Spatial transcriptomic analysis of tumour-immune cell interactions in melanoma arising from congenital melanocytic nevus.

BACKGROUND: Studies on the interaction between tumour-infiltrating immune cells (TIICs) and tumour cells in melanoma arising from congenital melanocytic nevus (CMN) are lacking. OBJECTIVE: The aim of this study was to determine the intratumoral immune landscape of TIICs and tumour cells during invasion and metastasis. METHODS: Tissue specimens were obtained from patients with melanoma originating from CMN. Differential gene expression in melanoma cells and TIICs during invasion and metastasis was determined using spatial transcriptomics. RESULTS: As invasion depth increased, the expression of LGALS3, known to induce tumour-driven immunosuppression, increased in melanoma cells. In T cells, the expression of genes that inhibit T-cell activation increased with increasing invasion depth. In macrophages, the expression of genes related to the anti-inflammatory M2 phenotype was upregulated with increasing invasion depth. Compared to primary tumour cells, melanoma cells in metastatic lesions showed upregulated expression of genes associated with cancer immune evasion, including AXL and EPHA2, which impede T-cell recruitment, and BST2, associated with M2 polarization. Furthermore, T cells showed increased expression of genes related to immunosuppression, and macrophages exhibited increased expression of genes associated with the M2 phenotype. CONCLUSIONS: The interaction between melanomas arising from CMN and TIICs may be important for tumour progression and metastasis.

Mesenchymal stem cell-derived extracellular vesicles subvert Th17 cells by destabilizing RORγt through posttranslational modification.

Mesenchymal stem cell (MSC)-derived small extracellular vesicles (MSC-sEVs) are known to exert immunosuppressive functions. This study showed that MSC-sEVs specifically convert T helper 17 (Th17) cells into IL-17 low-producer (ex-Th17) cells by degrading RAR-related orphan receptor γt (RORγt) at the protein level. In experimental autoimmune encephalomyelitis (EAE)-induced mice, treatment with MSC-sEVs was found to not only ameliorate clinical symptoms but also to reduce the number of Th17 cells in draining lymph nodes and the central nervous system. MSC-sEVs were found to destabilize RORγt by K63 deubiquitination and deacetylation, which was attributed to the EP300-interacting inhibitor of differentiation 3 (Eid3) contained in the MSC-sEVs. Small extracellular vesicles isolated from the Eid3 knockdown MSCs by Eid3-shRNA failed to downregulate RORγt. Moreover, forced expression of Eid3 by gene transfection was found to significantly decrease the protein level of RORγt in Th17 cells. Altogether, this study reveals the novel immunosuppressive mechanisms of MSC-sEVs, which suggests the feasibility of MSC-sEVs as an attractive therapeutic tool for curing Th17-mediated inflammatory diseases.

Comparing the Benefits and Drawbacks of Stem Cell Therapy Based on the Cell Origin or Manipulation Process: Addressing Immunogenicity.

Mesenchymal stem cells (MSCs) are effective in treating autoimmune diseases and managing various conditions, such as engraftment of allogeneic islets. Additionally, autologous and HLA-matched allogeneic MSCs can aid in the engraftment of human allogeneic kidneys with or without low doses of tacrolimus, respectively. However, HLA alloantigens are problematic because cell therapy uses more HLA-mismatched allogeneic cells than autologous for convenience and standardization. In particular, HLA-mismatched MSCs showed increased Ag-specific T/B cells and reduced viability faster than HLA-matched MSCs. In CRISPR/Cas9-based cell therapy, Cas9 induce T cell activation in the recipient's immune system. Interestingly, despite their immunogenicity being limited to the cells with foreign Ags, the accumulation of HLA alloantigen-sensitized T/B cells may lead to allograft rejection, suggesting that alloantigens may have a greater scope of adverse effects than foreign Ags. To avoid alloantigen recognition, the ß2-microglobulin knockout (B2MKO) system, eliminating class-I MHC, was able to avoid rejection by alloreactive CD8 T cells compared to controls. Moreover, universal donor cells in which both B2M and Class II MHC transactivator (CIITA) were knocked out was more effective in avoiding immune rejection than single KO. However, B2MKO and CIITA KO system remain to be controlled and validated for adverse effects such as the development of tumorigenicity due to deficient Ag recognition by CD8 T and CD4 T cells, respectively. Overall, better HLA-matching or depletion of HLA alloantigens prior to cell therapy can reduce repetitive transplantation through the long-term survival of allogeneic cell therapy, which may be especially important for patients seeking allogeneic transplantation.

