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. Author manuscript; available in PMC: 2015 Feb 1.
Published in final edited form as: Curr Opin Organ Transplant. 2014 Feb;19(1):33–40. doi: 10.1097/MOT.0000000000000040

Antibody-Mediated Graft Injury: Complement-Dependent and Complement-Independent Mechanisms

Nicole M Valenzuela 1, Jeffrey T McNamara 1, Elaine F Reed 1
PMCID: PMC4080796  NIHMSID: NIHMS597423  PMID: 24316758

Abstract

Purpose of Review

Antibody-mediated rejection (AMR) is emerging as the leading cause of chronic rejection and allograft failure. Traditionally, the mechanisms of graft injury mediated by donor specific antibodies beyond complement activation were not well appreciated. However, an evolving paradigm of Fc-independent antibody functions, along with clinical recognition of C4d negative AMR, has increased awareness of the action of antibodies leading to endothelial activation and dysfunction.

Recent Findings

Herein, we address current clinical trends, including the signature of microvascular inflammation in biopsies of grafts undergoing AMR, the prevalence of antibodies to HLA-DQ and non-HLA targets, and the functional characterization of HLA IgG subclasses and complement fixing capacity. We also discuss recent experimental evidence revealing new mechanisms of endothelial and smooth muscle cell activation by HLA antibodies, which may contribute to vascular inflammation and chronic rejection. Finally, we touch upon novel discoveries of the interplay between antibodies, the complement system and CD4 T cell-mediated alloimmunity.

Summary

The current literature suggests that, although complement fixing antibodies may have some prognostic value for graft outcome, complement-independent mechanisms of graft injury are increasingly relevant. Therapeutic strategies which target endothelial activation induced by antibodies may ameliorate vascular inflammation and mononuclear cell infiltration characteristic of AMR.

Keywords: Microvascular Inflammation, Endothelial Cell Activation, Complement, Antibody-mediated Rejection

Introduction

Antibody-mediated rejection (AMR) is an increasingly problematic entity in solid organ transplantation that leads to graft dysfunction and loss. AMR often occurs late (>1 year) after transplantation with an incidence of approximately 30% (1), and was recently attributed to cause half of all renal allograft failures (2). An understanding of how antibodies cause graft injury and promote acute and chronic rejection is critical for the management of sensitized recipients and improvement of therapeutics. Studies from our group and others have previously demonstrated that HLA I antibodies act as agonists, inducing molecular aggregation of HLA I class I molecules to trigger intracellular signaling pathways that are critical in the regulation of cell survival, proliferation and migration in graft vascular cells (reviewed in (3)). Recent investigations have revealed additional functional responses of vascular cells to antibodies, including induction of inflammatory mediators by endothelial cells and recruitment of immune cells. Moreover, the relevance of complement fixing capacity of donor specific antibodies has received increasing attention. Herein, we discuss the implications of recent work in these areas.

Characteristics of AMR and Drawbacks of Current Histological Criteria

Consensus guidelines for the histological diagnosis of antibody-mediated rejection center around circulating DSA, vascular injury, microvascular changes and complement (C4d) deposition. However, AMR criteria have in some cases proved inadequate because of the reliance on complement staining (1, 2), and, while C4d appears to be a specific marker in cardiac and renal transplantation, it is estimated that half of AMR events are missed due to the lack of sensitivity of C4d staining (4). To date, no consensus AMR diagnostic criteria exist in lung and liver (5, 6), due to controversy over the clinical significance of DSA in liver transplantation and a lack of specificity of C4d staining in lung allografts (5-8).

Recent efforts have aimed at discovery of superior histological indicators that can discriminate antibody-mediated rejection from acute cellular rejection, infection, drug toxicity and stable graft function, and identify C4d-negative AMR. Extracellular matrix regulators matrix metalloproteinase-2 (MMP2) and TIMP1 were upregulated in renal biopsies during AMR (9), which may play a role in fibrosis during chronic rejection. Several groups have recently reported that glomerulitis and peritubular capillaritis (microcirculation or microvascular inflammation) correlate with DSA and graft failure in renal transplants (10-12). Moreover, activation of capillary endothelial cell signaling correlates closely with AMR severity, DSA and microcirculation inflammation, and is a more sensitive AMR marker than C4d deposition (13, 14) cardiac transplantation. A “molecular phenotyping” approach, predominantly composed of endothelial-associated transcripts expressed in the renal allograft, could also identify C4d negative AMR (15). A new sub-classification of AMR with vasculitis was recently proposed, which increased predictive value for renal allograft loss (16).

Additionally, intravascular macrophages, identified by CD68 staining, appear to be a common feature of AMR (17), and provide a more sensitive histological marker compared with C4d in cardiac allografts (18). Intragraft macrophages also increase the risk of renal allograft dysfunction (11), although macrophage staining is not currently included in the Banff criteria. Natural killer (NK) cells have also been implicated in antibody-mediated graft injury, particularly in late AMR (19). These clinical results are paralleled by observations in experimental models, where depletion of NK cells ameliorated MHC I antibody-induced chronic rejection in murine cardiac allografts (20). Taken together, these clinical observations highlight the central role of endothelial activation and innate immune infiltration in graft injury by donor specific antibodies.

New Trends

There is a wealth of literature regarding the incidence and deleterious impact of donor specific HLA antibodies, and this topic has been thoroughly reviewed elsewhere (21, 22).

HLA-DQ Antibodies

HLA I antibodies appear early, while HLA II DSA tends to prevail late after transplantation and associates with chronic graft dysfunction (23, 24). An emerging trend is the intriguing but little understood phenomenon of predominant DSA to HLA-DQ in lung (25), cardiac (26), renal (27) and liver transplant recipients (28), particularly during chronic rejection (28). The possible pathogenic mechanisms of antibodies to HLA-DQ are not yet clear, although recipients tend to have worse outcome than when anti-DQ antibodies are absent (29). Interestingly, DQ mismatch increases the risk of de novo DSA development (27). It is yet unknown whether antibodies to HLA-DQ are a marker of end-stage graft injury and/or nonadherence; whether they are increased in circulation due to low HLA-DQ expression in the graft; or whether they represent an independent pathogenic actor.

