Therapeutic Effects and Functional Mechanism of Intravenous Immunoglobulin in Preclinical Rat Renal Transplant Model of Antibody-Mediated Rejection.

BACKGROUND: Intravenous immunoglobulin (Ig) therapy is used based on empirical findings for treatment of antibody-mediated rejection (AMR) in cases of renal transplantation, although its therapeutic efficacy has not been proven and the functional mechanism of an administered Ig remains elusive. In this study, the therapeutic effects of an Ig were examined in a preclinical rat renal transplant model of AMR to investigate this mechanism. METHODS: To establish an AMR renal graft model, skin graft specimens were obtained from Brown-Norway (BN) rats and transplanted to Lewis rats to produce donor-specific antibodies (DSAs), after which kidney transplantation from the Brown-Norway to Lewis rats was performed. AMR model rats were administered the Ig at a dose of 2 g/kg or saline as a control. Survival period, renal graft histopathology, complement factors, and DSA levels were assessed. RESULTS: The survival period of the group administered the Ig was significantly prolonged. Histopathological examinations of renal grafts also showed significant suppression of glomerulitis and peritubular capillaritis in the Ig group, and real-time polymerase chain reaction analysis results demonstrated significantly lower levels of expression of the complement factors C1q and C3. When the Ig was given to rats that underwent skin grafting but not renal transplantation, DSA was decreased after 6 hours and remained lower than the baseline level for at least 7 days. CONCLUSION: Ig administration suppressed DSA production. The present results showed that suppression of the complement system contributed to effective Ig treatment for AMR.

Single fixed low-dose rituximab as induction therapy suppresses de novo donor-specific anti-HLA antibody production in ABO compatible living kidney transplant recipients.

This study was conducted to evaluate de novo donor-specific anti-human leukocyte antigen (HLA) antibody (dnDSA) production leading to antibody-mediated rejection (ABMR) after rituximab induction in non-sensitized ABO-compatible living kidney transplantation (ABO-CLKTx). During 2008-2015, 318 ABO-CLKTx were performed at the Department of Surgery III at Tokyo Women's Medical University Hospital. To reduce confounding factors, we adopted a propensity score analysis, which was applied with adjustment for age, gender, duration of pretransplant dialysis, HLA mismatch count, preformed DSA, non-insulin-dependent diabetes mellitus, immunosuppressive treatment, and estimated glomerular filtration rate (eGFR) on postoperative day 7. Using a propensity score matching model (1:1, 115 pairs), we analyzed the long-term outcomes of 230 ABO-CLKTx recipients retrospectively. Recipients were classified into a rituximab-treated (RTX-KTx, N = 115) group and a control group not treated with rituximab (C-KTx, N = 115). During five years, adverse events, survival rates for grafts and patients, and incidence of biopsy-proven acute rejection (BPAR) and dnDSA production for the two groups were monitored and compared. All recipients in the RTX-KTx group received rituximab induction on preoperative day 4 at a single fixed low dose of 100 mg; the CD19+ B cells were eliminated completely before surgery. Of those recipients, 13 (11.3%) developed BPAR; 1 (0.8%) experienced graft loss. By contrast, of C-KTx group recipients, 25 (21.7%) developed BPAR; 3 (2.6%) experienced graft loss. The RTX-KTx group exhibited a significantly lower incidence of BPAR (P = .041) and dnDSA production (13.9% in the RTX-KTx group vs. 26.9% in the C-RTx group, P = .005). Furthermore, lower incidence of CMV infection was detected in the RTX-KTx group than in the C-KTx group (13.9% in the RTX-KTx group vs. 27.0% in the C-KTx group, P = .014). No significant difference was found between groups for several other factors: renal function (P = .384), graft and patient survival (P = .458 and P = .119, respectively), and the respective incidences of BK virus infection (P = .722) and leukopenia (P = .207). During five-year follow-up, single fixed low-dose rituximab therapy is sufficient for ensuring safety, reducing rejection, and suppressing dnDSA production for immunological low-risk non-sensitized ABO-CLKTx.

Clearance of Intra-graft Donor Specific Anti-HLA Antibodies in the Early Stage of Antibody-Mediated Rejection Following Rituximab and Apheresis Therapy in Renal Transplantation.

