Rationale: Club cell secretory protein (CC16) is a pneumoprotein produced predominantly by pulmonary club cells. Circulating CC16 is associated with protection from the inception and progression of the two most common obstructive lung diseases: asthma and COPD. Objective: While exact mechanisms remain elusive, studies consistently suggest a causal role of CC16 in mediating anti-inflammatory and antioxidant functions in the lung. We sought to determine any novel receptor systems that could participate in CC16's role in obstructive lung diseases. Methods: Protein alignment of CC16 across species led to the discovery of a highly conserved sequence of amino acids, Leucine-Valine-Aspartic Acid (LVD), a known integrin binding motif. Recombinant CC16 was generated with and without the putative integrin binding site. A Mycoplasma pneumoniae mouse model and a flourescent cellular adhesion assay were used to determine the impact of the LVD site in regards to CC16 function during live infection and on cellular adhesion during inflammatory conditions. Results: CC16 bound to integrin alpha 4 and beta 1 (α4β1), also known as the adhesion molecule very late antigen-4 (VLA-4), dependent on the presence of the LVD integrin binding motif. During infection, rCC16 rescued lung function parameters both when administered to the lung and intravenously, but only when the LVD integrin binding site is intact; likewise, neutrophil recruitment during infection and leukocyte adhesion were both impacted by the loss of the LVD site. Conclusions: We discovered a novel receptor for CC16, VLA-4, which has important mechanistic implications for the role of CC16 in circulation as well as in the lung compartment.

Obliterative bronchiolitis (OB) is a major cause of morbidity and mortality after lung transplantation (LTx). Club cells are bronchiolar epithelial progenitor cells that produce club cell secretory protein (CCSP), which has anti-inflammatory functions. We previously showed that depleting grafts of club cells using naphthalene (NA) results in OB in both syngeneic and allogeneic murine LTx. We aimed to determine whether post-transplant treatment with CCSP decreases acute rejection or OB in club cell-depleted grafts.

Club cell secretory protein-16 (CC16) is the major secreted product of airway club cells, but its role in the pathogenesis of chronic obstructive pulmonary disease (COPD) is unclear. We measured CC16 airway expression in humans with and without COPD and CC16 function in a cigarette smoke (CS)-induced COPD murine model.Airway CC16 expression was measured in COPD patients, smokers without COPD and non-smokers. We exposed wildtype (WT) and CC16(-/-)mice to CS or air for up to 6 months, and measured airway CC16 expression, pulmonary inflammation, alveolar septal cell apoptosis, airspace enlargement, airway mucin 5AC (MUC5AC) expression, small airway remodelling and pulmonary function.Smokers and COPD patients had reduced airway CC16 immunostaining that decreased with increasing COPD severity. Exposing mice to CS reduced airway CC16 expression. CC16(-/-) mice had greater CS-induced emphysema, airway remodelling, pulmonary inflammation, alveolar cell apoptosis, airway MUC5AC expression, and more compliant lungs than WT mice. These changes were associated with increased nuclear factor-kappa B (NF-kappa B) activation in CC16(-/-) lungs. CS-induced acute pulmonary changes were reversed by adenoviral-mediated over-expression of CC16.CC16 protects lungs from CS-induced injury by reducing lung NF-kappa B activation. CS-induced airway CC16 deficiency increases CS-induced pulmonary inflammation and injury and likely contributes to the pathogenesis of COPD.

By using adoptive transfer of Ag-loaded bone marrow-derived dendritic cells (BMDC), we have established an in vivo model of CTL priming. Activation of CTL in these experiments required both CD4(+) T cells and CD154, demonstrating that this model reflects CD4(+) T cell-dependent dendritic cell (DC) licensing. Because IL-12 has been suggested to play an important role in CTL activation by DC, we examined the ability of BMDC to prime CTL in the complete absence of IL-12 using p40-deficient mice. We observed that the absence of IL-12 does not affect the phenotype or allostimulatory function of BMDC after in vitro maturation. Moreover, there was no difference in the ability of Ag-loaded DC to elicit CTL cytotoxicity, whether the Ag was delivered by virus infection or peptide pulsing. Equal frequencies of Ag-specific, IFN-gamma-secreting CD8(+) T cells developed in both wild-type and IL-12-deficient backgrounds. Finally, CTL generated in the IL-12-deficient environment were capable of protecting immunized mice against tumor challenge, demonstrating that these CTL were fully functional, despite the absence of IL-12 during the maturation process in vivo. These results indicate that IL-12 is not critical for the development of IFN-gamma secreting, CD8(+) T cells and that another mechanism must be used by licensed DC to prime and activate CTL.

