Sunil Daga

Human leukocyte antigen antibody-incompatible renal transplantation: excellent medium-term outcomes with negative cytotoxic crossmatch.

Background. Human leukocyte antigen (HLA) antibody-incompatible renal transplantation has been increasingly performed since 2000 but with few data on the medium-term outcomes. Methods. Between 2003 and 2011, 84 patients received renal transplants with a pretreatment donor-specific antibody (DSA) level of more than 500 in a microbead assay. Seventeen patients had positive complement-dependent cytotoxic (CDC) crossmatch (XM), 44 had negative CDC XM and positive flow cytometric XM, and 23 had DSA detectable by microbead only. We also reviewed 28 patients with HLA antibodies but no DSA at transplant. DSAs were removed with plasmapheresis pretransplant, and patients did not routinely receive antithymocyte globulin posttransplant. Results. Mean follow-up posttransplantation was 39.6 (range 2–91) months. Patient survival after the first year was 93.8%. Death-censored graft survival at 1, 3, and 5 years was 97.5%, 94.2%, and 80.4%, respectively, in all DSA+ve patients, worse at 5 years in the CDC+ve than in the CDC−ve/DSA+ve group at 45.6% and 88.6%, respectively (P<0.03). Five-year graft survival in the DSA−ve group was 82.1%. Rejection occurred in 53.1% of DSA+ve patients in the first year compared with 22% in the DSA−ve patients (P<0.003). Conclusions. HLA antibody-incompatible renal transplantation had a high success rate if the CDC XM was negative. Further work is required to predict which CDC+ve XM grafts will be successful and to treat slowly progressive graft damage because of DSA in the first few years after transplantation.

Subclass analysis of donor HLA-specific IgG in antibody-incompatible renal transplantation reveals a significant association of IgG4 with rejection and graft failure.

Donor HLA‐specific antibodies (DSAs) can cause rejection and graft loss after renal transplantation, but their levels measured by the current assays are not fully predictive of outcomes. We investigated whether IgG subclasses of DSA were associated with early rejection and graft failure. DSA levels were determined pretreatment, at the day of peak pan‐IgG level and at 30 days post‐transplantation in eighty HLA antibody‐incompatible kidney transplant recipients using a modified microbead assay. Pretreatment IgG4 levels were predictive of acute antibody‐mediated rejection (P = 0.003) in the first 30 days post‐transplant. Pre‐treatment presence of IgG4 DSA (P = 0.008) and day 30 IgG3 DSA (P = 0.03) was associated with poor graft survival. Multivariate regression analysis showed that in addition to pan‐IgG levels, total IgG4 levels were an independent risk factor for early rejection when measured pretreatment, and the presence of pretreatment IgG4 DSA was also an independent risk factor for graft failure. Pretreatment IgG4 DSA levels correlated independently with higher risk of early rejection episodes and medium‐term death‐censored graft survival. Thus, pretreatment IgG4 DSA may be used as a biomarker to predict and risk stratify cases with higher levels of pan‐IgG DSA in HLA antibody‐incompatible transplantation. Further investigations are needed to confirm our results.

Pregnancy-induced HLA antibodies respond more vigorously after renal transplantation than antibodies induced by prior transplantation

Acute antibody mediated rejection after HLA-specific antibody incompatible renal transplantation is related to donor specific HLA antibody (DSA) levels. DSA levels may rise sharply after transplant, and aim of this study was to examine changes in DSA levels, particularly according to the primary sensitising event. Changes in 220 HLA specificities in 64 patients over the first 30days after transplantation were evaluated using microbead assays. The greatest increase from pre-treatment to peak DSA levels was seen in pregnancy-stimulated specificities, median (IQR) increase in MFI of 1981 (94-5870). The next highest increase was for those sensitised by transplant with repeat HLA epitope mismatch, at 546 (-308-2698) (p<0.01). The difference was especially marked when the pre-treatment antibody level was low; with pre-treatment MFI <1000, peak level was >1000 in 19/26 (73%) of pregnancy stimulated specificities, compared with 9/29 (31%) for all others (p<0.001). DSA production to specificities stimulated by previous pregnancy was marked, even from very low pre-transplant levels. By contrast, there was a lower rate of antibody resynthesis to specificities repeated from previous transplants, both at antigen and epitope levels.

Profiling antibodies to class II HLA in transplant patient sera.

