BACKGROUND
dendritic cells  are specialized antigen presenting cells that can initiate a primary immune response on encountering foreign antigens  <cit> .
there has been much focus aimed at harnessing their potency in several clinical applications including cancer, infectious and inflammatory diseases  <cit> .
however, there has been limited success in the treatment of many cancers using dendritic cell based immunotherapy  <cit> .
dc are capable of ingesting dead and dying  tumour cells which potentially expose the dc to an array of tumour-associated antigens for processing and presentation to t cells via hla class i and ii pathways  <cit> .
whilst several methods for loading dc ex vivo with tumour antigens are currently used, including dna, rna, peptides and apoptotic tumour cells, the optimal approach has yet to be determined.
the reasons why individual loading strategies may fail to successfully induce anti-tumour immunity are still not fully understood.
however, an understanding of the natural mechanisms by which dc acquire tumour antigens and mature in situ has resulted in the improvement of several dc-based immunotherapy strategies.
it has been proposed that in situ destruction of tumour cells using chemotherapy, radiotherapy or other physical methods releases suitable antigenic material which can lead to enhanced antigen acquisition and stimulation of an immune response  <cit> .
the chemotherapeutic agent paclitaxel  induces cancer cell death by promoting the polymerisation of tubulin, thereby causing cell death and apoptosis by disrupting the normal microtubule dynamics required for cell division  <cit> .
paclitaxel has been shown to be highly immunosuppressive at cytotoxic doses.
there is clinical evidence to show that systemic administration of cytotoxic compounds such as paclitaxel can have a detrimental effect on the number of systemic dc  <cit> .
however, at lower concentrations there is also evidence to suggest that paclitaxel may be immunostimulatory which may contribute to the overall antitumour effects in the clinical setting  <cit> .
several murine cancer models have demonstrated that combined chemotherapy and dc-based immunotherapy can lead to complete tumour regression in contrast to partial regression in response to each element used individually  <cit> .
the maturation status of dc is a key factor required for the induction of a specific immune response and is reliant on the presentation of antigens by fully mature dc.
paclitaxel has been shown to interact with tlr- <dig>  a know receptor for lipopolysaccharide , on murine myeloid cells  <cit> .
however, the effects of paclitaxel on dc maturation remain to be clarified.
the aims of this study were to evaluate the immunomodulatory effects of paclitaxel applied to ex vivo generated dc in terms of phenotype, function and cytokine expression.
the findings of this study may have important implications in treating malignancies with chemotherapy and concomitant administration of ex vivo dc.
RESULTS
mts assay of melanoma cell and dc mitochondrial activity
in clinical practice chemotherapy exposes both tumour cells and the cells of the immune system to the cytotoxic potential of the drug.
a common target in either cell type is cytoplasmic mitochondrial function.
the potency for inhibition of this activity can be reliably demonstrated using the mts/pms assay.
the human breast adenocarcinoma cell line mcf7-pr has been shown to be sensitive to paclitaxel both in vitro and in vivo in tumour models  <cit> .
the cytoxicity of this and other chemotherapeutic compounds on the newly established melanoma cell line mjt- <dig> has been previously demonstrated in our laboratory .
figure  <dig> shows that both mcf7-pr and mjt- <dig> cell lines are sensitive to paclitaxel over a wide range on concentrations from  <dig>  to  <dig> μmol.
dc were less sensitive to paclitaxel than mcf7-pr, with around 60% difference in mitochondrial activity, and marginally more sensitive than mjt- <dig> tumour cell lines with a difference of nearly 20% when treated with  <dig>  μmol.
correlation of dc viability by tb and pi staining
dc were incubated with paclitaxel as in the mts/pms assay but were analysed for viability and membrane integrity by employing tb dye exclusion by microscopy and pi exclusion by flow cytometry .
as can be seen in figure 2a, dc viability by pi exclusion was maintained even when the dc were exposed to paclitaxel for  <dig> h at concentrations up to  <dig> μm and returned to culture for up to  <dig> days.
