Epitope editing enables targeted immunotherapy of acute myeloid leukaemia.

Despite the considerable efficacy observed when targeting a dispensable lineage antigen, such as CD19 in B cell acute lymphoblastic leukaemia1,2, the broader applicability of adoptive immunotherapies is hampered by the absence of tumour-restricted antigens3-5. Acute myeloid leukaemia immunotherapies target genes expressed by haematopoietic stem/progenitor cells (HSPCs) or differentiated myeloid cells, resulting in intolerable on-target/off-tumour toxicity. Here we show that epitope engineering of donor HSPCs used for bone marrow transplantation endows haematopoietic lineages with selective resistance to chimeric antigen receptor (CAR) T cells or monoclonal antibodies, without affecting protein function or regulation. This strategy enables the targeting of genes that are essential for leukaemia survival regardless of shared expression on HSPCs, reducing the risk of tumour immune escape. By performing epitope mapping and library screenings, we identified amino acid changes that abrogate the binding of therapeutic monoclonal antibodies targeting FLT3, CD123 and KIT, and optimized a base-editing approach to introduce them into CD34+ HSPCs, which retain long-term engraftment and multilineage differentiation ability. After CAR T cell treatment, we confirmed resistance of epitope-edited haematopoiesis and concomitant eradication of patient-derived acute myeloid leukaemia xenografts. Furthermore, we show that multiplex epitope engineering of HSPCs is feasible and enables more effective immunotherapies against multiple targets without incurring overlapping off-tumour toxicities. We envision that this approach will provide opportunities to treat relapsed/refractory acute myeloid leukaemia and enable safer non-genotoxic conditioning.

Cytotoxic T lymphocyte antigen-4 regulates development of xenogenic graft versus host disease in mice via modulation of host immune responses induced by changes in human T cell engraftment and gene expression.

Graft versus host disease (GvHD) is a major clinical problem with a significant unmet medical need. We examined the role of cytotoxic T lymphocyte antigen-4 (CTLA-4) in a xenogenic GvHD (xeno-GvHD) model induced by injection of human peripheral mononuclear cells (hPBMC) into irradiated non-obese diabetic (NOD) SCID gamma (NSG) mice. Targeting the CTLA-4 pathway by treatment with CTLA-4 immunoglobulin (Ig) prevented xeno-GvHD, while anti-CTLA-4 antibody treatment exacerbated the lethality and morbidity associated with GvHD. Xeno-GvHD is associated with infiltration of hPBMCs into the lungs, spleen, stomach, liver and colon and an increase in human proinflammatory cytokines, including interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-5. Infiltration of donor cells and increases in cytokines were attenuated by treatment with CTLA-4 Ig, but remained either unaffected or enhanced by anti-CTLA-4 antibody. Further, splenic human T cell phenotyping showed that CTLA-4 Ig treatment prevented the engraftment of human CD45+ cells, while anti-CTLA-4 antibody enhanced donor T cell expansion, particularly CD4+ (CD45RO+ ) subsets, including T box transcription factor TBX21 (Tbet)+ CXCR3+ and CD25+ forkhead box protein 3 (FoxP3) cells. Comprehensive analysis of transcriptional profiling of human cells isolated from mouse spleen identified a set of 417 differentially expressed genes (DEGs) by CTLA-4 Ig treatment and 13 DEGs by anti-CTLA-4 antibody treatment. The CTLA-4 Ig regulated DEGs mapped to down-regulated apoptosis, inflammasome, T helper type 17 (Th17) and regulatory T cell (Treg ) pathways and enhanced Toll-like receptor (TLR) receptor signaling, TNF family signaling, complement system and epigenetic and transcriptional regulation, whereas anti-CTLA-4 antibody produced minimal to no impact on these gene pathways. Our results show an important role of co-inhibitory CTLA-4 signaling in xeno-GvHD and suggest the therapeutic utility of other immune checkpoint co-inhibitory pathways in the treatment of immune-mediated diseases driven by hyperactive T cells.

Natural killer cells and cytotoxic T lymphocytes are required to clear solid tumor in a patient-derived xenograft.

Existing patient-derived xenograft (PDX) mouse models of solid tumors lack a fully tumor donor-matched, syngeneic, and functional immune system. We developed a model that overcomes these limitations by engrafting lymphopenic recipient mice with a fresh, undisrupted piece of solid tumor, whereby tumor-infiltrating lymphocytes (TILs) persisted in the recipient mice for several weeks. Successful tumor engraftment was achieved in 83% to 89% of TIL-PDX mice, and these were seen to harbor exhausted immuno-effector as well as functional immunoregulatory cells persisting for at least 6 months postengraftment. Combined treatment with interleukin-15 stimulation and immune checkpoint inhibition resulted in complete or partial tumor response in this model. Further, depletion of cytotoxic T lymphocytes and/or natural killer cells before combined immunotherapy revealed that both cell types were required for maximal tumor regression. Our TIL-PDX model provides a valuable resource for powerful mechanistic and therapeutic studies in solid tumors.

Preclinical evaluation for engraftment of CD34+ cells gene-edited at the sickle cell disease locus in xenograft mouse and non-human primate models.

Sickle cell disease (SCD) is caused by a 20A > T mutation in the ß-globin gene. Genome-editing technologies have the potential to correct the SCD mutation in hematopoietic stem cells (HSCs), producing adult hemoglobin while simultaneously eliminating sickle hemoglobin. Here, we developed high-efficiency viral vector-free non-footprint gene correction in SCD CD34+ cells with electroporation to deliver SCD mutation-targeting guide RNA, Cas9 endonuclease, and 100-mer single-strand donor DNA encoding intact ß-globin sequence, achieving therapeutic-level gene correction at DNA (∼30%) and protein (∼80%) levels. Gene-edited SCD CD34+ cells contributed corrected cells 6 months post-xenograft mouse transplant without off-target δ-globin editing. We then developed a rhesus ß-to-ßs-globin gene conversion strategy to model HSC-targeted genome editing for SCD and demonstrate the engraftment of gene-edited CD34+ cells 10-12 months post-transplant in rhesus macaques. In summary, gene-corrected CD34+ HSCs are engraftable in xenograft mice and non-human primates. These findings are helpful in designing HSC-targeted gene correction trials.

NF-kappa-B activation unveils the presence of inflammatory hotspots in human gut xenografts.

The single-epithelial cell layer of the gut mucosa serves as an essential barrier between the host and luminal microflora and plays a major role in innate immunity against invading pathogens. Nuclear factor kB (NF-κB), a central component of the cellular signaling machinery, regulates immune response and inflammation. NF-κB proteins are activated by signaling pathways downstream to microbial recognition receptors and cytokines receptors. Highly regulated NF-κB activity in intestinal epithelial cells (IEC) is essential for normal gut homeostasis; dysregulated activity has been linked to a number of disease states, including inflammatory bowel diseases (IBD) such as Crohn's Disease (CD). Our aim was to visualize and quantify spatial and temporal dynamics of NF-κB activity in steady state and inflamed human gut. Lentivirus technology was used to transduce the IEC of human gut xenografts in SCID mice with a NF-κB luminescence reporter system. NF-κB signaling was visualized and quantified using low resolution, intravital imaging of the whole body and high resolution, immunofluorescence microscopic imaging of the tissues. We show that NF-κB is activated in select subset of IEC with low "leaky" NF-κB activity. These unique inflammatory epithelial cells are clustered in the gut into discrete hotspots of NF-κB activity that are visible in steady state and selectively activated by systemic LPS and human TNFα or luminal bacteria. The presence of inflammatory hotspots in the normal and inflamed gut might explain the patchy mucosal lesions characterizing CD and thus could have important implications for diagnosis and therapy.

MLL5 improves ATRA driven differentiation and promotes xenotransplant engraftment in acute promyelocytic leukemia model.

Although the mixed lineage leukemia 5 (MLL5) gene has prognostic implications in acute promyelocyte leukemia (APL), the underlying mechanism remains to be elucidated. Here, we demonstrate the critical role exerted by MLL5 in APL regarding cell proliferation and resistance to drug-induced apoptosis, through mtROS regulation. Additionally, MLL5 overexpression increased the responsiveness of APL leukemic cells to all-trans retinoic acid (ATRA)-induced differentiation, via regulation of the epigenetic modifiers SETD7 and LSD1. In silico analysis indicated that APL blasts with MLL5high transcript levels were associated with retinoic acid binding and downstream signaling, while MLL5low blasts displayed decreased expression of epigenetic modifiers (such as KMT2C, PHF8 and ARID4A). Finally, APL xenograft transplants demonstrated improved engraftment of MLL5-expressing cells and increased myeloid differentiation over time. Concordantly, evaluation of engrafted blasts revealed increased responsiveness of MLL5-expressing cells to ATRA-induced granulocytic differentiation. Together, we describe the epigenetic changes triggered by the interaction of MLL5 and ATRA resulting in enhanced granulocytic differentiation.

Patient Derived Xenografts for Genome-Driven Therapy of Osteosarcoma.

Osteosarcoma (OS) is a rare malignant primary tumor of mesenchymal origin affecting bone. It is characterized by a complex genotype, mainly due to the high frequency of chromothripsis, which leads to multiple somatic copy number alterations and structural rearrangements. Any effort to design genome-driven therapies must therefore consider such high inter- and intra-tumor heterogeneity. Therefore, many laboratories and international networks are developing and sharing OS patient-derived xenografts (OS PDX) to broaden the availability of models that reproduce OS complex clinical heterogeneity. OS PDXs, and new cell lines derived from PDXs, faithfully preserve tumor heterogeneity, genetic, and epigenetic features and are thus valuable tools for predicting drug responses. Here, we review recent achievements concerning OS PDXs, summarizing the methods used to obtain ectopic and orthotopic xenografts and to fully characterize these models. The availability of OS PDXs across the many international PDX platforms and their possible use in PDX clinical trials are also described. We recommend the coupling of next-generation sequencing (NGS) data analysis with functional studies in OS PDXs, as well as the setup of OS PDX clinical trials and co-clinical trials, to enhance the predictive power of experimental evidence and to accelerate the clinical translation of effective genome-guided therapies for this aggressive disease.

Eomes promotes esophageal carcinoma progression by recruiting Treg cells through the CCL20-CCR6 pathway.

Eomesodermin (Eomes) is a T-box transcription factor that drives the differentiation and function of cytotoxic lymphocytes. However, the underlying function and mechanism of Eomes in tumor cells remains elusive. Here, we studied the role of Eomes in human esophageal squamous cell carcinoma (ESCC). Using 2 human ESCC cell lines, we found that Eomes knockdown reduced esophageal cancer cell proliferation and that the esophageal cancer cell cycle was blocked in the G2/M phase. Mechanistically, we identified CCL20 as the main downstream target of Eomes. Furthermore, we found that CCL20 could chemoregulate regulatory T cells (Tregs) through their specific receptor CCR6, then promoting the proliferation of esophageal cancer cells. Eomes knockdown also delayed the growth of human ESCC xenografts in BALB/c nude mice. Importantly, in 133 human ESCC tissues, high Eomes levels were associated with poor clinical prognosis. Overall, our findings suggested that the Eomes-CCL20-CCR6 pathway plays a vital role in human ESCC progress. Therefore, targeting this pathway may represent a promising strategy for controlling human ESCC.

Patient-derived organoids and orthotopic xenografts of primary and recurrent gliomas represent relevant patient avatars for precision oncology.

Patient-based cancer models are essential tools for studying tumor biology and for the assessment of drug responses in a translational context. We report the establishment a large cohort of unique organoids and patient-derived orthotopic xenografts (PDOX) of various glioma subtypes, including gliomas with mutations in IDH1, and paired longitudinal PDOX from primary and recurrent tumors of the same patient. We show that glioma PDOXs enable long-term propagation of patient tumors and represent clinically relevant patient avatars that retain histopathological, genetic, epigenetic, and transcriptomic features of parental tumors. We find no evidence of mouse-specific clonal evolution in glioma PDOXs. Our cohort captures individual molecular genotypes for precision medicine including mutations in IDH1, ATRX, TP53, MDM2/4, amplification of EGFR, PDGFRA, MET, CDK4/6, MDM2/4, and deletion of CDKN2A/B, PTCH, and PTEN. Matched longitudinal PDOX recapitulate the limited genetic evolution of gliomas observed in patients following treatment. At the histological level, we observe increased vascularization in the rat host as compared to mice. PDOX-derived standardized glioma organoids are amenable to high-throughput drug screens that can be validated in mice. We show clinically relevant responses to temozolomide (TMZ) and to targeted treatments, such as EGFR and CDK4/6 inhibitors in (epi)genetically defined subgroups, according to MGMT promoter and EGFR/CDK status, respectively. Dianhydrogalactitol (VAL-083), a promising bifunctional alkylating agent in the current clinical trial, displayed high therapeutic efficacy, and was able to overcome TMZ resistance in glioblastoma. Our work underscores the clinical relevance of glioma organoids and PDOX models for translational research and personalized treatment studies and represents a unique publicly available resource for precision oncology.

Does expression of a human complement-regulatory protein on xenograft cells protect them from systemic complement activation?

BACKGROUND: There are many causes of systemic complement activation, which may have detrimental effects on a pig xenograft. Transgenic expression of one or more human complement-regulatory proteins (hCRPs), e.g., hCD46, provides some protection to the xenograft, but it is not known whether it protects the xenograft from the effects of systemic complement activation. We used wild-type (WT) pig aortic endothelial cells (pAECs) to activate complement, and determined whether the expression of hCD46 on a1,3galactosyltransferase gene-knockout (GTKO) pAECs protected them from injury. METHODS: CFSE-labeled and non-labeled pAECs from a WT, a GTKO, or a GTKO/hCD46 pig were separately incubated with heat-inactivated pooled human serum in vitro. Antibody pre-bonded CFSE-labeled and non-labeled pAECs were mixed, and then incubated with rabbit complement. The complement-dependent cytotoxicity was measured by flow cytometry. RESULTS: There was significantly less lysis of GTKO/CD46 pAECs (6%) by 50% human serum compared to that of WT (91%, p<0.001) or GTKO (32%, p<0.01) pAECs. The lysis of GTKO pAECs was significantly increased when mixed with WT pAECs (p<0.05). In contrast, there was no significant change in cytotoxicity of GTKO/CD46 pAECs when mixed with WT pAECs. CONCLUSIONS: The expression of hCD46 protected pAECs from systemic complement activation.

Autologously Humanized Mice for Immune-Oncologic Studies.

With the rapid approval of immune checkpoint inhibitors for lung, melanoma, breast, genitourinary, and hematological malignancies, the hematopoietic cells in the tumor microenvironment (TME) are now considered an important, if not essential, consideration for cancer scientists. In many instances, syngeneic murine models have not been highly predictive for responsiveness in clinical trials. Our limited understanding of the human TME have, therefore, precluded a rational translation of immunotherapeutic combinations. This has led to the adoption of hematopoietic humanized murine models for the study of human tumor immunology in vivo. However, concerns about chimerism rates, HLA mismatching, and incomplete reconstitution of the innate immune system have driven a quest for improvements in these allogeneic humanized murine systems. Presented in this article is a completely autologous xenotransplantation method for reconstituting the human tumor immune microenvironment in vivo without the use of a patient's peripheral blood which is known to be associated with low engraftment rates. With this new approach, the current limitations of allogeneic humanized models are avoided by using matched bone marrow cells (BMCs) and derived tumor xenoplants (PDXs) from solid tumors in cancer patients. This autologous system provides a platform for studying endogenous lymphocytic and myeloid cell infiltration into the human tumor in vivo. © 2020 Wiley Periodicals LLC. Basic Protocol: Autologous reconstitution of human tumors Support Protocol 1: Transduction of BMCs and/or tumor cells prior to autologous reconstitution Support Protocol 2: Modeling immunotherapeutic agents in an autologously humanized model.

Human CD31 on Swine Endothelial Cells Induces SHP-1 Phosphorylation in Macrophages.

BACKGROUND: Innate immunity by natural killer (NK) cells, macrophages, and neutrophils cause severe rejections in xenotransplantation. Therefore, the development of strategies for suppressing macrophages has considerable potential in practical applications of xenotransplantation. Recently, we found that human CD31 on swine endothelial cells (SECs) suppresses neutrophil-mediated xenogeneic rejection through homophilic binding. Since a significant amount of CD31 is expressed not only on neutrophils but also on macrophages, we studied the function of human CD31 in macrophage-mediated cytotoxicity. METHODS: SECs and hCD31-transfected SECs (SEC/hCD31) were co-cultured with macrophages and cytotoxicity by macrophages was evaluated with water-soluble tetrazolium salt, or WST-8, assay. To confirm whether or not inhibitory signals are induced by hCD31 homophilic binding, the phosphorylation of the enzyme SHP-1 was investigated with Western blotting. RESULTS: No suppression of cytotoxicity was induced in macrophages that had been co-cultured with SEC/CD31. However, phosphorylation of SHP-1 was induced in macrophages that had been co-cultured with SEC/hCD31. CONCLUSIONS: Human CD31 on SEC may induce not only inhibitory signals but also activation signals via the binding to other receptors for hCD31.

Human CD200 Suppresses the HL-60 Mediated Xenocytotoxicity.

BACKGROUND: Neutrophils play an important role in xenogeneic rejection and represent a major obstacle in clinical application of xenografts. CD200 and its receptor CD200R are both type-1 membrane glycoproteins, which are members of the immunoglobulin superfamily (IgSF) and the ligation of CD200 with CD200R induces inhibitory NPXY signaling. The expression of CD200R appears in myeloid cells such as macrophages and granulocytes. Thus, we hypothesized that human CD200 expression on porcine cells might suppress the xenogeneic neutrophil-mediated cytotoxicity against porcine cells. METHODS: To prove our hypothesis, the suppressive effect of human CD200 in neutrophil-like human cell line 60 (HL-60)-mediated xenogeneic cytotoxicity against swine endothelial cells (SECs) was examined. Cytotoxicity was assessed with water-soluble tetrazolium salt 8 (WST-8) assay. RESULTS: HL-60 cells differentiated into CD66b+ CD200R+ neutrophil-like cells in the presence of dimethyl sulfoxide (DMSO). HL-60-mediated cytotoxicity against SECs was significantly suppressed by human CD200 on SECs. CONCLUSIONS: The findings in this study indicate that human CD200 may suppress neutrophil-mediated xenogeneic rejection.

Surgical Treatment of Pyoderma Gangrenosum with Negative Pressure Wound Therapy and Skin Grafting, Including Xenografts: Personal Experience and Comprehensive Review on 161 Cases.

Significance: Pyoderma gangrenosum (PG) is a rare debilitating autoinflammatory ulcerative skin disease. No gold standard has been established for the treatment of PG. The role of surgical interventions and negative pressure wound therapy (NPWT) was discussed controversially until recently as these procedures might pose a trigger to further aggravate the condition. Recent Advances: Recent advances confirm the paradigm change that a surgical approach of PG with split thickness skin grafting (STSG) secured by NPWT is a safe and valuable treatment if performed under adequate immunosuppression. We elaborate this on the hand of a broad literature search retrieving 101 relevant articles describing 138 patients complemented with our personal experience on 23 patients, including 2 patients treated with a porcine xenodressing. Critical Issues: A wide range of surgical approaches have been reported, including xenografts. Treatment was finally successful in 86%, including the xenotransplant cases. Ten percent improved and failures were mainly reported without immunosuppression. Despite halting the inflammatory process, NPWT alone, without skin grafting, does not much accelerate healing time. The best surgical approach appears to be STSG fixed with NPWT as this leads to higher skin graft take. There remains the problem of the chronic nature of PG and the recurrence after tapering of immunosuppression or trauma; therefore, a sustained immunosuppressive treatment is suggested. Future Directions: While surgical treatment is supported by the published data, the exact immunosuppression is still evolving. Due to deeper insights into pathogenesis and growing clinical reports, a broader utilization of biologic treatments and a shift from tumor necrosis factor (TNF)-alpha to interleukin (IL)-12/23 or IL-23 antibodies alone are predictable, as IL-12/23 antibodies show good clinical responses with fewer side effects. The positive results with porcine xenodressings might be due to immunological effects of the xenomaterial; they appear promising, but are preliminary and should be confirmed in a larger patient collective.

Interleukin-27 Enforces Regulatory T Cell Functions to Prevent Graft-versus-Host Disease.

Graft-versus-host disease (GvHD) remains a significant complication of allogeneic hematopoietic cell transplantation (HCT), associated with significant morbidity and mortality. GvHD is characterized by dysregulated immune responses and resulting tissue damage of target organs. Recent investigations have focused on Foxp3+ regulatory T cells (Tregs) as a therapeutic tool, based on its regulatory functions in GvHD pathogenesis and their instrumental role in mitigating GvHD severity while preserving graft-versus-leukemia (GvL) activity. There are several challenges to its clinical application, including their paucity, impaired suppressive activity, and instability in vivo. Herein, we report that IL-27 pre-stimulation enhances suppressive functions of both mouse and human Tregs. In a complete MHC mismatched murine bone marrow transplant model, IL-27 pre-stimulated polyclonal iTregs diminish acute (a)GvHD lethality, while preserving the GvL effect. Allo-antigen specificity further improves suppressive functions when combined with IL-27 pre-stimulation. In a xenogeneic (human to mouse) GvHD model, IL-27 pre-stimulated human iTregs are superior in protecting recipients from GvHD. Lastly, we compared gene expression profiles of circulating Tregs isolated from HCT recipients with and without aGvHD and found that Tregs from aGvHD patients express distinct gene signatures enriched in immune activation and inflammation. Therefore, these results highlight a novel function of IL-27 in enforcing Treg functions to prevent aGvHD mediated lethality, proposing the hypothesis that dysregulated Treg functions may account for the potential mechanisms underlying GvHD development.

Xenoantibodies and Complement Activity Determinations by Flow Cytometry in Pig-to-Primate Xenotransplantation.

In pig-to-primate xenotransplantation, flow cytometry assays allow the examination of antibody reactivity to intact antigens in their natural conformation and location on cell membranes. Here we describe in detail the procedures of two flow cytometry assays to measure the antibody-mediated complement-dependent cytotoxicity (CDC) response or serum levels of IgG and IgM xenoantibodies. This information is key for understanding the rejection process of vascularized xenografts and finding strategies to overcome it.

Cell-Based Assays for Modeling Xenogeneic Immune Responses.

Research in xenotransplantation implies a high experimental complexity comprising molecular, cellular, and in vivo studies to investigate the mechanisms of xenograft immune rejection and functional failure, as well as the strategies to counteract them. After major advances associated with the identification of the carbohydrate xenoantigens and their elimination through genomic edition of the source pigs, the study of the cellular immune response against the xenograft is gaining particular attention. Xenogeneic cell-based assays that put together pig cells and human leukocytes such as monocytes, NK cells, and T cells are relevant to address this hurdle. Thus, we describe here coculture, co-stimulatory, and cytotoxicity assays for investigating the cellular and molecular mechanisms of xenograft rejection. These techniques allow elucidating the key pathways that take place during the xenogeneic immune response in a simplified setting. Treatment with either pro-inflammatory or anti-inflammatory cytokines can be used for studying the regulation of adhesion, co-stimulatory molecules, and receptors involved in triggering the immune response under various conditions. Furthermore, these assays can be used for the follow-up of the immune response of in vivo studies as well as for the development of tolerogenic approaches that promote xenograft survival.

Studying Xenograft Rejection of Bioprosthetic Heart Valves.

Millions of patients with valvular heart disease have benefitted from heart valve replacement since the procedure was first introduced in the 1960s; however, there are still many patients who get early structural valve deterioration (SVD) of their bioprosthetic heart valves (BHV). BHV are porcine, bovine, or equine tissues that have been glutaraldehyde fixed to preserve the tissue and presumably make the tissue immunologically inert. These glutaraldehyde-fixed BHV with anti-calcification treatments last long periods of time in older adults but develop early SVD in younger patients. The consensus at present is that the early SVD in younger patients is due to more "wear and tear" of the valves and higher calcium turnover in younger patients. However, as younger patients likely have a more robust immune system than older adults, there is a new hypothesis that BHV xenografts may undergo xenograft rejection, and this may contribute to the early SVD seen in younger patients.At present, the technology to noninvasively study in vivo whether an implanted BHV in a human patient is undergoing rejection is not available. Thus, a small animal discordant xenotransplant model in young rodents (to match the young patient getting a pig/bovine/equine BHV) was developed to study whether the hypothesis that glutaraldehyde-fixed BHV undergo xenograft rejection had any merit. In this chapter, we describe our model and its merits and the results of our investigations. Our work provides clear evidence of xenograft rejection in glutaraldehyde-fixed tissue, and our small animal model offers an opportunity to study this process in detail.

Intra-bone Bone Marrow Transplantation in Pig-to-Nonhuman Primates for the Induction of Tolerance Across Xenogeneic Barriers.

Mixed chimerism and thymic tissue transplantation strategies have achieved xenogeneic tolerance in pig-to-mouse models, and both have been extended to pig-to-baboon models. A mixed chimerism strategy has shown promise toward inducing tolerance in allogeneic models in mice, pigs, nonhuman primates (NHP), humans, and a rat-to-mouse small animal xeno-model. However, even though α-1,3-galactosyltransferase gene knockout (GalTKO) pigs have been used as bone marrow (BM) donors, direct intravenous injection of porcine BM cells was detected for only up to 4 days (peripheral macro-chimerism) in one case, and the rest lost chimerism within 2 days.Recent data in allogeneic models demonstrated that direct injection of donor BM cells into recipient BM spaces (intra-bone bone marrow transplantation: IBBMTx) produces rapid reconstitution and a higher survival rate compared to i.v. injection. In order to minimize the loss of injected porcine BM peripherally before reaching the BM space, Yamada developed a xeno-specific regimen including IBBMTx coated with a collagen gel matrix in a preclinical pig-to-baboon model (Yamada IBBMTx). This strategy aims to achieve improved, persistent macro-chimerism as well as engraftment of BM across a xenogeneic barrier. The initial study published in 2015 demonstrated that this IBBMTx strategy leads to markedly prolonged peripheral macro-chimerism detectable for up to 23 days. Furthermore, a more recent study using human CD47-transgenic (Tg) GalTKO pigs as xeno-donors achieved long-lasting macro-chimerism >60 days with evidence of reduction of anti-pig natural antibodies (nAb). This is the longest macro-chimerism that has ever been achieved in a preclinical large animal xenotransplant model to date. In this chapter, we introduce a brief summary of our achievements in regard to successful tolerance induction by utilizing our novel strategy of IBBMTx as well as describe the step-by-step methodology of surgical and in vitro procedures that are required for this project.

Epigenetic biomarkers indicate islet cell death in xenotransplantation.

BACKGROUND: Xenotransplantation of porcine islets has emerged in recent decades as a potential treatment for type 1 diabetes (T1D). Current methods of detection, indicative of successful engraftment, occur downstream of actual islet death. Epigenetic biomarkers can be detected in circulating cell-free DNA (cfDNA) to provide an earlier indication of graft dysfunction. AIMS: The present study identified a biomarker of islet death using differential methylation of the insulin gene, INS, originating from ß-cells in porcine islets. MATERIALS & METHODS: Pyrosequencing primers specific for porcine INS were designed to quantify hypomethylation along 12 cysteine-guanine dinucleotide (CpG) sites, including three sites in the cyclic adenosine monophosphate (cAMP) response element (CRE) binding protein 2 (CRE2) binding region of the 5' untranslated region (UTR) and nine sites within intron 2. RESULTS: PCR amplification of bisulfite-converted DNA combined with pyrosequencing data support the conclusion that hypomethylated porcine INS is specific to islet origin. CONCLUSION: Moreover, the results of this study indicate a highly specific epigenetic biomarker, capable of detecting a single islet, supporting the measurement of cfDNA as a biomarker for transplanted islet death. Defining the epigenetic characteristics of porcine-derived islets within cfDNA will be crucial to develop a better understanding of graft survival immunology for transplantation.