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Respiratory infections are a leading cause of morbidity and mortality. The presence of multiple heterologous virus infections is routinely observed in a subset of individuals screened for the presence of respiratory viruses. However, the impact overlapping infections has on disease severity and the host immune response is not well understood. Respiratory syncytial virus (RSV) and influenza A virus (IAV) are two of the most common respiratory infections observed in hospitalized patients, particularly in the very young and aged populations. In this study, we examined how the order in which BALB/c mice were infected with both RSV and IAV impacts disease severity. RSV infection prior to an IAV infection was associated with decreased weight loss and increased survival as compared with IAV infection alone. In contrast, IAV infection prior to an RSV infection was associated with similar morbidity and mortality as compared with an IAV infection alone. Our results suggest that the order in which viral infections are acquired plays a critical role in the outcome of disease severity and the host immune response.

Background Cognitive dysfunction after surgery is a major issue in older adults. Here, we determined the effect of APOE4 on perioperative neurocognitive function in older patients. Methods We enrolled 140 English-speaking patients ≥60 yr old scheduled for noncardiac surgery under general anaesthesia in an observational cohort study, of whom 52 underwent neuroimaging. We measured cognition; Aβ, tau, p-tau levels in CSF; and resting-state intrinsic functional connectivity in six Alzheimer's disease-risk regions before and 6 weeks after surgery. Results There were no significant APOE4-related differences in cognition or CSF biomarkers, except APOE4 carriers had lower CSF Aβ levels than non-carriers (preoperative median CSF Aβ [median absolute deviation], APOE4 305 pg ml−1 [65] vs 378 pg ml−1 [38], respectively; P=0.001). Controlling for age, APOE4 carriers had significantly greater preoperative functional connectivity than non-carriers between several brain regions implicated in Alzheimer's disease, including between the left posterior cingulate cortex and left angular gyrus (β [95% confidence interval, CI], 0.218 [0.137–0.230]; PFWE=0.016). APOE4 carriers, but not non-carriers, experienced significant connectivity decreases from before to 6 weeks after surgery between several brain regions including between the left posterior cingulate cortex and left angular gyrus (β [95% CI], –0.196 [–0.256 to –0.136]; PFWE=0.001). Most preoperative and postoperative functional connectivity differences did not change after controlling for preoperative CSF Aβ levels. Conclusions Postoperative change trajectories for cognition and CSF Aβ, tau or p-tau levels did not differ between community dwelling older APOE4 carriers and non-carriers. APOE4 carriers showed greater preoperative functional connectivity and greater postoperative decreases in functional connectivity in key Alzheimer's disease-risk regions, which occur via Aβ-independent mechanisms.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in young children. The T cell response plays a critical role in facilitating clearance of an acute RSV infection, and memory T cell responses are vital for protection against secondary RSV exposures. Tissue-resident memory (TRM) T cells have been identified as a subset of memory T cells that reside in nonlymphoid tissues and are critical for providing long-term immunity. There is currently limited information regarding the establishment and longevity of TRM T cell responses elicited following an acute RSV infection as well as their role in protection against repeated RSV infections. In this study, we examined the magnitude, phenotype, and protective capacity of TRM CD4 and CD8 T cells in the lungs of BALB/c mice following an acute RSV infection. TRM CD4 and CD8 T cells were established within the lungs and waned by 149 d following RSV infection. To determine the protective capacity of TRMs, FTY720 administration was used to prevent trafficking of peripheral memory T cells into the lungs prior to challenge of RSV-immune mice, with a recombinant influenza virus expressing either an RSV-derived CD4 or CD8 T cell epitope. We observed enhanced viral clearance in RSV-immune mice, suggesting that TRM CD8 T cells can contribute to protection against a secondary RSV infection. Given the protective capacity of TRMs, future RSV vaccine candidates should focus on the generation of these cell populations within the lung to induce effective immunity against RSV infection.

