plasmacytoid dendritic cells ( pdcs ) are important sensors of non - self - nucleic acids derived from bacteria or viruses and are crucial mediators of innate anti - microbial responses through the production of inflammatory cytokines and type - i ifns , . in addition , pdcs have been implicated in the development of several autoimmune diseases , including lupus , psoriasis , multiple sclerosis ( ms ) and type-1 diabetes , , , , . following abnormal release of self - dna during inflammatory processes importantly , a few years ago , the notion emerged that pdcs act not only as innate sensors but can also function as bona fide antigen ( ag ) presenting cells ( apcs ) and directly impact t cell responses . it was shown that pdcs capture and process ags , and load antigenic peptides onto mhc class i ( mhci )   and mhc class ii ( mhcii ) molecules , , . the modulation of ag - presenting pdc functions led to important consequences on t cell immunity , the outcome being highly dependent on the cytokine microenvironment . many studies , including those investigating oral tolerance and allograft models , suggest that steady - state ag - presenting pdcs exclusively promote t cell tolerance , , . although the nature of the factors controlling distinct pdc functions remains to be established , once activated , pdcs exhibit both immunogenic and tolerogenic functions . for example , using mice exhibiting a specific loss of mhcii expression by pdcs , we showed that cpg - b activated pdcs present ag and promote effector th17  cell differentiation , a property that can be exploited for anti - tumor vaccines . pro - pathogenic ag - presenting pdcs were similarly described in a mouse model of atherosclerosis in which pdcs induced pathogenic th1 cells . in addition , bst-2 mediated specific ag delivery to cpg - activated pdcs led to cytotoxic t lymphocyte ( ctl ) and th1 cell differentiation and triggered protective immunity against viral infection and tumor growth . in contrast , in the context of eae , ag targeting to pdcs via siglec - h promoted cd4 t cell anergy and inhibited cns inflammation . we previously demonstrated that in eae , pdcs present myelin ags on mhcii molecules to induce the expansion of suppressive tregs , a phenomenon correlated with disease amelioration . indoleamine 2,3-dioxygenase ( ido ) is an immunomodulatory enzyme involved in the initial and the rate - limiting step of tryptophan catabolism . upon inflammation , ido production has been shown to compromise t cell proliferation , promote t cell anergy and tregs , , . depending on the experimental context , ido can be induced either by ifn- , ifn-/ , or tgf-. ctla-4 binding to cell - surface expressed costimulatory molecules promotes ido production by pdcs through ifn- or ifn-/ signalling , , , . furthermore cd200-ig binding to his cognate receptor induces ido in an ifn-/ dependent signalling pathway . both ifn- and ifn-/ pathways result in ido immunosuppressive effects which are closely dependent on the catalytic activity of the enzyme . ctla-4-binding also promote ido in tumor contexts , but the enzyme has reveal activity in only a minor dc subpopulation expressing the marker cd19 , but none of the pdc classical markers , . ido enzymatic functions in tumor dln - sorted pdcs have been correlated to in  vitro treg differentiation and suppressive functions , . more recently , pallotta and colleagues described that ido pdcs induced long - lived tregs by using a tgf--dependent pathway distinct from the catalytic activity of the enzyme . in mouse a model of skin delayed - type hypersensitivity , they shown that whereas ifn--dependent ido enzymatic activity in pdcs leads to t cell anergy , tgf- induced ido phosphorylation results in increased treg frequencies . it is so far unknown whether ido expression in nave pdcs pre - exists , and how it would be regulated in steady - state lns . in contrast , recent work has implicated ido expression in pdc immunoregulatory functions , including treg induction , in inflamed lns . furthermore , ido production by tumor - associated pdcs has been correlated to in  vitro treg - mediated suppression . however , the nature of the cells expressing ido , as well as the impact on treg functions in chronic inflammatory diseases , such as autoimmune disorders , remain undetermined . here we show that in steady - state lymph nodes ( lns ) , ido is highly expressed by pdcs compared to other ln resident cells . we further established that ido expression is positively regulated