The presence of antibodies (Abs) against human leukocyte antigen (HLA) antigens before transplantation is associated with early, hyperacute, antibody-mediated rejection (AMR) of kidney grafts. Alloantibodies are now appreciated as important mediators of acute and chronic AMR (1). Acute AMR often arises in the first few weeks after transplantation and can also develop years after transplantation, often triggered by a decrease in immunosuppression. Evidence has accumulated that argues for a significant role of alloantibodies in the pathogenesis of slowly progressive graft injury and dysfunction such as chronic allograft nephropathy (2–4). The contribution of humoral alloreactivity to the rejection of renal allografts is yet not well defined because humoral antigraft reactions are not easily detectable in transplant biopsies, and serial measurements of circulating alloantibodies in the posttransplantation period are not routinely performed.
Patients awaiting renal transplantation are routinely tested for lymphocytotoxic panel-reactive antibodies, and graft allocation depends on the current T- and B-cell complement-dependent cytotoxicity (CDC) crossmatch. A positive T-cell crossmatch reflects the presence of anti-HLA class I Abs and is a contraindication to transplantation. A positive B-cell crossmatch reflects the presence of anti-HLA class II or weak anti-HLA class I, and its clinical significance remains controversial (5, 6). Although pretransplant-negative CDC deeply reduces the risk for hyperacute rejection, early AMR does occur in the presence of a negative CDC indicating that CDC may lack sensitivity and/or specificity to detect alloantibodies.
Currently, HLA Abs are increasingly measured by solid-phase tests with purified HLA molecules bound to either microtiter plates for enzyme immunoassays or microparticles (beads) for flow cytometry. These tests have better sensitivity and specificity and also add complexities to the interpretation of results. Flow cytometry-based solid phase assays, especially beads coated with single antigens, which, in contrast to the CDC technique, permit cell-independent HLA-specific alloantibody detection, represent a major advance in HLA Ab characterization (7). However, these highly sensitive assays cannot distinguish between complement fixing and presumably less harmful noncomplement-fixing anti-HLA Abs. Deposition of complement split product C4d is a valuable marker for AMR and often, but not always, correlates with detection of circulating anti-HLA Abs (8). Recently, the simultaneous measurement of panel-reactive and donor-specific Abs (DSAs) binding and complement fixation was evaluated (9) but did not improve the predictive value of posttransplant serology. Thus, optimal strategies for both monitoring and diagnostic still have to be defined.
Most alloantibodies are directed against HLA antigens. In addition to anti-HLA class I and class II, Abs directed to polymorphic nonclassical major histocompatibility complex (MHC) molecules, such as MHC class I-related chain A, may also contribute to graft rejection (10–12). We and others previously demonstrate that graft's endothelial cells (ECs) express and regulate both donor class I and class II HLA and nonclassical HLA molecules (13–15). Moreover, non-HLA type, EC-reactive Abs have been also identified (16, 17). ECs are the main cellular target for preformed and induced alloantibodies (1). Alloantibody binding may activate complement cascade that induce EC lysis and graft thrombosis as it occurs in hyperacute rejection. According to specificity and titer, alloantibody binding may also contribute to EC dysfunction (i.e., activation and apoptosis), which concur to accelerated allograft arteriosclerosis (18) or EC accommodation (19, 20). Therefore, an effective monitoring and control of alloantibody production is needed to improve allograft long-term survival.
We speculate that detection and characterization of posttransplant circulating alloantibodies that can really bind to graft EC may provide new advance in the knowledge of alloimmune response. In this study, an experimental donor-specific EC crossmatch (ECXM) by flow cytometry was performed using primary cultures of vascular ECs, prospectively isolated from deceased transplant donors, at the time of transplantation. Retrospective analysis was performed on pretransplant and posttransplant sera (n=256) from an historical cohort of 22 kidney allograft recipients transplanted in our center from 1999 to 2001 (patient's follow-up: ≥10 years). Time course and titer of EC-reactive alloantibody production were defined, and Ab IgG subclass was determined in regard to complement-binding capacity. Biological data (Ab time course, titer, IgG subclass, and specificity) were analyzed in respect to clinical data and graft outcome.