Immunizing events including pregnancy, transfusions, and transplantation promote strong alloantibody responses to HLA. Such alloantibodies to HLA preclude organ transplantation, foster hyperacute rejection, and contribute to chronic transplant failure. Diagnostic antibody-screening assays detect alloreactive antibodies, yet key attributes including antibody concentration and isotype remain largely unexplored. The goal here was to provide a detailed profile of allogeneic antibodies to class II HLA. Methodologically, alloantibodies were purified from sensitized patient sera using an HLA-DR11 immunoaffinity column and subsequently categorized. Antibodies to DR11 were found to fix complement, exist at a median serum concentration of 2.3μg/mL, consist of all isotypes, and isotypes IgG2, IgM, and IgE were elevated. Because multimeric isotypes can confound diagnostic determinations of antibody concentration, IgM and IgA isotypes were removed and DR11-IgG tested alone. Despite removal of multimeric isotypes, patient-to-patient antibody concentrations did not correlate with MFI values. In conclusion, allogeneic antibody responses to DR11 are comprised of all antibody isotypes at differing proportions, these combined isotypes fix complement at nominal serum concentrations, and enhancements other than the removal of IgM and IgA multimeric isotypes may be required if MFI is to be used as a means of determining anti-HLA serum antibody concentrations in diagnostic clinical assays.

Direct quantitative measurement of the kinetics of HLA-specific antibody interactions with isolated HLA proteins

HLA specific antibodies vary in their pathogenicity and this is likely to be the net effect of constant chain usage, quantity, specificity, and affinity. Here we have measured the affinity of human monoclonal antibodies for a range of HLA proteins. Purified antibodies and ligands allowed dynamic interactions to be measured directly by surface plasmon resonance. Physiochemical differences between pairs of ligands were quantified using electrostatic mismatch and hydrophobic mismatch scores. All antibodies were characterized by fast on-rates and slow off rates but with a wide range of association rates (kon, 3.63-24.25 × 105 per mol per second) and dissociation rates (koff, 0.99-10.93 × 10-3 per second). Dissociation constants (KD) ranged from 5.9 × 10-10 M to 3.0 × 10-8 M. SN320G6 has approximately a twenty-fold greater affinity for HLA A2 compared with SN607D8, but has a similar affinity for HLA-A2 and B57. In contrast, SN607D8 has greater than a twofold greater affinity for HLA-A2 compared with A68. Similarly, WK1D12 has about a threefold greater affinity for HLA-B27 compared with B7. The higher affinity interactions correlate with the specificity of stimulating antigen. This is the first study to directly measure the binding kinetics and affinity constants for human alloantibodies against HLA.

Antibody-incompatible kidney transplantation in 2015 and beyond

Rejection caused by donor-specific antibodies (principally ABO and HLA antibodies) has become one of the major barriers to successful long-term transplantation. This review focuses on clinical outcomes in antibody-incompatible transplantation, the current state of the science underpinning clinical observations, and how these may be translated into further novel therapies. The clinical outcomes for allografts facing donor-specific antibodies are at present determined largely by the use of agents developed in the 20th century for the treatment of T-lymphocyte-mediated cellular rejection, such as interleukin-2 agents and anti-thymocyte globulin. These treatments are partially effective, because acute antibody-mediated rejection is mediated to a considerable extent by T lymphocytes. However these treatments are essentially ineffective in chronic antibody-mediated rejection. Future therapies for the prevention and treatment of antibody-mediated rejection are likely to fall into the categories of those that reduce antibody production, extracorporeal antibody removal and disruption of the effector arms of antibody-mediated tissue damage.

Behaviour of non-donor specific antibodies during rapid re-synthesis of donor specific HLA antibodies after antibody incompatible renal transplantation