Protective lung tissue-resident memory CD8+T cells (Trm) form after influenza A virus (IAV) infection. We show that IAV infection of mice generates CD69+CD103+and other memory CD8+T cell populations in lung-draining mediastinal lymph nodes (mLNs) from circulating naive or memory CD8+T cells. Repeated antigen exposure, mimicking seasonal IAV infections, generates quaternary memory (4M) CD8+T cells that protect mLN from viral infection better than 1M CD8+T cells. Better protection by 4M CD8+T cells associates with enhanced granzyme A/B expression and stable maintenance of mLN CD69+CD103+4M CD8+T cells, vs the steady decline of CD69+CD103+1M CD8+T cells, paralleling the durability of protective CD69+CD103+4M vs 1M in the lung after IAV infection. Coordinated upregulation in canonical Trm-associated genes occurs in circulating 4M vs 1M populations without the enrichment of canonical downregulated Trm genes. Thus, repeated antigen exposure arms circulating memory CD8+T cells with enhanced capacity to form long-lived populations of Trm that enhance control of viral infections of the mLN.

Individuals with autism spectrum disorder (ASD) may exhibit chronic autonomic nervous system (ANS) hyperarousal (e.g., lower respiratory sinus arrhythmia and higher heart rate) compared to their typically developing peers, reflecting a chronic biological threat response. The sustained nature of this cardiac threat suggests tonic nervous system perception of threat in safe contexts. Herein, the cardiac literature in adult and child populations with ASD is reviewed and placed within a continuum of functioning in order to inform the relationship between cardiac response and functioning in ASD. Findings from this review support the relationship between chronic autonomic hyperarousal and emotional and behavioral difficulties observed in individuals with ASD.

Summary Obesity prevalence rises fastest during young adulthood when weight, diet, and physical activity may be influenced by life events, including becoming a parent, but the impact is uncertain. We searched six electronic databases to July 2019 for longitudinal studies (both sexes) aged 15 to 35 years with a prospective pre‐pregnancy/parenthood and post‐delivery outcome. Of 11 studies (across 15 papers), six studies (women only) were eligible for meta‐analysis of the difference in change in body mass index (BMI; kg/m2) between remaining without children and becoming a parent. Mean (±SD) BMI gain for non‐mothers was 2.8 ± 1.3 kg/m2 (~7.5 kg for 164‐cm woman) over 5.6 ± 3.1 years; 12.3% of baseline BMI (22.8 ± 2.5 kg/m2). Becoming a mother was associated with an additional BMI increase of 0.47 ± 0.26 kg/m2 (~1.3 kg), 4.3% of baseline BMI (22.8 ± 5.6 kg/m2); the one study including men reported no difference in change. Physical activity results were equivocal; 2/4 studies (women) and 2/2 (men) showed a greater decline in parents versus non‐parents; diet (three studies) varied by dietary measure, mostly indicating no difference. Becoming a mother is associated with 17% greater absolute BMI gain than remaining childless. Motherhood BMI gain is additional to an alarming BMI increase among young women, highlighting the need for obesity prevention among all young women, including mothers.

