Tammy T. Chang

Antigen-specific regulatory T cells develop via the ICOS-ICOS-ligand pathway and inhibit allergen-induced airway hyperreactivity.

Asthma is caused by T-helper cell 2 (Th2)-driven immune responses, but the immunological mechanisms that protect against asthma development are poorly understood. T-cell tolerance, induced by respiratory exposure to allergen, can inhibit the development of airway hyperreactivity (AHR), a cardinal feature of asthma, and we show here that regulatory T (TR) cells can mediate this protective effect. Mature pulmonary dendritic cells in the bronchial lymph nodes of mice exposed to respiratory allergen induced the development of TR cells, in a process that required T-cell costimulation via the inducible costimulator (ICOS)–ICOS-ligand pathway. The TR cells produced IL-10, and had potent inhibitory activity; when adoptively transferred into sensitized mice,* TR cells blocked the development of AHR. Both the development and the inhibitory function of regulatory cells were dependent on the presence of IL-10 and on ICOS–ICOS-ligand interactions. These studies demonstrate that TR cells and the ICOS–ICOS-ligand signaling pathway are critically involved in respiratory tolerance and in downregulating pulmonary inflammation in asthma.*There was an error in the AOP version of this article. The sentence in the abstract that read The TR cells produced IL-10, and had potent inhibitory activity; when adoptively transferred into sensitized mouse TR cells, blocked the development of AHR was worded incorrectly. The following sentence is correct: The TR cells produced IL-10, and had potent inhibitory activity; when adoptively transferred into sensitized mice, TR cells blocked the development of AHR. This has been corrected in the HTML and the PDF. We regret this error.

Mouse Inducible Costimulatory Molecule (ICOS) Expression Is Enhanced by CD28 Costimulation and Regulates Differentiation of CD4+ T Cells

The inducible costimulatory (ICOS) molecule is expressed by activated T cells and has homology to CD28 and CD152. ICOS binds B7h, a molecule expressed by APC with homology to CD80 and CD86. To investigate regulation of ICOS expression and its role in Th responses we developed anti-mouse ICOS mAbs and ICOS-Ig fusion protein. Little ICOS is expressed by freshly isolated mouse T cells, but ICOS is rapidly up-regulated on most CD4+ and CD8+ T cells following stimulation of the TCR. Strikingly, ICOS up-regulation is significantly reduced in the absence of CD80 and CD86 and can be restored by CD28 stimulation, suggesting that CD28-CD80/CD86 interactions may optimize ICOS expression. Interestingly, TCR-transgenic T cells differentiated into Th2 expressed significantly more ICOS than cells differentiated into Th1. We used two methods to investigate the role of ICOS in activation of CD4+ T cells. First, CD4+ cells were stimulated with beads coated with anti-CD3 and either B7h-Ig fusion protein or control Ig fusion protein. ICOS stimulation enhanced proliferation of CD4+ cells and production of IFN-γ, IL-4, and IL-10, but not IL-2. Second, TCR-transgenic CD4+ T cells were stimulated with peptide and APC in the presence of ICOS-Ig or control Ig. When the ICOS:B7h interaction was blocked by ICOS-Ig, CD4+ T cells produced more IFN-γ and less IL-4 and IL-10 than CD4+ cells differentiated with control Ig. These results demonstrate that ICOS stimulation is important in T cell activation and that ICOS may have a particularly important role in development of Th2 cells.

Monolayer and Spheroid Culture of Human Liver Hepatocellular Carcinoma Cell Line Cells Demonstrate Distinct Global Gene Expression Patterns and Functional Phenotypes

Understanding cell biology of three-dimensional (3D) biological structures is important for more complete appreciation of in vivo tissue function and advancing ex vivo organ engineering efforts. To elucidate how 3D structure may affect hepatocyte cellular responses, we compared global gene expression of human liver hepatocellular carcinoma cell line (HepG2) cells cultured as monolayers on tissue culture dishes (TCDs) or as spheroids within rotating wall vessel (RWV) bioreactors. HepG2 cells grown in RWVs form spheroids up to 100 mum in diameter within 72 h and up to 1 mm with long-term culture. The actin cytoskeleton in monolayer cells show stress fiber formation while spheroids have cortical actin organization. Global gene expression analysis demonstrates upregulation of structural genes such as extracellular matrix, cytoskeletal, and adhesion molecules in monolayers, whereas RWV spheroids show upregulation of metabolic and synthetic genes, suggesting functional differences. Indeed, liver-specific functions of cytochrome P450 activity and albumin production are higher in the spheroids. Enhanced liver functions require maintenance of 3D structure and environment, because transfer of spheroids to a TCD results in spheroid disintegration and subsequent loss of function. These findings illustrate the importance of physical environment on cellular organization and its effects on hepatocyte processes.

