dc are the professional antigen - presenting cells ( apc ) of the immune system that instruct and control the activation of b and t lymphocytes , the mediators of specific immunity .
dc are highly mobile cells and by their sequential migration from peripheral tissues to lymphoid organs they serve as sentinels of the immune system .
immature dc are very efficient in antigen uptake , mediated by high endocytotic activity and expression of an array of cell surface receptors capable of capturing antigens [ 34 , 53 ] .
inflammatory mediators and  danger signals promote maturation and re - routing of dc to the secondary lymphoid organs [ 5 , 43 ] . in the secondary lymphoid tissues ,
dc are mature and well equipped to attract , interact and activate naive t cells to initiate a primary immune response [ 1 , 5 ] .
dc are also able to directly activate nk cells   and can produce large amounts of interferon upon encounter with viral pathogens , thus , providing a link between the adaptive and innate immune system . in murine tumor models , protective immunity as well as regression of established tumors have been observed after vaccination with dc loaded with tumor antigens [ 13 , 44 , 66 ] . their unique capacity to initiate and modulate immune responses
is currently exploited by many groups , including ours , to fight infectious diseases and cancer .
one aspect of dc biology that is rapidly evolving is the apparent diversity of dc - subsets .
at least two distinct ontogenic pathways for dc development have been reported , the myeloid progenitor- and the lymphoid progenitor - derived dc .
part of these different dc - subsets may also be explained by differences in the maturation stage of dc and the local cytokine environment .
the geographical localization of the dc - subsets in secondary lymphoid tissues is distinct , myeloid derived dc mainly migrate to or reside in the marginal zone ( a primary entry point for blood - born antigens ) , whereas the lymphoid dc mainly reside in the t - cell areas .
this supports distinct functions for the dc - subsets , as shown in murine studies [ 5 , 40 ] .
it is now well appreciated that the dc subset , its maturation state and the microenvironment or type of pathogen a dc encounters in the periphery , determine the type of immune response that is induced , ranging from a th1 or th2 response to immune tolerance [ 38 , 40 , 51 ] .
data are now accumulating that immature dc can induce tolerance and are able to induce regulatory t cells in vitro [ 35 , 54 ] and in vivo .
regulatory t cells are involved in the control of peripheral tolerance   and the prevention of vigorous inflammatory reactions .
these regulatory t cells affect immune responses at the level of antigen - presentation and during the effector phase of t cells at the site of the tumor .
although the exact mechanisms by which regulatory t cells exert their suppressive functions are not yet elucidated , direct cell 
our data on vaccination of melanoma patients also demonstrate that mature dc , but not immature dc , induce strong immune responses in vivo .
another aspect in the evolving field of immunotherapy is the re - acknowledgement of the role of the innate immune system .
the eradication of a malignancy is the result of a concerted action of adaptive and innate immunity , in which natural killer ( nk ) cells and natural killer t ( nkt ) cells are important effector cells .
next to the direct cytotoxic effect on tumor cells , nk cells produce type i interferons that contribute to a great extent to a proinflammatory microenvironment .
clinical studies on adoptive nk - cell immunotherapy have shown that nk cells can target human tumors [ 45 , 52 ] .
over 60 different clinical studies have been carried out between 1996 and 2004 , applying tumor antigen - loaded dc - based vaccines .
the vast majority of these studies have been performed in melanoma patients [ 27 , 49 ] . in our studies , as well as in other groups , immunological and , notably long - lasting ,
clinical responses have consistently been observed following cellular therapy [ 6 , 27 , 46 ] . in several patients these clinical responses coincide with the induction of specific cytotoxic t - cell responses . we have explored vaccination of cancer patients with monocyte - derived dc loaded with peptides derived from tumor - associated antigens . in our current culture protocol , we routinely generate large amounts of clinical grade mature and immature dc .
the first dc trial at our institution has been initiated in 1998 . herein , the safety of dc - based vaccines and the efficacy of immature dc versus mature dc was studied .
