The perfect storm: HLA antibodies,complement, FcgRs, and endotheliumin transplant rejectionKimberly A. Thomas*, Nicole M. Valenzuela*, and Elaine F. Reed

Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles,

Los Angeles, CA 90095, USA

Review

Glossary

Acute rejection: commonly refers to rejection that arises rapidly and causes

graft dysfunction within days to weeks, often occurring in the early post-

transplantation period (less than 1 year). May be mediated primarily by T cells

[TCMR or acute cellular rejection (ACR)] or primarily by antibodies (humoral

rejection or AMR) and can often be reversed with aggressive treatment.

Allograft: transplanted cells or solid organ from a genetically disparate

member of the same species.

Alloimmunity: adaptive immune responses against non-self cells or tissue

from members of the same species as a result of polymorphisms in proteins

that are then recognized as foreign antigens.

Chronic rejection: also called transplant allograft vasculopathy (TAV), trans-

plant arteriopathy, or transplant arteriosclerosis (TA) in cardiac allografts,

transplant glomerulopathy (TG) in renal allografts, bronchiolitis obliterans

syndrome (BOS) in lung allografts, and vanishing bile duct syndrome in liver

allografts. Progressive and irreversible fibrosis and occlusion of the donor

vasculature, distinct from native atherosclerosis in that it is concentric rather

than focal and affects only the vessels of the allograft. Thought to result from

repair mechanisms in response to successive insults or indolent, ongoing

injury from antibodies and/or CD4 T cells; manifests as an expanded

subendothelial layer comprising ECs and smooth muscle cells that have

migrated and proliferated in the neointima, as well as CD4 T cells and

macrophages.

Classical complement pathway: a system of proteases that consecutively

cleave downstream components to generate catalytically active or inflamma-

tory and cytolytic products. The classical pathway is activated by immunoglo-

bulin and initiated by binding of C1 complex to the Fc region of IgM or IgG.

Donor-specific HLA-Abs (DSAs): antibodies directed against polymorphic HLA

molecules expressed by donor tissue.

Fc receptors: receptors for the crystallizable fragment (Fc) of immunoglobulin,

The pathophysiology of antibody-mediated rejection(AMR) in solid organ transplants is multifaceted andpredominantly caused by antibodies directed againstpolymorphic donor human leukocyte antigens (HLAs).Despite the clearly detrimental impact of HLA antibodies(HLA-Abs) on graft function and survival, the prevention,diagnosis, and treatment of AMR remain a challenge.The histological manifestations of AMR reflect thesignatures of HLA-Ab-triggered injury, specifically endo-thelial changes, recipient leukocytic infiltrate, and com-plement deposition. We review the interconnectedmechanisms of HLA-Ab-mediated injury that mightsynergize in a ‘perfect storm’ of inflammation. Charac-terization of antibody features that are critical for effec-tor functions may help to identify HLA-Abs that are morelikely to cause rejection. We also highlight recentadvances that may pave the way for new, more effectivetherapies.

Rejection of solid organ transplants challenges long-term allograft survivalOrgan failure is an immense human and economic burdenthat can be successfully reversed with transplantation,substantially improving quality of life and life expectancy.In the USA, more than 100 000 patients currently awaittransplantation of major solid organs. Significant advancesin histocompatibility and immunosuppression have dra-matically improved short-term graft and patient survivalrates. Recipient recognition of donor HLA (see Glossary)present in the allograft induces an allogeneic immuneresponse resulting in the production of donor-specificHLA-Abs (DSAs). These antibodies, through many differ-ent effector functions, are responsible for the damage, andultimately graft rejection, that occurs in AMR. AMR hasemerged as a leading cause of graft dysfunction and re-duced outcomes but is often unresponsive to current ther-apies [1]. Histological markers of AMR are frequentlyunreliable and it is controversial whether intervention is

1471-4914/

� 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molmed.2015.02.004

Corresponding author: Reed, E.F. (ereed@mednet.ucla.edu).Keywords: organ transplantation; antibody-mediated rejection; HLA antibodies;classical complement pathway.*These authors contributed equally to this work.

required for patients with DSAs but no graft dysfunction.Clinical evidence suggests that DSAs alone, in the absenceof histological or molecular evidence of antibody-mediatedinjury, is not detrimental to renal allograft survival[2,3]. However, long-term follow-up studies of asymptom-atic or subclinical AMR in cardiac [4,5] and renal [6]transplantation have demonstrated increased risk forchronic rejection. Consequently, AMR remains a diagnos-tic and therapeutic challenge. Here we highlight recentdevelopments in our understanding of how HLA-Abs func-tion to cause graft injury, emphasizing the multiple effec-tor mechanisms of HLA-Abs, specifically IgG, and how theyrelate to the risk for and manifestations of AMR.

expressed by myeloid and some lymphoid cells. Link the innate immune

system with adaptive immunity; binding to complexed or immobilized

antibody triggers intracellular signaling leading to activation and inflammatory

effector functions.

Human leukocyte antigen (HLA): genes encoded by the MHC. These proteins

function in antigen presentation of peptides to T cells and are the most

polymorphic loci in the human genome.

Transplant rejection: alloimmune response of the recipient against trans-

planted donor cells, tissues, or organs.

