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.

BackgroundDonor-specific antibodies are associated with increased risk of antibody-mediated rejection and decreased allograft survival. Therefore, reducing the risk of these antibodies remains a clinical need in transplantation. Plasma cells are a logical target of therapy given their critical role in antibody production.MethodsTo target plasma cells, we treated sensitized rhesus macaques with daratumumab (anti-CD38 mAb). Before transplant, we sensitized eight macaques with two sequential skin grafts from MHC-mismatched donors; four of them were also desensitized with daratumumab and plerixafor (anti-CXCR4). We also treated two patients with daratumumab in the context of transplant.ResultsThe animals treated with daratumumab had significantly reduced donor-specific antibody levels compared with untreated controls (57.9% versus 13% reduction; P<0.05) and prolonged renal graft survival (28.0 days versus 5.2 days; P<0.01). However, the reduction in donor-specific antibodies was not maintained because all recipients demonstrated rapid rebound of antibodies, with profound T cell–mediated rejection. In the two clinical patients, a combined heart and kidney transplant recipient with refractory antibody-mediated rejection and a highly sensitized heart transplant candidate, we also observed a significant decrease in class 1 and 2 donor-specific antibodies that led to clinical improvement of antibody-mediated rejection and to heart graft access.ConclusionsTargeting CD38 with daratumumab significantly reduced anti-HLA antibodies and anti-HLA donor-specific antibodies in a nonhuman primate model and in two transplant clinical cases before and after transplant. This supports investigation of daratumumab as a potential therapeutic strategy; however, further research is needed regarding its use for both antibody-mediated rejection and desensitization.

SummaryABO 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 Guy's 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 posttransplant, 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.

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/m per dose) clearly reduced the numbers of antibody-producing cells and CD38CD19CD20 plasma cells in the bone marrow (<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.

UK National Health Service Blood & Transplant and Guy's & St Thomas' National Institute for Health Research Biomedical Research Centre.

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 (CD19CD20CD38), Tfh cells (CD4ICOSPD-1), and GC B cells (BCL-6CD20); 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.

We have established a model of sensitization in non-human primates and tested two immunosuppressive regimens. Animals underwent fully mismatched skin transplantation, donor-specific antibody (DSA) response was monitored by flow crossmatch. Sensitized animals subsequently underwent kidney transplantation from their skin donor. Immunosuppression included tacrolimus, mycophenolate and methylprednisolone. Three animals received basiliximab induction, compared to non-sensitized animals they showed a shorter mean survival time (MST, 4.7±3.1 vs. 187±88 days). Six animals were treated with T-cell depletion (anti-CD4/CD8 mAbs), which prolonged survival (MST=21.6±19.0 days). All pre-sensitized animals showed antibody-mediated rejection (AMR). In 2/3 basiliximab animals cellular rejection (ACR) was prominent. After T cell depletion, 3/6 monkeys experienced early acute rejection within 8 days with histological evidence of thrombotic microangiopathy and AMR. The remaining three survived 27 to 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 response. Depletion controlled cell-mediated rejection in sensitized non-human primates better than basiliximab, yet grafts were rejected with concomitant DSA rise. This model provides an opportunity to test novel desensitization strategies.

Cross-talk 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 following depletional induction, and the subsequent effect of immunosuppressive manipulation of T-B cell interactions. Additionally, ectopic GCs are seen in transplantation, but their role is not fully understood. Therapeutic options to target T-B cell interactions are of considerable interest, both as immunosuppressive tools, and to aid further understanding of these important alloimmune mechanisms.