slc / ccl21 , normally expressed in high endothelial venules and in t cell zones of spleen and lymph nodes , strongly attracts t cells and dendritic cells ( dc ) [ 1 - 8 ] .
slc / ccl21 recruits both nave lymphocytes and antigen stimulated dc into t cell zones of secondary lymphoid organs , co - localizing these early immune response constituents that culminate in cognate t cell activation .
the capacity to facilitate the co - localization of both dc and t cells forms a strong rationale for the use of slc / ccl21 in cancer therapy . in previous studies
we have shown that the slc / ccl21-mediated anti - tumor responses are accompanied by the enhanced elaboration of ifn , ip-10/cxcl10 , mig / cxcl9 , gm - csf and il-12 , but decreased pge2 , vegf , il-10 , and tgf- at the tumor site .
in addition , there was a marked increase in cd4and cd8cells secreting ifn and gm - csf as well as dc expressing cd11c infiltrating the tumors .
mig / cxcl 9 and ip-10/cxcl10 are glutamine - leucine - arginine ( elr)-negative cxc chemokines that are potent chemoattractants for activated t cells [ 11 - 13 ]
. these chemokines also have in common the ability to signal through a g protein - coupled receptor , cxcr3 , expressed on activated t lymphocytes .
ip-10/cxcl10 , mig / cxcl9 and ifn are known to have potent anti - tumor activities in vivo [ 14 - 17 ] .
both mig / cxcl9 and ip-10/cxcl10 are important for the anti - tumor activity mediated by ifn inducing cytokines such as il-12 .
based on these findings we hypothesized that augmentation in ifn , mig / cxcl9 , and ip-10/cxcl10 would play an important role in the slc / ccl21-mediated anti - tumor response . here
we show that the cytokines ifn , ip-10/cxcl10 and mig / cxcl9 serve as effector molecules in slc / ccl21-mediated anti - tumor responses .
host antigen presenting cells ( apc ) are critical for the cross - presentation of tumor antigens .
however tumors have the capacity to limit apc maturation , function and infiltration of the tumor site [ 21 - 24 ] .
thus molecules that attract host apc and t cells could serve as potent agents for cancer immunotherapy .
a potentially effective pathway to restore ag presentation is the establishment of a chemotactic gradient that favors localization of both activated dc and type 1 cytokine producing lymphocytes at the tumor site .
slc / ccl21 , a cc chemokine expressed in high endothelial venules and in t cell zones of spleen and lymph nodes , strongly attracts naive t cells and dcs [ 1 - 8 ] . because dcs are potent apcs that function as principal activators of t cells , the capacity to facilitate the co - localization of both dc and t cells may reverse tumor - mediated immune suppression and orchestrate effective cell - mediated immune responses [ 9,10,25 - 27 ] .
in addition to its immunotherapeutic potential , slc / ccl21 has been found to have potent angiostatic effects , thus adding further support for its use in cancer therapy . based on these dual capacities ,
we as well as others have begun pre - clinical evaluation of slc / ccl21 in several tumor models [ 9,10,25 - 27 ] . utilizing two transplantable murine lung cancer models
, we have previously shown that the anti - tumor efficacy of slc / ccl21 is t cell - dependent . in both models recombinant
slc / ccl21 administered intratumorally led to complete tumor eradication in 40 % of the treated mice .
studies performed in cd4 and cd8 knockout mice also revealed a requirement for both cd4 and cd8 lymphocytes subsets for slc / ccl21 mediated tumor regression .
consistent with these findings we found that slc therapy did not alter tumor growth in scid mice . in immune competent mice ,
intratumoral injection of slc / ccl21 led to a significant increase in cd4 and cd8 t lymphocytes as well as dc infiltrating the tumor and the draining lymph nodes .
the cellular infiltrates were accompanied by an enhanced elaboration of il-12 , ifn , ip-10/cxcl10 and mig / cxcl9 at the tumor site .
