Proposed Diagnostic Criteria for Chronic Antibody-Mediated Rejection in Liver Allografts.

Donor-specific alloantibodies (DSA) can cause acute antibody-mediated rejection (AMR) in all solid organ allografts. However, long-term outcome in patients with posttransplant DSA needs further study. We retrospectively evaluated prospectively collected paired serum, tissue, and data on 45 matched DSA- positive [DSA+; mean florescence intensity (MFI) ≥10,000] and -negative (DSA-) recipients of a primary liver-only allograft from January 2000 to April 2009. Blinded histopathologic evaluation demonstrated that DSA+ versus DSA- patients were more likely to have subtle inflammation and unique patterns of fibrosis, despite normal or near-normal liver function tests. Stepwise multivariable modeling developed a score (putatively named the chronic AMR [cAMR] score) that included interface activity, lobular inflammation, portal tract collagenization, portal venopathy, sinusoidal fibrosis, and hepatitis C virus status. The score was developed (c = 0.811) and cross-validated (c = 0.704) to predict allograft failure. Two cutoffs were employed to optimize sensitivity and specificity (80% each); a value >27.5 predicted 50% 10-year allograft failure. We propose chronic AMR as a potential new entity defined by (1) a high cAMR score, (2) DSA, and (3) elimination of other potential causes of a similar injury pattern. In conclusion, cAMR score calculation identified liver allograft recipients with DSA at highest risk for allograft loss, although independent validation is needed.

Serum antibodies to human leucocyte antigen (HLA)-E, HLA-F and HLA-G in patients with systemic lupus erythematosus (SLE) during disease flares: Clinical relevance of HLA-F autoantibodies.

T lymphocyte hyperactivity and progressive inflammation in systemic lupus erythematosus (SLE) patients results in over-expression of human leucocyte antigen (HLA)-Ib on the surface of lymphocytes. These are shed into the circulation upon inflammation, and may augment production of antibodies promoting pathogenicity of the disease. The objective was to evaluate the association of HLA-Ib (HLA-E, HLA-F and HLA-G) antibodies to the disease activity of SLE. The immunoglobulin (Ig)G/IgM reactivity to HLA-Ib and ß2m in the sera of 69 German, 29 Mexican female SLE patients and 17 German female controls was measured by multiplex Luminex(®)-based flow cytometry. The values were expressed as mean flourescence intensity (MFI). Only the German SLE cohort was analysed in relation to the clinical disease activity. In the controls, anti-HLA-G IgG predominated over other HLA-Ib antibodies, whereas SLE patients had a preponderance of anti-HLA-F IgG over the other HLA-Ib antibodies. The disease activity index, Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)-2000, was reflected only in the levels of anti-HLA-F IgG. Anti-HLA-F IgG with MFI level of 500-1999 was associated with active SLE, whereas inactive SLE revealed higher MFI (>2000). When anti-HLA-F IgG were cross-reactive with other HLA-Ib alleles, their reactivity was reflected in the levels of anti-HLA-E and -G IgG. The prevalence of HLA-F-monospecific antibodies in SLE patients was also associated with the clinical disease activity. Anti-HLA-F IgG is possibly involved in the clearance of HLA-F shed from lymphocytes and inflamed tissues to lessen the disease's severity, and thus emerges as a beneficial immune biomarker. Therefore, anti-HLA-Ib IgG should be considered as a biomarker in standard SLE diagnostics.

Impact of IgG3 subclass and C1q-fixing donor-specific HLA alloantibodies on rejection and survival in liver transplantation.

Recent literature confirms donor-specific HLA alloantibodies (DSA) impair 5-year survival in some but not all liver transplant recipients. In an effort to improve DSA testing's association with rejection and death, we retrospectively evaluated 1270 liver transplant recipients for the presence of IgG3 and C1q-fixing DSA. In patients with preformed DSA, 29 and 51% had IgG3 and C1q-fixing DSA, respectively. In patients with de novo DSA, 62% and 67% had IgG3 and C1q-fixing DSA, respectively. When different types of DSA positive patients were compared to DSA negative patients, multivariable analysis showed that IgG3 DSA positivity had the highest numerical hazard ratio for death (IgG3: HR = 2.4, p < 0.001; C1q: HR = 1.9, p < 0.001; standard DSA: HR = 1.6, p < 0.001). Similarly, multivariable analysis demonstrated de novo IgG3 DSA positivity compared to no DSA had the highest hazard ratio for death (IgG3: HR = 2.1, p = 0.004; C1q: HR = 1.9, p = 0.02; standard DSA: HR = 1.8, p = 0.007). Preformed C1q-fixing class II DSA showed the strongest correlation with early rejection. In conclusion, preformed and de novo IgG3 subclass DSA positive patients had the highest absolute HR for death in side-by-side comparison with C1q and standard DSA positive versus DSA negative patients; however, IgG3 negative DSA positive patients still had inferior outcomes compared to DSA negative patients.

