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.     

CD4+ T-cell-independent rejection of corneal allografts

BACKGROUND: Several studies suggest that a significant number of corneal allografts undergo rejection in the absence of CD4 T cells. This study examined the role of CD4 T cell-independent mechanisms of corneal allograft rejection. METHODS: BALB/c corneal allografts were transplanted to C57BL/6 beige nude mice that received either CD8 or CD8 T cells from C57BL/6 CD4 knockout (KO) mice that had rejected BALB/c corneal allografts. Immune effector functions of CD8 or CD8 T cells from C57BL/6 CD4 KO mice were assessed using delayed-type hypersensitivity assays and Annexin V apoptosis assays respectively. RESULTS.: Both CD8 and CD8 T cells from CD4 KO corneal allograft rejector mice mediated corneal allograft rejection following adoptive transfer to nude mice. CD8 T cells, but not CD8 T cells, from CD4 KO mice adoptively transferred donor-specific DTH and induced apoptosis of BALB/c corneal endothelial cells in vitro. Apoptosis of BALB/c corneal endothelial cells was mediated by double negative (DN) T cells, as treatment of CD8 cells from CD4 KO mice with anti-Thy 1.2 plus complement abolished their effector function. CONCLUSION: The results support the proposition that CD4 T cell-independent rejection of corneal allografts can be mediated by either CD8 or CD8 T cells. The CD8 T cells represent a unique DN T cell population that might mediate rejection by either direct cytolysis or by inducing apoptosis of the donor corneal endothelium.     

Humoral immunity to heterotopic corneal allografts in the rat

Despite indications that humoral immune responses may be involved in corneal allograft rejection, few definitive reports of such responses exist, and the antigens responsible for evoking and targeting these responses remain unknown. We report the development of donor-specific humoral immunity in PVG (RT1c) rats given fully allogeneic ACI (RT1a) corneal grafts heterotopically. PVG anti-ACI cornea serum lysed ACI, but not PVG, target cells in complement-dependent cytotoxic assays. Target cells from PVG.1A or PVG.R1 congenic rats, which respectively share the entire rat major histocompatibility complex (MHC) region (i.e., RT1) or only class I MHC antigens (RT1.A region) with ACI (on PVG backgrounds) were also lysed by this serum. Lysis of PVG.1A targets was significantly more extensive than lysis of PVG.R1 cells, indicating that both RT1.A and non-RT1.A antigens act as targets for cell lysis. Levels of PVG.1A and ACI target lysis were equivalent, suggesting that minor transplantation antigens were not significantly involved in this humoral response.     

Cellular immunity to heterotopic corneal allografts in the rat

Although the immune nature of corneal allograft rejection has been recognized for over thirty years, the specific mechanisms involved in such reactions remain obscure. We investigated the cellular immune responses of PVG (RT1c) rats that were grafted with fully allogeneic ACI (RT1a) skin, ACI cornea, or PVG cornea to the chest wall or given sham grafts. Cell-mediated lymphocytotoxicity (CML) was tested at 10 days posttransplant by placing recipient spleen cells in culture with irradiated ACI stimulator cells, and six days later measuring specific lysis of 51Cr-labeled target lymphoblasts at several effector-to-target ratios. Effector cells from animals receiving allogeneic skin or cornea grafts lysed targets from the donor (ACI) strain at levels significantly (P less than 0.01) above those obtained using effectors from control (sham grafted or syngeneic corneal graft recipient) animals. Significant lysis was also seen using target cells from PVG.1A (RT1a) or PVG.R1 (RT1r1) congenic rats, which differ from recipients only at the RT1 complex and the RT1.A (class I antigen) region, respectively. Stimulator cells from PVG.1A and PVG.R1 animals also permitted detection of specific responses in secondary CML, but syngeneic PVG stimulators did not, indicating that in vitro restimulation of effector cells can be met by using stimulator cells bearing only allogeneic class I major histocompatibility complex (MHC) antigens. These results indicate that corneal allografts evoke specific cellular immune responses in the rat, and that class I MHC antigens act as effective targets for these responses.     

