Non-HLA Antibodies and Epitope Mismatches in Kidney Transplant Recipients With Histological Antibody-Mediated Rejection

Background: Correlation between antibody-mediated rejection (ABMR) and circulating HLA donor-specific antibodies (HLA-DSA) is strong but imperfect in kidney transplant (KT) recipients, raising the possibility of undetected HLA-DSA or non-HLA antibodies contributing to ABMR. Detailed evaluation of the degree of HLA matching together with the identification of non-HLA antibodies in KT may help to decipher the antibody involved.

Methods: We retrospectively assessed patients with transplant biopsies scored following Banff’15 classification. Pre- and post-transplant serum samples were checked for HLA and non-HLA antibodies [MICA-Ab, angiotensin-II type-1-receptor (AT₁R)-Ab, endothelin-1 type-A-receptor (ETAR)-Ab and crossmatches with primary aortic endothelial cells (EC-XM)]. We also analyzed HLA epitope mismatches (HLA-EM) between donors and recipients to explore their role in ABMR histology (ABMR_h) with and without HLA-DSA.

Results: One-hundred eighteen patients with normal histology (n = 19), ABMR_h (n = 52) or IFTA (n = 47) were studied. ABMR_h patients were HLA-DSA_pos (n = 38, 73%) or HLA-DSA_neg (n = 14, 27%). Pre-transplant HLA-DSA and AT₁R-Ab were more frequent in ABMR_h compared with IFTA and normal histology cases (p = 0.006 and 0.003), without differences in other non-HLA antibodies. Only three ABMR_hDSA_neg cases showed non-HLA antibodies. ABMR_hDSA_neg and ABMR_hDSA_pos cases showed similar biopsy changes and graft-survival. Both total class II and DRB1 HLA-EM were associated with ABMR_hDSA_pos but not with ABMR_hDSA_neg. Multivariate analysis showed that pre-transplant HLA-DSA (OR: 3.69 [1.31–10.37], p = 0.013) and AT₁R-Ab (OR: 5.47 [1.78–16.76], p = 0.003) were independent predictors of ABMR_hDSA_pos.

Conclusions: In conclusion, pre-transplant AT₁R-Ab is frequently found in ABMR_hDSA_pos patients. However, AT₁R-Ab, MICA-Ab, ETAR-Ab or EC-XM⁺ are rarely found among ABMR_hDSA_neg patients. Pre-transplant AT₁R-Ab may act synergistically with preformed or de novo HLA-DSA to produce ABMR_hDSA_pos but not ABMR_hDSA_neg. HLA epitope mismatch associates with ABMR_hDSA_pos compared with ABMR_hDSA_neg, suggesting factors other than HLA are responsible for the damage.

Introduction

Correlation between the detection of HLA donor-specific antibodies (HLA-DSA) and antibody-mediated rejection (ABMR) is strong but imperfect in kidney transplant (KT) recipients (1–7). Not all patients with pre- or post-transplant HLA-DSA have ABMR damage in their biopsies (8). Different groups have tried to identify characteristics of HLA-DSA that may predict ABMR (9–12). There is also an active search for other invasive or non-invasive biomarkers for ABMR diagnosis (13–15). In the other hand, some patients have biopsies with histological findings suggestive of ABMR (ABMR_h) without circulating HLA-DSA (16), generating the concept of the existence of ABMR_hDSA_pos and ABMR_hDSA_neg cases. There is still limited literature describing the incidence of this type of ABMR without HLA-DSA, evaluating if these cases collectively show different clinical or histological characteristics or if non-HLA antibodies may explain the damage. Besides, controversial results in outcomes comparing ABMR_hDSA_pos and ABMR_hDSA_neg cases have been reported (17, 18).

