CD8+ T cell priming that is required for curative intratumorally anchored anti-4-1BB immunotherapy is constrained by Tregs.

Although co-stimulation of T cells with agonist antibodies targeting 4-1BB (CD137) improves antitumor immune responses in preclinical studies, clinical success has been limited by on-target, off-tumor activity. Here, we report the development of a tumor-anchored ɑ4-1BB agonist (ɑ4-1BB-LAIR), which consists of a ɑ4-1BB antibody fused to the collagen-binding protein LAIR. While combination treatment with an antitumor antibody (TA99) shows only modest efficacy, simultaneous depletion of CD4+ T cells boosts cure rates to over 90% of mice. Mechanistically, this synergy depends on ɑCD4 eliminating tumor draining lymph node regulatory T cells, resulting in priming and activation of CD8+ T cells which then infiltrate the tumor microenvironment. The cytotoxic program of these newly primed CD8+ T cells is then supported by the combined effect of TA99 and ɑ4-1BB-LAIR. The combination of TA99 and ɑ4-1BB-LAIR with a clinically approved ɑCTLA-4 antibody known for enhancing T cell priming results in equivalent cure rates, which validates the mechanistic principle, while the addition of ɑCTLA-4 also generates robust immunological memory against secondary tumor rechallenge. Thus, our study establishes the proof of principle for a clinically translatable cancer immunotherapy.

Early cellular mechanisms of type I interferon-driven susceptibility to tuberculosis.

Mycobacterium tuberculosis (Mtb) causes 1.6 million deaths annually. Active tuberculosis correlates with a neutrophil-driven type I interferon (IFN) signature, but the cellular mechanisms underlying tuberculosis pathogenesis remain poorly understood. We found that interstitial macrophages (IMs) and plasmacytoid dendritic cells (pDCs) are dominant producers of type I IFN during Mtb infection in mice and non-human primates, and pDCs localize near human Mtb granulomas. Depletion of pDCs reduces Mtb burdens, implicating pDCs in tuberculosis pathogenesis. During IFN-driven disease, we observe abundant DNA-containing neutrophil extracellular traps (NETs) described to activate pDCs. Cell-type-specific disruption of the type I IFN receptor suggests that IFNs act on IMs to inhibit Mtb control. Single-cell RNA sequencing (scRNA-seq) indicates that type I IFN-responsive cells are defective in their response to IFNγ, a cytokine critical for Mtb control. We propose that pDC-derived type I IFNs act on IMs to permit bacterial replication, driving further neutrophil recruitment and active tuberculosis disease.

Protective intravenous BCG vaccination induces enhanced immune signaling in the airways.

Intradermal (ID) Bacillus Calmette-Guérin (BCG) is the most widely administered vaccine in the world. However, ID-BCG fails to achieve the level of protection needed in adults to alter the course of the tuberculosis epidemic. Recent studies in non-human primates have demonstrated high levels of protection against Mycobacterium tuberculosis ( Mtb ) following intravenous (IV) administration of BCG. However, the protective immune features that emerge following IV BCG vaccination remain incompletely defined. Here we used single-cell RNA-sequencing (scRNAseq) to transcriptionally profile 157,114 unstimulated and purified protein derivative (PPD)-stimulated bronchoalveolar lavage (BAL) cells from 29 rhesus macaques immunized with BCG across routes of administration and doses to uncover cell composition-, gene expression-, and biological network-level signatures associated with IV BCG-mediated protection. Our analyses revealed that high-dose IV BCG drove an influx of polyfunctional T cells and macrophages into the airways. These macrophages exhibited a basal activation phenotype even in the absence of PPD-stimulation, defined in part by IFN and TNF-α signaling up to 6 months following BCG immunization. Furthermore, intercellular immune signaling pathways between key myeloid and T cell subsets were enhanced following PPD-stimulation in high-dose IV BCG-vaccinated macaques. High-dose IV BCG also engendered quantitatively and qualitatively stronger transcriptional responses to PPD-stimulation, with a robust Th1-Th17 transcriptional phenotype in T cells, and augmented transcriptional signatures of reactive oxygen species production, hypoxia, and IFN-γ response within alveolar macrophages. Collectively, this work supports that IV BCG immunization creates a unique cellular ecosystem in the airways, which primes and enables local myeloid cells to effectively clear Mtb upon challenge.

TLR2 is non-redundant in the population and subpopulation responses to Mycobacterium tuberculosis in macrophages and in vivo.

