Expanding role of antibodies in kidney transplantation

Abstract

The role of antibodies in kidney transplant (KT) has evolved significantly over the past few decades. This role of antibodies in KT is multifaceted, encompassing both the challenges they pose in terms of antibody-mediated rejection (AMR) and the opportunities for improving transplant outcomes through better detection, prevention, and treatment strategies. As our understanding of the immunological mechanisms continues to evolve, so too will the approaches to managing and harnessing the power of antibodies in KT, ultimately leading to improved patient and graft survival. This narrative review explores the multifaceted roles of antibodies in KT, including their involvement in rejection mechanisms, advancements in desensitization protocols, AMR treatments, and their potential role in monitoring and improving graft survival.

Key Words: Antibodies; Humoral theory; Kidney transplantation; Rejection

Core Tip: Antibodies are of crucial importance in kidney transplantation (KT), posing challenges such as antibody-mediated rejection (AMR) but also offering opportunities for better transplant outcomes through advanced detection, prevention, and treatment strategies. This review explores the role of antibodies in rejection, progress in desensitization, AMR treatments, and their potential to enhance graft survival, reflecting evolving strategies to manage and leverage antibodies in KT.

INTRODUCTION

Kidney transplant (KT) is the preferred form of treatment for patients with end-stage kidney failure[1,2]. Considerable advancements have been made in the field of KT since its inception in the 1950s[3]. The success rates of KT have significantly improved over the years, largely due to advances in surgical techniques, immunosuppressive therapy, and the understanding of the immune responses involved in transplant rejection[3,4]. Two competing theories, the cellular and humoral, became popular as researchers sought to understand the mechanisms behind graft rejection. Among these, antibodies play key roles in damaging the graft. However, their crucial role in alloimmune responses was not fully recognized, particularly during the early days of transplantation[5,6]. In the recent past, significant advances have been made in understanding and expanding the role of antibodies in both the success and challenges of KT. From the early days of graft rejection to the development of more sensitive assays such as Luminex flow bead assays to detect low-titer antibodies, high-resolution sequence-based human leukocyte antigens (HLA) typing, a better understanding of immune mechanisms involved in the rejection processes, and the development of newer immunosuppressive drugs, the history of antibodies in KT reflects the broader advances in immunology and transplant medicine[7-10].

This review aims to provide a comprehensive overview of antibodies' evolving and multifaceted roles in KT, highlighting their detrimental and beneficial effects on the grafts.

ANTIBODIES AND KIDNEY ALLOGRAFT REJECTION

Antibodies play a crucial role in the immune response against transplanted kidneys. Historically, donor-specific antibodies (DSAs) have been associated with hyperacute (HAR) and accelerated acute rejections. HAR, occurring minutes to hours post-transplantation, is primarily mediated by high titer pre-formed DSAs binding to donor antigens on the vascular endothelium, leading to immediate graft loss. Accelerated acute rejection, a less severe form of HAR, occurs within 2-3 days of transplantation. It is also caused by pre-formed DSAs albeit in low titer. Acute antibody-mediated rejection (AMR) occurs days to weeks post-transplantation and is a result of DSAs, that may either be pre-formed or develop de-novo after transplantation. Chronic AMR, which develops over months to years, is a major cause of late graft failure and involves progressive fibrosis and vascular changes[11-15]. Clinically, the manifestations of the latter form of AMR range from asymptomatic in the early stages to nephrotic range-proteinuria, hypertension, and allograft dysfunction in the advanced stages.

The long-term effects of chronic AMR on kidney grafts include slow but progressive graft dysfunction, chronic allograft injury [manifested as transplant glomerulopathy (TG), interstitial fibrosis and tubular atrophy (IF/TA), and basement membrane multi layering (BMML)], vascular damage, and increased risk of graft loss. Indeed, chronic AMR has emerged as one of the leading causes of kidney graft loss in the long term.

HISTORICAL BACKGROUND AND EVOLUTION

The humoral theory of rejection, rooted in early immunological studies, suggests that antibodies play a crucial role in the rejection of transplanted organs. Its discussion needs to encompass at least three aspects: The discovery of antigens, methods for detecting antibodies, and the clinical KT activity. Emerging in the mid-20th century, humoral theory was based on the discovery of blood group antigens by Landsteiner[16] in 1901 and the identification of HLA by Dausset[17] in 1958. Dausset's discovery earned him a Nobel Prize and provided a critical link between immune recognition and graft rejection[17].

The history of the role of antibodies in KT dates back to the earliest attempts at KT. On December 25, 1952, Oeconomos and Hamburger performed a relatively less publicized KT at Necker Hospital in Paris, France, where a mother donated her kidney to her son. This was the first kidney graft that was removed from a voluntary living donor. No immunosuppression was used. The graft functioned for 21 days and was eventually rejected. A retrospective review of the archived slides revealed mixed acute cellular and humoral rejection[18]. In 1954, the first successful human KT was performed between identical twins by Murray and Merrill, which naturally avoided the problem of immune rejection[19]. Joseph Murray won the Nobel Prize for this great achievement. These examples of KTs demonstrated that immune compatibility was crucial for the success of this procedure and that even genetic proximity was unable to prevent the rejection of a transplanted organ in the absence of immunosuppression. As a consequence, no allotransplantations were performed until 1959 when immunosuppression started to be developed[20]. Subsequently, when an increasing number of allotransplants were attempted, graft rejection emerged as a common and often insurmountable challenge.

