Despite the advancement of improved immunosuppression regimens and optimized patient management, antibody-mediated rejection remains a major obstacle for long-term graft survival (1). Initial emphasis has been on the identification of human leukocyte antigen (HLA)-specific antibodies directed against the donor HLA class I and/or class II antigens. With the introduction of commercially available solid phase assay reagents, a greater understanding of the specificity, strength, and function of these antibodies has been made possible. Further, the wide acceptance of these reagents in the clinical transplant testing setting, along with proficiency testing programs has allowed for increased consistency in the definition of the antibodies detected. Correlating the antibody information obtained together with the clinical graft outcome has revealed that patients with antibodies against donor HLA are at a higher risk of antibody-mediated rejection and poorer graft outcome (2, 3). However, for many patients who have allograft dysfunction and show histological characteristics of antibody-mediated rejection on biopsy, no donor HLA-specific antibodies are detected (2, 4). In heart transplant, it has been shown that 40% of patients diagnosed with biopsy-proven antibody-mediated rejection did not show donor HLA-specific antibodies in the peripheral blood (5). These studies initiated investigation into non-HLA-specific antigens, many of which are expressed by the vascular endothelium and often revealed after stress or graft injury. Even though the detection of many of these non-HLA antibodies remains elusive, antibodies to the following non-HLA antigens have been shown to be associated with graft dysfunction or rejection: major histocompatibility complex class I chain-related gene A (MICA), angiotensin II type 1 receptor (AT₁R), endothelin type A receptor (ET_AR), heat shock protein, phospholipid, K-α-tubulin, vimentin, and endothelial cell antigens (6). Recently, commercial reagents have become available to define antibodies to AT₁R, ET_AR, MICA, and endothelial cell antigens. These results have been used in the clinical testing setting to investigate their impact on graft outcome. Studies of antibodies to other non-HLA antigens have been largely single center results. The results for anti-HLA and non-HLA-specific antibodies indicate an interplay between alloimmunity and autoimmunity, impacting graft outcome. This review focuses on the contribution on antibodies against these non-HLA antigens in kidney and heart transplantation. For lung transplant outcomes, please refer to the comprehensive review by Dr. Mohanakumar in this same issue.

Major histocompatibility complex class I chain-related gene A gene is localized in the HLA gene cluster. The MICA protein shares similar structure with HLA class I but does not associate with β2 microglobulin at the cell surface and cannot bind peptides. MICA can activate natural killer (NK) cells via interaction with activating immunoreceptor NKG2D (7). Of the non-HLA antigens listed above, MICA is highly polymorphic with around 100 alleles identified as of July 2016. Similar to HLA molecules, the recipient and donor may carry different MICA alleles. The recipient’s immune system may develop antibodies against the donor-specific MICA allele (8). It has been reported that 5–9% of renal recipients display MICA antibodies (9). The contribution of MICA antibody to pathogenesis of antibody-mediated rejection was first demonstrated in kidney transplantation (10) and was later found to be associated with rejection in pancreas and heart transplant (11, 12). Further, the patient with antibodies against donor-specific MICA is at higher risk of antibody-mediated rejection (5). The expression of MICA is not detectable on the quiescent endothelial cells which lie at the interface of the allograft and recipient blood and are directly targeted by immune response. The expression of MICA can be induced by stress and cytokines, such as TNF-α (13). The development of MICA antibodies may indicate an underlying inflammatory status which exists in these conditions.

Studies have demonstrated that expression of MICA in tumor cells leads to the activation of NK cells via MICA/NKG2D interaction, which in turn release cytotoxic proteins and INF-γ (7). Binding of MICA antibody on endothelial cells may block interaction between MICA and NKG2D, and thereby dampen NKG2D-mediated cytotoxicity. However, NK cells may still be activated mainly through Fc receptor-dependent cytotoxicity.

Vimentin is a subunit of an intermediate filament. As a cytoskeletal element, vimentin is important for stabilizing the architecture of the cytoplasm. However, vimentin is also found to be secreted by macrophages, endothelial cells, vascular smooth muscle cells, activated platelets, apoptotic T cells, and neutrophils (33). Secretion is increased by the pro-inflammatory cytokine TNF-α, but inhibited by the anti-inflammatory cytokine IL10, suggesting that vimentin may be involved in the immune response (34). Antibodies against citrullinated vimentin have been detected in sera of patients with rheumatoid arthritis (35). Vimentin antibodies also exist in pretransplant sera of patients with end-stage kidney disease (36). In this study, titers of IgM antibodies against vimentin increased every year posttransplant compared with pretransplant titers, but no difference was found in patients with interstitial fibrosis and tubular atrophy compared with a kidney recipient control group. By contrast, patients diagnosed with interstitial fibrosis and tubular atrophy have higher levels of IgG antibodies against vimentin. These results suggest that IgG, but not IgM, antibodies against vimentin may play a role in the pathology of interstitial fibrosis and tubular atrophy (37). Similarly, in heart transplant, patients with vimentin antibodies within the first 2 years of transplantation are at higher risk for cardiac allograft vasculopathy at 5 years posttransplant (38). The presence of vimentin antibodies is rather common in heart transplant recipients. Young et al. have shown 34% of patients display vimentin antibodies, but no difference in the rates of early rejection and graft survival was observed (39).

