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There is an unmet need for robust and clinically validated biomarkers of kidney allograft rejection. Here we present the KTD-Innov study (ClinicalTrials.gov, NCT03582436), an unselected deeply phenotyped cohort of kidney transplant recipients with a holistic approach to validate the clinical utility of precision diagnostic biomarkers. In 2018-2019, we prospectively enrolled consecutive adult patients who received a kidney allograft at seven French centers and followed them for a year. We performed multimodal phenotyping at follow-up visits, by collecting clinical, biological, immunological, and histological parameters, and analyzing a panel of 147 blood, urinary and kidney tissue biomarkers. The primary outcome was allograft rejection, assessed at each visit according to the international Banff 2019 classification. We evaluated the representativeness of participants by comparing them with patients from French, European, and American transplant programs transplanted during the same period. A total of 733 kidney transplant recipients (64.1% male and 35.9% female) were included during the study. The median follow-up after transplantation was 12.3 months (interquartile range, 11.9-13.1 months). The cumulative incidence of rejection was 9.7% at one year post-transplant. We developed a distributed and secured data repository in compliance with the general data protection regulation. We established a multimodal biomarker biobank of 16,736 samples, including 9331 blood, 4425 urinary and 2980 kidney tissue samples, managed and secured in a collaborative network involving 7 clinical centers, 4 analytical platforms and 2 industrial partners. Patients' characteristics, immune profiles and treatments closely resembled those of 41,238 French, European and American kidney transplant recipients. The KTD-Innov study is a unique holistic and multidimensional biomarker validation cohort of kidney transplant recipients representative of the real-world transplant population. Future findings from this cohort are likely to be robust and generalizable., (© 2024. Springer Nature B.V.)

Background: After kidney transplantation, donor-specific antibodies against human leukocyte antigen donor-specific antibodies (HLA-DSAs) drive antibody-mediated rejection (ABMR) and are associated with poor transplant outcomes. However, ABMR histology (ABMRh) is increasingly reported in kidney transplant recipients (KTRs) without HLA-DSAs, highlighting the emerging role of non-HLA antibodies (Abs)., Methods: W e designed a non-HLA Ab detection immunoassay (NHADIA) using HLA class I and II-deficient glomerular endothelial cells (CiGEnC Δ HLA) that had been previously generated through CRISPR/Cas9-induced B2M and CIITA gene disruption. Flow cytometry assessed the reactivity to non-HLA antigens of pretransplantation serum samples from 389 consecutive KTRs. The intensity of the signal observed with the NHADIA was associated with post-transplant graft histology assessed in 951 adequate biopsy specimens., Results: W e sequentially applied CRISPR/Cas9 to delete the B2M and CIITA genes to obtain a CiGEnC Δ HLA clone. CiGEnC Δ HLA cells remained indistinguishable from the parental cell line, CiGEnC, in terms of morphology and phenotype. Previous transplantation was the main determinant of the pretransplantation NHADIA result ( P <0.001). Stratification of 3-month allograft biopsy specimens ( n =298) according to pretransplantation NHADIA tertiles demonstrated that higher levels of non-HLA Abs positively correlated with increased glomerulitis ( P =0.002), microvascular inflammation ( P =0.003), and ABMRh ( P =0.03). A pretransplantation NHADIA threshold of 1.87 strongly discriminated the KTRs with the highest risk of ABMRh ( P =0.005, log-rank test). A multivariate Cox model confirmed that NHADIA status and HLA-DSAs were independent, yet synergistic, predictors of ABMRh., Conclusion: The NHADIA identifies non-HLA Abs and strongly predicts graft endothelial injury independent of HLA-DSAs., (Copyright © 2021 by the American Society of Nephrology.)