in steady - state pdcs following mhcii - mediated interactions with tregs . during autoimmune disorders , such as eae , ido expression by mhcii competent pdcs is mandatory to confer suppressive functions to pdc - induced tregs . ido - competent ag - presenting pdcs promote tregs that inhibit autoimmune effector t cell responses in lns , resulting in reduced disease severity . therefore , we have identified a bidirectional interaction between pdcs and tregs that favours self - tolerance . all mice had a pure c57bl/6 background and were bred and maintained under spf conditions at geneva medical school animal facility and under eops conditions at charles river , france or at the national institutes of health , bethesda , us . dereg , ubiquitin - egfp , piii  +  iv 
 , ido 
 , bdca2-dtr , marilyn rag2 , otii rag2 
 , and rag2 
 , smarta rag1 
 , rag2 , scurfy , cd45.1 ( charles river , france ) , and 2d2   mice have been previously described . wt c57bl/6 mice were purchased from harlan laboratories ( france ) or taconic ( us ) . all procedures were approved by and performed in accordance with the guidelines of the animal research committee of geneva or of the nih . 5 to 7    10  cells were injected intravenously into sub - lethally irradiated recipient mice ( two consecutive doses of 450  cgy ) . reconstitution was assessed by analysing blood cells by flow cytometry after 68 weeks . for mixed bm chimeras , cd45.2 wt egfp and cd45.2 piii  +  iv bm cells were simultaneously transferred into irradiated cd45.1 wt recipient mice in a 1:1 ratio . active eae was induced by immunizing mice , subcutaneously in both flanks , with 100  g of mog3555 peptide ( mevgwyrspfsrvvhlyrngk , biotrend ) emulsified in incomplete freund 's adjuvant ( bd diagnosis ) supplemented with 500  g / ml mycobacterium tuberculosis h37ra ( bd diagnosis ) . at the time of immunization and 48  h later , mice also received 300  ng of pertussis toxin ( sigma - aldrich ) into the tail vein . mice were monitored daily for disease clinical symptoms , and blindly scored as follows . 1 , flaccid tail ; 2 , impaired righting reflex and hind limb weakness ; 3 , complete hind limb paralysis ; 4 , complete hind limb paralysis with partial fore limb paralysis ; 5 , moribund . for passive eae induction , encephalitogenic cd4 t cells were generated in  vitro from ln and spleen cells of 2d2 mice as described . mice received 67  ng of pertussis toxin at the day of cell injection and 48  h later . wt    wt , piii  +  iv    wt and ido    wt chimeric mice were immunized or not with mog3555  +  cfa . cd4 cd25 t cells were harvested from total skin lns ( nave ) or dlns ( day 10 after eae immunization ) and 15    10 cd4 cd25 t cells were injected intravenously into tail vein of recipient mice . eae was induced by active immunization the day after treg transfer . in some experiments , cd45.1 mice were used as recipients , and treg migration was assessed in dlns at day 3 and in dlns and sc at day 15 after eae immunization . 1.21.5    10 bm derived pdcs loaded with 10  g / ml of mog3555 were injected intravenously into tail vein of recipient mice , and eae was induced by active immunization the day after . in some experiments , eae mice were treated i.p . at indicated time points with dt ( 100  ng / mouse for bdca2-dtr and 1  g / mouse for dereg ) . treg cells were isolated from total skin lns of nave mice or from dlns of eae mice ( day 10 after immunization ) . lns were scratched and ln cells were subjected to cd4 t cell enrichment using cd4 t cell isolation kit ( miltenyi biotec ) . cd4 cd25 treg cells were next sorted using a moflowastrios ( beckman coulter ) . for qpcr experiments , pdcs , cdcs , b cells and macrophages were recovered from ln after digestion with an enzymatic mix containing collagenase d ( 1  mg / ml ) and dnase i ( 10  g / ml ) ( roche ) in hbss . b cells were isolated using anti - cd19 beads ( miltenyi biotech ) , macrophages , cdcs and pdcs were then purified , after cd3 cells depletion ( miltenyi biotech ) , by flow cytometry as cd11ccd11bly6c for macrophages , cd11cpdca1 for cdcs and cd11cpdca1siglech for pdcs , using a moflowastrios ( beckman coulter ) or bd facsaria ( bd biosciences ) . pdcs were generated from bm of wt , piii  +  iv and ido mice as previously described . briefly , bm cells were recovered from tibia and femurs of mice and cultured , after red cell lysis , for 7 days in complete rpmi medium ( 10% heat - inactivated fetal bovine serum , 50  mm 2-mercaptoethanol , 100  mm sodium