Miriam Manook

Tailored desensitization strategies in ABO blood group antibody incompatible renal transplantation

ABO blood group incompatible renal transplantation, using desensitization procedures, is an effective strategy. Efforts have been made to reduce desensitization: these are usually applied to all patients indiscriminately. The Guys Hospital ABO blood group incompatible desensitization regimen uses a tiered approach, tailoring strategy according to initial antibody titres. Sixty‐two ABO blood group incompatible living donor transplant recipients were compared with 167 recipients of blood group compatible living donor renal transplants. There were no statistically significant differences in allograft survival rates at 1 or 3 years post‐transplant, rejection in the first year post‐transplant or renal function in the first 3 years post‐transplant. There was a higher rate of death in ABO blood group incompatible transplant recipients – this could be associated with differences in age and HLA mismatch between the two groups. Four ABO blood group incompatible patients experienced antibody‐mediated rejection (no episode was associated with a rise in ABO blood group antibodies). Of the patients who received no desensitization, or rituximab alone, none has experienced antibody mediated rejection or experienced allograft loss. Tailoring the use of desensitization in ABO blood group incompatible renal transplantation according to initial ABO blood group antibody titres led to comparable results to blood group compatible transplantation.

Antibody-Mediated Rejection in Sensitized Nonhuman Primates: Modeling Human Biology.

We have established a model of sensitization in nonhuman primates and tested two immunosuppressive regimens. Animals underwent fully mismatched skin transplantation, and donor‐specific antibody (DSA) response was monitored by flow cross‐match. Sensitized animals subsequently underwent kidney transplantation from their skin donor. Immunosuppression included tacrolimus, mycophenolate, and methylprednisolone. Three animals received basiliximab induction; compared with nonsensitized animals, they showed a shorter mean survival time (4.7 ± 3.1 vs. 187 ± 88 days). Six animals were treated with T cell depletion (anti‐CD4/CD8 mAbs), which prolonged survival (mean survival time 21.6 ± 19.0 days). All presensitized animals showed antibody‐mediated rejection (AMR). In two of three basiliximab‐injected animals, cellular rejection (ACR) was prominent. After T cell depletion, three of six monkeys experienced early acute rejection within 8 days with histological evidence of thrombotic microangiopathy and AMR. The remaining three monkeys survived 27–44 days, with mixed AMR and ACR. Most T cell–depleted animals experienced a rebound of DSA that correlated with deteriorating kidney function. We also found an increase in proliferating memory B cells (CD20+CD27+IgD−Ki67+), lymph node follicular helper T cells (ICOS+PD‐1hiCXCR5+CD4+), and germinal center (GC) response. Depletion controlled cell‐mediated rejection in sensitized nonhuman primates better than basiliximab, yet grafts were rejected with concomitant DSA rise. This model provides an opportunity to test novel desensitization strategies.

Crosstalk Between T and B Cells in the Germinal Center After Transplantation

Crosstalk between B and T cells in transplantation is increasingly recognized as being important in the alloimmune response. T cell activation of B cells occurs by a 3-stage pathway, culminating with costimulation signals. We review the distinct T cell subtypes required for B-cell activation and discuss the formation of the germinal center (GC) after transplantation, with particular reference to the repopulation of the GC after depletional induction, and the subsequent effect of immunosuppressive manipulation of T cell-B cell interactions. In addition, ectopic GCs are seen in transplantation, but their role is not fully understood. Therapeutic options to target T cell-B cell interactions are of considerable interest, both as immunosuppressive tools, and to aid in the further understanding of these important alloimmune mechanisms.

Humoral Compensation after Bortezomib Treatment of Allosensitized Recipients

The efficacy of bortezomib monotherapy in desensitizing kidney transplant candidates with preformed donor-specific antibodies remains unclear. We evaluated the effect of bortezomib on preformed antibodies and upstream components of the B cell response in a primate model sensitized by fully mismatched allogeneic skin transplants to provide mechanistic insights regarding the use of bortezomib as a means of desensitization. Bortezomib treatment given intravenously twice weekly for 1 month (1.3 mg/m2 per dose) clearly reduced the numbers of antibody-producing cells and CD38+CD19+CD20- plasma cells in the bone marrow (P<0.05), but donor-specific alloantibody levels did not decrease. We observed a rapid but transient induction of circulating IgG+ B cells and an increased number of proliferating B cells in the lymph nodes after 1 month of treatment. Notably, bortezomib treatment induced germinal center B cell and follicular helper T cell expansion in the lymph nodes. These data suggest that bortezomib-induced plasma cell depletion triggers humoral compensation.