hla - a2.1 + , gp100 + , tyrosinase+ metastatic melanoma patients were treated with peptide - pulsed immature or mature dc .
as peptides we used two hla - a2.1 restricted gp100 peptides ( either native or modified peptides to improve their hla - a2.1 binding affinity ) [ 3 , 4 ] and a tyrosinase peptide .
all dc vaccines were co - loaded with the foreign protein klh ( calbiochem , darmstadt , germany ) that serves as a control for immune competence and stimulation of a t - helper response .
vaccinations were given 3 times with 2-week intervals , followed by injections with the peptides alone .
immune monitoring consisted of ( 1 ) dth responses with pulsed dc and unpulsed dc , ( 2 ) elispot - assays [ 31 , 55 ] and ( 3 ) tetramer assays [ 2 , 22 , 30 ] .
in addition to peripheral blood , immune monitoring was also performed using biopsies taken from dth sites ( see later ) .
the results of the first clinical trial have been published , and unequivocally demonstrated that mature but not immature dc are capable of inducing potent anti - klh - specific t - cell , and b - cell responses .
clinical results demonstrated that 3/20 objective remissions were observed in stage iv melanoma patients vaccinated with mature dc . in this group
10 patients were vaccinated with mature dc , of these patients 1 was not evaluable because of detoriating condition and of the remaining 9 patients , 3 showed stable disease > 4  months ( respectively , 4.5 , 7.5 and 22 ) 1 patient showed a mixed response and 1 patient achieved a partial response with complete remission after surgical intervention , that now lasts > 7  years .
all patients in this study were melanoma patients with metastatic disease ( m1c , 1 patient m1a ) in who performance status 0 .
moreover , clinical results correlated with the presence of vaccine - induced immune responses against the tumor peptides ( see later ) .   to date ,
> 200 melanoma patients have been vaccinated in our ongoing dc - trials , we observed no clinical benefit from vaccinations in patients with high - tumor load ( as judged by the clinician ) , elevated serum ldh , brain metastases or rapid progressive disease .
therefore , we excluded these patients , with a life expectancy less than 3  months , in ongoing studies . while on the down side the vaccine is not yet very effective , with an objective clinical response rate ( i.e. > 1  year sd or better , stage iv melanoma patients ) of approximately 1015% , the positive message is that we clearly find t cell - mediated immunological responses : 60% in patients with regional lymph node metastasis and 30% in patients with metastatic disease .
nevertheless , a number of variables need to be evaluated and controlled to further improve clinical outcome in cellular therapies , among these are generation of dc , use of different dc - subsets , route of administration , optimal activation stimuli for dc , antigen loading of dendritic cells , selection of tumor - derived antigens and so on .
these variables are in ongoing debate , but one can conclude that the full potential of dc - based cellular therapy has not yet fully been exploited
. however , the current consensus is to continue cellular therapy in well - designed small trials that meet a standardized list of quality criteria .
this consensus list should at least describe quality - control criteria for ex vivo generated dc , patient characteristics , trial design including the different variables that are investigated , and tests for clinical and immunological responses [ 27 , 33 ] .
significant progress in cellular therapy against cancer , including dc vaccination , is only to be expected by careful immune monitoring studies in order to obtain detailed insight of the underlying ( pathological ) physiological processes that determine the success or failure of treatment .
different compartments and modalities are considered to monitor induced immune responses ; e.g. accuracy of delivery , immune responses in peripheral blood , tumor and delayed type hypersensitivity ( dth ) test biopsies , and clinical evaluation .
recently , it was shown that the modality of vaccination with a tumor - specific antigen influences the differentiation pathway of the anti - vaccine cd8 t cells , which may have an effect on their capacity to trigger a tumor rejection response .
observed that patients with a high - baseline level of melanoma antigen - specific immunity more often show an immune response to the vaccine .
