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Donor-specific antibodies, glomerulitis, and human leukocyte antigen
B eplet mismatch are risk factors for peritubular capillary C4d
deposition in renal allografts
Jin Zheng
1
, Hui Guo
2
, Hui-Lin Gong
3
, Ping Lan
4
, Chen-Guang Ding
1
,YangLi
1
, Xiao-Ming Ding
1
, Wu-Jun Xue
1
1
Department of Kidney Transplantation, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi 710061, China;
2
Institute of Organ Transplantation, Tongji Hospital affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China;
3
Department of Pathology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi 710061, China;
4
Department of Nephrology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi 710061, China.
Abstract
Background: The complement system plays an important role in the immune response to transplantation, and the diagnostic
significance of peritubular capillary (PTC) C4d deposition (C4d+) in grafts is controversial. The study aimed to fully investigate the
risk factors for PTC C4d+ and analyze its significance in biopsy pathology of kidney transplantation.
Methods: This retrospective study included 124 cases of kidney transplant with graft biopsy and donor-specific antibody (DSA)
testing from January 2017 to December 2019 in a single center. The effects of recipient pathological indicators, eplet mismatch
(MM), and DSAs on PTC C4d+ were examined using univariate and multivariate logistic regression analyses.
Results: In total, 35/124 (28%) were PTC C4d+, including 21 with antibody-mediated rejection (AMR), eight with renal tubular
injury, three with T cell-mediated rejection, one with glomerular disease, and two others. Univariate analysis revealed that DSAs
(P<0.001), glomerulitis (P<0.001), peritubular capillaritis (P<0.001), and human leukocyte antigen (HLA) B eplet MM
(P=0.010) were the influencing factors of PTC C4d+. According to multivariate analysis, DSAs (odds ratio [OR]: 9.608, 95%
confidence interval [CI]: 2.742–33.668, P<0.001), glomerulitis (OR: 3.581, 95%CI: 1.246–10.289, P=0.018), and HLA B eplet
MM (OR: 1.166, 95%CI: 1.005–1.353, P=0.042) were the independent risk factors for PTC C4d+. In receiver operating
characteristic curve analysis, the area under the curve was increased to 0.831 for predicting PTC C4d+ when considering
glomerulitis, DSAs, and HLA B eplet MM. The proportions of HLA I DSAs and PTC C4d+ in active antibody-mediated rejection
were 12/17 and 15/17, respectively; the proportions of HLA class II DSAs and PTC C4d+ in chronic AMR were 8/12 and 7/12,
respectively. Furthermore, the higher the PTC C4d+ score was, the more serious the urinary occult blood and proteinuria of
recipients at the time of biopsy.
Conclusions: PTC C4d+ was mainly observed in AMR cases. DSAs, glomerulitis, and HLA B eplet MM are the independent risk
factors for PTC C4d+.
Keywords: Kidney transplantation; C4d deposition; donor-specific antibody; Glomerulitis; human leukocyte antigen eplet
Introduction
The role of the complement system in the immune response
to transplantation is very complex and involves many
aspects.
[1]
In complement activation, C4d is a common
marker of the classical antibody-mediated pathway and
non-antibody-mediated lectin pathway. Peritubular capil-
lary C4d deposition (PTC C4d+) is used as a specific
marker of alloantibody-dependent graft injury in kidney
allografts and is a marker for antibody-mediated rejection
(AMR) in Banff 2003 diagnosis criteria.
[2,3]
The lectin
pathway is activated in transplant biopsies with delayed
graft function (DGF), AMR, and T cell-mediated rejection
(TCMR).
[4]
Thus, C4d is not only an indicator of the
interaction between antibodies and tissues but also a
marker of tissue damage, whether caused by ischemia-
reperfusion injury or by other immune factors.
Human leukocyte antigen (HLA) eplet mismatch (MM)
load has been suggested as improving HLA antigen MM
determination for organ selection. Indeed, several recent
publications show that HLA eplet matching associated
Correspondence to: Wu-Jun Xue, Department of Renal Transplantation, Hospital of
Nephrology, The First Affiliated Hospital of Medical College, Xi’an Jiaotong University,
277 Yanta West Road, Xi’an, Shaanxi 710061, China
E-Mail: xwujun126@mail.xjtu.edu.cn
Copyright ©2021 The Chinese Medical Association, produced by Wolters Kluwer, Inc. under the
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permissible to download and share the work provided it is properly cited. The work cannot be
changed in any way or used commercially without permission from the journal.