Immunoglobulin (Ig)G purified from human sera mirrors intravenous Ig human leucocyte antigen (HLA) reactivity and recognizes one's own HLA types, but may be masked by Fab complementarity-determining region peptide in the native sera.

Intravenous immunoglobulin (IVIg) reacted with a wide array of human leucocyte antigen (HLA) alleles, in contrast to normal sera, due possibly to the purification of IgG from the pooled plasma. The reactivity of IgG purified from normal sera was compared with that of native sera to determine whether any serum factors mask the HLA reactivity of anti-HLA IgG and whether IgG purified from sera can recognize the HLA types of the corresponding donors. The purified IgG, unlike native sera, mirrored IVIg reactivity to a wide array of HLA-I/-II alleles, indicating that anti-HLA IgG may be masked in normal sera - either by peptides derived from soluble HLA or by those from antibodies. A < 3 kDa peptide from the complementarity-determining region (CDR) of the Fab region of IgG (but not the HLA peptides) masked HLA recognition by the purified IgG. Most importantly, some of the anti-HLA IgG purified from normal sera - and serum IgG from a few donors - indeed recognized the HLA types of the corresponding donors, confirming the presence of auto-HLA antibodies. Comparison of HLA types with the profile of HLA antibodies showed auto-HLA IgG to the donors' HLA antigens in this order of frequency: DPA (80%), DQA (71%), DRB345 (67%), DQB (57%), Cw (50%), DBP (43%), DRB1 (21%), A (14%) and B (7%). The auto-HLA antibodies, when unmasked in vivo, may perform immunoregulatory functions similar to those of therapeutic preparations of IVIg.

Onset and progression of de novo donor-specific anti-human leukocyte antigen antibodies after BK polyomavirus and preemptive immunosuppression reduction.

BACKGROUND: BK polyomavirus (BKPyV) viremia/nephropathy and reduction in immunosuppression following viremia may increase the risk of alloimmune activation and allograft rejection. This study investigates the impact of BKPyV viremia on de novo donor anti-human leukocyte antigen (HLA)-specific antibodies (dnDSA). PATIENTS AND METHODS: All primary renal transplants at East Carolina University from March 1999 to December 2010, with at least 1 post-transplant BKPyV viral load testing, were analyzed. Patients were negative for anti-HLA antibodies to donor antigens (tested via single antigen beads) at transplantation and at first BKPyV testing. RESULTS: Nineteen of 174 patients (11%) tested positive for BKPyV viremia. Within 24 months of BKPyV viremia detection, 79% of BKPyV-viremic patients developed dnDSA. Only 20% of BKPyV viremia-persistent cases, compared to 86% of BKPyV viremia-resolved cases, developed dnDSA (P = 0.03). Poor allograft survival was evident in BKPyV viremia-persistent patients (60% failure by 2 years post BKPyV diagnosis) and in BKPyV viremia-resolved patients with dnDSA (5-year post BKPyV diagnosis allograft survival of 48%). CONCLUSIONS: Post-transplant BKPyV viremia and preemptive immunosuppression reduction is associated with high rates of dnDSA. When preemptively treating BKPyV viremia, dnDSA should be monitored to prevent allograft consequences.

The role of donor-specific HLA alloantibodies in liver transplantation.

The impact of donor-specific HLA alloantibodies (DSA) on short- and long-term liver transplant outcome is not clearly defined. While it is clear that not all levels of allosensitization produce overt clinical injury, and that liver allografts possess some degree of alloantibody resistance, alloantibody-mediated adverse consequences are increasingly being recognized. To better define the current state of this topic, we assembled experts to provide insights, explore controversies and develop recommendations for future research on the consequences of DSA in liver transplantation. This article summarizes the proceedings of this inaugural meeting. Several insights emerged. Acute antibody-mediated rejection (AMR), although rarely diagnosed, is increasingly understood to overlap with T cell-mediated rejection. Isolated liver allograft recipients are at increased risk of early allograft immunologic injury when preformed DSA are high titer and persist posttransplantation. Persons who undergo simultaneous liver-kidney transplantation are at risk of renal AMR when Class II DSA persist posttransplantation. Other under-appreciated DSA associations include ductopenia and fibrosis, plasma cell hepatitis, biliary strictures and accelerated fibrosis associated with recurrent liver disease. Standardized DSA testing and diagnostic criteria for both acute and chronic AMR are needed to distil existing associations into etiological processes in order to develop responsive therapeutic strategies.

