mononuclear cell recruitment , one of the hallmarks of allograft rejection 12 , is thought , conceptually , to follow the now classic sequence of leukocyte rolling along vascular endothelium , followed by stimulus - dependent attachment , triggering and transmigration , and directed migration along a chemotactic gradient 3 .
however , the chemokine - dependent phase of leukocyte recruitment , especially the mechanisms leading to graft infiltration by host t cells , which are key to allograft rejection , is still the least understood , with little in vivo data available 4 .
indeed , in contrast to most other areas of medicine , knowledge of the involvement of chemokine pathways is somewhat better established in humans than in experimental animals due to a paucity of appropriate tools for in vivo studies , leading to insufficient mechanistic and interventional studies 56 . our preliminary data 7 from the blinded molecular and immunohistologic evaluation of endomyocardial biopsies from patients with cardiac allografts showed that acute rejection was associated with intragraft expression of cxc chemokine receptor 3 ( cxcr3 [ 8 , 9 ] ) and its ligands , which include ifn-inducible protein of 10 kd ( ip-10 ; reference 10 ) , monokine induced by ifn- ( mig [ 11 , 12 ] ) , and ifn - inducible t cell  chemoattractant ( i - tac [ 9 , 13 ] ) .
these clinical findings have led to our serial analysis of intragraft chemokine and chemokine receptor expression using mhc fully mismatched mouse cardiac allografts , including expression of cxcr3 and its ligands during cardiac rejection .
our studies demonstrate that compared with control cxcr3 mice , cxcr3 mice show significantly delayed , or in some cases an absence of , acute or chronic rejection , and that anti - cxcr3 mab can reverse the course of developing rejection such that targeting of cxcr3 may prove to be of clinical therapeutic significance .
a mouse 129 genomic library ( genome systems ) was screened with mouse cxcr3 cdna 14 . a 13-kb genomic fragment containing exon 2 of the gene
was used to construct the targeting vector ; a 2.5-kb hindiii and xbali fragment was deleted and replaced by pgk - neo .
the targeted vector was linearized and electroporated into esi-1 embryonic stem cells ( genome systems ) .
chimeric males were bred to balb / c females to yield germline transmission of the targeted allele .
mice used in this study were backcrossed at least six generations onto the c57bl/6 strain .
cardiac allografting ( 15 ; n = 10/group ) was performed using balb / c ( h-2 ) donors and fully mhc - mismatched c57bl/6 ( h-2 ) recipients ; b6 mice were cxcr3 ( f6 ) versus littermate or commercial cxcr3 mice ( the jackson laboratory ) .
cyclosporine a ( csa ; sigma - aldrich ) was dissolved in olive oil and administered daily ( 10 mg / kg intraperitoneally ) , and mab therapy , using reagents described below , involved intraperitoneal injection of 500 g every second day ; in each case , therapy was begun at transplantation and stopped at 14 d after transplant . in additional experiments , the effects of mab administration beginning at day 4 after transplant were also tested .
mouse mabs ( bd pharmingen [ 16 , 17 ] ) , plus quantitative morphometry 15 . preparation and use of the cxcr3 probe for in situ hybridization was performed as described 15 .
spleen cells were stimulated with con a and cultured in il-2 for 812 d to generate cxcr3 t cell blasts 8 for use in chemotaxis and flow cytometry studies 18 .
northern blot analyses of t cells and t cell clones used cd3 mab  activated spleen cells supplemented with il-12 plus anti 
northern blots and rnase protection assays for cytokines , chemokines , and their receptors ( bd pharmingen ) were undertaken , with normalization to glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) or l32 genes 1518 .
mouse cxcr3 mab ( 4c4 , igm ) was generated by immunization with cxcr3 l1.2 cell transfectants , detection of binding to transfectants by flow cytometry , and screening for inhibition of chemotaxis of transfectants and t cell blasts to mouse recombinant cxcr3 ligands , as described 18 .
a mouse 129 genomic library ( genome systems ) was screened with mouse cxcr3 cdna 14 . a 13-kb genomic fragment containing exon 2 of the gene
was used to construct the targeting vector ; a 2.5-kb hindiii and xbali fragment was deleted and replaced by pgk - neo .