Chinese Medical Journal 2021;134(23)
Received: 01-01-2021 Edited by: Yuan-Yuan Ji
Access this article online
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DOI:
10.1097/CM9.0000000000001685
Original Article
2874
with allograft outcomes prevents donor-specific antibody
(DSA) development and graft rejection.
[5,6]
Nevertheless, it
remains unknown whether there is a relationship between
HLA MM and PTC C4d+.
In this study, 124 cases with pathological biopsy and HLA
eplet matching data as well as DSA detection were
collected to analyze the impact of PTC C4d+ in trans-
planted kidneys. The main objective of the study was to
reveal the risk factors for PTC C4d+ in transplanted
kidneys.
Methods
Ethics approval and consent to participate
This retrospective study was approved by the Institutional
Review Board/Ethics of The First Affiliated Hospital
of Xi’an Jiaotong University, Xi’an, China (No.
XJTU1AF2015LSL-058). This study was performed in
accordance with the ethical standards of the Declaration of
Helsinki. All patients signed an informed consent form,
were informed about the study, and agreed to have their
clinical information used in the reported research.
Study cohort
This study reviewed the records of 124 recipients of a
single kidney transplant in a single center from January
2017 to December 2019 for which biopsy pathology and
DSA testing were available. Deceased donation (DD)
organs were obtained by the Organ Procurement Organi-
zation of the First Affiliated Hospital of Xi’an Jiaotong
University and were allocated by the China Organ
Transplant Response System (COTRS, version 2.0,
Healthcare Commission, Xizhimen Wai Nan Lu, Xicheng
District, Beijing, China).
HLA typing and HLA eplet MM assessment
High-resolution HLA typing was performed using se-
quence-specific primer technology (LABType HD SSO,
Micro SSP; One Lambda, Canoga Park, CA, USA).
HLAMatchmaker software (version 3.1, http://www.
epitopes.net/downloads.html, University of Pittsburgh
Medical Center: 200 Lothrop St, Pittsburgh, PA 15213,
USA) was used to define eplet MM between donor and
recipient HLA alleles. First, donor and recipient HLA
eplets were analyzed with HLAMatchmaker software and
compared, and then the number of different HLA eplets
was calculated. The HLA eplet MM numbers of donors
and recipients were obtained.
Anti HLA antibody monitoring
Posttransplant monitoring of DSAs was implemented for
all kidney transplant patients. As routine clinical practice
in our program since 2017, serum samples were collected
at 0, 1, 3, 6, 9, and 12 months and then yearly or at the time
of biopsy for graft dysfunction. DSA screening was
performed using flow cytometry panel reactive antibody
(PRA) beads representing HLA-A, -B, -Cw, -DR, -DQ, and
-DP antigens (One Lambda). HLA antibody specificities
were validated by LABScreen single-antigen beads (one
lambda) using a threshold mean fluorescence intensity
(MFI) ≥1000.
Immunosuppression and postoperative management
The patients were treated with rabbit anti-human thymocyte
globulin (rabbit anti-thymocyte globulin [rATG], Sanofi:
Bridgewater, NJ, USA) (1.25–1.50 mg·kg
1
·d
1
,intrave-
nously) for induction therapy on the day of surgery and were
then tapered until discontinuation on postoperative day 5.
Methylprednisolone (Pfizer, Burtt Rd, Andover, MA 01810,
USA) was administered on the day of surgery, tapered along
(500, 250, 120, and 80 mg after the operation, respectively)
with the rATG and then replaced by prednisone (10 mg/day).
The basic immunosuppressive regimen was Tacrolimus
(TAC, Astellas Pharma Inc. Tokyo: 2-5-1, Nihonbashi-
Honcho, Chuo-Ku, Tokyo, Japan) (0.06–0.08 mg·kg
1
·d
1
)
or cyclosporine A (CsA, Novartis, 181 Massachusetts
Ave, Cambridge, MA 02139, USA) (4.0–4.5 mg·kg
1
·d
1
),
mycophenolate mofetil (MMF, Roche, Switzerland) (1500–
2000 mg/d), and prednisone (10 mg/d).
Biopsy pathology
Clinically indicated allograft biopsies were performed on
patients whose proteinuria was >0.5 g/d or whose serum
creatinine (sCr) rose >25% from baseline without a
known cause. Ultrasound-guided percutaneous biopsy was
performed with an 18G puncture needle to puncture two
tissues. To be qualified for adequate puncture tissue, >10
glomeruli and >2 arterioles with smooth muscle layers are
required. All tissues were routinely fixed, embedded, sliced,
and treated with immunofluorescence antibodies, hema-
toxylin and Eosin, periodic acid–Schiff, Masson, periodic
Schiff-Methenamine, and immunohistochemical staining.