The optimized core peptide derived from CABIN1 efficiently inhibits calcineurin-mediated T-cell activation.

The C-terminal fragment of CABIN1 interacts with calcineurin and represses the transcriptional activity of the nuclear factor of activated T cells (NFAT). However, the specific sequences and mechanisms through which it binds to calcineurin are unclear. This study determined that decameric peptide (CABIN1 residues 2146-2155) is minimally required for binding to calcineurin. This peptide contains a unique "PPTP" C-terminal sequence and a "PxIxIT" N-terminal motif. Furthermore, p38MAPK phosphorylated the threonine residue of the "PPTP" sequence under physiological conditions, dramatically enhancing the peptide's binding affinity to calcineurin. Therefore, the CABIN1 peptide inhibited the calcineurin-NFAT pathway and the activation of T cells more efficiently than the VIVIT peptide without affecting calcineurin's phosphatase activity. The CABIN1 peptide could thus be a more potent calcineurin inhibitor and provide therapeutic opportunities for various diseases caused by the calcineurin-NFAT pathway.

Deconvolution of Adult T-Cell Leukemia/Lymphoma With Single-Cell RNA-Seq Using Frozen Archived Skin Tissue Reveals New Subset of Cancer-Associated Fibroblast.

Adult T-cell Leukemia/Lymphoma (ATLL) is a rare aggressive T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1) infection. However, little is known about the underlying activated molecular pathways at the single cell level. Moreover, the intercellular communications between the tumor microenvironment (TME) and tumor cells in this malignancy are currently unknown. Difficulties in harvesting fresh tissue in a clinical setting have hampered our deeper understanding of this malignancy. Herein, we examined ATLL using archived fresh frozen tissue after biopsy using single-cell RNA sequencing (scRNA-seq) with T-cell receptor (TCR) clonal analysis. Highly clonal tumor cells showed multiple activating pathways, suggesting dynamic evolution of the malignancy. By dissecting diverse cell types comprising the TME, we identified a novel subset of cancer-associated fibroblast, which showed enriched epidermal growth factor receptor (EGFR)-related transcripts including early growth response 1 and 2 (EGR1 and EGR2). Cancer associated fibroblasts (CAFs) of ATLL play an important role for CD4 T-cell proliferation via FGF7-FGF1 and PDGFA-PDGFRA/B signaling, and CAFs, particularly EGR-enriched, are also associated with CD8 and NKT expansion by EGFR. These findings suggest a potential targeted therapeutic pathway to better treat this neoplasm.

Novel Immunomodulatory Approaches for Porcine Islet Xenotransplantation.

PURPOSE OF REVIEW: Porcine islet xenotransplantation is a promising alternative to overcome the shortage of organ donors. For the successful application of islet xenotransplantation, robust immune/inflammatory responses against porcine islets should be thoroughly controlled. Over the last few decades, there have been numerous attempts to surmount xenogeneic immune barriers. In this review, we summarize the current progress in immunomodulatory therapy for the clinical application of porcine islet xenotransplantation. RECENT FINDINGS: Long-term graft survival of porcine islets was achieved by using anti-CD154 Ab-based regimens in a preclinical non-human primate (NHP) model. However, owing to a serious complication of thromboembolism in clinical trials, the development of an anti-CD154 Ab-sparing immunosuppressant procedure is required. The efficacy of new immunosuppressive practices that employ anti-CD40 Abs or other immunosuppressive reagents has been tested in a NHP model to realize their utility in porcine islet xenotransplantation. The recent progress in the development of immunomodulatory approaches, including the immunosuppressive regimen, which enables long-term graft survival in a pig-to-non-human primate islet xenotransplantation model, with their potential clinical applicability was reviewed.

Contrasting Prognostic Effects of Tumor-Infiltrating Lymphocyte Density in Cardia and Non-cardia Gastric Adenocarcinomas.