Background HLA directed antibodies play an important role in acute and chronic allograft rejection. During viral infection of a patient with HLA antibodies, the HLA antibody levels may rise even though there is no new immunization with antigen. However it is not known whether the converse occurs, and whether changes on non-donor specific antibodies are associated with any outcomes following HLA antibody incompatible renal transplantation. Methods 55 patients, 31 women and 24 men, who underwent HLAi renal transplant in our center from September 2005 to September 2010 were included in the studies. We analysed the data using two different approaches, based on; i) DSA levels and ii) rejection episode post transplant. HLA antibody levels were measured during the early post transplant period and corresponding CMV, VZV and Anti-HBs IgG antibody levels and blood group IgG, IgM and IgA antibodies were quantified. Results Despite a significant DSA antibody rise no significant non-donor specific HLA antibody, viral or blood group antibody rise was found. In rejection episode analyses, multiple logistic regression modelling showed that change in the DSA was significantly associated with rejection (p = 0.002), even when adjusted for other antibody levels. No other antibody levels were predictive of rejection. Increase in DSA from pre treatment to a post transplant peak of 1000 was equivalent to an increased chance of rejection with an odds ratio of 1.47 (1.08, 2.00). Conclusion In spite of increases or decreases in the DSA levels, there were no changes in the viral or the blood group antibodies in these patients. Thus the DSA rise is specific in contrast to the viral, blood group or third party antibodies post transplantation. Increases in the DSA post transplant in comparison to pre-treatment are strongly associated with occurrence of rejection.

Meeting report: 3rd international transplant conference: how much risk can you take?

The 3rd International Transplant Conference took place on 31st October and 1st November 2014 at the University of Warwick, Coventry, UK. Key focal points of the meeting were the exploration of the molecular basis of antibody–antigen interactions and their relation to clinical practice and to share experiences and knowledge regarding strategies to transplant the ‘high‐risk’ patient. In addition, lively debate sessions were hosted where controversial clinical and immunological themes were discussed by leading experts in the field.

A new data-driven model for post-transplant antibody dynamics in high risk kidney transplantation

The dynamics of donor specific human leukocyte antigen antibodies during early stage after kidney transplantation are of great clinical interest as these antibodies are considered to be associated with short and long term clinical outcomes. The limited number of antibody time series and their diverse patterns have made the task of modelling difficult. Focusing on one typical post-transplant dynamic pattern with rapid falls and stable settling levels, a novel data-driven model has been developed for the first time. A variational Bayesian inference method has been applied to select the best model and learn its parameters for 39 time series from two groups of graft recipients, i.e. patients with and without acute antibody-mediated rejection (AMR) episodes. Linear and nonlinear dynamic models of different order were attempted to fit the time series, and the third order linear model provided the best description of the common features in both groups. Both deterministic and stochastic parameters are found to be significantly different in the AMR and no-AMR groups showing that the time series in the AMR group have significantly higher frequency of oscillations and faster dissipation rates. This research may potentially lead to better understanding of the immunological mechanisms involved in kidney transplantation.

Refining The Prediction of Early Antibody Mediated Rejection After Antibody Incompatible Renal Transplantation; Importance of Pregnancy-Induced Sensitisation.: Abstract# 1455

