Article Type: Research
Received : 03/07/2018
Accepted : 26/07/2018
Published : 02/01/2019
*Correspondence to: Hidetaka Hara, Department of Surgery, University of Alabama at Birmingham, USA,
Article Type: Research
Received : 03/07/2018
Accepted : 26/07/2018
Published : 02/01/2019
Objective: To investigate the immune/inflammatory responses in monkeys after corneal xenotransplantation.
Materials and methods: Rhesus monkeys were carried out penetrating keratoplasty (PKP) or Descemet's stripping endothelial keratoplasty (DSEK)from wild-type pigs (WT, n=4) or α1,3-galactosyltransferase gene-knockout pigs expressing the human complement-regulatory protein CD46 (GTKO/CD46, n=3). The monkeys received local and systemic corticosteroid therapy. Follow-up was for 3 months. Monitoring included observation for clinical features of rejection, anti-Gal and anti-pig IgM/IgG antibodies, memory phenotypes, and intracellular cytokine production of T and B cells, and direct/indirect MLRs. Plasma C-reactive protein, and proinflammatory cytokines in plasma and aqueous humor were measured, as were cytokines in supernatants from MLRs.
Results: PKP/DSEK xenograftslost transparency early from the development of a retro corneal membrane or graft detachment, respectively. In the recipient, we documented increased(i) CD4+central and CD8+effector memory T cells,(ii)switched B cells of memory phenotype, (iii) TNF-α producing T and B cells, and (iv) anti-pig antibody production. Although most recipients did not show increased T cell proliferation in direct MLR, increased levels of TNF-α in the supernatants were detected. IFN-γ and IL-17 were increased in indirect MLR. IL-6, IL-8, TGF-β and MCP-1 were increased in aqueous humor. There were no significant increases of C-reactive protein or serum cytokines. WT and GTKO/CD46 pig corneas induced similar immune responses, except for the development of anti-Gal antibody after WT corneal transplantation.
Conclusions: Pig corneal xenografts in monkeys induce both systemic humoral and cellular immune responses (T and B cell sensitization), suggesting that systemic immunosuppressive therapy may be required to prevent immune/inflammatory responses.
KEY WORDS: Antibodies; Cornea; Cytokines; Inflammation; Pig, Genetically-Engineered; Xenotransplantation
ABBREVIATIONS: CECs: Corneal Endothelial Cells; DSEK: Descemet's Stripping Endothelial Keratoplasty; Gal: Galactose-Α1, 3-Galactose; GTKO: α1, 3-Galactosyltransferase Gene-Knockout; PAECs: Porcine Aortic Endothelial Cells; PBMCs: Peripheral Blood Mononuclear Cells; PKP: Penetrating Keratoplasty; RBCs: Red Blood Cells; SLA: Swine Leukocyte Antigen; WT: Wild-Type
The most critical factor restricting the widespread application of corneal transplantation is the shortage of human donor corneas (1). The pig could provide an alternative source of corneas for transplantation (1-3).Because the cornea (which is non-vascularized) is an immune-privileged tissue (4), its success in xenotransplantation could be greater than that of vascularized pig organs (5, 6). Indeed, several encouraging reports of wild-type (WT) pig-to-monkey corneal xenotransplantation have been published (7-9).In addition, decellularized anterior lamellar WT pig corneal transplants have been carried out to treat corneal ulcers in human patients, again with encouraging results (10). Both humoral and cellular immune responses are involved in pig corneal xenograft rejection in primates (5).Although hyper acute rejection has not been documented in either rodent or primate corneal xenotransplantation models (5), it has been suggested that both preformed anti-donor antibodies, e.g., anti-galactose-α1,3-galactose (Gal) antibodies, and complement activation play a role in graft rejection (8, 9, 11, 12). In nonhuman primate models, CD4+, CD8+ T cells, B cells, and macrophages infiltrate into the pig cornea (8, 9, 13). Furthermore, the number of CD8+T cells, especially of effect or phenotype, increase in the blood, and also play a role in xenograft rejection (8, 9, 14) . Recently, our group has reported that sustained systemic inflammation occurs in recipient nonhuman primates after pig organ xenotransplantation (and that this precedes coagulation dysfunction) (15, 16). Corneas are a vascular but, if neovascularized, are vascularized by host vessels; it is therefore unlikely that coagulation dysfunction will prove problematic. However, whether corneal xenografts will induce a systemic inflammatory response is unknown. Therefore, we investigated the inflammatory response in addition to the humoral and cellular immune responses.
In the clinic, corneal allotransplantation has usually involved full-thickness corneal transplantation (penetrating keratoplasty [PKP]). In the last decade, however, new surgical techniques of endothelial keratoplasty have been developed, e.g., Descemet's stripping endothelial keratoplasty (DSEK) and Descemet's membrane endothelial keratoplasty (17, 18). In these procedures, only the posterior cornea (including the endothelium) is replaced. These surgical procedures are suture less, and therefore might reduce any inflammatory response.
Although long-term corneal xenograft (full-thickness and anterior lamellar) survival has been reported following WT pig corneal transplantation in monkeys (7-9, 19), we speculated that corneas from genetically-engineered pigs, e.g., α1,3-galactosyltransferase gene-knockout pigs expressing the human complement-regulatory protein CD46 (GTKO/CD46), might be associated with reduced xenograft rejection (20).
We hypothesized that a pig corneal xenograft would induce systemic immune and inflammatory reactions in the recipient regardless of (i) whether the graft was from a WT or a GTKO/CD46 pig and (ii) whether the graft was a PKP or EKP. However, we hypothesized that the extent of these reactions would be different depending on the nature of the graft (WT or GTKO/CD46) and on the type of the graft (PKP or EKP).
The specific aim of the present study was to investigate the extent and nature of the immune and inflammatory responses following pig-to-monkey corneal xenotransplantation. To our knowledge, this is the first study in which humoral and cellular immune monitoring has included cytokine production from immune cells following pig corneal xenotransplantation in monkeys. It is also the first study of EKP in the pig-to-primate model, and one of the first to investigate genetically-engineered pigs as sources of corneas. Our results demonstrated that a pig corneal xenograft, regardless of whether the graft was from a WT or GTKO/CD46 pig, induces a systemic immune response in recipients and can also produce a proinflammatory cytokine response.