Graft endothelial cell (EC) injury is central to the pathogenesis of antibody-mediated rejection (AMR). The ability of donor-specific antibodies (DSA) to bind C1q and activate the classical complement pathway is an efficient predictor of graft rejection highlighting complement-dependent cytotoxicity as a key process operating during AMR. In the past 5 y, clinical studies further established the cellular and molecular signatures of AMR revealing the key contribution of other, IgG-dependent and -independent, effector mechanisms mediated by infiltrating NK cells and macrophages. Beyond binding to alloantigens, DSA IgG can activate NK cells and mediate antibody-dependent cell cytotoxicity through interacting with Fcγ receptors (FcγRs) such as FcγRIIIa (CD16a). FcRn, a nonconventional FcγR that allows IgG recycling, is highly expressed on ECs and may contribute to the long-term persistence of DSA in blood. Activation of NK cells and macrophages results in the production of proinflammatory cytokines such as TNF and IFNγ that induce transient and reversible changes in the EC phenotype and functions promoting coagulation, inflammation, vascular permeability, leukocyte trafficking. MHC class I mismatch between transplant donor and recipient can create a situation of "missing self" allowing NK cells to kill graft ECs. Depending on the microenvironment, cellular proximity with ECs may participate in macrophage polarization toward an M1 proinflammatory or an M2 phenotype favoring inflammation or vascular repair. Monocytes/macrophages participate in the loss of endothelial specificity in the process of endothelial-to-mesenchymal transition involved in renal and cardiac fibrosis and AMR and may differentiate into ECs enabling vessel and graft (re)-endothelialization., Competing Interests: The author declares no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Binding of donor-specific antibodies (DSAs) to kidney allograft endothelial cells that does not activate the classic complement cascade can trigger the recruitment of innate immune effectors, including NK cells. Activated NK cells contribute to microvascular inflammation leading to chronic antibody-mediated rejection (AMR). Recipient NK cells can also trigger antibody-independent microvascular inflammation by sensing the absence of self HLA class I molecules ("missing self") on allograft endothelial cells. This translational study investigated whether the condition of missing self amplifies DSA-dependent NK cell activation to worsen chronic AMR., Methods and Results: Among 1682 kidney transplant recipients who underwent an allograft biopsy at Lyon University Hospital between 2004 and 2017, 135 fulfilled the diagnostic criteria for AMR and were enrolled in the study. Patients with complement-fixing DSAs identified by a positive C3d binding assay ( n =73, 54%) had a higher risk of transplant failure ( P =0.002). Among the remaining patients with complement-independent chronic AMR ( n =62, 46%), those in whom missing self was identified through donor and recipient genotyping exhibited worse allograft survival ( P =0.02). In multivariable analysis, only proteinuria (HR: 7.24; P =0.01) and the presence of missing self (HR: 3.57; P =0.04) were independent predictors for transplant failure following diagnosis of chronic AMR. Cocultures of human NK cells and endothelial cells confirmed that addition of missing self to DSA-induced NK cell activation increased endothelial damage., Conclusions: The assessment of missing self at the time of diagnosis of chronic AMR identifies patients at higher risk for kidney transplant failure., (Copyright © 2021 by the American Society of Nephrology.)

Immune response against human cytomegalovirus (HCMV) includes a set of persistent cytotoxic NK and CD8 T cells devoted to eliminate infected cells and to prevent reactivation. CD8 T cells against HCMV antigens (pp65, IE1) presented by HLA class-I molecules are well characterized and they associate with efficient virus control. HLA-E-restricted CD8 T cells targeting HCMV UL40 signal peptides (HLA-EUL40) have recently emerged as a non-conventional T-cell response also observed in some hosts. The occurrence, specificity and features of HLA-EUL40 CD8 T-cell responses remain mostly unknown. Here, we detected and quantified these responses in blood samples from healthy blood donors (n = 25) and kidney transplant recipients (n = 121) and we investigated the biological determinants involved in their occurrence. Longitudinal and phenotype ex vivo analyses were performed in comparison to HLA-A*02/pp65-specific CD8 T cells. Using a set of 11 HLA-E/UL40 peptide tetramers we demonstrated the presence of HLA-EUL40 CD8 αβT cells in up to 32% of seropositive HCMV+ hosts that may represent up to 38% of total circulating CD8 T-cells at a time point suggesting a strong expansion post-infection. Host's HLA-A*02 allele, HLA-E *01:01/*01:03 genotype and sequence of the UL40 peptide from the infecting strain are major factors affecting the incidence of HLA-EUL40 CD8 T cells. These cells are effector memory CD8 (CD45RAhighROlow, CCR7-, CD27-, CD28-) characterized by a low level of PD-1 expression. HLA-EUL40 responses appear early post-infection and display a broad, unbiased, Vβ repertoire. Although induced in HCMV strain-dependent, UL4015-23-specific manner, HLA-EUL40 CD8 T cells are reactive toward a broader set of nonapeptides varying in 1-3 residues including most HLA-I signal peptides. Thus, HCMV induces strong and life-long lasting HLA-EUL40 CD8 T cells with potential allogeneic or/and autologous reactivity that take place selectively in at least a third of infections according to virus strain and host HLA concordance.

