Donor-specific Alloantibodies Research Articles

Regulatory Dendritic Cell Infusion Prolongs Kidney Allograft Survival in Nonhuman Primates

We examined the influence of regulatory dendritic cells (DCreg), generated from cytokine-mobilized donor blood monocytes in vitamin D3 and IL-10, on renal allograft survival in a clinically relevant rhesus macaque model. DCreg expressed low MHC class II and costimulatory molecules, but comparatively high levels of programmed death ligand-1 (B7-H1), and were resistant to pro-inflammatory cytokine-induced maturation. They were infused intravenously (3.5–10 × 106/kg), together with the B7-CD28 costimulation blocking agent CTLA4Ig, 7 days before renal transplantation. CTLA4Ig was given for up to 8 weeks and rapamycin, started on Day −2, was maintained with tapering of blood levels until full withdrawal at 6 months. Median graft survival time was 39.5 days in control monkeys (no DC infusion; n = 6) and 113.5 days (p < 0.05) in DCreg-treated animals (n = 6). No adverse events were associated with DCreg infusion, and there was no evidence of induction of host sensitization based on circulating donor-specific alloantibody levels. Immunologic monitoring also revealed regulation of donor-reactive memory CD95+ T cells and reduced memory/regulatory T cell ratios in DCreg-treated monkeys compared with controls. Termination allograft histology showed moderate combined T cell- and Ab-mediated rejection in both groups. These findings justify further preclinical evaluation of DCreg therapy and their therapeutic potential in organ transplantation.

Pathology of C4d-negative antibody-mediated rejection in renal allografts

To summarize current evidence supporting the existence of C4d-negative antibody-mediated rejection (AMR) in renal allografts, its potential to cause chronic graft injury, and whether histopathologic features of C4d-negative AMR differ from those of C4d-positive AMR. Recently published molecular, clinicopathologic, and ultrastructural studies provide strong evidence that microvascular injury in the presence of donor-specific alloantibodies (DSA) has the potential to cause interstitial fibrosis/tubular atrophy, transplant glomerulopathy, and graft loss, whether or not peritubular capillary (PTC) C4d is present. Although C4d-positive AMR may represent a more severe form of AMR, recent studies have found that in patients with DSA, microvascular injury (glomerulitis, peritubular capillaritis) is more strongly associated with graft loss than C4d deposition. Our data suggest that C4d-positive and C4d-negative AMR show similar degrees of glomerulitis and peritubular capillaritis, similar frequencies of concurrent cell-mediated rejection, and that both may occur early or late posttransplantation. In renal allografts, microvascular injury in the presence of DSA but with negative C4d staining in PTC nonetheless is indicative of humorally mediated graft injury that has the potential to cause tubular atrophy/interstitial fibrosis, transplant glomerulopathy, and graft loss. Prompt treatment for AMR may prevent or at least delay subsequent development of transplant glomerulopathy.

Alloantibody and Autoantibody Monitoring Predicts Islet Transplantation Outcome in Human Type 1 Diabetes

Long-term clinical outcome of islet transplantation is hampered by the rejection and recurrence of autoimmunity. Accurate monitoring may allow for early detection and treatment of these potentially compromising immune events. Islet transplant outcome was analyzed in 59 consecutive pancreatic islet recipients in whom baseline and de novo posttransplant autoantibodies (GAD antibody, insulinoma-associated protein 2 antigen, zinc transporter type 8 antigen) and donor-specific alloantibodies (DSA) were quantified. Thirty-nine recipients (66%) showed DSA or autoantibody increases (de novo expression or titer increase) after islet transplantation. Recipients who had a posttransplant antibody increase showed similar initial performance but significantly lower graft survival than patients without an increase (islet autoantibodies P < 0.001, DSA P < 0.001). Posttransplant DSA or autoantibody increases were associated with HLA-DR mismatches (P = 0.008), induction with antithymocyte globulin (P = 0.0001), and pretransplant panel reactive alloantibody >15% in either class I or class II (P = 0.024) as independent risk factors and with rapamycin as protective (P = 0.006) against antibody increases. DSA or autoantibody increases after islet transplantation are important prognostic markers, and their identification could potentially lead to improved islet cell transplant outcomes.