PURPOSE: First line treatment with platinum-based chemotherapy with or without immunotherapy improves survival in metastatic non-small cell lung cancer (NSCLC). Preclinical studies suggest that pharmacological ascorbate (P-AscH-) enhances tumor response to platinum therapy. Hence, we conducted a single-arm phase II study to evaluate the efficacy of P-AscH- in combination with platinum-doublet chemotherapy in patients with advanced stage NSCLC (NCT02420314). METHODS: Chemotherapy naïve advanced stage NSCLC patients with an ECOG PS of 0-2 were enrolled to receive 4-cycles of carboplatin (AUC 6) and paclitaxel (200 mg/m2) every 3 weeks (wks). Ascorbate (75 g) infusions were given twice a wk for 12-wks. The primary endpoint was to assess tumor objective response per RECIST v1.1. The trial was conducted as an optimal Simon two-stage design. After initial therapy, patients could receive maintenance or consolidation treatment. Secondary endpoints were to evaluate tolerability, progression-free survival (PFS) and overall survival (OS). Serum cytokines and chemokines were measured at baseline, C2d1, and C4d21± 7d. RESULTS: Forty subjects were enrolled. The study met its primary endpoint with 38 efficacy evaluable subjects. The objective response rate was 34.2%. All were confirmed partial responses (cPR). Disease control rate was 84.2% (stable disease + cPR). Median duration of follow up was 11.7 months (mo), mPFS was 5.7 mo (95% CI:4.2-6.7), and mOS was 12.5 mo (95% CI:7.5-21.4). Treatment-related adverse events (TRAE) included one grade 5 (neutropenic fever) and five grade 4 (cytopenia) events. These events were not attributed to P-AscH-. Common (≥5%) grade 3 TRAE included transient hypertension (27.5%), lymphopenia (22.5%), fatigue (7.5%), anemia (7.5%) and hypokalemia (5%). Cytokine and chemokines data suggest that protocol regimen elicited an immune response with multiple distinct cytokine signatures. Immunophenotyping of peripheral blood mononuclear cells (n=7) demonstrated a mean fold increase in effector CD8 T cells of 4.9 in patients with PFS ≥ 6 mo compare to 1.6 in patients with PFS < 6 mo. Assessments of serum iron profile and somatic alterations in KRAS, KEAP1, NFE2L2 and STK11 genes are underway. CONCLUSIONS: This phase II trial met the primary objective of improving the tumor response rate in advanced stage NSCLC by adding P-AscH- to platinum-based chemotherapy. P-AscH- appears to alter the host immune response. These promising findings warrant further investigation. Citation Format: Muhammad Furqan, Taher Abu-Hejleh, Kellie L. Bodeker, Laura M. Pietrok, Stacey M. Hartwig, Mikaela M. Tremblay, Micaela G. Fosdick, Jon Houtman, Steven Varga, Casey F. Pulliam, Michael Petronek, Melissa A. Fath, Sarah L. Mott, Aaron D. Bossler, Andrew M. Bellizzi, Jun Zhang, Hariharasudan Mani, Varun Monga, Brian J. Smith, Joseph Cullen, Brett A. Wagner, Garry R. Buettner, John M. Buatti, Douglas R. Spitz, Bryan G. Allen. Pharmacological ascorbate enhances platinum-based chemotherapy responses in metastatic non-small cell lung cancer (NSCLC): A phase II clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT164.

Activated CD8+ T cells differentiate into cytotoxic effector (TEFF) cells that eliminate target cells. How TEFF cell identity is established and maintained is not fully understood. We found that Runx3 deficiency limited clonal expansion and impaired upregulation of cytotoxic molecules in TEFF cells. Runx3-deficient CD8+ TEFF cells aberrantly upregulated genes characteristic of follicular helper T (TFH) cell lineage, including Bcl6, Tcf7 and Cxcr5. Mechanistically, the Runx3-CBFβ transcription factor complex deployed H3K27me3 to Bcl6 and Tcf7 genes to suppress the TFH program. Ablating Tcf7 in Runx3-deficient CD8+ TEFF cells prevented the upregulation of TFH genes and ameliorated their defective induction of cytotoxic genes. As such, Runx3-mediated Tcf7 repression coordinately enforced acquisition of cytotoxic functions and protected the cytotoxic lineage integrity by preventing TFH-lineage deviation.

BACKGROUND: The Lung Volume Reduction Coil Treatment in Patients With Emphysema (RENEW) trial reported improvements in quality of life, pulmonary function, and exercise performance following endobronchial coil treatment.OBJECTIVES: The purpose of this post hoc analysis was to identify baseline predictors, including quantitative CT measures, that identify patients most likely to significantly benefit from endobronchial coil therapy.METHODS: Quantitative CT analysis by an independent radiology laboratory and a qualitative evaluation by five blinded experts of the baseline thoracic CT imaging were performed. Univariate and multivariate logistic regression analyses were performed to elucidate characteristics associated with clinical response.RESULTS: In total, 125 patients underwent coil treatment and had evaluable 12-month follow-up results. Of these, 78 patients received treatment of lobes with the highest emphysematous destruction determined by quantitative CT analysis (quantitative visual match [QVM]+), and 47 received treatment in at least one lobe that was not the most destroyed (QVM-). From the 78 patients with QVM+ treatment, a subgroup of 50 patients (64%) was identified with baseline residual volume > 200% predicted, emphysema score > 20% low attenuation area, and absence of airway disease. In this subgroup, greater lobar residual volume reduction in the treated lobes was achieved, which was associated with significant mean +/- SE improvement in FEV1 (15.2 +/- 3.1%), St. George's Respiratory Questionnaire (-12 +/- 2 points), and residual volume (-0.57 +/- 0.13 L).DISCUSSION: This post hoc analysis found that both significant hyperinflation (residual volume >= 200% predicted) and CT analysis are critical for patient selection and treatment planning for endobronchial coil therapy. Quantitative CT analysis is important to identify optimal lobar treatment and to exclude patients with insufficient emphysema (

