Alice Mui

Local expression of B7-H4 by recombinant adenovirus transduction in mouse islets prolongs allograft survival.

Background. Allogeneic pancreatic islet transplantation has the potential to cure type 1 diabetes. One of the barriers to islet transplantation is the alloreactive T-cell response between donors and recipients. Costimulatory molecules, which play a major role in the regulation of the immune response to antigens during graft rejection, may be used to inhibit allograft destruction. B7-H4 is one such member in the costimulatory family, which has established negative regulatory function of T-cell responses. Methods. To determine whether local expression of B7-H4 protein can protect &bgr; cells from damage in islet allotransplantation, we have constructed a recombinant adenovirus expressing a B7-H4 complementary deoxyribonucleic acid (Ad-B7-H4). To study the in vivo effects of B7-H4 expression on islet graft survival, adenovirus-transduced islets from donor Balb/c mice were transplanted into streptozotocin-diabetic C57BL/6 mice (n=12). Results. Expression of B7-H4 in islets by Ad-B7-H4 transduction at an optimized condition did not inhibit glucose-stimulated insulin secretion of the treated islets. The recipient mice transplanted with Ad-B7-H4–transduced islets established euglycemia for a longer time (mean 56.5 days), compared with control mice transplanted with Ad-LacZ–transduced islets (mean 14.5 days, [n=12, P<0.001]). Splenocytes isolated from the recipients of Ad-B7-H4–transduced islets showed hyporesponsiveness to alloantigenic stimulation, compared with control recipients. CD45 and insulin staining of the graft transplanted with Ad-B7-H4–transduced islets indicated the preservation of &bgr; cells and decrease of infiltrating immune cells. Conclusions. Local expression of B7-H4 prolongs islet allograft survival in vivo, suggesting translational potential for &bgr;-cell replacement with reduced immune injury.

B7-H4 Treatment of T Cells Inhibits ERK, JNK, p38, and AKT Activation.

B7-H4 is a newly identified B7 homolog that plays an important role in maintaining T-cell homeostasis by inhibiting T-cell proliferation and lymphokine-secretion. In this study, we investigated the signal transduction pathways inhibited by B7-H4 engagement in mouse T cells. We found that treatment of CD3+ T cells with a B7-H4.Ig fusion protein inhibits anti-CD3 elicited T-cell receptor (TCR)/CD28 signaling events, including phosphorylation of the MAP kinases, ERK, p38, and JNK. B7-H4.Ig treatment also inhibited the phosphorylation of AKT kinase and impaired its kinase activity as assessed by the phosphorylation of its endogenous substrate GSK-3. Expression of IL-2 is also reduced by B7-H4. In contrast, the phosphorylation state of the TCR proximal tyrosine kinases ZAP70 and lymphocyte-specific protein tyrosine kinase (LCK) are not affected by B7-H4 ligation. These results indicate that B7-H4 inhibits T-cell proliferation and IL-2 production through interfering with activation of ERK, JNK, and AKT, but not of ZAP70 or LCK.

Hair Follicle Mesenchyme-Associated PD-L1 Regulates T-Cell Activation Induced Apoptosis: A Potential Mechanism of Immune Privilege

The immune privilege (IP) of hair follicles (HFs) has been well established in previous studies. However, whether cultured HF cells still exhibit IP properties, the individual factors involved in this process, and the detailed mechanisms that drive and maintain IP, are largely unidentified. We found preferential expression of IP-associated genes in cultured HF dermal papilla and dermal sheath cup cells (DSCCs) compared with non-follicular fibroblasts (FBs) at passage 4, suggesting a potential for functional IP. Notably, programmed cell death 1 ligand 1 (PD-L1) was significantly upregulated in DSCCs and dermal papilla cells relative to FBs. IFNγ secretion from peripheral blood mononuclear cells (PBMCs) co-cultured with histoincompatible DSCCs was significantly lower than with FB and higher percentages of early apoptotic, Annexin V+ cells were observed in PBMC co-cultured with DSCCs. Knockdown of PD-L1 translation by silencing interfering RNA in DSCCs enabled increased IFNγ secretion by PBMCs, whereas transfection of pCMV6-XL4/hPD-L1 in FB significantly reduced IFNγ secretion and increased apoptosis in co-cultured PBMCs. We also found that apoptosis in allogeneic T cells induced by DSCCs was largely dependent on the mitochondrial pathway. Our study suggests IP properties are exhibited in cultured DSCCs in part through expression of negative co-signaling molecule PD-L1.

