BACKGROUND
a defective host anti-tumour immune response is an important mechanism enabling tumours to evade control and lead to progressive tumour growth  <cit> .
induction of an effective anti-tumour immune response requires the active and integrated participation of host antigen presenting cells  responsible for the presentation of tumour-associated antigens   <cit> .
dendritic cells  are the most potent apcs.
they are derived from haematopoietic progenitors and play a crucial role in anti-tumour immunity by taking up taags and stimulating ag-specific t cells  <cit> .
the transition of dcs from ag-processing to ag-presenting cells is accompanied by defined alterations in their surface membrane protein expression  <cit> .
up-regulation of class i and class ii major histocompatibility  proteins, cd <dig> and cd <dig> co-stimulatory molecules, and cd <dig> adhesion molecule expression enhances the ability of dcs to present ag to naïve t lymphocytes in secondary lymphoid compartments and induce their activation and generation of peptide-specific cytotoxic t lymphocytes   <cit> .
activated and mature dcs also produce interleukin- <dig> , which stimulates cd4+ t helper  <dig>  cell activation and development, which is important for the in situ  expansion of induced anti-tumour ctls  <cit> .
several groups have described defective function of dcs in tumour bearing mice and in patients with different types of solid cancers  <cit> .
the major findings from these studies were the lack of expression of class ii mhc proteins, and co-stimulatory molecules on dcs, consistent with an immature phenotype and functionally inactive status.
these dcs demonstrate a reduced capability to secrete key lymphocytic immunoregulatory and stimulatory cytokines  <cit> .
immature dcs also directly suppress antigen-specific t cell responses, thereby, inducing immune tolerance to taags and t cell anergy <cit> .
dendritic cells isolated from tumour bearing mice also demonstrate a decreased expression of cd <dig> and mhc class ii molecules, as well as certain adhesion molecules; in parallel with these changes is a reduced capacity to stimulate allogeneic t lymphocytes in the mixed lymphocyte dendritic cell reaction  assay in vitro.
these cells lack the capability to induce effective, peptide-specific and anti-tumour cytotoxic immune responses  <cit> .
we have previously shown in women with operable breast cancer, that both peripheral blood  and tumour-draining axillary lymph node dcs  were dysfunctional and switched off, as assessed by phenotypic profile  and functional assays in vitro  stimulation assay)  <cit> .
the data presented in this article shows that it is possible to reverse this defective dc function by generating dcs by in vitro cultures of peripheral blood monocytes  and lymph node monocytes  from these patients, with specific combinations of cytokines.
reversal of dc dysfunction  and the establishment of optimal dc activity is a key element in any strategy employed to generate an effective and clinically relevant anti-cancer immune response.
this immunotherapeutic approach requires the generation in vitro of large quantities of functionally and phenotypically active dcs, or, more practically in humans, through recovery of dysfunctional dcs in vitro and their subsequent pulsing with peptides/taags and used in immunotherapeutic vaccination  <cit> .
a number of autologous dc vaccination approaches have been used recently but with limited success  <cit> .
the generation of human dcs from plastic-adherent peripheral blood monocytic cultures has traditionally relied on two crucial cytokines, granulocyte-monocyte colony stimulating factor  and interleukin- <dig>   <cit> .
however, this regimen generates mostly immature dcs, with a cd <dig> and cd86low/int, cd1ahigh phenotype after one week of in vitro culture, even when tumour necrosis factor-α  is included in the culture medium  <cit> .
thus, additional cytokines are needed to facilitate the further activation and maturation of the cells into cd1alow, cd40high, cd 80high, cd86high and hla-drhighcells.
cytokines required for dc terminal differentiation from healthy volunteers have been described previously, which includes interferon-α  but there is very little published on its role in dc generation from monocytes of operable breast cancer patients  <cit> .
macrophage-conditioned medium has been shown to be the most potent inducer of the human dc maturation from immature precursors; however, no single cytokine has emerged to be indispensable in this process, as assessed by using cytokine-neutralizing antibodies  <cit> .
interferon-α is a potent immunoregulatory cytokine.
it is secreted early during an immune response by monocytes/macrophages and other less-defined monocytes of myeloid origin resembling immature dcs  <cit> .
it inhibits viral replication and aids in stimulating t cell-mediated immune responses against foreign pathogens <cit> .
interferon-α has also been used clinically for the treatment of chronic viral infections such as hepatitis and in patients with solid and haematopoietic tumours  <cit> .
type i ifn  may enhance the terminal differentiation of cd1a+ immature dcs, derived from human blood cd34+ progenitor cells  <cit> .
in the study reported here, we investigated the effect of low levels of ifn-α , used concurrently with other cytokines, on the activity and maturation of dcs.
RESULTS
the immunophenotype of dcs grown in different cytokine conditioned media
the starting population of mononuclear cells from blood and lymph nodes were heterogeneous, consisting of cd14+ cells , cd3+ cells , cd20+ cells , and cd56+ cells .
peripheral blood monocytes and lnms grown in various ccm;  <dig> ccm  or  <dig> ccm , expressed high levels of hla class ii proteins, cd <dig>  consistent with their differentiation into activated dcs compared with dcs cultured with  <dig> ccm  or medium alone .
the expression of these immunoregulatory proteins was highest on pbdcs  and lndcs  cultured in the presence of  <dig> ccm compared with  <dig> ccm and medium only cultured dcs .
the immunophenotypic profile of pbdcs generated from monocyte cultures of healthy donors demonstrated a pattern, comparable to the optimal immunophenotypic profile obtained with  <dig> ccm  .
cd <dig> expression  was expressed by a small number of dcs in each ccm culture, even with dc generated in the presence of  <dig> ccm.