Based on the hypothesis that other antibodies may play a lead role in the case of ABMR histological damage with or without HLA-DSA, some groups have evaluated non-HLA antibodies in KT recipients (19, 20). Although first reports connecting non-HLA antibodies and graft outcomes were published in 2005 (19, 21), evidence is still weak and debated. Antibodies against specific alloantigens such as MICA (MICA-Ab) or MICB, or against autoantigens like angiotensin II type 1 receptor (AT₁R-Ab), endothelin-1 type A receptor (ETAR-Ab), perlecan, agrin or vimentin, among others, have been reviewed recently (22). Some groups focused into the analysis of pathogenic antibodies directed against endothelial cells—which express some of those but also other antigens—with endothelial cell crossmatches (23–25). The increased evidence that the prevalence of non-HLA antibodies in KT recipients is high (26), together with the heterogeneous post-KT clinical course of patients included in these studies (25) hamper the correct identification of deleterious non-HLA antibodies. On the other hand, HLA epitope mismatch (HLA-EM) assessment has gained interest as an added immune monitoring tool to provide a more precise evaluation of HLA matching (27–29). HLA-EM has been previously associated with the development of de novo HLA-DSA (30) and ABMR (31). The clinical relevance of HLA-EM analysis remains under discussion and its application is not generalized yet.

Here, we systematically explored pre- and post-KT serum samples for HLA and different types of non-HLA antibodies: MICA-Ab, AT₁R-Ab and ETAR-Ab, and other non-HLA antibodies performing crossmatches with primary aortic endothelial cells (EC-XM). Additionally, we evaluated pre-KT HLA-EM load. We focused on KT patients with biopsies with Banff category 2 diagnosis and compared them with two other Banff diagnosis: category 1 or no abnormalities (normal), as a usual control group, and category 5 or interstitial fibrosis and tubular atrophy (IFTA), damage with not clear pathogenicity to evaluate the potential role of non-HLA antibodies in this case (32).

Materials and Methods

Study Population and Design

Prospective observational study performed in KT patients active at our transplant program in Hospital del Mar. A total of 234 consecutive clinical and surveillance renal biopsies were performed in ABO compatible KT after a negative CDC crossmatch (February 2011–June 2015). Ninety-two biopsies fulfilling Banff 2015 categories 3, 4 and 6 were excluded and 142 biopsies achieving categories 1, 2 or 5 were selected. From these 142 biopsies, we selected only one biopsy per patient according to these criteria: the first biopsy obtained after 3 months post-transplantation, unless a biopsy with category 2 diagnosis was available. Five biopsies were excluded due to unsuitable serum samples. Finally, 118 biopsies corresponding to 118 patients were included in the study (Supplementary Figure 1). Demographical and clinical data were collected as previously described (33), and follow-up was done until graft-loss, death, 96 months post biopsy or July/2020. The study was approved by the Parc de Salut Mar Ethical Research Board (2010/3904/I) and all patients signed informed consents. All clinical and research activities reported are consistent with the Declarations of Istanbul and Helsinki.

Histological Scoring and Classification of the Biopsies

Biopsies were performed for indication or follow-up (including HLA-DSA detection without graft dysfunction). Processing was undertaken as previously described (33). All biopsies were scored by a pathologist following Banff 2015 classification and assigned to any of the six Banff categories (33). Category 2 included biopsies that met the first two Banff 2015–2019 criteria for ABMR histology, fulfilling the suspicious or full diagnosis of ABMR in Banff 2015 classification.

Sera Collection and Detection of HLA and Non-HLA Antibodies

One-hundred one available pre-KT and 118 post-KT serum samples collected contemporaneously to biopsies were retrospectively analyzed. HLA antibody testing (HLA-A, B, C, DRB and DQB) was performed as previously described (34) using Luminex HLA Single Antigen Bead assays (LABScreen, One Lambda, Canoga Park, CA). Antibodies against MICA antigens (*001, *002, *004, *007, *009, *012, *017, *018, *019, *027) were determined using LABScreen assay by Luminex Technology, according to the manufacturer’s specifications (One Lambda, CA). MICA-Ab were considered positive if mean fluorescence intensity >1,000. MICA typing for donors and recipients was not available. AT₁R-Ab and ETAR-Ab were measured using enzyme-linked immunosorbent-based assays (35) (One Lambda, CA), diluted 1:100, tested in duplicate and read on an Epoch Microplate Spectrophotometer (Bio-Tek, Winooski, VT). Samples with AT₁R-Ab or ETAR ≥10 U/ml were considered positive based on previous studies and our receiver operating curve analysis.

