Strategies to Induce Marked Prolongation of Secondary Skin Allograft Survival in Alloantigen-Primed Mice

Alloreactive memory T cells mediate accelerated rejection. We investigated the effect of polyclonal anti-T-cell antibody (ALS) and rapamycin (RAPA) on skin allograft survival in naïve or alloantigen-primed mice. ALS prolonged graft survival in both naïve and alloantigen-primed mice. T-cell depletion by ALS was associated with increased CD4⁺CD44^hiOX40⁺ and CD8⁺CD44^hiCD122⁺ memory T cells. Addition of RAPA to ALS extended graft survival in naïve mice, but had no effect on secondary allograft survival in alloantigen-primed mice. In adoptive transfer experiments, RAPA inhibited alloantigen-stimulated proliferation and allograft rejection by naïve T cells. In contrast, alloantigen-primed memory T cells, particularly CD4⁺CD44^hiOX40⁺ and CD8⁺CD44^hiCD122⁺ T cells, were resistant to RAPA in response to alloantigen and mediated accelerated rejection in the presence of RAPA. Resistance to RAPA by alloantigen-primed mice was overcome by the use of high-dose ALS, which achieved marked prolongation of secondary skin allograft survival (>100 days). Inhibition of CD122⁺ T cells and/or OX40/OX40L costimulation blockade, combined with low-dose ALS and RAPA, was also effective. These results demonstrate that tolerance may be achieved in allosensitized individuals by T-cell depletion- and RAPA-based strategies employing high-dose ALS or targeting CD122⁺CD8⁺ T cells and/or the OX40/OX40L costimulatory pathway.     

Effect of Inflammation on Costimulation Blockade-Resistant Allograft Rejection

Previously, we reported that allogeneic skin grafts were rapidly rejected by CD28 and CD40 ligand double deficient mice mediated by CD8⁺ T cells. These results indicated that some elements in addition to CD28- and CD40-mediated costimulation provide stimulatory signals for the activation of donor-specific CD8⁺ T cells. In this report, we investigated the role of inflammation associated with transplantation on costimulation-independent priming of CD8⁺ T cell during graft rejection. B6 RAG1 KO mice were transplanted with BALB/c-skin and adoptively transferred with syngeneic CD8⁺ T cells the same day or 50 days after transplantation. When blockade of CD28- and CD40-mediated costimulation failed to prevent acute rejection of freshly transplanted skin grafts, it efficiently delayed rejection of well-healed skin grafts. These results showed that factors associated with transplantation have essential roles in inducing costimulation blockade-resistant allograft rejection. Costimulation blockade failed to prevent acute graft-infiltration of NK cells and increasing expression of intragraft IL-12 and IL-15. These factors may trigger the graft-infiltration and priming of CD8⁺ T cells to induce costimulation blockade-resistant allograft rejection.     

CCR5 Is Required for Regulation of Alloreactive T-Cell Responses to Single Class II MHC-Mismatched Murine Cardiac Grafts

The effector CD4 T-cell response in wild-type C57BL/6 recipients of single class II MHC-disparate B6.H-2^bm12 cardiac allografts is restricted by CD4⁺CD25⁺ regulatory T cells (Tregs) resulting in long-term allograft survival. To investigate the role chemokine receptors might play in Treg function, this study tested the requirement for CCR5 on Tregs to suppress the alloimmune response in C57BL/6 recipients of B6.H-2^bm12 cardiac allografts. In contrast to the long-term survival of B6.H-2^bm12 allografts in wild-type recipients (>100 days), the allografts were acutely rejected within 25 days in CCR5^−/− recipients with intense infiltration of CD4 T cells. Numbers and duration of donor-reactive CD4 T cells producing IFN-γ and IL-4 were markedly increased in spleens of B6.CCR5^−/− versus wild-type recipients. Wild-type and B6.CCR5^−/− mice had equivalent numbers of splenic FoxP3⁺ Tregs before and following transplantation, and these Tregs were equivalently suppressive in vitro . However, diminished numbers of FoxP3⁺ Tregs infiltrated B6.H-2^bm12 allografts in B6.CCR5^−/− recipients. Adoptive transfer of wild-type, but not CCR5-deficient, CD4⁺CD25⁺ Tregs to CCR5^−/− recipients restored long-term survival of B6.H-2^bm12 cardiac grafts. Collectively, these results indicate that CCR5 expression is required for the regulatory functions of Tregs that restrict alloreactive CD4 T-cell responses to single class II MHC-mismatched cardiac allografts.     