Materials and Methods
All procedures complied with the ARVO Statement Regarding the Use of Animals in Ophthalmic and Vision Research and the University of Pittsburgh Institutional Animal Care and Use Committee guidance for laboratory animals (IACUC # 12070448).
Seven rhesus monkeys (3-4 years-old) were used as recipients (Alpha Genesis, Yamassee, SC). Donor corneas were prepared from Large White (genetically-unmodified [WT]) pigs (Wally Whippo, EnonVally, PA; n=4) and from GTKO/CD46 pigs on a Large White background (Revivicor, Blacksburg, VA; n=3) (Table 1, Supplementary Figure 1). All donor pigs were of non-A (O) blood type. To match corneal thickness for PKP between donors and recipients, donor pigs were used at 1-3 months-old. Our preliminary studies demonstrated that corneas from young WT pigs (e.g.,
Recipients were electively euthanized after 3 months (except M89 DSEK recipient which was euthanized earlier because of progressive graft rejection),with an intravenous injection of pentobarbital (Beuthanasia-D, Merck Animal Health, Madison, NJ)and the enucleated eyes were examined by confocal microscopy to visualize the endothelial cells and stromal keratocytes, as previously described (21), and by light microscopy for histopathology.
1.1.2 Immunosuppressive/anti-inflammatory therapy
Triamcinolone 20mg/0.5mL (Bristol-Myers Squibb, New York, NY) was injected subconjunctivally on post-operative days 0, 4, 7, and then every two weeks for 3 months. Methylprednisolone (Solu-medrol, Pfizer, New York, and NY) was injected intramuscularly at an initial dose of 2mg/kg/day, tapered over 12 weeks, and discontinued at a final dose of 0.25mg/kg.
1.1.3 Monitoring of white blood cell and platelet counts and C-reactive protein
Whole blood samples were collected from recipient monkeys before and serially after transplantation. Blood samples were tested for complete and differential white blood cell counts, platelet count, and C-reactive protein (CRP) (Central Laboratory, Presbyterian Hospital, and Pittsburgh).
1.1.4 Monitoring of CD3+T cells and CD20+B cell memory phenotypes in blood
Blood samples were collected before and after transplantation, and stained to determine the percentage of T and B cell naïve and memory phenotypes by flow cytometric analysis, as previously described (22). Data acquisition was performed with a LSR II flow cytometer (BD Bioscience, San Jose, CA), and data were analyzed using FlowJo software (Tree Star, Ashland, OR).Absolute numbers of CD3+T cells (including CD4+and CD8+T cells), CD20+B cells, and CD3-CD8+ natural killer (NK) cells were calculated based on white blood cell counts and percentages obtained by flow cytometry. Cell suspensions were stained with fluoresce in-conjugated antibodies specific for the following human cell surface markers: anti-CD3-Alexa Fluor 700 (clone SP34-2), anti-CD4-FITC(clone L200), anit-CD8-PE-Cy7 (clone RPA-T8), anti-CD20-PE (clone 2H7), anti-CD21-V450 (clone B-ly4), anti-CD27-PerCP-Cy5.5 (clone O323), anti-CD28-PerCP-Cy5.5 (clone CD28.2), anti-CD95-APC (clone DX2), anti-IgM-APC (clone G20-127), and anti-IgG-PE-Cy7 (clone G18-145) (all from BD), and goat anti-humanIgD (δ chain)-FITC(from Southern Biotech, Birmingham, AL).
In nonhuman primates, CD95 has been shown to be a marker of memory T cells (23).Using multicolor flow cytometry, we distinguished CD3+CD4+ or CD8+T cells into different subsets on the basis of CD28 and CD95 expression (naive as CD28+/CD95-, central memory as CD28+/CD95+, and effectors memory as CD28-/CD95+) (8, 9, 14).
CD27 has been shown as a marker of memory B cells (23, 24). CD20+B cell memory phenotypes in the blood were determined on the basis of CD27 and IgD expression by flow cytometry. CD3-CD20+B cells were classified asIgD+/CD27- naïve(which express predominantly IgM), IgD+/CD27+non-switched memory (which express predominantly IgM), IgD-/CD27+ switched memory (which express predominantly IgG), and IgD-/CD27- double-negative (which express both IgM and IgG) (data not shown).
1.1.5 Intracellular cytokine staining in blood
To investigate the inflammatory cytokine production from immune cells (monocytes, T and B cells), heparinized whole blood was used for intracellular cytokine staining, as previously described (25). Briefly, 200µL of blood was incubated with Golgistop (BD), 50ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO), and 4μg/ml Ionomycin (Sigma) or 400ng/mL lipopolysaccharides (LPS) (Sigma) for 4h at 37°C. After washing with the lysing buffer (BD) followed by PBS, live/dead staining was carried out using a live/dead fixable staining kit (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. Cell suspensions were further stained with fluoresce in-conjugated antibodies specific for the following human cell surface markers: anti-CD3-Alexa Fluor 700, anti-CD4-APC-H7, anti-CD8-PE-Cy7, and anti-CD20-FITC, all from BD, and anti-CD14-PerCP/Cy5.5 (clone M5E2) from Sony Biotechnology (San Jose, CA). Intracellular cytokine staining was carried out using a Cytofix/Cytoperm plus Fixation/Permeabilization Kit (BD), according to the manufacturer’s instructions. Samples were fixed and permeabilized with Cytofix/Cytoperm buffer for 20min at 4°C. Cells were washed with Perm/wash buffer. Intracellular cytokines were stained with the following antibodies: anti-IFN-γ-APC (clone 4S.B3), and anti-IL-6-PE (clone MQ2-6A3) from BD, and anti-TNF-α-pacific blue (clone MAb11) and anti-IL-4-PE (clone 8D4-8) from Sony Biotechnology.
1.1.6 Statistical methods
The statistical significance of differences was determined by Student’s t-test, including paired t-test or nonparametric tests as appropriate, using Graph Pad Prism version 5(Graph Pad Software, San Diego, CA). Values are presented as mean ± SEM. Differences were considered to be significant at p
Although (i) two different surgical procedures were carried out and (ii) corneas were obtained from two different types of pig in the seven recipients, the results have been considered together (as the numbers of recipients in each subgroup was too small to warrant statistical analysis). In any case, if the results are examined in individual monkeys, there appears to be no observable difference in the immune or inflammatory responses between any of the subgroups.
2.1.1 Clinical course and graft survival
All recipient monkeys remained healthy without weight loss or infection throughout the period of follow-up. Regardless of the source of the corneal graft (WT or GTKO/CD46 pig), all PKP recipients developed retro corneal membranes within 3 to 11 days (which greatly reduced graft transparency) that were not responsive to local/systemic corticosteroid therapy (Supplementary Figure 2A, B). In contrast to PKP, DSEK grafts did not develop retro corneal membranes at this early time-point. However, DSEK grafts were either partially or completely detached from the internal surface of the host cornea, and the graft eventually became fibrotic (Supplementary Figure 2A,B).One DSEK graft from a GTKO/CD46 pig was dislocated and attached to the iris, resulting in anterior synechiae and the development of neovascularizationof the graft (Supplementary Figure 2A, B).
The rejection index was determined every week (Supplementary Figure 2C). None of the PKP recipients satisfied the criteria for rejection, but graft clarity was greatly reduced due to the presence of the retro corneal membrane. No persistent severe stromal edema or neovascularization developed in PKP grafts. In DSEK grafts, two DSEK grafts from WT and GTKO/CD46 pigs, respectively, developed neovascularization of the graft, and were considered (by the scoring system) to have been rejected (Supplementary Figure 2C). Although the recipient that rejected a WT DSEK graft was followed for 3 months, the recipient that rejected a GTKO/CD46 DSEK graft was electively euthanized at 6 weeks.
2.1.2 Post-transplantation changes in numbers of white blood cells (WBCs), lymphocytes, T cells and B cells
There were no significant changes in WBC counts throughout the period of follow-up (Figure1A). However, there were significantly reduced numbers of lymphocytes (p<0.01) (Figure 1A). This reduction of lymphocytes includedCD3+T cells (p<0.05) and CD20+B cells (p<0.01), but not CD3-CD8+NK cells (Figure 1A). (The results of individual recipients are shown in (Supplementary Figure 3). These results suggested that local and systemic steroid therapy reduced the number of lymphocytes.
2.1.3 The numbers of CD4+central memory and CD8+effector memory T cells, and switched memory B cells increased
CD3+T cells were gated, and naïve, central memory (CM), and effector memory (EM) phenotypes in the CD4+CD8- and CD4-CD8+T cell subsets were investigated based on the expression of CD28 and CD95 (Supplementary Figure 4A). The absolute numbers of CD3+CD4+T cells (Figure 1A and B, Supplementary Figure 4B and C)and CD3+CD8+T cells (Figure 1A and C, Supplementary Figure 4D and E) as well as naïve and memory phenotypes were also calculated before and after transplantation. The absolute number of CD3+CD4+T cells (p<0.05) (Figure 1A), but not CD3+CD8+T cells (Figure 1A), significantly decreased after transplantation. Both absolute number and percentage of naïve CD4+T cells were significantly decreased after transplantation (p<0.01, and p<0.05, respectively) (Figures1A and B, Supplementary Figure 5A). Although there was no significant change of the absolute number of CD4+CM T cells, there was a significant increase in the percentage of CM in CD4+T cells (p<0.05) (Figure 1B, Supplementary Figure 5B). In contrast, there was no significant difference of the absolute number of CD8+T cells (Figure 1A). However, the percentage of CD8+EM T cells was significantly increased after transplantation (p<0.05) (Figure 1C, Supplementary Figure 5C).
CD3-CD20+B cells were gated (Supplementary Figure 6A), and the absolute numbers (Figure 1D left, Supplementary Figure 6B) and percentages (Figure 1D right, Supplementary Figure 6C) of naïve, non-switched, switched, and double-negative phenotypes were investigated based on the expression of CD27 and IgD. Although the absolute number of CD20+B cells was significantly reduced (p<0.01) (Figure 1A), there was a significant increase in the percentage of switched memory B cells (p<0.05) (Figure 1D), as well as a reduction of naïve B cell phenotype (p<0.01).
These results indicated that pig corneal xenografts could increase the frequency of T cells (predominantlyCD4+central memory and CD8+effector T cells), and B cell memory (predominantly switched memory phenotypes) although the absolute numbers of these memory phenotypes did not increase.
2.1.4 Pig corneal xenografts induce the production of proinflammatory cytokines from CD4+T cells and CD20+B cells
To investigate the cytokine production from CD3+(including CD4+ and CD8+) T cells, CD20+Bcells, and CD3-CD8+NK cells, intracellular cytokine staining using agonists was carried out before and after transplantation (Figure 2, Supplementary Figure 7). There were significant increases in both IFN-γ cytokine production from CD4+ T cells and NK cells (p<0.05) (Figure 2A). TNF-α cytokine-producing cells significantly increased in CD4+and CD8+ T cells and B cells (Figure 2B). Although the absolute number of CD20+B cells significantly decreased (Figure 1A), the remaining B cells produced significantly more TNF-α compared to pre transplantation (p<0.05) (Figure 2B), but there was no production of IFN-γ (Figure 2A). There was no significant increase in IL-6-producing cells after transplantation in any recipient (Figure 2C).
These results suggested that, although the absolute number of lymphocytes was significantly reduced by local and systemic steroid therapy in the recipient monkeys, the remaining immune cells were capable of producing inflammatory cytokines.
2.1.5 Significant production of proinflammatory cytokines in supernatant obtained from MLR
To investigate T cell sensitization, CFSE-MLRs (as direct and indirect MLRs) were carried out at three time-points (before, 6 weeks and 12 weeks after transplantation) (Figure 3, Supplementary Figure 8). In direct MLR, most recipients showed no increase in CD4+and CD8+T cell proliferation after transplantation (Figure 3A), except two recipients (M87 an M88) who received GTKO/CD46 PKP xenografts (Supplementary Figure 8A). These two recipients showed increased CD4+ and/or CD8+T cell proliferation against both donor and third-party pig PBMC stimulators after transplantation.
Supernatant obtained from direct MLRs (Figure 3B, Supplementary Figure 8B) and indirect MLRs (Figure 3C, Supplementary Figure 8C) showed different characteristics of cytokine production after transplantation. The concentration of TNF-α significantly increased in direct MLR (p<0.05) (Figure 3B). In contrast, the concentration of IL-17 significantly increased in indirect MLR (p<0.05) (Figure 3C), where as the concentration of IFN-γ and IL-2 also increased (but not significantly) (Figure 3C). In direct and indirect MLR, both increases in T cell proliferation (e.g., M87, M88) and production of TNF-α and IFN-γ were found after stimulation with third-party pig cells (Supplementary Figure 8C). In contrast, increases in IL-17 production by indirect MLR were only found when donor stimulating cells were used (Figure 3C) suggesting IL-17 might play a role in a donor-specific immune response through the indirect pathway after pig corneal xenotransplantation in monkeys.