Class II Alloantibody and Mortality in Simultaneous Liver-Kidney Transplantation

Hyperacute kidney rejection is unusual in crossmatch positive recipients of simultaneous liver-kidney transplants (SLKT). However, recent data suggest that these patients remain at risk for antibody-mediated kidney rejection. To further investigate the risk associated with donor-specific alloantibodies (DSA) in SLKT, we studied 86 consecutive SLKT patients with an available pre-SLKT serum sample. Serum samples were analyzed in a blinded fashion for HLA DSA using single antigen beads (median florescence intensity≥2,000=positive). Post-SLKT samples were analyzed when available (76%). Thirty patients had preformed DSA, and nine developed de novo DSA. Preformed class I DSA did not change the risk of rejection, patient or allograft survival. In contrast, preformed class II DSA was associated with a markedly increased risk of renal antibody mediated rejection (AMR) (p=0.006), liver allograft rejection (p=0.002), patient death (p=0.02), liver allograft loss (p=0.02) and renal allograft loss (p=0.045). Multivariable modeling showed class II DSA (preformed or de novo) to be an independent predictor of patient death (HR=2.2; p=0.043) and liver allograft loss (HR=2.2; p=0.044). These data warrant reconsideration of the approach to DSA in SLKT.

Incidence and Impact of De Novo Donor-Specific Alloantibody in Primary Renal Allografts

To date, limited information is available describing the incidence and impact of de novo donor-specific anti-human leukocyte antigen (HLA) antibodies (dnDSA) in the primary renal transplant patient. This report details the dnDSA incidence and actual 3-year post-dnDSA graft outcomes. The study includes 189 consecutive nonsensitized, non-HLA-identical patients who received a primary kidney transplant between March 1999 and March 2006. Protocol testing for DSA via LABScreen single antigen beads (One Lambda) was done before transplantation and at 1, 3, 6, 9, and 12 months after transplantation then annually and when clinically indicated. Of 189 patients, 47 (25%) developed dnDSA within 10 years. The 5-year posttransplantation cumulative incidence was 20%, with the largest proportion of patients developing dnDSA in the first posttransplantation year (11%). Young patients (18-35 years old at transplantation), deceased-donor transplant recipients, pretransplantation HLA (non-DSA)-positive patients, and patients with a DQ mismatch were the most likely to develop dnDSA. From DSA appearance, 9% of patients lost their graft at 1 year. Actual 3-year death-censored post-dnDSA graft loss was 24%. We conclude that 11% of the patients without detectable DSA at transplantation will have detectable DSA at 1 year, and over the next 4 years, the incidence of dnDSA will increase to 20%. After dnDSA development, 24% of the patients will fail within 3 years. Given these findings, future trials are warranted to determine if treatment of dnDSA-positive patients can prevent allograft failure.

Belatacept and Sirolimus Prolong Nonhuman Primate Islet Allograft Survival: Adverse Consequences of Concomitant Alefacept Therapy

Calcineurin inhibitors (CNI) and steroids are known to promote insulin resistance, and their avoidance after islet transplantation is preferred from a metabolic standpoint. Belatacept, a B7-specific mediator of costimulation blockade (CoB), is clinically indicated as a CNI alternative in renal transplantation, and we have endeavored to develop a clinically translatable, belatacept-based regimen that could obviate the need for both CNIs and steroids. Based on the known synergy between CoB and mTOR inhibition, we studied rhesus monkeys undergoing MHC-mismatched islet allotransplants treated with belatacept and the mTOR inhibitor, sirolimus. To extend prior work on CoB-resistant rejection, some animals also received CD2 blockade with alefacept (LFA3-Ig). Nine rhesus macaques were rendered diabetic with streptozotocin and underwent islet allotransplantation. All received belatacept and sirolimus; six also received alefacept. Belatacept and sirolimus significantly prolonged rejection-free graft survival (median 225 days compared to 8 days in controls receiving basiliximab and sirolimus; p = 0.022). The addition of alefacept provided no additional survival benefit, but was associated with Cytomegalovirus reactivation in four of six animals. No recipients produced donor-specific alloantibodies. The combination of belatacept and sirolimus successfully prevents islet allograft survival in rhesus monkeys, but induction with alefacept provides no survival benefit and increases the risk of viral reactivation.