CD4+ T cells, without CD8+ or B lymphocytes, can reject xenogeneic skin grafts

Abstract: Previous experiments have shown that rejection of xenogeneic skin grafts by mice is particularly dependent on CD4⁺ T cells. There are two possible explantations for this finding: either 1) "help" provided by CD4⁺ T cells is essential for CD8⁺ T cell-, B cell-, or NK cell-mediated effector mechanisms of rejection, or 2) CD4⁺ cells are themselves responsible for rejection, perhaps by some nonspecific effector mechanism. To examine these two hypotheses, we transplanted pig skin onto SCID mice and then reconstituted the mice with selected subpopulations of lymphocytes. Mice that did not received CD4⁺ T cells were unable to reject their xenografts, whereas those receiving CD4⁺ cells could do so in the absence of CD8⁺ cells or B cells and even when additionally depleted of NK cells by treatment with anti-Asialo GM1 antibody. Additional experiments were performed both in vivo and vitro to confirm the absence in test mice of CD4⁺ or CD8⁺ and B lymphocytes, respectively. These results suggest that CD4⁺ T cells are not only necessary for rejection of xenogeneic skin grafts by mice, but that they can do so without CD8⁺ cells or B cells, and probably without NK cells. Since CD4⁺ cells in mice have been shown to recognize xenogeneic antigens indirectly, this suggests that a nonspecific effector mechanism may be involved in the rejection of xenografts. In these experiments allogeneic skin grafts behave quite differently as they could not be rejected by this mechanism.     

Ex Vivo-Expanded Natural CD4+CD25+ Regulatory T Cells Synergize With Host T-Cell Depletion to Promote Long-Term Survival of Allografts

Foxp3⁺CD4⁺CD25⁺ natural regulatory T (nT_reg) cells have been shown in immunodeficient mice to suppress allograft rejection after adoptive cotransfer. We hypothesized that immunotherapy using ex vivo -expanded nT_reg could suppress allograft rejection in wild-type mice. Donor alloantigen (alloAg) specificity of naive splenic nT_reg was enriched in vitro by culturing with anti-CD3/CD28-coated Dynabeads plus bone marrow-derived dendritic cells (BM-DC) in the presence of interleukin (IL)-2 or IL-2 plus transforming growth factor (TGF)-β. On average, 96.2% fresh CD4⁺CD25⁺ nT_reg were intracellular Foxp3⁺. By d+20 in culture, 6.4% nT_reg were Foxp3⁺ following expansion with IL-2 alone, and 14.4% or 19.7% nT_reg were Foxp3⁺ when expanded with IL-2 plus 0.5 or 2.5 ng/mL TGF-β, respectively. In vitro , alloAg-enriched, TGF-β/IL-2-conditioned nT_reg exerted stronger donor alloAg-specific suppression than cells with IL-2 alone in mixed lymphocyte reaction (MLR) assays. In vivo , alloAg-enriched, TGF-β/IL-2-conditioned nT_reg expressed high-level Foxp3 following infusion, effectively overcame acute rejection and induced long-term survival of donor but not third-party heart allografts in peritransplant host T-cell-depleted mice. Long-term surviving allografts were noted to possess Foxp3⁺ graft-infiltrating cells of exogenous and endogenous origins. In conjunction with transient host T-cell depletion, therapeutic use of ex vivo -expanded nT_reg may be a practical means of preventing acute allograft rejection.     

Homeostatic Repopulation by CD28−CD8+ T Cells in Alemtuzumab-Depleted Kidney Transplant Recipients Treated With Reduced Immunosuppression

Alemtuzumab (CAMPATH-1H) is a depleting agent introduced recently in transplantation and often used with reduced maintenance immunosuppression. In the current study we investigated the immune response of 13 kidney allograft recipients treated with alemtuzumab followed by weaned immunosuppression with reduced dose of mycophenolate mofetil (MMF) and tacrolimus. Tacrolimus was switched to sirolimus at 6 months and MMF withdrawn at 12 months after transplantation.
We found that after alemtuzumab induction the recovery of CD8⁺ T cells was much faster than that of CD4⁺ T cells. It was complete 6 months posttransplant while CD4⁺ T cells did not fully recover even 15 months posttransplant. Repopulating CD8⁺ T cells were mainly of immunosenescent CD28⁻CD8⁺ phenotype. In a series of in vitro experiments we showed that CD28⁻CD8⁺ T cells might suppress proliferation of CD4⁺ T cells. There were three successfully treated acute rejections during the study (first at +70 day, two others +12 months) that occurred in patients with the lowest level of CD28⁻CD8⁺ T cells.
We hypothesize that expanded CD28⁻CD8⁺ T cells might compete for 'immune space' with CD4⁺ T cells suppressing their proliferation and therefore delaying CD4⁺ T-cells recovery. This delay might be associated with the clinical outcome as CD4⁺ T cells, notably CD4⁺ T effector memory cells, were shown to be associated with rejection.     

