November 2001
Volume 42, Issue 12
Free
Immunology and Microbiology  |   November 2001
Acute Rejection of Orthotopic Corneal Xenografts in Mice Depends on CD4+ T Cells and Self-Antigen–Presenting Cells
Author Affiliations
  • Kazumi Tanaka
    From the Department of Ophthalmology, Yokohama City University of Medicine, Japan; the
  • Koh-hei Sonoda
    Department of Ophthalmology, Kyusyu University Graduate School of Medical Sciences, Fukuoka, Japan; and
  • J. Wayne Streilein
    Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
Investigative Ophthalmology & Visual Science November 2001, Vol.42, 2878-2884. doi:
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      Kazumi Tanaka, Koh-hei Sonoda, J. Wayne Streilein; Acute Rejection of Orthotopic Corneal Xenografts in Mice Depends on CD4+ T Cells and Self-Antigen–Presenting Cells. Invest. Ophthalmol. Vis. Sci. 2001;42(12):2878-2884.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Guinea pig corneal xenografts have been reported to be rejected acutely in eyes of normal adult mice. Rejection of this type is independent of xenoreactive antibodies, and mice deficient in CD8+ and NK T cells are unable to reject guinea pig corneal grafts acutely. Therefore, a study was conducted to determine the extent and manner by which CD4+ T cells are responsible for rejection of orthotopic corneal xenografts.

methods. Xenogeneic corneas were prepared from eyes of normal guinea pigs and grafted orthotopically into normal eyes of C57BL/6 mice, class II major histocompatibility complex (MHC) knockout (KO) mice, and class II MHC KO mice reconstituted with syngeneic (C57BL/6) CD4+ T cells and/or bone marrow cells. Graft survival was assessed clinically, and success of cellular reconstitution was assayed using flow cytometric analysis of peripheral blood leukocytes. T cells from rejector mice were analyzed for proliferative responses to guinea pig xenoantigens in vitro.

results. Median survival times (MST) of corneal xenografts in MHC class II KO mice was significantly delayed (31 days) compared with grafts in wild-type C57BL/6 eyes (9 days). Acute rejection was restored almost completely when MHC class II KO mice were reconstituted simultaneously with C57BL/6 bone marrow and CD4+ T cells, but not when the KO mice were reconstituted with either CD4+ T cells or bone marrow cells alone. Mice that rejected guinea pig corneas possessed only CD4+ T cells capable of responding to guinea pig xenoantigens in vitro.

conclusions. Acute rejection of orthotopic corneal xenografts in mice is mediated by CD4+ T cells that detect guinea pig xenoantigens that are presented on MHC class II+ syngeneic antigen-presenting cells. These results strongly suggest that rejection occurs exclusively through the indirect pathway of T-cell activation.