Induction of Antinuclear Antibodies by De Novo Autoimmune Hepatitis Regulates Alloimmune Responses in Rat Liver Transplantation

Concanavalin A (Con A) is a lectin originating from the jack-bean and well known for its ability to stimulate T cells and induce autoimmune hepatitis. We previously demonstrated the induction of immunosuppressive antinuclear autoantibody in the course of Con A-induced transient autoimmune hepatitis. This study aimed to clarify the effects of Con A-induced hepatitis on liver allograft rejection and acceptance. In this study, we observed the unique phenomenon that the induction of transient de novo autoimmune hepatitis by Con A injection paradoxically overcomes the rejection without any immunosuppressive drug and exhibits significantly prolonged survival after orthotopic liver transplantation (OLT). Significantly increased titers of anti-nuclear Abs against histone H1 and high-mobility group box 1 (HMGB1) and reduced donor specific alloantibody response were observed in Con A-injected recipients. Induction of Foxp3 and IL-10 in OLT livers of Con A-injected recipients suggested the involvement of regulatory T cells in this unique phenomenon. Our present data suggest the significance of autoimmune responses against nuclear histone H1 and HMGB1 for competing allogeneic immune responses, resulting in the acceptance of liver allografts in experimental liver transplantation.

The Natural History of Clinical Operational Tolerance After Kidney Transplantation Through Twenty-Seven Cases

We report here on a European cohort of 27 kidney transplant recipients displaying operational tolerance, compared to two cohorts of matched kidney transplant recipients under immunosuppression and patients who stopped immunosuppressive drugs and presented with rejection. We report that a lower proportion of operationally tolerant patients received induction therapy (52% without induction therapy vs. 78.3%[p = 0.0455] and 96.7%[p = 0.0001], respectively), a difference likely due to the higher proportion (18.5%) of HLA matched recipients in the tolerant cohort. These patients were also significantly older at the time of transplantation (p = 0.0211) and immunosuppression withdrawal (p = 0.0002) than recipients who rejected their graft after weaning. Finally, these patients were at lower risk of infectious disease. Among the 27 patients defined as operationally tolerant at the time of inclusion, 19 still display stable graft function (mean 9 ± 4 years after transplantation) whereas 30% presented slow deterioration of graft function. Six of these patients tested positive for pre-graft anti-HLA antibodies. Biopsy histology studies revealed an active immunologically driven mechanism for half of them, associated with DSA in the absence of C4d. This study suggests that operational tolerance can persist as a robust phenomenon, although eventual graft loss does occur in some patients, particularly in the setting of donor-specific alloantibody.

The Role of Donor Specific Antibodies in Liver Transplantation and Impact on Graft Survival on Positive Crossmatchs