Kidney Transplant Recipients and Sera
An historical cohort of 22 transplant recipients was selected for this study. These patients were transplanted in our center between 1999 and 2001 and they received transplant (kidney or kidney and pancreas) from 11 deceased organ donors. Patient's demographic data are listed in Table 1. A total of 256 sera from the 22 recipients (a mean of 11.6±2.8 sera/recipient) were retrospectively screened for the presence of Abs reactive to donor's EC. Time course of posttransplant sera tested in this study is shown in SDC 2 (http://links.lww.com/TP/A571).
Donor-Specific ECXM by Flow Cytometry: Methodological Aspects
Eleven cultures of donor EC corresponding to 22 paired renal transplantations have been selected for this study. A retrospective ECXM was performed by incubating recipient sera collected pretransplantation and posttransplantation with EC from their own graft donor. Ab binding was analyzed by flow cytometry using fluorescence-labeled anti-human IgG or IgM for detection. Controls include a pool of sera from healthy donor (n=20) and an isotype-matched IgG control. A positive ECXM (ECXM+) was defined by Ab binding leading to a median of fluorescence intensity at least equal to twice the median obtained with controls. An example is provided in Figure 1A. To assess the donor specificity of ECXM, sera reacting with donor-specific ECs were incubated with an irrelevant (third party) EC culture. Third-party ECs were carefully selected within our EC culture bank (n=104 samples) to avoid any cross alloreactivity with known anti-HLA Abs in the tested sera (Fig. 1B).
ECXM+: Strong Correlation With Posttransplant HLA Immunization
Among the 22 recipients, 6 (27.3%) have at least one serum that reacts with their donor EC and then elicit a ECXM+ according to the above criteria. Table 2 shows ECXM findings according to HLA matching, HLA sensitization, and Ab reactivity against lymphocytes. There was a strong association between anti-HLA Abs and ECXM+. All the six recipients with a ECXM+ also had detectable anti-HLA Abs posttransplantation. In contrast, among the 16 patients without EC-reactive Abs, only one developed anti-HLA Abs after transplantation. Thus, there is a positive and significant (P<0.0001) correlation between alloreactivity against EC, as reflected by an ECXM+, and the detection of anti-HLA Abs (Fig. 1C).
ECXM was performed using resting and cytokine-activated EC to ensure both optimal and differential expression for HLA class I and II molecules. To this aim, donor ECs were stimulated with interferon (IFN)γ that elicits both up-regulation of class I and de novo expression of class II or tumor necrosis factor (TNF)-α that only increases HLA class I level (see Figure, SDC 3,http://links.lww.com/TP/A572). Using these conditions, most of ECXM+ sera react with IFNγ- and/or TNF-α-treated EC (Fig. 1D). These data indicate that ECXM revealed Abs directed either to HLA or to EC surface molecules up-regulated or inducible on inflammatory conditions.
ECXM+: Donor Specificity and Target Antigens
Next, donor-specific alloreactivity of ECXM was determined using a third-party EC. Among the six ECXM+ recipients, we found that three patients (n=5, 7, and 15) have a donor-specific ECXM+ as determined by the lack of sera reactivity for a third-party EC as illustrated in Fig. 1B. Posttransplant HLA monitoring indicates that these three recipients developed anti-HLA class II DSA (anti-DQ1, -DQ3, and -DR53 for patients n=5, 7, and 15, respectively) strongly suggesting that these DSA account for ECXM positivity. Consistently, sera from recipients 5, 7, and 15 only react with IFNγ-treated EC strongly supporting the hypothesis of anti-class II DSA. Sera from the three other ECXM+ patients (n=12, 18, and 22) consistently bind to all third-party ECs tested indicating that non-donor-specific non-HLA Abs are involved. These sera bind to EC activated with either TNF-α or IFNγ or even to resting EC targets (patient 18). Importantly, these recipients also exhibit a broad panel of non-DSA anti-HLA classes I and II (Table 2).
