The contributions of different subsets of memory CD8+ T cells to recall responses at mucosal sites of infection are poorly understood. Here, we analyzed the CD8+ T cell recall responses to respiratory virus infection in mice and demonstrate that activation markers, such as CD27 and CD43, define three distinct subpopulations of memory CD8+ T cells that differ in their capacities to mount recall responses. These subpopulations are distinct from effector– and central–memory subsets, coordinately express other markers associated with activation status, including CXCR3, CD127, and killer cell lectin-like receptor G1, and are superior to CD62L in predicting the capacity of memory T cells to mediate recall responses. Furthermore, the capacity of vaccines to elicit these memory T cell subpopulations predicted the efficacy of the recall response. These findings extend our understanding of how recall responses are generated and suggest that activation and migration markers define distinct, and unrelated, characteristics of memory T cells.

The respiratory tract is characterized by its large surface area and the close association of an extensive vasculature with the external environment. As such, the respiratory tract is a major portal of entry for many pathogens. The immune system is able to effectively control most pulmonary pathogens and establish immunological memory that is capable of mediating an accelerated and enhanced recall response to secondary pathogen challenge. A key component of the recall response in the lung involves the rapid response of antigen-specific memory CD8+ T cells. Recent studies have shown that memory CD8+ T cells are extremely heterogeneous in terms of phenotype, function, anatomical distribution, and longevity. However, we have little understanding of how the different subsets of memory cells actually contribute to the recall response, especially with respect to peripheral or mucosal sites, such as the lung. Since immunological memory is the cornerstone of vaccination, it is essential that we understand how different memory CD8+ T-cell subsets are initially generated, maintained over time, and contribute to recall responses. This review focuses on memory T cells that mediate recall responses to influenza and parainfluenza virus infections in the lung.

Developing antiviral therapies for influenza A virus (IAV) infection is an ongoing process because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. The ideal strategy is to develop drugs that target well-conserved, functionally restricted, and unique surface structures without affecting host cell function. We recently identified the antiviral compound, RK424, by screening a library of 50,000 compounds using cell-based infection assays. RK424 showed potent antiviral activity against many different subtypes of IAV in vitro and partially protected mice from a lethal dose of A/WSN/1933 (H1N1) virus in vivo. Here, we show that RK424 inhibits viral ribonucleoprotein complex (vRNP) activity, causing the viral nucleoprotein (NP) to accumulate in the cell nucleus. In silico docking analysis revealed that RK424 bound to a small pocket in the viral NP. This pocket was surrounded by three functionally important domains: the RNA binding groove, the NP dimer interface, and nuclear export signal (NES) 3, indicating that it may be involved in the RNA binding, oligomerization, and nuclear export functions of NP. The accuracy of this binding model was confirmed in a NP-RK424 binding assay incorporating photo-cross-linked RK424 affinity beads and in a plaque assay evaluating the structure-activity relationship of RK424. Surface plasmon resonance (SPR) and pull-down assays showed that RK424 inhibited both the NP-RNA and NP-NP interactions, whereas size exclusion chromatography showed that RK424 disrupted viral RNA-induced NP oligomerization. In addition, in vitro nuclear export assays confirmed that RK424 inhibited nuclear export of NP. The amino acid residues comprising the NP pocket play a crucial role in viral replication and are highly conserved in more than 7,000 NP sequences from avian, human, and swine influenza viruses. Furthermore, we found that the NP pocket has a surface structure different from that of the pocket in host molecules. Taken together, these results describe a promising new approach to developing influenza virus drugs that target a novel pocket structure within NP.

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