Myosins are a large family of proteins which bind actin cytoskeleton and move cargo proteins through ATP hydrolysis. The thymus of mice and humans does not express myosin heavy chain proteins, thus CD4+ T cells are not negatively selected for myosin in the maturation process (40). The autoantibodies frequently associated with autoimmune myocarditis may be the result of this mechanism (40, 41). The presence of myosin antibodies has been associated with antibody-mediated rejection and development of chronic allograft vasculopathy in heart transplantation (42, 43). Three hundred single-nucleotide polymorphisms have been identified in the myosin motor domain of cardiac myosin (44), but it is not yet known whether myosin antibodies detected in the patients are donor specific. The impact of cardiac myosin antibodies on heart transplantation was confirmed by a swine minor antigen-mismatch model, in which animals with strongly binding myosin antibodies after immunization of myosin pretransplant rejected grafts acutely, while animals with low and transient binding myosin antibodies had long-term allograft survival (45). The presence of donor HLA-specific antibodies in patients with antibody-mediated rejection and cardiac allograft vasculopathy precedes the detection of myosin and vimentin antibodies (43), thus again suggesting the interplay of allo- and autoimmune responses.

Perlecan is a critical component of the endothelial basement membrane and serves as a barrier between the circulating blood and the surrounding tissue (46). Perlecan is a large, extracellular matrix proteoglycan with many functions. Perlecan can act as a coreceptor for fibroblast growth factor 2 to stimulate cell proliferation as demonstrated in a rat transplant model (47). Injection of LG3, a C-terminal fragment of perlecan, increases cell migration and accumulation of recipient-derived α smooth muscle actin-positive cells in neointima in a MHC-mismatched allogeneic aortic segment transplant mouse model (48). Elevated levels of LG3 have been found in the sera of kidney-transplant recipients with acute vascular rejection (49). Studies have shown that vascular injury prompts release of apoptotic exosome-like vesicles which trigger the production of antibodies to LG3 (50). Pre or posttransplant, higher levels of LG3 antibodies have been found to be associated with acute vascular rejection in kidney-transplant recipients (51). Anti-perlecan antibodies have been detected in the sera of transplant patients with glomerulopathy (52). Pretransplant LG3 antibodies have also been shown to be associated with an increased risk of delayed graft function. Passive transfer of LG3 antibodies can cause microvascular injury in a kidney ischemia-reperfusion injury mouse model (53).

Vigorous efforts to investigate non-HLA antibodies, their impact on graft outcome, and influence on the alloimmune and autoimmune processes are still ongoing. With the availability of commercial reagents, the study of several of these antibodies to non-HLA antigens has moved to the clinical setting. As more studies populate the literature, there are common themes that appear consistently. The antibodies to these non-HLA-specific antigens do appear to be part of the overall graft dysfunction response (Table 1). These non-HLA antibodies are often checked when antibody-mediated rejection is suspected, but no donor-specific HLA antibody is detected. The presence of non-HLA antibodies may also be tested for patients who are deemed to have a high risk of antibody-mediated rejection. Commercial available Luminex bead-based reagents, which can provide detection of non-HLA antibodies in a standardized manner, may promote more transplant programs to evaluate non-HLA antibodies. Some antigens targeted by non-HLA antibodies are not polymorphic at the amino acid sequence. These antibodies may also bind the recipient’s tissue and have a global effect in the recipient as happened in patients with AT₁R antibodies. It is unclear if other non-HLA antibodies also have a broader effect or if the effect is limited to the allograft because the graft expresses these antigens at the higher level. The presence of non-HLA antibodies may predispose patients to an increased risk to develop donor-specific HLA antibodies and rejection. Whether the antibodies to non-HLA antigens appear before the donor HLA-specific antibodies or the reverse is unknown at this time. The presence of these non-HLA antibodies may indicate underlying immune status. Lupus patients who usually display autoantibodies have higher level pro-inflammatory cytokines, which may promote immune response to allo-antigens. Autoantibodies may also cause allograft injury through complement-dependent cytotoxicity or antibody-dependent cell-mediated cytotoxicity. Donor HLA antigens released from injured cells may be captured by the recipient dendritic cells and trigger donor-specific HLA antibody production. Alternately, tissue injury caused by donor-specific HLA antibodies may expose neoantigens which lead to auto-antibody production. Certainly each scenario may be part of different overall immune responses in different organs, with different immunosuppressive regimes, and in different genetically disparate settings. The identity of some non-HLA antigens targeted by these antibodies remains elusive. Nonetheless, a continuing theme of these studies is that both alloimmune and autoimmune mechanisms impact graft outcome and may be synergistic to each other.

XZ and NLR participated in literature review and writing.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer, DW, and handling editor declared their shared affiliation, and the handling editor states that the process nevertheless met the standards of a fair and objective review.