Incidence and Outcome of C4d Staining With Tubulointerstitial Inflammation in Blood Group-incompatible Kidney Transplantation.

Background The last Banff 2013 report recognizes acute/active antibody-mediated rejection (ABMR) and C4d staining without evidence of rejection. The goal of our study was to analyze the incidence of C4d deposition after ABO-incompatible transplantation and assess outcomes in patients with ABMR, C4d staining without evidence of rejection (all acute Banff scores = 0), and C4d staining with tubulointerstitial inflammation (i > 0 with or without tubulitis). Methods Three-months ‘For cause’ or protocol biopsies in 50 ABO-incompatible patients were rescored and were correlated with clinical outcomes and antibody titres. Results Active/acute ABMR was found in 23 patients (46%), C4d staining without evidence of rejection in 7 patients (14%), C4d staining with tubulointerstitial inflammation in 6 patients (12%), tubulointerstitial inflammation in 6 patients (12%), and no evidence of rejection in 8 patients (16%). Patients with active/acute ABMR had a 3-month estimated glomerular filtration rate (median,: 43 mL/min) lower than patients with no evidence of rejection (median, 61 mL/min; P = 0.01). However, after 3 months, a progressively declining estimated glomerular filtration rate was observed more frequently in patients with C4d staining and tubulointerstitial inflammation when compared to patients with no evidence of rejection (100% vs 25%, P = 0.03). Finally, independently of C4d status, interstitial inflammation occurred more frequently in patients with a pretransplant ABO antibody titre higher than 16 and/or posttransplant ABO antibody increase. Conclusions Whereas isolated C4d deposition and isolated interstitial inflammation appear to be benign lesions, C4d deposition in association with interstitial inflammation is the biopsy finding most strongly associated with the development of chronic graft dysfunction.

Successful desensitization with proteasome inhibition and costimulation blockade in sensitized nonhuman primates

The detrimental effects of donor-directed antibodies in sensitized transplant patients remain a difficult immunologic barrier to successful organ transplantation. Antibody removal is often followed by rebound. Proteasome inhibitors (PIs) deplete antibody-producing plasma cells (PCs) but have shown marginal benefit for desensitization. In an allosensitized nonhuman primate (NHP) model, we observed increased germinal center (GC) formation after PI monotherapy, suggesting a compensatory PC repopulation mediated via GC activation. Here we show that costimulation blockade (CoB) targets GC follicular helper T (Tfh) cells in allosensitized NHPs. Combined PI and CoB significantly reduces bone marrow PCs (CD19+CD20-CD38+), Tfh cells (CD4+ICOS+PD-1hi), and GC B cells (BCL-6+CD20+); controls the homeostatic GC response to PC depletion; and sustains alloantibody decline. Importantly, dual PC and CoB therapy prolongs rejection-free graft survival in major histocompatibility complex incompatible kidney transplantation without alloantibody rebound. Our study illustrates a translatable desensitization method and provides mechanistic insight into maintenance of alloantibody sensitization.

Thrombalexin: Use of a Cytotopic Anticoagulant to Reduce Thrombotic Microangiopathy in a Highly Sensitized Model of Kidney Transplantation

Early activation of coagulation is an important factor in the initiation of innate immunity, as characterized by thrombotic microangiopathy (TMA). In transplantation, systemic anticoagulation is difficult due to bleeding. A novel “cytotopic” agent, thrombalexin (TLN), combines a cell‐membrane‐bound (myristoyl tail) anti‐thrombin (hirudin‐like peptide [HLL]), which can be perfused directly to the donor organ or cells. Thromboelastography was used to measure time to clot formation (r‐time) in both rhesus and human blood, comparing TLN versus HLL (without cytotopic tail) versus negative control. Both TLN‐ and HLL‐treated rhesus or human whole blood result in significantly prolonged r‐time compared to kaolin controls. Only TLN‐treated human endothelial cells and neonatal porcine islets prolonged time to clot formation. Detection of membrane‐bound TLN was confirmed by immunohistochemistry and fluorescence activated cell sorter. In vivo, perfusion of a nonhuman primate kidney TLN‐supplemented preservation solution in a sensitized model of transplantation demonstrated no evidence of TLN systemically. Histologically, TLN was shown to be present up to 4 days after transplantation. There was no platelet deposition, and TMA severity, as well as microvascular injury scores (glomerulitis + peritubular capillaritis), were less in the TLN‐treated animals. Despite promising evidence of localized efficacy, no survival benefit was demonstrated.