Non-HLA Antibodies

There is growing evidence to suggest that antibodies against non-HLA antigens may contribute to AMR in solid organ transplantation. Reports show that 10-23% of recipients are pre-sensitized to non-HLA antigens (30, 31), while 22% form non-HLA antibodies after transplant (32). Endothelial reactive antibodies have been used clinically to identify cases of AMR (33) and the nature of these targets is starting to be elucidated. Non-HLA antibodies are found in renal, lung and cardiac transplant recipients, but the targets appear to be unique to each solid organ. A recent study revealed that endothelial-reactive antibodies may recognize nonpolymorphic and polymorphic antigens on the endothelial surface (34). However, conflicting reports suggest either that non-HLA antibodies independently reduce graft outcome (35-37) or that they have no significant impact (38). The issue is complicated by observations that autoantibodies can occur independently of or concurrently with donor specific HLA and MICA antibodies. In lung transplants, non-HLA antibodies against K-α tubulin and collagen are associated with rejection, but these antibodies were preceded by HLA DSA and persisted after elevated HLA antibody levels subsided (39), suggesting that non-HLA antibodies are markers of alloimmune damage. Anti-endothelial cell antibody subclasses are reportedly IgG2 and IgG4 predominant, which are non-complement fixing, suggesting a complement-independent mechanism of action (30). However, antibodies against vimentin, K-α tubulin, and collagen have been associated with C4d deposition on erythrocytes, suggesting they could contribute to classical complement activation (40). It is also likely that the pathogenic potential of non-HLA antibodies depends upon the target. For example, anti-angiotensin II type 1 receptor (AT1R) antibodies contribute to hypertension and vasculopathy in renal and cardiac transplantation (41) and increase the risk of graft failure (42). In summary, the targets of antibodies to non-HLA endothelial proteins are only beginning to be identified, and, for the most part, their mechanism of action beyond Fc-mediated functions remain to be further elucidated.

The Mechanisms of Antibody-Mediated Graft Injury

Antibody-mediated acute rejection is primarily driven by the effector functions of the Fc fragment of HLA antibodies, while experimental evidence indicates that the Fc-independent effects of antibodies promote chronic inflammation and proliferation.

The Significance of Complement

IgG1 and IgG3 are efficient activators of the classical complement cascade, as well as high affinity ligands for Fc gamma receptors expressed on myeloid cells including macrophages and NK cells (reviewed in (43)). In recent years, new assays have been developed to characterize the functional deposition of complement components, including C1q, C3d and C4d, by HLA antibody in solid phase assays or cell-based protocols (1, 44-46). However, contradictory conclusions have been reached regarding the predictive significance of complement fixing DSA detected by these assays (47, 48), and the results often do not correlate with lymphocytotoxicity (CDC-XM) results (46). Moreover, despite the predominance of complement-fixing IgG1 DSA in recipients (28, 49), only certain specificities exhibit cytotoxicity or C1q deposition. Antigen density or synergism of multiple low-level allele-specific antibodies may explain the differences (reviewed in (50)). Additionally, complement fixation appears to be closely dependent upon antibody titer (51) and confounded by the presence of IgM (52). Indeed, poorer outcomes associated with complement fixing antibodies in some studies may be in fact reflective of high donor specific antibody levels (48). Duquesnoy et al. proposed the theory that complement fixation is a function of the epitope itself, which induces conformational changes in the antibody to confer binding to the C1 complex (53). Additionally, it is possible that differences in Fc glycan composition may lead to increased cytotoxicity of certain HLA DSA (reviewed in (54)). While interesting, experimental evidence addressing these putative mechanisms remains to be reported.

The utility of C4d staining has recently been questioned (16), particularly late in transplantation (23). Antibody-mediated chronic injury does not always correlate with or require complement activation in clinical studies (55) or experimental models (20). Whereas HLA antibodies trigger complement-dependent cytotoxicity of target lymphocytes, endothelial cells—the primary target of antibody-mediated alloimmunity—are generally resistant to complement-mediated cell lysis in response to HLA antibodies. Despite evidence of terminal complement activation at the endothelial cell surface (44, 56), endothelial cells may upregulate cytoprotective factors and survival signaling to prevent HLA antibody-induced cytotoxicity (57, 58). Therefore, the physiological relevance of complement activating antibodies detected by C1q or C3d-fixing assays remains unclear, and the mechanisms of antibody-mediated graft injury in C4d negative AMR have yet to be fully elucidated.

Complement-Independent Antibody-Mediated Endothelial and Smooth Muscle Cell Activation

HLA antibodies also act as agonists to induce intracellular signaling in endothelial and smooth muscle cells. Notably, these effects are independent of the Fc fragment, and may therefore be elicited by any IgG subclass. HLA I antibodies promote focal adhesion kinase (FAK)-dependent proliferation and migration of smooth muscle cells (59), which may contribute to the neointimal changes seen in chronic rejection. Recent evidence has also implicated matrix metalloproteinase-2 (MMP2) and neutral sphingomyelinase-2 (nSMase2) in HLA I antibody-induced smooth muscle cell proliferation in vitro and in vivo (60), which is interesting in light of clinical observations of increased intragraft expression of MMP2 during AMR (9). Active MMP2 acts on nSMase2 to stimulate, among other effects, production of ceramide. Ceramide is itself a proinflammatory stimulus, and can be converted to sphingosine-1-phosphate (S1P), which is mitogenic through MAPK/ERK pathways (61). In this study, the authors speculated that nSMase2 activation generates S1P, but as yet no evidence has linked HLA I antibodies with ceramide and/or S1P production. Further studies are needed in this novel area to dissect the proximal signaling leading to MMP2 and nSMase2 activation, to confirm the role of S1P in smooth muscle cell proliferation. If S1P is indeed produced in response to HLA I crosslinking, it would be of interest to examine other phenotypic changes triggered by this mediator, such as regulation of cell motility, control of barrier function, and modulation of endothelial inflammatory activation.