ifn-α increases the cd <dig> expression on immature dcs
we were interested in whether low levels of ifn-α , added at the initiation of dc development along with gm-csf, il- <dig>  and tnf-α, had any effect on the dc maturation process.
during a 5– <dig> day culture period of adherent monocytes, maturing dcs become non-adherent and appeared as aggregates of floating cells.
the majority of cells expressed high levels of hla class ii as well as cd <dig>  a well-documented marker for activated dcs  <cit> .
in the experiment shown in figure 1a,b and 1c, monocytes were cultured in the presence or absence of ifn-α  for  <dig> days and were then stained for hla-dr and cd <dig> expression.
cells exhibiting a high forward scatter and increased side scatter were gated.
this subset contained all the class ii, and cd <dig> co-expressing cells found in the cultures.
the increase in the cd <dig> expression during dc induction from monocytes was reproducible, both from the blood and regional tumour draining lymph nodes of patients with operable breast cancer.
flowcytometric histograms representative of phenotypic profiles of dcs generated using different ccm are illustrated in figure 2a and 2b.
the inclusion of ifn-α  at the beginning of the culture led to a 2– <dig> fold increase in the total number of activated cd86high dcs.
dcs generated with ifn-α are capable of enhanced dextran uptake via mannose receptors
dcs from peripheral blood of  <dig> breast cancer patients were evaluated for their ability to employ mannose receptors in putative ag uptake by incubating them with cascade blue-conjugated dextran and analysing them by flow cytometry.
the true fluorescence produced by dextran uptake was background fluorescence at 4°c subtracted from the fluorescence at 37°c.
flowcytometric histograms representative of this are illustrated in figure 3a, b, c and 3d.
dcs cultured using  <dig> ccm or  <dig> ccm, demonstrated significantly higher levels of dextran uptake than those dcs prepared using  <dig> ccm or with medium alone  .
cytokine production
the levels of pro-inflammatory or inhibitory cytokines may markedly influence the activation of t lymphocytes.
to address this issue, the production of the pro-inflammatory cytokine il- <dig> p <dig> and the inhibitory cytokine il- <dig> by pbdcs from patients with operable breast cancer was investigated .
the production and secretion of il- <dig> was highest from cultures containing  <dig> ccm.
using gm-csf/il-4/tnf-α ± ifn-α , however, did not lead to enhanced production and secretion of il- <dig>  the production of il- <dig> p <dig> was significantly increased in dcs generated with , when compared with il-4/gm-csf alone and control cultures.
the highest level was seen in cultures containing ifn-α.
we also analysed the levels of il- <dig> and il- <dig> p <dig> in culture supernatants of pbdcs generated from normal healthy donors with the optimal  <dig> ccm .
il- <dig>  was not produced by pbdcs from healthy donors in contrast to pbdcs from patients with operable breast cancer.
moreover, il- <dig> p <dig> was greater in pbdcs from patients with early breast cancer, generated by ifn-α containing  <dig> ccm compared with pbdcs generated identically from healthy donors .
ifn-α matured monocyte-derived dcs from healthy donors are known to have impaired production of il- <dig> p <dig>  <cit> .
a further  <dig> patients with operable breast cancer were recruited and pbdcs were generated with the optimal  <dig> ccm containing ifn-α, which optimally activates dcs as shown before.
the production and secretion of other cytokines  in response to lps stimulation were studied.
il-12p <dig> was consistently undetectable after lps stimulation of pbdcs from all  <dig> patients.
however, il- <dig>  was significantly increased after lps stimulation .
the dcs generated with ifn-α are potent activators of an allogeneic mldcr
the dcs produced by culturing monocytes in a variety of ccm were evaluated for their ability to stimulate t cells in an allogeneic mldcr.
pbdcs from  <dig> breast cancer patients generated in the presence of  <dig> and  <dig> ccms significantly stimulated allogeneic t lymphocyte proliferation in vitro in the mldcr compared with  <dig> ccm and medium only generated dcs .
however, the highest level of mldcr stimulation by pbdcs was seen when all four cytokines  were used .
in contrast, lndcs cultured in  <dig> ccm did not significantly enhance further, the level of stimulation seen with  <dig> ccm in the mldcr assays .
these results demonstrate that dcs, isolated from the circulation and secondary lymphoid compartments of patients with breast cancer, can be activated in vitro with a  <dig> ccm containing ifn-α, and are highly functional.
the activity of these cultured dcs in the allogeneic mldcr correlates closely with their expression of hla-dr, cd <dig>  and cd <dig> immunoregulatory proteins.
discussion
a rapidly evolving cancer immunotherapy approach is the induction of taag specific, cd8+ ctl-mediated anti-cancer immune response, generated by vaccination with functional and active autologous dcs, pulsed with taags.
the host and the tumour milieu are hostile to dc maturation and activation, necessitating ex vivo manipulation of these cells to resuscitate their impaired function and optimally activate them.
after pulsing with taag in vitro, they can be used in vaccination  <cit> .
sources of dcs for clinical trials include cd34+ blood or bone marrow-derived precursors, blood dcs and cd14+ monocytes.
cd14+monocyte-derived dcs are the most widely used in clinical trials.
the 'gold standard' approach, commonly used in many pilot studies, to derive mature dcs from cd14+ cells, is to culture these cells in the presence of il- <dig>  gm-csf, il-1β, tnf-α and pge <dig>  <cit> .
however, this approach has been criticized due to low or absent production of il- <dig>  <cit> .
moreover, there is speculation that lack of response may be due to terminally mature dcs , incapable of inducing efficient ctls against taags  <cit> .
