Faculty Bibliography

BACKGROUND:Xenografts from genetically modified pigs have become one of the most promising solutions to the dearth of human organs available for transplantation. The challenge in this model has been hyperacute rejection. To avoid this, pigs have been bred with a knockout of the alpha-1,3-galactosyltransferase gene and with subcapsular autologous thymic tissue. METHODS:We transplanted kidneys from these genetically modified pigs into two brain-dead human recipients whose circulatory and respiratory activity was maintained on ventilators for the duration of the study. We performed serial biopsies and monitored the urine output and kinetic estimated glomerular filtration rate (eGFR) to assess renal function and xenograft rejection. RESULTS:in Recipient 2. In both recipients, the creatinine level, which had been at a steady state, decreased after implantation of the xenograft, from 1.97 to 0.82 mg per deciliter in Recipient 1 and from 1.10 to 0.57 mg per deciliter in Recipient 2. The transplanted kidneys remained pink and well-perfused, continuing to make urine throughout the study. Biopsies that were performed at 6, 24, 48, and 54 hours revealed no signs of hyperacute or antibody-mediated rejection. Hourly urine output with the xenograft was more than double the output with the native kidneys. CONCLUSIONS:Genetically modified kidney xenografts from pigs remained viable and functioning in brain-dead human recipients for 54 hours, without signs of hyperacute rejection. (Funded by Lung Biotechnology.).

The therapeutic applications of regulatory T cells (T_regs ) include treating autoimmune diseases, graft-versus-host disease and induction of transplantation tolerance. For ex-vivo expanded T_regs to be used in deceased donor transplantation, they must be able to suppress T cell responses to a broad range of human leukocyte antigen (HLA). Here, we present a novel approach for the expansion of polyspecific T_regs in cynomolgus macaques that was adapted from a good manufacturing practice-compliant protocol. T_regs were isolated by fluorescence-activated cell sorting (FACS) and expanded in the presence of a panel of CD40L-stimulated B cells (CD40L-sBc). Prior to T_reg culture, CD40L-sBc were expanded in vitro from multiple major histocompatibility complex (MHC)-disparate macaques. Expanded T_regs expressed high levels of forkhead box protein 3 (FoxP3) and Helios, a high percentage of T_reg -specific demethylated region (TSDR) demethylation and strong suppression of naïve T cell responses in vitro. In addition, these T_regs produced low levels of inflammatory cytokines and were able to expand post-cryopreservation. Specificity assays confirmed that these T_regs were suppressive upon activation by any antigen-presenting cells (APCs) whose MHC was shared by CD40L-sBc used during expansion, proving that they are polyspecific. We developed an approach for the expansion of highly suppressive cynomolgus macaque polyspecific T_regs through the use of a combination of CD40L-engineered B cells with the potential to be translated to clinical studies. To our knowledge, this is the first report that uses a pool of MHC-mismatched CD40L-sBc to create polyspecific T_regs suitable for use in deceased-donor transplants.