pyruvate and 100  m of penicillin / streptomycin ) supplemented with 100  ng / ml of murine flt3l ( peprotech ) . cdcs were generated as pdcs from bm of wt mice , by supplementing complete rpmi medium with 20  ng / ml granulocyte - macrophage colony - stimulating factor ( peprotech ) , and activated for 24  h with 5  ng / ml lps ( enzo life sciences , inc . ) . cd4 t cells were recovered from scratched total skin lns of 2d2 mice and purified with cd4 t cell isolation kit ( miltenyi biotec ) according to manufacturer 's instructions . both cell types purity was assessed by flow cytometry using a cyan adp ( beckman coulter ) and exceeded 90% . wt and rag2 pdcs were recovered from total skin ln and spleen after digestion with the enzymatic mix described above . pdcs were isolated using the plasmacytoid dendritic cell isolation kit ii ( miltenyi biotec ) according to manufacturer 's instructions . pdcs were loaded or not with mog3555 peptide ( 10  g / ml ) . 300 000 pdcs were seeded in 48 well plates with 200 000 2d2 cd4 t cells . cells were co - cultured in complete rpmi medium for 16  h and pdcs were isolated again using the plasmacytoid dendritic cell isolation kit ii ( myltenyi biotech ) according to manufacturer 's instructions . wt bm - derived pdcs were generated in  vitro for 78 days and purified using the plasmacytoid dendritic cell isolation kit ii ( miltenyi biotec ) according to manufacturer 's instructions . cell purity was assessed by flow cytometry using a cyan adp ( beckman coulter ) and exceeded 90% . pdcs were loaded or not with mog3555 peptide ( 10  g / ml ) . 500  000 pdcs were seeded in 24 well plates with 300  000 2d2 cd4 t cells or 300  000 pdcs were seeded in 48 well plates with 100  000 2d2 cd4 t cells or 100  000 2d2 cd4 cd25 cells or 100  000 2d2 cd4 cd25 cells . cells were co - cultured in complete rpmi medium for 16  h and pdcs were isolated again using the plasmacytoid dendritic cell isolation kit ii ( miltenyi biotec ) according to manufacturer 's instructions . in  vitro treg suppressive assays wt    wt and ido    wt chimeric mice were immunized or not with mog3555  +  cfa . cd4 cd25 t cells were harvested from total skin lns ( nave ) or dlns ( day 10 after eae immunization ) and 17  000  cells were incubated with 50  000 proliferation dye - labeled 2d2 cd4 t cells ( treg:2d2 , ratio 1:3 ) and 50  000 lps activated , mog3555 loaded , bm derived cdcs for 5 days . the percentage of treg - mediated suppression was related to 0% of proliferation ( relates to 100% of suppression ) in absence of cognate mog3555 peptide . monoclonal antibodies used for flow cytometry were from : biolegend ; anti - cd11c ( n418 ) , anti - ly6c ( hk1.4 ) , anti - v11 ( kt11 ) , anti - cd45.2 ( 104 ) ; from ebioscience : anti - cd4 ( gk1.5 and rm4 - 5 ) , anti - cd69 ( h1.2f3 ) , anti - cd25 ( pc61.5 ) , anti - il10 ( jes5 - 16e3 ) anti - ter119 ( ter-119 ) , anti - foxp3 ( fjk-16s ) , anti  il-17 ( ebio17b7 ) , anti - ifn- ( xmg1.2 ) anti - siglec - h ( ebio440c ) , anti - ki67 ( sola15 ) , anti - cd45.1 ( a20 ) , anti - cd11b ( m1/70 ) , anti - cd11c ( n418 ) , anti - tcr ( h57 - 597 ) , anti - icos ( c398.4a ) , anti - cd103 ( 2e7 ) , anti cd16/32 ( 93 ) , anti - cd5 ( 537.3 ) ; from bd : anti - cd25 ( pc61 ) , anti - cd19 ( 1d3 ) , anti - cd3 ( 145 - 2c11 ) , anti - cd4 ( rm54 - 5 ) , anti - ki67 ( b56 ) and anti  ifn- ( xmg1.2 ) . gating on viable cells was performed using the fixable viability dye efluor 780 ( ebioscience ) . for flow cytometry analysis , single cell suspensions were incubated with fcblock ( anti - cd16/32 fcrii - riii ) for 10  min , at 4  c and stained with antibodies . intracellular cytokine stainings were done using the intracellular fixation & permeabilisation buffer set ( ebioscience ) or with the fix & perm kit ( bd biosciences ) for il-10 . cell proliferation was assessed by flow cytometry using anti  mouse ki67 and respective isotype control ( rat igg2a , kappa ) . for ifn- , il-17 and il-10 staining , cells were re - stimulated in complete rpmi containing pma / ionomycin , and incubated 4  h at 37  c , 5% co2 . golgi stop solution ( bd biosciences ) was added to the last 2.5  h of culture . data were acquired with a cyan adp or a gallios ( beckman coulter ) and analysed using flowjo software ( flowjo company ) . total rna was isolated and prepared with trizol reagent ( invitrogen ) and rt - pcr were performed as described . cdna was synthesized with random hexamers and m - mlv reverse transcriptase ( promega ) . pcr were