alternative dc activation and maturation approaches have been described using il- <dig> instead of il- <dig>  cd40l, poly i:c and flt3-ligand  <cit> .
the relatively low clinical responses with all these approaches, underpins the current need for novel ex vivo dc reactivation techniques, yielding optimally functional and mature dcs.
the aims of our study were two-fold: firstly, to establish that ex vivo dysfunctional/switched off dcs from women with early operable breast cancer can be replaced by fully functional dcs generated from monocytes, originating from peripheral blood and the lymph nodes, and secondly, to optimize the in vitro conditions to ensure the successful generation of dcs capable of inducing an effective anti-cancer cell-mediated immune response in vivo  <cit> .
our findings demonstrate a very effective in vitro approach, to both reversing and substantially activating, the dysfunctional and switched off dcs derived from blood and lymph node monocytes of patients with operable breast cancer.
this induction of dc maturation and activation has significant relevance in dc-based immunotherapy for cancer.
previous reports have shown that ifn-α can induce the expression of cd <dig> and icam- <dig> in murine mac- <dig> monocytes/macrophages in a model of diabetes induction in vivo  <cit> .
in addition, a number of hepatocellular carcinoma cell lines were shown to respond to ifn-α by inducing cd <dig> expression  <cit> .
our results show that the addition of low doses of ifn-α substantially enhanced the expression of cd40high, cd86high, hla class iihigh in dcs generated from adherent cultures of monocytes obtained from blood and lymph nodes of patients with operable breast cancer.
dendritic cells that mature in the presence of ifn-α also exhibit maximal ability to stimulate t cell proliferation in mldcr and show enhanced macropinocytic function.
to the best of our knowledge, this is the first time dcs have been generated and matured from monocytes from patients with operable breast cancer, using a combination of gm-csf+il-4+tnf-α and ifn-α.
our current and previous studies emphasize the necessity of defining the stage of dc activation/maturation when designing strategies and protocols for immunotherapy of cancer, using dc-based immunization regimens.
published data suggests that the capacity to induce a tumour-specific immune response by monocyte-derived dcs in vivo, is very much dependent on the degree of dc activation and/or maturation, when used for anti-cancer vaccination  <cit> .
in our study, pbms and lnms cultured with ifn-α containing  <dig> ccm optimally differentiated into activated dcs as demonstrated by immunophenotypic profile and mldcr,.
enhanced macropinocytosis and il-12p <dig> cytokine secretion was seen with the  <dig> ccm containing ifn-α.
the critical role of dcs as apcs is reflected by their high-level of expression of class i and ii hla molecules, adhesion molecules  and the co-stimulatory molecules cd <dig> and cd <dig>  <cit> .
dendritic cells generated with the  <dig> ccm exhibited the highest percentage of cells with these immunoregulatory proteins.
the key adhesion molecules , expressed on dcs are critical for the superfamily tnfr  cd40l  on cd4+t cells.
the latter are vital for cd4+t cell licensing and activation through dcs  <cit> .
co-stimulatory molecules  are crucial for optimally enhanced interaction of dcs with cd <dig> receptor on t lymphocytes and their resultant activation and proliferation.
these molecules were also expressed at high  levels by  <dig> ccm treated-dcs.
the cd <dig> molecule, a marker for mature terminally differentiated dcs, was expressed by only a minority of the dcs prepared using all four cytokines   <cit> .
these findings suggest that most of the dcs were not terminally differentiated and, therefore, were likely to be biologically effective in vivo.
moreover, cells grown in tnf-α, with or without ifn-α  had lobulated nuclei and numerous fine cytoplasmic projections consistent with the morphological features of mature and activated dcs.
cells cultured in  <dig> ccm only, had fewer fine cytoplasmic processes and were smaller with round nuclei, which are features of immature and inactivated dcs.
different combinations of cytokines using ifn-α have been described in generating dcs from pbms of healthy volunteers and combinations with this cytokine have been shown to produce dcs of mature phenotype and enhanced function  <cit> .
our results are at variance with previous studies, which describe dcs derived and matured using ifn-α from pbms of healthy subjects and patients, when cultured in the presence of tnf-α or other dc-activating agents   <cit> .
the immunophenotypic characteristics of the dcs predicted their functional capabilities.
for example, pbdcs and lndcs generated from pbms and lnms in different ccm, stimulated allogeneic t lymphocyte proliferation to a variable degree in the mldcr assay, depending on the cytokine combinations used.
dcs concurrently demonstrated higher and differential levels of cells expressing hla-dr, cd <dig>  and cd <dig>  depending on the ccm used.
as in the functional assays, maximal numbers of activated dcs were obtained using the  <dig> ccm.
moreover, the pbm-generated dcs using  <dig> or  <dig> ccm secreted significantly more il- <dig> p <dig> than dcs generated using  <dig> ccm or just medium alone .
in contrast to svane et al we have demonstrated that il-12p <dig> secretion was increased in pbm-generated dcs from patients with early malignant breast disease using ifn-α  <cit> .
this may possibly be due to dcs from patients with early  breast cancer being functionally different and responding differentially to cytokine stimulation compared with dcs from healthy volunteers.
in advanced breast cancer, tnf-α and polyi:c have also been used before as maturation stimuli and to induce secretion of il- <dig>  <cit> .
interferon-α cultured dcs from adherent pbms of healthy donors behave differently depending on the timing of exposure to this cytokine.
early exposure before dc differentiation depletes dc precursors, whereas late exposure after differentation confers dc maturity  <cit> .
in contrast to this, we have shown that early exposure of pbm-derived dcs from patients with early  breast cancer, to ifn-α, leads to a better phenotype and function.
