B lymphocytes migrate among varied microenvironmental niches during diversification, selection, and conversion to memory or Ab-secreting plasma cells. Aspects of the nutrient milieu differ within these lymphoid microenvironments and can influence signaling molecules such as the mechanistic target of rapamycin (mTOR). However, much remains to be elucidated as to the B cell-intrinsic functions of nutrient-sensing signal transducers that modulate B cell differentiation or Ab affinity. We now show that the amino acid-sensing mTOR complex 1 (mTORC1) is vital for induction of Bcl6-a key transcriptional regulator of the germinal center (GC) fate-in activated B lymphocytes. Accordingly, disruption of mTORC1 after B cell development and activation led to reduced populations of Ag-specific memory B cells as well as plasma cells and GC B cells. In addition, induction of the germ line transcript that guides activation-induced deaminase in selection of the IgG1 H chain region during class switching required mTORC1. Expression of the somatic mutator activation-induced deaminase was reduced by a lack of mTORC1 in B cells, whereas point mutation frequencies in Ag-specific GC-phenotype B cells were only halved. These effects culminated in a B cell-intrinsic defect that impacted an antiviral Ab response and drastically impaired generation of high-affinity IgG1. Collectively, these data establish that mTORC1 governs critical B cell-intrinsic mechanisms essential for establishment of GC differentiation and effective Ab production.

Reinfections with respiratory viruses are common and cause significant clinical illness, yet precise mechanisms governing this susceptibility are ill defined. Lung Ag-specific CD8+ T cells (TCD8) are impaired during acute viral lower respiratory infection by the inhibitory receptor PD-1. To determine if PD-1 contributes to recurrent infection, we first established a model of reinfection by challenging B cell-deficient mice with human metapneumovirus (HMPV) several weeks after primary infection, and found that HMPV replicated to high titers in the lungs. A robust secondary effector lung TCD8 response was generated during reinfection, but these cells were more impaired and more highly expressed the inhibitory receptors PD-1, LAG-3, and 2B4 than primary TCD8. In vitro blockade demonstrated that PD-1 was the most dominant inhibitory receptor early after reinfection. In vivo therapeutic PD-1 blockade during HMPV reinfection restored lung TCD8 effector functions (i.e. degranulation and cytokine production) and enhanced viral clearance. PD-1 also limited the protective efficacy of HMPV epitope-specific peptide vaccination and impaired lung TCD8 during heterotypic influenza virus challenge infection. Our results indicate that PD-1 signaling may contribute to respiratory virus reinfection and evasion of vaccine-elicited immune responses. These results have important implications for the design of effective vaccines against respiratory viruses.

Viruses are frequent causes of lower respiratory infection (LRI). Programmed cell death-1 (PD-1) signaling contributes to pulmonary CD8+ T cell (TCD8) functional impairment during acute viral LRI, but the role of TCD8 impairment in viral clearance and immunopathology is unclear. We now find that human metapneumovirus (HMPV) infection induces virus-specific lung TCD8 that fail to produce effector cytokines or degranulate late after infection, with minimally increased function even in the absence of PD-1 signaling. Impaired lung TCD8 upregulated multiple inhibitory receptors, including PD-1, LAG-3, TIM-3, and 2B4. Moreover, co-expression of these receptors continued to increase even after viral clearance, with most virus-specific lung TCD8 expressing ≥3 inhibitory receptors on day 14 post-infection. Viral infection also increased expression of inhibitory ligands by both airway epithelial cells and antigen presenting cells, further establishing an inhibitory environment. In vitro antibody blockade revealed that multiple inhibitory receptors contribute to TCD8 impairment induced by either HMPV or influenza virus infection. In vivo blockade of TIM-3 signaling failed to enhance TCD8 function or reduce viral titers. However, blockade of LAG-3 in PD-1-deficient mice restored TCD8 effector functions but increased lung pathology, indicating that LAG-3 mediates lung TCD8 impairment in vivo and contributes to protection from immunopathology during viral clearance. These results demonstrate that an orchestrated network of pathways modifies lung TCD8 functionality during viral LRI, with PD-1 and LAG-3 serving prominent roles. Lung TCD8 impairment may prevent immunopathology but also contribute to recurrent lung infections.

