SUMMARY Lung-resident primary memory CD8+ T cell populations (Trm) induced by a single influenza infection decline within months, rendering the host susceptible to new heterosubtypic influenza infections. Here, we demonstrate that, relative to single virus exposure, repeated antigen exposure dramatically alters the dynamics of influenza-specific lung Trm populations. Lung Trm derived from repeatedly stimulated circulating memory CD8+ T cells exhibit extended durability and protective heterosubtypic immunity relative to primary lung Trm. Parabiosis studies reveal that the enhanced durability of lung Trm after multiple antigen encounters resulted from the generation of long-lasting circulating effector memory (Tem) populations, which maintained the ability to be recruited to the lung parenchyma and converted to Trm, in combination with enhanced survival of these cells in the lung. Thus, generating a long-lasting Trm precursor pool through repeated intranasal immunizations might be a promising strategy to establish long-lasting lung Trm-mediated heterosubtypic immunity against influenza.

The impact of respiratory syncytial virus (RSV) on morbidity and mortality is significant in that it causes bronchiolitis in infants, exacerbations in patients with obstructive lung disease, and pneumonia in immunocompromised hosts. RSV activates protein kinase R (PKR) Respiratory syncytial virus (RSV) 2 is a ubiquitous pathogen that causes upper respiratory infections in healthy adults, bronchiolitis and pneumonia in young children, exacerbations in patients with obstructive lung disease, and life-threatening pneumonia in immunosuppressed patients. RSV infection early in life has been associated with the subsequent development of asthma (2-9). RSV is a member of the Paramyxoviridae family and consists of a negative strand RNA genome in a nucleocapsid surrounded by an envelope (10). Production of dsRNA is part of the replicative cycle for RNA viruses like RSV, and RNA serves as a template for both transcription and replication (11). Entry into the host respiratory epithelium is by cell surface fusion, and infection leads to viral replication and subsequent host inflammatory responses (12-17).,We previously showed that RSV increases the amount of protein kinase R (PKR) in airway epithelial cells (1). When activated by phosphorylation, PKR inhibits cellular translation through its ability to phosphorylate eIF2␣ on the Ser-51 regulatory site (18). eIF2␣ is a GTP-binding protein that delivers the initiator methionyl-tRNA to the small ribosomal subunit in translation initiation. Phosphorylation of eIF2␣ converts eIF2 from a substrate to an inhibitor of its GDP-GTP exchange factor eIF2B, blocking protein synthesis (19). If the phosphorylation of PKR during RSV infection triggers activation of eIF2␣, then cellular and viral protein translation should markedly decrease. However, viral protein translation occurs in a robust fashion during RSV infection.Viruses have different strategies to maintain viral protein translation, and PKR plays a limited role with some viruses and cell types. Subgenomic hepatitis C virus RNA replicates more efficiently in PKR knock-out mouse embryonic fibroblasts than in wild type mouse embryonic fibroblasts. The suppression of PKR activity by small interfering RNA enhances the level of hepatitis C virus RNA replication, suggesting PKR controls hepatitis C virus replication, likely via eIF2␣ phosphorylation (20). These findings were confirmed in PKR knockdown cells (21). However, inhibition of PKR by antisense peptide-conjugated phosphorodiamidate morpholino oligomers has no effect on severe acute respiratory syndrome virus titers nor does it affect the severe acute respiratory syndrome-induced phosphorylation of eIF2␣ (22). These observations suggest that another eIF2␣ kinase may regulate its activation or the severe acute respiratory syndrome virus has direct inhibitory effects on eIF2␣. Additionally, cells void of PKR protein by RNA interference do not alter the growth of adenovirus, reovirus, or measles virus (22,23). Studies using alphavirus demonstrate a decrease in viral titers in PKR Ϫ/...

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BackgroundSoft-tissue sarcomas (STS) in the extremities and trunk treated with standard-of-care preoperative external beam radiation therapy (EBRT) followed by surgical resection are associated with local and distant relapses. In preclinical studies, oncolytic virotherapy in sarcoma has demonstrated antitumor effects via direct intratumoral oncolysis and cytotoxic T-cell–mediated immune responses. Talimogene laherparepvec (TVEC) is a replication-competent, immune-enhanced, oncolytic herpes simplex virus type 1 engineered for intratumoral injection; it has been approved by the FDA for the treatment of locally advanced and metastatic melanoma.MethodsWe explored a novel combination of TVEC with standard-of-care EBRT administered preoperatively in patients with locally advanced STS of the extremities and trunk in a phase IB/II clinical trial. Thirty patients with primary STS >5 cm for which EBRT was indicated to achieve negative margins were enrolled. FDA-approved TVEC doses were used. Immune correlative studies in peripheral blood, biopsy and resected tumor tissues were performed.ResultsNo dose-limiting toxicity was observed. Adverse events were similar to those reported in prior studies with TVEC. One patient with myxoid liposarcoma exhibited a partial response. Seven of the 29 (24%) evaluable patients achieved 95% pathological necrosis. None of the patients developed a herpes infection due to the treatment. Eight of the 29 (27%) patients developed postoperative wound complications, which is consistent with previous studies. None of the patients developed local recurrence after surgical resection of the primary sarcoma. 2-year progression-free and overall survival were 57% and 88%, respectively. Caspase-3 demonstrated increased expression of both in TVEC-treated tissue samples as compared with control samples treated with radiation alone.ConclusionPreoperative intratumoral TVEC with concurrent EBRT for locally advanced STS is safe and well-tolerated. This combination treatment may enhance immune responses in some cases but did not increase the proposed rate of pathological necrosis. The Caspase-3 biomarker may be associated with a positive effect of TVEC in sarcoma tumor tissue and should be explored in future studies.Trial registration numberNCT02453191.

