The B7/CD28 family has profound modulatory effects in immune responses and constitutes important targets for the development of novel therapeutic drugs against human diseases. Here we describe a new CD28 homolog (CD28H) that has unique functions in the regulation of the human immune response and is absent in mice. CD28H is constitutively expressed on all naive T cells. Repetitive antigenic exposure, however, induces a complete loss of CD28H on many T cells, and CD28H-negative T cells have a phenotype of terminal differentiation and senescence. After extensive screening in a receptor array, a B7-like molecule, B7 homolog 5 (B7-H5), was identified as a specific ligand for CD28H. B7-H5 is constitutively found in macrophages and could be induced on dendritic cells. The B7-H5/CD28H interaction co-stimulates human T cell growth and cytokine production, selectively via an AKT-dependent signaling cascade. Our study identifies a novel co-stimulatory pathway regulating human T cell responses.

All animals are ecosystems, home to diverse microbial populations. Animal-associated microbes play important roles in the normal development and physiology of their hosts, but can also be agents of infectious disease. Traditionally, mice have been used to study pathogenic and beneficial associations between microbes and vertebrate animals. The zebrafish is emerging as a valuable new model system for host-microbe interaction studies, affording researchers with the opportunity to survey large populations of hosts and to visualize microbe-host associations at a cellular level in living animals. This chapter provides detailed protocols for the analysis of zebrafish-associated microbial communities, the derivation and husbandry of germ-free zebrafish, and the modeling of infectious disease in different stages of zebrafish development via different routes of inoculation. These protocols offer a starting point for researchers to address a multitude of questions about animals’ coexistence with microorganisms.

Cystic fibrosis (CF) is a genetic disease caused by recessive mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is associated with prevalent and chronic Pseudomonas aeruginosa lung infections. Despite numerous studies that have sought to elucidate the role of CFTR in the innate immune response, the links between CFTR, innate immunity, and P. aeruginosa infection remain unclear. The present work highlights the zebrafish as a powerful model organism for human infectious disease, particularly infection by P. aeruginosa. Zebrafish embryos with reduced expression of the cftr gene (Cftr morphants) exhibited reduced respiratory burst response and directed neutrophil migration, supporting a connection between cftr and the innate immune response. Cftr morphants were infected with P. aeruginosa or other bacterial species that are commonly associated with infections in CF patients, including Burkholderia cenocepacia, Haemophilus influenzae, and Staphylococcus aureus. Intriguingly, the bacterial burden of P. aeruginosa was found to be significantly higher in zebrafish Cftr morphants than in controls, but this phenomenon was not observed with the other bacterial species. Bacterial burden in Cftr morphants infected with a P. aeruginosa ⌬LasR mutant, a quorum sensing-deficient strain, was comparable to that in control fish, indicating that the regulation of virulence factors through LasR is required for enhancement of infection in the absence of Cftr. The zebrafish system provides a multitude of advantages for studying the pathogenesis of P. aeruginosa and for understanding the role that innate immune cells, such as neutrophils, play in the host response to acute bacterial infections commonly associated with cystic fibrosis.Cystic fibrosis (CF) is a multiorgan genetic disorder characterized by chronic pulmonary infections as well as gastrointestinal and reproductive abnormalities. In 1989, it was discovered that CF results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (25), which encodes a chloride ion channel present in most epithelial cells. Today, 80 to 95% of CF patients succumb to respiratory failure resulting from chronic lung infections that begin in early childhood and that are caused primarily by Pseudomonas aeruginosa (7, 32). Chronic infection with the bacterium leads to hyperinflammation characterized by the excessive recruitment of neutrophils to the airways and the subsequent release of oxidants and enzymes from activated neutrophils, which damages the lung tissue over time (32).Pseudomonas aeruginosa is a flagellated Gram-negative opportunistic pathogen of plants and animals and is found naturally in soil and water. As a nosocomial pathogen, it is the third most commonly isolated organism, leading primarily to urinary tract or wound infections in immunocompromised patients (17). Due to its production of alginate, the low permeability of its outer membrane and the presence of multidrug efflux pumps, P. aeruginosa exhibits a high degree of ...

To observe real-time interactions between green fluorescent protein-labeled immune cells and invading bacteria in the zebrafish (Danio rerio), a series of plasmids was constructed for the red fluorescent protein (RFP) labeling of a variety of fish and human pathogens. The aim of this study was to create a collection of plasmids that would express RFP pigments both constitutively and under tac promoter regulation and that would be nontoxic and broadly transmissible to a variety of Gram-negative bacteria. DNA fragments encoding the RFP dimeric (d), monomeric (m), and tandem dimeric (td) derivatives d-Tomato, td-Tomato, m-Orange, and m-Cherry were cloned into the IncQ-based vector pMMB66EH in Escherichia coli. Plasmids were mobilized into recipient strains by conjugal mating. Pigment production was inducible in Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, and Vibrio (Listonella) anguillarum strains by isopropyl-␤-D-thiogalactopyranoside (IPTG) treatment. A spontaneous mutant exconjugant of P. aeruginosa PA14 was isolated that expressed td-Tomato constitutively. Complementation analysis revealed that the constitutive phenotype likely was due to a mutation in lacI q carried on pMMB66EH. DNA sequence analysis confirmed the presence of five transitions, four transversions, and a 2-bp addition within a 14-bp region of lacI. Vector DNA was purified from this constitutive mutant, and structural DNA sequences for RFP pigments were cloned into the constitutive vector. Exconjugants of P. aeruginosa, E. tarda, and V. anguillarum expressed all pigments in an IPTG-independent fashion. Results from zebrafish infectivity studies indicate that RFP-labeled pathogens will be useful for the study of real-time interactions between host cells of the innate immune system and the infecting pathogen.

T lymphocytes require signals from self-peptides and cytokines, most notably interleukins 7 and 15 (IL-7, IL-15), for survival. While mouse T cells die rapidly if IL-7 or IL-15 is withdrawn, human T cells can survive prolonged withdrawal of IL-7 and IL-15. Here we show that IL-7 and IL-15 are required to maintain human T cell proliferative capacity through the STAT5 signaling pathway. T cells from humanized mice proliferate better if stimulated in the presence of human IL-7 or IL-15 or if T cells are exposed to human IL-7 or IL-15 in mice. Freshly isolated T cells from human peripheral blood lose proliferative capacity if cultured for 24 hours in the absence of IL-7 or IL-15. We further show that phosphorylation of STAT5 correlates with proliferation and inhibition of STAT5 reduces proliferation. These results reveal a novel role of IL-7 and IL-15 in maintaining human T cell function, provide an explanation for T cell dysfunction in humanized mice, and have significant implications for in vitro studies with human T cells.