BACKGROUND: The management of acute or, in particular, chronic antibody-mediated rejection (AMR) resulting from donor-specific HLA antibodies (DSA) is a critical barrier to obtaining better long-term graft survival. To ascertain the efficacy of anti-AMR therapies, the transition of intra-graft DSA (g-DSA) was assessed. METHODS: Allograft biopsy specimens were analyzed by graft immunocomplex capture fluorescence analysis, as previously described. One hundred recipients who underwent graft biopsies between April 2016 and December 2017 were enrolled for this study. Fifteen recipients diagnosed with g-DSA positive (+) received anti-humoral treatments and underwent follow-up biopsies. g-DSA levels were assessed again by a follow-up biopsy at 6-12 months following the treatments. RESULTS: With anti-humoral treatments, 9 out of 15 recipients comprised a g-DSA negative (-) (3.59 ± 2.82-.58 ± .25): g-DSA6-12- group, while the remaining 6 recipients comprised a g-DSA +(20.6 ± 17.0-14.9 ± 14.1): g-DSA6-12+ group. The initial g-DSA scores were significantly higher in the g-DSA6-12+ group (P = .01). All samples were diagnosed as chronic AMR in the g-DSA+ groups, whereas there were 3 chronic AMR, 4 acute AMR, and 2 incomplete AMR samples in the g-DSA- group. Interestingly, the frequency of responsible DSA belonging to class II tended to be higher in the g-DSA6-12+ group (4/6) compared to the g-DSA6-12- group (2/9) (P = .14). CONCLUSION: These results imply that chronic exposure to DSA causes significant and irreversible damage to the allograft. Timely and adequate anti-humoral intervention might reverse the early phase of AMR with complete clearance of g-DSA.

Everolimus-based Immunosuppression Possibly Suppresses Mean Fluorescence Intensity Values of De Novo Donor-specific Antibodies After Primary Kidney Transplantation.

PURPOSE: We evaluated de novo donor-specific antibody (DSA) production of everolimus (EVR)-based immunosuppression for primary kidney transplant recipients involved in the A1202 study at our institute. METHODS: From March 2008 to August 2009, 24 recipients were prospectively randomized into 2 groups. The EVR group received reduced cyclosporin A and EVR. The standard protocol (STD) group received standard cyclosporin A and mycophenolate mofetil. Both groups received basiliximab and steroids. De novo DSA was identified using LABScreen single antigen beads (One Lambda, Canoga Park, Calif., United States). Mean fluorescence intensity (MFI) values > 1000 were considered positive. P < .05 was considered significant. RESULTS: Graft survival was 100% in the EVR group and 90.9% in the STD group. All patients remained on the primary protocol in the EVR group, but 3 patients in the STD group (27.3%) were converted to tacrolimus due to DSA and non-adherence. Estimated glomerular filtration rate was similar in both groups. No EVR group recipients and 9.1% of STD group recipients were treated for T-cell-mediated rejection. No recipients of the EVR group exhibited peritubular capillaritis, while 9.1% in STD group developed chronic active antibody-mediated rejection. LABScreen revealed an accumulative class II DSA production rate of 15.4% in the EVR group and 18.3% in the STD group at 10 years. When the MFI cut-off level was set to 6000, anti-HLA antibody and de novo DSA-free survival was significantly better in the EVR group. CONCLUSIONS: EVR-based immunosuppression provided equivalent or even better clinical outcomes. EVR suppressed de novo DSA production at 10 years follow-up; however, further follow-up is inevitable.

Response to treatment and long-term outcomes in kidney transplant recipients with acute T cell-mediated rejection.

