x c57bl/6 or cd11c.dog x 4get il-4-egfp ( mohrs et al . , 2001 ) f1 mice , which were maintained under specific pathogen  free conditions at the university of edinburgh animal facilities and used at 812 wk of age . biomphalaria glabrata snails infected with s. mansoni were obtained from f. lewis ( biomedical research institute , rockville , md ) . mice were immunized s.c . in each rear footpad with 2,500 eggs in 50 l pbs . for cd11c depletion , mice were injected i.p . daily with 8 ng / g diptheria toxin ( sigma - aldrich ) in pbs or with pbs alone . for basophil depletion , 1 , 1 , and 3 with 10 g hamster igg or anti - fcr1 ( mar-1 ; ebioscience ) ab . endotoxin - free soluble egg ag ( sea ) from s. mansoni was prepared in - house as previously described ( macdonald et al . , 2001 ) . all experiments were approved under a project license granted by the home office ( uk ) and conducted in accordance with local guidelines . spleens and lns were diced and digested at 37c for 15 min with 1.75 wunsch units / ml liberase ci ( roche ) and 80 kunitz units / ml dnase i type vi ( sigma - aldrich ) in hbss ( sigma - aldrich ) containing 50 u / ml penicillin and 50 g / ml streptomycin ( invitrogen ) . 100 l 0.1 m , ph 7.3 , edta ( ambion ) stop solution per milliliter was then added , and the tube was topped off with dme containing 50 u / ml penicillin and 50 g / ml streptomycin . the resulting suspension was then passed through a 70-m cell strainer to obtain a single - cell suspension . for splenocytes , livers were perfused , diced , and digested at 37c for 30 min using the aforementioned method . the digested liver was then passed through a 100-m cell strainer with the aid of a syringe plunger . leukocytes were separated from other liver cells by resuspension in 33% isotonic percoll ( ge healthcare ) and centrifugated at 700 g. pelleted cells were resuspended and passed through a 40-m cell strainer to obtain a single - cell suspension and remove s. mansoni eggs . single - cell suspensions of splenocytes ( 2  10 cell / ml ) or ln or liver cells ( 10 cell / ml ) were cultured in x - vivo 15 medium ( biowhittaker ) containing 2 mm l - glutamine and 50 m 2-me ( invitrogen ) in 96-well plates at 37c in a humidified atmosphere of 5% co2 with or without 15 g / ml sea . supernatants were harvested from the cultures after 72 h. purified cd4 t cell were restimulated with irradiated splenocytes ( 1 cd4 t:10 splenocytes ) . purifications were performed using miltenyi biotec cd4 beads and ls columns according to the manufacturer s instructions ( > 90% purity ) . after fcr - block ( 2.4g2 ) , cell surface markers for several different cell populations were analyzed . staining for cd11c subsets was performed using the following mab conjugations : cd11c - apc , mhcii - percp / cy5.5 , b220-apc / efluor780 , cd11b - pe , and cd8-pe / cy7 . in pln and spleen , dcs were defined as cd11cmhcii , and in the liver as cd11cmhciicd11b . for egg injection experiments , il-4-egfp basophils were identified by exclusion of cells expressing cd4-apc , ccr3-pe , cd117-percp / cy5.5 , or b220-apc / efluor780 . for infection experiments , fcr1-fitc basophils were identified after surface staining to exclude cells expressing siglec - f - pe , cd19-alexa fluor 700 or efluor450 , and cd117-percp / cy5.5 . macrophages were identified using f4/80-fitc , cd11b - pe , and cd11c - apc , and eosinophils with siglec - f - pe and gr-1-apc . il-4-egfp was measured on ln cells directly ex vivo , with naive mice expressing negligible levels of egfp ( 0.51.5% of cd4 t cells ) . intracellular cytokine staining of splenocytes or liver leukocytes ex vivo was set up as follows : cells were rested overnight , stimulated with 10 ng / ml pma and 1 g / ml ionomycin ( sigma - aldrich ) for 2 h , and then stimulated with golgi stop ( bd ) for an additional 3 h. after restimulation , intracellular cytokine staining was set - up as follows : after 72 h , 10 ng / ml murine ril-2 ( peprotech ) was added to pln cell cultures . after an additional 18 h , cells were stimulated with pma and ionomycin and treated with golgistop . after fcr - block , cells were stained with cd4-fitc or cd4-apc / efluor780 , fixed with 1% isotonic formaldehyde , permeabilized with bd perm / wash buffer ( bd ) , and stained with il-4-apc , ifn--alexa fluor 488 , or ifn--apc in bd perm / wash buffer . samples were acquired using a facs lsr ii or facscanto ii flow cytometer using bd facsdiva software and analyzed with flowjo v.8 software ( tree star , inc . ) . cytokines and serum antibodies were measured by elisa . paired capture and detection abs ( produced from hybridomas in - house or purchased from r&d systems , bd , or ebioscience ) were used for analysis of murine il-4 , il-5 , il-10 , il-13 , and ifn-. plates ( nunc maxisorp ) were washed with 0.05% tween 20 in pbs and blocked with 10% ncs / pbs . recombinant cytokine standards ( peprotech or bd ) were used to determine quantity using a standard curve . plates were developed by incubation with 50 l 1:1,000 hr - peroxidase  labeled streptavidin ( kpl ) , and absorbance was read at 450 nm after addition of 100 l tmb substrate solution ( sigma - aldrich ) and 100 l 0.18 m h2so4 using a laboratory systems multiskan ascent plate reader . total ige was measured using paired capture and detection antibodies ( bd ) and recombinant murine ige ( bd ) to assess quantity using a standard curve . sea - specific igg1 , igg2c , and igm ab titers were determined using endpoint dilutions measured by elisa . plates were coated with 0.25 g / well of sea in 0.1 m , ph 9.6 , carbonate / bicarbonate buffer , washed , and blocked with 1% bsa / pbs . absorbance at 405 nm was determined as above after addition of 50 l pnpp substrate ( southernbiotech ) . total rna was extracted from spleen , liver , or gut ( illeal ) tissue samples that had been snap frozen individually in 0.5 ml of trizol reagent ( invitrogen ) . samples were defrosted , homogenized using a qiagen tissuelyser , and total rna was extracted as per manufacturer instructions . cdna was then generated from individual rna samples using superscript iii reverse transcriptase and oligo ( dt ) ( invitrogen ) . quantitative rt - pcr was performed using a light cycler 480 ii real - time pcr machine and software 1.5.0 sp3 ( roche ) . the relative concentration of mrna for the genes of interest was assessed using lightcycler - dna master sybr green i ( roche ) and by comparison to a serially diluted standard of pooled cdna . the mean concentration for each sample was determined from two technical repeats , with mrna levels for each sample normalized to gapdh to give a value for the gene of interest in arbitrary units . were as follows : cd11c , sense 5-atggagcctcaagacaggac-3 , antisense 5-ggatctgggatgctgaaatc-3 ; il-4 , sense 5-gagagatcatcggcattttga-3 , anti - sense 5-tctgtggtgttcttcgttgc-3 ; gapdh , sense 5-aatgtgtccgtcgtggatct-3 , anti - sense 5-cccagctctccccatacata-3. statistical analyses were performed using graphpad prism 4 software . experimental groups were compared by one - way anova for the absence of significant variance before collation . student s t tests were used where necessary to determine if there were any significant differences between sample groups . * , p < 0.05 ; * * , p < 0.01 ; * * * , p < 0.001 . fig . s1 shows reduced cd4 t cell ifn- production after cd11c depletion of schistosome egg - injected or infected mice . s3 depicts impaired il-4 mrna expression in spleens and livers of infected mice after cd11c depletion . s4 demonstrates that cd11c depletion impacts both effector and foxp3 regulatory t cells during infection . s5 shows that cd11c depletion during infection alters the proportions of splenic b cells , and reduces serum levels of ige . s6 illustrates that the proportions of macrophages , eosinophils , and basophils found during infection are not reduced after cd11c depletion . x c57bl/6 or cd11c.dog x 4get il-4-egfp ( mohrs et al . , 2001 ) f1 mice , which were maintained under specific pathogen  free conditions at the university of edinburgh animal facilities and used at 812 wk of age . biomphalaria glabrata snails infected with s. mansoni were obtained from f. lewis ( biomedical research institute , rockville , md ) . mice were immunized s.c . in each rear footpad with 2,500 eggs in 50 l pbs . for cd11c depletion , mice were injected i.p . daily with 8 ng / g diptheria toxin ( sigma - aldrich ) in pbs or with pbs alone . for basophil depletion , 1 , 1 , and 3 with 10 g hamster igg or anti - fcr1 ( mar-1 ; ebioscience ) ab . endotoxin - free soluble egg ag ( sea ) from s. mansoni was prepared in - house as previously described ( macdonald et al . , 2001 ) . all experiments were approved under a project license granted by the home office ( uk ) and conducted in accordance with local guidelines . spleens and lns were diced and digested at 37c for 15 min with 1.75 wunsch units / ml liberase ci ( roche ) and 80 kunitz units / ml dnase i type vi ( sigma - aldrich ) in hbss ( sigma - aldrich ) containing 50 u / ml penicillin and 50 g / ml streptomycin ( invitrogen ) . 100 l 0.1 m , ph 7.3 , edta ( ambion ) stop solution per milliliter was then added , and the tube was topped off with dme containing 50 u / ml penicillin and 50 g / ml streptomycin . the resulting suspension was then passed through a 70-m cell strainer to obtain a single - cell suspension . for splenocytes , livers were perfused , diced , and digested at 37c for 30 min using the aforementioned method . the digested liver was then passed through a 100-m cell strainer with the aid of a syringe plunger . leukocytes were separated from other liver cells by resuspension in 33% isotonic percoll ( ge healthcare ) and centrifugated at 700 g. pelleted cells were resuspended and passed through a 40-m cell strainer to obtain a single - cell suspension and remove s. mansoni eggs . single - cell suspensions of splenocytes ( 2  10 cell / ml ) or ln or liver cells ( 10 cell / ml ) were cultured in x - vivo 15 medium ( biowhittaker ) containing 2 mm l - glutamine and 50 m 2-me ( invitrogen ) in 96-well plates at 37c in a humidified atmosphere of 5% co2 with or without 15 g / ml sea . supernatants were harvested from the cultures after 72 h. purified cd4 t cell were restimulated with irradiated splenocytes ( 1 cd4 t:10 splenocytes ) . purifications were performed using miltenyi biotec cd4 beads and ls columns according to the manufacturer s instructions ( > 90% purity ) . after fcr - block ( 2.4g2 ) , cell surface markers for several different cell populations were analyzed . staining for cd11c subsets was performed using the following mab conjugations : cd11c - apc , mhcii - percp / cy5.5 , b220-apc / efluor780 , cd11b - pe , and cd8-pe / cy7 . in pln and spleen , dcs were defined as cd11cmhcii , and in the liver as cd11cmhciicd11b . for egg injection experiments , il-4-egfp basophils were identified by exclusion of cells expressing cd4-apc , ccr3-pe , cd117-percp / cy5.5 , or b220-apc / efluor780 . for infection experiments , fcr1-fitc basophils were identified after surface staining to exclude cells expressing siglec - f - pe , cd19-alexa fluor 700 or efluor450 , and cd117-percp / cy5.5 . macrophages were identified using f4/80-fitc , cd11b - pe , and cd11c - apc , and eosinophils with siglec - f - pe and gr-1-apc . il-4-egfp was measured on ln cells directly ex vivo , with naive mice expressing negligible levels of egfp ( 0.51.5% of cd4 t cells ) . intracellular cytokine staining of splenocytes or liver leukocytes ex vivo was set up as follows : cells were rested overnight , stimulated with 10 ng / ml pma and 1 g / ml ionomycin ( sigma - aldrich ) for 2 h , and then stimulated with golgi stop ( bd ) for an additional 3 h. after restimulation , intracellular cytokine staining was set - up as follows : after 72 h , 10 ng / ml murine ril-2 ( peprotech ) was added to pln cell cultures . after an additional 18 h , cells were stimulated with pma and ionomycin and treated with golgistop . after fcr - block , cells were stained with cd4-fitc or cd4-apc / efluor780 , fixed with 1% isotonic formaldehyde , permeabilized with bd perm / wash buffer ( bd ) , and stained with il-4-apc , ifn--alexa fluor 488 , or ifn--apc in bd perm / wash buffer . samples were acquired using a facs lsr ii or facscanto ii flow cytometer using bd facsdiva software and analyzed with flowjo v.8 software ( tree star , inc . ) . cytokines and serum antibodies were measured by elisa . paired capture and detection abs ( produced from hybridomas in - house or purchased from r&d systems , bd , or ebioscience ) were used for analysis of murine il-4 , il-5 , il-10 , il-13 , and ifn-. plates ( nunc maxisorp ) were washed with 0.05% tween 20 in pbs and blocked with 10% ncs / pbs . recombinant cytokine standards ( peprotech or bd ) were used to determine quantity using a standard curve . plates were developed by incubation with 50 l 1:1,000 hr - peroxidase  labeled streptavidin ( kpl ) , and absorbance was read at 450 nm after addition of 100 l tmb substrate solution ( sigma - aldrich ) and 100 l 0.18 m h2so4 using a laboratory systems multiskan ascent plate reader . total ige was measured using paired capture and detection antibodies ( bd ) and recombinant murine ige ( bd ) to assess quantity using a standard curve . sea - specific igg1 , igg2c , and igm ab titers were determined using endpoint dilutions measured by elisa . plates were coated with 0.25 g / well of sea in 0.1 m , ph 9.6 , carbonate / bicarbonate buffer , washed , and blocked with 1% bsa / pbs . absorbance at 405 nm was determined as above after addition of 50 l pnpp substrate ( southernbiotech ) . total rna was extracted from spleen , liver , or gut ( illeal ) tissue samples that had been snap frozen individually in 0.5 ml of trizol reagent ( invitrogen ) . samples were defrosted , homogenized using a qiagen tissuelyser , and total rna was extracted as per manufacturer instructions . cdna was then generated from individual rna samples using superscript iii reverse transcriptase and oligo ( dt ) ( invitrogen ) . quantitative rt - pcr was performed using a light cycler 480 ii real - time pcr machine and software 1.5.0 sp3 ( roche ) . the relative concentration of mrna for the genes of interest was assessed using lightcycler - dna master sybr green i ( roche ) and by comparison to a serially diluted standard of pooled cdna . the mean concentration for each sample was determined from two technical repeats , with mrna levels for each sample normalized to gapdh to give a value for the gene of interest in arbitrary units . were as follows : cd11c , sense 5-atggagcctcaagacaggac-3 , antisense 5-ggatctgggatgctgaaatc-3 ; il-4 , sense 5-gagagatcatcggcattttga-3 , anti - sense 5-tctgtggtgttcttcgttgc-3 ; gapdh , sense 5-aatgtgtccgtcgtggatct-3 , anti - sense 5-cccagctctccccatacata-3. statistical analyses were performed using graphpad prism 4 software . experimental groups were compared by one - way anova for the absence of significant variance before collation . student s t tests were used where necessary to determine if there were any significant differences between sample groups . * , p < 0.05 ; * * , p < 0.01 ; * * * , p < 0.001 . fig . s1 shows reduced cd4 t cell ifn- production after cd11c depletion of schistosome egg - injected or infected mice . s3 depicts impaired il-4 mrna expression in spleens and livers of infected mice after cd11c depletion . s4 demonstrates that cd11c depletion impacts both effector and foxp3 regulatory t cells during infection . s5 shows that cd11c depletion during infection alters the proportions of splenic b cells , and reduces serum levels of ige . s6 illustrates that the proportions of macrophages , eosinophils , and basophils found during infection are not reduced after cd11c depletion . x c57bl/6 or cd11c.dog x 4get il-4-egfp ( mohrs et al . , 2001 ) f1 mice , which were maintained under specific pathogen  free conditions at the university of edinburgh animal facilities and used at 812 wk of age . biomphalaria glabrata snails infected with s. mansoni were obtained from f. lewis ( biomedical research institute , rockville , md ) . mice were immunized s.c . in each rear footpad with 2,500 eggs in 50 l pbs . for cd11c depletion , mice were injected i.p . daily with 8 ng / g diptheria toxin ( sigma - aldrich ) in pbs or with pbs alone . for basophil depletion , 1 , 1 , and 3 with 10 g hamster igg or anti - fcr1 ( mar-1 ; ebioscience ) ab . endotoxin - free soluble egg ag ( sea ) from s. mansoni was prepared in - house as previously described ( macdonald et al . , 2001 ) . all experiments were approved under a project license granted by the home office ( uk ) and conducted in accordance with local guidelines . were diced and digested at 37c for 15 min with 1.75 wunsch units / ml liberase ci ( roche ) and 80 kunitz units / ml dnase i type vi ( sigma - aldrich ) in hbss ( sigma - aldrich ) containing 50 u / ml penicillin and 50 g / ml streptomycin ( invitrogen ) . 