furthermore , they show that patients who survived longer are those who showed immune response against two melanoma antigens presented on the dc vaccine .
although sometimes correlations between tumor regression and t - cell responses are observed [ 25 , 41 ] , the immunological studies performed so far are too diverse in their setup to pool them in a meta - analysis .
for example , an intact and proper functioning immune system seems to have a higher potential to react on immune therapy . from our immune monitoring data ,
mentioned below , we might not only conclude that the presence of tetramer - specific t cells is correlated with an improved progression free survival .
another conclusion should be that in the end - stage melanoma patients these tetramer - specific t cells are less frequently induced ( 8 of 26 patients ) than in melanoma patients with regional lymph node metastasis ( 24 of 31 patients ) .
secondly , upon induction of tumor antigen - specific t cells , the next hurdle to take is the local immune suppressive environment created by the tumor . in the end - stage melanoma patients ,
we have seen that in some patients tetramer - specific t cells are present after dc vaccination , but still experience progression .
it became clear that these tetramer - specific t cells did not produce interferon- nor showed cytotoxic activity upon tumor challenge .
apparently , these effector cells were not capable of breaking the local suppressive tumor - environment .
we might take better advantage of the unique capacity of dc to direct the immune response by exploiting dc - based cellular therapy earlier in the disease course .
it has been demonstrated that already in sentinel nodes melanoma - specific t cells are present , together with antigen - presenting cells . in this window between primary tumor and metastasis
it might be at this turning point in the development of melanoma , that ex vivo generated dc can assist the immune system .
for dc to induce potent immune responses their migration towards lymph nodes is essential . in mice , we have demonstrated that major differences can be found in numbers of migrating dc depending on the route of administration ( subcutaneous gave the best results ) and the maturation state ( mature gave the best results ) .
with respect to the latter we were able to confirm these data in stage iii melanoma patients with lymph node metastases who were scheduled for radical lymph node resection . during the first vaccination these patients received an injection of
indium - labeled mature or immature dc to allow scintigraphic imaging to study in vivo migration .
regardless of the route of administration ( intradermal or intranodal ) mature dc were more efficient than immature dc in reaching the draining lymph node in vivo .
the results described earlier were obtained by our developed method of radioactive labeling of dc ( fig .  1 ) [ 18 , 23 ] .
dc have therefore been labeled with radionuclides for scintigraphic imaging of cell trafficking , which is until to date the only fda - approved clinical cellular imaging modality [ 18 , 42 ] .
a major drawback of scintigraphy , however , is the lack of anatomical detail allowing only gross anatomical determination of migration between lns without the ability to assess the intranodal distribution pattern of dc within each ln .
furthermore , due to its low spatial distribution , accurate delivery of cells which may be essential for subsequent migration into nearby lymph nodes can not be properly evaluated using scintigrapy .
in contrast , mr imaging is well suited to obtain 3d whole body high - resolution images and is widely used in clinical practice .
the currently most sensitive markers to label cells for mr detection are ( ultrasmall ) superparamagnetic iron oxide [ ( u)spio ] particles .
we took advantage of the fact that dc naturally endocytose clinically applied , fda - approved spio - labels in significant amounts , obviating these concerns .
this provided us with the opportunity to label cells with high efficiency without affecting their function and use these cells in humans .fig .  1monitoring the accuracy of delivery of spio - labeled cells using mr imaging and scintigraphy .
monocytes are obtained by cytopheresis from stage iii melanoma patients ( a ) , they are cultured and labeled with spio particles and indium ( b ) .
the cells are then injected intranodally into the lymph node basin that is to be resected and their biodistribution is monitored in vivo by scintigraphy ( c ) and mri ( d ) .
the lymph node basin is resected ( e ) and separate lymph nodes are visualized with high - resolution mri at 7  tesla ( f ) and histology ( g ) monitoring the accuracy of delivery of spio - labeled cells using mr imaging and scintigraphy .
