**5. Graft rejection**

induce tolerance. Animal models have improved our understanding of this mechanism that ranges from tolerance induction through the injection of allogeneic cells into newborn mice to the use of adult irradiated animals injected with allogeneic donor bone marrow [34]. Tolerance induction at the peripheral systemic level needs to target mature T cells by blocking T cell molecules located on its surface, which have important roles in the activation of signaling pathways that impact cell function directly. To this end, antibodies directed against CD4 or CD8 or costimulatory molecules have been used. CD28 receptor blockade prevents proinflammatory cytokine production, as well as T cell survival and proliferation. It is also possible to interfere with the signaling pathways involved in T cell survival and proliferation, which is the case of the mTOR pathway inhibitor rapamycin. Clinical studies in humans have focused on graft tolerance induction by pretransplanting donor hematopoietic cells in human leukocyte antigen (HLA)-matched and mismatched

The discovery of effective immunosuppressive drugs has had great impact in the field of transplantation. Currently available immunosuppressive therapies focus on three main objectives: induction, maintenance, and treatment of rejection. For induction therapy three types of antibodies are used, the lymphocytes depleting agents, antithymocyte globulin and alemtuzumab, and basiliximab (nondepleting) [37]. Basiliximab, an IL-2 receptor antagonist and it is used in combination with other immunosuppressants, significantly reduces acute rejection in large clinical studies [38]. The use of antithymocyte antibodies in diseased donor recipients also reduces early acute rejection incidence. Nevertheless, its use has been associated with reversible leukopenia, thrombocytopenia, and cytomegalovirus

The use of calcineurin inhibitors cyclosporine and tacrolimus has been key in reducing the risk of rejection and has greatly improved short-term graft survival outcomes. Unfortunately, in the long run they also help develop histological changes typical of nephropathy that diminish kidney graft function and increase risk of graft loss [40, 41]. A T cell costimulatory inhibitor called belatacept was introduced to avoid the deleterious effects of calcineurin inhibitors. In several studies, belatacept prevents acute rejection in renal transplantation comparable to cyclosporine [42]. Since the 1980s, several options have been developed to reduce kidney transplant rejection. Monoclonal muromonab-CD3/ OKT3, monoclonal interleukin-2 receptor (IL-2R) antibodies (daclizumab and zenapax), and antiproliferative agents (mycophenolate mofetil) are part of a large list of options currently available in renal transplantation [43]. Nevertheless, some transplant experts propose a reduction in the use of immunosuppressive drugs in order to reduce the nephrotoxicity that can also end in fibrosis and graft rejection. Additionally, some transplant recipients develop diabetes, cardiovascular disease, dyslipidemia associated to immunosuppressant therapies. For this reason, it is estimated that kidney grafts can function on

kidney transplant recipients [35, 36].

148 Pathophysiology - Altered Physiological States

infection [39].

**4.3. Immunosuppressive therapy in renal transplantation**

average 10 years after the transplantation [44, 45].

Current immunosuppressant therapies have drastically reduced acute rejection events in kidney transplant recipients [46, 47]. Unfortunately, there is still a high percentage of short- and long-term kidney graft loses secondary to ABMR [2]. Here, we will discuss the contribution of adaptive and innate immune cells; as well as antibodies, molecules from the complement system and chemokines to disease states that lead to kidney graft rejection (**Figure 1**).

#### **5.1. T cell–mediated rejection**

T cell mediated rejection (TCMR) is characterized by infiltration of the donor graft interstitium by host CD4 and CD8 T effector and memory cells, macrophages, and dendritic cells; followed by epithelial dedifferentiation and tubulitis [48, 49] (see histopathological findings in **Figure 2**). TCMR is the predominant phenotype found in kidney transplant recipients with early rejection and it is still an important cause of graft dysfunction that when left untreated causes fibrosis, tubular atrophy, and irreversible nephron loss [50]. Genes expressed by effector T cells, APCs, and macrophages stimulated with IFN-*γ* are abundant in the transcriptomic signature of TCMR. These transcripts are mostly related to T cell receptor signaling, T helper differentiation and communication between adaptive and innate immune cells; highlighting the importance of these pathways in TCMR [51]. Cytotoxic molecules like perforin, granulysin,

**Figure 1.** Mechanisms of ABMR and TCMR in kidney transplantation. Preformed and *de novo* DSAs, complement proteins, and antibody-dependent NK cell-mediated IFN-*γ* release and cytotoxicity have emerged as key immune players in the development of the microvascular damage characteristic of ABMR. Meanwhile, in TCMR the interaction of infiltrating T cells with intragraft APCs and macrophages triggers an inflammatory response dependent on TCR synapse and subsequent activation, and characterized by chemokine (CCL5) and cytokine (CXCL9, CXCL10, and CXCL11) release.

