The improved ability to detect the presence or absence of human leukocyte antigen (HLA)-antibodies using sensitive bead immunoassays has facilitated investigation into the clinical relevance of non-HLA, anti-endothelial cell antibodies (AECAs). AECAs have been implicated in hyperacute, acute, and chronic rejections of kidney allografts (1–5). Moreover, the finding that there is decreased long-term graft survival in sensitized recipients transplanted with kidneys from HLA identical siblings provides evidence that sensitization to antigenic targets beyond HLA and ABO contribute to the alloimmune response (6).
Potential targets of AECAs that have been associated with rejection in renal allografts include antibodies specific for angiotensin II type 1 receptor 1, vimentin, the glomerular basement membrane protein agrin, and the major histocompatibility complex class I chain-related gene A (7–11). Sophisticated approaches combining proteomics and genomics are uncovering additional tissue specific non-HLA antigens capable of initiating de novo humoral responses in renal transplant recipients (12).
More recently, an endothelial cell crossmatch test (ECXM) has been described that uses endothelial cell precursors (ECPs) isolated from the blood of potential organ donors as targets to identify donor-reactive AECAs (13). In a multicenter clinical trial, 24% of patients (35/147) tested positive for donor-reactive ECP antibodies and had significantly higher incidences of early rejection (P=0.00005) compared with patients for whom no AECAs were detected (14). Although this clinical trial provided data with important predictive value, analysis of clinical outcome was limited to the first 6 months posttransplant.
We report here on the renal function and incidence of rejection in 60 live donor kidney recipients who were tested pretransplant for non-HLA, AECAs using donor-derived ECPs and for whom we obtained clinical follow-up data up to 4 years posttransplant. We also determined the immunoglobulin G (IgG) subclasses of ECP reactive antibodies and compared these with HLA antibodies.
Correlation of ECXM and Clinical Outcomes
Sixty live donor renal transplant recipients were enrolled in this study between 2005 and 2007. Early allograft outcomes (≤6 months) for 30 of these patients have been reported previously (14). All recipients tested negative in lymphocyte flow cytometric crossmatches (FCXMs) with their respective donors and no donor HLA-specific antibody (HLA-DSA) was detected by multianalyte bead immunoassays. Flow cytometric ECXM tests were performed on the day of transplant using ECPs isolated from donor blood. Fourteen patients (23%) tested positive for donor-reactive IgG AECAs, 13 (31%) for immunoglobulin M (IgM) AECAs, and four patients tested positive for both IgM and IgG AECAs (Table 1). Because of positive control failures, 18 IgM ECXM tests were excluded from the study. Demographics including recipient age, gender, race, HLA sensitization, donor age, and HLA mismatch were comparable in both the ECXM+ and ECXM− groups. The majority of the ECXM+ patients (79% IgG+, 11/14 and 85% IgM+, 11/13) had no detectable sensitization to HLA.
Serum creatinine (SCr) values within the first 100 days posttransplant were significantly higher in patients testing positive for IgG donor-reactive AECAs compared with patients testing negative (1.5 vs. 1.2, P=0.014) with an average posttransplant clinical follow-up of 45 and 55 days, respectively (Table 2). Because of a protocol change during the course of this study, not all patients received induction therapy with an anti-thymocyte antibody. However, among those who did, the SCr values within the first 100 days posttransplant were still higher in IgG+ ECXM patients compared with IgG− ECXM patients (P=0.034). This difference in SCr was no longer observed at more than 100 days posttransplant (1.5 vs. 1.4, P=NS), regardless of induction therapy, with an average posttransplant clinical follow-up of 788 and 843 days for IgG+ ECXM and IgG− ECXM patients, respectively. The incidences of biopsies performed due to renal dysfunction (64% vs. 35%, P<0.025) and of biopsy-proven rejection (36% vs. 17%, P<0.05) within the first 100 days posttransplant was higher in the IgG+ ECXM patients compared to IgG− ECXM patients. Induction therapy significantly reduced rejection in the IgG− ECXM patients (P<0.01); however, due to small numbers in the IgG+ ECXM cohort the benefit of induction therapy could not be statistically determined. All 13 early rejections in IgG+ ECXM and IgG− ECXM patients were cellular rejections according to Banff '97 criteria (15). Late rejection episodes (>100 days posttransplant) were diagnosed in five patients, one in the IgG+ ECXM group (414 days posttransplant) and four in the IgG− ECXM group (mean of 663 days posttransplant).