PURPOSE: This study sought to investigate the prognostic significance of tumor-infiltrating lymphocytes (TILs) in relation to tumor location within the stomach. MATERIALS AND METHODS: The densities and prognostic significance of TIL subsets were evaluated in 542 gastric cancer patients who underwent gastrectomy. Immunohistochemical staining for CD3, CD4, CD8, forkhead/winged helix transcription factor (Foxp3), and granzyme B was performed. RESULTS: Cardia cancer was associated with significantly lower densities of CD8 T-cells and higher densities of Foxp3 and granzyme B T-cells than non-cardia tumors. Multivariate analysis showed that advanced age (hazard ratio [HR], 1.023; 95% confidence interval [CI], 1.006-1.040), advanced T classification (HR, 2.029; 95% CI, 1.106-3.721), lymph node metastasis (HR, 3.319; 95% CI, 1.947-5.658), low CD3 expression (HR, 0.997; 95% CI, 0.994-0.999), and a high Foxp3/CD4 ratio (HR, 1.007; 95% CI, 1.001-1.012) were independent predictors of poor overall survival in cardia cancer patients. In non-cardia cancer patients, total gastrectomy (HR, 2.147; 95% CI, 1.507-3.059), advanced T classification (HR, 2.158; 95% CI, 1.425-3.266), lymph node metastasis (HR, 1.854; 95% CI, 1.250-2.750), and a low Foxp3/CD4 ratio (HR, 0.978; 95% CI, 0.959-0.997) were poor prognostic factors for survival. CONCLUSIONS: The densities and prognostic effects of TILs differed in relation to the location of tumors within the stomach. The contrasting prognostic effects of Foxp3/CD4 ratio in cardia and non-cardia gastric cancer patients suggests that clinicians ought to consider tumor location when determining treatment strategies.

Mesenchymal stem cell-derived exosomes suppress proliferation of T cells by inducing cell cycle arrest through p27kip1/Cdk2 signaling.

Mouse mesenchymal stem cells (MSCs) have been shown to suppress T cells. Especially, MSC-cultured media have shown suppressive functions against various immune cells including the T cells. However, the underlying immunosuppressive mechanisms of the MSC-cultured medium are not yet fully understood. In this study, we confirmed the T cell-suppression capacity of MSC culture supernatant (MSC-CS) through both apoptosis and cell cycle arrest, and hypothesized that the exosomes were the major immunosuppressive agents in the MSC-CS. MSC-derived exosomes (MSC-exo) exhibited potent suppressive effects on T cell proliferation while the rest of the supernatant fraction did not. Interestingly, the exosomes derived from MSC only induced the cell cycle arrest, and it was through the upregulation of p27kip1 protein and downregulation of Cdk2 protein. In conclusion, the exosomes secreted from MSCs could suppress the activated T cell proliferation through the induction of cell cycle arrest.

Allogeneic ADSCs Induce the Production of Alloreactive Memory-CD8 T Cells through HLA-ABC Antigens.

We investigated the immunogenicity of allogeneic human adipose-derived mesenchymal stem cells (ADSCs) through the production of alloreactive-CD8 T and -memory CD8 T cells, based on their human leukocyte antigen (HLA) expression. In surface antigen analysis, ADSCs do not express co-stimulatory molecules, but expresses HLA-ABC, which is further increased by exposure to the pro-inflammatory cytokines as well as IFN-γ alone. For immunogenicity analysis, allogeneic ADSCs cultured in xenofree medium (XF-ADSCs) were incubated with the recipient immune cells for allogeneic-antigen stimulation. As a result, XF-ADSCs induced IFN-γ and IL-17A release by alloreactive-CD8 T cells and the production of alloreactive-CD8 T cell through a direct pathway, although they have immunomodulatory activity. In the analysis of alloreactive memory CD8 T cells, XF-ADSCs also significantly induced the production of CFSE-low-CD8 TEM and -CD8 TCM cells. However, HLA-blocking antibodies significantly inhibited the production of CFSE-low memory-CD8 T cells, indicating that HLAs are the main antigens responsible for the development of allogeneic ADSCs' immunogenicity. These results suggested that HLA surface antigens expressed in allogeneic MSCs should be solved in order to address concerns related to the immunogenicity problem.

The effect of preexisting HMGB1 within fetal bovine serum on murine pancreatic beta cell biology.

High-mobility group box 1 (HMGB1) can act as a structural protein of the chromatin and at the same time as a mediator of the immune system. Its high correlation with the graft acceptance in pancreatic islet recipients makes it a biomarker in islet transplantation. With the suspicion that preexisting HMGB1 in the fetal bovine serum (FBS) would be detrimental to the viability and function of murine beta cells, HMGB1 was removed from FBS and its impact was investigated. Interestingly, the elimination of HMGB1 from FBS seemed unfavorable to the viability and function of cultured murine beta cells, suggesting that the preexisting HMGB1 in the FBS may be an indispensable component of islet cell culture.

A combination regimen of low-dose bortezomib and rapamycin prolonged the graft survival in a murine allogeneic islet transplantation model.

As the first FDA-approved proteasome inhibitor drug, bortezomib has been used for the treatment of multiple myeloma and lymphoma. However, its effects alone or in combination with other immunosuppressants on allogeneic islet transplantation have not been reported so far. In this study, we showed that the short-term combination treatment of low-dose bortezomib and rapamycin significantly prolonged the survival of islet allografts. Short-term treatment of low-dose (0.05 mg/kg or 0.1 mg/kg) bortezomib reduced the MHC class II expression in dendritic cells (DCs) of alloantigen-sensitized mice, and prolonged the islet allograft survival for up to 50 days in diabetic mice. Notably, when bortezomib was combined with rapamycin, it induced islet-specific immunological tolerance which allowed the acceptance of a second graft without additional immunosuppression. This regimen dramatically reduced the alloantigen-specific IFN-γ-producing T cells in the spleen, and increased regulatory T cells both at the graft site and in the spleen. Therefore, we propose that short-term treatment of low-dose bortezomib and rapamycin could be a new tolerance-promoting immunosuppressive regimen for allogeneic islet transplantation.

JAK3 inhibitor-based immunosuppression in allogeneic islet transplantation in cynomolgus monkeys.

Islet transplantation is efficacious to prevent severe hypoglycemia and glycemic liability of selected patients of type 1 diabetes. However, since calcineurin inhibitor (CNI) causes ß-cell and nephrotoxicity, alternative drug(s) with similar potency and safety profile to CNI will be highly desirable. Here we tested whether JAK3 inhibitor, tofacitinib could be used instead of tacrolimus in CIT07 immunosuppression regimen in cynomolgus nonhuman primate (NHP) model. Five independent streptozotocin (STZ)-induced diabetic monkeys were transplanted with MHC-mismatched allogeneic islets and three animals were further re-transplanted upon insufficient glycemic control or early islet graft rejection. After islet transplantation, blood glucose levels were quickly stabilized and maximal islet graft survival as measured by serum C-peptide concentration was >330, 98, >134, 31, or 22 days, respectively, after transplantation (median survival day; 98 days). Cellular and humoral immune responses were efficiently suppressed by JAK3 inhibitor-based immunosuppression during the follow-up periods. Although intermittent increases of the genome copy number of cynomolgus cytomegalovirus (CMV) were detected by quantitative real-time PCR analyses, serious infections or posttransplant lymphoproliferative disease (PTLD) was not found in all animals. Taken together, we have shown that JAK3 inhibitor could be used in replacement of tacrolimus in a highly translatable NHP islet transplantation model and these results suggest that JAK3 inhibitor will be potentially incorporated in human allogeneic islet transplantation.

Phc2 controls hematopoietic stem and progenitor cell mobilization from bone marrow by repressing Vcam1 expression.

The timely mobilization of hematopoietic stem and progenitor cells (HSPCs) is essential for maintaining hematopoietic and tissue leukocyte homeostasis. Understanding how HSPCs migrate between bone marrow (BM) and peripheral tissues is of great significance in the clinical setting, where therapeutic strategies for modulating their migration capacity determine the clinical outcome. Here, we identify an epigenetic regulator, Phc2, as a critical modulator of HSPC trafficking. The genetic ablation of Phc2 in mice causes a severe defect in HSPC mobilization through the derepression of Vcam1 in bone marrow stromal cells (BMSCs), ultimately leading to a systemic immunodeficiency. Moreover, the pharmacological inhibition of VCAM-1 in Phc2-deficient mice reverses the symptoms. We further determine that Phc2-dependent Vcam1 repression in BMSCs is mediated by the epigenetic regulation of H3K27me3 and H2AK119ub. Together, our data demonstrate a cell-extrinsic role for Phc2 in controlling the mobilization of HSPCs by finely tuning their bone marrow niche.

Peri-graft porcine-specific CD4+ FoxP3+ regulatory T cells by CD40-CD154 blockade prevented the rejection of porcine islet graft in diabetic mice.

BACKGROUND: Anti-CD154 monoclonal antibody (mAb) treatment has been known to have potential to induce immune tolerance in organ transplantation. Several studies have suggested the involvement of CD4+ regulatory T cells (Treg s) in xeno-immune tolerance. However, the characteristics of Treg s and the mechanisms of their regulatory functions in islet xenotransplantation have not been clearly defined. METHOD: Adult porcine islet cells were isolated and purified, and were transplanted under the kidney capsule of diabetic C57BL/6J mice with the administration of 0.5 mg/mouse of anti-CD154 mAb on 0, 1, 3, 5, and 7 days post-transplantation (DPT). The graft survival was monitored by blood glucose level. The islet graft and recipients' cells were analyzed by immunohistochemistry (IHC), flow cytometry, enzyme-linked immunosorbent spot (ELISPOT) assay, and mixed-lymphocyte reaction. RESULTS: Short-term anti-CD154 mAb monotherapy enabled the porcine islet graft to survive indefinitely in diabetic mice (n = 18). Immunohistochemical staining showed significantly higher ratio of CD4+ Foxp3+ Treg s in the peri-graft site, but not in the spleen and kidney-draining lymph node of the recipient mice. Depletion of Treg s evoked graft rejection, and adoptive transfer of Treg s from anti-CD154 mAb-treated recipients provided protection to the graft from rejection. These Treg s showed more potent suppressive capacity than those from the untreated control and were found to be porcine antigen-specific. CONCLUSIONS: In this study, we showed that anti-CD154 mAb monotherapy resulted in indefinite porcine islet graft survival in mice. The porcine-specific CD4+ Foxp3+ Treg s in the peri-graft site played the critical role in the protection of islet graft from rejection, which suggests a prospective immunosuppressive strategy for islet xenotransplantation.

Allogeneic ADSCs induce CD8 T cell-mediated cytotoxicity and faster cell death after exposure to xenogeneic serum or proinflammatory cytokines.

This study examined the induction of recipient T-cell cytotoxicity after exposure to allogeneic adipose-derived mesenchymal stem cells (ADSCs). ADSCs pre-exposed to xenogeneic serum significantly induced cytotoxicity through CD8 T-cell granzyme B secretion after allogeneic antigen stimulation, and this effect was increased with prolonged reaction time. ADSCs pretreated with proinflammatory cytokines also induced cytotoxicity through granzyme B secretion and significantly increased human leukocyte antigen (HLA)-ABC expression. T-cell cytotoxicity towards ADSCs grown in xeno-free medium (XF-ADSCs) was lower than that towards ADSCs exposed to xenogeneic serum or proinflammatory cytokines, but XF-ADSCs still induced cytotoxicity. We further investigated the causes of T-cell cytotoxicity towards XF-ADSCs. XF-ADSC death was effectively inhibited by HLA-blocking antibodies, suggesting that ADSC HLAs are a major cause of alloreactive T-cell generation. These results indicated that culturing of allogeneic ADSCs with recipient serum may alleviate alloreactive CD8 T-cell cytotoxicity. Ultimately, development of therapeutic agents using autologous ADSCs would be a suitable way to avoid immunogenicity and CD8 T cell-mediated cytotoxicity, but more attention should be paid to the potential immunogenicity of allogeneic ADSCs, which could perhaps be mitigated through the use of immunosuppressants.

Mismatch Repair Status of Gastric Cancer and Its Association with the Local and Systemic Immune Response.

BACKGROUND: Microsatellite instability (MSI)-high (MSI-H) colorectal cancer is known to be associated with increased tumor-infiltrating lymphocytes (TILs), elevated host systemic immune response, and a favorable prognosis. In gastric cancer, however, MSI status has rarely been evaluated in the context of TILs and systemic immune response. MATERIALS AND METHODS: We evaluated data for 345 patients with gastric cancer who underwent gastrectomy with MSI typing. The numbers of TILs were counted after immunohistochemical staining with anti-CD3, CD4, CD8, forkhead box P3 (Foxp3), and granzyme B to quantify the subsets of TILs. To evaluate the systemic immune response, the differential white blood cell count and prognostic nutritional index (PNI) were obtained. RESULTS: Of the 345 patients, 57 demonstrated MSI-H tumors and 288 demonstrated non-MSI-H tumors. MSI-H tumors carried significantly higher densities of CD8+ T cells, Foxp3+ T cells, and granzyme B+ T cells and a higher ratio of Foxp3/CD4 and granzyme B/CD8. The prognostic impact of TILs differed between patients with MSI-H tumors and those with non-MSI-H tumors. The TIL subsets were not found to be significant prognostic factors for recurrence-free survival (RFS) or overall survival (OS) in the MSI-H tumor group. In the non-MSI-H tumor group, multivariate analysis showed that stage, PNI, and CD4+ T cells were independent prognostic factors for RFS, and stage, PNI, and the Foxp3/CD4 ratio were independent prognostic factors for OS. CONCLUSIONS: The association between systemic/local immune response and prognosis differed according to MSI status. Different tumor characteristics and prognoses according to MSI status could be associated with the immunogenicity caused by microsatellite instability and subsequent host immune response. IMPLICATIONS FOR PRACTICE: This study demonstrates that the density of each subset of tumor-infiltrating lymphocytes (TILs) differed between microsatellite instability (MSI)-high and non-MSI-high tumors. Moreover, the prognostic effect of the preoperative systemic immune response status and TILs differed between the MSI-high (MSI-H) and non-MSI-H tumor groups. The present study may help to identify the mechanisms of cancer progression and develop treatment strategies for MSI-high gastric cancer.

Vascularization of PLGA-based bio-artificial beds by hypoxia-preconditioned mesenchymal stem cells for subcutaneous xenogeneic islet transplantation.

BACKGROUND: Subcutaneous tissue is an attractive extra-hepatic heterotopic site for islet transplantation; however, poor oxygen tension and blood supply during early engraftment of implanted islets have limited the use of this site in clinical applications. METHODS: This study investigated the vascularization potential of hypoxia-preconditioned mesenchymal stem cells (3% O2 ; hypo-MSCs) in PLGA-based bio-artificial beds for subsequent subcutaneous islet transplantation. Sheet-typed polymeric PLGA scaffolds coated with hypo-MSCs or normo-MSCs (MSCs cultured under normoxia conditions, 21% O2 ) were implanted subcutaneously in mice. RESULTS: Compared to normo-MSCs, hypo-MSCs significantly enhanced vasculogenesis, both on the interior and exterior surfaces of the implanted PLGA devices, which peaked 4 weeks after implantation. Further, infusion of porcine islets inside the prevascularized PLGA bed restored normal glycemic control in 6 of 6 STZ-induced diabetic mice. The mass of the marginal islet was approximately 2000 IEQs, which is comparable to that required for the renal subcapsular space, a highly vascularized site. CONCLUSIONS: Therefore, PLGA-based bio-artificial devices prevascularized with hypo-MSCs could be a useful modality for successful subcutaneous islet transplantation, which is of high clinical relevance.

Comparative efficacy of anti-CD40 antibody-mediated costimulation blockade on long-term survival of full-thickness porcine corneal grafts in nonhuman primates.

Porcine corneas may be good substitutes for human corneas in donor shortage. Therefore, we evaluated the efficacy and safety of an anti-CD40 antibody-based regimen compared with an anti-CD20 antibody-based regimen on the survival of full-thickness corneas in pig-to-rhesus xenotransplant. Thirteen Chinese rhesuses underwent full-thickness corneal xenotransplant. Six were administered anti-CD40 antibody, and the others were administered anti-CD20 antibody, basiliximab, and tacrolimus. Graft survival and changes in lymphocyte, donor-specific and anti-Galα1,3Galß1,4GlcNAc-R (αGal) antibody, and aqueous complement levels were evaluated. Treatment with the anti-CD40 antibody (>511, >422, >273, >203, >196, 41 days) and anti-CD20 antibody (>470, 297, >260, >210, >184, 134, >97 days) resulted in long-term survival of grafts. In the anti-CD20 group, the number of activated B cells was significantly lower than that in the anti-CD40 group, and the level of aqueous complements at 6 months was significantly higher than the preoperative level. There were no differences in the levels of T cells or donor-specific and anti-αGal antibodies between the 2 groups. In the anti-CD20 group, 3 primates had adverse reactions. In conclusion, both the anti-CD40 antibody- and the anti-CD20 antibody-based protocols were effective for the long-term survival of full-thickness corneal xenografts, but the anti-CD40 antibody-based treatment had fewer adverse effects.

Construction of EMSC-islet co-localizing composites for xenogeneic porcine islet transplantation.

Pancreatic islet transplantation is an ultimate solution for treating patients with type 1 diabetes (T1D). The pig is an ideal donor of islets for replacing scarce human islets. Besides immunological hurdles, non-immunological hurdles including fragmentation and delayed engraftment of porcine islets need solutions to succeed in porcine islet xenotransplantation. In this study, we suggest a simple but effective modality, a cell/islet co-localizing composite, to overcome these challenges. Endothelial-like mesenchymal stem cells (EMSCs), differentiated from bone-marrow derived mouse mesenchymal stem cells (MSCs), and MSCs evenly coated the surface of porcine islets (>85%) through optimized culture conditions. Both MSCs and EMSCs significantly reduced the fragmentation of porcine islets and increased the islet masses, designated as islet equivalents (IEQs). In fibrin in vitro and in vivo angiogenesis analysis, constructed EMSC-islet composites showed higher angiogenic potentials than naked islets, MSC-islet composites, or human endothelial cell-islet composites. This novel delivery method of porcine islets may have beneficial effects on the engraftment of transplanted islets by prevention of fragmentation and enhancement of revascularization.

Delayed revascularization of islets after transplantation by IL-6 blockade in pig to non-human primate islet xenotransplantation model.

BACKGROUND: Pancreatic islet transplantation is currently proven as a promising treatment for type 1 diabetes patients with labile glycemic control and severe hypoglycemia unawareness. Upon islet transplantation, revascularization is essential for proper functioning of the transplanted islets. As IL-6 is important for endothelial cell survival and systemic inflammation related to xenograft, the effect of IL-6 receptor antagonist, tocilizumab, on revascularization of the transplanted islets was examined in pig to non-human primate islet xenotransplantation model. Also, the endothelial cell origin in a new vessel of the transplanted pig islets was determined. METHODS: Pig islets were isolated from designated pathogen-free (DPF) SNU miniature pigs and transplanted via portal vein into five streptozotocin-induced diabetic monkeys. One group (n = 2, basal group) was treated with anti-thymoglobulin (ATG), anti-CD40 antibody (2C10R4), sirolimus, and tacrolimus, and the other group was additionally given tocilizumab on top of basal immunosuppression (n = 3, Tocilizumab group). To confirm IL-6 blocking effect, C-reactive protein (CRP) levels and serum IL-6 concentration were measured. Scheduled biopsy of the margin of the posterior segment right lobe inferior of the liver was performed at 3 weeks after transplantation to assess the degree of revascularization of the transplanted islets. Immunohistochemical staining using anti-insulin, anti-CD31 antibodies, and lectin IB4 was conducted to find the origin of endothelial cells in the islet graft. RESULTS: CRP significantly increased at 1~2 days after transplantation in Basal group, but not in Tocilizumab group, and higher serum IL-6 concentration was measured in latter group, showing the biological potency of tocilizumab. In Basal group, well-developed endothelial cells were observed on the peri- and intraislet area, whereas the number of CD31+ cells in the intraislet space was significantly reduced in Tocilizumab group. Finally, new endothelial cells in the pig islet graft were positive for CD31, but not for lectin IB4, suggesting that they are originated from the recipient monkey. CONCLUSIONS: Our results demonstrated that tocilizumab can delay revascularization of the transplanted islet, although this effect had no significant correlation to the overall islet graft survival. In the pig to NHP islet xenotransplantation model, the endothelial cells from recipient monkey form new blood vessels in and around pig islets.