Donor-Specific Antibodies in Kidney

Transplant Recipients
Rubin Zhang

Abstract
Donor-specific antibodies have become an established biomarker predicting antibody-mediated rejection.
Antibody-mediated rejection is the leading cause of graft loss after kidney transplant. There are several phenotypes Section of
of antibody-mediated rejection along post-transplant course that are determined by the timing and extent of Nephrology,
humoral response and the various characteristics of donor-specific antibodies, such as antigen classes, specificity, Department of
Medicine, Tulane
antibody strength, IgG subclasses, and complement binding capacity. Preformed donor-specific antibodies in
University School of
sensitized patients can trigger hyperacute rejection, accelerated acute rejection, and early acute antibody- Medicine, New
mediated rejection. De novo donor-specific antibodies are associated with late acute antibody-mediated rejection, Orleans, Louisiana
chronic antibody-mediated rejection, and transplant glomerulopathy. The pathogeneses of antibody-mediated
rejection include not only complement-dependent cytotoxicity, but also complement-independent pathways of Correspondence: Dr.
antibody-mediated cellular cytotoxicity and direct endothelial activation and proliferation. The novel assay for Rubin Zhang, Section
of Nephrology,
complement binding capacity has improved our ability to predict antibody-mediated rejection phenotypes. C1q
Department of
binding donor-specific antibodies are closely associated with acute antibody-mediated rejection, more severe graft Medicine, Tulane
injuries, and early graft failure, whereas C1q nonbinding donor-specific antibodies correlate with subclinical or Transplant Institute,
chronic antibody-mediated rejection and late graft loss. IgG subclasses have various abilities to activate Tulane University
complement and recruit effector cells through the Fc receptor. Complement binding IgG3 donor-specific School of Medicine,
1430 Tulane Avenue,
antibodies are frequently associated with acute antibody-mediated rejection and severe graft injury, whereas SL-45, New Orleans,
noncomplement binding IgG4 donor-specific antibodies are more correlated with subclinical or chronic antibody- LA 70112. Email:
mediated rejection and transplant glomerulopathy. Our in-depth knowledge of complex characteristics of rzhang@tulane.edu
donor-specific antibodies can stratify the patient’s immunologic risk, can predict distinct phenotypes of antibody-
mediated rejection, and hopefully, will guide our clinical practice to improve the transplant outcomes.
Clin J Am Soc Nephrol 13: 182–192, 2018. doi: https://doi.org/10.2215/CJN.00700117

Introduction Sensitization and DSA Identification

Antibody-mediated rejection has been recognized as
the leading cause of graft dysfunction and graft loss
after kidney transplant (1–4). Donor-specific anti-
bodies (DSAs) identified before kidney transplant
(preformed DSAs) can cause early rejection, such as
hyperacute rejection, accelerated acute rejection, early
acute antibody-mediated rejection, and graft loss
(1–6). Alternatively, de novo developed DSAs after
transplant are associated with late acute antibody-
mediated rejection, chronic antibody-mediated rejection,
and transplant glomerulopathy (3,4,7,8). However, there
are also “benign” DSAs that may not be clinically
relevant, because they are not associated with anti-
body-mediated rejection or graft failure (8–11). This
paper reviews the identification of DSAs and dis-
cusses their complex characteristics, including anti-
body classes, specificity, strength, IgG subclasses,
and complement binding capacity, as well as the
different phenotypes of antibody-mediated rejection
after kidney transplant. The advance in our under-
standing of DSA pathogenicity can help clinicians to
stratify patient’s immunologic risk, predict the phe-
notypes of antibody-mediated rejection, and guide our
clinical management.
Sensitization is defined by the presence of anti-
bodies in the recipient’s blood against a panel of
selected HLAs representing donor population. It is
reported as the percentage panel reactive antibody.
Panel reactive antibody estimates the likelihood of
positive crossmatch to potential donors (11). Sensiti-
zation is caused by previous exposure to HLA anti-
gens, usually through organ transplant, pregnancy, or
blood transfusion (5,6). Particularly relevant is the
exposure of a woman to her partner’s HLA during
pregnancy. This results in direct sensitization against
the partner, potentially making the partner and/or her
child an unsuitable donor (11). The technology of
screening antibodies has been advanced from the
complement-dependent cytotoxicity assay, the enzyme-
linked immunoabsorption, to multiplexed particle-based
flow cytometry (Luminex). Single antigen beads are
used to characterize the preformed DSAs before trans-
plant as well as any de novo development of DSAs after
transplant (4,11).
Luminex assay can characterize the preformed HLA
antibodies in sensitized patients awaiting trans-
plant. The recurrent antibodies or highly expressed
antibodies are considered clinically significant. The