Antibody-mediated rejection (ABMR) is associated with poor transplant outcome. Pathogenic alloantibodies are usually directed against human leukocyte antigens (HLAs). Histological findings suggestive of ABMR usually demonstrate an anti-HLA donor-specific antibody (DSA)-mediated injury, while a small subset of patients develop acute dysfunction with histological lesions suggestive of ABMR in the absence of anti-HLA DSAs. Although this non-HLA ABMR is not well recognized by current diagnostic classifications, it is associated with graft dysfunction and allograft loss. These clinical descriptions suggest a pathogenic role for non-HLA anti-endothelial cell antibodies. Diverse antigenic targets have been described during the last decade. This review discusses recent findings in the field and addresses the clinical relevance of anti-endothelial cell antibodies (AECAs)., (Copyright © 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Background: BK polyomavirus (BKPyV) frequently reactivates in kidney transplant recipients during immunosuppressive therapy and triggers BKPyV-associated nephropathy and graft rejection. Determining effective risk factors for BKPyV reactivation is required to achieve efficient prevention., Methods: This study investigated the role of major histocompatibility complex (MHC) class I-related chain A (MICA) in BKPyV reactivation in a cohort of 144 transplant donor/recipient pairs, including recipients with no reactivation (controllers) and those with mild (virurics) or severe (viremics) BKPyV reactivation after graft receipt., Results: We show that, in the kidney, MICA is predominantly expressed in tubule epithelial cells, the natural targets of BKPyV, questioning a role for MICA in the immune control of BKPyV infection. Focusing on MICA genotype, we found a lower incidence of BKPyV reactivation in recipients of a renal graft from a donor carrying the MICA A5.1 mutant, which encodes a truncated nonconventional MICA. We established that a mismatch for MICA A5.1 between transplant donor and recipient is critical for BKPyV reactivation and BKPyV-associated nephropathy. Functionally, we found that a low prevalence of BKPyV reactivation was associated with elevated anti-MICA sensitization and reduced plasma level of soluble MICA in recipients, 2 potential effector mechanisms., Discussions: These findings identify the MHC-related MICA as an immunogenetic factor that may functionally influence anti-BKPyV immune responses and infection outcomes., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Background: Rabbit-generated antithymocyte globulins (ATGs), which target human T cells, are widely used as immunosuppressive agents during treatment of kidney allograft recipients. However, ATGs can induce immune complex diseases, including serum sickness disease (SSD). Rabbit and human IgGs have various antigenic differences, including expression of the sialic acid Neu5Gc and α-1-3-Gal (Gal), which are not synthesized by human beings. Moreover, anti-Neu5Gc antibodies have been shown to preexist and be elicited by immunization in human subjects. This study aimed to assess the effect of SSD on long-term kidney allograft outcome and to compare the immunization status of grafted patients presenting with SSD following ATG induction treatment., Methods: We analyzed data from a cohort of 889 first kidney graft recipients with ATG induction (86 with SSD [SSD(+)] and 803 without SSD [SSD(-)]) from the Données Informatisées et Validées en Transplantation data bank. Two subgroups of SSD(+) and SSD(-) patients that had received ATG induction treatment were then assessed for total anti-ATG, anti-Neu5Gc, and anti-Gal antibodies using ELISA assays on sera before and after transplantation., Results: SSD was significantly associated with long-term graft loss (>10 years, P = 0.02). Moreover, SSD(+) patients exhibited significantly elevated titers of anti-ATG (P = 0.043) and anti-Neu5Gc (P = 0.007) IgGs in late post-graft samples compared with SSD(-) recipients., Conclusion: In conclusion, our data indicate that SSD is a major contributing factor of late graft loss following ATG induction and that anti-Neu5Gc antibodies increase over time in SSD(+) patients., Funding: This study was funded by Société d'Accélération du Transfert de Technologies Ouest Valorisation, the European FP7 "Translink" research program, the French National Agency of Research, Labex Transplantex, the Natural Science and Engineering Research Council of Canada, and the Canadian Foundation for Innovation.