Cell proliferation and migration are dependent upon reorganization of the cytoskeleton. Recently, our group characterized HLA I-induced cytoskeletal rearrangement in greater detail, demonstrating that HLA I crosslinking activated Rho kinase, myosin light chain kinase and ERK1/2 in a calcium-independent manner to stimulate stress fiber formation. This study also revealed a novel molecular association of ERK1/2 with mTORC2, and showed that Rictor and ERK relocalize to the plasma membrane after HLA I antibody stimulation. These results suggest that ERK1/2 is a critical regulator of endothelial cytoskeletal changes, migration and motility (62).

In addition to cell motility, the intact cytoskeleton is required for activation of critical kinases downstream of HLA I crosslinking. Proteomic analysis demonstrated that HLA I crosslinking induced the association of many phosphorylated proteins, including eukaryotic initiation factor 4A1 (eIF4A1), with the actin cytoskeletal fraction (63). eIF4A1 is a target of mTORC1 and is required for protein translation and cell proliferation. The cytoskeleton is actively involved in and required for translation, through spatial organization of the protein synthesis machinery, delivery of tRNAs to polysomes, and support of compartment-restricted localized translation of specific RNA (64). Taken together, these studies highlight the central role of cytoskeleton in mediating the cell signaling process and raise fascinating questions regarding the functional link between the cytoskeleton and protein synthesis machinery in HLA I signaling.

Intracapillary macrophages are an important hallmark of AMR in cardiac allografts (17), and macrophages are key immune mediators of graft injury. Endothelial cells actively regulate infiltration of leukocytes from the bloodstream into the tissue. Type 1 endothelial cell activation involves rapid and transient induction of adhesion molecules and chemoattractants, while type 2 activation requires protein synthesis resulting in chemokine and adhesion molecule expression over the course of hours or days. Emerging evidence indicates that HLA crosslinking can trigger both type 1 and type 2 endothelial cell activation, providing some insight into the mechanisms of macrophage recruitment to the allograft during HLA antibody-mediated injury. We and others reported that HLA I crosslinking by antibodies triggers rapid endothelial exocytosis (<30min, type 1 activation) leading to cell surface presentation of the adhesion molecule P-selectin and release of vWF (65, 66) from aortic, venous and microvascular endothelial cells (67). This induction did not require the Fc portion of the antibody, suggesting that all IgG subclasses can elicit pathogenic effects by direct endothelial cell activation. P-selectin was indispensable for increased adhesion of monocytes in vitro and for accumulation of macrophages in murine cardiac allografts in vivo in response to MHC I antibodies (65). Interestingly, we observed that complement fixing HLA I antibody dramatically augmented basal P-selectin-mediated monocyte adhesion through engagement of monocyte Fc gamma receptors (FcγRs) (67), indicating that DSA subclasses that interact efficiently with FcγRs elicit enhanced recruitment of monocytes through dual activation of endothelium and monocytes.

In addition to early endothelial activation, HLA I antibodies induce late phase adhesion molecules and synthesis of chemoattractants and inflammatory cytokines that promote inflammation. For example, HLA I crosslinking activated the transcription factor cAMP response element binding protein (CREB), which lead to increased adhesion molecule and chemokine expression by microvascular endothelium, and monocyte adhesion (68). Similar studies found increased synthesis of cytokines and chemokines by endothelial cells from other vascular beds (69) upon exposure to HLA antibodies (Table 1).

Table 1.

Production of inflammatory mediators by endothelial cell antibodies stimulated with HLA antibodies.

Cell Type HLA Antibody
Source		 Protein Induced by
HLA antibody		 Function Target Cell Reference
HAEC
HMEC-1
HUVEC		 Murine monoclonal
W6/32 and broadly
reactive HLA I anti-
serum		 P-selectin rapidly induced initiator of
leukocyte tethering		 all leukocytes (65-67)
HMEC-1 Murine monoclonal
W6/32		 ICAM-1 endothelial adhesion
molecule		 all leukocytes (68)
VCAM-1 endothelial adhesion
molecule		 lymphocytes and monocytes
IL-6 acute phase inflammatory
cytokine		 T cells (activation and proliferation)
monocytes (differentiation)
IL-8 (CXCL8) chemoattractant neutrophils and monocytes
Glomerular MVEC Broadly reactive HLA
I and II anti-serum		 ICAM-1 endothelial adhesion
molecule		 all leukocytes (69)
VCAM-1 endothelial adhesion
molecule		 lymphocytes and monocytes
GM-CSF growth factor and
chemoattractant		 granulocyte and monocyte
TNFα inflammatory cytokine all leukocytes and EC
MCP-1 (CCL2) chemoattractant monocyte and basophil
RANTES (CCL5) chemoattractant T cell and monocyte
IL-11 acute phase inflammatory
cytokine
extracellular matrix regulator		 endothelial cells
IL-1β inflammatory cytokine endothelial cells, monocytes
Fractalkine chemoattractant NK cells, T cells and monocytes
HUVEC Broadly reactive HLA
I and II anti-serum

(with complement)		 E-selectin late phase initiator of
leukocyte tethering		 all leukocytes (56)
VCAM-1 endothelial adhesion
molecule
RANTES chemoattractant T cells and monocytes
IL-6 acute phase inflammatory
cytokine		 T cells (activation and proliferation)
monocytes (differentiation)
MIP3α (CCL20) chemoattractant immature dendritic cells
memory CD4 T cells
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HAEC: human aortic endothelial cells, primary

HMEC-1: human microvascular endothelial cell line

MVEC: microvascular endothelial cells, primary

HUVEC: human umbilical vein endothelial cells

The Relationship between Donor Specific Antibodies and T Cell-Mediated Alloimmunity