182 Copyright © 2018 by the American Society of Nephrology www.cjasn.org Vol 13 January, 2018
Clin J Am Soc Nephrol 13: 182–192, January, 2018
corresponding antigens are regarded as unacceptable for
that patient, and in the United States, they are listed into the
United Network of Organ Sharing database. A patient will
not be offered a kidney from the deceased donor who
expresses an unacceptable HLA antigen (positive virtual
crossmatch). Only those patients whose HLA antibodies are
not donor directed will appear on the match run (negative
virtual crossmatch). Such “virtual crossmatch” improves
efficiency of organ allocation (12). When a potential donor is
identified, a final crossmatch with fresh serum from re-
cipient and lymphocytes from donor is performed to rule
out preformed DSAs before transplant surgery. The com-
monly used tests are complement-dependent cytotoxicity
crossmatch and flow cytometry crossmatch (11,12).
DSA Pathogenesis
Presence of DSA, either preformed or de novo, has become a
well established biomarker predicting poor transplant out-
comes, including high incidence of antibody-mediated re-
jection, graft dysfunction, and inferior graft survival. The
development of de novo DSAs after kidney transplant was
reported in 13%–30% of previously nonsensitized patients
(13–20). The risk factors for de novo DSA include the
following: (1) high HLA mismatches (especially DQ mis-
matches), (2) inadequate immunosuppression and nonad-
herence, and (3) graft inflammation, such as viral infection,
cellular rejection, or ischemia injury, which can increase
graft immunogenicity (18–20). De novo DSAs are predom-
inantly directed to donor HLA class 2 mismatches and
usually occur during the first year of kidney transplant, but
they can appear anytime, even several years later (18–20).
Binding of DSA to antigen expressed on allograft endothe-
lial cells can activate classic complement pathway, a key
pathologic process of acute antibody-mediated rejection
phenotypes (4,11). Even in absence of complement activa-
tion, some DSAs can cause graft damage through antibody-
dependent cellular cytotoxicity. The innate immune cells,
including neutrophils, macrophages, and natural killer cells,
can bind to Fc fragments of DSAs, trigger degranulation,
and release lytic enzymes, which cause tissue injury and cell
death. This process can mediate smoldering damages to the
endothelial cells and is proposed as an important patho-
genesis in subclinical and chronic antibody-mediated re-
jection phenotypes (21–23). Furthermore, DSAs can cause
graft injury by direct activation of endothelial proliferation
through increasing vascular endothelial growth factor pro-
duction, upregulating fibroblast growth factor receptor, and
increasing its ligand binding as well as other signaling
pathways for cellular recruitment (8,23–25). This pathogenesis
may contribute to transplant glomerulopathy and vasculop-
athy that feature vascular intima thickness with smooth
muscle cell invasion. The latter two complement-independent
mechanisms can explain the clinical phenotypes of antibody-
mediated rejection with negative C4d staining in peritubular
capillaries. Figure 1 illustrates the three proposed pathogen-
eses of DSA in antibody-mediated rejection (4,8,11,21–24).
DSA Classes and Specificity
DSAs target specific epitopes in the polymorphic regions
of HLA antigens. HLA class 1 antigens (A, B, and C) are
Donor-Specific Antibodies, Zhang 183
expressed on all nucleated cells, and each antigen consists
of one a-chain and one b2-microglobulin. The epitopes
reside only in the polymorphic a-chain (6,7,11). HLA class 2
antigens (DR, DQ, and DP) are normally restricted to
antigen-presenting cells (dendritic cells, B cells, and mac-
rophages), but they can be upregulated and expressed after
inflammatory insults, such as ischemia-reperfusion injury,
infection, and rejection (6–10). Each class 2 antigen consists of
one a-chain and one b-chain, and both chains are poly-
morphic. The b-chain of DQ is particularly polymorphic,
which adds clinical complexity of DQ antibodies (25).
Preformed DSAs in sensitized patients can be class 1, class
2, or both, and they may target either private or public
epitopes (26,27). Presence of preformed DSAs directly affects
the transplant decision making. Kidney transplant should not
proceed if there is a positive T cell crossmatch secondary to
cytotoxic IgG antibody, which is usually complement bind-
ing IgG1 or IgG3 subclass (6,8,27,28). The majority of de novo
DSAs after kidney transplant are class 2 antibodies, especially
DQ. Class 1 de novo DSAs are usually detected sooner after
transplant and more likely IgG1 and IgG3 subclasses. They
are associated with acute antibody-mediated rejection and
early graft loss (7,8). Class 2 de novo DSAs appear later and
are commonly noncomplement binding IgG2 or IgG4 sub-
class. They tend to be persistent and are associated with
chronic antibody-mediated rejection and transplant glomer-
ulopathy (6,8,29,30). Trying aggressively to eliminate class 2
DSA, especially the DQ, may not be successful, and it can put
patients at great risk of excessive immunosuppression
without much benefit (27). Table 1 compares the predomi-
nant characteristics of classes 1 and 2 DSAs, although certain
overlaps do exist between the two classes (6–10,25–30).
DSA Strength
The DSA strength (or titer) is usually expressed as the
mean fluorescence intensity by Luminex solid-phase assay.
Early studies have shown the clinical correlation between
mean fluorescence intensity and risk of antibody-mediated
rejection. High titer of DSA has been correlated with
complement binding capability and more severe tissue
injuries (28–34). Theoretically, high DSA titer provides a
sufficient number of IgG molecules that can bind to anti-
gens close together to form hexametric complexes that
activate complement more effectively (28,31). However,
the correlation between DSA strength and clinical out-
come is far from perfect. DSAs with similar mean fluores-
cence intensity do not always activate the complement
cascade. There are patients with transplants with high levels
of circulating DSAs who escape rejection or graft dys-
function (6,8,11). The ability of DSAs to bind on beads
may not be the same as that to bind on HLA antigens of
endothelial cells. Currently, there is no standardization and
normalization of the solid-phase bead assays. The thresh-
olds reported for clinically significant mean fluorescence
intensity vary widely between studies from 1000 to 10,000
depending on the antigen specificities (6,8,27,28,35). Our
HLA laboratory uses the common cutoffs of 3000 for class 1
DSAs and 5000 for class 2 DSAs as clinically significant
mean fluorescence intensity (1,6). Several technical limita-
tions of solid-phase bead assays have been captured. False
positive or high titers may be reported due to the presence
184 Clinical Journal of the American Society of Nephrology

