The underlying mechanisms of antibody-mediated rejection (AMR) have been intensively investigated over the past decade to identify potential therapeutic targets and biomarkers for patient follow-up.1,2 By expressing donor-specific HLA class I and class II antigens3 as well as other non-HLA antigens4,5 targeted by alloantibodies in the context of organ transplantation, graft endothelium is central to the pathogenesis of AMR.6 Whereas the detection of complement-activating donor-specific alloantibodies (DSA) has proven utility for AMR diagnosis and patient stratification according to risk of transplant failure, the specific effects of complement-activating DSA on the pathogenesis of AMR are still undefined at the molecular level. The final outcome of complement activation on vascular lesions is uncertain. Beyond DSA and complement, as primary effectors of allorecognition and of endothelial cell (EC) injury, recent advances consistently identified inflammation, NK cells, and macrophages as major contributors of AMR. Histology and immunohistopathology analyses of allograft biopsies together with transcriptomic analyses have been used to further decipher the mechanisms associated with vascular and graft injury mediated by DSA and complement and established molecular and cellular signatures of AMR.7-11 The specific role played by infiltrating NK cells and monocytes/macrophages, their activation by DSA and their crosstalk with the endothelium are emerging as the next targets of investigation. The present review focuses on highlighting both our basic knowledge and recent clinical insights on the effector cells, molecules, and mechanisms that may contribute to AMR in response to DSA binding to graft ECs. The important topics of the elucidation of non-HLA donor-specific antigens targeted by DSA4,5,12 as well as intracellular signaling mediated by anti-HLA DSA13 that may directly affect the graft’s EC functions are not developed in the present review.
DSA and IgG-Fc-ENGAGING EFFECTOR MOLECULES
The Fc domain of IgG is the target for many proteins, including receptors expressed on myeloid and immune cells, and ECs and thereby serves as a ligand for a set of effector molecules. Many biological activities of IgG are dependent on the interaction with these effector molecules, which may vary according to IgG subclasses. In human sera, IgG1 is the most abundant IgG (~60%), followed by IgG2 (~30%), IgG3 (~5%–7%), and IgG4 (~3%–5%), respectively. IgG3 is usually the first subclass to form, which is followed by IgG1 responses that later dominate. The development of IgG4 responses is often the outcome of repeated or prolonged antigen exposure. IgG4 contains unique structural features in the hinge, CH2 and CH3 domains, which are responsible for its binding characteristics and reduced effector function, compared with other IgG subclasses. Anti-HLA alloantibodies eluted from explanted renal allografts display all IgG subclasses (IgG1, IgG2, IgG3, IgG4).14 Among circulating DSA, IgG1 represents the predominant anti-HLA DSA subclass reported in the solid organ transplantation literature,15 which is variably associated with the other IgG subclasses (IgG2, 3, and 4).16-19 The majority of allograft recipients present a mixture of IgG subclasses. IgG3 and IgG4 subclasses of DSA are highly associated with antibody-mediated damage, whereas the presence of IgG3 DSA was associated with a greater risk of graft loss.15,20 The implication of IgG4 DSA in AMR remains controversial. Several studies reported that patients with exclusively weak complement-activating DSAs (mostly IgG1 and IgG4) tend to experience less acute AMR and better outcomes.21-23 Major effector molecules and receptors for IgG Fc comprise complement components (C1q), Fc gamma receptors (FcγRs), neonatal Fc receptor (FcRn) (Figure 1).24
Binding of C1q to monomeric IgG initiates complement activation. Functionally, IgG1 and IgG3 are strong inducers of complement cascade activation and complement-dependent cytotoxicity. A conserved proline residue is found at position 331 in the CH2 domains of human IgG subclasses which fix complement. This residue is replaced by a serine in IgG4, which is inactive.25 Binding and activation of C1q is in general more efficient with IgG3 compared with IgG1.
The 3 pathways by which the complement system can become activated, namely, the classical, lectin, and alternative pathway, converge at the level of C3 and generate C3 convertase. The C3 convertase promotes the cleavage of C3 into C3a and C3b. After cleavage of C3, C3b molecule combines with the C3 convertase to form C4bC2aC3b complex in classical and lectin pathways and to the formation of C3bBbC3b complex in the alternative pathway. Both complexes are known as C5 convertase and cleave C5 into C5a and C5b molecules. The generated C5b participates in the assembly of the membrane attack complex (C5b-9 or MAC) causing direct tissue injury by perforation of the cell membrane.26
Since the first descriptions of cytotoxic anti-HLA antibodies,27 activation of the complement cascade has been considered to be a key component of AMR. In the past decades, C4d deposition in graft capillaries has been proposed for the diagnosis of antibody-mediated allograft damage.28-30 As a marker, C4d staining allows visualization of the direct interaction between DSA and tissue injury in the graft. Although the use of C4d staining suggested a strong association with acute AMR and poor graft outcome, C4d is a less specific and sensitive marker than initially thought. C4d can be a marker of acute AMR but also of accommodation.31 Indeed, C4d on the endothelium does not preclude the protective action of the complement regulatory proteins DAF, MCP, and CD59, expressed by ECs, and thus does not always reflect cell damage.26 Thus, there are many instances in which transplant biopsies show evidence of chronic AMR but lack C4d deposition.32,33 Consequently, the capacity of anti-HLA antibodies to bind complement fractions C1q or C3d, which are the proximal steps in activation of the classic complement cascade has been proposed as a way to determine the cytotoxic potential of DSA, leading to the idea that an assessment of antibody complement-binding capacity may be useful for diagnosis of AMR and for risk stratification of transplant recipients. Studies integrating the analysis of clinical, functional, histologic, and immunologic factors with graft loss, functionally investigated the clinical impact of complement-binding DSA using a sensitive detection method for C1q-binding anti-HLA antibodies in kidney transplant recipients.34,35 Patients with C1q-binding anti-HLA DSA display the lowest 5-y graft survival after transplantation, as compared with patients with non C1q-binding DSA and patients without anti-HLA DSA.34 Circulating C1q-binding anti-HLA DSA are strong and independent predictors of renal allograft AMR.35 The presence of C3d-binding DSA at the time of AMR is another strong independent predictor of allograft loss.36 C3d is a cleavage product of C3 positioned downstream of C1q in the complement cascade. Thus, C1q, C3d, and C4d do not provide equivalent predictive information. C1q and C3d testing may help identify patients at risk, despite a lack of C4d staining. The clinical relevance of complement-activating anti-HLA DSA across all solid organ (kidney, liver, heart, and lung) transplant patients was confirmed in a meta-analysis of 37 studies on 7936 patients.37 This study provides evidence that circulating complement-activating anti-HLA DSA are a major determinant of long-term allograft rejection and allograft failure and a possible biomarker for the prevention and treatment of AMR.