Endothelial Cell Crossmatches

Primary human aortic endothelial cells (ECs) were isolated from aortic rings of explanted donor hearts (36). EC were cultured in M199 medium supplemented with 20% (vol/vol) FBS, penicillin–streptomycin (100 U/ml and 100 ug/ml; Invitrogen Life Technologies), sodium pyruvate (1 mM), heparin (90 ug/ml; Sigma-Aldrich) and EC growth supplement (20 mcg/ml; Fisher Scientific). ECs from passages 7–8 were frozen and used in the EC-XM. Two different ECs (phenotyped as follows, donor CAR: HLA A2, A68, B60, B65; and donor Y126: HLA A1, A11, B35, B37) were employed avoiding for each KT recipient any HLA class I match with the kidney graft which could yield a reaction towards donor-specific HLA antigens. A total of 2 ×10⁵ ECs were incubated 30 min with 100 ul patient serum on ice. ECs were washed three times and incubated with 50 mc of 1:400 diluted FITC-AffiniPure F(ab’)₂ Fragment Goat Anti-Human IgG Fc fragment (Jackson ImmunoResearch Laboratories) for 30 min on ice. After three washes, cell fluorescence was analyzed on a FACSCalibur flow cytometer using CellQuest software (BD Biosciences). Gates for forward and side scatter measurements were set on EC, and a minimum of 10,000 events was acquired. Positive EC-XM threshold was set at two standard deviations (50 Median Channel Shift) above the mean of negative control serum tests. EC-XM were only performed in 83 pre and 103 post-KT cases due to insufficient sample.

HLA Epitope Mismatch Characterization

HLAMatchmaker software according to the July 2020 update (ABC and DRDQDP Eplet Matching Program V3.1, http://www.epitopes.net) was used to define potential HLA-EM between donors and recipients (37). High-resolution typing for all donors and recipients was performed or inferred using the HaploStats tool (www.haplostats.org) selecting the most likely high-resolution typing for HLA-A, B, C, DR and DQ according to three-five highest haplotype frequencies in the population of each one (Caucasian, African American, Asian or Hispanic).

Statistics

Data are presented as mean ( ± standard deviation), median, interquartile range, or number (percentage) based on data distribution. Comparisons between clinical variables were carried out using Student’s T test for parametric continuous variables and U Mann–Whitney or Kruskal–Wallis test for non-parametric data. Chi-squared or Fisher’s exact tests were used to test categorical variables. Survival analyses were performed using the Kaplan–Meier method using the log-rank test. Logistic regression analysis was used to estimate the odds ratio (OR) for ABMR_hDSA_pos development. All variables with a p-value <0.10 in the univariate analysis were included in the multivariate analysis. Statistical analysis was performed using SPSS v.27.0 (IBM Corp., Armonk, NY, USA) and p-values <0.05 were considered statistically significant.

Results

Clinical Characteristics and Graft Survival

The selected 118 patients were grouped according to Banff diagnostic categories: category 1 or normal biopsy (n = 19), category 2 or ABMR histology (ABMR_h, n = 52) and category 5 or IFTA (n = 47). Thirty patients (25.4%) lost their grafts and 13 died with a functioning graft (11%) during the study period. Death-censored graft survival 68 months after the biopsy [IQR 48–80] was worse in ABMR_h cases than in those with IFTA or normal biopsies (Supplementary Figure 2). Baseline characteristics showed that normal histology patients were more frequently males, whereas ABMR_h patients received grafts from younger donors and were more frequently retransplanted. ABMR_h biopsies were less frequently surveillance biopsies and were performed later post-KT. At biopsy time, ABMR_h patients had worse glomerular filtration rate (GFR) and higher proteinuria. Finally, IFTA patients were more frequently receiving calcineurin inhibitors and less on mTOR inhibitors (Table 1).

TABLE 1

Table 1 Demographics and clinical characteristics of all included patients.

Pretransplant HLA-DSA and Non-HLA Antibodies

Pre-Transplant HLA-DSA

Pre-transplant serum samples were available for 101 patients (19 normal histology, 39 ABMR_h and 43 IFTA). We found pre-transplant HLA-DSA in 18 ABMR_h (46.2%), 9 IFTA (20.9%) and in two normal histology cases (10.5%) (p = 0.006) (Figure 1A). In ABMR_h pre-transplant HLA-DSA were more frequently class I&II combined (38.9%, p = 0.087) and less isolated class I (27.8%).