C4d+ was determined using immunohistochemistry with
rabbit anti-human C4d monoclonal antibody (ab136921,
Abcam, 152 Grove Street Waltham, MA 02453, USA).
Histology was evaluated based on Banff criteria 2017
[7]
by
two experienced renal transplant pathologists (Hui-Lin
Gong and Yan-Xia Sui).
Graft rejection diagnosis and treatment
Graft rejection was identified on biopsy and classified
according to the Banff 2017 criteria.
[7]
Recipients with
DSAs and/or graft rejection were treated via optimization
of TAC trough levels and mycophenolate doses. A steroid
bolus with a taper was given when clinical or subclinical
TCMR and/or AMR was present on biopsy. Occasionally,
rATG was administered to patients with severe TCMR.
Recipients with active antibody-mediated rejection
(aAMR) received high-dose (2 g/kg) intravenous immuno-
globulin (IVIG) and plasmapheresis combined with
rituximab (375 mg/m
2
BSA) or bortezomib (1.3 mg/m
2
).
For chronic active AMR (caAMR), IVIG and plasmaphe-
resis were given.
Quantitative criteria for C4d scores
According to the Banff criteria, PTC C4d+ is linear C4d
staining in peritubular capillaries (C4d2 or C4d3 by
Chinese Medical Journal 2021;134(23) www.cmj.org
2875
immunofluorescence in frozen sections or C4d >0by
immunohistochemistry in paraffin sections). Quantitative
criteria for the C4d score used in the study were according
to Banff 2015 criteria as follows
[8]
: C4d0, no staining of
PTCs (0); C4d1, minimal C4d staining (>0 but <10% of
PTCs); C4d2, focal C4d staining (10%–50% of PTCs);
and C4d3, diffuse C4d staining (>50% of PTCs).
Statistical analysis
The results are expressed as numerical values and
percentages for categorical variables and as the mean ±
standard error for continuous variables. Differences in the
clinical characteristics of recipients and donors were
examined using Student ttest if data match normal
distribution and homogenous variance. If the normal
distribution is not followed, the Mann-Whitney Utest is
used. Univariate and multivariate logistic regression
analyses were applied to analyze the influencing factors
for PTC C4d+ in grafts. Receiver operating characteristic
(ROC) curves were produced to compare the predictive
value of variables for PTC C4d+. All statistical analyses
were performed using SPSS for Windows (version 20.0,
IBM Corp., Armonk, NY, USA). A Pvalue of <0.050 was
considered statistically significant.
Results
Cohort characteristics
During the study period, 954 patients received a kidney
allograft at our center. We excluded 830 cases without
pathological biopsy and DSA testing, cases of ABO blood
group (ABO) incompatible (ABOi) transplants, and cases
with comorbidities (infection, hepatitis, diabetes, autoim-
mune disease, and tumor). The final cohort consisted of
124 patients, including 108 cases of DD kidney transplant
and 16 cases of living relative kidney transplant. The study
cohort included 33 TCMRs, 31 renal tubular injuries (TIs),
28 AMRs (including 17 aAMRs and 11 chronic active
antibody-mediated rejection [caAMRs]), 12 glomerular
diseases (GDs), 12 BK virus nephritis, and eight others
[Figure 1A]. There was a total of 35 cases of PTC C4d+,
including 21 AMRs, eight TIs, three TCMRs, one GD, and
two other cases (diabetic kidney injury and thrombotic
microangiopathy) [Figure 1B]. The PTC C4d+ scores of the
AMR cases were significantly higher than those of the
other diagnoses (P<0.001) [Figure 1C].
Clinical information for PTC C4d+ and PTC C4d0 cases
The characteristics of the cohort grouped by C4d+ and
C4d0 are summarized in Table 1. There were no significant
differences between the PTC C4d+ and PTC C4dgroups
regarding recipient age, sex, body mass index (BMI),
primary disease, dialysis type and duration, induction
therapy, maintenance immunosuppression, stability of
drug concentration, or time from kidney transplantation to
biopsy. Additionally, no significant differences were found
in donor age, BMI, cause of death, type of donation,
terminal creatinine, hypertension, or diabetes history.