Most recently, a novel paradigm has emerged in which antibody-mediated graft injury may potentiate cell-mediated alloimmune responses. Jordan Pober’s group demonstrated that complement activation can augment HLA I and II antibody activation of endothelium, which increased endothelial immunogenicity to T cells. Sublytic MAC deposition on endothelial cells in response to HLA antibodies exacerbated proinflammatory signaling through non-canonical NFκB, which in turn triggered CD4 T cell capture under shear stress. Moreover, allogeneic CD4 T cell activation was increased upon coculture with HLA antibody-activated endothelium (56). In another study, complement split products augmented costimulation of alloreactive CD4 T cell by dendritic cells (70). Taken together, these findings provoke interesting questions about the interplay between antibodies and alloreactive T cells, and highlight a role for complement in graft injury beyond lytic vascular damage. Indeed, a complete appreciation of the multifaceted mechanisms of antibody-mediated graft injury should consider the relationships between the humoral, cell-mediated and innate immune systems, which do not act in isolation under physiological conditions.

Concluding Remarks

In summary, vasculitis and microvascular inflammation have emerged as promising histological markers, underscoring the central role of endothelial injury in AMR. Experimental evidence suggests that HLA antibody-induced endothelial cell inflammatory activation may promote the microcirculation inflammation and innate immune infiltration characteristic of AMR. Moreover, Fc-mediated functions such as complement activation and engagement of Fc receptors act synergistically with endothelial activation to enhance inflammation and alloimmune damage. Many questions remain, including the physiological relevance of prevalent anti-DQ antibodies, non-HLA antibodies, and antibodies that fix complement in vitro; and the effects of HLA II antibodies on vascular cells.

Key Points.

  • HLA antibodies may be pre-existing or develop at any time after transplant; however, most de novo antibodies form 6 months or later post-transplant. HLA I antibodies appear earlier, while HLA II antibodies (particularly anti-HLA-DQ antibodies) develop in the late post-transplant period.

  • Donor specific HLA antibodies stimulate endothelial cell signaling, including rapid exocytosis of P-selectin and production of chemokines, leading to inflammatory activation and leukocyte recruitment.

  • Engagement of Fc receptors on monocytes by certain subclasses of HLA I antibodies enhances monocyte recruitment by activated endothelial cells.

  • It is yet unclear whether antibodies that fix complement in in vitro deposition assays have definitive clinical relevance. However, activation of complement by HLA antibodies in experimental models triggers production of complement split products that boost T cell-mediated alloimmune responses.

Acknowledgements

NMV has received the Ruth L. Kirschstein Post-Doctoral NRSA Training Grant (5 T32 HL069766-12). JTM has received the Regenerative Musculoskeletal Medicine Training Grant (5T32AR059033-03). EFR is currently receiving investigator-initiated research grant funding from Ipierian, Novartis, Gen-Probe, and Pfizer ARTS. EFR is also receiving funding from NIH and NIAID/NHLBI (R01 AI042819; U01 AI077821; U01 AI63594-06).

Abbreviations

AMR

antibody-mediated rejection

AT1R

angiotensin II type 1 receptor

CDC-XM

complement-dependent cytotoxicity crossmatch

DSA

donor specific (HLA) antibodies

FcγR

Fc gamma receptor

HLA

human leukocyte antigen

MICA

major histocompatibility complex class I-related chain A

Footnotes

Disclosure: The authors have no relevant conflicts of interest to declare.

