dendritic cells ( dcs ) are professional antigen - presenting cells that possess the unique capacity to activate and prime naive cd4 and cd8 t cells .
they form a heterogeneous population consisting of specialized dc subsets that differ in their surface marker expression , molecular phenotype , and antigen - processing and antigen - presentation capacity [ 24 ] . in peripheral blood
, at least two major types of dcs can be distinguished , namely , myeloid dcs ( mdcs ) and plasmacytoid dcs ( pdcs ) [ 5 , 6 ] .
myeloid dcs express high levels of cd11c and can further be subdivided based on the differential expression of either cd1c ( blood dendritic cell antigen 1 = bdca1 ) or cd141 ( bdca3 ) .
each dc subset has its own repertoire of toll - like receptors ( tlrs ) , underlining their functional specialization [ 3 , 7 ] .
both mdc subsets express tlr3 and tlr8 among others , although expression levels of tlr3 are much higher in cd141 mdcs .
plasmacytoid dcs are key effectors of innate immune responses due to their capacity to produce large amounts of type i ifns in response to bacterial or viral infections ; this production can also be induced by tlr agonists such as r848 and oligodeoxynucleotides class c ( cpg ) [ 8 , 9 ] . besides their role in the innate immune system ,
pdcs also participate in priming t helper ( th ) cells , depending on the stimulus they receive ( summarized in ) .
myeloid dcs , on the other hand , have the capacity to produce the th1 skewing cytokine interleukin- ( il- ) 12 . for both pdcs and mdcs , it has been shown that they induce proliferation in an allogeneic setting and that they can cross - present exogenous antigens to prime cd8 t cells [ 1016 ] . as a result of their unique capacity to orchestrate adaptive immune responses ,
recently , more advanced examination of primary blood dcs has come within reach through the availability of efficient isolation techniques .
primary dcs are hypothesized to be stronger inducers of anticancer responses than monocyte derived dcs in cell - based vaccination strategies since they differentiate in vivo and require only short ex vivo handling .
the first clinical studies utilizing primary blood dcs have recently been conducted by our group , demonstrating the safety and efficacy of cd1c mdcs and pdcs in cancer immunotherapy [ 17 , 18 ] . in order for dc - based immunotherapy to elicit potent antitumor t cell responses , the administered dcs need to raise an immune - stimulatory rather than tolerogenic t cell response .
naive t cells will proliferate upon encounter with antigen - presenting cells presenting their specific antigen in the presence of costimulatory signals .
the nature of costimulation and cytokines from the dc will influence the polarization of the t cells into different t helper phenotypes such as th1 , th2 , and th17 or regulatory t cells ( tregs ) .
for example , the presence of il-12 promotes the induction of th1 cells , whereas il-10 inhibits induction of th1 cells and promotes the differentiation of tregs [ 20 , 21 ] . in antiviral responses , th1 cells and antigen - specific cytotoxic cd8 t cells
this type of immune response is also highly desirable in antitumor strategies , in which the aim is to eradicate tumor cells .
toll - like receptor ligands such as polyinosinic : polycytidylic acid ( polyi : c ) , r848 , and cpg have been shown to possess th1 polarizing capacity when used as adjuvants or maturation agents for dcs [ 2226 ] . to be able to successfully manipulate t cell responses for therapeutic strategies , a better understanding of the functional specialization of human dc subsets is needed . in this study , we compared the cd4 t cell stimulatory and polarizing capacity of human blood mdcs and pdcs side by side  especially the capacity to induce th1 responses upon differential stimulation .
human blood dcs were isolated from buffy coats ( sanquin ) obtained from healthy volunteers after written informed consent and according to institutional guidelines .
pbmcs were purified via ficoll density gradient centrifugation ( lymphoprep by axis - shield ) .
for this , lineage positive cells were depleted from pbmcs either with dynabeads human dc enrichment kit ( invitrogen by life technologies ) or with anti - fitc microbeads ( miltenyi biotec ) after incubation with fitc - conjugated anti - lin1 antibody cocktail ( cd3cd14cd16cd19cd20cd56 ) ( bd biosciences ) .