2.1.6 Pig corneal xenografts induce sensitization in recipients
To investigate Gal and non Gal as well as donor-specific swine leukocyte antigen (SLA) class I and class II antibody responses in recipients after corneal xenotransplantation, recipient plasma was tested for anti-Gal antibody levels by ELISA (Figure 4A, Supplementary Figure 9A) and anti-pig antibody levels by flow cytometry (Figure 4B, C and D, Supplementary Figure 9B, C and D). Pig RBCs do not express SLA class I and class II on their surface. Therefore, antibody binding to pig RBCs is associated with Gal (if WT pRBCs are used) and non Gal antigens (but not to SDLA). In addition, to investigate anti-donor-specific sensitization, donor-derived pAECs that had been activated to up-regulate the expression of SLA class I and class II (data not shown) were used for antibody binding assay by flow cytometry. Antibody binding to donor-derived pAEC sis associated with sensitization (that includes to both SLA class I and II, and non-SLA class I and class II antigens e.g., Gal and/or non Gal antigens).
Recipients of WT grafts showed an increase in anti-Gal IgM/IgG antibodies after transplantation whereas GTKO/CD46 pig xenograft recipients showed no or minimal increase in anti-Gal IgM/IgG antibodies (Figure 4A). Anti-donor IgM/IgG antibodies also increased in all recipients, but the level was variable (Supplementary Figure 9B).
WT xenograft recipients increased anti-Gal (Figure 4A, Supplementary Figure 9A), anti-donor (Figure 4B, Supplementary Figure 9B), and anti-WT pRBC (Figure 4C, Supplementary Figure 9C) IgM/IgG antibodies in plasma after transplantation. Interestingly, there was no increase in anti-non Gal IgG antibodies and only a minimal increase in anti-non Gal IgM antibodies (Figure 4D, Supplementary Figure 9D). These results suggested that these recipients were mainly sensitized to Gal, even though WT xenografts should express both Gal and non Gal antigens.
We speculated that GTKO/CD46 xenograft recipients would show less sensitization compared to WT xenograft recipients because of the reduce dantigenicity of GTKO/CD46 xenografts. GTKO/CD46 pig xenograft recipients showed no or minimal increase in anti-Gal IgM/igG antibodies (Figure 4A, Supplementary Figure 9A). However, minimal increased anti-donor (Supplementary Figure 9B) and anti-non Gal antibodies (Figure 4D, Supplementary Figure 9D) were measured in plasma obtained from GTKO/CD46 recipients (but not all recipients) after transplantation, indicating that GTKO/CD46 pig corneal xenografts express non Gal antigens that are capable of inducing sensitization in the recipients.
Because two of three DSEK grafts had become neovascularized and had been rejected, we could not determine whether DSEK induced less sensitization compared to PKP grafts.
2.1.7 Cytokine/ chemokine concentrations in plasma
Proinflammatory cytokines/ chemokines (IFN-γ, TNF-α, IL-17, IL-6, IL-1β, IL-8, and MCP-1) in plasma were measured before and after transplantation. The concentration of proinflammatory cytokines in plasma fluctuated after transplantation, with no obvious continuous upward or downward trend (Supplementary Figure 10).
2.1.8 Cytokine/chemokine concentration in aqueous humor
Twelve weeks after transplantation, there were significantly higher levels of proinflammatory cytokines/chemokines (IL-6, IL-1β, IL-8, and MCP-1) in the aqueous humor obtained from the eye with the transplanted cornea compared to those from the non-grafted eye (Figure 5). These results indicated that a pig xenograft could induce local inflammation even under local steroid therapy.
2.1.9 C-reactive protein in plasma
The concentration of C-reactive protein in plasma fluctuated after transplantation, with no obvious continuous upward or downward trend (Data not shown).
It is well-known that WT pig corneas express Gal at the limbal area, but there is no expression of Gal on the epithelium or endothelium of the central cornea (20). There are some Gal-positive keratocytes in the anterior stroma, and stromal collagen also has a low expression of Gal (20).
Because the cornea is a vascular, our present study and those of other groups (7-9) have shown no hyper acute rejection after corneal xenotransplantation in primates, even when a WT pig cornea has been transplanted. Choi et al. showed that significant numbers of Gal-positive cells were detected in rejected WT pig corneas, and that anti-Gal antibodies in the recipient serum increased when a pig corneal xenograft was rejected (9). They also documented increased complement activity in the aqueous humor and serum during and after rejection (8, 9). These results suggested that preformed natural antibodies, especially anti-Gal antibodies, and complement activation play an important role in pig corneal xenograft failure.
However, there have been several encouraging reports even when WT xenografts were transplanted. Pan et al. reported that corneal xenografts (both full-thickness and anterior lamellar) from Wuzhishan miniature pigs survived for 6 months in monkeys, even when only local steroid therapy was administered (7, 19). Choi et al also demonstrated that WT corneal xenografts (both full-thickness and anterior lamellar) from Seoul National University (SNU) miniature pigs survived in monkeys when treated with local and systemic immunosuppressive therapy (8, 9). These results indicated that natural antibodies, e.g., anti-Gal, and complement activation can be regulated when adequate immunosuppressive therapy is administered.
We have previously demonstrated in vitro that the human humoral and cellular immune responses are significantly weaker to GTKO/CD46 pig corneal cells than to WT cells (20). There is a report that anterior lamellar xenografts from genetically-engineered (e.g., GTKO/hCD55/hC59/hCD39/hCTLA4-Ig) pigs were not associated with such long-term graft survival when compared with WT pig xenografts in monkeys (13).However, whether PKP grafts from GTKO/CD46 pigs could provide longer-term survival compared to survival of WT pig grafts remains unknown (26, 27).
Although the corneal graft is transplanted into a vascular bed, an immune reaction was clearly detected not only locally (i.e., in the aqueous humor) but also systemically (e.g., in the serum). Increases in anti-pig antibodies (e.g., anti-Gal) and complement activity in the serum, and effector CD8+T cells in the blood have been detected in recipient monkeys after corneal rejection (following both PKP and anterior lamellar xenotransplantation) (8, 9, 14).
The B cell response is a critical factor in xenograft rejection. Therefore, it will be important to investigate the B cell memory phenotype and anti-pig antibody development (sensitization) in addition to T cell memory phenotype after corneal xenotransplantation. Changes in T and B cell phenol types were found following corneal xenotransplantation. Under local/systemic steroid therapy, there was no increase in the number of any memory phenotypes associated with the reduced number of lymphocytes, CD4+T cells, and CD20+B cells. However, the percentages of CD4+ CM and CD8+EM T cells and of switched memory B cells were all increased after transplantation. These results suggested that monitoring the percentages of memory phenotypes in addition to the absolute numbers may be important.
Non-switched memory B cells (which predominantly express IgM) have been considered as innate B cells (24, 28), and these cells might produce preformed natural anti-pig IgM antibody, e.g., anti-Gal IgM antibody. We considered that switched memory B cells (which predominantly express IgG) might be increased if recipients become sensitized to pig antigens (24, 29). Although IgD-/CD27- double-negative B cells are considered as memory B cells in certain diseases, e.g., systemic lupus erythematosis (30), the role of these double-positive cells on graft function after transplantation still needs to be understood (31).
In all of our recipients, anti-pig IgM and/or IgG antibodies were increased. In addition to T cell memory phenotypes, investigating B cell memory phenotypes might be necessary to monitor the immune response since the relevance of non-switched and switched memory phenotypes in B cells remains uncertain.
In the mouse model, corneal graft rejection in low-risk recipients occurs predominantly through the indirect pathway rather than the direct pathway (32). However, in high-risk recipients with neovascularization or inflammation in the host graft, the direct pathway will also be involved in rejection (33).In the present study, to investigate T cell sensitization, we carried out both conventional CFSE-MLR (direct MLR) and indirect MLR. Differences in cytokine production were found between direct and indirect MLR.
Although not all recipients showed increased T cell proliferation in direct MLR, increased TNF-α was detected in the supernatant, especially in WT PKP recipients. In contrast to direct MLR, increased levels of IFN-γ and IL-17 were detected in the indirect MLR. IFN-γ has been shown to be an important cytokine in T cell sensitization as well as inducing delayed-type hypersensitivity (34-36). IL-17 has been considered as an inflammatory cytokine, and is involved in organ allograft rejection (37, 38). However, there still remains controversy regarding the role of IL-17 in corneal graft rejection. Mouse studies have demonstrated that IL-17 is involved in early acute corneal allograft acceptance whereas a Th1 response was predominant in the late stage (39, 40). In addition, IL-17 is important in the induction of regulatory T cells after corneal transplantation in mice (41). In contrast, a late phase of corneal allograft rejection is likely mediated by Th17 cells because therapeutic neutralization of IL-17A reverses graft rejection (40). In vitro monitoring showed that some recipients increased their direct T cell proliferation, as well as the levels of TNF-α and IFN-γ in the culture supernatant obtained from direct and/or indirect MLR. However, these finding were also found in the response to third-party pig cells, suggesting responses might not be donor-specific. In contrast, a donor-specific IL-17 response was detected in the indirect MLR, indicating that IL-17 might be an important marker to investigate donor-specific sensitization after corneal xenotransplantation.
The cornea is considered an immune-privileged tissue (4, 36). Antigen placed into the anterior chamber of the eye elicits deviant systemic immune responses, termed anterior chamber-associated immune deviation (42), which suppress delayed-type hypersensitivity, but preserve humoral immunity (i.e., antibody response) and the primed cytotoxic T cell response (42). The present study showed that, although the corneal graft was not clinically rejected, recipients became sensitized to pig antigens in the anterior chamber through T and B cell immune responses.
Recently, it has been demonstrated that sustained systemic inflammation occurs in xenograft recipients after pig organ/aortic patch transplantation in baboons (15, 43-45). Previous studies by others have demonstrated that inflammatory cytokines were detected in the aqueous humor during rejection of a corneal xenograft (7, 9). However, whether a pig corneal xenograft adjacent to the anterior chamber can induce systemic inflammation is unknown. Proinflammatory cytokines in the serum and in the aqueous humor were measured. There were significantly increased levels of proinflammatory cytokines/chemokines (i.e., IL-6, IL-8, MCP-1 and TGF-β) in the aqueous humor obtained from transplanted cornea, suggesting local inflammation. We also investigated cytokine production from T, NK, and B cells in the blood after transplantation. Some recipients (M88 and M89), but not all, demonstrated increased levels of proinflammatory cytokines in the plasma. In contrast, proinflammatory cytokines, especially TNF-α in T cells and/or B cells, increased after transplantation, although the absolute numbers of these cells were decreased. These results indicated that corneal xenografts can induce systemic inflammation in the recipients.
A limitation of the present study was that the number of animals in each group was small. Increasing the sample size would be necessary to draw significant conclusions. However, we believe that the data provide valuable information on the incidence of the immune/inflammatory responses in monkeys after pig corneal xenotransplantation.
In conclusion, in addition to the conventional immune assays, e.g., measuring anti-pig antibody and MLR, immune monitoring that includes memory phenotype and inflammatory cytokine production in T and B cells might be beneficial in understanding the recipient immune status after corneal xenotransplantation. Corneal xenografts, regardless of whether WT or GTKO/CD46 or PKP or DSEK, induce T and B cell sensitization and systemic inflammation in monkeys, even under local/systemic steroid therapy. Local and systemic immunosuppressive therapy (e.g., costimulation blockade in combination with steroid therapy) might be necessary to prevent both inflammatory and immune responses when a pig corneal xenograft is transplanted into a primate. If sensitization and the inflammatory response can be prevented, genetically-engineered pigs (which can reduce the human immune response) might be an alternative source of corneas to deceased humans for clinical transplantation, especially in high-risk patients, and may provide long-term graft survival compared to WT pig corneal xenografts.
HH1 participated in research design, in the performance of the research, interpretation of the results, and in writing of the manuscript. HH1 is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. WL1, YM1, and CL1 participated in the performance of the research, and in review of the manuscript. AM2, and DKD2 participated in the performance of the surgery, and in review of the manuscript. DA3 provided the genetically engineered pigs, and reviewed the paper. DKCC1 participated in research design, in interpretation of the results, and in writing of the manuscript.