Background As there has been a demonstrated link between sleep disturbance and suicide, the present study investigated the relationship between self-reported sleep disturbance during inpatient psychiatric treatment and clinical outcomes at discharge and six months post-discharge. Methods Participants were 2,970 adults receiving care in a long-term inpatient psychiatric hospital. Greater than 90% of inpatients reported at least mild sleep disturbance throughout treatment. Group-based trajectory modeling was used to determine patterns of sleep disturbance changes over time. Results Participants fit into distinct categories based on their trajectories of sleep problems across treatment: No Sleep Problems (10.3% of participants, no sleep problems endorsed over the course of inpatient psychiatric treatment), Resolvers (sleep problems completely resolved over the course of inpatient psychiatric treatment; 10.6%), Non-Responders (did not respond to inpatient psychiatric treatment; 35.7%) and Responders (sleep problems decreased but did not fully resolve over the course of inpatient psychiatric treatment; 43.4%). Individuals with comorbid major depressive disorder and generalized anxiety disorder were significantly more likely to demonstrate higher rates of sleep disturbance throughout inpatient psychiatric treatment and their sleep problems did not respond to treatment as usual (Non-Responders). Further, patients in the Non-Responder group had significantly more suicidal ideation and worse clinical outcomes (higher anxiety, more disability, and lower well-being) at discharge and six months post-discharge, as well as were on more medication including hypnotics throughout treatment. Conclusions Findings indicate the urgent need to design and implement inpatient psychiatry sleep protocols to not only improve sleep and clinical outcomes, but also reduce the risk for suicide post-discharge.

Respiratory syncytial virus (RSV) and the common commensal and opportunistic pathogen, non-typeable Haemophilus influenzae (NTHi) both serve as a frequent cause of respiratory infection in children. Although it is well established that some respiratory viruses can increase host susceptibility to secondary bacterial infections, few studies have examined how commensal bacteria could influence a secondary viral response. Here, we examined the impact of NTHi exposure on a subsequent RSV infection of human bronchial epithelial cells (16HBE14o-). Co-culture of 16HBE14o- cells with NTHi 2019 resulted in inhibition of viral gene expression following RSV infection. 16HBE14o- cells co-cultured with heat-killed NTHi 2019 failed to protect against an RSV infection, indicating that protection requires live bacteria. However, NTHi did not inhibit influenza A virus replication, indicating that NTHi-mediated protection was RSV-specific. Our data demonstrates that prior exposure to a commensal bacterium such as NTHi can elicit protection against a subsequent RSV infection.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in young children worldwide. RSV-induced disease ranges from mild to severe, with severe disease correlated with elevated levels of pro-inflammatory cytokines, including the Th17-associated cytokine, IL-17. To better understand the viral factors that influence the induction of a Th17 response, we compared the inflammatory responses elicited by different RSV strains. In contrast to the commonly used RSV A2 strain, RSV 2-20 infection resulted in increased protein levels of IL-6 and IL-1β, which were required to induce a pathogenic Th17 response. RSV 2–20 infection increased mucus production and airway hyperreactivity indicating strain-specific disease induction. RSV 2–20 and A2 induced similar protein levels of caspase-1, however, 2–20 infection resulted in increased protein levels of pro-IL-1β suggesting differential activation of inflammasome signal 1. Increased production of IL-1β following RSV 2–20 infection was dependent on TLR4 expression and signaling, a canonical inflammasome signal 1 activator. A recombinant A2 virus expressing the 2–20 fusion (F) protein induced IL-1β to similar levels as wild-type 2–20 indicating the 2–20 F protein is sufficient to increase pro-inflammatory cytokine production. Reversion of three-point mutations in a region likely to interact with host cells in the RSV A2/2–20 F recombinant virus to the A2 sequence significantly reduced IL-6 and IL-1β protein production. Our results demonstrate that variation in the RSV F protein plays a crucial role in the induction of pro-inflammatory cytokines and modulating disease severity in the animal model and may contribute to differential RSV pathogenesis.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children, accounting for nearly 7% of deaths in those less than one year of age. Despite the morbidity, mortality, and high economic burden caused by RSV worldwide, no licensed vaccine is available. Thus, there is a critical need for a safe, and highly efficacious vaccine to prevent RSV-induced disease. We have developed a novel vaccine utilizing a prefusion-stabilized variant of the RSV fusion (F) protein (DS-Cav1) encapsulated within polymer nanoparticles with a CpG1668 oligodeoxynucleotide adjuvant. The prefusion conformation of RSV F allows for better exposure of the major antigenic site, termed site Ø, a primary target of neutralizing antibody responses in human serum. To evaluate this vaccine candidate, BALB/c mice were vaccinated intranasally and then boosted 4 weeks later. Vaccination with the RSV nanovaccine significantly ameliorated weight loss and pulmonary dysfunction compared to unvaccinated mice in response to an RSV challenge. Additionally, vaccinated mice significantly reduced virus titers in the lungs as early as day 2 post-infection. Protection from disease and enhanced viral clearance remained until 100 days post prime vaccination. Vaccination also established an elevated number of tissue-resident memory CD4 and CD8 T cells in the lungs. The vaccine-induced protection was mediated by both humoral and cellular immune responses, as serum transfers from vaccinated mice provided only partial protection from weight loss and pulmonary dysfunction. Based on the robust immune response elicited and the high level of protection observed, our prefusion RSV F nanoparticle formulation may be a promising RSV vaccine candidate.

