Cutting Edge: First Lung Infection Permanently Enlarges Lymph Nodes and Enhances New T Cell Responses.

Humans experience frequent respiratory infections. Immunology and vaccinology studies in mice are typically performed in naive specific pathogen-free animals responding to their very first respiratory challenge. We found that the first respiratory infection induces lifelong enlargement of the lung-draining mediastinal lymph nodes (medLNs). Furthermore, infection-experienced medLNs supported better naive T cell surveillance and effector responses to new unrelated infections that exhibited more biased accumulation and memory establishment within the lung. Moreover, we observed that weight loss induced by influenza infection was substantially reduced in mice that had recovered from a previous unrelated respiratory viral challenge. These data show that the lack of infectious history and corresponding medLN hypoplasia in specific pathogen-free mice alter their immune response to lung infections. Preclinical vaccination and immunology studies should consider the previous infectious experience of the model organism.

Chronic antigen in solid tumors drives a distinct program of T cell residence.

Analyses of healthy tissue reveal signatures that identify resident memory CD8+ T cells (TRM), which survey tissues without recirculating. The density of TRM phenotype cells within solid tumors correlates favorably with prognosis, suggesting that intratumoral residents control cancer. However, residence has not been directly tested, and intratumoral TRM phenotype cells could instead reflect aspects of the microenvironment that correlate with prognosis. Using a breast cancer model in mice, we found that conventional TRM markers do not inform the tumor residence of either bystander or tumor-specific cells, which exhibit further distinct phenotypes in the tumor microenvironment and healthy mammary tissue. Rather, tumor-specific, stem progenitor CD8+ T cells migrate to tumors and become resident while acquiring select markers of exhaustion. These data indicate that tonic antigen stimulation and the tumor environment drive distinct programs of residence compared with healthy tissues and that tumor immunity is sustained by continued migration of tumor-specific stem cells.

Route of self-amplifying mRNA vaccination modulates the establishment of pulmonary resident memory CD8 and CD4 T cells.

Respiratory tract resident memory T cells (TRM), typically generated by local vaccination or infection, can accelerate control of pulmonary infections that evade neutralizing antibody. It is unknown whether mRNA vaccination establishes respiratory TRM. We generated a self-amplifying mRNA vaccine encoding the influenza A virus nucleoprotein that is encapsulated in modified dendron-based nanoparticles. Here, we report how routes of immunization in mice, including contralateral versus ipsilateral intramuscular boosts, or intravenous and intranasal routes, influenced influenza-specific cell-mediated and humoral immunity. Parabiotic surgeries revealed that intramuscular immunization was sufficient to establish CD8 TRM in the lung and draining lymph nodes. Contralateral, compared with ipsilateral, intramuscular boosting broadened the distribution of lymph node TRM and T follicular helper cells but slightly diminished resulting levels of serum antibody. Intranasal mRNA delivery established modest circulating CD8 and CD4 T cell memory but augmented distribution to the respiratory mucosa. Combining intramuscular immunizations with an intranasal mRNA boost achieved high levels of both circulating T cell memory and lung TRM. Thus, routes of mRNA vaccination influence humoral and cell-mediated immunity, and intramuscular prime-boosting establishes lung TRM that can be further expanded by an additional intranasal immunization.

P2RX7 Enhances Tumor Control by CD8+ T Cells in Adoptive Cell Therapy.

Expression of the purinergic receptor P2RX7 by CD8+ T cells promotes the generation of memory populations following acute infections. However, data suggest that P2RX7 may limit the efficacy of antitumor responses. Herein, we show that P2RX7 is beneficial for optimal melanoma control in a mouse CD8+ T-cell adoptive transfer model. Tumor-specific P2rx7-/- CD8+ T cells exhibited impaired mitochondrial maintenance and function but did not display signs of overt exhaustion early in the antitumor response. However, as the tumor burden increased, the relative frequency of P2RX7-deficient CD8+ T cells declined within the tumor; this correlated with reduced proliferation, increased apoptosis, and mitochondrial dysfunction. Extending these studies, we found that the transient in vitro stimulation of P2RX7 using the ATP analogue BzATP led to enhanced B16 melanoma control by CD8+ T cells. These findings are in keeping with the concept that extracellular ATP (eATP) sensing by P2RX7 on CD8+ T cells is required for their ability to efficiently eliminate tumors by promoting mitochondrial fitness and underscore the potential for P2RX7 stimulation as a novel therapeutic treatment to enhance tumor immunotherapy.