Introduction: The role of donor specific antibodies in liver transplantation is unclear. Though donor-specific alloantibodies (DSA) has been shown to no immediate impact via hyperacute rejection, the long term impact on graft survival is not well elucidated. Methods: We followed 42 liver transplants (LT) patients with positive DSA since 1989. The HLA specificities of the antibodies were characterized using a Luminex-based, CDC DSA and CDC PRA. Flow cytometry was also obtained in 32 of the 42 patients. Graft survival was measured at 1, 3, 5 and 10 years and compared to a control group from our institution with negative cross match. A liver biopsy was performed when Acute Cellular Rejection (ACR) was suspected Results: A retrospective analysis shows that 76% of the grafts survived after 1 year. 71%, 54% and 37% graft survival was seen in 3, 5 and 10 years. The degree of PRA/DSA did not influence in terms of the long term survival of the graft. (Table 1)[Table 1. Graft survival comparison between positiv]Our control group showed a graft survival of 84%, 76%, 65% and 45% at 1, 3, 5 and 10 years follow up. ACR was biopsy positive in 8 of 11 patients with 100% of the rejection episodes before completing the first year after the transplant. Conclusion: Hyperacute rejection of a transplanted liver is rare even when the recipient has circulating donor-specific alloantibodies (DSA). Comparing to our control group, LT from positive DSA grafts has a decrease graft survival short and long term. ACR is common in the positive group especially during the first year after LT. Close attention during the first year of transplant is needed in order to prevent decrease in long term graft function on LT patients with positive crossmatch.

A Novel Concept of Phased Desensitization Using Bortezomib and Rituximab for Highly HLA-Sensitized Kidney Transplantation

Introduction: Donor specific alloantibody producing plasma cells (DSA-PCs) appear resistant to conventional immunosuppressive agents. The recent studies demonstrated the impact of pretransplant desensitization with the proteasome inhibitor bortezomib on DSA-PCs in sensitized renal allograft candidates. However, other studies have showed that one cycle of bortezomib alone does not decrease DSA levels in sensitized kidney transplant recipients. We developed a concept of phased desensitization with rituximab and bortezomib, and applied it in to a highly sensitized kidney transplant candidate. Methods: We assessed the activity of bortezomib against various B cell subpopulations and plasma cells and suppressive effect of that to produce anti-blood group antibodies (Abs) DSA in mice. To elicit Abs against allopeptide (DSA), B6 mice were immunized with thymocytes from Balb/c mice. After the immunization, the mice received intravenous injections of bortezomib (0.75 mg/kg body weight) 3 times a week for 1 week. On the basis of the results, we have established a novel concept of desensitization for a sensitized patient. Results: Bortezomib led to a significant reduction in the subpopulations of CD21-/IgMhigh plasmablasts, and CD138+/CD25-/IgG κ-light chain+ plasma cells in the spleen of Balb/c mice, but not in that of CD21+/IgMhigh marginal-zone B cells and the CD21low/IgMlow follicular B cells. Flow cytometry analyses revealed that bortezomib did not reduce the B cells with receptors for either A-carbohydrates. Thus, bortezomib reduced not only IgG producing plasma cells but also IgM producing cells, although their precursor memory B cells were resistant to this reagent. Taken together with a known fact that rituximab is able to deplete memory B cells, we have proposed a concept of phased desensitization using bortezomib and rituximab, and applied it in to a highly sensitized kidney transplant patient. A 56-year-old man with renal graft failure was referred to our hospital. His first renal graft from his brother remained functioning for 16 years but was lost due to chronic rejection. This allograft loss resulted in sensitization of the recipient against HLA antigens. Unfortunately, his wife who volunteered as a second donor shared the same A2 antigen with the first donor, which might be a target of the preformed anti-HLA Abs. Single antigen flow-based DSA testing revealed 11 anti-HLA class I Abs including anti-donor HLA A2 Ab. He received a desensitization regimen consisting of a single dose of rituximab (375 mg/m2) combined with double-filtration plasmapheresis (DFPP) followed by low doses of intravenous immuno-globulin (DFPP/IVIG treatment). Tacrolimus and mycophenolate mofetil were started 2 weeks before the DFPP/IVIG treatment. Despite 3 DFPP/IVIG sessions, the crossmatch remained positive. Thereafter, he was treated with one cycle of bortezomib (1.3mg/m2 × 4 doses). After 3 DFPP/IVIG sessions, the crossmatch turned negative and renal transplantation was successfully performed. The induction quadruple immunosuppression protocol comprised tacrolimus, mycophenolate mofetil and methylprednisolone. After the transplantation, neither Ab-mediated rejection nor severe acute cellular rejection was encountered in this patient. Conclusions: The current study suggests that phased desensitization with rituximab and bortezomib may be needed for highly HLA-sensitized kidney transplant candidates.