In the early days of KT, the primary focus was on cellular mechanisms of rejection, predominantly T-cell mediated responses as these were readily visualized in biopsy tissues under the microscope[5,6]. However, early observations also noted that some patients experienced rapid graft rejection, which could not be fully explained by cellular responses alone. During the same time, researchers also observed that KTs often failed despite the absence of cellular rejection markers on allograft biopsies, suggesting a potential humoral component in the rejection process. In 1960, Kissmeyer-Nielsen et al[11] were the first to observe the deleterious impact of allo-antibodies in kidney grafts. Other researchers observed that recipients with pre-existing antibodies against donor HLAs experienced higher rejection rates. Building on these findings, Paul Terasaki, in 1964, developed the microcytotoxicity test, which allowed for the detection of anti-HLA antibodies in recipients, revealing the correlation between these antibodies and transplant outcomes[21]. In 1969, Terasaki and Cai[22] reported on the significance of a positive complement-dependent cytotoxicity (CDC) assay in 225 transplants. Immediate graft failure occurred in up to 80% (24 out of 30) of grafts carried out in patients with positive cross-match results. This led to the understanding that antibodies, through complement activation and antibody-dependent cellular cytotoxicity (ADCC), can damage graft tissues. These years also saw further advancements in serological techniques, such as the CDC assay, which enabled the detection of DSAs in transplant recipients. These tools allowed researchers to correlate the presence of DSAs with graft outcomes, reinforcing the notion that antibodies played a critical role in rejection[23,24].

The 1980s marked a significant period in transplant immunology with increased recognition of the role of antibodies in graft rejection. Researchers began to understand that antibodies against donor HLA could lead to graft injury. By the 1990s, the clinical significance of antibody-mediated mechanisms in kidney allograft rejection became widely accepted. Pathological criteria for AMR were established, including the identification of peritubular capillaritis, C4d deposition in peritubular capillaries (PTCs), and the presence of DSAs in the circulation[25]. The Banff classification of kidney allograft pathology, first convened in 1991, provided a standardized framework for diagnosing and categorizing rejection processes, including AMR[26,27].

In the 2000s and beyond, research into the molecular and cellular pathways of AMR expanded. Studies revealed the complex interactions between antibodies, complement activation, and endothelial injury[28-30]. Advances in immunosuppressive therapies aimed at reducing antibody production and mitigating complement-mediated damage improved graft survival rates[31-34].

The theory has since evolved with advancements in immunology, leading to improved organ-matching techniques and the development of therapies targeting AMR. Overall, the humoral theory of rejection, spanning over a century of research, underscores the importance of antibodies in transplant immunology and has significantly influenced modern transplantation practices and patient management strategies (Table 1).

Table 1 The main milestones in the history of role of antibodies in kidney transplantation.

Period	Key discoveries	Researchers
Early 20th century	Discovery of blood group antigens	Karl Landsteiner
1950s	Identification of human leukocyte antigens (HLA)	Jean Dausset
1952	First kidney transplant between living donor (mother) and son	N. Oeconomas, J. Hamburger
1954	First successful kidney transplant between identical twins	J P Merrill, J Murray
1964	Development of microcytotoxicity assay for detecting anti-HLA antibodies	Paul Terasaki
1970s-1980s	Elucidation of antibody-mediated graft damage mechanisms	Fritz Bach, John D. Dingell
1983	Introduction of flow cytometry and solid-phase assays	Margaret R Garovoy
1990s	Use of C4d in kidney transplant biopsies	H E Feuchet
2005	Virtual cross-match in kidney transplantation

ADVANCES IN THE DETECTION OF DSAS

Significant advancements have been made in the field of DSA detection, leading to improved sensitivity, specificity, and clinical utility of these biomarkers. These advancements have significantly improved the management of organ transplantation and the understanding of allorecognition. The key developments include high-throughput screening techniques, improved assay sensitivity, solid-phase assays, and the use of next-generation sequencing (NGS) to identify DSAs at a molecular level, allowing for a more precise understanding of the antibody repertoire and its implications for graft rejection and survival. In addition, advanced bioinformatics tools are being developed to analyze DSA data more effectively, integrating it with other clinical and immunological parameters to predict transplant outcomes. Innovations in wearable technology and point-of-care testing are enabling real-time monitoring of DSAs, facilitating timely interventions in transplant recipients. Understanding the role of DSAs in transplant rejection has led to the development of personalized immunosuppression strategies, aiming to tailor treatment based on individual DSA profiles. Research continues into identifying new biomarkers associated with DSAs and transplant outcomes, improving the ability to predict rejection and graft survival. These advancements contribute to more effective monitoring and management of transplant recipients, leading to better graft survival rates and overall patient outcomes[35].

