Memory T cells exhibit superior responses to pathogens and tumors compared with their naive counterparts. Memory is typically generated via an immune response to a foreign antigen, but functional memory T cells can also be produced from naive cells by homeostatic mechanisms. Using a recently developed method, we studied CD8 T cells, which are specific for model (ovalbumin) and viral (HSV, vaccinia) antigens, in unimmunized mice and found a subpopulation bearing markers of memory cells. Based on their phenotypic markers and by their presence in germ-free mice, these preexisting memory-like CD44hi CD8 T cells are likely to arise via physiological homeostatic proliferation rather than a response to environmental microbes. These antigen-inexperienced memory phenotype CD8 T cells display several functions that distinguish them from their CD44lo counterparts, including a rapid initiation of proliferation after T cell stimulation and rapid IFN-γ production after exposure to proinflammatory cytokines. Collectively, these data indicate that the unprimed antigen-specific CD8 T cell repertoire contains antigen-inexperienced cells that display phenotypic and functional traits of memory cells.

Traditionally, the identification and quantification of eosinophils in inflammatory tissues and exudates has been primarily based upon morphologic criteria and manual counting. In this study, we describe a new flow cytometry-based assay to enumerate eosinophils present in murine bronchoalveolar lavage fluid (BAL) and lung parenchyma obtained from the normal/non-inflamed respiratory tract, following experimentally-induced allergic pulmonary inflammation, and during experimental infection with respiratory syncytial virus (RSV). By using a murine Siglec-F-specific antibody in combination with antibodies directed to CD45 and CD11c, we demonstrate that eosinophils can be distinguished from other cell types in the BAL fluid and lung parenchyma based upon their distinct CD45 + Siglec-F + and CD11c low/− staining profile. In the BAL fluid, this flow cytometry-based method of eosinophil identification/quantitation yields results comparable to the standard morphology-based method without the potential observer bias or staining artifacts inherent in morphology-based quantitation. Furthermore, this flow cytometry-based method can be directly adapted to enumerate eosinophils infiltrating the inflamed lung parenchyma, thereby obviating the need for quantitative morphometry of tissue sections.

Influenza virus infections are a global public health problem, with a significant impact of morbidity and mortality from both annual epidemics and pandemics. The current strategy for preventing annual influenza is to develop a new vaccine each year against specific circulating virus strains. Because these vaccines are unlikely to protect against an antigenically divergent strain or a new pandemic virus with a novel hemagglutinin (HA) subtype, there is a critical need for vaccines that protect against all influenza A viruses, a so-called “universal” vaccine. Here we show that mice were broadly protected against challenge with a wide variety of lethal influenza A virus infections (94% aggregate survival following vaccination) with a virus-like particle (VLP) vaccine cocktail. The vaccine consisted of a mixture of VLPs individually displaying H1, H3, H5, or H7 HAs, and vaccinated mice showed significant protection following challenge with influenza viruses expressing 1918 H1, 1957 H2, and avian H5, H6, H7, H10, and H11 hemagglutinin subtypes. These experiments suggest a promising and practical strategy for developing a broadly protective “universal” influenza vaccine.

Lysophospholipids have emerged as biologically important chemoattractants capable of directing lymphocyte development, trafficking and localization. Lysophosphatidic acid (LPA) is a major lysophospholipid found systemically and whose levels are elevated in certain pathological settings such as cancer and infections. Here, we demonstrate that BCR signal transduction by mature murine B cells is inhibited upon LPA engagement of the LPA5 (GPR92) receptor via a Gα12/13 – Arhgef1 pathway. The inhibition of BCR signaling by LPA5 manifests by impaired intracellular calcium store release and most likely by interfering with inositol 1,4,5-trisphosphate receptor activity. We further show that LPA5 also limits antigen-specific induction of CD69 and CD86 expression and that LPA5-deficient B cells display enhanced antibody responses. Thus, these data show that LPA5 negatively regulates BCR signaling, B cell activation and immune response. Our findings extend the influence of lysophospholipids on immune function and suggest that alterations in LPA levels likely influence adaptive humoral immunity.

