The development of DNA vaccines has been hindered by inadequate immune responses observed in clinical trials. Wang et al. (p. 2860) identified a potential cause of these poor responses by characterizing human serum amyloid P (hSAP) as a negative regulator of human responses to DNA vaccines. hSAP and murine SAP (mSAP) have been previously identified as plasma proteins that bind to genomic DNA in a species-specific manner, but mSAP typically binds more weakly to DNA. The relevance of these binding differences was tested in the present study with a humanized mouse model engineered to express hSAP. Vaccination of hSAP transgenic mice with a DNA vaccine resulted in significantly lower Ab and CTL responses compared with those in wild-type mice. In vitro, a monocyte-derived macrophage cell line had diminished innate immune responses upon treatment with hSAP-complexed DNA compared with DNA alone or mSAP-complexed DNA. Further analysis revealed that formation of hSAP–DNA complexes competitively inhibited the binding of DNA to the DNA sensing molecules high mobility group B protein 1 and LL37, and hSAP-complexed DNA underwent endocytosis and degradation. A chemical SAP inhibitor partially restored innate immune responses to DNA in hSAP-treated macrophages in vitro and in vaccinated hSAP transgenic mice. Thus, hSAP appears to be a suppressor of innate responses to DNA, and hSAP suppression may have the potential to optimize human DNA vaccination.

The development of passive Ab therapies against Mycobacterium tuberculosis infection is being sought as a potential treatment for immunocompromised individuals and those infected with multi-drug–resistant strains. Earlier studies in mice showed that passive intranasal immunization with a murine IgA specific for the M. tuberculosis α-crystallin (Acr) Ag reduced pulmonary infection. In this issue, Balu et al. (p. 3113) characterized a human IgA1 mAb (2E9IgA1) against Acr that was generated from a single-chain variable fragment selected by Ag panning. In vitro, 2E9IgA1 bound to Acr and the human FcαR1 (CD89) with high affinity. Transgenic mice expressing human CD89 were used to test the immunotherapeutic properties of 2E9IgA1. Intranasal inoculation of 2E9IgA1 together with murine IFN-γ significantly reduced the bacterial load in the lungs of CD89 transgenic mice infected with M. tuberculosis strain H37Rv, compared with littermate controls. Moreover, 2E9IgA1 was able to inhibit H37Rv infection in some whole blood and monocyte samples from human volunteers. Taken together, these results indicate that 2E9IgA1 could be a prospective therapeutic mAb, which may be due to the effects of interactions with CD89.

Negative regulation of T cell activation is initiated in part through engagement of programmed cell death-1 (PD-1) on the surface of T cells with its cognate ligand, PD ligand-1. Terawaki et al. (p. 2772) carried out extensive PD-1 promoter analysis to clarify the transcription elements involved in PD-1 expression. Analysis of the 5′ region of PD-1 in the 2B4.11 T cell line revealed that a well-conserved cis-regulatory IFN-stimulated response element (ISRE) was required for PD-1 expression in activated T cells. ISRE-mediated gene expression is usually boosted by type I IFN signaling, and PD-1 expression was significantly increased in TCR-stimulated T cells exposed to IFN-α in vitro or in vivo. A chromatin immunoprecipitation assay indicated that IFN-α treatment of activated T cells induced binding of IFN responsive factor-9, a component of the IFN-stimulated gene factor 3 complex, to the PD-1 ISRE. IFN-α treatment alone was insufficient to induce PD-1 expression, which required TCR stimulation. In addition, treatment of mice carrying tumors with both IFN-α and an anti–PD-1 Ab elicited significant antitumor activity compared with IFN-α treatment alone. These results reveal that IFN-α signaling influences PD-1 expression, and antitumor therapy with type I IFN may be enhanced by PD-1 blockade.

Natural killer (NK) cells adhere to target cells using an array of activating and inhibitory receptors that are expressed on the NK cell surface, but the biophysical nature of this adhesion is not well understood. Hoffmann et al. (p. 2757) analyzed the biophysical force between NK cells and tumor cells at the single-cell level using single-cell force spectroscopy (SCFS) to measure adhesion forces generated by attachment of the NK cell receptor 2B4 to its ligand CD48. The SCFS technique permits measurement of the forces generated when two cells adhere to and detach from each other. Cells from the 2B4-expressing human NK cell line NK92-C1 rapidly adhered to HeLa cells expressing CD48, and a significant increase in force was required to detach the cells from each other as early as 120 s to 300 s after attachment. This rapid adhesion event also involved NK cell-expressed surface LFA-1 and an intact actin cytoskeleton, and is consistent with rapid inside-out signaling events triggered by 2B4 engagement. Overall, these findings reveal the rapid rate at which NK cells adhere to tumor target cells, which is critical to their function as innate cytotoxic cells.

