Hugo Kaneku

Abrogation of anti-HLA antibodies via proteasome inhibition.

Background. Current treatments for autoantibody-mediated diseases (i.e., systemic lupus erythematosus) and alloantibodies (in transplant) are minimally effective. Although they deplete naïve B cells, plasmablasts, and transiently reduce antibody concentrations, they are minimally effective against long-lived, antibody-producing plasma cells. In transplantation, plasma cells produce antibodies directed against human leukocyte antigen (HLA) antigens causing poor allograft survival. We report the first clinical experience with a plasma cell depleting therapy, bortezomib, to abrogate anti-HLA antibodies in transplantation (outside of rejection) in an attempt to improve long-term allograft survival. Methods. Eleven patients with anti-HLA alloantibodies were treated with bortezomib. All patients underwent plasmapheresis to aid in removal of antibodies and to determine the effect of bortezomib. Serial measurements of anti-HLA antibody levels were conducted weekly by single antigen bead on Luminex platform. Results. Bortezomib treatment elicited substantial reduction in both donor-specific antibody (DSA) and non-DSA levels. Antibodies were directed against DSA in 8 of 11 cases. Mean time to antibody appearance was 2 months posttransplant. Within 22 days (median) from treatment initiation, 9 of 11 patients’ antibody levels dropped to less than 1000 mean fluorescence intensity. Of two patients without successful depletion, all had peak mean fluorescence intensity more than 10,000. At a mean follow-up of approximately 4 months posttreatment, all patients have stable graft function. Minimal transient side effects were noticed with bortezomib in the form of gastrointestinal toxicity, thrombocytopenia, and paresthesias. Conclusions. Bortezomib therapy effectively abrogates anti-HLA antibodies. Hence, removal of antibodies, by proteasome inhibition, represents a new treatment strategy for transplantation and may have benefit in autoimmune-related disease.

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.

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.

Donor‐specific human leukocyte antigen antibodies of the immunoglobulin G3 subclass are associated with Chronic rejection and graft loss after liver transplantation

In a previous study, we found that 92% of patients with chronic rejection had donor‐specific human leukocyte antigen antibodies (DSAs), but surprisingly, 61% of comparator patients without rejection also had DSAs. We hypothesized that immunoglobulin G (IgG) subclasses were differentially distributed between the 2 groups. A modified single‐antigen bead assay was used to detect the presence of individual IgG subclasses against human leukocyte antigen in 39 chronic rejection patients and 66 comparator patients. DSAs of the IgG1 subclass were most common and were found in 45% of all patients; they were followed by IgG3 DSAs (21%), IgG4 DSAs (14%), and IgG2 DSAs (13%). The percentage of patients with multiple IgG subclasses was significantly higher in the chronic rejection group versus the comparator group (50% versus 14%, P < 0.001). Patients with normal graft function in the presence of DSAs mostly had isolated IgG1, whereas patients with chronic rejection had a combination of IgG subclasses. Patients who developed DSAs of the IgG3 subclass showed an increased risk of graft loss (hazard ratio = 3.35, 95% confidence interval = 1.39‐8.05) in comparison with patients with DSAs of other IgG subclasses or without DSAs. Although further study is needed, the determination of the IgG subclass in DSA‐positive patients may help us to identify patients with a higher risk of chronic rejection and graft loss. Liver Transpl, 2012.

Preformed class II donor-specific antibodies are associated with an increased risk of early rejection after liver transplantation

Preformed donor‐specific human leukocyte antigen antibodies (DSAs) are considered a contraindication to the transplantation of most solid organs other than the liver. Conflicting data currently exist on the importance of preformed DSAs in rejection and patient survival after liver transplantation (LT). To evaluate preformed DSAs in LT, we retrospectively analyzed prospectively collected samples from all adult recipients of primary LT without another organ from January 1, 2000 to May 31, 2009 with a pre‐LT sample available (95.8% of the patients). Fourteen percent of the patients had preformed class I and/or II DSAs with a mean fluorescence intensity (MFI) ≥ 5000. Preformed class I DSAs with an MFI ≥ 5000 remained persistent in only 5% of patients and were not associated with rejection. Preformed class II DSAs with an MFI of 5000 to 10,000 remained persistent in 23% of patients, and this rate increased to 33% for patients whose MFI was ≥10,000 (P < 0.001). Preformed class II DSAs in multivariable Cox proportional hazards modeling were associated with an increased risk of early rejection [hazard ratio (HR) = 1.58; p = 0.004]. In addition, multivariate modeling showed that in comparison with no DSAs (MFI < 1000), preformed class I and/or II DSAs with an MFI ≥ 5000 were independently correlated with the risk of death (HR = 1.51; p = 0.02). Liver Transpl 19:973–980, 2013.

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 testings 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.