Cutting Edge: Nucleocapsid Vaccine Elicits Spike-Independent SARS-CoV-2 Protective Immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Neutralizing Abs target the receptor binding domain of the spike (S) protein, a focus of successful vaccine efforts. Concerns have arisen that S-specific vaccine immunity may fail to neutralize emerging variants. We show that vaccination with a human adenovirus type 5 vector expressing the SARS-CoV-2 nucleocapsid (N) protein can establish protective immunity, defined by reduced weight loss and viral load, in both Syrian hamsters and K18-hACE2 mice. Challenge of vaccinated mice was associated with rapid N-specific T cell recall responses in the respiratory mucosa. This study supports the rationale for including additional viral Ags in SARS-CoV-2 vaccines, even if they are not a target of neutralizing Abs, to broaden epitope coverage and immune effector mechanisms.

Irreversible electroporation augments checkpoint immunotherapy in prostate cancer and promotes tumor antigen-specific tissue-resident memory CD8+ T cells.

Memory CD8+ T cells populate non-lymphoid tissues (NLTs) following pathogen infection, but little is known about the establishment of endogenous tumor-specific tissue-resident memory T cells (TRM) during cancer immunotherapy. Using a transplantable mouse model of prostate carcinoma, here we report that tumor challenge leads to expansion of naïve neoantigen-specific CD8+ T cells and formation of a small population of non-recirculating TRM in several NLTs. Primary tumor destruction by irreversible electroporation (IRE), followed by anti-CTLA-4 immune checkpoint inhibitor (ICI), promotes robust expansion of tumor-specific CD8+ T cells in blood, tumor, and NLTs. Parabiosis studies confirm that TRM establishment following dual therapy is associated with tumor remission in a subset of cases and protection from subsequent tumor challenge. Addition of anti-PD-1 following dual IRE + anti-CTLA-4 treatment blocks tumor growth in non-responsive cases. This work indicates that focal tumor destruction using IRE combined with ICI is a potent in situ tumor vaccination strategy that generates protective tumor-specific TRM.

Nucleocapsid vaccine elicits spike-independent SARS-CoV-2 protective immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Neutralizing antibodies target the receptor binding domain of the spike (S) protein, a focus of successful vaccine efforts. Concerns have arisen that S-specific vaccine immunity may fail to neutralize emerging variants. We show that vaccination with HAd5 expressing the nucleocapsid (N) protein can establish protective immunity, defined by reduced weight loss and viral load, in both Syrian hamsters and k18-hACE2 mice. Challenge of vaccinated mice was associated with rapid N-specific T cell recall responses in the respiratory mucosa. This study supports the rationale for including additional viral antigens, even if they are not a target of neutralizing antibodies, to broaden epitope coverage and immune effector mechanisms.

CD8+ T cell self-tolerance permits responsiveness but limits tissue damage.

Self-specific CD8+T cells can escape clonal deletion, but the properties and capabilities of such cells in a physiological setting are unclear. We characterized polyclonal CD8+ T cells specific for the melanocyte antigen tyrosinase-related protein 2 (Trp2) in mice expressing or lacking this enzyme (due to deficiency in Dct, which encodes Trp2). Phenotypic and gene expression profiles of pre-immune Trp2/Kb-specific cells were similar; the size of this population was only slightly reduced in wild-type (WT) compared to Dct-deficient (Dct-/-) mice. Despite comparable initial responses to Trp2 immunization, WT Trp2/Kb-specific cells showed blunted expansion and less readily differentiated into a CD25+proliferative population. Functional self-tolerance clearly emerged when assessing immunopathology: adoptively transferred WT Trp2/Kb-specific cells mediated vitiligo much less efficiently. Hence, CD8+ T cell self-specificity is poorly predicted by precursor frequency, phenotype, or even initial responsiveness, while deficient activation-induced CD25 expression and other gene expression characteristics may help to identify functionally tolerant cells.

Cutting Edge: Mouse SARS-CoV-2 Epitope Reveals Infection and Vaccine-Elicited CD8 T Cell Responses.

