In the above-mentioned article, Fig. 8 D showed the addition of recombinant human (rh) interleukin (IL)-10 with human peripheral blood mononuclear cells (PBMC) in NSG (NOD-SCID …

BACKGROUND:We investigated whether graft produced anti-human CD2, mediated by adenovirus (Adv) transduction of pig neonatal islet cell clusters (pNICC), would protect xenografts in a humanized mouse model from immune attack and whether such immunosuppression would remain local.METHODS:A mouse anti-human CD2 Ab (CD2hb11) previously generated by us was genetically engineered to produce chimeric and humanized versions. The three forms of CD2hb11 were named dilimomab (mouse), diliximab (chimeric) and dilizumab (humanized). All 3 forms of CD2hb11 Ab were tested for their ability to bind CD3(+) human T cells and to inhibit a human anti-pig xenogeneic mixed lymphocyte reaction (MLR). They were administered systemically in a humanized mouse model in order to test their ability to deplete human CD3(+) T cells and whether they induced a cytokine storm. An adenoviral vector expressing diliximab was generated for transduction of pNICC. Humanized mice were transplanted with either control-transduced pNICC or diliximab-transduced pNICC and human T cells within grafts and spleens were enumerated by flow cytometry.RESULTS:Dilimomab and diliximab inhibited a human anti-pig xenogeneic response but dilizumab did not. All 3 forms of CD2hb11 Ab bound human T cells in vitro though dilimomab and diliximab exhibited 300-fold higher avidity than dilizumab. All 3 anti-CD2 Abs could deplete human CD3(+) T cells in vivo in a humanized mouse model without inducing upregulation of activation markers or significant release of cytokines. Humanized mice transplanted with diliximab-transduced pNICC afforded depletion of CD3(+) T cells at the graft site leaving the peripheral immune system intact.CONCLUSIONS:Local production of a single Ab against T cells can reduce graft infiltration at the xenograft site and may reduce the need for conventional, systemic immunosuppression.

Porcine neonatal islet‐like cell clusters (NICC) are being considered as a source of β‐cell replacement. However, the lag time to full function due to hormonal immaturity remains a problem. This study aimed to determine whether time in culture was important for NICC function in vivo.

The instant blood-mediated inflammatory reaction (IBMIR) is a major obstacle to the engraftment of intraportal pig islet xenografts in primates. Higher expression of the galactose-α1,3-galactose (αGal) xenoantigen on neonatal islet cell clusters (NICC) than on adult pig islets may provoke a stronger reaction, but this has not been tested in the baboon model. Here, we report that WT pig NICC xenografts triggered profound IBMIR in baboons, with intravascular clotting and graft destruction occurring within hours, which was not prevented by anti-thrombin treatment. In contrast, IBMIR was minimal when recipients were immunosuppressed with a clinically relevant protocol and transplanted with NICC from αGal-deficient pigs transgenic for the human complement regulators CD55 and CD59. These genetically modified (GM) NICC were less susceptible to humoral injury in vitro than WT NICC, inducing significantly less complement activation and thrombin generation when incubated with baboon platelet-poor plasma. Recipients of GM NICC developed a variable anti-pig antibody response, and examination of the grafts 1 month after transplant revealed significant cell-mediated rejection, although scattered insulin-positive cells were still present. Our results indicate that IBMIR can be attenuated in this model, but long-term graft survival may require more effective immunosuppression or further donor genetic modification.

Aim: Transgenic expression of human complement regulatory proteins or deletion of aGal (GTKO) can protect porcine adult islet and neonatal islet cell cluster (NICC) xenografts, respectively, in monkeys. We investigated the combined effects of these modifications on the outcome of intraportal NICC transplantation in immunosuppressed baboons. Method: 1-5 day old GTKO piglets transgenic for human CD55, CD59 and H-transferasewere used as donors. Recipient baboons receivedGTKO/CD55-CD59-HT NICC under standard (ATG, tacrolimus, mycophenolate mofetil; n=5) or costimulation blockade-based immunosuppression (anti CD2, anti CD154, belatacept, tacrolimus; n=3). The early inflammatory/thrombotic response was compared to that induced by wild type (WT) NICC (n=4). Graft survival was evaluated by immunohistochemical analysis up to 3 months post-transplant. Results:GTKO/CD55-CD59-HT xenografts exhibited no signs of early thrombosis or infiltrate, and recipient platelet counts, fibrinogen and D-dimer levels were unchanged from baseline. In contrast, WT xenografts triggered widespread thrombosis within 12 hrs, with substantial neutrophil and mononuclear cell infiltrate, accompanied by transient decreases in platelet count and fibrinogen and increased D-dimer levels. Analysis of liver biopsies from recipients under standard immunosuppression revealed loss of GTKO/CD55-CD59-HT NICC within one month, with heavy T and B cell infiltrates. However, the change to costimulation blockade-based immunosuppression reduced cellular infiltration, and cells staining positive for insulin, glucagon and somatostatin were present in all GTKO/CD55-CD59-HT xenograftsat three months. Conclusions: Deletion of aGal and expression of human CD55 and CD59 prevent early thrombotic destruction of porcine NICCs in the baboon model. Costimulation blockade-based immunosuppression appears to be more effective than standard immunosuppression in prolonging the survival of genetically modified porcine NICC xenografts.

Aim: The aim of this study was to determine the mechanisms of co-stimulation blockade induced tolerance(Tol) to islet xenografts. Specifically, whether 1)Fox3+ Tregs were involved and 2)were xenospecific Tregs from the Tol-mice capable of transferring dominant tolerance to naïve recipients. Methods:Balb/c mice transplanted(Tx) with porcine-NICCs were given a single dose of CTLA4-Fc(500ug) and 4 doses of MR-1(500ug). At >100 days post Tx, CD4+CD25+ Tregs isolated from spleen of tolerised mice(Tol-Tregs) were co-transferred with naïve BALB/c spenocytes(Teff) into NOD-SCID recipients of NICCs xenografts. Tregs from the spleen of naïve BABL/c mice were used as controls. Islet graft function was assessed by histology and C-peptide. Results: Histology of NICC xenografts 100 days after Tx in CTLA4-Fc/MR1 treated recipients showed intact islets and positive insulin staining. The proportion of Tregs in the graft, spleen and draining lymph node(DLN) of Tol-recipients at 100 day post Tx was higher than in rejecting(Rej) and non-Tx mice. Graft infiltrating cells from Tol-xenografts expressed high levels of TGF-β(p<0.001), IFN-γ(p<0.05) and IL-10 compared to the Rej-group. B220+ T cells were identified in the tolerant grafts but not in the spleen, LN, or DLN suggesting this may be a maker of T cell apoptosis. Tol-Tregs but not naïve Tregs or CD4+FoxP3- Tcells expressed MHC-II. The xenografts in NOD-SCID mice cotransferred with Tol-Tregs:Teffs at a ratio of 1:10 and 1:20 remained intact up to 70 days and mice were porcine c-peptide positive. In contrast, xenografts in NOD-SCID mice transferred with naïve Treg at a ratio of 1:20 were rejected. Graft infiltrating cells from NOD-SCID mice expressed high levels of IL-10. Conclusions Tregs play an important role in maintaining xenograft tolerance following co-stimulation blockade. Xenospecific Tol-Tregs expressed MHC class II, produced high levels of IL-10 and were capable of transferring dominant tolerance.

T cell-mediated rejection remains a barrier to the clinical application of islet xenotransplantation. Regulatory T cells (Treg) regulate immune responses by suppressing effector T cells. This study aimed to determine the ability of human Treg to prevent islet xenograft rejection and the mechanism(s) involved. Neonatal porcine islet transplanted NOD-SCID IL2rγ−/− mice received human peripheral blood mononuclear cells (PBMC) with in vitro expanded autologous Treg in the absence or presence of anti-human interleukin-10 (IL-10) monoclonal antibody. In addition, human PBMC-reconstituted recipient mice received recombinant human IL-10 (rhIL-10). Adoptive transfer with expanded autologous Treg prevented islet xenograft rejection in human PBMC-reconstituted mice by inhibiting graft infiltration of effector cells and their function. Neutralization of human IL-10 shortened xenograft survival in mice receiving human PBMC and Treg. In addition, rhIL-10 treatment led to prolonged xenograft survival in human PBMC-reconstituted mice. This study demonstrates the ability of human Treg to prevent T-cell effector function and the importance of IL-10 in this response. In vitro Treg expansion was a simple and effective strategy for generating autologous Treg and highlighted a potential adoptive Treg cell therapy to suppress antigraft T-cell responses and reduce the requirement for immunosuppression in islet xenotransplantation.

BACKGROUND: A major barrier to the clinical application of xenotransplantation as a treatment option for patients is T cell-mediated rejection. Studies based on experimental rodent models of xenograft tolerance or rejection in vivo have provided useful information about the role of T cell immune response in xenotransplantation. However not all observations seen in rodents faithfully recapitulate the human situation This study aimed to establish a humanized mouse model of xenotransplantation, which mimics xenograft rejection in the context of the human immune system. METHODS: NOD-SCID IL2rγ -/- mice were transplanted with neonatal porcine islet cell clusters (NICC) followed by reconstitution of human peripheral blood mononuclear cells (PBMC). Human leukocyte engraftment and islet xenograft rejection were confirmed by flow cytometric and histological analyses. RESULTS: In the absence of human PBMC, porcine NICC transplanted into NOD-SCID IL2rγ -/- mice revealed excellent graft integrity and endocrine function. Human PBMC demonstrated a high level of engraftment in NOD-SCID IL2rγ -/- mice. Reconstitution of NICC recipient NOD-SCID IL2rγ -/- mice with human PBMC led to the rapid destruction of NICC xenografts in a PBMC number-dependent manner. CONCLUSIONS: Human PBMC-reconstituted NOD-SCID IL2rγ -/- mice provide an ideal model to study human immune responses in xenotransplantation. Studies based on this humanized mouse model will provide insight for improving the outcomes of clinical xenotransplantation.