**4. Classification and effector mechanisms of allograft rejection**

Allograft rejection mainly involves host-versus-graft reaction in liver transplantation, which is the rejection of the transplant by the recipient's body. The recipient's lymphocyte mediated reactions to allogeneic or xenogeneic cells – acquired as a graft or otherwise –lead to damage and/or the destruction of the grafted cells. The graft rejection has been divided into three groups: hyperacute rejection, acute rejection and chronic rejection (Table 1).


Table 1. Different types of graft rejection

#### **4.1 Hyperacute rejection**

82 Liver Transplantation – Basic Issues

The CD40/CD154 co-stimulatory pathway is a second important co-stimulatory pathway that is critical in the immune response of allotransplantation. CD40 is mainly expressed on APCs (including DCs, B cells, and macrophages) but it can also be expressed on nonimmune cells (including endothelial cells, mast cells, platelets and epithelial cells). However, CD154 is mainly expressed on CD4 T cells following activation, and to a lesser extent on NK cells, B cells, and CD8 T cells. CD154 combines with CD40, which is critical for the activation of DCs, B cells, and macrophages. In DCs, CD40 upregulates interleukin12 (IL-12) production, and in macrophages it results in the production of various proinflammatory cytokines. CD154 was also detected on Kupffer cells and on sinusoidal macrophages in livers during chronic rejection, but not in stable liver allografts or normal liver (Gaweco A S et al., 1999). The most widely-used measure to block CD28-B7 interactions has been CTLAimmunoglobulin (Ig). In the orthotopic rat liver transplantation model, repeated administration of CTLA-Ig – beginning with CTLA-Ig in combination with donor splenocytes – leads to extended graft survival of >100 days, whereas the delayed administration of CTLA4-Ig alone or donor splenocytes alone did not (Neumann U P, et al., 2002). In recent years, many studies have shown that B7 cross-linking on APCs by CTLA4-Ig induces indoleamine 2, 3-dioxygenase (IDO), which itself inhibits local T cell activation (Mellor A L et al., 2003; Li W et al., 2009). Gene therapy approaches to deliver CTLA4-Ig to liver allografts have been successfully used in some animal experiments. Adenoviral-mediated gene delivery of CTLA4-Ig through ex vivo perfusion of cold preserved livers resulted in indefinite survival of rat liver allografts and in the generation of donor-specific unresponsiveness (Olthoff K et al., 1998). An interesting report suggests that CD154/CD40 interaction plays a role in promoting dendritic cell-maturation in the absence of CD4+CD25+ regulatory lymphocytes, whilst these cells promote the maintenance of immaturity (Serra P et al., 2003; Misra N et al., 2004). This accounts for the importance of DC activation, not only by innate immune mechanisms but also by activated T cells. The efficacy of anti-CD154 in a rat liver allograft model not only prolongs allograft survival but it was also associated with fewer complications (Bartlett AS et al., 2002). These roles underline the significant beneficial effects of CTLA4-Ig and anti-CD154 in pre-clinical models of transplantation; however, its clinical application has a long way to go for liver transplantation.

**4. Classification and effector mechanisms of allograft rejection** 

Acute Days – weeks Primary activation of T cells

Type of rejection Time taken Cause

Table 1. Different types of graft rejection

Allograft rejection mainly involves host-versus-graft reaction in liver transplantation, which is the rejection of the transplant by the recipient's body. The recipient's lymphocyte mediated reactions to allogeneic or xenogeneic cells – acquired as a graft or otherwise –lead to damage and/or the destruction of the grafted cells. The graft rejection has been divided into three groups: hyperacute rejection, acute rejection and chronic rejection (Table 1).

Hyperacute Minutes-hours Pre-existing anti-donor antibodies and complement activation

Chronic Months – years Causes unclear: antibodies, slow cellular reactions,

immune complexes, recurrence of disease.

Hyperacute rejection often occurs within minutes to hours after the host blood vessels are anastomosed to graft vessels. The rejection is mediated by pre-existing antibodies specific to the graft antigens (including ABO blood type antigens, VEC antigens and HLA antigens). Furthermore, these different antigens can activate the complement of the host and lead to damage to the endothelial cell. Studies have reported that the process is often accompanied with platelets activation and results in thrombosis and vascular occlusion (Fiane A E et al., 1999). In addition, the massive recruitment of neutrophils occurs, followed by rapid inflammation after transplantation. The pathological changes of hyperacute rejection are thrombotic occlusion of the graft vasculature ischemia, denaturation and necrosis (Figure 4). This rejection is relatively rare in liver transplantation.

