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CD4+ T-Cell–Independent Rejection of Corneal Allografts

Niederkorn, Jerry Y.; Stevens, Christina; Mellon, Jessamee; Mayhew, Elizabeth

doi: 10.1097/01.tp.0000203140.70742.cb
Original Articles: Experimental Transplantation
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Background. Several studies suggest that a significant number of corneal allografts undergo rejection in the absence of CD4+ T cells. This study examined the role of CD4+ T cell-independent mechanisms of corneal allograft rejection.

Methods. BALB/c corneal allografts were transplanted to C57BL/6 beige nude mice that received either CD8 or CD8+ T cells from C57BL/6 CD4 knockout (KO) mice that had rejected BALB/c corneal allografts. Immune effector functions of CD8 or CD8+ T cells from C57BL/6 CD4 KO mice were assessed using delayed-type hypersensitivity assays and Annexin V apoptosis assays respectively.

Results. Both CD8 and CD8+ T cells from CD4 KO corneal allograft rejector mice mediated corneal allograft rejection following adoptive transfer to nude mice. CD8 T cells, but not CD8+ T cells, from CD4 KO mice adoptively transferred donor-specific DTH and induced apoptosis of BALB/c corneal endothelial cells in vitro. Apoptosis of BALB/c corneal endothelial cells was mediated by double negative (DN) T cells, as treatment of CD8 cells from CD4 KO mice with anti-Thy 1.2 plus complement abolished their effector function.

Conclusion. The results support the proposition that CD4+ T cell-independent rejection of corneal allografts can be mediated by either CD8+ or CD8 T cells. The CD8 T cells represent a unique DN T cell population that might mediate rejection by either direct cytolysis or by inducing apoptosis of the donor corneal endothelium.

Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX.

This work supported by National Institutes of Health Grants EY07641 and EY016664 and an unrestricted grant from Research to Prevent Blindness, New York, NY.

Address correspondence to: Jerry Y. Niederkorn, Ph.D., Department of Ophthalmology, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9057. E-mail: jerry.niederkorn@utsouthwestern.edu

Received 26 April 2005. Revision requested 13 May 2005. Accepted 15 December 2005.

Keratoplasty is the oldest, most common, and arguably, the most successful form of tissue transplantation. In the United States alone, over 33,000 corneal transplants are performed each year (1). Although 90% or more of the first time corneal transplants will succeed, approximately 10% will fail due to immune rejection (2). The precise immune mechanisms that mediate corneal graft rejection remain poorly understood even after 50 years of research in laboratory animals. T lymphocytes are required for the rejection of corneal allografts, as athymic, T cell-deficient nude mice do not reject corneal allografts (3). Histopathological analysis of rejected corneal grafts in both human and animal subjects reveals the presence of a mixed inflammatory infiltrate containing both CD4+ and CD8+ T lymphocytes (4–7). Studies in CD8 knockout (KO) and perforin KO mice demonstrated that CD8+ cytotoxic T lymphocytes (CTL) are unnecessary for the rejection of corneal allografts in mice (8, 9). The notion that CD4+ T cells might be the pivotal mediators of corneal graft rejection arose from studies demonstrating the close correlation between delayed-type hypersensitivity (DTH), a classical CD4+ T cell-mediated immune process, and corneal graft rejection in rodents (10, 11). Depletion of CD4+ T cells by in vivo administration of anti-CD4 monoclonal antibodies (12, 13) or deletion of the CD4 gene (14, 15) have demonstrated the importance of CD4+ T cells in corneal graft rejection. However, closer scrutiny of these studies raises questions about the role of CD4+ T cells as the sole mediators of corneal graft rejection, as corneal allografts undergo immune rejection in 33% of the mice and 64% of the rats treated with anti-CD4 monoclonal antibody (12, 13) and in 45% of the CD4 KO mice (14).

Until recently, the dogma in transplantation immunology held that allograft rejection was a Th1 CD4+ cell-mediated process. However, some studies have demonstrated a role for CD8+ T cell-mediated rejection of skin and cardiac allografts (16, 17). Moreover, we have recently shown that interferon-γ (IFN-γ) KO mice, which cannot mount classical Th1 immune responses, are nonetheless capable of rejecting corneal allografts (18, 19). Moreover, the immune rejection of skin, cardiac, and tumor allografts occurs in CD4 KO mice (20, 21). In the present study we sought to determine the role and mechanisms of CD4-independent rejection of corneal allografts.