Macrophages in TCMR exert dual functions by promoting initiation and progression of kidney injury through secretion of proinflammatory mediators and interaction with other cells in the graft; whilst also in charge of tissue remodeling and repair during the recovery phase [66, 67]. Interestingly, reversible rejection episodes with monocytic infiltrates and scant T cells have been described in severely T cell-depleted patients, emphasizing the central role of mac-

Immunopathology of Kidney Transplantation http://dx.doi.org/10.5772/intechopen.70596 151

An increase in IFN-*γ* induces CCL5, CXCL9, CXCL10, CXCL11, and MHC class I and II expression; and is an important feature of TCMR [68, 69]. In the context of TCMR, IFN-*γ* has protective and proinflammatory functions as evidenced by IFN-*γ*-deficient recipient animals

Evidence form multiple studies supports the humoral theory of transplantation strongly advocated by Dr. Paul Terasaki, in which antibodies are not only responsible for immediate hyperacute allograft rejection but can produce chronic vascular damage, fibrosis, and graft rejection months or even years posttransplantation [71]. Hyperacute allograft rejection occurs soon after the graft is perfused with blood of the recipient due to preformed

**Figure 3.** Interstitial hemorrhage in hyperacute allograft rejection. Microphotography shows severe capillary injury with subsequent peritubular capillary disruption. Hematoxylin & eosin 10×. Courtesy of Dr. Claudia Mendoza-Cerpa,

Laboratory of Pathology, IMSS-CMNO, Guadalajara; México.

or donors lacking IFN-*γ* receptors or IFN-*γ*-regulated factor 1 [68, 70].

rophages in allograft rejection [65].

**5.2. Antibody-mediated rejection**

**Figure 2.** T cell-mediated rejection. The hallmark of TCMR is the infiltration of mononuclear cells to the interstitium and tubules. (A) Prominent intersticial inflamatory cell infiltrate. PAS 5×. (B) Higher magnification reveals infiltration of the tubular epithelium by mononuclear cells (tubulitis). PAS 40×. Courtesy of Dr. Claudia Mendoza-Cerpa, Laboratory of Pathology, IMSS-CMNO, Guadalajara; México.

Fas ligand, and granzyme A and B are also expressed in TCMR; though it has been demonstrated that they are not directly linked to the mechanism of injury [48, 52]. Instead, TCMR is considered an inflammatory reaction initiated by the engagement of TCR on cognate T cells with its antigen on APCs [53, 54]. It has been suggested that a very small proportion of infiltrating T cells are able to establish a TCR-mediated interaction with the allograft [55]. However, this interaction is important in the establishment of TCMR inflammatory phenotype since it activates the effector T cell and APC, induces INF-*γ* secretion and further promotes myeloid and T cell recruitment by inducing chemokines and adhesion molecules [56]. Interestingly, increased expression of immune checkpoints responsible for modulating T cell activation such as cytotoxic T-lymphocyte antigen 4, programmed death-ligand 1 and 2 have also been associated with TCMR; suggesting these molecules might be regulating some of the interactions between T cells and APCs within the graft microenvironment [51]. Moreover, evidence that the programmed cell death protein 1 (PD1) pathway may be critical in maintaining tolerance and preventing TCMR comes from a report case in which administration of an anti-PD1 antibody to a kidney transplant recipient with metastatic cutaneous squamouscell carcinoma resulted in allograft rejection [57].

B cells are robust APCs that can readily capture, process and present antigen for T cell recognition. Still, the role of B cells in TCMR development was initially overlooked since studies in B cell-deficient mice reported similar rejection rates in skin and heart transplants, as well as efficient CD4 T cell priming [58, 59]. The first evidence of a possible role of B cells in TCMR came from a systematic study of gene expression patterns using DNA microarrays in biopsy samples from renal allografts that found a surprising association between dense CD20+ B cell infiltrates and both, steroid resistance and graft loss [60]. Although the prognostic significance of CD20+ B cell infiltrates in acute cellular rejection is a matter of debate, the presence of these B cell clusters in cases of pure TCMR and their close proximity to CD4+ T cells suggests they might have antibodyindependent functions in allograft rejection by acting as APCs [61–64]. Interestingly, reversible rejection episodes with monocytic infiltrates and scant T cells have been described in severely T cell-depleted patients, emphasizing the central role of macrophages in allograft rejection [65].