dc maturation with this cytokine combination and effects has not been previously reported in this group of cancer patients, nor has re-activation of lnm-derived dcs, from local tumour draining lymph nodes, been demonstrated.
dendritic cells have the ability to take up, process and present soluble ags to autologous t lymphocytes.
antigen uptake by dcs grown in  <dig> ccm is mediated predominantly by macropinocytosis or binding to mannose receptors  <cit> .
the ability of the activated dcs generated with ifn-α to retain macropinocytosis for antigen uptake is new and contrasts with published evidence that mature dcs lose this ability  <cit> .
despite a high percentage of our dcs expressing hla-dr, cd <dig> and cd <dig>  the expression of cd <dig> was negligible, suggesting that they are not terminally mature and were likely to retain their macropinocytic activity.
interferon-α, may confer preservation of macropinocytosis, despite enhanced maturity.
this is advantageous for immunotherapy, as these dcs are capable of antigen uptake before being inoculated into patients.
the allogeneic t cell responses generated by ifn-α containing  <dig> ccm -treated dcs was significantly better than that documented by dcs grown in  <dig> ccm only.
this is probably due to enhanced numbers of dcs expressing hla class ii proteins, cd <dig> adhesion molecules and co-stimulatory molecule cd <dig>  when prepared with ifn-α.
secretion of il- <dig> was significantly less with the  <dig> ccm generated dcs, compared with the  <dig> ccm generated dcs .
in a related study, dcs from cancer patients generated from cd14+monocytes by culturing with gm-csf and ifn-α, but without il- <dig> secreted higher levels of il- <dig> , compared with il- <dig> and gm-csf generated dcs  <cit> .
moreover, the activation/maturation induced by tnf-α, with or without ifn-α, was further underscored by the strongly enhanced production of the pro-inflammatory cytokine il- <dig> p <dig>  however, il-12p <dig> production was not observed after lps stimulation in dcs generated with  <dig> ccm milieu, but significantly enhanced il- <dig> production and secretion was seen.
this may represent il- <dig> exhaustion of dcs which inversely correlates with maturity as shown previously <cit> .
the il-12p <dig> subunit exists extracellularly as a monomer  or dimer 2) and can antagonize the action of il12p <dig>  the concentration of il12p <dig> alone is not indicative of il- <dig> bioactivity; rather, the bioactivity of il- <dig> produced by activated dcs depends on il12p <dig>  il12p <dig>  and il <dig> production and their competitive interaction with the il- <dig> receptor <cit> .
this may reflect an underestimation of the il- <dig> bioactivity or may be true il- <dig> exhaustion of these dcs.
this will be clarified as il- <dig> bio-activity assessment methods evolve.
interleukin- <dig> is an important licensing cytokine that mediates the polarization of activated cd4+ t helper cells to a th <dig> phenotype for the generation of potent cd8+ ctl responses.
dendritic cells can be induced in vitro and in vivo to secrete il- <dig>  but il- <dig> production and secretion is transient and dcs become refractive to subsequent induction of il- <dig> release, termed "exhaustion" or "paralysis"  <cit> .
these observations strongly suggest that ex vivo dc maturation protocols should avoid conditions that induce dcs to produce and secrete il- <dig>  and should instead use conditions that enable the dcs to acquire responsiveness to il- <dig> induction  <cit> .
despite different  biological activities, ifn-α and il- <dig> share similar but complementary actions on dc development.
the activity of ifn-α documented in our study appears to be distinct from that of previously described effects on dc development.
the first preparation of human dcs from haematopoietic progenitors in vitro was achieved by growing cd34+ cells in gm-csf and other cytokines  <cit> .
it was found that the substitution of tnf-α for ifn-α in pbm cultures containing gm-csf failed to result in generation of cells with a dc immunophenotype.
the addition of anti-tnf-α antibodies to cultures containing ifn-α and gm-csf did not affect the generation of dcs  <cit> .
ligation of cd <dig> on cd34+ haematopoietic progenitor cells was shown to induce them to differentiate into dcs  <cit> .
the ability of il- <dig> and gm-csf to differentiate monocytes into dcs in vitro is well established  <cit> .
transforming growth factor-β is required for the generation of dcs from cd34+ progenitor cells, but only in serum-free conditions  <cit> .
the addition of either c-kit ligand or flt- <dig> ligand to cd34+ cell cultures containing gm-csf and tnf-α can augment the yield of dcs but neither affects dc immunophenotype or function  <cit> .
other cytokines have been shown to play a role in dc development in vitro.
interleukin- <dig>  like gm-csf, can cooperate with tnf-α to generate dcs from cd34+ cells  <cit> .
this activity is distinct from that described in this study with ifn-α.
finally, extensive purification of monocytes before culture in ifn-α+gm-csf had only a minor effect on cell immunophenotype, suggesting that the effect of ifn-α on dc development from monocytes was a direct one  <cit> .
cutaneous recurrences of primary breast cancers when treated with.
injections of natural ifn α and γ, delivered in combination or separately, resulted in complete pathological regression of some of these lesions and was shown to correlate with enhanced expression of hla-dr in the dcs and consequent increased activation and infiltration of t cells locally.
such an enhanced dc maturation and activation response supports our findings that ifn-α is a pre-requisite for optimal dc maturation and activation in patients with breast cancer  <cit> .