However, significant differences between the PTC C4d+
and PTC C4d0 groups were found with regard to Banff
diagnosis criteria, including glomerulitis (g) (P<0.001),
tubulitis (t) (P=0.001), interstitial inflammation (i)
(P=0.003), and perivascular capillaritis (ptc) (P<0.001).
Regardless, no difference in intimal arteritis (v) (P=0.578)
was found between the two groups. The positive
percentage rate of PRA and DSAs in the PTC C4d+ group
was significantly higher than that of PTC C4d0 group
(P<0.001). For HLA eplet MM, only the HLA B eplet
MM in the PTC C4d+ group was markedly higher than
that of PTC C4d0 group (P=0.015).
Risk factors for PTC C4d+
According to univariate analysis of the influencing factors
for PTC C4d+, recipient ptc, g, PRA, DSAs, and HLA B
eplet MM were associated with a higher risk of PTC C4d+,
especially ptc (odds ratio [OR]: 6.594, 95% confidence
interval [CI]: 2.319–18.746, P<0.001), g (OR: 7.915,
95% CI: 3.023–20.725, P<0.001), and DSAs (OR:
4.038, 95% CI: 2.120–7.693, P<0.001) [Table 2].
Variables with Pvalues <0.1 and with the highest OR
values for similar variables were selected for multivariate
analysis, and as shown in Table 3, independent risk factors
for PTC C4d+ included DSAs (OR: 9.608, 95% CI: 2.742–
33.668, P<0.001), g (OR: 3.581, 95% CI: 1.246–10.289,
P=0.018), and HLA B eplet MM (OR: 1.166, 95% CI:
1.005–1.353, P=0.042).
Predictive value of composite parameters for PTC C4d+
The coevaluation of PTC C4d+ was based on g, DSAs, and
HLA B eplet MM using ROC curves, with calculated area
under the curves (AUCs) of 0.686, 0.738, and 0.640.
However, the AUC increased to 0.831 when all variables
Figure 1: Distribution of PTC C4d+ in grafts after kidney transplantation. (A) Diagnosis classification of graft pathology after kidney transplantation. (B) Distribution of PTC C4d+ in graft
pathology diagnosis. (C) The degree of PTC C4d+ in graft pathology diagnosis.
∗
P<0.001, AMR vs. TCMR, GD, TI, and others. AMR: Antibody-mediated rejection; BKVN: BK virus nephritis;
C4d+: C4d deposition; GD: Glomerular disease; GN: glomerulonephritis; PTC C4d+: Peritubular capillary C4d deposition; TCMR: T cell-mediated rejection; TI: Tubular injury.
Chinese Medical Journal 2021;134(23) www.cmj.org
2876
Table 1: Clinical information for PTC C4d+ and PTC C4d0 recipients.
Characteristics C4d+ (n=35) C4d0 (n=89) Statistics Pvalue
Recipient-related information
Age (years) 35.8 ±10.4 35.6 ±9.1 0.132
∗
0.895
Male/female 21/14 68/21 2.576
†
0.108
BMI (kg/m
2
) 20.19 ±3.33 21.24 ±3.30 1.573
∗
0.118
Primary disease (%) 0.426
†
0.980
Glomerulonephritis 65.7 64.0
Hypertensive nephropathy 11.4 13.5
Membranous nephropathy 11.4 11.2
IgA nephropathy 8.6 9.0
Diabetes 0 0
Others 2.9 2.3
Dialysis type (%) 0.358
†
0.549
Hemodialysis 94.9 88.8
Peritoneal dialysis 5.1 11.2
Dialysis duration (months) 25.45 ±24.79 19.17 ±19.43 1.230
‡
0.165
Induction therapy (%) 0.027
†
0.871
Anti-thymocyte globulin 85.7 80.9
Basiliximab 14.3 19.1
Maintenance immunosuppression (%) 0.039
†
0.844
FK506/MPA/Pred 91.4 94.4
CsA/MPA/Pred 8.6 5.6
Stability of drug concentration (stability %) 42.9 38.2 0.543
†
0.461
Time from KTx to biopsy (days) 163.37 ±62.24 298.63 ±37.81 4.015