Figure 1. | The three proposed pathogeneses of donor-specific antibodies (DSAs) in antibody-mediated rejection. Donor antigen-presenting
cells include macrophages, dendritic cells, and B cells. Complement binding DSAs target the class 1 HLA on endothelial cells, activate the classic
complement cascade, and deliver complement-dependent cytotoxicity in acute antibody-mediated rejection. Complement nonbinding DSAs
recruit innate immune cells (NK cells, macrophages, and neutrophils) through Fc receptors and lead to antibody-dependent cellular toxicity. In
addition, complement nonbinding DSAs have direct stimulation and pleotrophic effects that cause tissue injury, cellular recruitment, and
endothelial proliferation. The latter two mechanisms play an important role in acute antibody-mediated rejection with negative C4d deposit in
peritubular capillaries as well as chronic antibody-mediated rejection, transplant glomerulopathy, and vasculopathy (4,8,11,21–24). APC,
antigen-presenting cells; NK, natural killer cells.

of antibodies to denatured HLA molecules. DSAs targeting high levels of DSAs (26–28,35). Tambur et al. (28) noted that
one of the shared epitopes may be diluted across the beads. the “prozone effect” was very common (71%) in patients
False negative or low titers can also occur in the presence of with multiple DSAs, and serial dilution of sera before assay
inhibitors or “prozone effects,” affecting the assay of very provided more accurate measure of DSA strength.
Clin J Am Soc Nephrol 13: 182–192, January, 2018 Donor-Specific Antibodies, Zhang 185

Table 1. Comparison of the dominant characteristics of classes 1 and 2 donor-specific antibodies (6–10,25–30)

Class 1 Donor-Specific Class 2 Donor-Specific

Antibodies Antibodies

HLA
Antigens A, B, and C DR, DQ, and DP
Epitopes location a-chain a- and b-chains
Expression All nucleated cells Antigen-presenting cells
Preformed donor-specific antibodies
Important Very Less
Positive crossmatch T cells B cells
Transplant decision No transplant Permissible
De novo donor-specific antibodies
Detection Sooner Later
IgG subclasses IgG1, IgG3 IgG2, IgG4
Complement binding Strong Weak/no
Frequency Fewer Common, especially DQ
Antibody-mediated rejection
Phenotypes Acute Chronic, subclinical
Presentation Early Later
Graft dysfunction Rapidly Slowly
C4d deposit Positive Negative
Treatment More responsive Less responsive
Graft loss Early Later

Complement Binding DSA The incidences of chronic antibody-mediated rejection were