The vulnerability of solid-tissue xenografts to immune rejection exceeds that of solid-tissue allografts, in part because so-called natural antibodies are often present in the sera of normal animals, and these antibodies recognize xenoantigens on the grafts. 1 2 3 As a consequence, xenografts undergo hyperacute vascular rejection within minutes to hours of engraftment, due to the binding of complement-fixing, xenoreactive antibodies to vascular endothelium, or they undergo acute vascular rejection (delayed xenograft rejection) that is mediated within 2 to 3 days by antibodies that focus macrophages, NK cells, and/or neutrophils on the graft vasculature, leading to endothelial cell activation and eventual destruction. 4 5 6 The cornea differs from other types of solid-tissue xenografts in that it is avascular and therefore, it may not be vulnerable to hyperacute rejection. In addition, the graft forms the anterior wall of the anterior chamber of the eye, and this compartment is filled with aqueous humor that contains several potent inhibitors of complement activation. 7 These special features of the cornea probably account for the experimental evidence that antibody-mediated hyperacute rejection does not occur when guinea pig corneas are grafted into eyes of normal mice. 8 It is pertinent that the guinea pig–mouse donor–recipient combination is discordant, 9 10 —that is, sera of normal mice contain complement-fixing, xenoreactive anti-guinea pig antibodies. 
Although corneal xenografts are not vulnerable to antibody-mediated rejection, they are highly susceptible to acute, cell-mediated xenograft rejection. 3 Guinea pig corneas grafted orthotopically to eyes of C57BL/6 mice are rapidly destroyed, with a median survival time (MST) of approximately 9 days, and similar grafts placed in eyes of BALB/c recipients have an MST of approximately 16 days. 8 Formal proof of the irrelevancy of antibodies to corneal xenograft rejection in this system was reported by Tanaka et al. 8 who showed that the time and tempo of corneal xenograft rejection is identical in normal mice compared with mice in which the μ chain of immunoglobulin had been disrupted. In the study by Tanaka et al., mice with a disrupted β-2 microglobulin (β2μ) gene also rejected guinea pig corneal grafts in an acute manner similar to normal mice. This finding strongly suggests that the acute phase of cell-mediated xenograft rejection in mice is neither mediated by CD8+ cytotoxic T cells nor by NK T cells (both of which are depleted in β2μ knockout mice). Alternatively, Tanaka et al. showed that mice deficient in CD4+ T cells no longer reject guinea pig corneal grafts acutely (MST, 27days), implying that mice reject corneal xenografts acutely, using xenoreactive CD4+ T cells. 8  
Another feature of the cornea that distinguishes it, as a graft, from other solid tissues is the virtual absence of bone marrow–derived cells, termed passenger leukocytes. 11 12 13 14 In solid organ allografts, such as skin, heart, and kidney, class II major histocompatibility complex (MHC)+ passenger leukocytes (dendritic cells, macrophages) make the definitive contribution to the graft’s capacity to sensitize its recipient, especially alloreactive T cells of the so-called direct type—T cells that directly recognize MHC class I and II alloantigens. Because existing evidence indicates that CD4+ T cells are primarily responsible for the acute phase of cell-mediated xenograft rejection of orthotopic corneal xenografts in mice, it is of interest to know whether xenodestructive CD4+ T cells are activated through the direct or indirect pathways of antigen recognition. This critical matter was addressed by experiments in the current study. 
Using C57BL/6 mice with a targeted disruption of class II MHC genes, our results indicate that the ability of these mice to reject orthotopic guinea pig corneal grafts acutely (within 2 weeks) requires that the recipients be reconstituted with both CD4+ T cells and class II–expressing bone marrow–derived cells. These results indicate that acute cellular rejection of guinea pig corneal grafts is mediated by CD4+ T cells that recognize guinea pig antigens exclusively by the indirect pathway. 
Materials and Methods
Animals and Anesthesia
Hartley guinea pigs (450–550 g) were purchased from Elm Hill Breeding Laboratories (Cheslmsford, MA), and inbred strain 13 guinea pigs (550–600 g) were purchased from Crest Caviary (Prunedale, CA). C57BL/6 mice were obtained from our animal facility or purchased from Taconic Farm (Germantown, NY). MHC class II–deficient mice (C57BL/6Tac-[KO]Abb N5) were purchased from Taconic Farm. All mice were males, 8 to 12 weeks old, and were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. In all experiments, mice were used as recipients and guinea pigs were used as donors. Corneal grafts were prepared from eyes enucleated after the donor guinea pigs were killed. For experimental manipulations, mice were deeply anesthetized with an intraperitoneal injection of 3 mg ketamine and 0.0075 mg xylazine. 
Orthotopic Corneal Grafting
Donor guinea pig corneas were excised by a 2.0-mm diameter trephine and placed in Hanks’ balanced salt solution until grafting. The graft bed was prepared by excising with Vannas scissors a 1.5-mm site from the central cornea of the right eye. The graft was placed in the recipient bed and secured with 12 interrupted 11-0 nylon sutures (Sharpoint; Vanguard, Houston, TX) such that the epithelial surfaces of the donor and the recipient corneas were juxtaposed. Antibiotic ointment was applied to the corneal surface, and the lids were closed with an 8-0 nylon tarsorrhaphy. Tarsorrhaphy was maintained (except for clinical inspection purposes) until graft rejection was documented. Corneal sutures were removed on day 8. 
Assessment of Graft Survival
Grafts were evaluated by slit lamp biomicroscopy three times a week. The day of rejection was defined as that when graft transparency was lost—that is, iris margin and iris structure were not visible through the graft, and graft clarity never recovered subsequently. 
CD4+ T Cell Reconstitution of Class II MHC KO Mice
Cervical, inguinal, and mesenteric lymph nodes and spleen were obtained from naive C57BL/6 mice. Spleen cells, depleted of red blood cells by lysis with Tris-NH4Cl, and lymph node cells were pressed through nylon mesh to produce a single-cell suspension. To obtain primed CD4+ T cells, draining lymph nodes were obtained from C57BL/6 mice immunized by subcutaneous injected of guinea pig spleen cells (1 × 107) 2 weeks previously. T cells from these lymph nodes were purified to more than 90% by a T-cell enrichment column. After staining with rat anti-mouse CD8 antibody, CD4+ T cells were purified to more than 90% by a CD4 T-cell separation kit (Immulan; cat. no. BL-7154; Biotecx Laboratories, Inc., Houston, TX). Purified CD4+ T cells were injected intravenously into MHC class II KO mice. Corneal xenotransplantation was performed on the day after reconstitution with CD4+ T cells. 
Bone Marrow Transplantation into Class II KO Mice
Untreated bone marrow cells (30–50 × 106) from naive C57BL/6 mice were injected intravenously into MHC class II KO mice on four consecutive days (total, 150–200 × 106 bone marrow cells). 
Flow Cytometric Analysis of Lymphoid Cells from Reconstituted Mice
At 2 weeks after CD4+ T cell reconstitution, peripheral blood cells, depleted of red blood cells by lysis with Tris-NH4Cl, were triple-stained with Cy-Chrome-5–conjugated anti-TCRβ chain mAb (H57-597), phycoerythrin-conjugated anti-mouse CD4 mAb (RM 4.5) and FITC-conjugated anti-mouse CD8 mAb (53-6.7; all from PharMingen, San Diego, CA). At 4 weeks after bone marrow transplantation, peripheral blood cells, depleted of red blood cells, were double-stained with phycoerythrin-conjugated anti-mouse CD45 mAb (30-F11) and FITC-conjugated anti-mouse I-Ab mAb (25-9-17; PharMingen). The stained cells were analyzed by flow cytometry (Epics XL Analyzer; Coulter, Inc., Hialeah, FL). 
In Vitro Proliferation Assay
