dendritic cells ( dcs ) have been found to be the pivotal antigen presenting cells ( apcs ) in regulation of immune response .
activation through the t cell receptor in the absence of costimulation is proposed to render responder t cells anergic or tolerant .
the costimulatory signal is delivered through interactions between the t cells and apcs and results from ligation of molecules such as cd28 and cd154 ( cd40l ) , expressed on the t cells with their ligands cd80/cd86 and cd40 respectively , on apcs .
co - stimulation blockade targeting cd80/cd86 on dcs efficiently prevents acute heart or kidney rejection in many mouse and rat models [ 37 ] .
first clinical trial has been conducted to assess co - stimulation blockade strategy in renal transplantation .
it may allow patients to avoid the adverse effects of calcineurin inhibitors , whilst providing equally effective immunosuppression .
there are direct and indirect pathways of allorecognition , and both of which have been postulated to have roles in allograft immunity .
direct alloantigen ( ag ) presentation , mediated by donor apcs , leads to vigorous t cell proliferation and is mainly involved in acute rejection . as
then recipient dcs that can infiltrate to the graft become the predominant apcs , and they present alloag indirectly to t cells .
however , more data showed that indirect recognition might play a more important role in whole allograft rejection [ 1214 ] and indirect recognition also can initiate rapid skin graft rejection .
in addition , dcs can not be obtained from deceased donors ; so recipient dcs would potentially be taken into account in clinical transplantation .  
rna interference ( rnai ) is a recently identified phenomenon in which small interference rna ( sirna ) interacts with mrna containing homologous sequences , and ultimately this interaction results in degradation of the target mrna .
hill et al .   reported that transfection of dcs with sirna specific for il-12 p35 gene resulted in potent suppression of gene expression and blockade of bioactive il-12 p70 production .
based on the attractive methodology of rnai for silencing a particular gene expression , we used lentivirus mediated rnai to suppress cd80 and cd86 expression on host dcs .  
endoplasmic reticulum is the organelle in which newly synthesized secretory and transmembrane proteins form their proper tertiary structure by posttranslational modification , folding , and oligomerization .
however , many of these proteins are unfolded or misfolded by extracellular or intracellular stimuli .
eukaryotic cells possess several mechanisms to adapt to endoplasmic reticulum stress ( ers ) and thereby survive .
if the cells are exposed to prolonged or strong ers , the cells are destroyed by apoptosis .
recent evidence indicates that ers signaling pathways play an important role in the pathogenesis of neurodegenerative disorders and diabetes .
increasing evidences suggest ers is involved in allograft injury .   at present , it is not known whether ers is involved in peripheral tolerance . in this study
, we detected that these dcs - pulsed alloag could elicit lower proliferative responses and prolong heart allograft survival .
for the first time , our study demonstrates that ers - mediated apoptosis signal pathway is involved in t cell apoptosis after indirect recognition pathway blockade .
c3h / hej ( c3h ; h-2k ) , c57bl/6 ( b6 ; h-2k ) , and balb / c ( h-2k ) mice were purchased from shanghai laboratory animal center of chinese academy of sciences ( shanghai , china ) and were maintained in pathogen - free facility at fudan university ( shanghai , china ) .
animals were fed with standard chow ad libitum and were used at 79 weeks of age .
briefly , the bm cells were removed from femurs and tibias of c3h mice and depleted of erythrocytes by hypotonic lysis .
the cells were cultured in 24-well plates ( 110/well ) in 1  ml rpmi 1640 ( gibco , gaithersburg ,
md ) supplemented with 10%  v / v fetal bovine serum ( fbs ) and 10  ng / ml recombinant gm - csf ( r&d systems , minneapolis , mn ) .
after 6 days of culture , 1  g / ml lps ( sigma , st .
louis , mo ) was added to the culture for 18 hours to allow for maturation .
the purity of dc preparations was routinely monitored by flow cytometric analysis using anti - cd11c monoclonal antibody ( mab ) ( ebioscience , san diego , ca ) .
the recombinant lentivirus was constructed as previously described , with some modifications . in brief ,
short hairpin rna ( shrna ) fragments ( table 1 ) were hybridized with synthesized sense and antisense oligonucleotides .