Whayoung Lee supported by an Ocular Tissue Engineering and Regenerative Ophthalmology (OTERO) Postdoctoral Fellowship. Work on xenotransplantation in the Thomas E. Starzl Transplantation Institute at the University of Pittsburgh has been supported in part by NIH grants U01 AI068642, and 2U19 AI090959 and by Sponsored Research Agreements between the University of Pittsburgh and Revivicor, Inc., Blacksburg, VA. The cytokine assays carried out in the present study were supported in part by the University of Pittsburgh Cancer Institute Biomarkers Facility/Luminex Core Laboratory at the University of Pittsburgh Cancer Institute and the NIH Cancer Center by Grant P30CA047904.
Conflict of Interest
David Ayares is an employee of Revivicor, Inc, and receives a salary from Revivicor, Inc. However, David Ayares had no a role in the study design, data collection and analysis, decision to publish the manuscript as well as preparation of the manuscript, except reviewed the manuscript. The other authors have no conflict of interest.
Figure 1: Kinetics of absolute numbers of leukocytes and memory T and B cell phenotypes in the blood after corneal xenotransplantation.
Absolute numbers of (A) WBCs, lymphocytes, and T cells(including subtypes), B cells and NK cells in the blood were measured after corneal transplantation in all recipients (n=7). There were significantly reduced absolute numbers of lymphocytes, CD3+ (CD3+CD4+) T cells, and CD3-20+B cells after transplantation. Absolute numbers and percentages of naïve, central memory (CM), effector memory (EM) CD4+T cells (B), and CD8+T cells (C)(all CD3+T cells) and of naïve, non-switched, switched memory, and double-negative cells (all CD3-CD20+B cells) were measured. There were significant increases in the percentages of memory T cells and memory B cells after transplantation.(* p<0.05)
Figure 2: Kinetics of cytokine-producing cells in the blood after corneal transplantation.
Intracellular cytokine staining of (A) IFN-γ,(B) TNF-α, and (C) IL-6 was carried out. Cytokine-producing CD4+ and CD8+ T cells, CD3-CD8+NK cells, and CD3-CD20+B cells were investigated by flow cytometry (n=7). There were significant increases in IFN-γ (CD4+ and CD8+T cells, and NK cells), and TNF-α (CD4+ and CD8+T cells, and B cells)-producing cells in the blood after transplantation. (* p<0.05)
Figure 3: Proliferation of T cells on CFSE-MLR (direct MLR) and cytokine profiles of supernatant obtained from direct and indirect MLR
(A)CFSE-labeled recipients PBMCs were stimulated with donor and third-party pig PBMCs for 6 days. The percentages of proliferation of CD3+CD4+ and CD3+CD8+T cells were detected by flow cytometry (n=7). There was no significant increase in T cell proliferation against donor and third-party pig stimulators compared to pre-transplantation (n=7). Supernatants were obtained from (B) direct MLR (6 days culture) and(C) indirect MLR (5 days culture) for measurement of cytokines (n=7). In direct MLR, there was a significantly increased level of TNF-α in the supernatants obtained after donor and third-party stimulation12 weeks after transplantation. In indirect MLR, there was a significantly increased level of IL-17A in the supernatant obtained after donor stimulation 6 weeks after transplantation. (* p<0.05)
Figure 4: Kinetics of anti-Gal and anti-pig antibodies in plasma after corneal transplantation
(A) Anti-Gal IgM and IgG antibody levels (by ELISA), (B) anti-donor, (C) anti-WT pig RBCs, and (D) anti-non Gal (anti-GTKO/CD46 pig RBCs) IgM and IgG antibody levels (by flow cytometry) in plasma from recipients that received either WT (n=4) or GTKO/CD46 (n=3) pig corneal xenografts were measured after transplantation. Data are shown as fold increases in IgM and IgG antibody responses (with pre-transplantation = 1). Increased anti-Gal and/or anti-pig antibody production were found in all recipients.
Figure 5: Profile of cytokines/chemokines in aqueous humor after corneal xenotransplantation.
Aqueous humor was obtained from eyes with or without corneal transplants at 12 weeks (or 6weeks for M89) after corneal xenotransplantation (n=7). There were significantly higher levels of IL6, IL-8, TGF-β, and MCP-1 in the aqueous humor or eyes with corneal grafts compared to those from the non-transplanted eyes. In contrast, the levels of IL-17A in the aqueous humor were lower in the eyes with corneal grafts than in the non-transplanted eyes.
Supplementary Materials and Methods
Corneal graft evaluation
After euthanizing the pigs, corneal endothelial cell (CEC) density and corneal thickness were measured using confocal microscopy (Confoscan 3, Nidek Technologies, Fremont, CA) and a pachymeter (Tomey, Nagoya, Japan), respectively. The donor corneoscleral tissues were dissected out and stored in Optisol GS solution (Bausch & Lomb, Rochester, NY) for 2-3 days before transplantation. Information on the corneas of the donors and recipients is shown in Table 1.
Pig-to-monkey penetrating keratoplasty
PKP (full-thickness) was carried out, as described previously (1, 2). Briefly, the donor cornea was excised using a 6.5mm-diameter Barron corneal punch (Katena Products, Denville, NJ). The central cornea was excised from the recipient’s eye using a 6mm-diameter Hess burg-Barron vacuum trephine (Jed med, St. Louis, MO). The graft was placed and secured with 16 interrupted 10-0 nylon sutures (Ethicon, Somerville, NJ). Triamcinolone 20mg/0.5mL (Kenalog-40®, Bristol-Myers Squibb, New York, NY) was injected subconjunctivally, and the eyelids were closed with a 6-0 black silk suture. The tarsorrhaphy was maintained for 3 or 4 days, and then opened for clinical evaluation. Corneal transplantation was carried out in only one eye of each monkey.