ECXM+ Identifies Selective Profiles of Posttransplant Immunization
Detection of EC-reactive Abs was follow-up for the time period running from the day of transplantation to more than 5 years posttransplantation. This kinetic analysis of alloantibodies detected by ECXM is depicted in Figure 2A. ECXM identifies two typical patterns of recipient's sensitization. For patients n=12, 18, and 22, Ab response was both early and transient. Non-DSA reacting with donor EC were detected in pretransplant sera for patients 18 and 22 and during the first month posttransplantation for recipient 12. In two cases (patients 12 and 22), Abs were not detected after the first year of transplantation, whereas for recipient 18, Abs were consistently detected up to year 4. In contrast, in the second group of recipients (5, 7, and 15), anti-HLA class II Abs detected by ECXM appear later (from month 1 to year 2) but remain present throughout the posttransplantation area.
EC-Reactive Abs: Titer and IgG Subclass
Next, ECXM were repeated with serial dilutions (1/2 to 1/1024) of selected ECXM+ sera to determine Ab titer. Results shown in Table 3 indicate that Ab titers vary from 1/8 (patient 22) to 1/1024 (patient 18). Variation in titers was not related to patient's subgroup because a similar variability occurs among DSA-ECXM and non-DSA-ECXM. Moreover, in most cases, Abs that give raise to ECXM+ are IgG1 (patients 5, 12, 15, and 22; Table 3). Thus, IgG1 features both DS-ECXM (patients 5 and 15) and non-DS-ECXM (patients 12 and 22) Abs. For patient 5 who acquired DS (anti-DQ3) Abs posttransplantation, an IgG4 was detected at M60 in addition to the IgG1 present at M24. Together, these data demonstrate the occurrence in HLA-sensitized patients of EC-reactive, complement-fixing Abs with significant (1/8) and even high (1/1024) titers. ECXM was performed to detect both IgG and IgM in parallel assays. As a result, IgM were found in only one patient (patient 12). IgM were present posttransplantation and reacted with nonstimulated EC at D32 and IFNγ-treated EC at D74.
EC-Reactive Abs Trigger Selective Gene Transcription in Graft EC
TaqMan Low Density Arrays targeting immune and apoptosis pathways (96 transcripts each) were performed to further define the selective regulatory action mediated in EC by EC-reactive DSA and non-DSA found in our study. To this aim, ECs were treated with the respective patient's sera for 2 hr. ECs treated with IFNγ, irrelevant IgG (i.e., a pool of normal AB sera), culture medium, and also TNF, anti-HLA class I (W6/32) and class II (L243) monoclonal Abs were included as negative and positive controls, respectively (see detailed Materials and Methods in supplement). Transcript levels were expressed as fold changes after normalization to housekeeping genes and as a ratio to untreated controls (see Figure, SDC 4,http://links.lww.com/TP/A573). As a result, two transcripts discriminate the impact of the respective Abs on EC: interleukin (IL)-1β and CCR4. As shown in Figures 2(B and C), DSA selectively regulates CCR4, whereas non-HLA non-DSA regulate IL-1β, providing evidence for selective regulatory pathways initiated by anti-HLA class II and non-HLA non-DSA in graft EC.
ECXM+ and Clinical Outcomes
The clinical impact of an ECXM+ was then evaluated by comparing graft function of the six ECXM+ recipients with their respective paired mate kidney recipients which were ECXM−. Graft function was assessed at the time of detection (t0) of a ECXM+, 5 and 10 years post t0 by comparing serum creatinine and proteinuria (see Figure, SDC 5,http://links.lww.com/TP/A574). A trend toward higher creatinine (162.2±71.5 μmol/L) and proteinuria (1.25±1.34g/24 hr) was observed in ECXM+ compared with ECXM− (105.3±27.8 μmol/L and 0.66±0.89g/24 hr) recipients at t0. However, these differences were not significant probably because of the small sample sizes. At 5 years, renal functions were roughly similar in both ECXM+ and ECXM− groups. At 10 years, a trend toward higher creatinine was found in ECXM+ recipients but with no statistical significance. However, examination of graft and patient survival at 10 years indicated that patients with EC-reactive DSA experienced biopsy-proven Ab-mediated rejection and graft loss for two patients (data not shown). For recipient 5, AMR associated at M114 with C4d deposition in the graft. In contrast, kidney transplant in patients with EC-reactive non-DSA had no biopsy and are still functioning with a normal serum creatinine (mean value for serum creatinine 133.1±44.2 μmol/L for recipients 12, 18, and 22) and no significant proteinuria (0.13±0.06 g/24 hr) at 10 years posttransplantation (Table 3). Thus, clinical data suggest that EC-reactive DSA, but not non-DSA, impaired long-term outcome of graft and patients.