The recent recognition of complex and chronic phenotypes of T cell-mediated rejection (TCMR) has fostered the need to better evaluate the response of acute TCMR-a condition previously considered to lack relevant consequences for allograft survival-to the standard of care. In a prospective cohort of kidney recipients (n = 256) with biopsy-proven acute TCMR receiving corticosteroids, we investigated clinical, histological, and immunological phenotypes at the time of acute TCMR diagnosis and 3 months posttreatment. Independent posttreatment determinants of allograft loss included the glomerular filtration rate (GFR) (HR = 0.94; 95% CI = 0.92-0.96; P < .001), proteinuria (HR = 1.40; 95% CI = 1.10-1.79; P = .007), time since transplantation (HR = 1.02; 95% CI = 1.00-1.03; P = .016), peritubular capillaritis (HR = 2.27; 95% CI = 1.13-4.55; P = .022), interstitial inflammation in sclerotic cortical parenchyma (i-IF/TA) (HR = 1.87; 95% CI = 1.08-3.25; P = .025), and donor-specific anti-HLA antibodies (DSAs) (HR = 2.67; 95% CI = 1.46-4.88; P = .001). Prognostic value was improved using a composite evaluation of response to treatment versus clinical parameters only (cNRI = 0.68; 95% CI = 0.41-0.95; P < .001). A classification tree for allograft loss identified five patterns of response to treatment based on the posttreatment GFR, i-IF/TA, and anti-HLA DSAs (cross-validated accuracy = 0.80). Compared with responders (n = 155, 60.5%), nonresponders (n = 101, 39.5%) had a higher incidence of de novo DSAs, antibody-mediated rejection, and allograft loss at 10 years (P < .001 for all comparisons). Thus, clinical, histological, and immunological assessment of response to treatment of acute TCMR revealed different profiles of the response to treatment with distinct outcomes.

Outcomes of Prolonged Treatment With Intravenous Immunoglobulin Infusions for Acute Antibody-mediated Rejection in Kidney Transplant Recipients.

BACKGROUND: Treatment of antibody-mediated rejection (AMR) is one of the main problems after kidney transplantation (KTx). The results of intensive AMR treatment with plasmapheresis (PF) and repeated infusions of intravenous immunoglobulin (IVIg) are presented. METHODS: Diagnosis of AMR was based on graft biopsy and the presence of donor-specific antibodies (DSAs). AMR therapy consisted of 5 PF and IVIg infusions given after the last PF. Subsequent IVIg doses were given every 4 weeks for 6 months. Graft biopsy and DSA assessment were repeated at the end of the treatment (ET). RESULTS: Four women and 10 men were included in our study; mean time from KTx to AMR was 79 (range, 3-193) months. During the treatment, 4 patients had graft failure. Graft function at baseline was significantly worse (P = .02) in this group compared with patients who completed the therapy. At baseline, mean flourescence intensity (MFI) was 6574 (range, 852-15,917) in the whole group, 7088 (range, 1054-15,917) in patients who completed treatment, and 4828 (range, 852-11,797) in patients who restarted hemodialysis. At ET, DSA MFI decreased in 8 of 10 patients (80%) who completed the therapy. The MFI decrease was 3946 (range, 959-11,203). Control graft biopsies revealed decreased intensity of C4d deposits in peritubular capillaries in 7 patients (78%) and decreased peritubular capillaritis in 2 patients (22%). CONCLUSION: Intensive, prolonged AMR therapy with PF and IVIg resulted in a decrease in DSA titer and intensity of C4d deposits, but was not associated with reduction of microcirculation inflammation. Treatment was ineffective in patients with baseline advanced graft insufficiency.

Extending shelf life of dithiothreitol-treated panel RBCs to 28 days.

BACKGROUND: Daratumumab (DARA) causes non-specific results in indirect agglutination testing (IAT). Dithiothreitol (DTT) treatment of panel red blood cells (RBCs) abolishes DARA interference. The objective of our study was to extend stability of DTT-treated panel RBCs to 28 days through application of a commercially available panel RBC stabilizer. MATERIALS AND METHODS: Serological antigen typing and IAT using DARA sera and DARA plasma spiked with weakly reacting alloantibodies was performed up to 28 days after DTT treatment and stabilization. RESULTS: DTT treatment resulted in loss of Fy-antigen expression on some panel RBCs. Antigen profiles of stabilized, DTT-treated panel RBCs remained stable. Alloantibodies in DARA sera and DARA plasma were reliably detected. CONCLUSIONS: Application of a commercially available RBC stabilizer extends shelf life of DTT-treated panel RBCs to 28 days.

Triptolide inhibits donor-specific antibody production and attenuates mixed antibody-mediated renal allograft injury.