Fc Gamma Receptors
In addition to their role in binding antigen and C1q, antibodies can regulate immune responses through interacting with Fc gamma receptors (FcγRs). FcγRs are broadly expressed by hematopoietic cells and consist of several activating and 1 inhibitory receptors that differ in their affinity and specificity for IgG subclasses. The pattern of FcγRs expressed on innate effector cells determines a threshold for cell activation by immune complexes that regulate phagocytosis, antibody-dependent cell cytotoxicity (ADCC) and release of inflammatory mediators such as TNF and IFNγ. Classical FcγRs include an inhibitory receptor (FcγRIIb) and several activating receptors (FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb). The complexity in the FcγR family is mirrored by the presence of the 4 different IgG subclasses in humans (IgG1–IgG4), which bind with varying affinity and specificity to different FcγRs. FcγRI (CD64) is expressed on macrophages and neutrophils and mediates phagocytosis of target cells. FcγRI is the only known high-affinity receptor in humans; it binds IgG1 and IgG3 in humans with an affinity of 108–109 M, while other receptors (type II, FcγRII, or CD32, and type III, FcγRIII, or CD16) have lower affinity and exhibit less IgG subclass specificity. The FcγRII (CD32) class comprises activating low affinity FcγRIIa (which binds human IgG1, IgG2, and IgG3) and inhibitory FcγRIIb (which recognizes human IgG1 and IgG3 with low affinity) and may attenuate signaling from activating receptors if engaged, as does FcγRI. IgG4 binds to the activating receptors with a low affinity but displays similar or even higher affinity for the inhibitory FcγRIIb than other subclasses suggesting that IgG4 may have unique immunoregulatory properties. Most activating FcγRs associate with an intracellular immunoreceptor tyrosine-based activation motif (ITAM), which is either found directly in the cytoplasmic domain (FcγRIIa and FcγRIIc) or through the associated FcRγ-chain (FcγRIa and FcγRIIIa). The exceptions are FcγRIIIb, which is glycosylphosphatidylinositol (GPI)-linked, and the inhibitory FcγRIIb, which has an immunoreceptor tyrosine-based inhibition motif (ITIM). All FcγR-expressing cells, except NK cells, are tightly controlled by the balance between activating and inhibitory FcγRs.38
FcRn: pH-dependent IgG Recycling and Transcytosis
Although not involved in complement activation or cytolysis of target cells, the neonatal Fc receptor (FcRn), an atypical FcR, plays an important role in IgG biology. FcRn functions as a recycling or transcytosis receptor that is responsible for maintaining IgG and also albumin in the circulation.39 FcRn structurally and functionally differs from the classical FcγRs. Similar to MHC-I, the FcRn heavy chain consists of 3 extracellular domains (α1, α2, and α3), a transmembrane domain and a cytoplasmic tail that bind to β-2-microglobulin (β2m) but is unable to present antigenic peptides to T cells.40 Functions of FcRn are pH dependent and account for the very long half-life of human IgG and albumin in serum of around 19–21 d compared with only hours or a few days for most circulating proteins. FcRn is intracellular and predominantly located within acidified endosomes, where the low pH (5.0–6.0) allows binding of IgG. FcRn then recycles its IgG to the cell surface for release into the circulation upon exposure to the neutral pH of the blood. Although FcRn displays a ubiquitous expression pattern, FcRn is expressed in functionally active form in ECs, indicating that ECs are a possible site at which serum IgG homeostasis is maintained.41 The kidney is one of the important FcRn sites in the body together with the liver. FcRn is highly expressed on renal ECs, podocytes, and proximal tubular epithelial cells. FcRn is emerging as a promising target to significantly reduce the half-life of pathogenic antibodies such as DSA or to extend the current half-life of therapeutic antibodies.42 Effective approaches to treat IgG-mediated autoimmune diseases by blocking FcRn/IgG recycling have been reported in autoimmune arthritis mice43 and in myasthenia gravis in humans.44 The therapeutic saturation of FcRn by high-dose IVIg ameliorated arthritis43 and has been proposed to reduce pathogenic alloantibodies in solid organ transplantation.42
IgG-INDEPENDENT EFFECTORS OF THE COMPLEMENT SYSTEM
There are other mechanisms by which complement can mediate EC injury or death. In addition to expressing FcγRs, NK cells, and macrophages express the complement receptors CR3 and CR445 that bind to the C3 degradation fragments (iC3b, C3dg, and C3d).46 Until now, CR3 and CR4 were thought to have only a minor role, by acting mostly as an enhancer for FcγR-mediated ADCC.47 However, Lee et al showed that CR3 and CR4 can mediate the killing of cells by NK cells and macrophages independently of FcγR by a mechanism of complement-dependent cell-mediated cytotoxicity (CDCC).48
The complement system also directly establishes multiple interactions with the ECs leading to expression of adhesion molecules,49 leukocyte mobilization, monocyte adhesion,50 secretion of proinflammatory cytokines, and chemokines: IL-8, monocyte chemoattractant protein-1 (MCP-1), and IL-6.51,52 C1q can target ECs by binding to the receptors for the collagen-like tail (cC1qR) and the globular head of the molecule (gC1qR) as well as to other ligands such as heparan sulfate. As a result, C1q induces adhesion and spreading of ECs, stimulates EC permeability, proliferation and migration,53 recognition, and clearance of apoptotic ECs.54 Molecular networks involving C1q interactions with cells are emerging as mechanisms involved in modulation of immunity by C1q.55,56 C1q is produced by ECs and interacts with specific receptors on the EC surface57 to promote angiogenesis and vascular repair.
The small cationic peptides, C3a and C5a, also called anaphylatoxins, induce chemotaxis, cell activation, and inflammatory signaling by binding to their respective G-protein-coupled receptors (GPCR) C3aR and C5aR1. In human monocytes/macrophages, engagement of Ca3R or C5aR induces secretion of proinflammatory cytokines such as IL-1β, IL-6, and TNF, while C5a serves as a chemoattractant for monocytes. Activation of endothelium by C3aR and C5aR requires a preliminary stimulation of the endothelium by IL-1α. Similarly, insertion of the MAC into endothelium induces procoagulant and proinflammatory changes typical of EC activation. This effect is not direct but is mediated, at least in part, by the transcription, production, and secretion by ECs of IL-1α.58
NK CELL RECOGNITION AND INTERACTIONS WITH ALLOGENEIC ENDOTHELIAL CELLS
NK Cells: Definition and Functions
NK cells comprise 5%–15% of all circulating lymphocytes, and they represent an integral part of cellular innate immunity.59-61 NK cells are defined by the expression of the cell adhesion marker CD56 and by the lack of the T-cell receptor CD3 (CD56+CD3−). They can be divided into 2 functionally distinct subsets, CD56dim CD16+ and CD56bright CD16−, which possess different effector functions. The CD56dim CD16+ subset (90% of all blood NK cells) mediates an early response via direct cellular cytotoxicity and cytokine production. The CD56bright CD16− subset mediates a late but sustained effector function via potent proinflammatory cytokine and chemokine release of mainly IFN-γ but is poorly cytotoxic.62 These effector functions are tightly regulated through a large panel of activating and inhibitory receptors.63 Activating NK cell receptors generally recognize stress ligands on cells, whereas inhibitory receptors mainly interact with HLA class I molecules expressed by host cells, thereby preventing autoreactivity of NK cells. Following activation, NK cells also produce and secrete chemokines and cytokines that play a key role in boosting inflammatory responses and in priming other cells of the immune system such as dendritic cells or macrophages thus linking innate with adaptive immunity.
Various data have established that NK cells play a central role in the pathophysiology of AMR and graft failure after kidney transplantation.64 First, NK cell transcripts were found to be associated with AMR.1,8,65 The presence of infiltrating NK cells was subsequently demonstrated in transplant biopsies and correlated with graft function and outcome. NK cell infiltration was validated as a discriminating feature of AMR in renal transplantation, and specifically associated with glomerulitis and peritubular capillaritis.66 On kidney transplant biopsies, activated NK cell infiltration was recently reported as the best predictor of graft failure among all other immune cell subtypes and even outperformed a histologic diagnosis of acute rejection.66 In humans, NK cells have also been associated with lung allograft injury.67,68
NK Effector Molecules
One major characteristic of NK cells is their ability to respond immediately through the polarized delivery of apoptosis-inducing enzymes after formation of a lytic synapse between the NK cell and a target cell.69 Mechanistically, NK cells can kill target cells via the directed release of lytic granules or by inducing death receptor-mediated apoptosis via the expression of Fas ligand or TRAIL.70 The secretory granules contain the cytolytic proteins granulysin, perforin, and the serine protease family of proteins called granzymes. Perforin and granulysin are pore-forming proteins, which induce membrane permeability through independent mechanisms. Perforin is present in the granules of NK and CD8+ T cells. In contrast to T cells, NK cells constitutively express perforin. Perforin is a glycoprotein with a hydrophobic domain that is able to insert into cholesterol containing membranes and in conjunction with 20 other perforin molecules, generates a pore in the membrane. In contrast, granulysin binds to and preferentially permeabilizes negatively charged, cholesterol-free membranes.71 Among granzymes, granzyme A and B are the most abundant and trigger the induction of cell apoptosis via caspase-dependent and -independent pathways.72 Granzyme B plays an important role in cytotoxicity and mediates more rapid cytolysis than receptor-mediated death pathways.73
Donor/recipient Missing Self and NK Activation in Transplantation
NK cell activation relies on the ability to distinguish “self” from “non-self” via the recognition of MHC class I molecules by a large family of iNKRs.74 The interaction of these receptors with specific haplotypes of HLA class I molecules on target cell surface are key in determining NK cell alloreactivity against “non-self” leading to the “missing-self” hypothesis.75 The recognition of “self” is mediated mainly by 2 classes of receptors: the Killer Immunoglobulin-like Receptors (KIRs) and the C-type lectin receptors.76,77 KIRs are extremely polymorphic receptors able to distinguish among different HLA-A, -B, and -C allotypes.78 Activating and inhibitory KIRs are highly homologous in the extracellular domain, but they differ in the cytoplasmic domain. Inhibitory KIRs are characterized by a long cytoplasmic tail containing the ITIM, while the short intracellular domain of activating KIRs interacts with the adaptor signaling molecule DAP12, carrying the ITAM domain.79 C-type lectin HLA-I-specific receptors are represented by the CD94/NKG2 heterodimers of the C-type lectin receptor family: CD94/NKG2A is the inhibitory receptor and it contains an ITIM in the cytoplasmic domain, while, similar to activating KIRs, CD94/NKG2C is the activating receptor that lacks ITIM and it is associated to ITAM-containing DAP12 adaptor molecule.76 CD94/NKG2A/C receptors interact with the nonclassical HLA class I molecules HLA-E.80 HLA-E is highly expressed by ECs and upregulated upon inflammation in response to TNF and IFNγ.81 The inhibitory receptor CD94/NKG2A shows a higher binding affinity for HLA-E than CD94/NKG2C,82 and via its ligand HLA-E acts as a sensor to assess the net overall expression of HLA-I molecules on a target cell. Exposure to human cytomegalovirus (HCMV) induces the emergence of an NK cell subpopulation coexpressing the activating receptor CD94/NKG2C and CD57 that is referred to as “adaptive NK cells.”83,84 HLA-E is necessary for the expansion of NKG2C+CD57+ adaptive NK cells in vitro.85 Functionally, adaptive NK cell populations display lower cytotoxic activity but a higher capability for mediating ADCC. ADCC responses are regulated in a HLA-E-restricted and peptide-specific manner via the activating NK cell receptor CD94/NKG2C.84 The role of adaptive NK cells, in particular those induced in response to HCMV infection, remains to be evaluated in the context of AMR.
