**4. Role of B cells and DSA**

Based on the principles of immunology, B cells are known to play vital roles, from antigen presentation, immune regulation, to their most characteristic role of differentiating into plasma cells that secrete antibodies. The secretion of antibodies and their role in the pathogenesis of CAN is what makes B cells of great clinical significance in the long term survival of the renal graft.

The Allo-Immunological Injury in Chronic Allograft Nephropathy 407

develop slowly and sub clinically, and eventually lead to the dysfunction of the allograft

Fig. 5. Peritubular capillary deposition of C4d (right) in acute antibody mediated

With the development of a diagnostic criterion for CAMR, many of the pathological findings that had known to exist have been tied in and explain the underlying mechanism of renal injury. However, there are few caveats that still make the accurate recognition of this clinical

One factor is that under certain circumstances, C4d deposition might not be seen. This could happen when the DSAs induce damage via a non-complement fixing mechanism (Collins et al., 2008). Further, in advanced stages of CAMR, when tubular atrophy has already developed, it may well be hard to recognize positive C4d staining. Conversely, when typical changes such as PTC multi-lamination are seen in the absence of C4d deposition and circulating DSA, there could be a possibility of another diagnosis such as chronic or resolving thrombotic micro-angiopathy or these lesions could be assumed to be from a previous episode of acute antibody-mediated injury.Laboratory studies have demonstrated that complement, although relied on in order to make a clinical diagnosis, is not necessary in the pathogenesis of CAMR. Induction of DSAs that are non-complement fixing can have the same pathological changes as DSA that fix complement. Also, in animals that are selectively deficient in C3 (RAG1 -/-), introduction of complement fixing antibodies results in similar pathological changes and poor outcomes of the graft (Jin et al. 2005). What has been found to have a more pronounced role in the development of allograft arteriopathy characteristic of CAMR, is a host of changes in the endothelium brought about by the infiltration of natural killer (NK) cells that express FcγRIII, which is a receptor for the Fc (Fragment crystallizable) or the constant region of antibodies. Hence it is believed that in the pathogenesis of CAN mediated by circulating DSAs, NK cells have much more of a role

mediated by a slow inflammatory process.

rejection.(*Courtesy of Suzanne Meleg-Smith, MD.*)

than complement (Hirohasha et al., 2008).

entity challenging.

Fig. 3. Infiltration of arterial intima with T cells (*Courtesy of Suzanne Meleg-Smith, MD.*)

Fig. 4. Fibrinoid necrosis of arterial wall and transmural infiltration of T cells (*Courtesy of Suzanne Meleg-Smith, MD.*)

The association between graft dysfunction with antibodies produced against donor human leukocyte antigens (HLA) has long been recognized (Jeannet et al., 1970; Terasaki et al. 2007). Their role in acute AMR is well defined and they have been popularly referred to as DSA. Their pathogenesis became evident with the discovery of deposition of complement fragments (C4d) along peritubular capillaries (PTC) in grafts of patients suffering from graft dysfunction and known to have circulating DSAs (Feuch et al., 1991). (Figure 5)

In the context of CAN, arteriopathy or glomerulopathy in the transplanted kidney is also linked to C4d deposition in the PTCs and to DSAs (Mauiyyedi et al., 2001). The pattern of renal injury in these circumstances was further elaborated with the evidence that when chronic failure occurs in the renal allograft and circulating DSAs are present along with C4d deposition in the PTCs, capillaritis and basement membrane multi-lamination was seen (Regele et al.,2002). Other features described and attributed to this pathology include duplication of the glomerular basement membrane, mononuclear cell infiltration in the glomeruli and the PTCs along with loss of normal glomerular capillary endothelial fenestrations (Colvin et al. 2006). With well-described morphological features, along with association of DSA and C4d deposition, "chronic antibody mediated rejection (CAMR)" gained its place in the revision of the BANFF classification in 2005 (Solez et al., 2007). The presence of proteinuria is not pathognomonic but can be seen and graft function may seem quite stable for years. While the pathogenesis is strongly linked to circulating DSAs, prior sensitization or an episode of acute AMR are not essential pre-requisites. Instead, DSAs may

Fig. 3. Infiltration of arterial intima with T cells (*Courtesy of Suzanne Meleg-Smith, MD.*)

