Direct Correlation of Soluble HLA and HLA-Containing Exosomes and Inverse Correlation of Tolerance Marker-Containing Exosomes With Antibody-Mediated Rejection After Simultaneous Liver-Kidney Transplantation: A Case Study.

There is a lower incidence of antibody-mediated rejection (AMR) after simultaneous liver-kidney transplantation (SLKT) than after kidney-only transplantation. It has been suggested that soluble human leukocyte antigen (sHLA) produced by the liver protects the kidney from AMR. However, this hypothesis has not been tested after SLKT. We present a case of SLKT with 2 donor-specific antibodies (DSAs) (DR53, 12,364 mean fluorescence intensity [MFI]; DQ7, 1253 MFI) that displayed a decrease by day 7 (DR53, 2747 MFI; DQ7, 107 MFI). On day 351, the patient was diagnosed with kidney AMR associated with high levels of DSA (DR53, 18,542 MFI; DQ7, 22,007 MFI) that persisted until day 531. High levels of sHLA-DR/DQ and HLA-DR/DQ-containing exosomes were also detected on day 398. Consequently, the patient underwent treatment with plasmapheresis, intravenous immunoglobulin, prednisone, and rituximab. On day 752, biopsy results were negative for AMR. Moderate levels of DSA (DR53, 9798 MFI; DQ7, 1271 MFI), and baseline levels of sHLA-DR/DQ and HLA-DR/DQ-containing exosomes were observed. Increases in CD4+CD25+FOXP3+ regulatory T cell marker-containing exosomes (CD73, programmed death-ligand 1) were observed on day 752 compared to day 398. These data show a direct correlation between sHLA and HLA-containing exosomes and an inverse correlation between tolerance marker-containing exosomes and kidney AMR after SLKT.

Impact of Glomerulitis on Long-term Outcomes After Kidney Transplantation.

The Banff classification scheme provides a framework for interpreting transplant kidney biopsies and has undergone various updates in the past 2 decades especially related to antibody-mediated rejection. The clinical significance of early glomerulitis seen within 4 mo on protocol biopsies has received limited attention. We hypothesized that early glomerulitis seen on protocol biopsies will lead to significant adverse outcomes as assessed by histopathology and allograft outcome. Methods: A single-center retrospective study of a cohort of patients who underwent protocol biopsies within 4 mo after transplantation with timely follow-up protocol biopsies were assessed. Patients with recurrent glomerulonephritis were excluded. Results: We calculated glomerulitis (g) scores for 2212 biopsy specimens and identified 186 patients with glomerulitis (g > 0) and 2026 patients without glomerulitis (g = 0). The progression to chronic transplant glomerulopathy at 1 and 2 y was higher in patients with g > 0 as compared with g = 0 (year 1, 10.7% versus 2.3% [P < 0.001]' respectively; year 2, 17.2% versus 4.3% [P < 0.001], respectively) with no difference in other chronic lesions. The death-censored graft failure rate was higher in patients with g > 0 as compared with g = 0 (hazard ratio, 1.68 [95% CI, 1.07-2.65]; P = 0.02). We did not find any difference in outcomes in glomerulitis group based on donor-specific antibody. Conclusion: Our findings suggest that early glomerulitis (seen within 4 mo after transplantation) may lead to clinically significant long-term changes and thus could be a target for early intervention therapies.

Aspectos técnicos y clínicos de la prueba cruzada de histocompatibilidad en el trasplante de órganos solidos / Technical and clinical aspects of the histocompatibility crossmatch assay in solid organ transplantation

La presencia de anticuerpos dirigidos contra los antígenos leucocitarios humanos (Human Leukocyte Antigens, HLA) que se expresan en las células del donante, es uno de los factores de riesgo más importantes asociados con las complicaciones clínicas después del trasplante. La prueba cruzada es una de las pruebas de histocompatibilidad más eficaces para la detección de anticuerpos específicos contra el donante en los receptores de injertos. En los primeros métodos de la prueba cruzada, se utilizaba la citotoxicidad dependiente del complemento, que es útil para detectar dichos anticuerpos responsables del rechazo hiperagudo del injerto, pero carece de la sensibilidad adecuada. Por ello, se desarrollaron métodos de pruebas cruzadas más sensibles, entre ellas, la prueba cruzada por citometría de flujo que hoy se considera el método preferido. En este artículo se revisa la evolución de la prueba cruzada y los factores más importantes que deben tenerse en cuenta al realizarla y al interpretar los resultados de esta prueba fundamental para la supervivencia a largo plazo del injerto.

The presence of antibodies directed against human leukocyte antigens (HLA) expressed on donor cells is a significant risk factor for serious clinical complications after transplantation. The crossmatch assay is one of the most important tests available for the detection of donor-specific antibodies in potential allograft recipients. Early crossmatch methods utilized complement-dependent cytotoxicity, which is useful for detecting the donor-specific anti- HLA antibodies responsible for hyperacute allograft rejection but lacks adequate sensitivity. Consequently, more sensitive crossmatch methods have been developed, ultimately leading to the flow cytometry crossmatch as the currently preferred methodology. Herein, we review the evolution of the crossmatch assay and the most important factors to consider when performing and interpreting the results of this fundamental assay for ensuring the long-term survival of the transplanted organ.