ADVANCES IN COMPATIBILITY TESTING

Advancements in tissue compatibility testing have significantly improved KT outcomes by reducing the risk of organ rejection and increasing the success and longevity of transplants. Improved HLA typing has become crucial for transplant success, with advances in molecular techniques such as polymerase chain reaction and NGS allowing for more precise HLA matching. This better matching of donor and recipient HLA types reduces rejection risks and improves long-term graft survival. Additionally, crossmatching improvements using flow cytometry and Luminex-based assays have enhanced the detection of pre-existing antibodies in the recipient, such as DSAs, helping predict acute rejection and determine whether a transplant can safely proceed. Virtual crossmatch, a computerized system that simulates donor and recipient tissue interaction, offers faster, more accurate predictions of rejection likelihood, reducing unnecessary transplants and wait times. The introduction of single-antigen bead assays has further helped detect specific anti-HLA antibodies before transplants, allowing the selection of more compatible donors and reducing the risk of AMR. Desensitization protocols, including plasmapheresis and intravenous immunoglobulin (IVIG), are used for patients with high anti-HLA antibody levels, lowering antibody levels and enabling transplantation from less compatible donors, which expands the donor pool and shortens waiting times. Advances in immunosuppressive therapy, tailored to the individual’s immunological risk profile, also complement these compatibility tests, further reducing the risk of rejection. Together, these advancements have led to higher KT success rates, longer graft survival, and improved patient outcomes, transforming KT into a more reliable and accessible treatment for end-stage kidney disease[36].

NON-HLA ANTIBODIES

The presence of non-HLA antibodies has been associated with poorer graft survival and increased rejection episodes. Their detection and management are becoming increasingly important in the field of transplantation to improve long-term outcomes and graft longevity[37-40]. These antibodies can be categorized as alloantibodies, which target polymorphic antigens that differ between the recipients and donors, or, more commonly, as autoantibodies which recognize cryptic self-antigens. These antibodies target various non-HLA antigens found in various cells and tissues such as endothelial cells and smooth muscle cells of blood vessels, immune cells, tubular epithelial cells, mesangial cells, and podocytes. As the endothelial layer of the blood vessels is the main structure interjected between the immune system of the recipient and the grafted kidney, most of the antigens that trigger non-HLA autoantibody formation are endothelial autoantigens. Some of the well-studied non-HLA antibodies include anti-endothelial cell antibodies, which target endothelial cells of the transplanted kidney, potentially causing vascular inflammation and injury. Anti-angiotensin II type 1 receptor antibodies (AT1R) were described for the first time by Dragun et al[40] in 2005, and has generated the most interest in the KT community for the last 20 years. AT1Rs bind to the angiotensin II receptors on kidney cells and can lead to increased blood pressure and contribute to chronic rejection. Antibodies targeting the major histocompatibility complex class I-related chain A (MICA) glycoproteins were the first non-HLA antibodies described in KTs. Anti-MICA antibodies target the MICA molecules on the surface of endothelial cells and can be associated with graft rejection. Zou et al[41] were among the earliest and most important group of researchers to study the role of anti-MICA antibodies in KTs. The possible mechanism of anti-MICA antibody-mediated graft injury involves NKG2D-mediated cytotoxicity and complement-mediated cytotoxicity. Anti-vimentin antibodies target vimentin expressed in vascular endothelial cells, smooth muscle cells, T cells, neutrophils, platelets, epithelial cells, and fibroblasts. These antibodies bind and trigger the complement cascade as evidenced by the positivity of C4d in PTCs in those with chronic AMR. Patients with failing grafts have an increased prevalence of anti-vimentin antibodies in the circulation. Anti-vimentin antibodies have been reported in the context of two major histomorphological lesions: Chronic AMR and IFTA[42]. Anti-perlecan antibodies target perlecan, a heparan sulfate proteoglycan in the kidney's basement membrane, potentially leading to tissue damage and fibrosis[43,44].

SOLID PHASE ASSAYS FOR HLA ANTIBODY DETECTION IN CLINICAL KT

The CDC assay has been used for over 50 years for detecting HLA antibodies in KT recipients. This method has been replaced recently by more sensitive solid phase assays such as ELISA and bead-based technology including the Luminex method. The introduction of these techniques into clinical practice has revealed previously undetected sensitization in some patients and allowed the accurate assignment of antibody specificities directed at HLA-DQ and HLA-DP, which was not previously possible. Luminex flow bead assays offer several advantages over traditional methods for detecting low-titer antibodies, especially in terms of sensitivity, multiplexing capabilities, and the ability to detect a broad range of antibody specificities in a single sample. By combining high sensitivity, multiplexing, and the ability to quantify results accurately, Luminex flow bead assays represent a significant advancement over older methods in detecting low-titer antibodies, particularly in research and clinical settings where precision is critical[45].

