The major hurdle to successful organ transplantation is graft rejection, a process orchestrated by sophisticated cell and antibody-mediated defense mechanisms, which has evolved primarily to combat invading microbes or diseased and damaged cells. The T cell-targeted immunosuppressive regimens (including T cell-specific antibodies, calcineurin inhibitors, mycophenolic acid, rapamycin, and prednisone) have effectively reduced the incidence of cell-mediated transplant rejection and have substantially improved 1-year graft survival to 88% following renal transplantation (1). Nevertheless, alloantibodies mediate a substantial proportion of the remaining graft rejection episodes, contributing to both early and late graft loss, particularly in sensitized populations such as patients with previous transplants and patients who have previously had multiple pregnancies or multiple blood transfusions (2).

Antibody-mediated rejection (ABMR) is recognized to be a key problem in organ transplantation and a major cause of late graft loss (3). Based on time course, the ABMR is classified as hyperacute, acute, or chronic (1). Hyperacute rejection, the first rejection phenotype observed in human organ transplantation, occurs immediately on perfusion of the transplanted organ with the blood of the recipient (4). Preformed donor-specific antibodies (DSAs) in recipient’s blood bind to antigens expressed on vascular endothelium of the transplanted allograft [such as human leukocyte antigens (HLAs), ABO blood group antigens, and other endothelial antigens] and trigger a cascade of complement activation, which results in tissue injury involving blood vessel wall damage, hemorrhage, neutrophil infiltration, platelet, and fibrin deposition. Reliable cross-matching methods and screening recipients for preformed circulating HLA antibodies to the prospective donor have almost eliminated the incidence of this devastating phenotype (5, 6).

Acute ABMR occurs at any time from days to years following transplantation, and results from DSA that may be preexisting or develop de novo after transplantation (7). At present, acute ABMR is defined by four criteria: clinical evidence of acute graft dysfunction, histologic evidence of acute tissue injury, immunohistologic evidence for the action of DSAs (C4d deposition in peritubular capillaries), and DSAs detected in the serum (8). ABMR occurs in 6.7% of renal transplant patients and is present in approximately one-third of renal transplant patients diagnosed with acute rejection (9–11). Acute ABMR is characterized by a rapid rise in serum creatinine and is resistant to therapy with steroids or T cell-specific reagents.

Chronic ABMR develops over months or years before there are signs of graft dysfunction and is mediated by antibodies that develop de novo. The features of chronic ABMR in renal allografts include the following: duplication of the glomerular basement membrane, intimal cell proliferation of arterioles and infiltration with mononuclear cells, and lamination of the peritubular capillary basement membrane, which occurs together with the deposition of C4d in peritubular capillaries and glomeruli. Chronic ABMR is the result of cumulative damage to the kidney and over 50% of recipients develop chronic ABMR at 10 years after transplantation (12). A further category of rejection, subclinical rejection, has recently been recognized, and this refers to pathological injury in the graft that has been caused by antibody and/or T cells, but which has not yet resulted in graft dysfunction.

The Y-shaped structure of IgG antibodies provides a bifunctional capacity to initiate and regulate host defense mechanisms in the following ways: antigen binding through the Fab (antigen-binding fragment) portion of the antibody and the interaction with immune cells and complement proteins (fragment crystallizable or Fc). Following DSA binding to the allograft endothelium, at least four distinct cellular and humoral mechanisms exert significant graft injury and failure (Figure 1).