References

  • 1 *.Lawrence C, Willicombe M, Brookes PA, Santos-Nunez E, et al. Preformed complement-activating low-level donor-specific antibody predicts early antibody-mediated rejection in renal allografts. Transplantation. 2013;95(2):341–6. doi: 10.1097/TP.0b013e3182743cfa. This study examined the clinical significance of CDC and flow-crossmatch negative DSA, and characterized DSA by C4d deposition assay. C4d depositing capacity by low titer CDC negative DSA was predictive of AMR events.
  • 2.Sellares J, de Freitas DG, Mengel M, Reeve J, et al. Understanding the causes of kidney transplant failure: the dominant role of antibody-mediated rejection and nonadherence. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2012;12(2):388–99. doi: 10.1111/j.1600-6143.2011.03840.x. [DOI] [PubMed] [Google Scholar]
  • 3.Valenzuela NM, Reed EF. Antibodies in transplantation: the effects of HLA and non-HLA antibody binding and mechanisms of injury. Methods in molecular biology. 2013;1034:41–70. doi: 10.1007/978-1-62703-493-7_2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Sis B, Jhangri GS, Bunnag S, Allanach K, et al. Endothelial gene expression in kidney transplants with alloantibody indicates antibody-mediated damage despite lack of C4d staining. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2009;9(10):2312–23. doi: 10.1111/j.1600-6143.2009.02761.x. [DOI] [PubMed] [Google Scholar]
  • 5.Berry G, Burke M, Andersen C, Angelini A, et al. Pathology of pulmonary antibody-mediated rejection: 2012 update from the Pathology Council of the ISHLT. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation. 2013;32(1):14–21. doi: 10.1016/j.healun.2012.11.005. [DOI] [PubMed] [Google Scholar]
  • 6.Kozlowski T, Andreoni K, Schmitz J, Hayashi PH, et al. Sinusoidal C4d deposits in liver allografts indicate an antibody-mediated response: diagnostic considerations in the evaluation of liver allografts. Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society. 2012;18(6):641–58. doi: 10.1002/lt.23403. [DOI] [PubMed] [Google Scholar]
  • 7.Ali S, Ormsby A, Shah V, Segovia MC, et al. Significance of complement split product C4d in ABO-compatible liver allograft: diagnosing utility in acute antibody mediated rejection. Transplant immunology. 2012;26(1):62–9. doi: 10.1016/j.trim.2011.08.005. [DOI] [PubMed] [Google Scholar]
  • 8.O’Leary JG, Klintmalm GB. Impact of donor-specific antibodies on results of liver transplantation. Current opinion in organ transplantation. 2013;18(3):279–84. doi: 10.1097/MOT.0b013e3283614a10. [DOI] [PubMed] [Google Scholar]
  • 9.Yan Q, Sui W, Wang B, Zou H, et al. Expression of MMP-2 and TIMP-1 in renal tissue of patients with chronic active antibody-mediated renal graft rejection. Diagnostic pathology. 2012;7:141. doi: 10.1186/1746-1596-7-141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Sis B, Jhangri GS, Riopel J, Chang J, et al. A new diagnostic algorithm for antibody-mediated microcirculation inflammation in kidney transplants. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2012;12(5):1168–79. doi: 10.1111/j.1600-6143.2011.03931.x. [DOI] [PubMed] [Google Scholar]
  • 11.Papadimitriou JC, Drachenberg CB, Ramos E, Kukuruga D, et al. Antibody-mediated allograft rejection: morphologic spectrum and serologic correlations in surveillance and for cause biopsies. Transplantation. 2013;95(1):128–36. doi: 10.1097/TP.0b013e3182777f28. [DOI] [PubMed] [Google Scholar]
  • 12.de Kort H, Willicombe M, Brookes P, Dominy KM, et al. Microcirculation inflammation associates with outcome in renal transplant patients with de novo donor-specific antibodies. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2013;13(2):485–92. doi: 10.1111/j.1600-6143.2012.04325.x. [DOI] [PubMed] [Google Scholar]
  • 13 *.Tible M, Loupy A, Vernerey D, Suberbielle C, et al. Pathologic classification of antibody-mediated rejection correlates with donor-specific antibodies and endothelial cell activation. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation. 2013;32(8):769–76. doi: 10.1016/j.healun.2013.05.012. This study confirms and extends previous work (Lepin AJT 2006) demonstrating that phosphorylation of S6 ribosomal protein and S6 kinase in capillary endothelial cells of cardiac allografts correlates with DSA and microvascular inflammation, and is a superior marker of AMR compared with C4d. These results parallel in vitro findings in which HLA I crosslinking stimulates phosphorylation of mTOR-dependent targets, including S6 kinase and S6 ribosomal protein.
  • 14.Lepin EJ, Zhang Q, Zhang X, Jindra PT, et al. Phosphorylated S6 ribosomal protein: a novel biomarker of antibody-mediated rejection in heart allografts. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2006;6(7):1560–71. doi: 10.1111/j.1600-6143.2006.01355.x. [DOI] [PubMed] [Google Scholar]
  • 15.Sellares J, Reeve J, Loupy A, Mengel M, et al. Molecular diagnosis of antibody-mediated rejection in human kidney transplants. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2013;13(4):971–83. doi: 10.1111/ajt.12150. [DOI] [PubMed] [Google Scholar]
  • 16 **.Lefaucheur C, Loupy A, Vernerey D, Duong-Van-Huyen JP, et al. Antibody-mediated vascular rejection of kidney allografts: a population-based study. Lancet. 2013;381(9863):313–9. doi: 10.1016/S0140-6736(12)61265-3. In this large study of renal transplant recipients, the investigators propose a subclassification of AMR distinguishing biopsies with and without vasculitis. Stratification of AMR patients based on endarteritis revealed a significantly poorer outcome than when vasculitis was absent. Interestingly, only half of AMR biopsies were C4d+, irrespective of vascular rejection.
  • 17.Fishbein GA, Fishbein MC. Morphologic and immunohistochemical findings in antibody-mediated rejection of the cardiac allograft. Human immunology. 2012;73(12):1213–7. doi: 10.1016/j.humimm.2012.07.011. [DOI] [PubMed] [Google Scholar]
  • 18.Fedrigo M, Feltrin G, Poli F, Frigo AC, et al. Intravascular macrophages in cardiac allograft biopsies for diagnosis of early and late antibody-mediated rejection. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation. 2013;32(4):404–9. doi: 10.1016/j.healun.2012.12.017. [DOI] [PubMed] [Google Scholar]