Contribution of Naïve and Memory T-Cell Populations to the Human Alloimmune Response

T-cell alloimmunity plays a dominant role in allograft rejection. The precise contribution of naïve and memory T cells to this response however remains unclear. To address this question, we established an ex vivo flow-cytometric assay that simultaneously measures proliferation, precursor frequency and effector molecule (IFNγ, granzyme B/perforin) production of alloreactive T cells. By applying this assay to peripheral blood mononuclear cells from healthy volunteers, we demonstrate that the CD4⁺ and CD8⁺ populations mount similar proliferative responses and contain comparable frequencies of alloreactive precursors. Effector molecule expression, however, was significantly higher among CD8⁺ T cells. Analysis of sorted naïve and memory T cells showed that alloreactive precursors were equally present in both populations. The CD8⁺ effector and terminally differentiated effector memory subsets contained the highest proportion of granzyme B/perforin after allostimulation, suggesting that these cells present a significant threat to transplanted organs. Finally, we demonstrate that virus-specific lymphocytes contribute significantly to the alloresponse in certain responder–stimulator HLA combinations, underscoring the importance of T-cell cross-reactivity in alloimmunity. These results provide a quantitative assessment of the roles of naïve and memory T-cell subsets in the normal human alloimmune response and establish a platform for measuring T-cell alloreactivity pre- and posttransplantation.     

Alloreactive (CD4-Independent) CD8+ T Cells Jeopardize Long-Term Survival of Intrahepatic Islet Allografts

Despite success of early islet allograft engraftment and survival in humans, late islet allograft loss has emerged as an important clinical problem. CD8⁺ T cells that are independent of CD4⁺ T cell help can damage allograft tissues and are resistant to conventional immunosuppressive therapies. Previous work demonstrates that islet allografts do not primarily initiate rejection by the (CD4-independent) CD8-dependent pathway. This study was performed to determine if activation of alloreactive CD4-independent, CD8⁺ T cells, by exogenous stimuli, can precipitate late loss of islet allografts. Recipients were induced to accept intrahepatic islet allografts (islet 'acceptors') by short-term immunotherapy with donor-specific transfusion (DST) and anti-CD154 mAb. Following the establishment of stable long-term islet allograft function for 60–90 days, recipients were challenged with donor-matched hepatocellular allografts, which are known to activate (CD4-independent) CD8⁺ T cells. Allogeneic islets engrafted long-term were vulnerable to damage when challenged locally with donor-matched hepatocytes. Islet allograft loss was due to allo specific immune damage, which was CD8- but not CD4-dependent. Selection of specific immunotherapy to suppress both CD4- and CD8-dependent immune pathways at the time of transplant protects islet allografts from both early and late immune damage.     

Donor-specific Immune Regulation by CD8+ Lymphocytes Expanded from Rejecting Human Cardiac Allografts

To assess whether regulatory T cells are present in rejecting human cardiac allografts, we performed functional analyses of graft lymphocytes (GLs) expanded from endomyocardial biopsies (EMB; n = 5) with histological signs of acute cellular rejection. The GL cultures were tested for their proliferative capacity and regulatory activity on allogeneic-stimulated peripheral blood mononuclear cells (PBMC) of the patient (ratio PBMC:GLs = 5:1). Three of these GL cultures were hyporesponsive to donor antigens and suppressed the antidonor proliferative T-cell response of PBMC, but not the anti-third-party response. Interestingly, it was the CD8⁺ GL subset of these cultures that inhibited the antidonor response (65–91% inhibition of the proportion of proliferating cells); the CD4⁺ GLs of the expanded GL cultures were not suppressive. In conclusion, CD8⁺ GLs expanded from rejecting human cardiac allografts can exhibit donor-specific immune regulatory activities in vitro . We suggest that during acute cellular rejection, GLs may not only consist of graft-destructing effector T cells, but also of cells of the CD8⁺ type with the potential to specifically inhibit antidonor immune reactivity.     

