Viruses are leading causes of severe acute lower respiratory infections (LRIs). These infections evoke incomplete immunity, as individuals can be repeatedly reinfected throughout life. We report that acute viral LRI causes rapid pulmonary CD8 + cytotoxic T lymphocyte (T CD8 ) functional impairment via programmed death-1/ programmed death ligand-1 (PD-1/PD-L1) signaling, a pathway previously associated with prolonged antigenic stimulation during chronic infections and cancer. PD-1-mediated T CD8 impairment occurred acutely in mice following infection with human metapneumovirus or influenza virus. Viral antigen was sufficient for PD-1 upregulation, but induction of PD-L1 was required for impairment. During secondary viral infection or epitope-only challenge, memory T CD8 rapidly reexpressed PD-1 and exhibited severe functional impairment. Inhibition of PD-1 signaling using monoclonal antibody blockade prevented T CD8 impairment, reduced viral titers during primary infection, and enhanced protection of immunized mice against challenge infection. Additionally, PD-1 and PD-L1 were upregulated in the lungs of patients with 2009 H1N1 influenza virus, respiratory syncytial virus, or parainfluenza virus infection. These results indicate that PD-1 mediates T CD8 functional impairment during acute viral infection and may contribute to recurrent viral LRIs. Therefore, the PD-1/PD-L1 pathway may represent a therapeutic target in the treatment of respiratory viruses.

CD8 + T cells (TCD8) confer protective immunity against many infectious diseases, suggesting that microbial TCD8 determinants are promising vaccine targets. Nevertheless, current T cell antigen identification approaches do not discern which epitopes drive protective immunity during active infection -information that is critical for the rational design of TCD8-targeted vaccines. We employed a proteomics-based approach for large-scale discovery of naturally processed determinants derived from a complex pathogen, vaccinia virus (VACV), that are presented by the most frequent representatives of four major HLA class I supertypes. Immunologic characterization revealed that many previously unidentified VACV determinants were recognized by smallpox-vaccinated human peripheral blood cells in a variegated manner. Many such determinants were recognized by HLA class I-transgenic mouse immune TCD8 too and elicited protective TCD8 immunity against lethal intranasal VACV infection. Notably, efficient processing and stable presentation of immune determinants as well as the availability of naive TCD8 precursors were sufficient to drive a multifunctional, protective TCD8 response. Our approach uses fundamental insights into T cell epitope processing and presentation to define targets of protective TCD8 immunity within human pathogens that have complex proteomes, suggesting that this approach has general applicability in vaccine sciences.

The complex life cycle of Plasmodium falciparum (Pf) makes it difficult to limit infections and reduce the risk of severe malaria. Improved understanding of Pf blood-stage growth and development would provide new opportunities to evaluate and interfere with successful completion of the parasite's life cycle. Cultured blood stage Pf was incubated with Hoechst 33342 (HO) and thiazole orange (TO) to stain DNA and total nucleic acids, respectively. Correlated HO and TO fluorescence emissions were then measured by flow cytometry. Complex bivariate data patterns were analyzed by manual cluster gating to quantify parasite life cycle stages. The permutations of viable staining with both reagents were tested for optimal detection of parasitized RBC (pRBC). Pf cultures were exposed to HO and TO simultaneously to achieve optimal staining of pRBC and consistent quantification of early and late stages of the replicative cycle (rings through schizonts). Staining of Pf nucleic acids allows for analysis of parasite development in the absence of fixatives, lysis, or radioactivity to enable examination of erythrocytes from parasite invasion through schizont rupture using sensitive and rapid assay procedures. Investigation of the mechanisms by which anti-malarial drugs and antibodies act against different Pf lifecycle stages will be aided by this cytometric strategy. '

The capacity to achieve sufficient concentrations of Ag-specific Ab of the appropriate isotypes is a critical component of immunity that requires efficient differentiation and interactions of Ag-specific B and T helper (Th) cells along with dendritic cells. Numerous bacterial toxins catalyze mono-(ADP-ribosyl)ation of mammalian proteins to impact cell physiology and adaptive immunity. However, little is known about biological functions of intracellular mammalian mono-(ADP-ribosyl)transferases (mART), such as any ability to regulate Ab responses. Poly-(ADP-Ribose) Polymerase 14 (PARP14), an intracellular protein highly expressed in lymphoid cells, binds to STAT6 and encodes a catalytic domain with mART activity. Here we show that recall IgA as well as the STAT6-dependent IgE Ab responses are impaired in PARP14-deficient mice. Whereas PARP14 regulation of IgE involved a B cell intrinsic process, the predominant impact on IgA was B cell-extrinsic. Of note, PARP14 deficiency reduced the levels of Th17 cells and CD103+ DCs which are implicated in IgA regulation. PARP14 enhanced the expression of RORα, Runx1 and Smad3 after T cell activation, and, importantly, its catalytic activity of PARP14 promoted Th17 differentiation. Collectively, the findings show that PARP14 influences the class distribution, affinity repertoire, and recall capacity of antibody responses in mice, and provide direct evidence of requirement for protein mono-ADP-ribosylation in T helper differentiation.