ROLE OF ALLOANTIBODIES VS AUTOANTIBODIES IN KT

Although most of the literature on the role of antibodies in KT is related to alloantibodies, more recently, many autoantibodies against non-HLA targets have been incriminated in renal allograft injury (Table 2). The list of autoantigens against which these antibodies are directed is growing steadily. In the context of KT, alloantibodies are produced by the recipient's immune cells against non-self antigens from the donor, specifically targeting the donor kidney cells. These antigens are often HLA or blood group antigens. These antibodies often lead to rejection of the allograft kidney because they recognize the donor's kidney as foreign and attack it. They play a significant role in HAR, as well as acute and chronic rejection. They typically form in response to prior sensitization events such as previous transplants, blood transfusions, or pregnancy[46].

Table 2 Common examples of allo- and autoantibodies in kidney transplantation.

Alloantibodies	ABO blood group antibodies
	Human leukocyte antigen antibodies
	MICA
Autoantibodies	Anti-AT1R
	Anti-ETAR
	Anti-perlecan
	Anti-agrin
	Anti-collagen type IV, III, and I
	Anti-fibronectin
	Anti-vimentin
	Anti-H-Y
	Anti-ARHGDIB
	Anti-PECR
	Anti-PRKCZ
	Anti-Phospholipid antibodies
	Anti-Phospholipase A2 receptors

ATIR: Angiotensin II type 1 receptor; ETAR: Endothelin A receptor; MICA: Major histocompatibility complex class I-related chain antigens; ARHGDIB: Rho guanine nucleotide exchange factor 2; PECR: Peroxisomal trans-2-enoyl-CoA reductase; PRKCZ: Protein kinase C zeta type.

On the other hand, autoantibodies are directed against the recipient's own tissues and antigens (i.e., self-antigens). They are usually involved in autoimmune diseases. In the context of a KT, autoantibodies may be relevant if the recipient has a pre-existing autoimmune condition like lupus nephritis that led to kidney failure. After transplantation, such conditions may reoccur and affect the transplanted kidney. While autoantibodies do not directly cause rejection of a transplanted kidney, they may play a synergistic or additive role with anti-HLA DSAs in causing allograft rejection. However, their role in isolation in the absence of HLA DSA in causing allograft damage remains unknown. These antibodies are formed as a result of a dysfunction of the immune system regulatory pathways or expression of cryptic self-antigens exposed as a result of allograft injury. The concept of kidney damage-mediated autoantibody production leading to augmented kidney injury is not new. In the 1970s, Halloran et al[47] reported that rabbits that had rejected mismatched kidney allografts developed antibody-mediated injury in their native kidneys. This early work showed that an alloimmune attack on a kidney graft can result in the development of autoantibodies, possibly through the release or increased immunogenicity of kidney-specific autoantigens.

MECHANISM OF ANTIBODY ACTION IN AMR

AMR is a complex immunological response where DSAs target and attack the graft causing graft dysfunction and ultimately failure. It is now established that immunologic reactions associated with AMR can be triggered by circulating antibodies against donor HLA, non-HLA, or ABO blood group antigens, i.e. DSAs. The major mechanism involved in AMR is the activation of the classical complement pathway by the binding of DSAs to HLAs and subsequent binding of the C1 complex, which ultimately leads to the formation of the membrane attack complex (C5b-C9), which destroys the membrane of vascular endothelial cells (Figure 1). Complement activation also generates anaphylatoxins (C3a, C5a) that promote the migration of inflammatory cells, natural killer cells (NK), monocytes, and neutrophils, through the innate immune responses and amplify the inflammatory response[12-17]. Antigen-antibody interaction on endothelial cells is also known to increase von Willebrand factor along with externalization of P-selectin molecules resulting in increased platelet activation and leukocyte trafficking. This activation of the coagulation cascade leads to widespread microvascular injury evident as peritubular capillaritis, glomerulitis, and microvascular thrombosis (Figure 2). Antibodies can also mediate injury via complement-independent mechanisms such as ADCC. This is mediated through cells of the innate immune system (NK cells and macrophages). NK cells bind IgG-coated cells through Fcγ RIII receptors (Figure 1). Cytokines released from NK cells like granulysin and granzymes A and B damage endothelial cells[48].

Figure 1 Mechanisms of antibody-mediated graft injury. The chief target of antibodies consists of vascular endothelial cells. A: Antibodies recognize class I and II human leucocyte antigens (HLA) as foreign and bind to them; B: This antibody binding to donor HLA antigens leads to complement-fixing and generation of anaphylatoxins and assembly of the membrane attack complex; C: Antibodies can also damage the graft tissue by antibody-dependent cellular cytotoxicity by engaging inflammatory cells. HLA: Human leucocyte antigen. This figure was created by BioRender.com (Supplementary material).