The recent advance of Luminex-based assay for comple-
ment binding DSAs has significantly improved our ability
to predict antibody-mediated rejection phenotypes and
graft failure (13–17,29–37). Several studies have shown
that, compared with C1q nonbinding DSAs, C1q binding
DSAs are associated with significantly higher risk of
antibody-mediated rejection, severe tissue injury, and graft
loss (13–18,33–38). In the large cohort study from Loupy
et al. (33), C1q binding DSA detected at 1 year after kidney
transplant or during antibody-mediated rejection was an
independent factor of graft loss. It increased the risk of graft
loss by 4.78-fold (95% confidence interval [95% CI], 2.69 to
8.49). C1q binding DSA remained independently associ-
ated with the risk of loss of the kidney allograft after
adjustment for the mean fluorescence intensity (hazard
ratio [HR], 4.5; 95% CI, 2.2 to 9.0). Compared with patients
with C1q nonbinding DSAs, C1q binding DSAs were
associated with higher incidence of antibody-mediated
rejection with more severe graft injuries that included more
extensive inflammation, microvasculitis, endarteritis,
transplant glomerulopathy, and C4d deposits (33). Re-
cently, Guidicelli et al. (18) compared the long-term effects
of C1q binding versus C1q nonbinding DSAs on graft
survivals. They found that both are associated with graft
loss. However, C1q binding DSAs were associated with
graft loss occurring quickly after their appearances, and
C1q nonbinding DSAs led to graft loss in the long term.
Calp-Inal et al. (36) studied C1q binding DSA in 284
patients prospectively (group 1) as well as 405 patients
retrospectively (group 2). The incidences of acute antibody-
mediated rejection were significantly higher in patients
with C1q-positive DSAs in groups 1 (45%) and 2 (15%)
compared with patients with C1q-negative DSAs (5% and
2%, respectively) and patients who were DSA negative (1%
and 3%, respectively; P,0.001 and P=0.001, respectively).
also significantly higher in patients with C1q-positive DSAs
in groups 1 (36%) and 2 (51%) compared with patients with
C1q-negative DSAs (5% and 25%, respectively) and pa-
tients who were DSA negative (2% and 6%, respec-
tively; P,0.001). Graft survival in group 1 was numerically
lower in patients with C1q-positive DSAs (73%) compared
with patients with C1q-negative DSAs (95%) and patients
who were DSA negative (94%; P=0.21). Presence of C1q
binding DSAs was associated with both acute and chronic
phenotypes of antibody-mediated rejection in the study
(36). Yabu et al. (37) showed that, although testing DSA
by IgG level was very sensitive for positive C4d deposit,
positive C1q binding DSA was more specific for trans-
plant glomerulopathy and graft loss. In conclusion, these
studies indicate that C1q binding DSAs are more detri-
mental than C1q nonbinding DSAs. Positive C1q binding
DSA is an independent risk of antibody-mediated re-
jection and graft loss beyond the traditional DSA mean
fluorescence intensity.
There are preliminary data suggesting C3d or C4d bind-
ing DSA as a predictor of antibody-mediated rejection.
Sicard et al. (34) compared both C1q and C3d binding DSA
assays at the time of rejection diagnosis. They found that
presence of C3d binding DSA was associated with a higher
risk of graft loss independent of mean fluorescence in-
tensity. C3d binding DSA was a better predictor of graft
loss than C1q binding DSA (34). Comoli et al. (38) reported
similar results in the pediatric population: C3d binding
DSA was better than C1q binding DSA in stratifying the
risk of antibody-mediated rejection and graft loss. Theo-
retically, these two assays analyze different steps of the
complement activation. C1q is the first step of the classic
pathway, and it may not be necessarily indicative of full
activation of the complement cascade. C3d is more down-
stream and more likely indicates complete complement
 186

Table 2. Recent studies of complement binding donor-specific antibodies in patients with kidney transplants

Positive Complement Binding DSA-

Ref. Design Patient/DSA Sera Collecting C Binding DSA Follow-Up
Associated Outcomes

37 Retrospective 274/31 Pre, biopsy 12 C1q 10 Predict transplant glomerulopathy

and graft loss
36 Prospective 284/31 De novo 11 C1q 4 Predict acute and chronic antibody-
mediated rejection; low graft
survival
36 Retrospective 405/77 De novo 33 C1q 4 Predict acute and chronic antibody-
mediated rejection; low graft
survival
29 Retrospective 284/54 De novo 34 C1q 5 DQ-DSA studied; predict acute
antibody-mediated rejection and
graft loss; more likely IgG1/IgG3
subclasses
31 Prospective 34/34 Biopsy 12 C1q 8 Reflect high DSA strength; predict
Clinical Journal of the American Society of Nephrology

