TRM integrins CD103 and CD49a differentially support adherence and motility after resolution of influenza virus infection.

Tissue-resident memory CD8 T (TRM) cells are a unique immune memory subset that develops and remains in peripheral tissues at the site of infection, providing future host resistance upon reexposure to that pathogen. In the pulmonary system, TRM are identified through S1P antagonist CD69 and expression of integrins CD103/ß7 and CD49a/CD29(ß1). Contrary to the established role of CD69 on CD8 T cells, the functions of CD103 and CD49a on this population are not well defined. This study examines the expression patterns and functions of CD103 and CD49a with a specific focus on their impact on T cell motility during influenza virus infection. We show that the TRM cell surface phenotype develops by 2 wk postinfection, with the majority of the population expressing CD49a and a subset that is also positive for CD103. Despite a previously established role in retaining TRM in peripheral tissues, CD49a facilitates locomotion of virus-specific CD8 T cells, both in vitro and in vivo. These results demonstrate that CD49a may contribute to local surveillance mechanisms of the TRM population.

Live Imaging of Influenza Infection of the Trachea Reveals Dynamic Regulation of CD8+ T Cell Motility by Antigen.

During a primary influenza infection, cytotoxic CD8+ T cells need to infiltrate the infected airways and engage virus-infected epithelial cells. The factors that regulate T cell motility in the infected airway tissue are not well known. To more precisely study T cell infiltration of the airways, we developed an experimental model system using the trachea as a site where live imaging can be performed. CD8+ T cell motility was dynamic with marked changes in motility on different days of the infection. In particular, significant changes in average cell velocity and confinement were evident on days 8-10 during which the T cells abruptly but transiently increase velocity on day 9. Experiments to distinguish whether infection itself or antigen affect motility revealed that it is antigen, not active infection per se that likely affects these changes as blockade of peptide/MHC resulted in increased velocity. These observations demonstrate that influenza tracheitis provides a robust experimental foundation to study molecular regulation of T cell motility during acute virus infection.

NS1 Protein Mutation I64T Affects Interferon Responses and Virulence of Circulating H3N2 Human Influenza A Viruses.

Influenza NS1 protein is the main viral protein counteracting host innate immune responses, allowing the virus to efficiently replicate in interferon (IFN)-competent systems. In this study, we analyzed NS1 protein variability within influenza A (IAV) H3N2 viruses infecting humans during the 2012-2013 season. We also evaluated the impact of the mutations on the ability of NS1 proteins to inhibit host innate immune responses and general gene expression. Surprisingly, a previously unidentified mutation in the double-stranded RNA (dsRNA)-binding domain (I64T) decreased NS1-mediated general inhibition of host protein synthesis by decreasing its interaction with cleavage and polyadenylation specificity factor 30 (CPSF30), leading to increased innate immune responses after viral infection. Notably, a recombinant A/Puerto Rico/8/34 H1N1 virus encoding the H3N2 NS1-T64 protein was highly attenuated in mice, most likely because of its ability to induce higher antiviral IFN responses at early times after infection and because this virus is highly sensitive to the IFN-induced antiviral state. Interestingly, using peripheral blood mononuclear cells (PBMCs) collected at the acute visit (2 to 3 days after infection), we show that the subject infected with the NS1-T64 attenuated virus has diminished responses to interferon and to interferon induction, suggesting why this subject could be infected with this highly IFN-sensitive virus. These data demonstrate the importance of influenza virus surveillance in identifying new mutations in the NS1 protein, affecting its ability to inhibit innate immune responses and, as a consequence, the pathogenicity of the virus. IMPORTANCE: Influenza A and B viruses are one of the most common causes of respiratory infections in humans, causing 1 billion infections and between 300,000 and 500,000 deaths annually. Influenza virus surveillance to identify new mutations in the NS1 protein affecting innate immune responses and, as a consequence, the pathogenicity of the circulating viruses is highly relevant. Here, we analyzed amino acid variability in the NS1 proteins from human seasonal viruses and the effect of the mutations in innate immune responses and virus pathogenesis. A previously unidentified mutation in the dsRNA-binding domain decreased NS1-mediated general inhibition of host protein synthesis and the interaction of the protein with CPSF30. This mutation led to increased innate immune responses after viral infection, augmented IFN sensitivity, and virus attenuation in mice. Interestingly, using PBMCs, the subject infected with the virus encoding the attenuating mutation induced decreased antiviral responses, suggesting why this subject could be infected with this virus.

Mouse Memory CD8 T Cell Subsets Defined by Tissue-Resident Memory Integrin Expression Exhibit Distinct Metabolic Profiles.

Tissue-resident memory CD8 T cells (TRM) principally reside in peripheral nonlymphoid tissues, such as lung and skin, and confer protection against a variety of illnesses ranging from infections to cancers. The functions of different memory CD8 T cell subsets have been linked with distinct metabolic pathways and differ from other CD8 T cell subsets. For example, skin-derived memory T cells undergo fatty acid oxidation and oxidative phosphorylation to a greater degree than circulating memory and naive cells. Lung TRMs defined by the cell-surface expression of integrins exist as distinct subsets that differ in gene expression and function. We hypothesize that TRM subsets with different integrin profiles will use unique metabolic programs. To test this, differential expression and pathway analysis were conducted on RNA sequencing datasets from mouse lung TRMs yielding significant differences related to metabolism. Next, metabolic models were constructed, and the predictions were interrogated using functional metabolite uptake assays. The levels of oxidative phosphorylation, mitochondrial mass, and neutral lipids were measured. Furthermore, to investigate the potential relationships to TRM development, T cell differentiation studies were conducted in vitro with varying concentrations of metabolites. These demonstrated that lipid conditions impact T cell survival, and that glucose concentration impacts the expression of canonical TRM marker CD49a, with no effect on central memory-like T cell marker CCR7. In summary, it is demonstrated that mouse resident memory T cell subsets defined by integrin expression in the lung have unique metabolic profiles, and that nutrient abundance can alter differentiation.