Figure 2 Mechanisms of action of non-human leucocyte antigen antibodies. A: The targets of these antibodies are non-human leucocyte antigens (HLA) molecules expressed on endothelial cells. Non-HLA antibodies recognize various antigens such as angiotensin type 1 receptor and endothelin A receptor on endothelial cells and bind to them; B: This antibody binding to donor non-HLA antigens leads to complement-fixation and generation of anaphylatoxins (C3a and C5a) and recruitment of inflammatory cells. These antibodies can also activate the endothelial cells resulting in increased expression of HLA, non-HLA, and adhesion molecules such as intercellular adhesion molecule and vascular cell adhesion molecule; C: This results in damage to the endothelial cells, initiation of thrombogenesis, and induction of apoptosis of these cells, ultimately resulting in microvascular injury, thrombosis, and inflammation. HLA: Human leucocyte antigen; ICAM: Intercellular adhesion molecule; VCAM: Vascular cell adhesion molecule. This figure was created by BioRender.com (Supplementary material).

ROLE OF NK CELLS IN AMR

The NK cells play a crucial role in mediating allograft injury by alloantibodies by recognizing IgG-coated target cells, inducing cell lysis through ADCC, secreting pro-inflammatory cytokines, and interacting with other immune cells. Their role is crucial in amplifying the immune response against the transplanted organ, leading to tissue injury and dysfunction. NK cells can directly damage endothelial cells in the transplanted organ, which is critical since these cells are often the primary targets in AMR. Damage to the endothelium can lead to thrombosis, inflammation, and graft dysfunction. Understanding the precise mechanisms of NK cell involvement in AMR can help in developing strategies to modulate their activity and improve transplant outcomes[49].

MECHANISM OF ACTION OF NON-HLA ANTIBODIES

Although the literature abounds on the mechanisms of HLA DSAs in inducing graft damage, data are scarce on the specific mechanisms or pathways of action of non-HLA antibodies in this context (Figure 2). Increasing evidence suggests that these antibodies can act in both an alloimmune and autoimmune manner and may participate in allograft rejection. Several studies have shown that genetic mismatches of non-HLA molecules between the recipient and donor and the alloimmune responses against these molecules are significant predictors of kidney allograft outcome. Reindl-Schwaighofer et al[50] reported that genetic mismatches in the immune-accessible transmembrane non-HLA proteins can form DSAs and lead to graft damage. In addition, Steers et al[51] have shown that the genomic mismatches at the LIMS1 locus may result in allosensitization in hypoxia-induced conditions and kidney allograft rejection. These discoveries question the significance of non-HLA autoimmunity and suggest that the clinically pertinent non-HLA antibodies in KT may inflict allograft injury through alloimmune mechanisms. As the number of these antibodies is increasing, additional pathways are being explored for their pathogenicity. Non-HLA antibodies can activate the complement cascade, leading to the formation of the membrane attack complex and direct cell lysis. These antibodies can bind to target cells, such as endothelial cells in the graft, and recruit immune cells (e.g., NK cells, macrophages) through their Fc receptors, leading to target cell destruction. In addition, non-HLA antibodies can directly activate endothelial cells, leading to the upregulation of adhesion molecules, pro-inflammatory cytokines, and chemokines, promoting inflammation and immune cell infiltration into the graft. Non-HLA antibodies also induce endothelial cell apoptosis, impairing the integrity of the blood vessels, causing capillary leakiness, and increasing the likelihood of graft thrombosis (Figure 2). It is important to note that the specific mechanisms of action of non-HLA antibodies can vary depending on the target antigen, antibody isotype, and individual patient factors.

PATHOLOGY OF AMR

The pathology of AMR, as for all types of rejection, is closely linked to the history and evolution of the Banff classification (Table 3). The Banff classification is a critical framework in the field of KT, specifically for assessing and categorizing KT pathology. Established during the Banff Conference on Allograft Pathology in 1991 and subsequently immensely updated and modified at consensus meetings held every two years, it provides a standardized system for diagnosing and grading the various forms of transplant-related kidney injuries, particularly acute rejection, and chronic allograft injury. Its key roles include standardization of diagnosis, classification of rejection types, guiding treatment decisions, serving as a benchmark for clinical trials and research, monitoring allograft health, and ultimately improving patient outcomes. Overall, the Banff classification plays a vital role in improving patient outcomes in KT by guiding diagnosis, treatment, and research in transplant pathology[52].

Table 3 Main updates in the nomenclature and classification of antibody-mediated rejection in the Banff classification (1991 to 2019).

Banff meetings, years	Category 2 Antibody-mediated rejection1	Basis of diagnosis and classification
Banff ‘93	Hyperacute rejection	Clinical criteria only
Banff ‘97	Antibody-mediated rejection (AMR)
	Hyperacute
	Accelerated acute
Banff ’97 update (2001)	Diagnostic criteria for acute antibody-mediated rejection were developed	Histological criteria
	Three types were described as: Types I. ATN-like; II. Capillary; III. Arterial	Immunohistopathological criteria
		Serological criteria
Banff ‘05	Diagnostic criteria for chronic antibody-mediated rejection were developed
Banff ‘07	Antibody-mediated changes2
	C4d deposition without rejection
	Acute antibody-mediated rejection
	Chronic active antibody-mediated rejection
Banff ‘13	Antibody-mediated changes
	Acute/active antibody-mediated rejection	Histological
	Chronic active antibody-mediated rejection	Immunohistopathological
	C4d-negative antibody-mediated rejection	Serological
Banff ‘15	Antibody-mediated changes	Molecular criteria
	Acute/active antibody-mediated rejection
	Chronic active antibody-mediated rejection
	C4d staining without evidence of rejection
	Transplant arteriopathy may be seen in chronic
	AMR
Banff ‘17	Antibody-mediated changes
	Active AMR
	Chronic active AMR
	C4d staining without evidence of rejection
	3 criteria for AMR diagnosis remain but C4d
	can substitute for DSA
	DSA testing still advised
	Suspicious for AMR eliminated
Banff ‘19	Category 2: Antibody-mediated changes	Histological
	Active ABMR	Immunohistopathological
	Chronic active ABMR	Serological
	Chronic (inactive) ABMR	Molecular criteria
	C4d staining without evidence of rejection	Electron microscopy