CONCLUSIONS
the ability of ifn-α to induce peripheral blood and lymph node monocyte differentiation into dcs and recovery of tumour-induced dc anergy appears to be distinct from that of previously studied cytokine combinations.
we have shown that ex vivo dc generation and maturation from monocytes of breast cancer patients is optimally possible in a system of  <dig> cytokines including ifn-α, which can potentially be used in dc immunotherapy of operable breast cancer patients.
ex vivo generated dcs prepared by the system described are currently being administered to patients and immunological responses  monitored in an ongoing study which has ethical and mhra approval  <cit> .

 a defective host anti-tumour immune response is an important mechanism enabling tumours to evade control and lead to progressive tumour growth. induction of an effective anti-tumour immune response requires the active and integrated participation of host antigen presenting cells responsible for the presentation of tumour-associated antigens. dendritic cells are the most potent apcs. they are derived from haematopoietic progenitors and play a crucial role in anti-tumour immunity by taking up taags and stimulating ag-specific t cells. the transition of dcs from ag-processing to ag-presenting cells is accompanied by defined alterations in their surface membrane protein expression. up-regulation of class i and class ii major histocompatibility proteins, cd and cd co-stimulatory molecules, and cd adhesion molecule expression enhances the ability of dcs to present ag to naïve t lymphocytes in secondary lymphoid compartments and induce their activation and generation of peptide-specific cytotoxic t lymphocytes. activated and mature dcs also produce interleukin-, which stimulates cd4+ t helper cell activation and development, which is important for the in situ expansion of induced anti-tumour ctls. several groups have described defective function of dcs in tumour bearing mice and in patients with different types of solid cancers. the major findings from these studies were the lack of expression of class ii mhc proteins, and co-stimulatory molecules on dcs, consistent with an immature phenotype and functionally inactive status. these dcs demonstrate a reduced capability to secrete key lymphocytic immunoregulatory and stimulatory cytokines. immature dcs also directly suppress antigen-specific t cell responses, thereby, inducing immune tolerance to taags and t cell anergy. dendritic cells isolated from tumour bearing mice also demonstrate a decreased expression of cd and mhc class ii molecules, as well as certain adhesion molecules; in parallel with these changes is a reduced capacity to stimulate allogeneic t lymphocytes in the mixed lymphocyte dendritic cell reaction assay in vitro. these cells lack the capability to induce effective, peptide-specific and anti-tumour cytotoxic immune responses. we have previously shown in women with operable breast cancer, that both peripheral blood and tumour-draining axillary lymph node dcs were dysfunctional and switched off, as assessed by phenotypic profile and functional assays in vitro stimulation assay). the data presented in this article shows that it is possible to reverse this defective dc function by generating dcs by in vitro cultures of peripheral blood monocytes and lymph node monocytes from these patients, with specific combinations of cytokines. reversal of dc dysfunction and the establishment of optimal dc activity is a key element in any strategy employed to generate an effective and clinically relevant anti-cancer immune response. this immunotherapeutic approach requires the generation in vitro of large quantities of functionally and phenotypically active dcs, or, more practically in humans, through recovery of dysfunctional dcs in vitro and their subsequent pulsing with peptides/taags and used in immunotherapeutic vaccination. a number of autologous dc vaccination approaches have been used recently but with limited success. the generation of human dcs from plastic-adherent peripheral blood monocytic cultures has traditionally relied on two crucial cytokines, granulocyte-monocyte colony stimulating factor and interleukin-. however, this regimen generates mostly immature dcs, with a cd and cd86low/int, cd1ahigh phenotype after one week of in vitro culture, even when tumour necrosis factor-α is included in the culture medium. thus, additional cytokines are needed to facilitate the further activation and maturation of the cells into cd1alow, cd40high, cd 80high, cd86high and hla-drhighcells. cytokines required for dc terminal differentiation from healthy volunteers have been described previously, which includes interferon-α but there is very little published on its role in dc generation from monocytes of operable breast cancer patients. macrophage-conditioned medium has been shown to be the most potent inducer of the human dc maturation from immature precursors; however, no single cytokine has emerged to be indispensable in this process, as assessed by using cytokine-neutralizing antibodies. interferon-α is a potent immunoregulatory cytokine. it is secreted early during an immune response by monocytes/macrophages and other less-defined monocytes of myeloid origin resembling immature dcs. it inhibits viral replication and aids in stimulating t cell-mediated immune responses against foreign pathogens. interferon-α has also been used clinically for the treatment of chronic viral infections such as hepatitis and in patients with solid and haematopoietic tumours. type i ifn may enhance the terminal differentiation of cd1a+ immature dcs, derived from human blood cd34+ progenitor cells. in the study reported here, we investigated the effect of low levels of ifn-α, used concurrently with other cytokines, on the activity and maturation of dcs.  the immunophenotype of dcs grown in different cytokine conditioned media the starting population of mononuclear cells from blood and lymph nodes were heterogeneous, consisting of cd14+ cells, cd3+ cells, cd20+ cells, and cd56+ cells. peripheral blood monocytes and lnms grown in various ccm; ccm or ccm, expressed high levels of hla class ii proteins, cd consistent with their differentiation into activated dcs compared with dcs cultured with ccm or medium alone. the expression of these immunoregulatory proteins was highest on pbdcs and lndcs cultured in the presence of ccm compared with ccm and medium only cultured dcs. the immunophenotypic profile of pbdcs generated from monocyte cultures of healthy donors demonstrated a pattern, comparable to the optimal immunophenotypic profile obtained with ccm. cd expression was expressed by a small number of dcs in each ccm culture, even with dc