**3.4 Co-stimulatory pathways and the immunology of allografts**

Evidently, T cells must receive two distinct but coordinated signals in order to achieve optimal activation. The first signal is provided by the TCR engagement with recognition of peptide/ MHC I or II on APCs, and the second signal is achieved by the interaction of costimulatory molecules on the T cells and their ligands on APCs. The importance of costimulation was found through experimental models in which its inhibition was achieved by some means, Signal 1 in the absence of signal 2 – as likely occurs in the liver – leads to a state of T cell non-responsiveness (or anergy) in which T cells can recognise cognate antigens through the TCR, but fail to mount a functional response upon reencounters with the antigen. So, there have been significant efforts to inhibit or block co-stimulatory pathways as a means of achieving allograft tolerance. There are two co-stimulatory pathways that are important in the generation of a complete T cell response are CD28/B7 and CD40/CD154 in the co-stimulatory field.

The role of CD28 has perhaps been that most intensively investigated in the co-stimulatory field. CD28 represents the prototypical T cell co-stimulatory molecule. In humans, CD28 is expressed on 90% of CD4 T cells and 50% of CD8 T cells; moreover, ligands for CD28, B7-1 (CD80) and B7-2 (CD86) are found on a variety of APCs including DCs, B cells and macrophages. The expression of CD86 is greater than for CD80 on APCs, although CD80 expression is enhanced during APC activation. The expression of CD80 and CD86 has been examined by immunohistochemistry or real-time polymerase chain reaction in livers following transplantation (Kwekkeboom J et al., 2003). CD80 was expressed only sporadically on normal liver but was present on at least 25% of the Kupffer cells in 45% of the transplanted livers. CD86 was found on the majority of Kupffer cells in all transplanted liver tissue and in normal liver tissue. The effect of ligation of CD28 by either CD86 or CD80 appears to be increased cytokine synthesis and proliferation by various intracellular signalling. Immunohistochemical analysis of CD86 expression in biopsies of liver recipients demonstrated an association with the increased expression of CD86 in the graft during severe acute cellular rejection (Bartlett A S et al., 2003). CTLA4 (CD152) is a CD28-related protein that binds to CD86 and CD80. Whereas CD28 delivers a positive co-stimulatory signal to T cells, CD152 delivers a negative signal that attenuates T cell function. CD152 expression is enhanced after T cell activation, and it has a higher affinity for CD86 or CD80 than does CD28; it has been proposed that the physiologic function of CD152 is to downregulate T cell responses. Therefore, specific activation of CD152 could potentially yield immunoinhibitory function and achieve allograft tolerance, but this ideal approach has been reached by the lack of suitable reagents.

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.
