British Journal ofOphthalmology 1992; 76: 602-606

Prolongation of rat corneal graft survival bytreatment with anti-CD4 monoclonal antibody

W Ayliffe, Y Alam, E B Bell, D McLeod, I V Hutchinson

AbstractA ratmodel oforthotopic corneal graft rejectionwas used to investigate the effect of depletionof subpopulations of immune cells by treat-ment with monoclonal antibodies. ThoughCD4+ cells were not eliminated completely byanti-CD4 monoclonal antibodies there was aprofound delay in the rejection times oforthotopic corneal allografts. Furthermore athird of the CD4+ depleted animals failed toreject corneal ailografts by 100 days postgrafting. Despite an almost complete depletionof circulating CD8+ cells, the anti-CD8 anti-body treated animals rejected corneal aliograftsin a similar time course to aliografted controlstreated with a non-reactive control antibodyOX21. These results demonstrate that CD8+T-celis are not required for rejection ofcornealaliografts whereas CD4+ T-celis play a criticalrole in the rejection response. Treatment withanti-CD4 antibodies may have a useful clinicalapplication.(BrJ7 Ophthalmol 1992; 76: 602-606)

University Department ofOphthalmology,Manchester Royal EyeHospital, Oxford Road,ManchesterW AyliffeD McLeod

Immunology Group,Department of Cell andStructural Biology,Stopford Building,University ofManchesterW AyliffeY AlamE B BellI V HutchinsonCorrespondence to:Mr W Ayliffe, FRCSFCOphth, Manchester RoyalEye Hospital, Oxford Road,Manchester M 13 9WH.

Accepted for publication23 April 1992

Human corneal grafts are not rejected in themajority of recipients unlike allografts of mostother organs, and this lower susceptibility torejection is termed 'immunological privilege'.'However privilege is relative and corneal graftsin eyes that have suffered inflammation have ahigh failure rate.2 The main cause of graft failureis immunological rejection3 but the mechanismsof corneal graft rejection are poorly understood.It has been proposed that CD4+ T-helper cells,CD8+ T-cytotoxic cells, and inflammatory cellsare involved in solid organ graft rejection.4Though recognition of foreign antigens via theafferent limb of the immune system by specificT-cells is important in mediating corneal graftfailure' the precise effector mechanism isunknown. There is little direct evidence tosupport the role of cytotoxic CD8+ T-cells as theeffector cells of corneal graft rejection, thoughanti-donor splenic CD8+ T-cells have beengenerated in vivo from corneal grafted animals.6However the association of a particular in vitroimmune response with graft rejection does notnecessarily imply that it is a requirement forrejection.'

It would seem from observations of therejection of other organs that the CD4+ T-cellplays a critical role in allograft rejection, whereasthe CD8+ T-cell does not.8-" Indirect evidencederived from immunohistochemical analysis ofcells infiltrating rejecting corneal allografts inrats failed to identify significant infiltration byCD8+ cells though a heavy infiltration of macro-phages was identified." 2 That CD8+ cells areneither necessary nor sufficient to cause corneal

graft rejection in mice was recently suggested bydepletion experiments in mice.'3 We studied thesurvival of corneal allografts in adult euthymicrats depleted of either CD8+ or CD4+ cells andprolonged by regular monoclonal antibody(mAb) therapy. We found that the rat cornealallograft rejection response was not affected byprofound CD8+ depletion whereas even onlypartial CD4+ depletion led to delayed rejection orindefinite survival.

Materials and methods

ANIMALSInbred male DA (RTJa) and Lewis (RT1') ratsaged 8-14 weeks were obtained from ManchesterUniversity Medical School Animal unit. BALB/cmice aged 6-8 weeks were bred in the sameunit.