‡
<0.001
Donor-related information
Age (years) 51.65 ±13.76 49.92 ±15.19 0.577
∗
0.565
BMI (kg/m
2
) 22.65 ±2.93 21.73 ±2.83 1.731
∗
0.112
Cause of death (%) 1.932
†
0.748
Trauma 27.1 40.5
Hematencephalon 34.3 40.4
Hypoxic encephalopathy 5.7 2.3
Tumor 5.7 5.6
Others 17.1 11.2
Type of donation (%) 0.343
†
0.558
Deceased donation 82.9 88.8
Living relative donation 17.1 11.3
Terminal creatinine (mmoI/L) 87.21 ±8.44 99.76 ±7.00 0.848
‡
0.396
Hypertension (%) 34.3 38.2 0.040
†
0.842
Diabetes (%) 5.7 3.4 0.205
†
0.650
Banff criteria
g 0.51 ±0.10 0.09 ±0.03 4.697
‡
<0.001
t 0.32 ±0.11 0.73 ±0.07 3.279
∗
0.001
i 0.54 ±0.10 0.95 ±0.07 3.036
∗
0.003
v 0.14 ±0.08 0.09 ±0.05 0.559
∗
0.578
ptc 0.40 ±0.09 0.07 ±0.03 4.426
‡
<0.001
Anti-HLA antibody (%)
PRA 60.0 10.1 31.423
†
<0.001
DSA 54.3 6.7 32.386
†
<0.001
HLA mismatches (eplet)
A 5.77 ±0.85 6.89 ±0.59 0.956
‡
0.339
B 6.57 ±0.64 4.82 ±0.34 2.425
‡
0.015
C 2.71 ±0.33 2.56 ±0.20 0.411
‡
0.681
DRB1 5.45 ±0.62 5.01 ±0.37 0.465
‡
0.642
DQB1 2.77 ±0.53 2.08 ±0.24 0.761
‡
0.447
HLA I 15.06 ±1.29 14.28 ±0.80 0.406
‡
0.685
HLA II 8.23 ±0.93 7.10 ±0.45 0.732
‡
0.464
HLA I +II 23.29 ±1.85 21.38 ±0.99 0.659
‡
0.510
Values were shown as mean ±standard error, n, or percentage.
∗
Student ttest.
†
Chi-square test.
‡
Mann-Whitney Utest. BMI: Body mass index; C4d+:
C4d deposition; CsA: Cyclosporine A; DD: Deceased donation; DSA: Donor-specific antibody; g: Glomerulitis; HLA: Human lymphocyte antigen; i:
Interstitial inflammation; MPA: mycophenolic acid; MMs: Mismatches; MN: Membranous nephropathy; PRA: Panel reactive antibody; PTC C4d+:
Peritubular capillary C4d deposition; ptc: Peritubular capillaritis; PTC: Peritubular capillary; Pred: prednisone; t: Tubulitis; v: Intimal arteritis; KTx:
Kidney transplantation.
Chinese Medical Journal 2021;134(23) www.cmj.org
2877
were combined [Table 4]. Furthermore, the specificity and
sensitivity of predictive PTC C4d+ were 0.816 and 0.818,
respectively.
PTC C4d+ and HLA antibody type in aAMR and caAMR
PTC C4d+ and the type of serum DSA in aAMR and
caAMR were also analyzed. In total, 15/17 of the aAMR
cases were PTC C4d+, but only 7/12 of caAMR cases were
PTC C4d+. Moreover, the PTC C4d+ grade in aAMR was
significantly higher than that of caAMR (2.16 ±0.25 vs.
0.88 ±0.35, Z=2.522, P=0.013). HLA class I DSAs
were found in 12/17 of aAMR cases, whereas HLA class II
DSAs were mainly detected in 8/12 of caAMR cases.
Moreover, the average MFI of DSAs in aAMR cases was
lower than that of caAMR cases (t=1.152, P=0.265),
but there was no significant difference.
Effect of PTC C4d+ on allograft function at the time of
biopsy
It has been reported that PTC C4d+ is associated with
poorer graft function.
[9,10]
Therefore, this study also
analyzed renal function at the time of biopsy, including
sCr, the estimated glomerular filtration rate (eGFR),
urinary occult blood, and urinary protein. The urinary
occult blood (t=4.315, P<0.001) and urinary protein
(t=2.900, P=0.004) levels of PTC C4d+ cases were
significantly higher than that of PTC C4d0 cases, but no
Table 2: Univariate analysis of risk factors affecting PTC C4d+ (N=124)
∗
.