Detection of pretransplant donor-reactive IgM AECAs was not predictive of the posttransplant clinical course in our patient cohort. Mean SCr values within the first 100 days posttransplant were 1.3 and 1.2 for IgM+ and IgM− ECXM patients (P=0.5) with mean posttransplant clinical follow-up of 59 and 51 days, respectively. Similarly, no statistical difference in SCr values at more than 100 days posttransplant was observed among patients testing IgM+ versus IgM− in the ECXM (1.3 vs. 1.4, P=0.67) with mean posttransplant follow-up of 788 and 842 days, respectively. Patients with IgM AECAs did have more biopsies performed due to early graft dysfunction (69% IgM+ vs. 21% IgM−, P<0.025), but only two (15%) had biopsy-proven rejection and these two patients also tested positive for donor-reactive IgG AECAs.
IgG Subclass Determination of HLA and Non-HLA Endothelial Cell Reactive Antibodies
Human IgG is divided into four subclasses, IgG1 to IgG4, each having different serum concentrations, half lives, and effector functions (16). IgG subclass determination of ECP reactive antibodies was performed in six IgG+ ECXM patients described above for whom there was sufficient pretransplant serum. Pretransplant sera from six additional IgG+ ECXM kidney recipients with no sensitization to HLA were also analyzed. Because blood specimens from the actual kidney donors were unavailable, IgG subclass testing was performed using seven surrogate donors that produced positive ECXM tests with patient sera but for whom there was no detectable HLA-DSA. ECXM tests were performed using fluorescently conjugated antibodies specific for IgG subclasses: IgG1, IgG2, IgG3, and IgG4. Using cell surface markers specific for ECPs and lymphocytes, IgG subclass data were acquired for both cell types within the same crossmatch sample.
IgG subclass distribution in human serum is reflected by the subclass number, with IgG1 being most abundant (65%), followed by IgG2 (24%), IgG3 (7%), and IgG4 (4%) (16). To control for both non-specific IgG binding and differences in the affinities of the IgG subclass-specific antibodies, we normalized the IgG1 subclass median fluorescence values for each donor-serum combination to those for the donor tested with negative control serum. Figure 1 shows the mean of the normalized values for sera from 12 IgG+ ECXM patients tested with multiple surrogate donors. When lymphocytes were used as targets, there was no significant AECA binding over background. In contrast, AECAs bound to ECPs were significantly enriched for IgG2 (2.2 vs. 1.1, P=0.005) and IgG4 (1.5 vs. 0.9, P=0.0005) when compared with lymphocytes.
As a comparison, we performed IgG subclass determination of HLA antibodies contained in pooled sera from patients sensitized to HLA. This pooled serum was tested with cells isolated from 10 donors that expressed the appropriate HLA antigens. The median fluorescence values for each IgG subclass for each donor-serum combination were normalized to those of the donor tested with negative control serum. As expected, HLA antibodies bound to HLA antigens expressed on both ECPs and lymphocytes, and were significantly enriched for subclasses IgG1, IgG2, and IgG3 when compared with negative control serum (Fig. 2). Reduced HLA-specific antibody binding to ECP targets reflects lower HLA class I and class II expression compared with lymphocytes and inherently higher background fluorescence (data not shown and Fig. 3).
Limited Crossmatch Sensitivity When Using ECPs Versus Lymphocytes as Target Cells
To determine the dynamic range of IgG AECA titers in the serum of ECXM+ patients, tests using serial serum dilutions were performed with five patients. AECA titers sufficient to yield a positive IgG ECXM varied between individuals, ranging from 1:2 to 1:8 (data not shown). However, ECPs possess inherently high background fluorescence when compared with lymphocytes, which limits the sensitivity of the ECXM test. To illustrate this, we have performed parallel FCXM tests using T lymphocytes and ECPs from the same donor and serial dilutions of sera containing HLA antibodies specific for donor class I HLA antigens (Fig. 3 and data not shown). Using identical cytometer settings and anti-IgG detection antibodies, the median fluorescence value for target cells alone in the absence of serum is 78 for T cells and 7046 for ECPs. This high level of non-specific fluorescence with ECPs creates a narrow window of reactivity between negative and positive tests. The median fluorescence of the test serum (neat) is 41 times stronger than the negative control serum when using T lymphocyte as targets but two times stronger when using ECPs. Therefore, reduced sensitivity in the ECXM may limit accurate AECA titer determination.