Lymph nodes cells were removed from naive C57BL/6 mice or from C57BL/6 mice that had received orthotopic strain 13 guinea pig corneal xenotransplants 2 weeks previously and had already rejected the grafts. Culture conditions were chosen based on the preliminary experiments (data not shown). Murine responder lymph node cells (5 × 105 cells) and x-irradiated (2000 rads) 13 guinea pig stimulator spleen cells (5 × 105 cells) were added in a final volume of 200 μl of culture medium composed of RPMI 1640 medium, 10 mM HEPES, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin (all from BioWhitaker, Walkersville, MD), and 1 × 10−5 M 2-ME (Sigma Chemical Co., St. Louis, MO), supplemented with heat-inactivated 10% fetal calf serum (Sigma) to triplicate wells of 96-well flat-bottomed microculture plates (Corning, Corning, NY). The cultures were incubated at 37°C in humidified air containing 5% CO2 for varying lengths of time. Cultures were pulsed with 0.5 μCi of[ 3H]thymidine 8 hours before termination, and the samples were harvested onto glass filters using an automated well harvester (Tomtec, Orange, CT). Radioactivity was assessed by liquid scintillation spectrometry (Wallac, Gaithersburg, MD), and the amount was expressed as counts per minute. For anti-CD4 blocking experiments, azide-free, low-endotoxin purified anti-mouse L3T4 (GK1.5) was used, and purified rat IgG2bκ was used as an isotype control (both from PharMingen). Antibodies were added to responder cells (lymph node cells from C57BL/6 mice that had rejected orthotopic strain 13 corneal xenotransplants), and the mixture was incubated on ice for 1 hour. After incubation, stimulator cells (13 guinea pig spleen cells) were added and managed the same as the other cultures. 
Statistical Analysis
Statistical analysis of graft survival, enabling comparison of MSTs, was performed using the Mantel-Cox rank test. 
Results
Fate of Orthotopic Guinea Pig Corneal Grafts in Eyes of Mice Deficient in Class II MHC Expression
C57BL/6 mice in which the MHC class II A β b gene had been disrupted (class II KO) received orthotopic grafts of guinea pig cornea. These mice were chosen because they are not only deficient in MHC class II expression, but they also have been reported to by profoundly deficient in CD4+ T cells. 15 Flow cytometry was performed to confirm that these mice were deficient in CD4+ T cells (Fig. 1A) . Normal C57BL/6 mice served as controls. The fate of these grafts was evaluated clinically, as described previously, 8 and the results are displayed in Figure 1B . Whereas normal C57BL/6 mice rejected guinea pig corneal grafts acutely (MST, 9 days), mice deficient in class II expression failed to reject guinea pig corneas at a similar tempo. Instead, a high proportion of guinea pig corneal grafts placed in eyes of class II–deficient mice remained clear for a protracted interval (MST, 31days). One graft remained permanently clear (the experiment was terminated at 8 weeks). Thus, mice deficient in class II MHC expression have a grossly impaired capacity to reject guinea pig corneal grafts acutely. This pattern of rejection strongly resembles that reported previously in mice genetically deficient in CD4 expression (CD4 KO mice). 8  
Attempts to Restore Acute Corneal Xenograft Rejection in Class II–Deficient Mice with Syngeneic CD4+ T Cells
Class II–deficient mice display a profound deficit of CD4+ T cells, suggesting a reason that these mice fail to reject guinea pig corneal grafts acutely. To test this possibility, CD4+ T cells were harvested from normal C57BL/6 mice and transfused intravenously (15 × 106, 20 × 106, or 25 × 106) into class II KO mice the day before orthotopic guinea pig corneal grafting. To determine the extent to which exogenous CD4+ T cells were present in these mice subsequently, peripheral blood was obtained at 2 weeks after CD4+ T cells injection, and analyzed by flow cytometry for content of T-cell receptor (TCR)–positive lymphocytes that also expressed CD4. The result of a representative experiment is presented in Figure 2A . At 2 weeks after infusion, 7% of TCR-positive cells in class II KO mice reconstituted with 25 × 106 CD4+ T cells expressed CD4. By contrast, virtually no CD4+ T cells were detected in the blood of untreated class II–deficient mice. Orthotopic guinea pig corneas were grafted into eyes of CD4-reconstituted mice the day after infusion, and the fate of the grafts was evaluated clinically. Class II KO mice reconstituted with CD4+ wild-type T cells did not reject the grafts acutely (MST, 29 days; Fig. 2B ). In fact, there was no difference between the rate of corneal xenograft rejection in these mice and the rate observed in nonreconstituted class II KO mice. 
The failure of naive, syngeneic CD4+ T cells to cause class II KO mice to reject guinea pig corneal grafts acutely can be explained in more than one way. One possibility is that the transfused CD4+ cells did not become sensitized to guinea pig antigens in class II KO mice, because these mice contain no class II–bearing antigen-presenting cells (APCs) to present guinea pig xenoantigens (i.e., an indirect pathway). To test this possibility, we attempted to reconstitute class II KO mice with presensitized CD4+ T cells. Normal C57BL/6 mice were sensitized to guinea pig antigens by subcutaneous immunization with strain 13 guinea pig spleen cells. Two weeks later, draining lymph nodes were removed, rendered into single-cell suspensions, and fractionated into a CD4-enriched population. These guinea pig-sensitized CD4+ T cells were then infused (10 × 106 or 20 × 106) into class II KO mice. When the peripheral blood of these mice was analyzed at 2 weeks by flow cytometry for content of CD4+ T cells, 8.6% of TCR positive cells expressed CD4 (Fig. 2A) . Accordingly, the day after infusion, these mice received orthotopic strain 13 guinea pig corneal grafts. The fates of these grafts in class II KO and reconstituted mice are displayed in Figure 2C . Whether reconstituted with specifically sensitized CD4+ T cells or not, class II KO mice did not reject strain 13 corneas acutely, although they rejected the grafts in a chronic manner similar to unreconstituted class II KO mice. 
Attempt to Restore Acute Corneal Xenograft Rejection in Class II–Deficient Mice with Class II–Bearing Hematopoietic Cells
One explanation for the failure of specifically sensitized CD4+ T cells to reconstitute acute corneal xenograft rejection in class II KO mice is that murine T cells can only recognize guinea pig xenoantigens through the indirect pathway. As such, activation of CD4+ T cells would require the presentation of guinea pig xenoantigens on self–class II molecules. In an effort to reconstitute the indirect pathway in class II KO mice, bone marrow cells were harvested from normal C57BL/6 mice and injected (150–200 × 106) intravenously into class II KO recipients. A similar experimental strategy has been reported 16 17 to produce significant hematopoietic chimerism in unirradiated normal mice. At 4 weeks after this injection, the blood of recipient mice was analyzed by flow cytometry and found to contain a significant number (7.3%) of CD45+ leukocytes that expressed I-Ab. For comparison, only 2.1% of CD45+ blood cells expressed I-Ab in class II KO mice (Fig. 3A) , whereas 65% of CD45+ blood cells expressed class II MHC molecules in wild-type C57BL/6 mice. 
We considered this to be evidence of successful reconstitution of class II KO mice with hematopoietic cells expressing class II MHC molecules. Therefore, at 4 weeks after hematopoietic reconstitution, guinea pig corneas were grafted to the eyes of these mice, and the clinical course of the grafts was observed. As the results displayed in Figure 3B indicate, enhanced levels of class II–bearing leukocytes in reconstituted class II–deficient mice failed to promote acute rejection of guinea pig corneal grafts. Once again, these mice rejected guinea pig corneal grafts in a chronic fashion. 