The effect of water-soluble vitamin E on cyclosporine pharmacokinetics in healthy volunteers

We evaluated the effect of water‐soluble vitamin E (d‐α‐tocopheryl polyethylene glycol 1000 succinate [TPGS]; Liqui‐E) on the oral pharmacokinetics of the cyclosporine, a poorly available (~30%) drug, in healthy volunteers. Ten healthy subjects were given two doses of oral cyclosporine (10 mg/kg) separated by a 7‐day washout period. Oral TPGS (2.6 IU/kg) was administered concomitantly with one of the cyclosporine doses in a randomized order. A significant increase was observed in area under the blood concentration‐time curve (AUC; mean ± SD) with concomitant TPGS administration (3908 ± 2601 versus 6296 ± 5102 ng · hr/ml). Significant decreases were observed in apparent oral clearance (0.24 ± 0.14 versus 0.15 ± 0.08 L/hr/kg) and apparent oral steady‐state volume of distribution (1.57 ± 0.95 versus 1.07 ± 0.73 L/kg). No significant changes were observed in the ratios of metabolites to parent drug AUC values. The comparable relative decreases in apparent oral clearance (38%) and apparent oral steady‐state volume of distribution (30%) with TPGS are most likely explained by enhanced absorption, decreased counter transport back into the intestine by P‐glycoprotein, or some unknown mechanism by which cyclosporine is protected from metabolism in the gut, thereby increasing bioavailability.

The Rel/NF‐κB pathway and transcription of immediate early genes in T cell activation are inhibited by microgravity

This study tested the hypothesis that transcription of immediate early genes is inhibited in T cells activated in μg. Immunosuppression during spaceflight is a major barrier to safe, long‐term human space habitation and travel. The goals of these experiments were to prove that μg was the cause of impaired T cell activation during spaceflight, as well as understand the mechanisms controlling early T cell activation. T cells from four human donors were stimulated with Con A and anti‐CD28 on board the ISS. An on‐board centrifuge was used to generate a 1g simultaneous control to isolate the effects of μg from other variables of spaceflight. Microarray expression analysis after 1.5 h of activation demonstrated that μg‐ and 1g‐activated T cells had distinct patterns of global gene expression and identified 47 genes that were significantly, differentially down‐regulated in μg. Importantly, several key immediate early genes were inhibited in μg. In particular, transactivation of Rel/NF‐κB, CREB, and SRF gene targets were down‐regulated. Expression of cREL gene targets were significantly inhibited, and transcription of cREL itself was reduced significantly in μg and upon anti‐CD3/anti‐CD28 stimulation in simulated μg. Analysis of gene connectivity indicated that the TNF pathway is a major early downstream effector pathway inhibited in μg and may lead to ineffective proinflammatory host defenses against infectious pathogens during spaceflight. Results from these experiments indicate that μg was the causative factor for impaired T cell activation during spaceflight by inhibiting transactivation of key immediate early genes.

Recovery from EAE is associated with decreased survival of encephalitogenic T cells in the CNS of B7-1/B7-2-deficient mice.

Adoptive transfer experiments using C57BL/6 mice lacking B7‐1 and B7‐2 as recipients of wt (wt) encephalitogenic T cells demonstrate a key role for B7 costimulation during the effector phase of experimental autoimmune encephalomyelitis (EAE). Following transfer of encephalitogenic T cells, B7‐1/B7‐2‐deficient (–/–) recipients develop a transient and mild disease as compared to wt recipients. To understand the mechanism by which B7‐1/B7‐2 may influence the effector phase of EAE, we analyzed T cells, pro‐inflammatory cytokines and chemokines within the CNS of wt and B7‐1/B7‐2–/– recipients at different times after adoptive transfer of activated myelin specific T cells. There was a marked decline in T cells and inflammatory mediators in the CNS of B7‐1/B7‐2–/– recipients by day 30 post transfer. B7‐1/B7‐2–/– mice developed more TUNEL+ apoptotic cells in the parenchyma and greater ratios of TUNEL+ cells/parenchymal foci than wt mice resulting in virtual disappearance of parenchymal foci. Therefore, without B7‐1 and B7‐2 costimulation in the target organ, there is increased T cell apoptosis and attenuation of inflammation. Theseresults indicate that B7‐1 and B7‐2 provide critical costimulatory signals for sustaining survival of pathogenic T cells within the central nervous system parenchyma during the effector phase of EAE and suggest novel treatment approaches in the effector phase of autoimmune diseases.

Implementation of a multidisciplinary treatment team for hepatocellular cancer at a Veterans Affairs Medical Center improves survival

Several methods of treatment for hepatocellular carcinoma (HCC) are often used in combination for either palliation or cure. We established a multidisciplinary treatment team (MDTT) at the San Francisco Veterans Affairs Medical Center in November 2003 and assessed whether aggressive multimodality treatment strategies may affect survival. A prospective database was established and follow-up information from patients with presumed HCC was collected up to November 2006. Information from the American College of Surgeons (ACS) cancer registry from January 2000 to November 2003 identified patients with HCC that were evaluated at the same institution prior to the establishment of the MDTT. The establishment of a MDTT resulted in the doubling of patient referrals for treatment. Significantly more patients were evaluated at earlier stages of disease and received either palliative or curative therapies. The overall survival (p<0.0001) and length of follow-up (p<0.05) were significantly improved after the establishment of the MDTT. Stage-by-stage comparisons indicate that aggressive multimodality therapy conferred significant survival advantage to patients with American Joint Commission on Cancer (AJCC) stage II HCC (odds ratio 15.50, p<0.001). Multidisciplinary collaboration and multimodality treatment approaches are important in the management of hepatocellular carcinoma and improves patient survival.

