September 2016
Volume 57, Issue 11
Open Access
Cornea  |   September 2016
Allogeneic Sensitization and Tolerance Induction After Corneal Endothelial Cell Transplantation in Mice
Author Affiliations & Notes
  • Jun Yamada
    Department of Ophthalmology Kyoto Prefectural University of Medicine, Kyoto, Japan
    Department of Ophthalmology, Meiji University of Integrative Medicine, Kyoto, Japan
    Department of Mechanism of Aging, Research Institute, National Center for Geriatrics and Gerontology, Aichi, Japan
  • Morio Ueno
    Department of Ophthalmology Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Munetoyo Toda
    Department of Ophthalmology Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Katsuhiko Shinomiya
    Department of Ophthalmology Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Chie Sotozono
    Department of Ophthalmology Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Shigeru Kinoshita
    Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Junji Hamuro
    Department of Ophthalmology Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Correspondence: Jun Yamada, Department of Ophthalmology, Kyoto Prefectural University of Medicine, 465 Kajiicho Hirokoji-agaru, Kawaramachi-dori Kamigyo-ku, Kyoto 602-0841, Japan; jyamada@koto.kpu-m.ac.jp
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 4572-4580. doi:10.1167/iovs.15-19020
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Jun Yamada, Morio Ueno, Munetoyo Toda, Katsuhiko Shinomiya, Chie Sotozono, Shigeru Kinoshita, Junji Hamuro; Allogeneic Sensitization and Tolerance Induction After Corneal Endothelial Cell Transplantation in Mice. Invest. Ophthalmol. Vis. Sci. 2016;57(11):4572-4580. doi: 10.1167/iovs.15-19020.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose: We evaluated the allogeneic response after corneal endothelial cell transplantation in the anterior chamber (AC) in a new mouse model by examining the acquisition of a delayed-type hypersensitivity (DTH) response, induction of allogeneic AC-associated immune deviation (ACAID), and acquisition of delayed transplantation tolerance.

Method: The corneal eyecups from C57BL/6 mice were prepared. The epithelial layer was detached with EDTA solution and treated with trypsin to release mouse-derived primary corneal endothelial cells (mpCECs). The mpCECs (1 × 104 cells) were transplanted into the AC of the eye or subcutaneously (SC) into the neck of BALB/c mice. In the mouse model of endothelial cell transplantation, the endothelial cells in a 2-mm central area of the cornea were eliminated by cryoinjury. The mpCEC transplant model was evaluated by measuring allogeneic cell survival and corneal thickness. The allospecific DTH response and ACAID induction were evaluated 1 week after transplantation. The long-term transplantation tolerance was evaluated by observing a secondary penetrating keratoplasty (PKP) performed on the same donor C57BL/6 mice.

Results: The SC injection of mpCECs induced a DTH response, whereas the AC injection induced ACAID. However, eyes inflamed by cryoinjury showed neither the DTH response nor ACAID following AC injection. The mpCECs survived for at least 1 week after injection. Penetrating keratoplasty allografts at 8 weeks after mpCEC transplantation survived indefinitely (100%).

Conclusions: The mpCECs display low allogenicity in the AC and are capable of inducing allogeneic tolerance. Corneal endothelial cell transplantation into the AC may represent a safe technique for allogeneic transplantation.