New Insights into Mechanisms of Spontaneous Liver Transplant Tolerance: The Role of Foxp3-Expressing CD25+CD4+ Regulatory T Cells

Liver allografts in mice are accepted across MHC barriers without requirement for immunosuppressive therapy. The mechanisms underlying this phenomenon remain largely undefined. In this study, we investigated the role of Foxp3-expressing CD25⁺CD4⁺ regulatory T cells (Treg) in the induction of murine liver transplant tolerance. Foxp3⁺CD25⁺CD4⁺ T cells were increased in liver grafts and recipient spleens from day 5 to day 100 posttransplantation, associated with enhanced CTLA4 and TGF-β expression and IL-4 production. Depletion of recipient CD25⁺CD4⁺ T cells using anti-CD25 mAb (250 μg/day) induced acute liver allograft rejection. This was associated with a decreased ratio of Foxp3⁺ Treg: T effector cells, decreased IL-4 and elevated IL-10 and IL-2 production by graft-infiltrating T cells, and reduced apoptotic activity of graft-infiltrating CD4⁺ and CD8⁺ T cells in anti-CD25-mAb-treated recipients. Thus, the data suggest that Foxp3⁺CD25⁺CD4⁺Treg are involved in spontaneous acceptance of liver allografts in mice. The ratio of Treg to T effector cells appears to determine liver transplant outcome. CTLA4, IL-4, TGF-β and apoptosis of graft-infiltrating T cells are also associated with liver transplant tolerance and may contribute, at least in part, to the mechanisms of Treg-mediated immune regulation in this model.     

Primed CD8+ T-Cell Responses to Allogeneic Endothelial Cells Are Controlled by Local Complement Activation

CD8 T cells primed by transplantation recognize allogeneic class I MHC molecules expressed on graft vascular endothelium and contribute to allograft injury. We previously showed that immune cell-derived complement activation fragments are integral to T cell activation/expansion. Herein we tested the impact of local complement production/activation on T cell/endothelial cell (EC) interactions. We found that proinflammatory cytokines upregulated alternative pathway complement production by ECs, yielding C5a. We further found that ECs deficient in the cell surface C3/C5 convertase regulator decay accelerating factor (DAF, CD55) induced greater CD8 T-cell proliferation and more IFNγ⁺ and perforin⁺ effector cells than wild-type (WT) ECs. Allogeneic C3^−/− EC induced little or no CD8 responses. Abrogation of responses following C5a receptor (C5aR) blockade, or augmentation following addition of recombinant C5a demonstrated that the effects were mediated through T-cell-expressed-C5aR interactions. Analyses of in vivo CD8 cell responses to transplanted heart grafts deficient in EC DAF showed similar augmentation. The findings reveal that EC-derived complement triggers secondary CD8 T-cell differentiation and expansion and argue that targeting complement and/or C5aR could limit T-cell-mediated graft injury.     

Aging Augments IL-17 T-Cell Alloimmune Responses

As increasing numbers of elderly patients require solid organ transplantation, the need to better understand how aging modifies alloimmune responses increases. Here, we examined whether aged mice exhibit augmented, donor-specific memory responses prior to transplantation. We found that elevated donor-specific IL-17, but not IFN-γ, responses were observed in aged mice compared to young mice prior to transplantation. Further characterization of the heightened IL-17 alloimmune response with aging demonstrated that memory CD4⁺ T cells were required. Reduced IL-2 alloimmune responses with age contributed to the elevated IL-17 phenotype in vitro , and treatment with an anti-IL-17 antibody delayed the onset of acute allograft rejection. In conclusion, aging leads to augmented, donor-specific IL-17 immune responses that are important for the timing of acute allograft rejection in aged recipients. IL-17 targeting therapies may be useful for averting transplant rejection responses in older transplant recipients.     

An Anti-CD103 Immunotoxin Promotes Long-Term Survival of Pancreatic Islet Allografts

Previous studies using knockout mice document a key role for the integrin CD103 in promoting organ allograft rejection and graft-versus-host disease. However, a determination of whether blockade of the CD103 pathway represents a viable therapeutic strategy for intervention in these processes has proven problematic due to the lack of reagents that efficiently deplete CD103⁺ cells from wild type hosts. To circumvent this problem, we conjugated the nondepleting anti-CD103 monoclonal antibody, M290, to the toxin, saporin, to produce an immunotoxin (M290-SAP) that efficiently depletes CD103⁺ cells in vivo . Herein, we show that M290-SAP dramatically reduces the frequency and absolute numbers of CD103-expressing leukocytes in the blood, spleen, mesenteric lymph nodes and intestinal epithelium of treated mice. We further demonstrate that M290-SAP promotes indefinite islet allograft survival in a fully MHC mismatched mouse model. The prolonged islet allograft survival resulting from M290-SAP treatment was associated with multiple effects in the host immune system including not only depletion of CD103-expressing leukocytes, but also an increase in CD4⁺CD25⁺FoxP3⁺ T regulatory cells and a predominance of effector-memory CD8 T cells. Regardless of the underlying mechanisms, these data document that depletion of CD103-expressing cells represents a viable strategy for therapeutic intervention in allograft rejection.     

