Shutdown of immunological priming and presentation after in vivo administration of adenovirus.

Adenoviral (Adv) vectors are widely used in both experimental and clinical trials for vaccination and gene therapy. Recombinant Adv can evoke potent innate immune responses and adaptive immune responses to encoded antigens. However, how Adv infection affects the response to subsequently encountered antigens is poorly understood. We show that intravenously administered replication defective (E1 and E3 deleted) Adv educes functional changes in dendritic cells (DC) resulting in impaired priming of cytotoxic T lymphocytes (CTL) more than 7 days after Adv treatment. Generalized DC activation was indicated by transient upregulation of CD86 and reduced endocytosis of fluorescent beads. It is known that CD8+ DC are predominantly responsible for uptake and presentation (cross-presentation) of exogenous antigens to CD8+ CTL. Hence, impaired endocytosis in CD8+, but not CD8-, DC at 7 days after Adv administration provided an explanation for the impaired CTL response to antigen at this time. Shutdown of cross-presentation was confirmed using cytochrome c (cytc), an agent that selectively depletes cross-presenting DC. Adv-infection rendered CD8+ DC resistant to depletion by cytc. As the cross-presentation pathway underlies CD8 T-cell responses to many cancers and to vaccines or viruses that do not directly infect DC, systemic Adv administration may impair these responses.

Without CD4 help, CD8 rejection of pig xenografts requires CD28 costimulation but not perforin killing.

Although CD4 cells are major mediators in cellular rejection of fetal pig pancreas (FPP) in the mouse, rejection still occurs in the absence of CD4 cells, albeit with delayed kinetics. CD4 cell-independent mechanisms of cellular rejection are poorly understood. To investigate the involvement of CD8 T cells in FPP rejection and their activation requirements, we used mice transgenic for anti-CD4 Ab; this is the most complete model of CD4 cell deficiency. We showed that in such mice FPP was infiltrated with CD8 cells starting from 2 wk posttransplantation and FPP was eventually rejected 8 wk posttransplantation. Ab depletion of CD8 cells greatly improved the survival of FPP and reduced cell infiltration at the graft site. This suggests that CD8 cells can mediate the rejection of porcine xenografts in the absence of CD4 cells. This CD8-mediated rejection of FPP is independent of their perforin-mediated lytic function, as graft survival was not affected in mice deficient in perforin. The production of IFN-gamma and IL-5 by the graft infiltrates indicates that CD8 cells may act through cytokine-mediated mechanisms. Remarkably, in the absence of CD4 cells, lymphocyte infiltration at the graft site was absent in mice transgenic for CTLA4Ig such that the islet grafts flourished beyond 24 wk. In contrast, rejection was little affected by CD40 ligand deficiency. Therefore, we show that CD8 cells are activated to mediate FPP rejection independent of perforin and that this CD4-independent activation of CD8 cells critically depends on B7/CD28 costimulation.

Cell-associated ovalbumin is cross-presented much more efficiently than soluble ovalbumin in vivo.

To better understand the antigenic requirements for cross-presentation, we compared the in vivo efficiency of presentation of cell-associated vs soluble OVA with the OT-I (CD8) and OT-II (CD4) TCR transgenic lines. Cross-presentation of cell-associated OVA was very efficient, requiring as little as 21 ng of OVA to activate OT-II cells and 100-fold less to activate OT-I cells. In contrast, soluble OVA was presented inefficiently, requiring at least 10,000 ng OVA for activation of either T cell subset. Thus, cell-associated OVA was presented 500-fold more efficiently than soluble OVA to CD4 T cells and 50,000-fold more efficiently to CD8 T cells. These data, which represent the first quantitative in vivo analysis of cross-presentation, show that cell-associated OVA is very efficiently presented via the class I pathway.

Overcoming the poor immunogenicity of a protein by DNA immunization as a fusion construct.