the hybridized cd80 shrna fragment was cloned into the plasmid pgcl - gfp ( figure 1 ) .
the 293 t cells were cotransduced with 20  g of pgcl - gfp , 15  g of phelper1.0 , and 10  g of phelper2.0 by 100  l of lipofectamine 2000 ( invitrogen , garlsbad , ca ) .
the titers of cd80 lenti were determined with a green fluorescent protein ( gfp ) assay .
nc lenti contained scrambled shrna fragment , an irrelevant shrna with random nucleotides , and a gc ratio close to above - mentioned shrna .
lentivirus transduction was conducted when dcs were 30%50% confluent , lentivirus was added at different mois in serum - free medium at 37c in 5% humidified co2 .
after 4 hours , complete medium was added to the cells , and there was complete replacement of medium after 48 hours .
then , 4 days post transduction , marker gene expression was examined using fluorescent microscopy . on day 6 of dcs culture
, allogeneic splenocyte lysates were added to the culture at a dc : splenocyte equivalent ratio of 1  :  10 for 18 hours at 37c in 5% humidified co2 .
b6 splenocyte lysates were obtained by six cycles of freeze / thaw exposure in pbs .
grafts were harvested , minced , and homogenized with 5% collagenase type ii ( worthington , lakewood , nj ) for 30 minutes at 37c .
lymphocytes were purified by lymphocyte separation medium ( mediatech , herndon , va ) and then subjected to flow cytometry .
expression of cell surface ag was detected on a facscan ( bd biosciences , san jose , ca ) and analyzed using cellquest software ( bd biosciences ) .
cells were stained with the following mab : pe - iak , pe - cd40 , pe - cd80 , pe - cd86 ( ebioscience ) , and fitc - cd3 ( bd biosciences ) .
apoptosis was assessed by pe - annexin v staining ( bd biosciences ) . for primary mlr ,
nylon wool - eluted spleen t cells ( 210 ) from c3h mice were used as responders . -irradiated ( 20  gy )
isolated splenic t cells from dcs - injected c3h mice were responders and restimulated with -irradiated b6 , c3h , balb / c ag - pulsed c3h - derived dcs .
cultures were maintained in complete medium for 3 days at 37c in 5% humidified co2 .
[ h]-tdr ( 0.5  ci / well ) was added for the final 18 hours of culture .
cells were harvested onto glass fiber disks using an automated system , and incorporation of [ h]-tdr into dna was assessed by wallac 1450 liquid scintillation counter ( perkinelmer , boston , ma ) .
cervical vascularized heart transplantation was performed from b6 donors to size - matched c3h recipients using a cuff technique .
rejection was defined as total cessation of cardiac contraction . to assess the effect of equipped dcs on allograft survival , animals received 210 cells administered intravenously through the lateral tail vein 7 days prior to heart transplantation in the absence of immunosuppression .
total rna of splenic t cells was extracted using trizol reagent according to the manufacturer 's instructions ( invitrogen ) .
rna was then reverse - transcribed into cdna , using random primers and superscript ii reverse transcriptase ( invitrogen ) . for semiquantitative rt - pcr
statistical analysis was performed with stata 8.0 software ( stata , college station , tx ) .
statistical comparisons between groups were preformed using a one - way anova followed by a scheff 's test , as appropriate .
graft survival between groups of transplanted animals was compared using the log - rank test for comparison of survival curves .
to determine the lentiviral transduction efficiency in dcs , gfp expression was examined by microscopy and flow cytometry at different mois on day 4 after transduction .
lentivirus demonstrated a high ( 79.8%    5.0% ) transduction efficiency in dcs at moi of 20 ( figure 2 ) .
the percentage of gfp - expressing cells remained minor changes when moi is > 20 .
therefore , in the following experiments all viral titers were at moi of 20 . to blockade both cd80 and cd86 expression , dcs
as measured by flow cytometry , we found that cd80 lenti and cd86 lenti together could enhance suppressive effect on cd80 and cd86 expression ( figure 3(a ) ) .