Suppression of blastogenesis and proliferation of activated CD4(+) T cells: intravenous immunoglobulin (IVIg) versus novel anti-human leucocyte antigen (HLA)-E monoclonal antibodies mimicking HLA-I reactivity of IVIg.

Activated CD4(+) T cells undergo blastogenesis and proliferation and they express several surface receptors, including ß2-microglobulin-free human leucocyte antigen (HLA) heavy chains (open conformers). Intravenous immunoglobulin (IVIg) suppresses activated T cells, but the mechanism is unclear. IVIg reacts with HLA-Ia/Ib antigens but its reactivity is lost when the anti-HLA-E Ab is adsorbed out. Anti-HLA-E antibodies may bind to the peptides shared by HLA-E and the HLA-I alleles. These shared peptides are cryptic in intact HLA, but exposed in open conformers. The hypothesis that anti-HLA-E monoclonal antibodies (mAbs) that mimic HLA-I reactivity of IVIg may suppress activated T cells by binding to the shared peptides of the open conformers on the T cell surface was tested by examining the relative binding affinity of those mAbs for open conformers coated on regular beads and for intact HLA coated on iBeads, and by comparing the effects on the suppression of phytohaemagglutinin (PHA)-activated T cells of three entities: IVIg, anti-HLA-E mAbs that mimic IVIg [Terasaki Foundation Laboratory (TFL)-006 and (TFL)-007]; and anti-HLA-E antibodies that do not mimic IVIg (TFL-033 and TFL-037). Suppression of blastogenesis and proliferation of those T cells by both IVIg and the anti-HLA-E mAbs was dose-dependent, the dose required with mAbs 50-150-fold lower than with IVIg. TFL-006 and TFL-007 significantly suppressed blastogenesis and proliferation of activated CD4(+) T cells, but neither the non-IVIg-mimicking mAbs nor control antibodies did so. The suppression may be mediated by Fab-binding of TFL-006/TFL-007 to the exposed shared peptides. The mAb binding to the open conformer may signal T cell deactivation because the open conformers have an elongated cytoplasmic tail with phosphorylation sites (tryosine(320)/serine(335)).

Suppression of allo-human leucocyte antigen (HLA) antibodies secreted by B memory cells in vitro: intravenous immunoglobulin (IVIg)†versus a monoclonal anti-HLA-E IgG that mimics HLA-I reactivities of IVIg.

B memory cells remain in circulation and secrete alloantibodies without antigen exposure > 20 years after alloimmunization postpartum or by transplantation. These long-lived B cells are resistant to cytostatic drugs. Therapeutically, intravenous immunoglobulin (IVIg) is administered to reduce allo-human leucocyte antigen (HLA) antibodies pre- and post-transplantation, but the mechanism of reduction remains unclear. Recently, we reported that IVIg reacts with several HLA-I alleles and the HLA reactivity of IVIg is lost after its HLA-E reactivity is adsorbed out. Therefore, we have generated an anti-HLA-E monoclonal antibody that mimics the HLA-reactivity of IVIg to investigate whether this antibody suppresses IgG secretion, as does IVIg. B cells were purified from the blood of a woman in whose blood the B memory cells remained without antigen exposure > 20 years after postpartum alloimmunization. The B cells were stimulated with cytokines using a well-defined culture system. The anti-HLA-E monoclonal antibody (mAb) significantly suppressed the allo-HLA class-II IgG produced by the B cells, and that this suppression was far superior to that by IVIg. These findings were confirmed with HLA-I antibody secreted by the immortalized B cell line, developed from the blood of another alloimmunized woman. The binding affinity of the anti-HLA-E mAb for peptide sequences shared (i.e. shared epitopes) between HLA-E and other ß2-microglobulin-free HLA heavy chains (open conformers) on the cell surface of B cells may act as a ligand and signal suppression of IgG production of activated B memory cells. We propose that anti-HLA-E monoclonal antibody may also be useful to suppress allo-HLA IgG production in vivo.

De novo donor-specific HLA antibodies decrease patient and graft survival in liver transplant recipients.

