John J. Erickson

Viral acute lower respiratory infections impair CD8+ T cells through PD-1

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 (TCD8) 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 TCD8 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 TCD8 rapidly reexpressed PD-1 and exhibited severe functional impairment. Inhibition of PD-1 signaling using monoclonal antibody blockade prevented TCD8 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 TCD8 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.

Discovering naturally processed antigenic determinants that confer protective T cell immunity

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.

Human Metapneumovirus Virus-Like Particles Induce Protective B and T Cell Responses in a Mouse Model

ABSTRACT Human metapneumovirus (HMPV) is a leading cause of respiratory disease in infants, children, and the elderly worldwide, yet no licensed vaccines exist. Live-attenuated vaccines present safety challenges, and protein subunit vaccines induce primarily antibody responses. Virus-like particles (VLPs) are an attractive alternative vaccine approach because of reduced safety concerns compared with live vaccines. We generated HMPV VLPs by expressing viral proteins in suspension-adapted human embryonic kidney epithelial (293-F) cells and found that the viral matrix (M) and fusion (F) proteins were sufficient to form VLPs. We previously reported that the VLPs resemble virus morphology and incorporate fusion-competent F protein (R. G. Cox, S. B. Livesay, M. Johnson, M. D. Ohi, and J. V. Williams, J. Virol. 86:12148–12160, 2012), which we hypothesized would elicit F-specific antibody and T cell responses. In this study, we tested whether VLP immunization could induce protective immunity to HMPV by using a mouse model. C57BL/6 mice were injected twice intraperitoneally with VLPs alone or with adjuvant and subsequently challenged with HMPV. Mice were euthanized 5 days postinfection, and virus titers, levels of neutralizing antibodies, and numbers of CD3+ T cells were quantified. Mice immunized with VLPs mounted an F-specific antibody response and generated CD8+ T cells recognizing an F protein-derived epitope. VLP immunization induced a neutralizing-antibody response that was enhanced by the addition of either TiterMax Gold or α-galactosylceramide adjuvant, though adjuvant reduced cellular immune responses. Two doses of VLPs conferred complete protection from HMPV replication in the lungs of mice and were not associated with a Th2-skewed cytokine response. These results suggest that nonreplicating VLPs are a promising vaccine candidate for HMPV. IMPORTANCE Human metapneumovirus (HMPV) is a leading cause of acute respiratory infection in infants, children, and the elderly worldwide, yet no licensed vaccines exist. Live-attenuated vaccines present safety challenges, and protein subunit vaccines induce primarily antibody responses. Virus-like particles (VLPs) are an attractive alternative vaccine approach. We generated HMPV VLPs by expressing the viral matrix (M) and fusion (F) proteins in mammalian cells. We found that mice immunized with VLPs mounted an F-specific antibody response and generated CD8+ T cells recognizing an F protein-derived epitope. VLP immunization induced a neutralizing-antibody response that was enhanced by the addition of either TiterMax Gold or α-galactosylceramide adjuvant. Two doses of VLPs conferred complete protection against HMPV replication in the lungs of mice and were not associated with a Th2-skewed cytokine response. These results suggest that nonreplicating VLPs are a promising vaccine candidate for HMPV.

Programmed Death-1 Impairs Secondary Effector Lung CD8+ T Cells during Respiratory Virus Reinfection

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 programmed death-1 (PD-1). To determine whether 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 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.

B Cell-Intrinsic and -Extrinsic Regulation of Antibody Responses by PARP14, an Intracellular (ADP-Ribosyl)Transferase

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 Th cells along with dendritic cells. Numerous bacterial toxins catalyze mono(ADP-ribosyl)ation of mammalian proteins to influence cell physiology and adaptive immunity. However, little is known about biological functions of intracellular mammalian mono(ADP-ribosyl)transferases, 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 mammalian mono(ADP-ribosyl)transferase activity. In this article, 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 Ab responses in mice, as well as provide direct evidence of the requirement for protein mono-ADP-ribosylation in Th cell differentiation.

