit.  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 d further reduces effector t cell activation by both allergen extracts, and may be a useful adjuvant for sit. 
depigmented-polymerised allergen extracts stimulated less proliferation of pbmc, and reduced effector cell numbers after 7 days in culture than did native extracts. however, the frequency of foxp3+ tregs in cultures were similar to those seen with native extract so that ratios of regulatory to effector t cells were significantly increased in cultures stimulated with depigmented-polymerised extracts. we compared the capacity of modified versus unmodified allergen extracts of grass pollen and house dust mite to stimulate proliferation/cytokine production and to modulate treg/effector t cell frequency in cultures of peripheral blood mononuclear cells, from volunteers sensitised to both allergens in vitro. depigmented-polymerised allergen extracts appear to favour treg expansion over activation of effector t cells and this may relate to their demonstrated efficacy and safety in sit. 1α, 25-dihydroxyvitamin d further reduces effector t cell activation by allergen extracts and may be a useful adjuvant for sit. it is suggested that these allergoid extracts also have reduced t cell activity, which could compromise clinical efficacy. allergen immunotherapy is the only treatment for allergic disease capable of modifying disease long term. effective sit is thought to act through regulatory t cells rather than activation of effector t cells. addition of 1α, 25-dihydroxyvitamin d further favoured treg, and reduced effector cytokine production, but not interleukin- 
 allergen immunotherapy is the only treatment for allergic disease capable of modifying disease long term. to reduce the risk of anaphylaxis from sit, allergen-extracts have been modified by polymerisation with glutaraldehyde to reduce ige binding. it is suggested that these allergoid extracts also have reduced t cell activity, which could compromise clinical efficacy. effective sit is thought to act through regulatory t cells rather than activation of effector t cells. there is no published data on the activity of modified extracts on tregs.  we compared the capacity of modified versus unmodified allergen extracts of grass pollen and house dust mite to stimulate proliferation/cytokine production and to modulate treg/effector t cell frequency in cultures of peripheral blood mononuclear cells, from volunteers sensitised to both allergens in vitro. depigmented-polymerised allergen extracts stimulated less proliferation of pbmc, and reduced effector cell numbers after 7 days in culture than did native extracts. however, the frequency of foxp3+ tregs in cultures were similar to those seen with native extract so that ratios of regulatory to effector t cells were significantly increased in cultures stimulated with depigmented-polymerised extracts. addition of 1α, 25-dihydroxyvitamin d further favoured treg, and reduced effector cytokine production, but not interleukin-  depigmented-polymerised allergen extracts appear to favour treg expansion over activation of effector t cells and this may relate to their demonstrated efficacy and safety in sit. 1α, 25-dihydroxyvitamin d further reduces effector t cell activation by allergen extracts and may be a useful adjuvant for sit. allergen extractdepigmented-polymerisedimmunotherapyregulatory t cellvitamin d 