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. 2015 May;26(5):1161-71.
doi: 10.1681/ASN.2013121277. Epub 2014 Nov 7.

Endothelial cell antibodies associated with novel targets and increased rejection

Annette M Jackson  1 , Tara K Sigdel  2 , Marianne Delville  3 , Szu-Chuan Hsieh  2 , Hong Dai  2 , Serena Bagnasco  4 , Robert A Montgomery  5 , Minnie M Sarwal  6
Affiliations

Endothelial cell antibodies associated with novel targets and increased rejection

Annette M Jackson et al. J Am Soc Nephrol. 2015 May.

Abstract

The initial contact point between a recipient's immune system and a transplanted graft is the vascular endothelium. Clinical studies suggest a pathogenic role for non-HLA antiendothelial cell antibodies (AECAs) in allograft rejection; however, evidence linking AECAs of known specificity to in vivo vascular injury is lacking. Here, we used high-density protein arrays to identify target antigens for AECAs isolated from the sera of recipients of kidney transplants experiencing antibody-mediated rejection in the absence of donor-specific HLA antibodies. Four antigenic targets expressed on endothelial cells were identified: endoglin, Fms-like tyrosine kinase-3 ligand, EGF-like repeats and discoidin I-like domains 3, and intercellular adhesion molecule 4; the first three have been implicated in endothelial cell activation and leukocyte extravasation. To validate these findings, ELISAs were constructed, and sera from an additional 150 renal recipients were tested. All four AECAs were detected in 24% of pretransplant sera, and they were associated with post-transplant donor-specific HLA antibodies, antibody-mediated rejection, and early transplant glomerulopathy. AECA stimulation of endothelial cell cultures increased adhesion molecule expression and production of inflammatory cytokines: regulated on activation, normal T cell expressed and secreted PDGF and RESISTIN. These correlations between in vitro experiments and in vivo histopathology suggest that AECAs activate the vascular endothelium, amplifying the alloimmune response and increasing microvascular damage. Given the growing number of transplant candidates, a better understanding of the antigenic targets, beyond HLA, and mechanisms of immune injury will be essential for improving long-term allograft survival.

Keywords: acute allograft rejection; endothelial cells; immunology and pathology; kidney transplantation.

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Figures

Figure 1.
Figure 1.
Antibody mediated injury observed in the AECA positive Discovery Cohort. Shown are renal biopsies with positive histologic scores>1 acquired during the 1.5 years post-transplantation according to protocol or at time of dysfunction. Histologic scoring (0–3) was performed using updated Banff 1997–2007 criteria.– Shown are grades for glomerulitis (g), interstitial (i) and tubular (t) inflammation, vasculitis (v), and peritubular capillaritis (ptc). C4d staining was performed on frozen tissue by indirect immunofluorescence. Transplant glomerulopathy (cg) was defined as duplication of the glomerular basement membrane as observed on electron and light microscopy. Low-level DR52 HLA-DSA (median fluorescent intensity<1000) was detected in one recipient at the time of biopsy.
Figure 2.
Figure 2.
Identification of antigenic targets for AECAs and the effect of desensitization treatment. (A) Protein array analysis of 14 AECA eluates derived from endothelial cell crossmatch-positive sera. Relative fluorescence units (RFUs) for antibodies specific for EDIL3, endoglin (ENG), ICAM4, and FLT3 Ligand (FLT3LG) were significant compared with the mean signal intensity of low-abundance antibodies. We defined a positive threshold of 2000 RFU as a cutoff for abundance. The error bars represent SEM. (B) Effect of plasmapheresis/intravenous Ig (PP) on AECA levels. AECAs specific for ENG, FLT3LG, EDIL3, and ICAM4 decreased after PP treatments and rebounded in a post-transplant serum that yielded a positive endothelial cell crossmatch test. Data shown are from a single renal recipient, and values were normalized to total IgG. EC, endothelial cell; Txn, transplantation.
Figure 3.
Figure 3.
Expression of endoglin and FLT3 on renal endothelium. Immunohistochemistry performed on biopsies taken at time of rejection shows expression of (A) endoglin and (B) FLT3 on glomerular and peritubular microvasculature and arteries. Data shown are representative of biopsies tested from nine Discovery Cohort recipients.
Figure 4.
Figure 4.
Endothelial cell cultures stimulated with AECA eluates upregulate markers of activation. Primary endothelial cell cultures were stimulated with AECA-positive sera, AECA eluates, TNF-α (10 ng/well), or an HLA antibody-positive serum. Cell surface phenotype analysis was performed 24 hours poststimulation using flow cytometry. Comparisons of median fluorescence values were made between cells stimulated with AECA eluates versus recipient sera (P values shown). Cumulative data were obtained from seven independent experiments, and AECA eluates from five Discovery Cohort recipients were tested. Aby, antibody; PECAM1, platelet/endothelial cell adhesion molecule 1.
Figure 5.
Figure 5.
Endothelial cell cultures stimulated with AECA eluates differentially affect production of inflammatory cytokines and chemokines. Primary endothelial cell cultures were stimulated with culture media alone, AECA eluates, an HLA antibody-positive serum, or TNF-α (10 ng/well). Culture supernatant was tested 72 hours poststimulation using a Procarta human 54 analyte immunoassay acquired on a Luminex xMAP multiplex platform. Median fluorescent intensities (MFIs) for differentially expressed analytes and positive control standards are shown. Data are representative of four independent experiments, and testing AECA eluates were derived from five Discovery Cohort recipients. Production of inflammatory cytokines PDGF, regulated on activation normal T cell expressed and presumably secreted (RANTES; also known as CCL5), and RESISTIN were increased after stimulation with the AECA eluates compared with negative controls (P<0.001, P<0.001, and P<0.001, respectively) or cells stimulated with TNF-α or HLA antibodies (P<0.001, P<0.001, and P=0.002, respectively). In contrast, chemokines CCL3, CCL4, CXCL5, and CXCL decreased in cultures stimulated with the AECA eluates compared with stimulation with TNF-α or HLA antibodies (P<0.001, P=0.05, P=0.07, and P=0.04, respectively). Pos, positive; Std, standard.
Figure 6.
Figure 6.
Histologic scores for recipients who were AECA ELISA strongly positive (n=28) and negative (n=24). Renal biopsies with positive histologic scores>1 were acquired during the first year post-transplantation according to protocol or at the time of dysfunction. Shown are scores from all biopsies or a subset of biopsies when HLA-DSA was not detected at time of biopsy or detected at a negative FCXM level. Histologic scoring (0–3) was performed using updated Banff 1997–2007 criteria.– Shown are grades for glomerulitis (g), interstitial (i) and tubular (t) inflammation, vasculitis (v), and peritubular capillaritis (ptc). C4d staining was performed on frozen tissue by indirect immunofluorescence. Transplant glomerulopathy (cg) was defined by duplication of the glomerular basement membrane as observed on electron and light microscopy. Additional details and statistics are provided in Table 2.
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Comment in

  • Renal allograft rejection: pieces of the puzzle.
    Racusen L, Lefaucheur C. Racusen L, et al. J Am Soc Nephrol. 2015 May;26(5):1004-5. doi: 10.1681/ASN.2014090932. Epub 2014 Nov 7. J Am Soc Nephrol. 2015. PMID: 25381428 Free PMC article. No abstract available.

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