The corneal endothelial cells (CECs) are important for their pump and barrier functions, which maintain corneal transparency.1 The loss of a significant number of CECs results in a type of corneal edema called bullous keratopathy, which can lead to severe visual impairment. Because CECs in humans are severely limited in their proliferative ability,24 restoration to clear vision has required corneal transplantation in the form of full-thickness corneal transplantation, Descemet's stripping automated endothelial keratoplasty (DSAEK), or Descemet's membrane endothelial keratoplasty (DMEK).57 Tissue transplantation usually requires the use of immunosuppressive treatments to suppress ocular inflammation and allograft rejection. Thus, less-invasive techniques for CEC transplantation are highly desired. 
Recently, many groups have established new treatment methods using CEC sheets, prepared by cultivating CECs on amniotic membranes,8 collagen sheets,914 or without carrier materials,15 as practical clinical interventions to repair corneal endothelial dysfunctions. However, the artificial or biological sheet substrate may introduce several problems, including substrate nontransparency, detachment of the cell sheet from the cornea, or technical difficulties in transplantation. Recently, the cultivation of CECs in combination with a Rho kinase (ROCK) inhibitor has been shown to enhance the adhesion of cells injected onto the recipient corneal tissue without the use of a substrate. This technique resulted in the successful recovery of corneal transparency in animal models16 of corneal endothelial dysfunction. The transplantation of cultivated CECs is now undergoing a course of clinical trials. 
Allograft rejection is always the main complication after allogeneic transplants, especially after penetrating keratoplasty.17,18 The donor-derived graft epithelium, but not the endothelium, has been implicated in the induction of alloimmunity, because chimeric allografts consisting of the host corneal epithelium over an epithelial-denuded allogeneic cornea showed no graft rejection in mice.19,20 In fact, the clinical rejection ratio is very much lower following DSAEK and DMEK transplantation, in which donor-derived epithelial cells are not transplanted to the recipients. However, endothelial cell rejection still can be observed even after DSAEK and DMEK transplants.21 Therefore, the allogeneic reactions occurring subsequent to CEC transplantation require more research. 
Unlike solid tissue transplantations, many CECs injected into the anterior chamber (AC) do not adhere to the inner corneal surface. Anterior chamber–associated immune deviation (ACAID), characterized by a selective deficiency of antigen-specific delayed-type hypersensitivity (DTH) following AC administration of antigens or allogeneic splenocytes, is one process that contributes to ocular immune privilege.22,23 This finding has raised questions concerning the ability of alloantigens expressed on corneal endothelial cells to induce ACAID and to contribute to the survival of transplanted CECs. In fact, corneal allograft survival was promoted by the induction of ACAID through the injection into AC of either allogeneic splenocytes or corneal endothelial cell lines.24 
The mouse penetrating keratoplasty model confirmed that allografted acceptor mice displaying no rejection episodes for 8 weeks had acquired donor-specific tolerance25 (i.e., those acceptors never rejected secondary corneal allografts from the same donor). Induction of this kind of delayed tolerance after CEC transplantation would be highly advantageous for avoiding allorejection. For this reason, we examined the alloimmunogenicity of CECs injected into the AC by excising murine primary CECs (mpCECs) and creating a mouse CEC transplantation model. The fates of the donor mpCECs were examined clinically and histologically, and the ability of recipient mice to develop DTH was assessed over time. In addition, we investigated the ability of eyes transplanted with mpCECs to support ACAID and to display the induction of chronic tolerance. 
Materials and Methods
Animals
Male BALB/c (H-2d), C57BL/6 (H-2b), and C3H/HeN (H-2k) mice (SLC, Osaka, Japan), aged between 8 and 12 weeks, were used in the experiments. All animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All experiments were approved by the Committee for Animal Research, Kyoto Prefectural University of Medicine. Before any surgical procedures, all animals were deeply anesthetized with an intraperitoneal injection of 3 mg ketamine. 
Preparation of mpCECs
The corneal eyecups, excluding the corneal limbus, were dissected from donor C57BL/6 mice and incubated in 20 mM EDTA at 37°C for 20 minutes, followed by a wash with sterile Hanks's balanced salt solution (HBSS). Under a dissecting microscope, the epithelial layer was peeled off to prepare the subjacent stromal endothelium. The resulting stroma-endothelium eyecups were incubated in Tryple Express (Invitrogen Co., Carlsbad, CA, USA), containing EDTA-Trypsin, at 37°C for 30 minutes. The released mpCECs were collected by gentle pipetting. Almost 1.5 × 105 mpCECs could be collected from 10 corneas. In some experiments, the mpCECs were labeled using a PKH26 Red Fluorescent cell linker kit (Sigma-Aldrich Corp., St. Louis, MO, USA) before transplantation. 
Cryoinjury Treatment and CEC Transplantation
Endothelial cells were eliminated by cryoinjury inflicted on the right eye of BALB/c recipients.26,27 Briefly, a topical mydriatic agent (Mydrin-P; Santen Pharmaceutical Co., Inc., Osaka, Japan) was applied and transcorneal freezing was initiated by gently placing a stainless steel cryoprobe (2-mm diameter), precooled to −196°C in liquid nitrogen, onto the central cornea. No pressure was applied to avoid damage to adjacent tissues, including the lens and the trabecular meshwork. The cryoprobe was maintained on the corneal surface for approximately 10 seconds, the time required for an ice ball to form on the cornea and to cover the entire corneal surface, because the defect on the endothelium was the same size as the ice ball.28 Immediately after freezing, the cryoprobe was freed from the corneal surface by irrigation with a balanced salt solution, and the cornea was allowed to thaw spontaneously. No topical medication was applied during the study period. 
After eliminating the endothelial cells by cryoinjury, an oblique incision was made at the central cornea of the host BALB/c mice with a microknife and 3 μL aqueous humor was removed with a glass needle. Then, 1 × 104 mpCECs in 3 μL HBSS were injected into the AC without any leakage. This cell number was calculated based on the 5 × 105 cells typically used in human cases. A control group with injection of 3 μL HBSS, without endothelial cell injection, was compared with the experimental groups. The mice were kept immobile as the corneal side down for 3 hours by an additional injection of 1 mg ketamine per hour to allow the mpCECs to precipitate onto the endothelium surface. 
Clinical and Histopathologic Evaluation
Clinical evaluation consisted of assessing the eyes by slit-lamp biomicroscopy twice a week. Corneal rejection and opacity were scored in accordance with a previous scoring system.29 Corneal thicknesses were measured with a pachymeter (SP-100; Tomey, Nagoya, Japan) at the appropriate time intervals. 
For histologic evaluation, eyes were enucleated at specified times, fixed in 4% paraformaldehyde at 4°C for 3 days, dissected to make corneal eyecups, flat-mounted after making four incisions, and mounted with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Sections were evaluated by fluorescence microscopy (OLYMPUS, Tokyo, Japan). 
Assessment of Delayed Hypersensitivity and ACAID Induction
The donor-specific DTH against alloantigens was assessed by injecting 10 μL HBSS containing 1 × 106 Mitomycin C (MMC)-treated spleen cells from C57BL/6 mice into the right ear pinnae at specific time points subsequent to mpCEC injection. As a positive control, similar numbers of spleen cells were injected into the ear pinnae of BALB/c mice immunized 1 week previously by a subcutaneous injection of 1 × 106 C57BL/6 spleen cells. At 24 and 48 hours after this ear challenge, the ear thickness was measured with a low-pressure micrometer (Mitutoyo; MTI Corp., Paramus, NJ, USA). 
The donor-specific ACAID induction after intracameral injection of mpCECs was evaluated by immunizing mpCECs transplanted, positive, and ACAID control mice with a subcutaneous injection of 1 × 106 C57BL/6 splenocytes. The ACAID control group of mice received an AC injection of 1 × 105 C57BL/6 spleen cells 1 week before immunization.30 Seven days later, 10 μL HBSS containing 1 × 106 MMC-treated C57BL/6 spleen cells was injected into the right pinnae. At 24 and 48 hours after the ear challenge, ear thickness was measured as described above. 
Orthotopic Corneal Transplantation and Assessment of Graft Rejection
To determine the capacity for the mpCECs transplanted recipient to reject allogeneic mpCECs, we transplanted secondary healthy C57BL/6 corneas onto the eyes of BALB/c mice that had previously received C57BL/6 mpCECs into the AC or intravenously. The corneal transplantation technique has been described elsewhere.29 Briefly, the central 2 mm of the donor cornea was excised and secured in a recipient graft bed with eight interrupted 11–0 nylon sutures (Sharppoint; Vanguard, Houston, TX, USA). All transplant sutures were removed on day 7. All grafts were examined twice a week with a slit-lamp biomicroscope. At each time point, grafts were scored from 0 to 5+ for opacification, using a previously described scoring system.29 Grafts exhibiting an opacity score of 3+ or greater (moderate stromal opacity with only pupil margin visible) at 2 weeks, or 2+ or greater (mild deep [stromal] opacity with pupil margin and iris vessels visible) after 3 weeks, were considered as rejected (immunological failure). 
Statistical Methods
We constructed Kaplan-Meier survival curves and used the Breslow-Gehan-Wilcoxon test to compare the probabilities of allograft survival. Student's t-test was used to compare ear-swelling responses. P values less than 0.05 were deemed statistically significant. 
Results
The Preparation of the Allogeneic mpCEC Transplantation Model and the Corneal Outcome
We have developed a new corneal endothelial cell transplantation model, as described above. The cryoinjured corneas with corneal endothelial cell damage at the central cornea were opacified and edematous at 1 day after injury. When the mpCEC-transplanted group (mpCEC Tp; n = 10) was compared with the nontransplanted group (HBSS vehicle–only injection) (no Tp; n = 10), the opacity was significantly recovered in the mpCEC-Tp group at postoperative days 1 and 3 (Fig. 1A). This corneal opacity was gradually recovered and the corneas became transparent at approximately postoperative day 7 in both groups. All the corneal shapes were then identical between the two groups for the next 56 days. Particularly, the corneas of mpCEC-Tp eyes did not display any clinical abnormalities, including corneal opacity, edema, and others that are associated with the criteria of mice allograft rejection in penetrating keratoplasty. The corneal thickness, as an evaluation of corneal edema, was significantly recovered by postoperative day 3 in the mpCEC-Tp group, when compared with the no-Tp group (Fig. 1B, P < 0.01). This edema subsided by postoperative day 7 in both groups. 
Figure 1
 