In the experiment just described, enhanced numbers of MHC class II–bearing leukocytes in the blood were not accompanied by increased levels of CD4+ T cells. Because CD4+ T cells appear to be required for acute corneal xenograft rejection, we attempted to reconstitute class II KO mice with both class II–bearing hematopoietic cells and mature CD4+ T cells. Class II KO mice received intravenously an inoculum of C57BL/6 bone marrow cells (150–200 × 106). Four weeks later, these mice received an infusion of CD4+ T cells (30 × 106) from normal C57BL/6 donors. One day later, they received orthotopic guinea pig corneal grafts. Doubly reconstituted class II KO mice mounted acute and intense rejection reactions against corneal xenografts (Fig. 4A) . All grafts were destroyed acutely (MST, 11 days). Thus, that class II KO mice did not reject orthotopic guinea pig corneal grafts acutely rests, on the one hand, on the absence of class II–expressing bone marrow–derived cells and, on the other hand, on the deficiency of CD4+ T cells. The need for class II-bearing cells in these mice implies that recipient APCs are required both for sensitization and for expression of immunity directed at guinea pig xenoantigens. 
Capacity of Murine T Cells to Proliferate in Response to Guinea Pig Xenoantigens In Vitro
Our remaining experiments addressed the questions of whether guinea pig xenoantigens are recognized by murine T cells through the indirect pathway and whether the responding T cells are CD4+ or CD8+. For these experiments strain 13 guinea pigs served as donors of corneal xenografts and as the source of spleen cells used for immunization of mice and for stimulation of T cells in proliferative assays in vitro. Purified responder lymph node cells were prepared from naïve C57BL/6 mice and from C57Bl/6 mice that had received an orthotopic strain 13 guinea pig cornea 2 weeks previously. At the time of lymph node harvest, these mice had already rejected the corneal xenografts. The responder cells were cultured with x-irradiated (2000 rads) 13 guinea pig spleen cells and assayed for proliferation. Naïve murine T cells failed to proliferate at any time point in response to guinea pig stimulator cells (Fig. 5A) . By contrast, sensitized responder cells proliferated at each examined time point, with peak proliferation observed on days 4 and 5 of culture. This result supports our claim that the direct pathway of recognition of guinea pig xenoantigens is not available in mice. Moreover, this result supports our hypothesis that the indirect pathway of xenoantigen recognition is open, because only T cells from presensitized mice were capable of proliferation in these experiments. 
To identify the type of proliferating T cell in these in vitro stimulation assays, we repeated these experiments and included additional cultures in which antibodies directed at CD4+ or at CD8+ T cells were included. The results of a representative experiment are displayed in Figure 5B . Sensitized murine T cells proliferated when stimulated with guinea pig spleen cells in the presence of isotype control antibodies to an extent similar to sensitized T cells stimulated in the absence of antibodies. By contrast, little if any proliferation was observed in cultures to which anti-CD4 antibodies had been added. In companion experiments, no inhibition of T-cell proliferation was observed when anti-CD8 antibodies were present (data not shown). This finding indicates that CD4+ T cells are sensitized in mice that receive and reject orthotopic guinea pig corneas. We conclude that the CD4+ T cells activated by orthotopic guinea pig grafts, which are responsible for graft rejection, recognize guinea pig xenoantigens almost exclusively by the indirect pathway. 
Discussion
Immune privilege protects corneal xenografts placed in mouse eyes from destruction by antibody-dependent mechanisms. This privilege does not extend, however, to protection of these grafts from T-cell–dependent rejection. Previous studies have revealed that orthotopic guinea pig corneal grafts in normal mice undergo acute rejection (within 2–3 weeks) by a mechanism that is almost exclusively dependent on CD4+ T cells. 8 In orthotopic murine allografts, rejection is mediated largely (but not completely) by T cells that recognize donor antigens through the indirect pathway of allorecognition. 18 19 Recipient APCs (especially Langerhans cells) that migrate into the donor allografts have been shown to be central to presentation of graft-derived antigens, and recipient APCs are required for graft rejection by (primarily) CD4+ T cells. The current studies in the guinea pig corneal xenotransplantation model tested the hypothesis that acute rejection of xenogeneic corneal grafts is also dictated by T cells that recognize xenoantigens after presentation by recipient APCs—that is, the indirect pathway of antigen recognition. In large measure, our results support the validity of this hypothesis. 
We have previously reported that acute rejection of orthotopic guinea pig corneal grafts failed to occur in mice in which the CD4 gene had been disrupted experimentally. 8 Now, in class II KO mice that were also profoundly deprived of CD4+ T cells as recipients, 15 acute rejection of guinea pig corneal grafts failed once again to occur. However, this deficit could not be restored simply by providing class II KO mice with exogenous CD4+ T cells. In fact, even exogenous CD4+ T cells specifically sensitized in vivo to guinea pig xenoantigens failed to promote acute rejection of corneal xenografts in class II KO mice. These results further support the contention that the CD4+ T cells that mediate rejection of guinea pig corneal grafts do not recognize guinea pig class II MHC molecules directly. Moreover, the observation that naïve murine T cells infused into class II KO mice reconstituted with normal hematopoietic cells promoted acute rejection of orthotopic corneal xenografts confirms that bone marrow–derived recipient cells are necessary for presentation of guinea pig xenoantigens to murine xenoreactive T cells. In aggregate, these results lead us to conclude that the vulnerability of orthotopic guinea pig corneal grafts to acute cellular rejection is dependent almost exclusively on CD4+ T cells that recognize xenoantigens presented by recipient APCs—that is, the indirect pathway of antigen recognition. 
Support for this interpretation of the in vivo experiments comes from our in vitro studies of the proliferative capacity of normal and presensitized murine lymph node cells. First, normal mouse lymph node cells failed to proliferate when stimulated with guinea pig spleen cells in vitro. Because normal C57BL/6 mouse lymph node cells proliferate vigorously when stimulated with MHC-incompatible allogeneic spleen cells in vitro, and because this type of proliferation is believed to reflect direct recognition of MHC-encoded alloantigens, 20 our results strongly suggest that the direct pathway of recognition of xenoantigens does not exist in C57BL/6 mice. However, lymph node cells from C57Bl/6 mice that had rejected orthotopic strain 13 guinea pig corneas proliferated strongly when stimulated with strain 13 spleen cells in vitro. This type of proliferation correlates with the indirect pathway of recognition of alloantigens. By analogy, we conclude that murine T cells can recognize guinea pig xenoantigens through the indirect pathway. The finding that anti-CD4, but not anti-CD8, antibodies inhibited proliferation of presensitized mouse T cells in vitro confirms that the responding, sensitized T cells are CD4+
It is important to reconsider the ultimate fate of guinea pig corneas in mouse eyes. Elimination of CD4+ T cells enables these grafts to avoid the acute phase of cell-mediated xenograft rejection. However, most of these grafts are eventually destroyed in a chronic fashion. Thus, the final solution to the corneal xenograft problem must avoid chronic as well as acute rejection. The mediator(s) of chronic rejection, observed in CD4 and/or class II–deficient mice, remain to be identified. At the very least, we can exclude CD4+ T cells from further consideration. Our previous experiments using various KO mice eliminated antibodies, CD8+ T cells, NK T cells, and complement from consideration as the potential effectors of acute rejection of corneal xenografts. 8 In future experiments, we will reconsider these effectors as mediators of chronic corneal xenograft rejection, and we will extend our survey to include NK cells and macrophages. 
 