Preparation of Descemet’s Stripping Endothelial Keratoplasty (DSEK) grafts
A Moria Evolution 3 Microkeratome (Moria, Doylestown, PA) was used for preparation of DSEK grafts one day before transplantation (Supplementary Figure 1A and B). The pig cornea was placed on the artificial chamber (Moria), and fixed with ametal helmet. One line from the artificial chamber was connected to a balanced salt solution (Alcon, Fort Worth, TX) infusion, and the other was connected to a syringe containing Optisol GS solution. The artificial chamber was filled with Optisol until it reached the optimum pressure. The corneal epithelium was removed with an eye spear (Beaver-Visitec International, Waltham, MA). This step helped to reduce the thickness of the cornea (by approximately 50-80μm). The central area of the reduced corneal thickness was measured by pachymeter x3. Before cutting the anterior part of the corneal graft, the intraocular pressure was adjusted to approximately90 mmHg by measuring with a tonometer. A 350μm microtome blade (Moria) was used to cut the surface of the cornea. The anterior chamber cap was removed and set aside (Supplementary Figure 1A, middle picture).The residual tissue was again measured by pachymeter. If necessary (depending residual graft thickness), a second (refinement) cut was made with a 110μm microkeratome head.
(If the DSEK graft is too thick, it can easily be detached from the recipient cornea after transplantation. Ideally, the goal is to fashion a graft of approximately 100μm thickness or less.)
The anterior cap was replaced on to the cornea (to protect the graft from swelling). The whole cornea was carefully removed from the artificial chamber, and placed in a container of Optisol GS (with endothelial surface uppermost) and kept at 4°C until use.
Pig-to-monkey Descemet’s Stripping Endothelial Keratoplasty (DSEK)
Under general anesthesia, DSEK was carried out in the monkey within 24h after DSEK graft preparation (Supplementary Figure 1C). First, the recipient’s cornea was marked to measure the size of the DSEK graft (6.5 mm-diameters).The recipient’s Descemet’s membrane with endothelium was removed using a 90° spatula for Descemet stripping (Moria). (Roughening of the anterior Descemet's membrane improves adherence of the DSEK graft.)
The DSEK graft was placed on a 6.5mm-diameter Barron corneal punch (Katena Products, Denville, NJ) with the endothelial side up, and punched out. Graft quality was confirmed by staining with trypan blue. Thestromal side was marked to facilitate correct intra operative orientation of the graft. The DSEK graft (healthy endothelial cells facing down) was put on a glide spatula (Moria), and the graft was pulled through the glidespatula as the edges curl in. The graft was pulled across the anterior chamber using Bus in inserting forceps (Moria). After injection of air deep to the graft, the recipient monkey was maintained supine for at least one hour. The tarsorrhaphy was maintained for 3 or 4 days.
All monkeys received topical tobramycin/dexamethasone eye ointment (Tobradex®, Alcon, Fort Worth, TX) x2-3 weekly. Enrofloxacin 5mg/kg (Baytryl® 100, Leverkusen, Germany) was administered once a day intramuscularly for 3 days.
Clinical evaluation of the grafts
After sedation of the monkeys, the grafts were examined by portable slit-lamp biomicroscopy (Keeler Ophthalmic Instruments, Broomall, PA) to evaluate graft clarity, edema, and neovascularization. A scoring system (providing a ‘rejection index’) was used, as previously described by Pan et al (1, 2). (In this scoring system, each factor is scored on a scale from 1 to 4, and if the sum of the scores is greater than 6 for 2 consecutive weeks, the graft is considered to be rejected.) Corneal thickness was measured with a pachymeter.
Preparation of peripheral blood mononuclear cells
Peripheral blood mononuclear cells (PBMCs) were isolated, as previously described (3). Isolated donor PBMCs were resuspended in ice-cold storing medium, composed of RPMI (Invitrogen, Carlsbad, CA), 20% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO), and 10% dimethyl-sulfoxide (Sigma). Cells were distributed in cryogenic vials (Sigma) and transferred overnight to −80°C in a freezing container (Mr. Frosty, Thermo Fisher Scientific, Waltham, MA), allowing a controlled freezing rate of about 1°C/min, and then transferred into a nitrogen container.
These stored cells were thawed rapidly in a 37°C water bath, transferred on ice, diluted drop by drop into chilled RPMI containing 10% FBS, 10,000 U/mL penicillin-streptomycin (Invitrogen) and HEPES (Invitrogen). After washing x2, cells were resuspended in AIM-V Medium (Life Technologies, Carlsbad, CA) and used as stimulators or third-parties in MLR.
5-(and 6)-carboxyfluorescein diacetate succinimidyl ester-mixed lymphocyte reaction (CFSE-direct MLR)
To investigate the direct T cell responses, CFSE-MLR was carried out before transplantation and 6 weeks and 12 weeks after transplantation, as previously described (4, 5). Recipient monkey PBMCs were isolated from heparinized blood and labeled with CFSE (Molecular Probes, Eugene, OR) at a final concentration of 5µM. Responder PBMCs (2x106 cells/ml) from monkey recipients were co-cultured with irradiated (2,800cGy) PBMCs prepared as stimulator cells (2x106 cells/ml) from the autologous monkey or donor or third-party pigs. The responder: stimulator ratio was 1:1. Mitogen stimulation using 5µg/ml phytohemagglutinin (PHA, Roche, Basel, Switzerland) of responder PBMCs was used as a positive control. Supernatants from culture medium were collected after 3 and 6 days of culture, and stored at -20°C until measurement of cytokines. After 6 days culture, cells were harvested for staining with live/dead fixable staining kit (Invitrogen) followed by staining with anti-CD4-PE-Cy7 (clone SK3), anti-CD8-PE (clone RPA-T8), andanti-CD3-pacific blue (clone SP34-2) antibodies (BD). The data were analyzed as percentage of CD3+CD4+ and CD3+CD8+T cell proliferation.
An indirect MLR was used to determine sensitization to pig antigens through indirect T cell responses, as described elsewhere (6, 7). T-cell reactivity was measured through cytokine secretion. Stored donor and third-party pig PBMCs were irradiated (2,800cGy) and re suspended with PBS (Invitrogen) at a 10x106 cells/50μL concentration into cryogenic vials (Sigma). Cell lysates were generated using four rounds of freeze-thawing. For indirect MLR using membrane-bound cell-free pig antigens, no intact cells were visible microscopically. Cell lysate (50μL) obtained from donor or third-party pig PBMCs was added to responder PBMCs (2x106 cells/ml with AIM-V medium), and incubated for 5 days. Supernatant was collected to measure the cytokines after 3 and 5 days of culture.
