Adenovirus-mediated interferon alpha gene transfer induces regional direct cytotoxicity and possible systemic immunity against pancreatic cancer.

We previously demonstrated a characteristically high sensitivity of pancreatic cancer cells to interferon alpha (IFN-alpha) gene transfer, which induced a more prominent growth suppression and cell death in pancreatic cancer cells than in other types of cancers and normal cells. The IFN-alpha protein can exhibit both direct cytotoxicity and indirect immunological antitumour activity. Here, we dissected and examined the two mechanisms, taking advantage of the fact that IFN-alpha did not show any cross-species activity in its in vivo effect. When a human IFN-alpha adenovirus was injected into subcutaneous xenografts of human pancreatic cancer cells in nude mice, tumour growth was significantly suppressed due to cell death in an adenoviral dose-dependent manner. The IFN-alpha protein concentration was markedly increased in the injected subcutaneous tumour, but leakage of the potent cytokine into the systemic blood circulation was minimal. When a mouse IFN-alpha adenovirus was injected into the same subcutaneous tumour system, all mice showed significant tumour inhibition, an effect that was dependent on the indirect antitumour activities of IFN-alpha, notably a stimulation of natural killer cells. Moreover, in this case, tumour regression was observed not only for the injected subcutaneous tumours but also for the untreated tumours at distant sites. This study suggested that a local IFN-alpha gene therapy is a promising therapeutic strategy for pancreatic cancer, due to its dual mechanisms of antitumour activities and lack of significant toxicity.

Murine xenogeneic immune responses to the human testis: a presumed immune-privileged tissue.

INTRODUCTION: Immune privilege provides a natural paradigm for potentially down-regulating allogeneic and xenogeneic inflammatory immune responses. Fas ligand has been suggested as a general underlying mechanism of immune privilege; the human Fas ligand has been shown to ligate murine Fas in vitro. METHODS: In this study, we examined whether the human testicular xenograft, a presumed immune-privileged tissue would have prolonged survival in mice. In addition, in vitro and in vivo murine xenogeneic immune responses to the human testicular xenografts were characterized using MHC class I, MHC class II, CD4, CD8, CD4/8 knockout mice. RESULTS: Unlike in rodent testis, Fas ligand mRNA is not expressed and Fas is highly expressed in human testis. Human testicular xenografts are immunogenic, and do not induce any preferential pattern of recipient systemic Th1 or Th2 cytokine bias. Interestingly, an indefinite survival of the human testicular xenografts is observed in murine MHC class II knockout mice, whereas the human skin xenografts were rejected without a delay. In vivo murine immune responses to human testicular xenografts require a recipient MHC class II-dependent CD4 T cell-mediated process that appears to depend on B7-1/B7-2 costimulatory signals. CONCLUSIONS: Our results demonstrate that the concept of immune privilege, as defined by the expression of Fas ligand and prolonged survival after transplantation, cannot be extended to human testis. The stringent restriction of murine xenogeneic immune responses to discordant human testicular xenografts to the indirect MHC class II-dependent CD4 T cell-mediated pathway suggests a potential venue for immune modulation to induce tolerance across a discordant species barrier.

Dendritic cells genetically engineered to express Fas ligand induce donor-specific hyporesponsiveness and prolong allograft survival.

Polarization of an immune response toward tolerance or immunity is dictated by the interactions between T cells and dendritic cells (DC), which in turn are modulated by the expression of distinct cell surface molecules, and the cytokine milieu in which these interactions are taking place. Genetic modification of DC with genes coding for specific immunoregulatory cell surface molecules and cytokines offers the potential of inhibiting immune responses by selectively targeting Ag-specific T cells. In this study, the immunomodulatory effects of transfecting murine bone marrow-derived DC with Fas ligand (FasL) were investigated. In this study, we show that FasL transfection of DC markedly augmented their capacity to induce apoptosis of Fas+ cells. FasL-transfected DC inhibited allogeneic MLR in vitro, and induced hyporesponsiveness to alloantigen in vivo. The induction of hyporesponsiveness was Ag specific and was dependent on the interaction between FasL on DC and Fas on T cells. Finally, we show that transfusion of FasL-DC significantly prolonged the survival of fully MHC-mismatched vascularized cardiac allografts. Our findings suggest that DC transduced with FasL may facilitate the development of Ag-specific unresponsiveness for the prevention of organ rejection. Moreover, they highlight the potential of genetically engineering DC to express other genes that affect immune responses.

T cells are necessary and critical for xenograft rejection in new concordant cardiac xenotransplant model.

BACKGROUND: A new vascularized concordant xenotransplant model using the Chinese hamster as donor and mouse as recipient species is reported. This model takes advantage of the wealth of informative immune reagents and knockout and transgenic backgrounds available for the mouse. METHODS: Heterotopic auxillary cardiac transplantation was performed. The mean survival time was assessed by daily palpation. Xenoreactive antibody production was measured by flow cytometry, and cardiac xenografts were examined by light microscopy. RESULTS: The tempo of xenograft rejection in this model is consistent with concordant species combination. IgM and IgG3 responses were not critical for the concordant xenograft rejection. Long-term survival (>100 days) of the concordant cardiac xenografts was observed without any immunosuppression in nude mice. Reconstitution of nude mice with CD3+ T cells induced the xenograft rejection in 5.7 days (P<0.01). CONCLUSION: This new concordant cardiac xenotransplant model demonstrates that T-dependent xenogeneic immune response is necessary and critical for the xenograft rejection.

A 150-base pair 5' region of the MHC class I HLA-B7 gene is sufficient to direct tissue-specific expression and locus control region activity: the alpha site determines efficient expression and in vivo occupancy at multiple cis-active sites throughout this region.

To characterize cis- and trans-acting mechanisms that regulate MHC class I transcription during development and in adult tissues, we have used transgenic mice to study a series of human MHC (HLA)-B7 class I gene constructs. Previous studies identified the 5' -0.66-kb to -0.075-kb region as sufficient to direct appropriate and efficient tissue-specific levels of HLA-B7 RNA relative to H-2 class I. Results here show that DNA 5' of -0.26 kb is not required for any aspect of expression. As the expression level correlated with the transgene copy number, was comparable to H-2 or a per-gene copy basis and was independent of integration site, the -0.075 to -0.26-kb segment also functions as a locus control region. With this region, sequences 3' of -0.075 kb, possibly at the promoter, appear to direct the appropriate tissue distribution. Of conserved sequences in the -0.075 to -0.26-kb region, enhancer B box is nonessential. In contrast, in vivo "footprinting" implicated region I/ enhancer A/NF-kappaB, IFN consensus/response sequence, and alpha in class I regulation as they are "occupied" in a tissue-specific pattern that correlates with expression. Mutation of alpha leads to decreased expression and loss of occupancy not only at alpha but also at region I/enhancer A/NF-kappaB and IFN consensus/response sequence. Thus, site alpha is an essential class I regulatory element, the dominant function of which is to mediate tissue-specific occupancy at multiple adjacent cis-active sites, possibly by facilitating stable synergistic interactions between factors at these distinct elements.