generated in the presence of ccm. ifn-α increases the cd expression on immature dcs we were interested in whether low levels of ifn-α, added at the initiation of dc development along with gm-csf, il- and tnf-α, had any effect on the dc maturation process. during a 5– day culture period of adherent monocytes, maturing dcs become non-adherent and appeared as aggregates of floating cells. the majority of cells expressed high levels of hla class ii as well as cd a well-documented marker for activated dcs. in the experiment shown in figure 1a,b and 1c, monocytes were cultured in the presence or absence of ifn-α for days and were then stained for hla-dr and cd expression. cells exhibiting a high forward scatter and increased side scatter were gated. this subset contained all the class ii, and cd co-expressing cells found in the cultures. the increase in the cd expression during dc induction from monocytes was reproducible, both from the blood and regional tumour draining lymph nodes of patients with operable breast cancer. flowcytometric histograms representative of phenotypic profiles of dcs generated using different ccm are illustrated in figure 2a and 2b. the inclusion of ifn-α at the beginning of the culture led to a 2– fold increase in the total number of activated cd86high dcs. dcs generated with ifn-α are capable of enhanced dextran uptake via mannose receptors dcs from peripheral blood of breast cancer patients were evaluated for their ability to employ mannose receptors in putative ag uptake by incubating them with cascade blue-conjugated dextran and analysing them by flow cytometry. the true fluorescence produced by dextran uptake was background fluorescence at 4°c subtracted from the fluorescence at 37°c. flowcytometric histograms representative of this are illustrated in figure 3a, b, c and 3d. dcs cultured using ccm or ccm, demonstrated significantly higher levels of dextran uptake than those dcs prepared using ccm or with medium alone. cytokine production the levels of pro-inflammatory or inhibitory cytokines may markedly influence the activation of t lymphocytes. to address this issue, the production of the pro-inflammatory cytokine il- p and the inhibitory cytokine il- by pbdcs from patients with operable breast cancer was investigated. the production and secretion of il- was highest from cultures containing ccm. using gm-csf/il-4/tnf-α ± ifn-α, however, did not lead to enhanced production and secretion of il- the production of il- p was significantly increased in dcs generated with, when compared with il-4/gm-csf alone and control cultures. the highest level was seen in cultures containing ifn-α. we also analysed the levels of il- and il- p in culture supernatants of pbdcs generated from normal healthy donors with the optimal ccm. il- was not produced by pbdcs from healthy donors in contrast to pbdcs from patients with operable breast cancer. moreover, il- p was greater in pbdcs from patients with early breast cancer, generated by ifn-α containing ccm compared with pbdcs generated identically from healthy donors. ifn-α matured monocyte-derived dcs from healthy donors are known to have impaired production of il- p. a further patients with operable breast cancer were recruited and pbdcs were generated with the optimal ccm containing ifn-α, which optimally activates dcs as shown before. the production and secretion of other cytokines in response to lps stimulation were studied. il-12p was consistently undetectable after lps stimulation of pbdcs from all patients. however, il- was significantly increased after lps stimulation. the dcs generated with ifn-α are potent activators of an allogeneic mldcr the dcs produced by culturing monocytes in a variety of ccm were evaluated for their ability to stimulate t cells in an allogeneic mldcr. pbdcs from breast cancer patients generated in the presence of and ccms significantly stimulated allogeneic t lymphocyte proliferation in vitro in the mldcr compared with ccm and medium only generated dcs. however, the highest level of mldcr stimulation by pbdcs was seen when all four cytokines were used. in contrast, lndcs cultured in ccm did not significantly enhance further, the level of stimulation seen with ccm in the mldcr assays. these results demonstrate that dcs, isolated from the circulation and secondary lymphoid compartments of patients with breast cancer, can be activated in vitro with a ccm containing ifn-α, and are highly functional. the activity of these cultured dcs in the allogeneic mldcr correlates closely with their expression of hla-dr, cd and cd immunoregulatory proteins. discussion a rapidly evolving cancer immunotherapy approach is the induction of taag specific, cd8+ ctl-mediated anti-cancer immune response, generated by vaccination with functional and active autologous dcs, pulsed with taags. the host and the tumour milieu are hostile to dc maturation and activation, necessitating ex vivo manipulation of these cells to resuscitate their impaired function and optimally activate them. after pulsing with taag in vitro, they can be used in vaccination. sources of dcs for clinical trials include cd34+ blood or bone marrow-derived precursors, blood dcs and cd14+ monocytes. cd14+monocyte-derived dcs are the most widely used in clinical trials. the'gold standard' approach, commonly used in many pilot studies, to derive mature dcs from cd14+ cells, is to culture these cells in the presence of il- gm-csf, il-1β, tnf-α and pge. however, this approach has been criticized due to low or absent production of il-. moreover, there is speculation that lack of response may be due to terminally mature dcs, incapable of inducing efficient ctls against taags. alternative dc activation and maturation approaches have been described using il- instead of il- cd40l, poly i:c and flt3-ligand. the relatively low clinical responses with all these approaches, underpins the current need for novel ex vivo dc reactivation techniques, yielding optimally functional and mature dcs. the aims of our study were two-fold: firstly, to establish that ex vivo dysfunctional/switched off dcs from women with early operable breast cancer can be replaced by fully functional dcs generated from monocytes, originating from peripheral blood and the lymph nodes, and secondly, to optimize the in vitro conditions to ensure the successful generation of dcs capable of inducing an effective anti-cancer cell-mediated immune response in vivo. our findings demonstrate a very effective in vitro approach, to both reversing and substantially activating, the dysfunctional and switched off dcs derived from blood and lymph node monocytes of patients with operable breast cancer. this induction of dc maturation and activation has significant