Sensitized patients are difficult to transplant due to pre-formed anti-donor immunity. We have previously reported successful desensitization using carfilzomib and belatacept in a non-human primate (NHP) model. Here we evaluated selective blockade of the co-stimulatory signal (CD28-B7) with Lulizumab, which preserves the co-inhibitory signal (CTLA4-B7). Five maximally MHC-mismatched pairs of NHPs were sensitized to each other with two sequential skin transplants. Individuals from each pair were randomized to either desensitization with once-weekly Carfilzomib (27mg/m2 IV) and Lulizumab (12.5mg/kg SC) over four weeks, or no desensitization (Control). NHPs then underwent life-sustaining kidney transplantation from their previous skin donor. Rhesus-specific anti-thymocyte globulin was used as induction therapy and immunosuppression maintained with tacrolimus, mycophenolate, and methylprednisolone. Desensitized subjects demonstrated a significant reduction in donor-specific antibody, follicular helper T cells (CD4+PD-1+ICOS+), and proliferating B cells (CD20+Ki67+) in the lymph nodes. Interestingly, regulatory T cell (CD4+CD25+CD127lo) frequency was maintained after desensitization in addition to increased frequency of naïve CD4 T cells (CCR7+CD45RA+) and naïve B cells (IgD+CD27-CD20+) in circulation. This was associated with significant prolongation in graft survival (MST = 5.8 ± 4.0 vs. 64.8 ± 36.3; p<0.05) and lower antibody-mediated rejection scores compared to control animals. However, all desensitized animals eventually developed AMR and graft failure. Desensitization with CFZ and Lulizumab improves allograft survival in allosensitized NHPs, by transient control of the germinal center and shifting of the immune system to a more naive phenotype. This regimen may translate into clinical practice to improve outcomes of highly sensitized transplant patients.

Porcine islet xenotransplantation is a viable strategy to treat diabetes. Its translation has been limited by the pre-clinical development of a clinically available immunosuppressive regimen. We tested two clinically relevant induction agents in a non-human primate (NHP) islet xenotransplantation model to compare depletional versus nondepletional induction immunosuppression. Neonatal porcine islets were isolated from GKO or hCD46/GKO transgenic piglets and transplanted via portal vein infusion in diabetic rhesus macaques. Induction therapy consisted of either basiliximab (n = 6) or rhesus-specific anti-thymocyte globulin (rhATG, n = 6), combined with a maintenance regimen using B7 costimulation blockade, tacrolimus with a delayed transition to sirolimus, and mycophenolate mofetil. Xenografts were monitored by blood glucose levels and porcine C-peptide measurements. Of the six receiving basiliximab induction, engraftment was achieved in 4 with median graft survival of 14 days. All six receiving rhATG induction engrafted with significantly longer xenograft survival at 40.5 days (P = 0.03). These data suggest that depletional induction provides superior xenograft survival to nondepletional induction, in the setting of a costimulation blockade-based maintenance regimen.

Introduction: Sensitized patients are difficult to transplant due to pre-formed immunity against MHC antigens. We previously reported the benefit of desensitization using carfilzomib and belatacept in a non-human primate (NHP) model. Here we evaluated a desensitization strategy combining carfilzomib (CFZ) with lulizumab (CD28dAb) which, conceptually, has greater immunoregulatory benefit over belatacept by selectively blocking the co-stimulating signal (CD28-B7) whilst preserving the co-inhibitory signal (CTLA4-B7). Methods: Five maximally MHC-mismatched pairs of NHPs were sensitized to each other with two sequential skin transplants. Individuals from each pair were randomized to receive either desensitization with once weekly treatments of CFZ (27mg/m2 IV) and CD28dAb (12.5mg/kg SC) over four weeks, or no desensitization (Control). This was followed by a life-sustaining kidney transplant between the NHP pairs. All primates received induction therapy with rhesus-specific antithymocyte globulin and maintenance immunosuppression with tacrolimus, mycophenolate, and methylprednisolone. Results: The desensitized group demonstrated a significant reduction in donor specific antibody (DSA) (ΔMFI -708±83, p<0.05; Fig 1A), Tfh cells (CD4+PD-1+ICOS+; Fig 1B), and proliferating B cells (CD20+Ki67+; Fig1C) in the lymph nodes. Bone marrow plasma cells (CD3-CD14-CD20-CD19+CD38+ or CD3-CD20-CD38+CD138+BLIMP-1+) showed only a marginal reduction. Interestingly, regulatory T cell (CD4+CD25+CD127lo) frequency was maintained after desesnsitization in addition to an increased circulating frequency of naïve CD4 T cells (CCR7+CD45RA+) and naïve B cells (IgD+CD27-CD20+). Treatment with CFZ and CD28dAb significantly prolonged graft survival (MST = 5.8 ± 4.0 vs. 64.8 ± 36.3; p<0.05; FIg 1D) with lower AMR scores (g+ptc; Fig 1E).We did not observe clinically relevant CMV reactivation in either group. After kidney transplantation, LN-Tfh cells and proliferating B cells remained reduced in the treatment group. Furthermore, we observed an increase in Tregs. Ultimately, however, all animals showed a rebound of DSA and experienced antibody-mediated rejection. Conclusions: Desensitization with CFZ and CD28dAb significantly prolongs allograft survival in allosensitized NHPs by reducing DSA and the germinal center response, likely by promoting a more naive lymphocyte repertoire and increasing the frequency of Tregs. Despite this, the strategy failed to promote durable desensitization, suggesting the need for continuous control of the humoral immune response post-transplantation.

Background Membrane cofactor protein CD46 attenuates the complement cascade by facilitating cleavage of C3b and C4b. In solid organ xenotransplantation, organs expressing CD46 have been shown to resist hyperacute rejection. However, the incremental value of human CD46 expression for islet xenotransplantation remains poorly defined. Methods This study attempted to delineate the role of CD46 in early neonatal porcine islet engraftment by comparing Gal-knocked out (GKO) and hCD46-transgenic (GKO/CD46) islets in a dual transplant model. Seven rhesus macaques underwent dual transplant and were sacrificed at 1 hour (n = 4) or 24 hours (n = 3). Both hemilivers were recovered and fixed for immunohistochemistry (CD46, insulin, neutrophil elastase, platelet, IgM, IgG, C3d, C4d, CD68, Caspase 3). Quantitative immunohistochemical analysis was performed using the Aperio Imagescope. Results Within 1 hour of intraportal infusion of xenografts, no differences were observed between the two types of islets in terms of platelet, antibody, or complement deposition. Cellular infiltration and islet apoptotic activity were also similar at 1 hour. At 24 hours, GKO/CD46 islets demonstrated significantly less platelet deposition (P = 0.01) and neutrophil infiltration (P = 0.01) compared to GKO islets. In contrast, C3d (P = 0.38) and C4d (P = 0.45) deposition was equal between the two genotypes. Conclusions Our findings suggest that expression of hCD46 on NPIs potentially provides a measurable incremental survival advantage in vivo by reducing early thrombo-inflammatory events associated with instant blood-mediated inflammatory reaction (IBMIR) following intraportal islet infusion.

One opportunity to reduce hernia occurrence and recurrence rates (currently estimated to be 30% at 10 years postoperatively) is by enhancing the ability of hernia meshes to anchor into tissue to prevent mesh migration, mesh contraction, and mesh tearing away from tissue. To address this, we developed a novel moderate-weight, macroporous, polypropylene mesh (termed the T-line mesh) with mesh extensions to optimize anchoring. We examined the physical properties, biomechanical performance, and biocompatibility of this novel mesh versus a predicate mesh anchored with #0-suture. The physical properties of the T-line mesh and predicate mesh were measured using American Society for Testing and Materials (ASTM) standards. Meshes were implanted into a swine hernia model and harvested after one day to determine anchoring strength of the meshes in the perioperative period. A separate group was implanted into a swine hernia model and harvested at 30 days and 90 days for semiquantitative histological analysis of biocompatibility. T-line mesh physical properties were similar to commonly used moderate-weight meshes in thickness and areal density. The T-line mesh outperformed the predicate mesh in all mechanical testing (P < 0.05). In the perioperative period, the T-line mesh was ∼275% stronger (P < 0.001) than the standard of care. Histological analysis of biocompatibility demonstrated no significant difference between the T-line mesh and predicate mesh (P > 0.05). The T-line mesh is a novel hernia mesh that outperforms a predicate mesh in mechanical and biomechanical performance testing while exhibiting similar biocompatibility. The T-line mesh has the potential to reduce hernia occurrence and recurrence caused by mechanical failure.

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.