Flexible and time efficient commissioning, sorting and packing of materials provide the access to individualized products goods worldwide. At the same time, these demands and the ongoing digitalization of work can result in working conditions that are not ideal, especially from a psychological work science point of view. To get an overview of psychologic health relevant aspects in modern intralogistics jobs, this paper analyses the results from the working time survey from the German Institute for Occupational Safety and Health regarding intralogistics employees. In order to identify which psychological demands in terms of working conditions lead to high job satisfaction as well as better health status, descriptive analyses and bivariate correlations were conducted, indicating significant differences between intralogistics and other workplaces. Consistent correlations between working conditions and indicators for mental health also highlight the importance for psychological research and interventions to improve working conditions and in turn mental health and wellbeing of employees in the business.

BACKGROUND The purpose of this study was to explore the role of perinatal vitamin-D intake on the development and characterization of hyperkyphosis in a porcine model. METHODS The spines of 16 pigs were assessed at 9, 13, and 17 weeks of age with radiography and at 17 weeks with computed tomography (CT), magnetic resonance imaging (MRI), histology, and bone-density testing. An additional 169 pigs exposed to 1 of 3 maternal dietary vitamin-D levels from conception through the entire lactation period were fed 1 of 4 nursery diets supplying different levels of vitamin D, calcium, and phosphorus. When the animals were 13 weeks of age, upright lateral spinal radiography was performed with use of a custom porcine lift and sagittal Cobb angles were measured in triplicate to determine the degree of kyphosis in each pig. RESULTS The experimental animals had significantly greater kyphotic sagittal Cobb angles at all time points when compared with the control animals. These hyperkyphotic deformities demonstrated no significant differences in Hounsfield units, contained a slightly lower ash content (46.7% ± 1.1% compared with 50.9% ± 1.6%; p < 0.001), and demonstrated more physeal irregularities. Linear mixed model analysis of the measured kyphosis demonstrated that maternal diet had a greater effect on sagittal Cobb angle than did nursery diet and that postnatal supplementation did not completely eliminate the risk of hyperkyphosis. CONCLUSIONS Maternal diets deficient in vitamin D increased the development of hyperkyphosis in offspring in this model. CLINICAL RELEVANCE This study demonstrates that decreased maternal dietary vitamin-D intake during pregnancy increases the risk of spinal deformity in offspring. In addition, these data show the feasibility of generating a large-animal spinal-deformity model through dietary manipulation alone.