relevance in dc-based immunotherapy for cancer. previous reports have shown that ifn-α can induce the expression of cd and icam- in murine mac- monocytes/macrophages in a model of diabetes induction in vivo. in addition, a number of hepatocellular carcinoma cell lines were shown to respond to ifn-α by inducing cd expression. our results show that the addition of low doses of ifn-α substantially enhanced the expression of cd40high, cd86high, hla class iihigh in dcs generated from adherent cultures of monocytes obtained from blood and lymph nodes of patients with operable breast cancer. dendritic cells that mature in the presence of ifn-α also exhibit maximal ability to stimulate t cell proliferation in mldcr and show enhanced macropinocytic function. to the best of our knowledge, this is the first time dcs have been generated and matured from monocytes from patients with operable breast cancer, using a combination of gm-csf+il-4+tnf-α and ifn-α. our current and previous studies emphasize the necessity of defining the stage of dc activation/maturation when designing strategies and protocols for immunotherapy of cancer, using dc-based immunization regimens. published data suggests that the capacity to induce a tumour-specific immune response by monocyte-derived dcs in vivo, is very much dependent on the degree of dc activation and/or maturation, when used for anti-cancer vaccination. in our study, pbms and lnms cultured with ifn-α containing ccm optimally differentiated into activated dcs as demonstrated by immunophenotypic profile and mldcr,. enhanced macropinocytosis and il-12p cytokine secretion was seen with the ccm containing ifn-α. the critical role of dcs as apcs is reflected by their high-level of expression of class i and ii hla molecules, adhesion molecules and the co-stimulatory molecules cd and cd. dendritic cells generated with the ccm exhibited the highest percentage of cells with these immunoregulatory proteins. the key adhesion molecules, expressed on dcs are critical for the superfamily tnfr cd40l on cd4+t cells. the latter are vital for cd4+t cell licensing and activation through dcs. co-stimulatory molecules are crucial for optimally enhanced interaction of dcs with cd receptor on t lymphocytes and their resultant activation and proliferation. these molecules were also expressed at high levels by ccm treated-dcs. the cd molecule, a marker for mature terminally differentiated dcs, was expressed by only a minority of the dcs prepared using all four cytokines. these findings suggest that most of the dcs were not terminally differentiated and, therefore, were likely to be biologically effective in vivo. moreover, cells grown in tnf-α, with or without ifn-α had lobulated nuclei and numerous fine cytoplasmic projections consistent with the morphological features of mature and activated dcs. cells cultured in ccm only, had fewer fine cytoplasmic processes and were smaller with round nuclei, which are features of immature and inactivated dcs. different combinations of cytokines using ifn-α have been described in generating dcs from pbms of healthy volunteers and combinations with this cytokine have been shown to produce dcs of mature phenotype and enhanced function. our results are at variance with previous studies, which describe dcs derived and matured using ifn-α from pbms of healthy subjects and patients, when cultured in the presence of tnf-α or other dc-activating agents. the immunophenotypic characteristics of the dcs predicted their functional capabilities. for example, pbdcs and lndcs generated from pbms and lnms in different ccm, stimulated allogeneic t lymphocyte proliferation to a variable degree in the mldcr assay, depending on the cytokine combinations used. dcs concurrently demonstrated higher and differential levels of cells expressing hla-dr, cd and cd depending on the ccm used. as in the functional assays, maximal numbers of activated dcs were obtained using the ccm. moreover, the pbm-generated dcs using or ccm secreted significantly more il- p than dcs generated using ccm or just medium alone. in contrast to svane et al we have demonstrated that il-12p secretion was increased in pbm-generated dcs from patients with early malignant breast disease using ifn-α. this may possibly be due to dcs from patients with early breast cancer being functionally different and responding differentially to cytokine stimulation compared with dcs from healthy volunteers. in advanced breast cancer, tnf-α and polyi:c have also been used before as maturation stimuli and to induce secretion of il-. interferon-α cultured dcs from adherent pbms of healthy donors behave differently depending on the timing of exposure to this cytokine. early exposure before dc differentiation depletes dc precursors, whereas late exposure after differentation confers dc maturity. in contrast to this, we have shown that early exposure of pbm-derived dcs from patients with early breast cancer, to ifn-α, leads to a better phenotype and function. dc maturation with this cytokine combination and effects has not been previously reported in this group of cancer patients, nor has re-activation of lnm-derived dcs, from local tumour draining lymph nodes, been demonstrated. dendritic cells have the ability to take up, process and present soluble ags to autologous t lymphocytes. antigen uptake by dcs grown in ccm is mediated predominantly by macropinocytosis or binding to mannose receptors. the ability of the activated dcs generated with ifn-α to retain macropinocytosis for antigen uptake is new and contrasts with published evidence that mature dcs lose this ability. despite a high percentage of our dcs expressing hla-dr, cd and cd the expression of cd was negligible, suggesting that they are not terminally mature and were likely to retain their macropinocytic activity. interferon-α, may confer preservation of macropinocytosis, despite enhanced maturity. this is advantageous for immunotherapy, as these dcs are capable of antigen uptake before being inoculated into patients. the allogeneic t cell responses generated by ifn-α containing ccm -treated dcs was significantly better than that documented by dcs grown in ccm only. this is probably due to enhanced numbers of dcs expressing hla class ii proteins, cd adhesion molecules and co-stimulatory molecule cd when prepared with ifn-α. secretion of il- was significantly less with the ccm generated dcs, compared with the ccm generated dcs. in a related study, dcs from cancer patients generated from cd14+monocytes by culturing with gm-csf and ifn-α, but without il- secreted higher levels of il-, compared with il- and gm-csf generated dcs. moreover, the activation/maturation induced by tnf-α, with or without ifn-α, was