  • 19.Hidalgo LG, Sellares J, Sis B, Mengel M, et al. Interpreting NK cell transcripts versus T cell transcripts in renal transplant biopsies. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2012;12(5):1180–91. doi: 10.1111/j.1600-6143.2011.03970.x. [DOI] [PubMed] [Google Scholar]
  • 20 **.Hirohashi T, Chase CM, Della Pelle P, Sebastian D, et al. A novel pathway of chronic allograft rejection mediated by NK cells and alloantibody. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2012;12(2):313–21. doi: 10.1111/j.1600-6143.2011.03836.x. In an experimental model of chronic cardiac allograft rejection, donor specific MHC I antibodies were sufficient to induce vascular lesions characteristic of vasculopathy. Depletion of NK cells ameliorated this response, indicating that NK cells play an important role in antibody-mediated chronic rejection.
  • 21.Kaneku H. Annual literature review of donor-specific HLA antibodies after organ transplantation. Clinical transplants. 2011:311–8. [PubMed] [Google Scholar]
  • 22.Kittleson MM, Kobashigawa JA. Antibody-mediated rejection. Current opinion in organ transplantation. 2012;17(5):551–7. doi: 10.1097/MOT.0b013e3283577fef. [DOI] [PubMed] [Google Scholar]
  • 23 *.Frank R, Molina MR, Wald JW, Goldberg LR, et al. Correlation of circulating donor-specific anti-HLA antibodies and presence of C4d in endomyocardial biopsy with heart allograft outcomes: a single-center, retrospective study. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation. 2013;32(4):410–7. doi: 10.1016/j.healun.2012.12.005. The authors identified a correlation between C4d staining and HLA I DSA (with or without concurrent HLA II DSA) in cardiac transplantation, but not with HLA II DSA alone. However, HLA II DSA alone was an independent risk factor for cardiac allograft vasculopathy. Therefore, the utility of C4d staining in late transplantation and in patients with HLA II DSA alone is questioned.
  • 24 *.Wiebe C, Gibson IW, Blydt-Hansen TD, Karpinski M, et al. Evolution and clinical pathologic correlations of de novo donor-specific HLA antibody post kidney transplant. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2012;12(5):1157–67. doi: 10.1111/j.1600-6143.2012.04013.x. This paper characterized the “natural history” of de novo DSA, including the timing of antibody development in renal transplant recipients. The authors found that 15% of low risk recipients develop DSA after transplant, and these patients have significantly lower 10 year graft survival compared with patients without DSA.
  • 25.Lobo LJ, Aris RM, Schmitz J, Neuringer IP. Donor-specific antibodies are associated with antibody-mediated rejection, acute cellular rejection, bronchiolitis obliterans syndrome, and cystic fibrosis after lung transplantation. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation. 2013;32(1):70–7. doi: 10.1016/j.healun.2012.10.007. [DOI] [PubMed] [Google Scholar]
  • 26.Ticehurst EH, Molina MR, Frank R, Kearns J, et al. Antibody-mediated rejection in heart transplant patients: long-term follow up of patients with high levels of donor-directed anti-DQ antibodies. Clinical transplants. 2011:409–14. [PubMed] [Google Scholar]
  • 27.Everly MJ, Rebellato LM, Haisch CE, Ozawa M, et al. Incidence and impact of de novo donor-specific alloantibody in primary renal allografts. Transplantation. 2013;95(3):410–7. doi: 10.1097/TP.0b013e31827d62e3. [DOI] [PubMed] [Google Scholar]
  • 28.Kaneku H, O’Leary JG, Banuelos N, Jennings LW, et al. De novo donor-specific HLA antibodies decrease patient and graft survival in liver transplant recipients. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2013;13(6):1541–8. doi: 10.1002/ajt.12212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29 *.Freitas MC, Rebellato LM, Ozawa M, Nguyen A, et al. The role of immunoglobulin-G subclasses and C1q in de novo HLA-DQ donor-specific antibody kidney transplantation outcomes. Transplantation. 2013;95(9):1113–9. doi: 10.1097/TP.0b013e3182888db6. In this study of renal transplant patients, the presence of DSA to HLA-DQ significantly increased the risk of rejection and graft loss, whether DQ antibodies were preformed or de novo. These authors were the first to characterize the subclass distribution and complement fixing capacity of HLA-DQ DSA, and found that they were primarily IgG1 and IgG3. Predominance of IgG1/IgG3 DQ antibodies was especially detrimental to outcome.
  • 30.Jackson AM, Lucas DP, Melancon JK, Desai NM. Clinical relevance and IgG subclass determination of non-HLA antibodies identified using endothelial cell precursors isolated from donor blood. Transplantation. 2011;92(1):54–60. doi: 10.1097/TP.0b013e31821b60e9. [DOI] [PubMed] [Google Scholar]
  • 31.Qin Z, Lavingia B, Zou Y, Stastny P. Antibodies against nucleolin in recipients of organ transplants. Transplantation. 2011;92(7):829–35. doi: 10.1097/TP.0b013e31822d0977. [DOI] [PubMed] [Google Scholar]
  • 32.Sigdel TK, Li L, Tran TQ, Khatri P, et al. Non-HLA antibodies to immunogenic epitopes predict the evolution of chronic renal allograft injury. Journal of the American Society of Nephrology : JASN. 2012;23(4):750–63. doi: 10.1681/ASN.2011060596. [DOI] [PubMed] [Google Scholar]
  • 33.Jackson AM, Lucas DP, Badders JL. A flow cytometric crossmatch test using endothelial precursor cells isolated from peripheral blood. Methods in molecular biology. 2013;1034:319–29. doi: 10.1007/978-1-62703-493-7_18. [DOI] [PubMed] [Google Scholar]
  • 34.Qin Z, Zou Y, Lavingia B, Stastny P. Identification of endothelial cell surface antigens encoded by genes other than HLA. A combined immunoprecipitation and proteomic approach for the identification of antigens recognized by antibodies against endothelial cells in transplant recipients. Human immunology. 2013 doi: 10.1016/j.humimm.2013.05.002. [DOI] [PubMed] [Google Scholar]
  • 35.Giral M, Foucher Y, Dufay A, Van Huyen JP, et al. Pretransplant Sensitization Against Angiotensin II Type 1 Receptor Is a Risk Factor for Acute Rejection and Graft Loss. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2013 doi: 10.1111/ajt.12397. [DOI] [PubMed] [Google Scholar]
  • 36.Jackson AM, Kuperman MB, Montgomery RA. Multiple hyperacute rejections in the absence of detectable complement activation in a patient with endothelial cell reactive antibody. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2012;12(6):1643–9. doi: 10.1111/j.1600-6143.2011.03955.x. [DOI] [PubMed] [Google Scholar]