The Effects of Acute Neutrophil Depletion on Resolution of Acute Influenza Infection, Establishment of Tissue Resident Memory (TRM), and Heterosubtypic Immunity.

After disease resolution, a small subset of influenza specific CD8+ T cells can remain in the airways of the lung as a tissue resident memory population (TRM). These cells are critical for protection from subsequent infections with heterosubtypic influenza viruses. Although it is well established that expression of the collagen IV binding integrin alpha 1 is necessary for the retention and maintenance of TRM cells, other requirements allowing them to localize to the airways and persist are less well understood. We recently demonstrated that inhibition of neutrophils or neutrophil derived chemokine CXCL12 during acute influenza virus infection reduces the effector T cell response and affects the ability of these cells to localize to the airways. We therefore sought to determine whether the defects that occur in the absence of neutrophils would persist throughout resolution of the disease and impact the development of the TRM population. Interestingly, the early alterations in the CD8+ T cell response recover by two weeks post-infection, and mice form a protective population of TRM cells. Overall, these observations show that acute neutrophil depletion results in a delay in the effector CD8+ T cell response, but does not adversely impact the development of TRM.

Visualization of integrin Mac-1 in vivo.

ß2 integrins play critical roles in migration of immune cells and in the interaction with other cells, pathogens, and the extracellular matrix. Among the ß2 integrins, Mac-1 (Macrophage antigen-1), composed of CD11b and CD18, is mainly expressed in innate immune cells and plays a major role in cell migration and trafficking. In order to image Mac-1-expressing cells both in live cells and mouse, we generated a knock-in (KI) mouse strain expressing CD11b conjugated with monomeric yellow fluorescent protein (mYFP). Expression of CD11b-mYFP protein was confirmed by Western blot and silver staining of CD11b-immunoprecipitates and total cell lysates from the mouse splenocytes. Mac-1-mediated functions of the KI neutrophils were comparable with those in WT cells. The fluorescence intensity of CD11b-mYFP was sufficient to image CD11b expressing cells in live mice using intravital two-photon microscopy. In vitro, dynamic changes in the intracellular localization of CD11b molecules could be measured by epifluorescent microscopy. Finally, CD11b-expressing immune cells from tissue were easily detected by flow cytometry without anti-CD11b antibody staining.

Neutrophil trails guide influenza-specific CD8⁺ T cells in the airways.

During viral infections, chemokines guide activated effector T cells to infection sites. However, the cells responsible for producing these chemokines and how such chemokines recruit T cells are unknown. Here, we show that the early recruitment of neutrophils into influenza-infected trachea is essential for CD8(+) T cell-mediated immune protection in mice. We observed that migrating neutrophils leave behind long-lasting trails that are enriched in the chemokine CXCL12. Experiments with granulocyte-specific CXCL12 conditionally depleted mice and a CXCR4 antagonist revealed that CXCL12 derived from neutrophil trails is critical for virus-specific CD8(+) T cell recruitment and effector functions. Collectively, these results suggest that neutrophils deposit long-lasting, chemokine-containing trails, which may provide both chemotactic and haptotactic cues for efficient CD8(+) T cell migration and localization in influenza-infected tissues.

Antigenic and immunogenic properties of recombinant hemagglutinin proteins from H1N1 A/Brisbane/59/07 and B/Florida/04/06 when produced in various protein expression systems.

Antibodies directed against the influenza hemagglutinin (HA) protein largely mediate virus neutralization and confer protection against infection. Consequently, many studies and assays of influenza vaccines are focused on HA-specific immune responses. Recombinant HA (rHA) proteins can be produced in a number of protein expression and cell culture systems. These range from baculovirus infection of insect cell cultures, to transient transfection of plants, to stably transfected human cell lines. Furthermore, the rHA proteins may contain genetic modifications, such as histidine tags or trimerization domains, intended to ease purification or enhance protein stability. However, no systematic study of these different forms of the HA protein have been conducted. It is not clear which, if any, of these different protein expression systems or structural modifications improve or diminish the biological behavior of the proteins as immunogens or antigens in immune assays. Therefore we set out to perform systematic evaluation of rHA produced in different proteins expression systems and with varied modifications. Five rHA proteins based on recent strains of seasonal influenza A and five based on influenza B HA were kindly provided by the Biodefense and Emerging Infections Reagent Repository (BEIR). These proteins were evaluated in a combination of biochemical and structural assays, in vitro humoral and cellular immune assays, and in an animal vaccination model. Marked differences in the behavior of the individual proteins was evident suggesting that they are not equal when being used to detect an immune response. They were, nevertheless, similar at eliciting neutralizing antibody responses.