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MATERIALS AND METHODS

Animals

C57BL/6 (H-2b) and CD4 knockout (C57BL/6-Cd4 <tm1Mak>, H-2b) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in pathogen-free animal facilities. BALB/c (H-2d) mice were purchased from Taconic Farms (Germantown, NY). Beige nude (Cr NIH-bg-nu-xid, H-2b) mice were purchased from the NIH (Bethesda, MD). C.B.-17 SCID/beige mice were purchased from Charles River (Willmington, MA) and Taconic Farms (Germantown, NY). Animals used in grafting experiments were female, 8–12 weeks in age. All animals used in these experiments were housed and cared for in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.

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Surgical Technique

Full-thickness penetrating orthotopic corneal grafts were performed as previously described (22), with a few modifications. Mice were anesthetized systemically with an intraperitoneal (i.p.) injection of 80 mg/kg of ketamine HCl (Fort Dodge Laboratories, Fort Dodge, IA) and 8.0 mg/kg of xylazine (Bayer Corporation, Shawnee Mission, KS). Proparacaine HCl ophthalmic solution (USP 0.5%; Alcon Laboratories, Ft. Worth, TX) was used as a topical anesthetic. Donor grafts and recipient graft beds were scored with 2.5 mm and 2.0 mm trephines respectively and the corneas were excised with Vannas scissors. Donor grafts were sewn into place using running 11-0 nylon sutures (Ethicon, Sommerville, NJ), and sutures were removed on day seven posttransplantation. Topical antibiotic (Akorn, Decatur, IL) was applied immediately after surgery as well as immediately after removal of sutures. No immunosuppressive drugs were used, either topically or systemically.

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Clinical Evaluation of Grafted Corneas

Corneal grafts were examined 2–3 times a week with a slit-lamp biomicroscope (Carl Zeiss, Oberkochen, Germany). Graft opacity was scored using a scale of 1–4. Degree of graft opacity was scored as follows: 0=clear; 1+=minimal superficial opacity; 2+=mild deep stromal opacity with pupil margin and iris visible; 3+=moderate stromal opacity with pupil margin visible, but iris structure obscured; and 4+=complete opacity, with pupil and iris totally obscured. Corneal grafts were considered rejected upon two successive scores of 3+.

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Cell Lines

BALB/c corneal epithelial and endothelial cell cultures were established as described previously (23). Briefly, corneal cell cultures were established from freshly dissected corneal explants (24, 25) and propagated in MEM (Bio Whittaker, Walkersville, MD) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) (Hyclone Laboratories, Logan, UT) at 37°C and 10% CO2. Once primary cultures were established, the cells were immortalized with human papilloma virus E6 and E7 oncogenes using the disabled recombinant retroviral vector pLXSN16E6/E7 (26). The transformed corneal cells proliferate indefinitely, maintaining their original morphologic characteristics and expressing the same histocompatibility antigens as their nontransformed counterparts (27). Cell lines were then maintained in complete MEM (CMEM) at 37°C and 10% CO2. CMEM consisted of MEM supplemented with 10% FBS, 2 mM l-glutamine (BioWhittaker), 1 mM sodium pyruvate (BioWhittaker), 2 mM MEM vitamins (Bio Whittaker), and 1% penicillin-streptomycin-fungizone solution (BioWhittaker). Tissue-cultured corneal cells were used for in vitro studies rather than the usual lymphoid cells since corneal cells are the relevant target cells in vivo during corneal allograft rejection.

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In Vitro Boost of Effector Splenocytes with Alloantigen