Clinical and histologic appearance after allogeneic mpCEC transplantation. (A) The clinical appearance in a cryoinjury-only control (no Tp) at 1 day (a) and 3 days (c) and mpCEC transplantation (mpCEC Tp) at 1 day (b) and 3 days (d) subsequent to cryoinjury. (B) Corneal thickness at 3 days and 1 week after cryoinjury. The mean thickness was compared between the no-Tp and mpCEC-Tp groups. *P < 0.01. (C) The presence of PKH-labeled mpCECs 7 days after injection (b, c, d, e). Flat-mounted corneas were observed by fluorescence microscopy. The white dotted line shows the estimated endothelial defect area created by cryoinjury. Cryoinjured corneas without mpCEC injection were used as a control (a).
Figure 1
 
Clinical and histologic appearance after allogeneic mpCEC transplantation. (A) The clinical appearance in a cryoinjury-only control (no Tp) at 1 day (a) and 3 days (c) and mpCEC transplantation (mpCEC Tp) at 1 day (b) and 3 days (d) subsequent to cryoinjury. (B) Corneal thickness at 3 days and 1 week after cryoinjury. The mean thickness was compared between the no-Tp and mpCEC-Tp groups. *P < 0.01. (C) The presence of PKH-labeled mpCECs 7 days after injection (b, c, d, e). Flat-mounted corneas were observed by fluorescence microscopy. The white dotted line shows the estimated endothelial defect area created by cryoinjury. Cryoinjured corneas without mpCEC injection were used as a control (a).
The survival of the donor-derived mpCECs was confirmed by transplanting PKH-labeled mpCECs and then evaluating them histologically in flat-mounted corneas at postoperative day 7 (Fig. 1C). The PKH-labeled mpCECs were present and healthy at postoperative day 7 (Fig. 1Cb), but the PKH-labeled negative cells were also chimerically present (Figs. 1Cc–e). Interestingly, PKH-labeled mpCECs were located only at the central area of the endothelial layer corresponding to the cryoinjured area. 
Failure of Allogeneic Sensitization in mpCEC Transplantation
Several observations confirmed that the transplanted mpCECs could adhere to the endothelial cell defect areas and survive for more than 7 days. Thus, these transplanted cells did not induce the typical corneal allograft rejection, characterized by corneal opacity and inflammation. The apparent allogenicity of the mpCECs was confirmed by injecting 1 × 104 mpCECs subcutaneously into the necks of mice and assessing the subsequent allogeneic DTH response. One week after mpCEC injection, the mice showed a significant DTH response, indicating that the mpCECs were allogeneic (Fig. 2a). 
Figure 2
 
Capacity of mpCECs to induce donor-specific DTH: (a) 1 × 104 mpCECs were injected subcutaneously; (b) 1 × 104 C57BL/6 splenocytes or 1 × 104 mpCECs were injected into the AC of “normal” eyes; (c) 1 × 104 C57BL/6 splenocytes or 1 × 104 mpCECs were injected into the AC of the eyes subsequent to cryoinjury. Positive controls received subcutaneous injection of 1 × 106 spleen cells. One week later, delayed hypersensitivity was assessed. Mean responses compared with negative controls, *P < 0.01.
Figure 2
 
Capacity of mpCECs to induce donor-specific DTH: (a) 1 × 104 mpCECs were injected subcutaneously; (b) 1 × 104 C57BL/6 splenocytes or 1 × 104 mpCECs were injected into the AC of “normal” eyes; (c) 1 × 104 C57BL/6 splenocytes or 1 × 104 mpCECs were injected into the AC of the eyes subsequent to cryoinjury. Positive controls received subcutaneous injection of 1 × 106 spleen cells. One week later, delayed hypersensitivity was assessed. Mean responses compared with negative controls, *P < 0.01.
The AC of the eye is a known immune-privileged site. Therefore, we wished to confirm the capacity for acquiring the DTH response following mpCEC injection into the AC. Intracameral injection of 1 × 104 mpCECs into nontreated BALB/c eyes did not induce an allogeneic DTH response in those mice, nor did injection of 1 × 104 C57BL/6 splenocytes (Fig. 2b). The intracameral injection of allogeneic splenocytes is well known to elicit allospecific ACAID and to suppress the DTH response when the eyes are noninflamed normal eyes. Thus, a similar experiment was performed to examine cryoinjured inflamed eyes. When 1 × 104 C57BL/6 splenocytes were injected into the AC immediately after cryoinjury, the mice showed the DTH response. Surprisingly, however, injection of 1 × 104 mpCECs into the ACs of cryoinjured inflamed eyes did not elicit the allogeneic DTH response in those mice (Fig. 2c). Those results clearly indicate that mpCECs lose the capacity for allogeneic DTH induction. 
The Loss of DTH Acquisition Is Not Due to the ACAID Induction
The AC injection of allogeneic cells is well known to suppress the donor-specific DTH response called ACAID, which may correlate with rejection episodes. Thus, the capacity to induce ACAID by mpCECs was examined. First, 1 × 104 mpCECs or C57BL/6 splenocytes were injected into the ACs of normal BALB/c eyes. One week later, the mice and positive control mice were immunized by subcutaneous injection of 1 × 106 C57BL/6 splenocytes. An additional week later, the DTH response was measured. The intracameral injection of splenocytes significantly suppressed the DTH response, indicating ACAID induction. Intracameral injection of 1 × 104 mpCECs also suppressed the DTH response, indicating that mpCECs also have the capacity to induce ACAID (Fig. 3a). 
Figure 3
 