The magnitude of SARS-CoV-2-specific T cell responses correlates inversely with human disease severity, suggesting T cell involvement in primary control. Whereas many COVID-19 vaccines focus on establishing humoral immunity to viral spike protein, vaccine-elicited T cell immunity may bolster durable protection or cross-reactivity with viral variants. To better enable mechanistic and vaccination studies in mice, we identified a dominant CD8 T cell SARS-CoV-2 nucleoprotein epitope. Infection of human ACE2 transgenic mice with SARS-CoV-2 elicited robust responses to H2-Db/N219-227, and 40% of HLA-A*02+ COVID-19 PBMC samples isolated from hospitalized patients responded to this peptide in culture. In mice, i.m. prime-boost nucleoprotein vaccination with heterologous vectors favored systemic CD8 T cell responses, whereas intranasal boosting favored respiratory immunity. In contrast, a single i.v. immunization with recombinant adenovirus established robust CD8 T cell memory both systemically and in the respiratory mucosa.

Responding to measles outbreaks in underserved Roma and Romanian populations in England: the critical role of community understanding and engagement.

Since 2016, the European Region has experienced large-scale measles outbreaks. Several measles outbreaks in England during 2017/18 specifically affected Romanian and Romanian Roma communities. In this qualitative interview study, we looked at the effectiveness of outbreak responses and efforts to promote vaccination uptake amongst these underserved communities in three English cities: Birmingham, Leeds and Liverpool. Semi-structured in-depth interviews were conducted with 33 providers involved in vaccination delivery and outbreak management in these cities. Interviews were analysed thematically and factors that influenced the effectiveness of responses were categorised into five themes: (1) the ability to identify the communities, (2) provider knowledge and understanding of the communities, (3) the co-ordination of response efforts and partnership working, (4) links to communities and approaches to community engagement and (5) resource constraints. We found that effective partnership working and community engagement were key to the prevention and management of vaccine-preventable disease outbreaks in the communities. Effective engagement was found to be compromised by cuts to public health spending and services for underserved communities. To increase uptake in under-vaccinated communities, local knowledge and engagement are vital to build trust and relationships. Local partners must work proactively to identify, understand and build connections with communities.

Optimizing Integrated Electrode Design for Irreversible Electroporation of Implanted Polymer Scaffolds.

Irreversible electroporation (IRE) is an emerging technology for non-thermal ablation of solid tumors. This study sought to integrate electrodes into microporous poly(caprolactone) (PCL) scaffolds previously shown to recruit metastasizing cancer cells in vivo in order to facilitate application of IRE to disseminating cancer cells. As the ideal parallel plate geometry would render much of the porous scaffold surface inaccessible to infiltrating cells, numerical modeling was utilized to predict the spatial profile of electric field strength within the scaffold for alternative electrode designs. Metal mesh electrodes with 0.35 mm aperture and 0.16 mm wire diameter established electric fields with similar spatial uniformity as the parallel plate geometry. Composite PCL-IRE scaffolds were fabricated by placing cylindrical porous PCL scaffolds between two PCL dip-coated stainless steel wire meshes. PCL-IRE scaffolds exhibited no difference in cell infiltration in vivo compared to PCL scaffolds. In addition, upon application of IRE in vivo, cells infiltrating the PCL-IRE scaffolds were successfully ablated, as determined by histological analysis 3 days post-treatment. The ability to establish homogeneous electric fields within a biomaterial that can recruit metastatic cancer cells, especially when combined with immunotherapy, may further advance IRE technology beyond solid tumors to the treatment of systemic cancer.

Engineering T cell response to cancer antigens by choice of focal therapeutic conditions.

Focal thermal therapy (Heat), cryosurgery (Cryo) and irreversible electroporation (IRE) are increasingly used to treat cancer. However, local recurrence and systemic spread are persistent negative outcomes. Nevertheless, emerging work with immunotherapies (i.e., checkpoint blockade or dendritic cell (DC) vaccination) in concert with focal therapies may improve outcomes. To understand the role of focal therapy in priming the immune system for immunotherapy, an in vitro model of T cell response after exposure to B16 melanoma cell lysates after lethal exposures was designed. Exposure included: Heat (50 °C, 30 min), Cryo (-80 °C, 30 min) and IRE (1250 V/cm, 99 pulses, 50 µs pulses with 1 Hz intervals). After viability assessment (CCK-8 assay), cell lysates were collected and assessed for protein release (BCA assay), protein denaturation (FTIR-spectroscopy), TRP-2 antigen release (western blot), and T cell activation (antigen-specific CD8 T cell proliferation). Results showed IRE released the most protein and antigen (TRP-2), followed by Cryo and Heat. In contrast, Cryo released the most native (not denatured) protein, compared to IRE and Heat. Finally, IRE dramatically outperformed both Cryo and Heat in T cell activation while Cryo modestly outperformed Heat. This study demonstrates that despite all focal therapies ability to destroy cells, the 'quantity' (i.e., amount) and 'quality' (i.e., molecular state) of tumor protein (including antigen) released can dramatically change the ensuing priming of the immune system. This suggests protein-based metrics whereby focal therapies can be designed to prime the immune system in concert with immunotherapies to eventually achieve improved and durable cancer treatment in vivo.