Reduction of Transplant Arteriosclerosis After Treatment With Mycophenolate Mofetil and Ganciclovir in a Mouse Aortic Allograft Model

Transplant arteriosclerosis is a major obstacle for long-term allograft survival in heart transplant. The aim of this study was to investigate potential synergistic effects of combined treatment with mycophenolate mofetil and ganciclovir on the development of transplant arteriosclerosis, presence of regulatory T cells, and expression of donor specific alloantibodies. Donor aortas from C57BL/6 (H2b) mice that were fully mismatched to the major histocompatibility complex were transplanted into CBA (H2k) mouse recipients. Groups of mice received mycophenolate mofetil (100 or 300 mg/kg, oral), ganciclovir (10 or 72 mg/kg, intraperitoneal), or a mycophenolate mofetil and ganciclovir combination. Grafts were analyzed by histology and morphometry on day 30 after transplant. Numbers of regulatory T cells and donor-specific alloantibodies were examined by fluorescence-activated cell sorting analysis of splenic tissue and peripheral blood. Mycophenolate mofetil (100 mg/kg) and ganciclovir (10 mg/kg and 72 mg/kg) did not show effects on transplant arteriosclerosis formation or alloantibody production. However, groups treated with mycophenolate mofetil (300 mg/kg) or a low- or high-dose mycophenolate mofetil and ganciclovir combination had significantly reduced transplant arteriosclerosis and alloantibody levels. Expression of regulatory T cells within the spleen was similar between all experimental groups and untreated controls. The combination of mycophenolate mofetil and ganciclovir significantly reduced the development of transplant arteriosclerosis in a mouse abdominal aortic allograft model. This effect may be a result of decreased alloantibody production.

The role of complement in antibody-mediated rejection in kidney transplantation

Over the past decade, several studies have suggested that the complement system has an active role in both acute and chronic allograft rejection. These studies have been facilitated by improved techniques to detect antibody-mediated organ rejection, including immunohistological staining for C4d deposition in the allograft and solid-phase assays that identify donor-specific alloantibodies (DSAs) in the serum of transplant recipients. Studies with eculizumab, a humanized monoclonal antibody directed against complement component C5, have shown that activation of the terminal complement pathway is necessary for the development of acute antibody-mediated rejection in recipients of living-donor kidney allografts who have high levels of DSAs. The extent to which complement activation drives chronic antibody-mediated injury leading to organ rejection is less clear. In chronic antibody-mediated injury, early complement activation might facilitate chemotaxis of inflammatory cells into the allograft in a process that later becomes somewhat independent of DSA levels and complement factors. In this Review, we discuss the different roles that the complement system might have in antibody-mediated allograft rejection, with specific emphasis on renal transplantation.

NK Cells are Required for Costimulatory Blockade Induced Tolerance to Vascularized Allografts

The role of natural killer (NK) cells in organ transplantation is poorly understood because studies link these cells to both regulatory and inflammatory functions. NK cells exacerbate inflammation and adaptive immunity under conditions of allograft rejection, but little is known regarding their roles in allograft tolerance. We test the hypothesis that NK cells have regulatory function and promote tolerance induction to murine cardiac allografts. Murine hearts were transplanted as fully vascularized heterotopic grafts from BALB/c donors into C57BL/6 recipients. Allograft tolerance was achieved using donor splenocyte transfusion + anti-CD40L monoclonal antibody (mAb) before transplantation. The requirement for NK cells in tolerance induction was tested by administering anti-NK1.1-depleting mAb or anti-NKG2D-blocking mAb. Intragraft and peripheral immune cell populations were determined by flow cytometry and immunohistochemistry. CD4 T-cell alloantigen-specific responses and donor-specific alloantibody were also determined. NK cell-depleted recipients acutely reject allografts despite anti-CD40L blockade, but rejecting recipients lacked alloantibody and alloantigen-specific CD4 T-cell responses. NK cell depletion resulted in elevated numbers of graft-infiltrating macrophages. NKG2D blockade in tolerized recipients did not cause acute rejection but increased macrophage graft infiltration and increased the expression of NKG2D ligand Rae-1γ on these cells. Our data show that NK cells are required for tolerance induction in recipients given donor splenocyte transfusion + anti-CD40L mAb. Our data suggest NK cells regulate monocyte or macrophage activation and infiltration into allografts by a mechanism partially dependent on NKG2D receptor-ligand interactions between NK cells and monocytes/macrophages.