95% CI for mean
Variables Odds ratios Lower bound Upper bound Pvalue
Recipients’variables
Age 1.003 0.962 1.045 0.894
Sex 2.159 0.937 4.974 0.071
BMI 0.905 0.798 1.026 0.120
Primary disease 1.013 0.685 1.498 0.950
Dialysis type 2.082 0.404 10.726 0.381
Dialysis duration 1.013 0.994 1.033 0.171
Induction therapy 0.823 0.481 1.141 0.478
Maintenance immunosuppression 1.449 0.388 5.409 0.581
Stability of drug concentration 0.742 0.291 1.892 0.537
Time from KTx to biopsy 0.999 0.997 1.000 0.073
Donors’variables
Age 1.008 0.981 1.037 0.562
BMI 1.120 0.973 1.290 0.114
Cause of death 0.867 0.575 1.305 0.493
Type of donation 1.468 0.779 2.766 0.235
Terminal creatinine 0.996 0.989 1.004 0.316
Hypertension 0.805 0.355 1.823 0.603
Diabetes 1.717 0.274 10.745 0.563
Banff variables
ptc 6.594 2.319 18.746 <0.001
g 7.915 3.023 20.725 <0.001
t 0.327 0.160 0.669 0.002
i 0.397 0.211 0.747 0.004
v 1.243 0.578 2.673 0.578
HLA antibody variables
PRA 3.180 1.976 5.118 <0.001
DSA 4.038 2.120 7.693 <0.001
HLA MM variables
HLA A eplet MM 0.961 0.892 1.036 0.300
HLA B eplet MM 1.163 1.033 1.308 0.010
HLA C eplet MM 1.042 0.851 1.277 0.689
HLA DRB1 eplet MM 1.036 0.928 1.157 0.526
HLA DQB1 eplet MM 1.104 0.953 1.279 0.186
HLA I eplet MM 1.014 0.963 1.068 0.605
HLA II eplet MM 1.053 0.968 1.144 0.227
HLA I+II eplet MM 1.020 0.980 1.062 0.332
∗
Logistic regressions were performed; odds ratios and 95% confidence intervals were reported. BMI: Body mass index; CI: Confidence interval; DSA:
Donor-specific antibody; g: Glomerulitis; HLA: Human lymphocyte antigen; i: Interstitial inflammation; MM: Mismatch; PRA: Panel reactive antibody;
PTC C4d+: Peritubular capillary C4d deposition; ptc: Peritubular capillaritis; PTC: Peritubular capillary; t: Tubulitis; v: Intimal arteritis; KTx: Kidney
transplantation.
Chinese Medical Journal 2021;134(23) www.cmj.org
2878
significant differences were found in sCr (Z=1.807,
P=0.071) and eGFR (t=1.381, P=0.170) between the
two groups.
Discussion
Our study included 124 cases of kidney transplant with
graft biopsy and DSA testing. We identified that DSAs,
glomerulitis, and HLA B eplet MM are the risk factors for
PTC C4d+. The proportion of PTC C4d+ was higher in
aAMR than that of caAMR. In addition, HLA class I DSAs
were mostly associated with aAMR, whereas HLA class II
DSAs were associated with caAMR. PTC C4d+ obviously
affected graft sCr, urinary occult blood, and urinary
protein; and the PTC C4d+ score was higher.
Complement deposition strongly correlates with histo-
pathological changes observed in renal transplants, and all
three complement recognition pathways are involved.
Regarding organ ischemia-reperfusion injury that activates
the lectin pathway, antibodies, such as IgM, immunoglob-
ulin G (IgG)1, IgG2, and IgG3, activate the classical
pathway associated with most AMRs; and C4d is a
common marker of both pathways. On the other hand,
IgA, IgE, IgG4, and lipopolysaccharide (LPS) activate the
alternative pathway of the complement system, which is
related to some cases of chronic AMR.
[11]
There is evidence that all three complement pathways are
activated in TCMR, AMR, and DGF. Collectin-11 is also
an initiator of the lectin-mediated complement pathway,
and C1q is a marker for classical complement pathway
activation. Moreover, complement factor D is an early
marker for activation of the alternative complement
pathway. All three markers have been detected in biopsies
of transplant cases with DGF, AMR, and TCMR.
[4]
In the
present study, AMR and TI accounted for 60% and 22.9%
of PTC C4d+ cases, respectively. We also found three
TCMR cases to be PTC C4d+. In general, AMR was most
common among the PTC C4d+ cases, and the PTC C4d
scores of AMR cases were significantly higher than that of
TI and TCMR. Hence, the main factors causing graft
injury after transplantation are immune factors.