Macrophages in TCMR exert dual functions by promoting initiation and progression of kidney injury through secretion of proinflammatory mediators and interaction with other cells in the graft; whilst also in charge of tissue remodeling and repair during the recovery phase [66, 67]. Interestingly, reversible rejection episodes with monocytic infiltrates and scant T cells have been described in severely T cell-depleted patients, emphasizing the central role of macrophages in allograft rejection [65].

An increase in IFN-*γ* induces CCL5, CXCL9, CXCL10, CXCL11, and MHC class I and II expression; and is an important feature of TCMR [68, 69]. In the context of TCMR, IFN-*γ* has protective and proinflammatory functions as evidenced by IFN-*γ*-deficient recipient animals or donors lacking IFN-*γ* receptors or IFN-*γ*-regulated factor 1 [68, 70].

#### **5.2. Antibody-mediated rejection**

Fas ligand, and granzyme A and B are also expressed in TCMR; though it has been demonstrated that they are not directly linked to the mechanism of injury [48, 52]. Instead, TCMR is considered an inflammatory reaction initiated by the engagement of TCR on cognate T cells with its antigen on APCs [53, 54]. It has been suggested that a very small proportion of infiltrating T cells are able to establish a TCR-mediated interaction with the allograft [55]. However, this interaction is important in the establishment of TCMR inflammatory phenotype since it activates the effector T cell and APC, induces INF-*γ* secretion and further promotes myeloid and T cell recruitment by inducing chemokines and adhesion molecules [56]. Interestingly, increased expression of immune checkpoints responsible for modulating T cell activation such as cytotoxic T-lymphocyte antigen 4, programmed death-ligand 1 and 2 have also been associated with TCMR; suggesting these molecules might be regulating some of the interactions between T cells and APCs within the graft microenvironment [51]. Moreover, evidence that the programmed cell death protein 1 (PD1) pathway may be critical in maintaining tolerance and preventing TCMR comes from a report case in which administration of an anti-PD1 antibody to a kidney transplant recipient with metastatic cutaneous squamous-

**Figure 2.** T cell-mediated rejection. The hallmark of TCMR is the infiltration of mononuclear cells to the interstitium and tubules. (A) Prominent intersticial inflamatory cell infiltrate. PAS 5×. (B) Higher magnification reveals infiltration of the tubular epithelium by mononuclear cells (tubulitis). PAS 40×. Courtesy of Dr. Claudia Mendoza-Cerpa, Laboratory of

B cells are robust APCs that can readily capture, process and present antigen for T cell recognition. Still, the role of B cells in TCMR development was initially overlooked since studies in B cell-deficient mice reported similar rejection rates in skin and heart transplants, as well as efficient CD4 T cell priming [58, 59]. The first evidence of a possible role of B cells in TCMR came from a systematic study of gene expression patterns using DNA microarrays in biopsy samples from renal allografts that found a surprising association between dense CD20+ B cell infiltrates and both, steroid resistance and graft loss [60]. Although the prognostic significance of CD20+ B cell infiltrates in acute cellular rejection is a matter of debate, the presence of these B cell clusters in cases of pure TCMR and their close proximity to CD4+ T cells suggests they might have antibodyindependent functions in allograft rejection by acting as APCs [61–64]. Interestingly, reversible rejection episodes with monocytic infiltrates and scant T cells have been described in severely T cell-depleted patients, emphasizing the central role of macrophages in allograft rejection [65].

cell carcinoma resulted in allograft rejection [57].

Pathology, IMSS-CMNO, Guadalajara; México.

150 Pathophysiology - Altered Physiological States

Evidence form multiple studies supports the humoral theory of transplantation strongly advocated by Dr. Paul Terasaki, in which antibodies are not only responsible for immediate hyperacute allograft rejection but can produce chronic vascular damage, fibrosis, and graft rejection months or even years posttransplantation [71]. Hyperacute allograft rejection occurs soon after the graft is perfused with blood of the recipient due to preformed

**Figure 3.** Interstitial hemorrhage in hyperacute allograft rejection. Microphotography shows severe capillary injury with subsequent peritubular capillary disruption. Hematoxylin & eosin 10×. Courtesy of Dr. Claudia Mendoza-Cerpa, Laboratory of Pathology, IMSS-CMNO, Guadalajara; México.

antibodies directed primarily at the vasculature of the donor organ [72]. These antibodies activate the complement cascade and induce neutrophil infiltration, endothelial damage, interstitial hemorrhage (**Figure 3**), edema, fibrin deposition, platelet aggregation, and thrombosis; causing the organ to fail within a few hours after transplantation. Hyperacute rejection used to be a frequent occurrence in transplantation before cross-match tests were designed to screen potential recipients for circulating anti-HLA antibodies to the prospective donor [73].