2-P

Aim For the past two years, we have compared retrospective solid phase crossmatches (DSAXM) with flow crossmatching on all patients who were either transplanted or exhibited positive flow cytometry crossmatches (FXM) with deceased donors to determine the realtionship between the two XM assays. Methods An MFI cut-off of 4000 MFI was used in SAB testing for unacceptable antigens. Flow cytometry crossmatches were analyzed on a BD FacsCanto II flow cytometer (pronase-treated cells). DSAXM was according to the manufacturer’s procedure. Results We analyzed 734 solid phase crossmatch/flow crossmatch pairs: 421 TFXM/DSAIXM and 313 BFXM/DSAXM. 108 TFXM+ crossmatches were eliminated for the BFXM comparison to minimize the interference of Class I on the BFXM/DSAXM comparisons. We observed that for TFXM/DSA1XM there was a 77% agreement. 22% of the discrepancies were mediated by a false positive TFXM crossmatch, based on lack of DSA. 21 of the 75 FXMs in this group had channel shifts above 1000 (our cut-off is 120). The remaining 1% of the discrepancies were mediated by false positive DSA1XM (n = 5). We observed that for BFXM/DSAXM there was a 73% agreement. As seen with the TFXM/DSA1XM the majority of the discrepancies were due to false positive BFXM (19.5%). There were also more discrepant false positive DSAXMs (7.7%). Conclusions These data demonstrate that the DSA1XM reliably detects Class I antibodies, (however the DSA2XM does not reliably detect DQ antibodies.) In this study, the incidences of unexplained positive TFXM and BFXM were high: 22% and 20%, respectively. For BFXM/DSAXM comparisons, neither assay demonstrated a competitive edge. Since neither the flow assay nor the solid phase assay reliably predicts crossmatch outcomes, we continue to use both assays in a complementary fashion. Combining the solid phase technology (single antigen beads and the DSAXM) and flow cytometry crossmatching provides a better assessment of risk due to preformed donor-specific alloantibodies than either assay alone.

The Spleen Is the Major Source of Antidonor Antibody-Secreting Cells in Murine Heart Allograft Recipients

Antibody-mediated allograft rejection is an increasingly recognized problem in clinical transplantation. However, the primary location of donor-specific alloantibody (DSA)-producing cells after transplantation have not been identified. The purpose of this study was to test the contribution of allospecific antibody-secreting cells (ASCs) from different anatomical compartments in a mouse transplantation model. Fully MHC-mismatched heart allografts were transplanted into three groups of recipients: nonsensitized wild type, alloantigen-sensitized wild-type and CCR5(-/-) mice that have exaggerated alloantibody responses. We found that previous sensitization to donor alloantigens resulted in the development of antidonor alloantibody (alloAb) with accelerated kinetics. Nevertheless, the numbers of alloantibody-secreting cells and the serum titers of antidonor IgG alloantibody were equivalent in sensitized and nonsensitized recipients 6 weeks after transplantation. Regardless of recipient sensitization status, the spleen contained higher numbers of donor-reactive ASCs than bone marrow at days 7-21 after transplantation. Furthermore, individual spleen ASCs produced more antidonor IgG alloantibody than bone marrow ASCs. Taken together, our results indicate that the spleen rather than bone marrow is the major source of donor-reactive alloAb early after transplantation in both sensitized and nonsensitized recipients.