This study was carried out to investigate the humoral response posttransplantation using an endothelial-based detection assay of DSA and to evaluate the impact of circulating EC-reactive Abs on renal allograft dysfunction. The first finding is the tight association of HLA sensitization with the detection of EC-reactive Abs. In the absence of HLA sensitization, no ECXM+ was found (15/22 recipients). DSA were identified in three of seven anti-HLA Ab-containing kidney recipients (42.8%). The donor-specific and EC-reactive Abs were identified as anti-class II HLA (anti-DQ1, -DQ3, and -DR53). We showed that, in ECXM, sera from these patients bind only to IFNγ-treated EC but not to resting or TNFα-activated EC. Consistent with these data, we and others previously demonstrated that IFNγ is required to maintain HLA class II expression in cultured vascular human EC (16, 21). ECXM indicates that DS anti-HLA class II Abs arise from month 1 to year 2 posttransplantation and then consistently detectable all along the posttransplantation period (up to 5 years). Features of these DS- and EC-reactive Abs are a high titer (ranging from 1/46 to 1/256) and their subclass, mostly IgG1.
ECXM identifies a second group of HLA-sensitized kidney transplant recipients. These patients (3/7) possess, non-DS, EC-reactive Abs not directed against HLA. These Abs are either preformed (2/3) or rapidly induced (1/3) within a month posttransplantation. In contrast to DS anti-HLA class II, non-DS response is transient, and Abs are not detected at year 1 (2/3) or year 4 (1/4) posttransplantation. ECXM indicates that these Abs bind to both resting and cytokine-activated EC without HLA restriction. Interestingly, although these Abs only bind transiently to EC, they are persistently detected by CDC thus questioning the biological relevance of CDC in these cases. Similar to anti-class II sensitization, the hallmark of non-HLA non-DS humoral response also includes high titer of Abs (ranging from 1/8 to 1/1024) and the predominance of IgG1 isotype.
The biological function of both DSA and non-DSA detected by ECXM remains to be elucidated. Remarkably, most of these Abs belong to complement-fixing IgG subclass. Besides the presence of complement-fixing IgG subclasses (mostly IgG1), Ab-binding density (which relies on Ab concentration, avidity, and/or antigen density) may be a critical determinant of complement fixation (8). It could be speculated that endogenous endothelial expression of targeted HLA antigens, in particular HLA-DQ, may be not sufficient for complement activation as suggested in earlier studies (9, 22). On another hand, complement activation is modulated not only by Ab concentration, antigen density, IgG subclass, and combination but also by Ab specificity (23–25). In our study, IgM reacting with EC were transiently detected in only one patient with non-DSA (patient n=12). A recent study demonstrated that pretransplant IgM non-HLA Abs constitute a significant risk factor for poor survival; the main effect of this IgM non-HLA Ab occurring in the first year after transplantation (26) suggesting a reemerging role for the IgM autoantibodies in the pathogenesis of allograft rejection (27).
Previous reports demonstrated that, beyond complement activation, anti-HLA alloantibody binding to vascular EC may impair graft outcome (28) and also controls EC proliferation and survival by triggering specific intracellular signaling (22, 29, 30). In particular, we previously showed that anti-HLA-DR Abs elicit a protective pathway in EC through activation of both the protein kinase C and PI3 Kinase pathways (22). Nevertheless, HLA-DQ signaling is still to be determined. Gene transcription analysis suggests that anti-MHC class II and non-DSA reacting with donor EC trigger different pathways leading to specific gene transcription patterns as illustrated by the regulation of CCR4 and IL-1β. CCR4 expression has been associated with regulatory T cells (Tregs) and is shown to control migration and immune suppression in skin (31, 32) and in immunologically tolerant cardiac allografts (33). On another hand, elevated expression of IL-1β is associated with inflammation and transplant injury (34); IL-1β also differentiates T-helper 17 cells at the expense of Tregs (35, 36). Together, these data suggest that, according to their specificities, circulating Abs selectively affect EC phenotype and may locally impact on the inflammatory Tregs/Th17 balance and migration and recruitment within the graft. This hypothesis is supported by a recent study showing a role for EC in Tregs/Th17 polarization (37).