Role of Type I Interferon Signaling in Human Metapneumovirus Pathogenesis and Control of Viral Replication

ABSTRACT Type I IFN signaling, which is initiated through activation of the alpha interferon receptor (IFNAR), regulates the expression of proteins that are crucial contributors to immune responses. Paramyxoviruses, including human metapneumovirus (HMPV), have evolved mechanisms to inhibit IFNAR signaling, but the specific contribution of IFNAR signaling to the control of HMPV replication, pathogenesis, and adaptive immunity is unknown. We used IFNAR-deficient (IFNAR−/−) mice to assess the effect of IFNAR signaling on HMPV replication and the CD8+ T cell response. HMPV-infected IFNAR−/− mice had a higher peak of early viral replication but cleared the virus with kinetics similar to those of wild-type (WT) mice. However, IFNAR−/− mice infected with HMPV displayed less airway dysfunction and lung inflammation. CD8+ T cells of IFNAR−/− mice after HMPV infection expressed levels of the inhibitory receptor programmed death 1 (PD-1) similar to those of WT mice. However, despite lower expression of inhibitory programmed death ligand 1 (PD-L1), HMPV-specific CD8+ T cells of IFNAR−/− mice were more functionally impaired than those of WT mice and upregulated the inhibitory receptor Tim-3. Analysis of the antigen-presenting cell subsets in the lungs revealed that the expansion of PD-L1low dendritic cells (DCs), but not PD-L1high alveolar macrophages, was dependent on IFNAR signaling. Collectively, our results indicate a role for IFNAR signaling in the early control of HMPV replication, disease progression, and the development of an optimal adaptive immune response. Moreover, our findings suggest an IFNAR-independent mechanism of lung CD8+ T cell impairment. IMPORTANCE Human metapneumovirus (HMPV) is a leading cause of acute respiratory illness. CD8+ T cells are critical for clearing viral infection, yet recent evidence shows that HMPV and other respiratory viruses induce CD8+ T cell impairment via PD-1–PD-L1 signaling. We sought to understand the role of type I interferon (IFN) in the innate and adaptive immune responses to HMPV by using a mouse model lacking IFN signaling. Although HMPV titers were higher in the absence of type I IFN, virus was nonetheless cleared and mice were less ill, indicating that type I IFN is not required to resolve HMPV infection but contributes to pathogenesis. Further, despite lower levels of the inhibitory ligand PD-L1 in mice lacking type I IFN, CD8+ T cells were more impaired in these mice than in WT mice. Our data suggest that specific antigen-presenting cell subsets and the inhibitory receptor Tim-3 may contribute to CD8+ T cell impairment.

Acute Viral Respiratory Infection Rapidly Induces a CD8+ T Cell Exhaustion–like Phenotype

Acute viral infections typically generate functional effector CD8+ T cells (TCD8) that aid in pathogen clearance. However, during acute viral lower respiratory infection, lung TCD8 are functionally impaired and do not optimally control viral replication. T cells also become unresponsive to Ag during chronic infections and cancer via signaling by inhibitory receptors such as programmed cell death-1 (PD-1). PD-1 also contributes to TCD8 impairment during viral lower respiratory infection, but how it regulates TCD8 impairment and the connection between this state and T cell exhaustion during chronic infections are unknown. In this study, we show that PD-1 operates in a cell-intrinsic manner to impair lung TCD8. In light of this, we compared global gene expression profiles of impaired epitope-specific lung TCD8 to functional spleen TCD8 in the same human metapneumovirus–infected mice. These two populations differentially regulate hundreds of genes, including the upregulation of numerous inhibitory receptors by lung TCD8. We then compared the gene expression of TCD8 during human metapneumovirus infection to those in acute or chronic lymphocytic choriomeningitis virus infection. We find that the immunophenotype of lung TCD8 more closely resembles T cell exhaustion late into chronic infection than do functional effector T cells arising early in acute infection. Finally, we demonstrate that trafficking to the infected lung alone is insufficient for TCD8 impairment or inhibitory receptor upregulation, but that viral Ag–induced TCR signaling is also required. Our results indicate that viral Ag in infected lungs rapidly induces an exhaustion-like state in lung TCD8 characterized by progressive functional impairment and upregulation of numerous inhibitory receptors.

Multiple Inhibitory Pathways Contribute to Lung CD8+ T Cell Impairment and Protect against Immunopathology during Acute Viral Respiratory Infection

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 infection induces virus-specific lung TCD8 that fail to produce effector cytokines or degranulate late postinfection, with minimally increased function even in the absence of PD-1 signaling. Impaired lung TCD8 upregulated multiple inhibitory receptors, including PD-1, lymphocyte activation gene 3 (LAG-3), T cell Ig mucin 3, and 2B4. Moreover, coexpression of these receptors continued to increase even after viral clearance, with most virus-specific lung TCD8 expressing three or more inhibitory receptors on day 14 postinfection. Viral infection also increased expression of inhibitory ligands by both airway epithelial cells and APCs, further establishing an inhibitory environment. In vitro Ab blockade revealed that multiple inhibitory receptors contribute to TCD8 impairment induced by either human metapneumovirus or influenza virus infection. In vivo blockade of T cell Ig mucin 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 contributes to recurrent lung infections.