Fig. 4. Fibrinoid necrosis of arterial wall and transmural infiltration of T cells (*Courtesy of* 

dysfunction and known to have circulating DSAs (Feuch et al., 1991). (Figure 5)

The association between graft dysfunction with antibodies produced against donor human leukocyte antigens (HLA) has long been recognized (Jeannet et al., 1970; Terasaki et al. 2007). Their role in acute AMR is well defined and they have been popularly referred to as DSA. Their pathogenesis became evident with the discovery of deposition of complement fragments (C4d) along peritubular capillaries (PTC) in grafts of patients suffering from graft

In the context of CAN, arteriopathy or glomerulopathy in the transplanted kidney is also linked to C4d deposition in the PTCs and to DSAs (Mauiyyedi et al., 2001). The pattern of renal injury in these circumstances was further elaborated with the evidence that when chronic failure occurs in the renal allograft and circulating DSAs are present along with C4d deposition in the PTCs, capillaritis and basement membrane multi-lamination was seen (Regele et al.,2002). Other features described and attributed to this pathology include duplication of the glomerular basement membrane, mononuclear cell infiltration in the glomeruli and the PTCs along with loss of normal glomerular capillary endothelial fenestrations (Colvin et al. 2006). With well-described morphological features, along with association of DSA and C4d deposition, "chronic antibody mediated rejection (CAMR)" gained its place in the revision of the BANFF classification in 2005 (Solez et al., 2007). The presence of proteinuria is not pathognomonic but can be seen and graft function may seem quite stable for years. While the pathogenesis is strongly linked to circulating DSAs, prior sensitization or an episode of acute AMR are not essential pre-requisites. Instead, DSAs may

*Suzanne Meleg-Smith, MD.*)

develop slowly and sub clinically, and eventually lead to the dysfunction of the allograft mediated by a slow inflammatory process.

Fig. 5. Peritubular capillary deposition of C4d (right) in acute antibody mediated rejection.(*Courtesy of Suzanne Meleg-Smith, MD.*)

With the development of a diagnostic criterion for CAMR, many of the pathological findings that had known to exist have been tied in and explain the underlying mechanism of renal injury. However, there are few caveats that still make the accurate recognition of this clinical entity challenging.

One factor is that under certain circumstances, C4d deposition might not be seen. This could happen when the DSAs induce damage via a non-complement fixing mechanism (Collins et al., 2008). Further, in advanced stages of CAMR, when tubular atrophy has already developed, it may well be hard to recognize positive C4d staining. Conversely, when typical changes such as PTC multi-lamination are seen in the absence of C4d deposition and circulating DSA, there could be a possibility of another diagnosis such as chronic or resolving thrombotic micro-angiopathy or these lesions could be assumed to be from a previous episode of acute antibody-mediated injury.Laboratory studies have demonstrated that complement, although relied on in order to make a clinical diagnosis, is not necessary in the pathogenesis of CAMR. Induction of DSAs that are non-complement fixing can have the same pathological changes as DSA that fix complement. Also, in animals that are selectively deficient in C3 (RAG1 -/-), introduction of complement fixing antibodies results in similar pathological changes and poor outcomes of the graft (Jin et al. 2005). What has been found to have a more pronounced role in the development of allograft arteriopathy characteristic of CAMR, is a host of changes in the endothelium brought about by the infiltration of natural killer (NK) cells that express FcγRIII, which is a receptor for the Fc (Fragment crystallizable) or the constant region of antibodies. Hence it is believed that in the pathogenesis of CAN mediated by circulating DSAs, NK cells have much more of a role than complement (Hirohasha et al., 2008).

The Allo-Immunological Injury in Chronic Allograft Nephropathy 409

inflammatory response, which is not very severe, but in most cases chronic does occur and over time results in graft loss. There has been a clear demonstration that subclinical rejection leads to an early development of CAN and graft loss particularly if there is coexisting interstitial fibrosis and tubular atrophy (Cosio et al, 2005; Nankivell et al., 2004, Moreso et al., 2006; Shishido et al., 2003; Veronese et al. 2004). It is also important to stress that while there may not be a significant functional deterioration at the time sub-clinical rejection is diagnosed, many times the actual injury as demonstrated by protocol biopsies may be of high grade. One study categorized the results of a cohort of protocol biopsies and revealed that 1 out of 3 of these cases has interstitial acute rejection Grade 1 and 2 out of 3 were classified as borderline changes (Nankivell et al., 2004). There has also been a repeated demonstration that the degree of infiltration seen in protocol biopsies revealing subclinical rejection has correlated to the degree of HLA incompatibility further proving that this infiltration is driven by an immunological phenomenon. There are instances when there is clear histological demonstration of infiltration in the renal parenchyma with no rise in serum creatinine implying that there is no functional decline. This further elaborates the unreliability and underestimation of renal dysfunction offered by measuring serum