at least 80% viability was maintained when dc were continuously exposed to paclitaxel for  <dig> h and  <dig> h at concentration below  <dig> μm.
however, when dc were exposed to paclitaxel at  <dig> μm for  <dig> h there is a significant decrease of viability to around 60%, figure 2b.
in comparison, dc exposed to lps for the same duration of the experiment maintained their viability, consistent with reports of lps inducing a survival signal to dc  <cit> .
phenotypic changes of dc treated with paclitaxel
the resistance of dc to paclitaxel at concentrations up to  <dig> μm was also reflected in surface marker expression by flow cytometry.
as outlined in table  <dig> the addition of paclitaxel led to the dose dependent increase in the expression of hla class ii molecules  i.e.
the mfi for the cells treated with  <dig>   <dig> or  <dig> μm was  <dig>   <dig>  <dig> respectively.
the expression levels of class ii on paclitaxel-treated dc at  <dig> μm were equivalent to that induced by lps.
higher concentrations of paclitaxel  did not permit analysis of class ii expression due to dc viability being too low for analysis .
there were no significant effects on co-stimulatory molecule expression or on cd <dig> or cd <dig>
dc were incubated with paclitaxel at various concentrations or lps  for  <dig> h before being washed and returned to culture for a further  <dig> h. surface expression of various molecules was assessed by flow cytometry as outlined in the materials and methods.
the panel reports the delta mfi  mean fluorescence intensity .
data representative of  <dig> independent experiments.
enhanced dc stimulatory capacity at low dose paclitaxel
the immunostimulatory properties of paclitaxel-treated dc were assessed by studying their ability to induce the proliferation of allogeneic t cells.
the addition of paclitaxel at a concentration of  <dig> μm did not significantly alter the ability of dc to induce proliferation when compared to non-treated counterparts.
higher concentrations of paclitaxel  totally abolished proliferation indicative of toxicity to the dc at the higher dose .
the stimulatory effect of  <dig> μm and  <dig> μm doses of paclitaxel on dc was significantly higher  when compared to lps matured dc and untreated controls.
at a 80: <dig> t:dc ratio the biggest increase was seen with a 3-fold increase in proliferation with dcs treated with  <dig> μm and 2-fold increase with dcs treated with  <dig> μm.
this enhancement in proliferation was maintained at all ratios of t cell:dc tested.
paclitaxel reduces receptor-mediated endocytosis
as already demonstrated, the exposure of dc to paclitaxel induced hla class ii expression but not other markers including the mannose receptor cd <dig> .
this was reflected by similar levels of dextran-fitc binding to the cell surface receptor of quiescent dc incubated at 4°c figure  <dig>  however, during active endocytosis at 37°c an increase in the concentration of paclitaxel caused a decrease in the endocytic capacity of treated dc.
at concentrations above  <dig> μm the level of uptake was significantly reduced by at least a  <dig> -fold change in mfi when compared to untreated dc , figure  <dig>
microarray analysis
the effect of paclitaxel and lps on day  <dig> dc were examined by microarray.
all microarray data was deposited in array express .
duplicate samples were compared.
fold changes in gene expression compared to untreated cells are shown in table  <dig>  only significant differences with p <  <dig>  are shown.
as expected the most significant effects were observed with lps with induction of th <dig> cytokine and chemokine gene expression, stat <dig> activation and cd <dig>  a marker of t cell activation.
other findings included an increase in cell cycle gene p <dig>  fas and fas ligand, receptors such as icam <dig> and cxcr <dig>  interferon alpha response genes ifi <dig> and nfκb.
in contrast, paclitaxel reduced the expression of most of these genes apart from il-1alpha.
lps induced tlr <dig> expression but resulted in marked reduction in tlr <dig> expression: paclitaxel was associated with a moderate reduction in expression of tlr <dig>
data shown is for samples where gene expression differed from untreated
cells .
all microarray data was deposited in array express; accession number e-mexp- <dig>  experiment name pandha dc taxol.
differential effect of paclitaxel on cytokine secretion by lps-treated dc
dc produced an array of inflammatory cytokines in response to lps stimulation.
