adoptive immunotherapy , particularly adoptive transfer of antigen - specific cytotoxic t lymphocytes , has shown some success in clinical trials for treating both cancer and viral infections [ 15 ] .   however , due to resident tolerance within the tumor environment the most robust ctl must be stimulated for highest efficacy .  
delivery of antigen genes into dendritic cells ( dcs ) or precursor monocytes ( mo ) allows for the stimulation of robust antigen - specific ctl . however , there are a variety of improvements that might be made to further enhance ctl stimulation .  
one obvious course is the delivery of th1-response interferons / cytokine genes into immune cells for their continuous expression .
interferon gamma ( ifn- ) is an important th1 response interferon / cytokine involved in ctl generation and function and might be used to enhance ctl stimulation .  
in addition , ifn- may have other non - t cell - associated attributes as it is believed to stimulate expression of hla class i and ii molecules .   however , overall ifn- is strongly linked to the induction of th1 response , the generation of ctl .  
of all the th1 response interferons / cytokines ifn- correlates most strongly with the th1 response [ 911 ] as , in fact , many laboratories study t cell expression of ifn- as a substitute for carrying out ctl killing / chromium release assays [ 12 , 13 ] .  
moreover , ifn- plus il-12 appears to act cooperatively in the generation of a very strong th1 response [ 14 , 15 ] .  
ifn- may also partially overcome low activation and expansion rates of low - avidity ctl [ 16 , 17 ] .   as with most of the th1-response - associated chemokines ,
thus , gene delivery of ifn- will likely be very useful in generating a robust th1 ctl response .
while ifn- gene delivery would seem to be beneficial for generating robust ctl , it is unclear which cells should specifically express this cytokine for maximum ctl stimulation .  
while activated t cells naturally express ifn- , it has been shown that ifn- is able to significantly affect dc maturity and function as well as dc precursor mo and macrophage ( m ) phenotypes [ 1921 ] .  
thus it is unclear which immune cell type should express this cytokine during the initial stimulation of the cd8 + ctl .  
two general approaches are available , transducing the dc ( paracrine delivery ) which stimulate the responder t cells , or the t cell themselves ( autocrine delivery ) .   here
we demonstrate that ifn- autocrine gene delivery resulted in significantly higher cea - specific ctl killing compared with paracrine delivery or exogenous ifn-.
the sw480 colorectal adenocarcinoma cell line was obtained from the american tissue culture collection ( atcc ) .  
a carcinoembryonic antigen- ( cea- ) positive lymphoblastoid cell line ( cea+ lcl ) was generated by transfecting an hla - a2 positive lcl cell line with a cea plus neomycin resistance gene ( neo ) expression plasmid and selection with 1  mg / ml g418 for two weeks .  
peripheral blood mononuclear cells ( pbmcs ) from five hla - a2 positive healthy donors were separated by routine ficoll gradient method .  
all blood donors gave informed consent in writing , and the study protocol conformed to the ethical guidelines of the 1975 declaration of helsinki as reflected in a priori approval by our human research internal review board .  
the hla haplotype of all donors was compatible with sw480 cells ( hla a2 ) and other cells used as controls .
human cea and ifn- cdnas were amplified by reverse - transcription polymerase chain reaction ( rt - pcr ) .  
trizol reagent ( invitrogen ) was employed to isolate total rna from sw480 cells and pha - stimulated primary human t lymphocytes , respectively .  
subsequently , the total mrna was separate from the total rna using oligotex mrna isolation kit ( qiagen ) .  
after the first - strand cdna was generated , pcr amplification for each of the cdna was carried out using the following primer pair : cea : 5-accatggagtctccctcg-3 and 5-ctatatcagagcaacccc-3 that amplify the sequence from nucleotides 112 to 2223 ; ifn- : 5-ttctctcggaaacgatg-3 and 5-ggcaggacaacca ttac-3 that amplify the sequence from nt 94 to 622 .  
cea and ifn- cdna were inserted in the downstream of p5 promoter of an aav vector , respectively , as described previously .
the raavs were generated , purified , and tittered as described previously [ 24 , 25 ] .  
after freshly isolated pbmcs ( 5  10 ) were cultured for two hours in aim - v medium , the nonadherent cells were removed .  
the remaining adherent mos were infected immediately with 1  10 encapsidated genomes ( eg)/ml of aav / cea virus or aav / cea plus aav / ifn- virus .  