Trends in Molecular Medicine, May 2015, Vol. 21, No. 5 319

Ac�vatedendothelium

Pericyte

CD4T

CD4T

C4d

C1q

Mono NK

NK

HLA-I AbKey: C5a receptor HLA I

HLA II

P-selec�n

C3a receptor

PSGL-1

HLA-II Ab

FcγRIIa

MacCD68

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Figure 1. Human leukocyte antigen (HLA) antibodies cause graft injury by inducing

phenotypic changes in the donor vasculature. HLA crosslinking by antibodies of

any subclass causes intracellular signaling leading to endothelial cell (EC)

activation. Activated ECs express P-selectin, which promotes recruitment of

leukocytes via interactions with P-selectin glycoprotein ligand-1 (PSGL-1).

Recruited monocytes differentiate into CD68+ macrophages, which can be

detected histologically in the capillaries and subendothelial space. Crosslinking

of HLA molecules also enhances EC immunogenicity to recipient CD4 T cells,

which proliferate and differentiate in response to alloantigen HLA class II.

Complement-activating antibodies trigger the classical pathway through binding

of C1q, resulting in production of the anaphylatoxins C3a and C5a, which have the

potential to directly augment leukocyte recruitment and T cell alloresponses.

Complement activation can be detected by immunohistochemical staining for C4d.

Monocytes, neutrophils, and natural killer (NK) cells also express Fc receptors

(FcgRs), which can interact with the heavy chain of HLA antibodies bound to donor

ECs. FcgR functions augment leukocyte recruitment and mediate phagocytosis and

antibody-dependent cellular cytotoxicity. Taken together, the pleiotropic functions

of HLA antibodies on the allograft ECs cause the microvascular inflammation

characteristic of antibody-mediated rejection. Antibodies in the figure with the

same coloration as the Fc region are of the same subclass, whereas the various

colors within the F(ab’)2 denote unique antigenic specificities.

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The alloimmune responseImmunity to alloantigens is surprisingly robust, mediatedby MHC molecules, and based on exposure to allogeneictissues. The MHC locus covers almost 4000 kb on humanchromosome 6 and is polygenic, containing three loci ofHLA class I (HLA-A, -B, and -C) and six to nine functionalHLA class II loci (a and b chains of HLA-DR, -DP, and -DQ), as well as many nonclassical MHC, minor histocom-patibility antigen, and immune-related genes. Balancingselection has resulted in extreme polymorphism withinHLA class I and class II genes. To date, over 10 000 nucleo-tide sequences encoding more than 6000 class I and2000 class II unique proteins have been reported[7]. The high allelic diversity of MHC genes is advanta-geous for protection of populations against pathogens butis highly unfavorable for cell and organ transplantation.

Immune sensitization to HLA occurs after exposure toallogeneic tissue through pregnancy, transfusion, or trans-plantation. Twenty percent of healthy individuals [8,9] andup to 30% of transplantation candidates have HLA-Abs.Another 8–25% of recipients develop de novo DSAs afterreceiving a graft [10–12]. Half of pre-sensitized patientsand a third of patients with de novo DSAs will experienceAMR within the first year after transplantation [10,12]. An-tibody responses against donor HLA proteins are notwell controlled by current immunosuppression regimens[1]. Therefore, AMR can occur at any time and is a commonoccurrence more than 1 year post-transplantation[13]. DSAs and subsequent rejection episodes are stronglyassociated with risk of chronic rejection and late graftfailure [13–15].

Histological manifestations and diagnostic criteria ofAMRAMR is best defined in renal [16], cardiac [17], and pan-creas [18] transplantation, although the diagnostic histo-logical criteria for AMR differ somewhat from organto organ. Central features include endothelial cell (EC)swelling, microvascular inflammation (subendothelialmononuclear cell infiltration), and intravascular CD68+

macrophages, with or without complement C4d deposition,often in the presence of circulating DSAs (Figure 1)[17,19,20]. While HLA-Abs are detrimental to liver [21],lung [22], and small bowel [23] allograft survival, clearpathological definitions of AMR remain contentious [17], asthe utility of C4d and other histological markers remainsunclear in these tissues.

The donor vasculature present at the interface betweendonor tissue and the recipient immune system is theprimary target of the alloimmune response. AMR is in-creasingly viewed as predominant endothelial injury andvascular inflammation [24,25] and the principal involve-ment of the endothelium in AMR has been revealed bygene-profiling studies of renal biopsies undergoing AMR[2,3,26].

HLA-Abs and subclass biologyThe fact that some patients with DSAs do not experienceAMR suggests that other factors influence susceptibility orrisk of rejection in the presence of antibodies that bind thegraft. The histological manifestations of AMR reflect the

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injurious functions of HLA-Ab binding to the vasculature,causing endothelial signaling and inflammation, activa-tion of the classical complement cascade, and recruitmentof effector cells. IgG is the most common isotype of circu-lating immunoglobulin and is divided into four subclasseswith unique patterns of biological activity. IgG3 is thestrongest activator of complement, followed closely byIgG1, and, to a far lesser extent, by IgG2 [27]. IgG4 hasno detectable complement activity and is often linked withIgG2 as ‘non-complement fixing’. However, it should benoted that, under unique conditions such as high antigen/epitope density or increased concentrations of complement