Capacity of mpCECs to induce donor-specific ACAID as revealed by subsequent subcutaneous immunization with 1 × 106 spleen cells 1 week after AC injection. Cells (1 × 104 splenocytes or 1 × 104 mpCECs) were injected into the AC of BALB/c mice with (a) “normal” eyes or (b) cryoinjured eyes. One week after subcutaneous immunization, delayed hypersensitivity was assessed. Mean responses were compared with the responses of positive controls that received subcutaneous injection of 1 × 106 spleen cells 1 week before an ear challenge, *P < 0.01.
Figure 3
 
Capacity of mpCECs to induce donor-specific ACAID as revealed by subsequent subcutaneous immunization with 1 × 106 spleen cells 1 week after AC injection. Cells (1 × 104 splenocytes or 1 × 104 mpCECs) were injected into the AC of BALB/c mice with (a) “normal” eyes or (b) cryoinjured eyes. One week after subcutaneous immunization, delayed hypersensitivity was assessed. Mean responses were compared with the responses of positive controls that received subcutaneous injection of 1 × 106 spleen cells 1 week before an ear challenge, *P < 0.01.
We confirmed that the loss of the DTH response, shown in Figure 2c, was due to ACAID induction by performing a similar experiment on cryoinjured eyes. Immediately after cryoinjury, 1 × 104 mpCECs or C57BL/6 splenocytes were injected into the ACs of normal BALB/c eyes and the DTH response was measured as described above. No induction of ACAID was observed following the intracameral injection of splenocytes or of mpCECs into the inflamed eyes (Fig. 3b). Thus, the loss of the DTH response due to mpCECs was dependent on the site of immunization, but was not regulated by ACAID. 
Induction of Delayed-Type Allogeneic Tolerance 8 Weeks After mpCEC Transplantation
A serious clinical concern is that allogeneic CECs might be rejected in the long term after transplantation. A penetrating keratoplasty model in mice showed that the donor-specific DTH response was suppressed at 8 weeks postoperatively, as no mice showed rejection episodes and graft rejection was never observed after postoperative week 8.31 Moreover, corneal regrafts from the same donor onto 8 weeks acceptor mice were never rejected, indicating that long-term acceptors acquired allogeneic tolerance.25 We clarified the induction of delayed-type tolerance after mpCEC transplantation by transplanting penetrating C57BL/6 corneal allografts into the eyes that had received mpCEC transplants 8 weeks earlier. As shown in Figure 4a, none of the allografts onto cryoinjury-only corneas (HBSS vehicle was injected into the AC) survived, but 40% of the allografts placed onto normal eyes survived indefinitely. This result indicates that the cryoinjured eyes were high-risk eyes even 8 weeks after the operation. In contrast, 100% of the C57BL/6 allografts onto mpCEC-transplanted eyes survived indefinitely. To clarify the capacity of allograft acceptance at the earlier time point after CEC transplantation, penetrating C57BL/6 corneal allografts were performed at 1 week after mpCEC transplantation. As shown in Figure 4b, none of the allografts onto the eyes that had received mpCEC transplants 1 week earlier survived, similarly as the allografts onto cryoinjured-only corneas. Moreover, this incidence of rejection was significantly higher than normal control eyes. This result indicates that a 1-week interval is not enough to induce the delayed-type tolerance, unlike ACAID induction. Second, the donor-specificity was evaluated. The mpCEC-transplanted mice received corneal allografts from C3H mice that shared no major histocompatibility complex molecules with either C57BL/6 or BALB/c mice (third-party allogeneic). All C3H corneal allografts onto the cryoinjured eyes with or without injection of mpCEC intracamerally were rejected within 4 weeks (n = 10 each). In contrast, all of C57BL/6 allografts survived indefinitely (Fig. 4c). These results indicate that the donor-specific corneal acceptance was acquired by the mpCEC-transplanted mice in an antigen-specific manner. To confirm the role of immune cells in mpCEC-transplanted mice, we examined the ability of mpCEC-transplanted mice–derived lymphocytes to transfer corneal graft acceptance to naive mice. We intravenously injected naive BALB/c mice with 5 × 107 mpCEC-transplanted mice–derived splenocytes and grafted C57BL/6 corneas onto their eyes. As shown in Figure 4d, splenocytes from mpCEC-transplanted mice promoted the survival of the C57BL/6 allografts (n = 9). In contrast, in BALB/c mice adoptively transferred with splenocytes from naive BALB/c mice, allograft survival was not prolonged (n = 9, P < 0.001). These results suggest that corneal allograft acceptance is not due to immune ignorance and splenocytes are involved in the immune response to corneal allografts. Therefore, the mpCEC transplantation was also able to induce delayed-type tolerance and suppress further rejection. 
Figure 4
 
Fate of C57BL/6 full-thickness corneal allografts in BALB/c mice. (a, b) The corneas of BALB/c mice were subjected to cryoinjury and then the ACs were injected with 1 × 104 C57BL/6 mpCECs (open circle) or HBSS vehicle (closed square). Eight weeks (a) or 1 week (b) after AC injection, these mice and naive control mice (closed circle) received C57BL/6 corneal allografts. *P < 0.0001. (c) The cornel cryoinjured BALB/c mice with AC injection of 1 × 104 C57BL/6 mpCECs were prepared. Eight weeks after AC injection, these mice received C57BL/6 (open circle) or C3H/HeN (closed square) corneal allografts. The control mice without AC injection also received C3H/HeN allografts (closed circle). *P < 0.0001. (d) Fate of C57BL/6 corneal allografts after adoptive transfer of 5 × 107 splenocytes of C57BL/6 mpCEC-transplanted BALB/c mice (open circle). Control BALB/c mice received 5 × 107 splenocytes of naive BALB/c mice intravenously (closed circle). *P < 0.001.
Figure 4
 