after four hours the medium / virus solution was removed and the cells were finally fed with the medium containing recombinant human gm - csf ( immunex , 800  iu / ml ) .   at day 2 , to induce the maturation of mo into dc , recombinant human il-4 and tnf- ( r & d systems . ) were added to the medium at 1000  iu / ml and 20  ng / ml , respectively .  
finally , at day 6 the dcs were mixed with cd3 + t cells .  
briefly , pan t cell isolation kit ii ( miltenyi biotec ) was employed to isolate cd3 + t cells from the nonadherent cells from the pbmc according to the kit instruction .   at day 5
the cd3 + t cells ( 1  10 ) were infected with 1  10  eg / ml of aav / ifn- virus and cultured with 20  ng / ml of il-2 .
the total dnas were isolated from the raav - infected or uninfected dc or t cells using dnazol reagent ( invitrogen ) according to supplier 's protocol .  
chromosomal integration of the aav / cea genome was studied by vector - chromosome junction pcr amplification and southern blot analysis , as previously described . at day 4 of mo / dc culture and dc - t cell culture , isolation and amplification of the mrna
pcr amplification for the tfiib cdna was performed using the primer pair : 5-tgtctgttgtgtcttgttgc-3 and 5-taggctatgtacaacagg c-3 that amplify the sequence from nt 319 to 1310 .    at day 6 of mo / dc culture and day 4 of raav infection of cd3 + t cells ,
briefly , cells were incubated with the fitc- or pe - labeled monoclonal antibodies recognizing the following antigens , respectively : cea , ifn- , and il-2 ( bd pharmingen ) .
control irrelevant isotype - matched fitc- or pe - conjugated monoclonal antibodies were also obtained from bd pharmingen .  
a facscalibur flow cytometer ( becton - dickinson ) was used for data acquisitions .  
at least ten thousand events were counted for each sample . at day 5 - 6 of dc culture
the mature dcs were harvested and mixed with cd3 + t lymphocytes ( ratios from 20  :  1 , t  :  dc ) in aim - v medium , respectively .  
some t cells were untransduced while others were previously infected with aav / ifn-.   mixtures were cultured in aim - v containing il-2 ( 20  iu / ml ) and il-7 ( 20  ng / ml ) .  
exogenous ifn- was used at 100  u / ml . at 7 - 8 days postaddition to dc the cells were harvested and analyzed further .   
after 6 days the nonadherent dcs were harvested ( > 95% viable as assessed by trypan blue exclusion ) and the cells counted and distributed .  
for the analysis of dc a panel of fitc- or pe - labeled monoclonal antibodies recognizing the following antigens was used : cd14 , cd40 ( chemicon international ) , hla - dr , cd80 , cd8 , cd86 , and isotype - matched antibodies ( bd pharmingen ) .  
stained cells were assayed by fluorescence activated cell sorting ( facs ) for these cd markers according to the routine method .    for the analysis of activated t cells , at day 8 of the mixed cell culture
the stimulated t cell populations were analyzed for their surface markers with immunofluorescence staining by flow cytometry .  
a panel of fitc- or pe - labeled monoclonal antibodies recognizing the following antigens was used : cd4 , cd8 , cd25 , and cd69 ( bd pharmingen ) .   at day
intracellular staining assay was performed to analyze the expression of ifn- in the t cells according to the method described above .  
fitc - labeled anti - ifn- monoclonal antibody ( bd pharmingen ) was used .  
after the cd3 + t cells were mixed with the dc on day 6 , each group of mixed cells was inoculated into wells of a 96-well cell culture plate .  
there were 5  10 cells ( 200  l ) in each well .  
after the mixed cells were cultured for 8 hours in 37c , 5% co2 , h - tdr incorporation test was carried out according to the routine method .
ctls were generated from 3 donors .   at day 14 ( day 8 of t cell : dc coincubation ) , chromium-51 ( cr ) release assay was used to analyze the killing activity of ctl elicited by aav / cea - transduced and control dc against the target cells as previously described .   
briefly , the ctl cells and cr - labeled target cells were mixed ( 20  :  1 ) and incubated for 6 hours at 37c with 5% co2 .    to determine mhc / hla class i restriction of ctl killing anti - mhc class i monoclonal antibodies were used to block cytotoxicity .  
the cr - labeled targets were preincubated with mouse antihuman mhc class i antibody ( serotec ) for 1 hour before the cr release assay was performed .   