Galactosyl-transferase KO (GalT-KO) pigs represent a potential solution to xenograft rejection, particularly in the context of additional genetic modifications. We have performed life supporting kidney xenotransplantation into baboons utilizing GalT-KO pigs transgenic for human CD55/CD59/CD39/HT. Baboons received tacrolimus, mycophenolate mofetil, corticosteroids and recombinant human C1 inhibitor combined with cyclophosphamide or bortezomib with or without 2-3 plasma exchanges. One baboon received a control GalT-KO xenograft with the latter immunosuppression. All immunosuppressed baboons rejected the xenografts between days 9 and 15 with signs of acute humoral rejection, in contrast to untreated controls (n = 2) that lost their grafts on days 3 and 4. Immunofluorescence analyses showed deposition of IgM, C3, C5b-9 in rejected grafts, without C4d staining, indicating classical complement pathway blockade but alternate pathway activation. Moreover, rejected organs exhibited predominantly monocyte/macrophage infiltration with minimal lymphocyte representation. None of the recipients showed any signs of porcine endogenous retrovirus transmission but some showed evidence of porcine cytomegalovirus (PCMV) replication within the xenografts. Our work indicates that the addition of bortezomib and plasma exchange to the immunosuppressive regimen did not significantly prolong the survival of multi-transgenic GalT-KO renal xenografts. Non-Gal antibodies, the alternative complement pathway, innate mechanisms with monocyte activation and PCMV replication may have contributed to rejection., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

MHC class I-related chain A (MICA) antigens are surface glycoproteins strongly implicated in innate immunity, and the MICA gene is highly polymorphic. Clinical observations suggest a role for donor MICA antigens expressed on transplant endothelial cells in the alloimmune response, but the effect of MICA genotype is not well understood. Here, we investigated the immunologic effect of the A5.1 mutation, related to the common MICA*008 allele. Compared with wild-type endothelial cells (ECs), homozygosity for MICA A5.1 associated with an endothelial phenotype characterized by 7- to 10-fold higher levels of MICA mRNA and MICA proteins at the cell surface, as well as exclusive release in exosomes instead of enzymatic cleavage. Mechanistically, we did not detect quantitative changes in regulatory microRNAs. Functionally, A5.1 ECs enhanced NKG2D interaction and natural killer cell activation, promoting NKG2D-dependent lysis of ECs. In kidney transplant recipients, polyreactive anti-MICA sera bound preferentially to ECs from MICA A5.1 donors, suggesting that MICA*008(A5.1) molecules are the preferential antigenic determinants on ECs of grafts. Furthermore, the incidence of MICA A5.1 mismatch revealed a statistically significant association between donor MICA A5.1 and both anti-MICA sensitization and increased proteinuria in kidney recipients. Taken together, these results identify the A5.1 mutation as an immunodominant factor and a potential risk factor for transplant survival.

Background: Pathogenesis of antibody (Ab) responses to transplant are yet not well defined. This study aimed to detect and to analyze posttransplant circulating allo-Abs reacting toward graft endothelial cells (ECs) using primary EC cultures prospectively isolated from the transplant donor at the time of transplantation., Methods: This study shows a retrospective analysis performed using a dedicated EC crossmatch (ECXM) assay that we developed for the experimental assessment of donor-specific EC-reactive Abs. Donor-specific ECXM was performed by flow cytometry on posttransplant sera (n=256) from an historical cohort of 22 kidney allograft recipients., Results: In this study, we show that 27.3% (6/22) of recipients have a positive ECXM that strictly correlates (100%, 6/6) with the presence of anti-human leukocyte antigen (HLA) Abs posttransplantation. ECXM identifies both donor-specific Abs (DSA; 50%) and non-DSA (50%) reactive to EC. DSA and non-DSA are mostly IgG1 and exhibit peak titers ranging from 1/8 to 1/1024. ECXM indicates that DSA correspond to anti-HLA class II Abs; this immunization is late (M3-M60) but persistent (still detected at M60). In contrast, non-DSA are non-HLA-type Abs reacting with third-party EC and reflecting an early but transient immunization (ended at M3-M12). Our findings demonstrate selective regulatory pathways initiated by anti-HLA class II and non-DSA in graft EC reflected by CCR4 and interleukin 1β up-regulation, respectively., Conclusions: We provide evidence that circulating Abs in HLA-sensitized transplant recipients include both DSA and non-HLA/non-DSA able to bind to graft EC and induce specific gene transcription.