Natural killer cells are the third population of lymphocytes defined by the CD3⁻ CD56⁺ cell surface phenotype, and they represent 5–25% of the mononuclear cell fraction of normal human peripheral blood (35). NK cells share several features with CD8⁺ cytolytic T-lymphocytes in their development, morphology, cell surface phenotypes, killing mechanism, and cytokine production (36). NK cells were originally described as innate lymphocytes capable of lysing target cells quickly by direct cytotoxicity in an antigen-independent manner without the “priming” period required by T-cells (37). NK cells are recognized to express a sophisticated repertoire of activating and inhibitory receptors that are calibrated to ensure self-tolerance, while exerting early assaults against virus infection (38) and tumor transformation (39). In addition to cytolytic functions, NK cells produce high levels of IFN-γ and a wide range of pro-inflammatory cytokines and chemokines, which contribute to the shaping of adaptive immune responses (40). Recently, NK cells have been shown to mount antigen-specific immunologic memory, a hallmark characteristic of adaptive immunity (41). Having properties of both innate and adaptive immunity, NK cells spontaneously lyse target cells, as well as function as regulatory cells influencing subsequent antigen-specific T-cell and B-cell responses.

Experiments with rodent models clearly indicate a role for NK cells in acute and chronic allograft rejection (42–44). The most convincing evidence of NK cell-mediated rejection was observed with the heart allograft missing-self-MHC class I in CD28-deficient recipient mice; in this model, rejection is prevented by depletion of host NK cells (45). NK cells play a crucial role in mediating long-term kidney allograft injury (46). Currently, used clinical regimen of immunosuppressive agents such as cyclosporine A (47), FK506 (48), mycophenolate mofetil (49), azathioprine (50), and rapamycin (51) appears not to abrogate NK cell function. NK cell number and the cytotoxicity function were preserved to a greater extent in a regimen of tacrolimus and mycophenolate mofetil than they were with cyclosporine A and azathioprine 12 months after kidney transplantation (52). Even in the presence of polyclonal anti-thymoglobuline antibody that depleted T and NK cells transiently, the NK cell effector function is preserved after kidney transplantation (53).

Natural killer cells use very complex and specific receptor–ligand system that integrates signals triggered by an array of inhibitory and activating receptors, which trigger cytotoxicity and the secretion of chemokines and cytokines (54, 55). Unlike T- and B-lymphocytes, NK cells do not express receptors that require somatic gene rearrangements to generate receptor diversity and specificity. Instead, NK cells express a wide array of conventional germline-encoded receptor families with inhibitory or activating functions that scan for missing-self, induced-self, and altered-self on target cells. The well-characterized NK cell receptor gene families include killer cell immunoglobulin-like receptors (KIR), killer cell lectin-like receptors, leukocyte immunoglobulin-like receptors, and natural cytotoxicity receptors (56–59).

The KIRs are crucial for human NK cell development and function (56, 58, 60) (Figure 2). The KIR gene family does not exist in rodents and found only in primates, and therefore KIR genes are considered to be originated recently and evolved rapidly (61, 62). The KIR gene family consists of 16 highly homologous genes clustered at the leukocyte receptor complex on chromosome 19 (63, 64) (Figure 3). Seven of them encode inhibitory KIRs (3DL1–3, 2DL1–3, and 2DL5), six encode activating KIRs (3DS1, 2DS1–2DS5), one encode a KIR that can trigger both inhibitory and activating signals (2DL4), and two are pseudogenes (2DP1 and 3DP1) that do not encode a cell surface receptor. By recognizing specific HLA class I ligands, the inhibitory KIRs trigger signals that stop NK cell function, while the ligands for activating KIRs are not elucidated. Genetic association studies suggest the possibility of activating KIRs recognizing cell surface determinants expressed following infection or tumor transformation, or under certain physiological, stress such as transplantation (65).