the remaining cells were labeled with fitc - conjugated anti - lin1 antibody cocktail ( bd biosciences ) , pe - cy7-conjugated anti - hla - dr ( bd biosciences ) , bv421-conjugated anti - cd1c ( biolegend ) , apc - conjugated anti - cd141 ( miltenyi biotec ) , and pe - conjugated anti - bdca4 ( miltenyi biotec ) .
subsets were sorted to obtain cd1c mdcs , cd141 mdcs , or pdcs , respectively ( purity 98100% ) ( see suppl .
in some experiments , cd1c mdcs were isolated from pbmcs with a cd1c dc isolation kit ( miltenyi biotec ) .
cd141 mdcs and pdcs were isolated from pbls by positive selection with anti - cd141 ( cd141 ) and anti - bdca4 magnetic microbeads , respectively ( miltenyi biotec ) .
naive cd4 t cells were isolated from pbls by depleting cd4 cells with macs multisort beads and additional use of pe - conjugated anti - cd45ro ( dako ) and anti - pe beads ( miltenyi biotec ) for the depletion of cd45ro memory t cells ( purity > 95% ) .
all cells were cultured in x - vivo 15 medium ( lonza ) supplemented with 2% human serum ( sigma - aldrich ) .
the dcs were stimulated with the following tlr ligands : 4  g / ml r848 ( axxora ) , 2  g / ml polyi : c ( sigma ) ( figures 1 and 2 ) or 20  g / ml polyi : c ( enzo life sciences ) ( figures 3 and 4 ) , 450  u / ml gm - csf ( cellgenix ) , or 5  g / ml cpg - c ( designated cpg throughout text ; enzo life sciences ) .
for the control condition of pdcs , the medium was supplemented with 10  ng / ml recombinant human il-3 ( cellgenix ) to ensure pdc survival .
cell sorting was performed on a bd aria and flow cytometry on a bd calibur or bd verse .
the dc subsets were incubated overnight at 37c with different stimuli in triplicate ( cd1c mdcs , pdcs ) or in duplicate ( cd141 mdcs ) .
the next day , supernatants were taken and cells were labeled with pe - conjugated anti - mhc class i and fitc - conjugated anti - mhc class ii ( bd ) , pe - conjugated anti - cd80 ( bd biosciences ) , and pe - conjugated anti - cd86 ( bd biosciences ) .
supernatants were analyzed for il-10 ( ebioscience ) and il-12p70 ( m122 and m121b by pierce endogen , standard by bd biosciences ) by standard sandwich elisa .
depicted in figure 2 is the cytokine production by 50,000 dcs in a volume of 100  l . for cd141 mdcs , in some instances
cd1c mdcs , cd141 mdcs , or pdcs ( 1  10 cells ) were incubated overnight at 37c with different stimuli in triplicate .
the next day , allogeneic naive cd4 t cells were added to the dcs at a ratio of 1  :  5 ( dc  :  t cell ) .
proliferative responses were determined by adding 1  ci [ 0.037 mbq]/well of tritiated thymidine ( h ) ( mp biomedicals ) to the cells after three days of coculture .
h incorporation over a time course of 16 hours was measured with a scintillation counter .
dendritic cells ( 1  10 ) were stimulated overnight with the different stimuli in 100  l medium .
next , allogeneic naive cd4 t cells ( 4  10 ) were added at a ratio of 1  :  4 ( dc  :  t cell ) in a final volume of 200  l medium containing 10  pg / ml superantigen staphylococcus aureus enterotoxin b ( seb ) ( sigma ) . at day 5 , human ril-2 ( 20  iu / ml , novartis ) was added and the cultures expanded for the next 68 days . on days 1113 , resting t cells were counted and analyzed by flow cytometry with three panels .
panel 1 includes anti - cd25-apc ( bd bioscience ) , anti - cd127-pe ( ebioscience ) , and anti - foxp3-a488 ( ebioscience ) .