in keeping with the microarray findings, the addition of paclitaxel inhibited the expression of il-12p <dig>  tnfα, il- <dig>  il- <dig> and il-1β at all concentrations tested
paclitaxel induces actin remodelling of dc cytoskeleton
in the absence of lps or paclitaxel immature dc were characterised as being relatively large, irregular shaped bodies with a readily visible dense network of microtubules often focusing around the centrosome, figure  <dig>  the treatment of dc with  <dig> μm paclitaxel resulted in distinct alterations to both dc morphology and microtubule distribution.
many of the dc became rounded with evidence of microtubule stabilisation, figure  <dig>  the changes in dc cytoskeleton organisation were even more dramatic with  <dig> μm paclitaxel where all cells appeared small, rounded with microtubules that were no longer focused around the centrosome figure  <dig>  maturation in response to  <dig> μg/ml lps also induced a rapid reduction in the size of the dc to smaller rounded cells with many forming small clumps figure  <dig>  again, the microtubules were less distinct and paralleled than observed for the paclitaxel treated dc.
treatment of lps matured dc with various concentrations of paclitaxel had no distinguishable effects on morphology or microtubule stabilisation when compared to lps alone or high dose paclitaxel treated dc .
effects of paclitaxel are not mediated via tlr- <dig> binding
we have demonstrated that the treatment of dc with paclitaxel induces a dose dependent upregulation of surface hla-class ii expression.
in order to determine whether this is mediated through the binding of tlr- <dig> we employed a blocking antibody against the tlr- <dig> molecule.
whilst lps and paclitaxel induced the upregulation of surface class ii molecules, blocking of tlr- <dig> with a specific monoclonal antibody only partially inhibited the response to lps, figure  <dig>
discussion
several chemotherapeutic agents have been shown to have dose dependent immunostimulatory activity  <cit> .
however, there is conflicting data to suggest that these compounds are also capable of inhibiting the generation and maturation of cells capable of antitumour effects, especially in cancer patients who are already debilitated by their disease  <cit> .
paclitaxel is a widely used cytotoxic agent which targets microtubules and induces cellular death via apoptosis.
the doses of paclitaxel under evaluation in this study reflect clinical practice: paclitaxel disappearance from plasma after intravenous infusion is biphasic; half-lives of the first and second phases occur approximately  <dig>  and  <dig> hours, respectively.
the peak plasma concentration with a high dose such as  <dig> mg/m <dig>  occurs immediately post-infusion, and is approximately  <dig> μm  <cit> .
we have demonstrated that, at similar plasma levels as in clinical practice, the mitochondrial function of human dc is less susceptible to inhibition by paclitaxel when compared to cancer cell lines.
this is in accordance with data published on murine dc which were more resistant to paclitaxel compared to the murine melanoma cell line b <dig>  <cit> .
the same report demonstrated that there was no induction of necrosis or apoptosis in murine dc treated with paclitaxel for  <dig> hours.
a recent publication indicated significant effects of paclitaxel on dc at a dose range of 1- <dig> μm albeit on murine dcs  <cit> .
we found, by two different methods, that paclitaxel at  <dig> μm does not induce either immediate cellular death or reduce dc viability for up to  <dig> days in culture.
in contrast, it has been reported that human dc can undergo apoptosis following incubation with paclitaxel at concentrations above  <dig> μm when continually exposed for  <dig> hours but these cells still maintaining mitochondrial function  <cit> .
one possible mode of action by which chemotherapeutic agents may be immunomodulatory is by the induction of dc maturation by the upregulation of key molecules, especially those involved in antigen presentation such as hla class ii.
we found that paclitaxel selectively enhanced the expression of hla class ii on dc in a dose dependent manner.
interestingly, the upregulation of class ii did not correlate with allo-stimulatory activity.
enhancement of the allo-stimulatory activity was observed at low doses of paclitaxel which was superior to lps activated dc.
this would suggest that factors other than antigen presentation molecules are crucially affected by treatment with higher doses of paclitaxel.