NK cell alloreactivity relies on the pattern of iNKRs expressed on NK cell surface and on the strength of their binding with donor HLA-I complex. The implication of missing self in transplant rejection has been investigated only recently (Figure 2). HLA-KIR mismatch and NK cell alloreactivity have been reported to promote transplant survival through the selective killing of antigen presenting cells in mismatched hematopoietic transplants86 and in lung allografts.87 Koenig et al showed that histological lesions associated with microvascular inflammation in AMR in kidney transplant recipients were not due to DSA in 50% of grafts. The genetic analysis of these patients showed a higher prevalence of mismatches between donor HLA-I and recipient inhibitory KIRs supporting a role for missing self in microvascular inflammation and injury.88 This study demonstrated that the inability of graft ECs to provide HLA I-mediated inhibitory signals to recipient circulating NK cells triggers both NK activation and promotes NK-mediated endothelial and vascular damage. Thus missing self may trigger microvascular injury and allograft rejection. This study also established that missing self-induced NK cell activation is mTORC1-dependent and that rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), can prevent the development of this type of vascular rejection.88
Contribution of NK Cells to Vascular Injury Through ADCC
The ability of DSA to induce ADCC relies on the bifunctional format of IgG antibodies. After the antigen-recognizing fragment (Fab) binds to the donor antigens on graft ECs, the IgG-Fc domain is free to interact with FcγR expressed on NK cells (Figure 2).89 Unlike other effector cells, human NK cells express only activating FcγR (FcγRIIIa, also known as CD16a, and FcγRIIc, also known as CD32c) and do not coexpress an inhibitory FcγR, suggesting that they may be the predominant effector cells in ADCC.
NK cells localize in the microcirculation in AMR and have been postulated to be activated by anti-HLA DSAs triggering their CD16a Fc receptors. This was confirmed by gene expression analyses suggesting that ADCC plays a role in acute AMR.65 Functional assessment of DSA and NK triggering of ADCC was used to demonstrate that enhanced ADCC responsiveness associates with reduced graft function and could be used as a predictive factor of kidney transplant outcome.90 However, direct evidence for NK cell CD16a triggering in AMR was established only recently using molecular analyses. Parkes et al identified CD16a-inducible transcripts in NK cells and their expression was subsequently studied in human kidney transplant biopsies with AMR.91 This study shows that CD16a signaling upregulates a large panel of transcripts in human NK cells such as those encoding the cytokines (IFNγ, TNF), GM-CSF, chemokines (CCL3, CCL4, and XCL1) and modulators of NK cell effector functions (4-1BB, CRTAM, CD160). Investigations on biopsies confirmed that CD160 and XCL1 mRNAs were likely to be selective for NK cells in AMR together with CCL4, CCL3, CRTAM, FCRL3, STARD4, 4-1BB, CD16a-inducible transcripts highly associated with AMR. Transcriptome analyses of kidney transplant biopsies during AMR highlighted the upregulation of FcγRIIIa further supporting a role for ADCC in AMR.92
ADCC and CD16-related Signaling Pathway
CD16a (FCγRIIIa) binds to the Fc portion of IgG antibodies. CD16a is a transmembrane protein that colocalizes with CD3ζ (CD247) and Fc-εRI-γ that are both basally expressed on NK cells.93 Upon ligation, it induces a potent series of signals resulting in cytokine production and cytotoxic effector activity via ADCC.94 The second type of FCγRIIIb, CD16b, is only found on neutrophils. Most CD56bright NK cells in the peripheral blood express no or only slight CD16a. In contrast, most CD56dim NK cells uniformly express high levels of CD16a. The signaling of CD16 follows the classical ITAM pathway of Src-family kinase-mediated tyrosine phosphorylation, Syk association leading to PI3K, Vav1, and PLC-γ1 and PLC-γ2 activation.95 Signaling for ADCC involves a PI3K-dependent activation of the ADP-ribosylation factor (Arf)-6, which couples CD16 to the production of phosphatidylinositol-4,5-bisphosphate (PIP2) by a phosphatidylinositol-4-phosphate 5-kinase (PI5K) and the activation of phospholipase D.96 PI3K–Rac1–PAK1–MEK–ERK signaling is required for polarization and cytotoxicity of NK cells.97,98 PI5K activity in human NK cells is needed for degranulation but not for granule polarization.99 Exocytosis of lytic granules in NK cells is strongly dependent on PLC-γ phosphorylation and subsequent mobilization of Ca2+ from the endoplasmic reticulum.100 In contrast to other ITAM-coupled NK cell receptors, triggering of CD16 is by itself sufficient to activate cytotoxicity and cytokine production in resting human NK cells.101 Nevertheless, the coactivating receptor NKG2D also plays a role in the dynamic of cell target engagement and lysis by NK cells. After stimulation, a downregulation of CD16a occurs as a result of shedding by the metalloprotease ADAM17.102 This process is important for the rapid turnover of CD16 expression and for the sustained effector function of NK cells which is required for the serial killing of multiple target cells. Repeated activation via CD16 decreases the amount of perforin secreted. However, perforin secretion was restored upon subsequent activation via a different activating receptor, NKG2D. Repeated stimulation via NKG2D also decreases perforin secretion, but this was not rescued by stimulation via CD16.103 Srpan et al further demonstrated that CD16 shedding also increases NK cell motility and facilitates detachment of NK cells from target cells. Disassembly of the immune synapse caused by CD16 shedding aided NK cell survival and boosted serial engagement of target cells.103
NKG2D (CD314) is an important coactivating receptor expressed by NK and T cells.104,105 Human NKG2D associates with the adaptor protein DAP10, which carries a PI3K binding motif to carry its signaling. DAP10 also binds Grb2, which associates with Vav1. All 3 of these molecules are required to mediate the full signaling of NKG2D.106 Engagement of NKG2D stimulates the production of cytokines and cytotoxic molecules. NKG2D is a master regulator of activation thresholds for various receptors. In human NK cells, NKG2D promotes CD16 signaling and ADCC, since blocking of NKG2D receptors results in a reduced ability of NK cells to mediate ADCC.107
Human ECs constitutively express low levels of the NKG2D ligands, MICA, MICB, ULBP-1, -2, and -3 and their expression is regulated upon stimulation with IFNγ or TNF.108,109 MICA genetic variants such as MICA A5.1 (rs9279200) and MICA-129Val/Met dimorphism, caused by a SNP (rs1051792) may also contribute to alloimmune responses by altering the expression of MICA on graft ECs, the release of the circulating forms of MICA, anti-MICA antibodies and also affect NKG2D binding.110-112 MICA-129Met allelic as well as Met/Met genotypic frequencies dominated in recipients experiencing AMR.113 This effect could be the consequence of an altered NKG2D signaling by MICA-129Met variant.112
Polymorphism and Glycosylation of FcyRs
The CD16a encoding gene FCGR3A bears a SNP (rs396991) resulting in an amino acid change at position 158 of phenylalanine (Phe) to valine (Val). Human IgG1 binds more efficiently to NK cells expressing the FcγRIIIa-158Val than to the FcγRIIIa-158Phe proteins.114FCGR3A polymorphism does not affect the level of expression on NK cells but rather increases the affinity of IgG to FcγRIIIa-158Val.115 The adequate concentration of rituximab to achieve 50% of cell lysis has been shown to be significantly lower in FcγRIIIa-158 V/V donors compared with FcγRIIIa-158Phe/Phe donors.116 However, the clinical impact of the FCGR3A polymorphism remains controversial in some studies.117 In cardiac allografts, patients with an FcγRIIIa-158 V/V genotype had an enhanced FcγRIIIa expression and a higher risk of developing vasculopathy and rejection.118 Posttranslational modifications such as asparagine (N)-linked-glycosylation also cause a variability in protein composition and impact the function of CD16a and CD56 on NK cells. Changes in N-glycans at N162 can increase affinity by up to 50 fold while N45 glycosylation contributes to affinity by stabilizing CD16a structure.119 IgG, like CD16a, can be modified. N-glycosylation of the IgG Fc is required for receptor binding and defects in fucosylation of the core N-acetylglucosamine (GlcNAc) residue increases the affinity for CD16a.