Tuberculosis (TB), caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb), is a global health threat. Targeting host pathways that modulate protective or harmful components of inflammation has been proposed as a therapeutic strategy that could aid sterilization or mitigate TB-associated permanent tissue damage. In purified form, many Mtb components can activate innate immune pathways. However, knowledge of the pathways that contribute most to the observed response to live Mtb is incomplete, limiting the possibility of precise intervention. We took a systematic, unbiased approach to define the pathways that drive the earliest immune response to Mtb. Using a macrophage model of infection, we compared the bulk transcriptional response to infection with the response to a panel of Mtb-derived putative innate immune ligands. We identified two axes of response: an NF-kB-dependent response similarly elicited by all Mtb pathogen-associated molecular patterns (PAMPs) and a type I interferon axis unique to cells infected with live Mtb. Consistent with growing literature data pointing to TLR2 as a dominant Mtb-associated PAMP, the TLR2 ligand PIM6 most closely approximated the NF-kB-dependent response to the intact bacterium. Quantitatively, the macrophage response to Mtb was slower and weaker than the response to purified PIM6. On a subpopulation level, the TLR2-dependent response was heterogeneously induced, with only a subset of infected cells expressing key inflammatory genes known to contribute to the control of infection. Despite potential redundancies in Mtb ligand/innate immune receptor interactions during in vivo infection, loss of the TLR2/PIM6 interaction impacted the cellular composition of both the innate and adaptive compartments. IMPORTANCE Tuberculosis (TB) is a leading cause of death globally. Drug resistance is outpacing new antibiotic discovery, and even after successful treatment, individuals are often left with permanent lung damage from the negative consequences of inflammation. Targeting host inflammatory pathways has been proposed as an approach that could either improve sterilization or improve post-treatment lung health. However, our understanding of the inflammatory pathways triggered by Mycobacterium tuberculosis (Mtb) in infected cells and lungs is incomplete, in part because of the complex array of potential molecular interactions between bacterium and host. Here, we take an unbiased approach to identify the pathways most central to the host response to Mtb. We examine how individual pathways are triggered differently by purified Mtb products or infection with the live bacterium and consider how these pathways inform the emergence of subpopulation responses in cell culture and in infected mice. Understanding how individual interactions and immune pathways contribute to inflammation in TB opens the door to the possibility of developing precise therapeutic interventions.

Tregs constrain CD8+ T cell priming required for curative intratumorally anchored anti-4-1BB immunotherapy.

Although co-stimulation of T cells with agonist antibodies targeting 4-1BB (CD137) improves antitumor immune responses in preclinical studies, clinical development has been hampered by on-target, off-tumor toxicity. Here, we report the development of a tumor-anchored ɑ4-1BB agonist (ɑ4-1BB-LAIR), which consists of an ɑ4-1BB antibody fused to the collagen binding protein LAIR. While combination treatment with an antitumor antibody (TA99) displayed only modest efficacy, simultaneous depletion of CD4+ T cells boosted cure rates to over 90% of mice. We elucidated two mechanisms of action for this synergy: ɑCD4 eliminated tumor draining lymph node Tregs, enhancing priming and activation of CD8+ T cells, and TA99 + ɑ4-1BB-LAIR supported the cytotoxic program of these newly primed CD8+ T cells within the tumor microenvironment. Replacement of ɑCD4 with ɑCTLA-4, a clinically approved antibody that enhances T cell priming, produced equivalent cure rates while additionally generating robust immunological memory against secondary tumor rechallenge.

Characteristics of Active Duty Service Members Referred to the Navy's Weight-Management Program.

INTRODUCTION: Rates of overweight and obesity have increased in the military, particularly in the U.S. Navy. While the Navy has implemented weight-management programs like ShipShape, findings on the effectiveness of these programs are mixed. Further knowledge on the characteristics of service members (SMs) who participate in these programs may help inform course curricula and improve outcomes. This study aimed to (1) examine characteristics of SMs referred to the Navy's ShipShape program at a large military treatment facility, (2) compare these characteristics among SMs who did and did not enroll in a randomized clinical trial of ShipShape (ShipShape study participants), and (3) compare demographic and health characteristics of ShipShape study participants to that of a random and similarly sized sample of Navy SMs who responded to the 2015 DoD Health-Related Behaviors Survey (HRBS). MATERIALS AND METHODS: Data from active duty Navy SMs referred to the ShipShape program at a large military treatment facility were evaluated (n = 225). A subset of these SMs enrolled in the ShipShape study (n = 187). Among enrolled SMs, data from 147 who completed all measures were compared to that of HRBS respondents. Univariate ANOVA and chi-square analyses were used to examine (1) demographic and motivational differences between SMs who did and did not enroll in the ShipShape study and (2) differences in demographics and medical and mental health conditions between ShipShape study participants and Navy HRBS respondents. RESULTS: The majority of SMs referred to ShipShape were female with an average age of 28.3 years. Compared to SMs who did not enroll in the ShipShape study (n = 38), ShipShape study participants were more likely to be female, less likely to be Hispanic, and had higher motivation and emotional eating scores. Compared to Navy HRBS respondents (n = 164), ShipShape study participants (n = 147) were younger, more likely to be female, non-Hispanic, enlisted, and obese. Further, ShipShape study participants reported significantly fewer medical health conditions but higher rates of probable depression, anxiety, and PTSD and were more likely to report receiving current mental health treatment than HRBS respondents. CONCLUSION: Overweight or obese SMs seeking weight loss in the ShipShape study were relatively young, female, non-Hispanic, motivated, but with greater emotional eating. ShipShape study participants endorsed few medical health conditions but had higher rates of probable mental health conditions compared to the HRBS sample. These findings suggest that SMs referred to Navy weight-management programs are likely experiencing comorbid mental health conditions which may interfere with the effectiveness of their weight loss efforts. The descriptive nature of this study and the focus on Navy SMs in only one ShipShape program may decrease the generalizability of our findings to participants at other locations. Nonetheless, these findings demonstrate the potential need for Navy weight-management programs that incorporate mental health treatment and address the specific needs of female and diverse SMs. A more comprehensive curriculum could improve the results of weight-management efforts, increase SM quality of life and fitness and thereby operational readiness.