antibody-mediated rejection
33 Prospective 1016/316 Pre, biopsy, 1 yr 77 C1q 7 With lowest 4-yr graft survival;
predict acute antibody-mediated
rejection and more severe graft
injury
18 Retrospective 246/47 De novo 12 C1q 10 Predict both short- and long-term
graft loss; C1q nonbinding DSAs
predict long-term graft loss only
15 Retrospective 83/52 De novo 13 C1q N/A Increase risk of antibody-mediated
rejection and graft loss
13 Retrospective 517/60 Preformed 13 C1q 8 Predict graft loss and antibody-
mediated rejection better than
DSA strength
14 Retrospective 30/30 Rejection 30 C1q 3 Reduction of C1q binding DSA after
treatment predicts better graft
survival than the reduction of DSA
titer
16 Retrospective 193/35 De novo 15 C1q 7 Predict antibody-mediated rejection,
positive C4d deposit, and graft loss
17 Retrospective 62/26 Biopsy 9 C1q 4 Predict active chronic antibody-
mediated rejection, transplant
glomerulopathy, and graft loss
42 Prospective 851/Pre: 110; post: 186 Pre and 1 and C1q; pre: 35; post: 57 5 IgG3 subclass or C1q binding DSA
2 yr, biopsy detected pre- and post-transplant
predicted graft loss in .60% of
patients; also predicted acute and
chronic antibody-mediated
rejection
Clin J Am Soc Nephrol 13: 182–192, January, 2018 Donor-Specific Antibodies, Zhang 187

activation. In another study, preformed DSAs able to bind

negative pretransplant crossmatch

C3d binding DSAs predict graft loss;

C3d binding DSAs predict graft loss;

Patient/DSA, total numbers of study patients/number of patients with positive donor-specific antibody; C binding DSA, number of patients with positive complement (C1q, C3d, or C4d) binding
Positive Complement Binding DSA-

rejection and graft loss despite of

C4d were reported to predict antibody-mediated rejection

Predict acute antibody-mediated

and graft loss, although the crossmatch was negative before

donor-specific antibody; Follow-up, follow-up years; DSA, donor-specific antibody; pre, pretransplant; biopsy, at the time of kidney biopsy; N/A, not applicable; post, post-transplant.
transplant (5). More rigid and large studies are needed to
Associated Outcomes

C1q binding DSAs are less

C1q binding DSAs are less

elucidate the role of C3d and C4d binding DSAs in predicting
antibody-mediated rejection and graft loss. Table 2 summa-
rizes the recent studies of complement binding DSAs in
patients with kidney transplants.
predictive

predictive

DSA IgG Subclasses

IgG has several subclasses (IgG1, -2, -3, and -4), and they
have various abilities to activate complement and recruit
effector cells through the Fc receptor. Over the course of
immune response and antibody production, there is a
sequential IgG subclass switching, typically from IgG3 to
IgG1 to IgG2 to IgG4 (39–41). IgG1 and IgG3 are usually
Follow-Up

complement binding subclasses. IgG1 is the most abundant

6

5
10

subclass and mirrors the total IgG level, but IgG3 has the
greater binding efficiency to C1q and activate complement
cascade. Although IgG2 can activate complement weakly,
IgG4 does not activate complement at all, but both can
recruit effector cells through the Fc receptor. IgG4 is also
considered as a biomarker of matured alloresponse and
C Binding DSA

30 C1q, 40 C3d

associated with advanced stage of rejection (8,35,39).

25 C1q, 9 C3d

Therefore, the pathogenicities of DSA are dynamic and

10 C4d

vary according to IgG subclass profile. Lefaucheur et al. (41)

recently studied 125 patients with DSAs detected in the
first year after kidney transplants: 51 of them had acute
antibody-mediated rejection, 36 patients had subclini-
cal antibody-mediated rejection, and 38 were antibody-
mediated rejection free. Patients with antibody-mediated
rejection had multiple DSA IgG subclasses and C1q binding
immune-dominant donor-specific antibody (iDSA). Anti-
Sera Collecting

body-mediated rejection–free patients did not have C1q

Preformed

binding iDSAs or IgG3 or IgG4 iDSAs. Interestingly, they

De novo

found that acute antibody-mediated rejection was mainly

Biopsy

driven by IgG3 iDSA (91%), whereas subclinical antibody-

mediated rejection was driven by IgG4 iDSA (76%). IgG3
iDSA was associated with a shorter time to rejection,
increased microcirculation injury, and positive C4d de-
posits, which indicate the complement-dependent cytotox-
icity. IgG4 iDSA was associated with later graft injury with
increased transplant glomerulopathy and interstitial fibro-
Patient/DSA

sis/tubular atrophy. Further analysis indicated that IgG3

938/69

114/39

52/52

and C1q binding iDSAs were strongly and independently

associated with graft loss (HR, 4.8; 95% CI, 1.7 to 13.3
and HR, 3.6; 95% CI, 1.1 to 11.7, respectively). IgG4
iDSA–associated subacute and chronic phenotypes of
antibody-mediated rejection were consistent with complement-
independent pathogeneses (21–24,35). Viglietti et al. (42)
recently published a prospective study of 851 kidney
recipients. Systemic DSA screening and characterization
Retrospective

Retrospective
Prospective

(titer, C1q binding capacity, and IgG subclasses) were

Design
Table 2. (Continued)

performed at transplant, 1 and 2 years post-transplant, and

the time of clinical events. They found that the addition of
IgG3 subclass or C1q binding capacity to the conventional
approach of DSA strength improved the performance in
assessing the individual risk for allograft loss in .60% of
patients. The advances in complement binding DSA assay
Ref.