Figure 1.
 
(A) Detection of CD4+ and CD8 + T cells in class II MHC KO mice. Peripheral blood leukocytes were obtained from adult C57BL/6 and class II KO mice and analyzed by flow cytometry for expression, first, of the TCRβ chain, and then for expression of CD4 and CD8. Histograms of a representative experiment are presented. (B) Fate of guinea pig corneal xenografts placed orthotopically into eyes of normal and class II KO mice. Guinea pig corneas were grafted into normal eyes of wild-type C57BL/6 mice and class II KO mice. Grafts were considered to be rejected when opacity prevented visualization of the recipient pupil and iris through the graft. Results are presented as a Kaplan-Meier survival curve. MST of grafts were 31 days in class II KO mice and 9 days in wild-type mice (P < 0.0001).
Figure 1.
 
(A) Detection of CD4+ and CD8 + T cells in class II MHC KO mice. Peripheral blood leukocytes were obtained from adult C57BL/6 and class II KO mice and analyzed by flow cytometry for expression, first, of the TCRβ chain, and then for expression of CD4 and CD8. Histograms of a representative experiment are presented. (B) Fate of guinea pig corneal xenografts placed orthotopically into eyes of normal and class II KO mice. Guinea pig corneas were grafted into normal eyes of wild-type C57BL/6 mice and class II KO mice. Grafts were considered to be rejected when opacity prevented visualization of the recipient pupil and iris through the graft. Results are presented as a Kaplan-Meier survival curve. MST of grafts were 31 days in class II KO mice and 9 days in wild-type mice (P < 0.0001).
Figure 2.
 
(A) Detection of CD4+ T cells in class II MHC KO mice reconstituted with syngeneic wild-type CD4+ T cells. Class II KO mice received intravenous inocula of CD4+ T cells from normal C57BL/6 donors or from C57BL/6 donors presensitized to guinea pig xenoantigens. Peripheral blood leukocytes were collected 2 weeks later and analyzed by flow cytometry for proportion of cells expressing CD4 and CD8. Histograms of a representative experiment are shown. (B) Fate of guinea pig corneal xenografts placed orthotopically into eyes of class II KO mice reconstituted with naïve CD4+ T cells. Guinea pig corneas were grafted into normal eyes of class II KO mice or class II KO mice that had received CD4+ T cells from normal C57BL/6 donors 2 weeks previously. Results of graft outcome are presented as a Kaplan-Meier survival curve. MSTs of grafts were 31 days in class II KO mice and 29 days in class II KO mice reconstituted with naïve CD4+ T cells. There is no significant difference between these MSTs. (C) Fate of guinea pig corneal xenografts placed orthotopically into eyes of class II KO mice reconstituted with primed CD4+ T cells. Guinea pig corneas were grafted into normal eyes of class II KO mice (□) or class II KO mice that had received 2 weeks previously CD4+ T cells from C57BL/6 mice previously sensitized to guinea pig xenoantigens (○). Results of graft outcome are presented as a Kaplan-Meier survival curve. MSTs of grafts are 31 days in class II KO mice and 30 days in class II KO mice reconstituted with primed CD4+ T cells. There is no significant difference between these MSTs.
Figure 2.
 