the targeted vector was linearized and electroporated into esi-1 embryonic stem cells ( genome systems ) .
chimeric males were bred to balb / c females to yield germline transmission of the targeted allele .
mice used in this study were backcrossed at least six generations onto the c57bl/6 strain .
cardiac allografting ( 15 ; n = 10/group ) was performed using balb / c ( h-2 ) donors and fully mhc - mismatched c57bl/6 ( h-2 ) recipients ; b6 mice were cxcr3 ( f6 ) versus littermate or commercial cxcr3 mice ( the jackson laboratory ) .
cyclosporine a ( csa ; sigma - aldrich ) was dissolved in olive oil and administered daily ( 10 mg / kg intraperitoneally ) , and mab therapy , using reagents described below , involved intraperitoneal injection of 500 g every second day ; in each case , therapy was begun at transplantation and stopped at 14 d after transplant . in additional experiments , the effects of mab administration beginning at day 4 after transplant were also tested .
infiltrating cells were detected by immunoperoxidase staining of cryostat sections with rat anti  mouse mabs ( bd pharmingen [ 16 , 17 ] ) , plus quantitative morphometry 15 . preparation and use of the cxcr3 probe for in situ hybridization was performed as described 15 .
spleen cells were stimulated with con a and cultured in il-2 for 812 d to generate cxcr3 t cell blasts 8 for use in chemotaxis and flow cytometry studies 18 .
northern blot analyses of t cells and t cell clones used cd3 mab  activated spleen cells supplemented with il-12 plus anti 
northern blots and rnase protection assays for cytokines , chemokines , and their receptors ( bd pharmingen ) were undertaken , with normalization to glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) or l32 genes 1518 .
mouse cxcr3 mab ( 4c4 , igm ) was generated by immunization with cxcr3 l1.2 cell transfectants , detection of binding to transfectants by flow cytometry , and screening for inhibition of chemotaxis of transfectants and t cell blasts to mouse recombinant cxcr3 ligands , as described 18 .
as allograft rejection is a t lymphocyte  dependent event 2 , we undertook northern blot analysis of cxcr3 expression by primary t cells and t cell clones in culture , and assessed cxcr3 involvement in allograft rejection in vivo .
freshly isolated mouse splenocytes lacked cxcr3 expression , but upon activation and culture in conditions that promote th cell differentiation , th1 cells expressed cxcr3 mrna ( fig .
1 a ) . established th1 clones ( pl17 and of6 ) , but not th2 clones ( cdc35 and d10 ) , also expressed cxcr3 mrna ( fig .
heterotopic cardiac allografts in the fully mhc - mismatched balb / c to c57bl/6 combination survive 78 d 17 .
rnase protection assays showed that normal hearts and isografts lacked cxcr3 mrna , whereas allografts showed progressive cxcr3 mrna expression , peaking at day 6 after transplant , just before end - stage rejection ( fig .
cxcr3 mrna expression closely followed intragraft mrna levels of the three known cxcr3 ligands ip-10 , mig , and i - tac , plus ifn- ( fig .
expression of cxcr3 mrna was localized to infiltrating leukocytes by in situ hybridization ( fig . 1 d ) , and cxcr3 mononuclear cell infiltration was confirmed by immunohistology ( fig
these results show that activated t cells , especially th1 cells , express cxcr3 and infiltrate cardiac allografts in conjunction with graft expression of the ligands ip-10 , mig , and i - tac 19 . to establish the role of cxcr3 expression in allograft rejection , we used homologous recombination to disrupt exon 2 of the x chromosome  linked 20 cxcr3 gene ( fig .
mice heterologous and homozygous for the cxcr3 mutation were normal in appearance , growth , and fertility .
cxcr3 deficiency was transmitted in a mendelian fashion , as identified by southern blot analysis of tail dna ( fig .
t cell blasts from homozygous cxcr3-deficient ( cxcr3 ) mice lacked cxcr3 protein expression ( fig .
2 c ) , and failed to respond in chemotaxis assays to ip-10 ( fig .
2 d ) or mig , indicating that cxcr3 is the sole functional receptor for these chemokines . with relevance to subsequent in vivo studies
, t cell blasts from cxcr3 but not cxcr3 mice were also labeled using the cxcr3-specific mab ( fig .
the addition of anti - cxcr3 mab reduced the mlr of wild - type mice ( fig .
3 c ) , but had no effect on mitogen responses ( data not shown ) .
whereas cxcr3 mice rejected balb / c cardiac allografts within 1 wk , cxcr3 mice accepted corresponding allografts for a mean of 58  3 d ( p < 0.001 ) ; no benefit was seen when hearts from cxcr3 mice were used as donor organs in balb / c mice .
targeting of cxcr3 resulted in strong synergy with the immunosuppressive agent , csa , consistent with data that csa does not affect cxcr3 ligand induction 1521 . 2 wk of therapy with csa ( 10 mg / kg ) prolonged allograft survival by only 3 d in wild - type recipients , but induced permanent engraftment ( > 100 d , p < 0.001 ) in cxcr3 mice ( fig . 3 d ) ;
evaluation of the latter allografts harvested at day 100 after transplant showed normal morphology , with a lack of graft leukocyte infiltration , myocardial injury , or evidence of transplant arteriosclerosis ( data not shown ) .
use of the cxcr3 mab in cxcr3 mice significantly prolonged allograft survival when used from the time of transplantation , and was also effective when commenced once rejection had begun , i.e. , at day 4 after transplant ( fig .
these findings demonstrate a profound effect of targeting cxcr3 on the development of cardiac allograft rejection .
histologic analysis of allografts harvested at day 5 after transplant showed that in contrast to control grafts , grafts in cxcr3 or cxcr3 mab 
had significantly reduced numbers of cd4 and cd8 t cells and macrophages , and no il-2r ( cd25 ) cells indicative of immune activation ( fig . 4 e ) .
allografts in cxcr3 recipients contained decreased ifn- mrna ( fig . 4 f ) , and decreased expression of the chemokines macrophage inflammatory protein ( mip)-1 and regulated upon activation , normal t cell expressed and secreted ( rantes ; fig .
consistent with the latter findings , allografts in cxcr3 mice showed decreased expression of the corresponding chemokine receptors ccr1 , ccr2 , and ccr5 , produced by t cells and macrophages ( fig . 4 h ) . in conclusion ,
this effect is associated with significantly decreased intragraft accumulation of activated t cells producing ifn- and consequent impairment of chemokine - dependent recruitment of multiple effector cell types , suggesting a rationale for targeting of cxcr3 , in synergy with conventional immunosuppression , in clinical transplantation , and potentially in the management of acute allograft rejection .
 chemokines provide signals for activation and recruitment of effector cells into sites of inflammation , acting via specific g protein  coupled receptors . 
 however , in vitro data demonstrating the presence of multiple ligands for a given chemokine receptor , and often multiple receptors for a given chemokine , have led to concerns of biologic redundancy . here 
 we show that acute cardiac allograft rejection is accompanied by progressive intragraft production of the chemokines interferon ( ifn)-inducible protein of 10 kd ( ip-10 ) , monokine induced by ifn- ( mig ) , and ifn - inducible t cell  chemoattractant ( i - tac ) , and by infiltration of activated t cells bearing the corresponding chemokine receptor , cxcr3 . 
 we used three in vivo models to demonstrate a role for cxcr3 in the development of transplant rejection . 
 first , cxcr3-deficient ( cxcr3/ ) mice showed profound resistance to development of acute allograft rejection . 
 second , cxcr3/ allograft recipients treated with a brief , subtherapeutic course of cyclosporin a maintained their allografts permanently and without evidence of chronic rejection . 
 third , cxcr+/+ mice treated with an anti - cxcr3 monoclonal antibody showed prolongation of allograft survival , even if begun after the onset of rejection . taken in conjunction with our findings of cxcr3 expression in rejecting human cardiac allografts , 
 we conclude that cxcr3 plays a key role in t cell activation , recruitment , and allograft destruction . 
Introduction
Materials and Methods
Generation of CXCR3
Transplantation.
Immunopathology.
Cellular and Molecular Studies.
Results and Discussion