the mouse antihuman mhc class ii antibody ( serotec ) was also used as a control .
the cea gene , a common adenocarcinoma tumor marker , was cloned from colorectal adenocarcinoma cell line sw480 and ligated down - stream of the aav p5 promoter within a fully gutted aav2-based vector ( dl3 - 97 ) .  
raavs were generated as described previously [ 24 , 25 ] and used to transduce dc and t cells at efficiencies above 85% .  
our approach for transducing dc has been to infect freshly adherent peripheral blood monocytes with raav , to treat these cells with gm - csf alone for one day , and then add il-4 to induce their differentiation into dc [ 24 , 25 , 28 ] .  
this technique has proven to be very effective in generating specific antigen - presenting dc and cytokine - expressing dc [ 24 , 25 , 28 ] .
one issue in the field of aav - based gene therapy is the form of latency of the aav proviral dna within the transduced primary cells .   in vitro tissue culture
transduced cell lines often display a chromosomally integrated provirus , while in vivo transduced cells often show the latent raav dna as an episomal element .  
to address this issue chromosomal dna from   the transduced t cells and dc were analyzed for integrated aav / ifn- provirus by pcr amplification of vector - chromosome junctions by using one primer directed towards the vector and another directed towards the alu i repetitive chromosomal element .  
products were then agarose gel electrophoresed , southern blotted , and p - dna probed for vector sequences .    as shown in figures 2(a ) and
2(b ) this technique clearly demonstrates some level of chromosomal latency in both dc and cd3 + t cells .  
.   figures 3(a ) and 3(b ) show that the resulting raav / ifn- provirus does express by rt - pcr analysis , in both dc and t cells .  
to observe both the transduction efficiency and protein expression of ifn- we carried out an intracellular staining analysis of transduced and untransduced dc and t cells .  
the transduction efficiency of dc by aav / ifn- , as shown in figure 4(a ) ( dc ) and figure 4(b ) ( t cells ) , approached 90% .  
this agrees with our earlier studies with other transgenes , but it must be noted that t cells which were not transduced with aav / ifn- displayed a high background expression of 48% .   in any case ,
we infect dc with either the aav / cytokine vector at day 0 or the t cells on day 5 just before their addition to the cea antigen loaded dc .
dcs ( mo ) were always loaded by infection with aav / cea on day 0 .   
we examined the dc on day 6 , as shown in table 1 , for surface expression of cd14 , cd40 , cd80 , cd83 , and cd86 by facs and found that cd80 , cd86 , and cd83 were upregulated by raav infection as shown previously .  
the addition of exogenous ifn- further upregulated these markers , but the use of aav / ifn- had a more profound effect .
we further observed the resulting expression level of il-12 and il-10 in dc by these various treatments , as shown in figure 5 .   
dcs were treated with exogenous ifn- or aav / ifn-.   a high il-12/il-10 ratio reflects the likelihood that these dcs would stimulate a more robust th1 ctl response ; however both treatments gave a similar ratio .  
secretion of ifn- from dc ( and t cells ) was also measured by elisa .  
ifn- secretion from aav / ifn- transduced dc , with or without aav / cea transduction , shown in figure 6 , was similar .  
expression rose from 68 to 80 hours postinfection and then remained stable out to 92 hours .  
however , it is noteworthy that aav / ifn--infected t cells secrete more ifn- than aav / ifn--infected dc . a robust mhc / hla class
i - restricted th1 ctl response is most consistent with a high cd8  :  cd4 ratio .  
the resulting cell population stimulated by the various dc treatments was analyzed by facs and the results listed in table 2 upper panel .  
the cd8/cd4 ratio in t cell populations was higher when these cells were stimulated by aav - transduced dc than with mock - treated dc ( table 2 ) .   
however t cells derived from aav / ifn--treated dc had a slightly higher cd8/cd4 ratio , consistent these cells having slightly higher cd80 , cd83 , and cd86 expression ( table 1 ) , and slightly lower il-10 expression ( figure 5 ) .  
however , the direct treatment of t cells by aav / ifn- , stimulated by aav / cea - loaded dc , resulted in t cell populations with the highest cd8/cd4 ratio ( 7.9 ) ( table 2 lower panel ) .  
a robust th1 ctl response is also consistent with a high ifn-/il-4 expression ratio .  
