Islet Xenograft Survival Research Articles

A bioengineered artificial interstitium supports long-term islet xenograft survival in nonhuman primates without immunosuppression.

Cell-based therapies hold promise for many chronic conditions; however, the continued need for immunosuppression along with challenges in replacing cells to improve durability or retrieving cells for safety are major obstacles. We subcutaneously implanted a device engineered to exploit the innate transcapillary hydrostatic and colloid osmotic pressure generating ultrafiltrate to mimic interstitium. Long-term stable accumulation of ultrafiltrate was achieved in both rodents and nonhuman primates (NHPs) that was chemically similar to serum and achieved capillary blood oxygen concentration. The majority of adult pig islet grafts transplanted in non-immunosuppressed NHPs resulted in xenograft survival >100 days. Stable cytokine levels, normal neutrophil to lymphocyte ratio, and a lack of immune cell infiltration demonstrated successful immunoprotection and averted typical systemic changes related to xenograft transplant, especially inflammation. This approach eliminates the need for immunosuppression and permits percutaneous access for loading, reloading, biopsy, and recovery to de-risk the use of "unlimited" xenogeneic cell sources to realize widespread clinical translation of cell-based therapies.

401.1: Surface modification of alginate-based microcapsules using ethylamine-bridged EGCG dimer improves the structural stability of microcapsules and supports islet-xenograft survival

401.1: Surface modification of alginate-based microcapsules using ethylamine-bridged EGCG dimer improves the structural stability of microcapsules and supports islet-xenograft survival

CTLA4Ig/VISTAIg combination therapy selectively induces CD4+ T cell-mediated immune tolerance by targeting the SOCS1 signaling pathway in porcine islet xenotransplantation.

T cell inhibitory receptors can regulate the proliferation or function of T cells by binding to their ligands and present a unique opportunity to manage destructive immune responses during porcine islet xenotransplantation. We applied ex vivo porcine islet xenotransplantation and in vitro mixed lymphocyte-islet reaction models to assess immune checkpoint receptor expression profiles in recipient T cells, investigated whether CTLA4 or VISTA immunoglobulin (Ig) combination therapy alone could suppress porcine islet xenograft rejection and further analyzed its potential immune tolerance mechanism. Recipient T cells expressed moderate to high levels of CTLA4, PD-1, TIGIT and VISTA, and the frequency of CTLA4+ CD4+ , TIGIT+ CD4+ , VISTA+ CD4+ and VISTA+ CD8+ T cells was positively correlated with porcine islet xenograft survival time in xenotransplant recipients. Combined treatment with CTLA4Ig and VISTAIg selectively inhibited recipient CD4+ T cell hyper-responsiveness and proinflammatory cytokine production and significantly delayed xenograft rejection. SOCS1 deficiency in CD4+ T cells stimulated by xenogeneic islets facilitated hyper-responsiveness and abolished the suppressive effect of combination therapy on recipient T cell-mediated porcine islet damage in vivo and in vitro. Further mechanistic studies revealed that combined treatment significantly induced SOCS1 expression and inhibited the Jak-STAT signalling pathway in wild-type recipient CD4+ T cells stimulated by xenogeneic islets, whereas SOCS1 deficiency resulted in Jak-STAT signalling pathway activation in recipient CD4+ T cells. We demonstrated a major role for CTLA4 and VISTA as key targets in CD4+ T cell hyper-responsiveness and porcine islet xenograft rejection. The selective inhibition of CD4+ T cell immunity by CTLA4Ig/VISTAIg is based on SOCS1-dependent signalling.

HLA-G1+ Expression in GGTA1KO Pigs Suppresses Human and Monkey Anti-Pig T, B and NK Cell Responses.

