**5.1 Graft selection**

The majority of liver transplant centres regard blood group compatibility as the primarily immunological selection criterion. A liver from a donor with a compatible ABO and Rh blood group is easy and feasible, with well-documented reports of this being performed in urgent situations (Gordon R D et al., 1986). In recent years, many transplantation centres have also carried out the operation with ABO-incompatible grafts, and the outcomes of ABO-incompatible liver transplantations have been similar to that of blood-type-matched transplantations in some centres. However, infection is the major cause of morbidity and mortality after ABO-incompatible liver transplantation (Tanabe M et al., 2010). At present, the transplantation of compatible but not identical livers is common practice, especially for recipients with the less common blood groups. Interestingly, the results of ABO identical grafts were slightly better than the ABO compatible but non-identical grafts (Gugenheim J et al., 1990). An occasional complication with compatible, non-identical grafts is the occurrence of allograft rejection, due to the immunocompetent passenger lymphocytes within the transplanted liver producing antibodies against the recipient erythrocytes. It is wellestablished that renal transplantation in the presence of donor-specific cytotoxic antibodies – demonstrated by a positive cross-match – will result in rapid graft loss. However, the liver behaves in a totally contrary manner. In addition, the major histocompatibility antigens have a well-documented role in renal transplantation. However, early studies of liver transplantation in pigs implied that the liver may be a privileged organ exhibiting minimal rejection, with some grafts surviving without immunosuppression. This special feature prompted surgeons to ignore HLA-matching in patient selection for donor shortages. Retrospective data has not shown any clear survival advantages associated with good HLAmatching (Navarro V et al., 2006). Interestingly, some studies suggest that there is a clear disadvantage with certain aspects of HLA-matching. The largest series from Pittsburgh, involving more than 500 transplants, concludes that overall graft survival is actually reduced in grafts matched for HLA (Markus BH et al., 1988).

#### **5.2 Immunosuppressive therapy**

The liver is a privileged organ with a lower incidence of rejection than other organs, but immunosuppressive regimens are nonetheless required to control the alloreactive Tlymphocyte response after transplantation. In the 1990s, acute liver rejection occurred in up to 60% [1] of patients, without compromising graft or and patient survival (Neuberger J, 1999). Since 2000, the incidence of acute liver rejection has decreased to 15% of recipients. The incidence of chronic rejection is also declining, and most centres report current rates of 4% to 8%, whereas in the 1990s, rates of 15% to 20% were observed (Neuberger J, 1999).This decrease correlated with the use of new immunosuppressive drugs and improvements in treatment-management.

Over the last three decades, the number and types of immunosuppressive agents available to clinicians have increased considerably. The immunosuppressive therapy used in liver transplantation includes agents such as corticosteroids, calcineurin inhibitor (CNI), antimetabolites, inhibitors of TOR, and monoclonal and polyclonal antibodies which have different patterns of action (Figure 9) (Beaudreuil S et al., 2007). Corticosteroids are a class of steroid hormones that are produced in the adrenal cortex. Corticosteroids are involved in a wide range of physiological systems, including stress-response, immune-response and the regulation of inflammation. The drugs are hydrophobic, which enables them to enter the cell by membrane diffusion. They then form complexes with cytosolic receptors, leading to their translocation to the nucleus where they bind to glucocorticoid-response elements in the promoter regions of cytokine genes, thereby blocking T cell-mediated cytokine expression. Thus, corticosteroids have been a mainstay of treatment during the early days after transplantation, but as immunosuppressive agents they are often accompanied by many side-effects within a few years. Calcineurin inhibitor is the first routinely employed immunosuppressive agent, including cyclosporine A (CyA) and tacrolimus (FK-506). CyA selectively inhibits T lymphocyte proliferation by forming a complex with cyclophilin. This complex can inhibit the calcium and calmodulin-dependent phosphatase calcineurin.