CD8 T Cells Can Reject Major Histocompatibility Complex Class I-Deficient Skin Allografts

Following transplantation, recipient T cells can recognize and respond to donor antigens expressed directly on donor cells, and can respond to donor-derived peptides that have been processed and presented in the context of recipient MHC through the indirect pathway. Indirectly primed CD4⁺ T cells have been well studied in transplantation, but little information is available regarding whether indirectly primed CD8⁺ T cells participate in rejection. To address this, we placed MHC class I-deficient D^bK^b knockout skin grafts onto allogeneic H-2 ^k SCID recipients followed by adoptive transfer of purified H-2 ^k CD8⁺ T cells. The MHC class I-deficient grafts were rejected and only CD8⁺ T cells were detectable in the recipient lymphoid organs and in the skin grafts. Immunohistochemical analysis showed that CD8⁺ T cells were found in close proximity to vascular endothelial cells and to recipient infiltrating macrophages, suggesting specific interactions. The data demonstrate that cross-primed polyclonal CD8⁺ T cells can function as active participants in the effector phase of rejection. The findings confirm and extend previous studies using a monoclonal TCR transgenic T cell and shed light on mechanisms of acute and chronic graft injury that are potentially relevant to human transplant recipients.     

Aqueous Humor Alloreactive Cell Phenotypes, Cytokines and Chemokines in Corneal Allograft Rejection

As biopsies are not taken at the time of human corneal allograft rejection, most information on the early cellular changes in rejection is from animal models. We examined the phenotype of alloreactive cells present in the human anterior chamber during corneal graft rejection by flow cytometry and quantified aqueous humor levels of cytokines and chemokines using cytometric bead array. Aqueous and peripheral blood samples were taken from patients with graft endothelial rejection (n = 11) and from control patients undergoing cataract surgery (n = 8). CD45⁺CD4⁺, CD45⁺CD8⁺ and CD45⁺CD14⁺ cells were found in aqueous during rejection; no CD45⁺ cells were seen in control samples. Higher proportions of CD45⁺ cells found in aqueous during rejection were CD14⁺, denoting monocyte/macrophage lineage, than were CD4⁺ or CD8⁺. Large elevations were seen in aqueous levels of IL-6, MCP-1 and IP-10 during rejection compared with controls; smaller but still statistically significant increases were seen in MIP-1α and eotaxin. The role of CD14⁺ cells in allorejection is unclear as is the potential of these chemokines and their receptors as therapeutic targets. Aqueous humor samples offer a unique opportunity to analyze components of the allogeneic response in direct contact with donor tissue but without artifacts inherent in examination of tissue.     

Selective Immunomodulation: Utilization of CD29/VLA Molecules

Abstract: Accumulating evidence suggests that the VLA/ CD29 molecule plays an important role in T-cell costim-ulation, and CD4 ⁺ CD29/VLA ⁺ memory T cells play a key role in induction of CD8 killer effector T cells which are considered to be a major population involved in graft rejection. To target limited elements of the T-lymphocyte population, we have described the preparation of a bi-specific antibody-toxin conjugate designed to target CD4 ⁺ CD29 ⁺ memory T cells. We also showed that the solid-phase crosslinking of VLA-4 by the antibody against this molecule or by its ligand, the CS-1 region of fibronectin, stimulates tyrosine phosphorylation of 140, 120-105, 80-70, 60-55, 50, and 45 kilodalton proteins. In addition, we identified the ppl40 protein as PLCγ, ppl20 protein as ppl25^FAK, pp70 and pp50 proteins as paxillin, and pp60-55 proteins as pp59^fyn and pp56^lck, and pp45 as MAP kinase, respectively. Moreover, we demonstrated that ppl25^FAK is directly associated with paxillin. The paxillin binding domain of ppl25^FAK is homologous to the paxillin binding domain of vinculin. Mutations in the conserved amino acid residues between ppl25^FAK and vinculin result in the loss of paxillin-binding activity. Because VLA/CD29 is preferentially expressed on CD4 memory T cells, the above described system will be used to develop a novel drug design for providing selective immunosuppression useful for organ transplantation. Key Words: Immunomodulation–Graft rejection– Transplantation.     