Table 1. Summary of the biological properties of human FcgRs and IgG subclasses

FcgRI, CD64 FcgRIIa, CD32a FcgRIIb, CD32b FcgRIIIa, CD16a FcgRIIIb, CD16b

Expression Monoa, Macb,activated PMNc

Mono, Mac, PMN,DCd, platelet

All immune cellsexcept T and NK

APCe (mono,DC, B), NK cell

PMN, some mono

Activating or inhibitory Activating Activating Inhibitory Activating Activating

Polymorphism None known R131 H131 I232T F158 V158 NA1/NA2

Affinity for:

IgG1 ++++ + ++ � + + �IgG2 � + + � � � �IgG3 ++++ + + � ++ +++ +

IgG4 +++ + + � � � �Murine counterpart FcgRI FcgRIII FcgRIIb FcgRIV

Adapted from [33] and [75].

aMonocyte.

bMacrophage.

cPolymorphonuclear leukocyte.

dDendritic cell.

eAntigen-presenting cell.

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and IgG [28,29], all subclasses, including IgG2 and IgG4,effectively activate complement. In addition, work withmurine MHC antibodies has demonstrated synergism be-tween high- and low-complement-fixing IgG subclasses[30–32]. While this has not yet been explored using humanIgG and complement, it is pertinent given that most anti-body responses are polyclonal and HLA is often recognizedby an admixture of subclasses.

IgG subclass interaction with Fc receptors (FcgRs) ismore complex (Table 1). In general, IgG3 and IgG1 havethe highest affinity for most FcgRs, while IgG2 and IgG4are bound by a more restricted repertoire of FcgRs. Unfor-tunately the disparity between murine and human immu-noglobulin systems limits the translation of in vivomechanistic studies of IgG subclass effector functions inmurine models of AMR to human disease [33].

After transplantation, IgG1 antibodies are directedagainst approximately 90% of HLA specificities whereasthose of IgG2/3/4 recognize about 40% or less [34–36]. Theseresults indicate a polyclonal response wherein each donorHLA antigen is recognized by multiple subclasses, mostcommonly including IgG1. It has been difficult to reconcilethe apparently conflicting results regarding the associationof DSA subclass and clinical outcome, despite reports ofIgG1/3 dominating the alloantibody responses [37]. IgG3DSAs were associated with increased risk of allograft loss inliver [35] and renal [38] transplantation. By contrast, othershave reported no correlation between DSA subclass and riskof AMR or graft loss, although one study found a trendtoward lower AMR in patients with only IgG2/4 DSAs [39].

HLA-Abs and complement activationThe historical paradigm of AMR was one of complement-mediated damage caused by classical pathway activation byFc regions of DSAs bound to the allograft [30]. In recentyears, complement-fixing DSAs have become a controversialtopic. C4d-negative AMR is becoming increasingly recog-nized and the diagnostic schemata for heart and renal AMRhave been updated to reflect this entity [20]. Experimentalmouse models of AMR suggest that acute rejection is de-pendent on complement fixation [40]. By contrast, intimalthickening during antibody-induced chronic rejection

occurred in complement-deficient murine recipients, sug-gesting that there was no requirement for complement inthis process [41]. Importantly, local production of comple-ment by donor ECs could not be excluded [42]. These resultsfrom animal models are consistent with clinical observa-tions that terminal complement inhibitors could not preventchronic rejection [43,44]. Furthermore, studies using meth-ods to define DSAs that are complement fixing, and deter-mine whether complement fixation translates to graftdamage, have had conflicting results [45–49].

Of the three complement pathways [50], the classicalpathway is primarily responsible for DSA-mediated com-plement activation. Early activation results in the produc-tion of soluble mediators such as the anaphylatoxins C3aand C5a, which are potent chemoattractants for leukocytesand alter the microvasculature by increasing vascularpermeability and inducing expression of adhesion mole-cules. The later stages are characterized by membraneattack complex (MAC) formation, which causes osmoticlysis of the target. Given the general resistance of ECs tocomplement-mediated lysis due to high expression of com-plement regulatory proteins, the physiological relevance oflytic terminal MAC formation during rejection is unclear[30]. Early complement proteins, rather than terminalMAC formation, are likely to be the mediators of mostcomplement-associated damage to the graft (Figure 2A).

Factors that dictate complement activationMany components modulate complement fixation by IgG.Of these, three are intrinsic to the antibody itself: IgGsubclass, glycosylation, and affinity (Figure 2B,C). Multi-ple studies have defined the importance of antibody affinityin dictating the level of complement activation [51]. Repeat-ed injury and consistent antigen exposure may increasethe affinity of DSAs over time, resulting in HLA-Abs thatare more inflammatory and cause robust complementinduction.

Additionally, extrinsic factors such as antigen density/epitopes and complement concentration also regulate an-tibody-induced complement activation [52]. Despite consti-tutive allograft endothelium expression of HLA class I andII [53], these levels are altered in response to inflammatory

321

(A)

(B)

(C)

IgG3

IgG3

EC

HLA I

HLA-I Ab

C1qC3a

C5a

C4dsIMAC

IgG3+Gal

IgG4

(D)

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Figure 2. Complement activation by antibody/antigen determinants. (A) Activation

of the classical complement pathway by human leukocyte antigen (HLA)

antibodies (HLA-Abs) is mediated by C1q recognition of the Fc region of IgG.