panel 2 includes anti - t - bet - a488 , anti - gata-3-pe , and anti - rort - apc ( all ebioscience ) .
panel 3 includes anti - cd45ro - apc ( bd bioscience ) , anti - cd197 ( r&d systems ) with goat - anti - mouse igg2a - a488 ( life technologies ) , and anti - cd62-l ( ebioscience ) with rat - anti - mouse igg1-pe ( bd pharmingen ) .
the population of tregs was determined by selecting cd25 cd127 cells and subsequently gating on the foxp3 population ( suppl .
tcm were determined by further gating on cd197/cd62-l and tem were determined by further gating on cd197 cells .
both populations are shown as percentage of live cells ( forward - sideward scatter ) ( suppl .
furthermore , 5  10 of the t cells of each condition were restimulated with 5  10 anti - cd3/anti - cd28 beads ( dynabeads gibco by life technologies ) in triplicate and supernatants from 24-hour cultures were analyzed for levels of ifn- ( pierce endogen ) , il-5 , and il-10 ( ebioscience ) by standard sandwich elisa .
data were analyzed by kruskal - wallis test followed by dunn 's testing , by a 1-way anova followed by tukey testing or with paired student 's t - test using prism5 ( graphpad prism5 ) .
statistical significance was defined as < 0.05 ( p < .05 ; p < .01 ; p < .001 ) .
high expression of mhc molecules is a hallmark of dcs , underlining their antigen - presenting capacities .
accordingly , we found high levels of both mhc class i and mhc class ii molecules on all three dc subsets ( figure 1(a ) ) .
the levels of both mhc class i and mhc class ii molecules were highest for cd141 mdcs and comparable for cd1c mdcs and pdcs , both on freshly isolated cells and after tlr activation .
the expression of costimulatory molecules by dcs is essential to activate t cells and can be induced by tlr ligands . throughout the study , cd1c and cd141 mdc maturation
cd1c mdcs were also stimulated with granulocyte - macrophage colony - stimulating factor ( gm - csf ) .
plasmacytoid dcs were stimulated with r848 or cpg and il-3 used for the control to secure pdc survival .
on cd1c mdcs , the costimulatory molecule cd86 was already highly expressed after overnight culture in medium alone ; on cd141 mdcs , this holds true for the expression of both cd80 and cd86 ( figure 1(b ) ) . in comparison ,
cd141 mdcs showed the highest expression of cd80 and cd86 , both after culturing in medium alone or after tlr ligation ( figure 1(b ) ) .
although cd80 and cd86 molecules were expressed already at high levels on immature dcs , expression of both molecules was significantly increased upon culture with tlr ligands on all dcs .
dendritic cell - derived il-10 is known to inhibit th1 cells and induce type 1 tregs ( tregs producing il-10 ) , whereas il-12 is a th1-inducing cytokine and therefore desirable in the context of anticancer therapy [ 20 , 21 ] .
plasmacytoid dcs did not secrete il-10 or il-12 at detectable levels , whereas cd1c and cd141 mdcs secreted both il-10 and il-12 at differential levels depending on the stimulus ( figure 2 ) .
r848 and polyi : c , alone or in combination , induced il-10 production in cd1c mdcs , while only the combination of both tlr ligands induced a significant increase in the secretion of il-12 .
cd141 mdcs secreted low amounts of il-10 , irrespective of the stimulus and at lower levels than cd1c mdcs .
we observed a significant increase in il-12 production by cd141 mdcs after stimulation with both polyi : c and r848 , which is higher than the production by cd1c mdcs .
we can therefore conclude that cd141 mdcs produce less il-10 and more il-12 than cd1c mdcs .
a primary immune response constitutes the activation of naive t cells in response to antigen and their subsequent proliferation and differentiation . besides recognition of their cognate antigen ,
naive t cells depend on costimulation by the antigen - presenting cell to start such a primary response .