Role of natural killer cells in the rejection process of corneal allografts in rats

BACKGROUND: The exact mechanism of human corneal allograft rejection, which is the major cause of corneal transplant failure, remains unclear. We investigated the role of natural killer (NK) cells in rat corneal allograft rejection by examining the aqueous humor (AH) cell infiltrate on different postoperative days. METHODS: Flow cytometric analysis was performed on the AH and submandibular draining lymph node (DLN) cells before transplantation and at different time points thereafter. In addition, we performed functional cytotoxicity assays with cells present in the AH during corneal rejection. RESULTS: We demonstrated a gradual increase in the absolute cell number of different hematopoietic subpopulations in the AH after allogeneic cornea transplantation. CD3CD4 cells, mainly monocytes and macrophages, were the predominant subpopulation 2 days after transplantation, followed by a successive relative increase of CD4 T cells, CD8 T cells, CD161 T cells, and NK cells. NK and CD161 T cells were present at a 10- to 15-fold higher percentage than in the DLN, suggestive of local expansion of these cells. A higher percentage of NK cells were CD8-negative compared with DLN NK cells. AH cells specifically lysed allogeneic cells, and this cytotoxicity was mainly attributable to NK cells but not to CD4 or CD8 T lymphocytes. CONCLUSION: These results confirm the crucial role of CD4 cells in the allogeneic corneal graft rejection process and implicate NK cells as possible mediators of the rejection.     

Analysis of primed donor-specific T cells in recipient mice bearing orthotopic corneal allografts

BACKGROUND: Orthotopic corneal allografts placed in normal eyes of mice are often not rejected, whereas grafts placed in high-risk (neovascularized) eyes are routinely destroyed. Because rejection of solid tissue allografts is usually mediated by donor-specific T cells, we wished to determine the extent to which donor-specific T cells become primed in mice bearing orthotopic corneal allografts in normal and "high-risk" eyes. METHODS AND RESULTS: Our data indicate corneal allografts placed in neovascularized eyes were rejected within 2 weeks, and lymph nodes draining these grafts contained primed donor-specific T cells that proliferated in vitro and displayed cytotoxic activity. By contrast, only 50% of corneal allografts placed in normal eyes experienced rejection. Lymphoid cells from all of these mice displayed donor-specific proliferative activity, irrespective of whether the graft was accepted or rejected. At no time were donor-specific cytotoxic T cells detected. Failure to detect primed cytotoxic T cells was not the result of anergy or deletion of unprimed donor-specific precursors of CTL. CONCLUSIONS: We conclude that primed donor-specific proliferative and cytotoxic T cells directed at MHC alloantigens correlate well with rejection of orthotopic corneal allografts in neovascularized high-risk eyes. However, rejection of cornea allografts in normal eyes does not correlate well with proliferative T cells, nor are donor MHC-specific cytotoxic T cells detected. The possibility is discussed that graft rejection in normal eyes is not mediated by T cells that recognize MHC alloantigens via the direct pathway, but via T cells that recognize donor alloantigens presented by recipient MHC molecules (indirect pathway).     

Vascular rejection and graft eosinophilia in rat lung allografts

Allogeneic lung transplantation was performed using a rat model in order to assess the pathologic changes that developed during the process of rejection. The left lungs of 38 BN rats (RT-1n) were orthotopically transplanted into LEW rats (RT-1). The allografts developed the well-known changes of perivascular, peribronchial, and interstitial lymphocytic infiltrates resulting in necrosis of the pulmonary parenchyma at 7-8 days after transplantation. In addition, we document two findings that have not been reported previously in lung transplantation: vasculitis and eosinophilic infiltrates. Vasculitis with swelling and vacuolization of the endothelial cells was observed in transplants as early as 3 days following transplantation. Vasculitis with fibrinoid necrosis of the vessel wall was prominent at 7-8 days after grafting. The allografts also exhibited eosinophilia at 2 to 4 days following transplantation. The density of eosinophils in the inflammatory infiltrate reached a peak of 20% on Day 3 post-transplantation. These findings suggest an important role of humoral immunity and a possible involvement of eosinophils in lung allograft rejection.     