performed with cfx connect real - time system ( bio rad ) and iq sybr green super - mix ( bio - rad labolatories ) . primer sequences were as follows : ido , forward , 5-ggg atg acg atg ttc gaa ag-3 and reverse 5-cag gac aca gtc tgc ata ag-3 ; gapdh , forward , 5- ccc gta gac aaa atg gtg aag -3 and reverse 5- agg tca atg aag ggg tcg ttg -3. significance was assessed by two - tailed mann - whitney test or by one - way anova with bonferroni post hoc test . * p  <  0.05 , * * p  <  0.01 , * * * p  < 0.001 , * * * * p  <  0.0001 , ns  =  non significant . we analysed ido mrna expression in different cell subtypes sorted from steady - state lns . ido mrna was expressed to far greater levels ( > 5 fold ) by pdcs ( cd11cpdca-1 ) , than by cdcs ( cd11cpdca-1 ) , b cells ( cd11ccd19 ) , and macrophages ( cd11ccd11blyc6 ) ( fig . consistently , ido preferential expression by steady - state ln pdcs compared to cdcs was also observed in balb / c mice , ruling out a mouse c57bl/6 strain restricted effect ( supplementary fig . ido expression by pdcs was shown to be induced by t - cell produced cytokines , such as ifn- and tgf- , . to determine whether , in steady state , t cells are involved in the regulation of ido expression by pdcs , we measured ido mrna in rag2 mice , which are devoid of t cells . compared to wt mice , pdcs from steady - state lns of rag2 exhibited a significant reduction of ido mrna , which was in fine comparable to levels obtained in ido pdcs ( fig . we next tested whether interactions with t cell were required for ido induction in pdcs of nave mice . we examined ido expression in pdcs from lns of mice expressing a monoclonal population of cd4 t cells specific to ova peptide , ot - ii rag2 mice . ido expression was significantly reduced and was comparable to what was seen in rag2 mice ( fig . thus , ot - ii transgenic cd4 t cells were insufficient at inducing ido in pdcs . this result might be explained by a low tcr self - reactivity for mhcii molecules of the otii transgenic cd4 t cells . to test the possibility that distinct tcr self - reactivity would exhibit differential ability to induce ido expression in pdcs , we isolated pdcs from different cd4 tcr transgenic rag1 mice that express distinct levels of cd5 , which reflects clone - specific strength self - reactivity . each tcr transgenic t cell population display a specific surface amount of cd5 that covered a range from low ( marilyn , tcr specific for hy ) , medium ( smarta , tcr specific for lcmv ) , to high ( and , tcr specific for pigeon cytochrome c ) ( fig . ido mrna expression was reduced in ln pdcs isolated from all tcr transgenic rag mice compared to wt mice ( fig . 1c ) , showing that ido induction in pdcs is independent of tcr self - reactivity . an alternative hypothesis which might explain why ido expression in negligible is a potential specific requirement of tregs in regulating ido expression in pdcs , this population being almost absent in lns of the tcr transgenic t cell mice tested ( fig . consistently , mog3555-loaded rag2 pdcs , exhibit a significant restoration of ido expression when co - cultured with 2d2 tcr tg cd4 t cells , which contain a low but significant frequency of foxp3 cells ( fig . moreover , pdcs isolated from lns of scurfy mice , which are devoid of foxp3 tregs , presented a significant reduction of ido mrna expression compared to wt pdcs ( fig . thus , our data suggest that tregs significantly contribute to induce ido expression by pdcs in steady - state ln . we next evaluated whether ag - specific pdc - t cell contacts were required to promote ido expression in pdcs . to do so , cultures were done using in  vitro generated bm - derived pdcs , for which ido mrna levels were found to be negligible , reinforcing the idea that a crosstalk with t cells is indeed required for pdcs to competently express ido ( fig . 1 g ) . when co - cultured with 2d2 tcr tg cd4 t cells , a modest increase in ido mrna expression was observed in bm - derived pdcs ( fig . ido expression was further increased by 2 fold when pdcs were previously loaded with mog3555 peptide , reaching similar levels of expression as pdcs isolated from wt lns ( fig . 