Three different complement pathways can be activated in response to infection, but the influence of each of these pathways during sepsis is not well understood. Dahlke et al. (p. 3066) examined the role of the classical and alternative complement pathways in mice in response to sepsis induced by cecal ligation/puncture (CLP). Survival was significantly reduced, compared with that in wild-type mice, following CLP in C1q−/− or factor D (fD)−/− mice, which have defects in the classical and alternative pathways, respectively. Both C1q−/− and fD−/− mice exhibited a significant decrease in C3 activation, but C1q−/− mice also had significantly higher bacterial loads in the blood, liver, and lung, compared with those in wild-type and fD−/− mice 6 h post-CLP. Monocytes and granulocytes from C1q−/− and fD−/− mice collected 6 h post-CLP were less effective at phagocytosing opsonized Escherichia coli in vitro than were the same cells from wild-type mice. In spite of this defect, fD−/− mice were able to clear bacteria, but these mice also had a significant increase in neutrophil recruitment in the blood and lungs and a dramatic rise in inflammatory cytokine production 24 h post-CLP. These results suggest that the ability of fD−/− mice to control bacterial loads during sepsis comes at the cost of a destructive, uncontrolled inflammatory response and provide evidence that the alternative and classical complement pathways generate distinct responses to sepsis.

Cystic fibrosis (CF) is a devastating lung disease caused by a mutation in the CF transmembrane conductance regulator (CFTR), and is associated with chronic neutrophil-mediated pulmonary inflammation due to recurrent bacterial infections. The T cell Ig and mucin-domain–containing molecules (TIMs) are immunomodulatory molecules that have been linked to asthma and other pulmonary inflammatory conditions. Vega-Carrascal et al. (p. 2897) examined the role of TIM-3 in the inflammatory responses associated with CF. TIM-3 and its ligand, galectin-9, were expressed at a constitutively higher level in CF bronchial epithelial (CFBE) cells compared with normal human bronchial epithelial (HBE) cells. In addition, treatment of HBE cells with a CFTR inhibitor induced a significant increase in TIM-3 and galectin-9 expression. TIM-3 localized to the plasma membrane in CFBE cells, and galectin-9 was able to activate TIM-3 signaling and induce secretion of the neutrophil chemoattractant IL-8, which confirmed that CFBE-expressed TIM-3 was functional. Interestingly, TIM-3 and galectin-9 were shown to be degraded in the bronchoalveolar lavage fluid from CF patients via serine proteases, including neutrophil elastase. These observations provide a new insight into a potential role for TIM-3/galectin-9 in the pathology associated with CF.

CD8+ T cell responses are fundamental to controlling acute viral infections, but the signals essential to effector CD8+ T cell survival and memory CD8+ T cell formation are not completely known. Previous studies have repeatedly demonstrated that CD4+ T cell help supports CD8+ T cell survival, and this present study by Novy et al. (p. 2729) demonstrates that IL-21 produced by activated CD4+ T cells significantly enhances CD8+ T cell survival in vitro. IL-21 signaling was also required in vivo for mice to generate effective virus-specific CD8+ T cell responses upon vaccinia virus infection. Whereas effector CD8+ T cell activation, proliferation, and differentiation did not require IL-21, survival of these activated cells and memory CD8+ T cell formation were dependent on intrinsic IL-21 signaling. Additional studies in vivo confirmed that CD4+ T cell help supported CD8+ T cell survival through IL-21–driven activation of STAT1 and STAT3 and increased expression of Bcl-2 and Bcl-xL transcripts. These observations elucidate the importance of IL-21 signaling in CD8+ T cells for the development of effective antiviral responses, which supports further exploration into the use of IL-21 to augment vaccine and cancer immunotherapy strategies.

Neutrophils can curb pulmonary Aspergillus fumigatus infection by phagocytosis of fungal conidia and reactive oxygen species (ROS)-mediated killing of fungal hyphae. Boyle et al. (p. 2978) dissected the signaling mechanisms essential to NADPH oxidase production of ROS by hyphae-exposed neutrophils. Treatment of murine bone marrow neutrophils (BMNs) with a variety of PI3K inhibitors upon exposure to A. fumigatus hyphae revealed that NADPH oxidase activity required the class IA PI3Ks, PI3Kβ and PI3Kδ. These kinases also supported neutrophil spreading on the hyphal surface, and the accumulation of class I PI3K lipid products was observed in the region of the neutrophil plasma membrane in contact with the hyphal interface. ROS activity was significantly lower in BMNs from mice lacking the β2-integrin subunit CD18 in response to A. fumigatus hyphae, compared with wild-type BMNs. Further analysis revealed that the tyrosine kinase Syk and the Vav family of Rac guanine nucleotide exchange factors were required for activation of neutrophil ROS responses. These findings better define the signaling events required for ROS production in response to A. fumigatus hyphae, but further studies are needed to understand the relationship between these different signaling components.

Summaries written by Christiana N. Fogg, Ph.D.