monocytes are obtained by cytopheresis from stage iii melanoma patients ( a ) , they are cultured and labeled with spio particles and indium ( b ) .
the cells are then injected intranodally into the lymph node basin that is to be resected and their biodistribution is monitored in vivo by scintigraphy ( c ) and mri ( d ) .
the lymph node basin is resected ( e ) and separate lymph nodes are visualized with high - resolution mri at 7  tesla ( f ) and histology ( g ) we investigated the biodistribution of these spio - prelabeled dc applied as cancer vaccines in melanoma patients using mr imaging . in our dc vaccination protocols , in vitro generated dc loaded with tumor - derived antigenic peptides were administered to stage iii melanoma patients as outlined in fig .  1 [ 18 , 19 ] .
dc were labeled with in - oxine and spio ( endorem ) separately and co - injected in ln in the lymph node basin to be resected .
this provided the unique opportunity to not only obtain mr scans at 3  tesla ( t ) before surgery but also to generate high - resolution mr images at 7  t of individual resected lns , and to correlate the results with scintigraphy and ( immunology ) histopathology .
dc were correctly injected into the ln , despite ultrasound guidance of the injection needle by a highly experienced radiologist .
subsequent migration could be observed only when dc were correctly injected into the lymph node , demonstrating not only the importance of accurate delivery but also of careful monitoring of cell tracking in cellular therapy .
inadequate delivery may be an important reason why only a limited proportion of patients respond in ongoing clinical trials using dc vaccines .
we found that the accuracy of mr imaging to visualize truly dc - positive lns was significantly better than scintigraphy .
these findings illustrate the power of additional anatomical information , which can also be of value for other fields of biomedical research
we observed immunohistologically that the spio - labeled dc that do migrate enter the lymph nodes via the sinuses and reach the t - cell areas where the actual dc 
we were able to demonstrate that intranodally injected spio - labeled dc , electroporated with rna encoding the tumor antigen gp100 , express the gp100 protein . from resected lymph nodes rosettes , containing spio - labeled dc surrounded by enlarged and activated t cells ,
immune monitoring is most straightforward after vaccination with defined antigens , however , responses have also been detected after lysate or total rna - loaded dc vaccines .
fortunately , many novel tools are now available to detect immune responses against known and unknown tumor antigens , including mhc - tetramers , eli - spot assays and cytokine release / catch assays [ 2 , 22 , 31 , 55 ] .
we developed a novel approach to efficiently monitor dc vaccine related t - cell responses in vaccinated patients using biopsies derived from dth sites .
the results of this monitoring method correlated with the clinical outcome in stage iii and iv melanoma patients .
dth challenges consisting of peptide - loaded dc plus or minus klh , dc loaded with klh , and unloaded dc revealed that essentially all patients mounted a positive dth response with indurations up to 33  mm .
as both unloaded dc and dc loaded with klh and/or peptides were positive , indurations at the dth site were not predictive of vaccine - related t - cell responses in our setting .
however , as no dth was detected after the first intradermal injection of the vaccine , the occurrence of a positive dth reaction should be directly related to the vaccination .
the reason for the dth response to unloaded dc is not clear but could be explained by the vast amount of chemokines produced by mature dc .
punch biopsies ( 6  mm ) were taken from positive dth sites and divided in half .
one part was used for histochemistry and the other part was used to isolate dth - infiltrating leukocytes ( dil ) .
immune staining showed clusters of cd2 + and cd3 + infiltrating cells of which 5070% were cd4 + and 5030% were cd8 + t cells .
dil were generated by cutting the biopsies in pieces and culturing of the outgrowing cells for 23  weeks in the presence of low dose il-2 ( 100 u / ml ) without restimulation .
interestingly , dil specific for klh could easily be found in biopsies from klh - pulsed dc , not in dil from peptide or unpulsed dth biopsies .