Through a subsequent series of enzymatic cleavages, the complement pathway

yields the soluble anaphylatoxins C3a and C5a, which are potent chemoattractants

and stimulators of immune responses. C4d is covalently linked to the cell surface

and is a defining marker of antibody-mediated rejection (AMR) in renal and cardiac

transplants. Additionally, sublytic membrane attack complex (slMAC), the terminal

complex bound to cells but unable to induce lysis, is proving to be an important

mediator of endothelial cell (EC) activation. Differences in antibody clonality, as

demonstrated by the antibodies of varying specificity [red or blue F(ab’)2 region],

allow increased ratios of IgG:HLA, allowing more C1q binding. (B) Antibody

subclass determines the propensity for C1q binding, as IgG3, a prominent

complement fixer, is recognized by C1q whereas the structure of IgG4 makes it a

poor C1q binding partner. (C) Differential patterning of the N297 glycan (blue

square) of IgG also modulates the level of C1q interaction. Terminal galactose

residues confer maximal C1q binding to antibodies. (D) The density of HLA antigen

on the surface of the cell, as well as the number of epitopes, strongly dictates the

level of complement activation. The proximity of antibody Fc regions is increased

when multiple antibodies can bind the same molecule of HLA. Patients with high-

titer polyclonal donor-specific antibodies (DSAs) may be predisposed to

exacerbated complement activation during times of heightened inflammation,

such as infection, when HLA expression is increased on the surface of

endothelium.

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cues [54]. Many in vitro studies have shown that increasedalloantibody bound to cells resulted in enhanced comple-ment deposition, and this was augmented under inflamma-tory conditions [55,56]. Moreover, binding of multipleantibodies with distinct epitopes to a single HLA moleculesynergistically enhanced complement activation [36]. If an-tibody subclass and antigen density/epitopes coordinate todetermine complement activation by DSAs, polyclonal anti-bodies should elicit more complement activation than mono-clonal antibodies. Sera with >80% panel reactive antibody(PRA) are strong inducers of complement activation [55,56],

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supporting the notion that differing levels of HLA antigen/epitopes determine both the quantity and quality of DSAsbound to the graft (Figure 2D).

Lastly, variations in complement can determine thedegree of activation, as some complement proteins arelocated in the MHC locus (C2, C4) and are also polymorphic[57]. Genetic predisposition to specific polymorphisms maybe useful for risk stratifying patients, and polymorphismsin complement C4 [58] but not C3 [59] have been shown toinfluence renal allograft outcome. In addition, complementconcentration is potentiated in response to local inflamma-tion. Renal epithelium, macrophages, cardiocytes, andvascular endothelium [42] are sources of extrahepaticcomplement production during episodes of rejection. Aslow-lytic antibodies have enhanced activity when comple-ment is elevated, and IgG4 activates complement whenantigen density and complement levels are increased [28],patients with minimal complement-fixing DSAs may havea higher degree of damage during rejection episodes, whencomplement and antigens are more abundant.

Measuring DSA-induced complement activationComplement activation by DSAs is a highly dynamic pro-cess responsible for mediating damage to the allograft;therefore, clinical assays that discern the complement-fixing potential of DSAs are in high demand. The lympho-cytotoxicity cross-match (CDC-XM) assay developed byMcClelland and Terasaki [60] was established for highlysensitive detection of DSAs to recipient HLAs. Althoughthis assay utilizes complement fixation as a read-out, it isnot fully reflective of the potential physiological capacity ofDSAs to activate human complement due to the use ofrabbit serum as a source of complement. It should also benoted that human IgG2 is highly effective in the activationof rabbit complement [61]; consequently, DSA subclass andCDC-XM results may not always correlate.

The development of high-throughput, single-antigen,bead-based assays has been an important tool for riskstratifying patients with complement-fixing DSAs [45,62,63]. Specifically, the C1q assay measures HLA-Abs thatbind C1q and, although informative, recognizes bindingonly,not physiologicalcomplementactivation[62]. Recently,a new assay measuring DSA-induced complement deposi-tion (C3d) reported that C3d+ DSAs were significant pre-dictors of allograft loss [64]. Collectively, these in vitrodiagnostics attempt to measure the pathogenicity of HLA-Abs with regard to their complement-fixing potential. How-ever, results differ regarding the predictive value of detect-ing complement-fixing HLA-Abs in vitro with respect toclinical outcomes [39,45,49,62,65–67], and new diagnosticcriteria for AMR include rejection without histological evi-dence of complement activation (C4d deposition) [17,20,68].