slc / ccl21 is documented to have direct anti - angiogenic effects ; the tumor reductions observed in this model could have been due to participation by t cells secreting ifn leading to inhibition of angiogenesis via mig / cxcl9 and ip-10/cxcl10 , as well as t cell - dependent immunity .
both mig / cxcl9 and ip-10/cxcl10 are chemotactic for stimulated cxcr3-expressing activated t lymphocytes that could further amplify ifn at the tumor site .
hence we postulated that in addition to ifn , ip-10/cxcl10 and mig / cxcl9 would also be important contributors to the tumor reduction in the context of slc / ccl21 therapy .
to determine the importance of mig / cxcl9 , ip-10/cxcl10 and ifn in the slc / ccl21 mediated antitumor response , these cytokines were depleted in slc / ccl21-treated mice .
anti - ip-10/cxcl10 , mig / cxcl9 and ifn antibodies each partially but yet significantly inhibited the anti - tumor efficacy of slc / ccl21 ( figure 1 * p < 0.01 compared to the control antibody group ) .
neutralization of ifn caused a significant decrease in both mig / cxcl9 and ip-10/cxcl10 indicating that these chemokines are largely ifn dependent .
thus , an increase in ifn at the tumor site of slc / ccl21-treated mice could explain the relative increases in ip-10/cxcl10 and mig / cxcl9 .
the converse was also observed ; ifn production at the tumor site was found to be mig / cxcl9- and ip-10/cxcl10-dependent as indicated by the fact that neutralization of these cytokines caused a significant decrease in ifn(figure 2 ) .
neutralization of any one of these cytokines caused a concomitant decrease in all three cytokines , thus indicating that ifn , mig / cxcl9 and ip-10/cxcl10 are interdependent in the slc / ccl21-mediated anti - tumor responses . based on these findings , we speculated that the decrease of ip-10/cxcl10 and mig / cxcl9 led to a decrease in ifn by limiting the influx of t cells producing ifn at the tumor site .
because mig / cxcl9 and ip-10/cxcl10 are chemotactic for stimulated cxcr3 expressing activated t lymphocytes , we determined if neutralizing these chemokines in vivo in the slc / ccl21-treated mice would decrease the frequency of cxcr3 expressing t cells at the tumor sites .
compared to slc / ccl21-treated mice receiving control antibody , flow cytometric evaluation of single - cell suspensions of non - necrotic tumors from slc / ccl21-treated mice receiving neutralizing antibodies to mig / cxcl9 , ip-10/cxcl10 and ifn showed 10 , 12 and 31 % decreases , respectively , in the gated cd3 cxcr3 t cell populations ( table 1 ) .
the fact that neutralization of ifn was the most efficient at decreasing the total number of cxcr3-activated t cells may be due to the decrease in the ifn-dependent cxcr3 ligands mig / cxcl9 and ip-10/cxcl10 .
individually , neutralizing mig / cxcl9 and/or ip-10/cxcl10 only led to a partial decrease in the total number of cxcr3-activated t cells . because mig / cxcl9 and ip-10/cxcl10 share the same receptor ( cxcr3 ) , one possible explanation of a partial decrease in cxcr3-activated t cells is that neutralization of one ligand may overexpose the receptor to the other ligand .
furthermore , because murine slc / ccl21 binds cxcr3 receptor , slc / ccl21 may be recruiting cxcr3 t cells directly .
an alternative explanation is that residual cytokines such as ifn - inducible t cell  chemoattractant ( itac / cxcl11 ) present after in vivo neutralization of ifn , mig , ip-10 may have remaining activity and attract cxcr3 t cells .
our results imply that all 3 cytokines , ifn , mig and ip-10 play interrelated roles in the recruitment of cxcr3-activated t cells in slc / ccl21-mediated anti - tumor responses . while the number of cd4 and cd8 cells remained unaltered ,
compared to slc / ccl21-treated mice receiving control antibody , mice administered neutralizing antibodies to mig / cxcl9 , ip-10/cxcl10 and ifn showed respective decreases of 38 , 28 and 16% in the number of cd11cdec205dc infiltrating the tumor ( table 1 ) . at present
it is not clear why there is a decrease in dc following neutralization of mig / cxcl9 , ip-10/cxcl10 and ifn. a plausible explanation is that these cytokines may be involved in complex interactions with other molecules that are chemotactic for dc .
further studies are required to delineate the mechanisms responsible for mig / cxcl9 , ip-10/cxcl10 and ifn mediated localization of dc at the tumor site .