low concentrations of paclitaxel have been shown to have a cytostatic effect on cells by effecting both cellular mitochondrial membrane and reducing potential.
however, the apoptotic pathway is stopped upstream of mitochondria permeabilization and therefore does not lead to cell death <cit> .
the interactions of mhc class ii and the mitochondria are central during the induction of mature dc death via cross linking of the surface molecules  <cit> .
the expression of mhc class ii in blood mononuclear cells is also associated with the presence of myosin-v which is itself frequently associated with the central microtubules  <cit> .
therefore, alteration in mhc class ii expression may be due to either paclitaxel induced disruption of mitochondrial function or the effect on microtubule arrangement via the co-localization with myosin-v.
a reduction in the endocytic function of dc was also observed in response to paclitaxel despite no overall change in the expression of the specific surface receptor cd <dig>  phagocytosis of listeria monocytongenes by macrophages has been shown to be dependent on functional actin filaments.
depolymerising drugs such as colchicine and nocodazole greatly reduced the cells ability to phagocytose l. monocytogenes while the stabilization of microtubles with paclitaxel had no significant effect on uptake  <cit> .
paclitaxel may therefore inhibit receptor mediated endocytosis by preventing mitochondrial activity from facilitating this active process.
we have demonstrated that paclitaxel was very potent at altering both dc morphology and microtubule organization.
paclitaxel treatment in patients results in the release of a spectrum of pro-and anti-inflammatory cytokines, but most significantly il- <dig>  il- <dig> and il- <dig>  <cit> .
microarray analysis of dc after paclitaxel exposure showed downregulation of most cytokine gene expression; the overall effects of paclitaxel appeared to be anti-inflammatory.
cytokine analysis from supernatants reflected these findings.
in contrast, as expected, lps effects on dc were profoundly pro-inflammatory.
of note also was the reduction in il- <dig> production after exposure to paclitaxel suggests that paclitaxel-treated dc may induce a th <dig>  cell bias with implications regarding their use therapeutically.
similarly, the lack of chemokine secretion after paclitaxel exposure implies a reduction in dc migration to lymph nodes thereby reducing the possibility of priming naive t cells in a dc therapy context.
the reduction of mitochondrial potential in dc responding to glucocorticoid has been shown to inhibit dc maturation and il- <dig> production  <cit> .
as with mhc class ii expression, depolarisation of the microtubule network can severely inhibit dc production of il- <dig> in response to a tlr- <dig> agonist due to the disruption of intracellular tlr- <dig>  <cit> .
therefore, yet again paclitaxel-induced disruption of mitochondrial activity or microtubule arrangement may explain the inhibition of cytokine production.
paclitaxel has been shown to mimic lps activation of murine macrophages in a cell cycle-independent manner.
stimulation of these primary cells with paclitaxel is a tlr- <dig> and myd88- dependent pathway which augments the release of tnf and nitric oxide  <cit> .
tlr- <dig> has been shown to physically associate with the md- <dig> and it is this molecule that confers lps-responsiveness on tlr- <dig>  the species-specific activity of paclitaxel is determined by murine md- <dig> and not human md- <dig>  suggesting that md- <dig> is the crucial element for recognition  <cit> .
however, both we and others have demonstrated that human and murine myeloid cells respond to paclitaxel in a tlr- <dig> independent manner.
CONCLUSIONS
in conclusion, this study has increased our understanding of the effects of a potentially immunomodulatory chemotherapeutic agent on the modulation of dc.
paclitaxel has been shown to both enhance and inhibit many functional capabilities of dc via inhibiting mitochondrial function and microtubule polymerization.
our preliminary data suggests that these mechanisms may occur in the absence of signaling through tlr- <dig>  in the clinical scenario, overall, we would anticipate that concomitant administration of paclitaxel would enhance dc-based vaccine through upregulation chemokine expression and increased allostimulatory activity of dc although the precise time of administration of vaccine and paclitaxel need to be carefully considered in view of paclitaxel's rapid distribution post-infusion.
combined paclitaxel and dc-based vaccines are currently under investigation in a murine model.