recipients with the less common blood groups. Interestingly, the results of ABO identical grafts were slightly better than the ABO compatible but non-identical grafts (Gugenheim J et al., 1990). An occasional complication with compatible, non-identical grafts is the occurrence of allograft rejection, due to the immunocompetent passenger lymphocytes within the transplanted liver producing antibodies against the recipient erythrocytes. It is wellestablished that renal transplantation in the presence of donor-specific cytotoxic antibodies – demonstrated by a positive cross-match – will result in rapid graft loss. However, the liver behaves in a totally contrary manner. In addition, the major histocompatibility antigens have a well-documented role in renal transplantation. However, early studies of liver transplantation in pigs implied that the liver may be a privileged organ exhibiting minimal rejection, with some grafts surviving without immunosuppression. This special feature prompted surgeons to ignore HLA-matching in patient selection for donor shortages. Retrospective data has not shown any clear survival advantages associated with good HLAmatching (Navarro V et al., 2006). Interestingly, some studies suggest that there is a clear disadvantage with certain aspects of HLA-matching. The largest series from Pittsburgh, involving more than 500 transplants, concludes that overall graft survival is actually

The liver is a privileged organ with a lower incidence of rejection than other organs, but immunosuppressive regimens are nonetheless required to control the alloreactive Tlymphocyte response after transplantation. In the 1990s, acute liver rejection occurred in up to 60% [1] of patients, without compromising graft or and patient survival (Neuberger J, 1999). Since 2000, the incidence of acute liver rejection has decreased to 15% of recipients. The incidence of chronic rejection is also declining, and most centres report current rates of 4% to 8%, whereas in the 1990s, rates of 15% to 20% were observed (Neuberger J, 1999).This decrease correlated with the use of new immunosuppressive drugs and improvements in

Over the last three decades, the number and types of immunosuppressive agents available to clinicians have increased considerably. The immunosuppressive therapy used in liver transplantation includes agents such as corticosteroids, calcineurin inhibitor (CNI), antimetabolites, inhibitors of TOR, and monoclonal and polyclonal antibodies which have different patterns of action (Figure 9) (Beaudreuil S et al., 2007). Corticosteroids are a class of steroid hormones that are produced in the adrenal cortex. Corticosteroids are involved in a wide range of physiological systems, including stress-response, immune-response and the regulation of inflammation. The drugs are hydrophobic, which enables them to enter the cell by membrane diffusion. They then form complexes with cytosolic receptors, leading to their translocation to the nucleus where they bind to glucocorticoid-response elements in the promoter regions of cytokine genes, thereby blocking T cell-mediated cytokine expression. Thus, corticosteroids have been a mainstay of treatment during the early days after transplantation, but as immunosuppressive agents they are often accompanied by many side-effects within a few years. Calcineurin inhibitor is the first routinely employed immunosuppressive agent, including cyclosporine A (CyA) and tacrolimus (FK-506). CyA selectively inhibits T lymphocyte proliferation by forming a complex with cyclophilin. This complex can inhibit the calcium and calmodulin-dependent phosphatase calcineurin.

reduced in grafts matched for HLA (Markus BH et al., 1988).

**5.2 Immunosuppressive therapy** 

treatment-management.

Fig. 9. Pattern of T cell activation and targeting of mainly immunosuppressors. Signals 1, 2 and 3 for T cell activation are shown with a number. The drug targets used in tolerance protocols are shown with blue arrow

