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.