Pharmacological ascorbate (i.e., intravenous infusions of vitamin C reaching ~ 20 mM in plasma) is under active investigation as an adjuvant to standard of care anti-cancer treatments due to its dual redox roles as an antioxidant in normal tissues and as a prooxidant in malignant tissues. Immune checkpoint inhibitors (ICIs) are highly promising therapies for many cancer patients but face several challenges including low response rates, primary or acquired resistance, and toxicity. Ascorbate modulates both innate and adaptive immune functions and plays a key role in maintaining the balance between pro and anti-inflammatory states. Furthermore, the success of pharmacological ascorbate as a radiosensitizer and a chemosensitizer in pre-clinical studies and early phase clinical trials suggests that it may also enhance the efficacy and expand the benefits of ICIs.

Respiratory syncytial virus (RSV) is the leading cause of severe respiratory tract infection in infants and young children. CD8 T cells play a critical role in mediating viral clearance following an acute RSV infection. However the role of memory CD8 T cells in providing protection against RSV remains understudied. To generate high magnitude CD8 T cell memory in the absence of CD4 T cell memory and antibodies, we immunized naïve mice with dendritic cells pulsed with an RSV-derived peptide followed by a boost with a recombinant Listeria monocytogenes expressing the same RSV-derived epitope. Memory CD8 T cells significantly reduced viral titers following RSV challenge, but did so at the expense of increased airway dysfunction, weight loss, and mortality compared to controls. Importantly, the severe immunopathology and mortality observed was specific to the context of an RSV infection, as prime-boosted mice challenged with a recombinant influenza virus expressing the same RSV-derived epitope did not exhibit enhanced disease. The induction of a pro-inflammatory cytokine storm mediated by TNF-α and IFN-γ was observed in the serum of prime-boosted mice following RSV challenge. Additionally, RSV-specific memory CD8 T cells produced large amounts of IFN-γ locally within the lung, and adoptive transfer of wild-type but not IFN-γ-deficient memory CD8 T cells resulted in enhanced airway dysfunction and weight loss. Our results indicate that memory CD8 T cells are able to mediate protection against RSV infection. However, memory CD8 T cells acting alone in the absence of antibodies and memory CD4 T cells induce significant immunopathology and mortality through the induction of a systemic pro-inflammatory cytokine storm and local IFN-γ production.

ObjectiveViral acute rhinosinusitis (ARS) is the leading cause of work and school absence and antibiotic over‐prescription. There are limited treatment options available to ameliorate the symptoms caused by viral ARS. We have previously demonstrated that topical adenosine treatment enhances mucociliary clearance in the sino‐nasal tract. Here, we assessed the therapeutic potential of topical adenosine in a mouse model of viral ARS.MethodsThe effect of topical adenosine on inflammatory response and mucin gene expression was examined in a mouse model of viral ARS induced by respiratory syncytial virus (RSV) nasal‐only infection. We also investigated the inflammatory effect of both endogenous and exogenous adenosine in the sino‐nasal tract.ResultsTopical adenosine significantly inhibited the expression of pro‐inflammatory cytokines, goblet hyperplasia, mucin expression, and cell damage in the nose of mice with viral ARS. This treatment did not prolong virus clearance. This inhibitory effect was primarily mediated by the A2A adenosine receptor (AR). Although previous studies have shown that adenosine induces a robust inflammatory response in the lungs, neither endogenous nor exogenous adenosine produced inflammation in the sino‐nasal tract. Instead, exogenous adenosine inhibited the baseline expression of TNF and IL‐1β in the nose. Additionally, baseline expression of ARs was lower in the nose than that in the trachea and lungs.ConclusionWe demonstrated that intranasal adenosine administration effectively decreased inflammation and mucus production in a mouse model of viral ARS.Level of EvidenceN/A Laryngoscope, 133:2095–2103, 2023