The production of antibodies against poorly immunogenic proteins is problematic. Often there is a failure to generate such antibodies. Furthermore, antibodies against other specificities are frequently induced. We describe a simple approach, analogous to conjugation to a protein carrier, whereby immunization with naked DNA was used to raise antibody to a highly homologous and poorly immunogenic allotypic protein. Deoxyribonucleic acid encoding the protein of interest was fused to DNA encoding the Fc region of a foreign Ig, resulting in increased immunogenicity. The potential applications of this approach include the production of antisera and mAb to allotypic variants, mutant proteins, and proteins that are highly conserved between species.

Delayed rejection of fetal pig pancreas in CD4 cell deficient mice was correlated with residual helper activity.

CD4 cells have been shown to play a dominant role in the rejection of xenografts. Depletion of murine CD4 cells by injecting anti-CD4 antibody prolongs the graft survival, but does not prevent its rejection. For a more stable phenotype, we used genetically modified mice. To test whether the delayed rejection is caused by incomplete depletion of CD4 cells, we evaluated the response to fetal pig pancreas (FPP) xenografts in three types of CD4 cell deficient mice. They are MHC class II deficient mice (MHC II(o/o), CD4 deficient mice (CD4(o/o)) and a novel type of CD4 cell deficient mice (designated GK). GK mice were rendered permanently and completely CD4 deficient by transgenic expression of anti-CD4 antibody, whereas both MHC II(o/o) and CD4(o/o) mice have a residual helper cell population. FPP grafts in wild type mice were rejected within a week, whereas FPP grafts survived up to 4 weeks in MHC II(o/o) and CD4(o/o) mice. Survival of grafts in GK mice was even longer (8 weeks). Differences in histology were also noted. Rejecting grafts in MHC II(o/o) and wild-type mice were infiltrated with both eosinophils and mononuclear cells, whereas the infiltrates in CD4(o/o) and GK mice were exclusively mononuclear cells. Immunohistochemistry showed that they were primarily CD8 cells. The immune response to FPP was clearly different in the three types of CD4 cell deficient mice. Splenocytes of MHC II(o/o) 3 weeks post-transplant with FPP produced substantial amounts of IFN-gamma and IL-5, whereas splenocytes of CD4(o/o) mice produced low levels of IFN-gamma but no detectable IL-5. At similar times, these cytokines were not detected in GK mice. Furthermore, CD4(o/o) mice were capable of mounting helper dependent, although reduced, IgG responses to FPP antigens, while GK mice were not. The above results indicate that residual helper activity in some types of CD4 cell deficient mice could still contribute to xenograft rejection. Caution needs to be exercised where such mice are used as models of CD4 cell deficiency. Also, because there is eventual rejection of xenograft FPP in GK mice which lack detectable helper activity, we argue that these mice are a better model to investigate the involvement of CD4-independent rejection mechanisms.

Local production of anti-CD4 antibody by transgenic allogeneic grafts affords partial protection.

BACKGROUND: Immunosuppressive drugs and anti-lymphocyte antibody are used clinically to suppress cellular rejection responses. However, these systemic regimens often led to general immunodeficiency and thus increased susceptibility to opportunistic infection and neoplasia. Immunosuppressive molecules delivered locally may be a way of inhibiting rejection responses, whereas systemic immunity is preserved. To achieve protective local immunosuppression, we produced a graft secreting its own immunomodulator, by deriving transgenic mice expressing a chimeric anti-CD4 antibody (GK2c) in the pancreas. METHODS AND RESULTS: Transgenic mice in bml genetic background expressing a modified anti-mouse CD4 antibody (GK2c) under two promoters have been produced. Tissue expression of GK2c was detected by immunoperoxidase staining. Under the cytomegalovirus promoter, there was abundant GK2c expression in pancreatic exocrine tissue. Under the rat preproinsulin II promoter, there was abundant GK2c expression in pancreatic endocrine tissue only. High-expression transgenic lines had 10-100 microg/ml GK2c in blood plasma. By flow cytometry, these transgenic mice were devoid of CD4+ cells in their peripheral lymphoid organs. To test transgenic mice as donors, fetal pancreata from transgenic mice were grafted into fully allogeneic CBA mice under the kidney capsule, transgenic grafts had prolonged survival compared with control non-transgenic grafts. Furthermore, GK2c transgenic grafts had reduced infiltration with an absence of CD4+ cells at the graft site without any effect on the cell composition in lymphatic tissues. CONCLUSION: Transgenic grafts that secrete anti-CD4 antibody can afford some protection against graft rejection, while only affecting the CD4 population at the graft site.

CD4 help-independent induction of cytotoxic CD8 cells to allogeneic P815 tumor cells is absolutely dependent on costimulation.

Mice made transgenic (Tg) for a rat anti-mouse CD4 Ab (GK mice) represent a novel CD4-deficient model. They not only lack canonical CD4 cells in the periphery, but also lack the residual aberrant Th cells that are found in CD4-/- mice and MHC class II-/- mice. To analyze the role of CD4 help and costimulation for CTL induction against alloantigens, we have assessed the surface and functional phenotype of CD8 cells in vivo (e.g., clearance of allogeneic P815 cells) and in vitro. In our CD4-deficient GK mice, CTL responses to allogeneic P815 cells were induced, albeit delayed, and were sufficient to eliminate P815 cells. Induction of CTL and elimination of allogeneic P815 cells were inhibited both in the presence and absence of CD4 cells by temporary CD40 ligand blockade. This indicated that direct interaction of CD40/CD40L between APCs and CD8 cells may be an accessory signal in CTL induction (as well as the indirect pathway via APC/CD4 interaction). Furthermore, whereas in CTLA4Ig single Tg mice P815 cells were rejected promptly, in the double Tg GK/CTLA4Ig mice CTL were not induced and allogeneic P815 cells were not rejected. These findings suggest that CD40/CD40L is involved in both CD4-dependent and CD4-independent pathways, and that B7/CD28 is pivotal in the CD4-independent pathway of CTL induction against allogeneic P815 cells.

Protective effect of CTLA4Ig secreted by transgenic fetal pancreas allografts.

BACKGROUND: Pancreas allotransplantation offers a cure for insulin-dependent diabetes mellitus. Systemic immunosuppression used to prevent immune destruction of the graft has side-effects, including increased susceptibility to infection and neoplasia. These unwanted effects may be limited by engineering the graft to secrete immunomodulatory molecules, to achieve local immunosuppression. Several studies have shown that transient local CTLA4Ig results in partial protection of allogeneic grafts. Our intent has been to determine whether sustained secretion of transgenic CTLA4Ig from pancreatic islets is able to protect against allograft rejection. METHODS AND RESULTS: Mouse CTLA4 (test=CTLA4Ig) or CD5 leader sequence (control=CD5LIg) was fused to the Fc of mouse IgG2c, and expressed transgenically under the control of the rat insulin promoter in C57BL/6 mice carrying the bml mutation of H-2K(b) (B6.C-H-2(bm1)). This resulted in expression in pancreatic islets. We used ELISA quantification of transgene products secreted into the supernatants of cultured fetal pancreata to select high (CTLA4Ig(hi)) and low (CTLA4Ig(lo)) expresser transgenic mice. Cultured fetal pancreata were transplanted under the kidney capsule of wholly allogeneic CBA recipient mice. CTLA4Ig(hi) but not CTLA4Ig(lo) expresser grafts showed enhanced survival compared with control CD5LIg grafts at 6 weeks posttransplant, provided the recipient mice were transiently depleted of CD4 T cells (by a single low-dose injection of GK1.5) before transplantation. CONCLUSIONS: Sustained local secretion of CTLA4Ig from transgenic grafts in combination with transient systemic CD4 T-cell depletion can enhance allograft acceptance.

CD8 T cell ignorance or tolerance to islet antigens depends on antigen dose.

There are two major mechanisms reported to prevent the autoreactivity of islet-specific CD8(+) T cells: ignorance and tolerance. When ignorance is operative, naïve autoreactive CD8(+) T cells ignore islet antigens and recirculate without causing damage, unless activated by an external stimulus. In the case of tolerance, CD8(+) T cells are deleted. Which factor(s) contributes to each particular outcome was previously unknown. Here, we demonstrate that the concentration of self antigen determines which mechanism operates. When ovalbumin (OVA) was expressed at a relatively low concentration in the pancreatic islets of transgenic mice, there was no detectable cross-presentation, and the CD8(+) T cell compartment remained ignorant of OVA. In mice expressing higher doses of OVA, cross-presentation was detectable and led to peripheral deletion of OVA-specific CD8(+) T cells. When cross-presentation was prevented by reconstituting the bone marrow compartment with cells incapable of presenting OVA, deletional tolerance was converted to ignorance. Thus, the immune system uses two strategies to avoid CD8(+) T cell-mediated autoimmunity: for high dose antigens, it deletes autoreactive T cells, whereas for lower dose antigens, it relies on ignorance.

Activation of metallothionein gene expression by hypoxia involves metal response elements and metal transcription factor-1.

Metallothioneins (MTs) are a family of stress-induced proteins with diverse physiological functions, including protection against metal toxicity and oxidants. They may also contribute to the regulation of cellular proliferation, apoptosis, and malignant progression. We reported previously that the human (h)MT-IIA isoform is induced in carcinoma cells (A431, SiHa, and HT29) exposed to low oxygen, conditions commonly found in solid tumors. The present study demonstrates that the genes for hMT-IIA and mouse (m)MT-I are transcriptionally activated by hypoxia through metal response elements (MREs) in their proximal promoter regions. These elements bind metal transcription factor-1 (MTF-1). Deletion and mutational analyses of the hMT-IIA promoter indicated that the hMRE-a element is essential for basal promoter activity and for induction by hypoxia, but that other elements contribute to the full transcriptional response. Functional studies of the mMT-I promoter demonstrated that at least two other MREs (mMRE-d and mMRE-c) are responsive to hypoxia. Multiple copies of either hMRE-a or mMRE-d conferred hypoxia responsiveness to a minimal MT promoter. Mouse MT-I gene transcripts in fibroblasts with targeted deletions of both MTF-1 alleles (MTF-1(-/-); dko7 cells) were not induced by zinc and showed low responsiveness to hypoxia. A transiently transfected MT promoter was unresponsive to hypoxia or zinc in dko7 cells, but inductions were restored by cotransfecting a mouse MTF-1 expression vector. Electrophoretic mobility shift assays detected a specific protein-DNA complex containing MTF-1 in nuclear extracts from hypoxic cells. Together, these results demonstrate that hypoxia activates MT gene expression through MREs and that this activation involves MTF-1.

Tumor hypoxia and gene expression--implications for malignant progression and therapy.

Most histopathological classifications of human cancers include significant numbers of hypoxic cells. There is increasing evidence that, at least in certain types of human solid tumors, there is a positive relationship between the presence of hypoxia and poor outcome after radiation therapy alone or radiation combined with other therapies. Hypoxia appears to be an independent prognostic factor. There is evidence for enhanced malignant progression associated with hypoxia, including locoregional invasion and distant metastases. The presence of hypoxia may negatively affect outcome by induction of radiation resistance by the classical oxygen effect and/or by effects on gene expression and malignant progression, causing more aggressive locoregional and distant disease. It is now clear that hypoxia has the potential to influence expression of genes and activities of associated proteins that regulate growth and tissue homeostasis, resulting in cellular phenotypic heterogeneity. The molecular pathways involved in signaling and regulating changes in gene activities in response to external stresses such as hypoxia are becoming known. Identification of patients with hypoxic tumors will lead to improved selective therapy.

The redox-sensitive human antioxidant responsive element induces gene expression under low oxygen conditions.

Transient transfection studies of human HepG2 and mouse Hepa hepatocarcinoma cells with a reporter gene construct regulated by a human antioxidant responsive element (ARE) from the NQO1 gene demonstrated that the element is responsive to low oxygen conditions. The antioxidant N-acetyl L-cysteine (NAC) strongly inhibited basal aerobic reporter gene activity in HepG2 cells without obviously affecting the hypoxic induction, as is consistent with ARE sensitivity to oxidative stress in aerobic cultures. Electrophoretic mobility shift (EMS) assays of nuclear extracts of HepG2 and Hepa cells lysed under aerobic or hypoxic conditions or after exposure to the phenolic compound 3-(2)-tert-butyl-4-hydroxyanisole (BHA), showed specific and constitutive protein binding to the ARE under all of these conditions. Taken together, these findings show that the ARE can mediate gene expression in response to low oxygen conditions. Co-ordinately regulated expression of ARE-dependent genes, such as phase II detoxification enzymes, may be an important phenotype of solid tumors containing significant regions of pathophysiological hypoxia.

CD4+ and CD8+ T cells are not major effectors of mouse hepatitis virus A59-induced demyelinating disease.

We examined murine hepatitis virus strain A59 (MHV-A59)-induced demyelinating disease in C57BL/6 mice which had previously been thymectomized at 25 days of age. Demyelination was observed in 51-96% of spinal cord quadrants examined 30 or 60 days post infection (dpi), indicating that neither an intact thymus nor thymic infection is a prerequisite to demyelination. Depletion of CD4+ or CD8+ T cells at 5, 7 or 10 dpi did not influence the extent of demyelination indicating that neither T cell subset is a major effector of demyelination. However, these findings do not exclude the possibility that T cells are involved in initiating demyelinating disease very early in infection.

Temporal discontinuities in progression of NOD autoimmune diabetes.

Consideration of the pathophysiology of insulin-dependent diabetes mellitus in the nonobese diabetic (NOD) mouse can be viewed from a temporal perspective. We argue that there are discontinuous phases and each phase may reflect a phenotype educed by a particular set of genetic and epigenetic events. Therefore, temporal dissection may be a useful platform for causal dissection and we have set out this article as follows: 1. Introduction. 2. "Pre-time." a. Genetics. b. Parental effects. 3. Development of insulitis. a. Development of autoimmunity vs waning of or failure to establish tolerance. b. Importance of beta cell mass. c. Homing. 4. Onset of beta cell destruction. 5. Further Discussion.

Regulation of mucosal IgA responses in vivo: cytokines and adjuvants.

The predominance of IgA plasma cells at mucosal sites reflects a combination of the selective localisation and vigorous proliferation after extravasation of IgA plasma cell precursors. Experiments are described here which demonstrate that post-extravasation events leading to IgA precursor cell retention, proliferation and antibody secretion are under cytokine control. This has led to investigation of therapeutic interventions to modify cytokine availability to maximise mucosal vaccination responses and correct IgA deficiencies.

Expression of the ATDC (ataxia telangiectasia group D-complementing) gene in A431 human squamous carcinoma cells.

The ATDC gene was originally identified by its ability to complement the radiosensitivity defect of an ataxia telangiectasia (AT) fibroblast cell line. Because hypersensitivity to ionizing radiation is an important feature of the AT phenotype, we reasoned that ATDC may function generally in the suppression of radiosensitivity. Previous work in our laboratory focused on radiosensitization mechanisms in human squamous carcinoma (SC) cells, especially A431 cells. To establish a basis for investigating the role of ATDC in radiation-responsive signaling pathways in human SC cells, we characterized ATDC message and protein expressions in A431 cells. ATDC message expression was also compared among human epidermoid cells (A431 cells, HaCaT spontaneously immortalized human keratinocytes and normal human epidermal keratinocytes) and a normal human fibroblast cell line (LM217). We made the following major observations: (i) the relative abundance of ATDC message is substantially higher in the epidermoid cells than in the fibroblast cell line, which has a message level comparable to those reported for other fibroblast lines; (ii) ATDC is constitutively phosphorylated on serine/threonine in A431 cells; (iii) in A431 cells, ATDC is a substrate for the serine/threonine protein kinase C (PKC) but not the epidermal growth factor (EGF) receptor tyrosine kinase; and (iv) EGF decreases ATDC message and protein expressions in A431 cells after a 24-hr exposure. The phosphorylation studies suggest that the ability of ATDC to modulate cellular radiosensitivity may be mediated in part through a PKC signaling pathway.

Exploitation of the Vbeta8.2 T cell receptor in protection against experimental autoimmune encephalomyelitis using a live vaccinia virus vector.

This study takes advantage of the predominant usage of Vbeta8.2 by the TCRs of encephalitogenic T cells specific for myelin basic protein. Vaccinia virus recombinants expressing Vbeta8.2 (VVbeta8.2) 8.2) and Vbeta3 (VVbeta 3) proteins were constructed, and their abilities to confer protection against experimental autoimmune encephalomyelitis (EAE) induction in H-2u mice were examined. Mice immunized with VVbeta8.2 developed very mild EAE by comparison with mice that were vaccinated with VVbeta3, which developed severe clinical symptoms. This reduction in EAE correlated with a diminished T cell proliferative response to myelin basic protein in the mice that received VVbeta8.2 compared with that in mice receiving VVbeta3.

Fused pyrazine mono-n-oxides as bioreductive drugs. II Cytotoxicity in human cells and oncogenicity in a rodent transformation assay.

PURPOSE: To determine what structural moieties of the fused pyrazine mono-N-oxides are determining factors in their in vitro cytotoxicity and oncogenicity. METHODS AND MATERIALS: A new series of experimental bioreductive drugs, fused pyrazine mono-N-oxides, was evaluated in vitro for aerobic and hypoxic cytotoxicity in the HT29 human colon adenocarcinoma cell line by using clonogenic assays. The relative oncogenicities of these compounds were also determined in aerobic cultures of C3H 10T1/2 mouse embryo fibroblasts by using a standard transformation assay. RESULTS: Removal of the 4-methyl piperazine side chain from the parent compound, RB 90740, reduced the potency of the hypoxic cytotoxin. Reduction of the N-oxide function increased the aerobic cytotoxicity and eliminated most of the hypoxic/aerobic cytotoxic differential. The reduced N-oxide also had significant oncogenicity, consistent with a mechanism of genotoxicity following bioreduction of RB 90740. CONCLUSION: This new series of bioreductive compounds may be effective in cancer therapy, particularly the lead compound RB 90740. The oncogenic potential of these compounds is similar to that for other cancer therapies. Further studies should include evaluation of these compounds in vivo and the development of analogs with reduced oncogenic potential and retention of the hypoxic/aerobic cytotoxicity differential.

Mapping of the vascular endothelial growth factor-producing hypoxic cells in multicellular tumor spheroids using a hypoxia-specific marker.

We have investigated the hypoxia inducibility of vascular endothelial growth factor (VEGF) in multicellular tumor spheroids of HT29 cells using a monoclonal antibody to a fluorinated bioreductive drug, EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)aceta mide], a chemical probe for hypoxia. We have shown that VEGF expression is predominantly localized in interior spheroid cells that are sufficiently hypoxic to bioreductively activate the 2-nitroimidazole and produce immunologically detectable adducts of the EF5 compound. Northern blotting analyses demonstrated that VEGF165 is the predominant form of VEGF produced by HT29 cells and that the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate did not induce VEGF expression. This study demonstrates that VEGF expression is up-regulated in response to hypoxia and in the microenvironments found in human multicellular tumor spheroids. This investigation also illustrates the utility of the EF5 binding in multi-cellular tumor spheroids as a means of studying the expression and regulation of hypoxia-inducible genes.

Cytokine mediated effects in mucosal immunity.

The predominance of IgA antibodies in mucosal sites reflects a combination of high rate IgA isotype switching among precursor cells in induction sites, their selective localization in mucosal effector tissues and vigorous proliferation of these cells after extravasation. Each of these steps leading to IgA expression at the mucosa is under cytokine control. This paper will address the role of cytokines in induction and expression of IgA responses and strategies for manipulating cytokine expression. Therapeutic interventions based on this information may not only lead to improved vaccination responses and correction of immunodeficiencies but also, by invoking the phenomenon of oral tolerance, may assist in the management of autoimmune, allergic and alloreactive disease.