The Histopathology of Lung Allograft Dysfunction Associated With the Development of Donor-specific HLA Alloantibodies

The histopathology of antibody-mediated rejection in lung allografts, outside of classical hyperacute rejection, has been poorly defined, in part because of the difficulty in identifying potential alloantibodies and in separating alloreactive and nonspecific complement-mediated reactions. In this study, we looked at lung biopsies coinciding with the development of anti-human leukocyte antigen (anti-HLA) antibodies and lung dysfunction in a diverse group of lung transplant recipients over a 3-year period. We identified 23 patients and found that 17 had coexistent high-grade acute cellular rejection, 5 had patchy acute lung injury, and 1 had bronchiolitis at the time that anti-HLA alloantibodies appeared. Capillaritis was seen in 4/22 (18%) cases, usually in the context of cellular rejection. When the 17 cases of acute cellular rejection with coexistent anti-HLA antibodies were compared with a matched group of 26 patients with equivalent cellular rejection grade without anti-HLA antibodies, the only morphologic feature that separated the 2 groups was capillaritis seen in a minority of cases. C4d deposits were seen in both groups, although more frequently in those cases with anti-HLA antibodies (76% vs. 24%). The development of anti-HLA alloantibodies often occurs in the setting of acute cellular rejection, which in only a minority of cases can be identified as likely having an antibody-mediated component. C4d staining does not consistently separate the 2 groups. Identifying and ascribing allograft dysfunction to antibody-mediated rejection related to the development of anti-HLA antibodies is a difficult task that cannot be solved exclusively by morphology and requires significant clinicopathologic correlation.

Sinusoidal C4d deposits in liver allografts indicate an antibody-mediated response: Diagnostic considerations in the evaluation of liver allografts

There is a paucity of data concerning the correlation of complement component 4d (C4d) staining in liver allografts and antibody-mediated rejection. Data about the location and character of C4d deposits in native and allograft liver tissues are inconsistent. We performed C4d immunofluorescence (IF) on 141 fresh-frozen liver allograft biopsy samples and native livers, documented the pattern of C4d IF staining, and correlated the findings with the presence of donor-specific alloantibodies (DSAs). A linear/granular sinusoidal pattern of C4d IF was noted in 18 of 28 biopsy samples obtained after transplantation from patients with positive crossmatch and detectable donor-specific alloantibody (pos-XM/DSA) findings. None of the 59 tested biopsy samples from patients with negative crossmatch and detectable donor-specific alloantibody (neg-XM/DSA) findings were C4d-positive (P < 0.001). No significant association was found between pos-XM/DSA and C4d IF staining in other nonsinusoidal liver compartments. To compare the results of sinusoidal C4d staining with IF and 2 immunohistochemistry (IHC) techniques, C4d IHC was performed on 19 liver allograft biopsy samples in which a sinusoidal pattern of C4d IF had been noted. Sinusoidal C4d IHC findings were negative for 17 of the 19 biopsy samples; 2 showed weak and focal staining, and both patients had pos-XM/DSA findings. Portal vein endothelium staining was present in only 1 IF-stained biopsy sample (pos-XM/DSA) but in 11 IHC-stained biopsy samples (2 of the 11 samples had neg-XM/DSA findings). We conclude that sinusoidal C4d deposits detected by IF in frozen tissue samples from liver allograft recipients correlate with the presence of DSAs and an antibody-mediated alloresponse. These observations are similar to findings reported for other solid organ transplants and can provide relevant information for patient management. Further validation of IHC techniques for C4d detection in liver allograft tissue is required.