further underscored by the strongly enhanced production of the pro-inflammatory cytokine il- p however, il-12p production was not observed after lps stimulation in dcs generated with ccm milieu, but significantly enhanced il- production and secretion was seen. this may represent il- exhaustion of dcs which inversely correlates with maturity as shown previously. the il-12p subunit exists extracellularly as a monomer or dimer 2) and can antagonize the action of il12p the concentration of il12p alone is not indicative of il- bioactivity; rather, the bioactivity of il- produced by activated dcs depends on il12p il12p and il production and their competitive interaction with the il- receptor. this may reflect an underestimation of the il- bioactivity or may be true il- exhaustion of these dcs. this will be clarified as il- bio-activity assessment methods evolve. interleukin- is an important licensing cytokine that mediates the polarization of activated cd4+ t helper cells to a th phenotype for the generation of potent cd8+ ctl responses. dendritic cells can be induced in vitro and in vivo to secrete il- but il- production and secretion is transient and dcs become refractive to subsequent induction of il- release, termed "exhaustion" or "paralysis". these observations strongly suggest that ex vivo dc maturation protocols should avoid conditions that induce dcs to produce and secrete il- and should instead use conditions that enable the dcs to acquire responsiveness to il- induction. despite different biological activities, ifn-α and il- share similar but complementary actions on dc development. the activity of ifn-α documented in our study appears to be distinct from that of previously described effects on dc development. the first preparation of human dcs from haematopoietic progenitors in vitro was achieved by growing cd34+ cells in gm-csf and other cytokines. it was found that the substitution of tnf-α for ifn-α in pbm cultures containing gm-csf failed to result in generation of cells with a dc immunophenotype. the addition of anti-tnf-α antibodies to cultures containing ifn-α and gm-csf did not affect the generation of dcs. ligation of cd on cd34+ haematopoietic progenitor cells was shown to induce them to differentiate into dcs. the ability of il- and gm-csf to differentiate monocytes into dcs in vitro is well established. transforming growth factor-β is required for the generation of dcs from cd34+ progenitor cells, but only in serum-free conditions. the addition of either c-kit ligand or flt- ligand to cd34+ cell cultures containing gm-csf and tnf-α can augment the yield of dcs but neither affects dc immunophenotype or function. other cytokines have been shown to play a role in dc development in vitro. interleukin- like gm-csf, can cooperate with tnf-α to generate dcs from cd34+ cells. this activity is distinct from that described in this study with ifn-α. finally, extensive purification of monocytes before culture in ifn-α+gm-csf had only a minor effect on cell immunophenotype, suggesting that the effect of ifn-α on dc development from monocytes was a direct one. cutaneous recurrences of primary breast cancers when treated with. injections of natural ifn α and γ, delivered in combination or separately, resulted in complete pathological regression of some of these lesions and was shown to correlate with enhanced expression of hla-dr in the dcs and consequent increased activation and infiltration of t cells locally. such an enhanced dc maturation and activation response supports our findings that ifn-α is a pre-requisite for optimal dc maturation and activation in patients with breast cancer.  the ability of ifn-α to induce peripheral blood and lymph node monocyte differentiation into dcs and recovery of tumour-induced dc anergy appears to be distinct from that of previously studied cytokine combinations. we have shown that ex vivo dc generation and maturation from monocytes of breast cancer patients is optimally possible in a system of cytokines including ifn-α, which can potentially be used in dc immunotherapy of operable breast cancer patients. ex vivo generated dcs prepared by the system described are currently being administered to patients and immunological responses monitored in an ongoing study which has ethical and mhra approval. 
dendritic cells play a crucial role in initiating effective cell-mediated immune responses, but are dysfunctional and anergic in breast cancer. dysfunctional and anergic pbdcs and lndcs from patients with operable breast cancer can be optimally reversed by ex vivo culturing of precursor adherent monocytes using a ccm containing ifn-α. peripheral blood dcs and lymph node dcs generated in vitro from adherent cultures of peripheral blood monocytes and lymph node monocytes, respectively, using the cytokine conditioned medium or ccm demonstrated a significantly higher degree of recovery and functional capacity in a mixed lymphocyte dc reaction, expressed significantly higher levels of hla-dr, cd compared with ccm or medium alone generated dcs from pbms and lnms. reversal of this dysfunction and establishment of optimal dc function is a key prerequisite for the induction of effective anti-cancer immune responses.
 dendritic cells play a crucial role in initiating effective cell-mediated immune responses, but are dysfunctional and anergic in breast cancer. reversal of this dysfunction and establishment of optimal dc function is a key prerequisite for the induction of effective anti-cancer immune responses.  peripheral blood dcs and lymph node dcs generated in vitro from adherent cultures of peripheral blood monocytes and lymph node monocytes, respectively, using the cytokine conditioned medium or ccm demonstrated a significantly higher degree of recovery and functional capacity in a mixed lymphocyte dc reaction, expressed significantly higher levels of hla-dr, cd compared with ccm or medium alone generated dcs from pbms and lnms. the pbdcs generated with ccm or ccm showed a significantly enhanced macropinocytotic capability and induced increased production and secretion of interleukin-12p in vitro, compared with pbdcs generated from monocytes using ccm or medium alone. lipopolysaccharide stimulation of pbdcs generated with ccm demonstrated enhanced secretion of il- but not il-12p compared with control dcs unstimulated with lps.  dysfunctional and anergic pbdcs and lndcs from patients with operable breast cancer can be optimally reversed by ex vivo culturing of precursor adherent monocytes using a ccm containing ifn-α. maximal immunophenotypic recovery and functional reactivation of dcs is seen in the presence of ifn-α. however, ccm containing ifn-α generated-pbdcs, do not produce and secrete il-12p in vitro. 