Genetic immunization through ex vivo transduction of dendritic cells has been suggested as an effective approach to enhance antitumor immunity by activating both CD4+ and CD8+ T cells. Immunizing mice with dendritic cells transduced with an adenovirus expressing the human melanoma antigen glycoprotein 100 (DCAdhgp100) as a cancer vaccine, we demonstrated complete protective immunity and a potent CTL response against melanomas expressing murine glycoprotein 100 in a CD4+ cell-dependent manner. Surprisingly, however, effective tumor rejection was not the result of cooperation between CD4+ and CD8+ T cells. Protective immunity was completely lost when CD4+ cells were depleted immediately before tumor challenge, whereas it was unaffected by removal of CD8+ cells, establishing a principal role for CD4+ cells in the effector phase of tumor rejection. Neither protective immunity nor CTL generation in this model required interleukin 12, in spite of high levels of IFN-gamma secretion by tumor-reactive T cells. Most notably, the DCAdhgp100 vaccine could elicit protective antitumor CD4+ cells in the absence of CD40 ligand, although it does not bypass the need for CD40-mediated signals to generate melanoma-reactive CTLs. Thus, in contrast to the current thinking that the optimal cancer vaccine should include determinants for both CD4+ and CD8+ cells, the potency of the DCAdhgp100 vaccine appears to be a result of its ability to directly prime autoreactive CD4+ cells through a process that does not require interleukin 12 and CD40 signals.

Glycoprotein 100 (gp100) is one of a series of well-characterized human melanoma-associated antigens expressed by most melanoma cells. Immunization of C57BL/6 mice with an adenovirus (Ad) vector encoding human gp100 (Adhgp100) has been shown to induce limited protective immunity against challenge with murine melanoma B16 cells. In the current study we determined whether gp100-specific immunity can be enhanced using bone-marrow-derived dendritic cells (DCs) transduced with Adhgp100 ex vivo. Subcutaneous injection of Adhgp100-infected DCs resulted in potent T-cell-mediated protective immunity and a greater than 80% reduction of established tumors when administered to B16 tumor-bearing hosts. Compared to direct injection of Adhgp100 vector alone, immunization with Adhgp100-infected DCs induced markedly greater antitumor activity. In vitro CTL analysis demonstrated that DC-Adhgp100 immunization activated both CD4(+) and CD8(+) CTLs, while no lytic activity was generated by vaccination with Adhgp100 alone. In vivo depletion of CD4(+) T cells, but not CD8(+) T cells, completely abrogated CTL activity, suggesting that Adhgp100-transduced DCs result in activation of both CD4(+) and CD8(+) CTLs via a CD4(+)-dependent mechanism. We speculate that this improved efficacy of Adhgp100-transduced DCs compared to direct immunization with Adhgp100 may be the result of direct DC-mediated CD4(+) T cell activation. These results emphasize the importance of CD4(+) T cells in the development of therapeutic antigen-specific cancer vaccines.

Ubiquitin fusion technology represents an emerging method for economically producing peptides and small proteins in the bacterium Escherichia coli . Our focus is on peptide production where the need for cost‐effective, scaleable processes has recently been highlighted by Kelley (1996) . There are two principal features: (1) the expression system consists of a suitable E. coli host strain paired with a plasmid that encodes the ubiquitin fusion and (2) an ubiquitin‐specific protease, UCH‐L3, which cleaves only C‐terminal extensions from ubiquitin. In this work, multigram yields were obtained of four ubiquitin fusions derived from cell paste generated in single 10‐L fermentations. All were expressed intracellularly and remained soluble at extremely high levels of expression. Bacterial freeze−thaw lysates contained over 95% pure ubiquitin fusion protein. All four fusions were efficiently cleaved to ubiquitin and the peptide products. In one case, the final yield of peptide was 1.08 g from 3 L of low cell density bacterial culture. The combination of exceptional overexpression of the ubiqutin−peptide fusion proteins and a robust and specific protease are unique advantages contributing to a cost‐effective, scaleable, and generic bioprocess for peptide production.