FIGURE 1

Figure 1 Pre-transplant HLA and non-HLA antibodies. (A) HLA, AT₁R-Ab, ETAR-Ab, MICA-Ab and EC-XM before transplantation in the three groups of study. (B) Pre-transplant AT₁R-Ab positive and negative patients with preformed persistent HLA-DSA, preformed cleared HLA-DSA, de novo HLA-DSA or without HLA-DSA at any time. (C) Detection of pre-transplant HLA-DSA and/or AT₁R-Ab in the three study groups. (D) Post-transplant HLA and non-HLA antibodies. HLA, AT₁R-Ab, ETAR-Ab, MICA-Ab and EC-XM after transplantation in the three groups of study. ABMR_h, antibody-mediated rejection histology; AT₁R-Ab, antibodies against angiotensin II type 1 receptor; EC-XM, crossmatch with primary aortic endothelial cells; ETAR-Ab, antibodies against endothelin-1 type A receptor; IFTA, interstitial fibrosis and tubular atrophy; KT, kidney transplantation; MICA-Ab, antibodies against major histocompatibility complex class I related chain A. ns, non-significant.

Pre-Transplant AT₁R-Ab

Pre-transplant AT₁R-Ab strongly associated with ABMR_h diagnosis (16/39 ABMR_h (41%) vs. 2/19 normal histology (10.5%) and 5/43 IFTA (11.6%), p = 0.003) (Figure 1A). All 16 ABMR_h patients with pre-transplant AT₁R-Ab developed ABMR_hDSA_pos, whereas no ABMR_hDSA_neg patient showed pre-transplant AT₁R-Ab (p = 0.029). Detection of pre-transplant AT₁R-Ab correlated with both persistent preformed HLA-DSA (12/23, 52%) and de novo HLA-DSA detection (8/18, 44%), but not with preformed HLA-DSA which cleared after transplant (1/6, 17%) or no HLA-DSA (2/54, 4%, p <0.001) (Figure 1B). The median MFI of preformed HLA-DSA coexistent with AT₁R-Ab was not significantly different than preformed HLA-DSA without AT₁R-Ab (8898 vs 2874, p = 0.083).

Other Non-HLA Antibodies

Neither pre-transplant ETAR-Ab nor MICA-Ab associated with ABMR_h. Pre-transplant ETAR-Ab and MICA-Ab were present similarly in normal histology, ABMR_h and IFTA cases (31.6, 25.6 and 18.6%, p = 0.51; 10.5, 7.7 and 9.3%, p = 1.00). Of 83 KT recipients tested with EC-XM, only 3/29 ABMR_h (10.3%) and 3/39 IF/TA cases (7.7%) had a pre-transplant positive EC-XM (Figure 1A).

Pre-Transplant Combination of HLA-DSA and Non-HLA Antibodies

Detection of pre-transplant HLA-DSA and/or AT₁R-Ab were highly associated with ABMR_h compared with IFTA and normal biopsies (66.7 vs. 25.6 vs. 21%, p <0.001, Figure 1C). Nine ABMR_h cases presented with simultaneous HLA-DSA and AT₁R-Ab (23.1%), 17 with either HLA-DSA or AT₁R-Ab (43.6%) and the remaining 13 did not present any of these antibodies (33.3%).

Post-Transplant HLA-DSA and Non-HLA Antibodies

Post-Transplant HLA-DSA

At the time of biopsy, HLA-DSA was detectable in 38/52 ABMR_h patients [73.1%, 17 preformed (44.7%) and 21 de novo (55.3%)]. Among them, 7.7% were class I, 53.8% class II and 11.5% combined class I&II. HLA-DSA were also detected in 17% IFTA and 15.8% normal histology cases (Figure 1D).

Post-Transplant AT₁R-Ab

Post-transplant AT₁R-Ab showed no association with ABMR_h (23.1% in ABMR_h vs. 26.3% in normal histology and 27.7% in IFTA cases, p = 0.85, Figure 1D). Detection of post-transplant AT₁R-Ab did not correlate with the detection of HLA-DSA [15/49 HLA-DSA_pos cases had AT₁R-Ab at biopsy (30.6%) vs. 15/69 HLA-DSA_neg cases (21.7%), p = 0.28].