(A) Detection of CD4+ T cells in class II MHC KO mice reconstituted with syngeneic wild-type CD4+ T cells. Class II KO mice received intravenous inocula of CD4+ T cells from normal C57BL/6 donors or from C57BL/6 donors presensitized to guinea pig xenoantigens. Peripheral blood leukocytes were collected 2 weeks later and analyzed by flow cytometry for proportion of cells expressing CD4 and CD8. Histograms of a representative experiment are shown. (B) Fate of guinea pig corneal xenografts placed orthotopically into eyes of class II KO mice reconstituted with naïve CD4+ T cells. Guinea pig corneas were grafted into normal eyes of class II KO mice or class II KO mice that had received CD4+ T cells from normal C57BL/6 donors 2 weeks previously. Results of graft outcome are presented as a Kaplan-Meier survival curve. MSTs of grafts were 31 days in class II KO mice and 29 days in class II KO mice reconstituted with naïve CD4+ T cells. There is no significant difference between these MSTs. (C) Fate of guinea pig corneal xenografts placed orthotopically into eyes of class II KO mice reconstituted with primed CD4+ T cells. Guinea pig corneas were grafted into normal eyes of class II KO mice (□) or class II KO mice that had received 2 weeks previously CD4+ T cells from C57BL/6 mice previously sensitized to guinea pig xenoantigens (○). Results of graft outcome are presented as a Kaplan-Meier survival curve. MSTs of grafts are 31 days in class II KO mice and 30 days in class II KO mice reconstituted with primed CD4+ T cells. There is no significant difference between these MSTs.
Figure 3.
 
(A) Detection of class II MHC+ cells in class II MHC KO mice reconstituted with syngeneic wild-type bone marrow. Class II KO mice received intravenous inocula of hematopoietic cells from normal C57BL/6 donors. Peripheral blood leukocytes were collected 4 weeks later from these mice, from class II KO mice, and from wild-type C57BL/6 donors and then separated by flow cytometry into a CD45+ population that was then analyzed for the proportion of cells expressing I-Ab molecules. Histograms of a representative experiment are presented. (B) Fate of guinea pig corneal xenografts placed orthotopically into eyes of class II KO mice reconstituted with syngeneic bone marrow cells. Guinea pig corneas were grafted into normal eyes of class II KO mice or class II KO mice that had received bone marrow cells from normal C57BL/6 donors 4 weeks previously. Results of graft outcome are presented as a Kaplan-Meier survival curve. MSTs of grafts are 31 days in class II KO mice and 32 days in class II KO mice reconstituted with C57BL/6 bone marrow cells. There is no significant difference between these MSTs.
Figure 3.
 
(A) Detection of class II MHC+ cells in class II MHC KO mice reconstituted with syngeneic wild-type bone marrow. Class II KO mice received intravenous inocula of hematopoietic cells from normal C57BL/6 donors. Peripheral blood leukocytes were collected 4 weeks later from these mice, from class II KO mice, and from wild-type C57BL/6 donors and then separated by flow cytometry into a CD45+ population that was then analyzed for the proportion of cells expressing I-Ab molecules. Histograms of a representative experiment are presented. (B) Fate of guinea pig corneal xenografts placed orthotopically into eyes of class II KO mice reconstituted with syngeneic bone marrow cells. Guinea pig corneas were grafted into normal eyes of class II KO mice or class II KO mice that had received bone marrow cells from normal C57BL/6 donors 4 weeks previously. Results of graft outcome are presented as a Kaplan-Meier survival curve. MSTs of grafts are 31 days in class II KO mice and 32 days in class II KO mice reconstituted with C57BL/6 bone marrow cells. There is no significant difference between these MSTs.
Figure 4.
 
(A) Fate of guinea pig corneal xenografts placed orthotopically into eyes of class II KO mice reconstituted with syngeneic CD4+ T cells and bone marrow cells. Guinea pig corneas were grafted into normal eyes of class II KO mice or class II KO mice that had received normal C57BL/6 CD4+ T cells 2 weeks previously and bone marrow cells 4 weeks previously. Results of graft outcome are presented as a Kaplan-Meier survival curve. MSTs of grafts were 31 days in class II KO mice and 11 days in class II KO mice reconstituted with C57BL/6 CD4+ T cells and bone marrow cells (P < 0.0001). (B) Detection of CD4+ and CD8+ T cells in class II MHC KO mice reconstituted with syngeneic CD4+ T cells and bone marrow cells. Class II KO mice received intravenous inocula of hematopoietic cells and CD4+ T cells from normal C57BL/6 donors 4 and 2 weeks, respectively, before death. Peripheral blood leukocytes were then collected from these mice and from class II KO mice and were separated by flow cytometry into a TCRβ-chain+ population that was then analyzed for the proportion of cells expressing CD4 and CD8 molecules. Histograms of a representative experiment are presented.
Figure 4.
 
(A) Fate of guinea pig corneal xenografts placed orthotopically into eyes of class II KO mice reconstituted with syngeneic CD4+ T cells and bone marrow cells. Guinea pig corneas were grafted into normal eyes of class II KO mice or class II KO mice that had received normal C57BL/6 CD4+ T cells 2 weeks previously and bone marrow cells 4 weeks previously. Results of graft outcome are presented as a Kaplan-Meier survival curve. MSTs of grafts were 31 days in class II KO mice and 11 days in class II KO mice reconstituted with C57BL/6 CD4+ T cells and bone marrow cells (P < 0.0001). (B) Detection of CD4+ and CD8+ T cells in class II MHC KO mice reconstituted with syngeneic CD4+ T cells and bone marrow cells. Class II KO mice received intravenous inocula of hematopoietic cells and CD4+ T cells from normal C57BL/6 donors 4 and 2 weeks, respectively, before death. Peripheral blood leukocytes were then collected from these mice and from class II KO mice and were separated by flow cytometry into a TCRβ-chain+ population that was then analyzed for the proportion of cells expressing CD4 and CD8 molecules. Histograms of a representative experiment are presented.
Figure 5.
 
Capacity of murine T cells to proliferate in vitro in response to guinea pig xenoantigens. (A) Lymph node cells from naive C57BL/6 and from C57BL/6 mice that rejected orthotopic strain 13 guinea pig corneal grafts were stimulated in vitro with x-irradiated strain 13 guinea pig spleen cells and cultured for 3 to 6 days.[ 3H]thymidine was added during the terminal 8 hours of culture. Results are presented as mean counts per minute ± SD for triplicate cultures. (B) Cultures similar to those described in (A) were established. Anti-CD4 or isotype control antibodies were added to some cultures. After 3 to 7 days of culture, proliferation was assessed as described. Results are presented as mean counts per minute ± SD for triplicate cultures.
Figure 5.
 