This raises the question of whether protocol biopsies should be performed on a regular interval. While some studies have demonstrated a clear benefit in terms of a decreased incidence of AR and lower serum creatinine at two years after the kidney transplant (Rush et al., 1998), there have been other studies that indicate that treatment of subclinical infiltration on the basis of a protocol biopsy may not have significant improvement in the long term and may further expose the patient to increased amounts of immunosuppression and further the risk of CNI toxicity. Therefore, with the currently existing data, most centers do not perform protocol biopsies on all patients; however, experts do recommend performing protocol biopsies on at least some of the patients that are considered high risk where an inflammatory infiltrate likely means a clinical rejection. If left untreated, it will likely result in an accelerated course towards CAN and the eventual loss of function of the

Three pairs of human leukocyte antigens (HLA) loci A, B and DR are traditionally used for organ allocation. They exist on chromosome 6 with both alleles inherited from either parent are co-expressed, resulting in any individual having 6 antigens. There is tremendous amount of variation in the actual antigen that is coded by each of these loci among individuals as this gene exhibits what is known as polymorphism. With advances in molecular biology more than 230 polymorphisms have been identified for HLA-A, more than 470 for HLA-B and more than 380 for HLA-DR. Their relevance stems from the fact that these antigens are expressed on the surface of all cells and are the major barrier to transplantation. Because of the way our immunological system is designed, the recognition of self versus foreign antigens is mediated through these HLA antigens. Hence when foreign tissue is introduced to the immune system of a host and it is recognized as foreign, it is due to lack of tolerance that the host has developed towards its own variety of HLA antigens.

As these antigens are carried on fixed loci, their inheritance follows a Mendelian pattern, and a combination of HLA-A, B and DR is inherited by an individual from both parents.

creatinine level (Kaplan et al., 2003; Levey et al., 1999).

allograft.

**6. Degree of HLA mismatch** 

Yet another phenomenon observed in the context of circulating antibodies is that sometimes, there may be no evidence of graft destruction at all, even with varying degree of C4d deposition. This process, termed accommodation, has been the focus of research in recent years, with a wealth of insight provided by transplantation of organs across the barrier of ABO incompatibility. Though anti-A or B isoagglutinin reappear after transplant, they can co-exist without precipitating rejection (Gonzalez-Strawinski et al., 2008). Interestingly, C4d deposition can be observed but, compatible with other observations, does not necessarily mean that CAMR is taking place. Understanding the mechanism by which the graft attains this ability to remain "non-reactive" despite the presence of antibodies circulating against it is of great interest as it can be therapeutically mimicked when DSAs are known to exist that would otherwise lead to an immunologically mediated rejection of the graft. Accommodated grafts have been found to have changes in the cells of the endothelium and that are believed to help in the adaptation to the presence of antibodies. These changes include increased expression of bcl-xL, (Salama et al. 2001), increased muc-1 expression (Park et al. 2003) and increase in the expression of indolamine-2,3-dioxygenase (Minnei et al., 2008) in the glomerular and PTC endothelium.

In conclusion, CAMR occurs slowly, with the first step being the development of DSA, followed by an immunological reaction that *may* result in the deposition of C4d, the resultant development of visible pathological changes characteristic of CAMR and then eventual graft loss. The speed at which these events occur is variable and the challenge is not just limited to the difficulty in diagnosis, but also in terms of therapy. In the future, the main strategies to counteract the risk for CAMR will be focused on screening for the development of new DSA, following the titers of known DSAs and correlating them with the function of the transplanted kidney. Also, as we learn more about the adaptive capabilities that lead to accommodation, strategies will likely be developed to mimic them in vivo to prolong the renal graft survival.