CORNEAL TRANSPLANTATIONLewis strain donor rats were killed by cervicaldislocation and 3 mm central corneal discs werescored by trephine. The removal was completedwith corneoscleral scissors and the button,covered by balanced salt solution, was left in situuntil required in order to minimise endothelialdamage caused by excessive manipulation.The technique used throughout the study was

a modification of that described by Coster'4 andallowed retention of a non-irritant single suturewith a buried knot. Recipient DA rats wereanaesthetised with ether and 0-2 ml of diazepamsolution (2 mg/l) was injected intraperitoneally.The right eye was anaesthetised with a drop ofbenoxinate 0-4% before a 3 mm trephine markedthe recipient bed. The button was removed withmicroforceps and corneoscleral scissors. Theprepared donor button was carefullymanipulatedinto the bed and secured with 8-10 bites of acontinuous 10/0 monofilament nylon sutureplaced intrastromally and tied into the woundwith a triplicate knot cut flush to the surface. Asingle drop of chloramphenicol 0 5% wasinstilled.

POSTOPERATIVE MANAGEMENTAnimals were examined on the first postoperativeday; those with surgical failures due to wounddehiscence, iris prolapse, and hyphaema wereexcluded from the study and killed. Theremaining animals received a drop ofchloramphenicol and were thereafter examinedon alternate days. On day 3 postoperatively anyopaque grafts were eliminated from the studybecause they were more likely to representexcessive mechanical damage to the donor

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Prolongation ofrat corneal graft survival by treatment with anti-CD4 monoclonal antibody

material rather than immunological rejection. Afinal drop of chloramphenicol was then instilled.

CLINICAL OBSERVATIONGrafted animals were examined under anaes-thesia on alternate days using a portable slit-lamp. The grafts were graded from 0-4 foropacity, oedema, and vascularity as previouslydescribed."2 The day of rejection was recorded asthat on which the combined score reached 6 ormore.

STATISTICAL ANALYSISThe survival times from transplantation untilrejection were compared using the MannWhitney test.

MONOCLONAL ANTIBODIES (mAb)Ascites fluid from BALB/c mice injected withone of the following monoclonal producinghybridoma cell lines was collected in thislaboratory: anti-CD4 (W3/25, OX35, OX38),anti-CD8 (OX8), anti-alpha/beta T-cell receptor(R73) (a kind gift of Professor Thomas Hunig,Wurzburg). Radial immunodiffusion assayswere used to estimate the immunoglobin (Ig)concentrations. For injection mAbs were dilutedin phosphate buffered saline (PBS) and stored at-70°C in 1 ml aliquots.

CELLULAR DEPLETION IN VIVODA rats aged 10-12 weeks old received intra-peritoneal injections of 1 ml of ascites containing6 mg of OX8 (anti-CD8) mAb three times aweek. A second group was treated three times aweek with 1 ml of a cocktail containing two non-competing anti-CD4 mAbs, OX35 (0 75 mg) andOX38 (2 5 mg) which deplete CD4+ T-cells.'sThe third group was injected three times a weekwith 1 ml of a non-reactive control mAb OX21(anti-human C3b) that does not react with ratantigens. Treatment was continued for 48 days.On day 16 after the initial injection of mAb theanimals received a penetrating allogeneic Lewiscorneal graft into the right eye.

MONITORING OF DEPLETIONThe ability of the antibody injections to depletecirculating cells was monitored at 7-10 dayintervals by flow cytometry. Blood samples of 1ml were obtained from the tail vein of each CD8-and CD4- depleted rat. After separation ofleucocytes on a Ficoll density gradient (specificgravity 1 083), cells were washed and surfacephenotyped. Leucocytes from the anti-CD8treated rats were stained using a primary mouseanti-CD8 mAb (OX8) followed by fluoresceinconjugated FITC-F(ab')2 rabbit anti-mouse IgG,which fluoresces green (Dako Ltd, HighWycombe, UK), containing 1% normal ratserum to block anti-rat Ig cross-reactivity.Leucocytes prepared from the anti-CD4 treatedrats were double stained using a five-stagestaining protocol (1) mAb R73 (anti-T-cellreceptor); (2) FITC-F(ab')2 anti-mouse IgG in1% normal rat serum; (3) blocked with W6/32

(mouse mAb non-reactive with rat); (4)biotinylated anti-CD4 mAb (b-W3/25); (5)phycoerythrin-streptavidin, which fluorescesred. Cells were held on ice and washed at eachstage with PBS containing 2% fetal calf serumplus 0-02 M sodium azide to prevent capping ofsurface molecules, and finally fixed in 1%formaldehyde for flow cytometric analysis usingthe Beckton Dickinson FACS cell sorter. Usingan optical to electronic coupling system, the flowcytometer records how the cell interacts with alaser beam in terms of the ability of the cell toscatter the incident light and to emit fluorescence.Dead cells and cell debris were excluded byelectronic gating on forward and side angle lightscatter. Analysis of 101 events was performedusing the Consort 30 program.