the ability of blood dcs to induce proliferation of naive t cells was directly compared by coculturing overnight stimulated pdcs and cd1c and cd141 mdcs of the same donors with allogeneic naive cd4 t cells .
all primary blood dc subsets showed the ability to induce proliferation of naive cd4 t cells ( figure 3 ) .
even so , r848-matured mdcs induced the highest levels of proliferation , while polyi : c maturation did not further increase proliferation as compared to unstimulated mdcs . for pdcs , r848 and il-3 ( control )
treatment stimulate similar levels of naive cd4 t cell proliferation , while the levels for cpg - treated pdcs tend to be lower than for r848 or il-3 . besides providing effector
t cells , a primary immune response can generate immunological memory in the form of memory t cells .
while central memory t cells ( tcm ) are responsible for rapid clonal expansion after reexposure to antigen and localize in lymphoid organs , effector memory t cells ( tem ) localize in mucosal tissue and mediate rapid effector functions there .
although the formation and longevity of such memory cells can only be accurately measured in vivo , we wanted to get an idea of the individual capacities of the different dc subsets to induce them .
for this , we cocultured naive , allogeneic cd4 t cells with the differentially activated blood dc subsets until the t cells had ceased to proliferate . at this resting state ( after ~12 days ) , we analyzed their cd45ro , ccr7 ( cd197 ) , and l - selectin ( cd62l ) expression .
the percentages of cd45ro ccr7 ( tem ) and cd45ro l - selectin ccr7 ( tcm ) among the t cells did not differ significantly between the subsets or different stimuli ( suppl .
at the time point measured , the t cells comprise a larger proportion of tem ( mean 47.14%71.51% ) than tcm ( mean 13.47%24% )
. taken together , all subsets can effectively induce proliferation of naive t cells and are probably able to induce memory t cells .
however , mdcs induce significantly higher proliferation when matured with r848 in comparison to polyi : c maturation or culturing alone .
dendritic cells play a critical role in the polarization of naive cd4 t cells into different t helper phenotypes or tregs . in a th1 response ,
cytotoxic cd8 t cells that are able to kill cells bearing their specific antigen are elicited .
therefore , this type of immune response is highly desirable in antitumor strategies . to compare the differential t cell stimulatory and polarizing capacity  especially the capacity to induce th1 responses upon differential stimulation with polyi : c , r848 , and cpg  and possible treg induction by human blood mdcs and pdcs
, naive cd4 t cells were cocultured with the dc subsets until they reached resting state .
importantly , analysis of the resting t cells did not show a large fraction of tregs for any dc subset or condition ( mean 3%10% ) ( figure 4(a ) ; suppl .
2a ) . although the differences are small , it is interesting to note that the percentages of these cells were lowest for polyi : c and r848-matured mdcs and were highest for gm - csf - stimulated cd1c mdcs ( mean 3% and 10% , resp . ) . for the pdc cocultures ,
there is a tendency of a higher proportion of tregs being induced after r848 or cpg stimulation compared to the control ( il-3-treated cells ) ( mean 7% , 7% , and 4% , resp . ) .
furthermore , we analyzed the induction of th subset - specifying transcription factors t - bet , gata-3 , and rort ( figure 4(b ) ) .
we found pronounced populations expressing t - bet across the subsets and stimuli ( cd1c mdcs : 9%35% , cd141 mdcs : 23%35% , and pdcs : 32%42% ) , indicating th1 polarization by all subsets .
cd141 mdcs showed the most pronounced gata-3 expression for control and r848 stimulation ( mean 5.1% and 4.25% , resp . ) , which was significantly reduced with polyi : c or combined r848 and polyi : c stimulation of cd141 mdcs ( mean 1.87% for both ) .
furthermore , rort expression was only detected in a very small population of cd4 t cells across the subsets ( 0.08%0.72% ) , indicating th1 rather than th17 polarization of these cells .
resting cd4 t cells were also restimulated with anti - cd3/anti - cd28 beads and their supernatants analyzed for il-5 , il-10 , il-17 , and ifn- production to determine the th1 polarization capacity of the dc subsets .