Memory CD8 T cells can provide protection from re-infection by respiratory viruses such as influenza and SARS. However, the relative contribution of memory CD8 T cells in providing protection against respiratory syncytial virus (RSV) infection is currently unclear. To address this knowledge gap, we utilized a prime-boost immunization approach to induce robust memory CD8 T cell responses in the absence of RSV-specific CD4 T cells and antibodies. Unexpectedly, RSV infection of mice with pre-existing CD8 T cell memory led to exacerbated weight loss, pulmonary disease, and lethal immunopathology. The exacerbated disease in immunized mice was not epitope-dependent and occurred despite a significant reduction in RSV viral titers. In addition, the lethal immunopathology was unique to the context of an RSV infection as mice were protected from a normally lethal challenge with a recombinant influenza virus expressing an RSV epitope. Memory CD8 T cells rapidly produced IFN-γ following RSV infection resulting in elevated protein levels in the lung and periphery. Neutralization of IFN-γ in the respiratory tract reduced morbidity and prevented mortality. These results demonstrate that in contrast to other respiratory viruses, RSV-specific memory CD8 T cells can induce lethal immunopathology despite mediating enhanced viral clearance., Author summary Memory CD8 T cells have been shown to provide protection against many respiratory viruses. However, the ability of memory CD8 T cells to provide protection against RSV has not been extensively examined. Unexpectedly, mice with pre-existing CD8 T cell memory, in the absence of memory CD4 T cells and antibodies, exhibited exacerbated morbidity and mortality following RSV infection. We demonstrate that the immunopathology is the result of early and excessive production of IFN-γ by memory CD8 T cells in the lung. Our research provides important new insight into the mechanisms of how memory T cells induce immunopathology. In addition, our findings serve as an important cautionary tale against the use of epitope-based T cell vaccines against RSV.

Recent work has suggested that current mouse models may underrepresent the complexity of human immune responses. While most mouse immunology studies utilize inbred mouse strains, it is unclear if conclusions drawn from inbred mice can be extended to all mouse strains or generalized to humans. We recently described a "surrogate activation marker" approach that could be used to track polyclonal CD8 T cell responses in inbred and outbred mice and noted substantial discord in the magnitude and kinetics of CD8 T cell responses in individual outbred mice following infection. However, how the memory CD8 T cell response develops following infection and the correlates of memory CD8 T cell-mediated protection against re-infection in outbred mice remains unknown. In this study, we investigated development of pathogen-specific memory CD8 T cell responses in inbred C57B/6 and outbred National Institutes of Health Swiss mice following lymphocytic choriomeningitis virus or L. monocytogenes infection. Interestingly, the size of the memory CD8 T cell pool generated and rate of phenotypic progression was considerably more variable in individual outbred compared to inbred mice. Importantly, while prior infection provided both inbred and outbred cohorts of mice with protection against re-infection that was dependent on the dose of primary infection, levels of memory CD8 T cells generated and degree of protection against re-infection did not correlate with primary infection dose in all outbred mice. While variation in CD8 T cell responses to infection is not entirely surprising due to the genetic diversity present, analysis of infection-induced immunity in outbred hosts may reveal hidden complexity in CD8 T cell responses in genetically diverse populations and might help us further bridge the gap between mouse and human studies.

Background Inhibitors of mTOR, such as sirolimus, have been shown to induce thymus involution and inflammatory lung disease in mice. The latter effect supports the role of this serine/threonine kinase in ameliorating lung inflammation. Other studies have shown sirolimus reduces/delays lung disease associated with various strains of influenza A virus (IAV). Thus, the effects of mTOR inhibitors on influenza infection deserve further studies. Methods Here, we examined the changes in lung viral copies, pathology and pulmonary function associated with IAV (A/PR/8/34) infection in mice treated with sirolimus. Results Body weight loss peaked between days 6–11 post-infection and was more severe in IAV-infected mice that were administered sirolimus as compared to mice that received IAV alone (p = 0.030). Natural log viral gene copies, mean ± SD per mg lung tissue, in IAV-infected mice that were administered sirolimus were 17.31 ± 1.27 on day 4, 19.31 ± 7.46 on day 10, and 0 on day 25. The corresponding number of copies in mice that received IAV alone were 18.56 ± 0.95 on day 4 (p = 0.132), 1.52 ± 1.39 on day 10 (p = 0.008), and 0 on day 25. Lung pathology was evident on days 4, 10, and 25 post infection, with mean ± SD inflammatory score of 9.0 ± 4.5 in IAV-infected mice that were administered sirolimus, as compared to 11.5 ± 4.5 (p = 0.335) in mice received IAV alone (maximum score, 26.0). Impaired lung function was evident in IAV-infected mice on days 4 and 10, as demonstrated by increased airway resistance and decreased compliance. Conclusions In this model, the effects of sirolimus on influenza infection included severe weight loss and modified viral replication, respiratory function and lung inflammation. The adverse events associated with sirolimus treatment are consistent with its potent immunosuppressive activity and, thus, preclude its use in IAV infection.