MACROPHAGES-ENDOTHELIUM CROSSTALK IN TRANSPLANTATION
Macrophage Infiltrate and Transplant Rejection
The monocyte/macrophage cell lineage is increasingly recognized as a major player in acute and chronic allograft immunopathology.120,121 Both acute and chronic AMR are characterized among others by an accumulation of monocyte/macrophage transcripts92 and cells.122 The quantity of interstitial macrophages was identified as a predictive factor for allograft loss in a group of AMR patients.123 Similarly, macrophage density in early surveillance biopsies had a predictive value for allograft loss.124 Monocytes and macrophages contribute to alloimmunity via diverse pathways such as antigen processing and presentation, costimulation, and proinflammatory cytokine production. Interaction between monocytes/macrophages and the endothelium is critical for the trafficking and homing of macrophages, as well as for activation and recruitment of other inflammatory cells to specific tissue sites.125 Macrophages have also been showed to regulate vessel permeability and repair. Recent evidence also supports the concept that the crosstalk between vascular ECs and macrophages impacts not only EC function and integrity but also the differentiation of monocytes and polarization of macrophages (Figure 3).
Macrophages Phenotypic Diversity
Macrophages exhibit a wide and dynamic range of phenotypes and functions including the M1 and M2 stages that can be considered as extremes on a continuum of macrophage stages.126,127 M1 macrophages express surface markers: MHCII, CD40, CD80, CD86, and CD11b. They can produce inflammatory cytokines and chemokines such as TNF-α, IL-1, IL-6, IL-8, IL-12, CCL2, CXCL9, and CXCL10. M2 macrophages show immunomodulatory functions and express surface markers: CD163, CD206, and CD209. M2 macrophages produce IL-10 and TGF-β mainly leading to tissue repair and angiogenesis. They can be subdivided into M2a, M2b, M2c, and M2d. M2a macrophages are generated in response to IL-4 and IL-13. Immune complexes and toll-like receptor (TLR)/IL-1R ligands activate M2b macrophages, whereas M2c macrophages are activated by IL-10, TGF-β, and glucocorticoids. Regulatory macrophages (M2d or Mregs) express several molecules such as MHCII, FCγR, IFNγR, TLR-4, and PD-L1 and have potent T-cell suppressive function. Plasticity and flexibility are key features of macrophages and the phenotype of polarized M1/M2 cells can, to some extent, be reversed.128 Functionally, inflammation and resolution of the inflammation, tissue, and vascular remodeling and repair occur dynamically during AMR, and these processes are orchestrated by macrophages. Data concerning the differentiation state of graft infiltrating macrophages or the dynamics of macrophage polarization postgraft still remain rare. The M1 and M2 profiles of the inflammatory infiltrates in diagnostic biopsies were only recently investigated in kidney transplants and suggest that changes may happen in M1/M2 distribution according to nature of rejection and time posttransplantation and could be markers of successful anti-rejection therapy.129
Macrophages and EC Activation
M1 production of inflammatory cytokines such as TNF and IL-1β is a key player in EC activation defined by coordinated changes in endothelial function that promote coagulation, inflammation, vasoconstriction, and also alloantigen presentation.130 After binding to their respective receptors, TNF and IL-1β activate the transient transcriptional activation of a large variety of procoagulant and proinflammatory mediators in ECs. ECs may increase platelet adhesion to the vessel wall through release of preformed von Willebrand factor polymers. Damaged ECs initiate coagulation by expression of tissue factor and by delivery of microparticles that serve for assembly of coagulation factors. Activated ECs may lose their capacities to inhibit coagulation and platelet activation leading to microvascular thrombosis. Leukocyte recruitment is limited under basal conditions because EC–leukocyte interaction is prevented by absent or limited expression of leukocyte-binding adhesion molecules such as E-selectin (CD62E), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). In response to localized inflammation, ECs transiently increase their surface expression of adhesion molecules and express chemokines to locally recruit leucocytes. The activation of NF-κB, but also of PI3K and MAPK pathways, play a role in the tightly controlled induction of the proinflammatory genes that are a feature of the activated EC phenotype and functions but also the return to a resting state.130
Bidirectional Relationship Between Vascular ECs and Macrophages for Differentiation
ECs Promote Macrophage Polarization
In human cardiac transplants microvascular inflammation and macrophage infiltration are key features and histological markers of AMR, which correlate with alterations in Notch signaling pathway at the endothelium/macrophage interface. These changes include a reduced expression of the receptor Notch4 on ECs and the acquisition of the Notch ligand Delta like-4 (DLL4) in both ECs and macrophages.122 The endothelial expression of DLL4 allows circulating monocytes to polarize into a M1 proinflammatory fate (CD40highCD64highCD200Rlow HLA-DRlowCD11blow) eliciting the production of IL-6. The expression of DLL4 and IL-6 is Notch-dependent and is required for macrophage polarization through a selective down and upregulation of M2- and M1-type markers, respectively. These findings suggest that upon AMR the graft endothelium may contribute to macrophage recruitment and differentiation via Notch signaling.122 DLL4 expressed on activated ECs in response to inflammation also selectively impairs the polarization and induces a caspase3/7-dependent apoptosis of M2 macrophages.131 Thus, these findings indicate that targeting the Notch signaling pathway could be helpful to manipulate and change the phenotype and functions of macrophages according to the pathological context.