Boosting cultures are frequently used to maximize the in vitro detection of cell-mediated immune responses that are initially induced in vivo (28). Accordingly, spleens were collected from either naïve C57BL/6 CD4 KO mice or CD4 KO mice that had rejected BALB/c corneal allografts 7 to 14 days earlier. Positive controls consisted of C57BL/6 mice that had been subcutaneously (SC) immunized with 1×107 BALB/c spleen cells 14 days earlier. Negative controls consisted of naïve C57BL/6 mice that had been exposed to BALB/c alloantigens in vivo. Single spleen cell suspensions from all three categories of mice were prepared in complete RPMI 1640 medium (CRPMI) containing 5×10−5 M 2mercaptoethanol (Sigma). CRPMI was made up of: RPMI-1640 medium (BioWhittaker) supplemented with 10% heatinactivated fetal bovine serum (FBS), 2 mM l-glutamine, 1 mM sodium pyruvate, 1% penicillin-streptomycin-fungizone, 1% non-essential amino acids (BioWhittaker), and 1% HEPES buffer (BioWhittaker). Spleen cells (3×107) from either experimental or control effector lymphocytes were dispensed into 75-cm2 tissue culture flasks along with 20 ml of boosting medium and 6×105 γ-irradiated (3,000 rad) BALB/c spleen cells (erythrocytes lysed). The flasks were incubated upright at 37°C and 5% CO2 for 96 hr. The in vitro boosted effector cells were washed once with HBSS and then resuspended in CRPMI and used as effector cells in the cytotoxicity and apoptosis assays (below). Such boosting cultures contain C57BL/6 antigen presenting cells that could back stimulate the γ-irradiated BALB/c T cells used in the culture system, resulting in the release of cytokines, such as interferon-γ. However, this in vitro boosting culture protocol did not induce de novo CTL activity in cells from naïve mice.

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CD8+ T-cell Enrichment

CD8+ T cells were harvested by positive selection using rat anti-mouse CD8-conjugated magnetic microbeads (MACS system; Miltenyi Biotec Inc., Auburn, CA). Briefly, single-cell suspensions of splenocytes were prepared from either experimental or control host strain mice, as described earlier. Lymphocytes were washed once in PBS and then resuspended in 90 μl ice cold 0.5% BSA in PBS (=buffer solution) per 107 cells. Ten microliters of the MicroBeads were then added per 107 cells, mixed well, and then incubated for 15 min at 4°C. Cells were washed once, resuspended in degassed buffer solution, and run through a MACS mini-separation column on a magnetic field. Cells not attached to anti-CD8-conjugated beads passed through the column, while cells that were retained in the column were attached to anti-CD8-conjugated beads. To eliminate any CD4+ cells that may have been retained in the column, cells were extracted from the column, resuspended in 10 μg/ml anti-mouse CD4 (BD PharMingen) in RPMI containing 0.3% BSA and 1% HEPES, and incubated for 30 min at 4°C. Cells were washed once with HBSS, resuspended in low-tox rabbit complement (Cedarlane, Hornby, ON) diluted 1:10 in RPMI containing 0.3% BSA and 1% HEPES, and incubated for 30 min at 37°C. Cells were washed three times in HBSS and counted. CD8 cells that were not retained in the column were purged of CD8+ cells using anti-mouse CD8 monoclonal antibody (BD PharMingen) plus complement as described above. Depletions of CD8+ and CD4+ cells were >95% as determined by flow cytometry.

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Flow Cytometry

Spleen cell suspensions that were enriched or depleted of CD8+ cells were examined by flow cytometry for the relative percent of αβTCR+, γδTCR+, and Thy 1+ cells. Spleen cells (1×106) were incubated with hamster anti-murine αβTCR (Accurate Chemical, Westbury, NY), hamster anti-murine γδTCR (GL3; kindly provided by Leo LeFrancois, University of Connecticut, Farmington, CT), or PE-conjugated rat anti-Thy 1.2 (PharMingen, San Diego, CA) at a concentration of 1 μg/ml for 30 min on ice, and washed three times in HBSS. Cells treated with hamster anti-αβTCR or hamster murine γδTCR were washed three times in HBSS and incubated in PE-labeled Armenian hamster antihamster IgG (PharMingen) secondary antibody for 20 min at 4°C, washed three additional times in PBS, fixed in 1% paraformaldehyde, and assessed for fluorescence in a FACScan flow cytometer (Becton Dickinson). All events were analyzed using CellQuest software.

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Adoptive Transfer of CD8+ and CD8 Cells

Cell suspensions were enriched or depleted of CD8+ cells as described above and one donor-equivalent was transferred intravenously (i.v.) to each C57BL/6 nude mouse. For CD8 cells, one donor-equivalent was 20±5×106 cells per recipient and for CD8+ cells, one donor-equivalent was 4.4±1.5×106 cells per recipient. In some experiments, one donor-equivalent of unfractionated spleen cells (5–8×107 cells/mouse) was transferred to each C57BL/6 nude mouse. Nude mice were challenged with BALB/c corneal allografts one day after the adoptive cell transfers.