Other Non-HLA Antibodies

Neither post-transplant ETAR-Ab nor MICA-Ab was related with ABMR_h. Post-transplant ETAR-Ab were found in 3/19 normal histology (15.8%), 7/52 ABMR_h (13.5%) and 9/47 IFTA cases (19.1%, p = 0.80). MICA-Ab were detectable in 1/19 normal histology (5.3%), 8/52 ABMR_h (15.4%) and 6/47 IFTA cases (12.8%, p = 0.62). Two normal histology (11.1%), four ABMR_h (9.3%) and two IFTA cases (4.8%) had a positive EC-XM (p = 0.70, Figure 1D).

Patients With ABMR_h With and Without HLA-DSA

From 52 patients with ABMR_h 14 (26.9%) had no peri-biopsy HLA-DSA. ABMR_hDSA_pos cases were more frequently HLA sensitized, less well DR-matched with their donors and received more frequently a graft from a deceased donor than those ABMR_hDSA_neg. No differences were found in graft function or immunosuppression at biopsy (Table 2). Patients showed similar microvascular inflammation, but diffuse C4d was more frequent in ABMR_hDSA_pos cases (27% vs 0%, p = 0.07, Table 2). Graft survival was similar between both groups (Figure 2). We assessed pre- and post-transplant non-HLA antibodies in ABMR_hDSA_neg cases. Of 7 cases with pre-transplant sample, two had EC-XM⁺ but none showed MICA-Ab, AT₁R-Ab or ETAR-Ab (Table 3A). After KT, one had coexistent MICA-Ab, AT₁R-Ab and ETAR-Ab; one had MICA-Ab and a third one AT₁R-Ab (Supplementary Table 1A). In 9/14 ABMR_hDSA_neg patients (64.3%) we could not identify any of the non-HLA antibodies studied.

TABLE 2

Table 2 Characteristics of patients with and without HLA-DSA.

FIGURE 2

Figure 2 Death censored graft survival in ABMR_h patients with and without HLA-DSA. Kaplan–Meier survival curves representing death censored graft survival. ABMR_h, antibody-mediated rejection histology; DSA, donor-specific antibodies.

TABLE 3A

Table 3A Comparison of pre-transplant non-HLA antibodies between ABMR_hDSA_pos and ABMR_hDSA_neg cases.

HLA Epitope Mismatch Characterization

The median number of class I and class II HLA-EM in our cohort were 16 (0–36) and 18 (0–46) respectively. Among them, 10 class I and 7 class II HLA-EM were antibody-verified (HLA-EM^ver). We observed similar class I and class II HLA-EM^ver in all three groups of study (data not shown). We compared the load of HLA-EM^ver between ABMR_hDSA_pos and ABMR_hDSA_neg patients, finding similar class I but significantly higher class II and DRB HLA-EM^ver in ABMR_hDSA_pos cases (8 vs 4.5, p = 0.046; 5 vs. 0.5, p = 0.044, Figure 3). We compared HLA-EM and HLA antigen mismatch (HLA-AM) for de novo DSA (dnDSA) development prediction. Neither class I HLA-EM^ver nor HLA-AM were useful tools for class I dnDSA prediction. Class II HLA-EM^ver were significantly associated with class II dnDSA (8 vs. 7, p = 0.031), but not class II HLA-AM (p = 0.26). The extent of DRB HLA-EM^ver associated with DRB dnDSA (6 vs. 4, p = 0.024), and the rate of DQB HLA-EM^ver showed a weak association with DQB dnDSA (4 vs. 2, p = 0.077). Neither DRB nor DQB HLA-AM predicted DRB or DQB dnDSA (p = 0.27, p = 0.21).

FIGURE 3

Figure 3 HLA epitope mismatch analysis in ABMR_hDSA_pos and ABMR_hDSA_neg cases. (A) Number of antibody-verified class I and class II epitope mismatches and (B) Number of antibody-verified DRB and DQB epitope mismatches in ABMR_hDSA_pos (black and white hexagons) and ABMR_hDSA_neg (black squares) cases. All plots show median and interquartile range (IQR).

Risk Factors for Post-Transplant ABMR_hDSA_pos Development

ABMR_hDSA_pos patients showed higher rates of pre-transplant HLA-DSA and AT₁R-Ab (p <0.001, Table 3B), but regarding post-transplant antibodies, only HLA-DSA was associated with ABMR_hDSA_pos (p <0.001, Supplementary Table 1B). In order to assess the role of each factor, we adjusted a multivariate model which showed that both pre-transplant HLA-DSA (OR: 3.69 [1.31–10.37], p = 0.013) and AT₁R-Ab (OR: 5.47 [1.78–16.76], p = 0.003) were independent ABMR_hDSA_pos predictors. DRB HLA-EM^ver also showed a weak association with ABMR_hDSA_pos (p = 0.071, Table 4).