but as to those transduced dcs lps failed to bring great changes of the cd80 and cd86 expression . on the other hand , cd80 lenti and cd86 lenti had no effect on expression of unrelated proteins ( such as mhc - ii and cd40 ) ( figure 3(b ) ) .
our study indicates that recombinant lentivirus can provide highly efficient and specific cd80 and cd86 knockdown in dcs . to explore the influence of these dcs on t cell response
cd80 lenti and cd86 lenti - transduced dcs were poor stimulators of t cells compared to nc lenti - transduced dcs or no treated dcs ( figure 4(a ) ) .
as shown in figure 4(b ) , t cells from c3h mice primed by injection of cd80 lenti and cd86 lenti - transduced dcs exhibited marked reduced responses to alloag ( b6 ) versus to third party unrelated ag ( balb / c ) pulsed c3h - derived dcs . in contrast , t cells from c3h mice primed by injection of nc lenti - transduced or no treated dcs exhibited markedly increased proliferative responses to donor alloag and third party ag - pulsed c3h - derived dcs . however , t cells from c3h mice primed by injection of various dcs all exhibited significant low proliferative response to syngeneic ag ( c3h ) pulsed dcs . to investigate the therapeutic potential of these equipped dcs in murine cardiac transplant model , recipient mice ( c3h ) were pretreated with 210 dcs in nc lenti group , or cd80 lenti and cd86 lenti group , 7 days before heterotopic heart transplantation from b6 to c3h .
median survival time ( mst ) in the control group was 9.3 days , while nc lenti - transduced dcs pretreatment accelerated heart allograft rejection ( mst 6 days compared to control group ) .
pretreatment with cd80 lenti and cd86 lenti - transduced dcs significantly prolonged the mst to 21.8 days but had no effect on pulsing third party ag ( mst 7.5 days ) .
these findings suggest that infusion of cd80 lenti and cd86 lenti - transduced dcs - pulsed alloag prior to transplantation prolongs heart allograft survival , and the prolongation is ag specific ( figure 5 ) .
the most striking finding from the current study was the possible mechanism of equipped dcs prolonging graft survival .
lymphocytes were isolated from spleens , mesenteric lymph nodes , and grafts and were assessed by annexin v staining .
apoptotic cell in cd3 + t cell population from mice receiving cd80 lenti and cd86 lenti - transduced dcs was markedly higher than the t cells in nc lenti group ( figure 6 ) .
to better characterize the mechanism of t cell apoptosis after costimulatory blockade , we used rt - pcr to analyze a number of apoptosis signaling pathway molecules in recipient spleens .
as shown in figure 7 , a striking upregulation of bax and grp78 was found in recipients treated with cd80 lenti and cd86 lenti - transduced dcs compared to nc lenti - transduced dcs or pbs .
these changes in endoplasmic reticulum and mitochondrial pathway were enhanced by upregulation of chop and suppression of bcl - xl expression in these cells .
the success of organ transplantation depends on the continuous administration of nonspecific immunosuppressive agents , immunosuppression therapies present risks for opportunistic infection , malignancy , and a variety of agent side effects .
in particular , several dc - based therapies in blocking the costimulation pathways have achieved encouraging results [ 38 ] .
however , antibodies or fusion proteins have a limited half - life and require multiple antibody treatments .
our previous studies have indicated that chemically synthesized sirna can specifically and effectively knock down cd80 and cd86 gene expression in bm - derived dcs .
then we choose recombinant lentiviruses that contain stem - loop constructs encoding that hairpin rnas lend to the intracellular generation of sirna - like species .
lentivirus can infect noncycling and postmitotic cells ; reverse transcribed dna sequences can subsequently integrate into the genome of target cells and are stably expressed at high levels for extended times .   
our data clearly demonstrate that lentivirus transduced dcs induce t cell hyporesponsiveness and t cells primed by alloag - pulsed dcs later inhibit allogeneic responses , in an ag - specific manner .
the findings are similar with taner et al .   who showed that t cells cross - primed by alloag - pulsed , rapamycin treated dcs were hyporesponsive to subsequent challenge .
recipient dcs can acquire intact mhc molecules from donor cells or tissues and stimulate direct antidonor alloimmune responses .
