for studies performed in the united states , female wild - type ( wt ) and cd4 t cell  deficient balb / c ( cd4 ) mice were bred in an association for assessment and accreditation of laboratory animal care - accredited university of pittsburgh ( pittsburgh , pa , usa ) animal facility and used as corneal transplant recipients . wild - type balb / c ( h-2 ; jackson laboratory , bar harbor , me , usa ) and c57bl/6j ( h-2 ; jackson laboratory ) mice served as syngeneic and allogeneic corneal donors , respectively . all animal studies were approved by and conducted in accordance with the university of pittsburgh institutional animal care and use committee . for studies performed in scotland , mice were bred and housed in the uk home office accredited medical research facility of the university of aberdeen ( aberdeen , scotland , uk ) . wild - type donor ( c57bl/6j ) and recipient ( balb / c ) pairs were used as described above . an additional group of animals was treated with systemic acyclovir ( acv ) at a dose of 10 mg / kg body weight in the drinking water . all animal studies were performed in accordance with guidelines described in the arvo statement for the use of animals in vision and ophthalmic research and animal license act ( uk ) . a central 1.5 mm of the host cornea was trephined and the corneal button secured to the host bed using eight interrupted 11 - 0 nylon sutures ( pittsburgh ) or one continuous suture ( aberdeen ) as described previously . six- to 10-week - old female wt or cd4 balb / c were anesthetized , the corneas scarified with a sterile 30-g needle and 3 l of rpmi containing 1  10 pfu of the re strain of hsv-1 applied as previously described . wild - type and cd4 mice were infected for more than 30 and 10 days , respectively , prior to transplantation . cd4 t cells were isolated from spleens of wt balb / c mice by negative selection using easysep mouse cd4 t cell enrichment kit ( stemcell technologies , vancouver , bc , canada ) . purification was assessed by flow cytometry and was greater than 90% in all experiments . where indicated , 2.5  10 isolated cells in 200-l sterile hanks ' balanced salt solution ( hbss ) were transferred per mouse via tail vein injection . mice were killed with an overdose of co2 , the eyes were immediately immersed in oct medium , snap frozen in cooled isopentane on dry ice , and stored at 80c until used . primary antibody was placed for 1 hour at room temperature on 6-m thick eye sections , followed by secondary biotinylated rabbit anti - rat ( dakocytomation , glostrup , denmark ) or biotinylated mouse anti - hamster ig cocktail ( bd pharmingen , oxford , uk ) for 30 minutes at room temperature followed by vectastain abc - ap kit ( vector laboratories , peterborough , uk ) for 30 minutes . for confocal imaging secondary fluorescent - labelled antibodies alexa fluor 555 goat anti - rat igg ( invitrogen , waltham , ma , usa ) and alexa fluor 488 goat anti - rat igg ( invitrogen ) were used together with nuclear staining 4,6-diamidino-2-phenylindole ( dapi , vector laboratories , peterborough , uk ) . the following primary monoclonal antibodies ( mab ) were used in concentrations as suggested by the manufacturer : rat anti - mouse cd11b ( clone m1/70 ) ; rat anti - mouse cd4 ( clone h129.19 ) , rat anti - mouse cd8 ( clone 53 - 6.7 ) , rat anti - mouse gr-1 ( clone rb6 - 8c5 ) , hamster anti - mouse cd11c ( clone hl3 ; all from bd pharmingen ) and rat anti mouse f4/80 ( clone c1:a3 - 1 ; abd serotec , kidlington , uk ) . corneas were excised , washed in pbs with 4% fetal bovine serum ( fbs ) for 10 minutes at 4c prior to fixation in cytofix / cytoperm for 1 hour at 4c . the tissue was washed three times in pbs with 4% fbs prior to incubation with primary fluorochrome - conjugated antibodies overnight at 4c . following three washes , the tissue was mounted in immu - mount ( thermo scientific , waltham , ma , usa ) or vectashield mounting medium ( vector laboratories , burlingame , ca , usa ) with dapi ( vector laboratories , burlingame , ca , usa ) prior to confocal imaging . antibodies used were pacific blue - conjugated anti - cd4 ( bd ; clone : rm4 - 5 ) , pacific blue- or allophycocyanin-(apc)-conjugated anti - cd11b ( ebioscience ; clone : m1/70 ) , apc - conjugated anti - mhc class ii ( i - a / i - e ; clone : m5/114.15.2 ; ebioscience , san diego , ca , usa ) . images were acquired by sequential scanning to avoid fluorescence crossover on an olympus fluoview 1000 confocal microscope ( center valley , pa , usa ) . all image reconstructions were made using metamorph ( version : 7.5.4.0 ; molecular devices , sunnyvale , ca , usa ) . corneal buttons and tg were removed at the specific time points after corneal allografting and snap frozen in liquid nitrogen . dna was extracted with dneasy blood and tissue kit ( qiagen , manchester , uk ) and quantified with nanodrop 1000 spectrophotometer ( thermo scientific ) . twenty - five nanograms dna or control water only was mixed in taqman pcr master mix ( applied biosystems , thermo scientific , waltham , ma , usa ) with an hsv-1 glycoprotein h ( gh)-specific primer . ipsilateral eye draining ( submandibular ) ln ( dln ) were identified and removed at different times after infection of the cornea with hsv-1 ( see above ) . as a control we used inguinal , non - eye dln . in a preliminary analysis , samples from nongrafted mice were analyzed for hsv-1 dna at various times up 42 days post infection ( dpi ) . samples harvested from grafted mice were taken 42 dpi plus 7 days post graft ( i.e. , 49 days in total post infection ) and were compared with positive control samples taken from nongrafted hsv-1infected mice 5 dpi and tested in the same assay . lymphocytes were seeded ( 1.5  10 cells / well ) into 96-well plates in triplicates and stimulated with b6 antigen ( lysate of splenocytes from c57bl/6 mice ; 6 g / well ) or with hsv-1 protein ( lysate of infected vero cells ; 6 g / well ) with appropriate controls . cells were cultured for 96 hours , last 16 hours spiked with [ h]thymidine . for statistical analysis a log - rank test ( mantel - cox ) was used for comparing survival curves ( graphpad prism software , inc . , version 5.04 , la jolla , ca , usa ) . a p value of less than 0.05 for studies performed in the united states , female wild - type ( wt ) and cd4 t cell  deficient balb / c ( cd4 ) mice were bred in an association for assessment and accreditation of laboratory animal care - accredited university of pittsburgh ( pittsburgh , pa , usa ) animal facility and used as corneal transplant recipients . wild - type balb / c ( h-2 ; jackson laboratory , bar harbor , me , usa ) and c57bl/6j ( h-2 ; jackson laboratory ) mice served as syngeneic and allogeneic corneal donors , respectively . all animal studies were approved by and conducted in accordance with the university of pittsburgh institutional animal care and use committee . for studies performed in scotland , mice were bred and housed in the uk home office accredited medical research facility of the university of aberdeen ( aberdeen , scotland , uk ) . wild - type donor ( c57bl/6j ) and recipient ( balb / c ) pairs were used as described above . an additional group of animals was treated with systemic acyclovir ( acv ) at a dose of 10 mg / kg body weight in the drinking water . all animal studies were performed in accordance with guidelines described in the arvo statement for the use of animals in vision and ophthalmic research and animal license act ( uk ) . a central 1.5 mm of the host cornea was trephined and the corneal button secured to the host bed using eight interrupted 11 - 0 nylon sutures ( pittsburgh ) or one continuous suture ( aberdeen ) as described previously . six- to 10-week - old female wt or cd4 balb / c were anesthetized , the corneas scarified with a sterile 30-g needle and 3 l of rpmi containing 1  10 pfu of the re strain of hsv-1 applied as previously described . wild - type and cd4 mice were infected for more than 30 and 10 days , respectively , prior to transplantation . cd4 t cells were isolated from spleens of wt balb / c mice by negative selection using easysep mouse cd4 t cell enrichment kit ( stemcell technologies , vancouver , bc , canada ) . purification was assessed by flow cytometry and was greater than 90% in all experiments . where indicated , 2.5  10 isolated cells in 200-l sterile hanks ' balanced salt solution ( hbss ) were transferred per mouse via tail vein injection . mice were killed with an overdose of co2 , the eyes were immediately immersed in oct medium , snap frozen in cooled isopentane on dry ice , and stored at 80c until used . primary antibody was placed for 1 hour at room temperature on 6-m thick eye sections , followed by secondary biotinylated rabbit anti - rat ( dakocytomation , glostrup , denmark ) or biotinylated mouse anti - hamster ig cocktail ( bd pharmingen , oxford , uk ) for 30 minutes at room temperature followed by vectastain abc - ap kit ( vector laboratories , peterborough , uk ) for 30 minutes . for confocal imaging secondary fluorescent - labelled antibodies alexa fluor 555 goat anti - rat igg ( invitrogen , waltham , ma , usa ) and alexa fluor 488 goat anti - rat igg ( invitrogen ) were used together with nuclear staining 4,6-diamidino-2-phenylindole ( dapi , vector laboratories , peterborough , uk ) . the following primary monoclonal antibodies ( mab ) were used in concentrations as suggested by the manufacturer : rat anti - mouse cd11b ( clone m1/70 ) ; rat anti - mouse cd4 ( clone h129.19 ) , rat anti - mouse cd8 ( clone 53 - 6.7 ) , rat anti - mouse gr-1 ( clone rb6 - 8c5 ) , hamster anti - mouse cd11c ( clone hl3 ; all from bd pharmingen ) and rat anti  mouse f4/80 ( clone c1:a3 - 1 ; abd serotec , kidlington , uk ) . corneas were excised , washed in pbs with 4% fetal bovine serum ( fbs ) for 10 minutes at 4c prior to fixation in cytofix / cytoperm for 1 hour at 4c . the tissue was washed three times in pbs with 4% fbs prior to incubation with primary fluorochrome - conjugated antibodies overnight at 4c . following three washes , the tissue was mounted in immu - mount ( thermo scientific , waltham , ma , usa ) or vectashield mounting medium ( vector laboratories , burlingame , ca , usa ) with dapi ( vector laboratories , burlingame , ca , usa ) prior to confocal imaging . antibodies used were pacific blue - conjugated anti - cd4 ( bd ; clone : rm4 - 5 ) , pacific blue- or allophycocyanin-(apc)-conjugated anti - cd11b ( ebioscience ; clone : m1/70 ) , apc - conjugated anti - mhc class ii ( i - a / i - e ; clone : m5/114.15.2 ; ebioscience , san diego , ca , usa ) . images were acquired by sequential scanning to avoid fluorescence crossover on an olympus fluoview 1000 confocal microscope ( center valley , pa , usa ) . all image reconstructions were made using metamorph ( version : 7.5.4.0 ; molecular devices , sunnyvale , ca , usa ) . corneal buttons and tg were removed at the specific time points after corneal allografting and snap frozen in liquid nitrogen . dna was extracted with dneasy blood and tissue kit ( qiagen , manchester , uk ) and quantified with nanodrop 1000 spectrophotometer ( thermo scientific ) . twenty - five nanograms dna or control water only was mixed in taqman pcr master mix ( applied biosystems , thermo scientific , waltham , ma , usa ) with an hsv-1 glycoprotein h ( gh)-specific primer . ipsilateral eye draining ( submandibular ) ln ( dln ) were identified and removed at different times after infection of the cornea with hsv-1 ( see above ) . as a control we used inguinal , non - eye dln . in a preliminary analysis , samples from nongrafted mice were analyzed for hsv-1 dna at various times up 42 days post infection ( dpi ) . samples harvested from grafted mice were taken 42 dpi plus 7 days post graft ( i.e. , 49 days in total post infection ) and were compared with positive control samples taken from nongrafted hsv-1infected mice 5 dpi and tested in the same assay . lymphocytes were seeded ( 1.5  10 cells / well ) into 96-well plates in triplicates and stimulated with b6 antigen ( lysate of splenocytes from c57bl/6 mice ; 6 g / well ) or with hsv-1 protein ( lysate of infected vero cells ; 6 g / well ) with appropriate controls . for statistical analysis a log - rank test ( mantel - cox ) was used for comparing survival curves ( graphpad prism software , inc . , version 5.04 , la herpes simplex virus-1 applied to scarified balb / c mouse corneas produces an initial dendritic / geographic lesion that heals around 3 dpi , with clearance of infectious virus by 6 to 8 dpi . most of the mice ( 80%90% ) , over the next 2 to 3 weeks , develop chronic hsk with vascularization and variable ulceration . ten percent to 20% mice clear the virus without inflammatory sequelae and have clear nonvascularized corneas . to determine acceptance rates , allografts were performed in chronically hsv - infected ( 6 weeks post infection ) wt balb / c mice , 80% to 90% of which had hsk grade 2 to 3 or above ( fig . all grafts in active hsk recipients failed ( onset 3 days , maximal 15 days post grafting ; fig . survival curves of previously infected recipients ( allo - hsk or syn - hsk ) were compared with noninfected allografts ( allo ) and showed some striking differences with p values as follows : allo - hsk versus allo , p less than 0.0001 ; allo - hsk versus syn - hsk , p = 0.9053 . failed grafts showed extensive vascularization with stromal opacification ( see fig . interestingly , in recipients with severe hsk and 360 vascularization , syngeneic grafts failed as rapidly as allografts ( fig . we therefore assessed the effect of acv therapy on the survival of corneal allografts in hsv - infected recipients . herpes simplex virus  infected animals were treated with acv ( 10 mg / kg ) in the drinking water immediately after surgical procedure for the duration of the study . the survival of allografts was not different from the survival of allografts in hsv - infected , untreated animals ( fig . 1b ; allo - hsk versus allo - hsk+acv p = 0.6692 ; allo - hsk+acv versus allo p = 0.0002 ) . in contrast , hsv - infected allogeneic grafts , where the inflammation had subsided and the cornea was clear without vascularization at the time of grafting , underwent accelerated graft rejection ( fig.1d ; nonvascularized corneal graft bed : allo - hsk versus syn - hsk , p = 0.0012 ; allo - hsk versus allo p = 0.2285 ; syn - hsk versus allo p = 0.0356 ) . wild - type balb / c mice were infected with hsv-1 6 weeks ( 42 days ) prior to placement of corneal grafts , which were secured with a continuous suture in this set of experiments . ( a ) clinical appearance of the vascularized corneal graft bed at 6 week post hsv-1 infection ; ( b ) corneal graft survival in vascularized corneal beds in syngraft hsk ( n = 15 ) , allograft hsk ( n = 20 ) , and allograft hsk treated with acv ( n = 10 ) groups compared with wt , noninfected balb / c recipients transplanted with allograft ( n = 20 ) ; ( c ) clinical appearance of rejecting corneal allograft placed into vascularized corneal graft bed ( day 3 post allograft ) ; ( d ) corneal graft survival in previously infected but not vascularized ( at 6 weeks post infection [ p.i . ] ) graft beds for both allo- and syngrafts compared with wt , noninfected balb / c recipient transplanted with allograft . endothelial cell loss is the main cause of graft failure and may result from immune ( allograft ) or nonimmune ( allograft and syngraft ) damage . the above results suggested that failure of both syngeneic and allogeneic grafts following hsv infection may have been due to innate ( hsv - induced inflammation ) rather than adaptive ( allo ) immunity . we hypothesized therefore that allografts , but not syngrafts , in hsk recipients would fail through alloimmune damage only if the grafts were performed before hsv - induced inflammation / neovascularization ( i.e. , clinically overt hsk ) occurred . we therefore performed grafts in hsv - infected corneas before they developed overt hsk ( i.e. , 10 dpi ) when the corneas were still clear and had no vascularization . however , when the grafts were performed in hsv - infected recipients , both syn- and allo - grafts rapidly failed ( p = 0.1211 ; fig . we then assessed the contribution of adaptive immunity ( specifically the cd4 t - cell response ) directly by performing grafts in hsv infected cd4 mice , which do not develop significant inflammation or vascularization following hsv-1 corneal infection but do establish a latent infection in the tg by 10 dpi . as shown in figure 2b , only approximately 25% of infected cd4 balb / c mice developed graft failure , while 100% of infected cd4 balb / c mice that were adoptively transferred with nave wt cd4 t cells , rejected their allografts by 14 days post graft . in contrast , only 20% of syngrafts in cd4 mice failed after adoptive transfer of nave wt cd4 t cells , which was not significantly different from the 100% acceptance of syngeneic grafts in nonreconstituted mice ( fig . 2c ) . ( a ) wild - type balb / c mice were infected with hsv-1 and corneal grafts were performed 10 dpi ( grafts secured with continuous suture ) when corneas typically do not have corneal neovascularization or corneal scar . ( b , c ) cd4 balb / c mice were infected with hsv-1 for 10 days prior to placement of corneal allografts ( grafts secured with interrupted sutures ; [ b ] c57bl/6j donors ) or syngrafts ( grafts secured with interrupted sutures ; [ c ] wt balb / c donors ) . mice received 2  10 nave wt balb / c cd4 t cells intravenously ( with cd4s ) at the time of transplant or not ( no cd4s ) , and grafts were monitored for rejection at various times post transplant . data in figure 3 confirm the lack of blood or lymphatic vessels in hsv-1infected corneas of cd4 mice 10 dpi as well as in hsv - infected corneas of cd4 mice after grafting . however , allografted corneas from mice that received cd4 t cells at the time of grafting had extensive blood and lymphatic vessel ingrowth into both the graft bed and the rejecting graft ( figs . thus , while hsk - associated high - risk graft failure is accompanied by an ingrowth of blood and lymphatic vessels , preexisting vessels did not directly influence either the frequency or tempo of allograft rejection . infiltration of blood and lymph vessels as well as antigen - presenting cells into transplanted corneas . corneas from the indicated mice at the indicated times were excised , stained for lymph vessels ( lyve-1 ; green ) , blood vessels ( cd31 ; red ) , and mhc class ii+ cells ( blue ) , and imaged by confocal microscopy . ( a ) representative montages acquired via wide - field microscopy of a nongrafted cd4 cornea obtained 10 days after hsv-1 corneal infection ( top ) , cd4 cornea infected with hsv-1 10 days prior to corneal allotransplantation and adoptive transfer of nave wt balb / c cd4 t cells ( middle ) , and cd4 cornea infected 10 days prior to corneal allograft without receiving cd4 t cells ( bottom ; all above results generated from grafts secured with interrupted sutures ) . ( b ) high magnification images ( left : xy ; right : xz ) of a cornea from a cd4 mouse infected with hsv-1 10 days prior to corneal allotransplantation and adoptive transfer of nave wt balb / c cd4 t cells ( [ a ] , middle panel ) . the inflammatory cell infiltrate also correlated with the clinical findings . in wt mice that were hsv infected and grafted with syn- or allograft , there was a heavy corneal cellular infiltration of cd11b and gr1 cells with few f4/80 cells ( figs . similar findings of inflammatory cell infiltration in hsv - infected and grafted eyes with syngeneic corneas were found , while cd4 t cells were not detected in hsv - infected corneas which had received either a syn- or allograft ( data not shown ) . in contrast , corneas from cd4 hsv - infected allografted mice exhibited minimal infiltration of inflammatory cells , which was mainly cd11b gr1 ( presumed macrophages ) with a few cd11b gr1 ( presumed neutrophils ) observed near the sutures ( black arrows , fig . 5a ) unlike rejecting allografts in hsv-1infected cd4 recipients that were reconstituted with cd4 t cells , which had a heavy graft cd11b gr1 and cd11b gr1 cell infiltrate ( fig . 5b ) . infiltration of macrophages and neutrophils into corneal grafts in wt hsk mice . corneal allografts stained with gr1 and cd11b antibodies in early phase post corneal graft , day 3 ( a  e ) and day 7 ( f ) in hsk recipients show massive neutrophil ( gr1 ) and macrophage ( cd11b ) infiltration in the recipient cornea as well as in the donor corneal graft and anterior chamber of the eye . dual staining of confocal microscopy images of day 7 grafts ( g  i ) show that majority of these cells are neutrophils ( gr1cd11bf4/80 ) and macrophages ( gr1cd11b ) ; white arrow indicating multilobed nucleus at day 7 post grafting ( h ) . the f4/80 cells ( g , i ) were infrequently observed cells in the inflammatory cell infiltrate and did not colocalize with gr1cells ( g ) . ten days after hsv-1 corneal infection , cd4 mice received an allogeneic corneal transplant without cd4 t - cell transfer ( a ) or with adoptive transfer of nave wt balb / c cd4 t cells ( b ) . transplanted corneas were excised and stained with dapi ( blue ) , cd11b ( green ) , and gr1 ( red ) prior to imaging with confocal microscopy . full - thickness reconstructions are shown in the xy top and xz ( middle ) planes . the xz image is displayed with the corneal epithelium facing up and the endothelial side facing down . the bottom panels depict representative montages of full - thickness reconstructions acquired with a 40 objective in the xy plane . we next explored the possibility that latent hsv reactivation was the cause of graft failure in hsk recipients . periocular herpetic vesicles accompany hsv keratitis during the early stages of infection , but clear after 2 to 3 weeks and do not normally recur . in wt mice grafted 42 days post infection ( i.e. , long after viral clearance ) , 10% of allografted mice were observed to develop periocular herpetic vesicles at various times post grafting ( 222 days post graft , equal to 4466 dpi ; fig . peak incidence for vesicle occurrence in allografted mice was seven days post grafting ( 4/17 mice ) , some mice having more than one recurrence ( fig . a corneal allograft was placed on a recipient bed with previous hsk but no vascularization at the time of grafting . at the time of grafting ( a ) an obvious periocular vesicular skin lesion appeared 7 days after placement of the allograft although the graft remains clear at this early time post grafting . ( b ) table showing the incidence of reactivation of periocular hsv vesicles in hsk recipients ( allo- and syngrafts ) . ( c ) dna detection of gh protein in corneal tissue and tg in noninfected and hsv - infected ( infected with hsv 42 days previously ) recipients of corneal allografts . herpes simplex virus gh bands were detected only in hsv - infected animals and only in the tg , but not in the cornea of transplanted mice . controls for the tg upper panel are as follows : ( 1 ) hsv+ tg 7 dpi , ( 2 ) dh20 , ( 3 ) hsv+ vero cell extract , ( 4)/(5 ) hsv+ corneas 3 dpi , ( 6 ) ladder . controls for the middle cornea panel are as follows : ( 1 ) dh20 , ( 2 ) hsv+ tg 7 dpi , ( 3 ) hsv+ vero cell extract , ( 4)/(5 ) hsv+ corneas 3 dpi , ( 6 ) ladder . we also sought evidence of viral reactivation / shedding in tears , cultured tg explants and in samples of corneal tissue ( by rt - pcr [ data not shown ] and by pcr techniques , see methods ) from wt hsk recipient mice after grafting . herpes simplex virus negative control samples were prepared from nave , not infected balb / c animals grafted with b6 cornea . no evidence for viral shedding in tear samples was observed nor was virus cultured from tg explants directly ex vivo ( data not shown ) . herpes simplex virus type 1 gh rna ( hsv-1 glycoprotein h ) was detected in tg samples by pcr from hsv - infected , allografted mice at days 3 and 7 post grafting . in contrast , no hsv gh rna was detected at any time point from samples of corneal grafts ( fig . both tg and corneal grafts from not infected , control group were negative for gh pcr product . finally , we examined peripheral t - cell responses to hsv antigen , b6 alloantigen , and to nonrelated ova antigen in allografted hsk recipients and compared the responses to hsv - infected , nongrafted mice . herpes simplex virus type 1 antigen was prepared from protein extracts of hsv - infected vero cells while b6 alloantigen was prepared from c57bl/6 spleen homogenates . lymphocyte ( predominantly t cell ) proliferative responses from individual eye - dln from each mouse were assessed by [ h]-thymidine incorporation ( see methods ) . a variable but robust t - cell response in eye - draining ( submandibular ) lymph node ( dln ) cells from nongrafted hsk mice was detected 3 dpi , which persisted for several weeks ( the duration of the experiment ; fig . a strong anti - hsv lymphocyte t - cell response was detected in allografted hsk mice ( 7 days post transplant , i.e. , 49 dpi ) of similar magnitude to the anti - hsv response 5 dpi in nongrafted hsk mice ( fig . 7b , middle panel ) . importantly , dln cells from allografted hsk recipients failed to respond in the absence of hsv antigen ( fig . syngeneic mice produced a low but variable anti - hsv t - cell response ( p = 0.11 ) . ( a ) lymphocyte proliferation from eye - dln at different times after infection with hsv-1 . note consistent anti - hsv response even at the time - point when clinically the cornea is healed ( i.e. , day 42 dpi = time - point of corneal grafting procedure ) . ( b ) lymphocyte proliferation responses to hsv antigen and b6 alloantigen day 7 post corneal allo- and syngraft ( i.e. , = day 49 pi ) . the response to hsv antigen after allografting procedure ( middle panel ) was equal to the lymphocyte response to hsv antigen 5 dpi in the ungrafted cornea . the difference between the allo- and syngraft response is not significant ( see main text ) . herpes simplex virus-1 applied to scarified balb / c mouse corneas produces an initial dendritic / geographic lesion that heals around 3 dpi , with clearance of infectious virus by 6 to 8 dpi . most of the mice ( 80%90% ) , over the next 2 to 3 weeks , develop chronic hsk with vascularization and variable ulceration . ten percent to 20% mice clear the virus without inflammatory sequelae and have clear nonvascularized corneas . to determine acceptance rates , allografts were performed in chronically hsv - infected ( 6 weeks post infection ) wt balb / c mice , 80% to 90% of which had hsk grade 2 to 3 or above ( fig . all grafts in active hsk recipients failed ( onset 3 days , maximal 15 days post grafting ; fig . survival curves of previously infected recipients ( allo - hsk or syn - hsk ) were compared with noninfected allografts ( allo ) and showed some striking differences with p values as follows : allo - hsk versus allo , p less than 0.0001 ; allo - hsk versus syn - hsk , p = 0.9053 . failed grafts showed extensive vascularization with stromal opacification ( see fig . interestingly , in recipients with severe hsk and 360 vascularization , syngeneic grafts failed as rapidly as allografts ( fig . we therefore assessed the effect of acv therapy on the survival of corneal allografts in hsv - infected recipients . herpes simplex virus  infected animals were treated with acv ( 10 mg / kg ) in the drinking water immediately after surgical procedure for the duration of the study . the survival of allografts was not different from the survival of allografts in hsv - infected , untreated animals ( fig . 1b ; allo - hsk versus allo - hsk+acv p = 0.6692 ; allo - hsk+acv versus allo p = 0.0002 ) . in contrast , hsv - infected allogeneic grafts , where the inflammation had subsided and the cornea was clear without vascularization at the time of grafting , underwent accelerated graft rejection ( fig.1d ; nonvascularized corneal graft bed : allo - hsk versus syn - hsk , p = 0.0012 ; allo - hsk versus allo p = 0.2285 ; syn - hsk versus allo p = 0.0356 ) . wild - type balb / c mice were infected with hsv-1 6 weeks ( 42 days ) prior to placement of corneal grafts , which were secured with a continuous suture in this set of experiments . ( a ) clinical appearance of the vascularized corneal graft bed at 6 week post hsv-1 infection ; ( b ) corneal graft survival in vascularized corneal beds in syngraft hsk ( n = 15 ) , allograft hsk ( n = 20 ) , and allograft hsk treated with acv ( n = 10 ) groups compared with wt , noninfected balb / c recipients transplanted with allograft ( n = 20 ) ; ( c ) clinical appearance of rejecting corneal allograft placed into vascularized corneal graft bed ( day 3 post allograft ) ; ( d ) corneal graft survival in previously infected but not vascularized ( at 6 weeks post infection [ p.i . ] ) graft beds for both allo- and syngrafts compared with wt , noninfected balb / c recipient transplanted with allograft . endothelial cell loss is the main cause of graft failure and may result from immune ( allograft ) or nonimmune ( allograft and syngraft ) damage . the above results suggested that failure of both syngeneic and allogeneic grafts following hsv infection may have been due to innate ( hsv - induced inflammation ) rather than adaptive ( allo ) immunity . we hypothesized therefore that allografts , but not syngrafts , in hsk recipients would fail through alloimmune damage only if the grafts were performed before hsv - induced inflammation / neovascularization ( i.e. , clinically overt hsk ) occurred . we therefore performed grafts in hsv - infected corneas before they developed overt hsk ( i.e. , 10 dpi ) when the corneas were still clear and had no vascularization . however , when the grafts were performed in hsv - infected recipients , both syn- and allo - grafts rapidly failed ( p = 0.1211 ; fig . we then assessed the contribution of adaptive immunity ( specifically the cd4 t - cell response ) directly by performing grafts in hsv infected cd4 mice , which do not develop significant inflammation or vascularization following hsv-1 corneal infection but do establish a latent infection in the tg by 10 dpi . as shown in figure 2b , only approximately 25% of infected cd4 balb / c mice developed graft failure , while 100% of infected cd4 balb / c mice that were adoptively transferred with nave wt cd4 t cells , rejected their allografts by 14 days post graft . in contrast , only 20% of syngrafts in cd4 mice failed after adoptive transfer of nave wt cd4 t cells , which was not significantly different from the 100% acceptance of syngeneic grafts in nonreconstituted mice ( fig . 2c ) . ( a ) wild - type balb / c mice were infected with hsv-1 and corneal grafts were performed 10 dpi ( grafts secured with continuous suture ) when corneas typically do not have corneal neovascularization or corneal scar . ( b , c ) cd4 balb / c mice were infected with hsv-1 for 10 days prior to placement of corneal allografts ( grafts secured with interrupted sutures ; [ b ] c57bl/6j donors ) or syngrafts ( grafts secured with interrupted sutures ; [ c ] wt balb / c donors ) . mice received 2  10 nave wt balb / c cd4 t cells intravenously ( with cd4s ) at the time of transplant or not ( no cd4s ) , and grafts were monitored for rejection at various times post transplant . data in figure 3 confirm the lack of blood or lymphatic vessels in hsv-1infected corneas of cd4 mice 10 dpi as well as in hsv - infected corneas of cd4 mice after grafting . however , allografted corneas from mice that received cd4 t cells at the time of grafting had extensive blood and lymphatic vessel ingrowth into both the graft bed and the rejecting graft ( figs . thus , while hsk - associated high - risk graft failure is accompanied by an ingrowth of blood and lymphatic vessels , preexisting vessels did not directly influence either the frequency or tempo of allograft rejection . infiltration of blood and lymph vessels as well as antigen - presenting cells into transplanted corneas . corneas from the indicated mice at the indicated times were excised , stained for lymph vessels ( lyve-1 ; green ) , blood vessels ( cd31 ; red ) , and mhc class ii+ cells ( blue ) , and imaged by confocal microscopy . ( a ) representative montages acquired via wide - field microscopy of a nongrafted cd4 cornea obtained 10 days after hsv-1 corneal infection ( top ) , cd4 cornea infected with hsv-1 10 days prior to corneal allotransplantation and adoptive transfer of nave wt balb / c cd4 t cells ( middle ) , and cd4 cornea infected 10 days prior to corneal allograft without receiving cd4 t cells ( bottom ; all above results generated from grafts secured with interrupted sutures ) . ( b ) high magnification images ( left : xy ; right : xz ) of a cornea from a cd4 mouse infected with hsv-1 10 days prior to corneal allotransplantation and adoptive transfer of nave wt balb / c cd4 t cells ( [ a ] , middle panel ) . the inflammatory cell infiltrate also correlated with the clinical findings . in wt mice that were hsv infected and grafted with syn- or allograft , there was a heavy corneal cellular infiltration of cd11b and gr1 cells with few f4/80 cells ( figs . similar findings of inflammatory cell infiltration in hsv - infected and grafted eyes with syngeneic corneas were found , while cd4 t cells were not detected in hsv - infected corneas which had received either a syn- or allograft ( data not shown ) . in contrast , corneas from cd4 hsv - infected allografted mice exhibited minimal infiltration of inflammatory cells , which was mainly cd11b gr1 ( presumed macrophages ) with a few cd11b gr1 ( presumed neutrophils ) observed near the sutures ( black arrows , fig . 5a ) unlike rejecting allografts in hsv-1infected cd4 recipients that were reconstituted with cd4 t cells , which had a heavy graft cd11b gr1 and cd11b gr1 cell infiltrate ( fig . 5b ) . infiltration of macrophages and neutrophils into corneal grafts in wt hsk mice . corneal allografts stained with gr1 and cd11b antibodies in early phase post corneal graft , day 3 ( a  e ) and day 7 ( f ) in hsk recipients show massive neutrophil ( gr1 ) and macrophage ( cd11b ) infiltration in the recipient cornea as well as in the donor corneal graft and anterior chamber of the eye . dual staining of confocal microscopy images of day 7 grafts ( g  i ) show that majority of these cells are neutrophils ( gr1cd11bf4/80 ) and macrophages ( gr1cd11b ) ; white arrow indicating multilobed nucleus at day 7 post grafting ( h ) . the f4/80 cells ( g , i ) were infrequently observed cells in the inflammatory cell infiltrate and did not colocalize with gr1cells ( g ) . ten days after hsv-1 corneal infection , cd4 mice received an allogeneic corneal transplant without cd4 t - cell transfer ( a ) or with adoptive transfer of nave wt balb / c cd4 t cells ( b ) . transplanted corneas were excised and stained with dapi ( blue ) , cd11b ( green ) , and gr1 ( red ) prior to imaging with confocal microscopy . full - thickness reconstructions are shown in the xy top and xz ( middle ) planes . the xz image is displayed with the corneal epithelium facing up and the endothelial side facing down . the bottom panels depict representative montages of full - thickness reconstructions acquired with a 40 objective in the xy plane . we next explored the possibility that latent hsv reactivation was the cause of graft failure in hsk recipients . periocular herpetic vesicles accompany hsv keratitis during the early stages of infection , but clear after 2 to 3 weeks and do not normally recur . in wt mice grafted 42 days post infection ( i.e. , long after viral clearance ) , 10% of allografted mice were observed to develop periocular herpetic vesicles at various times post grafting ( 222 days post graft , equal to 4466 dpi ; fig . peak incidence for vesicle occurrence in allografted mice was seven days post grafting ( 4/17 mice ) , some mice having more than one recurrence ( fig a corneal allograft was placed on a recipient bed with previous hsk but no vascularization at the time of grafting . at the time of grafting ( a ) an obvious periocular vesicular skin lesion appeared 7 days after placement of the allograft although the graft remains clear at this early time post grafting . ( b ) table showing the incidence of reactivation of periocular hsv vesicles in hsk recipients ( allo- and syngrafts ) . ( c ) dna detection of gh protein in corneal tissue and tg in noninfected and hsv - infected ( infected with hsv 42 days previously ) recipients of corneal allografts . herpes simplex virus gh bands were detected only in hsv - infected animals and only in the tg , but not in the cornea of transplanted mice . controls for the tg upper panel are as follows : ( 1 ) hsv+ tg 7 dpi , ( 2 ) dh20 , ( 3 ) hsv+ vero cell extract , ( 4)/(5 ) hsv+ corneas 3 dpi , ( 6 ) ladder . controls for the middle cornea panel are as follows : ( 1 ) dh20 , ( 2 ) hsv+ tg 7 dpi , ( 3 ) hsv+ vero cell extract , ( 4)/(5 ) hsv+ corneas 3 dpi , ( 6 ) ladder . we also sought evidence of viral reactivation / shedding in tears , cultured tg explants and in samples of corneal tissue ( by rt - pcr [ data not shown ] and by pcr techniques , see methods ) from wt hsk recipient mice after grafting . herpes simplex virus negative control samples were prepared from nave , not infected balb / c animals grafted with b6 cornea . no evidence for viral shedding in tear samples was observed nor was virus cultured from tg explants directly ex vivo ( data not shown ) . herpes simplex virus type 1 gh rna ( hsv-1 glycoprotein h ) was detected in tg samples by pcr from hsv - infected , allografted mice at days 3 and 7 post grafting . in contrast , no hsv gh rna was detected at any time point from samples of corneal grafts ( fig . both tg and corneal grafts from not infected , control group were negative for gh pcr product . finally , we examined peripheral t - cell responses to hsv antigen , b6 alloantigen , and to nonrelated ova antigen in allografted hsk recipients and compared the responses to hsv - infected , nongrafted mice . herpes simplex virus type 1 antigen was prepared from protein extracts of hsv - infected vero cells while b6 alloantigen was prepared from c57bl/6 spleen homogenates . lymphocyte ( predominantly t cell ) proliferative responses from individual eye - dln from each mouse were assessed by [ h]-thymidine incorporation ( see methods ) . a variable but robust t - cell response in eye - draining ( submandibular ) lymph node ( dln ) cells from nongrafted hsk mice was detected 3 dpi , which persisted for several weeks ( the duration of the experiment ; fig . a strong anti - hsv lymphocyte t - cell response was detected in allografted hsk mice ( 7 days post transplant , i.e. , 49 dpi ) of similar magnitude to the anti - hsv response 5 dpi in nongrafted hsk mice ( fig . 7b , middle panel ) . importantly , dln cells from allografted hsk recipients failed to respond in the absence of hsv antigen ( fig . syngeneic mice produced a low but variable anti - hsv t - cell response ( p = 0.11 ) . ( a ) lymphocyte proliferation from eye - dln at different times after infection with hsv-1 . note consistent anti - hsv response even at the time - point when clinically the cornea is healed ( i.e. , day 42 dpi = time - point of corneal grafting procedure ) . ( b ) lymphocyte proliferation responses to hsv antigen and b6 alloantigen day 7 post corneal allo- and syngraft ( i.e. , = day 49 pi ) . the response to hsv antigen after allografting procedure ( middle panel ) was equal to the lymphocyte response to hsv antigen 5 dpi in the ungrafted cornea . the difference between the allo- and syngraft response is not significant ( see main text ) . the most recent epidemiologic evidence from the unites states documents an overall incidence of 6.8% per 100,000 person years and a prevalence of 5.7% with an increasing prevalence with age ( 18.8% in people > 75 years ) . these data are not greatly dissimilar from data in previous studies dating back more than 50 years . the most common form of ocular disease in this study was epithelial keratitis ( 72% ) while stromal keratitis ( hsk ) accounted for 36% of cases overall . stromal keratitis is considered to be a significant cause of blindness and is the most common cause of infectious corneal blindness in the united states . global estimates are difficult to obtain but a recent review estimates that hsk causes serious blindness ( visual acuity < 20/200 ) in approximately 1.5% of cases of hsk . blindness in hsk is due to corneal stromal opacification for which the only treatment is corneal transplantation . however , inflammation of the cornea , and particularly hsk , constitutes a high risk for graft failure (  high - risk graft  ) with 10-year survival rates as low as 50% to 60% . the increased risk of graft failure / rejection in hsk recipients has been attributed to corneal vascularization of the host bed . stromal keratitis not only induces blood vessel growth but also induces extensive lymphangiogenesis , which provides the transit for a robust anti - hsv t - cell response initially generated in the eye draining lymph node ( fig . neovascularization from any cause is considered to be an independent risk factor for corneal allograft rejection and this has been experimentally validated in the commonly used suture - induced neovascularization  high - risk  graft rejection model . syngeneic grafts in the suture - injury model are not at risk of rejection and survive normally . the data in the present study confirm the strong association of allograft rejection with neovascularization ( fig . 1 ) . in addition , in the hsk model reported here , graft  rejection  extended to syngeneic grafts and was more akin to graft  failure ,  because it developed very early after grafting ( 3 days ) and was associated with a heavy inflammatory myeloid cell infiltrate ( figs . 1 , 4 ) . herpetic stromal keratitis is a well - recognized cause of graft failure in humans in which hsv antigen has been detected in the stroma and even in the endothelial cells . however , the presence of subclinical inflammation independently of neovascularization has been identified in hsk as a significant predictor of graft failure in humans . in the present experimental study , we also observed that after hsv infection there was an accelerated tempo of allograft rejection that occurred in mice in which neovascularization and opacity were absent ( fig . this effect was lost in cd4 t cell  deficient mice and could be restored when the mice were reconstituted with nave cd4 t cells , although at a slightly slower tempo than wt mice ( fig . in contrast , most syngeneic grafts were accepted in cd4 mice reconstituted with nave t cells ( fig . this indicates that the transferred t cells might require some degree of specificity to induce graft rejection or failure . in the case of hsk - allografts however , in hsk - syngrafts allospecificity is not operational and t - cell mediated graft rejection or failure would probably require adoptive transfer of anti - hsv specific t cells ( figs . in fact , the data in this study provide evidence for a significant anti - hsv response in both hsk - allografts and hsk - syngrafts in that the graft failure / rejection data correlated with the systemic anti - hsv response . following hsv infection of the cornea , a robust anti - hsv t - cell response developed , which was variable but persisted for the duration of the experiment ( 42 days ) . a similar , strong response was observed in the dln of allografted hsk mice and at a significantly lower level in syngrafted hsk mice . in both cases , the anti - hsv response was considerably stronger than the allospecific t - cell response in allografted mice ( fig . a t - cell response is necessary for the development of hsk , and cd4 t cells are particularly important for induction of hsk in mice by the re strain of hsv . the data in this study also implicate the cd4 t - cell response in allograft rejection in hsk mice and point toward a specific anti - hsv response as a major contributor to graft failure / rejection . 1specific cd4 t cells have been detected in hsv - infected human corneas and hsv antigens are present in recipient corneal buttons from patients undergoing corneal graft surgery . given that hsv - infected corneas appear to harbor persistent chronic infection it is likely that at least part of this cell infiltrate will include hsv - specific resident effector memory t cells . the question then arises : does graft in hsk induce activation of latent virus from the tg or does the surgical procedure itself activate locally resident hsv - specific resident effector memory t cells in a bystander fashion ? in the present study we have detected viral dna in tg but not in the corneal graft in hsk recipients . however , given that nerve endings to the graft will be severed during the procedure and are not likely to have regenerated in the immediate post graft period , anterograde passage of reactivated hsv may not be possible and so it is not unexpected that hsv dna was not detected in the graft . however , we noted that there was a significant induction of periocular skin vesicles in allografted mice , which we interpret as reactivation of hsv in the intact skin branches of the tg neurones . furthermore , there was a strong anti - hsv t - cell response associated with allografting hsk recipients , less so in syngeneic recipients , which would be consistent with viral reactivation and increased levels of viral antigen to activate hsv - specific effector memory t cells . in addition , other immune cells , including neutrophils and macrophages , heavily infiltrated rejected grafts . infiltration of neutrophils is a feature of hsk , further suggesting that hsv - driven mechanisms are accelerating graft rejection . in summary , the data in this report are consistent with the concept that three components contribute to allograft rejection in the hsv-1infected corneas , preexisting activated innate immunity ( persistent inflammation and vascularization ) , an hsv-1 specific cd4 t - cell response and a weak allo - response . syngeneic grafts appear to fail when there is a combination of preexisting innate immune response and an hsv-1 specific cd4 t - cell response . however , in the absence of preexisting innate immunity both an hsv-1 cd4 t cell and an allo - specific response are necessary for the graft to fail . these data suggest that both the innate and the adaptive immune responses must be controlled if corneal grafts are to survive in patients with hsk .
purposethe  high - risk phenotype  of corneal graft recipients is considered to be related to preexisting vascularization such as that associated with herpes simplex virus-1 ( hsv-1 ) keratitis ( hsk ) . 
 the purpose of this study was to investigate the immunologic mechanisms underlying accelerated corneal graft rejection using a mouse model of hsk.methodsherpes simplex virus type 1 keratitis was induced in balb / c mice . 
 syngeneic and allogeneic ( c57bl/6 mice ) corneal grafts were performed in mice with hsk at different times after infection . 
 some grafts were performed on hsv - infected cd4 t cell  deficient balb / c mice . 
 clinical , histologic , immunologic , and virus detection studies were performed on samples of cornea , draining lymph node ( ln ) , and trigeminal ganglion ( tg ) cells.resultscorneal grafts in mice with hsk rejected with higher frequency and more rapid tempo compared with grafts in uninfected mice . in corneas with hsk and vascularization at the time of grafting , both syngeneic and allogeneic corneal grafts failed with similar frequency and tempo . however , in the absence of preexisting inflammation and vascularization , syngeneic grafts were accepted when the grafts were performed at a late time point after hsv infection ( 42 days ) , whereas allografts were rejected at this time . in contrast , syngeneic grafts in nonvascularized hsv - infected recipients failed if they were performed within 10 days of hsv infection , an effect that was dependent on cd4 t cells , as demonstrated using cd4 deficient mice . 
 importantly , a variably sustained but strongly positive anti - hsv t - cell response was detected in allografted hsk recipients with a similar but lesser response in syngeneic hosts.conclusionsa previous hsv-1 corneal infection predisposes donor grafts to a high risk of failure by both innate and adaptive immune mechanisms in which an anti - hsv cd4 t - cell response plays a prominent role .