B7-H4 Pathway in Islet Transplantation and β-Cell Replacement Therapies

Type 1 diabetes (T1D) is a chronic autoimmune disease and characterized by absolute insulin deficiency. β-cell replacement by islet cell transplantation has been established as a feasible treatment option for T1D. The two main obstacles after islet transplantation are alloreactive T-cell-mediated graft rejection and recurrence of autoimmune diabetes mellitus in recipients. T cells play a central role in determining the outcome of both autoimmune responses and allograft survival. B7-H4, a newly identified B7 homolog, plays a key role in maintaining T-cell homeostasis by reducing T-cell proliferation and cytokine production. The relationship between B7-H4 and allograft survival/autoimmunity has been investigated recently in both islet transplantation and the nonobese diabetic (NOD) mouse models. B7-H4 protects allograft survival and generates donor-specific tolerance. It also prevents the development of autoimmune diabetes. More importantly, B7-H4 plays an indispensable role in alloimmunity in the absence of the classic CD28/CTLA-4 : B7 pathway, suggesting a synergistic/additive effect with other agents such as CTLA-4 on inhibition of unwanted immune responses.

Endogenous expression of B7-H4 improves long-term murine islet allograft survival.

Background Allograft rejection is one of the main obstacles for islet transplantation. B7-H4 plays a key role in maintaining T-cell homeostasis by reducing T-cell proliferation and cytokine production. In this study, we investigated whether the endogenous expression of B7-H4 in &bgr; cells from B7-H4 transgenic mice enhances islet allograft survival. Methods B7-H4 transgenic C57BL/6 (B6) mice (RIP.B7-H4) were developed by inserting the entire B7-H4 open reading frame under the rat insulin promoter (RIP). B7-H4 protein expression was examined by flow cytometric analysis and immunohistochemical staining. Islet allograft survival was investigated in streptozotocin-induced diabetic recipient BALB/c (H-2d) mice transplanted with 400 islets from RIP.B7-H4 (H-2b) mice under the kidney capsule. The recipient control group received islets from wild-type B6 donors. Results B7-H4 protein was significantly up-regulated in isolated islets from RIP.B7-H4 compared with wild-type B6 mice (56%±23% vs. 3%±1.2%). B7-H4 was coexpressed with insulin, but not glucagon, suggesting that B7-H4 is expressed in a &bgr;-cell–specific manner. Recipient BALB/c mice transplanted with RIP.B7-H4 islets established euglycemia for 42.3±18.4 days (mean±SD; n=9) compared with controls at 23.1±7.8 days (mean±SD; n=12; P<0.004, log-rank test). Conclusions The endogenous expression of B7-H4 in donor &bgr; cells from transgenic mice prolongs islet allograft survival, confirming the negative role of B7-H4 in regulating alloreactive T-cell responses.

Improving Islet Engraftment by Gene Therapy

Islet cell transplantation is currently the only feasible long-term treatment option for patients with type 1 diabetes. However, the majority of transplanted islets experience damage and apoptosis during the isolation process, a blood-mediated inflammatory microenvironment in the portal vein upon islet infusion, hypoxia induced by the low oxygenated milieu, and poor-revascularization-mediated lack of nutrients, and impaired hormone modulation in the local transplanted site. Strategies using genetic modification methods through overexpression or silencing of those proteins involved in promoting new formation of blood vessels or inhibition of apoptosis may overcome these hurdles and improve islet engraftment outcomes.

Blockade of both B7-H4 and CTLA-4 co-signaling pathways enhances mouse islet allograft survival

Costimulation blockade is an effective way to prevent allograft rejection. In this study, we tested the efficacy of two negative co-signaling molecules in protecting islet allograft function. We used local expression of B7-H4 by adenoviral transduction of islets (Ad-B7-H4) and systemic administration of CTLA-4.Ig to investigate the outcomes of allograft survival. Five groups of streptozotocin-induced diabetic C57BL/6 mice received 400 islets each from BALB/c donors. The groups consisted of control (G1); CTLA-4.Ig (G2); Ad-LacZ (G3); Ad-B7-H4 (G4); and Ad-B7-H4 and CTLA-4.Ig combined (G5). G1 and G3 developed graft failure on average of two weeks. G2, G4 and G5 survived for 43.8 ± 34.8, 54.7 ± 31.2 and 77.8 ± 21.5 d, respectively. Activated T and B cells in the lymph nodes were significantly controlled by CTLA-4.Ig treatment. Significantly reduced infiltrates were also detected in the allografts of G2 compared with G1. By contrast, B7-H4 significantly inhibited Th1-associated IFN-gamma secretion in the early stage and increased Foxp3+ T cells in the long-term surviving allografts. Our study suggests that CTLA-4 and B7-H4 inhibit alloimmune responses through distinct mechanisms, and that combination therapy which activates two negative co-signaling pathways can further enhance islet allograft survival.

OVER-EXPRESSION OF B7-H4 IN BETA CELLS FROM TRANSGENIC MICE PROLONGS ISLET ALLOGRAFT SURVIVAL IN TRANSPLANTATION: 3365

X. Wang1, J. Hao1, D. Metzger2, A. Mui1, Z. Ao3, C.B. Verchere4, L. Chen5, D. Ou6, G.L. Warnock1 1Surgery, UBC, Vancouver/BC/CANADA, 2Pediatrics, University of British Columbia, vancouver/BC/CANADA, 3Department Of Surgery, University Of British Columbia, Human Islet Transplant Laboratory, Vancouver/CANADA, 4Pathology And Laboratory Medicine, Child and Family Research Institute, University of British Columbia, Vancouver/ BC/CANADA, 5Oncology, Johns Hopkins University School of Medicine, Baltimore/MD/UNITED STATES OF AMERICA, 6Surgery, University of British Columbia, Vancouver/BC/CANADA

Local Expression of B7-H4 by Transducing Recombinant Adenovirus in Islet Grafts Improves Allotransplant Outcomes.

Genetic Deficiency for Proprotein Convertase 2 (PC2) in Mice is Protective form Age and Diet-induced Obesity. Younes Anini, Janice Mayne, Francine Sirois, Jeffrey Gagnon, Andrew Chen, Nadine Kaefer, Mary-Ellen Harper, Michel Chretien, Majambu Mbikay, Dalhousie University, Halifax, and Ottawa Health Research Institute, Ottawa. Proprotein convertase 2 (PC2) is a member of the subtilase family of proteinases. PC2 is primarily expressed in endocrine and neroendocrine tissues where it mediates the production of neuropeptides and hormones known to influence glucose and energy homeostasis. In this study, we examined the importance of PC2 in food intake, fuel preference, energy expenditure and body mass. Relative to their wild type counterparts, 16 weeks old PC2 mice carried significantly less epididymal fat, (PC2, 0.386 ± 0.102 g vs PC2, 2.276 ± 0.156, P < 0.0001) with no significant difference in the lean mass (gastroc muscle). Visceral and perikidney fat mass was also significantly reduced in PC2. These observation was corroborated by the reduced level of circulating leptin in PC2 mice. There was no significant difference between PC2 and PC2 mice in the rate of food intake nor in the level of the orexigenic hormone ghrelin. Mice of both genotypes were subjected to either lowfat (LFD) or high-fat diet (HFD), for 10 weeks. PC2 mice gained more weight under the HFD. In contrast, PC2 mice gained similar amount of weight under HFD and LFD. Indirect calorimetry was conducted on aged mice indicated that PC2 mice use carbohydrates more than lipids as energy source. We have shown that PC2-deficient mice are insensitive to age and diet-induced body weight gain. These data indicate that PC2 level in endocrine tissue is a quantitative trait controlling diet-induced obesity. ABSTRACT #141