1 g ) , therefore , mhcii - restricted ag - specific interactions with cd4 t cells significantly contribute to the induction of ido expression in pdcs . to test a role for tregs in this process , we repeated co - culture experiments using 2d2 tcr tg cd4 t cells that were separated into in cd25 and cd25 populations . while 2d2 tcr tg cd4cd25 cells were incompetent at inducing ido , 2d2 tcr tg cd4cd25 cells significantly enhanced ido mrna levels in pdcs loaded with mog3555 ( fig . pdcs co - cultured with total 2d2 tcr tg cd4 t cells exhibit a slight , but not significant increase in ido mrna levels compared to pdcs co - cultured with 2d2 tcr tg cd4cd25 cells , suggesting that although tregs are required to promote ido , non - treg cd4 t cells might also contribute to this process ( fig . 1h ) . to clearly demonstrate that mhcii - restricted ag specific interactions between pdcs and t cells are necessary to induce ido in pdcs , we used genetically deficient mice that selectively lack mhcii expression by pdcs . these mice have been described before and are deficient for the promoters iii and iv ( piii  +  iv ) of ciita , the master regulator for mhcii expression . in mice , ciita is under the control of cell specific promoters , pi , piii and piv ,   ( supplementary fig .  2 ) . absence of piv leads to mhcii abrogation on cortical thymic epithelial cells ( ctecs ) , resulting in the lack of cd4 t cell positive selection . to restore cd4 t cell thymic positive selection by mhcii competent ctecs , bone marrow ( bm ) cell precursors from piii +  iv mice need to be injected into irradiated wt recipients ( piii  +  iv    wt ) and compared to wt    wt controls . piii  +  iv    wt mice exhibiting genetic deficiencies compared to wt    wt , it is possible that distinct immunological environments will affect ido expression by pdcs . therefore , to immerse mhcii competent and deficient pdcs in an identical milieu , we performed mixed bm chimeric mice using bm cells from ubi - egfp wt and piii  +  iv mice ( ratio 1:1 ) that were co - injected into irradiated recipient mice expressing the congenic marker cd45.1 ( fig . ln cells were sorted 2 months later as pdcs and cdcs from donor bm cells ( gated on cd45.2 ) and further separated as wt ( egfp ) or piii  +  iv ( egfp ) cells ( fig . ido expression by mhcii deficient pdcs was impaired compared to mhcii competent pdcs in mixed bm chimeric mice ( fig . since cells were isolated from the same lns , decreased ido expression in absence of mhcii expression by pdcs was not related to different cytokine expression profiles , but linked to a defective mhcii expression by pdcs . an alternative explanation is that ciita directly acts as a transcription factor regulating ido gene expression . however , we observed a similar reduction of ido mrna in pdcs isolated from h2-db mice ( not shown ) , ruling out this hypothesis . altogether , our data demonstrated that mhcii - restricted ag specific interactions with tregs are required for the induction of ido expression by pdcs in steady - state lns . we next evaluated whether , as in steady - state lns , pdcs remains the major expressors of ido in lns in a model of chronic inflammation . ido has been shown to exert a protective role in eae using either ido mice or ido blocking antibodies , . therefore , we have quantified ido mrna expression in different cell types sorted from lns draining the site of eae immunization ( day 10 ) in wild type ( wt ) mice . ido was predominantly ( > 5 fold ) expressed by pdcs compared to other ln cells ( fig . levels of expression were comparable to steady - state ln pdcs ( supplementary fig . pdcs and cdcs were sorted from draining lns of wt    wt and piii  +  iv    wt mice 10 days after eae induction . we observed a substantial reduction in ido expression by pdcs sorted from piii  +  iv    wt compared to wt    wt chimeras ( fig . differential ido expression by mhcii competent and mhcii deficient pdcs might be explained by distinct inflammatory cytokinic environments in wt    wt and piii  +  iv    wt mice , as the latter developed more severe eae . therefore , we performed mixed bm chimeric mice using as before bm cells from ubi - egfp wt and piii  +  iv mice ( ratio 1:1 ) that were injected into irradiated cd45.1 recipient mice . eae was induced in mixed bm chimeric mice , and ln cells were sorted 10 days after immunization as pdcs and cdcs from donor bm cells ( gated on cd45.2 ) and further separated as wt ( egfp ) or piii  +  iv ( egfp ) cells . ido expression was significantly impaired in mhcii deficient compared to mhcii competent pdcs isolated from ln of eae mice ( fig . therefore , decreased ido expression in absence of mhcii expression by pdcs was not related to different cytokine expression profiles between wt    wt and piii  +  iv    wt chimeras during eae , but rather linked to a defective mhcii expression by pdcs . altogether , our data suggest that ido expression by ln pdcs is dependent on their expression of mhcii molecules , independent on the inflammatory status of the mice , and reflects a more general regulation of ido protein expression at the mrna level . to investigate the role of pdc - induced treg in eae pathogenesis , we backcrossed dereg mice , in which foxp3 tregs can be selectively depleted using diphtheria toxin ( dt ) injection , with piii  +  iv mice and generated deregxpiii  +  iv    wt chimeras ( supplementary fig .  