Molecular mechanisms underlying the enhanced functions of three-dimensional hepatocyte aggregates

Three-dimensional (3D) culture of hepatocytes leads to improved and prolonged synthetic and metabolic functions, but the underlying molecular mechanisms are unknown. In order to investigate the role of 3D cell-cell interactions in maintaining hepatocyte differentiated functions ex vivo, primary mouse hepatocytes were cultured either as monolayers on tissue culture dishes (TCD) or as 3D aggregates in rotating wall vessel (RWV) bioreactors. Global gene expression analyses revealed that genes upregulated in 3D culture were distinct from those upregulated during liver development and liver regeneration. Instead, they represented a diverse array of hepatocyte-specific functional genes with significant over-representation of hepatocyte nuclear factor 4α (Hnf4a) binding sites in their promoters. Expression of Hnf4a and many of its downstream target genes were significantly increased in RWV cultures as compared to TCD. Conversely, there was concomitant suppression of mesenchymal and cytoskeletal genes in RWV cultures that were induced in TCDs. These findings illustrate the importance of 3D cell-cell interactions in maintaining fundamental molecular pathways of hepatocyte function and serve as a basis for rational design of biomaterials that aim to optimize hepatocyte functions ex vivo for biomedical applications.

Physiological Ranges of Matrix Rigidity Modulate Primary Mouse Hepatocyte Function In Part Through Hepatocyte Nuclear Factor 4 Alpha

Matrix rigidity has important effects on cell behavior and is increased during liver fibrosis; however, its effect on primary hepatocyte function is unknown. We hypothesized that increased matrix rigidity in fibrotic livers would activate mechanotransduction in hepatocytes and lead to inhibition of liver‐specific functions. To determine the physiologically relevant ranges of matrix stiffness at the cellular level, we performed detailed atomic force microscopy analysis across liver lobules from normal and fibrotic livers. We determined that normal liver matrix stiffness was around 150 Pa and increased to 1‐6 kPa in areas near fibrillar collagen deposition in fibrotic livers. In vitro culture of primary hepatocytes on collagen matrix of tunable rigidity demonstrated that fibrotic levels of matrix stiffness had profound effects on cytoskeletal tension and significantly inhibited hepatocyte‐specific functions. Normal liver stiffness maintained functional gene regulation by hepatocyte nuclear factor 4 alpha (HNF4α), whereas fibrotic matrix stiffness inhibited the HNF4α transcriptional network. Fibrotic levels of matrix stiffness activated mechanotransduction in primary hepatocytes through focal adhesion kinase. In addition, blockade of the Rho/Rho‐associated protein kinase pathway rescued HNF4α expression from hepatocytes cultured on stiff matrix. Conclusion: Fibrotic levels of matrix stiffness significantly inhibit hepatocyte‐specific functions in part by inhibiting the HNF4α transcriptional network mediated through the Rho/Rho‐associated protein kinase pathway. Increased appreciation of the role of matrix rigidity in modulating hepatocyte function will advance our understanding of the mechanisms of hepatocyte dysfunction in liver cirrhosis and spur development of novel treatments for chronic liver disease. (Hepatology 2016;64:261–275)

Genetic background determines the requirement for B7 costimulation in induction of autoimmunity.

B7 costimulatory molecules play an important role in inducing autoimmunity, tumor immunity, and transplant rejection, and therapeutic manipulation of B7 is being investigated in human diseases. To determine whether B7 costimulation is essential for inducing autoimmunity on different genetic backgrounds, we backcrossed B7.1/B7.2 deficient (–/–) mice on to the C57BL/6 (B6) and SJLbackgrounds and induced experimental autoimmune encephalomyelitis (EAE) in these mice. B7.1/B7.2–/– mice on the B6 background were resistant to EAE induced with MOG 35–55, whereas the SJL B7.1/B7.2–/– mice were susceptible to PLP 139–151 or PLP 178–191‐induced EAE. The SJL B7.1/B7.2–/– mice had a qualitatively different lesion pattern in that they showed increased white matter vacuolation compared to wild‐type SJL mice when immunized with either PLP 139–151 or PLP 178–191. (B6×SJL)F1 B7.1/B7.2+/+ mice were susceptible to EAE whereas (B6×SJL)F1 B7.1/B7.2–/– mice were resistant to EAE induced with either encephalitogenic peptide. Thus, genetic background determines the B7 requirement for inducing autoimmunity. These data have important implications for developing B7‐based immunotherapies for human diseases.