Antibody-mediated rejection caused by preformed DSAs manifests as hyper acute rejection immediately after transplantation, leading to failure of the transplanted organ within several hours. In these cases, DSAs immediately bind to all capillary endothelium surfaces in the transplanted organ, and concomitant complement activation leads to the formation of fibrin clots and acceleration of blood coagulation. Subsequently, rapid peripheral circulation incompetence causes necrosis of vascular walls, intense bleeding of the transplant, and necrosis in neighboring tissues. Finally, inflammatory cells, such as neutrophils, infiltrate capillary endothelial surfaces, and further undermine the transplant (6, 77).

Improvements in desensitization therapy have enabled management of high risk recipients, such as those with cross match-positive phenotypes and high organ transplantation sensitivity; as a result, the prevalence of severe hyper acute rejection by preformed DSAs has decreased significantly (7). Accordingly, Ng et al. summarized desensitization protocols and complications using rituximab, bortezomib, eculizumab, and alemtuzumab, and reported promising graft survival in patients across various institutes (78). However, complications included anemia and thrombocytopenia, likely reflecting myelosuppression by these agents. In addition, various infections in some cases were detected, including cytomegalovirus (CMV), BK virus, and Epstein–Barr virus (EBV), indicating that desensitization therapy disposes patients to an increased risk of opportunistic viral infections. In addition, it was reported that induction with T-cell depleting agents (anti-thymocyte globulin) was closely associated with CMV, EBV, and BK polyomavirus (BKV) infections in comparison with IL-2a receptor antagonists (anti-CD25) (79). Therefore, the use of T-cell depleting agents should be avoided as an immunosuppressive reagent or induction. If possible, the use of IL-2a receptor antagonists or no induction should be considered (79, 80). Additionally, it was expected that these virus infection may contribute to the activation of immune responses in transplanted organs, and dose reductions of immunosuppressive agents may activate immune reactions to graft antigens.

To address this issue, prediction and early detection of viral infections is critical, and could be used to inform doses reductions of immunosuppressive agents. Concomitant administration of preventive and therapeutic antiviral agents is also critical in the management of these patients.

Prior to the introduction of rituximab, plasmapheresis and splenectomy were long recommended as desensitization therapies for patients with ABO incompatible kidney transplants. Subsequently, rituximab was shown to inhibit the onset of ABMR without splenectomy. Rituximab is a monoclonal antibody (mAb) against the protein CD20, which is expressed in immature and mature B cells. However, because CD20 is not expressed on PCs, rituximab may not inhibit the production of DSAs by PCs. In addition, recent studies show varying effects of rituximab on B cell phenotypes, with higher sensitivity of naïve B cells than memory B cells (81). Thus, although rituximab suppresses immune activation and may not provide protection from infection, memory B cells may remain viable.

In addition, posterior reversible encephalopathy syndrome (PRES) and acute respiratory distress syndrome (ARDS) was reportedly increased in patients treated with rituximab as severe adversity effect (82). These data warrant further clarification of the depletion mechanisms of rituximab in B cells.

Bortezomib is a proteasome inhibitor that was developed as a treatment for multiple myeloma, and the effectiveness of this agent against transplant rejection was reported in 2008. These studies showed downregulated immune responses to donor antigens, recovery of graft function, and long-term suppression of serum antibody levels. However, inhibition of the proteasome by bortezomib may be detrimental to healthy cells (83–88).

As an alternative, eculizumab is a recombinant humanized monoclonal IgG2/4 antibody that suppresses complement activation and inhibits production of C5, which is the final product of the complement pathway and activates inflammatory responses and ultimately results in apoptosis of infected cells (89). Accordingly, treatments with this agent led to severe infectious diseases, including meningitis (90).

Finally, alemtuzumab is a recombinant DNA-derived humanized IgG1 kappa mAb that is directed toward CD52 and is used is used to treat B-cell chronic lymphocytic leukemia (B-CLL) and multiple sclerosis patients, warranting consideration for the treatment of ABMR. As adverse effect, it has been associated with infusion-related events (91, 92).

Determination of HLA antibodies in supernatants of cultured B cells can better inform ABMR management than those in sera. Therefore, some researchers have suggested that if peripheral B cells could be differentiated into PCs in vitro, then the in vitro differentiation of peripheral B cells into PCs may facilitate the control of ABMR. However, unlike in vitro T cell assays that have long been used to control T cell-mediated rejection (TCMR), primary cultures of B cells are difficult to maintain and in vitro B cells assays have not long been established. Although memory B cells were reportedly differentiated into APC in vitro (120), these short comings require further improvements in the ease and convenience of B cell culture, and subsequent assay development that can be used to detect HLA antibodies in B cell culture supernatants to ultimately prevent transplant rejection.