HLA-Abs and FcgRsAn almost universal histological feature of AMR is theinfiltration of CD68+ macrophages in the microvascularand perivascular spaces of heart and renal allografts[17,19,69,70] and neutrophils in lung transplants [22],which is predictive of worse outcome [69]. In addition,gene-expression profiling studies have uncovered a naturalkiller (NK) cell signature during AMR [2,71,72], findings

(A)

(B)

Mono

NK

NK

NK

NK

Cell injury

HLA-I AbKey: HLA I

HLA II

P-selec�n

HLA-II Ab

FcγRIIa PSGL-1

Perforin

C5a receptor

MacCD68

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Figure 3. Fc-mediated functions that contribute to graft injury. (A) Increased

leukocytic infiltrate is a hallmark of antibody-mediated rejection (AMR) and this

occurrence is mediated by the Fc region of donor-specific antibodies (DSAs). On

DSA binding to human leukocyte antigens (HLAs), DSA Fc is recognized by Fc

receptors (FcgRs) expressed on myeloid and natural killer (NK) cells. Additionally,

monocytes are able to interact with the endothelium through HLA-induced P-

selectin to enhance tethering and extravasation. (B) An important feature of FcgRs

is their role in antibody-dependent cell-mediated cytotoxicity (ADCC). DSAs bind to

HLAs on the surface of the endothelium, facilitating Fc interaction with FcgRs

expressed by myeloid and NK cells. This can lead to perforin-mediated lysis of

target cells – in this case, in the endothelium – resulting in damage to the allograft.

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that were paralleled by experimental animal models ofAMR implicating NK cells in chronic antibody-mediatedrejection [73]. Monocytes, macrophages, neutrophils, andNK cells express receptors for the Fc region of antibodies(Table 1) [74] and FcgRs mediate innate immune cellfunctions such as leukocyte recruitment, cytotoxicity,and phagocytosis that are highly relevant to the etiologyof AMR (Figure 3).

FcgR families and allelesFcgR families and alleles have distinct subclass specifici-ties and divergent activities (Table 1) [33,75]. Moreover,functional polymorphic variants of FcgRIIa (H131R),FcgRIIIa (F158V), and FcgRIIIb (NA1/NA2 alleles) areassociated with differential phenotypes in response toantibody-based antitumor therapies, susceptibility to in-fection, and risk of autoimmune disease [76]. In the context

of transplantation, the low-affinity FcgRIIa-R131 allelewas associated with increased risk of acute T cell-mediatedrejection (TCMR) [77], but this is likely a reflection ofreduced responsiveness to antibody-based leukocyte deple-tion induction regimens rather than predisposition to re-jection per se. However, the effect of transplant recipientFcgR polymorphism on risk of AMR has not yet beenstudied and warrants investigation.

As with IgG subclasses, the human FcgR system isdissimilar from the murine system, complicating the studyof FcgRs in vivo and confounding the translation of experi-mental results in murine models of AMR to the humansetting. A recently described novel transgenic mouse car-rying the full repertoire of human FcgRs [78] may enablefuture studies. Several important caveats, however, in-cluding cross-reactivity of human FcgRs with endogenousmurine IgG and the representation of only one FcgRgenotype, may limit findings [33].

FcgR functions relevant to graft injuryFcgRs on monocytes, macrophages, and NK cells facilitateantibody-dependent cell-mediated cytotoxicity (ADCC).While HLA-Abs trigger NK cell degranulation and cyto-toxicity against allogeneic target cells in vitro [79], andmacrophages also perform ADCC, currently there is nodirect evidence that these cells cause cytotoxicity in thegraft. However, murine models of chronic AMR haverevealed a novel role for NK cells in MHC antibody-inducedtransplant vasculopathy [73], through undefined FcgR-dependent mechanisms. An elegant study imaging thetrafficking of recipient immune cells into murine cardiacallografts revealed elevated phagocytic activity duringrejection, mediated by recipient macrophages [80]. HLA-Abs may provoke antibody-dependent cellular phagocyto-sis (ADCP) by macrophages and neutrophils, contributingto enhanced presentation of alloantigen to T cells, but thepathophysiological relevance of phagocytosis during rejec-tion remains to be explored.

Finally, FcgRs are involved in the capture of leukocytesby immune complexes (ICs) and monomeric anti-endothe-lial cell antibodies, and enhanced trafficking of neutrophilsto inflamed endothelium in autoimmune settings [81]. It isnotable that there was a prerequisite for tumor necrosisfactor alpha (TNFa) activation of endothelium, as deposi-tion of antibody on resting cells did not cause efficientneutrophil adhesion. Moreover, concurrent expression ofchemokines was required for efficient neutrophil adhesionto ECs coated with monomeric IgG but not with ICs. It wasrecently demonstrated that monocyte recruitment to HLA-Ab-activated endothelium was augmented by the interac-tion of monocyte FcgRs with the Fc portion of the HLA-Abs[82,83]. This interaction was subclass dependent, influ-enced by monocyte FcgRIIa allelic variants, and abrogatedby enzymatic modulation of antibody Fc regions usingEndoS or IdeS [83]. In contrast to reports using murineanti-endothelial cell antibodies [81], efficient recruitmentwas observed using HLA-Abs without pre-activating ECswith inflammatory cytokines, and it has been hypothesizedthat HLA-Abs are unique in their capacity to trigger directendothelial activation and expression of selectins as wellas stimulate FcgRs [82]. Interestingly, monocytes from

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donors who expressed the high-affinity FcgRIIa-H131 al-lele exhibited significantly greater FcgR-dependent adhe-sion to ECs activated with HLA-Abs of both IgG1 and IgG2subclasses compared with monocytes expressing onlyFcgRIIa-R131. These results suggest that transplant re-cipients carrying high-affinity FcgR alleles may experienceexacerbated leukocyte infiltration in response to HLA-Abs,predisposing them to AMR.