Pretransplant ECXMs were performed on 60 live donor kidney recipients using ECPs isolated from donor blood. Absence of HLA-DSA was determined by FCXM using donor lymphocytes and multianalyte bead immunoassays before transplant. Fourteen patients (23%) tested positive for non-HLA, donor-reactive IgG AECAs and had statistically higher SCr values, higher incidences of biopsy, and cellular rejection in the early posttransplant period (≤100 days) compared with 46 patients who tested negative. As part of the study protocol, transplant clinicians were blinded to the ECXM results and testing for HLA-DSA at the time of biopsy was performed for only one of the five IgG+ patients with rejection. In this one case, HLA-DSA was negative. Nevertheless, biopsy findings in all five patients were consistent with cell-mediated rejection and in all cases elevated SCr values were reduced to baseline after treatment with a steroid bolus or T-cell depleting antibodies. A higher percentage of patients (42%, 13/42) tested positive for IgM+ AECAs but unlike the XM-ONE multicenter clinical trial study (14), we did not see a correlation between IgM+ AECAs and increased incidences of rejection or increased SCr values during the early posttransplant period (<100 days).
Our finding that patients with donor-reactive IgG AECAs had higher incidences of cellular rejection prompted us to investigate their IgG subtype and compare them to HLA antibodies. Consistent with other reports, our data show HLA-specific antibodies to be comprised of IgG subclass 1 to 3 (Fig. 3). Our experiments used a cell-based method to determine IgG subclasses, whereas others have confirmed this finding using solid phase immunoassays and solubilized HLA molecules (17, 18). The deleterious nature of HLA antibodies lie in their ability to activate complement, resulting in cell lysis and amplification of the immune response through the creation of complement split products with chemotactic and anaphylactic properties (19, 20). Donor-reactive ECP antibodies identified in 12 ECXM+ patients within our study were enriched for IgG2 and IgG4, subclasses that activate complement poorly or not at all (21). AECAs have been shown to exert their action through endothelial cell activation, proliferation, or apoptosis (22–24). Increased incidences of cellular rejection in our IgG ECXM+ patients and in patients evaluated in the XM-ONE multicenter clinical trial may reflect the ability of ECP reactive antibodies to disrupt the integrity of the vascular endothelium or activate the endothelium thereby increasing transmigration of recipient leukocytes into the underlying allograft (14). Endothelial cell activation has been shown to increase expression of adhesion molecules such as intercellular adhesion molecule 1 and vascular cell adhesion molecule-1, and decrease Fas-ligand expression allowing safe transmigration of activated T cells (25, 26). In addition, transmigration through activated endothelium alters the leukocytes themselves, up-regulating costimulatory markers and enhancing effector functions (27–31). Thus, AECAs differ from HLA-specific antibodies with regard to IgG subclass and possible mechanisms for eliciting rejection.
AECAs have been shown to bind more readily to activated endothelial cells after incubation with inflammatory cytokines (26, 32). This finding suggests that inflammation itself may play an important role in the deleterious action of AECAs in transplanted allografts. Inflammation from surgery and ischemic injury may increase antigen expression on the vascular endothelium providing transient binding sites for ACEAs (33, 34). The majority of our IgG+ ECXM patients displayed higher SCr values and increased incidences of rejection in the early posttransplant period only, suggesting that ACEAs may not pose a problem once inflammation subsides. However, failure to see an effect in the late posttransplant period may be impacted by the increased percentage of ECXM+ patients treated with induction therapy. Although the benefit of induction therapy was not fully achieved in the ECXM+ patients compared to ECXM- patients, the small number of ECXM+ patients transplanted without induction prevented statistical comparisons. Nevertheless, these data imply that current protocols including immune cell depletion are insufficient to eliminate the correlation between donor-specific AECAs and cell mediated rejection.
Given the strong immunogenicity of HLA, we found it curious that the majority of ECXM+ patients in our study and in the XM-ONE multicenter trial had developed antibodies to endothelial cell antigens but not HLA (14). This could easily be explained if the ECP reactive AECAs were specific for autoantigens and arose in the absence of sensitizing events. Interestingly, only one of the 14 IgG+ patients and none of the IgM+ patients had a history of autoimmune disease. Our finding that sera from ECXM+ patients displayed different reactivity patterns with individual surrogate donors suggests that some of the antigenic targets may be polymorphic or may be expressed at different levels among donors (Fig. 1). Further investigation into the antigenic targets expressed on renal vascular endothelium may elucidate the route of sensitization for these AECAs, and provide insight as to why they differ with regards to IgG subclass from that of HLA antibodies. Humoral responses to protein antigens such as HLA normally result in the formation of IgG1 and IgG3 antibodies, while carbohydrate moieties or chronic antigen stimulation are thought to generate antibodies of subclass IgG2 and IgG4, respectively (16, 35). Further characterization of AECAs and their targets will reveal, which antigens elicit the most deleterious antibodies and possible avenues for developing therapeutic interventions.