Activated CD8+ T cells differentiate into cytotoxic effector (TEFF) cells that eliminate target cells. How TEFF cell identity is established and maintained remains less understood. Here we show Runx3 deficiency limits clonal expansion and impairs upregulation of cytotoxic molecules in TEFF cells. Runx3-deficient CD8+ TEFF cells aberrantly upregulate genes characteristic of follicular helper T (TFH) cell lineage, including Bcl6, Tcf7 and Cxcr5. Mechanistically, the Runx3-CBFβ complex deploys H3K27me3 to Bcl6 and Tcf7 genes to suppress the TFH program. Ablating Tcf7 in Runx3-deficient CD8+ TEFF cells prevents the upregulation of TFH genes and ameliorates their defective induction of cytotoxic genes. As such, Runx3-mediated Tcf7 repression coordinately enforces acquisition of cytotoxic functions and protects the cytotoxic lineage integrity by preventing TFH-lineage deviation.

Unlike systemic infections, little is known about the role of repeated localized infections on (re)shaping pathogen-specific memory CD8 T cell responses. Here, we used primary (1°) and secondary (2°) intranasal influenza virus infections of mice as a model to study intrinsic memory CD8 T cell properties. We show that secondary antigen exposure, relative to a single infection, generates memory CD8 T cell responses of superior magnitude in multiple tissue compartments including blood, spleen, draining lymph nodes, and lung. Unexpectedly, regardless of the significantly higher number of 2° memory CD8 T cells, similar degree of protection against pulmonary challenge was observed in both groups of mice containing 1° or 2° memory CD8 T cells. Mechanistically, using pertussis toxin-induced migration block, we showed that superior antigen-driven proliferation and ability to relocate to the site of infection allowed 1° memory CD8 T cells to accumulate in the infected lung during the first few days after challenge, compensating for the initially lower cell numbers. Taken together, the history of antigen exposures to localized pulmonary infections, through altering basic cell biology, dictates dynamic properties of protective memory CD8 T cell responses. This knowledge has important implications for a design of novel and an improvement of existing vaccines and immunization strategies.

The migration of pathogen-specific T cells into nonlymphoid tissues, such as the lung, is critical to control peripheral infections. Use of in vivo intravascular labeling of leukocytes has allowed for improved discrimination between cells located in the blood from cells present within peripheral tissues, such as the lung. This is particularly important in the lung, which is comprised of an intricate network of blood vessels that harbors a large proportion of the total blood volume at any given time. Recent work has demonstrated that >80% of antigen-specific effector CD8 T cells remain in the pulmonary vasculature following an intratracheal infection with a systemic viral pathogen. However, it remains unclear what proportion of effector CD8 T cells are located within lung tissue following a localized respiratory viral infection. We confirm that most effector and memory CD8 T cells are found in the vasculature after an intranasal infection with the systemic pathogens lymphocytic choriomeningitis virus (LCMV) or vaccinia virus (VACV). In contrast, following pulmonary viral infections with either respiratory syncytial virus (RSV) or influenza A virus (IAV), 80 to 90% of the antigen-specific effector CD8 T cells were located within lung tissue. Similarly, the majority of antigen-specific CD4 T cells were present within lung tissue during a pulmonary viral infection. Furthermore, a greater proportion of gamma interferon-positive (IFN-γ + ) effector CD8 and CD4 T cells were located within lung tissue following a localized respiratory viral infection. Our results indicate that T cells exhibit significantly altered distribution patterns dependent upon the tissue tropism of the infection. IMPORTANCE The migration of T cells to nonlymphoid sites, such as the lung, is critical to mediate clearance of viral infections. The highly vascularized lung holds up to 40% of blood, and thus, the T cell response may be a reflection of lymphocytes localized to the pulmonary vasculature instead of lung tissue. We examined the localization of T cell responses within the lung following either a localized or systemic viral infection. We demonstrate that following intranasal infection with a systemic pathogen, most T cells are localized to the pulmonary vasculature. In contrast, T cells are primarily localized to lung tissue following a respiratory viral infection. Our results demonstrate vast differences in the localization of T cell responses within the lung parenchyma between pathogens that can replicate locally versus systemically and that intravascular antibody labeling can be utilized to assess the localization patterns of T cell responses in nonlymphoid organs.