Fate of C57BL/6 full-thickness corneal allografts in BALB/c mice. (a, b) The corneas of BALB/c mice were subjected to cryoinjury and then the ACs were injected with 1 × 104 C57BL/6 mpCECs (open circle) or HBSS vehicle (closed square). Eight weeks (a) or 1 week (b) after AC injection, these mice and naive control mice (closed circle) received C57BL/6 corneal allografts. *P < 0.0001. (c) The cornel cryoinjured BALB/c mice with AC injection of 1 × 104 C57BL/6 mpCECs were prepared. Eight weeks after AC injection, these mice received C57BL/6 (open circle) or C3H/HeN (closed square) corneal allografts. The control mice without AC injection also received C3H/HeN allografts (closed circle). *P < 0.0001. (d) Fate of C57BL/6 corneal allografts after adoptive transfer of 5 × 107 splenocytes of C57BL/6 mpCEC-transplanted BALB/c mice (open circle). Control BALB/c mice received 5 × 107 splenocytes of naive BALB/c mice intravenously (closed circle). *P < 0.001.
Lack of DTH, ACAID, or Tolerance Induction Following Intravenous mpCEC Injection
Many of the transplanted mpCECs will probably leak into vascular circulation from the eyes, as well as adhere to the intracameral region of the eye. We investigated the potential immunological effect of mpCECs leaking into the circulation by assessing the DTH response after intravenous injection of 1 × 104 mpCECs. One week after injection, the mpCEC-injected mice showed no DTH response, similar to the response of mice with normal eyes that underwent AC injection with 1 × 104 mpCECs (Fig. 5A). However, subcutaneous immunization with 10 × 106 splenocytes 1 week after intravenous injection of 1 × 104 mpCECs did not suppress allospecific DTH, whereas injection of mpCECs into the AC did suppress it (Fig. 5B). These results clearly indicate that intravenously injected mpCECs showed immunological ignorance. 
Figure 5
 
Capacity of intravenously injected mpCECs to induce DTH, ACAID, or delayed tolerance in corneal allografts. The mpCECs (1 × 104 cells) were injected intravenously or directly into the AC of the “normal” eye. Positive controls received a subcutaneous injection of 1 × 106 C57BL/6 spleen cells and naive mice were prepared as a negative control. (A) Assessment of the donor-specific DTH response 1 week after injection. Mean responses were compared with negative controls, *P < 0.01. (B) The mpCEC-injected mice underwent subcutaneous immunization with 1 × 106 spleen cells 1 week after the mpCEC injection. Delayed hypersensitivity was assessed after an additional week. The mean responses were compared with positive controls, *P < 0.01. (C) BALB/c mice received C57BL/6 full-thickness corneas 1 week (a) or 8 weeks (b) after injection of mpCECs intravenously (n = 10; open circle) or into the AC (n = 10; open square) (these eyes were not subjected to cryoinjury). Positive (n = 10; closed square) and negative controls (n = 10; closed circle) also are shown.
Figure 5
 