  • 37.Breimer ME, Rydberg L, Jackson AM, Lucas DP, et al. Multicenter evaluation of a novel endothelial cell crossmatch test in kidney transplantation. Transplantation. 2009;87(4):549–56. doi: 10.1097/TP.0b013e3181949d4e. [DOI] [PubMed] [Google Scholar]
  • 38 *.Zitzner JR, Shah S, Jie C, Wegner W, et al. A prospective study evaluating the role of donor-specific anti-endothelial crossmatch (XM-ONE assay) in predicting living donor kidney transplant outcome. Human immunology. 2013 doi: 10.1016/j.humimm.2013.06.007. In this study, the authors could not find any prognostic value on outcome for donor specific anti-endothelial cell antibodies in renal transplant recipients. This conclusion conflicts with previous reports (Breimer Transpl 2009).
  • 39.Saini D, Weber J, Ramachandran S, Phelan D, et al. Alloimmunity-induced autoimmunity as a potential mechanism in the pathogenesis of chronic rejection of human lung allografts. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation. 2011;30(6):624–31. doi: 10.1016/j.healun.2011.01.708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Golocheikine A, Nath DS, Basha HI, Saini D, et al. Increased erythrocyte C4D is associated with known alloantibody and autoantibody markers of antibody-mediated rejection in human lung transplant recipients. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation. 2010;29(4):410–6. doi: 10.1016/j.healun.2009.10.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Lukitsch I, Kehr J, Chaykovska L, Wallukat G, et al. Renal ischemia and transplantation predispose to vascular constriction mediated by angiotensin II type 1 receptor-activating antibodies. Transplantation. 2012;94(1):8–13. doi: 10.1097/TP.0b013e3182529bb7. [DOI] [PubMed] [Google Scholar]
  • 42.Taniguchi M, Rebellato LM, Cai J, Hopfield J, et al. Higher Risk of Kidney Graft Failure in the Presence of Anti-Angiotensin II Type-1 Receptor Antibodies. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2013 doi: 10.1111/ajt.12395. [DOI] [PubMed] [Google Scholar]
  • 43.Akiyoshi T, Hirohashi T, Alessandrini A, Chase CM, et al. Role of complement and NK cells in antibody mediated rejection. Human immunology. 2012;73(12):1226–32. doi: 10.1016/j.humimm.2012.07.330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.AlMahri A, Holgersson J, Alheim M. Detection of complement-fixing and non-fixing antibodies specific for endothelial precursor cells and lymphocytes using flow cytometry. Tissue antigens. 2012;80(5):404–15. doi: 10.1111/j.1399-0039.2012.01954.x. [DOI] [PubMed] [Google Scholar]
  • 45.Bartel G, Wahrmann M, Schwaiger E, Kikic Z, et al. Solid phase detection of C4d-fixing HLA antibodies to predict rejection in high immunological risk kidney transplant recipients. Transplant international : official journal of the European Society for Organ Transplantation. 2013;26(2):121–30. doi: 10.1111/tri.12000. [DOI] [PubMed] [Google Scholar]
  • 46.Chen G, Sequeira F, Tyan DB. Novel C1q assay reveals a clinically relevant subset of human leukocyte antigen antibodies independent of immunoglobulin G strength on single antigen beads. Human immunology. 2011;72(10):849–58. doi: 10.1016/j.humimm.2011.07.001. [DOI] [PubMed] [Google Scholar]
  • 47.Crespo M, Torio A, Mas V, Redondo D, et al. Clinical relevance of pretransplant anti-HLA donor-specific antibodies: Does C1q-fixation matter? Transplant immunology. 2013 doi: 10.1016/j.trim.2013.07.002. [DOI] [PubMed] [Google Scholar]
  • 48 **.Loupy A, Lefaucheur C, Vernerey D, Prugger C, et al. Complement-binding anti-HLA antibodies and kidney-allograft survival. The New England journal of medicine. 2013;369(13):1215–26. doi: 10.1056/NEJMoa1302506. These investigators sought to characterize DSA using the C1q Screen to determine whether complement fixing antibody play a more significant role in renal allograft failure in presensitized patients. Here, C1q+ DSA predicted worse survival compared with non-complement fixing DSA, and the authors propose that patients can be further risk stratified based on C1q binding assays. Notably, most of the C1q+ DSA were also high strength (MFI >6000) on single antigen flow beads.
  • 49 **.Lowe D, Higgins R, Zehnder D, Briggs DC. Significant IgG subclass heterogeneity in HLA-specific antibodies: Implications for pathogenicity, prognosis, and the rejection response. Human immunology. 2013;74(5):666–72. doi: 10.1016/j.humimm.2013.01.008. This study evaluated the DSA IgG subclass profiles in renal transplant recipients. While there was no clear association of subclass distribution with specificity, the authors found that the initial sensitizing event skewed subsequent heavy chain usage. IgG1 predominated in HLA antibodies triggered by transfusion, while pregnancy and failed transplant stimulated a mixture of IgG subclasses.
  • 50.Bohmig GA, Bartel G, Wahrmann M. Antibodies, isotypes and complement in allograft rejection. Current opinion in organ transplantation. 2008;13(4):411–8. doi: 10.1097/MOT.0b013e3283028312. [DOI] [PubMed] [Google Scholar]
  • 51.Zeevi A, Lunz J, Feingold B, Shullo M, et al. Persistent strong anti-HLA antibody at high titer is complement binding and associated with increased risk of antibody-mediated rejection in heart transplant recipients. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation. 2013;32(1):98–105. doi: 10.1016/j.healun.2012.09.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Llorente S, Boix F, Eguia J, Lopez M, et al. C1q-fixing human leukocyte antigen assay in immunized renal patients: correlation between Luminex SAB-C1q and SAB-IgG. Transplantation proceedings. 2012;44(9):2535–7. doi: 10.1016/j.transproceed.2012.09.084. [DOI] [PubMed] [Google Scholar]
  • 53.Duquesnoy RJ, Marrari M, Jelenik L, Zeevi A, et al. Structural aspects of HLA class I epitopes reacting with human monoclonal antibodies in Ig-binding, C1q-binding and lymphocytotoxicity assays. Human immunology. 2013;74(10):1271–9. doi: 10.1016/j.humimm.2013.05.016. [DOI] [PubMed] [Google Scholar]
  • 54.Anthony RM, Wermeling F, Ravetch JV. Novel roles for the IgG Fc glycan. Annals of the New York Academy of Sciences. 2012;1253:170–80. doi: 10.1111/j.1749-6632.2011.06305.x. [DOI] [PubMed] [Google Scholar]