In general, humans have two copies of each autosomal gene, one per chromosome. However, due to deletion and duplication, the basic diploid rule does not apply to KIR gene family. The number and type of KIR genes vary substantially between haplotypes, and all KIR genes display sequence polymorphism (66) (Figure 3). On the basis of gene content, KIR haplotypes are broadly classified into two groups (67). Group A haplotypes have a fixed gene content (KIR3DL3–2DL3–2DP1–2DL1–3DP1–2DL4–3DL1–2DS4–3DL2) that encode four inhibitory KIRs, 2DL1, 2DL3, 3DL1, and 3DL2, specific for four major HLA class I ligands, C2, C1, Bw4, and A3/A11, respectively, and an activating KIR 2DS4, which is weakly specific for some HLA-C allotypes (C1 or C2 epitope), as well as the HLA-A3/11 epitope (Figure 2). In contrast, group B haplotypes are variable both in numbers and combinations of KIR genes, and comprising several genes (2DL2, 2DL5, 2DS1, 2DS2, 2DS3, 2DS5, and 3DS1) that are not part of the A haplotype (63, 68, 69). Moreover, B haplotypes possess KIRs that have no binding to HLA class I ligands, such as KIR2DL5, 2DS2, 2DS3, and 2DS5. While group A haplotypes contain only KIR2DS4 as an activating gene, group B haplotypes contain up to five activating KIRs – KIR2DS1, 2DS2, 2DS3, 2DS5, and 3DS1. Inheritance of paternal and maternal haplotypes comprising different KIR gene contents generates human diversity in KIR genotypes (70). For example, homozygotes for group A haplotypes have only seven functional KIR genes, while the heterozygotes for group A and certain group B haplotypes may have all 14 functional KIR genes. All human populations have both group A and B KIR haplotypes, but their incidences vary substantially among populations (71–74). The A and B haplotypes are equally distributed in Africans and Caucasians, while the A haplotype is overrepresented in Northeast Asians (Chinese, Japanese, and Koreans) and the B haplotype occurred most frequently in the indigenous populations of India, Australia, and America (75).

The recipient NK cells can recognize and respond against the allograft by three possible mechanisms: missing-self recognition, induced-self recognition, and ADCC (Figure 4) (76). Because NK cells circulate in a state that can spontaneously deliver effector function, it is critical that they do not attack surrounding healthy cells. To prevent such detrimental autoreactivity, NK cells express an array of inhibitory receptors recognizing self-HLA class I molecules (Figure 4A). Expression of four distinct HLA class I molecules (HLA-A, -B, -C, and -E) on normal healthy cells provides ligands for various inhibitory receptors of NK cells and, consequently, are resistant to NK cell attack. Downregulation of HLA class I expression due to certain viral infections, neoplastic transformations, or absence of relevant HLA class I ligands on the allograft at the setting of allogeneic transplantation, alleviates inhibitory signals, permitting NK cells to eliminate these unhealthy or allogeneic target cells, a phenomenon originally described as the “missing-self” hypothesis (77) (Figure 4B, i). In addition to the “missing-self” mechanism, the expression of ligands for activating receptors on stressed target cell surface might also contribute to NK cell attack, known as “induced-self” recognition (Figure 4B, ii). The activation receptors can directly recognize stress-induced ligands associated with certain physiological conditions, such as infection, tumor transformation, and transplanted allograft (58, 78). The third mechanism is mediated via an ADCC (Figure 4B, iii), in which NK cells are activated through the low-affinity Fc receptor for IgG FcγRIIIA (CD16) by binding to the Fc portion of DSA. In summary, the NK cells discriminate the stressed unhealthy cells or allograft from the healthy self by gauging the net input of activating and inhibitory signals perceived from the NK cell receptors upon their interactions with target cell ligands.

Similar to T- and B-lymphocytes, NK cells are developed from CD34⁺ hematopoietic stem cells in the bone marrow and undergo terminal maturation in secondary lymphoid tissues (79–81). A signature feature of KIR is their clonal expression on NK cells, so that each NK cell clone in a person expresses only a portion of the genes within their KIR genotype (82–84). Stochastic expression of different combinations of receptors by NK cells results in this repertoire of NK clones with various ligand specificities. Once a given KIR is expressed on an NK cell clone, it is maintained in a stable way in the progeny of the clone. The process that establishes these clonal patterns is based on epigenetic regulation by DNA methylation and histone modifications (85–87).