Although association between CMV infection and allograft rejection is well admitted, the precise mechanisms involved remain uncertain. Here, we report the characterization of an alloreactive HLA-E-restricted CD8 T cell population that was detected in the PBL of a kidney transplant patient after its CMV conversion. This monoclonal CD8 T cell population represents a sizable fraction in the blood (3% of PBL) and is characterized by an effector-memory phenotype and the expression of multiple NK receptors. Interestingly, these unconventional T cells display HLA-E-dependent reactivity against peptides derived from the leader sequences of both various HCMV-UL40 and allogeneic classical HLA-I molecules. Consequently, while HLA-E-restricted CD8 T cells have potential to contribute to the control of CMV infection in vivo, they may also directly mediate graft rejection through recognition of peptides derived from allogeneic HLA-I molecules on graft cells. Therefore, as HLA-E expression in nonlymphoid organs is mainly restricted to endothelial cells, we investigated the reactivity of this HLA-E-restricted T cell population towards allogeneic endothelial cells. We clearly demonstrated that CMV-associated HLA-E-restricted T cells efficiently recognized and killed allogeneic endothelial cells in vitro. Moreover, our data indicate that this alloreactivity is tightly regulated by NK receptors, especially by inhibitory KIR2DL2 that strongly prevents TCR-induced activation through recognition of HLA-C molecules. Hence, a better evaluation of the role of CMV-associated HLA-E-restricted T cells in transplantation and of the impact of HLA-genotype, especially HLA-C, on their alloreactivity may determine whether they indeed represent a risk factor following organ transplantation.

Galactosyl-transferase knockout (GT-KO) pigs represent the latest major progress to reduce immune reactions in xenotransplantation. However, their organs are still subject to rapid humoral rejection involving complement activation requiring the ongoing development of further genetic modifications in the pig. In a pig-to-baboon renal transplantation setting, we have used donor pigs that are not only GT-KO, but also transgenic for human CD55 (hCD55), hCD59, hCD39, and fucosyl-transferase (hHT). We studied kidney xenograft survival, physiological and immunologic parameters, xenogeneic rejection characteristics, as well as viral transmission aspects among two groups of baboons: control animals (n = 2), versus those (n = 4) treated with a cocktail of cyclophosphamide, tacrolimus, mycophenolate mofetil, steroids, and a recombinant human C1 inhibitor. Whereas control animals showed clear acute humoral rejection at around day 4, the treated animals showed moderately improved graft survival with rejection at around 2 weeks posttransplantation. Biopsies showed signs of acute vascular rejection (interstitial hemorrhage, glomerular thrombi, and acute tubular necrosis) as well as immunoglobulin (Ig)M and complement deposition in the glomerular and peritubular capillaries. The low level of preformed non-Gal-α1.3Gal IgM detected prior to transplantation increased at 6 days posttransplantation, whereas induced IgG appeared after day 6. No porcine endogenous retrovirus (PERV) transmission was detected in any transplanted baboon. Thus, surprisingly, organs from the GT-KO, hCD55, hCD59, hCD39, and hHT transgenic donors did not appear to convey significant protection against baboon anti-pig antibodies and complement activation, which obviously continue to be significant factors under a suboptimal immunosuppression regimen. The association, timing, and doses of immunosuppressive drugs remain critical. They will have to be optimized to achieve longer graft survivals., (Copyright © 2011 Elsevier Inc. All rights reserved.)