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Three-Color Annexin V Staining to Evaluate T-cell–Induced Apoptosis In Vitro

CD8+ and CD8 T cells were collected from either C57BL/6 mice or adoptive cell transfer recipients one week after the rejection of BALB/c corneal allografts. Following in vitro boosting (described above), the CD8 and CD8 + T cells were incubated with 5 μM Syto-59 red fluorescent nucleic acid stain (Molecular Probes, Eugene, OR) in CRPMI for 45 min at 37°C. Syto-59-stained C57BL/6 CD4+ effector cells were cultured with BALB/c corneal epithelial or endothelial cells overnight. Apoptosis of the corneal cells was then measured at various time-points with the TACS Annexin V-FITC apoptosis detection system (R&D Systems, Minneapolis, MN). Briefly the target cells were harvested with 0.05% trypsin/EDTA and washed once with ice-cold PBS. The cells were then resuspended in 100 μl Annexin V Incubation Reagent (10 μl 10× Binding Buffer, 10 μl propidium iodide (PI), 1 μl Annexin V-FITC, and 79 μl of de-ionized water) and incubated in the dark at room temperature for 15 min. 300 μl of ice-cold 1× binding buffer were then added to each sample and the samples were analyzed by fluorescence-activated cell sorting (FACS) within one hour for maximal signal. During FACS analysis, Syto-59-stained cells were gated out to allow only host strain corneal cell apoptosis to be detected. Annexin V-FITC staining was used as a marker for apoptotic cells, while PI staining was used as an indicator of necrotic cells that had lost membrane integrity. Cells that stained positively with both PI and Annexin V-FITC were considered necrotic. A standard 4-hour 51Cr-release assay was used to detect allospecific CTL as previously described (24). Briefly, single-cell suspensions of C57BL/6 KO and wild-type lymphocytes, which had undergone a 96 hr in vitro boost with γ-irradiated (3,000 rad) BALB/c stimulator lymphocytes, were added to 96-well microtiter plates (Corning Inc., Corning, NY) along with 2×10451Cr-labeled BALB/c corneal epithelial or endothelial cells in a total volume of 200 μl/well. Assays were performed in triplicate using effector to target cell ratios ranging from 50:1 to 200:1. Plates were centrifuged at 500 rpm (25×G) for 3 min before incubating at 37°C for 4 hr. Plates were centrifuged at 800 rpm (100×G) for 6 min before harvesting 100 μl of the supernatant from each well and counting in a gamma counter (Packard BioScience, Meriden, CT). Cytotoxicity was determined by the amount of 51Cr released by the target cells and the specific lysis was calculated as follows:

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Delayed Type Hypersensitivity (DTH) Assay

DTH was measured using a conventional footpad swelling assay (3). An eliciting dose of 1×107 γ-irradiated (3,000 rad) BALB/c spleen cells in 25 μl of Hanks’ balanced salt solution (HBSS) was inoculated into the subcutaneous tissue of the right hind footpad of C57BL/6 mice. The left hind footpad served as a negative control and was injected with 25 μl of HBSS without cells. Results were expressed as specific footpad swelling = (24-hr measurement − 0-hr measurement) for experimental footpad − (24-hr measurement − 0-hr measurement) for negative control footpad.

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RESULTS

Corneal Allograft Rejection in CD4 KO Mice

Previous studies have reported the importance of CD4+ T cells in corneal allograft rejection in rodent models of keratoplasty, yet rejection occurred in 33% to 64% of the CD4 T cell-deficient hosts (12–14). Experiments were performed to confirm that a significant number of corneal allografts underwent rejection in the absence of CD4+ T cells. BALB/c corneal allografts were transplanted to either normal C57BL/6 mice (n=10) or CD4 KO mice (n=70). The results indicated that 54% (38/70) of the BALB/c corneal allografts underwent rejection in the CD4 KO hosts, while 100% of the grafts were rejected in wild-type C57BL/6 mice (Figure 1).

FIGURE 1.

FIGURE 1.

An adoptive transfer experiment was performed to confirm that the high incidence of corneal allograft rejection was immune-mediated. Spleen cell suspensions were collected 7–14 days after CD4 KO mice rejected BALB/c corneal allografts. Spleen cells (5×107) were injected intraperitoneally (i.p.) into C57BL/6 beige nude mice. BALB/c corneal allografts were transplanted 24 hr later and the fate of the corneal allografts determined. None of the ten C57BL/6 beige nude mice rejected BALB/c corneal allografts (Figure 2A). By contrast, 71% (5/7) of the beige nude mice that received spleen cells from CD4 KO mice rejected their BALB/c corneal allografts.