TABLE 3B

Table 3B Pre-transplant HLA and non-HLA antibodies: comparison between ABMR_hDSA_pos and non-ABMR_hDSA_pos cases (normal histology, IFTA and ABMR_hDSA_neg cases).

TABLE 4

Table 4 Logistic regression analysis of ABMR_hDSA_pos risk factors.

Discussion

We report here that ABMR damage in KT recipients occurs in a significant proportion of cases without the detection of HLA-DSA at biopsy. We have evaluated the role of non-HLA antibodies, such as AT₁R-Ab, ETAR-Ab, MICA-Ab or anti-EC antibodies detected with crossmatches and found they could not explain ABMR_hDSA_neg. Our results suggest a synergistic interaction between pre-transplant AT₁R-Ab and HLA-DSA to produce ABMR_hDSA_pos or facilitate de novo appearance of HLA-DSA, but not to induce ABMR_hDSA_neg. Interestingly, it appears more strongly associated with ABMR_h than incompatibility evaluated through HLA-EM analysis.

The relationship between ABMR_h and HLA-DSA has been described in KT recipients for over 20 years (1, 2). However, there is increased evidence that ABMR compatible histological lesions may be present in the graft without detectable circulating HLA-DSA (18, 38). Up to 27% of our ABMR_h patients did not show circulating HLA-DSA at the time of biopsy. This could be attributed to the inability of current techniques to detect these HLA antibodies or due to the participation of a different set of antibodies in graft damage. ABMR_hDSA_neg patients presented significantly lower class II and DRB HLA-EM compared with ABMR_hDSA_pos cases. This finding strengthens the hypothesis of the participation of other mechanisms of damage in these cases rather than non-detected HLA-DSA. However, neither AT₁R-Ab, ETAR-Ab, MICA-Ab nor antibodies identified with EC-XM before or after KT were able to explain the ABMR_hDSA_neg cases in our study. We describe here that ABMR_h patients without HLA-DSA showed similar graft function, immunosuppressive treatment, histological features at biopsy and graft survival at the end of follow-up compared with ABMR_hDSA_pos cases. Like us, Sablik et al. (17) reported a similar histological phenotype in ABMR_hDSA_pos and ABMR_hDSA_neg patients, but a larger study by Senev et al. (18) found that ABMR_hDSA_pos biopsies were more frequently C4d positive compared with ABMR_hDSA_neg cases, as the unique histological difference between the groups. In our series, although C4d positivity was similar between both groups, C4d intensity was higher in the ABMR_hDSA_pos group. In our cohort, graft survival was similar between both groups, in agreement with results reported by Sablik et al. (17) but in contrast with the study from Senev et al. (18), which included mostly active ABMR cases without chronicity, unlike our cohort.

KT recipients may produce immune responses through indirect recognition against foreign proteins or even against own proteins expressed by the donor graft acting as autoantigens due to different factors that induce graft damage during the transplant process. These antibodies may then react against polymorphic alloantigens, like HLA related MICA or MICB, or against autoantigens like AT₁R, ETAR, agrin, vimentin, perlecan, K-tubulin, etc. (39–41) which may be prevalent in KT recipients. Some of these autoantibodies and new ones recently validated (42) have not been evaluated in our cohort yet. They might explain some ABMR_hDSA_neg cases. Some groups have evaluated the relationship between antibodies directed against ECs—the barrier between donor and recipient—and graft survival (43), and exploratory studies have employed array techniques in limited series with antibodies against ECs validating potential target proteins with ELISA (44, 45). Jackson and col. were able to identify four antigenic targets expressed on ECs in nine patients with ABMR_hDSA_neg (44). They found that antibodies against these proteins in pre-transplant sera predicted ABMR_hDSA_pos. In our cohort, of seven ABMR_hDSA_neg cases with pre-transplant samples, two had a positive EC-XM⁺, but none showed MICA-Ab, AT₁R-Ab or ETAR-Ab. In line with our results, a recent report from Delville et al. (23) found that only 26% of patients with early acute ABMR_hDSA_neg had pre-transplant AT₁R-Ab using our same threshold of 10 UI/ml. Moreover, MICA-Ab were only detected in two of these ABMR_hDSA_neg cases. However, these cases had preformed IgG antibodies against constitutively expressed antigens of microvascular glomerular cells (23). Of note, our two cases with pre-transplant EC-XM⁺ developed ABMR_h within the first 12 months of KT, while the other twelve developed ABMR_h later on. Unlike Lefaucheur et al. (46), despite employing the same threshold for AT₁R antibodies, the presence of these antibodies in our ABMR_hDSA_neg cohort is negligible. Nevertheless, our overall prevalence of 25% in post-transplant AT₁R-Ab is not different from theirs. Unfortunately, these authors do not analyze the relation between pre-transplant AT₁R-Ab and ABMR.

We report here a strong and independent association between pre-transplant AT₁R-Ab and ABMR_hDSA_pos development. AT₁R can be found in several cell types such as vascular endothelial cells and binds to angiotensin II (39, 47). First report linking AT₁R-Ab and kidney allograft rejection suggested a potential relationship between AT₁R agonistic antibodies and vascular injury (19, 39). Subsequently, pre- or post-transplant AT₁R-Ab detection have been linked to both rejection and allograft failure (19, 48). Philogene et al. (24) described higher post-transplant AT₁R-Ab levels in patients with ABMR compared with patients with cellular rejection or those without rejection, however, they provided no data regarding pre-transplant AT₁R-Ab. In another report (49), pre- and post-transplant AT₁R-Ab were strongly associated with biopsy-proven rejection, not specifically ABMR. Some reports suggest that non-HLA and HLA-DSA antibodies may function in synergy (24, 49). Taniguchi et al. (49) reported lower graft survival mainly in the presence of de novo AT₁R-Ab and HLA-DSA at biopsy with lesions compared with those cases with HLA-DSA alone. Here we show a strong association of pre-transplant AT₁R-Ab with post-transplant HLA-DSA, either persistent preformed or de novo, and with ABMR_hDSA_pos development. This association may be of utmost importance for KT outcomes. We previously reported the strong association among persistent preformed HLA-DSA and lower ABMR free survival, only surpassed by the development of de novo HLA-DSA (34). Moreover, here we show that all 16 ABMR_h patients with pre-transplant AT₁R-Ab had HLA-DSA at biopsy, nine of them maintained the preformed HLA-DSA and seven developed de novo HLA-DSA. We found no association between pre-transplant AT₁R-Ab and graft survival, in line with other reports (26, 49). In our multivariate analysis, pre-transplant HLA-DSA and AT₁R-Ab were independent predictors for ABMR_h. Our study may not be powered enough to assess the relationship between AT₁R-Ab and graft loss. Given the strong and already known association between ABMR and increased risk of kidney allograft loss (34, 50–52), our data supports that pre-transplant AT₁R-Ab assessment should be carefully considered in KT candidates.

In the last years, HLA-EM analysis has been proposed as a better strategy to prevent HLA-DSA development than antigen matching (29). Here we confirm that class II and DRB dnDSA development may be predicted with HLA-EM, as previously reported (30), however, only a weak association was observed with DQB dnDSA, probably due to the limited number of cases included. Interestingly, neither class II, DRB or DQB HLA-AM were able to predict dnDSA. As mentioned, the detection of lower number of class II and DRB HLA-EM in ABMR_hDSA_neg cases may contradict the idea of undetected HLA-DSA responsible for the damage. Class II and DRB HLA-EM associated with ABMR_hDSA_pos, although the existence of preformed HLA-DSA or AT₁R-Ab are more potent predictors of ABMR_hDSA_pos in our experience. In our study, ABMR_hDSA_neg could not be explained by higher HLA-EM or by the non-HLA antibodies evaluated. Interestingly, an alternative mechanism to produce ABMR_h termed “the missing-self hypothesis” has been proposed. According to it, the inability of graft EC to provide HLA I-mediated inhibitory signals to recipient circulating NK cells may trigger NK cell activation, resulting in endothelial damage and chronic vascular rejection (53).

The main limitation of our study is the restricted number of ABMR_hDSA_neg cases in the whole cohort. In order to further increase its number and the significance of the study, a multicenter trial is advisable. Besides, it is based on a mix of indication and surveillance biopsies which introduces heterogeneity in the timing and clinical picture of patients. Of note, EC-XM were performed with aortic cells which may not express the same proteins as a renal EC. Last, another limitation may be the use of inferred four-digit HLA typing for HLA-EM analysis. Despite careful estimation of second field HLA typing, we cannot rule out the possibility that some rare HLA genotypes are not correctly assigned, as recently suggested (54). However, ours is a large well characterized cohort of KT recipients, reflecting clinical practice, with thorough analysis of biopsies, including electron microscopy, crucial to detect some cases of ABMR_h and with systematical study of HLA-DSA and a known set of non-HLA antibodies.

In summary, although the majority of patients with HLA-DSA at the time of biopsy show ABMR_h, almost 30% of ABMR_h patients did not show evidence of circulating HLA-DSA. These patients were more frequently HLA unsensitized pretransplant and less HLA matched but did not show other specific characteristics at transplantation or at biopsy. Neither AT₁R-Ab, ETAR-Ab, MICA-Ab nor antibodies identified with EC-XM before or after KT were able to explain ABMR_hDSA_neg cases. Importantly AT₁R-Ab with or without HLA-DSA before KT clearly increased the risk of ABMR_hDSA_pos, suggesting it should be included in the pre-transplant immune assessment together with HLA-DSA.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics Statement

The studies involving human participants were reviewed and approved by the Parc de Salut Mar Ethical Research Board. The patients/participants provided their written informed consent to participate in this study.

Author Contributions

MC designed the study, coordinated logistics, analyzed the results, and drafted the manuscript. LL-M analyzed the results and drafted the manuscript. DR-P analyzed the results and revised the manuscript. CBut coordinated lab procedures and revised the manuscript. JG contributed with the assessment of the graft biopsies. MP-S, CBur, AB, CA-C, and SS-U revised the manuscript. MF coordinated sample drawing and storage. NV supervised HLA and non-HLA antibody interpretation. ER and JP evaluated the design of the study and revised the manuscript. All authors contributed to the article and approved the submitted version.

Funding

This study was performed with funding from projects PI13/00598, PI16/00617, and PI20/00090 (Spanish Ministry of Health ISCIII FIS-FEDER); RD16/0009/0013 (ISCIII FEDER REDinREN) and 201822-10 (Fundació la Marató de TV3). MC received grants of the Sociedad Española de Nefrología and Parc de Salut Mar for a research stay at UCLA Immunogenetics Center (Los Angeles, USA). ER is supported by National Institute of Allergy and Infectious Diseases Grants RO1 AI135201. One Lambda provided reagents but had no role in the design of the study or the analyses and writing of the manuscript.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We are indebted to Anna Faura, Rosa Causadias and Anna Bach for their assistance with patients. We thank Duska Dragun for her personal encouragement to perform the study the way it is. We also extend our gratitude to the staff of the UCLA Immunogenetics Center for technical assistance with non-HLA antibody tests and EC-XM. Finally, the authors hereby express their thanks to the organ donors and their families for giving the gift of life and, together with the recipients, the gift of knowledge. LL-M did this study as part of her doctoral thesis program at the Department of Medicine from the Universitat Autonoma of Barcelona (UAB).

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2021.703457/full#supplementary-material

Abbreviations

ABMR, antibody-mediated rejection; ABMR_h, antibody-mediated rejection histology; ABMR_hDSA_pos, antibody-mediated rejection histology with HLA-DSA; ABMR_hDSA_neg, antibody-mediated rejection histology without HLA-DSA; AT₁R-Ab, antibodies against angiotensin II type 1 receptor; CDC, complement-dependent cytotoxicity; CTG, chronic transplant glomerulopathy; ECs, primary human aortic endothelial cells; EC-XM, crossmatch with primary human aortic endothelial cells; EM, electron microscopy; ETAR-Ab, antibodies against endothelin-1 type A receptor; GFR, glomerular filtration rate; HLA-DSA, HLA donor-specific antibodies; HLA-AM, HLA antigen mismatches; HLA-EM, HLA epitope mismatches; IFTA, interstitial fibrosis and tubular atrophy; IQR, interquartile range; KT, kidney transplant; MICA-Ab, antibodies against major histocompatibility complex class I related chain A; PRA, panel-reactive antibody; PTCML, peritubular capillary multilayering; SAB, Single Antigen Bead assays; SD, standard deviation.

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