It is likely that the polymorphic MICA (MHC class I related chain A) molecules on graft endothelial cells (ECs) may be a target for specific antibodies and T cells directed against solid organ grafts. Although there is evidence for a role of MICA in vascular and transplant biology, genotyping is not performed routinely, and thus there are few correlations between polymorphism and endothelial phenotype. The present study examined the frequency of the various alleles for the nonclassical MHCI MICA gene among a cohort of kidney transplant donors, particularly MICA genetic variants (MICA A5.1 and MICA-129) that may affect MICA expression and function on graft EC. Genotyping was performed on genomic DNA derived from primary cultures of EC established from transplant donors at the time of transplantation. Herein we have reported that among 106 alleles analyzed, 28/69 MICA alleles were distributed among 7 major variants (*00804, *00801, *004, *00201, *00901*, *011, *010), representing 70% of the donors. MICA*008 the most abundant allele (31.1%) was associated with the MICA A5.1 mutation. The majority of donors (52.8%) had at least one MICA A5.l allele, with 13.2% homozygous for this mutation. The MICA-129 val/val genotype, which encodes a low-affinity ligand, was predominant (49%), while the MICA-129 met/met, corresponding to a high-affinity ligand, was observed in 11.3% of the transplants. Our findings highlighted the MICA gene polymorphism that produces functional diversity in transplant recipients with variable interactions between MICA and its receptor NKG2D., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Diagnosis of the specific cause of late allograft injury is necessary if more personalized and efficient immunosuppressive regimens are to be introduced. This study sought previously unrecognized biomarkers for specific histologic diagnoses of late graft scarring by comparison of gene sets from published microarray studies. Tribbles-1 (TRIB1), a human homolog of Drosophila tribbles, was identified to be a potentially informative biomarker. For testing this, mRNA expression in 76 graft biopsies, 71 blood samples, and 11 urine samples were profiled from independent cohorts of renal transplant patients with different histologic diagnoses recruited at two European centers. TRIB1 but not TRIB2 or TRIB3 was found to be a potential blood and tissue biomarker of chronic antibody-mediated rejection, an active immune-mediated form of chronic allograft failure associated with a poor prognosis. TRIB1 mRNA levels in peripheral blood mononuclear cells discriminated patients with chronic antibody-mediated rejection from those with other types of late allograft injury with high sensitivity and specificity. TRIB1 was also upregulated in a rodent model of chronic cardiac vasculopathy, suggesting that this biomarker may be useful in other solid-organ transplants and across species. It was determined that TRIB1 is expressed primarily by antigen-presenting cells and activated endothelial cells. Overall, these data support the potential use of TRIB1 as a biomarker of chronic antibody-mediated allograft failure.

HLA class I ligation on graft endothelial cells (EC) has been shown to promote graft arteriosclerosis and chronic allograft nephropathy. This study investigated transcriptional and functional changes mediated by anti-HLA antibodies (Ab), developed by transplant recipient, on vascular renal EC. For mimicking interactions that occur between alloantibodies and graft endothelium, HLA-typed primary cultures of human EC were incubated in vitro in the presence of monomorphic or polymorphic anti-HLA class I Ab. Gene expression analysis identified the upregulation of several molecules involved in cell signaling and proliferation, including the GTP-binding protein RhoA. It was demonstrated further that HLA class I ligation on EC induced a rapid translocation of RhoA to the cell membrane associated with F-actin stress fiber formation and cytoskeleton reorganization. Western blot analysis showed that anti-HLA class I Ab induced, in addition to RhoA, the activation of phosphatidylinositol 3-kinase, reflected by the phosphorylation of Akt (Ser473) and GSK3beta (Ser9), in EC. C3 exoenzyme, an inhibitor of RhoA, inhibited RhoA translocation in response to HLA class I ligation and reduced phosphatidylinositol 3-kinase activation. EC proliferation and cell cycle progression, examined by 5,6-carboxyfluorescein diacetate succinimidyl ester staining, demonstrated that anti-HLA-induced EC proliferation was efficiently prevented by the 3-hydroxy-3-methylglutaryl CoA reductase inhibitor simvastatin (0.1 micromol/L) through inhibition of RhoA geranylgeranylation. Taken together, these findings support the conclusion that RhoA is a key mediator of signaling pathways that lead to cytoskeletal reorganization and EC proliferation in response to alloantibodies that bind to HLA class I and demonstrate the specific and potent inhibitory effect of simvastatin on allostimulated EC growth.

The vascular endothelium of transplanted organs represents an important target for allograft-directed immune responses. Although HLA antigens expressed on graft endothelial cells (EC) can become targets of the host immune response, the role of other, non-HLA-encoded EC antigens has been proposed but is still unclear. The aim of this study was to investigate the presence of and to characterize anti-EC antibodies (AECA) in 57 kidney transplant recipients according to their HLA-immunization status. Flow cytometry in pretransplant sera was used to detect AECA reactive with surface antigens on ABO and HLA-typed primary cultures of arterial ECs, stimulated or not with tumor necrosis factor-alpha (TNFalpha) or interferon-gamma (IFNgamma). FACS analysis revealed the presence of AECA in 47% of HLA-sensitized (PRA = 10%: mostly IgG) vs. 16.0% in nonsensitized patients (PRA < 10%) (p < 0.02). No significant correlation was found between the presence of AECA and acute rejection occurrence and graft outcome. Non-HLA reactive AECA are directed against TNFalpha- and IFNgamma-inducible membrane molecule(s), and react with two predominant antigens of approximately 35 kDa and approximately 50 kDa expressed on ECs but not on B cells. Binding of AECA decreases in vitro EC viability by 50-60% by promoting EC apoptosis, as demonstrated by DNA fragmentation assays.