Expression of CD44 in rat liver allografts during rejection

As CD44 is believed to be a homing receptor involved in lymphoid trafficking and inflammatory responses, it is expected to be closely linked to transplant rejection. In this study, the expression of CD44 during liver transplant rejection was compared with the expression of lymphocyte-function associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1), which play an essential role in cell interactions and the initiation of immune responses. Male Brown Norway (BN) and Lewis (LEW) rats were used as donors and recipients, respectively. Orthotopic liver transplantation (OLTX) was done using the cuff technique of Kamada and Calne. Animals were killed on days 3, 5, and 7 after OLTX, and a piece of tissue from each of the liver grafts was obtained. Immunohistochemical staining was used to investigate the expression of CD44, ICAM-1, and LFA-1. CD44 was strongly expressed in portal areas of the rejected liver, and LFA-1 and ICAM-1 were expressed mainly on sinusoids and hepatocytes. These findings indicate that CD44 is closely involved in lymphocyte infiltration, which is dominant in portal areas, and that lymphocyte infiltration during the rejection process may involve a homing mechanism. Received for publication on Oct. 17, 1997; accepted on Oct. 21, 1997     

肝硬化大鼠原位肝移植模型的制作及术后排斥反应的观察

目的 制备肝硬化大鼠的原位肝移植模型,观察术后排斥反应发生情况,为进行其它研究创建一个平台.方法 以皮下注射CCl4联合饮用苯巴比妥钠和乙醇溶液的方法制备大鼠肝硬化模型,应用改良的"二袖套"法建立大鼠原位肝移植模型,同系移植者的供、受者均为SD大鼠(SD实验组),以接受肝移植的正常SD大鼠为对照(SD对照组);同种移植者的供者为Lewis大鼠,受者为BN大鼠(BN实验组),以接受肝移植的正常BN大鼠为对照(BN对照组).术后观察受者的存活情况以及移植肝的组织学变化.结果 肝硬化大鼠门静脉压力为(182.0±10.7)mm H2O,显著高于正常大鼠的(70.8±5.5)mm H2O(P＜0.01),移植后7 d降至(82.7±10.7)mm H2O.同种移植组术后5～12 d,移植肝组织中均可见中、重度急性排斥反应病理改变.同系移植者存活时间中位数均＞100 d;同种移植者中,BN对照组和BN实验组受者肝移植后存活时间中位数均为10 d.结论 以Lewis大鼠为供者、肝硬化BN大鼠为受者制备的肝移植模型术后排斥反应的发生率较高,可作为肝移植后排斥反应相关研究的平台.     

The differential effects of donor versus host Langerhans cells in the rejection of MHC-matched corneal allografts

The fate of MHC-identical, multiple minor H-disparate corneal grafts was examined in the rat. Although skin grafts exchanged between LEW and F344 rats were invariably rejected, only 26% of the corresponding corneal grafts underwent rejection. The immunologic privilege of the minor H-disparate corneal grafts was due, at least in part, to the absence of donor-derived Langerhans cells. Corneal grafts were normally devoid of donor-derived Langerhans cells; however, grafts pretreated with latex beads became infiltrated with donor-derived Langerhans cells and were rejected by 59% of the naive minor H--compatible recipients. By contrast, untreated LEW corneal grafts underwent rejection in 26% of the naive F344 hosts even though the grafts became heavily infiltrated with host-derived Langerhans cells. The immunologic privilege of minor H-disparate corneal grafts was not the result of efferent blockade or suppression of the immune response. F344 hosts bearing long-term surviving LEW corneal allografts were challenged with LEW skin grafts. In all cases, orthotopic skin grafts were rejected acutely. Moreover, all previously clear corneal grafts underwent rejection following skin graft rejection. Thus, the unique absence of donor-derived Ia+ passenger cells and the avascular graft bed conspire to provide the primary minor H-disparate corneal graft with an immunologic privilege not shared by other organ grafts.     

Cyclosporin, toxicity and efficacy in rejection of liver allografts in the rat

52 orthotopic liver transplants were performed in DA to lewis rat strain combination, in order to appreciate cyclosporine toxicity, and efficacy at doses of 10 mg/kg day (G II) and 20 mg/kg/day (GIII) compared to liver allografts in DA/lewis rats. The first signs of cyclosporine hepatotoxicity are biological (increased plasma level of bilirubine and transaminase) that were noticed at the dose of 20 mg/kg/day. Histological signs (cells inclusion, hepatocytic necrosis) appeared late and were less constant as well as difficult to assert creatinine plasma level was the best reflect of cyclosporine nephrotoxicity. Renal toxicity was practically constant at the dose of 20 mg/kg/day. In spite of renal and hepatic toxicity, cyclosporin by itself, allows the abolition of the acute rejection of liver allografts in the rat.