 dendritic cells are specialized antigen presenting cells that can initiate a primary immune response on encountering foreign antigens. there has been much focus aimed at harnessing their potency in several clinical applications including cancer, infectious and inflammatory diseases. however, there has been limited success in the treatment of many cancers using dendritic cell based immunotherapy. dc are capable of ingesting dead and dying tumour cells which potentially expose the dc to an array of tumour-associated antigens for processing and presentation to t cells via hla class i and ii pathways. whilst several methods for loading dc ex vivo with tumour antigens are currently used, including dna, rna, peptides and apoptotic tumour cells, the optimal approach has yet to be determined. the reasons why individual loading strategies may fail to successfully induce anti-tumour immunity are still not fully understood. however, an understanding of the natural mechanisms by which dc acquire tumour antigens and mature in situ has resulted in the improvement of several dc-based immunotherapy strategies. it has been proposed that in situ destruction of tumour cells using chemotherapy, radiotherapy or other physical methods releases suitable antigenic material which can lead to enhanced antigen acquisition and stimulation of an immune response. the chemotherapeutic agent paclitaxel induces cancer cell death by promoting the polymerisation of tubulin, thereby causing cell death and apoptosis by disrupting the normal microtubule dynamics required for cell division. paclitaxel has been shown to be highly immunosuppressive at cytotoxic doses. there is clinical evidence to show that systemic administration of cytotoxic compounds such as paclitaxel can have a detrimental effect on the number of systemic dc. however, at lower concentrations there is also evidence to suggest that paclitaxel may be immunostimulatory which may contribute to the overall antitumour effects in the clinical setting. several murine cancer models have demonstrated that combined chemotherapy and dc-based immunotherapy can lead to complete tumour regression in contrast to partial regression in response to each element used individually. the maturation status of dc is a key factor required for the induction of a specific immune response and is reliant on the presentation of antigens by fully mature dc. paclitaxel has been shown to interact with tlr- a know receptor for lipopolysaccharide, on murine myeloid cells. however, the effects of paclitaxel on dc maturation remain to be clarified. the aims of this study were to evaluate the immunomodulatory effects of paclitaxel applied to ex vivo generated dc in terms of phenotype, function and cytokine expression. the findings of this study may have important implications in treating malignancies with chemotherapy and concomitant administration of ex vivo dc.  mts assay of melanoma cell and dc mitochondrial activity in clinical practice chemotherapy exposes both tumour cells and the cells of the immune system to the cytotoxic potential of the drug. a common target in either cell type is cytoplasmic mitochondrial function. the potency for inhibition of this activity can be reliably demonstrated using the mts/pms assay. the human breast adenocarcinoma cell line mcf7-pr has been shown to be sensitive to paclitaxel both in vitro and in vivo in tumour models. the cytoxicity of this and other chemotherapeutic compounds on the newly established melanoma cell line mjt- has been previously demonstrated in our laboratory. figure shows that both mcf7-pr and mjt- cell lines are sensitive to paclitaxel over a wide range on concentrations from to μmol. dc were less sensitive to paclitaxel than mcf7-pr, with around 60% difference in mitochondrial activity, and marginally more sensitive than mjt- tumour cell lines with a difference of nearly 20% when treated with μmol. correlation of dc viability by tb and pi staining dc were incubated with paclitaxel as in the mts/pms assay but were analysed for viability and membrane integrity by employing tb dye exclusion by microscopy and pi exclusion by flow cytometry. as can be seen in figure 2a, dc viability by pi exclusion was maintained even when the dc were exposed to paclitaxel for h at concentrations up to μm and returned to culture for up to days. at least 80% viability was maintained when dc were continuously exposed to paclitaxel for h and h at concentration below μm. however, when dc were exposed to paclitaxel at μm for h there is a significant decrease of viability to around 60%, figure 2b. in comparison, dc exposed to lps for the same duration of the experiment maintained their viability, consistent with reports of lps inducing a survival signal to dc. phenotypic changes of dc treated with paclitaxel the resistance of dc to paclitaxel at concentrations up to μm was also reflected in surface marker