¹May coincide with categories 3, 4 and 5 and 6.

²With the identification of C4d positivity without rejection, the category name was changed from rejection to changes.

Changes in the nomenclature or criteria are in bold face.

The Banff classification initially included alloantibodies but did not fully encompass their role in transplant injury for lack of evidence. Initially, it was believed that antibodies only affected the immediate post-transplant period[6]. However, over time, evidence showed that antibodies could cause various types of rejection over a longer period, leading to significant changes in the Banff classification (Table 3). Factors contributing to the increased focus on AMR included the identification of the C4d biomarker for AMR, improved detection techniques for anti-donor antibodies, more re-transplants, and higher rates of transplants across immunologic barriers due to organ shortages. AMR primarily targets the endothelial cells in blood vessels, with the severity and type of damage depending on the antibodies involved. The Banff classification's terminology and criteria for AMR have evolved significantly. Initially called HAR, the category was later renamed as AMR and subdivided based on clinical presentation in the Banff 1997 meeting. In 2001, the roles of antibodies beyond the immediate post-transplant period were recognized, and C4d staining and serology were included in diagnostics. For the first time, pathological criteria for diagnosing and classifying AMR were developed in this meeting (Figure 3). Since then, these have evolved and improved significantly over subsequent years to improve AMR's accuracy and clinical relevance. In 2005, criteria for chronic AMR, including specific pathological lesions, were established (Figure 4). In 2007, the category was renamed antibody-mediated changes to reflect the broader spectrum of antibody actions, and a subcategory for C4d deposition without evidence of active rejection was introduced. Further changes included the acceptance of focal C4d staining as a criterion for AMR in 2013, the inclusion of any degree of vascular inflammation (v > 0) in AMR diagnosis, and the incorporation of molecular diagnostics into the classification. In 2019, a subtype for chronic inactive AMR was added. Overall, the Banff classification has significantly advanced, particularly in the AMR category, incorporating molecular studies, removing the absolute requirement for C4d and DSAs for diagnosis, and eliminating the "suspicious for AMR" category[5,6,52]. The contemporary approach to diagnosing AMR now incorporates histology, immunohistochemistry (C4d), serology (DSAs), and molecular markers. Future directions include increasing emphasis on molecular diagnostics, the role of non-HLA antibodies, and ongoing refinement of criteria to incorporate new biomarkers and technologies, reflecting advancements in understanding the immunological mechanisms of rejection and the development of more sophisticated diagnostic tools to improve patient outcomes in KT[5,6,52].

Figure 3 Pathology of acute antibody-mediated graft injury. The principal target of antibodies is the microcirculation and larger blood vessels. A: Severe glomerulitis. Many of the glomerular capillary lumens are obliterated by inflammatory cell infiltration (HE, × 400); B: A glomerulus showing fibrin thrombi obliterating some of the capillary lumens (arrow) (HE, × 200); C: A focus of moderate peritubular capillaritis (ptc2) (arrow) (HE, × 400); D: Diffuse C4d positivity (C4d) (C4d3) in peritubular capillaries (FITC, IF for C4d, × 200).

Figure 4 Pathology of acute and chronic antibody-mediated graft injury. The principal target of antibodies is the microcirculation and larger blood vessels. A: Severe interstitial fibrosis and tubular atrophy. This lesion is entirely non-specific and may result from a variety of immune and non-immune causes [Periodic acid-Schiff (PAS) stain, × 200]; B: A glomerulus showing double contouring of capillary walls and an occasional inflammatory cell infiltration obliterating the capillary lumen (arrow) (PAS, × 400); C: A small artery exhibiting fibrinoid necrosis of the wall (v3). This lesion is strongly suggestive of active antibody-mediated rejection (AMR) (HE, × 400); D: Moderate fibrointimal thickening of an interlobular size artery. This lesion may be seen in chronic AMR as well as chronic T cell-mediated rejection (Trichrome stain, × 400).