MATERIALS AND METHODS
Patients and Immunosuppression
Sixty-five sequential live donor kidney transplant recipients and corresponding donors were entered into this study under a Johns Hopkins University institutional review board approved protocol, between July 2005 and December 2007. Five patients were excluded from the study, three due to failed positive controls in IgG and IgM ECXM tests and two patients due to the presence of low level HLA-DSA detected before transplant. Data collected less than or equal to 6 months posttransplant for 30 of these recipients has been previously reported as part of a multicenter clinical trial (14). Immunosuppression consisted of tacrolimus or sirolimus, mycophenolate, and steroids (36). Anti-thymocyte antibody was administered to 39 patients as induction therapy and 10 additional patients in response to a cellular rejection within the first 9 days posttransplant (Thymoglobulin, Sangstat, Fremont, CA; 1.5 mg/kg/day for 5 days). SCr was tested daily while hospitalized and twice weekly for the first 3 months after the transplant. Patient demographics are provided in Table 1.
HLA-Specific Antibody Analysis
The absence of HLA-DSA was determined following T- and B-lymphocyte FCXM tests and multianalyte bead assays performed on a Luminex platform using beads coated with HLA class I and class II phenotypes (Lifematch ID class I and II, Gen-Probe, San Diego, CA) (37). The presence of donor-specific HLA antibody is determined by analyzing the top ranking HLA phenotypes and organized stacking of HLA phenotypes within the panel.
Endothelial Cell Crossmatch Test
Flow cytometric XM-ONE crossmatch tests were performed using angiopoietin receptor positive ECPs isolated from donor blood according to manufacturer's instructions (Absorber AB, Stockholm, Sweden). Positive IgG and IgM ECXM tests were determined by shifts of 50 or 80 channels, respectively, above the negative control serum (14). Sera demonstrating a fluorescent shift below that of the negative control serum were considered negative unless retested after IgM depletion (38) (see Figure, Supplemental Digital Content1,http://links.lww.com/TP/A417). Tests were acquired and analyzed using a BD FACSCalibur and Cellquest software (BD Biosciences, Franklin Lakes, NJ). Crossmatch tests using ECPs and T cells as targets were performed using sera depleted of IgM through hypotonic dialysis and according to our clinical FCXM procedure (38, 39). Purity of Tie2+ ECPs were determined using a polyclonal rabbit anti-CD133 (Abcam Inc., Cambridge, MA), and allophycocyanin-conjugated goat anti-rabbit IgG (R&D Systems, Minneapolis, MN) (40). An optimal FITC voltage of 500 V was used for both target cell types and logarithmic data acquired using a BD FACSAria and FACSDIVA software (BD Biosciences, Franklin Lakes, NJ). Positive crossmatch tests were determined when the ratio of the median fluorescence of test serum to negative control serum was 1.3 or greater for ECP targets and 3.0 or greater for T-cell targets.
IgG Subclass Determination
Pretransplant sera from 12 IgG+ ECXM patients were depleted of IgM using hypotonic dialysis and tested in XM-ONE crossmatches using ECPs isolated from seven surrogate donors for whom there was no detectable HLA-DSA (39). IgG subclasses of the AECAs were determined using phycoerythrin-conjugated monoclonal antibodies specific for IgG1 (clone 4E3), IgG2 (clone 31-7-4), IgG3 (clone HP6050), and IgG4 (clone HP6025) (Southern Biotech, Birmingham, AL). Gating parameters for lymphocytes were determined using a peridinin chlorophyll protein complex (PerCP) conjugated anti-CD3 (clone SK7, BD Biosciences, Franklin Lakes, NJ) and for ECPs as described earlier. Cells were acquired and analyzed using a BD FACSAria and FACSDiva software (BD Biosciences, Franklin Lakes, NJ). Median fluorescence values for IgG subclasses identified in test serum were normalized to values obtained when cells were incubated with normal control AB serum (Atlanta Biologicals, Norcross, GA). IgG subclass analysis of HLA antibodies was assessed in the same manner using 10 ECP donors and pooled sera from high calculated panel reactive antibody (cPRA) transplant candidates.
Histopathology and Rejection
Biopsies were performed when clinically indicated and analyzed by microscopy using hematoxylin-eosin, periodic acid-Schiff, methenamine silver, and Masson's trichrome stains. C4d deposition was evaluated using indirect immunofluorescence. Clinical rejection was determined according to Banff '97 criteria (15).
Descriptive statistics included mean and standard deviation. Chi squared and Student's t tests (two tailed) were used to evaluate differences in the distribution of discreet variables and means, respectively.
The authors thank Drs. A.A. Zachary and M.S. Leffell for critical review of this manuscript. Reagents were provided in part by AbSorber AB.
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