Vascular remodeling and repair occur dynamically during inflammation, and these processes are orchestrated by macrophages. The diversity of macrophage phenotypes impacts their interaction and functional relationship with the endothelium. In particular, M1 macrophages have angiostatic properties and do not necessarily associate with vessels. In contrast, M2 macrophages tightly associate with ECs and promote angiogenesis. In this context, the findings that ECs promote the selective growth and differentiation of macrophages, especially the switch towards an M2-like phenotype, are consistent with a productive and symbiotic relationship between these 2 cell types.122,131-133 EC-induced M2 polarization requires direct contact with the endothelium and the regulation of various signaling pathways.132 Consistent with the concept that tissue-resident macrophages proliferate locally134 endothelial-induced M2-like macrophages also have proliferative capacity.132 Perivascular M2 macrophages provide a direct contribution to vessel barrier integrity by controlling vascular permeability via interaction with pericytes and ECs.135
Endothelial-to-mesenchymal transition (EndMT) is a complex biological process whereby ECs lose their specific endothelial biomarkers, such as VE-cadherin, von Willebrand factor, CD31, endothelial nitric oxide synthase NOS3, and acquire a mesenchymal or myofibroblastic phenotype and consequently express mesenchymal cell markers, including α-smooth muscle actin, collagen I, collagen III, fibronectin, calponin, SM22α, and versican.136 At molecular level, the TGF-β signaling network is considered a master regulator of EndMT.137 Active TGF-β signals through a canonical (Smad-mediated) pathway.138 TNF, IL-1β, and IFN-γ are also potent cytokines produced by the M1 macrophages that induce in vitro EndMT.139
EndMT plays a crucial role in the pathogenesis of cardiac fibrosis.140 EndMT also plays an important role in renal interstitial fibrosis that occurs in chronic kidney diseases as well as in the development and progression of pulmonary fibrosis, hepatic fibrosis, intestinal fibrosis, and wound healing.141,142 Wang et al showed that EndMT plays an important role in kidney interstitial fibrosis during the development of chronic allograft dysfunction through the TGF-β/Smad and Akt/mTOR/p70S6K signaling pathways in kidney transplant recipients.143 Downstream Akt, p70S6K regulates protein synthesis and proliferation and could be blocked by its upstream kinase mTOR. Thus EndMT-induced transplant renal interstitial fibrosis could therefore be a potential target for development and use of antifibrosis agents such as inhibitors of mTOR.144 Xu-Dubois et al have shown that 3 mesenchymal proteins, fascin1, vimentin, and hsp47, are upregulated in ECs from peritubular capillaries during AMR suggesting that EndMT is a feature of AMR.145 The expression of these EndMT markers in renal grafts was associated with the presence of DSA and predicts poor graft outcome. Consequently, detection of EndMT markers was proposed as a reliable diagnostic tool for detecting EC injury that may help to consolidate the diagnosis of AMR in renal grafts and to predict loss of allograft function. This study suggested a correlation of EndMT with the duration of ischemia reperfusion and the presence of DSA supporting a role for hypoxia and alloantibody binding to graft ECs.145
Pharmacological modulation of the signaling pathways underlying EndMT, such as inhibition of TGF-β signaling have been tested, with variable degrees of success, for their ability to inhibit EndMT.138,146 Interestingly, the changes in endothelial differentiation status and cell behavior during EndMT are illustrative of their inherent plasticity since their ability to transition is reversible (ie, mesenchymal-to-endothelial transition). Recently, RNA sequencing provided new insights into EC plasticity, which is the ability of an EC to switch its identity, including to additional phenotypes other than mesenchymal cells and also, having changed identity, to revert back to an EC state.147 Interestingly, EndMT could also be used in a different manner, wherein ECs may be exploited to derive multipotent mesenchymal stem cells, which can be readily redifferentiated into various distinct cell types148 for regenerative medicine.146
Re-endothelialization and Macrophages
Despite extensive experience with engineering vascular grafts, the mechanism of endothelialization remains unknown. Andreadis’s laboratory successfully developed an acellular vascular graft model with immobilized heparin and vascular endothelial growth factor (VEGF) on the graft lumen to capture VEGF receptor expressing cells from the blood. After implantation, the VEGF grafts were fully endothelialized and functional within 1 mo.149 More recently, these authors showed that the cells populating the lumen of grafts bearing immobilized VEGF were mostly VEGFR1-expressing monocytes that display a M2 phenotype (CD14+/ CD163+) and which differentiate into ECs in a timely manner. This mechanism supports a cellular plasticity between monocytes and ECs consistent with their common developmental origin.150 Mechanistically, shear stress and the Wnt pathway150 are both involved in monocyte-to-EC transition in these models.151 Therefore, re-endothelialization by an abundant cell population in the blood such as monocytes may be critical for the success of vascular grafts and perhaps also transplants. These findings suggest that monocytes may be even more plastic than originally thought, depending upon the microenvironment. Consequently it has been proposed that the multiple macrophage identities regrouped under the “M2” denomination, such as the already well established M2a, M2b, M2c, and M2d152-154 may also include an “EC-committed M2 type or M2-EC or M2e” that can differentiate into an EC for vascular repair after injury when re-endothelialization is needed. Recruitment, polarization, and activation of M2e depend on signals mediated by VEGF and WNT. Thus, dedicated sorting and programming of circulating monocytes could open the way to novel strategies for acellular graft endothelialization but also transplant re-endothelialization. Indeed, it has been postulated that endothelium that is damaged upon vascular rejection is repaired by cells from the recipient.155
Graft EC activation and injury are central to the pathogenesis of AMR. Although the usual suspects (DSA, complement, inflammation, NK cells, and macrophages) have been identified, their respective contributions to the different cellular and molecular mechanisms to AMR still remain difficult to establish. Qualitative and quantitative changes in the subclass of DSA IgG posttransplantation are most probably a crucial determinant for the local stoichiometry of DSA:complement and also for the nature of effector function (ie, complement-dependent cytotoxicity or ADCC). The role of NK cell activation mediated by CD16 signaling does not exclude the contribution of other cytotoxic pathways such as NK direct cytotoxicity resulting from missing self. Most probably, these effector mechanisms may coexist and may occur sequentially in the graft upon AMR depending on the subclasses IgG DSA (IgG1 and IgG3) and their relative affinity for CD16a and C1q and on MHC mismatch between transplant donor and recipient. The interactions between ECs and macrophages are probably more complex than initially thought. Beyond codriving inflammatory processes, both cells may engage bidirectional interplays affecting their differentiation involved in processes such as EndMT and re-endothelialization. In this context, the contribution of donor versus recipient as well as the dynamics of EC repopulation of the graft after vascular injury caused by AMR still remain unknown. These cellular crosstalks between ECs, NK cells, and macrophages are modulated by several processes including gene polymorphisms (IgG, FcγRs, NKG2DLs, HLA-E) but also N-glycosylation and fucosylation. A better understanding of the phenotype, the plasticity, and the dynamics of infiltrating NK cells and macrophages in the setting of organ transplantation may also allow the identification of new biomarkers and potential therapeutic targets.
The figures were drawn using biorender.com. We thank Jenny Greig for proofreading the article.
1. Sellarés J, Reeve J, Loupy A, et al. Molecular diagnosis of antibody-mediated rejection in human kidney transplants. Am J Transplant. 2013;13:971–983.
2. Montgomery RA, Loupy A, Segev DL. Antibody-mediated rejection: new approaches in prevention and management. Am J Transplant. 2018;18(Suppl 3):3–17.
3. Jin YP, Valenzuela NM, Zhang X, et al. HLA class II-triggered signaling cascades cause endothelial cell proliferation and migration: relevance to antibody-mediated transplant rejection. J Immunol. 2018;200:2372–2390.
4. Jackson AM, Sigdel TK, Delville M, et al. Endothelial cell antibodies associated with novel targets and increased rejection. J Am Soc Nephrol. 2015;26:1161–1171.
5. Delville M, Lamarthée B, Pagie S, et al. Early acute microvascular kidney transplant rejection in the absence of anti-HLA antibodies is associated with preformed IgG antibodies against diverse glomerular endothelial cell antigens. J Am Soc Nephrol. 2019;30:692–709.
6. Wehmeier C, Karahan GE, Krop J, et al.; Swiss Transplant Cohort Study. Donor-specific B cell memory in alloimmunized kidney transplant recipients: first clinical application of a novel method. Transplantation. 2020;104:1026–1032.
7. Sis B, Jhangri GS, Bunnag S, et al. Endothelial gene expression in kidney transplants with alloantibody indicates antibody-mediated damage despite lack of C4d staining. Am J Transplant. 2009;9:2312–2323.
8. Hidalgo LG, Sis B, Sellares J, et al. NK cell transcripts and NK cells in kidney biopsies from patients with donor-specific antibodies: evidence for NK cell involvement in antibody-mediated rejection. Am J Transplant. 2010;10:1812–1822.
9. Vitalone MJ, Sigdel TK, Salomonis N, et al. Transcriptional perturbations in graft rejection. Transplantation. 2015;99:1882–1893.
10. Loupy A, Duong Van Huyen JP, Hidalgo L, et al. Gene expression profiling for the identification and classification of antibody-mediated heart rejection. Circulation. 2017;135:917–935.
11. Lefaucheur C, Loupy A. Antibody-mediated rejection of solid-organ allografts. N Engl J Med. 2018;379:2580–2582.
12. Zhang X, Reed EF. Effect of antibodies on endothelium. Am J Transplant. 2009;9:2459–2465.
13. Tsai EW, Reed EF. MHC class I signaling: new functional perspectives for an old molecule. Tissue Antigens. 2014;83:375–381.
14. Heinemann FM, Roth I, Rebmann V, et al. Characterization of anti-HLA antibodies eluted from explanted renal allografts. Clin Transpl. 2006:371–378.
15. Lefaucheur C, Viglietti D, Bentlejewski C, et al. IgG donor-specific anti-human HLA antibody subclasses and kidney allograft antibody-mediated injury. J Am Soc Nephrol. 2016;27:293–304.
16. Griffiths EJ, Nelson RE, Dupont PJ, et al. Skewing of pretransplant anti-HLA class I antibodies of immunoglobulin G isotype solely toward immunoglobulin G1 subclass is associated with poorer renal allograft survival. Transplantation. 2004;77:1771–1773.
17. Bartel G, Wahrmann M, Exner M, et al. Determinants of the complement-fixing ability of recipient presensitization against HLA antigens. Transplantation. 2007;83:727–733.
18. Arnold ML, Ntokou IS, Doxiadis II, et al. Donor-specific HLA antibodies: evaluating the risk for graft loss in renal transplant recipients with isotype switch from complement fixing IgG1/IgG3 to noncomplement fixing IgG2/IgG4 anti-HLA alloantibodies. Transpl Int. 2014;27:253–261.
19. McAlister VC. Anti-donor immunoglobulin G subclass in liver transplantation. Hepatobiliary Surg Nutr. 2019;8:125–128.