5 days following tumor implantation , mice were treated intratumorally with recombinant murine slc / ccl21 ( 0.5 g ) 3x / week .
one day before slc / ccl21 administration , mice were given the respective cytokine antibody by i.p .
antibodies to : ip-10/cxcl10 , mig / cxcl9 and ifn significantly inhibited the antitumor efficacy of slc / ccl21 ( * p < 0.01 compared to the control antibody treated group ) .
n = 8 mice group slc / ccl21-treated mice had a significant induction in ifn , mig / cxcl9 and ip-10/cxcl10 at the tumor site compared to diluent treated control tumor bearing mice ( p < 0.001 ) .
assessment of cytokine production at the tumor site of slc / ccl21 treated mice receiving anti - ifn , anti mig / cxcl9 and anti ip-10/cxcl10 showed an interdependence of ifn , mig / cxcl9 and ip-10/cxcl10 : neutralization of any one of these cytokines in vivo caused a concomitant decrease in all three cytokines ( * p < 0.01 compared to the control antibody treated group ) .
n= 8 mice per group neutralization of ip-10 , mig and ifn reduces the frequency of cd3cxcr3 t lymphocytes and cd11cdec205dc at the tumor site of slc / ccl21-treated mice single cell suspensions of non - necrotic tumor nodules from slc / ccl21 treated mice receiving neutralizing antibodies to p-10/cxcl10 , mig / cxcl9 , ifn , control ab and diluent treated tumor bearing mice were prepared .
cell surface staining for t cell markers cd4 , cd8 , cd3 , and the chemokine receptor cxcr3 as well as dc markers cd11c and dec205 were evaluated by flow cytometry .
cells were identified as lymphocytes or dc by gating on the forward and side scatter profiles ; 10,000 gated events were collected and analyzed using cell quest software . within the gated t lymphocyte population , intratumoral injection of slc / ccl21 led to an increase in the frequency of cd4 , cd8 , cd3cxcr3events compared to the diluent control ( * p < 0.001 ) . within the gated dc population ,
intratumoral injection of slc / ccl21 led to an increase in the frequency of cd11cdec205events compared to the diluent control ( * p < 0.001 ) . compared to slc / ccl21-treated mice receiving control antibody , mice receiving neutralizing antibodies to ip-10/cxcl10 , mig / cxcl9 , and ifn showed decrease in the gated cd3cxcr3 t cell as well as cd11cdec205 dc events ( * * p < 0.01 ) ( n = 8 mice per group ) .
our results suggest that the anti - tumor properties of slc / ccl21 may be due to its chemotactic capacity in co - localization of dc and t cells as well as in part to the induction of key cytokines such as ifn , ip-10/cxcl10 and mig / cxcl9 .
abs to murine ifn , recombinant ifn were obtained from pharmingen ( san diego , ca ) .
abs to murine mig / cxcl9 , ip-10/cxcl10 and recombinant cytokine standards was purchased from r&d ( minneapolis , mn ) .
polyclonal goat anti - murine mig / cxcl9 and anti - murine ip-10/cxcl10 specific anti - serum were produced and characterized as previously described .
anti - mouse ifn monoclonal ( r4 - 462 , american type culture collection , rockville , maryland ,
md ) neutralizing antibody was purified by affinity chromatography from scid mice ascites , which was generated 34 weeks after intraperitoneal injection of 10r4 - 462 hybridoma cells per mouse . for flow cytometry , labeled antibodies ( fitc , pe , percp ) against the t cell surface markers to cd3 , cd4 , cd8 and against the dc markers , cd11c and dec 205 and the appropriate controls were purchased from pharmingen .