The human leukocyte antigen G1 (HLA-G1), a non-classical class I major histocompatibility complex (MHC-I) protein, is a potent immunomodulatory molecule at the maternal/fetal interface and other environments to regulate the cellular immune response. We created GGTA1-/HLAG1+ pigs to explore their use as organ and cell donors that may extend xenograft survival and function in both preclinical nonhuman primate (NHP) models and future clinical trials. In the present study, HLA-G1 was expressed from the porcine ROSA26 locus by homology directed repair (HDR) mediated knock-in (KI) with simultaneous deletion of α-1-3-galactotransferase gene (GGTA1; GTKO) using the clustered regularly interspersed palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas9) gene-editing system. GTKO/HLAG1+ pigs showing immune inhibitory functions were generated through somatic cell nuclear transfer (SCNT). The presence of HLA-G1 at the ROSA26 locus and the deletion of GGTA1 were confirmed by next generation sequencing (NGS) and Sanger’s sequencing. Fibroblasts from piglets, biopsies from transplantable organs, and islets were positive for HLA-G1 expression by confocal microscopy, flow cytometry, or q-PCR. The expression of cell surface HLA-G1 molecule associated with endogenous β2-microglobulin (β2m) was confirmed by staining genetically engineered cells with fluorescently labeled recombinant ILT2 protein. Fibroblasts obtained from GTKO/HLAG1+ pigs were shown to modulate the immune response by lowering IFN-γ production by T cells and proliferation of CD4+ and CD8+ T cells, B cells and natural killer (NK) cells, as well as by augmenting phosphorylation of Src homology region 2 domain-containing phosphatase-2 (SHP-2), which plays a central role in immune suppression. Islets isolated from GTKO/HLA-G1+ genetically engineered pigs and transplanted into streptozotocin-diabetic nude mice restored normoglycemia, suggesting that the expression of HLA-G1 did not interfere with their ability to reverse diabetes. The findings presented here suggest that the HLA-G1+ transgene can be stably expressed from the ROSA26 locus of non-fetal maternal tissue at the cell surface. By providing an immunomodulatory signal, expression of HLA-G1+ may extend survival of porcine pancreatic islet and organ xenografts.

Toll-like receptor 2-modulating pectin-polymers in alginate-based microcapsules attenuate immune responses and support islet-xenograft survival

Encapsulation of pancreatic islets in alginate-microcapsules is used to reduce or avoid the application of life-long immunosuppression in preventing rejection. Long-term graft function, however, is limited due to varying degrees of host tissue responses against the capsules. Major graft-longevity limiting responses include inflammatory responses provoked by biomaterials and islet-derived danger-associated molecular patterns (DAMPs). This paper reports on a novel strategy for engineering alginate microcapsules presenting immunomodulatory polymer pectin with varying degrees of methyl-esterification (DM) to reduce these host tissue responses. DM18-pectin/alginate microcapsules show a significant decrease of DAMP-induced Toll-Like Receptor-2 mediated immune activation in vitro, and reduce peri-capsular fibrosis in vivo in mice compared to higher DM-pectin/alginate microcapsules and conventional alginate microcapsules. By testing efficacy of DM18-pectin/alginate microcapsules in vivo, we demonstrate that low-DM pectin support long-term survival of xenotransplanted rat islets in diabetic mice. This study provides a novel strategy to attenuate host responses by creating immunomodulatory capsule surfaces that attenuate activation of specific pro-inflammatory immune receptors locally at the transplantation site.

2289-PUB: Comparison of GalT-KO Porcine Islet Xenograft Survival between Microcapsule against Hypoxia and Microcapsule against Fibrosis in Diabetic Mice Model

Hypoxia and fibrosis are one of the most important obstacles for encapsulated islet transplantation. Previously, we have developed alginate-based microcapsules improving fibrosis (alginate-chitosan) or hypoxia (alginate-perfluorodecalin) and proved their effects. In this study, we compared long-term graft survival of encapsulated islet with microcapsule against fibrosis or hypoxia in diabetic mice model, using 1,3-galactosyltransferase gene-knockout (GalT-KO) pigs. Islets were isolated form GalT-KO pigs and then encapsulated with alginate-chitosan or alginate-perfluorodecalin microcapsule. To magnify graft survival gap between different microcapsules, marginal dose of islets were transplanted into streoptozotocin-induced diabetic mice intraperitoneally. Blood glucose level were measured for a year. Intraperitoneal glucose tolerance test (IPGTT) was performed at 6 and 12 months after transplantation. In the alginate-chitosan group, the blood glucose level of diabetic mice decreased after encapsulated islet transplantation. However, blood glucose level increased again within two months in 3 out of 4 mice. In the alginate-perfluorodecalin group, the blood glucose level of diabetic mice decreased after transplantation and maintained normoglycemia more than 6 months. Although blood glucose level tended to increase 6 months after transplantation, it remained below 300 mg/dL by one year. The area under curve (AUC) of IPGTT was similar between two groups at baseline. At 6 months after transplantation, AUC of IPGTT in alginate-perflourodecalin group lower than AUC of alginate-chitosan group. At one year after transplantation, microcapsules were retrieved. Dithizone stain was positive in most retrieved islet and fibrosis was minimal in both groups. In conclusion, microcapsule against hypoxia showed a longer xenograft survival than microcapsule against fibrosis in diabetic mice. Disclosure E. Lee: None. Y. Yang: None. Y. Cho: None. S. Lee: None. J. Cho: None. K. Yoon: None. Funding Cooperative Research Program for Agriculture Science and Technology Development (PJ01345301); Rural Development Administration, Republic of Korea

Rejection of xenogeneic porcine islets in humanized mice is characterized by graft-infiltrating Th17 cells and activated B cells.