Antibody‐mediated rejection as a contributor to previously unexplained early liver allograft loss

We analyzed 60 patients with idiopathic early allograft loss (defined as death or retransplantation at <90 days) to determine the relative contribution of preformed donor‐specific human leukocyte antigen alloantibodies (DSAs) to this endpoint, and we defined strict criteria for the diagnosis of antibody‐mediated rejection (AMR) in liver allografts. The inclusion criteria encompassed the availability of a pretransplant serum sample and both postreperfusion and follow‐up tissue specimens for a blinded, retrospective re‐review of histology and complement component 4d (C4d) staining. AMR was diagnosed on the basis of the presence of all 4 of the following strict criteria: (1) DSAs in serum, (2) histopathological evidence of diffuse microvascular injury/microvasculitis consistent with antibody‐mediated injury, (3) diffuse C4d staining in the portal microvasculature with or without staining in the sinusoids or central veins in at least 1 sample, and (4) the exclusion of other causes of a similar type of injury. Patients thought to be experiencing definite AMR on the basis of routine histopathology alone showed the highest levels of DSA sensitization. Forty percent of patients with pretransplant DSAs with a pattern of bead saturation after serial dilutions developed AMR. Another multiparous female developed what appeared to be a strong recall response, which resulted in combined AMR and acute cellular rejection (ACR) causing graft failure. A contribution of DSAs to allograft failure could not be excluded for 3 additional patients who received marginal grafts. In conclusion, liver allograft recipients with preformed DSAs with a high mean fluorescence intensity despite dilution seem to be at risk for clinically significant allograft injury and possibly for loss from AMR, often in combination with ACR. Liver Transpl 20:218‐227, 2014.

Antibodies to HLA-E in Nonalloimmunized Males: Pattern of HLA-Ia Reactivity of Anti–HLA-E–Positive Sera

Natural anti-HLA Abs found in sera of healthy nonalloimmunized males recognize HLA-Ia alleles parallel to those recognized by anti–HLA-E mAbs (MEM-E/02/06/07). Therefore, some of the HLA-Ia Abs seen in healthy males could be due to anti–HLA-E Abs cross-reacting with HLA-Ia. If anti–HLA-E Abs occur in healthy nonalloimmunized males, it can be assessed whether they evoke HLA-Ia reactivity as do mouse HLA-E mAbs. IgG and IgM Abs to HLA-E and HLA-Ia alleles are identified in sera of healthy males using microbeads coated with recombinant denatured HLA-E or a panel of rHLA-Ia alleles. The pattern of allelic recognition is comparable to that of anti–HLA-E mAbs. Sixty-six percent of the sera with HLA-E IgG have a high level of HLA-Ia IgG, whereas 70% of those with no anti–HLA-E Abs have no HLA-Ia Abs. HLA-E IgM/IgG ratios of sera are divided into four groups: IgMLow/IgGLow, IgMHigh/IgGLow, IgMHigh/IgGHigh, and IgMLow/IgGHigh. These groups correspond to anti–HLA-Ia IgM/IgG ratio groups. When HLA-E IgM and IgG are absent or present in males, the IgM or IgG of HLA-Ia are similarly absent or present. The mean fluorescent intensity of HLA-Ia Abs correlates with that of anti–HLA-E Abs. Most importantly, HLA-E and HLA-Ia reactivities of the sera are inhibited by the shared, but cryptic, peptide sequences 117AYDGKDY123 and 137DTAAQIS143. Therefore, Abs to the H chain of HLA-E may be responsible for some of the HLA-Ia allele reactivity of the natural HLA-Ia Ab in human sera. Absence of any anti–HLA-Ia Abs in 112 nonvegans and the presence of the same in vegans suggest that dietary meat proteins might not have induced the natural allo-HLA Abs.

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.

Donor‐specific alloantibodies are associated with fibrosis progression after liver transplantation in hepatitis C virus–infected patients

Hepatitis C virus (HCV) fibrosis progression after liver transplantation (LT) is accelerated in comparison with fibrosis progression before transplantation. The vast majority of the risk factors for fibrosis progression after LT are not modifiable. With the goal of identifying modifiable risk factors for fibrosis progression, we evaluated the impact of preformed and de novo donor‐specific human leukocyte antigen alloantibodies (DSAs) on fibrosis progression after LT in HCV‐viremic patients. After blinding, we analyzed all 507 HCV‐viremic patients who underwent primary LT from January 2000 to May 2009 and had pretransplant and posttransplant samples available for analysis (86% of the total) for preformed and de novo class I and class II DSAs with a mean fluorescence intensity ≥ 5000 with single‐antigen bead technology. Fibrosis was assessed on the basis of indication and protocol liver biopsies; compliance with protocol liver biopsies at 1, 2, and 5 years was ≥80%. Preformed class I DSAs [hazard ratio (HR) = 1.44, P = 0.04] and class II DSAs (HR = 1.86, P < 0.001) were independent predictors of progression to stage 2‐4 fibrosis, and de novo DSAs (HR = 1.41, P = 0.07) had borderline significance. In addition, preformed class I DSAs (HR = 1.63, P = 0.03) and class II DSAs (HR = 1.72, P = 0.03) were statistically significantly associated with an increased risk of death. In conclusion, after we controlled for donor and recipient characteristics in multivariate modeling, DSAs were independently associated with fibrosis progression and death after LT in HCV‐viremic patients. Liver Transpl 20:655‐663, 2014.