Calcineurin is a key enzyme involved in controlling the transcription of IL-2 and other cytokines (Friman S et al., 1996). Therefore, impairing IL-2 transduction has a profound effect on the immune process of rejection by inhibiting calcineurin. However, the CyA metabolism is complex in liver transplant patients. Because it is metabolised primarily in the intestine and the liver, it increases the burden on the liver and even results in liver failure. Fk506 is very similar in action to CyA, but it is substantially more potent. It acts by binding to the FK-binding protein 12. The complex formed inhibits calcineurin, which regulates the transcription of the genes encoding IL-2, IL-3, IL-4, IL-8, as well as various chemotactic factors (Komolmit P et al., 1999). The side-effects of Fk506 are similar to those of CyA. In clinical practice, given the initial impairment of liver function and the frequent renal failure observed in the postoperative period, physicians should delay the administration of CyA or FK-506, with no impact on the outcome of liver transplantation or the occurrence of allograft rejection. Antimetabolites were not initially used in liver transplantation. Mycophenolate mofetil (MMF) – as a new antimetabolite molecule – has been shown to inhibit T and B cell proliferation, making it possible to reduce the rate of acute rejection in renal transplantation. These antimetabolites can be used together with an antibody against the IL2 receptor, delaying the introduction of CINs. These findings rapidly led to the use of these drugs in liver transplantation. Combination therapy with tacrolimus and MMF may significantly reduce the incidence of acute liver allograft rejection, allow a significant reduction in tacrolimus dosage, and decrease the incidence of nephrotoxicity (Eckhoff D E et al., 1998). In addition, the side-effects of MMF were relatively few. Inhibitors of TOR mainly include Rapamicine and Everolimus. Rapamicine is a macrocyclic triene antibiotic that is structurally similar to tacrolimus. It forms a complex with the FK506-binding protein but it does not inhibit calcineurin. The complex blocks the cytokine response to T cell and B cell activation, preventing cell cycle progression and proliferation. Its principal side-effects are leukopenia, thrombocytopenia, high serum cholesterol and triglyceride levels, anaemia, lymphocele, wound dehiscence and mouth ulcers (Levitsky J., 2011). The biggest advantage of Rapamicine is associated with the lack of any significant nephrotoxicity (Vivarelli M et al., 2010). Compared with Rapamicine, Everolimus has greater bioavailability and a shorter half-life. The antibodies used in transplantation may be monoclonal or polyclonal. At present, monoclonal antibodies primarily include IL-2R antibodies and anti-CD52 antibodies. Two humanised IL-2R antibodies have been put on the market: basiliximab and daclizumab, which inhibit T cell proliferation by the competitive antagonism of IL-2 induced T cell proliferation, and they are accompanied with very few side-effects. OK3 is also currently the most widely-used monoclonal antibody, which binds to part of the T cell receptor (CD3) complex. The major impact of OK3 has been in the reversal of steroidresistant, acute rejection (Cosimi A B et al., 1981). Polyclonal antibodies are IgG fractions from animals inoculated with human lymphocytes, thymocytes or cultured lymphoblast. Polyclonal antibodies have more profound and long-lasting biological depleting effects than other antibodies (Rebellato L M). However, polyclonal antibodies often induce the oversuppression of the immune system, increasing the risk of infectious diseases, lymphoproliferative syndrome and tumours.

There are significant variations in the regimens for immunosuppressive therapy used by different liver transplant centres. In general, most regimens include corticosteroids plus one calcineurin inhibitor, such as CyA or FK506. Anti-proliferative agents are often used in the first few months, with the patients also receiving bitoherapy with low doses of CIN and steroids. In liver transplantation, physicians tend to withdraw steroids within a few years due to their many side-effects. In addition, a study compared two groups of patients, one given induction therapy based on anti-thymocyte globulin (ATG) and FK506 without steroids, and the other treated with FK506, MMF and steroids. A graft survival of one-and-ahalf years was 89% in both groups. However, rejection-rates were significantly lower in the group that was treated without steroids than in the group that was treated with steroids (Eason JD et al., 2001). Calcineurin inhibitor treatment often caused renal failure by nephrotoxicity. Studies have shown that up to 21% of patients were found to have developed chronic renal failure within five years of receiving a non renal transplant (Ojo AO et al.,2003). A recent report has shown that Sirolimus-based immunosuppressive therapy is a safe, effective replacement agent for primary immunosuppressive therapy in liver transplant recipients with FK506-related chronic renal insufficiency (Yang YJ et al., 2008). Furthermore, the addition of MMF to the regimen, and the reduction of the dose of calcineurin inhibitor by more than 50%, has been shown to improve renal function.

#### **5.3 Prospective of using recipients T regulatory cell**

In fact, the immunosuppression regimens used in liver transplantation were historically derived from those used in renal transplantation. Immunosuppressive regimens are required to control the allogeneic response in clinical liver transplantation, but they may also lead to severe complications, such as infectious diseases, cancers, cardiovascular diseases and – for treatments involving calcineurin inhibitors – chronic renal insufficiency.