Moreover, there are important points to detect HLA antibodies from the B cell culture supernatant. Peripheral B cells include naïve B cells and memory B cells (121, 122); and these B cells derived from PCs survive for varying durations and produce antibodies. However, HLA antibodies that cause ABMR are mainly produced by memory B cells (123–125), warranting establishment of in vitro B cell assays in which memory B cells are selectively differentiated into PCs in vitro and are used to produce antibodies. In addition, many reports have showed that long-lived PCs, which produce antibodies in the bone marrow for long periods, play an important role in ABMR (126–129). Therefore, to monitor the progression of antibody-mediated diseases, in vitro culture systems, in which B cells are differentiated to their terminal stage (long-lived PCs), are urgently required. On using a clinical specimen, the volume of B cells in peripheral blood is very low following immunosuppression or particularly when we could collect B cells from recipients who experienced desensitization therapy.

However, feeder cells can strongly activate human B cells to proliferate and differentiate in a cell–cell contact-dependent manner in these cases. Thus, in vitro B cell assays of HLA antibodies from the cultured supernatants may lead to drug sensitivity tests that are similar to those for T cells, may contribute to clinical applications of personalized immunosuppression, and the development of new immunosuppressant agents that control ABMR.

We found a report about immunosuppressive agent susceptibility for the differentiation of human B (CD19⁺) cells in vitro with a combination of IL-21, phosphorothioate CpG-oligodeoxynucleotide (CpG-ODN), histidine-tagged soluble recombinant human CD40 L and anti-polyhistidine mAb (130). IL-21 is produced by follicular helper T cells (131), which synergistically induce maximum Blimp-1 upregulation and optimal PC differentiation with CD40 L (132). TLR9 agonist CpG-ODN activates B cells proliferation and promotes PCs differentiation (133). This culture system induced IgG production but could not sustain the survival of PCs for a long period. It might indicate that the other cytokines play an important role in human B cells differentiation into mature PCs in vitro, because other groups have reported that APRIL and the B cell activating factor (BAFF) would support the survival of PBs and PCs recently (134–137). In addition, a previous report has shown that CD27⁺ memory B cells could be differentiated into long-lived PCs with supernatants from bone marrow stromal cell line M2-10B4 (138), which support the long-term culture of human bone marrow stem cells. The mechanisms by which M2-10B4 cells contribute to PCs survival has yet to be revealed, but it is suggested that CD27⁺ memory B cells demand well-balanced support from stromal cells (139–142). In addition, different environments or signal transmission might be required for the differentiation of CD27⁻ naïve and CD27⁺ memory B cells into mature PCs. Therefore, we should improve the in vitro B cell assay to sustain CD27⁺ memory B cell-derived PC survival for a long-term selectively. For example, we should examine how any humoral factors, including growth factor or any cytokines from activated T cells could affect CD27⁺ memory B cell growth and survival in vitro, while referring to the reports that helper T-cells may mediate CD27⁺ memory B cell differentiation into PCs in vivo (143).

Not all DSAs participate in ABMR and transplanted organ prognosis (7), and although C1q binding DSAs are reported risk factors for ABMR onset, further studies of DSA characteristics are required to identify those with prognostic value. In addition, various other factors influence transplanted organ prognoses (ABMR onset, graft survival) and require further investigation. About the risk factors for graft loss, thrombotic microangiopathy (TMA), glomerulopathy, C4d deposition, and chronic injury change in histopathological diagnoses were reported.

As other factors besides histopathological findings, a history of subclinical ABMR and TCMR and a decline of graft function could be risk factors. This might indicate that a graft would fail with high probability when the humoral immune response toward a donor-specific antigen has proceeded to an irreversible stage.

Antibody-mediated rejection caused by de novo DSAs typically appears several weeks to months after transplantation, but can develop at any time as far as a graft engrafts afterward.

Following the absorption of HLA antibodies onto capillary endothelial donor antigens (mainly HLA antigen), activation of pro-complement solidification and accumulation of inflammatory cytokines, macrophages, and neutrophils are caused successively. Therefore, it leads to microangiopathy and gradual annual declines in graft function (52, 163–165). Under these conditions, ABMR-mediated microangiopathy is chronic and sustained, although moderate inflammatory activities result in slow and irreversible disease progression.

In a previous section, we suggested that immunosuppressive therapy limits differentiation of naïve B cells to germinal center B cells by controlling CD4⁺ effector T cell stimulation. However, stronger immunosuppressive therapy is required to control B cell growth and survival after differentiation of naive B cells to memory B cells in germinal centers. As a result, chronic use of immunosuppressive agents after differentiation of naïve B cells into memory B cells corresponding to HLA antibodies may no longer affect B cells in germinal centers.

Thus, further attention should be paid to pancytopenia, anemia, and viral infection as well as to those concerning B cell differentiation, because more strong immunosuppressive therapy might be necessary to inhibit memory B cell growth and survival in comparison with naïve B cells. In particular, rituximab administration induces CD20⁺ memory B cell apoptosis (166), bortezomib therapy inhibits the production of DSAs production from PCs (167), and IVIG can be used to reduce circulating DSAs (168) (Figure 3). Hence, the development of new immunosuppressive agents that inhibit memory B cell growth and survival is warranted.