The role of de novo donor-specific HLA antibodies (DSA) in liver transplantation remains unknown as most of the previous studies have only focused on preformed HLA antibodies. To understand the significance of de novo DSA, we designed a retrospective cohort study of 749 adult liver transplant recipients with pre- and posttransplant serum samples that were analyzed for DSA. We found that 8.1% of patients developed de novo DSA 1 year after transplant; almost all de novo DSAs were against HLA class II antigens, and the majority were against DQ antigens. In multivariable modeling, the use of cyclosporine (as opposed to tacrolimus) and low calcineurin inhibitor levels increased the risk of de novo DSA formation, while a calculated MELD score >15 at transplant and recipient age >60 years old reduced the risk. Multivariable analysis also demonstrated that patients with de novo DSA at 1-year had significantly lower patient and graft survival. In conclusion, we demonstrate that de novo DSA development after liver transplantation is an independent risk factor for patient death and graft loss.

Higher risk of kidney graft failure in the presence of anti-angiotensin II type-1 receptor antibodies.

Reports have associated non-HLA antibodies, specifically those against angiotensin II type-1 receptor (AT1R), with antibody-mediated kidney graft rejection. However, association of anti-AT1R with graft failure had not been demonstrated. We tested anti-AT1R and donor-specific HLA antibodies (DSA) in pre- and posttransplant sera from 351 consecutive kidney recipients: 134 with biopsy-proven rejection and/or lesions (abnormal biopsy group [ABG]) and 217 control group (CG) patients. The ABG's rate of anti-AT1R was significantly higher than the CG's (18% vs. 6%, p < 0.001). Moreover, 79% of ABG patients with anti-AT1R lost their grafts (vs. 0%, CG), anti-AT1R levels in 58% of those failed grafts increasing posttransplant. With anti-AT1R detectable before DSA, time to graft failure was 31 months-but 63 months with DSA detectable before anti-AT1R. Patients with both anti-AT1R and DSA had lower graft survival than those with DSA alone (log-rank p = 0.007). Multivariate analysis showed that de novo anti-AT1R was an independent predictor of graft failure in the ABG, alone (HR: 6.6), and in the entire population (HR: 5.4). In conclusion, this study found significant association of anti-AT1R with graft failure. Further study is needed to establish causality between anti-AT1R and graft failure and, thus, the importance of routine anti-AT1R monitoring and therapeutic targeting.

De novo donor HLA-specific antibodies after heart transplantation are an independent predictor of poor patient survival.

Preformed donor HLA-specific antibodies are a known indicator for poor patient survival after cardiac transplantation. The role of de novo donor-specific antibodies (DSA) formed after cardiac transplantation is less clear. Here we have retrospectively analyzed 243 cardiac transplant recipients, measuring HLA antibody production every year after transplantation up to 13 years post-transplant. Production of de novo DSA was analyzed in patients who had been negative for DSA prior to their transplant. DSA including transient antibodies were associated with poor patient survival (p = 0.0018, HR = 3.198). However, de novo and persistent DSA was strongly associated with poor patient survival (p = 0.0001 HR = 4.351). Although complement fixing persistent DSA correlated with poor patient survival, this was not increased compared to noncomplement fixing persistent DSA. Multivariable analysis indicated de novo persistent DSA to be an independent predictor of poor patient survival along with HLA-DR mismatch and donor age. Only increasing donor age was found to be an independent risk factor for earlier development of CAV. In conclusion, patients who are transplanted in the absence of pre-existing DSA make de novo DSA after transplantation which are associated with poor survival. Early and regular monitoring of post-transplant DSA is required to identify patients at risk of allograft failure.

High mean fluorescence intensity donor-specific anti-HLA antibodies associated with chronic rejection Postliver transplant.

In contrast to kidney transplantation where donor-specific anti-HLA antibodies (DSA) negatively impact graft survival, correlation of DSA with clinical outcomes in patients after orthotopic liver transplantation (OLT) has not been clearly established. We hypothesized that DSA are present in patients who develop chronic rejection after OLT. Prospectively collected serial serum samples on 39 primary OLT patients with biopsy-proven chronic rejection and 39 comparator patients were blinded and analyzed for DSA using LABScreen(®) single antigen beads test, where a 1000 mean fluorescence value was considered positive. In study patients, the median graft survival was 15 months, 74% received ≥ one retransplant, 20% remain alive and 87% had ≥ one episode of acute rejection. This is in contrast to comparator patients where 69% remain alive, and no patient needed retransplant or experienced rejection. Thirty-six chronic rejection patients (92%) and 24 (61%) comparator patients had DSA (p = 0.003). Chronic rejection versus comparator patients had higher mean fluorescence intensity (MFI) DSA. Although a further study with larger numbers of patients is needed to identify clinically significant thresholds, there is an association of high-MFI DSA with chronic rejection after OLT.