again , the resulting t cells generated from any aav - transduced dc had a higher ifn-/il-4 expression ratio than by mock treated dc ( table 2 upper panel ) .  
however , the direct treatment of t cells with aav / ifn- delivery , stimulated by aav / cea loaded dc , resulted in t cell populations with the highest ifn-/il-4 ratio ( 18.3 ) ( table 2 lower panel ) .  
moreover , a robust th1 ctl response is also consistent with a high proportion of cd69+/cd8 + t cells .   yet
again , the direct treatment of t cells by aav / ifn- , stimulated by aav / cea loaded dc , resulted in t cell populations with the highest percent of cd69+/cd8 + t cells ( 91.3% ) ( table 2 lower panel ) .  
finally , a robust th1 ctl would also be consistent with low cd25+/cd4 + treg cell numbers .  
again , the direct treatment of t cells by aav / ifn , stimulated by aav / cea loaded dc , resulted in t cell populations with the lowest percent of cd25+/cd4 + treg cells ( 17.1% ) ( table 2 lower panel ) .   taken together these data suggest that aav / ifn autocrine - delivery into t cells offers the most powerful th1-t cell population as measured by the cd8/cd4 ratio , ifn-/il-4 ratio , highest percentage cd69 + , cd8 + cells , and lowest percent cd25 + , cd4 + cells .
the generation of responder ctl involves both the proliferation of cd4 + helper t cells as well as proliferation of the cd8 + t cells themselves .   to test the level of t cell proliferation we carried out the standard protocol for the generation of antigen - specific ctl .   however , in addition to loading the dc with the antigen ( aav / cea ) we also added the delivery of aav / ifn- into dc or t cells .  
proliferation of cd3 + t cells was measured by the incorporation of 3h - tdr , and the results are shown in figure 7 .  
it appears that the use of ifn- in any form offered no advantage to that of the stimulation of proliferation by aav / cea - loaded dc .  
overall t cell proliferation is relatively equal among all treatments ( figure 7 ) , while the cd8/cd4 ratio increases ( table 2 ) when t cells are aav / ifn--infected .  
having characterized the aav / ifn- transduced dc and t cells we then assayed the resulting ctl for their ability to kill a genetically altered cea - positive lymphoblastoid cell line ( lcl ) which was hla a2-matched with blood donors .   to do this
we carried out the experiment depicted in figure 1 and tested for sw480 cell target killing using the standard cr release assay , and the results are shown in figure 8(a ) .  
as can be seen the highest level of cea - directed killing results from aav / ifn- autocrine delivery into t cells , a 49% increase over aav / cea - only control .  
killing was blocked by polymorphic anticlass i antibody , but not by anticlass ii .   the high killing that results from aav
/ ifn- autocrine treatment is fully consistent with the highest cd8/cd4 ratio , highest ifn-/il-4 ratio , highest percentage cd69 + , cd8 + cells , and lowest percent cd25 + , cd4 + cells .  
the higher killing may be attributed to the higher cd8 + t cell number due to higher cd8/cd4 ratio of t cells in aav / ifn--treated bulk t cell cultures versus the lower ratio generated from aav / ifn--treated dc , 84.6%/10.7% versus 56.5%/22.5% , respectively ( table 2 ) .  
we also utilized cea+ lcl cells as a target and , again , aav / ifn- autocrine delivery into t cells resulted in ctl with the highest killing of cea+ lcl , shown in figure 8(b ) .
while ifn- has multiple actions which promote th1 response [ 710 ] , it was unclear which immune cell type should best express this cytokine .  
this study demonstrates that only a specific approach of ifn- gene delivery , autocrine delivery , results in a cd3 + t cell population with higher killing ability .  
this was not fully anticipated as ifn- affects the phenotype of both dc and t cells
.   for dc ifn stimulates significantly higher mhc class ii , cd86 , and slightly higher cd80 levels [ 3032 ] .  
ifn- also stimulates higher il-12 expression and dc maturation [ 3032 ] .    comparing dc from ifn knockout mice and normal mice , ifn- expression by dc
normally ifn- expression in dc is dependent upon t - bet , but in this study we have circumvented this need by expressing it from the cmv promoter within the aav backbone ( aa ) .    moreover , nk and nkt cells are believed to be the major sources of ifn- for induction of macrophage maturation .  