Respiratory syncytial virus (RSV) is a single-stranded RNA virus that assembles into viral filaments at the cell surface. Virus assembly often depends on the ability of a virus to use host proteins to accomplish viral tasks. Since the fusion protein cytoplasmic tail (FCT) is critical for viral filamentous assembly, we hypothesized that host proteins important for viral assembly may be recruited by the FCT. Using a yeast two-hybrid screen, we found that filamin A interacted with FCT, and mammalian cell experiments showed it localized to viral filaments but did not affect viral replication. Furthermore, we found that a number of actin-associated proteins also were excluded from viral filaments. Actin or tubulin cytoskeletal rearrangement was not necessary for F trafficking to the cell surface or for viral assembly into filaments, but was necessary for optimal viral replication and may be important for anchoring viral filaments. These findings suggest that RSV assembly into filaments occurs independently of actin polymerization and that viral proteins are the principal drivers for the mechanical tasks involved with formation of complex, structured RSV filaments at the host cell plasma membrane.

Acute respiratory virus infection (ARI) induces CD8 T cells with diminished cytokine production and functional impairment. The role of cellular mediators of immune impairment, specifically CD4 regulatory T cells (Tregs), is incompletely understood in ARI. Tregs are known suppressors of effector T cell function, but whether they are detrimental or beneficial in ARI remains controversial. We show in this paper that Treg depletion leads to increased CD8 T cell function and lower virus titer in mice infected with human metapneumovirus. We further demonstrate that Tregs play a temporal role in the immune response to human metapneumovirus and influenza: Treg depletion before infection pathologically reduces virus-specific CD8 T cell numbers and delays virus clearance, whereas depletion 2 d postinoculation enhances CD8 T cell functionality without reducing virus-specific CD8 T cell numbers. Mechanistically, Treg depletion during immune priming led to impaired dendritic cell and CD8 T cell migration. Further, early Treg depletion was associated with immune skewing toward a type 2 phenotype characterized by increased type 2 innate lymphoid cells and T2 CD4 T cells, which was not observed when Treg depletion was delayed until after inoculation. These results indicate that the presence of Tregs at inoculation is critical for efficient priming of the CD8 T cell response to ARI, whereas later in infection, Tregs are dispensable for virus clearance.

Chitin (β-1,4-linked-N-acetylglucosamine) provides structural integrity to the nematode eggshell and pharyngeal lining. Chitin is synthesized in nematodes, but not in plants and vertebrates, which are often hosts to parasitic roundworms; hence, the chitin metabolism pathway is considered a potential target for selective interventions. Polysaccharide deacetylases (PDAs), including those that convert chitin to chitosan, have been previously demonstrated in protists, fungi and insects. We show that genes encoding PDAs are distributed throughout the phylum Nematoda, with the two paralogs F48E3.8 and C54G7.3 found in C. elegans. We confirm that the genes are somatically expressed and show that RNAi knockdown of these genes retards C. elegans development. Additionally, we show that proteins from the nematode deacetylate chitin in vitro, we quantify the substrate available in vivo as targets of these enzymes, and we show that Eosin Y (which specifically stains chitosan in fungal cells walls) stains the C. elegans pharynx. Our results suggest that one function of PDAs in nematodes may be deacetylation of the chitinous pharyngeal lining.

Viral lung infections are leading causes of morbidity and mortality. Effective immune responses to these infections require precise immune regulation to preserve lung function after viral clearance. One component of airway pathophysiology and lung injury associated with acute respiratory virus infection is effector T cells, yet these are the primary cells required for viral clearance. Accordingly, multiple immune mechanisms exist to regulate effector T cells, limiting immunopathology while permitting clearance of infection. Much has been learned in recent years about regulation of T cell function during chronic infection and cancer, and it is now clear that many of these mechanisms also control inflammation in acute lung infection. In this review, we focus on regulatory T cells, inhibitory receptors, and other cells and molecules that regulate cell-mediated immunity in the context of acute respiratory virus infection.

No abstract