SPECIFIC HISTOLOGICAL FEATURES OF CHRONIC AMR

Chronic AMR is characterized by specific histological features that differentiate it from other forms of rejection, particularly T cell-mediated rejection (TCMR) and acute AMR. One hallmark feature is TG, where there is duplication or splitting of the glomerular basement membrane, creating a double contour appearance, which reflects chronic endothelial damage. Another key feature is IF/TA, characterized by widespread fibrosis in the interstitial tissue and atrophy of renal tubules, often seen in chronic rejection but particularly prominent in chronic AMR when accompanied by other changes like TG and vascular involvement. The vascular changes in chronic AMR include peritubular capillary BMML and arterial intimal fibrosis, highlighting chronic antibody-mediated injury. Inflammation involving the PTC, is another important feature, often presenting with mononuclear cell infiltration and suggesting ongoing endothelial damage. Additionally, C4d deposition along the PTCs, detected through immunohistochemical staining, is a distinctive marker of antibody-mediated complement activation, often persistent in chronic AMR and less commonly seen in TCMR. These histological features, particularly the presence of TG, vascular lesions, PTC, and C4d positivity, are critical in identifying chronic AMR and distinguishing it from other types of graft rejection[52].

USE OF ANTIBODIES IN THE TREATMENT OF AMR

The management of AMR has evolved with the understanding of its immunopathology and therapeutic options have expanded markedly. Traditional treatments included plasmapheresis, IVIG, and high-dose steroids. The addition of anti-CD20 monoclonal antibodies which deplete B cells, has improved outcomes in refractory cases. Eculizumab is a monoclonal antibody that targets complement protein C5. Eculizumab binding to this protein prevents activation of the terminal complement complex (C5b-C9). It has been used to prevent complement-mediated damage during AMR. Moreover, proteasome inhibitors targeting antibody-producing plasma cells are being investigated for their potential to treat AMR. An alternative approach to B cell or plasma-cell depletion is to target key cytokine/cytokine receptors that support the activation/proliferation of these antibody-producing cells. In this context, there is an emerging focus on the interleukin 6 (IL-6)/IL-6R pathway, which has several roles related to AMR. Personalized treatment strategies based on the specific immunologic profile of each patient are becoming more common, aiming to improve graft survival and function[53-57].

USE OF ANTIBODIES AND NOVEL BIOMARKERS AS DIAGNOSTIC AND MONITORING TOOLS

Advances in diagnostic techniques such as the solid phase Luminex assay have improved the detection and monitoring of DSAs. The development of non-invasive biomarkers, such as donor-derived cell-free DNA (dd-cfDNA) and gene expression profiling, provides early detection of rejection and monitoring of graft wellbeing. These tools enable timely intervention and personalized treatment adjustments, enhancing the optimal management of KT recipients[58].

dd-cfDNA is a promising non-invasive biomarker that has significantly improved monitoring in KT. Studies have shown that dd-cfDNA can detect rejection earlier than traditional methods, with high sensitivity and specificity. The reported sensitivity rates of dd-cfDNA for the detection of early rejection have varied from 80% to 90%, which is higher than serum creatinine or proteinuria, especially for acute rejection cases. Similarly, the specificity rates have ranged between 80%-95%, which reduces the chances of false positives when compared to other non-invasive markers.

dd-cfDNA improves graft rejection surveillance by detecting the rejection early with increased sensitivity and may prevent the need for invasive biopsies. Elevated dd-cfDNA can help distinguish between antibody-mediated and T-cell mediated rejections, guiding tailored immunosuppressive therapies. dd-cfDNA levels can be monitored to assess the effectiveness of immunosuppressive treatments, allowing for personalized adjustments. Studies suggest that dd-cfDNA monitoring can contribute to improved long-term graft survival rates. Early detection and intervention facilitated by dd-cfDNA monitoring can potentially reduce the need for costly interventions and hospitalizations[59].

ADVANCEMENTS IN DESENSITIZATION PROTOCOLS

Desensitization protocols emerged in the early 1990s to deal with the large numbers of HLA-incompatible patients waiting on maintenance hemodialysis[60]. Two largely accepted desensitization protocols include high-dose IVIG from Cedars-Sinai Medical Center and low-dose IVIG from John Hopkins Hospital, the latter in combination with plasmapheresis. These protocols typically involve combinations of plasmapheresis, IVIG, and rituximab, with the aims of reducing antibody levels and reducing the risk of AMR. Other protocols are modifications of these two protocols. Low-dose IVIG and plasmapheresis are used in ABO-incompatible and HLA-incompatible transplants, while high-dose IVIG is used to desensitize both living donor cross-match positive and highly sensitized deceased donation recipients on the waiting list. Recent advancements include the use of Imlifidase, a cysteine protease novel enzyme derived from the bacterium Streptococcus pyogenes, designed to cleave IgG in a quick initial reaction and subsequently 2 F(ab')2 fragments and a fully separated Fc fragment in a slower subsequent reaction. A single dose is given within 24 hours before transplantation to obtain a negative cross-match. Imlifidase application in KT, in desensitization protocols, represents a significant advancement for patients with high levels of DSA especially those who are resistant to conventional desensitization therapy. Overall, imlifidase represents a promising advancement in the field of KT, offering hope to many patients who previously had limited options due to high levels of DSAs. These therapies target various aspects of the immune response, offering new hope for highly sensitized patients[61-65].