Donor-specific antibodies (DSAs) are major mediators of renal allograft injury, and strategies to inhibit DSAs are important in promoting long-term graft survival. Triptolide exhibits a wide spectrum of antiinflammatory and immunosuppressive activities, and in autoimmune diseases it inhibits autoantibody levels. In this study, we investigated the suppressive role of triptolide in the generation of DSAs in transplant recipients. We found that triptolide treatment of skin allograft recipients in mice significantly suppressed the development of circulating anti-donor-specific IgG and effectively alleviated DSA-mediated renal allograft injury, which led to prolonged allograft survival. In vitro studies revealed that triptolide inhibited the differentiation of B cells into CD138+ CD27++ plasma cells; reduced the levels of IgA, IgG, and IgM secreted by plasma cells; and repressed somatic hypermutation and class switch recombination of B cells. Moreover, triptolide-treated recipients showed reduced numbers of B cells, plasma cells, and memory B cells in spleens and decreased numbers of T, B, natural killer (NK) cells, and macrophages infiltrating grafts. These findings highlight the importance of triptolide in suppressing DSAs and establish triptolide as a novel therapeutic agent for antibody-mediated allograft rejection.

Belatacept Combined With Transient Calcineurin Inhibitor Therapy Prevents Rejection and Promotes Improved Long-Term Renal Allograft Function.

Belatacept, a T cell costimulation blocker, demonstrated superior renal function, lower cardiovascular risk, and improved graft and patient survival in renal transplant recipients. Despite the potential benefits, adoption of belatacept has been limited in part due to concerns regarding higher rates and grades of acute rejection in clinical trials. Since July 2011, we have utilized belatacept-based immunosuppression regimens in clinical practice. In this retrospective analysis of 745 patients undergoing renal transplantation at our center, we compared patients treated with belatacept (n = 535) with a historical cohort receiving a tacrolimus-based protocol (n = 205). Patient and graft survival were equivalent for all groups. An increased rate of acute rejection was observed in an initial cohort treated with a protocol similar to the low-intensity regimen from the BENEFIT trial versus the historical tacrolimus group (50.5% vs. 20.5%). The addition of a transient course of tacrolimus reduced rejection rates to acceptable levels (16%). Treatment with belatacept was associated with superior estimated GFR (belatacept 63.8 mL/min vs. tacrolimus 46.2 mL/min at 4 years, p < 0.0001). There were no differences in serious infections including rates of cytomegalovirus or BK viremia. We describe the development of a costimulatory blockade-based strategy that ultimately allows renal transplant recipients to achieve calcineurin inhibitor-free immunosuppression.

A systematic review of the use of rituximab for the treatment of antibody-mediated renal transplant rejection.

Rituximab is a B-lymphocyte depleting agent that is used to treat hematological malignancies and autoimmune diseases. Recently, it has gained interest as an immunomodulatory agent in renal transplantation. This systematic review evaluates the evidence for its use in the treatment of acute and chronic antibody-mediated renal transplant rejection (AAMR; CAMR). A systematic search of four databases and three trial registries was conducted. The small number and heterogeneous nature of included studies precluded meta-analysis and thus a narrative review was conducted. A total of 28 records met the inclusion criteria (AAMR, 18 records relating to 9 studies; CAMR, 10 records relating to 7 studies). Two systematic reviews were identified that had differing inclusion criteria to this current review. Of seven primary studies in the setting of AAMR, four reported increased graft survival and one reported improved graft function with rituximab. This contrasts with CAMR in which only one of seven studies reported improved graft outcomes with a rituximab-based regimen; three studies reported inferior outcomes and three reported no difference. Only one study reported that rituximab was associated with an increase in adverse effects. The included studies suggest that rituximab may be of some benefit in the setting of AAMR but a lack of high quality evidence precludes firm conclusions from being drawn. Rituximab does not appear to reliably improve outcomes in CAMR. Further well-conducted studies are required to better define the effects and long-term safety profile of rituximab in the treatment of antibody-mediated renal transplant rejection.

Bortezomib-based treatment of antibody mediated rejection in pancreas allograft recipients.

This report presents the first experience with plasma cell-targeted therapy in treating antibody mediated rejection in pancreas transplant recipients. In this experience, bortezomib provided results similar to those previously reported in kidney transplant recipients, with the exception that DSA responses were not quite as dramatic in pancreas transplant recipients. However, even in patients with antibody mediated rejection refractory to standard therapies, significant responses were obtained with the proteasome inhibitor, bortezomib. These results confirm the potential for bortezomib-based therapies in pancreas transplant recipients, and also demonstrate that rejection following pancreas transplantation may require innovative approaches to provide optimal results.

Use of bortezomib for prevention and treatment of rejection in sensitized patients.

We report 2 cases of sensitized patients who were successfully treated with bortezomib therapy resulting in reduction of donor-specific antibodies (DSA). Our cases illustrate the synergistic effects of combination therapy that includes bortezomib on prevention and treatment of AMR in highly sensitized patients. Moving forward, long-term data on sensitized patients treated with bortezomib are needed to fully evaluate the impact of this therapy.

Desensitization protocol using bortezomib for highly sensitized patients awaiting heart or lung transplants.

The use of bortezomib in combination with other desensitization therapies like plasmapheresis, IVIG, and rituximab has allowed the decrease of antibody levels during treatment in some patients. However, all patients described here have experienced rebound effects to the same or higher levels than the ones before therapy was started. Unfortunately, no donors became available to some of these patients when their antibodies were at lower levels. Three out of the four patients presented had adverse reactions to bortezomib which include nausea, vomiting, diarrhea, myalgias and severe neuropathy. In one patient (pre-heart transplant patient #1) we noticed that some clones were selectively more susceptible to the treatment with bortezomib than others. We will continue antibody monitoring in this patient and hopefully the possibility is there, even though the overall PRA is not reduced, that certain clones will be affected and no longer produced antibody allowing these patients a wider selection of acceptable donors.

The effect of antithymocyte globulin on anti-human leukocyte antigen antibody detection assays.

BACKGROUND: We evaluated the effect of antithymocyte globulin (ATG) on anti-human leukocyte antigen (HLA) antibody assays. METHODS: We tested sera from six in vivo ATG-treated kidney transplant patients after measuring serum concentrations, as well as six nonsensitized sera with ATG added in vitro. T- and B-cell complement-dependent cytotoxicity (CDC), flow cytometric (FXM), and solid-phase HLA class I and II assays based on antigen-coated microspheres and enzyme-linked immunosorbent assay (ELISA) were studied. Sera were then retested after treatment to remove ATG. RESULTS: We found that ATG affects test results differently depending on whether sera is obtained from in vivo treated patients or added in vitro. In vitro treated sera produced ATG concentration-dependent positive results for T/B CDC, FXM, and flow bead testing for HLA I/II, while the ELISA-based assay was unaffected. In vivo treated sera from ATG-treated patients produced positive test results for T CDC and T/B FXM, while the B-cell CDC crossmatch remained negative. Solid phase assays were minimally affected using in vivo treated sera. After ATG extraction, all tests became negative. CONCLUSION: We conclude that ATG produces positive results in anti-HLA antibody testing, and treatment to remove ATG abolishes this effect. This treatment allows ATG-treated patients to be monitored for anti-HLA antibodies.

Management of sensitized patients awaiting renal transplantation: does sequential therapy of intravenous immunoglobulin and simvastatin offer a solution?

The value of intravenous immunoglobulin and simvastatin as potential modalities for the treatment of sensitized patients was studied. We aimed to test their efficacy as solo agents to inhibit anti-human leukocyte antigen (HLA) antibodies. We tested samples from 11 adult hemodialysis patients who were waiting for renal allotransplantation at our center, all of whom had persistently positive crossmatches with their living related donors and panel reactive antibody titers more than 20%. All patients received intravenous immunoglobulin (500 mg/kg/day on alternate days for 6 doses). Panel reactive antibody titer measurement and crossmatch testing were carried out after each dose and before each subsequent one. Two months later, 8 patients received simvastatin (20 mg/day) for 2 months. Panel reactive antibody measurement titer and crossmatch testing were carried out every 2 weeks. Only 4 patients showed an insignificant reduction in panel reactive antibody activity (P=0.36). None of them attained a negative crossmatch. Furthermore, simvastatin also resulted in an insignificant reduction of HLA antibodies in 3 patients (P=0.32). We concluded that intravenous immunoglobulin or simvastatin alone cannot effectively inhibit preformed anti-HLA antibodies to allow successful renal transplantation. Further trials of the use of intravenous immunoglobulin and simvastatin with other modalities to desensitize these patients may be warranted.