a weakly immunogenic lung cancer , lewis lung carcinoma ( 3ll , h-2 ) obtained from atcc ( crl-1642 , llc ) was utilized for assessment of cytokines important for slc / ccl21  mediated anti - tumor responses in vivo .
the cells were routinely cultured as monolayers in 25 cmtissue culture flasks containing rpmi 1640 medium ( irvine scientific , santa anna , ca ) supplemented with 10% fetal bovine serum ( fbs ) ( gemini bioproducts , calabasas , ca ) , penicillin ( 100 u / ml ) , streptomycin ( 0.1 mg / ml ) , 2 mm glutamine ( jrh biosciences , lenexa , ks ) and maintained at 37c in a humidified atmosphere containing 5% co2 in air .
the cell lines were mycoplasma free and cells were utilized up to the tenth passage before thawing frozen stock cells from liquid n2 . for tumorigenesis experiments ,
injection in the right supra scapular area of c57bl/6 mice and tumor volumes were monitored three times each week .
five day established tumors were treated with intratumoral injection of 0.5 g of recombinant murine slc / ccl21 ( pepro tech , rocky hill , nj ) or murine serum albumin ( utilized as an irrelevant protein for control injections ) were administered three times per week for two weeks as previously described .
24 h prior to slc / ccl21 treatment , and then three times a week , mice were injected i.p . with 1 ml / dose of anti - ip-10/cxcl10 , or anti - mig / cxcl9 , or 100 g / dose of purified monoclonal anti - ifn or appropriate control antibodies ( goat igg and rat igg ) at equivalent doses for the duration of the experiment .
in response to these antibodies , there was a significant reduction of the respective cytokines in vivo ( figure 2 ) .
mig / cxcl9 , ip-10/cxcl10 and ifn were quantified using a modification of a double ligand method as previously described .
flow cytometric analyses for t cell and dc markers were performed on a facscan flow cytometer ( becton dickinson , san jose , ca ) in the university of california , los angeles , jonsson cancer center flow cytometry core facility . cells were identified as lymphocytes or dc by gating based on forward and side scatter profiles .
10,000-gated events were collected and analyzed using cell quest software ( becton dickinson ) . for staining , two or three fluorochromes ( pe , fitc , percp ) ( pharmingen ) were used to gate on the cd3 , cd4 and cd8 t lymphocyte population or cd11c dc in single cell suspensions from tumor nodule . for cxcr3 expression , t cells
statistical analyses of the data in figures 1 , 2 and in table 1 were performed using the paired student t test .
abs to murine ifn , recombinant ifn were obtained from pharmingen ( san diego , ca ) .
abs to murine mig / cxcl9 , ip-10/cxcl10 and recombinant cytokine standards was purchased from r&d ( minneapolis , mn ) .
polyclonal goat anti - murine mig / cxcl9 and anti - murine ip-10/cxcl10 specific anti - serum were produced and characterized as previously described .
anti - mouse ifn monoclonal ( r4 - 462 , american type culture collection , rockville , maryland ,
md ) neutralizing antibody was purified by affinity chromatography from scid mice ascites , which was generated 34 weeks after intraperitoneal injection of 10r4 - 462 hybridoma cells per mouse . for flow cytometry , labeled antibodies ( fitc , pe , percp ) against the t cell surface markers to cd3 , cd4 , cd8 and against the dc markers , cd11c and dec 205 and the appropriate controls were purchased from pharmingen .
a weakly immunogenic lung cancer , lewis lung carcinoma ( 3ll , h-2 ) obtained from atcc ( crl-1642 , llc ) was utilized for assessment of cytokines important for slc / ccl21  mediated anti - tumor responses in vivo .