Alloantibodies after simultaneous liver-kidney transplant: A story of primary nonfunction, retransplantation, and antibody-mediated rejection.

Simultaneous liver-kidney transplant (SLKT) in the presence of antihuman leukocyte antigen (HLA) donor-specific antibodies (DSA) is a well-accepted practice. Herein, we describe the evolution of alloantibodies in a patient who received an SLKT. The pre-SLKT serum sample showed multiple strong DSA. As expected, all DSA cleared in a sample collected 4 days after the SLKT. Because of the primary nonfunction of the liver in the SLKT, the patient had a second liver transplant 4 days later. An abrupt increase in DSA levels against the kidney was detected 10 days after the second liver transplant. These DSA were refractory to treatment, and the transplanted kidney was lost due to antibody-mediated rejection (AMR). A detailed study of the HLA epitopes recognized by DSA and, after normalization with third-party alloantibodies to address the effect of multiple transfusions and liver allograft neutralization, showed that the elimination of these antibodies depended on the HLA antigens expressed by the transplanted liver cells. The return of DSA after removal of the first transplanted liver was associated with AMR in the transplanted kidney.

Variations in HLA-B cell surface expression, half-life and extracellular antigen receptivity.

The highly polymorphic human leukocyte antigen (HLA) class I molecules present peptide antigens to CD8+ T cells, inducing immunity against infections and cancers. Quality control mediated by peptide loading complex (PLC) components is expected to ensure the cell surface expression of stable peptide-HLA class I complexes. This is exemplified by HLA-B*08:01 in primary human lymphocytes, with both expression level and half-life at the high end of the measured HLA-B expression and stability hierarchies. Conversely, low expression on lymphocytes is measured for three HLA-B allotypes that bind peptides with proline at position 2, which are disfavored by the transporter associated with antigen processing. Surprisingly, these lymphocyte-specific expression and stability differences become reversed or altered in monocytes, which display larger intracellular pools of HLA class I than lymphocytes. Together, the findings indicate that allele and cell-dependent variations in antigen acquisition pathways influence HLA-B surface expression levels, half-lives and receptivity to exogenous antigens.

Use of complement binding assays to assess the efficacy of antibody mediated rejection therapy and prediction of graft survival in kidney transplantation.

BACKGROUND: The Luminex® single antigen bead assay (SAB) is the method of choice for monitoring the treatment for antibody-mediated rejection (AMR). A ⩾50% reduction of the dominant donor-specific antibody (IgG-DSA) mean fluorescence intensity (MFI) has been associated with improved kidney allograft survival, and C1q-fixing DSA activity is associated with poor outcomes in patients with AMR. We aimed to investigate if C1q-DSA can be used as a reliable predictor of response to therapy and allograft survival in patients with biopsy-proven AMR. METHODS: We tested pre- and post-treatment sera of 30 kidney transplant patients receiving plasmapheresis and low-dose IVIG for biopsy-proven AMR. IgG-DSA and C1q-DSA MFI were measured and correlated with graft loss or survival. Patients were classified as nonresponders (NR) when treatment resulted in <50% reduction in MFI of IgG-DSA and/or C1q-DSA was detectable following therapy. RESULTS: Differences in the percentage of patients deemed NR depended upon the end-point criterion (73% by reduction in IgG-DSA MFI vs. 50% by persistent C1q-DSA activity). None of the seven patients with <50% reduction of IgG-DSA but non-detectable C1q-DSA-fixing activity after therapy experienced graft loss, suggesting that C1q-DSA activity may better correlate with response. Reduction of C1q-DSA activity predicted graft survival better than IgG-DSA in the univariate Cox analysis (20.1% vs. 5.9% in NR; log-rank P-value=0.0147). CONCLUSIONS: A rapid reduction of DSA concentration below the threshold required for complement activation is associated with better graft survival, and C1q-DSA is a better predictor of outcomes than IgG-DSA MFI reduction.

Beyond Criteria and Definitions: Outcome of a Standardized Antibody-Mediated Rejection Protocol with a Diagnostic Schema Different from the Banff 2009 Criteria.

A new clinical diagnostic schema is needed for the diagnosis of antibody-mediated rejection (AMR) in kidney transplant recipients due to the limited utility of C4d staining, lack of standardized quantitative tests for donor specific antibodies, and potential new diagnostic markers. The treatment of AMR remains controversial because previous studies included heterogeneous treatment modalities, small sample sizes, and short follow-up time. At the University of Michigan Transplant Center, 26 patients were diagnosed with AMR based on our diagnostic protocol including C4d-negative AMR in thesetting of graft dysfunction and Banff tissue injury type II (capillaritis) or type III (arteritis). After diagnosis, these patients received six sessions of plasmapheresis (PP) and IVIG (100 mg/kg after the first to fifth PP and 500 mg/kg with the last PP). Our novel finding in this analysis was the association between persistent C1q detection and graft loss. We confirmed that C4d positivity at diagnosis is associated with worse outcomes. Also, we found that response to our treatment protocol is dependent on C4d staining and Banff tissue injury type.