Innate networking: Thrombotic microangiopathy, the activation of coagulation and complement in the sensitized kidney transplant recipient

Thrombotic microangiopathy (TMA) is a histological feature of antibody-mediated rejection and has the potential to cause problematic graft dysfunction, particularly for highly sensitized cross-match positive kidney transplant recipients. Prompt recognition of pertinent histopathological and systemic features of TMA in kidney transplantation is necessary. Underlying mechanisms of this process involve the activation of both complement and coagulation systems as a response to HLA antibody. As serine proteases, coagulation and complement cascades exhibit similar characteristics with respect to homeostatic function. Increasing evidence now exists for the interaction between these innate defenses in both activation and regulation, lending scope for intervention. Understanding the complexities of these interactions remains a challenge. This review provides an overview of the current understanding, particularly with respect to the activation of coagulation and complement by HLA antibody in the setting of highly sensitized kidney transplantation.

Dual targeting: Combining costimulation blockade and bortezomib to permit kidney transplantation in sensitized recipients

Previous evidence suggests that a homeostatic germinal center (GC) response may limit bortezomib desensitization therapy. We evaluated the combination of costimulation blockade with bortezomib in a sensitized non‐human primate kidney transplant model. Sensitized animals were treated with bortezomib, belatacept, and anti‐CD40 mAb twice weekly for a month (n = 6) and compared to control animals (n = 7). Desensitization therapy–mediated DSA reductions approached statistical significance (P = .07) and significantly diminished bone marrow PCs, lymph node follicular helper T cells, and memory B cell proliferation. Graft survival was prolonged in the desensitization group (P = .073). All control animals (n = 6) experienced graft loss due to antibody‐mediated rejection (AMR) after kidney transplantation, compared to one desensitized animal (1/5). Overall, histological AMR scores were significantly lower in the treatment group (n = 5) compared to control (P = .020). However, CMV disease was common in the desensitized group (3/5). Desensitized animals were sacrificed after long‐term follow‐up with functioning grafts. Dual targeting of both plasma cells and upstream GC responses successfully prolongs graft survival in a sensitized NHP model despite significant infectious complications and drug toxicity. Further work is planned to dissect underlying mechanisms, and explore safety concerns.

Assessing the Feasibility of Deceased Donor ABO-Incompatible Transplantation in Wait-Listed Transplant Recipients.: Abstract# A163

A163 Assessing the Feasibility of Deceased Donor ABO-Incompatible Transplantation in Wait-Listed Transplant Recipients. M. Manook,1 D. Veniard,1 N. Barnett,2 O. Shaw,1 T. Maggs,1 N. Mamode.1 1Renal Transplant, Guys & St Thomas’ NHS Trust, London, United Kingdom; 2Colchester Hospital, London, United Kingdom. Introduction: Outcomes for living donor ABO-Incompatible are widely accepted as being favourable. In our centre, a tailored approach to desensitisation for ABOi is used, based on baseline ABO antibody titres.1 For patients with titres < 1:8, no additional treatment is given compared to ABO-compatible transplants. This offers the potential of deceased donor ABOi (DD ABOi) transplantation. This study was to determine the feasibility of DD ABOi in a single centre Methods: Prospective cohort study of ABO antibody titres & calculated reaction frequency (CRF) in active & suspended patients on the adult renal transplant waiting list in a single centre. Total immunoglobulin (Ig) Anti-A & Anti-B titres were measured by the Indirect Antiglobulin Test (IAT) using Biorad gel cards (Coombs anti-IgG) on untreated plasma capturing initial IgM binding & subsequent IgG. Results: Of the 124 patients tested, 3 (2.4%) were excluded from analysis due to the presence of atypical antibodies. 60% (n=72) were male. Blood group distribution was A 29.2% (n = 35); B 11.7% (n= 14); O 56.7% (n=68); AB 2.5% (n=3) Black ethnicity was the most common (50%), White (35.8%), Asian (7.5%). Of the patients screened, 3 had ‘neat’ anti-A or Anti-B titres. 25% (n=30) had either an anti-A or anti-B titre of 1:8 or less – ‘low titre’ (of these 10% were on immunosuppressive medication). Haemagglutinin titres varied with blood group: Grp A 21 ‘low titre’ vs14 high titre; Grp B 3 ‘low’ vs11 high, Grp O 3 ‘low’ vs 65 high, p = <0.02 .For Group O, Anti-A titres were higher than antiB (median anti-A titre 1:256, median anti-B titres 1:128), 26.5% of Group O’s had a greater than expected dilution difference (>2) between anti-A & Anti-B. Mean Calculated Reaction Frequency (CRF – a measure of anti-HLA antibody) was 43.5% (SD 43.2) Comparing a high CRF (>80%) to low there was no signifi cant difference in mean anti-A (p = 0.6) or anti-B titre (p=0.3). There was no correlation between anti-A or anti-B & CRF. Discussion: Preliminary results suggest that 25% of patients on the deceased donor waiting list would be suitable for DD ABOi transplantation without any additional treatment. Patients who are highly sensitised to HLA-antibody do not have high ABO antibody titres. Further work to follow up the cohort is planned DISCLOSURES: Manook, M.: Grant/Research Support, Glycorex. Mamode, N.: Grant/Research Support, Alexion, Astellas. Abstract# A164 Does Recipient Race Affect Alloantibody Presentation and Graft Outcome? P. Kimball, A. Sharma, F. McDougan, A. King. Transplant Surgery, Virginia Commonwealth Univ. Health Systems, Richmond. Purpose. African Americans (AA) are more likely to be sensitized against HLA antigens and have poorer renal transplant (RT) outcomes than Caucasian Americans (CA). We speculated that alloantibody presentation might differ between AA and CA and contribute to different outcomes. Methods. Among 253 RT recipients, 71 were targeted for preemptive desensitization due to PRA >50%, FCXM > 100 MCS and DGF. Alloantibody content was identifi ed by fl ow crossmatch (FC) and DSA using single antigen bead luminex. Postransplant antibody was tested quarterly. Results. More AA than CA needed desensitization (31% vs. 22%, p<0.05). Preoperatively, more AA than CA were FC+DSA+ (70 vs. 43%, p<0.05). CA tended to be FC+ only (57% vs. 30%, p<0.05) which is considered non-HLA. Multiple DSA were prevalent for AA than CA(78% vs. 28%, p<0.05). DSA against both Class I and II was prevalent among AA than CA (40% vs. 12%, p<0.05). DSA strength (MFI) against Class I was equivalent (p=ns) between AA and CA (6606 ±4472 vs. 8323 ±3471). MFI against Class II increased 2-fold among AA (10,247 ±5015, p=0.01) but not CA (8707 ±7045. p=ns). FC strength (MCS) against Class I was higher for AA than CA (114 ±90 vs. 50 ±72 MCS, p=0.01) but equivalent against Class II (117 ±70 vs. 137 ±77 MCS, p=ns). After RT, alloantibody clearance was equivalent between AA and CA for FC+DSA+ (31% vs. 37%, p=ns) and FC+ (42% vs. 33%, p=ns). Among patients with alloantibody elimination, graft outcomes were equivalent between AA and CA with 100% 2-yr graft survival. In contrast, AA with antibody persistence had more rejections (33% vs. 0%, p<0.05) and poorer 2 yr graft survival (60% vs. 100%, p<0.05) than CA. Similar results were seen with FC+ only. Conclusions. AA and CA variation in antibody content may impact outcome. AA had anti-HLA DSA whereas CA had non-HLA antibody. AA had multiple DSAs, higher DSA levels particularly against Class II. A164 Does Recipient Race Affect Alloantibody Presentation and Graft Outcome? P. Kimball, A. Sharma, F. McDougan, A. King. Transplant Surgery, Virginia Commonwealth Univ. Health Systems, Richmond. Purpose. African Americans (AA) are more likely to be sensitized against HLA antigens and have poorer renal transplant (RT) outcomes than Caucasian Americans (CA). We speculated that alloantibody presentation might differ between AA and CA and contribute to different outcomes. Methods. Among 253 RT recipients, 71 were targeted for preemptive desensitization due to PRA >50%, FCXM > 100 MCS and DGF. Alloantibody content was identifi ed by fl ow crossmatch (FC) and DSA using single antigen bead luminex. Postransplant antibody was tested quarterly. Results. More AA than CA needed desensitization (31% vs. 22%, p<0.05). Preoperatively, more AA than CA were FC+DSA+ (70 vs. 43%, p<0.05). CA tended to be FC+ only (57% vs. 30%, p<0.05) which is considered non-HLA. Multiple DSA were prevalent for AA than CA(78% vs. 28%, p<0.05). DSA against both Class I and II was prevalent among AA than CA (40% vs. 12%, p<0.05). DSA strength (MFI) against Class I was equivalent (p=ns) between AA and CA (6606 ±4472 vs. 8323 ±3471). MFI against Class II increased 2-fold among AA (10,247 ±5015, p=0.01) but not CA (8707 ±7045. p=ns). FC strength (MCS) against Class I was higher for AA than CA (114 ±90 vs. 50 ±72 MCS, p=0.01) but equivalent against Class II (117 ±70 vs. 137 ±77 MCS, p=ns). After RT, alloantibody clearance was equivalent between AA and CA for FC+DSA+ (31% vs. 37%, p=ns) and FC+ (42% vs. 33%, p=ns). Among patients with alloantibody elimination, graft outcomes were equivalent between AA and CA with 100% 2-yr graft survival. In contrast, AA with antibody persistence had more rejections (33% vs. 0%, p<0.05) and poorer 2 yr graft survival (60% vs. 100%, p<0.05) than CA. Similar results were seen with FC+ only. Conclusions. AA and CA variation in antibody content may impact outcome. AA had anti-HLA DSA whereas CA had non-HLA antibody. AA had multiple DSAs, higher DSA levels particularly against Class II. Abstract# A165 Anti-ABO Specific Assay Variability Affects Antibody Removal in ABO Incompatible Kidney Transplantation (ABOiKTx) UK Multicentre Study. A. Bentall,1 M. Kaur,1 N. Mamode,3 D. Briggs,2 N. Kessaris,3 S. Ball.1 1Department of Nephrology and Transplantation, University Hospitals Birmingham, Birmingham, United Kingdom; 2Department of Histocompatibility, NHSBT, Birmingham, United Kingdom; 3Department of Transplantation, Guys and St Thomas Hospital, London, United Kingdom. INTRODUCTION: ABOiKTx outcomes are reported better outcomes in single centres than in registry data. Intracentre and intercentre variability in ABO blood group antibody titres has been reported without clinical correlate. In a multi-centre observational study, we report the clinical outcomes and anti-ABO titre variations between centres and the impact on clinical practice. METHODS: 100 patients recruited received an ABOiKTx and data and samples were collected prospectively and analysed by blood group and extracorporeal antibody treatment (EART). Samples were analysed locally and central samples were retrospectively analysed. A single technician undertook analysis of all samples, initially demonstrating excellent reproducibility within same red cell batch; using different batches; freeze thaw repeats and on different days. RESULTS: Patient and graft survival at 1 year were 99% and 94% respectively. 70 patients were blood group ‘O’. 36 were pre-emptive transplants and 86 were 1st transplants. 80 patients had rituximab for induction. 55 patients had immunoadsorption(IA) and 31 had plasma exchange (PEx). There was no difference in clinical outcomes between IA and PEx in graft survival (Figure 1A). 29 patients experience acute rejection with 4 with antibody-mediated rejection with more rejection in blood group B recipients(Figure1B). CMV and BK nephropathy occurred in 3 and 5 patients respectively. ABO titres were higher in local assays in PEx cohort, and received more EART, however when analysed centrally, there was no signifi cant difference in ABO titres between IA and PEx. A165 Anti-ABO Specific Assay Variability Affects Antibody Removal in ABO Incompatible Kidney Transplantation (ABOiKTx) UK Multicentre Study. A. Bentall,1 M. Kaur,1 N. Mamode,3 D. Briggs,2 N. Kessaris,3 S. Ball.1 1Department of Nephrology and Transplantation, University Hospitals Birmingham, Birmingham, United Kingdom; 2Department of Histocompatibility, NHSBT, Birmingham, United Kingdom; 3Department of Transplantation, Guys and St Thomas Hospital, London, United Kingdom. INTRODUCTION: ABOiKTx outcomes are reported better outcomes in single centres than in registry data. Intracentre and intercentre variability in ABO blood group antibody titres has been reported without clinical correlate. In a multi-centre observational study, we report the clinical outcomes and anti-ABO titre variations between centres and the impact on clinical practice. METHODS: 100 patients recruited received an ABOiKTx and data and samples were collected prospectively and