B-lymphocyte alloantigens in Caucasians.

Human B lymphocytes have been shown to have at least five polymorphic specificities defined by 32 antisera. The antisera were produced by absorption with pooled platelets to remove HLA activity and were selected out of over 400 tested sera. The sera that defined the five specificities had high correlation coefficients within a group (generally in the range of 0.6-0.9). As shown by the fit in the Hardy-Weinberg equilibrium, the five specificities appear to be determined by alleles at one genetic locus. No association between these specificities and HLA was noted.

Autologous stimulation of human lymphocyte subpopulation.

The background stimulation universally seen when lymphocytes are cultured in vitro has been shown to be markedly lowered by reducing the proportion of B lymphocytes. B-rich fractions of lymphocytes had extremely high background stimulation. It is concluded that stimulation of T cells, probably by autologous B cells, provides the most probable explanation for the findings described.

Lymphocyte antigenicity loss with retention of responsiveness.

During culturing at 22 degrees C for more than 4 days, human peripheral blood lymphocytes lose their ability to stimulate allogeneic lymphocytes in mixed lymphocyte cultures. The cells retain their ability to respond to allogeneic lymphocytes or phytohemagglutinin for up to 10 days of culturing. The findings are relevant to reports on successful transplantation of cultured skin.

Cross-reactions between streptococcal M proteins and human transplantation antigens.

Allogeneic antiserums against human lymphocytes were specifically inhibited by M protein from beta hemolytic group A Streptococcus pyogenes, type 1. Analogous M proteins from streptococcus types 3, 4, 5, 6, 12, and 14 had little or no inhibitory activity. The specific inhibition by M protein is not a result of anticomplementary activity or of coating of the lymphocyte surface. Streptococcal polysaccharide and 73 other polysaccharides were inactive. Because all seven HL-A specificities tested were inhibited, it is inferred that M1 protein has a structure common to human histocompatibility antigens.

Prolonged survival of second human kidney transplants.

Rejection of kidney transplants in 264 patients, followed by retransplantation from cadaver donors, resulted in a 1-year survival rate of 51 +/- 3 percent (rate +/- standard error) as compared to 51 +/- 1 percent for first transplants. If the first transplant immunizes the patient or is rejected by immunologically responsive patients, second grafts into the same patients would be expected to be rejected at a higher rate. Only those reject who reject first grafts hyperacutely or between 1 to 3 months were found to have low second graft survival rates. Patients who rejected transplants after 3 months tended to have second transplant survival rates which were higher than their first graft survival rates.

Multiple sclerosis and high incidence of a B lymphocyte antigen.

Multiple sclerosis patients were tested for six new antigens present on human B lymphocytes. The group 4 specificity occurred in 83.9% of the 56 patients as compared to 32.5 percent in 72 healthy controls (P less than .003). The antiserums defining the five other B lymphocyte specificities reacted at a lower frequency to B cells from multiple sclerosis patients, showing that increased reactivity to group 4 antiserum was specific. Linkage of a hypothesized multiple sclerosis susceptibility gene with certain haplotypes of HLA-A3, HLA-B7 HLA-DW2, and the new B group 4 can be inferred.

Utilization of cultured human lymphoid cells for detection of humoral sensitization in prospective recipients of kidney transplants.

Prospective recipients of kidney transplants were tested for lymphocytotoxicity; from these we selected 102 sera that lacked cytotoxic antibodies against peripheral lymphocytes from at least 80 unrelated subjects. To detect humoral sensitization, we then reacted these with 17 cultured human lymphoid cell lines having different HL-A phenotypes. Cytotoxic antibodies reacting with these cultured cells were now detected in some of the sera. These antibodies were not directed against HL-A antigens, yet mediated lysis of target cells in the presence of rabbit but not of human or guinea pig complement. Furthermore, they activated the classical pathway of the rabbit complement system. Later, a significant association was found between occurrence of cytotoxic antibodies and rejection of the transplant. Thus, cultured human lymphoid cells, because of their great susceptibility to complement-mediated lysis, appear to be useful in detecting humoral sensitization in candidates for kidney grafts.