20. O’Leary JG, Demetris AJ, Friedman LS, et al. The role of donor-specific HLA alloantibodies in liver transplantation. Am J Transplant. 2014;14:779–787.
21. Wahrmann M, Exner M, Schillinger M, et al. Pivotal role of complement-fixing HLA alloantibodies in presensitized kidney allograft recipients. Am J Transplant. 2006;6:1033–1041.
22. Hönger G, Hopfer H, Arnold ML, et al. Pretransplant IgG subclasses of donor-specific human leukocyte antigen antibodies and development of antibody-mediated rejection. Transplantation. 2011;92:41–47.
23. Cicciarelli JC, Kasahara N, Lemp NA, et al. Immunoglobulin G subclass analysis of HLA donor specific antibodies in heart and renal transplant recipients. Clin Transpl. 2013:413–422. PMID: 25095537.
24. de Taeye SW, Rispens T, Vidarsson G. The ligands for human IgG and their effector functions. Antibodies (Basel). 2019;8:30.
25. Xu Y, Oomen R, Klein MH. Residue at position 331 in the IgG1 and IgG4 CH2 domains contributes to their differential ability to bind and activate complement. J Biol Chem. 1994;269:3469–3474.
26. Dunkelberger JR, Song WC. Complement and its role in innate and adaptive immune responses. Cell Res. 2010;20:34–50.
27. Patel R, Terasaki PI. Significance of the positive crossmatch test in kidney transplantation. N Engl J Med. 1969;280:735–739.
28. Feucht HE, Felber E, Gokel MJ, et al. Vascular deposition of complement-split products in kidney allografts with cell-mediated rejection. Clin Exp Immunol. 1991;86:464–470.
29. Collins AB, Schneeberger EE, Pascual MA, et al. Complement activation in acute humoral renal allograft rejection: diagnostic significance of C4d deposits in peritubular capillaries. J Am Soc Nephrol. 1999;10:2208–2214.
30. Böhmig GA, Exner M, Habicht A, et al. Capillary C4d deposition in kidney allografts: a specific marker of alloantibody-dependent graft injury. J Am Soc Nephrol. 2002;13:1091–1099.
31. Sis B, Kaplan B, Halloran PF. Histologic findings from positive crossmatch or ABO-incompatible renal allografts: accomodation or chronic allograft injury? Am J Transplant. 2006;6:1753–1754.
32. Haas M. Pathology of C4d-negative antibody-mediated rejection in renal allografts. Curr Opin Organ Transplant. 2013;18:319–326.
33. Haas M. Evolving criteria for the diagnosis of antibody-mediated rejection in renal allografts. Curr Opin Nephrol Hypertens. 2018;27:137–143.
34. Loupy A, Lefaucheur C, Vernerey D, et al. Complement-binding anti-HLA antibodies and kidney-allograft survival. N Engl J Med. 2013;369:1215–1226.
35. Viglietti D, Bouatou Y, Kheav VD, et al. Complement-binding anti-HLA antibodies are independent predictors of response to treatment in kidney recipients with antibody-mediated rejection. Kidney Int. 2018;94:773–787.
36. Sicard A, Ducreux S, Rabeyrin M, et al. Detection of C3d-binding donor-specific anti-HLA antibodies at diagnosis of humoral rejection predicts renal graft loss. J Am Soc Nephrol. 2015;26:457–467.
37. Bouquegneau A, Loheac C, Aubert O, et al. Complement-activating donor-specific anti-HLA antibodies and solid organ transplant survival: a systematic review and meta-analysis. Plos Med. 2018;15:e1002572.
38. Bryceson YT, March ME, Ljunggren HG, et al. Synergy among receptors on resting NK cells for the activation of natural cytotoxicity and cytokine secretion. Blood. 2006;107:159–166.
39. Pyzik M, Sand KMK, Hubbard JJ, et al. The neonatal Fc receptor (FcRn): a misnomer? Front Immunol. 2019;10:1540.
40. Roopenian DC, Akilesh S. FcRn: the neonatal Fc receptor comes of age. Nat Rev Immunol. 2007;7:715–725.
41. Borvak J, Richardson J, Medesan C, et al. Functional expression of the MHC class I-related receptor, FcRn, in endothelial cells of mice. Int Immunol. 1998;10:1289–1298.
42. Jordan SC, Ammerman N, Vo A. Implications of Fc neonatal receptor (FcRn) manipulations for transplant immunotherapeutics. Transplantation. 2020;104:17–23.
43. Akilesh S, Petkova S, Sproule TJ, et al. The MHC class I-like Fc receptor promotes humorally mediated autoimmune disease. J Clin Invest. 2004;113:1328–1333.
44. Howard JF Jr, Bril V, Burns TM, et al.; Efgartigimod MG Study Group. Randomized phase 2 study of FcRn antagonist efgartigimod in generalized myasthenia gravis. Neurology. 2019;92:e2661–e2673.
45. van der Poel CE, Carroll MC. Untangling Fc and complement receptors to kill tumors. Nat Immunol. 2017;18:874–875.
46. Grafals M, Thurman JM. The role of complement in organ transplantation. Front Immunol. 2019;10:2380.
47. Boross P, van Montfoort N, Stapels DA, et al. FcRγ-chain ITAM signaling is critically required for cross-presentation of soluble antibody-antigen complexes by dendritic cells. J Immunol. 2014;193:5506–5514.
48. Lee CH, Romain G, Yan W, et al. IgG Fc domains that bind C1q but not effector Fcγ receptors delineate the importance of complement-mediated effector functions. Nat Immunol. 2017;18:889–898.
49. Feng X, Tonnesen MG, Peerschke EI, et al. Cooperation of C1q receptors and integrins in C1q-mediated endothelial cell adhesion and spreading. J Immunol. 2002;168:2441–2448.
50. Valenzuela NM, Thomas KA, Mulder A, et al. Complement-mediated enhancement of monocyte adhesion to endothelial cells by HLA antibodies, and blockade by a specific inhibitor of the classical complement cascade, TNT003. Transplantation. 2017;101:1559–1572.
51. van den Berg RH, Faber-Krol MC, Sim RB, et al. The first subcomponent of complement, C1q, triggers the production of IL-8, IL-6, and monocyte chemoattractant peptide-1 by human umbilical vein endothelial cells. J Immunol. 1998;161:6924–6930.
52. Fischetti F, Tedesco F. Cross-talk between the complement system and endothelial cells in physiologic conditions and in vascular diseases. Autoimmunity. 2006;39:417–428.
53. Bossi F, Tripodo C, Rizzi L, et al. C1q as a unique player in angiogenesis with therapeutic implication in wound healing. Proc Natl Acad Sci U S A. 2014;111:4209–4214.
54. Navratil JS, Watkins SC, Wisnieski JJ, et al. The globular heads of C1q specifically recognize surface blebs of apoptotic vascular endothelial cells. J Immunol. 2001;166:3231–3239.
55. Son M, Diamond B, Santiago-Schwarz F. Fundamental role of C1q in autoimmunity and inflammation. Immunol Res. 2015;63:101–106.
56. Kouser L, Madhukaran SP, Shastri A, et al. Emerging and novel functions of complement protein C1q. Front Immunol. 2015;6:317.
57. Bulla R, Agostinis C, Bossi F, et al. Decidual endothelial cells express surface-bound C1q as a molecular bridge between endovascular trophoblast and decidual endothelium. Mol Immunol. 2008;45:2629–2640.
58. Brunn GJ, Saadi S, Platt JL. Differential regulation of endothelial cell activation by complement and interleukin 1alpha. Circ Res. 2006;98:793–800.
59. Lanier LL. NK cell recognition. Annu Rev Immunol. 2005;23:225–274.
60. Vivier E, Tomasello E, Baratin M, et al. Functions of natural killer cells. Nat Immunol. 2008;9:503–510.
61. Vivier E, Raulet DH, Moretta A, et al. Innate or adaptive immunity? The example of natural killer cells. Science. 2011;331:44–49.
62. De Maria A, Bozzano F, Cantoni C, et al. Revisiting human natural killer cell subset function revealed cytolytic CD56(dim)CD16+ NK cells as rapid producers of abundant IFN-gamma on activation. Proc Natl Acad Sci U S A. 2011;108:728–732.
63. Long EO, Kim HS, Liu D, et al. Controlling natural killer cell responses: integration of signals for activation and inhibition. Annu Rev Immunol. 2013;31:227–258.
64. Miyairi S, Baldwin WM 3rd, Valujskikh A, et al. Natural killer cells: critical effectors during antibody-mediated rejection of solid organ allografts. Transplantation. 2021;105:284–290.