100 l 0.1 m , ph 7.3 , edta ( ambion ) stop solution per milliliter was then added , and the tube was topped off with dme containing 50 u / ml penicillin and 50 g / ml streptomycin . the resulting suspension was then passed through a 70-m cell strainer to obtain a single - cell suspension . for splenocytes , livers were perfused , diced , and digested at 37c for 30 min using the aforementioned method . the digested liver was then passed through a 100-m cell strainer with the aid of a syringe plunger . leukocytes were separated from other liver cells by resuspension in 33% isotonic percoll ( ge healthcare ) and centrifugated at 700 g. pelleted cells were resuspended and passed through a 40-m cell strainer to obtain a single - cell suspension and remove s. mansoni eggs . single - cell suspensions of splenocytes ( 2  10 cell / ml ) or ln or liver cells ( 10 cell / ml ) were cultured in x - vivo 15 medium ( biowhittaker ) containing 2 mm l - glutamine and 50 m 2-me ( invitrogen ) in 96-well plates at 37c in a humidified atmosphere of 5% co2 with or without 15 g / ml sea . supernatants were harvested from the cultures after 72 h. purified cd4 t cell were restimulated with irradiated splenocytes ( 1 cd4 t:10 splenocytes ) . purifications were performed using miltenyi biotec cd4 beads and ls columns according to the manufacturer s instructions ( > 90% purity ) . after fcr - block ( 2.4g2 ) , cell surface markers for several different cell populations were analyzed . staining for cd11c subsets was performed using the following mab conjugations : cd11c - apc , mhcii - percp / cy5.5 , b220-apc / efluor780 , cd11b - pe , and cd8-pe / cy7 . in pln and spleen , dcs were defined as cd11cmhcii , and in the liver as cd11cmhciicd11b . for egg injection experiments , il-4-egfp basophils were identified by exclusion of cells expressing cd4-apc , ccr3-pe , cd117-percp / cy5.5 , or b220-apc / efluor780 . for infection experiments , fcr1-fitc basophils were identified after surface staining to exclude cells expressing siglec - f - pe , cd19-alexa fluor 700 or efluor450 , and cd117-percp / cy5.5 . macrophages were identified using f4/80-fitc , cd11b - pe , and cd11c - apc , and eosinophils with siglec - f - pe and gr-1-apc . il-4-egfp was measured on ln cells directly ex vivo , with naive mice expressing negligible levels of egfp ( 0.51.5% of cd4 t cells ) . intracellular cytokine staining of splenocytes or liver leukocytes ex vivo was set up as follows : cells were rested overnight , stimulated with 10 ng / ml pma and 1 g / ml ionomycin ( sigma - aldrich ) for 2 h , and then stimulated with golgi stop ( bd ) for an additional 3 h. after restimulation , intracellular cytokine staining was set - up as follows : after 72 h , 10 ng / ml murine ril-2 ( peprotech ) was added to pln cell cultures . after an additional 18 h , cells were stimulated with pma and ionomycin and treated with golgistop . after fcr - block , cells were stained with cd4-fitc or cd4-apc / efluor780 , fixed with 1% isotonic formaldehyde , permeabilized with bd perm / wash buffer ( bd ) , and stained with il-4-apc , ifn--alexa fluor 488 , or ifn--apc in bd perm / wash buffer . samples were acquired using a facs lsr ii or facscanto ii flow cytometer using bd facsdiva software and analyzed with flowjo v.8 software ( tree star , inc . ) . cytokines and serum antibodies were measured by elisa . paired capture and detection abs ( produced from hybridomas in - house or purchased from r&d systems , bd , or ebioscience ) were used for analysis of murine il-4 , il-5 , il-10 , il-13 , and ifn-. plates ( nunc maxisorp ) were washed with 0.05% tween 20 in pbs and blocked with 10% ncs / pbs . recombinant cytokine standards ( peprotech or bd ) were used to determine quantity using a standard curve . plates were developed by incubation with 50 l 1:1,000 hr - peroxidase  labeled streptavidin ( kpl ) , and absorbance was read at 450 nm after addition of 100 l tmb substrate solution ( sigma - aldrich ) and 100 l 0.18 m h2so4 using a laboratory systems multiskan ascent plate reader . total ige was measured using paired capture and detection antibodies ( bd ) and recombinant murine ige ( bd ) to assess quantity using a standard curve . sea - specific igg1 , igg2c , and igm ab titers were determined using endpoint dilutions measured by elisa . plates were coated with 0.25 g / well of sea in 0.1 m , ph 9.6 , carbonate / bicarbonate buffer , washed , and blocked with 1% bsa / pbs . absorbance at 405 nm was determined as above after addition of 50 l pnpp substrate ( southernbiotech ) . total rna was extracted from spleen , liver , or gut ( illeal ) tissue samples that had been snap frozen individually in 0.5 ml of trizol reagent ( invitrogen ) . samples were defrosted , homogenized using a qiagen tissuelyser , and total rna was extracted as per manufacturer instructions . cdna was then generated from individual rna samples using superscript iii reverse transcriptase and oligo ( dt ) ( invitrogen ) . quantitative rt - pcr was performed using a light cycler 480 ii real - time pcr machine and software 1.5.0 sp3 ( roche ) . the relative concentration of mrna for the genes of interest was assessed using lightcycler - dna master sybr green i ( roche ) and by comparison to a serially diluted standard of pooled cdna . the mean concentration for each sample was determined from two technical repeats , with mrna levels for each sample normalized to gapdh to give a value for the gene of interest in arbitrary units . primers used for the detection of gene expression were as follows : cd11c , sense 5-atggagcctcaagacaggac-3 , antisense 5-ggatctgggatgctgaaatc-3 ; il-4 , sense 5-gagagatcatcggcattttga-3 , anti - sense 5-tctgtggtgttcttcgttgc-3 ; gapdh , sense 5-aatgtgtccgtcgtggatct-3 , anti - sense 5-cccagctctccccatacata-3. experimental groups were compared by one - way anova for the absence of significant variance before collation . student s t tests were used where necessary to determine if there were any significant differences between sample groups . * , p < 0.05 ; * * , p < 0.01 ; * * * , p < 0.001 . s1 shows reduced cd4 t cell ifn- production after cd11c depletion of schistosome egg - injected or infected mice . s3 depicts impaired il-4 mrna expression in spleens and livers of infected mice after cd11c depletion . s4 demonstrates that cd11c depletion impacts both effector and foxp3 regulatory t cells during infection . s5 shows that cd11c depletion during infection alters the proportions of splenic b cells , and reduces serum levels of ige . s6 illustrates that the proportions of macrophages , eosinophils , and basophils found during infection are not reduced after cd11c depletion .
although dendritic cells ( dcs ) are adept initiators of cd4 + t cell responses , their fundamental importance in this regard in th2 settings remains to be demonstrated . 
 we have used cd11c  diphtheria toxin ( dtx ) receptor mice to deplete cd11c+ cells during the priming stage of the cd4 + th2 response against the parasitic helminth schistosoma mansoni . 
 dtx treatment significantly depleted cd11c+ dcs from all tissues tested , with 7080% efficacy . 
 even this incomplete depletion resulted in dramatically impaired cd4 + t cell production of th2 cytokines , altering the balance of the immune response and causing a shift toward ifn- production . 
 in contrast , basophil depletion using mar-1 antibody had no measurable effect on th2 induction in this system . 
 these data underline the vital role that cd11c+ antigen - presenting cells can play in orchestrating th2 development against helminth infection in vivo , a response that is ordinarily balanced so as to prevent the potentially damaging production of inflammatory cytokines .