4 a moreover , eae clinical course was similarly aggravated in dereg    wt treated with dt and deregxpiii  +  iv    wt chimeras injected or not with dt ( supplementary fig . encephalitogenic th1 and th17 cd4 t cell frequencies were increased in dt - injected dereg    wt and deregxpiii  +  iv    wt mice compared to un - injected dereg    wt mice ( supplementary fig . this demonstrates that the depletion of tregs and the absence of mhcii expression by pdcs similarly enhance pathogenic t cell priming and disease severity . altogether , our data show that pdc - instructed tregs inhibit the priming of encephalitogenic t cells in lns during eae . we next evaluated whether ido was implicated in the regulation of encephalitogenic t cell priming during eae . knowing that mhcii deficient pdcs exhibit impaired ido expression both in steady - state and during eae ( fig .  2 , fig . 3c ) , we analysed eae development in wt    wt , piii  +  iv    wt and ido    wt chimeric mice . as before , disease was exacerbated in mice lacking mhcii on pdcs ( piii  +  iv    wt ) compared to control wt    wt animals ( fig . this could be consequent to either an absence of ag - presentation by pdcs and/or a resulting defect in ido expression by pdcs from piii  +  iv    wt mice . consistently with previously published data , , mice deficient for ido ( ido    wt ) developed aggravated eae compared to wt controls . importantly , clinical scores of ido    wt were comparable to mice lacking mhcii on pdcs ( fig . notably , we did not notice any significant impact of ido deficiency during eae induced by transferring mog3555-specific 2d2 cd4 effector t cells ( fig . s5 ) , suggesting a critical role for ido in the modulation of pathogenic t cell priming in slos during eae . to confirm this hypothesis , we measured effector cytokine production by t cells before clinical symptom appearance ( day 9 ) in ido    wt , piii  +  iv    wt and wt    wt chimeras . elevated ifn- and il-17 producing encephalitogenic cd4 t cell frequencies were observed in lns of ido deficient mice ( ido    wt ) and mice lacking mhcii expression by pdcs ( piii  +  iv    wt ) compared to wt    wt controls , ( fig . foxp3cd25 treg proliferation was impaired in piii  +  iv    wt mice , whereas it was unaffected in ido    wt mice ( fig . 4c , left ) , showing that mhcii - mediated ag presentation , but not ido expression by pdcs , promoted foxp3 treg proliferation . the proliferation of foxp3 tregs exhibiting a suppressive phenotype ( cd103icos )   ( fig . 4c middle ) , as well as the frequency of il-10 expressing foxp3 tregs ( fig . 4c right ) , were significantly decreased in lns from both piii  +  iv    wt and ido    wt compared to wt    wt mice . our results identify a new role for ido in impacting the ability of tregs to suppress encephalitogenic t cells in lns . we have shown that ido mrna was preferentially expressed by pdcs compared to other ln cells both in steady - state and during eae ( fig .  1 , fig .  3a ) . however , whether restricted ido expression by pdcs is sufficient and required to inhibit disease development still need to be clarified . to address this , we used bdca-2 dtr mice in which endogenous pdcs were depleted following dt injection . it has been demonstrated that active immunization induces dt toxicity , and may therefore confound experiments in dtr transgenic mice for neuroinflammatory models , such as eae . to avoid this problem , we generated bm chimeric mice by injecting bm cells from bdca-2 dtr into irradiated wt recipients . in these chimeric mice , no signs of toxicity or lethality in dt - treated mice upon eae induction were observed . moreover , efficient depletion of endogenous pdcs in the blood and lns of bdca-2 dtr    wt chimeras was achieved after dt injection ( not shown ) . we transferred ( i.v . ) mog3555-loaded wt or ido pdcs into dt - treated bdca-2 dtr    wt chimeras one day prior to eae induction . in these settings , following eae induction , only wt , but not ido pdcs significantly inhibited disease development ( fig . foxp3cd25 treg proliferation was increased in ln upon pdc transfer compared to control bdca-2 dtr    wt eae mice , whether transferred pdcs expressed ido or not ( fig . 