structurally similar to tacrolimus. It forms a complex with the FK506-binding protein but it does not inhibit calcineurin. The complex blocks the cytokine response to T cell and B cell activation, preventing cell cycle progression and proliferation. Its principal side-effects are leukopenia, thrombocytopenia, high serum cholesterol and triglyceride levels, anaemia, lymphocele, wound dehiscence and mouth ulcers (Levitsky J., 2011). The biggest advantage of Rapamicine is associated with the lack of any significant nephrotoxicity (Vivarelli M et al., 2010). Compared with Rapamicine, Everolimus has greater bioavailability and a shorter half-life. The antibodies used in transplantation may be monoclonal or polyclonal. At present, monoclonal antibodies primarily include IL-2R antibodies and anti-CD52 antibodies. Two humanised IL-2R antibodies have been put on the market: basiliximab and daclizumab, which inhibit T cell proliferation by the competitive antagonism of IL-2 induced T cell proliferation, and they are accompanied with very few side-effects. OK3 is also currently the most widely-used monoclonal antibody, which binds to part of the T cell receptor (CD3) complex. The major impact of OK3 has been in the reversal of steroidresistant, acute rejection (Cosimi A B et al., 1981). Polyclonal antibodies are IgG fractions from animals inoculated with human lymphocytes, thymocytes or cultured lymphoblast. Polyclonal antibodies have more profound and long-lasting biological depleting effects than other antibodies (Rebellato L M). However, polyclonal antibodies often induce the oversuppression of the immune system, increasing the risk of infectious diseases,

There are significant variations in the regimens for immunosuppressive therapy used by different liver transplant centres. In general, most regimens include corticosteroids plus one calcineurin inhibitor, such as CyA or FK506. Anti-proliferative agents are often used in the first few months, with the patients also receiving bitoherapy with low doses of CIN and steroids. In liver transplantation, physicians tend to withdraw steroids within a few years due to their many side-effects. In addition, a study compared two groups of patients, one given induction therapy based on anti-thymocyte globulin (ATG) and FK506 without steroids, and the other treated with FK506, MMF and steroids. A graft survival of one-and-ahalf years was 89% in both groups. However, rejection-rates were significantly lower in the group that was treated without steroids than in the group that was treated with steroids (Eason JD et al., 2001). Calcineurin inhibitor treatment often caused renal failure by nephrotoxicity. Studies have shown that up to 21% of patients were found to have developed chronic renal failure within five years of receiving a non renal transplant (Ojo AO et al.,2003). A recent report has shown that Sirolimus-based immunosuppressive therapy is a safe, effective replacement agent for primary immunosuppressive therapy in liver transplant recipients with FK506-related chronic renal insufficiency (Yang YJ et al., 2008). Furthermore, the addition of MMF to the regimen, and the reduction of the dose of

calcineurin inhibitor by more than 50%, has been shown to improve renal function.

In fact, the immunosuppression regimens used in liver transplantation were historically derived from those used in renal transplantation. Immunosuppressive regimens are required to control the allogeneic response in clinical liver transplantation, but they may also lead to severe complications, such as infectious diseases, cancers, cardiovascular diseases and – for treatments involving calcineurin inhibitors – chronic renal insufficiency.

**5.3 Prospective of using recipients T regulatory cell** 

lymphoproliferative syndrome and tumours.

T regulatory cells (Tregs), a subset of CD4+CD25+Foxp3+ lymphocytes, have the functional ability to suppress alloimmune responses both *in vitro* and *in vivo*. Increasing evidence from animal transplant research shows that Tregs can play a key role in promoting immunological unresponsiveness to allograft transplants (Pilat N et al., 2010; Webster KE et al., 2009). Regulatory T cells are the key cell-types in the induction of immune tolerance, and so the modulation of such cells may provide new strategies in creating transplant tolerance. However, there are several challenges to translating Tregs into the clinic. Tregs only account for about 5-10% of the total CD4+ T cells in the periphery, the limitation of cell number restricted the clinical application. There are a number of studies demonstrating the functional instability of Tregs *in vivo*, which can become IL-17 producing T effector cells in the presence of IL-6 (Yang XO et al., 2008). Furthermore, T effector cells activated under inflammatory conditions are highly resistant to Tregs-mediated suppression (Korn T et al., 2007). Concerning the Tregs, there are two broad approaches to the use of Tregs to promote transplant tolerance. The first is to expand Tregs *in vitro* and then apply expanded Tregs as a cell therapy *in vivo*. The advantage of this approach is that antigen-specific Tregs can be created *in vitro* using donor antigens. The second approach is to selectively and specifically stimulate *in vivo*, by taking advantage of fundamental differences between the biology of Tregs and T effector cells. In general, Tregs are a promising substance for the achievement of transplant tolerance.