FIGURE 2.

FIGURE 2.

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Adoptive Transfer of Corneal Allograft Rejection with CD8+ and CD8 T Lymphocytes

Additional adoptive transfer experiments were performed to ascertain the role of CD8+ and CD8 T lymphocytes in CD4-independent rejection of corneal allografts. Spleen cells were collected from CD4 KO mice 7–14 days after they had rejected BALB/c corneal allografts. CD8+ and CD8 T lymphocytes were isolated using immunomagnetic beads. To ensure depletion of the respective cell populations, the enriched cell suspensions were treated with either anti-CD4 antibody or anti-CD8 antibody in the presence of complement. BALB/c corneal allografts underwent rejection in 46% (6/13; MST=46 days) of the recipients of CD8 T lymphocytes and in 95% (19/20; MST=15 days) of the recipients of CD8+ T lymphocytes (Figure 2B). Histopathological examination of rejected corneal allografts in CD4 KO mice revealed a mixed inflammatory infiltrate containing large numbers of neutrophils and mononuclear cells, which was indistinguishable from the infiltrate seen in rejected corneal allografts in wild-type mice (data not shown). Rejected corneal allografts in recipients of CD8+ and CD8 T lymphocytes from CD4 KO donor mice contained fewer numbers of neutrophils, with a predominance of mononuclear cells (data not shown). At the end of the experiment, spleens were removed from each group and analyzed by FACS for the presences of αβTCR+, γδTCR+, and Thy 1+ cells. Spleen cell suspensions from recipients of CD8-enriched cell suspensions contained 91.6±5.8% αβTCR+ cells, 8.1±6.6% γδTCR+ cells, 87±11.5% Thy 1+ cells, and 92.4 % CD8+ cells. By contrast, recipients of the CD8-depleted cell suspensions contained 39.0±21 % αβTCR+ cells, 1.78±2.4% γδTCR+ cells, 32.5%± 9.9% Thy 1+ cells, and <1 % CD8+ cells.

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CTL and DTH Responses in Recipients of CD8+ and CD8 T Lymphocytes from CD4 KO Donors

Allospecific CTL and DTH responses have been associated with the immune rejection of various categories of allografts. Accordingly, CD4 KO mice were examined for the development of CTL that lysed BALB/c corneal epithelial and endothelial cells. Spleen cells were isolated 7–14 days after C57BL/6 CD4 KO mice had rejected BALB/c corneal allografts and were tested for anti-BALB/c CTL in a conventional 4-hr 51Cr-release assay using BALB/c corneal epithelial and endothelial target cells. The results of a typical experiment are shown in Figure 3 and demonstrate that even though spleen cells from CD4 KO mice could adoptively transfer corneal allograft rejection, they did not display conventional CTL activity against either BALB/c corneal epithelial or endothelial cells. The absence of detectable CTL activity against BALB/c corneal cells was not due to poor sensitivity of the in vitro assay, as mice immunized subcutaneously with BALB/c spleen cells produced >75% cytolysis of BALB/c corneal epithelial and endothelial cells. By contrast, spleen cells from naïve C57BL/6 mice did not develop detectable CTL, even after a four-day in vitro boost with BALB/c stimulator cells. This is in keeping with previous reports indicating that orthotopic corneal allografts in mice do not normally induce detectable CTL responses to donor alloantigens in normal mice and corneal allograft rejection occurs in CD8 KO mice and perforin KO mice, which cannot generate allospecific CTL (8, 9).

FIGURE 3.

FIGURE 3.

Development of DTH responses to donor alloantigens has been correlated with corneal allograft rejection. Like wild-type mice, C57BL/6 CD4 KO mice developed DTH responses to BALB/c alloantigens following rejection of their BALB/c corneal allografts (data not shown). Additional experiments were performed to determine if both CD8+ and CD8 T lymphocytes from CD4 KO mice that had rejected BALB/c corneal allografts were capable of mounting DTH responses to BALB/c alloantigens. CD8+ and CD8 T lymphocyte suspensions were prepared using spleen cells taken from CD4 KO mice 7–14 days after they had rejected BALB/c corneal allografts. Lymphoid cells were adoptively transferred to C57BL/6 beige nude mice and DTH responses to BALB/c alloantigens were determined 24 hr later using a conventional footpad swelling assay. Interestingly, recipients of CD8+ T lymphocytes failed to mount DTH responses to BALB/c alloantigens (Figure 4), even though similar panels of mice rejected 95% of their BALB/c corneal allografts (Figure 2B). By contrast, recipients of CD8 T lymphocytes from CD4 KO donors developed positive DTH responses to BALB/c alloantigens that were significantly greater than the negative controls and the recipients of CD8+ T lymphocytes (P=0.01).

FIGURE 4.

FIGURE 4.

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Apoptosis of Corneal Cells By CD8+ and CD8 T Cells

The curious absence of both CTL and DTH activity in CD8+ T cell suspensions isolated from corneal allograft rejector mice begs the question as to the nature of the effector mechanisms that these cells invoke for the rejection of corneal allografts. Apoptosis has been implicated as a T cell-dependent immune effector mechanism in various forms of organ graft rejection (29–31). Immunohistochemical staining of rejected human corneal allografts has revealed the presence of apoptosis in corneal stromal cells (5). Apoptosis was also demonstrated in corneal epithelial and endothelial cells of rejected rat corneal allografts (32). With this in mind, we examined CD8+ and CD8 T cells from rejector mice for their capacity to induce apoptosis of corneal epithelial and endothelial cells in vitro. Spleen cells were enriched for CD8+ and CD8 T cells using magnetic beads. Following enrichment, the CD8+ T cell population was treated with anti-CD4 monoclonal antibody and complement, while the CD8 T cell population was treated with anti-CD8 monoclonal antibody and complement. The capacity of each cell population to induce apoptosis was tested using a 24-hr three-color apoptosis assay. The results indicated that CD8 T cells induced significant, albeit weak, apoptosis of allogeneic corneal epithelial cells (Figure 5A). By contrast, both CD8+ and CD8 T cells induced extensive apoptosis of allogeneic corneal epithelial and endothelial cells (Figure 5B). Importantly, the apoptosis was antigen specific, as spleen cells from hosts that rejected BALB/c corneal allografts did not induce apoptosis of C57BL/6 corneal endothelial cells (data not shown). Spleen cells from naïve C57BL/6 mice stimulated in vitro with γirradiated BALB/c spleen cells prior to the in vitro apoptosis assay did not induce apoptosis that was greater than the spontaneous apoptosis found in the medium control, thereby confirming that the in vitro boosting procedure did not induce de novo generation of anti-BALB/c effector cells.

FIGURE 5.

FIGURE 5.

The extensive apoptosis of allogeneic corneal endothelial cells by CD8 T cells from CD4 KO mice was perplexing, as this cell population should not contain CD4+ cells. Accordingly, this assay was repeated using similar CD4 KO donors that had rejected their BALB/c corneal allografts. However, in addition to depleting CD8+ and CD4+ cells, the cell suspensions were also treated with anti-Thy 1.2 monoclonal antibody plus complement to remove any putative CD4,8 T cells (i.e., double negative T cells). Flow cytometric analysis revealed that anti-Thy 1.2 antibody plus complement depleted >97% of the Thy 1.2+ cells (data not shown). Apoptosis assay were performed as before and the results indicated that treatment with anti-Thy 1.2 antibody in the presence of complement abolished the apoptosis-inducing population of CD4,8 cells, thus confirming that a double negative T cell was involved in the apoptosis of corneal endothelial cells (Figure 6).

FIGURE 6.

FIGURE 6.

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DISCUSSION

Although the immune basis for corneal allograft rejection was formally established in experimental animals by Maumenee (33) over 50 years ago, the exact immune mechanisms that lead to the corneal graft failure remain poorly understood. The results reported here reaffirm previous findings indicating that T cells are absolutely required for corneal allograft rejection in the mouse (3). As previously reported, CD4+ T cells play a major role in corneal graft rejection, yet approximately half of corneal allografts undergo rejection in the absence of CD4+ T cells (3, 12–14).On the surface one would conclude that the CD4+ T cell-independent rejection was mediated by allospecific CD8+ T cells, which can arise in the absence of CD4+ T helper cells (20, 21, 34, 35). Indeed, our results indicate that adoptive transfer of CD8+ T cells from CD4 KO mice that had rejected corneal allografts resulted in the rejection of 95% of the corneal allografts transplanted to athymic recipients. By contrast, adoptive transfer of CD8 spleens cells from similar donors resulted in the rejection of corneal allografts in almost half of the hosts.