Capacity of murine T cells to proliferate in vitro in response to guinea pig xenoantigens. (A) Lymph node cells from naive C57BL/6 and from C57BL/6 mice that rejected orthotopic strain 13 guinea pig corneal grafts were stimulated in vitro with x-irradiated strain 13 guinea pig spleen cells and cultured for 3 to 6 days.[ 3H]thymidine was added during the terminal 8 hours of culture. Results are presented as mean counts per minute ± SD for triplicate cultures. (B) Cultures similar to those described in (A) were established. Anti-CD4 or isotype control antibodies were added to some cultures. After 3 to 7 days of culture, proliferation was assessed as described. Results are presented as mean counts per minute ± SD for triplicate cultures.
The authors thank Marie Ortega for outstanding management of the vivarium and Jacqueline Doherty for excellent laboratory management. 
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Figure 1.
 
(A) Detection of CD4+ and CD8 + T cells in class II MHC KO mice. Peripheral blood leukocytes were obtained from adult C57BL/6 and class II KO mice and analyzed by flow cytometry for expression, first, of the TCRβ chain, and then for expression of CD4 and CD8. Histograms of a representative experiment are presented. (B) Fate of guinea pig corneal xenografts placed orthotopically into eyes of normal and class II KO mice. Guinea pig corneas were grafted into normal eyes of wild-type C57BL/6 mice and class II KO mice. Grafts were considered to be rejected when opacity prevented visualization of the recipient pupil and iris through the graft. Results are presented as a Kaplan-Meier survival curve. MST of grafts were 31 days in class II KO mice and 9 days in wild-type mice (P < 0.0001).
Figure 1.
 
(A) Detection of CD4+ and CD8 + T cells in class II MHC KO mice. Peripheral blood leukocytes were obtained from adult C57BL/6 and class II KO mice and analyzed by flow cytometry for expression, first, of the TCRβ chain, and then for expression of CD4 and CD8. Histograms of a representative experiment are presented. (B) Fate of guinea pig corneal xenografts placed orthotopically into eyes of normal and class II KO mice. Guinea pig corneas were grafted into normal eyes of wild-type C57BL/6 mice and class II KO mice. Grafts were considered to be rejected when opacity prevented visualization of the recipient pupil and iris through the graft. Results are presented as a Kaplan-Meier survival curve. MST of grafts were 31 days in class II KO mice and 9 days in wild-type mice (P < 0.0001).
Figure 2.
 
(A) Detection of CD4+ T cells in class II MHC KO mice reconstituted with syngeneic wild-type CD4+ T cells. Class II KO mice received intravenous inocula of CD4+ T cells from normal C57BL/6 donors or from C57BL/6 donors presensitized to guinea pig xenoantigens. Peripheral blood leukocytes were collected 2 weeks later and analyzed by flow cytometry for proportion of cells expressing CD4 and CD8. Histograms of a representative experiment are shown. (B) Fate of guinea pig corneal xenografts placed orthotopically into eyes of class II KO mice reconstituted with naïve CD4+ T cells. Guinea pig corneas were grafted into normal eyes of class II KO mice or class II KO mice that had received CD4+ T cells from normal C57BL/6 donors 2 weeks previously. Results of graft outcome are presented as a Kaplan-Meier survival curve. MSTs of grafts were 31 days in class II KO mice and 29 days in class II KO mice reconstituted with naïve CD4+ T cells. There is no significant difference between these MSTs. (C) Fate of guinea pig corneal xenografts placed orthotopically into eyes of class II KO mice reconstituted with primed CD4+ T cells. Guinea pig corneas were grafted into normal eyes of class II KO mice (□) or class II KO mice that had received 2 weeks previously CD4+ T cells from C57BL/6 mice previously sensitized to guinea pig xenoantigens (○). Results of graft outcome are presented as a Kaplan-Meier survival curve. MSTs of grafts are 31 days in class II KO mice and 30 days in class II KO mice reconstituted with primed CD4+ T cells. There is no significant difference between these MSTs.
Figure 2.
 
(A) Detection of CD4+ T cells in class II MHC KO mice reconstituted with syngeneic wild-type CD4+ T cells. Class II KO mice received intravenous inocula of CD4+ T cells from normal C57BL/6 donors or from C57BL/6 donors presensitized to guinea pig xenoantigens. Peripheral blood leukocytes were collected 2 weeks later and analyzed by flow cytometry for proportion of cells expressing CD4 and CD8. Histograms of a representative experiment are shown. (B) Fate of guinea pig corneal xenografts placed orthotopically into eyes of class II KO mice reconstituted with naïve CD4+ T cells. Guinea pig corneas were grafted into normal eyes of class II KO mice or class II KO mice that had received CD4+ T cells from normal C57BL/6 donors 2 weeks previously. Results of graft outcome are presented as a Kaplan-Meier survival curve. MSTs of grafts were 31 days in class II KO mice and 29 days in class II KO mice reconstituted with naïve CD4+ T cells. There is no significant difference between these MSTs. (C) Fate of guinea pig corneal xenografts placed orthotopically into eyes of class II KO mice reconstituted with primed CD4+ T cells. Guinea pig corneas were grafted into normal eyes of class II KO mice (□) or class II KO mice that had received 2 weeks previously CD4+ T cells from C57BL/6 mice previously sensitized to guinea pig xenoantigens (○). Results of graft outcome are presented as a Kaplan-Meier survival curve. MSTs of grafts are 31 days in class II KO mice and 30 days in class II KO mice reconstituted with primed CD4+ T cells. There is no significant difference between these MSTs.
Figure 3.
 