.   showed that mhc - ii/cd4 + recipients could reject cd80/cd86/ cardiac grafts as rapidly as wild - type grafts .
the observed acute graft rejection may have been due to the direct activation of recipient t cells by recipient apcs after acquiring intact donor mhc molecules .
the cd80/cd86-cd28 pathway is probably the most important and best characterized in t cell activation ; we have shown herein that blockade of cd80 and cd86 by lentivirus mediated rnai increases cardiac allograft survival but does not extend it indefinitely .
since co - stimulation blockade is less effective in memory t cells than in naive t cells , memory t cells may also play an active role .
several groups have addressed whether one or multiple injections will achieve a more significant effect .
the mechanism by which lentivirus transduced dcs prolong heart allograft survival remains to be determined .
the key requirement for long - term survival is that none of the activated effector t cells survives in a functional state .
these include death receptor pathway , mitochondrial pathway and most recently described the endoplasmic reticulum pathway .
the first pathway occurs in repetitively stimulated t cells and is largely mediated through fas and through related members of the tnf - receptor family .
bcl-2 family proteins control the release of cytochrome c and apoptosis - inducing factor from mitochondria .
sen 's group has found that alloreactive t cell apoptosis is induced via fas / fasl pathway in cardiac allograft [ 35 , 36 ] .
.   showed that the peripheral deletion of donor - reactive t cells in the early period after bm transplantation with costimulatory blockade using anti - cd154 and ctla4ig was mediated in part by fas and could be overcome by the constitutive expression of bcl - xl . under ers ,
unfolded proteins accumulate in the lumen of the endoplasmic reticulum , and then cells activate a self - protective mechanism by activation of genes encoding endoplasmic reticulum - resident chaperones such as bip / grp78 and grp94 .
if these adaptive responses are not sufficient to relieve cells from ers , cells undergo apoptosis associated with several molecules , including chop , caspase-12 , and map kinase cascade .
several investigators have reported that ers is involved in b cell differentiation and the innate immune response .
ers response influences ischemia - reperfusion in human liver transplantation , while the endoplasmic reticulum pathway - mediated apoptosis in t cells of the immune system has not been studied .
first we observed that , in recipient spleens , grp78 and chop were much higher in recombinant lentivirus group .
phagocytosis of apoptotic cells by macrophages often leads to the production of il-10 and tgf and may promote tolerance . nevertheless , it is conceivable that apoptosis of activated t cells not only reduces the mass of t cells but also promotes active immune regulation that is essential for long - lasting tolerance . in summary ,
rnai - treated , alloag - pulsed host dcs can induce ag - specific t cell hyporesponsiveness and prolong heart allograft survival .
ers - mediated apoptosis pathway may play a role in peripheral tolerance after heart transplantation with costimulatory blockade .
a detailed understanding of activated t cells apoptosis in this process will undoubtedly lead to the design of more effective strategies to achieve tolerance in the clinic .
 implementation of dendritic cell- ( dc- ) based therapies in organ transplantation can reduce dependency on nonspecific immunosuppression . despite extensive research 
 , mechanisms of equipped dcs inducing transplant tolerance remain incomplete . here 
 , we applied rna interference technique to inhibit cd80 and cd86 expression in host bone marrow - derived dcs . 
 this approach could specifically and effectively knock down cd80 and cd86 expression . 
 t cells primed by these dcs inhibited allogeneic responses . 
 administration of recipient dcs loaded with alloantigen after cd80 and cd86 blockade prolonged cardiac allograft survival . 
 we also found a higher percentage of apoptotic t cells in lymph tissues and grafts than that detected in control group . 
 in addition , these t cells expressed high expression of grp78 than controls , indicating activation of unfolded protein responses . 
 upregulation of chop expression among these cells suggested that the endoplasmic reticulum stress ( ers ) response switched to a proapoptotic response . 
 our results indicated that ers - induced apoptosis may be involved in allogeneic t - cell apoptosis , and the ers - mediated apoptosis pathway may be a novel target in clinical prevention and therapy of allograft rejection . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion