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Article

Correlation of Anti-HLA IgA Alloantibodies and Fc Receptor Motives with Kidney Allograft Survival

by
Marie-Luise Arnold
1,
Ulrike Steffen
1,
Michael Wiesener
2,
Christian Bach
3,
Bernd M. Spriewald
3 and
Monika Lindemann
4,*
1
Department of Internal Medicine 3—Rheumatology and Immunology, Friedrich-Alexander-University Erlangen-Nuremberg (FAU), University Hospital Erlangen, 91054 Erlangen, Germany
2
Department of Internal Medicine 4—Nephrology, Friedrich-Alexander-University Erlangen-Nuremberg (FAU), University Hospital Erlangen, 91054 Erlangen, Germany
3
Department of Internal Medicine 5—Hematology and Oncology, Friedrich-Alexander-University Erlangen-Nuremberg (FAU), University Hospital Erlangen, 91054 Erlangen, Germany
4
Institute for Transfusion Medicine, University of Duisburg-Essen, University Hospital Essen, 45147 Essen, Germany
*
Author to whom correspondence should be addressed.
Immuno 2022, 2(2), 372-386; https://doi.org/10.3390/immuno2020023
Submission received: 18 March 2022 / Revised: 20 April 2022 / Accepted: 28 April 2022 / Published: 29 April 2022
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Abstract

:
Immunoglobulin A (IgA) is the most abundant antibody isotype in humans and anti-HLA IgA was found in sera of transplant recipients. Focusing on patients awaiting kidney re-transplantation, we tested the impact of anti-HLA-class I/II IgA antibodies on graft survival. We analyzed 276 patients with and 238 without allograft failure. Eight motives of the Fcα receptor (FCAR) and Fcγ receptor were analyzed in patients with allograft failure. The distribution of anti-HLA IgA1/A2 and IgG antibodies differed significantly (p < 0.0001) between both patient groups, and IgA1 plus IgA2 antibodies were more abundant in patients with allograft failure. Allograft survival was significantly impaired if anti-HLA-class I plus II IgA was present, in the first 105 months (9 years) of follow-up (median of 43 vs. >105 months, p = 0.007). Patients with anti-HLA IgA and IgG vs. anti-HLA IgG only had a significantly shorter allograft survival within that follow-up period (88 vs. >105 months, p = 0.008). Moreover, allograft survival was shorter (p = 0.02) in carriers of GG vs. AA + AG genotypes of FCAR rs16986050. Thus, the presence of anti-HLA IgA plus IgG vs. IgG only was associated with shorter kidney allograft survival and FCAR motives may impact on graft survival.

1. Introduction

Chronic antibody-mediated graft rejection (AMR) is a major cause for the late loss of allograft function [1,2,3,4]. Accordingly, the presence of antibodies against allogeneic human leukocyte antigens (HLA) in the serum of recipients is strongly associated with an increased risk of graft failure. Monitoring of immunoglobulin G (IgG) antibodies against HLA is an important component of most current AMR risk stratification strategies after kidney transplantation. The impact of other complement-fixing and also non-complement-fixing antibody isotypes, such as immunoglobulin A (IgA)1 and IgA2, on graft survival has also gained attention [5]. In previous studies we have already reported the widespread occurrence of anti-HLA alloantibodies of the IgA isotype (anti-HLA IgA) in sera of solid organ transplant patients [6,7]. In addition to the damage caused by anti-HLA antibodies, the impact of Fcα receptor (FCAR) and Fcγ receptor (FCGR) polymorphisms on graft survival is still under debate. IgA can interact with the Fcα receptor (FcαRI), leading to pro- or anti-inflammatory responses [8].
The Janus-like nature of IgA is mainly due to the heterogeneity in molecular forms and the interaction with IgA receptors. IgA is a relatively “unique” antibody isotype in humans due to its heterogeneity in molecular forms, subclasses and glycosylation, and the unexpectedly low involvement of serum IgA in systemic immune responses. More IgA is produced per day (66 mg per kg and day) than all other classes combined [9].
In our retrospective study, we assessed the impact of the presence and specificity of anti-HLA IgA alloantibodies on graft survival in 514 kidney transplant patients from the Transplant Center in Erlangen-Nuremberg (Germany). Two hundred and seventy-six patients suffered from allograft failure, indicated by the need for dialysis after transplantation (group I), whereas in 238 transplanted patients the kidney allograft was still functioning (group II). The group I patients awaiting re-transplantation were additionally tested for five FCAR and three FCGR polymorphisms, in comparison to a cohort of 213 healthy, randomly selected, unrelated blood donors. In the current project, we demonstrate for the first time that the presence of anti-HLA IgA and motives of its corresponding receptor (FCAR rs16986050) appear as additional risk factors for allograft failure, alongside anti-HLA IgG.

2. Materials and Methods

2.1. Patients and Study Design

Sera of 514 patients from the Transplant Center in Erlangen-Nuremberg (Germany) were collected and divided into two groups.
Group I comprised 276 patients who were transplanted between 1987 and 2018 and suffered from kidney allograft failure (dysfunction and/or graft loss). They were selected in 2018 for this follow-up study based on the following criteria: (1) history of at least one previously failed kidney graft, (2) listed for kidney re-transplantation, and (3) availability of date of previous kidney transplantation and date of first dialysis after previous transplantation. Of note, inclusion of patients was irrespective of cause of rejection and previous screening results. We employed the median time to first dialysis after transplantation (TTD, Time-To-Dialysis) as a measure for graft failure. In this group, the median time between transplantation and blood sampling was 77 months. This timing was determined by the interval between transplantation, graft loss with return to dialysis and decision to re-list for a further transplantation (when the samples were drawn).
Group II consists of 238 patients who were transplanted between 1988 and 2019 and still had a functioning graft. We consecutively included all samples from patients who were kidney transplanted only once and who were screened routinely post-transplant during the period from August to December 2019. The median time between transplantation and blood sampling was 62 months.
Demographics and anti-HLA alloantibody status for both groups are displayed in Table 1. Due to the fact that the patient cohorts belonged to the same transplant center, the initial immunosuppressive therapy was comparable. The patients usually received induction therapy with an interleukin-2 receptor antagonist (basiliximab). Sensitized patients and patients of the European Senior Program (ESP) offered from Eurotransplant (Leiden, The Netherlands) usually received anti-thymocyte globulin (ATG). Initial baseline triple immunosuppression consisted of a calcineurin inhibitor (CNI, either tacrolimus or cyclosporine A), an anti-metabolite (mycophenolate sodium or mycophenolate mofetil) and steroids.
In the patients with and without allograft failure anti-HLA IgA1 and IgA2 and anti-HLA IgG antibodies were measured, and specificity against HLA class I and/or class II was determined. Furthermore, the patients with allograft failure and 213 randomly selected, healthy controls (HC) were tested for FCAR and FCGR polymorphisms. Of the healthy controls, 60% were female and 40% were male.
The study was approved by the local Ethics Committee, the participants provided written informed consent and the study was performed in accordance with the declaration of Helsinki.

2.2. Antibody Screening and Specification

After freezing and thawing, all patient sera were tested for the presence of anti-HLA IgA and IgG as previously described [6,7] by using Luminex mixed bead array (OneLambda, Canoga Park, CA, USA). The sera were tested according to the manufactures’ instructions and analyzed on a LABScanTM 200 Luminex flow analyzer (Luminex Corp., Austin, TC, USA). For the differentiation of the IgA1 and IgA2 isotypes within the anti-HLA antibodies, we modified the test by replacing the IgGall secondary antibody conjugate by two different mouse anti-human antibodies, secondary antibodies specific for IgA1 (clone B3506B4) and IgA2 isotypes (clone A9604D2). These secondary antibodies were obtained from Southern Biotech (Birmingham, AL, USA) and were R-phycoerythrin (R-PE)-conjugated. Due to the different sensitivity level in the generic IgG and IgA microsphere-based assay, the threshold for positive results was set at a decreased ratio of 2.0 for IgA1 and IgA2 isotype antibodies compared to a ratio of 5.0 for IgGall anti-HLA alloantibodies. The subclasses IgA1 and IgA2 are grouped together as IgA.

2.3. Genotyping of FCAR and FCGR Polymorphisms

Genomic DNA was isolated from 0.5 mL peripheral blood using the E.Z.N.A. Blood DNA Kit II (VWR, Darmstadt, Germany) according to the manufacturer’s protocol. Genotyping of four intron polymorphisms of the FCAR on chromosome 19 (rs10402324, rs11084377, rs1865097, rs4806608) and one exon 5 polymorphism on chromosome 19 (rs16986050, 844A/G, which changes codon 248 from AGC (Serine) to GGC (Glycine) in the cytoplasmic domain of the receptor) as well as of three polymorphisms of the FCGR (FCGRIIA (rs1801274, 519A/G, codon 131 histidine to arginine), FCGRIIIA (rs396991, 559G/A, codon 176 valine to phenylalanine), and FCGRIIIB (rs35139848, neutrophil antigen 1 (NA1)/NA2)) was carried out in a StepOnePlus real-time PCR detection system (Applied Biosystems, Darmstadt, Germany) using TaqMan SNP Genotyping Assay and TaqMan Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems, Darmstadt, Germany). TaqMan MGB probe labelled with VICTM dye detects the allele 1 and probe labelled with FAMTM dye detects the allele 2.

2.4. Statistical Analysis

Data were analyzed using GraphPad Prism version 8.4.2 for Windows (GraphPad Prism Software, La Jolla, San Diego, CA, USA) or IBM SPSS Statistics version 22 (Armonk, NY, USA). The distribution of anti-HLA IgA and IgG antibodies in patients with and without allograft failure or in patients with shorter and longer allograft survival was compared by chi-square test. Frequencies of various genotypes or alleles in kidney transplant recipients and healthy controls were compared by Fisher’s exact test. Allograft survival of various patient groups was shown by Kaplan–Meier curves and the respective groups were compared by Log Rank (Mantel–Cox) test. All analyses were performed 2-tailed and results were considered significant at p < 0.05. To adjust for multiple testing in cases of comparing graft survival of >2 subgroups a Bonferroni-corrected p-value was used to assert statistical significance.

3. Results

3.1. Anti-HLA IgA Antibodies Are Frequent in Kidney Transplant Recipients

In both groups, patients with allograft failure (n = 276) and patients with functioning graft (n = 238) we observed overall a high frequency of anti-HLA IgA isotype antibodies (Table 1).
Eighty-nine out of 276 patients with allograft failure (32%) had detectable anti-HLA IgA antibodies and 243 patients (88%) anti-HLA IgG antibodies in the serum. In total, 246 out of 276 patients (89%) displayed anti-HLA antibodies (IgA and/or IgG). One hundred and 24 out of 238 patients with functioning graft (52%) had detectable anti-HLA IgA and 107 patients (45%) anti-HLA IgG antibodies. Anti-HLA antibodies independent of isotypes were detectable in 162 patients (68%). The distribution of anti-HLA IgA and IgG antibodies in patients with and without allograft failure differed significantly (p < 0.0001) (Table 1, Figure 1). The overall frequency of IgA antibodies was lower in patients with allograft failure (Figure 1A) and the frequency of IgG antibodies was higher (Figure 1B). Antibodies either of the IgA1 or IgA2 subclass were more abundant in patients with a functioning graft; whereas IgA1 plus IgA2 antibodies were found more frequently in patients with graft failure (Figure 1A).

3.2. In Patients with Kidney Allograft Failure the Presence of Anti-HLA IgA Antibodies Correlates with Reduced Graft Survival

Based on anti-HLA antibodies of various isotypes (IgA/IgG), specificity (against HLA class I and class II), and subclasses (IgA1/IgA2), the 276 patients with allograft failure were divided into subgroups and graft survival was compared. In the group of patients with allograft failure and TTD below the median of 105 months (9 years), i.e., with shorter graft function, anti-HLA IgA antibodies were observed more frequently (p = 0.006) than in patients with allograft failure and longer graft function (65% vs. 35%, respectively) (Table 2). This association was observed for IgA but not for IgG antibodies. In patients with TTD below vs. above the median of 105 months, the frequency of anti-HLA IgG antibodies was similar (53 vs. 47%, respectively).
We hypothesized that IgA antibodies plus IgG antibodies against HLA class I or class II may have a higher impact on graft survival, compared to IgG antibodies only. Thus, we determined the TTD value with respect to IgG antibodies against HLA class I and/or class II (Table 3). The median TTD was shorter in patients with anti-HLA IgA antibodies in combination with anti-HLA class I IgG (87 months), anti-HLA class II IgG (90 months) or anti-HLA class I and class II IgG (88 months) compared to patients with exclusively anti-HLA IgG antibodies (116 months) as well as antibody negative patients (127 months).
Patients with IgA tended to have a shorter allograft survival compared to patients without IgA antibodies, as shown by Kaplan–Meier curves (Figure 2A). This effect was most pronounced when antibodies were directed against HLA class I and II. As already demonstrated in our previous publications on anti-HLA IgA antibodies [6,7], also in this study the presence of anti-HLA IgA antibodies strongly correlated with the presence of anti-HLA IgG antibodies. The vast majority of patients with IgA antibodies also had IgG antibodies (86/89). When considering not the complete time of follow up, i.e., a TTD of up to 348 months (29 years), but a shorter period (which was defined as the median follow-up of 105 months), differences with respect to IgA antibodies were highly significant (p = 0.01). The graft survival was shortest when IgA antibodies against HLA class I and II were present (Figure 2B). The TTD differed significantly between patients with HLA class I and II IgA antibodies and those without IgA antibodies (median allograft survival of 43 vs. > 105 months, p = 0.007).
Moreover, Kaplan–Meier curves indicated that the complete course and the earlier course depended significantly (p < 0.001) on IgA and IgG antibodies (Figure 2C,D). Graft survival was inferior if IgA and IgG antibodies were present, as compared to IgG antibodies alone. Censoring for the first 105 months (9 years) of follow-up, the median allograft survival was 88 vs. >105 months (p = 0.008; Figure 2D). The short graft survival in patients with only IgA antibodies (n = 3) can hardly be interpreted, because of the very small sample size.
Graft survival showed no significant dependency on the presence of IgG antibodies against HLA class I or II (Figure 3A). However, survival tended to be shorter if antibodies against HLA class I and II were present. When IgG plus IgA antibodies were present, the course was similar in the case of HLA class I, class II, or class I and II antibodies. The graft survival was shorter if patients had IgG plus IgA antibodies as compared to those with IgG antibodies only or without antibodies (Figure 3B). Of note, the survival curves were similar in patients with IgG antibodies only or without antibodies. In addition, the course of patients with both IgA1 and IgA2 showed no clear difference from those with either IgA1 or IgA2 antibodies (Figure 3C).
Univariate analyses revealed no significant association of either sex, underlying disease, or patient age with the presence of anti-HLA IgA antibodies. The limited number of patients in this study precluded multivariate analysis.

3.3. FCAR and FCGR Polymorphism and Allograft Survival

Kidney transplant recipients with allograft failure were genotyped for five FCAR polymorphisms (FCAR rs10402324, rs1184377, rs16986050, rs1865097, and rs4806608) and three FCGR polymorphisms (FCGR2A rs1801274, FCGR3A rs396991, FCGR3B, and rs35139848) [10]. The frequency of the respective genotypes and alleles was compared in these 276 patients with graft failure and 213 healthy controls (Figure 4). Fisher’s exact test showed that patients and controls differed significantly for the three dimorphisms FCAR rs16986050 (p < 0.0001), FCAR rs1865097 (p = 0.01), and FCGR3B rs35139848 (p = 0.002), considering genotype frequencies (Figure 4A). In patients we observed an increase in the frequency of the A allele of FCAR rs16986050 (p < 0.0001) and of the G allele of FCAR rs1865097 (p = 0.007) (Figure 4B).
Kaplan–Meier curves showed that the allograft survival (defined by TTD) correlates with the exon polymorphism FCAR rs16986050 (Figure 5). Patients carrying the genotype GG had the shortest median allograft survival (50 months), followed by carriers of the genotype AG (93 months) and AA (112 months). Log Rank (Mantel–Cox) test indicated that the graft survival differed significantly between carriers of AA and AG + GG genotypes (p = 0.02) and carriers of AA and AG genotypes (p = 0.04). The remaining FCAR and FCGR polymorphisms did not significantly correlate with allograft function.

4. Discussion

Screening and identification of anti-HLA IgG alloantibodies is currently the standard approach for risk stratification of kidney transplantations. Corroborating the validity of this approach, anti-HLA IgG is both prevalent and associated with poor prognosis in our patient cohort. However, we also demonstrate that alloantibodies of the IgA isotype can be found frequently in patients awaiting re-transplantation, often together with anti-HLA IgG. As previously shown [7], the specificity of anti-HLA IgA and IgG antibodies is similar. Hence, we did not further specify the target of anti-HLA IgA antibodies in this current study. Importantly, our data indicate that patients with both anti-HLA IgA and anti-HLA IgG display the shortest median graft survival of all subgroups. This implies that testing for anti-HLA IgA in addition to IgG may allow for the separation of subgroups of patients at intermediate (IgG+/IgA−) and high risk of graft failure (IgG+/IgA+). Of note, the occurrence of anti-HLA IgA without detectable anti-HLA IgG was rare within group I patients (1%), prohibiting definitive conclusions about the specific contribution of anti-HLA IgA only to transplantation outcome. Nevertheless, it can be speculated that the presence of IgA only indicates an overall weaker immune response, whereas both anti-HLA IgA and anti-HLA IgG is an indicator of a stronger immune response. This hypothesis is supported by the fact that a higher proportion of patients without allograft failure had IgA antibodies only (23%). We propose that testing for IgA alloantibodies can supplement routine IgG testing to improve risk stratification in kidney transplant recipients.
Effective treatment strategies of chronic AMR are still controversial and understudied [11]. Accordingly, the clinical impact of an improved stratification remains largely speculative. As our dataset did not include the cause for the loss of graft function, further studies are needed to examine whether IgA alloantibodies are specifically associated with any specific cause of graft loss such as AMR. The reasons for kidney allograft failure are still not well understood. Some authors have postulated that late deterioration results from dysregulated fibrosis, drug toxicity [12] or progressive “chronic allograft nephropathy” [12,13,14]. Another explanation is that kidney transplants are essentially stable after recovering from the stress of implantation until specific diseases or conditions develop, including AMR and recurrent renal diseases [4,15]. As shown in a landmark publication by Sellares et al. [4] histologic diagnoses change according to time post-transplantation. As of month 100 after transplantation, the probability of transplant atrophy/fibrosis and glomerulonephritis constantly increased, whereas the probability of antibody-mediated rejection remained constant and of T cell-mediated rejection decreased. It may thus be reasonable to split the course after kidney transplantation and to consider the earlier course separately. The separate analysis until month 105 showed that anti-HLA IgA antibodies had a significant impact on allograft survival, which was not detectable thereafter. In the later period, the effect of dysregulated fibrosis and drug toxicity may have covered the effect of HLA antibodies.
In the present study we tested sera from patients with a previously failed graft who were re-listed for transplantation (group I). The time between transplantation and blood sampling in this group was on average 77 months. The patient sera were collected after the first dialysis following transplantation. We unfortunately have no data on the initial time point of IgA and/or IgG alloantibody formation, during the course after transplantation. Previous data indicate that human IgA, the most prominent antibody class at mucosal surfaces, induces antibody-dependent cell-mediated cytotoxicity/phagocytosis (ADCC/P) after binding to activating Fc receptors [16,17]. In addition to monocytes, macrophages, and eosinophils as FcαRI expressing immune cells, neutrophils are especially vigorous in eliminating IgA opsonized cells [16]. Triggering of neutrophils by IgA was more efficient than by IgG and IgA engagement of neutrophils elicited stronger Fc receptor signaling than IgG [16]. It has been hypothesized that cross-linking of FcαRI by aberrant IgA-antigen complexes, albeit in patients with ulcerative colitis, may be a key process causing severe tissue damage [17]. It remains to be further studied if a similar phenomenon occurs when anti-HLA IgA is bound to mucosal sites or to the tissue of a kidney graft. However, it appears likely that IgA alloantibodies induce ADCC/P leading to allograft failure, because we observed that an exon polymorphism within FcαRI (FCAR rs16986050) correlated with allograft survival. However, IgA could also initiate the complement cascade via the nonclassical lectin pathway [18]. In detail, carbohydrate moieties on IgA could bind to the pattern-recognition molecule mannan-binding lectin (MBL) [19], which leads to the activation of MBL-associated serine proteases (MASPs) 1–3, as described in a review by Nauser et al. [20]. These proteases could activate C3 and C5 convertases, resulting in the formation of the membrane attack complex (C5b-9). Of note, antigen presenting cells (APC) express complement receptors, e.g., against C3a and C5a. Activation by C3a and C5a enhances APC priming of T cells by increasing the presentation of alloantigens and the expression of costimulatory molecules. Upon stimulation APC promote T cell differentiation and proliferation. CD4+ T cells ultimately stimulate antibody production, whereas CD8+ T cells can mediate cellular rejection. Of note, a study including but not limited to IgA nephropathy patients found an association of post-transplantation IgA deposition and graft dysfunction [21]. Therefore, prolonged IgA deposition is another plausible mechanism for impaired graft function eventually resulting in graft failure. In contrast to these studies, however, our analysis specifically focuses on anti-HLA IgA. Taken together, a functional impact of anti-HLA IgA on allograft survival is conceivable. The effect of IgA on graft function occurred rather shortly after transplantation, as depicted by Figure 2. It was detectable only when considering the first 105 months after transplantation, but not thereafter. Thus, IgA alloantibodies seem to be more dangerous in the earlier phase after transplantation. Apart from anti-HLA IgA, anti-HLA IgG leads to complement activation, which is used diagnostically in the Eurotransplant area for antibody screening and cross-matching-based on the complement-dependent cytotoxicity (CDC) assay-prior to transplantation [22]. In contrast to IgA, however, IgG activates the classical pathway, via formation of antigen-antibody complexes and binding of C1q to the Fc portion of IgG. Of note, only anti-HLA IgA but not total IgA had an impact on graft survival. Total IgA serum levels did not correlate significantly with graft survival, presumably because the fraction of antibodies with HLA specificity was too small [7].
Comparing anti-HLA IgA antibody results between patients with and without allograft failure, we could clearly see that anti-HLA IgA antibodies are detectable at lower numbers in patients with graft failure (32% vs. 52%, respectively). The decrease in patients with allograft failure might be due to the disappearance of IgA monomers through the vessels into the tissue. This mechanism is already described by Kerr and Phalipon [23,24]. Serum IgA, the second most abundant isotype in the circulation, mainly consists of monomers derived from bone marrow plasma cells [23], whereas secretory IgA is synthesized as dimers by local plasma cells before being transported to mucosal surfaces through epithelial cells by the polymeric Ig receptor [24]. It can be assumed that the monomeric anti-HLA IgA antibodies, which were detected in our study in sera of transplanted patients without graft failure, may be also lost due to a leakage into the urine especially in patients with allograft failure. In patient sera, these anti-HLA IgA antibodies are no longer visible. In the tissue, especially of the kidneys, dimeric anti-HLA IgA antibodies can deposit in the glomeruli and harm the organ, which can result in graft loss, as described recently by Perse [25]. The hallmark of IgA nephropathy is the deposition of IgA in the glomeruli. Deposits are composed mainly of IgA, sometimes together with IgG or complement components such as C3.
A major determinant of MFI values to single antigen beads testing is the HLA specificity of the beads, e.g., MFI values for HLA-DQ are much higher as compared to HLA-C, due to various density of the HLA molecules on the beads. Thus, the MFI level correlates poorly with antibody levels and is technically hard to compare between patients. Moreover, the major fraction of anti-HLA IgA is found in the tissue, whereas IgG is found in the blood. In the case of rejection, IgA and IgG can decline in the serum and increase in the graft.
IgA and IgA receptors play a significant role in vivo in maintaining the integrity of immune responses in systemic and mucosal compartments [9]. On the one hand, this functional balance may be altered in a variety of pathological conditions where a role for selected IgA receptors is well established. On the other hand, IgA and anti-FcαRI Fab may be used as therapeutic tools in human inflammatory diseases, by restoring this balance or dampening immune responses [9]. Recent data by Steffen et al. demonstrate an important role of the different glycosylation profiles of the IgA1 and IgA2 subclasses on effector cell functions [26]. IgA1 possesses more sialic acid than IgA2 and IgA2 acts more pro-inflammatory on neutrophils and macrophages than IgA1. Removal of sialic acid increases the pro-inflammatory capacity of IgA1, making it comparable to IgA2. Interestingly, disturbances in the IgA subclass balance are associated with autoimmune disease. It needs to be further analyzed if there is any correlation between the higher fraction of anti-HLA IgA1 plus IgA2 and allograft failure.
As compared to a previous study on 289 individuals with known alloantigen exposure through pregnancy (n = 91) or kidney transplantation (n = 198) [27], the frequency of anti-HLA IgA was considerably higher in our current cohort of kidney transplant recipients with and without allograft failure (32 or 52% vs. 3.5%). Most likely, these differences were caused by the fact that we used different antibody clones for IgA2 detection and tested allograft recipients on average later after transplantation. In our study, an antibody by One Lambda was applied, which does not cross-react with IgA1. Due to the Janus-like nature of IgA [9], a differentiation between IgA1 and IgA2 appears as essential and a pan-IgA secondary antibody may not be adequate. In the current study we tested serum samples for IgA1 and IgA2. However, concentrations of IgA1 and IgA2 in renal tissue are presumably a better predictor of transplant survival.
Nevertheless, recent data indicate that the immunological function of IgA is more extensive than previously thought and suggest that serum IgA-induced inflammation plays an important role in orchestrating host defense by different cell types in non-mucosal tissues [28]. In our cohort patients carrying the genotype GG of FCAR rs16986050 had the shortest median allograft survival (TTD of 50 months), followed by carriers of the genotype AG (TTD of 93 months) and AA (TTD 112 months). Thus, not only IgA concentrations in the serum or tissue but also the respective FCAR motives may have an impact on allograft survival.
Our study has several limitations. The major point is that it is not a longitudinal but a cross-sectional study. Because we performed it retrospectively, we cannot clarify whether the IgA alloantibodies were produced before, during, or even after allograft rejection. This precludes assessment, e.g., of the relevance of pre-formed IgA alloantibodies on graft survival. Moreover, as our dataset did not include the cause of graft loss, further studies are needed to examine whether IgA alloantibodies are associated with any specific cause of graft loss such as antibody or cell mediated rejection.
In summary, our data in kidney re-transplant candidates clearly demonstrate that the presence of anti-HLA IgA antibodies is an indicator of a significantly inferior outcome, in particular in combination with anti-HLA IgG. A larger study allowing multivariate analyses is needed to investigate the influence of various covariates, including IgA, on allograft loss. Alternatively, longitudinal studies could permit to assess the timing of IgA and IgG antibody development after transplantation. Furthermore, the IgA serum status should be determined also at baseline, i.e., prior to transplantation. It would be particularly interesting to compare the specificity of IgA and IgG antibodies and to determine their interaction with various motives of FCAR and FCGR.

5. Conclusions

In this single center study on patients awaiting kidney re-transplantation, we demonstrate for the first time that the presence of anti-HLA-IgA plus IgG vs. IgG alone correlated significantly with allograft failure and that the time-to-dialysis was associated with the anti-HLA IgA but not IgG status. Moreover, genotypes of an IgA receptor (FCAR rs16986050) were predictive of allograft survival and may therefore be a new prognostic marker. In conclusion, screening for anti-HLA IgA1 and IgA2 in serum and tissue could improve the risk stratification in this patient cohort.

Author Contributions

Conceptualization, M.-L.A., U.S., M.W., C.B. and B.M.S.; methodology, M.-L.A. and U.S.; validation, M.-L.A. and M.L.; formal analysis, M.-L.A. and M.L.; investigation, M.-L.A.; resources, M.W., B.M.S. and C.B.; data curation, M.L.; writing—original draft preparation, M.-L.A. and M.L.; writing—review and editing, M.L. and U.S.; visualization, M.L.; supervision, B.M.S.; project administration, M.-L.A.; funding acquisition, B.M.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Friedrich-Alexander-University Erlangen-Nuremberg, Germany (251_18B; 17 December 2020).

Informed Consent Statement

Written informed consent was obtained from all subjects involved in the study.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgments

We are grateful to the patients and healthy controls for their participation in this study.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Distribution of anti-HLA IgA and IgG antibodies in kidney transplant recipients with and without allograft failure. This analysis includes 276 patients with allograft failure, which again required dialysis. The control group of 238 kidney transplant recipients still had a functioning graft. Both groups were compared by chi-square test. Panel (A) shows data on IgA and its subclasses IgA1 and IgA2, panel (B) on IgG and IgA. Abs, antibodies.
Figure 1. Distribution of anti-HLA IgA and IgG antibodies in kidney transplant recipients with and without allograft failure. This analysis includes 276 patients with allograft failure, which again required dialysis. The control group of 238 kidney transplant recipients still had a functioning graft. Both groups were compared by chi-square test. Panel (A) shows data on IgA and its subclasses IgA1 and IgA2, panel (B) on IgG and IgA. Abs, antibodies.
Immuno 02 00023 g001
Immuno 02 00023 g002 550
Figure 2. Impact of anti-HLA IgA and IgG antibodies on kidney allograft survival. This analysis includes 276 patients with allograft failure. Panel (A,B) shows allograft survival stratified by the presence of IgA antibodies against HLA class I (I), HLA class II (II) or both (I+II). Panel (B) shows only the earlier course of IgA antibodies (<median time of 105 months), which was analyzed after censoring the data. Panel (C,D) shows allograft survival stratified by the presence of IgA and/or IgG antibodies against any HLA-antigen. Panel (D) shows the earlier course of IgA and/or IgG antibodies (< median time of 105 months). The time to first dialysis was used as a measure of the duration of allograft function. Graft survival of various groups was compared by Log Rank (Mantel–Cox) test. ** p < 0.01.
Figure 2. Impact of anti-HLA IgA and IgG antibodies on kidney allograft survival. This analysis includes 276 patients with allograft failure. Panel (A,B) shows allograft survival stratified by the presence of IgA antibodies against HLA class I (I), HLA class II (II) or both (I+II). Panel (B) shows only the earlier course of IgA antibodies (<median time of 105 months), which was analyzed after censoring the data. Panel (C,D) shows allograft survival stratified by the presence of IgA and/or IgG antibodies against any HLA-antigen. Panel (D) shows the earlier course of IgA and/or IgG antibodies (< median time of 105 months). The time to first dialysis was used as a measure of the duration of allograft function. Graft survival of various groups was compared by Log Rank (Mantel–Cox) test. ** p < 0.01.
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Immuno 02 00023 g003 550
Figure 3. Impact of anti-HLA IgG and IgA antibodies on kidney allograft survival, stratified by HLA-class and isotype, respectively. This analysis includes 276 patients with allograft failure, which again required dialysis. Panel (A) shows allograft survival stratified by the presence of IgG antibodies against HLA-class I (I), HLA-class II (II) or both (I+II). Panel (B) combines the HLA-specificity of IgG antibodies with the presence of any IgA antibody. Panel (C) stratifies survival by IgG antibodies, together with the isotype of IgA antibodies (IgA1/A2). The time to first dialysis was used as a measure of the duration of allograft function. Graft survival of various groups was compared by Log Rank (Mantel–Cox) test.
Figure 3. Impact of anti-HLA IgG and IgA antibodies on kidney allograft survival, stratified by HLA-class and isotype, respectively. This analysis includes 276 patients with allograft failure, which again required dialysis. Panel (A) shows allograft survival stratified by the presence of IgG antibodies against HLA-class I (I), HLA-class II (II) or both (I+II). Panel (B) combines the HLA-specificity of IgG antibodies with the presence of any IgA antibody. Panel (C) stratifies survival by IgG antibodies, together with the isotype of IgA antibodies (IgA1/A2). The time to first dialysis was used as a measure of the duration of allograft function. Graft survival of various groups was compared by Log Rank (Mantel–Cox) test.
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Figure 4. Genotype (A) and allele frequencies (B) of Fcα and Fcγ receptor polymorphisms (FCAR and FCGR, respectively) in 276 kidney transplant recipients with graft failure (KTX) and in 213 healthy controls. Data were compared by Fisher’s exact test. rs10x, rs10402324; rs11x, rs11084377; rs16x, rs16986050; rs18x, rs1865097; rs48x, rs4806608; 2A, rs1801274; 3A, rs396991; and 3B, rs35139848.
Figure 4. Genotype (A) and allele frequencies (B) of Fcα and Fcγ receptor polymorphisms (FCAR and FCGR, respectively) in 276 kidney transplant recipients with graft failure (KTX) and in 213 healthy controls. Data were compared by Fisher’s exact test. rs10x, rs10402324; rs11x, rs11084377; rs16x, rs16986050; rs18x, rs1865097; rs48x, rs4806608; 2A, rs1801274; 3A, rs396991; and 3B, rs35139848.
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Figure 5. Graft survival in 276 kidney transplant recipients with allograft failure, stratified by an Fcα receptor polymorphism (FCAR rs16986050, 844A/G). The time to first dialysis was used as a measure of the duration of allograft function. Graft survival of various groups was compared by Log Rank (Mantel–Cox) test. * p < 0.05.
Figure 5. Graft survival in 276 kidney transplant recipients with allograft failure, stratified by an Fcα receptor polymorphism (FCAR rs16986050, 844A/G). The time to first dialysis was used as a measure of the duration of allograft function. Graft survival of various groups was compared by Log Rank (Mantel–Cox) test. * p < 0.05.
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Table
Table 1. Demographics of kidney transplant recipients.
Table 1. Demographics of kidney transplant recipients.
Kidney Transplant PatientsWith Graft Failure Group I, n = 276Without Failure Group II, n = 238
Age at blood sampling, years, median (range)49 (14–75)56 (3–84)
Age at last tx, years, median (range)36 (1–74)48 (2–76)
Time between Tx and blood sampling, months, median (range)77 (0.5–446)62 (0.3–372)
TTD 1, months, median (range)105 (1–348)-
Sex, total, n (%)
Male167 (60.5%)177 (64%)
Female109 (39.5%)99 (36%)
No of previous transplants, total, n (%)
1233 (84%)238 (100%)
>143 (16%)0
Anti-HLA alloantibody status, total, n (%)
Presence of IgA and IgG antibodies *
Anti-HLA IgA antibodies89 (32%)124 (52%)
Anti-HLA IgG antibodies243 (88%)107 (45%)
Anti-HLA antibody positive246 (89%)162 (68%)
Combination of IgA and IgG antibodies *
IgG+/IgA+86 (31%)69 (29%)
IgG+/IgA−157 (57%)38 (16%)
IgG−/IgA+3 (1%)55 (23%)
IgG−/IgA−30 (11%)76 (32%)
IgA subclass antibodies *
IgA126 (9%)87 (37%)
IgA29 (3%)15 (6%)
IgA1 and IgA254 (20%)22 (9%)
IgA negative187 (68%)114 (48%)
1 Median time to first dialysis (TTD) after first transplantation (tx); * p < 0.0001 (chi2 test).
Table
Table 2. Association of anti-HLA IgA and IgG status and early 1 time-to-dialysis (TTD) in 276 patients with allograft failure.
Table 2. Association of anti-HLA IgA and IgG status and early 1 time-to-dialysis (TTD) in 276 patients with allograft failure.
n (%)IgA+IgA−Total
TTD < median (105 months)58 (65%) *89 (48%)147
TTD > median (105 months)31 (35%)98 (52%)129
Total89 (100%)187 (100%)276
n(%)IgG+IgG−Total
TTD < median (105 months)130 (53%)17 (52%)147
TTD > median (105 months)113 (47%)16 (48%)129
Total243 (100%)33 (100%)276
1 Early was defined as TTD < median (105 months). * p = 0.006 (chi2 test).
Table
Table 3. Time-to-dialysis (TTD) in patients with allograft failure and anti-HLA IgG specificity, combined with anti-HLA IgA.
Table 3. Time-to-dialysis (TTD) in patients with allograft failure and anti-HLA IgG specificity, combined with anti-HLA IgA.
IgG−
IgA−		IgG+
IgA−		IgG+
IgA+		IgG
HLA Class I + IgA+		IgG
HLA Class II + IgA+		IgG
HLA Class I+II + IgA+
Number of patients301578651467
TTD (months)12711688879088
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Arnold, M.-L.; Steffen, U.; Wiesener, M.; Bach, C.; Spriewald, B.M.; Lindemann, M. Correlation of Anti-HLA IgA Alloantibodies and Fc Receptor Motives with Kidney Allograft Survival. Immuno 2022, 2, 372-386. https://doi.org/10.3390/immuno2020023

AMA Style

Arnold M-L, Steffen U, Wiesener M, Bach C, Spriewald BM, Lindemann M. Correlation of Anti-HLA IgA Alloantibodies and Fc Receptor Motives with Kidney Allograft Survival. Immuno. 2022; 2(2):372-386. https://doi.org/10.3390/immuno2020023

Chicago/Turabian Style

Arnold, Marie-Luise, Ulrike Steffen, Michael Wiesener, Christian Bach, Bernd M. Spriewald, and Monika Lindemann. 2022. "Correlation of Anti-HLA IgA Alloantibodies and Fc Receptor Motives with Kidney Allograft Survival" Immuno 2, no. 2: 372-386. https://doi.org/10.3390/immuno2020023

APA Style

Arnold, M.-L., Steffen, U., Wiesener, M., Bach, C., Spriewald, B. M., & Lindemann, M. (2022). Correlation of Anti-HLA IgA Alloantibodies and Fc Receptor Motives with Kidney Allograft Survival. Immuno, 2(2), 372-386. https://doi.org/10.3390/immuno2020023

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