and IgG subclass analysis have improved our ability to

34

38

assess patient’s immunologic risk. They can help clinicians

188 Clinical Journal of the American Society of Nephrology
to identify more harmful DSAs, predict the distinct pheno-
types of antibody-mediated rejection, and guide our clinical
decision for kidney biopsy as well as immunosuppressive
management.
DSA and C4d Deposit
C4d is a degradation product of the classic complement
pathway. A unique feature of C4d is that it binds covalently
to the endothelial basement membrane, thereby avoiding
removal during tissue processing. Positive C4d deposit in
peritubular capillaries serves as an immunologic footprint
of antibody-mediated rejection. It is in a linear pattern and
best shown by immunofluorescence in frozen tissue section
(43,44). Some patients with antibody-mediated rejection
may not have positive C4d staining. Cases with positive
DSA but negative C4d staining may result from either
technique error (false negative) or noncomplement-
activating DSA (45). Also, antibody-mediated rejection
can be caused by non-HLA antibodies that may result in
positive C4d deposit without DSA against HLA (46–52).
Furthermore, Kiki
c et al. (53) reported that patients who
were C4d positive, with or without antibody-mediated
rejection histologic features, had worse death-censored 8-year
graft survival than patients who were C4d negative. Positive
C4d deposit was independently associated with a steeper
decline of kidney function and an independent risk factor
for graft failure. Recently, Orandi et al. (54) compared the
patient outcomes of C4d-positive versus -negative anti-
body-mediated rejection. They found that both phenotypes
were associated with significantly lower graft survival
compared with in patients who were rejection free, and
C4d-positive antibody-mediated rejection had the worst
graft survival. C4d-positive antibody-mediated rejection
was significantly more likely to have a clinical presentation
(85.3% versus 54.9%) and happen earlier (median =14
versus 46 days), and it was three times more common (7.8%
versus 2.5%). This supports the hypothesis that C4d-
positive antibody-mediated rejection is directly caused
by complement-dependent cytotoxicity and associated
with more acute and severe clinical presentation, whereas
C4d-negative antibody-mediated rejection may be medi-
ated by complement-independent mechanisms, which
are subclinical or chronic in nature and lead to late graft
dysfunction and graft loss.
Non-HLA Antibodies
Graft rejection can occur in HLA-identical sibling
transplants, indicating an immune response to non-HLA
antigens (47,48). Non-HLA antibodies can cause antibody-
mediated rejection and graft loss. ABO blood antigens are
expressed on not only red blood cells but also, vascular
endothelium and other cells. ABO-incompatible organ
transplant can cause hyperacute rejection due to presence
of the preformed hemagglutinin A and/or B antibody.
Rhesus factor and other red cell antigens are not that
relevant to organ transplant, because they are not ex-
pressed on endothelial cells (11). H-Y minor histocom-
patibility antigens are encoded by the Y chromosome in
men and can induce alloimmune response when an or-
gan from a man is transplanted into a woman (49). The
MHC1-related chains A and B are minor histocompatibility
molecules expressed on endothelial cells, and their anti-
bodies can trigger antibody-mediated rejection (46,51).
Several other antibodies have been also reported, such as
antiendothelial antibodies, antiglutathione S-transferase
T1, and antiangiotensin-2 receptor (47,50,52). Interestingly,
angiotensin-2 receptor antibody has an agonistic effect that
causes severe vascular constriction and hypertension. It
responds well to the treatment with angiotensin receptor
blocker (6,52). As our knowledge in transplant immu-
nology advances, there will likely be more alloreactive and
autoreactive non-HLA antibodies to be discovered.
Acute Phenotypes of Antibody-Mediated Rejection
There are several phenotypes of antibody-mediated
rejection along the post-transplant course that seem to be
determined by the extent, timing, and various character-
istics of DSA during humoral response. Hyperacute
rejection occurs immediately after transplant. It is medi-
ated by high titer of preformed antidonor antibodies:
anti-HLA, anti-ABO, or other non-HLA antibodies
(4,8,11). Hyperacute rejection results in an irreversible
vascular rejection, intravascular thrombosis, and graft
necrosis, and graft nephrectomy is usually indicated. The
routine pretransplant crossmatch and verification of ABO
compatibility should prevent the majority of hyperacute
rejection.
Accelerated acute rejection (or delayed hyperacute re-
jection) can occur within 24 hours to several days after
transplant. It represents an anamnestic response by mem-
ory B and plasma cells from prior sensitization (4,11,55,56).
Even a negative crossmatch before transplant may not
prevent it, because the preformed DSA titer may be too low
to be detected before transplant, but alloresponse from
memory cells can trigger a surge of DSA titer after trans-
plant. Indeed, with a novel HLA B cell enzyme-linked