HLA eplet MM load has been suggested as an improve-
ment of HLA antigen MM determination for organ
selection. More recently, the term “eplet MM load”was
introduced in publications.
[12,13]
Donor–recipient match-
ing based on predicted indirectly recognizable HLA eplets
independently predicts the incidence of de novo donor
specific antibody (dnDSA) following renal transplanta-
tion.
[13]
In this study, among all the HLA eplet MMs of the
HLA locus, we found that only HLA B eplet MM affects
PTC C4d+. Furthermore, when we combined DSAs,
glomerulitis, and HLA B eplet MM, the AUC of predicted
PTC C4d+ increased to 0.831. This indicates that HLA B
eplet MM also plays a certain role in PTC C4d+, even
though DSAs and glomerulitis were found to be the main
risk factors for PTC C4d+. We speculated the reasons that
are as follows: (1) this may be related to the limited sample
size and the short observation follow-up time of this study;
(2) as the HLA B locus has more antigens than other loci,
the probability of donor and recipient matching is
relatively small; and (3) the effect of HLA B eplet MM
on PTC C4d+ is still mediated by the immune response.
Because the risks of DSAs and AMR are higher in
recipients with a higher donor HLA eplet MM, the
probability of PTC C4d+ also increases correspondingly.
Based on the Banff diagnostic criteria, the only glomerulitis
was the independent risk factor for PTC C4d+ in this
study. Tubulitis and interstitial inflammation are the
important diagnostic indicators of TCMR according to the
Banff criteria and mainly induce tubular epithelial cell
damage.
[14]
Arteritis, mainly occurring in small arteries, is
also an important index of TCMR according to the Banff
diagnostic criteria, and glomerulonephritis and peritubular
capillaritis (ptc) are common in AMR. These phenomena
were also observed in our study. As shown in Supple-
mentary Table 1, http://links.lww.com/CM9/A726, the
glomerulonephritis and peritoneal capillaritis scores of
Table 3: Multivariate analysis of risk factors affecting PTC C4d+ (N=124)
∗
.
95% CI for mean
Variables Odds ratios Lower bound Upper bound Pvalue
DSA 9.608 2.742 33.668 <0.001
g 3.581 1.246 10.289 0.018
HLA B eplet MM 1.166 1.005 1.353 0.042
Time from KTx to biopsy 0.999 0.998 1.001 0.244
Recipient gender 0.780 0.237 2.569 0.684
∗
Logistic regressions were performed, odds ratios and the 95% confidence intervals were reported. CI: Confidence interval; DSA: Donor-specific
antibody; g: Glomerulitis; HLA: Human lymphocyte antigen; MM: Mismatch; PTC C4d+: Peritubular capillary C4d deposition; PTC: Peritubular
capillary; KTx: kidney transplantation.
Table 4: ROC curves for g, DSA, and HLA B eplet MM as predictors of
PTC C4d+.
Asymptotic 95% CI
Variables Area Lower bound Upper bound
g 0.686 0.572 0.800
DSA 0.738 0.628 0.847
HLA B eplet MM 0.640 0.527 0.753
CI: Confidence interval; DSA: Donor-specific antibody; g: Glomerulitis;
HLA: Human lymphocyte antigen; MM: Mismatch; PTC C4d+:
Peritubular capillary C4d deposition; ROC: Receiver operating char-
acteristic.
Chinese Medical Journal 2021;134(23) www.cmj.org
2879
AMR cases were significantly higher than that of all the
other diagnoses. It has been reported that the total number
of infiltrating cells in glomeruli and PTC are associated
with PTC C4d+ and that infiltrating cells in glomeruli and
PTC are predominantly macrophages and T cells with a
completely cytotoxic phenotype.
[15]
Both macrophages
and cytotoxic T cells can damage endothelial cells and
induce lectin-mediated complement pathway activa-
tion,
[11]
which findings support our results.
In our study, the proportion of PTC C4d+ in aAMR was
higher than that of caAMR. In general, HLA class I DSAs
were mostly associated with aAMR, whereas HLA class II
DSAs were associated with caAMR. It has been reported
that antibodies that activate the classical pathway of
complement include IgM, IgG1, IgG2, and IgG3, which are
mainly related to aAMR. IgA, IgE, IgG4, and LPS activate
the alternative pathway, which is associated with
aAMR.
[11]
HLA class I DSAs mostly include IgG1 and
IgG3, which are primarily involved in the classical
pathway of complement activation and PTC C4d+, which
is a reason why most cases of aAMR have PTC C4d+.
