γδ T Cells Provide Protective Function in Highly Pathogenic Avian H5N1 Influenza A Virus Infection

Given the high mortality rate (>50%) and potential danger of intrapersonal transmission, highly pathogenic avian influenza (HPAI) H5N1 epidemics still pose a significant threat to humans. γδ T cells, which participate on the front line of the host immune defense, demonstrate both innate, and adaptive characteristics in their immune response and have potent antiviral activity against various viruses. However, the roles of γδ T cells in HPAI H5N1 viral infection remain unclear. In this study, we found that γδ T cells provided a crucial protective function in the defense against HPAI H5N1 viral infection. HPAI H5N1 viruses could directly activate γδ T cells, leading to enhanced CD69 expression and IFN-γ secretion. Importantly, we found that the trimer but not the monomer of HPAI H5N1 virus hemagglutinin (HA) proteins could directly activate γδ T cells. HA-induced γδ T cell activation was dependent on both sialic acid receptors and HA glycosylation, and this activation could be inhibited by the phosphatase calcineurin inhibitor cyclosporin A but not by the phosphatidylinositol 3-kinase (PI3-K) inhibitors wortmannin and LY294002. Our findings provide a further understanding the mechanism underlying γδ T cell-mediated innate and adoptive immune responses against HPAI H5N1 viral infection, which helps to develop novel therapeutic strategies for the treatment of H5N1 infection in the future

Ambient atmospheric PM worsens mouse lung injury induced by influenza A virus through lysosomal dysfunction

Abstract Background Particulate matter (PM) air pollution poses a significant risk to respiratory health and is especially linked with various infectious respiratory diseases such as influenza. Our previous studies have shown that H5N1 virus infection could induce alveolar epithelial A549 cell death by enhancing lysosomal dysfunction. This study aims to investigate the mechanisms underlying the effects of PM on influenza virus infections, with a particular focus on lysosomal dysfunction. Results Here, we showed that PM nanoparticles such as silica and alumina could induce A549 cell death and lysosomal dysfunction, and degradation of lysosomal-associated membrane proteins (LAMPs), which are the most abundant lysosomal membrane proteins. The knockdown of LAMPs with siRNA facilitated cellular entry of both H1N1 and H5N1 influenza viruses. Furthermore, we demonstrated that silica and alumina synergistically increased alveolar epithelial cell death induced by H1N1 and H5N1 influenza viruses by enhancing lysosomal dysfunction via LAMP degradation and promoting viral entry. In vivo, lung injury in the H5N1 virus infection-induced model was exacerbated by pre-exposure to silica, resulting in an increase in the wet/dry ratio and histopathological score. Conclusions Our findings reveal the mechanism underlying the synergistic effect of nanoparticles in the early stage of the influenza virus life cycle and may explain the increased number of respiratory patients during periods of air pollution

A Rapid Method to Characterize Mouse IgG Antibodies and Isolate Native Antigen Binding IgG B Cell Hybridomas

<div><p>B cell hybridomas are an important source of monoclonal antibodies. In this paper, we developed a high-throughput method to characterize mouse IgG antibodies using surface plasmon resonance technology. This assay rapidly determines their sub-isotypes, whether they bind native antigen and their approximate affinities for the antigen using only 50 μl of hybridoma cell culture supernatant. Moreover, we found that mouse hybridomas secreting IgG antibodies also have membrane form IgG expression without Igα. Based on this surface IgG, we used flow cytometry to isolate rare γ2a isotype switched variants from a γ2b antibody secreting hybridoma cell line. Also, we used fluorescent antigen to single cell sort antigen binding hybridoma cells from bulk mixture of fused hybridoma cells instead of the traditional multi-microwell plate screening and limiting dilution sub-cloning thus saving time and labor. The IgG monoclonal antibodies specific for the native antigen identified with these methods are suitable for in vivo therapeutic uses, but also for sandwich ELISA assays, histology, flow cytometry, immune precipitation and x-ray crystallography.</p></div

Summary for single hybridoma cell sorting.

<p>^aDP: Double positive staining by both anti-IgG antibody and OVA.</p><p>^bSP: Single positive staining by anti-IgG antibody and negative staining by OVA.</p><p>“+” means antibody binds native OVA, while “-” means antibody does not bind native OVA in BIAcore testing.</p><p>* These two hybridomas secrete both IgG ɣ1 and ɣ2a antibodies, and both of them bind native OVA and have the same VH and VK.</p><p>Summary for single hybridoma cell sorting.</p

Sorting of native antigen binding IgG hybridomas from a fusion bulk mixture.

<p>(A) Staining of OVA specific hybridoma cell line with fluorescent OVA. (B) Sorting of OVA specific hybridoma clls from a fusion bulk mixture.</p

Hybridoma cell surface IgG expression correlates with antibody secretion.

<p>(A) Cell surface IgG staining of S161.1 hybridoma cell line. (B) Antibody secretion by the sorted surface IgG negative or positive cells from the S161.1 cell line. (C) Cell surface IgG staining of negative secretors or positive secretors derived from single cell colony by limiting dilution method.</p

Primers used to amplify secreted and membrane form of mouse IgG.

<p>Primers used to amplify secreted and membrane form of mouse IgG.</p

Two applications of detecting antibody-antigen interaction with BIAcore.

<p>(A) Detecting three native MBP binding antibodies separately using BIAcore. (B) Characterizing the interaction of these three antibodies with MBP using BIAcore. (C) A cartoon for the interaction of these three antibodies with MBP. (D) Pairing of MBP antibodies as coating and detection antibodies for detection of MBP. (E) Detection of interaction between two monoclonal SARS-CoV spike protein specific antibodies with spike and virus receptor ACE2.</p