the cells were routinely cultured as monolayers in 25 cmtissue culture flasks containing rpmi 1640 medium ( irvine scientific , santa anna , ca ) supplemented with 10% fetal bovine serum ( fbs ) ( gemini bioproducts , calabasas , ca ) , penicillin ( 100 u / ml ) , streptomycin ( 0.1 mg / ml ) , 2 mm glutamine ( jrh biosciences , lenexa , ks ) and maintained at 37c in a humidified atmosphere containing 5% co2 in air .
the cell lines were mycoplasma free and cells were utilized up to the tenth passage before thawing frozen stock cells from liquid n2 . for tumorigenesis experiments ,
injection in the right supra scapular area of c57bl/6 mice and tumor volumes were monitored three times each week .
five day established tumors were treated with intratumoral injection of 0.5 g of recombinant murine slc / ccl21 ( pepro tech , rocky hill , nj ) or murine serum albumin ( utilized as an irrelevant protein for control injections ) were administered three times per week for two weeks as previously described .
24 h prior to slc / ccl21 treatment , and then three times a week , mice were injected i.p . with 1 ml / dose of anti - ip-10/cxcl10 , or anti - mig / cxcl9 , or 100 g / dose of purified monoclonal anti - ifn or appropriate control antibodies ( goat igg and rat igg ) at equivalent doses for the duration of the experiment .
in response to these antibodies , there was a significant reduction of the respective cytokines in vivo ( figure 2 ) .
mig / cxcl9 , ip-10/cxcl10 and ifn were quantified using a modification of a double ligand method as previously described .
flow cytometric analyses for t cell and dc markers were performed on a facscan flow cytometer ( becton dickinson , san jose , ca ) in the university of california , los angeles , jonsson cancer center flow cytometry core facility .
cells were identified as lymphocytes or dc by gating based on forward and side scatter profiles .
10,000-gated events were collected and analyzed using cell quest software ( becton dickinson ) . for staining , two or three fluorochromes ( pe , fitc , percp ) ( pharmingen ) were used to gate on the cd3 , cd4 and cd8 t lymphocyte population or cd11c dc in single cell suspensions from tumor nodule . for cxcr3 expression ,
statistical analyses of the data in figures 1 , 2 and in table 1 were performed using the paired student t test .
authors sh , lxz , mh , rkb and mdb contributed to the discussion of the article .
authors rms and smd together with ss conceived of the study , participated in its design and drafted the manuscript .
we thank kimberly atianzar for excellent technical assistance in conducting the elisa measurements . national institutes of health grant r01 ca78654 , ca85686 , p50 ca90388 , medical research funds from the department of veterans affairs , the research enhancement award program in cancer gene medicine and the tobacco - related disease research program of the university of california supported this work .
 backgroundslc / ccl21 , normally expressed in high endothelial venules and in t cell zones of spleen and lymph nodes , strongly attracts t cells and dendritic cells ( dc ) . 
 we have previously shown that slc / ccl21-mediated anti - tumor responses are accompanied by significant induction of ifn and the cxc chemokines , monokine induced by ifn ( mig / cxcl9 ) and ifn-inducible protein-10 ( ip-10/cxcl10).resultswe assessed the importance of ifn , ip-10/cxcl10 and mig / cxcl9 in slc / ccl21 therapy . in vivo depletion of ip-10/cxcl10 , mig / cxcl9 or ifn significantly reduced the anti - tumor efficacy of slc / ccl21 . 
 assessment of cytokine production at the tumor site showed an interdependence of ifn , mig / cxcl9 and ip-10/cxcl10 ; neutralization of any one of these cytokines caused a concomitant decrease in all three cytokines . 
 similarly , neutralization of any one of these cytokines led to a decrease in the frequency of cxcr3+ve t cells and cd11c+ve dc at the tumor site.conclusionthese findings indicate that the full potency of slc / ccl21-mediated anti - tumor responses require in part the induction of ifn , mig / cxcl9 and ip-10/cxcl10 . 
Background
Results and Discussion
Conclusions
Materials and Methods
Reagents
Cell culture and tumorigenesis model
Cytokine ELISA
Flow Cytometry
Statistical Analysis
Author's contributions
Acknowledgements