1455 Refining The Prediction of Early Antibody Mediated Rejection After Antibody Incompatible Renal Transplantation; Importance of Pregnancy-Induced Sensitisation. R. Higgins,1 D. Lowe,2 M. Hathaway,2 C. Williams,2 C. Imray,1 N. Krishnan,1 S. Daga,3 D. Briggs,2 D. Zehnder.3 1Renal Transplantation, University Hospital, Coventry, United Kingdom; 2H&I Laboratory, NHS BT, Birmingham, United Kingdom; 3Warwick Medical School, University of Warwick, Coventry, United Kingdom. Background. Acute antibody mediated rejection (AMR) after HLA antibody incompatible renal transplantation is related to donor specifi c HLA antibody (DSA) levels. However, pre-treatment DSA levels have a low predictive value for AMR, partly because of rapid changes in DSA levels post-transplant. This study examined changes in DSA according to the primary sensitising event. Methods. Responses to 220 HLA specifi cities in 64 patients over the fi rst 30 days after transplantation were evaluated using single antigen microbead assays. The primary sensitising event for each specifi city was assigned according to the time of appearance of antibody in relation to pregnancy, transfusion or transplantation, taking into account the HLA type of the sensitisor and HLA epitope maps. Results. The greatest increase from pre-treatment to peak DSA level was seen in those stimulated by pregnancy, median (IQR) increase in MFI of 1981 (94-5870) u. The next highest increase was for those sensitised by transplant with repeat HLA epitope mismatch, at 546u (-308u-2698u) (p<0.01). Time to peak level was shorter in the pregnancy group, median 13 (9-19.75) days, compared to 15.5 (11-21) days in the transfusion group and 16 (12-23) days in the transplant group (p<0.01). For specifi cities with pre-treatment MFI <5000u, peak level of >5,000u occurred in 24/58 (41%) stimulated by pregnancy, compared to 8/119 (7%) in all other specifi cities (p<0.0001). With pre-treatment total DSA MFI <5000u, AMR occurred after primary sensitisation by pregnancy in 7/9 (78%) patients, compared to 2/5 (40%) for transfusion and 1/7 (14%) for transplantation. Conclusion. After transplantation rises in DSA were greatest, fastest and most likely to be associated with rejection for specifi cities where pregnancy was the primary sensitising event. In contrast, there were lower rises DSA corresponding to HLA exposure from previous transplants, at both antigen and epitope levels. Greater insight into the antibody response post-transplantation will enable better risk stratifi cation and cost-effective tailoring of therapy. DISCLOSURE: Higgins, R.: Grant/Research Support, Pure Transplant Solutions. Lowe, D.: Grant/Research Support, Pure Transplant Solutions. Daga, S.: Grant/ Research Support, Pure Transplant Solutions. Briggs, D.: Grant/Research Support, Pure Transplant Solutions. Zehnder, D.: Grant/Research Support, Pure Transplant Solutions. Abstract# 1456 A Phase I/II Trial of Tocilizumab (Anti-IL-6 Receptor) + Intravenous Immunoglobulin (IVIG) for Desensitization (DES) in Diffi cult to DES Patients. A. Vo,1 J. Choi,1 K. Cisneros,1 J. Kahwaji,1 M. Toyoda,2 S. Bongha,2 S. Ge,2 A. Peng,1 R. Villicana,1 S. Jordan.1 1Kidney Transplant, ; 2Transplant Immunology Laboratory, Cedars-Sinai Medical Center, Los Angeles, CA. Introduction: Approximately 25% of HS patients fail current DES and remain on dialysis for prolonged periods. Thus, newer, more effective approaches to DES that have limited side effects are desirable. Here, we undertook a phase I/II pilot study of DES using a novel drug (anti IL-6 receptor, Tocilizumab [TCZ]) + IVIG to assess the safety and limited effi cacy. Patients and Methods: From 7/2012 to 11/2013, 9 diffi cult to DES patients unresponsive to DES with IVIG + Rituximab were entered in an open label trial with IVIG+TCZ. Patients received IVIG on day 0 and day 30 at 2 g/ kg and TCZ 8 mg/kg on day 15 then monthly X6M. If transplanted, patients received IVIG on day 0; TCZ on day 2; then monthly x6M. Patients received Campath1H induction and were maintained on Prograf/Cellcept/Prednisone. Results: No differences in baseline characteristics were seen in patients not transplanted (G1) vs. transplanted (G2), including previous transplants as sensitizing events, female ratio and caucasian ethnicity. Two patients in each group developed SAEs: G1fl uid overload + pulmonary congestion as a result of under dialysis and hyperkalemia with epilepticus (likely not related to drug) vs. G2infective colitis with colonic perforation required bowel resection and Bells palsy (both possibly related). Five of 9 patients (67%; 4DD/1LD) were transplanted. Table 1 summarizes transplant characteristics. Mean time to transplant from 1st DES was 25±10.5M but after TCZ was 8.1±5.4M. No ABMR was seen; however, CMR was seen in 2 patients (40%). Mean DSA RIS pre-treatment was 7.8±1.9 and declined to 3.2±1.9 at Tx. Further DSA reductions were seen post-Tx. Renal function in transplanted patients has been excellent. Conclusions: TCZ and IVIG appears to be safe as DES agents and may have more durable effects on regulation of B-cells, DSA and Tregs. From this pilot trial, we are cautiously optimistic that TCZ may offer a novel alternative for diffi cult to DES patients. 1456 A Phase I/II Trial of Tocilizumab (Anti-IL-6 Receptor) + Intravenous Immunoglobulin (IVIG) for Desensitization (DES) in Diffi cult to DES Patients. A. Vo,1 J. Choi,1 K. Cisneros,1 J. Kahwaji,1 M. Toyoda,2 S. Bongha,2 S. Ge,2 A. Peng,1 R. Villicana,1 S. Jordan.1 1Kidney Transplant, ; 2Transplant Immunology Laboratory, Cedars-Sinai Medical Center, Los Angeles, CA. Introduction: Approximately 25% of HS patients fail current DES and remain on dialysis for prolonged periods. Thus, newer, more effective approaches to DES that have limited side effects are desirable. Here, we undertook a phase I/II pilot study of DES using a novel drug (anti IL-6 receptor, Tocilizumab [TCZ]) + IVIG to assess the safety and limited effi cacy. Patients and Methods: From 7/2012 to 11/2013, 9 diffi cult to DES patients unresponsive to DES with IVIG + Rituximab were entered in an open label trial with IVIG+TCZ. Patients received IVIG on day 0 and day 30 at 2 g/ kg and TCZ 8 mg/kg on day 15 then monthly X6M. If transplanted, patients received IVIG on day 0; TCZ on day 2; then monthly x6M. Patients received Campath1H induction and were maintained on Prograf/Cellcept/Prednisone. Results: No differences in baseline characteristics were seen in patients not transplanted (G1) vs. transplanted (G2), including previous transplants as sensitizing events, female ratio and caucasian ethnicity. Two patients in each group developed SAEs: G1fl uid overload + pulmonary congestion as a result of under dialysis and hyperkalemia with epilepticus (likely not related to drug) vs. G2infective colitis with colonic perforation required bowel resection and Bells palsy (both possibly related). Five of 9 patients (67%; 4DD/1LD) were transplanted. Table 1 summarizes transplant characteristics. Mean time to transplant from 1st DES was 25±10.5M but after TCZ was 8.1±5.4M. No ABMR was seen; however, CMR was seen in 2 patients (40%). Mean DSA RIS pre-treatment was 7.8±1.9 and declined to 3.2±1.9 at Tx. Further DSA reductions were seen post-Tx. Renal function in transplanted patients has been excellent. Conclusions: TCZ and IVIG appears to be safe as DES agents and may have more durable effects on regulation of B-cells, DSA and Tregs. From this pilot trial, we are cautiously optimistic that TCZ may offer a novel alternative for diffi cult to DES patients. IND#114362, NCT01594424. DISCLOSURE: Jordan, S.: Grant/Research Support, Genentech, Inc., Other, Genentech, Inc., Consulting. Abstract# 1457 Long-Term (More Than 20 Years) Outcome of ABO-Incompatible Living Donor Kidney Transplantation: A Single Center Experience. K. Tanabe, H. Ishida, K. Omoto, T. Shimizu, T. Nozaki, M. Inui, H. Shirakawa, M. Okumi, D. Toki. Urology, Tokyo Women’s Medical University Hospital. Background Since serious shortage of organ donation is still the biggest barrier to the increase in transplants in Japan, ABO-incompatible living kidney transplantation (ABOILKT) has been an effective alternative to deceased kidney transplantation. In this retrospective, single center study, we analyzed the long-term (more than 20 year outcome of ABO-ILKT. Methods: A total of 1122 pappptients with ESRD underwent living donor kidney transplantation at our institute between 1989 and 2013. Of them, 267 cases were ABO-ILKT and 855 cases ABO-compatible living kidney transplantation (ABOCLKT). The mean age of the ABO-ILKT group was 43.1 years (range 17 to 75), with 168 males and 99 females. Plasmapheresis was carried out to remove anti-AB antibodies before kidney transplantation in most of the cases. Most recipients received CNI (tacrolimus or cyclosporine)-based immunosuppression including methylprednisolone and mycophenolate mofetil. After 2002, all patients received basiliximab preoperatively. Between 1986 and 2004, splenectomy was performed at the time of ABO-ILKT. Thereafter, as an alternative to splenectomy, rituximab at a dose of 200mg/person was administered prior to ABO-ILKT. ABO-CLKT was employed as the control. Results: ABO-ILKT patient survival at 1, 5, 10, 15, and 20 years post-transplant was 98.1, 97.1, 92.8, 86.6, and 84.4%, respectively. ABO-ILKT graft survival at 1, 5, 10, 15, and 20 years post-transplant was 91.0, 86.0, 72.2, 59.1, and 53.0%, respectively. Although graft survival of ABO-CLKT at 1, 5, 10, 15, and 20 years post-transplant was 97.4, 88.7, 76.3, 65.3, and 55.6%, respectively, there was no signifi cant difference in patient survival from that of ABO-CLKT patients (p=0.740). The graft survival of ABO-ILKT at 20 years post-transplant was almost identical with ABO-CLKT, with no statistical difference between the two groups (p=0.0.144). The incidence of rejection was no different between the two groups as well. Furthermore, no signifi c