However, IgG sub-types among HLA class II DSAs are
mostly IgG2 and IgG4, and these IgGs mainly participate
in complement alternative pathways, which explains why
PTC C4d0 can occur in some cases of caAMR. In this
study, the proportions of HLA class I DSAs and PTC C4d+
in aAMR were 12/17 and 15/17, respectively; the
proportions of HLA class II DSAs and PTC C4d0 in
caAMR were 8/12 and 7/12, respectively. Although these
data are rather consistent with the reported findings,
further studies are needed to clarify the IgG sub-types
among DSAs.
It has been reported that PTC C4d+ is associated with poor
graft function and inferior graft survival.
[9,10,16]
In our
study, PTC C4d+ was associated with more severe urinary
occult blood and urinary protein, indicating a poor graft
function. On the other hand, some studies have shown that
C4d+ may be a manifestation of graft accommodation. For
example, anti-HLA-class I ligation may induce cytopro-
tective genes, but anti-A/B ligation enhances complement
regulatory molecules, such as CD55 and CD59, which may
inhibit the formation of membrane attack complexes.
[17]
Additionally, anti-A/B ligation may reduce the activation
of mitogen-activated protein kinase (MAPK) and mecha-
nistic target of rapamycin (mTOR) pathways, induce
programmed death factor 1 (PD-1) production, and inhibit
the allogenic immune response of CD4+ T cells.
[18]
Hence,
a small degree of anti-HLA-class I antibody and anti-A/B
antibody ligation has the potential to induce accommoda-
tion. There were no ABO-incompatible renal transplants
among the patients in the study, which is also a limitation.
Nonetheless, some cases of caAMR were included with
insignificant clinical symptoms, continuous DSA positivi-
ty, and pathological manifestations such as graft glomer-
ulopathy and PTC C4d+. The presence of graft
accommodation remains to be further studied and
discussed.
C4d+ in glomeruli is a common manifestation of renal
disease in a disguised form. Except for systemic lupus
erythematosus, PTC C4d+ is very rare in primary renal
diseases.
[17]
In fact, only one case of graft GD was PTC
C4d+ in this study. It has been reported that in addition to
C4d+ in glomeruli and tubules, C4d+ in PTC also occurs in
primary Sjögren’s syndrome-related membranous ne-
phropathy and IgA nephrosis.
[19]
Therefore, differential
diagnosis of recurrent or new nephropathy with PTC C4d+
should be considered when graft pathology is used for the
diagnosis of AMR.
All patients in this study underwent pathological biopsy,
HLA molecular genotyping, and high-resolution monitor-
ing of HLA antibodies. The collection of these data
provided comprehensive supporting materials for the
comprehensive analysis of the risk factors for PTC C4d+
in this study. However, as a clinical study, the cases in this
study were from a single center, and the number of
included cases was only 124, which was a limitation of this
study. Our next plan is to conduct a national multicenter
study to increase the sample size and extend the follow-up
time to reveal the influencing factors of PTC C4d+ and the
relationship between PTC C4d+ and renal graft function,
as well as long-term survival.
In summary, our study identified that DSAs, glomerulitis,
and HLA B eplet MM are risk factors for PTC C4d+ and
that DSAs are the main risk factors for PTC C4d+. The
AUC of predicted PTC C4d+ increased when combining
glomerulitis, DSAs, and HLA B eplet MM. PTC C4d + was
associated with more severe urinary occult blood and
urinary protein.
Availability of data and material
The data and material used and/or analyzed during the
current study are available from the corresponding author.
The contents of this article were communicated in the form
of oral speech at the organ transplantation annual meeting
of the Chinese Medical Association in 2020.
Acknowledgements
The authors very much appreciate the help of Mr. Jian
Yang who helped to analyze data and revise the
manuscript.
Funding
This article was supported by the Clinical Research Award
of the First Affiliated Hospital of Xi’an Jiaotong
University, China (No. XJTU1AF-CRF-2018-026) and
the Natural Science Foundation of China (No. 82070768).
Conflicts of interest
None.
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How to cite this article: Zheng J, Guo H, Gong HL, Lan P, Ding CG, Li Y,
Ding XM, Xue WJ. Donor-specific antibodies, glomerulitis, and human
leukocyte antigen B eplet mismatch are risk factors for peritubular
capillary C4d deposition in renal allografts. Chin Med J 2021;134:2874–
2881. doi: 10.1097/CM9.0000000000001685
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