interleukin-5 is a th2 cytokine , while il-10 inhibits th1 polarization and ifn- is a strong th1 inducer [ 20 , 21 , 28 ] .
notably , coculture with all three blood dc subsets induced t cells with prominent ifn- production after restimulation even without tlr maturation ( figure 4(c ) ) .
t cells primed by cd141 or cd1c mdcs induced prominent populations of t - bet expressing cells and secreted high levels of ifn- , indicating th1 skewing ( figure 4(c ) , lower panel ) .
gm - csf - stimulated cd1c mdcs induced smaller populations of t - bet expressing cells and lower levels of ifn- and similar levels of both il-5 and il-10 as the medium control or tlr - matured dcs ; therefore , gm - csf maturation of cd1c mdcs does not induce the most potent th1 response .
also pdcs induce a prominent population of t - bet expressing cells and ifn- release from restimulated cd4 t cells , although the levels of ifn- are lower than for optimally stimulated mdcs .
plasmacytoid dcs induce similar levels of il-5 in cocultured t cells as mdcs . however , the levels of il-10 are higher , especially for r848 and cpg stimulated pdcs , which coincides with a tendency for a bigger proportion of tregs ( cd25 cd127 foxp3 ) induced in these conditions .
we measured no il-17 for pdcs and modest levels for mdcs stimulated with r848 or cd1c mdcs stimulated with the combination of r848 and polyi : c ( suppl .
4 ) . together with the rort expression data we conclude a th1 rather than th17 polarization of the nave cd4 t cells . in sum ,
all subsets polarize naive cd4 t cells mainly towards th1 cells with a strong t - bet signature producing mainly ifn- after restimulation .
in order to manipulate t cell responses for dc - based cancer immunotherapy , a better understanding of the functional specialization of human dc subsets is needed . in this study , we compared the capacities of primary human blood mdcs and pdcs to activate and polarize cd4 t cells side by side .
we report that cd1c mdcs , cd141 mdcs , and pdcs all induce proliferation of naive cd4 t cells .
importantly , naive cd4 t cells are not skewed towards a regulatory phenotype by coculture with either mature mdcs or pdcs . despite differences in activation and cytokine profile , both cd141 and cd1c mdcs polarize naive cd4 t cells towards t cells with a strong ifn- signature ; also pdcs induce ifn- , although at lower levels and accompanied by a higher il-10 production . while all dc subsets mature upon tlr ligation , we observed distinct cytokine responses for different subsets and stimuli .
cd1c mdcs produced only a limited amount of il-12 after maturation with either r848 or polyi : c alone , but production was significantly increased with a combination of these tlr ligands .
even higher levels of il-12 are produced by cd141 mdcs when stimulated with the combination of polyi : c and r848 .
in contrast to our findings , nizzoli et al . did not find il-12 production for cd141 mdcs after combined polyi : c and r848 stimulation .
other studies have shown that , in order to induce strong il-12 responses in human and mouse dcs , both an innate trigger such as tlr ligation and a second trigger like ligation of cd40 by cd40l on t cells are needed [ 7 , 30 ] .
more recently , it has been shown for cd1c mdcs that the combination of the tlr ligands r848 and lps can trigger significant il-12 production . in the case of cd141 mdcs
, a cocktail of polyi : c together with the cytokines ifn- , tnf- , ifn- , and il-1 was shown to induce significant levels of il-12 .
showed that cd141 mdcs produced less il-12 as compared to cd1c mdcs for single tlr ligation but that a higher proportion produced il-12 after tlr1/2 or tlr3 ligation in a whole blood assay .
our data supports the notion that a single stimulus is not sufficient to induce high il-12 production and with polyi : c and r848 we describe a new combination that can trigger substantial il-12 secretion by both human mdc subsets . all dc subsets induced proliferation of naive cd4 t cells  regardless of the stimulus .