expressed by graft microvasculature and induce antibody-dependent cell cytotoxicity, complement activation and modulation of signaling pathways within vascular cells. These events promote the development of irreversible lesions that compromise graft function that eventu-

Immunopathology of Kidney Transplantation http://dx.doi.org/10.5772/intechopen.70596 153

Complement activation is a well-established mechanism of ABMR [22, 77–79]. Although in some models, a causal relationship between antibody-mediated complement activation and graft damage has not been demonstrated [80]. DSAs bind to their targets on donor endothelial cells where they cause complement activation, and membrane attack complex formation. Interestingly, DSAs also activate an endothelial proinflammatory gene program to support allograft injury through noncanonical NF-κB signaling [81]. The graft microvasculature limits antibody injury by inducing the expression of the complement inhibitors CD55 and CD59 [82]. IgG subclasses exhibit variability in their hinge region that controls Fc region affinity for FcγRs and complement components [83]. Transcriptomic studies of ABMR biopsies have revealed an enrichment of endothelial, NK cells and IFN-*γ*-inducible transcripts. NK cells secrete IFN-*γ* upon FcγR crosslinking, a positive feedback mechanism that enhances HLA expression on endothelial cells and results in more DSA deposition and activation of local

Improving kidney transplantation outcomes and patient survival is a challenging task. It is now clear that the cooperation between the innate and humoral arms of the immune system plays complex roles in graft tolerance and rejection. For this reason, understanding the immune mechanisms responsible for graft rejection in allotransplantation has becomes essential in our quest to develop better diagnostic tools and immunosuppressant therapies that can

This work was supported by the National Council of Science and Technology of Mexico

1 CONACyT-Centro de Investigacion Biomedica de Occidente, IMSS-Guadalajara, Mexico

, Claudia A. Mendoza-Cerpa2

and Raquel Echavarria<sup>1</sup>

ally lead to rejection [76].

immunity [82].

**6. Future directions**

**Acknowledgements**

**Author details**

(CONACYT project number 653).

Zesergio Melo1,2\*, Juan A. Ruiz-Pacheco1

\*Address all correspondence to: zesergiomelo@gmail.com

2 Laboratory of Pathology, IMSS-CMNO, Guadalajara, Mexico

successfully be translated into the clinic.

Antibody-mediated rejection (ABMR) pathogenesis involves mechanisms of graft injury caused by donor-specific anti-HLA antibodies (DSAs) and non-HLA antibodies; and has been associated with progressive decline in graft function and poor transplantation outcomes [74]. Molecular changes in the microvasculature characteristic of tissue remodeling and repair are common manifestations of ABMR, as well as neutrophilic infiltration and fibrosis (**Figure 4**) [50]. ABMR can be acute or chronic, and can also manifest in cases of mixed TCMR/AMBR rejection [2]. It is estimated that close to 20% of renal allograft recipients will develop *de novo* DSAs within 10 years posttransplant [75]. DSAs bind to allogenic HLA and non-HLA targets

**Figure 4.** Antibody-mediated rejection. Two of the three criteria required for AMR. (A to C) Microvascular inflammation: glomerulitis, peritubular capilaritis, and intimal arteritis. PAS and H&E 40×. (D) Linear staining of C4d in peritubular capillaries IP 40×. Courtesy of Dr. Claudia Mendoza-Cerpa, Laboratory of Pathology, IMSS-CMNO, Guadalajara; México.

expressed by graft microvasculature and induce antibody-dependent cell cytotoxicity, complement activation and modulation of signaling pathways within vascular cells. These events promote the development of irreversible lesions that compromise graft function that eventually lead to rejection [76].

Complement activation is a well-established mechanism of ABMR [22, 77–79]. Although in some models, a causal relationship between antibody-mediated complement activation and graft damage has not been demonstrated [80]. DSAs bind to their targets on donor endothelial cells where they cause complement activation, and membrane attack complex formation. Interestingly, DSAs also activate an endothelial proinflammatory gene program to support allograft injury through noncanonical NF-κB signaling [81]. The graft microvasculature limits antibody injury by inducing the expression of the complement inhibitors CD55 and CD59 [82]. IgG subclasses exhibit variability in their hinge region that controls Fc region affinity for FcγRs and complement components [83]. Transcriptomic studies of ABMR biopsies have revealed an enrichment of endothelial, NK cells and IFN-*γ*-inducible transcripts. NK cells secrete IFN-*γ* upon FcγR crosslinking, a positive feedback mechanism that enhances HLA expression on endothelial cells and results in more DSA deposition and activation of local immunity [82].