In addition, the development of a diagnostic method for predicting the development of DSA specific memory B cells as soon as possible has been required.

Therefore, the control of ABMR is very difficult; the disease state progresses irreversibly and severely and is unresponsive to increasing immunosuppression following diagnosis using currently available methods. Although graft tissue biopsies are the most reliable diagnostic method for ABMR, it was hard to perform frequently because it is very invasive. Therefore, less invasive diagnostic approaches are urgently required to predict the development of DSA-specific memory B cells.

Histological studies of ABMR following solid organ transplantation show that the classical complement pathway is activated after adhesion of DSAs to capillary endothelia, and that C4d produced and deposited as the final product of this pathway is an important ABMR diagnostic factor before (169). In late years, the number of reported cases of C4d-negative ABMR has recently increased (15) and some DSA-related mechanisms that are independent of the classical complement pathway have been identified. In each organ transplantation, these observations necessitate revision of histologic diagnostic criteria for all organ transplant patients, and improved the understanding of ABMR (170–172).

Pathological diagnoses play important clinical roles, and diagnostic criteria have been revised for all organ transplants. In particular, Banff score are widely used as histologic methods for kidney transplantations, although diagnostic criteria were substantively revised in 2013; indeed, the roles of ABMR, DSAs, and C4d deposition in grafts received greater emphasis in the previous diagnostic criteria before the meeting in 2013. In the 2013 revised edition (Table 2), C4d-negative ABMR became the diagnostic focus especially, reflecting on the increased numbers of reported cases. Thus, we listed the important diagnostic criteria for ABMR in the revised edition in 2013, including confirmation of microangiopathy, evidence for DSA-capillary endothelial reactions, and detection of DSA in the serum. In particular, diagnosis of DSA-capillary endothelial reactions requires at least one of the following observations; (i) C4d deposition in peritubular capillaries (PTC), (ii) evidence of more than moderate microangiopathy and microvascular inflammation (MVI; g + ptc ≥ 2), and (iii) expression of endothelial activation (ENDAT) and injury transcripts.

Microvascular inflammation scores and C4d deposition in PTC are currently the most commonly used diagnostic criteria. However, according to this standard, ABMR diagnoses are recommended in the presence of strong MVI, even in specimens that are C4d-negative.

Thus, C4d deposition has not been necessary for ABMR diagnoses after the meeting in 2013.

By contrast, prior to 2013, ABMR was considered reflective of injury to capillary endothelia (165, 173), and C1q was the assumed trigger of the classical complement pathway following binding of DSAs to capillary endothelia. Although C4d is the final product (Figure 4), this classical pathway was not necessarily activated during ABMR in C4d-negative patients.

About the other pathways, DSAs may directly promote vascular endothelial cell (EC) growth and proliferation, and inhibit apoptosis in capillary endothelia (Figure 4); DSAs have been shown to bind with Fcγ on the cell membrane surfaces of macrophages, natural killer cells, and neutrophils, and to induce inflammatory cytokine production and microangiopathy (Figure 4).

Furthermore, in the revised criteria, ABMR phenotypes have been classified as acute/active, chronic/active corresponding to the diagnostic criteria, which have been listed in detail.

Among these, evidence of acute tissue injury as the diagnostic criteria of acute/active ABMR, and morphologic evidence of chronic tissue injury as the diagnostic criteria of chronic/active ABMR, is considered central. Therefore, effective management of ABMR entails varied treatments for differing levels of pathological progress, and these diagnostic criteria identify ABMR phenotypes with sufficient accuracy to inform treatments.

Previous studies have reported the detection of nHLA antibodies using ELISA and FACS. However, the clinical utility of these assays remains unclear, because they fail to distinguish antibodies from autoantibodies. Additionally, nHLA antibodies may be detectable in sera from patients with failed grafts but no immunological factors (26, 187). Thus, specific detection of nHLA antibodies that activate humoral immune responses to grafts requires more sensitive methods. For example, by using high-density protein array platforms (26), serum nHLA antibodies in transplant recipients may have up to 9000 different target proteins/antigens and these antibodies were screened immediately, indicating the importance of high throughput screening. In addition, nHLA genotyping of donor and recipient to estimate the risk of ABMR in recipients, such as HLA may be required; the specificities of these antibodies to nHLA should be identified in more details. For example, we should examine if nHLA has the capacity to bound complement or not and if these antibodies could activate humoral immune response toward the donor antigen by using donor-specific ECs.

Mechanism of participation of nHLA antibodies in ABMR and graft loss has not been investigated sufficiently. However, we could find reports that C4d deposition is related to ABMR causing from nHLA antibodies with high probability, indicating that this type of ABMR was caused by the activation of the complement classical pathway. Moreover, further studies are warranted to establish effective immunosuppressive therapies thereby clarifying the mechanism.

In addition, we summarized the association between the representative nHLA antibodies and graft prognosis (Table 3).