IMMUNOHISTOCHEMISTRYCorneoscleral buttons were removed andimmediately frozen in liquid nitrogen. Serialsections of 5 ,um thickness were cut across thecentral area of the graft and stained using anindirect immunoperoxidase technique. Normalrabbit serum was added to the sections to blocknon-specific antibody binding prior to mAbincubation. A panel of mouse mAbs (producedas supernatants ofhybridoma cell cultures in thislaboratory) was used: W3/25, OX8, R73, OX1(anti-CD45, the leucocyte common antigen) orOX42 (anti-macrophage). Rabbit anti-mouseperoxidase conjugated secondary antibody(Dako Ltd) was added before development indiaminobenzidine and hydrogen peroxide andslides were counterstained with 50%haematoxylin solution.A non-reactive culture supernatant (OX2 1)

was applied to sections as a negative control andsections of normal rat spleen were used as apositive control.

Slides were examined by the same observer ina masked fashion on three separate occasions.Cell numbers were estimated in the mid stromaof the centre of the graft using a x 40 powerobjective and x 12 5 eye piece graticule.

Results

CELL DEPLETION STUDIES IN VIVO

Depletion ofCD4+ cellsCD4+ T-cells are the majority of circulating T-cells in DA rats comprising 36-3 (SD 3 4)% ofcirculating leucocytes in untreated animals (FigIA). Monoclonal antibody therapy reduced thisto 13-6 (SD 2 8)% within a week but furtherdepletion did not occur in the majority ofanimals, despite continuing antibody treatment,until day 51 when a sudden dramatic decline incell numbers to 4 9 (SD 1-0)% occurred (Table1A). However two animals demonstratedvirtually complete depletion ofCD4+ cells by day14 and double staining demonstrated that mostof the circulating T cells were CD8+ at this stage.

Depletion ofCD8+ cellsCD8+ T-cells comprise 23-9 (SD 0 7)% of the

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Ayliffe, Alam, Bell, McLeod, Hutchinson

A Day 0

B Day 0

25-1%

Day 14

TCR

Day 7

03%

Day 51

Day 40

CD8

Figure 1 Flow cytometry profiles ofperipheral blood leucocytes preparedfrom a DA rtreatment (day 0) and during treatment with mAb therapy. (A) Flow cytometry profilesperipheral blood leucocytes ofaDA rat double stainedfor the T-cell receptor, TCR, (vmAb, fluorescing green, x axis) and CD4 (with W3125 mAb, fluorescing red, y axis). Idistinct populations ofcells were identified on the basis ofthe intensity offluorescence, 2proportional to the density ofeach ofthe expressed cell surface antigens that were markocells (TCR +)fluorescing strongly in the green are located in quadrants 2 and 4. CD4+fluorescing red are located in quadrants I and 2. The double stained CD4+ T-cells arerepresented in quadrant 2 and comprise 38-9% ofthe peripheral blood leucocytes in the urat (day 0). Regular mAb therapy depleted the CD4+ T-cells to 14% on day 14 and 3-day 51. (B) Flow cytometry profiles ofleucocytesfrom a DA rat single stainedfor CThOX8 mAb). The number ofcells (y axis) is plotted against the amount offluorescence aaxis with a vertical line drawn at the point which no negatively stained cells were foun,non-staining cells are to the left of this line and cells stainingfor CD8 are found to the rpercentage ofthese positively staining cells is indicated on the diagram and shows CD8comprising 25 1% ofthe circulating leucocytes before anti-CD8 treatment (day 0) andcomplete depletion ofCD8+ cells on days 7 with a small recovery by day 40.

peripheral blood leucocytes in normal I(Fig IB). Thrice weekly monoclonal artherapy reduced this to 0-7 (+0*2)%week, but cell numbers recovered to rec(SD 2 9)% by day 40 despite continuiniment (Table iB).

CORNEAL ALLOGRAFT SURVIVAL IN DEPLET]RECIPIENTS

Untreated allograft recipientsOf 11 DA rats bearing Lewis allografttechnical failures were excluded from theAll the remaining eight grafts were rbetween 7 and 21 (median 12) days.

Control (OX21) treated recipientsOne graft was a technical failure and thewas killed. The other six rats rejected theiigraft between 7 and 21 (median 12) days a

Table 1 Depletion ofCD4+ and CD8+ cells in peripheral blood in mAb treated, conallografted DA rats

The percentage of blood leucocytes that stained were either CD4' or CD8' is shown against the day oftreatment. The day of the first treatment is day 0 and the rats were grafted on day 16.

difference compared to the survival times of2 grafts in injected recipients (Fig 2A).

32%

Anti-CD8 (OX8) treated recipientsThe six allografts were rejected between 7 and 27

4 days with a median rejection time of 12 days.20.4% There was no difference in the rejection times

compared with graft rejection in the OX2 1treated controls (Fig 2B).

Anti-CD4 (OX351OX38) treated recipientsSix technically successful allografts were followed

3.5% for 100 days. There was a significant delay inrejection times compared with the OX8 or OX2 1treated recipients (p 60-cells >~50-intreated 0 40-2% on?(with 30m the x 20 -d. Thus ..right, the 10 -V cells o .... ..,., ..lalmost 0 5 10 15 20 25 30 35 40 45 50

10090 - B

)A rats 80-atibody 70-after 1 , 60-ach 8-0 >g treat- 0 40

30 -

20 - .. ,ED 10-

0 .... .....0 5 10 15 20 25 30 35 40 45 50

100 ..........

:s three 90 Cstudy. 80..

-ejected 70

60

50 ..................50-.4-0

animal 30............

r Lewis 20with no 10 X

0 .. .. ..0 5 10 15 20 25 30 35 40 45 50

Days post graftFigure 2 (A) Survival ofallografted corneas in DA ratsuntreated or treated with non-reactive mAb OX21 (solid line)compared with allografts in untreated control recipients (dottedline). (B) The % survival ofcorneal allografts in anti-CD8treated recipients (dotted line) compared with cornealallografis in control (OX21) treated recipients (solid line). (C)The % survival ofcorneal allografts in anti-CD4 treated rats(dotted line) compared with allografts in control (OX21)treated recipients (solid line).

,0a

L-oq)

Ec

0

(A) The means (standard errors) of CD4+ staining cells in the anti-CD4 treated animals.Day of treatment: 0 7 14 23 37 51% ofCD4' cells: 36-3(3-4) 13 6(2-8) 10-8(2-4) 10-2(1 0) 7-5 (0 6) 4-9 (1-0)(B) The means (standard errors) ofCD8' cells in the anti-CD8 treated animals.Day of treatment: 0 8 14 22 40% of CD8' cells 23-9 (0-7) 0 7 (0 2) 3-5 (0 6) 6-2 (1-1) 8-0 (2 9)

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Prolongation ofrat corneal graft survival by treatment with anti-CD4 monoclonal antibody

r

Figure 3 (A)Vascularised, opaquerejected Lewis cornealallograft in an untreatedcontrolDA recipient. (B)Anon-rejected long termsurviving clear Lewiscorneal allograft in a DArecipient depleted ofCD4'cells by regular mAbtherapy. .. i. l

Fig3A

There was no correlation between the amount ofCD4+ cell depletion at the time of grafting andthe eventual graft survival.

Rejected allografts became opaque andvascularised and, though after several weeks theopacity partially cleared, the grafts remainedvascularised (Fig 3A). In contrast, the long termsurviving grafts remained clear and thoughvessels grew up to the wound they did not growonto the graft (Fig 3B).

Figure 4Immunoperoxidase stainingwith mAb OX42 in a sectionfrom the centre ofa rejectedgraft in the anti-CD8depleted group showingheavy infiltration ofmacrophages.

Histology ofgraftsSyngeneic grafts had low numbers of infiltratingleucocytes staining for the leucocyte commonantigen CD45 (OX1) and most of these appearedto be macrophages marked by OX42. Rejectedgrafts in the untreated, CD4+ depleted, andCD8+ depleted animals had a heavy infiltrationof macrophages staining with OX42 (Fig 4) butall showed low numbers of T-cells and very fewCD8 (OX8+) cells present. There was a lessheavy infiltration of macrophages in rejectedgrafts from the CD4-depleted animals comparedwith the untreated controls or CD8-depletedanimals.

DiscussionThe mechanism of destruction of allogeneic

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tissue is unknown4 and this applies to cornealgrafts. Although T-cells are required for theafferent limb of the corneal allograft response,5the precise effector mechanism of corneal graftrejection remains an enigma. Using selectedmAbs and frequent administration it was possibleto deplete T-cell subsets and this enabled anexamination of their roles in corneal allograftrejection in vivo.CD8+ cells are important effector cells in

immune responses, particularly viral infectionsand depletion of cells by anti-CD8 mAbs hasbeen shown to modify the outcome of viralencephalitis.'3 They have also been presumed tobe important in corneal allograft rejection;indeed, in vitro evidence using spleen cellsobtained from animals with rejected cornealallografts implicates the cytotoxic CD8+ T-cell asthe major effector cell.6 However the results ofthe present study suggest that this may notreflect the mechanism of corneal allograftrejection in vivo because animals depleted ofCD8+ cells rejected corneal allografts in the sametempo as controls. This result, although initiallysurprising, is in fact in keeping with otherexperimental models where depletion of CD8+cells failed to modify allograft rejection in skinand heart allograft models.7 161 7 Despite 96-100% depletion of circulating CD8+ cells at thetime of grafting we also failed to influencecorneal graft rejection.

In contrast, results presented in this studyshow that pretreatment ofDA rat recipients for 2weeks with murine anti-CD4 mAbs led tosignificantly prolonged corneal allograft survivalor even tolerance.

Depletion of CD4+ cells is notoriously dif-ficult'5 and different anti-CD4 mAbs vary intheir ability to deplete. The mAb W3/25 reactswith CD4 but fails to deplete, whereas a cocktailof OX35 and OX38 depletes CD4+ cells veryeffectively after 6 weeks of treatment.'5 Onedifficulty has been that the CD4 molecule isexpressed on other cells such as macrophages aswell as on T-cells.'" In the present investigationthe depletion of CD4 T-cells was determined bydouble staining for CD4 and the alpha-betaTCR. Depletion of CD4+ T-cells from 36% toY10% of peripheral blood lymphocytes cor-related with a delayed rejection of corneal

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allografts in this group. It may be that graftingafter 6 weeks of treatment when greaterdepletion of CD4+ cells is obtained may allowbetter survival of corneal allografts and this isbeing investigated.

If the CD8+ cytotoxic T-cell is not required forallograft rejection in vivo then several optionalcandidates remain. The possibilities that anti-bodies,'9 natural killer cells,20 or macrophages" 12are involved in the effector mechanism of cornealgraft rejection have received some attention. It isalso possible that cytotoxic CD4+ T-cells areinvolved but there is no direct evidence for this.Their low numbers in the rejecting corneal graftsand the overwhelming infiltration of macro-phages even in CD4+ and CD8+ depletedrecipients suggest that the latter may in fact playthe major role in tissue damage, in a localdelayed-type hypersensitivity reaction.2' Therole of the CD4+ T-cells is more likely to beconcerned with the recognition of alloantigenpresented on antigen presenting cells whichexplains the specificity of the rejection response.Antigen presentation may occur locally in thelimbus and ocular Langerhans cells have beenshown to be capable of presenting antigens toT-cells.22 Cytokine release by the alloreactive T-cells may activate effector cells, possibly macro-phages, locally within the graft leading to graftdamage and opacity. Failure to recognise allo-antigen in syngeneic grafts or depletion of CD4+cells by mAb does not lead to activation of theeffector mechanisms and the corneal graftsremain clear.As well as increasing understanding of corneal

allograft rejection this study suggests that treat-ment of high risk recipients with anti-CD4antibodies may have important clinicalapplications.

We thank the Royal National Institute for the Blind and the NorthWestern Regional Health Authority for grant support for thisstudy.

1 Niederkorn JY. Immune privilege and immune regulation inthe eye. Adv Immunol 1990; 48: 191-226.

2 Coster DJ. Mechanisms of corneal graft failure: the erosion ofcorneal privilege. Eye 1989; 3: 251-62.

3 Khodadoust AA. The allograft reaction: the leading cause oflate failure of clinical corneal grafts. Ciba FoundationSymposium 1973; 15: 151-67.

4 Dallman M, Morris PJ. The immunology of rejection. In:Morris PJ, ed. Kidney transplantation: principals and practice.Philadelphia: Saunders, 1988: 15-36.

5 Maumenee AE. The influence of donor recipient sensitisationon corneal grafts. AmJ Ophthalmol 1951; 34: 142-52.

6 Peeler JS, Niederkorn JY, Matoba AY. Corneal allograftsinduce cytotoxic T cell but not delayed type hypersensitivityresponses in mice. Invest Ophthalmol Vis Sci 1985; 26: 1516.

7 Hall BM, Dorsch S, Roser BJ. The cellular basis of allograftrejection in vivo.3J Exp Med 1978; 148: 878-89.

8 Dallman MJ, Mason DW, Webb M. The role of host anddonor cells in the rejection of skin allografts by T cell-deprived rats injected with syngeneic T cells. EurJ7 Immunol1982; 12: 511-18.

9 Bolton EM, Gracie JA, Briggs JD, Kampinga J, Bradley JA.Cellular requirements of renal allograft rejection in theathymic nude rat. JExp Med 1989; 169: 1931-46.

10 Whitby EH, Sparshott SM, Bell EB. Allograft rejection inathymic nude rats by transferred T-cell subsets. I. Theresponse ofnaive CD4+ and CD8+ thoracic duct lymphocytesto complete allogeneic incompatibilities. Immunology 1990;69: 78-84.

11 Brown R, Ayliffe W, Alam Y, McLeod D, Hutchinson IV.The effect of predsol on cells infiltrating rat cornealallografts. Invest Ophthalmol Vis Sci 1991; 32: Suppl 2209.

12 Holland EJ, Chi-Chao Chan, Wetzig P, Palestine AG,Nussenblatt RB. Clinical and immunohistologic studies ofcorneal rejection in the rat penetrating keratoplasty model.Cornea 1991; 5: 374-7.

13 He YuGuang, Ross J, Niederkorn JY. Promotion of murineorthotopic corneal allograft survival by systemic admini-stration of anti-CD4 mAb. Invest Ophthalmol Vis Sci 1991;32: 2723-8.

14 Williams KA, Coster DJ. Penetrating corneal transplantationin the inbred rat: a new model. Invest Ophthalmol Vis Sci1985;27: 1199-204.

15 Roser BJ. Cellular mechanisms in neonatal and adult tolerance.Immunol Rev 1989; 107: 179-202.

16 Sedgwick JD. Long term depletion of CD8+ T cells in vivo inthe rat. Eurl Immunol 1988; 18: 495-502.

17 Bradley JA, Sarawar SR, Porteous C, Wood PJ, Card S, AgerA, et al. Allograft rejection in CD4 T cell reconstitutedathymic nude rats: the non-essential role of host derivedCD8+ cells. Transplant (in press).

18 Jefferies WA, Green JR, Williams AF. Authentic T helperCD4 (W3.25) antigen on rat peritoneal macrophages. J ExpMed 1985; 162: 117-27.

19 Grunnet N, Kirstensen T, Kissmeyer Neilsen F, Ehlers N.Occurrence oflymphocytotoxic lymphocytes and antibodiesafter corneal transplantation. Acta Ophthalmol (Kbh) 1976;54: 167-73.

20 Opremcak EM, Whistler RL, Dangle ME. Natural killer cellsagainst the corneal endothelium. AmJ Ophthalmol 1985; 99:524-9.

21 Gebhardt BM. Cell mediated immunity in the cornea.Transplantation 1988; 46: 273-80.

22 Williams KA, Ash JK, Mann TS, Coster DJ. Antigen-presenting capabilities of cells infiltrating inflamed corneas.Transplant Proc 1987; 19: 255-6.

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