expression by flow cytometry. as outlined in table the addition of paclitaxel led to the dose dependent increase in the expression of hla class ii molecules i.e. the mfi for the cells treated with or μm was respectively. the expression levels of class ii on paclitaxel-treated dc at μm were equivalent to that induced by lps. higher concentrations of paclitaxel did not permit analysis of class ii expression due to dc viability being too low for analysis. there were no significant effects on co-stimulatory molecule expression or on cd or cd dc were incubated with paclitaxel at various concentrations or lps for h before being washed and returned to culture for a further h. surface expression of various molecules was assessed by flow cytometry as outlined in the materials and methods. the panel reports the delta mfi mean fluorescence intensity. data representative of independent experiments. enhanced dc stimulatory capacity at low dose paclitaxel the immunostimulatory properties of paclitaxel-treated dc were assessed by studying their ability to induce the proliferation of allogeneic t cells. the addition of paclitaxel at a concentration of μm did not significantly alter the ability of dc to induce proliferation when compared to non-treated counterparts. higher concentrations of paclitaxel totally abolished proliferation indicative of toxicity to the dc at the higher dose. the stimulatory effect of μm and μm doses of paclitaxel on dc was significantly higher when compared to lps matured dc and untreated controls. at a 80: t:dc ratio the biggest increase was seen with a 3-fold increase in proliferation with dcs treated with μm and 2-fold increase with dcs treated with μm. this enhancement in proliferation was maintained at all ratios of t cell:dc tested. paclitaxel reduces receptor-mediated endocytosis as already demonstrated, the exposure of dc to paclitaxel induced hla class ii expression but not other markers including the mannose receptor cd. this was reflected by similar levels of dextran-fitc binding to the cell surface receptor of quiescent dc incubated at 4°c figure however, during active endocytosis at 37°c an increase in the concentration of paclitaxel caused a decrease in the endocytic capacity of treated dc. at concentrations above μm the level of uptake was significantly reduced by at least a -fold change in mfi when compared to untreated dc, figure microarray analysis the effect of paclitaxel and lps on day dc were examined by microarray. all microarray data was deposited in array express. duplicate samples were compared. fold changes in gene expression compared to untreated cells are shown in table only significant differences with p < are shown. as expected the most significant effects were observed with lps with induction of th cytokine and chemokine gene expression, stat activation and cd a marker of t cell activation. other findings included an increase in cell cycle gene p fas and fas ligand, receptors such as icam and cxcr interferon alpha response genes ifi and nfκb. in contrast, paclitaxel reduced the expression of most of these genes apart from il-1alpha. lps induced tlr expression but resulted in marked reduction in tlr expression: paclitaxel was associated with a moderate reduction in expression of tlr data shown is for samples where gene expression differed from untreated cells. all microarray data was deposited in array express; accession number e-mexp- experiment name pandha dc taxol. differential effect of paclitaxel on cytokine secretion by lps-treated dc dc produced an array of inflammatory cytokines in response to lps stimulation. in keeping with the microarray findings, the addition of paclitaxel inhibited the expression of il-12p tnfα, il- il- and il-1β at all concentrations tested paclitaxel induces actin remodelling of dc cytoskeleton in the absence of lps or paclitaxel immature dc were characterised as being relatively large, irregular shaped bodies with a readily visible dense network of microtubules often focusing around the centrosome, figure the treatment of dc with μm paclitaxel resulted in distinct alterations to both dc morphology and microtubule distribution. many of the dc became rounded with evidence of microtubule stabilisation, figure the changes in dc cytoskeleton organisation were even more dramatic with μm paclitaxel where all cells appeared small, rounded with microtubules that were no longer focused around the centrosome figure maturation in response to μg/ml lps also induced a rapid reduction in the size of the dc to smaller rounded cells with many forming small clumps figure again, the microtubules were less distinct and paralleled than observed for the paclitaxel treated dc. treatment of lps matured dc with various concentrations of paclitaxel had no distinguishable effects on morphology or microtubule stabilisation when compared to lps alone or high dose paclitaxel treated dc. effects