HLA-Abs and glycosylationPatterns of antibody glycosylation strongly influence affin-ity of FcgRs [84]. The bulk of evidence comes from the fieldsof tumor immunology and recombinant therapeutic anti-bodies, through glycoengineering of antibodies to alterADCC and complement-dependent cytotoxicity (CDC)properties. In addition, several studies have correlatedthe degree of IgG-Fc glycosylation with the severity ofantibody-mediated disease [85]. A common theme appears:antibodies with agalactosylated Fc-glycans are more proin-flammatory than those containing glycans with terminalgalactosylation or sialic acid. As the properties of glycosyl-ation moieties modulate the inflammatory nature of IgG,the Fc-glycan may participate in determining the degree ofpathogenicity of DSAs with respect to AMR.

The conserved yet highly heterogeneous N297 glycanpresent on the Fc of all IgGs [27,86] contains a biantennarycore heptasaccharide that is further modified by the addi-tion of fucose (over 90% of IgGs), galactose, and sialic acidto greatly diversify the IgG glycoform pool. Variouschanges to this structure can completely alter the functionof IgG with respect to both FcgR and complement-depen-dent activities (reviewed elsewhere [27,87]). In brief, re-moval of fucose increases ADCC whereas removal ofgalactose residues reduces ADCC (mediated by FcgRIIIa)and CDC. Interestingly, sialic acid has been identified asthe mediator of the anti-inflammatory properties of intra-venous immunoglobulin (IVIg) [84], a common modality inthe treatment of AMR. Whereas all sialic acid linkagescontribute to decreased ADCC, the alpha-2,6 version isresponsible for the anti-inflammatory effects of sialylatedIgG, through direct binding of SIGN-R1/DC-SIGN, causingupregulation of inhibitory FcgRs. Although there is mini-mal literature regarding differential glycosylation pat-terns of DSAs, one would be remiss to disregard thepotential role of DSA glycan heterogeneity during thecourse of AMR.

Regulation of IgG glycosylationGiven that both complement activation and FcgR engage-ment are key effector functions of HLA-Abs in causingallograft injury, the Fc region of the antibody is a potentialtherapeutic target. The Gram-positive bacterium Strep-tococcus pyogenes expresses a battery of immunomodula-tory enzymes that aid in its pathogenicity, two of whichhave shown promise in preclinical autoimmune modelsthrough specific actions on IgG [88]. The peptidase IdeScleaves off the Fc fragment of human IgG, generating aF(ab’)2 fragment, while the endoglycosidase EndoS hydro-lyzes the N297-linked Fc glycan. Both ameliorate inflam-mation, complement activation, and FcgR-dependentleukocyte recruitment in several experimental models

324

and treatment of HLA-Abs with either EndoS or IdeSdramatically reduced the recruitment of monocytes toECs [83]. Clinical trials are currently under way testingthe efficacy of IdeS in sensitized kidney transplant recip-ients (NCT02224820).

Glycan analysis of antibodies produced during inflam-mation in response to pathogens or autoimmune diseasehave shown an increased proportion of agalactosylatedIgG. Moreover, IgGs from active immune responses havealtered glycan profiles that differ from normal serumIgGs [89,90]. The mechanism by which antibodies areglycosylated during immune responses is not well under-stood, although distinct glycan profiles from individualpatients suggest differential glycosylation by unique Bcell subsets [91,92]. This indicates that the ability toregulate levels of glycosylation relies on B cell-intrinsicfactors and would be subject to the immune milieu. Inthis regard, B cells presented with T-dependent antigensunder proinflammatory conditions produced antibodythat lacked galactose (proinflammatory) [89,93], whereasantibodies produced in response to T-dependent antigensbut under tolerogenic settings were heavily sialylated(anti-inflammatory) [89]. Finally, in the context ofT-independent antigens, regardless of the inflammatorysurroundings IgGs were sialylated and immunosuppres-sive [93].

This comprehensive understanding of the contributionof Fc-glycan to the immune function of IgG and thecircumstances modulating the production of these glyco-sylated antibodies allows conjecture regarding the patho-genic potential of DSAs. One could surmise that acuterejection episodes increase levels of agalactosylated DSAs,which would incur damage to the graft through bothcomplement and FcgR pathways, whereas DSAs presentin accommodated grafts may be heavily sialylatedand somewhat tolerogenic. Future work detailing theglycan profiles of DSAs would determine whether anti-body glycosylation status correlates with the severity ofAMR. Additionally, a new methodology described to si-multaneously measure both the subclass and glycosyla-tion of antigen-specific IgG [94] may be adapted totransplantation.

HLA-Abs and endothelial activation and regulation ofimmunogenicityThere has been resurgence in the appreciation of ECs asimportant regulators of the immune response. ECs canundergo acute (Type I) or chronic (Type II) activation,leading to expression of chemokines and adhesion moleculesand recruitment of leukocytes to sites of inflammation[95]. Past work showed that crosslinking of HLA by anti-bodies triggers intracellular signaling through focal adhe-sion kinase (FAK), Akt, mammalian target of rapamycin(mTOR), S6 kinase (S6K), S6 ribosomal protein (S6RP), andextracellular signal-regulated kinase (ERK1/2) in endothe-lial and smooth muscle cells leading to dynamic cytoskeletalreorganization and increased cell proliferation, migration,and survival [96]. Multiple groups recently confirmed theactivation of these signaling pathways in biopsies fromcardiac allografts undergoing AMR [97,98]. Importantly,the agonistic signaling capacity is an observed property of

Review Trends in Molecular Medicine May 2015, Vol. 21, No. 5

all HLA-Abs requiring the bivalent F(ab’)2 region of IgG anddoes not appear to depend on subclass, complement, orFcgRs. Alternatively, complement activation, antigenexpression, epitope density, and antibody affinity will allsignificantly impact the binding to and crosslinking of HLAson ECs, in turn affecting intracellular signaling.