Capacity of intravenously injected mpCECs to induce DTH, ACAID, or delayed tolerance in corneal allografts. The mpCECs (1 × 104 cells) were injected intravenously or directly into the AC of the “normal” eye. Positive controls received a subcutaneous injection of 1 × 106 C57BL/6 spleen cells and naive mice were prepared as a negative control. (A) Assessment of the donor-specific DTH response 1 week after injection. Mean responses were compared with negative controls, *P < 0.01. (B) The mpCEC-injected mice underwent subcutaneous immunization with 1 × 106 spleen cells 1 week after the mpCEC injection. Delayed hypersensitivity was assessed after an additional week. The mean responses were compared with positive controls, *P < 0.01. (C) BALB/c mice received C57BL/6 full-thickness corneas 1 week (a) or 8 weeks (b) after injection of mpCECs intravenously (n = 10; open circle) or into the AC (n = 10; open square) (these eyes were not subjected to cryoinjury). Positive (n = 10; closed square) and negative controls (n = 10; closed circle) also are shown.
The transplant rejection observed after intravenous mpCEC injection was also investigated. As shown in Figures 5Ca and 5Cb, C57BL/6 penetrating corneal allografts were performed on four groups of mice. The mpCECs (1 × 104 cells) were injected intravenously (experimental group) or into the AC of the “normal” eye (AC control). Positive controls received a subcutaneous injection of 1 × 106 C57BL/6 spleen cells and naive mice were prepared as a negative control. Then, BALB/c mice received C57BL/6 full-thickness corneas 1 week or 8 weeks after mpCEC injection. Importantly, these were noninflamed normal eyes and had not undergone cryoinjury. Only 30% of the C57BL/6 penetrating corneal allografts survived 1 week after mpCEC injection, either intravenously or into the AC of normal eyes. These rejection episodes were similar to those observed for allografts onto nonmanipulated normal eyes of naive mice (40% rejection), although allosensitized mice swiftly rejected 100% of the allografts (Fig. 5Ca). The capacity to induce delayed-type tolerance also was examined 8 weeks after intravenous injection of mpCECs; the results showed that intravenous injection of mpCECs did not increase the survival of penetrating corneal allografts (Fig. 5Cb; 30% survival). This incidence of rejection is similar to that seen for corneal allografts onto normal eyes (40% survival), although subcutaneously sensitized mice swiftly rejected allografts (0% survival). These results indicate that intravenous injection of mpCECs did not induce tolerance, even 8 weeks after the injection. Moreover, mice that had not experienced cryoinjury still showed no delayed-type tolerance following injection of mpCECs into the AC. This result indicates the necessity for the survival of the allogeneic mpCECs at the inside corneal surface for induction of delayed-type tolerance. 
Discussion
We have developed a new model of allogeneic CEC transplantation in mice. The experimental results indicate that mpCECs injected into the AC have no capacity to induce the allogeneic DTH response, even when the eyes are inflamed. Moreover, mpCECs that adhered to the inside surface of the cornea did not show graft rejection and did induce allograft tolerance 8 weeks after injection. These results indicate that mpCEC transplantation may be protected from allogeneic rejection, supporting the concept that human CEC transplantation may be a safe and ideal form of allotransplantation. 
This new murine model of mpCEC transplantation is somewhat different from CEC transplantation in clinical settings in humans. First, the mpCECs were able to proliferate in vivo. In fact, the adhered mpCECs and the proliferated host endothelial cells survived chimerically in this model. Second, human endothelial cells are eliminated manually by a scraping technique, rather than by cryoinjury. Eyes experiencing inflammation due to keratoplasty, corneal cauterization, and corneal sutures do not support ACAID induction.32 From this standpoint, scraping, as well as cryoinjury, could change the microenvironment of the eye to one that does not support ACAID induction. Third, immunosuppressive agents are always applied clinically as part of human therapies. These agents suppress regulatory cell induction as well as allogeneic immune responses. The mild allogeneic rejection that does not support delayed-type tolerance is also difficult to evaluate. Last, AC injection of CECs in combination with a ROCK inhibitor (Y-27632) enhances the adhesion of injected cells in humans. The influence of ROCK inhibitors on the induction of allosensitization and delayed tolerance should be examined in the near future. Modification of the vehicle solution, addition of ROCK inhibitor,33 or other changes34,35 could result in the development of a more robust mouse model. 
Our results revealed that recipients undergoing mpCEC transplantation did not acquire a donor-specific DTH response. This finding indicates that the minimum usage of immunosuppressive agents is the appropriate treatment in humans. No clear reason can yet explain why intracameral injections of mpCECs did not sensitize the hosts, whereas subcutaneous injections did. The observed suppression was not due to ACAID induction. One possibility is that the immunogenicity of CECs is extremely low in the ACs of eyes in which immunosuppressive TGF-ß2 and others are present. 
The intracameral injection of mice primary CECs, as well as the CEC cell lines,24 has the capacity to induce ACAID. Corneal rejection is believed to associate with the DTH response, and because ACAID can suppress the donor-specific DTH response, the donor-specific induction of ACAID may provide an interesting strategy for enhancing allograft acceptance.24,36 However, we observed that cryoinjury abolished the eye's capacity to support ACAID induction (Fig. 3) and that mpCEC-induced ACAID could not promote the acceptance of penetrating corneal allografts (Fig. 5Ca, 5Cb). We have not yet been able to show any benefit of CEC-induced ACAID on allograft acceptance. 
In contrast, delayed tolerance can perfectly suppress allogeneic reactions (100% acceptance) and this was induced in all mice who underwent mpCEC transplantation (100% induction). Delayed tolerance seems to generate a stronger regulation than ACAID induction. Cunnusamy et al.37 displayed that regulatory T cells (Tregs) induced by corneal allograft are different from Tregs induced by ACAID. The identification of the Tregs and the mechanism of delayed tolerance are the critical issues elusive in the future study. Confirmation of this delayed tolerance after transplants in humans will allow indefinite graft survival without a requirement for any immunosuppressants. Surprisingly, transplanted mpCECs were able to induce tolerance to a full-thickness cornea, including epithelial cells, stromal cells, and donor-derived immature antigen-presenting cells.38 The tissue-specific antigens seem to be the same among these layers. 
These findings have raised questions concerning tolerance induction without allosensitization. One possible explanation for the sensitization that supports delayed tolerance is that it takes approximately 3 weeks to induce the donor-specific DTH response, as seen with full-thickness corneal transplantation.39 Uncovering the mechanism of delayed tolerance induction is our next goal. 
Transplantation by CEC injection into the AC is clearly protected from allorejection. Even when the CECs directly leak out from the AC into veins, the CECs may not play any role in either sensitization or tolerance induction. Thus, CEC transplantation is one of the safest types of allogeneic transplantation. 
Acknowledgments
Supported in part by the Highway Program for Realization of Regenerative Medicine from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and the Program for the Promotion of Science from MEXT, Japan, and the Research Grant for Longevity Sciences from the Ministry of Health, Labour, and Welfare. 
Disclosure: J. Yamada, None; M. Ueno, None; M. Toda, None; K. Shinomiya, None; C. Sotozono, None; S. Kinoshita, None; J. Hamuro, None 
References
Bourne WM. Clinical estimation of corneal endothelial pump function. Trans Am Ophthalmol Soc. 1998; 96: 229–239. 239, discussion 239–242.
Engelmann K, Bohnke M, Friedl P. Isolation and long-term cultivation of human corneal endothelial cells. Invest Ophthalmol Vis Sci. 1988; 29: 1656–1662.
Joyce NC. Proliferative capacity of the corneal endothelium. Prog Retin Eye Res. 2003; 22: 359–389.
Yue BY, Sugar J, Gilboy JE, Elvart JL. Growth of human corneal endothelial cells in culture. Invest Ophthalmol Vis Sci. 1989; 30: 248–253.
Gorovoy MS. Descemet-stripping automated endothelial keratoplasty. Cornea. 2006; 25: 886–889.
Price FWJr, Price MO. Descemet's stripping with endothelial keratoplasty in 50 eyes: a refractive neutral corneal transplant. J Refract Surg. 2005; 21: 339–345.
Price FWJr, Price MO. Evolution of endothelial keratoplasty. Cornea. 2013; 32: S28–S32.
Ishino Y, Sano Y, Nakamura T, et al. Amniotic membrane as a carrier for cultivated human corneal endothelial cell transplantation. Invest Ophthalmol Vis Sci. 2004; 45: 800–806.
Shimmura S, Miyashita H, Konomi K, et al. Transplantation of corneal endothelium with Descemet's membrane using a hyroxyethyl methacrylate polymer as a carrier. Br J Ophthalmol. 2005; 89: 134–137.
Koizumi N, Sakamoto Y, Okumura N, et al. Cultivated corneal endothelial transplantation in a primate: possible future clinical application in corneal endothelial regenerative medicine. Cornea. 2008; 27: S48–S55.
Mimura T, Amano S, Usui T, et al. Transplantation of corneas reconstructed with cultured adult human corneal endothelial cells in nude rats. Exp Eye Res. 2004; 79: 231–237.
Hitani K, Yokoo S, Honda N, et al. Transplantation of a sheet of human corneal endothelial cell in a rabbit model. Mol Vis. 2008; 14: 1–9.
Lai JY, Chen KH, Hsiue GH. Tissue-engineered human corneal endothelial cell sheet transplantation in a rabbit model using functional biomaterials. Transplantation. 2007; 84: 1222–1232.
Mimura T, Yamagami S, Yokoo S, et al. Cultured human corneal endothelial cell transplantation with a collagen sheet in a rabbit model. Invest Ophthalmol Vis Sci. 2004; 45: 2992–2997.
Sumide T, Nishida K, Yamato M, et al. Functional human corneal endothelial cell sheets harvested from temperature-responsive culture surfaces. FASEB J. 2006; 20: 392–394.
Okumura N, Ueno M, Koizumi N, et al. Enhancement on primate corneal endothelial cell survival in vitro by a ROCK inhibitor. Invest Ophthalmol Vis Sci. 2009; 50: 3680–3687.
Maguire MG, Stark WJ, Gottsch JD, et al. Risk factors for corneal graft failure and rejection in the collaborative corneal transplantation studies. Collaborative Corneal Transplantation Studies Research Group. Ophthalmology. 1994; 101: 1536–1547.
The Collaborative Corneal Transplantation Studies (CCTS). Effectiveness of histocompatibility matching in high-risk corneal transplantation. The Collaborative Corneal Transplantation Studies Research Group. Arch Ophthalmol. 1992; 110: 1392–1403.
Saban DR, Chauhan SK, Zhang X, et al. ‘Chimeric' grafts assembled from multiple allodisparate donors enjoy enhanced transplant survival. Am J Transplant. 2009; 9: 473–482.
Hori J, Streilein JW. Role of recipient epithelium in promoting survival of orthotopic corneal allografts in mice. Invest Ophthalmol Vis Sci. 2001; 42: 720–726.
Monnereau C, Bruinsma M, Ham L, et al. Endothelial cell changes as an indicator for upcoming allograft rejection following descemet membrane endothelial keratoplasty. Am J Ophthalmol. 2014; 158: 485–495.
Niederkorn JY. Immune privilege and immune regulation in the eye. Adv Immunol. 1990; 48: 191–226.
Streilein JW. Immune regulation and the eye: a dangerous compromise. FASEB J. 1987; 1: 199–208.
Niederkorn JY, Mellon J. Anterior chamber–associated immune deviation promotes corneal allograft survival. Invest Ophthalmol Vis Sci. 1996; 37: 2700–2707.
Yamada J, Hamuro J, Sano Y, Maruyama K, Kinoshita S. Allogeneic corneal tolerance in rodents with long-term graft survival. Transplantation. 2005; 79: 1362–1369.
Han SB, Ang H, Balehosur D, et al. A mouse model of corneal endothelial decompensation using cryoinjury. Mol Vis. 2013; 19: 1222–1230.
Maumenee AE, Kornblueth W. Regeneration of the corneal stromal cells; technique for destruction of corneal corpuscles by application of solidified frozen, carbon dioxide. Am J Ophthalmol. 1948; 31: 699–702.
Khodadoust AA, Green K. Physiological function of regenerating endothelium. Invest Ophthalmol. 1976; 15: 96–101.
Sonoda Y, Streilein JW. Orthotopic corneal transplantation in mice––evidence that the immunogenetic rules of rejection do not apply. Transplantation. 1992; 54: 694–704.
Yamada J, Streilein JW. Induction of anterior chamber–associated immune deviation by corneal allografts placed in the anterior chamber. Invest Ophthalmol Vis Sci. 1997; 38: 2833–2843.
Sonoda Y, Streilein JW. Impaired cell-mediated immunity in mice bearing healthy orthotopic corneal allografts. J Immunol. 1993; 150: 1727–1734.
Streilein JW, Bradley D, Sano Y, Sonoda Y. Immunosuppressive properties of tissues obtained from eyes with experimentally manipulated corneas. Invest Ophthalmol Vis Sci. 1996; 37: 413–424.
Guo Y, Liu Q, Yang Y, et al. The effects of ROCK inhibitor Y-27632 on injectable spheroids of bovine corneal endothelial cells. Cell Reprogram. 2015; 17: 77–87.
Bartakova A, Kunzevitzky NJ, Goldberg JL. Regenerative cell therapy for corneal endothelium. Curr Ophthalmol Rep. 2014; 2: 81–90.
Moysidis SN, Alvarez–Delfin K, Peschansky VJ, et al. Magnetic field–guided cell delivery with nanoparticle-loaded human corneal endothelial cells. Nanomedicine. 2015; 11: 499–509.
Sano Y, Okamoto S, Streilein JW. Induction of donor-specific ACAID can prolong orthotopic corneal allograft survival in “high-risk” eyes. Curr Eye Res. 1997; 16: 1171–1174.
Cunnusamy K, Paunicka K, Reyes N, et al. Two different regulatory T cell populations that promote corneal allograft survival. Invest Ophthalmol Vis Sci. 2010; 51: 6566–6574.
Hamrah P, Liu Y, Zhang Q, Dana MR. Alterations in corneal stromal dendritic cell phenotype and distribution in inflammation. Arch Ophthalmol. 2003; 121: 1132–1140.
Sonoda Y, Sano Y, Ksander B, Streilein JW. Characterization of cell-mediated immune responses elicited by orthotopic corneal allografts in mice. Invest Ophthalmol Vis Sci. 1995; 36: 427–434.
Figure 1
 