65. Venner JM, Hidalgo LG, Famulski KS, et al. The molecular landscape of antibody-mediated kidney transplant rejection: evidence for NK involvement through CD16a Fc receptors. Am J Transplant. 2015;15:1336–1348.
66. Yazdani S, Callemeyn J, Gazut S, et al. Natural killer cell infiltration is discriminative for antibody-mediated rejection and predicts outcome after kidney transplantation. Kidney Int. 2019;95:188–198.
67. Fildes JE, Yonan N, Tunstall K, et al. Natural killer cells in peripheral blood and lung tissue are associated with chronic rejection after lung transplantation. J Heart Lung Transplant. 2008;27:203–207.
68. Calabrese DR, Lanier LL, Greenland JR. Natural killer cells in lung transplantation. Thorax. 2019;74:397–404.
69. Orange JS. Formation and function of the lytic NK-cell immunological synapse. Nat Rev Immunol. 2008;8:713–725.
70. Prager I, Watzl C. Mechanisms of natural killer cell-mediated cellular cytotoxicity. J Leukoc Biol. 2019;105:1319–1329.
71. Barman H, Walch M, Latinovic-Golic S, et al. Cholesterol in negatively charged lipid bilayers modulates the effect of the antimicrobial protein granulysin. J Membr Biol. 2006;212:29–39.
72. Andrade F, Roy S, Nicholson D, et al. Granzyme B directly and efficiently cleaves several downstream caspase substrates: implications for CTL-induced apoptosis. Immunity. 1998;8:451–460.
73. Ewen CL, Kane KP, Bleackley RC. A quarter century of granzymes. Cell Death Differ. 2012;19:28–35.
74. Lanier LL. NK cell receptors. Annu Rev Immunol. 1998;16:359–393.
75. Kim S, Sunwoo JB, Yang L, et al. HLA alleles determine differences in human natural killer cell responsiveness and potency. Proc Natl Acad Sci U S A. 2008;105:3053–3058.
76. Lanier LL, Corliss B, Wu J, et al. Association of DAP12 with activating CD94/NKG2C NK cell receptors. Immunity. 1998;8:693–701.
77. Cerwenka A, Lanier LL. Ligands for natural killer cell receptors: redundancy or specificity. Immunol Rev. 2001;181:158–169.
78. Middleton D, Gonzelez F. The extensive polymorphism of KIR genes. Immunology. 2010;129:8–19.
79. Takaki R, Watson SR, Lanier LL. DAP12: an adapter protein with dual functionality. Immunol Rev. 2006;214:118–129.
80. Braud VM, Allan DS, O’Callaghan CA, et al. HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature. 1998;391:795–799.
81. Coupel S, Moreau A, Hamidou M, et al. Expression and release of soluble HLA-E is an immunoregulatory feature of endothelial cell activation. Blood. 2007;109:2806–2814.
82. Valés-Gómez M, Reyburn HT, Erskine RA, et al. Kinetics and peptide dependency of the binding of the inhibitory NK receptor CD94/NKG2-A and the activating receptor CD94/NKG2-C to HLA-E. EMBO J. 1999;18:4250–4260.
83. Lopez-Vergès S, Milush JM, Schwartz BS, et al. Expansion of a unique CD57+
NKG2Chi natural killer cell subset during acute human cytomegalovirus infection. Proc Natl Acad Sci U S A. 2011;108:14725–14732.
84. Rölle A, Jäger D, Momburg F. HLA-E peptide repertoire and dimorphism-centerpieces in the adaptive NK Cell Puzzle? Front Immunol. 2018;9:2410.
85. Béziat V, Liu LL, Malmberg JA, et al. NK cell responses to cytomegalovirus infection lead to stable imprints in the human KIR repertoire and involve activating KIRs. Blood. 2013;121:2678–2688.
86. Ruggeri L, Capanni M, Urbani E, et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science. 2002;295:2097–2100.
87. Greenland JR, Sun H, Calabrese D, et al. HLA mismatching favoring host-versus-graft NK cell activity Via KIR3DL1 is associated with improved outcomes following lung transplantation. Am J Transplant. 2017;17:2192–2199.
88. Koenig A, Chen CC, Marçais A, et al. Missing self triggers NK cell-mediated chronic vascular rejection of solid organ transplants. Nat Commun. 2019;10:5350.
89. Seidel UJ, Schlegel P, Lang P. Natural killer cell mediated antibody-dependent cellular cytotoxicity in tumor immunotherapy with therapeutic antibodies. Front Immunol. 2013;4:76.
90. Legris T, Picard C, Todorova D, et al. Antibody-dependent NK cell activation is associated with late kidney allograft dysfunction and the complement-independent alloreactive potential of donor-specific antibodies. Front Immunol. 2016;7:288.
91. Parkes MD, Halloran PF, Hidalgo LG. Evidence for CD16a-mediated NK cell stimulation in antibody-mediated kidney transplant rejection. Transplantation. 2017;101:e102–e111.
92. Lefaucheur C, Viglietti D, Hidalgo LG, et al. Complement-activating anti-HLA antibodies in kidney transplantation: allograft gene expression profiling and response to treatment. J Am Soc Nephrol. 2018;29:620–635.
93. Blázquez-Moreno A, Park S, Im W, et al. Transmembrane features governing Fc receptor CD16A assembly with CD16A signaling adaptor molecules. Proc Natl Acad Sci U S A. 2017;114:E5645–E5654.
94. Watzl C, Urlaub D, Fasbender F, et al. Natural killer cell regulation - beyond the receptors. F1000Prime Rep. 2014;6:87.
95. Billadeau DD, Brumbaugh KM, Dick CJ, et al. The Vav-Rac1 pathway in cytotoxic lymphocytes regulates the generation of cell-mediated killing. J Exp Med. 1998;188:549–559.
96. Galandrini R, Micucci F, Tassi I, et al. Arf6: a new player in FcgammaRIIIA lymphocyte-mediated cytotoxicity. Blood. 2005;106:577–583.
97. Jiang K, Zhong B, Gilvary DL, et al. Pivotal role of phosphoinositide-3 kinase in regulation of cytotoxicity in natural killer cells. Nat Immunol. 2000;1:419–425.
98. Mace EM. Phosphoinositide-3-kinase signaling in human natural killer cells: new insights from primary immunodeficiency. Front Immunol. 2018;9:445.
99. Micucci F, Capuano C, Marchetti E, et al. PI5KI-dependent signals are critical regulators of the cytolytic secretory pathway. Blood. 2008;111:4165–4172.
100. Caraux A, Kim N, Bell SE, et al. Phospholipase C-gamma2 is essential for NK cell cytotoxicity and innate immunity to malignant and virally infected cells. Blood. 2006;107:994–1002.
101. Bryceson YT, March ME, Ljunggren HG, et al. Activation, coactivation, and costimulation of resting human natural killer cells. Immunol Rev. 2006;214:73–91.
102. Romee R, Foley B, Lenvik T, et al. NK cell CD16 surface expression and function is regulated by a disintegrin and metalloprotease-17 (ADAM17). Blood. 2013;121:3599–3608.
103. Srpan K, Ambrose A, Karampatzakis A, et al. Shedding of CD16 disassembles the NK cell immune synapse and boosts serial engagement of target cells. J Cell Biol. 2018;217:3267–3283.
104. Raulet DH. Roles of the NKG2D immunoreceptor and its ligands. Nat Rev Immunol. 2003;3:781–790.
105. Ogasawara K, Lanier LL. NKG2D in NK and T cell-mediated immunity. J Clin Immunol. 2005;25:534–540.
106. Upshaw JL, Arneson LN, Schoon RA, et al. NKG2D-mediated signaling requires a DAP10-bound Grb2-Vav1 intermediate and phosphatidylinositol-3-kinase in human natural killer cells. Nat Immunol. 2006;7:524–532.
107. Parsons MS, Richard J, Lee WS, et al. NKG2D Acts as a Co-receptor for natural killer cell-mediated anti-HIV-1 antibody-dependent cellular cytotoxicity. AIDS Res Hum Retroviruses. 2016;32:1089–1096.
108. Chauveau A, Tonnerre P, Pabois A, et al. Endothelial cell activation and proliferation modulate NKG2D activity by regulating MICA expression and shedding. J Innate Immun. 2014;6:89–104.
109. Gavlovsky PJ, Tonnerre P, Guitton C, et al. Expression of MHC class I-related molecules MICA, HLA-E and EPCR shape endothelial cells with unique functions in innate and adaptive immunity. Hum Immunol. 2016;77:1084–1091.