BENEFICIAL ROLES OF ANTIBODIES

Not all antibodies are detrimental to the transplanted organ. Some antibodies may play a role in the accommodation/acceptance of the transplanted organs. In addition, antibodies are being explored for their potential therapeutic benefits. For instance, IVIG, initially used for desensitization and AMR treatment, has immunomodulatory properties that can enhance immune regulation and promote tolerance. Monoclonal antibodies targeting specific immune pathways, such as anti-CD40 and anti-IL-6, are being used for their ability to prevent rejection while minimizing immunosuppression-related complications[66-68]. Some antibodies could suppress the activity of certain immune cells (e.g., T cells, NK cells) that would otherwise attack the graft. They might promote the production of molecules that protect the graft from immune damage. In some cases, the presence of certain antibodies might actually prevent the formation or effectiveness of other, more harmful antibodies. This could be through competition for binding sites, effectively neutralizing the harmful ones or regulation of immune response. Emerging research suggests that even in the presence of DSAs, certain antibody profiles might be associated with better graft survival. This suggests a complex interplay between different antibody types and their overall effect on the graft. It is important to note that the concept of beneficial antibodies in transplantation is still under investigation. Identifying and characterizing these antibodies is crucial for developing strategies to promote transplant tolerance. This area of research holds great promise for improving long-term transplant outcomes by harnessing the power of the immune system itself[67,68].

TREATMENT OF AMR

The treatment strategies in AMR aim to reduce or eliminate DSAs and control the immune response to minimize graft damage and prolong graft survival. Treatments for AMR can be classified into traditional approaches and emerging therapies (Table 4). Traditional treatments like plasmapheresis, IVIG, and rituximab remain the backbone of AMR management, but emerging therapies such as eculizumab and imlifidase are showing promise in refractory cases. In clinical practice, AMR is often treated using a multi-modality approach. Combining plasmapheresis with IVIG and rituximab, for example, can help control antibody levels while suppressing B cell activity. Ongoing research into these treatments continues to refine AMR management strategies, particularly for patients who do not respond to conventional therapies[69].

Table 4 Examples of some traditional and emerging therapies in kidney transplantation.

Traditional therapies	Plasmapheresis
	Immunoadsorption
	Intravenous immunoglobulin
	Anti-CD20 monoclonal antibody rituximab
	Complement inhibitors: Eculizumab, C1 INH
	Proteasome inhibitors: Bortezomib
Emerging therapies	Carfilzomib
	Tocilizumab
	Clazakizumab
	Daratumumab
	Belimumab
	Imlifidase
	Ofatumumab
	Obinutuzumab
	Inebilizumab

CURRENT STATUS OF ANTIBODIES IN KT

Today, the humoral theory of kidney allograft rejection is well-established and integral to transplant medicine. It underscores the importance of pre-transplant antibody screening, perioperative management, and post-transplant monitoring for the presence of DSAs[70]. Diagnostic techniques, such as solid-phase assays for DSA detection and molecular profiling of graft biopsies, have enhanced the precision of AMR diagnosis. Ongoing research focuses on novel therapeutic strategies to prevent and treat AMR, aiming to improve long-term outcomes for KT recipients[71-73].

In recent years, the role of antibodies in KT has continued to evolve with the advent of NGS and more sophisticated immunological assays. These technologies allow for even more precise matching and monitoring of donor-recipient pairs, reducing the risk of rejection and improving long-term outcomes[74-77].

Improvements in compatibility testing between donor and recipient may also decrease the risk of allograft rejection and increase survival of the transplant. The better matched the donor and the recipient are, the more tolerant the recipient’s immune system will be to the transplanted organ or tissue. Additionally, a greater understanding of the disparity between the donor and recipient will better inform treatment strategies after transplantation and help avoid repeated episodes of AR[78-81]. Immunological research has resulted in huge advancements in transplant medicine. However, immune rejection still remains the most formidable barrier to successful transplantation.

FUTURE PERSPECTIVES

Ongoing research into the mechanisms of antibody-mediated injury and the development of targeted therapies will further improve outcomes for KT recipients. The future of antibody-based therapies in KT lies in precision medicine and the development of more effective immunomodulatory therapies and the potential for tolerance induction. Understanding the unique immunologic landscape of each patient will enable tailored desensitization protocols and rejection treatments. Additionally, the integration of novel diagnostic tools and biomarkers will facilitate real-time monitoring and early intervention. Research into gene therapy, cellular therapies, and other innovative approaches is also ongoing, and aims to achieve long-term graft acceptance without the need for lifelong immunosuppression. Continual research is needed to find ways to decrease the risk of rejection, improve diagnosis, and maintain long-term survival of the transplant; all of which would have a significant impact on long-term graft and patient survival[82-86].

CONCLUSION

In summary, the history of the role of antibodies in KT reflects a journey of discovery, innovation, and clinical application. The humoral theory of kidney allograft rejection highlights the crucial role of antibodies in transplant rejection, from initial observations of HAR to sophisticated modern diagnostic and therapeutic approaches. The history of the role of antibodies in KT is a testament to the advances in immunology and medical science. These advances have significantly influenced the management and success of KT.