Clinical and histologic appearance after allogeneic mpCEC transplantation. (A) The clinical appearance in a cryoinjury-only control (no Tp) at 1 day (a) and 3 days (c) and mpCEC transplantation (mpCEC Tp) at 1 day (b) and 3 days (d) subsequent to cryoinjury. (B) Corneal thickness at 3 days and 1 week after cryoinjury. The mean thickness was compared between the no-Tp and mpCEC-Tp groups. *P < 0.01. (C) The presence of PKH-labeled mpCECs 7 days after injection (b, c, d, e). Flat-mounted corneas were observed by fluorescence microscopy. The white dotted line shows the estimated endothelial defect area created by cryoinjury. Cryoinjured corneas without mpCEC injection were used as a control (a).
Figure 1
 
Clinical and histologic appearance after allogeneic mpCEC transplantation. (A) The clinical appearance in a cryoinjury-only control (no Tp) at 1 day (a) and 3 days (c) and mpCEC transplantation (mpCEC Tp) at 1 day (b) and 3 days (d) subsequent to cryoinjury. (B) Corneal thickness at 3 days and 1 week after cryoinjury. The mean thickness was compared between the no-Tp and mpCEC-Tp groups. *P < 0.01. (C) The presence of PKH-labeled mpCECs 7 days after injection (b, c, d, e). Flat-mounted corneas were observed by fluorescence microscopy. The white dotted line shows the estimated endothelial defect area created by cryoinjury. Cryoinjured corneas without mpCEC injection were used as a control (a).
Figure 2
 