Recent studies demonstrated that HLA class I signalingtriggers Type I EC activation, resulting in a rapid increaseof cell surface P-selectin and the adhesion of neutrophilsand monocytes to endothelium [99,100]. Exocytosed vonWillebrand factor (vWF) and P-selectin also facilitated thecapture and activation of platelets, which aggregate in themicrovasculature and support the tethering of monocytes[101]. Platelets express FcgRIIa [102]; therefore, addition-al mechanisms of FcgR-dependent platelet adhesion can-not be excluded. HLA crosslinking also activated thetranscription factors cAMP response element-binding pro-tein (CREB) and noncanonical nuclear factor kappa B (NF-kB), resulting in increased protein expression of late phase

(A)

MonoMono

1

2

3

4

5

HLA-I AbKey: C5a receptor

C3a receptor

VCAM-1

IL-6

FcγRIIa

6a

MacCD68

6b

Figure 4. Proposed models of donor-specific antibody (DSA)-induced inflammatory loo

receptor (FcgR) pathways. DSA crosslinking of human leukocyte antigens (HLAs) on endo

platform for C1q (1). Classical complement activation produces C5a (2), which has two fu

P-selectin via P-selectin glycoprotein ligand-1 (PSGL-1), promoting graft infiltration a

macrophages and induce FcgR expression (4). These cells can recognize immune comp

C5a may signal in either an autocrine (6a) or a paracrine (6b) fashion, mediating furthe

from the periphery, respectively. (B) Recent studies have identified a novel role for ECs a

inflammatory conditions [such as interferon gamma (IFNg) activation], ECs express H

promoting allo-CD4 T cell proliferation (4) and differentiation into T helper 17 (Th17) and

modulate endothelial alloimmunogenicity through activation of the classical compleme

(2). MAC triggers noncanonical nuclear factor kappa B (NF-kB) signaling leading to infla

also express receptors for the complement split products C3a and C5a, which provide c

presence of these anaphylatoxins at the endothelial–T cell interface might enhance T c

adhesion molecules, cytokines, and chemokines [56,103],consistent with Type II EC activation.

An expanding paradigm of the role of vascular endothe-lium in directly stimulating adaptive immune responseshas gained attention [104,105]. HLA class II-expressingECs trigger allogeneic CD4 T cell proliferation and pro-mote generation of T helper 17 (Th17) and regulatory T cell(Treg) subsets [106,107]. Interestingly, rapamycin treat-ment of ECs resulted in selective expansion of Tregs viaprogrammed death ligand (PD-L) 1 and 2 [107], indicatinga role for mTOR in the regulation of endothelial alloim-munogenicity through modulation of costimulatory mole-cule expression. The mTOR inhibitors sirolimus andeverolimus also prevent HLA I antibody-induced endothe-lial migration and proliferation [108], suggesting thatrapalogs may be beneficial in preventing multiple mani-festations of graft injury by HLA-Abs. A recent studyshowed HLA-Abs increased expression of proinflammatorycytokines and the activation of noncanonical NF-kB [56],

1

2

3NFκB

Pro-inflammatorytranscrip�on

5

4CD4

T

C5a HLA I VLA-4

IL-6 receptor

Subly�c MAC

HLA II

PSGL-1

C3a

P-selec�n

(B)

TRENDS in Molecular Medicine

ps. (A) Macrophages perpetuate activation and recruitment via complement and Fc

thelium results in P-selectin mobilization to the cell surface and provides a binding

nctions: (i) it recruits monocytes to activated endothelial cells (ECs), which tether to

nd differentiation into macrophages (3); and (ii) it may act on intragraft CD68+

lexes (ICs) via FcgRs, which can upregulate C5a production (5). Newly synthesized

r activation of intragraft macrophages or recruitment and activation of monocytes

nd complement in antigen presentation and stimulation of allogeneic T cells. Under

LA class II as well as ICAM-1, VCAM-1, and IL-6 – molecules that are critical for

regulatory T cell (Treg) subsets. Preliminary work has shown that HLA antibodies

nt pathway (1) resulting in deposition of sublytic membrane attack complex (MAC)

mmatory gene expression (3) and stimulation of allogeneic CD4 T cells (4). T cells

ostimulatory signals and augment T cell proliferation. Therefore, it is likely that the

ell alloimmunity (5).

325

Determinants

Effectorfunc�ons

Mechanismsof injury

Histologicalmanifesta�ons

of AMR

Macrophageinfiltrate

CD68+

Leukocyterecruitment

Complementac�va�on Endothelial

ac�va�on

HLA crosslinkingC1q bindingFcyR binding

Subclass Glycosyla�on Concentra�onAn�gen densityepitopes

Complementdeposi�on

C4d+

Endothelialchanges

EC swellingphospho markers

TRENDS in Molecular Medicine

Figure 5. Mechanisms of donor-specific antibodies (DSAs) in graft pathogenesis.