Zoonotic avian influenza virus infections may lead to epidemics or pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its H1 hemagglutinin was identified as a key mammalian virulence factor. A chimeric 1918 virus expressing a contemporary avian H1 hemagglutinin, however, displayed murine pathogenicity indistinguishable from that of the 1918 virus. Here, isogenic chimeric avian influenza viruses were constructed on an avian influenza virus backbone, differing only by hemagglutinin subtype expressed. Viruses expressing the avian H1, H6, H7, H10, and H15 subtypes were pathogenic in mice and cytopathic in normal human bronchial epithelial cells, in contrast to H2-, H3-, H5-, H9-, H11-, H13-, H14-, and H16-expressing viruses. Mouse pathogenicity was associated with pulmonary macrophage and neutrophil recruitment. These data suggest that avian influenza virus hemagglutinins H1, H6, H7, H10, and H15 contain inherent mammalian virulence factors and likely share a key virulence property of the 1918 virus. Consequently, zoonotic infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals.

A novel avian-origin influenza A H7N9 virus emerged in China in 2013 and continues to cause sporadic human infections with mortality rates approaching 35%. Currently there are no approved human vaccines for H7N9 virus. Recombinant approaches including hemagglutinin (HA) and virus-like particles (VLPs) have resulted in experimental vaccines with advantageous safety and manufacturing characteristics. While high immunogenicity of VLP vaccines has been attributed to the native conformation of HA arranged in the regular repeated patterns within virus-like structures, there is limited data regarding molecular organization of HA within recombinant HA vaccine preparations. In this study, the full-length recombinant H7 protein (rH7) of A/Anhui/1/2013 (H7N9) virus was expressed in Sf9 cells. We showed that purified full-length rH7 retained functional ability to agglutinate red blood cells and formed oligomeric pleomorphic subviral particles (SVPs) of ~20 nm in diameter composed of approximately 10 HA0 molecules. No significant quantities of free monomeric HA0 were observed in rH7 preparation by size exclusion chromatography. Immunogenicity and protective efficacy of rH7 SVPs was confirmed in the mouse and ferret challenge models suggesting that SVPs can be used for vaccination against H7N9 virus.

A series of potent, broadly neutralizing HIV antibodies have been isolated from B cells of HIV-infected individuals. VRC01 represents a subset of these antibodies that mediate neutralization with a restricted set of IGHV genes. The memory B cells expressing these antibodies were isolated years after infection; thus, the B-cell subpopulation from which they originated and the extent of participation in the initial HIV antibody response, if any, are unclear. Here we evaluated the frequency of anti-gp120 B cells in follicular (FO) and marginal zone (MZ) B-cell compartments of naïve WT mice and comparable human populations in uninfected individuals. We found that in non-HIV-exposed humans and mice, the majority of gp120-reactive B cells are of naïve and FO phenotype, respectively. Murine FO B cells express a diverse antibody repertoire to recognize gp120. In contrast, mouse MZ B cells recognize gp120 less frequently but preferentially use IGHV1-53 to encode gp120-specific antibodies. Notably, IGHV1-53 shows high identity to human IGHV1-2*02, which has been repeatedly found to encode broadly neutralizing mutated HIV antibodies, such as VRC01. Finally, we show that human MZ-like B cells express IGHV1-2*02, and that IGHV1-53 expression is enriched in mouse MZ B cells. These data suggest that efforts toward developing an HIV vaccine might consider eliciting protective HIV antibody responses selectively from alternative B-cell populations harboring IGHV gene segments capable of producing protective antibodies.B-cell subsets | virus | germ line | polyreactive

This was the first nonhuman primate study of influenza and bacterial co-infection where high-resolution computed tomography scanning of the lungs was used to quantitatively assess pneumonia over the course of illness and where hypoxemia was correlated with pneumonia severity. With additional validation this model may serve as a pathway for regulatory approval of vaccines and therapeutics for the prevention and treatment of severe influenza pneumonia.