Capacity of mpCECs to induce donor-specific DTH: (a) 1 × 104 mpCECs were injected subcutaneously; (b) 1 × 104 C57BL/6 splenocytes or 1 × 104 mpCECs were injected into the AC of “normal” eyes; (c) 1 × 104 C57BL/6 splenocytes or 1 × 104 mpCECs were injected into the AC of the eyes subsequent to cryoinjury. Positive controls received subcutaneous injection of 1 × 106 spleen cells. One week later, delayed hypersensitivity was assessed. Mean responses compared with negative controls, *P < 0.01.
Figure 2
 
Capacity of mpCECs to induce donor-specific DTH: (a) 1 × 104 mpCECs were injected subcutaneously; (b) 1 × 104 C57BL/6 splenocytes or 1 × 104 mpCECs were injected into the AC of “normal” eyes; (c) 1 × 104 C57BL/6 splenocytes or 1 × 104 mpCECs were injected into the AC of the eyes subsequent to cryoinjury. Positive controls received subcutaneous injection of 1 × 106 spleen cells. One week later, delayed hypersensitivity was assessed. Mean responses compared with negative controls, *P < 0.01.
Figure 3
 
Capacity of mpCECs to induce donor-specific ACAID as revealed by subsequent subcutaneous immunization with 1 × 106 spleen cells 1 week after AC injection. Cells (1 × 104 splenocytes or 1 × 104 mpCECs) were injected into the AC of BALB/c mice with (a) “normal” eyes or (b) cryoinjured eyes. One week after subcutaneous immunization, delayed hypersensitivity was assessed. Mean responses were compared with the responses of positive controls that received subcutaneous injection of 1 × 106 spleen cells 1 week before an ear challenge, *P < 0.01.
Figure 3
 
Capacity of mpCECs to induce donor-specific ACAID as revealed by subsequent subcutaneous immunization with 1 × 106 spleen cells 1 week after AC injection. Cells (1 × 104 splenocytes or 1 × 104 mpCECs) were injected into the AC of BALB/c mice with (a) “normal” eyes or (b) cryoinjured eyes. One week after subcutaneous immunization, delayed hypersensitivity was assessed. Mean responses were compared with the responses of positive controls that received subcutaneous injection of 1 × 106 spleen cells 1 week before an ear challenge, *P < 0.01.
Figure 4
 
Fate of C57BL/6 full-thickness corneal allografts in BALB/c mice. (a, b) The corneas of BALB/c mice were subjected to cryoinjury and then the ACs were injected with 1 × 104 C57BL/6 mpCECs (open circle) or HBSS vehicle (closed square). Eight weeks (a) or 1 week (b) after AC injection, these mice and naive control mice (closed circle) received C57BL/6 corneal allografts. *P < 0.0001. (c) The cornel cryoinjured BALB/c mice with AC injection of 1 × 104 C57BL/6 mpCECs were prepared. Eight weeks after AC injection, these mice received C57BL/6 (open circle) or C3H/HeN (closed square) corneal allografts. The control mice without AC injection also received C3H/HeN allografts (closed circle). *P < 0.0001. (d) Fate of C57BL/6 corneal allografts after adoptive transfer of 5 × 107 splenocytes of C57BL/6 mpCEC-transplanted BALB/c mice (open circle). Control BALB/c mice received 5 × 107 splenocytes of naive BALB/c mice intravenously (closed circle). *P < 0.001.
Figure 4
 
Fate of C57BL/6 full-thickness corneal allografts in BALB/c mice. (a, b) The corneas of BALB/c mice were subjected to cryoinjury and then the ACs were injected with 1 × 104 C57BL/6 mpCECs (open circle) or HBSS vehicle (closed square). Eight weeks (a) or 1 week (b) after AC injection, these mice and naive control mice (closed circle) received C57BL/6 corneal allografts. *P < 0.0001. (c) The cornel cryoinjured BALB/c mice with AC injection of 1 × 104 C57BL/6 mpCECs were prepared. Eight weeks after AC injection, these mice received C57BL/6 (open circle) or C3H/HeN (closed square) corneal allografts. The control mice without AC injection also received C3H/HeN allografts (closed circle). *P < 0.0001. (d) Fate of C57BL/6 corneal allografts after adoptive transfer of 5 × 107 splenocytes of C57BL/6 mpCEC-transplanted BALB/c mice (open circle). Control BALB/c mice received 5 × 107 splenocytes of naive BALB/c mice intravenously (closed circle). *P < 0.001.
Figure 5
 
Capacity of intravenously injected mpCECs to induce DTH, ACAID, or delayed tolerance in corneal allografts. The mpCECs (1 × 104 cells) were injected intravenously or directly into the AC of the “normal” eye. Positive controls received a subcutaneous injection of 1 × 106 C57BL/6 spleen cells and naive mice were prepared as a negative control. (A) Assessment of the donor-specific DTH response 1 week after injection. Mean responses were compared with negative controls, *P < 0.01. (B) The mpCEC-injected mice underwent subcutaneous immunization with 1 × 106 spleen cells 1 week after the mpCEC injection. Delayed hypersensitivity was assessed after an additional week. The mean responses were compared with positive controls, *P < 0.01. (C) BALB/c mice received C57BL/6 full-thickness corneas 1 week (a) or 8 weeks (b) after injection of mpCECs intravenously (n = 10; open circle) or into the AC (n = 10; open square) (these eyes were not subjected to cryoinjury). Positive (n = 10; closed square) and negative controls (n = 10; closed circle) also are shown.
Figure 5
 
Capacity of intravenously injected mpCECs to induce DTH, ACAID, or delayed tolerance in corneal allografts. The mpCECs (1 × 104 cells) were injected intravenously or directly into the AC of the “normal” eye. Positive controls received a subcutaneous injection of 1 × 106 C57BL/6 spleen cells and naive mice were prepared as a negative control. (A) Assessment of the donor-specific DTH response 1 week after injection. Mean responses were compared with negative controls, *P < 0.01. (B) The mpCEC-injected mice underwent subcutaneous immunization with 1 × 106 spleen cells 1 week after the mpCEC injection. Delayed hypersensitivity was assessed after an additional week. The mean responses were compared with positive controls, *P < 0.01. (C) BALB/c mice received C57BL/6 full-thickness corneas 1 week (a) or 8 weeks (b) after injection of mpCECs intravenously (n = 10; open circle) or into the AC (n = 10; open square) (these eyes were not subjected to cryoinjury). Positive (n = 10; closed square) and negative controls (n = 10; closed circle) also are shown.
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×