(A) Detection of class II MHC+ cells in class II MHC KO mice reconstituted with syngeneic wild-type bone marrow. Class II KO mice received intravenous inocula of hematopoietic cells from normal C57BL/6 donors. Peripheral blood leukocytes were collected 4 weeks later from these mice, from class II KO mice, and from wild-type C57BL/6 donors and then separated by flow cytometry into a CD45+ population that was then analyzed for the proportion of cells expressing I-Ab molecules. Histograms of a representative experiment are presented. (B) Fate of guinea pig corneal xenografts placed orthotopically into eyes of class II KO mice reconstituted with syngeneic bone marrow cells. Guinea pig corneas were grafted into normal eyes of class II KO mice or class II KO mice that had received bone marrow cells from normal C57BL/6 donors 4 weeks previously. Results of graft outcome are presented as a Kaplan-Meier survival curve. MSTs of grafts are 31 days in class II KO mice and 32 days in class II KO mice reconstituted with C57BL/6 bone marrow cells. There is no significant difference between these MSTs.
Figure 3.
 
(A) Detection of class II MHC+ cells in class II MHC KO mice reconstituted with syngeneic wild-type bone marrow. Class II KO mice received intravenous inocula of hematopoietic cells from normal C57BL/6 donors. Peripheral blood leukocytes were collected 4 weeks later from these mice, from class II KO mice, and from wild-type C57BL/6 donors and then separated by flow cytometry into a CD45+ population that was then analyzed for the proportion of cells expressing I-Ab molecules. Histograms of a representative experiment are presented. (B) Fate of guinea pig corneal xenografts placed orthotopically into eyes of class II KO mice reconstituted with syngeneic bone marrow cells. Guinea pig corneas were grafted into normal eyes of class II KO mice or class II KO mice that had received bone marrow cells from normal C57BL/6 donors 4 weeks previously. Results of graft outcome are presented as a Kaplan-Meier survival curve. MSTs of grafts are 31 days in class II KO mice and 32 days in class II KO mice reconstituted with C57BL/6 bone marrow cells. There is no significant difference between these MSTs.
Figure 4.
 
(A) Fate of guinea pig corneal xenografts placed orthotopically into eyes of class II KO mice reconstituted with syngeneic CD4+ T cells and bone marrow cells. Guinea pig corneas were grafted into normal eyes of class II KO mice or class II KO mice that had received normal C57BL/6 CD4+ T cells 2 weeks previously and bone marrow cells 4 weeks previously. Results of graft outcome are presented as a Kaplan-Meier survival curve. MSTs of grafts were 31 days in class II KO mice and 11 days in class II KO mice reconstituted with C57BL/6 CD4+ T cells and bone marrow cells (P < 0.0001). (B) Detection of CD4+ and CD8+ T cells in class II MHC KO mice reconstituted with syngeneic CD4+ T cells and bone marrow cells. Class II KO mice received intravenous inocula of hematopoietic cells and CD4+ T cells from normal C57BL/6 donors 4 and 2 weeks, respectively, before death. Peripheral blood leukocytes were then collected from these mice and from class II KO mice and were separated by flow cytometry into a TCRβ-chain+ population that was then analyzed for the proportion of cells expressing CD4 and CD8 molecules. Histograms of a representative experiment are presented.
Figure 4.
 
(A) Fate of guinea pig corneal xenografts placed orthotopically into eyes of class II KO mice reconstituted with syngeneic CD4+ T cells and bone marrow cells. Guinea pig corneas were grafted into normal eyes of class II KO mice or class II KO mice that had received normal C57BL/6 CD4+ T cells 2 weeks previously and bone marrow cells 4 weeks previously. Results of graft outcome are presented as a Kaplan-Meier survival curve. MSTs of grafts were 31 days in class II KO mice and 11 days in class II KO mice reconstituted with C57BL/6 CD4+ T cells and bone marrow cells (P < 0.0001). (B) Detection of CD4+ and CD8+ T cells in class II MHC KO mice reconstituted with syngeneic CD4+ T cells and bone marrow cells. Class II KO mice received intravenous inocula of hematopoietic cells and CD4+ T cells from normal C57BL/6 donors 4 and 2 weeks, respectively, before death. Peripheral blood leukocytes were then collected from these mice and from class II KO mice and were separated by flow cytometry into a TCRβ-chain+ population that was then analyzed for the proportion of cells expressing CD4 and CD8 molecules. Histograms of a representative experiment are presented.
Figure 5.
 
Capacity of murine T cells to proliferate in vitro in response to guinea pig xenoantigens. (A) Lymph node cells from naive C57BL/6 and from C57BL/6 mice that rejected orthotopic strain 13 guinea pig corneal grafts were stimulated in vitro with x-irradiated strain 13 guinea pig spleen cells and cultured for 3 to 6 days.[ 3H]thymidine was added during the terminal 8 hours of culture. Results are presented as mean counts per minute ± SD for triplicate cultures. (B) Cultures similar to those described in (A) were established. Anti-CD4 or isotype control antibodies were added to some cultures. After 3 to 7 days of culture, proliferation was assessed as described. Results are presented as mean counts per minute ± SD for triplicate cultures.
Figure 5.
 
Capacity of murine T cells to proliferate in vitro in response to guinea pig xenoantigens. (A) Lymph node cells from naive C57BL/6 and from C57BL/6 mice that rejected orthotopic strain 13 guinea pig corneal grafts were stimulated in vitro with x-irradiated strain 13 guinea pig spleen cells and cultured for 3 to 6 days.[ 3H]thymidine was added during the terminal 8 hours of culture. Results are presented as mean counts per minute ± SD for triplicate cultures. (B) Cultures similar to those described in (A) were established. Anti-CD4 or isotype control antibodies were added to some cultures. After 3 to 7 days of culture, proliferation was assessed as described. Results are presented as mean counts per minute ± SD for triplicate cultures.
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