moreover , in 11 ( 6 stage iii , 5 stage iv ) of 22 patients tested , gp100/tyr tetramer - positive t - cell populations were readily detected . in 5 additional patients , antigen - specific cytotoxic t cells in dil cultures
no tetramer - positive t cells were detected in dth biopsies injected with unloaded dc or klh - loaded dc .
strikingly , in 6 of 7 patients in whom no tetramer - positive cells were found in freshly isolated pbmc , significant numbers ( up to 45% ) of tetramer - positive t cells were present in their cultured dth biopsies taken at the same time point .
cytokine production and cytotoxicity of dil upon co - culture with the appropriate target cells were fully correlated with the specificity in the tetramer analysis . moreover ,
dth reactions induced with dc pulsed with the gp100 peptides accumulated gp100-specific t cells and not tyrosinase and vice versa ( fig .  2 ) .
finally , in situ tetramer staining on cryo - sections revealed that gp100/tyr - specific tetramer - positive cells were specifically present in the infiltrating t - cell clusters .
collectively , these data not only indicate that significant numbers of tetramer - positive t cells accumulate in the dth site but also demonstrated that these t cells specifically produced cytokines ( fig .  2 ) and/or are cytotoxic for tumor antigen expressing target cells ( data not shown).fig . 
2specificity in dth - infiltrated leukocyte ( dil ) cultures derived from patients vaccinated with peptide - loaded dc . a in dil cultured from a dth performed with dc loaded with gp100 and klh only t cells positive for the gp100 tetramers were observed .
tyrosinase tetramer - positive cells were observed in the dil derived from a dth induced with tyrosinase peptide - loaded dc .
b dil derived from a dth site induced by dc loaded with gp100 and klh did not produce cytokines in response to the hla - a2.1-positive melanoma cell line blm transfected with control antigen g250 ( a ) whereas blm transfected with gp100 was recognized ( b ) .
the dil culture also recognized an hla - a2.1-positive melanoma cell line expressing both gp100 and tyrosinase ( c ) specificity in dth - infiltrated leukocyte ( dil ) cultures derived from patients vaccinated with peptide - loaded dc .
a in dil cultured from a dth performed with dc loaded with gp100 and klh only t cells positive for the gp100 tetramers were observed .
tyrosinase tetramer - positive cells were observed in the dil derived from a dth induced with tyrosinase peptide - loaded dc .
b dil derived from a dth site induced by dc loaded with gp100 and klh did not produce cytokines in response to the hla - a2.1-positive melanoma cell line blm transfected with control antigen g250 ( a ) whereas blm transfected with gp100 was recognized ( b ) .
the dil culture also recognized an hla - a2.1-positive melanoma cell line expressing both gp100 and tyrosinase ( c ) next , we compared the clinical and immunological data of 26 stage iv melanoma patients ( fig .  3 ) .
patients had documented progressive disease within 2  months before study entry , serum lactate dehydrogenase 2 the upper limit of normal , no prior chemotherapy or immunotherapy within 3  months before study entry , and no residual toxicity from prior treatments . of these patients , 15 patients had progressive disease ( pd ) , 9 patients had stable disease with > 4  months duration ( sd ) , and 2 patients , one with multiple liver metastases at time of inclusion , are in complete remission ( cr ) .
no tumor - reactive dil were found in 13 of the 15 patients with pd . of the 9 patients with sd , 4 patients with specific t cells had a progression free survival of > 42 , 22 , 12 , and 4.5  months ( median 12  months ) .
in the 5 patients with sd without tumor - reactive dil the median progression free survival was 6  months ( range 114.5 ) . both patients in cr ( > 60 and > 42  months ) had tumor - specific t cells .
although the number of patients in this study is limited , a statistically significant ( p  =  0.0012 ) correlation was observed between the presence of tumor - specific t - cell reactivity and progression free survival .