Donor-Specific Antibodies after Ceasing Immunosuppressive Therapy, with or without an Allograft Nephrectomy

Within the last few years, anti-human leukocyte antigen detection assays have significantly improved. This study asked, using the Luminex single-antigen assay, whether an allograft nephrectomy allowed donor-specific alloantibodies to appear that were not previously detected in the serum when the failed kidney was still in place. After losing the kidney allograft and stopping immunosuppressive therapy, the proportions of donor-specific alloantibodies and nondonor-specific alloantibodies were compared in patients who had (n=48; group I) and had not (n=21; group II) undergone an allograft nephrectomy. Allograft nephrectomies were performed at 150 days after kidney allograft loss, and the time between allograft nephrectomy and last follow-up was 538 ± 347 days. At kidney allograft loss, donor-specific alloantibodies were detected in three group II patients (14.2%) and six group I patients (12.5%). At last follow-up, donor-specific alloantibodies were detected in 11 patients (52.4%) without and 39 patients (81%) with an allograft nephrectomy (P=0.02). Anti-human leukocyte antigen class I donor-specific alloantibodies were positive in 23.8% of group II and 77% of group I patients (P<0.001); anti-human leukocyte antigen class II donor-specific alloantibodies were positive in 42.8% of group II and 62.5% of group I patients. Independent predictive factors for developing donor-specific alloantibodies after losing kidney allograft and stopping immunosuppressants were number of anti-human leukocyte antigen A/B mismatches at transplantation (zero versus one or more) and allograft nephrectomy. The development of donor-specific alloantibodies was significantly greater in patients with a failed kidney who had undergone an allograft nephrectomy compared with those patients who had not undergone allograft nephrectomy.

Accommodation

The term "accommodation" was invoked to describe endothelial cell resistance to antibody-mediated rejection after ABO-incompatible kidney or experimental xenograft transplantation. Currently, there is much interest in how to achieve successful human leukocyte antigen (HLA)-incompatible allograft transplantation in HLA-sensitized patients and how to treat antibody-mediated rejection after ABO-compatible HLA-incompatible allotransplantation. The term "accommodation" is often used interchangeably to describe patients who have donor-specific ABO or HLA alloantibodies in the absence of damage to their allograft. Here, we suggest that there are important differences between the immune responses to protein versus carbohydrate antigens and that graft HLA molecules may respond differently to antibodies (and antibody isotypes) than ABO antigens. Neither the mechanisms nor a phenotype of accommodation have been defined fully. Further research is needed to define mechanisms of both resistance and susceptibility to antibody-mediated injury and to predict under which circumstances allograft accommodation may occur.

General insufficiency of the classical CDC-based crossmatch to detect donor-specific anti-HLA antibodies leading to invalid results under recipients' medical treatment or underlying diseases.

Antibodies directed against HLA antigens of a given donor represent the most prominent cause for hyper-acute and acute rejections. In order to select recipients without donor-specific antibodies the complement-dependent cytotoxicity (CDC-) crossmatch as the standard procedure was established. As a functional assay it strongly depends on the availability of isolated donor lymphocytes and in particular on their vitality. However, due to several diseases or pharmacological treatment of a given recipient unexpected "false-positive" results of the CDC-crossmatch may arise. We here present three groups of patients which demonstrate the limits of the conventional crossmatch. 1) Kidney recipients before living donations exhibited positive CDC-reactions due to their conditioning using the therapeutical anti-CD20 mAb Rituximab (n=7), routinely used to deplete B-cells, or the anti-CD25 mAb basiliximab (n=2) to inhibit the proliferation of activated T-cells. 2) Recipients suffering from various leukaemias (n=5) exhibited "positive" CDC-crossmatches using PBL of the donors, although formerly these patients had never shown anti-HLA antibodies. Instead of donor-specific allo-antibodies, cytostatic agents such as 6-mercaptopurine led to an unspecific cell death. 3) Patients projected for post mortem or living kidney donations (n=44) exhibited "positive" CDC-crossmatch results which were not in accordance with their former antibody status and, partially, with high degrees of HLA-matching. These implausible results were due to underlying auto-immune diseases, mainly of the systemic immune complex type III such as lupus erythematosus, mainly leading to false-positive B-cell crossmatches by immune complexes binding to Fcγ-receptors. In all these 58 cases the alternatively performed ELISA-based "Antibody Monitoring System" (AMS-) crossmatch assay was not artifically affected, suggesting that this assay may be comprehensively established at least for the cases described.