Adoptive transfer: The role of perforin in mouse cytotoxic T lymphocyte rejection of human tumor xenografts in vivo

ABSTRACT: The popliteal lymph node cells of immunocompetent mice generated a strong in vitro cytotoxic response to footpad injection of several human tumor cell lines and the resulting mouse effector cells predominantly used a perforin-mediated cytotoxic mechanism. A relatively minor FasL-dependent cytotoxic response to CEM-CCRF and Jurkat leukemias, but not colon carcinoma COLO 205 cells, was also detected in immunized perforin-deficient mice. In vitro depletion of CD3⁺ CD8⁺ T cells, but not CD4⁺ T or NK1.1⁺ cells, completely inhibited lysis of human tumor cells, suggesting that CD3⁺ CD8⁺ T cells were effectors of perforin-mediated xenospecific cytotoxicity. Xenospecific cytotoxic T cells from wild-type mice were extremely efficient at rejecting tumor when adoptively transferred into scid mice bearing established COLO 205, CEM-CCRF, or Jurkat tumor xenografts. By contrast, cytotoxic T lymphocytes of perforin-deficient mice had no effect on the growth of established tumor xenografts. These data indicate that perforin, and hence direct cytotoxicity, plays a key role in the ability of adoptively transferred CD8⁺ cytotoxic T lymphocytes to eradicate established xenografts.     

RIP2 Is Required for NOD Signaling But Not for Th1 Cell Differentiation and Cellular Allograft Rejection

Two previous reports that receptor-interacting protein (RIP)-2 knockout (RIP2^–/–) mice had defective nuclear factor-kappa B (NF-κB) signaling and T helper (Th)1 immune responses had led us to believe that this putative serine-threonine kinase might be a possible target for transplant immunosuppression. Thus, we tested whether RIP2^–/– mice were able to reject vascularized allografts. Surprisingly, we found that T cells from RIP2^–/– mice proliferated and produced interferon (IFN)-γ after allostimulation in vitro . Moreover, naïve RIP2^–/– CD4⁺ T cells differentiated normally into Th1 or Th2 cells under appropriate cytokine microenvironments. Consistent with these findings, no difference in allograft survival was observed between wild-type and RIP2^–/– recipient mice, and rejection had similar pathology and cytokine profiles in both types of recipients. RIP2 deficiency was associated with defective NOD signaling, but this did not affect T-cell receptor (TCR)-dependent activation of the canonical NF-κB signaling or expression of NF-κB genes in rejecting allografts. Our data demonstrate that RIP2-deficient mice have intact canonical NF-κB signaling and can mount Th1-mediated alloresponses and reject vascularized allografts as efficiently as wild-type mice, thus arguing against RIP2 as a primary target for immunosuppression.     

Acute cell-mediated rejection in orthotopic pig-to-mouse corneal xenotransplantation

Abstract: Background:  To investigate the role of T cells and natural killer (NK) cells in mediating corneal xenograft rejection in a pig-to-mouse model.
Methods:  Pig corneas were orthotopically transplanted into BALB/c, C57BL/6, nude, severe combined immunodeficiency (SCID), and NOD/SCID/γc^null (NOG) mice. Graft survival was clinically assessed by slit-lamp biomicroscopy and median survival times (MST) were calculated. The rejected grafts were histologically evaluated using antibodies against CD4, CD8, NK1.1, and F4/80.
Results:  The pig corneal xenografts were acutely rejected by BALB/c and C57BL/6 mice (MST 9.0 days), while nude, SCID and NOG mice rejected pig corneas in a more delayed fashion (MST 16.0, 16.4, and 16.9 days, respectively). The majority of infiltrating cells found in rejected grafts in C57BL/6 mice were macrophages and CD4⁺ T cells, while CD8⁺ T cells and NK cells were rarely found. The grafts in nude mice had markedly decreased inflammatory infiltration with small numbers of macrophages and CD4⁺ T cells. Infiltration was even more modest in grafts in SCID and NOG mice.
Conclusions:  T cells play an important role in acute rejection of pig corneal xenografts in mice, although acute rejection is not solely the result of T-cell-mediated immunity. NK cells are less likely to be involved in the rejection process.