First use of whole-genome sequencing to investigate a cluster of Yersinia enterocolitica, Liverpool, United Kingdom, 2017.

PURPOSE: In May 2017 we were notified of a cluster of Yersinia enterocolitica-positive isolates from Liverpool. The purpose of this work was to investigate this cluster of cases and find a possible common source. We combined epidemiological information with whole-genome sequencing (WGS) results, which indicated that these cases were unlikely to be from the same source. This investigation provides evidence that WGS could be used to investigate future clusters of Y. enterocolitica cases. METHODS: A case was defined as a person with a laboratory-confirmed isolate of Y. enterocolitica, sampled in 2017, who is a resident in Liverpool local authority at the time of sampling. Faecal samples were cultured at the local laboratory and presumptive isolates of Yersinia sp. were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Positive isolates were whole-genome sequenced by the reference laboratory. RESULTS: Nine cases were identified, which was significantly greater (P<0.0001) than the average number of cases in this area from the last 10 years. Six cases were female (66.67 %) and the ages of the patients ranged from 20 to 81 (median 54). The sample dates ranged from 29 April to 1 August 2017. The WGS results showed that Y. enterocolitica isolates belonged to different sequence types. CONCLUSION: This was the first time that WGS was used to investigate a cluster of Y. enterocolitica cases; the cases were clustered in time, person and place, but the WGS results indicate that these cases were not from the same source. This result informed the Outbreak Control Team's decision-making and resulted in the investigation being closed.

Mosquito Larvae in Tires from Mississippi, United States: The Efficacy of Abiotic and Biotic Parameters in Predicting Spatial and Temporal Patterns of Mosquito Populations and Communities.

Container systems, including discarded vehicle tires, which support populations of mosquitoes, have been of interest for understanding the variables that produce biting adults that serve as both nuisances and as public health threats. We sampled tires in six sites at three times in 2012 across the state of Mississippi to understand the biotic and abiotic variables responsible for explaining patterns of larvae of common species, species richness, and total abundance of mosquitoes. From 498 tires sampled, we collected >58,000 immatures representing 16 species, with the most common species including Aedes albopictus (Skuse), Culex quinquefasciatus (L.), Orthopodomyia signifera (Coquillett), Aedes triseriatus (Say), Toxorhynchites rutilus septentrionalis (Coquillett), and Culex territans (Walker) accounting for ∼97% of all larvae. We also documented 32 new county records for resident species and recent arrivals in the state, including Aedes japonicus japonicus (Theobald) and Culex coronator (Dyar & Knab). Cluster analysis, which was used to associate sites and time periods based on similar mosquito composition, did reveal patterns across the state; however, there also were more general patterns between species and genera and environmental factors. Broadly, Aedes was often associated with factors related to detritus, whereas Culex was frequently associated with habitat variables (e.g., tire size and water volume) and microorganisms. Some Culex did lack factors connecting variation in early and late instars, suggesting differences between environmental determinants of oviposition and survival. General patterns between the tire environment and mosquito larvae do appear to exist, especially at the generic level, and point to inherent differences between genera that may aid in predicting vector locations and populations.

Preparing soldiers for the stress of combat.

UNLABELLED: Protracted use of stressors during military training courses does not necessarily enhance a Soldier?s ability to regulate stress on the battlefield. Extensive stress during training can be a contributing factor to suboptimal neurologic and overall long-term health. Prolonged high-stress military training programs, as well as extended duration combat deployments, should be comprehensively scrutinized for opportunities to preserve health and increase combat effectiveness. Contemporary research in neuroscience and psychology can provide insight into training techniques that can be used to control stress and optimize performance in combat. Physical fitness training programs can elevate the stress threshold. Extensive situational training can also inoculate Soldiers to specific combat stressors. Training methods such as these will enable Soldiers to achieve higher levels of performance while under enemy fire and are encouraged for units deploying to combat. KEYWORDS: combat stress, military training, military deployment, physical training, post-traumatic stress disorder, sleep deprivation, stress inoculation training.