3presence of tetramer - specific t cells highly correlates with progression free survival in melanoma patients .
correlation between the presence of specific t cells and clinical outcome is shown in this plot comparing the progression free survival of stage iv melanoma patients with ( closed line ) and without ( hatched line ) tumor - specific t cells in their dth - infiltrated lymphocytes presence of tetramer - specific t cells highly correlates with progression free survival in melanoma patients .
correlation between the presence of specific t cells and clinical outcome is shown in this plot comparing the progression free survival of stage iv melanoma patients with ( closed line ) and without ( hatched line ) tumor - specific t cells in their dth - infiltrated lymphocytes
besides the production of highly reproducible gmp quality controlled batches of dc vaccines , in vitro and in vivo tools to analyze b and t - cell responses are developed and injected dc can now be visualized in the patient by advanced imaging technology .
another major breakthrough is the application of rna transfected dc that express complete tumor antigens .
this has now proven to be effective and safe in patients and , in comparison to gene therapy , rna transfection is much more straight forward .
cellular therapy with dendritic cells is still in its infancy , but a number of variables are recognized that can contribute to exploit its full potential . in particular , monocyte - derived dc may not seem optimally equipped for their task in vivo . in this context
, rna technology is a promising new tool to achieve temporarily expression or suppression of specific proteins to optimize dc function .
recent findings have shown that dc transfected with rna encoding the full - length tumor antigens express the corresponding protein in vivo for a prolonged period of time . moreover , these rna transfected dc are able to induce anti - tumor responses in patients .
but one can think of a broad range of applications of this technology to improve the functional capabilities of monocyte - derived dendritic cells . during our investigations we observed that the majority of injected dc die early by apoptosis and necrosis .
after injection the cells encounter hypoxic conditions and it is known that hypoxia inhibits migration of dc , likely because it blocks the production of metalloproteases .
similarly , decoy receptor 3 was shown to be upregulated by dc that propagates apoptotic signals .
secondly , migration of current monocyte - derived dc in vivo is poor , their migration may be impaired due to limited chemokine receptor and cell adhesion receptor functioning . by exploiting rna technology
similarly , induced production of gm - csf and il-15   might directly or indirectly enhance dc migration .
next , rna technology can be exploited to improve dc maturation and t - cell stimulation .
toll - like receptors have been shown to be of key importance in dc maturation and subsequent induction of immunity through the upregulation of cytokines / chemokines and co - stimulatory molecules . to enhance dc maturation dc
might be transfected with rna encoding constitutively active tlrs or rnas encoding co - stimulatory molecules , like cd70 and cd40l , can be introduced .
expression of cd40l in dc has previously been reported to enhance the magnitude of cd4 + and cd8 + t - cell responses in preclinical models [ 21 , 28 ] .
recently , cd70 has emerged as a key molecule for priming of cd8 + t - cell responses .
rna interference to optimize t - cell stimulation by ex vivo generated dc by another means is to induce expression of pro - inflammatory cytokines like interleukin-12 and type i interferons .
aside expression of activatory molecules in dc , it is becoming more and more evident that down regulatory mechanisms are in place that limit the dc s potential as a vaccine adjuvant .
recent murine data have demonstrated that socs1 ( suppressor of cytokine signaling 1 ) expression restricts the dendritic cells ability to break tolerance and induce antitumor immunity [ 24 , 56 ] .
we have recently demonstrated that upon human dc maturation socs1 as well as socs3 are also rapidly upregulated .
therefore , silencing of inhibitory molecules like socs 1 and il-10 by rna interference might be another means to improve human dc - based cancer vaccines .
furthermore , one of the most potent factors limiting vaccine efficacy is the immune suppressive activity of the regulatory t cell ( treg ) .
remarkably , several reports indicate the rapid expansion of treg following immunotherapy , including following dc vaccination .
another treg stimulatory molecule / pathway in dc as a candidate to silence concerns the enzyme indoleamine-2,3-dioxygenase ( ido ) .
ido1 appears most crucial for tryptophan catabolism and immune suppression [ 32 , 50 ] .