5b ) , confirming that ido expression by pdcs is not involved in mhcii - dependent , pdc - mediated , treg expansion . in contrast , the frequencies of suppressive tregs co - expressing cd103 and icos ( fig . 5e ) , were significantly reduced in ln from bdca-2 dtr    wt eae mice transferred with ido pdcs compared to wt pdcs . these results demonstrate that during eae , ido expression by pdcs is required to elicit pdc - mediated suppressive tregs . we next tested the in  vivo suppressive activity of tregs primed during eae in piii  +  iv    wt , ido    wt and wt    wt chimeric mice . 6a ) , were transferred into wt hosts one day prior to eae induction ( fig . tregs primed in wt mice reduced eae incidence , delayed disease onset , and conferred significant protection as we have previously reported . in contrast , tregs primed in mice lacking mhcii expression by pdcs did not exhibit any suppressive activity ( fig . strikingly , tregs primed in absence of ido did not confer any delay in disease onset , nor reduced eae severity ( fig . frequencies of donor tregs primed in wt and ido mice were similar in dlns ( day 3 ) and dlns and sc ( day 15 ) of recipient mice . these results suggest that the absence of eae protection by tregs primed in ido deficient mice does not rely on an impaired migration of donor cells , but rather results from impaired intrinsic suppressive functions ( supplementary fig . accordingly , compared to tregs isolated from wt eae mice , tregs primed in ido deficient eae mice were significantly less efficient at suppressing mog3555 loaded dc induced 2d2 cd4 t cell proliferation in  vitro ( fig . furthermore , tregs isolated from nave wt and ido mice did not confer any suppression , further suggesting that ag - specific priming in ido context is required for the acquisition of treg suppressive functions ( fig . in  vivo , only tregs activated following eae immunization in wt mice efficiently inhibited eae incidence and severity ( day 14 ) , whereas neither tregs isolated from nave wt , nor tregs isolated from nave or immunized ido mice , conferred any protection upon transfer into eae mice ( fig . therefore , eae - mediated activation is mandatory to confer ido - dependent treg suppressive functions . altogether , our data demonstrate that ido provided by pdcs is critical for the acquisition of ag - specific treg in  vivo suppressive functions . whether pdcs exhibit tolerogenic or immunogenic apc functions in different immunogical context is an important matter of debate , and might depend on their own ability to produce inflammatory cytokines , as suggested by previous studies . pdc tolerogenicity has been correlated to their expression of ido in different settings , , . in a mouse model of skin inflammation , ido was induced in pdcs either after ifn- or tgf- stimulation , the first pathway promoting ido enzymatic activity - dependent tryptophan depletion , the second one implicating ido phosphorylation and subsequent treg induction . here we show that already in steady - state , pdcs are the major expressors of ido in lns . we further provide evidence that ido expression in nave pdcs is regulated through ag - specific mhcii - restricted interaction with foxp3 tregs . first , pdcs isolated from tcr tg mice in which cd4 t cells all recognize a non - expressed antigenic peptide , exhibited substantially reduced ido expression , independently of the self - reactivity of the tcr . second , in absence of an existing treg population in those tcr tg mice , ln pdcs exhibited a dramatic reduction in ido expression . in addition , pdcs isolated from ln of treg - deficient scurfy mice expressed very little ido mrna . we can not exclude that in scurfy mice , the non - treg cd4 t cell population , which is strongly biased towards a pro - inflammatory autoimmune repertoire compared to wt mice , might impact ido expression . nevertheless , ido expression by pdcs from t cell deficient mice was induced after co - culture with a cd4 t cells only when the later contained a descent treg population . in addition , ido restoration was further dependent on the presence of the cognate antigenic peptide . the contribution of treg in inducing ido in pdcs has been suggested before , however without