the level of t cell proliferation induced by polyi : c - matured mdcs is similar to nonstimulated mdcs . strikingly , gm - csf - stimulated cd1c mdcs and r848-matured cd1c and cd141 mdcs cause an extra boost in proliferation of naive cd4 t cells .
this is in accordance with an earlier study by jongbloed et al . , which described equally high induction of proliferation of naive cd4 t cells for nonstimulated or polyi : c stimulated cd1c and cd141 mdcs after six days .
because of the upregulation of the expression of costimulatory molecules with both stimuli compared to untreated dcs , one would expect a higher proliferation rate than with untreated dcs .
certainly , other cytokines and immunostimulatory , but also immunoinhibitory , molecules expressed by cultured dcs are integrated into a single response by the t cells and possible differences in these factors might cause the observed differences in t cell proliferation .
only a minor percentage of cd4 t cells that grew out of cocultures with the different dc subsets displayed a treg phenotype .
earlier studies suggest that pdcs can act as th1 , th2 , th17 , or even treg inducers in t cell priming , depending on the stimulus they receive ( summarized in ) .
one stimulus that can induce this regulatory t cell phenotype is cpg and the proposed mechanism is via the expression of inducible costimulator ligand ( icos - l ) .
icos - l upon cpg maturation , which triggers il-10 production of t cells but no production of il-4 , il-5 , or il-13 .
this is in accordance with our findings , where we observed higher levels of il-10 and a tendency of a higher proportion of tregs for pdcs matured with cpg or r848 compared to matured mdcs but no elevated levels of il-5 .
however , regardless of the stimulus we also found a strong t - bet expression and ifn- production by cd4 t cells that had grown out of cocultures .
plasmacytoid dcs secrete large amounts of type i ifns in response to bacterial or viral stimuli , including r848 and cpg .
type i ifns not only are important in innate responses , but can also help to skew t cells towards a th1 phenotype .
type i ifns secreted by pdcs might play a role in the observed ifn- induction .
regulatory t cell induction with functional effects on t cells has been described in one study for tissue mdcs of the skin .
we show here that primary blood mdcs induce only a low proportion of tregs and , importantly , the overall cd4 t cell population displays a th1 phenotype after coculture ( pronounced t - bet expression and high ifn- production ) and no th2 or th17 phenotype .
this is in line with the ability of mdcs to produce il-12 after combined polyi : c and r848 stimulation .
for the other conditions , one can speculate whether the addition of the cd4 t cells and therefore ligation of cd40 on the dcs give the needed second signal for il-12 production and help the th1 skewing .
different groups have shown that blood mdcs can induce ifn- production in naive cd4 and cd8 t cells [ 7 , 35 , 36 ] .
found that cd141 mdcs were more potent than cd1c dcs at inducing ifn- responses in total cd4 t cells , especially after polyi : c stimulation .
we found that although cd1c mdcs show a less mature phenotype than cd141 mdcs including higher il-10 and lower il-12 production , cd1c and cd141 mdcs induce similar ifn- responses after coculture with naive cd4 t cells .
however , there is a tendency for cd141 mdcs to induce less il-5 and il-10 than cd1c , also arguing for an overall stronger th1 skewing by this subset . for cd141 mdcs , high tlr3 expression and the ability to produce ifn- and cxcl10 ,
both known to induce antiviral responses , all suggest their capability to induce th1 skewing in t cells [ 7 , 37 , 38 ] . certainly , r848 and polyi : c can trigger distinct pathways as tlr3 signals through a trif - to - irf3 pathway , rather than an myd88-to - irf7 pathway that is used by tlr8 .
it is interesting to note that both mdc subsets react strongly and in a similar way to polyi : c , although the expression levels of tlr3 are much higher in cd141 mdcs than in cd1c mdcs .
likely , other receptors for polyi : c contribute to the response in one or both of the mdc subsets .
the synthetic dsrna analog is a ligand for multiple pathogen recognition receptors , and besides tlr3 also triggers cytosolic rig - i - like receptors that are expressed by mdcs [ 39 , 40 ] .