direct targeting of antigens to dc surface receptors in vivo might replace laborious and expensive ex vivo culturing , and facilitate large - scale application of dc - based vaccination therapies .
a major advantage of in vivo targeting strategies is that they can be produced in bulk quantities , whereas vaccines based on dc loaded with antigens ex vivo require tailor - made procedures for each individual . in addition , the opportunity to target natural dc - subsets and at multiple sites in vivo might be preferable above loading more artificial ex vivo cultured dc .
many of the receptors that are studied in targeting strategies belong to the c - type lectin receptor ( clr ) family .
the clrs are a family of calcium - dependent lectins that share primary structural homology in their carbohydrate domain . through this domain , clrs bind to specific self or non - self sugar residues and
are implicated in antigen capture and endocytosis . in our targeting studies we mainly investigated the targeting of dc - sign .
dc - sign is predominantly expressed on immature dc and at lower levels on mature dc and macrophages [ 9 , 29 , 58 ] . by cloning the hypervariable domains of a mouse antibody specific for human dc - sign into human framework regions
we demonstrated that this antibody efficiently targets myeloid apc in vivo and reached saturation with one single dose .
the binding of the humanized antibody to dc - sign showed high affinity and facilitated endocytosis .
furthermore , targeted delivery to human monocyte - derived dc of a model antigen conjugated to the humanized dc - sign - specific antibody leads to presentation of the antigen by mhc class i and ii molecules and elicits both naive and memory t - cell responses in vitro .
the clr targeting strategies that are most likely to enter the clinic in the near future target dc - sign , cd205 ( dec205 ) and the mannose receptor . of these
, dc - sign seems the most dc / macrophage lineage - specific receptor , which might be advantageous since the targeting vector will not be scavenged by other cell types that could result in lower targeting efficiencies and undesirable side - effects .
the expression of cd205 for example is in humans less restricted than in mice . although human cd205 expression levels are highest in mature dc , cd205 is also expressed by b cells , t cells , monocytes , macrophages and nk cells .
cd205 however seems to be more potent in mediating cross - presentation in vitro compared to the other two receptors .
furthermore , due to lack of direct control in these targeting strategies , the duration and stability of the vaccine following administration will be difficult to determine .
it has proven to be feasible , non - toxic and effective in some cancer patients , particularly if the dc are appropriately matured and activated .
one of the concerns related to ex vivo generated dc is how to ensure effective migration to the t - cell areas in the lymph node . in this context
, we are pursuing the enhancement of migration of ex vivo generated dc by preconditioning the skin with inflammatory cytokines .
the recent application of rna technology in cellular therapy has paved the way to the next generation of dendritic cell - based therapies .
different aspects of dc biology can now be optimized to enhance immunological and clinical responses .
one can think of exploiting ex vivo generated dc equipped with enhanced expression of chemokine receptors to locate lymph nodes , silenced apoptotic pathways to increase longevity and upregulated production of pro - inflammatory cytokines to skew naive t cells .
these multiple approaches need to be investigated in small two - armed principle of concept trials with thorough immune monitoring , in order to increase the clinical efficacy of dc - based cancer vaccines . a second promising approach that circumvents many of the posed hurdles with ex vivo
targeting studies using members of the clr family have paved the way to clinical studies .
 we exploited dendritic cells ( dc ) to vaccinate melanoma patients . 
 we recently demonstrated a statistical significant correlation between favorable clinical outcome and the presence of vaccine - related tumor antigen - specific t cells in delayed type hypersensitivity ( dth ) skin biopsies . 
 however , favorable clinical outcome is only observed in a minority of the treated patients . 
 therefore , it is obvious that current dc - based protocols need to be improved . for this reason , we study in small proof of principle trials the fate , interactions and effectiveness of the injected dc . 
Antigen-presenting dendritic cells
DC-based cancer vaccines: current status
DC migration in vivo
Immune monitoring in tissues
Optimizing DC capabilities by RNA technology
In vivo targeting of DC
Conclusions and future prospects