Recent human decedent model studies^1,2 and compassionate xenograft use³ have explored the promise of porcine organs for human transplantation. To proceed to human studies, a clinically ready porcine donor must be engineered and its xenograft successfully tested in nonhuman primates. Here we describe the design, creation and long-term life-supporting function of kidney grafts from a genetically engineered porcine donor transplanted into a cynomolgus monkey model. The porcine donor was engineered to carry 69 genomic edits, eliminating glycan antigens, overexpressing human transgenes and inactivating porcine endogenous retroviruses. In vitro functional analyses showed that the edited kidney endothelial cells modulated inflammation to an extent that was indistinguishable from that of human endothelial cells, suggesting that these edited cells acquired a high level of human immune compatibility. When transplanted into cynomolgus monkeys, the kidneys with three glycan antigen knockouts alone experienced poor graft survival, whereas those with glycan antigen knockouts and human transgene expression demonstrated significantly longer survival time, suggesting the benefit of human transgene expression in vivo. These results show that preclinical studies of renal xenotransplantation could be successfully conducted in nonhuman primates and bring us closer to clinical trials of genetically engineered porcine renal grafts.

De novo donor‐specific antibody (DSA) after organ transplantation promotes antibody‐mediated rejection (AMR) and causes late graft loss. Previously, we demonstrated that depletion using anti‐CD3 immunotoxin combined with tacrolimus and alefacept (AMR regimen) reliably induced early DSA production with AMR in a nonhuman primate kidney transplant model. Five animals were assigned as positive AMR controls, four received additional belatacept and four received additional anti‐CD40 mAb (2C10R4). Notably, production of early de novo DSA was completely attenuated with additional belatacept or 2C10R4 treatment. In accordance with this, while positive controls experienced a decrease in peripheral IgM⁺ B cells, bela‐ and 2C10R4‐added groups maintained a predominant population of IgM⁺ B cells, potentially indicating decreased isotype switching. Central memory T cells (CD4⁺CD28⁺CD95⁺) as well as PD‐1^hiCD4⁺ T cells were decreased in both bela‐added and 2C10R4‐added groups. In analyzing germinal center (GC) reactions in situ, lymph nodes further revealed a reduction of B cell clonal expansion, GC‐follicular helper T (Tfh) cells, and IL‐21 production inside GCs with additional belatacept or 2C10R4 treatment. Here we provide evidence that belatacept and 2C10R4 selectively suppresses the humoral response via regulating Tfh cells and prevents AMR in this nonhuman primate model.

Alemtuzumab is a monoclonal antibody that depletes T and B cells and is used as induction therapy for renal transplant recipients. Without long-term calcineurin inhibitor (CNI) therapy, alemtuzumab-treated patients have a propensity to develop alloantibody and may undergo antibody-mediated rejection (AMR). In pursuit of a mechanistic explanation, we analyzed peripheral B cells and serum of these patients for BAFF (Blys) and BAFF-R, factors known to be integral for B-cell activation, survival, and homeostasis. Serum BAFF levels of 22/24 alemtuzumab-treated patients were above normal range, with average levels of 1967 pg/mL compared to 775 pg/mL in healthy controls (p = 0.006). BAFF remained elevated 2 years posttransplant in 78% of these patients. BAFF-R on CD19⁺ B cells was significantly downregulated, suggesting ligand/receptor engagement. BAFF mRNA expression was increased 2–7-fold in CD14⁺ cells of depleted patients, possibly linking monocytes to the BAFF dysregulation. Addition of recombinant BAFF to mixed lymphocyte cultures increased B-cell activation to alloantigen, as measured by CD25 and CD69 coexpression on CD19⁺ cells. Of note, addition of sirolimus (SRL) augmented BAFF-enhanced B-cell activation whereas CNIs blocked it. These data suggest associations between BAFF/BAFF-R and AMR in alemtuzumab-treated patients.

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.

Background. Injuries in kidney transplant is currently diagnosed by needle biopsy. A noninvasive test that sensitively detects these injuries would benefit the patients. Methods. Urine samples were collected from healthy controls and kidney transplant recipients. Urine samples were screened first with an antibody array consisting of 120 chemokines and cytokines and then with a multiplex beads assay. Representative parameters, including macrophage inflammatory protein-1Δ, osteoprotegerin, monokine induced by interferon-γ (IFN), and IFN-γ–induced protein of 10 kDa, were simultaneously determined by a quadruplex assay in urine samples from 84 patients with renal allograft injury, 29 patients with stable graft function, and 19 healthy individuals. Results. Twenty-three cytokines/chemokines were found to be elevated in urine samples of patients with acute rejection by the antibody array. The second round of screening confirmed that 11 of the 23 parameters were elevated in the patients but not in the healthy controls. Induced protein of 10 kDa and monokine induced by IFN-γ were significantly elevated in urine samples of patients with acute renal injury, and macrophage inflammatory protein-1Δ and osteoprotegerin were significantly elevated in patients with both acute and chronic renal injuries. The combination of the four parameters had a high positive detection rate (97.6%) for renal transplant injury and could differentiate between acute and chronic injury. Conclusion. These results might indicate that the present multiplex assay provides a basis to establish a noninvasive method for the diagnosis and monitoring of renal transplant injury.

Spontaneous acceptance of liver allografts occurs in several species. However, tolerance is rare in human transplant patients even though rejection is relatively easily reversed. Histological features of acute rejection in liver transplantation are similar to those in other organs. Nevertheless, mechanisms of rejection of liver transplants may differ in degrees and cellular involvement. Liver-specific cell populations, such as Kupffer cells (KCs), liver sinusoidal epithelial cells (LSECs), and hepatic stellate cells (HSCs), may contribute to liver tolerogenicity. Other mechanisms, such as microchimerism, soluble major histocompatibility complex (MHC molecules), donor human leukocyte antigen (HLA)-C genotype, and regulatory T cells, may participate in inducing tolerance. The low incidence of hyperacute or antibody-mediated rejection in liver might be linked to the infrequency of chronic rejection of liver transplants. Understanding the mechanisms of liver transplant rejection/tolerance and the availability of better immune monitoring could help develop strategies to recognize tolerance and reduce rejection.