immunospot assay, Lúcia et al. (55) reported that circulat-
ing memory B cells against donor HLA antigens can be
identified in the sensitized patients on waiting list but
cannot be identified in the nonsensitized patients. Further-
more, memory B cells can be depleted by rituximab, a
chimeric anti-CD20 mAb; therefore, anamnestic response
may be abrogated by rituximab treatment before kid-
ney transplant (56). Presence of long-lived plasma cells
remains a challenge, because they are usually resistant to
current available therapeutics (4,55,56).
Acute antibody-mediated rejection is more common than
hyperacute rejection and accelerated acute rejection, and it
may be further divided into early and late subtypes. Early
acute antibody-mediated rejection occurs sooner after
transplant from rising titer of preformed DSA. Late acute
antibody-mediated rejection develops due to the emer-
gence of de novo DSA after kidney transplant (4). Antibody-
mediated rejection can also develop in a graft already
suffering from delayed graft function. This can be difficult
to be recognized if the patient remains anuric or oliguric
(57). Therefore, any new kidney transplant with delayed
graft function should have serial DSA monitoring and
protocol biopsies to detect covert rejection and be treated
properly (57). Early acute rejection is usually more re-
sponsive to treatment, whereas late acute rejection may not
Clin J Am Soc Nephrol 13: 182–192, January, 2018
respond to treatment well and is associated with inferior
graft function and poorer long-term outcome.
Treatment of Acute Antibody-Mediated Rejection
The optimal therapy for acute antibody-mediated re-
jection and accelerated acute rejection remains to be de-
fined but usually consists of a combination of several
modalities, such as plasmapheresis or immunoadsorption,
intravenous Igs, rituximab, bortezomib, antithymocyte
globulin, and corticosteroids (58–64). Plasmapheresis or im-
munoadsorption with protein A removes the circulating
DSA, but neither can suppress antibody production (58,59).
Intravenous Igs are often administrated after plasmaphe-
resis. Possible mechanisms of intravenous Igs include
anti-idiotypic antibodies neutralizing DSAs and immune-
modulatory molecules inhibiting complement binding or
activation and suppressing DSA production (58,60–62).
Rituximab depletes both pre-B and mature B cells, thus
reducing the transformation of plasma cell from B cells (61–
63). Bortezomib inhibits the proteasome and activates
apoptosis of antibody-producing plasma cells; therefore,
it can directly decrease DSA production. There are several
studies showing the benefit of bortezomib in treating
antibody-mediated rejection (63–66). Antithymocyte glob-
ulin remains beneficial, because it can control the B cell
response directly by inducing B cell apoptosis as well as
indirectly by depleting helper T cells (2,4). In addition,
there are several unconventional approaches that can be
considered, especially for difficult patients unresponsive to
the traditional therapies. Splenectomy was reported as a
rescue therapy for severe antibody-mediated rejection after
HLA-incompatible kidney transplant, and the combination
of splenectomy with eculizumab may be more effective
than either modality alone (67). Eculizumab is a humanized
mAb to C5, and it inhibits its cleavage to C5a and C5b.
Stegall et al. (68) studied it as part of a desensitization
protocol in patients with positive crossmatch. They found
that eculizumab treatment significantly decreased the in-
cidence of early acute antibody-mediated rejection
compared with a historical control group (6.7% versus
43.8%, respectively). However, after a 2-year follow-up,
eculizumab did not prevent the development of chronic
antibody-mediated rejection (69). This supports the
complement-dependent pathogenesis in acute antibody-
mediated rejection and complement-independent mech-
anisms in chronic antibody-mediated rejection. Recently,
Viglietti et al. (70) reported a pilot study of a C1 inhibitor
(berinert, a human plasma–derived C1 esterase inhibitor)
added to high-dose intravenous Igs for treating acute
antibody-mediated rejection that initially failed to re-
spond to conventional therapy. Berinert appeared to be
well tolerated. After a 6-month treatment, they found an
improvement in kidney function, a decrease in C4d de-
position rate, and a decrease in C1q binding DSA status.
Theoretically, C1 inhibitor should offer some benefits
over C5 inhibitor, because its block is proximal to C5,
preventing the production of C3a and C5a, which are
anaphylatoxins leading to further inflammatory re-
sponses. Formal clinical trials of complement inhibitors
will be needed to elucidate the efficacy in acute antibody-
mediated rejection.
Donor-Specific Antibodies, Zhang 189
Chronic Antibody-Mediated Rejection
Acute antibody-mediated rejection is a predictor of
developing chronic antibody-mediated rejection and trans-
plant glomerulopathy (2,4,7,11,71). Developing or worsen-
ing proteinuria after acute rejection episode suggests the
development of chronic rejection or transplant glomerulo-
pathy (4,11). Subclinical antibody-mediated rejection is
defined as immunohistologic evidence of antibody-