Preparation of pig aortic endothelial cells (PAECs)
PAECs were collected from WT and GTKO/CD46 donor pig aortas, and cultured as previously described (8). PAECs of passages 3 to 5 were used for antibody binding assay. The sub-confluent PAECs were activated for 36h by co-culture with recombinant porcine IFN-γ (50ng/mL, R&D Systems, Minneapolis, MN). Activation of the cells was evaluated by staining with swine leukocyte antigen (SLA) class I (clone JM1E3, Serotec, and Raleigh, NC) and SLA class II (DR) (clone 2E9/13, BD) using flow cytometry.
Measurement of anti-pig IgM and IgG antibodies
Anti-pig antibody levels were measured by flow cytometry using WT and GTKO/CD46 pig cells, including red blood cells (RBCs) and activated pAECs, as previously described (9). Briefly, 2.5µl and 20µl of recipient plasma, which had been heat-inactivated for 30min at 56ºC, were incubated with 1x106pig RBCs or 0.1x106PAECs, respectively, for 30min at 4ºC. Cells were washed and incubated with 10% heat-inactivated goat serum for 20min followed by incubation with secondary antibodies - anti-human FITC-IgM (μ-chain-specific) and FITC-IgG (γ-chain-specific) (Invitrogen) for 30min at 4ºC. Binding of IgM and IgG was measured using the relative geometric mean, which was calculated by dividing the geometric mean value for each sample by the negative control. Negative controls were obtained by incubating the target cells with secondary anti-human antibodies only (with no plasma). Data were shown as the fold IgM and IgG antibody responses compared to the relative geometric mean pre-transplantation (=1).
Measurement of plasma anti-Gal antibodies
Anti-Gal IgM and IgG antibodies in recipient plasma were measured by an enzyme-linked immunosorbent assay (ELISA), as previously described (10). ELISA data were assessed in terms of optical density, and shown as the fold IgM and IgG antibody responses (with pre-transplantation=1).
Measurement of cytokines
To measure cytokines, samples were obtained from plasma, aqueous humor and culture supernatant was obtained from direct and indirect MLR. To measure several cytokines, including TNF-α, IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17A, TGF-β, MCP-1 (Millipore, Billerica, MA), multiplexed plasma immunoassays were performed using the Luminexx MAP technology platform (Luminex Corporation, Austin, TX), as previously described (11).
In contrast to previous encouraging reports (1, 12), in the present study all PKP recipients developed retro corneal membranes regardless of whether the graft was from a WT or GTKO/CD46 pig (2). The possible causes of the development of a retro corneal membrane have been discussed elsewhere (2, 13). The comparative thickness of the graft and host corneas and mismatched corneal biomechanical properties (because a cornea from a young pig has different properties than a cornea from an older monkey) may play roles. These factors may result in abnormal wound healing. An inflammatory reaction might also be a factor. Therefore, prevention of the development of a retro corneal membrane will be essential to achieve successful corneal xenotransplantation.
In WT pig corneas, Gal is mainly expressed in the anterior stroma (3). However, our previous study demonstrated that human IgM/IgG antibody binding to both WT and GTKO/CD46 pig corneal tissue was mainly found in the epithelium with less binding to the posterior part of the cornea (i.e., the endothelium) (14, 15). DSEK grafts should therefore have less antigenicity compared to PKP grafts. The suture less technique might also reduce inflammation. Furthermore, a mismatching graft-host corneal thickness should not be problematic because a DSEK graft is transplanted internal to the host cornea. Although DSEK surgery was successfully carried out, there were some technical issues that could be improved in the future.
The first relates to DSEK graft preparation. Since the pig cornea, especially from young pigs, is extremely elastic, perforation/tearing is a risk during DSEK preparation. GTKO/CD46 pig corneas appeared to be more easily damaged than those from WT pigs. Therefore, we were unable to obtain the thin DSEK grafts (<100μm) we hoped for. The second issue related to dislocation of the DSEK graft after accurate insertion (because the graft was thicker than intended). Ideally, it would be necessary to use an older pig (>8 months-old) as the cornea would be less elastic.
Figure 1: (A) DSEK graft preparation, (B) histology of DSEK graft, and (C) DSEK operation in pig-to-monkey model.
Figure 2: Clinical evaluation of corneal grafts after transplantation. (A) Representative figures of the macroscopic appearance. (B) Representative features on slit-lamp microscopy. (C) Results of scoring system, indicating extent of rejection.
Figure 3: Absolute numbers of(A) WBCs, lymphocytes, and (B) T cells, B cells, and NK cells in the blood in individual recipients (n=7; WT n=4; GTKO/CD46 n=3).
Figure 4: Absolute numbers of CD3+CD4+T and of CD3+CD8+T cell and of naïve, central memory (CM), and effector memory (EM) cells in the blood in individual recipients. . (A) Gating for CD3+ cells by flow cytometry. (B) Absolute numbers ofCD4+T cells, and of (C) naïve, central memory, and effector memory CD4+T cells. (D) Absolute numbers ofCD8+T cells, and of (E) naïve, central memory, and effector memory CD8+T cells.
Figure 5: Percentages of naïve, central memory (CM), and effector memory (EM) T cells gated on CD3+CD4+CD8- or CD3+CD4-CD8+ T cells in the blood in individual recipients.
Figure 6: Absolute numbers and percentages of various B cells in the blood in individual recipients. (A) Gating for CD20+B cells by flow cytometry. B cell phenotypes, including naïve, non-switched memory, switched memory, and double-negative B cells, were investigated. Blood from individual recipients was examined to determine (B) the absolute numbers and (C) percentages of naïve, non-switched memory, switched memory, and double-negative B cells in the blood after transplantation.
Figure 7: Profiles of production of (A) IFN-γ and (B) TNF-α by CD4+CD8- and CD4-CD8+ T cells, CD3-CD8+ NK cells, and CD3-CD20`B cells in the blood in individual recipients.
Figure 8: (A) Proliferation of T cells on CFSE-MLR (direct MLR), and profiles of cytokines in supernatants obtained from (B) direct and (C) indirect MLR in individual recipients.
Figure 9: (A) Anti-Gal IgM and IgG antibody levels (by ELISA), (B) anti-donor, (C) anti-WT pig RBCs, and (D) anti-non Gal (anti-GTKO/CD46 pig RBC) IgM and IgG antibody levels (by flow cytometry) in plasma from individual recipients after corneal xenotransplantation. Data are shown as the fold increases in IgM and IgG antibody responses (with pre-transplantation = 1).
Figure 10: Pro inflammatory cytokine and chemokine levels in plasma from individual recipients after corneal xenotransplantation.
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