Xenogeneic porcine islet transplantation is a promising potential therapy for type 1 diabetes (T1D). Understanding human immune responses against porcine islets is crucial for the design of optimal immunomodulatory regimens for effective control of xenogeneic rejection of porcine islets in humans. Humanized mice are a valuable tool for studying human immune responses and therefore present an attractive alternative to human subject research. Here, by using a pig-to-humanized mouse model of xenogeneic islet transplantation, we described the human immune response to transplanted porcine islets, a process characterized by dense islet xenograft infiltration of human CD45+ cells comprising activated human B cells, CD4+ CD44+ IL-17+ Th17 cells, and CD68+ macrophages. In addition, we tested an experimental immunomodulatory regimen in promoting long-term islet xenograft survival, a triple therapy consisting of donor splenocytes treated with ethylcarbodiimide (ECDI-SP), and peri-transplant rituximab and rapamycin. We observed that the triple therapy effectively inhibited graft infiltration of T and B cells as well as macrophages, promoted transitional B cells both in the periphery and in the islet xenografts, and provided a superior islet xenograft protection. Our study therefore indicates an advantage of donor ECDI-SP treatment in controlling human immune cells in promoting long-term islet xenograft survival.

Overcoming Immunobiological Barriers Against Porcine Islet Xenografts: What Should Be Done?

Porcine islets might represent an ideal solution to the severe shortage of living donor islets available for transplantation and thus have great potential for the treatment of diabetes. Although tremendous progress has been achieved through recent experiments, the immune response remains a major obstacle. This review first describes the 3 major pathways of rejection: hyperacute rejection mediated by preformed natural antibodies and complement, instant blood-mediated inflammatory reactions, and acute cell-mediated rejection. Furthermore, this review examines immune-related strategies, including major advances, which have been shown to extend the life and/or function of porcine islets in vitro and in vivo: (1) genetic modification to make porcine islets more compatible with the recipient, (2) optimization of the newly defined biological agents that have been shown to promote long-term survival of xenografts in nonhuman primates, and (3) development of novel immunoisolation technologies that maintain the long-term survival of islet xenografts without the use of systemic immunosuppressive drugs. Finally, the clinical application of porcine islet transplantation is presented. Even though less clinical information is available, experimental data indicate that porcine islet xenografts are likely to become a standard treatment for patients with type 1 diabetes in the future.

Adoptive transfer of xenoantigen‑stimulated T cell receptor Vβ‑restricted human regulatory T cells prevents porcine islet xenograft rejection in humanized mice.

Polyclonal expansion of human regulatory T cells (Tregs) prevents xenogeneic rejection by suppressing effector T cell responses in vitro and in vivo. However, a major limitation to using polyclonally expanded Tregs is that they may cause pan-immunosuppressive effects. The present study was conducted to compare the ability of ex vivo expanded human xenoantigen-stimulated Tregs (Xeno-Treg) and polyclonal Tregs (Poly-Treg) to protect islet xenografts from rejection in NOD-SCID interleukin (IL)-2 receptor (IL2r)γ−/− mice. Human cluster of differentiation (CD)4+CD25+CD127lo Tregs, expanded either by stimulating with porcine peripheral blood mononuclear cells (PBMCs) or anti-CD3/CD28 beads, were characterized by immune cell phenotyping, T cell receptor (TCR) Vβ CDR3 spectratyping and performing suppressive activity assays in vitro. The efficiency of adoptively transferred ex vivo human Tregs was evaluated in vivo using neonatal porcine islet cell clusters (NICC) transplanted into NOD-SCID IL-2rγ−/− mice, which received human PBMCs with or without Xeno-Treg or Poly-Treg. Xeno-Treg, which expressed increased levels of human leukocyte antigen-DR and secreted higher levels of IL-10, demonstrated enhanced suppressive capacity in a pig-human mixed lymphocyte reaction. Spectratypes of TCR Vβ4, Vβ10, Vβ18 and Vβ20 in Xeno-Treg showed restriction and expanded clones at sizes of 205, 441, 332 and 196 respectively, compared to those of Poly-Treg. Reconstitution of mice with human PBMCs and Poly-Treg resulted in NICC xenograft rejection at 63 days. Adoptive transfer with human PBMCs and Xeno-Treg prolonged islet xenograft survival beyond 84 days, with grafts containing intact insulin-secreting cells surrounded by a small number of human CD45+ cells. This study demonstrated that adoptive transfer of ex vivo expanded human Xeno-Treg may potently prevent islet xenograft rejection in humanized NOD-SCID IL2rγ−/− mice compared with Poly-Treg. These findings suggested that adoptive Treg therapy may be used for immunomodulation in islet xenotransplantation by minimizing systemic immunosuppression.