Features of antibody–antigen and antibody–effector system interactions that

influence pathogenic functions and mechanisms of injury are shown. Variable

factors regulating antibody–antigen interactions (blue ovals) directly influence the

capacity of an antibody to trigger effector functions (yellow boxes) and

mechanisms causing graft injury (green boxes), which ultimately manifest in the

graft as common histological features (red circles). Linear effects are indicated by

solid arrows. The functional end points of antibody-mediated injury are

interrelated (with potential inflammatory loops indicated by broken arrows) and

are likely to synergize to cause maximal inflammation during antibody-mediated

rejection (AMR). For example, direct endothelial cell activation by human leukocyte

antigen (HLA) antibodies triggers adhesion of leukocytes, which can be enhanced

when those leukocytes bind antibody through Fc receptors (FcgRs). Activation of

complement at the endothelial cell surface may cause production of the

anaphylatoxins C3a and C5a, which can act on leukocytes as chemoattractants,

or enhance endothelial activation.

Box 1. Outstanding questions

� Which mechanisms of HLA-Abs are critical for rejection and graft

injury and how do these mechanisms vary depending on antibody

characteristics?

� What are the effector functions of NK cells during AMR? Do ADCC

and ADCP play a mechanistic role in AMR?

� Can we reliably define the HLA-Ab repertoire, including specifi-

city, glycosylation, complement-fixing capacity and subclass

distribution, of transplantation patients? In particular, do current

in vitro assays of complement detection reliably predict whether

HLA-Abs will cause complement-mediated injury?

� Are some patients predisposed to experience rejection in the

presence of antibodies? Does the glycan profile of DSAs or the

recipient FcgR genotype influence transplant outcome?

� Should patients be treated when they have DSAs but no evidence

of graft dysfunction?

� What is the significance of C4d-negative AMR? Does it represent

complement-independent graft injury by non-complement-fixing

antibodies or is it capturing AMR after complement is no longer

active?

Review Trends in Molecular Medicine May 2015, Vol. 21, No. 5

indicating that HLA-Abs modulate endothelial immunoge-nicity and antigen presentation to T cells.

Inflammatory loops and interplay between antibodyfunctionsConcurrent processes of EC activation, classical comple-ment activation, and FcgR-dependent immune cell func-tions are likely to independently and cumulativelypromote graft inflammation during AMR. Crosstalk be-tween FcgR and complement adds another level of com-plexity to IgG modulation of the immune response[109]. Abrogation of either Fc/FcgR or C5a/C5aR signalingabolished inflammation induced by ICs and it is knownthat both are necessary for robust immune responses. C5aacts directly on macrophages, simultaneously upregulat-ing activating FcgR and downregulating inhibitory FcgR[110,111]. Additionally, IC binding to macrophagesthrough FcgRIII induced C5a synthesis [112]. Further-more, binding of C5a to Kupffer cells triggered increasedexpression of activating FcgR, which bound ICs, therebystimulating C5a production and creating a proinflamma-tory loop [113]. This cycle could potentially translate toexacerbated AMR-associated pathophysiology. Local acti-vation of complement in the graft by DSAs can activatemacrophages and increase expression of FcgR, which maybind sequestered DSA ICs, thereby augmenting local C5aproduction (Figure 4A). In addition to the direct effects ofcomplement on macrophages, anaphylatoxins and subly-tic MAC enhance EC activation. Endothelial NF-kB sig-naling and inflammatory gene expression induced by DSAbinding was augmented in the presence of sublytic MACand led to increased T cell stimulation [56]. These findingsdemonstrate an additional mechanism of synergy betweencomplement and HLA-Abs in endothelial activation(Figure 4B). As C5a is a potent mediator of leukocyterecruitment as well as a novel modulator of T cell alloim-munity [114], this DSA-induced inflammatory loop couldexacerbate damage during episodes of AMR.

Concluding remarks and future perspectivesIn summary, graft injury results from the pleiotropic func-tion of antibodies (Figure 5), through canonical Fc-mediat-ed effector functions as well as novel agonistic actions onHLA molecules. The collective action of antibodies on donorvascular cells, including complement activation, FcgR-de-pendent macrophage and NK cell functions, and EC acti-vation, are likely to synergize, causing damage to theallograft. Features such as antibody subclass, Fc glycosyl-ation, and FcgR polymorphisms may be key determinantsof HLA-Ab pathogenicity and recipient risk of AMR. There-fore, characterization of the patient’s DSAs and immunerepertoire provides a foundation for individualized medi-cine as well as possible guidelines for the risk stratificationof transplantation patients. Highly tailored and specificimmunotherapies could be used in the transplantationfield to modulate patient immune responses according tothe details of an individual’s immune repertoire. Furtherexperimental dissection of alloimmunity variables (Box 1)will guide future practice in allocation/antigen avoidance,management in sensitized patients, and the developmentof new drugs to prevent and treat AMR.

326

AcknowledgmentsThis work was supported by the Ruth L. Kirschstein National ResearchService Award T32HL69766 (to K.A.T.) and the National Institute ofAllergy and Infectious Diseases Grant R01AI042819 (to E.F.R.). Theauthors thank A. Roux and M. Rossetti for critical review of the manuscript.

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