The small number of patient sera tested for EC activation also limits our ability to interpret the gene expression profiling data, which remains preliminary, and requires further validation using a larger number of HLA and non-HLA Abs before making general conclusions on specific patterns of gene expression.
A key feature of our study was the ability to detect Abs that bind to adequate levels of antigens (HLA or non HLA type) expressed on EC reflecting the clinical setting in contrast to the stable amount of antigen coated to the surface of beads or microplates as previously discussed (38). The contribution of HLA class II DSA to renal graft rejection was demonstrated relatively recently and studies showing that patients who develop HLA class II DSA have a higher risk of developing transplant glomerulopathy associated with reduced long-term graft survival (28,39–41) are consistent with our current findings. In contrast, the clinical significance of non-HLA/non-DSA is still to be explored. Identification of the target antigen(s) for non-DSA is under progress and would help to design new focused assays for transplant monitoring.
MATERIALS AND METHODS
A detailed Materials and Methods section is provided as SDC 6 (http://links.lww.com/TP/A575).
Patients and Sera
A total of 256 sera taken before and after transplantation from 22 transplanted patients receiving cadaveric kidney allografts at the CHU de Nantes from 1999 to 2001 (patient's follow-up: 6–8 years) were tested. Sera from healthy blood donors (n=9 individuals and a pool of 20 male AB donors) were provided by EFS (Nantes, France) and used as controls. For HLA typing, pretransplant crossmatch and clinical data, see SDC 6 (http://links.lww.com/TP/A575).
EC Isolation, Culture, and Activation
Human arterial ECs were isolated and cultured as described previously (16). For activation, confluent EC monolayers were starved overnight in endothelial cell basal medium supplemented with 2% fetal calf serum without growth factors and incubated with recombinant human TNFα (100 U/mL, kindly provided by Prof. P. Neuman, BASF, Ludwigshafen, Germany) or IFNγ (100 U/mL, Imukin, Boehringer Ingelheim, Germany) for the indicated period of time. ECs were used between passages 2 and 5. For details, see SDC 6 (http://links.lww.com/TP/A575).
Flow Cytometry and Donor-Specific ECXM Assays
For EC phenotype analysis, immunostaining was performed as reported previously (16, 42). Abs used in this study were as follows: anti-pan HLA class I (clone W6/32 from American Tissue Culture Collection, Manassas, VA) and anti-HLA-DR (Clone L243, ATCC). For ECXM, ECs were incubated with 25 μL of patient's sera (dilution 1/4 in PBS/BSA/NaN3), washed, and then incubated with a phycoerythrin- or fluorescein isothiocyanate-labeled goat anti-human F(ab′)2 IgG or IgM (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Negative controls were performed using a pool of normal human AB sera from 20 healthy male donors (EFS; Nantes, France) or isotype-matched IgG control (Jackson Laboratory). Experiments were repeated at least three times. For details, see SDC 6 (http://links.lww.com/TP/A575).
TaqMan Low Density Arrays and Quantitative Polymerase Chain Reaction Validation
Profiling of gene expression on EC was performed using the TaqMan Array Human Immune and Human apoptosis Gene Signature arrays (Applied Biosystems, CA) as described previously (43). Confirmatory quantitative polymerase chain reaction analysis was conducted with the following primers and probes from Applied Biosytems: IL-1β (Hs_00174097_m1), CCR4 (Hs_99999919_m1), and HPRT (H99999909_m1). The normalized expression level was then calculated as log2 2−ΔCt. For details, see SDC 6 (http://links.lww.com/TP/A575).
The statistical analysis was performed using GraphPad Prism Version 5.00 software. Comparison of median of fluorescence intensity obtained with sera on TNF- or IFN-activated EC was performed using a Wilcoxon test. Correlation between a ECXM+ and HLA immunization was investigated using a Fisher test. HLA classes I and II at cell surface according to culture condition was compared by means of nonparametric Kruskal-Wallis test. For clinical data, a Wilcoxon test was also used to compare creatinine and proteinuria between paired-mate kidney transplant recipients. Results with P values of less than 0.05 were considered statistically significant.
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