suggest in a study on mdcs and nk cells that both tlr3 stimulation and rig - i - like receptor ligation are needed for ifn- induction by mdcs .
in addition to their cd4 t cell activating capacities , all three subsets can cross - present exogenous antigens for cognate restimulation of previously activated cd8 t cells [ 1014 ] , making them promising candidates for dc - based vaccination strategies against cancer .
both cd1c mdcs and pdcs have generated promising results in first clinical studies utilizing these primary blood dc subsets as vaccines [ 17 , 18 ] .
these studies support their excellent in vivo functioning and mark them as the next generation of cancer vaccines . in this context
, we have learned from the current work that gm - csf is not the optimal stimulus for cd1c mdcs , since gm - csf stimulation showed an overall lower th1 skewing capacity and induced more tregs than other stimuli . while maturation with polyi : c or the combination of polyi : c and r848 induces the most pronounced th1 skewing ,
the number of t cells that grow out with these stimuli is lower than , for example , with r848 stimulation . considering the proliferation data and the similar polarization capacity by all subsets and with all stimuli including control dcs
, one can only speculate about a recommendation for a suited stimulation of dcs for dc - based vaccination strategies .
for example , tlr activation of dcs can lead to the upregulation of otherwise unexpressed tlr ligands in the dcs , making them sensitive to a broader range of danger signals .
furthermore , in an in vivo situation also other cell types might play a crucial role for the overall outcome of a therapy .
such cells include nk and cd8 t cells , for which type i ifns and il-12secreted at higher levels upon tlr maturation  are important [ 4346 ] .
as discussed above , cd141 mdcs certainly display promising properties for dc - based anticancer vaccination strategies .
besides their th1-inducing capacity , human cd141 mdcs are also excellent cross - presenters of exogenous antigens to cd8 t cells .
while some publications show superior cross - presentation capacity of cd141 mdcs [ 31 , 4749 ] and put them forward as the human counterparts of mouse cd8 dcs [ 31 , 4750 ] , other studies suggest that the different human dcs subsets bear similar cross - presentation capacities at least for soluble antigens delivered through early endosomes [ 14 , 15 , 51 ] . the type and size of the antigen as well as
the compartments they are targeted to probably underlie these differing outcomes ( reviewed in [ 52 , 53 ] ) .
in addition to using single subsets for therapeutic approaches , we hypothesize that using a combination of several dc subsets could further increase t cell activating properties , since earlier studies have shown that cell - cell interactions as well as soluble factors can act to cross - activate the different dcs ( summarized in ) . with this comparative study ,
we have reinforced the establishment of human circulating cd1c mdcs , cd141 mdcs , and pdcs as promising candidates for dc - based immunotherapy in the context of cancer .
 dendritic cells ( dcs ) are central players of immune responses ; they become activated upon infection or inflammation and migrate to lymph nodes , where they can initiate an antigen - specific immune response by activating naive t cells . 
 two major types of naturally occurring dcs circulate in peripheral blood , namely , myeloid and plasmacytoid dcs ( pdcs ) . 
 myeloid dcs ( mdcs ) can be subdivided based on the expression of either cd1c or cd141 . 
 these human dc subsets differ in surface marker expression , toll - like receptor ( tlr ) repertoire , and transcriptional profile , suggesting functional differences between them . here , we directly compared the capacity of human blood mdcs and pdcs to activate and polarize cd4 + t cells . 
 cd141 + mdcs show an overall more mature phenotype over cd1c+ mdc and pdcs ; they produce less il-10 and more il-12 than cd1c+ mdcs . despite these differences 
 , all subsets can induce the production of ifn- in naive cd4 + t cells . 
 cd1c+ and cd141 + mdcs especially induce a strong t helper 1 profile . 
 importantly , naive cd4 + t cells are not polarized towards regulatory t cells by any subset . 
 these findings further establish all three human blood dcs  despite their differences  as promising candidates for immunostimulatory effectors in cancer immunotherapy . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion