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Background: Regulatory T (Treg) cell-based immunotherapies have been studied as potential cell-based modalities for promoting transplant survival. However, the efficacy of local delivery of Treg cells in corneal transplantation has not been fully elucidated. Herein, we investigated the kinetics of migration of subconjunctivally injected Treg cells and their role in promoting corneal allograft survival., Methods: GFPCD4CD25Foxp3 Treg cells were isolated from draining lymph nodes (DLNs) of GFP transgenic mice and were subconjunctivally injected to corneal allograft recipients. Next, Treg cells, conventional T cells (Tconv) or a combination of both was locally injected to graft recipients, and graft survival was determined by evaluating opacity scores for 10 weeks. Transplanted mice without treatment served as controls. The frequencies of major histocompatibility complex-IICD11b antigen-presenting cells, IFNγCD4 Th1 cells, and CD45 cells in the DLNs and cornea were evaluated at week 2 posttransplantation using flow cytometry. Expressions of IFNγ, IL-10 and TGF-β in the grafts were assessed using reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay., Results: GFP Treg cells were detected in the ipsilateral cornea and DLNs of recipients 6 hours after injection. Subconjunctival injection of Treg cells significantly decreased the frequencies of mature antigen-presenting cells in the graft and DLNs, suppressed Th1 frequencies in DLNs, and inhibited CD45 cell infiltration to the graft. Finally, locally delivered Treg cells significantly reduced the expression of IFN-γ, enhanced the levels of IL-10 and TGF-β in the graft, and promoted long-term allograft survival., Conclusions: Our study elucidates the kinetics of migration of locally delivered Treg cells and shows their role in suppressing host immune response against the allograft.

Background: Corneal neovascularization increases the risk of T cell-mediated allograft rejection. Here, we investigate whether T cells promote angiogenesis in transplantation., Methods: Conventional effector T cells were collected from draining lymph nodes of allogeneic or syngeneic corneal transplanted BALB/c mice. T cells were either cocultured with vascular endothelial cells (VECs) to assess VEC proliferation or used in a mixed lymphocyte reaction assay. Messenger RNA (mRNA) expression of vascular endothelial growth factor (VEGF)-A, -C, and VEGF receptor 2 (VEGF-R2) in VECs was assessed by real-time PCR. VEGF-A protein expression was determined by enzyme-linked immunosorbent assay. Flow cytometry was used to analyze VEGF-R2 expression in corneal CD31 cells, and VEGF-A and IFNγ expression in corneal CD4 T cells., Results: Allogeneic T cells from high-risk (HR) grafted mice induced more VEC proliferation than those from syngeneic transplant recipients (P = 0.03). Vascular endothelial growth factor-A mRNA and protein expression were higher in T cells from draining lymph nodes (P = 0.03 and P = 0.04, respectively) and cornea (protein; P = 0.04) of HR compared with low-risk (LR) grafted hosts. Vascular endothelial growth factor-A, VEGF-C, and VEGF-R2 mRNA expression were increased in VECs when cocultured with T cells from HR transplants compared with LR transplants and naive mice. In addition, IFNγ blockade in T cell/VEC coculture increased VEC proliferation and VEGF-A protein expression, whereas blocking VEGF-A significantly reduced VEC proliferation (P = 0.04)., Conclusions: Allogeneic T cells from corneal transplant hosts promote VEC proliferation, probably via VEGF-A signaling, whereas IFNγ shows an antiangiogenic effect. Our data suggest that T cells are critical mediators of angiogenesis in transplantation.

Background: Immune rejection continues to threaten all tissue transplants. Here we sought to determine whether platelet (P)- and endothelial (E)-selectin mediate T cell recruitment in corneal transplantation and whether their blockade can reduce T cell graft infiltration and improve long-term corneal allograft survival., Methods: In a murine model of allogeneic corneal transplantation, we used PCR and immunohistochemistry to investigate expression of P- and E-selectin in rejected versus accepted allografts and lymph node flow cytometry to assess expression of selectin ligands by effector T cells. Using P- and E-selectin neutralizing antibodies, we evaluated the effect of blockade on CD4 T cell recruitment, as well as the effect of anti-E-selectin on long-term allograft survival., Results: The P- (93.3-fold, P < 0.05) and E-selectin (17.1-fold, P < 0.005) are upregulated in rejected versus accepted allogeneic transplants. Type 1 T helper cells from hosts with accepted and rejected grafts express high levels of P-selectin glycoprotein ligand 1 and glycosylated CD43. In vivo blockade of P (0.47 ± 0.03, P < 0.05) and E selectin (0.49 ± 0.1, P < 0.05) reduced the number of recruited T cells compared with IgG control (0.98 ± 0.1). Anti-E-selectin reduced the number of mature antigen-presenting cells trafficking to lymphoid tissue compared with control (6.96 ± 0.9 vs 12.67 ± 0.5, P < 0.05). Anti-E-selectin treatment delayed graft rejection and increased survival compared with control, although this difference did not reach statistical significance., Conclusions: In a model of corneal transplantation, P- and E-selectin mediate T cell recruitment to the graft, E-selectin mediates APC trafficking to lymphoid tissue, and blockade of E-selectin has a modest effect on improving long-term graft survival.

Background: Corneal allograft survival dramatically decreases in hosts with inflamed or vascularized recipient beds. We have previously shown that in rejected corneal allografts regulatory T cells (Treg) demonstrate diminished Foxp3 expression and immunoregulatory function. Treatment with low doses of IL-2 selectively expands Treg and has been proposed for the treatment of autoimmune diseases. In this study, we investigated the effect of low-dose IL-2 administration on Treg function and corneal allograft survival., Methods: Allogeneic corneal transplantation was performed on inflamed host beds. Low-dose systemic IL-2 was administered starting 3 days before grafting until 6 weeks after transplantation. Frequencies of Treg and their immunosuppressive function and antigen specificity were assessed using flow cytometry, in vitro proliferation assays, and adoptive transfer experiments. Frequencies of effector T cells (Teff) and graft infiltrating immune cells were measured at 2 weeks posttransplantation. Long-term allograft survival was evaluated for up to 9 weeks using Kaplan-Meier survival analysis., Results: Treatment with low-dose IL-2 significantly increased frequencies of CD4CD25Foxp3 Treg and their immunosuppressive function. It also suppressed alloimmune response as shown by the decreased CD4 IFNγ T cell frequencies and graft infiltration of CD45 and CD4 cells. Clinical evaluation of the grafts showed significant improvement in long-term corneal allograft survival in the IL-2 treated group compared with controls., Conclusions: Our study is the first to report that treatment with low-dose IL-2 increases survival of corneal allografts. We propose that IL-2-mediated Treg expansion can be an effective tool to prevent alloimmunity and to improve long-term allograft survival in transplantation.

Background: The survival rate of corneal allografts in high-risk vascularized corneal bed recipients is poor, similar to vascularized solid organ allografts. Although the early induction of selective chemokines in solid organs is required for the optimal recruitment of T cells into rejecting allografts, little is known about the role of these chemokines in high risk corneal allografts., Methods: Orthotopic corneal allotransplants were performed in low-risk (nonvascularized) and high-risk (vascularized) C57BL/6 (H-2b) recipients using Balb/c (H-2d) donors. Intragraft production of CXC chemokines was measured by Luminex and enzyme-linked immunosorbent assay on corneal transplant extracts at different times after surgery. Rabbit anti-KC serum was used to test its role in high risk corneal allograft survival., Results: Early upregulation of CXCL1/KC occurs 3 days after transplantation in high risk allograft only. Moreover, the T-cell chemoattractants, CXCL9/Mig and CXCL10/IP10, are produced late (day 10) after surgery and their production correlates with the recruitment of CD4 T cells into the graft. Furthermore, in vivo neutralization of CXCL1/KC with anti-KC sera results in increased graft survival and decreased recruitment of T cells into high-risk allografts., Conclusion: We propose that a high risk vascularized cornea behaves like a vascularized solid organ transplant. The early production of CXCL1/KC is crucial to the induction of T-cell chemoattractants necessary for the recruitment of allospecific CD4 T cells into the graft. In vivo neutralization of CXCL1/KC represents a potential novel therapy that could be used to increase the survival rate of high-risk vascularized corneal allografts.

Corneal allotransplantation is highly successful in the short term, but much less successful in the longer term. Many corneal grafts in recipients with corneal neovascularization or the sequelae of ocular inflammation undergo irreversible rejection, despite topical immunosuppression with glucocorticosteroids. Sensitization to cornea-derived alloantigen proceeds by both direct and indirect routes, but the anatomic location of sensitization remains unclear. Multiple and redundant mechanisms operate in the effector phase of corneal graft rejection, which is largely cell-mediated rather than antibody-mediated. Human leukocyte antigen matching may improve outcomes in high-risk patients but systemic immunosuppression is frequently ineffective and is seldom used.

Background: Allogeneic rejection is the most common cause of corneal graft failure. The aim of this work was to establish the kinetics of cytokine and chemokine mRNA expression before and after onset of corneal graft rejection., Methods: Intracorneal cytokine and chemokine mRNA levels were investigated in the Brown Norway-->Lewis inbred rat model in which rejection onset is observed at 8/9 days after grafting in all animals. Nongrafted corneas and syngeneic (Lewis-->Lewis) corneal transplants were used as controls. Donor and recipient cornea was examined by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) for hypoxyanthine phosphoribosyltransferase (HPRT), CD3, CD25, interleukin (IL)-1beta, IL-1RA, IL2, IL-6, IL-10, interferon-gamma (IFN-gamma), tumor necrosis factor (TNF), transforming growth factor (TGF)-beta1, and macrophage inflammatory protein (MIP)-II and by nonquantitative RT-PCR for IL4, IL-5, IL-12 p40, IL-13, TGF-beta2, monocyte chemotactic protein-1 (MCP-1), MIP-1alpha, MIP-1beta, and RANTES (for regulated upon activation normal T cell expressed and secreted)., Results: A biphasic expression of cytokine and chemokine mRNA was found after transplantation. During the early phase (days 3-9), there was an elevation of the majority of the cytokines examined, including IL-1beta, IL-6, IL-10, IL-12 p40, and MIP-II. There was no difference in cytokine expression patterns between allogeneic or syngeneic recipients at this time. In syngeneic recipients, cytokine levels reduced to pretransplant levels by day 13, whereas levels of all cytokines rose after observed rejection onset in the allografts, including TGF-beta1, TGF-beta2, and IL-1RA. The T cell-derived cytokines IL-4, IL-13, and IFN-gamma were detected only during the rejection phase in allogeneic recipients., Conclusions: There is an early cytokine and chemokine response to the transplantation process, evident in syngeneic and allogeneic grafts, that probably drives angiogenesis, leukocyte recruitment, and affects leukocyte functions. After an immune response has been generated, allogeneic rejection results in the expression of Th1 cytokines (IL-2, IL-12 p40, IFN-gamma), Th2 cytokines (IL-4, IL-6, IL-10, and IL-13), and antiinflammatory/Th3 cytokines (TGF-beta1/2 and IL-1RA).

Background: In this study, we determined the binding characteristics of F(ab')2 alloantibody fragments to corneal antigens and assessed the capacity of these antibody fragments to protect corneal allografts from immune attack., Methods: Goat anti-rabbit alloantibodies were pepsin-digested and labeled with 125I, and the time course of association and dissociation of the F(ab')2 fragments was determined. Corneal allografts were incubated in unlabeled F(ab')2 fragments and transplanted into allogeneic recipients, and the graft survival times were recorded., Results: Binding of radiolabeled F(ab')2 fragments to rabbit cornea cells reached a maximum at 12 hr. At 32 degrees C (rabbit corneal temperature), the radiolabel eluted rapidly from the cornea, reaching baseline at 72 hr. At 4 degrees C (corneal graft storage temperature), significant amounts remained associated with the cornea at 96 hr. Mean survival time for grafts incubated in F(ab')2 anti-rabbit fragments was significantly greater than that of grafts incubated in nonimmune F(ab')2 fragments. Three of the corneal allografts incubated in goat F(ab')2 anti-rabbit fragments survived for 100 days, whereas the longest surviving control allograft incubated in goat F(ab')2 nonimmune fragments was rejected on day 24. Preincubation of corneas in unlabeled, immune F(ab')2 fragments followed by incubation in radiolabeled, immune F(ab')2 fragments suggested that antigen masking was not a factor in the prolongation of graft survival., Conclusion: Based on the binding and release kinetics and the graft survival times, it appears that the protective effect of immune F(ab')2 fragments extends well beyond the binding interval of the antibody fragments to corneal cell membranes.

The present study examined the potential of orally induced tolerance for preventing immunological rejection of corneal allografts. Orthotopic corneal allografts were transplanted from either C3H (MHC + multiple minor H-mismatched) or NZB (multiple minor H-mismatched only) donors to CB6F1 recipients on day 0. Tissue cultured corneal epithelial and endothelial cells from relevant donor strains were administered orally from day -14 to day -4 on a daily basis, The incidence of graft rejection, graft mean survival time (MST), and alloimmune responses, and the antigen specificity of induced tolerance were studied. Oral immunization induced a remarkable tolerance such that only 55% of the orally immunized hosts rejected their fully allogeneic corneal grafts (MST = 43 days) compared with 100% rejection (MST = 18 days) in normal controls. Likewise, rejection of MHC-matched, multiple minor H-mismatched corneal grafts fell from 80% in untreated controls to 36% in orally immunized hosts. Oral immunization was effective in desensitizing previously immunized hosts. Rejection of MHC-matched, multiple H minor-mismatched corneal allografts fell from 93% in preimmune, unfed hosts to 36% in preimmune, orally tolerized mice. Thus, oral immunization is a safe and effective method for desensitizing high-risk, preimmune hosts and promoting corneal allograft survival.

A model of murine heterotopic allogeneic transplantation was used to study the rejection characteristics of three tissues--adult cornea, fetal pancreas, and fetal skin--for attributes that might explain their variation in rejection rates and help define the determinants of graft immunogenicity. Under identical conditions, tissues were transplanted to the renal subcapsular space and their base-line rejection rates compared. The expression of MHC class I and II and intercellular adhesion molecule-1 (ICAM-1), was determined for each tissue, as was their ability to produce interleukin-6, IL-3, interferon-gamma, and granulocyte-macrophage colony-stimulating factor in vitro. These studies were performed under basal conditions and after stimulation with concanavalin A-stimulated spleen cell supernatant (CAS) or INF gamma. Corneal grafts had a slow rejection rate compared with pancreas and skin. While all three tissues had low basal expression of MHC class II, both fetal skin and pancreas, but not adult cornea, were able to increase this under our experimental conditions. Pancreas and skin produced IL-6 under basal conditions and could be stimulated to increase production 2-3-fold but the cornea did not basally produce IL-6 and showed minimal upregulation. We postulate that delayed corneal rejection, compared with pancreas and skin, results from two compounding deficiencies: the relative lack of class II MHC-positive APC and the inability to overcome this deficiency by upregulating class II expression and producing accessory molecules for antigen presentation.

Corneal grafts are more likely to be rejected when placed in a vascularized rather than in a normal host cornea. Using immunohistochemical techniques, normal rabbit cornea was found to contain measurable numbers of cells of hemopoietic origin, probably of either macrophage or dendritic lineage. After the deliberate induction of corneal inflammation and neovascularization, the number of these accessory cells was found to increase significantly. There was also a marked increase in the number of T cells present. Enzyme staining indicated a degree of heterogeneity in the infiltrate. The process of rejection of rabbit corneal grafts was found to occur earlier when additional infiltrating cells were present in either donor button or graft bed, and earlier still when the load of infiltrating cells was increased in both donor and recipient. We hypothesize that resident accessory cells of recipient origin may be implicated in graft rejection in vascularized, inflamed corneas.

The well-characterized immunogenetic constitution of the rat has made it a valuable resource for the study of corneal transplantation. However, little is known about the expression of histocompatibility antigens in the rat cornea or about the populations of passenger leukocytes rat corneal grafts may contain. Thus the relevance of rat corneal transplantation to the human situation is unclear. We examined the expression of antigens of the histocompatibility complex (MHC) of the rat (RT1) and of antigens identifying rat leukocyte populations in corneas of selected rat strains using a sensitive immunoperoxidase technique. Class I MHC antigens were found in fairly uniform association with most cells of the rat corneal epithelium, endothelium, and stroma. In contrast, class II MHC antigens were not associated with most corneal parenchymal cells, but were clearly present on multiple isolated dendritic cells throughout the corneal stroma, especially near the limbus, and on very rare isolated cells in the limbal epithelium; these antigens were not detectable on the corneal endothelium. Cells expressing an antigen common to all rat leukocytes were present in a distribution similar to that of class II MHC antigen-bearing cells. There was no consistent expression of antigens associated with T lymphocytes, macrophages, neutrophils, or NK cells. It thus appears that the expression of MHC and leukocyte antigens by cells in the rat cornea is similar to that reported in the human, indicating that the rat may indeed provide a good model of human corneal transplantation.

Although the immune nature of corneal allograft rejection has been recognized for over thirty years, the specific mechanisms involved in such reactions remain obscure. We investigated the cellular immune responses of PVG (RT1c) rats that were grafted with fully allogeneic ACI (RT1a) skin, ACI cornea, or PVG cornea to the chest wall or given sham grafts. Cell-mediated lymphocytotoxicity (CML) was tested at 10 days posttransplant by placing recipient spleen cells in culture with irradiated ACI stimulator cells, and six days later measuring specific lysis of 51Cr-labeled target lymphoblasts at several effector-to-target ratios. Effector cells from animals receiving allogeneic skin or cornea grafts lysed targets from the donor (ACI) strain at levels significantly (P less than 0.01) above those obtained using effectors from control (sham grafted or syngeneic corneal graft recipient) animals. Significant lysis was also seen using target cells from PVG.1A (RT1a) or PVG.R1 (RT1r1) congenic rats, which differ from recipients only at the RT1 complex and the RT1.A (class I antigen) region, respectively. Stimulator cells from PVG.1A and PVG.R1 animals also permitted detection of specific responses in secondary CML, but syngeneic PVG stimulators did not, indicating that in vitro restimulation of effector cells can be met by using stimulator cells bearing only allogeneic class I major histocompatibility complex (MHC) antigens. These results indicate that corneal allografts evoke specific cellular immune responses in the rat, and that class I MHC antigens act as effective targets for these responses.

The purpose of this investigation was to compare the immunogenicity of corneal stromal keratocytes with that of splenic lymphocytes. The corneal stromal keratocytes of two inbred strains of rats were propagated in tissue culture in sufficient numbers to immunize allogeneic recipients and to determine the numbers of these cells required to elicit an allogenic immune response in the recipient. At least 30 x 10(6) tissue culture-propagated keratocytes injected intraperitoneally were required to elicit an alloantigenic response in the recipient strain. This alloantigenic response was measured by determining both the titer and specificity of serum alloantibody and by assessing the cellular immune response generated in the alloimmunized recipients. The results indicate that corneal stromal keratocytes can elicit both an antibody-mediated and a cell-mediated immune response, as indicated by the presence of serum antibody and cytotoxic lymphocytes in immunized recipients. The alloantibody and cellular immune responses were directed against the class I histocompatibility antigens of the immunizing strain. Class II antigens were not detected on the cultured keratocytes.

Despite indications that humoral immune responses may be involved in corneal allograft rejection, few definitive reports of such responses exist, and the antigens responsible for evoking and targeting these responses remain unknown. We report the development of donor-specific humoral immunity in PVG (RT1c) rats given fully allogeneic ACI (RT1a) corneal grafts heterotopically. PVG anti-ACI cornea serum lysed ACI, but not PVG, target cells in complement-dependent cytotoxic assays. Target cells from PVG.1A or PVG.R1 congenic rats, which respectively share the entire rat major histocompatibility complex (MHC) region (i.e., RT1) or only class I MHC antigens (RT1.A region) with ACI (on PVG backgrounds) were also lysed by this serum. Lysis of PVG.1A targets was significantly more extensive than lysis of PVG.R1 cells, indicating that both RT1.A and non-RT1.A antigens act as targets for cell lysis. Levels of PVG.1A and ACI target lysis were equivalent, suggesting that minor transplantation antigens were not significantly involved in this humoral response.

An animal model for corneal graft rejection is described in which corneal neovascularization is induced prior to keratoplasty. This model closely resembles high-risk human corneal grafting, and relies on rejection being initiated solely by the transplanted corneal tissue. All eleven allografts performed using this model rejected; median time to rejection was 17 days. Two treatment regimens were assessed using this rejection model. Topical cyclosporine produced a significant improvement in graft survival (P = 0.0025). When systemic cyclosporine was administered there was a highly significant improvement in graft survival (P = 0.0001).

Corneal allografts have been shown to give rise to immune responses, but the role and relative importance of individual corneal cell populations in evoking such responses remain unclear. We dissected ACI (RT1a) rat corneas into separate epithelial, stromal, and endothelial components by a method that yields pure cell populations in tissue culture, and grafted these components separately to groups of fully allogeneic PVG (RT1c) recipients). Grafts of corneal stroma elicited strong cellular cytotoxic immune responses in a cell-mediated lymphocytotoxic assay, but corneal epithelium failed to generate any detectable response. Grafts of corneal endothelium alone, however, evoked a potent cellular cytotoxic response. Using congenic rats, it was found that grafts from PVG.1A (RT1a) donors to PVG (RT1c) recipients (which differ at both the RT1.A and B loci) yielded identical results. However, no corneal component graft from PVG.R1 (RT1rl) donors to PVG recipients (which differ only at RT1.A) generated a detectable immune response. Use of target lymphoblasts from congenic strains established that at least part of all responses detected were directed against class I (RT1.A) major histocompatibility complex antigens. These findings indicate that there is differential immunogenicity of specific corneal tissue components in the rat that may be further influenced by the degree of MHC disparity between donor and recipient.

The outcome of clinical corneal transplantation depends on the degree of vascularization and inflammation present in the graft bed at the time of the operation, but the reason for this is unclear. Normal, diseased, and rejected human corneas have been examined with an immunoperoxidase staining procedure, employing monoclonal antibodies to class I and II major histocompatibility complex (MHC) antigens and to other leukocyte markers. In particular, departures from normal in the expression of MHC antigens and in the passenger cell distribution in the diseased or rejected corneas were sought. MHC antigen expression did not alter with inflammation, vascularization, or rejection. However, dendritic-like passenger cells, which were found in low numbers throughout the central stroma of normal cornea as well as in basal epithelium, significantly increased in number in vascularized corneas. We suggest that the breakdown of corneal privilege in vascularized eyes may reflect the increased number of accessory cells in the graft bed.

The relative contributions made by the major class I (RT1.A) and class II (RT1.B) antigens of the rat major histocompatibility complex (MHC) to the immunogenicity of corneal and skin allografts were investigated using congenic animals. PVG (RT1c) recipients were given skin or heterotopic cornea grafts from congenic PVG.1A (RT1a) or PVG.R1 (RT1r1) donors, which respectively share the entire RT1 complex or only the RT1.A (major class I MHC antigen) region with fully allogeneic ACI (RT1a) rats. Recipient splenocytes were tested at ten days posttransplant for their ability to lyse ACI, PVG.1A, PVG.R1, and PVG target cells in a secondary CML following 6 days in vitro stimulation with irradiated ACI spleen cells. Effector cells from PVG recipients of both RT1.A and B disparate (PVG.1A donor) and RT1.A disparate (PVG.R1) skin or cornea grafts lysed ACI, PVG.1A, and PVG.R1 (but not PVG) targets at levels significantly above controls given syngeneic grafts. However, the level of cytotoxicity against PVG.R1 as well as ACI and PVG.1A allogeneic targets was always significantly higher following PVG.1A grafts than following PVG.R1 grafts, indicating that the addition of a class II MHC antigen difference markedly augmented the immunogenicity of class I MHC antigen disparate cornea and skin grafts. Taken together with other recent evidence confirming the presence of Langerhans cells in the normal rat (and human) cornea, these results suggest that class II MHC-bearing cells make an important contribution to the immunogenicity of corneal allografts.

An alternative model for corneal allografting termed the reverse corneal allograft reaction (RCAR) was developed in this study. Spleen cells from an alloimmunized donor were injected into the corneal stroma of the immunizing donor strain or were restimulated in mixed lymphocyte culture and then injected into the corneal stroma of the immunizing strain. The reaction began as a circular opaque site that spread and became irregularly shaped during the first 5 days after cell injection. The epithelial surface of the cornea became uneven and epithelial cell erosions were noted. Histological examination revealed that corneal stromal keratocytes at the site of inoculation had undergone degeneration and the injected cells had migrated toward the epithelial-stromal boundary, wherein a disruption of the basement membrane and disintegration of the epithelial cells occurred. Purified spleen cell subsets injected separately did not mediate the reaction. A suspension of T lymphocytes and class II antigen-positive macrophagelike cells was required to cause the RCAR. This reaction, which mimics a delayed-type hypersensitivity response, was transient, reaching a peak by day 5 and waning by day 8. This experimental model of the corneal allograft reaction shows promise for the study of cells and mediators of the corneal allograft reaction and can be employed as a reproducible system in which to test drug therapies for the treatment of corneal allograft rejection.