Immunosuppressive effect of arsenic trioxide on islet xenotransplantation prolongs xenograft survival in mice

The role of arsenic trioxide (As2O3) in inhibiting immune rejection and prolonging islet allograft survival has been identified in islet allotransplantation. This study aims to explore the role of As2O3 in islet xenotransplantation and the action mechanism. The streptozotocin (STZ) was used in C57BL/6 mice to induce the type 1 diabetes mellitus (T1DM) for xenotransplantation models establishment. Donor islets were isolated by digesting. The flow cytometry (FCM) was used to analyze lymphocyte types. The blood sugar level was detected by using intraperitoneal glucose tolerance test (IPGTT). The serum level of cytokines was determined by the enzyme-linked immunosorbent assay (ELIZA). The cell proliferation was measured by MTT assay. The mRNA levels were quantified with qRT-PCR. As2O3 prolonged the survival of the recipient mice but had no influence on body weight. As2O3 protected the function of xenograft in insulin secretion and suppressed immune rejection of recipient. As2O3 inhibited proliferation of T lymphocyte and increased the proportion of Foxp3+ regulatory T cells in recipient mice. As2O3 inhibited activation and promoted clonal anergy of T lymphocyte. As2O3 decreased total number of B cells and reduced partial antibody levels in recipient mice. As2O3 and leflunomide showed a synergistic effect in suppressing islet xenotransplant rejection. As2O3 prolongs islet xenograft survival by inhibiting cellular immune response, and increasing Foxp3+ regulatory T cells, while decreasing partial antibody levels in serum.

The effect of epitope-based ligation of ICAM-1 on survival and retransplantation of pig islets in nonhuman primates.

Pig islet xenotransplantation is a promising alternative to allogeneic transplantation. However, the wide immunologic barrier between pigs and primates limits the long-term survival of the graft. MD-3, a novel monoclonal antibody (mAb) that recognizes a particular epitope of human ICAM-1, can render T cells tolerant to a xenograft by arresting dendritic cell maturation. We report the long-term survival of adult wild-type pig islets and successful retransplantation in nonhuman primates using a protocol comprising induction with MD-3 mAb and maintenance with anti-CD154 mAb and sirolimus. Eleven rhesus monkeys were assigned to three groups. Group 1 (n=4) involved treatment with MD-3 induction, short-term (<4months) administration of anti-CD154 mAb, and maintenance therapy with sirolimus. Group 2 (n=4) involved treatment with MD-3 induction and long-term maintenance therapy with anti-CD154 mAb and sirolimus. Group 3 (n=3) involved only maintenance therapy with anti-CD154 mAb and sirolimus. Diabetes was induced in monkeys by streptozotocin, and pig islets (61000-112000IEQ/kg for each transplant; up to 280000IEQ/kg per recipient) were infused through the portal vein. The in vivo functional potency of the isolated islets was tested by minimal model transplant in streptozotocin-induced diabetic NOD/SCID mice, and the mean AUC of blood glucose level divided by the number of follow-up days was calculated. The islet grafts survived more than 6months (between 225 and 727days) in nine of 12 transplants of MD-3-treated groups 1 and 2, whereas in the absence of MD-3 mAb, survival was <40days. In three transplants of the MD-3-treated Group 2, functional graft survival was only for 104, 125, and 154days. In these cases, a retrospective analysis suggested that the relatively short survival duration was associated with the relatively high AUC value in the NOD/SCID bioassay. Notably, when retransplantation was performed in Group 3, blood glucose control was extended up to 956days, which was supported by MD-3 mAb-based suppression of adaptive immunity. No replication of cytomegalovirus genes was observed. Long-term survival of pig islet xenografts and successful retransplantation were achieved with MD-3 mAb-based immunosuppression regimen in this pig-to-monkey transplantation model. It should be emphasized that these encouraging results were achieved following the transplantation of islets from pigs that had not been genetically modified. Considering that it is possible to further substantially reduce the destruction of grafted islet using genetically modified pig islet, the islet requirement could be reduced and much longer graft survival can be achieved.

Ex vivo-expanded baboon CD39+ regulatory T cells prevent rejection of porcine islet xenografts in NOD-SCID IL-2rγ-/- mice reconstituted with baboon peripheral blood mononuclear cells.

A high immunosuppressive burden is required for long-term islet xenograft survival in non-human primates even using genetically modified donor pigs. We aimed to investigate the capacity of baboon regulatory T cells (Treg) to suppress islet xenograft rejection, thereby developing a potential immunoregulatory or tolerance therapy that could be evaluated in NHP models of xenotransplantation. Baboon Treg expanded with stimulation by porcine peripheral blood mononuclear cells (PBMC) were characterized by cell phenotyping and suppressive activity assays in vitro. Their function in vivo was evaluated in neonatal porcine islet cell clusters (NICC) transplanted NOD-SCID IL-2rγ-/- (NSG) mice receiving baboon PBMC alone or with expanded autologous Treg. The majority of expanded Treg coexpressed Foxp3 and CD39 and were highly suppressive of the baboon anti-pig xenogeneic T cell response in vitro. Reconstitution of mice with baboon PBMC alone resulted in NICC xenograft rejection within 35days. Cotransfer with baboon PBMC and Treg prolonged islet xenograft survival beyond 100days, correlating with Treg engraftment, intragraft CD39 and Foxp3 gene expression, and reduced graft infiltrating effector T cells and reduced interferon-γ production. Our data supports the capacity of ex vivo expanded CD39+ baboon Treg to suppress islet xenograft rejection in primatized mice, suggesting it haspotential as an adjunctive immunotherapy in preclinical NHP models of xenotransplantation.

Differential Role of B Cells and IL-17 Versus IFN-γ During Early and Late Rejection of Pig Islet Xenografts in Mice.

Xenogeneic islet transplantation is an emerging therapeutic option for diabetic patients. However, immunological tolerance to xenogeneic islets remains a challenge. The current study used a pig-to-mouse discordant xenogeneic islet transplant model to examine antidonor xenogeneic immune responses during early and late rejection and to determine experimental therapeutic interventions that promote durable pig islet xenograft survival. We found that during early acute rejection of pig islet xenografts, the rejecting hosts exhibited a heavy graft infiltration with B220 B cells and a robust antipig antibody production. In addition, early donor-stimulated IL-17 production, but not IFN-γ production, dominated during early acute rejection. Recipient treatment with donor apoptotic 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide-treated splenocytes significantly inhibited antidonor IL-17 response, and when combined with B cell depletion and a short course of rapamycin led to survival of pig islet xenografts beyond 100 days in approximately 65% recipients. Interestingly, treated recipients in this model experienced late rejection between 100 and 200 days posttransplant, which coincided with B cell reconstitution and an ensuing emergence of a robust antidonor IFN-γ, but not IL-17, response. These findings reveal that early and late rejection of pig islet xenografts may be dominated by different immune responses and that maintenance of long-term xenogeneic tolerance will require strategies that target the temporal sequence of antixenogeneic immune responses.

Beneficial effects of the transgenic expression of human sTNF-αR-Fc and HO-1 on pig-to-mouse islet xenograft survival

Both human soluble tumor necrosis factor-α receptor-Fc (sTNF-αR-Fc) and heme oxygenase-1 (HO-1) transgenic pigs have been generated previously for xenotransplantation. Here, we investigated whether overexpression of sTNF-αR-Fc or HO-1 in pig islets prolongs islet xenograft survival. Adult porcine islets were isolated from human sTNF-αR-Fc or HO-1 transgenic and wild type pigs, and were transplanted into diabetic nude mice. Effects of the expression of both genes on islet apoptosis, chemokine expression, cellular infiltration, antibody production, and islet xenograft survival were analyzed. Human sTNF-αR-Fc transgenic pigs successfully expressed sTNF-αR-Fc in the islets; human HO-1 transgenic pigs expressed significant levels of HO-1 in the islets. Pig-to-mouse islet xenograft survival was significantly prolonged in both the sTNF-αR-Fc and HO-1 groups compared with that in the wild type group. Both the sTNF-αR-Fc and HO-1 groups exhibited suppressed intragraft expression of monocyte chemoattractant protein-1 (MCP-1) and decreased perigraft infiltration of immune cells. However, there was no difference in the anti-pig antibody levels between the groups. Apoptosis of islet cells during the early engraftment was suppressed only in the HO-1 group. Porcine islets from both sTNF-αR-Fc and HO-1 transgenic pigs prolonged xenograft survival by suppressing islet cell apoptosis or secondary inflammatory responses following islet death, indicating that these transgenic pigs might have applications in successful islet xenotransplantation.

The Introduction of Human Heme Oxygenase-1 and Soluble Tumor Necrosis Factor-α Receptor Type I With Human IgG1 Fc in Porcine Islets Prolongs Islet Xenograft Survival in Humanized Mice

Apoptosis during engraftment and inflammation induce poor islet xenograft survival. We aimed to determine whether overexpression of human heme oxygenase-1 (HO-1) or soluble tumor necrosis factor-α receptor type I with human IgG1 Fc (sTNF-αR-Fc) in porcine islets could improve islet xenograft survival. Adult porcine islets were transduced with adenovirus containing human HO-1, sTNF-αR-Fc, sTNF-αR-Fc/HO-1 or green fluorescent protein (control). Humanized mice were generated by injecting human cord blood-derived CD34(+) stem cells into NOD-scid-IL-2Rγ(null) mice. Both HO-1 and sTNF-αR-Fc reduced islet apoptosis under in vitro hypoxia or cytokine stimuli and suppressed RANTES induction without compromising insulin secretion. Introduction of either gene into islets prolonged islet xenograft survival in pig-to-humanized mice transplantation. The sTNF-αR-Fc/HO-1 group showed the best glucose tolerance. Target genes were successfully expressed in islet xenografts. Perigraft infiltration of macrophages and T cells was suppressed with decreased expression of RANTES, tumor necrosis factor-α and IL-6 in treatment groups; however, frequency of pig-specific interferon-γ-producing T cells was not decreased, and humoral response was not significant in any group. Early apoptosis of islet cells was suppressed in the treatment groups. In conclusion, overexpression of HO-1 or sTNF-αR-Fc in porcine islets improved islet xenograft survival by suppressing both apoptosis and inflammation. HO-1 or sTNF-αR-Fc transgenic pigs have potential for islet xenotransplantation.

CXCL12-delivery by double microcapsules and dual costimulatory blockade synergistically prolong the survival of porcine islet xenografts in diabetic NOD mice

Event Abstract Back to Event CXCL12-delivery by double microcapsules and dual costimulatory blockade synergistically prolong the survival of porcine islet xenografts in diabetic NOD mice Susan Safley1, Graham F. Barber1, Stephanie M. Duncanson2, Robert W. Holdcraft3, Lawrence S. Gazda3, Mark C. Poznansky4, Athanassios Sambanis2 and Collin J. Weber1 1 Emory University, Surgery, United States 2 Georgia Institute of Technology, School of Chemical and Biomolecular Engineering, United States 3 The Rogosin Institute, Xenia Division, United States 4 Massachusetts General Hospital, Vaccine and Immunotherapy Center, United States Introduction: Our goal is to improve islet transplantation for patients with Type I diabetes mellitus. Because human donor islets are in short supply, we are studying the efficacy of microencapsulated adult porcine islets (APIs) transplanted intraperitoneally in diabetic NOD mice. We have developed a biocompatible and durable double alginate microcapsule that excludes host IgG and that prevents islets from protruding through the capsule wall. The objective of this study was to test the effectiveness of our double microcapsule for therapeutic delivery of CXCL12 (SDF-1), a chemokine that provides pro-survival signals to islets and also recruits regulatory T cells while repelling effector T cells[1],[2]. In NOD mice, we have evaluated delivery of CXCL12 via our double microcapsule, alone and in combination with: (1) belatacept (CTLA4-Ig)[3], (2) anti-CD154 mAb, and (3) the combination of CTLA4-Ig plus anti-CD154 mAb. Since CD4+ T cells are the primary effectors in the rejection of islet xenografts[4],[5], we also have assessed the efficacy of a non-depleting anti-CD4 mAb (YTS177.9) that spares regulatory T cells[6], alone and in combination with CXCL12. Materials and Methods: APIs were isolated from adult pig pancreata, prepared as described[7], encapsulated in our double capsules as described[8] and transplanted i.p. in diabetic NOD mice (9,000 IEQ/mouse) given no immunosuppresssion, CTLA4-Ig (Orencia, Bristol-Myers Squibb, New York, NY,400 mg i.p. on day 0, QOD for 21 days, then once weekly), anti-CD154 mAb (BioXCell, West Lebanon, NH, 500 mg i.p. days 0, 2, 4, and 6 then once weekly), or non-depleting anti-CD4 mAb (YTS177.9, BioXCell, 250 mg i.p. days -3, -2, -1, then weekly). CXCL12 (Preprotech, Rocky Hill, NJ) was incorporated into the matrix of our double capsule (1μg/mL alginate). Graft function was monitored by measuring random blood glucose (BG) and histologic analyses of graft biopsies. Results and Discussion: APIs in double capsules functioned for 45 ± 33 days in diabetic NOD mice given no immunosuppression (n=24). Intracapsular CXCL12 alone prolonged graft survival for 90 ± 123 days (n=10), similar to the effects of CTLA4-Ig alone (77 ± 66, n=9), and not significantly longer than survival in untreated mice. The combination of CXCL12 and CTLA4-Ig (n=5) has augmented graft survival for 105 ± 52 days to date (n=5) with two animals still living with functioning grafts on day 146 post-transplant. Anti-CD4 mAb (YTS177.9) alone yielded significantly prolonged graft survival compared to untreated controls (217 ± 56, p<0.001, n=6). The addition of CTLA4-Ig to YTS177.9 did not further improve graft function (168 ± 44, n=6). Dual costimulatory blockade (CTLA4-Ig plus anti-CD154 mAb) (n=25) significantly prolonged graft function to 144 ± 74 days (p<0.001, vs no treatment). The most durable and consistent results to date were found when NOD mice were treated with CTLA4-Ig and anti-CD154 mAb plus CXCL12 (n=5), which resulted in encapsulated API xenograft function for 438 ± 133 days. Conclusions: Single therapy with a non-depleting anti-CD4 mAb (YTS177.9) significantly augmented API xenograft function in diabetic NOD mice for up to 301 days, suggesting that our double capsule may allow reduced immuno-suppression. Notably, CXCL12 plus CTLA4-Ig and anti-CD154 mAb has resulted in robust graft function for >300 days in all mice tested, up to 645 days in one animal. Both CXCL12 and YTS177.9 enhance regulatory T cell responses, and therefore we hypothesize that recruitment of these immunoregulatory T cells may be an important mechanism for the survival and function of encapsulated porcine islet xenografts. Our data suggest that combining CXCL12 or YTS177.9 with CTLA4-Ig and anti-CD154 mAb may be an effective means of augmenting the survival and function of encapsulated APIs. Funding from the Juvenile Diabetes Research Foundation

Progress in pig-to-non-human primate transplantation models (1998-2013): a comprehensive review of the literature.

The pig-to-non-human primate model is the standard choice for in vivo studies of organ and cell xenotransplantation. In 1998, Lambrigts and his colleagues surveyed the entire world literature and reported all experimental studies in this model. With the increasing number of genetically engineered pigs that have become available during the past few years, this model is being utilized ever more frequently. We have now reviewed the literature again and have compiled the data we have been able to find for the period January 1, 1998 to December 31, 2013, a period of 16 yr. The data are presented for transplants of the heart (heterotopic and orthotopic), kidney, liver, lung, islets, neuronal cells, hepatocytes, corneas, artery patches, and skin. Heart, kidney, and, particularly, islet xenograft survival have increased significantly since 1998. The reasons for this are briefly discussed. A comment on the limitations of the model has been made, particularly with regard to those that will affect progression of xenotransplantation toward the clinic.

A review of piscine islet xenotransplantation using wild-type tilapia donors and the production of transgenic tilapia expressing a "humanized" tilapia insulin.

Most islet xenotransplantation laboratories have focused on porcine islets, which are both costly and difficult to isolate. Teleost (bony) fish, such as tilapia, possess macroscopically visible distinct islet organs called Brockmann bodies which can be inexpensively harvested. When transplanted into diabetic nude mice, tilapia islets maintain long-term normoglycemia and provide human-like glucose tolerance profiles. Like porcine islets, when transplanted into euthymic mice, they are rejected in a CD4 T-cell-dependent manner. However, unlike pigs, tilapia are so phylogenetically primitive that their cells do not express α(1,3)Gal and, because tilapia are highly evolved to live in warm stagnant waters nearly devoid of dissolved oxygen, their islet cells are exceedingly resistant to hypoxia, making them ideal for transplantation within encapsulation devices. Encapsulation, especially when combined with co-stimulatory blockade, markedly prolongs tilapia islet xenograft survival in small animal recipients, and a collaborator has shown function in diabetic cynomolgus monkeys. In anticipation of preclinical xenotransplantation studies, we have extensively characterized tilapia islets (morphology, embryologic development, cell biology, peptides, etc.) and their regulation of glucose homeostasis. Because tilapia insulin differs structurally from human insulin by 17 amino acids, we have produced transgenic tilapia whose islets stably express physiological levels of humanized insulin and have now bred these to homozygosity. These transgenic fish can serve as a platform for further development into a cell therapy product for diabetes.

Human α1-Antitrypsin and Temporary T Cell Depletion Synergize to Expand Regulatory T Cells and to Extend Allogeneic Skin Graft Survival.

Introduction: Treatment with α1-antityrpsin (AAT), an anti-inflammatory tolerogenic circulating protein that lacks direct effect on T cells, prolongs rat-to-mouse pancreatic islet xenograft survival in combination with temporary T cell depletion, yet treatment with each drug separately results in graft failure. Aim: To elucidate the mechanism behind the synergy and examine its effect on skin allograft transplants. Methods: Wild type (WT) and human AAT transgenic mice (hAAT+/+) (both C57BL/6; H-2b background) were used as graft recipients. Islet xenotransplantation. Temporary T cell depletion was performed with either anti-CD4 or anti-CD8-depleting antibodies. Circulating T-cell subtypes were monitored. Rat islets were grafted into non-depleted untreated mice, as well as to depleted mice that were either untreated or treated with hAAT (240 mg/kg every 3 days). Graft survival was determined. Skin allotransplantation. Mice received a single dose of anti-CD4/CD8 depleting antibodies and then grafted with skin from BALB/c mice (H-2d). Graft survival was monitored. Results: T cell repopulation profile. hAAT group displayed significantly higher levels of CD4+ cells and lower levels of CD8+ after depletion (6.47±1.56 and 2.12±0.27-fold compared to WT mice, respectively), as well as a greater proportion of foxp3+ cells after depletion (6.18±0.86-fold compared to WT mice). Islet xenograft survival. Without depletion, untreated mice rejected islet xenografts on day 13.2 (mean, n=5); CD4-depleted mice rejected on day 26.4 (mean, n=5), and CD8-depeleted mice on day 14 (mean, n=5). None displayed graft survival after day 35. However, in the hAAT group, graft function was evident after day 35 in 75% CD8-depleted and 25% CD4-depleted mice. Skin allograft survival. Non-depleted, WT mice rejected skin allografts on day 11 (mean, n=5) and hAAT+/+ mice rejected on day 9.5 (mean, n=4). WT, T cell depleted mice rejected skin grafts on day 13.8 (mean, n=5). None displayed graft survival after day 20. However, 57% of the T cell depleted hAAT+/+ mice exhibited skin allograft survival after day 35. Conclusion: The combination between AAT and temporal T cell depletion appears to facilitate regulatory T cells expansion in favor of both islet-xenograft and skin-allograft transplantation outcomes. Considering the safety of both regimens, their use in treatment-resistant transplants should be considered.

Rat islets are not rejected by anti-islet antibodies in mice treated with costimulation blockade.

Costimulation blockade can prevent rejection of islet xenografts in naïve but not sensitized recipients. Donor-specific antibodies (DSA) may partly explain this observation. The effect of DSA on rat islet xenograft survival in mice receiving costimulation blockade was investigated. Naïve C57BL/6 mice with alloxan-induced diabetes were transplanted under the left kidney capsule with 100 Lewis rat islets. Recipients were divided into three groups receiving: (i) isotype control antibodies (Abs); (ii) anti-CD154 and CTLA4Ig; or (iii) anti-CD154, CTLA4Ig, and anti-LFA-1 every second day, day 0-8. At the time of transplantation (Tx), half of the animals in each group received naïve mouse serum and half xenoimmune serum derived from mice previously transplanted with rat islets. Non-fasting blood glucose levels and body weight were followed daily. Cured mice were examined by intraperitoneal glucose tolerance (IPGT) tests at 1 and 4 months after transplantation. Donor-specific antibodies were detected in immune serum-injected recipients up to at least 96 h post-Tx. Short term (≤96 h), there was no significant difference with regard to graft mass, infiltrating and apoptotic cells between groups of mice receiving naïve and immune sera. A moderate infiltration of polymorphonuclear and mononuclear cells was seen 96 h post-Tx in mice given control Abs, whether or not they received immune or naïve mouse serum. Mice given costimulation blockade had well-maintained endocrine tissue and very little cell infiltration. There was no significant difference in islet xenograft function and survival long term between groups receiving naïve and immune sera in combination with costimulation blockade. About half of the mice receiving costimulation blockade lost graft function within 110 days. The presence at Tx of DSA does not appear to negatively influence early and late islet xenograft survival in mice receiving costimulation blockade.