real evidence for a requirement of mhcii - restricted , ag - specific interactions between pdcs and tregs . whether a particular treg subpopulation would be specifically involved in this crosstalk with pdcs to induce ido expression , and possibly the expression of other genes as well , remains to be determined . therefore , it is possible that il-10 producing tregs might be more competent to induce ido up - regulation in pdcs . our results reinforce the idea that treg exhibit their ag - specific immunomodulatory roles by impacting different cellular targets , not only by inhibiting effector t cells , inducing tumor - associated dc death   but also by promoting pdc tolerogenic functions . future investigations will determine whether other pdc - specific tolerogenic features similarly depend on interactions with tregs . ido expression by dcs has also been correlated with high levels of icos   and ctla-4 expression by t cells , and might depend , or not , on ifn- production , , , , . in mixed bm chimeric mice containing both mhcii sufficient and deficient pdcs , ido is selectively abolished in mhcii deficient pdcs , whereas the same treg populations expressing a certain level of icos , ctla-4 or other molecules are present . therefore , although icos and ctla-4 might be required to promote ido in pdcs , cells nevertheless need to express mhcii molecules . during eae , mice lacking mhcii on pdcs developed exacerbated disease , with increased ifn- and reduced tgf- production in lns compared to wt mice . mhcii - deficient pdcs nevertheless still exhibit a strong impairment in ido expression upon eae . therefore , neither ifn- , nor tgf- , was sufficient to induce ido in absence of mhcii expression by pdcs . in addition , we show that pdcs tolerogenicity in eae context is dependent on their expression of ido . first , in ido deficient mice , encephalitogenic t cell frequency was increased , and as a consequence , disease was exacerbated . second , adoptively transferred tregs inhibited eae only when isolated from actively mog3555 immunized wt , but not ido mice , suggesting that tregs need to be primed in an ido sufficient microenvironment to acquire their suppressive functions . third , in lns , ido was predominantly expressed by pdcs , in both nave and eae mice . finally , in an experimental setting in which we controlled ido expression exclusively in pdcs , we observed an inhibition of eae , as well as an increase in suppressive tregs , only when pdcs expressed ido . notably , treg induced after ag - specific interaction with ido pdcs are necessary to inhibit pathogenic t cells in lns . altogether , our results demonstrate that ido expression by pdcs is required for the generation of suppressive tregs that , upon ag - specific activation , competently inhibit encephalitogenic t cells during eae priming phase . in conclusion , we showed that nave pdcs are high ido expressors in the ln , a property acquired after a cross - talk with foxp3 tregs . by expressing ido , pdcs become competent to confer suppressive functions to tregs in the context of eae , where ag - presenting ido pdcs promote suppressive tregs that inhibit the priming of encephalitogenic th1 and th17  cells and dampen cns autoimmunity . future investigations will determine whether other pdc features and functions , apart from ido expression , are impacted following interactions with tregs . together , our data have identified a new regulatory role for tregs in shaping ag - presenting pdc functions towards tolerogenicity in autoimmunity .
plasmacytoid dendritic cells ( pdcs ) have been shown to both mediate and prevent autoimmunity , and the regulation of their immunogenic versus tolerogenic functions remains incompletely understood . here 
 we demonstrate that , compared to other cells , pdcs are the major expressors of indoleamine-2,3-dioxygenase ( ido ) in steady - state lymph nodes ( lns ) . 
 ido expression by ln pdcs was closely dependent on mhcii - mediated , antigen - dependent , interactions with treg . 
 we further established that ido production by pdcs was necessary to confer suppressive function to tregs . during eae development , ido expression by pdcs 
 was required for the generation of tregs capable of dampening the priming of encephalitogenic t cell and disease severity . 
 thus , we describe a novel crosstalk between pdcs and tregs : tregs shape tolerogenic functions of pdcs prior to inflammation , such that pdcs in turn , promote treg suppressive functions during autoimmunity .