Fig. 4. Hyperacute rejection: complement activation, endothelial damage, inflammation, thrombosis and vascular occlusion

#### **4.2 Acute rejection**

Acute rejection occurs within days and up to three months after transplantation (80-90% of cases occur within one month). The rejection occurs due to donor HLA interaction with the host T cells, creating a cascade of immune responses initiated by that interface. After a solid organ transplant, there is an immunological milieu of activity. The mechanisms of the process involve abundant immune factors, such as humoral and/or cellular mechanisms (Figure 5). Antibodies can injure the graft by activating complement and mononuclear cells with Fc receptors that recognise alloantigens on the endothelial cell, resulting in vasculitis. Cytotoxic T cells (CD8+) will recognise alloantigens on an antigen presenting cell (APC) by direct presentation on the donor tissue and endothelial cells, which promotes the apoptosis of transplanted tissue. It has been shown that CD8+ cells alone are sufficient for the mediation of acute allograft rejection, but with the help of CD4+ cytokines secretion – such as IL-2 – clonal expansion and the expression of cytotoxic attack molecules will be upregulated (Kreisel D et al., 2002). The Fas/Fas ligand (FasL) pathway is another deathinducing pathway which is utilised by CD8+ cells. Whereas FasL is specifically induced upon CD8+ cells' activation, Fas is ubiquitously expressed on lymphoid and non-lymphoid tissue, including the liver. The Fas/FasL pathway is thought to play an important role in a variety of hepatic pathologies, and there is evidence that this pathway is also active during liver allograft rejection (Tannapel A et al., 1999; Ogura Y et al., 2001). Delayed hypersensitivity also has an important role in acute rejection, being initiated by alloantigenprimed CD4+ cells specific to the donor class II (Carrodeguas L et., 1999). CD4+ cells release IFN-γ by re-exposure to specific alloantigens, a proinflammatory cytokine that can cause the activation of macrophages and the subsequent release of a variety of inflammatory mediators. These inflammatory mediators can augment the cellular anti-graft response or else can cause direct tissue damage. The acute rejection relatively occurs after liver transplantation is rare. But it has been a challenging process to try to unravel the participation of specific effector pathways and their interrelationships in the acute rejection of liver transplantation. The pathological features of acute rejection are acute vasculitis and parenchymal cell necrosis, along with the infiltration of lymphocytes and marophages (Figure 6).

Fig. 5. Acute rejection: parenchymal cell damage and endotheliitis

Fig. 6. A higher magnification of the previous photomicrograph details the subendothelial localisation of lymphocytes and the slight extension of the infiltrate into the perivenular hepatic parenchyma (http://tpis.upmc.com/TPIShome/)

#### **4.3 Chronic rejection**

Chronic rejection is less well-defined than either hyperacute or acute rejection, developing months or years after acute rejection reactions have subsided. Chronic rejection is an indolent but progressive form of allograft injury that is usually irreversible and which eventually results in the failure of most vascularised solid organ allografts. It is the single most significant obstacle to morbidity-free long-term survival. By five years after transplantation, it affects as many as 30-50% of heart, lung, pancreas and kidney allograft recipients, but only 4-8% of patients who undergo liver replacement (Demetris, A J et al.,

primed CD4+ cells specific to the donor class II (Carrodeguas L et., 1999). CD4+ cells release IFN-γ by re-exposure to specific alloantigens, a proinflammatory cytokine that can cause the activation of macrophages and the subsequent release of a variety of inflammatory mediators. These inflammatory mediators can augment the cellular anti-graft response or else can cause direct tissue damage. The acute rejection relatively occurs after liver transplantation is rare. But it has been a challenging process to try to unravel the participation of specific effector pathways and their interrelationships in the acute rejection of liver transplantation. The pathological features of acute rejection are acute vasculitis and parenchymal cell

necrosis, along with the infiltration of lymphocytes and marophages (Figure 6).

Fig. 5. Acute rejection: parenchymal cell damage and endotheliitis

hepatic parenchyma (http://tpis.upmc.com/TPIShome/)

**4.3 Chronic rejection** 

Fig. 6. A higher magnification of the previous photomicrograph details the subendothelial localisation of lymphocytes and the slight extension of the infiltrate into the perivenular

Chronic rejection is less well-defined than either hyperacute or acute rejection, developing months or years after acute rejection reactions have subsided. Chronic rejection is an indolent but progressive form of allograft injury that is usually irreversible and which eventually results in the failure of most vascularised solid organ allografts. It is the single most significant obstacle to morbidity-free long-term survival. By five years after transplantation, it affects as many as 30-50% of heart, lung, pancreas and kidney allograft recipients, but only 4-8% of patients who undergo liver replacement (Demetris, A J et al., 1997). Liver allografts differ from other solid organs in that chronic rejection is potentially reversible. This feature has been mainly attributed to its unique immunobiological privilege and the regenerative capacity of the process. Livers with chronic rejection have a decreased number of bile ducts on biopsy. This is referred to as "vanishing bile duct syndrome" (Demetris A et al., 2000). Chronic rejection is characterised by vasculopathy, fibrosis and a progressive loss of organ function. It is probably caused by multiple factors, viz., antibodies as well as lymphocytes (Figure 7). Chronic rejection may be mediated by a low-grade, persistent, delayed hypersensitivity response in which activated macrophages secrete mesenchymal cell-growth factors. Of potential importance are the persistent viral infections which induce cellular immune responses which in turn may synergise with donor-specific alloreactive T cells within the allograft. Chronic rejection may also reflect chronic ischemia secondary to the injury of blood vessels by antibody or cell-mediated mechanisms. Vascular occlusion may also occur as a result of smooth muscle cell proliferation in the intimae of arterial walls.

Fig. 7. Severe or very late-stage chronic rejection can result in the loss of the small branches of the hepatic artery, in addition to the loss of bile ducts. Note the lack of bile ducts and lack of hepatic artery branches in this portal tract (http://tpis.upmc.com/TPIShome/)