The capacity of CD8+ T cells to mediate corneal allograft rejection could have been due to the CTL population that might have been present in the CD8+ T cell suspensions used in the adoptive transfer inocula. The CD8+ T cells were already primed, as they were collected from CD4 KO mice that had rejected corneal allografts and thus, may have functioned differently from CD8+ T cells from naïve CD4 KO mice. However, in vitro assays using spleen cells from CD4 KO mice that had rejected BALB/c corneal allografts failed to detect CTL activity against donor corneal epithelial or endothelial cells. These results are consistent with previous studies indicating that corneal allograft rejection does not normally elicit allospecific CTL responses in mice (8, 9) and occurs in both CD8 KO and perforin KO mice. Numerous studies have demonstrated a close correlation between the development of donor-specific DTH and corneal allograft rejection (3, 11, 14, 36). The present results indicate that athymic recipients of CD8+ T cells from CD4 KO donors that had rejected corneal allografts did not mount DTH responses to donor histocompatibility antigens. However, lymphocytes isolated from recipients of either CD8+ T cells or CD8 T cells produced significant in vitro apoptosis of donor-specific corneal endothelial cells. The present demonstration of T cell-mediated apoptosis of allogeneic corneal cells from CD4 KO mice is consistent with previous findings, which noted the presence of apoptotic keratocytes and corneal endothelial cells in rejected corneal allografts in humans and rats respectively (5, 32). In our study, CD8 T cells from CD4 KO rejector mice failed to display CTL or DTH activity, yet they were capable of inducing donor-specific apoptosis of corneal endothelial cells.

The capacity of CD8 T cells from CD4 KO donors to mediate corneal allograft rejection is puzzling and on the surface, counterintuitive, since these cells are presumably double negative (DN) T cells. The role of DN T cells in corneal allograft rejection was confirmed in two separate in vitro assays in which CD8 cells were isolated from CD4 KO donors that had rejected corneal allografts and were found to induce apoptosis of donor-specific corneal cells. The putative DN cells were isolated by negative selection using anti-CD8 magnetic bead isolation followed by in vitro treatment with anti-CD8 antibody and complement. Flow cytometric analysis of the cells following anti-CD8 antibody and complement treatment indicated that <2% were CD8+. These cells produced significant apoptosis of BALB/c corneal endothelial cells, but did not affect third-party corneal endothelial cells. Moreover, the allospecific apoptotic activity of the putative DN T cells was abolished in two separate experiments using in vitro treatment with anti-Thy 1.2 antibody plus complement, which removed >97% of the Thy 1+ cells, as determined by flow cytometry. The capacity of DN T cells to kill allogeneic target cells in vitro and mediate tumor allograft rejection in vivo has been previously reported by Young et al. (37). In their study, DN T cells displayed allospecific cytotoxicity in vitro and mediated tumor allograft rejection in vivo.

The present findings are derived from studies using CD4KO mice and thus, raise the question as to whether the CD4+ T cell-independent immune mechanisms in CD4 KO mice differ from those involved in corneal allograft rejection in wild-type mice whose CD4+ T cells population have been depleted with monoclonal antibodies. However, at the very least, these studies demonstrate that CD4+ T cell-independent mechanisms can mediate corneal allograft rejection. In CD4 KO mice, T cell-independent rejection can involve either CD8+ or CD8 T cells. Rejection by CD8+ T cells does not appear to involve either allospecific CTL or DTH effector mechanisms; however, CD8+ T cells are able to induce significant apoptosis of allogeneic corneal endothelial cells. By contrast, DN T cells are able to mediate DTH to donor alloantigens and induce apoptosis of donor-specific corneal endothelial cells. Either of these effector mechanisms might contribute to corneal allograft rejection. These findings also demonstrate the remarkable plasticity and redundancy in the immune mechanisms that mediate allograft rejection. Removing one immune effector element only reveals the presence of an ancillary pathway for allograft rejection.

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Keywords:

Apoptosis; CD4− T cells; Double-negative T cells; Corneal allograft

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