of paclitaxel are not mediated via tlr- binding we have demonstrated that the treatment of dc with paclitaxel induces a dose dependent upregulation of surface hla-class ii expression. in order to determine whether this is mediated through the binding of tlr- we employed a blocking antibody against the tlr- molecule. whilst lps and paclitaxel induced the upregulation of surface class ii molecules, blocking of tlr- with a specific monoclonal antibody only partially inhibited the response to lps, figure discussion several chemotherapeutic agents have been shown to have dose dependent immunostimulatory activity. however, there is conflicting data to suggest that these compounds are also capable of inhibiting the generation and maturation of cells capable of antitumour effects, especially in cancer patients who are already debilitated by their disease. paclitaxel is a widely used cytotoxic agent which targets microtubules and induces cellular death via apoptosis. the doses of paclitaxel under evaluation in this study reflect clinical practice: paclitaxel disappearance from plasma after intravenous infusion is biphasic; half-lives of the first and second phases occur approximately and hours, respectively. the peak plasma concentration with a high dose such as mg/m occurs immediately post-infusion, and is approximately μm. we have demonstrated that, at similar plasma levels as in clinical practice, the mitochondrial function of human dc is less susceptible to inhibition by paclitaxel when compared to cancer cell lines. this is in accordance with data published on murine dc which were more resistant to paclitaxel compared to the murine melanoma cell line b. the same report demonstrated that there was no induction of necrosis or apoptosis in murine dc treated with paclitaxel for hours. a recent publication indicated significant effects of paclitaxel on dc at a dose range of 1- μm albeit on murine dcs. we found, by two different methods, that paclitaxel at μm does not induce either immediate cellular death or reduce dc viability for up to days in culture. in contrast, it has been reported that human dc can undergo apoptosis following incubation with paclitaxel at concentrations above μm when continually exposed for hours but these cells still maintaining mitochondrial function. one possible mode of action by which chemotherapeutic agents may be immunomodulatory is by the induction of dc maturation by the upregulation of key molecules, especially those involved in antigen presentation such as hla class ii. we found that paclitaxel selectively enhanced the expression of hla class ii on dc in a dose dependent manner. interestingly, the upregulation of class ii did not correlate with allo-stimulatory activity. enhancement of the allo-stimulatory activity was observed at low doses of paclitaxel which was superior to lps activated dc. this would suggest that factors other than antigen presentation molecules are crucially affected by treatment with higher doses of paclitaxel. low concentrations of paclitaxel have been shown to have a cytostatic effect on cells by effecting both cellular mitochondrial membrane and reducing potential. however, the apoptotic pathway is stopped upstream of mitochondria permeabilization and therefore does not lead to cell death. the interactions of mhc class ii and the mitochondria are central during the induction of mature dc death via cross linking of the surface molecules. the expression of mhc class ii in blood mononuclear cells is also associated with the presence of myosin-v which is itself frequently associated with the central microtubules. therefore, alteration in mhc class ii expression may be due to either paclitaxel induced disruption of mitochondrial function or the effect on microtubule arrangement via the co-localization with myosin-v. a reduction in the endocytic function of dc was also observed in response to paclitaxel despite no overall change in the expression of the specific surface receptor cd phagocytosis of listeria monocytongenes by macrophages has been shown to be dependent on functional actin filaments. depolymerising drugs such as colchicine and nocodazole greatly reduced the cells ability to phagocytose l. monocytogenes while the stabilization of microtubles with paclitaxel had no significant effect on uptake. paclitaxel may therefore inhibit receptor mediated endocytosis by preventing mitochondrial activity from facilitating this active process. we have demonstrated that paclitaxel was very potent at altering both dc morphology and microtubule organization. paclitaxel treatment in patients results in the release of a spectrum of pro-and anti-inflammatory cytokines, but most significantly il- il- and il-. microarray analysis of dc after paclitaxel exposure showed downregulation of most cytokine gene expression; the overall effects of paclitaxel appeared to be anti-inflammatory. cytokine analysis from supernatants reflected these findings. in contrast, as expected, lps effects on dc were profoundly pro-inflammatory. of note also was the reduction in il- production after exposure to paclitaxel suggests that paclitaxel-treated dc may induce a th cell bias with implications regarding their use therapeutically. similarly, the lack of chemokine secretion after paclitaxel exposure implies a reduction in dc migration to lymph nodes thereby reducing the possibility of priming naive t cells in a dc therapy context. the reduction of mitochondrial potential in dc responding to glucocorticoid has been shown to inhibit dc maturation and il- production. as with mhc class ii expression, depolarisation of the microtubule network can severely inhibit dc production of il- in response to a tlr- agonist due to the disruption of intracellular tlr-. therefore, yet again paclitaxel-induced disruption of mitochondrial activity or microtubule arrangement may explain the inhibition of cytokine production. paclitaxel has been shown to mimic lps activation of murine macrophages in a cell cycle-independent manner. stimulation of these primary cells with paclitaxel is a tlr- and myd88- dependent pathway which augments the release of tnf and nitric oxide. tlr- has been shown to physically associate with the md- and it is this molecule that confers lps-responsiveness on tlr- the species-specific activity of paclitaxel is determined by murine md- and not human md- suggesting that md- is the crucial element for recognition. however, both we and others have demonstrated that human and murine myeloid cells respond to paclitaxel in a tlr- independent manner.  in conclusion, this study has increased our understanding of the effects of a potentially immunomodulatory chemotherapeutic agent on the modulation of dc. paclitaxel has been shown to both enhance and inhibit many functional capabilities of dc via inhibiting mitochondrial function and microtubule polymerization. our preliminary data suggests that these mechanisms may occur in the absence of signaling through tlr- in the clinical scenario, overall, we would anticipate that concomitant administration of paclitaxel would enhance dc-based vaccine through upregulation chemokine expression and increased allostimulatory activity of dc although the precise time of administration of vaccine and paclitaxel need to be carefully considered in view of paclitaxel's rapid distribution post-infusion. combined paclitaxel and dc-based vaccines are currently under investigation in a murine model. 
however, paclitaxel exposure reduced the endocytic capacity of dc but reduced the expression of key pro-inflammatory cytokines such as il- and tnfα. paclitaxel exposure led to a dose dependent increase in hla class ii expression equivalent to exposure to lipopolysaccharide, and a corresponding increase in proliferation of allogeneic t cells at the clinically relevant doses of paclitaxel. the potential utility of dendritic cells as cancer vaccines has been established in early trials in human cancers. the concomitant administration of cytotoxic agents and dc vaccines has been previously avoided due to potential immune suppression by chemotherapeutics.
 the potential utility of dendritic cells as cancer vaccines has been established in early trials in human cancers. the concomitant administration of cytotoxic agents and dc vaccines has been previously avoided due to potential immune suppression by chemotherapeutics. recent studies show that common chemotherapy agents positively influence adaptive and innate anti-tumour immune responses.  we investigated the effects of paclitaxel on human dc biology in vitro. dcs appear to sustain a significant level of resistance to paclitaxel and maintain normal viability at concentrations of up to μmol. in some cases this resistance against paclitaxel is significantly better than the level seen in tumour cell lines. paclitaxel exposure led to a dose dependent increase in hla class ii expression equivalent to exposure to lipopolysaccharide, and a corresponding increase in proliferation of allogeneic t cells at the clinically relevant doses of paclitaxel. increase in hla-class ii expression induced by paclitaxel was not blocked by anti tlr- antibody. however, paclitaxel exposure reduced the endocytic capacity of dc but reduced the expression of key pro-inflammatory cytokines such as il- and tnfα. key morphological changes occurred when immature dc were cultured with μmol paclitaxel. they became small rounded cells with stable microtubules, whereas there were little effects on lps-matured dc.  the effect of paclitaxel on human monocyte derived dc is complex, but in the clinical context of patients receiving preloaded and matured dc vaccines, its immunostimulatory potential and resistance to direct cytotoxicity by paclitaxel would indicate potential advantages to co-administration with vaccines. 