110. Boukouaci W, Busson M, Peffault de Latour R, et al. MICA-129 genotype, soluble MICA, and anti-MICA antibodies as biomarkers of chronic graft-versus-host disease. Blood. 2009;114:5216–5224.
111. Tonnerre P, Gérard N, Chatelais M, et al. MICA variant promotes allosensitization after kidney transplantation. J Am Soc Nephrol. 2013;24:954–966.
112. Isernhagen A, Schilling D, Monecke S, et al. The MICA-129Met/Val dimorphism affects plasma membrane expression and shedding of the NKG2D ligand MICA. Immunogenetics. 2016;68:109–123.
113. Baranwal AK, Goswami S, Bhat DK, et al. Soluble major histocompatibility complex class i related chain A (sMICA) levels influence graft outcome following renal transplantation. Hum Immunol. 2018;79:160–165.
114. Koene HR, Kleijer M, Algra J, et al. Fc gammaRIIIa-158V/F polymorphism influences the binding of IgG by natural killer cell Fc gammaRIIIa, independently of the Fc gammaRIIIa-48L/R/H phenotype. Blood. 1997;90:1109–1114.
115. Congy-Jolivet N, Bolzec A, Ternant D, et al. Fc gamma RIIIa expression is not increased on natural killer cells expressing the Fc gamma RIIIa-158V allotype. Cancer Res. 2008;68:976–980.
116. Dall’Ozzo S, Tartas S, Paintaud G, et al. Rituximab-dependent cytotoxicity by natural killer cells: influence of FCGR3A polymorphism on the concentration-effect relationship. Cancer Res. 2004;64:4664–4669.
117. Alduaij W, Illidge TM. The future of anti-CD20 monoclonal antibodies: are we making progress? Blood. 2011;117:2993–3001.
118. Paul P, Picard C, Sampol E, et al. Genetic and functional profiling of CD16-dependent natural killer activation identifies patients at higher risk of cardiac allograft vasculopathy. Circulation. 2018;137:1049–1059.
119. Subedi GP, Falconer DJ, Barb AW. Carbohydrate-polypeptide contacts in the antibody receptor CD16A identified through solution NMR spectroscopy. Biochemistry. 2017;56:3174–3177.
120. Mannon RB. Macrophages: contributors to allograft dysfunction, repair, or innocent bystanders? Curr Opin Organ Transplant. 2012;17:20–25.
121. van den Bosch TP, Kannegieter NM, Hesselink DA, et al. Targeting the monocyte-macrophage lineage in solid organ rransplantation. Front Immunol. 2017;8:153.
122. Pabois A, Pagie S, Gérard N, et al. Notch signaling mediates crosstalk between endothelial cells and macrophages via Dll4 and IL6 in cardiac microvascular inflammation. Biochem Pharmacol. 2016;104:95–107.
123. Sicard A, Meas-Yedid V, Rabeyrin M, et al. Computer-assisted topological analysis of renal allograft inflammation adds to risk evaluation at diagnosis of humoral rejection. Kidney Int. 2017;92:214–226.
124. Bräsen JH, Khalifa A, Schmitz J, et al. Macrophage density in early surveillance biopsies predicts future renal transplant function. Kidney Int. 2017;92479–489.
125. Ley K, Laudanna C, Cybulsky MI, et al. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol. 2007;7:678–689.
126. Locati M, Mantovani A, Sica A. Macrophage activation and polarization as an adaptive component of innate immunity. Adv Immunol. 2013;120:163–184.
127. Murray PJ, Allen JE, Biswas SK, et al. Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity. 2014;41:14–20.
128. Mantovani A, Biswas SK, Galdiero MR, et al. Macrophage plasticity and polarization in tissue repair and remodelling. J Pathol. 2013;229:176–185.
129. Aguado-Domínguez E, Cabrera-Pérez R, Suarez-Benjumea A, et al. Computer-assisted definition of the inflammatory infiltrates in patients with different categories of banff kidney allograft rejection. Front Immunol. 2019;10:2605.
130. Pober JS, Min W, Bradley JR. Mechanisms of endothelial dysfunction, injury, and death. Annu Rev Pathol. 2009;4:71–95.
131. Pagie S, Gérard N, Charreau B. Notch signaling triggered via the ligand DLL4 impedes M2 macrophage differentiation and promotes their apoptosis. Cell Commun Signal. 2018;16:4.
132. He H, Xu J, Warren CM, et al. Endothelial cells provide an instructive niche for the differentiation and functional polarization of M2-like macrophages. Blood. 2012;120:3152–3162.
133. Fantin A, Vieira JM, Gestri G, et al. Tissue macrophages act as cellular chaperones for vascular anastomosis downstream of VEGF-mediated endothelial tip cell induction. Blood. 2010;116:829–840.
134. Davies LC, Rosas M, Smith PJ, et al. A quantifiable proliferative burst of tissue macrophages restores homeostatic macrophage populations after acute inflammation. Eur J Immunol. 2011;41:2155–2164.
135. He H, Mack JJ, Güç E, et al. Perivascular macrophages limit permeability. Arterioscler Thromb Vasc Biol. 2016;36:2203–2212.
136. Kovacic JC, Dimmeler S, Harvey RP, et al. Endothelial to mesenchymal transition in cardiovascular disease: JACC state-of-the-art review. J Am Coll Cardiol. 2019;73:190–209.
137. Hata A, Chen YG. TGF-β signaling from receptors to smads. Cold Spring Harb Perspect Biol. 2016;8:a022061.
138. Xu X, Zheng L, Yuan Q, et al. Transforming growth factor-β in stem cells and tissue homeostasis. Bone Res. 2018;6:2.
139. Wu KQ, Muratore CS, So EY, et al. M1 macrophage-induced endothelial-to-mesenchymal transition promotes infantile hemangioma regression. Am J Pathol. 2017;187:2102–2111.
140. Zeisberg EM, Tarnavski O, Zeisberg M, et al. Endothelial-to-mesenchymal transition contributes to cardiac fibrosis. Nat Med. 2007;13:952–961.
141. Zeisberg EM, Potenta SE, Sugimoto H, et al. Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition. J Am Soc Nephrol. 2008;19:2282–2287.
142. He J, Xu Y, Koya D, et al. Role of the endothelial-to-mesenchymal transition in renal fibrosis of chronic kidney disease. Clin Exp Nephrol. 2013;17:488–497.
143. Wang Z, Han Z, Tao J, et al. Role of endothelial-to-mesenchymal transition induced by TGF-β1 in transplant kidney interstitial fibrosis. J Cell Mol Med. 2017;21:2359–2369.
144. Djamali A, Samaniego M. Fibrogenesis in kidney transplantation: potential targets for prevention and therapy. Transplantation. 2009;88:1149–1156.
145. Xu-Dubois YC, Peltier J, Brocheriou I, et al. Markers of endothelial-to-mesenchymal transition: evidence for antibody-endothelium interaction during antibody-mediated rejection in kidney recipients. J Am Soc Nephrol. 2016;27:324–332.
146. Man S, Sanchez Duffhues G, Ten Dijke P, et al. The therapeutic potential of targeting the endothelial-to-mesenchymal transition. Angiogenesis. 2019;22:3–13.
147. Dejana E, Hirschi KK, Simons M. The molecular basis of endothelial cell plasticity. Nat Commun. 2017;8:14361.
148. Medici D. Endothelial-Mesenchymal Transition in Regenerative Medicine. Stem Cells Int. 2016;2016:6962801.
149. Smith RJ Jr, Yi T, Nasiri B, et al. Implantation of VEGF-functionalized cell-free vascular grafts: regenerative and immunological response. Faseb J. 2019;33:5089–5100.
150. Long Y, Huang H. On signaling pathways: hematopoietic stem cell specification from hemogenic endothelium. Sci China Life Sci. 2015;58:1256–1261.
151. Smith RJ Jr, Nasiri B, Kann J, et al. Endothelialization of arterial vascular grafts by circulating monocytes. Nat Commun. 2020;11:1622.
152. Porta C, Rimoldi M, Raes G, et al. Tolerance and M2 (alternative) macrophage polarization are related processes orchestrated by p50 nuclear factor kappaB. Proc Natl Acad Sci U S A. 2009;106:14978–14983.
153. Mantovani A, Locati M. Orchestration of macrophage polarization. Blood. 2009;114:3135–3136.
154. Ferrante CJ, Leibovich SJ. Regulation of macrophage polarization and wound healing. Adv Wound Care (New Rochelle). 2012;1:10–16.
155. Lagaaij EL, Cramer-Knijnenburg GF, van Kemenade FJ, et al. Endothelial cell chimerism after renal transplantation and vascular rejection. Lancet. 2001;357:33–37.