paracrine ifn- expression by dc is believed to stimulate t cells to autocrine express their own ifn-.   however , ifn- also has pronounced effects on t cells as well .   while ifn- inhibits the formation of th2 cells , its expression directly correlates with the formation of th1 response and ctl killing , as mentioned earlier , so much so that ifn- secretion is used as a substitute for antigen specific killing assays [ 12 , 13 ] .    yet ifn- appears needed for the activity of unusual cd8(+)lap(+)foxp3(+ ) treg cells .  
thus , there is some evidence that ifn- may ultimately turn off the th1 response that produces it .   
our purpose for these studies was to further analyze the mechanism of action of ifn- and to optimize ex vivo generated anticancer ctl for adoptive therapies in cancer .  
while the adoptive transfer of ex vivo manipulated dendritic cells ( dcs ) appears mostly ineffective in anticancer therapies , the adoptive transfer of ex vivo generated anti - cancer cd8 + mhc class i - restricted cytotoxic t lymphocytes ( ctls ) seem to be a more promising treatment therapy [ 39 , 40 ] .  
while this study demonstrates that ifn- is best delivered and expressed via the autocrine route in the generation of antigen - specific cd8(+ ) ctl , it should be pointed out that the basal levels of natural ifn- expression also remain present and we are simply augmenting the level of ifn-.   thus , our interpretation of this data is that most likely the maximum effective ceiling of ifn- expression and effect in dc had been reached , while the effective ceiling of ifn- expression and effect in t cells had not been .  
another advantage of ifn- overexpression in ctl would be its known ability to upregulate mhc class i molecules , allowing for enhanced recognition of antigen expressing tumor cells .  
yet the practicality of the autocrine approach for certain th1 response cytokines is questionable as the endogenous expression of certain cytokines in th1 t cells may be problematic .   
while bone - related granulomatous lesions and degeneration of cartilage was observed , there were no serious hyperproliferative cellular elements seen in the bone marrow , spleen , or lymph nodes .   
thus , the adoptive transfer of ifn--over expressing t cells in clinical trials may be further considered .  
in fact we observed no advantageous proliferation by the t cells ( figure 7 ) .  
moreover , if further work suggests that there are serious side effects of ifn- overexpression in t cells , then the inclusion of a suicide gene within the vector , such as herpes thymidine kinase gene , would allow for the elimination of these cells when needed .  
this head - to - head comparison of ifn- overexpression in dc versus t cells was done with the purpose of enhancing th1 cd8(+ ) ctl response
.   however , these data also give us information as to the primary mechanism of action of ifn- on th1 immune response may be through endogenous t cell expression .  
yet further analysis is needed to determine whether it is the cd4(+ ) or cd8(+ ) t cell which is the primary vehicle through which ifn- acts .  
cell ratio studies ( table 2 ) suggest that it is the cd8(+ ) t cell .  
ultimately dna microarray analysis of the t cell 's transcriptome and proteomics analysis are warranted to fully understand how high ifn- expression affects the cell 's phenotype .
 the adoptive transfer of antigen - specific cytotoxic t lymphocytes ( ctl ) shows promise in the treatment of cancer and infectious diseases . 
 we utilize adeno - associated virus-(aav- ) based antigen gene - loaded dendritic cells ( dcs ) to stimulate such antigen - specific ctl . 
 yet further improvements in ctl stimulation and killing may result by gene delivery of various th1-response interferons / cytokines , such as interferon  ( ifn- ) , as the delivered gene can continuously produce that interferon . 
 however which immune cell type should optimally express ifn- is unclear as the phenotypes of both dc and t cells are enhanced by it . here , we used aav to compare and contrast ifn- gene delivery into dc or t cells , and versus the addition of exogenous ifn- , for stimulating carcinoembryonic antigen-(cea- ) specific ctl . 
 it was found that aav / ifn- delivery into t cells ( autocrine ) resulted in t cell populations with the highest cd8(+)/cd4(+ ) ratio , highest ifn-(+)/il-4(+ ) ratio , highest cd69(+),cd8(+ ) levels , and lowest cd4(+)/cd25(+ ) levels , all consistent with the strongest th1 response . 
 most importantly , aav / ifn- transduction of t cells resulted in antigen - specific t cell populations with the highest killing capabilities , 49% above other treatments . 
 these data strongly suggest that aav / ifn- autocrine gene delivery into t cells is worthy of further study towards maximizing the generation of antigen - specific anticancer ctl killers . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion