Inflammation promotes the loss of adeno-associated virus-mediated transgene expression in mouse liver.

BACKGROUND & AIMS: Non-self transgenes delivered to mouse liver via adeno-associated virus (AAV) are expressed stably due to the induction of immune tolerance. However, such transgene expression has been reported to be lost in higher-order primates. We investigated whether inflammatory processes, which likely differ between species, impact the stability of transgene expression. METHODS: We developed a mouse model that mimics a scenario in which a subject that has received hepatic AAV-mediated gene transfer develops subsequent, vector-unrelated, systemic inflammation. RESULTS: Inflammation eliminated previously stable expression of transgenes delivered by AAV; the limited tissue destruction and persistence of AAV genomes implicated pathways besides the cytotoxic T-cell response. Tumor necrosis factor-a down-regulated expression of the transgene from the AAV, indicating a role for similar inflammatory cytokines in such loss of transgene expression. CONCLUSIONS: Inflammation can block AAV-mediated expression of transgenes in mouse liver. The presence of inflammation might therefore affect hepatic expression of transgenes from viral vectors in humans.

Potential of immunotherapy for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with limited therapeutic options. Since HCC has been shown to be immunogenic, T cell-based immunotherapy is considered a promising treatment. In this review, we summarize current knowledge of T cell responses against tumour-associated antigens, as well as the mechanisms underlying the poor quality of these responses in patients with HCC. Insights into these important aspects of HCC immunology are crucial for the further development of novel immunotherapies.

Lack of ex vivo peripheral and intrahepatic α-fetoprotein-specific CD4+ responses in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignancies with a poor prognosis and limited therapeutic options that is often characterized by the expression of the tumor-associated antigen α-fetoprotein (AFP). CD4+ helper T cells are important in generating potent anticancer immunity as they prime and expand CD8+ T-cell memory and may also have direct antitumor activity. However, very little information is currently available about the relative frequency, immunodominance and peripheral versus intratumoral distribution of AFP-specific CD4+ T-cell responses in patients with HCC. We, therefore, analyzed AFP-specific CD4+ responses in blood and tumor tissue of patients with HCC by using overlapping peptides spanning the entire AFP protein and novel sensitive approaches such as antigen-specific upregulation of CD154. We found that AFP-specific CD4+ T-cell responses were not detectable in the peripheral blood ex vivo. However, after in vitro stimulation, AFP-specific CD4+ T-cell responses were detectable in a large fraction of patients targeting different previously unreported epitopes with no clear immunodominance. These results indicate that AFP-specific CD4+ T-cell responses are not completely deleted but only present at very low frequencies. Importantly, AFP-specific CD4+ T-cell responses were also rarely detectable in tumor tissue, suggesting that the relative absence of these cells in the circulation ex vivo is not due to a rapid accumulation to the tumor side. Taken together, these results suggest that the lack of sufficient CD4+ T-cell help, especially within the tumor tissue, may be one central mechanism responsible for the failure of AFP-specific immune responses to control HCC progression.

AAV vectors avoid inflammatory signals necessary to render transduced hepatocyte targets for destructive T cells.

Studies in mice indicate that gene transfer to liver with vectors based on adeno-associated viruses (AAVs) is characterized by immunological tolerance to antigenic transgene products. Mechanisms to explain host nonresponsiveness have focused on aberrant T-cell responses. We propose a distinct mechanism for conferring tolerance to AAV-transduced hepatocytes that relates to diminished sensitivity of the target organ to T cell-mediated effects. T cells to beta-galactosidase (beta-gal) were adoptively transferred into RAG(-/-) mice expressing beta-gal in hepatocytes due to prior administration of either Ad or AAV vectors. Adoptive transfer was associated with extinction of LacZ expression in Ad-LacZ-transduced RAG(-/-) mice and had no effect on liver LacZ expression in AAV-LacZ-transduced RAG(-/-) mice. Systemic administration of TLR ligands lipopolysaccharide (LPS) and CpG at the time of adoptive transfer did lead to extinction of LacZ expression. Systemic TLR ligands were associated with upregulation of major histocompatibility complex (MHC) class I and the cell adhesion molecules ICAM and VCAM as was seen with Ad-LacZ alone. These data indicate that AAV transduction lacks the inflammatory signals necessary to render hepatocyte targets for cytotoxic T lymphocytes (CTLs). Underlying liver pathology may confound vector performance and should be considered in the design of clinical trials.

BALB/c mice show impaired hepatic tolerogenic response following AAV gene transfer to the liver.

Following adeno-associated virus (AAV) gene transfer to the liver, both C57BL/6 and BALB/c mice show long-term expression of nonself transgene antigens along with the absence of a transgene-specific immune response. However, in this study, we report that despite the equal ability to induce T-cell tolerance to vector-encoded antigens, the underlying mechanisms are entirely different in these two strains. We have previously shown that in C57BL/6 mice, cytotoxic T lymphocyte (CTL) responses to systemic AAV-delivered antigens are suppressed by combined actions of hepatic regulatory T cells (Tregs), Kupffer cells, and hepatic suppressive cytokines. In stark contrast, our present findings reveal that such tolerogenic response is not induced in the liver of BALB/c mice systemically administered with AAV. As a result, these mice fail to suppress a transgene-specific CTL response induced by a strong immunogenic challenge and express dramatically reduced levels of AAV-encoded antigen. Interestingly, there was active B-cell tolerance to the transgene antigen, which was mediated by splenic Tregs. We conclude that lack of tolerance induction in the liver renders BALB/c mice susceptible to CTL-mediated clearance of transduced hepatocytes.

Hepatic regulatory T cells and Kupffer cells are crucial mediators of systemic T cell tolerance to antigens targeting murine liver.

UNLABELLED: The mechanisms of tolerance in the liver that limit susceptibility to food allergy and that mediate the acceptance of liver transplants, even with a complete major histocompatibility complex (MHC) mismatch, remain poorly defined. Here we report that in a model of liver-directed gene transfer, cytotoxic T lymphocyte (CTL) responses to non-self antigens are controlled by hepatic regulatory T cells (Tregs) that secrete the immunosuppressive cytokine interleukin (IL)-10 in response to the antigen. In addition, Kupffer cells (KCs), normally thought to initiate immune responses, are rendered tolerogenic in this context. The depletion of KCs results in a complete abrogation of IL-10 production by hepatic Tregs, indicating an interaction between Tregs and KCs in the induction of tolerance. CONCLUSION: Our study suggests that hepatic Tregs together with KCs create a local suppressive microenvironment that prevents the establishment of the CTL response. These mechanisms provide pivotal insights and may prove instrumental in the tolerization toward non-self therapeutic proteins delivered to the liver.

Promoter cloning and characterisation of the transcriptional regulation of the human thyrostimulin A2 subunit.

The novel heterodimeric glycoprotein hormone thyrostimulin consists of two unique subunits, A2 and B5. To understand its yet unknown transcriptional regulation, we characterised the 3.1-kb immediate 5'-flanking region of the human A2 gene localised on chromosome 11q13. In transient transfection assays this sequence exhibited promoter activity, which could be confined to nucleotides -506 to -347 relative to the ATG start codon. Interestingly, this minimal promoter appeared to be non-tissue-specific. Deletional, mutational and gel shift analyses revealed regulatory elements that are essential for the regulation of the A2 gene expression. Another noteworthy feature of this gene is the presence of silencer elements upstream and downstream of the promoter. To surmise, our results provide an initial step toward a detailed analysis of the underlying molecular mechanisms of the human thyrostimulin gene expression.

The promoter of the human sodium/iodide symporter responds to certain phthalate plasticisers.

The diesters of benzene-1,2-dicarboxylic (phthalic) acid, the phthalates, are used to make plastics flexible and can comprise 40% of the weight of plastic. Human exposure to phthalates can occur via ingestion, inhalation and dermal routes, as well as through parenteral exposure from medical devices containing phthalates. Since earlier morphological studies showed that some phthalates induced thyroid hyperactivity, we thought it important to investigate possible effects of six major phthalates on the transcriptional activity of sodium/iodide symporter (NIS). Di-isodecyl phthalate (DIDP), benzyl butyl phthalate (BBP) and di-octyl phthalate (DOP) increased the activity of the human NIS promoter construct 2.5-, 2.6- and 2.4-fold, respectively. Likewise, these phthalates also enhanced the rat NIS endogenous mRNA expression ca. 2-fold. No effect was observed for bis-(2-ethylhexyl) phthalate (DEHP) and di-isononyl phthalate (DINP), whereas dibutyl phthalate (DBP) appeared to down-regulate hNIS promoter. Although the demonstrated stimulation of NIS gene transcription by DIDP, BBP and DOP is not very strong, this finding is of great importance as humans are routinely exposed for long periods to phthalate plasticisers, the accumulation of which may contribute to thyroid hyperfunction.

Graves' IgG activate upstream enhancer of the sodium/iodide symporter.

Graves' thyroid tissue has been shown to express elevated levels of human sodium/iodide symporter (hNIS) mRNA and protein. In the present work, we demonstrate for the first time that hNIS gene expression in Graves' disease (GD) is up-regulated by Graves' IgG. Here, in transient transfection experiments using FRTL-5 cells, hNIS promoter and enhancer/luciferase construct showed an up to six-fold increase in transcriptional activity after incubation with purified Graves' IgG. Mutation of a CRE site in hNIS enhancer inhibited Graves' IgG response. In addition, mutation of a novel putative regulatory region in hNIS promoter reduced the stimulation three-fold. This discovered putative regulatory sequence might play a role in hNIS up-regulation by Graves' IgG and TSH. The data presented here complement our current knowledge of the pathogenesis of GD and will contribute to a better understanding of mechanisms regulating the thyroid iodide concentrating activity.