mediated rejection by protocol biopsy with normal graft
function, and it is also a predictor of chronic antibody-
mediated rejection if not treated (3,4,10,11). Chronic
antibody-mediated rejection is diagnosed by a combina-
tion of the typical chronic changes from biopsy (glomerular
double counters, multilayering of peritubular capillary
basement membrane, interstitial fibrosis/tubular atrophy,
and fibrous intimal thickening in arteries) and ongoing
humoral activity (positive DSA, non-HLA antibody, or C4d
staining). Chronic rejection is usually irreversible and pre-
dominantly caused by class 2 DSAs that tend to be persistent
and difficult to treat (68–70). Bortezomib and large doses of
intravenous Igs were shown to reduce class 2 DSA level
less effectively than class 1 DSA level (72,73). Attempts to
aggressively eliminate class 2 DSAs (especially the DQ) may
not be achievable, because they can put the patient at great
risk of overimmunosuppression (27). The treatment with
rituximab and/or intravenous Igs can provide partial control
of humoral activity and was reported to improve graft
survival (62,73,74). The maintenance immunosuppressive
drugs may also be increased or adjusted with more potent
ones with the hope to control humoral activity and stabilize
the graft function. Kulkarni et al. (75) recently reported a pilot
trial of 6-month eculizumab therapy for chronic antibody-
mediated rejection. The treatment group had a stabilization of
graft function compared with the slowly deteriorating kidney
function noted in the control group. It suggests a complement-
dependent pathogenesis in chronic antibody-mediated rejec-
tion, although complement-independent pathways are
commonly proposed. The combination of complement-
dependent and -independent mechanisms may also exist
in chronic antibody-mediated rejection.
Summary
DSAs, either preformed or de novo, have become a well
established biomarker predicting antibody-mediated re-
jection and graft loss. Preformed DSAs can cause instant
hyperacute rejection and graft necrosis, whereas memory B
and plasma cell response may trigger an accelerated acute
rejection within hours or days of kidney transplant. Preformed
DSAs are also responsible for early acute antibody-mediated
rejection after transplant. The development of de novo
DSAs in previously nonsensitized patients is associated
with late acute antibody-mediated rejection, chronic
antibody-mediated rejection, and transplant glomerul-
opathy. Clinical phenotypes of antibody-mediated rejec-
tion are determined by the complex characteristics of
DSAs, including HLA classes, specificity, strength, IgG
subclasses, and complement binding capacity. IgG sub-
classes have various abilities to activate complement and
recruit effector cells through the Fc receptor. C1q binding
IgG3 DSA is associated with acute antibody-mediated
rejection, more severe graft injury, and early graft loss.
190 Clinical Journal of the American Society of Nephrology
This indicates a predominant role of complement-dependent
pathogenesis. C1q nonbinding IgG4 DSA is more related with
subclinical or chronic antibody-mediated rejection and
transplant glomerulopathy, which suggests complement-
independent pathways as the underline pathogeneses.
In our practice, complement binding DSA and IgG
subclass assays are not routinely performed. They should
be considered and can be helpful in several clinical situa-
tions. HLA-incompatible kidney transplant is associated with
high incidence of antibody-mediated rejection and inferior
graft survival. The current practice is to reduce the preformed
DSA to an acceptable level by various desensitization protocols
before proceeding with the transplant. If we integrate the
complement binding and IgG subclass assays into risk assess-
ment and add disappearance of C1q binding IgG3 DSA as a
goal of desensitization protocols, then the risk of antibody-
mediated rejection and graft loss may be significantly reduced.
In the post-transplant setting, de novo DSAs are frequently
detected, even in stable transplant recipients. C1q binding and
IgG subclass assays can provide noninvasive tools to identify
those at high risk of antibody-mediated rejection, so that close
monitoring and early biopsy are indicated. These assays can
also guide our clinical treatment of different phenotypes of
antibody-mediated rejection. Complement inhibitors may be
considered as part of treatment for acute rejection mediated by
C1q binding IgG3 DSA, and they are unlikely to be beneficial in
treating rejection caused by C1q nonbinding IgG4 DSA. These
should also be taken into consideration when future clinical
trials are designed to evaluate the efficacy of complement
inhibitors on antibody-mediated rejection. The advances in
transplant immunology will provide us with more insights in
the complex pathogeneses of antibody-mediated rejection. Our
in-depth knowledge of DSA characteristics can stratify the
patient’s immunologic risk, distinguish harmful DSAs from
benign DSAs, and predict the phenotypes of antibody-
mediated rejection, and hopefully, they will guide our clinical
practice to improve the transplant outcomes.
Disclosures
None.
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