  • 55.Hayde N, Bao Y, Pullman J, Ye B, et al. The Clinical and Genomic Significance of Donor-Specific Antibody-Positive/C4d-Negative and Donor-Specific Antibody-Negative/C4d-Negative Transplant Glomerulopathy. Clinical journal of the American Society of Nephrology : CJASN. 2013 doi: 10.2215/CJN.04240413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56 **.Jane-Wit D, Manes TD, Yi T, Qin L, et al. Alloantibody and Complement Promote T Cell-Mediated Cardiac Allograft Vasculopathy through Non-Canonical NF-kappaB Signaling in Endothelial Cells. Circulation. 2013 doi: 10.1161/CIRCULATIONAHA.113.002972. This work showed that HLA alloantibody promotes endothelial cell activation, and that complement activation by HLA antibodies acts synergistically to trigger endothelial production of chemokines and adhesion molecules. Endothelial cell activation led to recruitment and activation of alloreactive CD4 T cells. These investigators are the first to examine the cooperative effects of HLA antibody and complement on endothelial cell activation. Moreover, their findings raise questions regarding the immunogenicity of endothelial cells and the role of the humoral response in augmenting cell-mediated alloimmunity.
  • 57.Fukami N, Ramachandran S, Narayanan K, Liu W, et al. Mechanism of accommodation in a sensitized human leukocyte antigen transgenic murine cardiac transplant model. Transplantation. 2012;93(4):364–72. doi: 10.1097/TP.0b013e3182406a6b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Jin YP, Fishbein MC, Said JW, Jindra PT, et al. Anti-HLA class I antibody-mediated activation of the PI3K/Akt signaling pathway and induction of Bcl-2 and Bcl-xL expression in endothelial cells. Human immunology. 2004;65(4):291–302. doi: 10.1016/j.humimm.2004.01.002. [DOI] [PubMed] [Google Scholar]
  • 59.Li F, Zhang X, Jin YP, Mulder A, et al. Antibody ligation of human leukocyte antigen class I molecules stimulates migration and proliferation of smooth muscle cells in a focal adhesion kinase-dependent manner. Human immunology. 2011;72(12):1150–9. doi: 10.1016/j.humimm.2011.09.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Galvani S, Trayssac M, Auge N, Thiers JC, et al. A key role for matrix metalloproteinases and neutral sphingomyelinase-2 in transplant vasculopathy triggered by anti-HLA antibody. Circulation. 2011;124(24):2725–34. doi: 10.1161/CIRCULATIONAHA.111.021790. [DOI] [PubMed] [Google Scholar]
  • 61.Bohm A, Flosser A, Ermler S, Fender AC, et al. Factor-Xa-induced mitogenesis and migration require sphingosine kinase activity and S1P formation in human vascular smooth muscle cells. Cardiovascular research. 2013;99(3):505–13. doi: 10.1093/cvr/cvt112. [DOI] [PubMed] [Google Scholar]
  • 62.Ziegler ME, Jin YP, Young SH, Rozengurt E, et al. HLA class I-mediated stress fiber formation requires ERK1/2 activation in the absence of an increase in intracellular Ca2+ in human aortic endothelial cells. American journal of physiology Cell physiology. 2012;303(8):C872–82. doi: 10.1152/ajpcell.00199.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63 *.Ziegler ME, Souda P, Jin YP, Whitelegge JP, et al. Characterization of the endothelial cell cytoskeleton following HLA class I ligation. PloS one. 2012;7(1):e29472. doi: 10.1371/journal.pone.0029472. In a phosphoproteomic analysis, HLA I crosslinking by antibodies stimulated molecular associations between the actin cytoskeleton and phosphorylated proteins involved in protein synthesis.
  • 64.Kim S, Coulombe PA. Emerging role for the cytoskeleton as an organizer and regulator of translation. Nature reviews Molecular cell biology. 2010;11(1):75–81. doi: 10.1038/nrm2818. [DOI] [PubMed] [Google Scholar]
  • 65 *.Valenzuela NM, Hong L, Shen XD, Gao F, et al. Blockade of p-selectin is sufficient to reduce MHC I antibody-elicited monocyte recruitment in vitro and in vivo. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2013;13(2):299–311. doi: 10.1111/ajt.12016. This work demonstrated that HLA I antibodies trigger rapid endothelial cell activation leading to monocyte recruitment. Concurrent engagement of monocyte Fc receptors by complement-fixing antibody significantly exacerbated monocyte adhesion to endothelial cells, suggesting that HLA I antibodies promote recruitment of monocytes into the allograft by two parallel mechanisms.
  • 66.Yamakuchi M, Kirkiles-Smith NC, Ferlito M, Cameron SJ, et al. Antibody to human leukocyte antigen triggers endothelial exocytosis. Proceedings of the National Academy of Sciences of the United States of America. 2007;104(4):1301–6. doi: 10.1073/pnas.0602035104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67.Valenzuela NM, Mulder A, Reed EF. HLA class I antibodies trigger increased adherence of monocytes to endothelial cells by eliciting an increase in endothelial P-selectin and, depending on subclass, by engaging FcgammaRs. Journal of immunology. 2013;190(12):6635–50. doi: 10.4049/jimmunol.1201434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 68.Naemi FM, Carter V, Kirby JA, Ali S. Anti-donor HLA class I antibodies: pathways to endothelial cell activation and cell-mediated allograft rejection. Transplantation. 2013;96(3):258–66. doi: 10.1097/TP.0b013e3182985504. [DOI] [PubMed] [Google Scholar]
  • 69.Mannam VK, Lewis RE, Cruse JM. The fate of renal allografts hinges on responses of the microvascular endothelium. Experimental and molecular pathology. 2013;94(2):398–411. doi: 10.1016/j.yexmp.2012.06.002. [DOI] [PubMed] [Google Scholar]
  • 70 **.Cravedi P, Leventhal J, Lakhani P, Ward SC, et al. Immune Cell-Derived C3a and C5a Costimulate Human T Cell Alloimmunity. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2013 doi: 10.1111/ajt.12405. This experimental study demonstrates that T cells express receptors for complement split products C3a and C5a, which potentiate activation of alloreactive CD4 T cells by dendritic cells. This work has important implications for the role of the complement system in cell-mediated alloimmunity, and suggests that complement activation during antibody-mediated rejection may potentiate alloreactive T cell activation.

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