Because KIR and HLA genes are located on different chromosomes (KIR on chromosome 19 and HLA on chromosome 6), KIR genes are inherited independently from HLA genes, and KIR may be expressed in the absence of their HLA ligands (88). Most, but not all, NK cell clones in peripheral blood express at least one inhibitory receptor for self-HLA class I (82). Only those NK cell clones expressing at least one inhibitory KIR specific for self-HLA class I molecule are “licensed,” or functionally active, to eliminate target cells that have downregulated or which are missing the respective HLA class I ligands (74, 78, 89–91). A conceivable explanation for NK cell licensing is that inhibitory KIRs, upon specific interaction with self-HLA class I allotypes, deliver a signal resulting in NK cell maturation and acquisition of effector function. NK cells lacking inhibitory receptors for self-HLA class I molecules are considered to be developmentally immature, “unlicensed,” and substantially hyporesponsive to HLA class I-negative targets (74, 89, 92, 93). Therefore, the NK cell responsiveness is most fundamentally distinguished by the presence or lack of inhibitory KIR for self-HLA class I. Licensed NK cells further vary in effector function quantitatively according to the strength of the inhibitory KIR and HLA interactions and the copy number of the corresponding inhibitory KIR and HLA genes (94–96). In summary, KIR receptor–HLA class I ligand interactions at the developmental stage set the functional threshold for NK cell and regulate NK cell effector function.

Polymorphic variation among the KIR and HLA class I genes and their resulting impact on the KIR and HLA interaction constitute a major source of variability in NK cell responsiveness (94–96). These differences influence clinical outcomes in diverse settings, including monoclonal antibody therapy for lymphoma (97), transplantation for hematological malignancies (98), kidney transplantation (99, 100), and other settings in which NK cell involvement contributes to disease control and clinical responses (101–105). However, not all KIR⁺ licensed NK cells are equivalent, as polymorphic diversity in the KIR and HLA genes underlie significant variation in binding strength and specificity, which quantitatively influence licensing, inhabitability, and ADCC (104, 106–111).

However, studies supporting a role for licensing in human ADCC are limited. A prominent role for KIR3DL1/HLA-Bw4 interactions in licensing NK cells for CD16-mediated effector function was published recently (112). When individuals expressed both inhibitory KIRs that interact with HLA-C and the corresponding HLA-C ligand, their NK cells exhibited greater general and Fc receptor-mediated effector functions than NK cells from those individuals lacking the relevant HLA-C ligand (74). Similarly, expression of KIR3DL1, an inhibitory KIR that interacts with the HLA-Bw4 public epitope, was associated with higher NK cell cytotoxicity and IFN-γ production upon exposure to HLA class I-deficient target cells when the NK cells were isolated from HLA-Bw4 donors (94, 112). Therefore, the interindividual differences in compound KIR and HLA class I ligand genotypes associated with differences in NK cell reactivity would impact DSA-mediated NK cell ADCC against the organ allograft. The individualized assessment of the recipient’s KIR, FcR, HLA types, HLA antibodies, and the donor’s HLA types at the molecular and functional levels have the potential to distinguish between mechanisms that could guide identification of new therapeutic targets for ABMR.

The complement-independent mechanisms that lead to the ABMR of kidney allografts remain poorly understood. Recent studies finding a link between ABMR and abundance of NK cell molecular signatures in transplant biopsy suggest relevance of NK cells as innate immune cytotoxic effectors to antibodies through ADCC. However, the direct pathogenic role of donor-specific HLA antibody-mediated NK cytotoxicy in transplant rejection remains poorly documented by mechanistic studies. NK cells use very complex and specific germline-encoded KIR receptor and HLA class I ligand system that integrate signals triggered by an array of inhibitory and activating receptors, which set NK cell maturation and acquisition of effector function. Moreover, the FcγRIIIA polymorphisms and expression levels can also modulate NK cell activation against allograft. Future studies that integrate both recipient factors (such as KIR receptors, HLA class I ligands, FcγRIIIA polymorphisms, and donor-specific HLA antibodies) and donor factors (such as HLA class I ligand compatibility with recipient) that establish variable KIR–HLA conditioned NK cell-FcγRIIIA-antibody–antigen interactions will identify potential interindividual variability of humoral alloimmune responses.

The author confirms being the sole contributor of this work and approved it for publication.

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.