Lessons Learned From Transition of an In-Person to a Virtual Randomized Controlled Trial for Weight and Fitness Concerns in Active-Duty Service Members: Survey Study.

BACKGROUND: This paper describes and discusses the transition of and modifications to a weight management randomized controlled trial among active-duty military personnel from an in-person to a virtual format as a result of the COVID-19 pandemic. The original pragmatic cohort-randomized controlled trial was designed to compare the effectiveness of an 8-week group weight management program, ShipShape, to a version of ShipShape enhanced with acceptance and commitment therapy. OBJECTIVE: The objective of our study was to assess potential differences between in-person and virtual participation in participants' demographics, motivation, confidence, credibility, expectations, and satisfaction with the interventions; we also examined the pragmatics of the technology and participants' experiences in virtual-format intervention groups. METHODS: A total of 178 active-duty personnel who had failed or were at risk of failing their physical fitness assessment or were overweight or obese were enrolled in the study. In-person (n=149) and virtual (n=29) participants reported demographics, motivation, confidence, credibility, expectations, and satisfaction. Interventionists recorded attendance and participation in the group sessions. Independent-sample 2-tailed t tests and chi-square tests were used to compare the characteristics of the in-person and virtual participants. Pragmatics of the technology and participants' experiences in the virtual format were assessed through surveys and open-ended questions. RESULTS: Participants were 29.7 (SD 6.9) years old on average, 61.8% (110/178) female, and 59.6% (106/178) White and had an average BMI of 33.1 (SD 3.9) kg/m2. Participants were highly motivated to participate and confident in their ability to complete a weight management program. A total of 82.6% (147/178) of all participants attended 5 of the 8 sessions, and participation was rated as "excellent" by interventionists in both formats. The interventions were found to be credible and to have adequate expectations for effectiveness and high satisfaction in both formats. There were no differences between in-person and virtual participants in any of these metrics, other than interventionist-rated participation, for which virtual participants had significantly higher ratings (P<.001). Technical satisfaction with the virtual sessions was rated as "good" to "very good," and participants were satisfied with the content of the virtual sessions. A word cloud of responses identified "mindfulness," "helpful," "different," "food," "binder," and "class" as concepts the virtual participants found most useful about the program. CONCLUSIONS: Modifications made in response to the COVID-19 pandemic were successful, given the recruitment of active-duty personnel with similar demographic characteristics, attendance levels, and indicators of credibility, expectancy, and satisfaction in the virtual format and the in-person format. This successful transition provides support for the use of virtual or digital weight management interventions to increase accessibility and reach among highly mobile active-duty personnel. TRIAL REGISTRATION: ClinicalTrials.gov NCT03029507; https://clinicaltrials.gov/ct2/show/NCT03029507.

Artificial neural networks enable genome-scale simulations of intracellular signaling.

Mammalian cells adapt their functional state in response to external signals in form of ligands that bind receptors on the cell-surface. Mechanistically, this involves signal-processing through a complex network of molecular interactions that govern transcription factor activity patterns. Computer simulations of the information flow through this network could help predict cellular responses in health and disease. Here we develop a recurrent neural network framework constrained by prior knowledge of the signaling network with ligand-concentrations as input and transcription factor-activity as output. Applied to synthetic data, it predicts unseen test-data (Pearson correlation r = 0.98) and the effects of gene knockouts (r = 0.8). We stimulate macrophages with 59 different ligands, with and without the addition of lipopolysaccharide, and collect transcriptomics data. The framework predicts this data under cross-validation (r = 0.8) and knockout simulations suggest a role for RIPK1 in modulating the lipopolysaccharide response. This work demonstrates the feasibility of genome-scale simulations of intracellular signaling.

Posttraumatic stress disorder symptom severity and functional impairment in the education domain.

To comprehensively understand and treat Posttraumatic Stress Disorder (PTSD), we need to accurately assess how PTSD symptoms affect people's daily functioning (e.g., in work, study, and relationships). However, the predominant use of self-report functional impairment measures-which are not validated against observable behavior-limits our understanding of this issue. To address this gap, we examined the relationship between posttraumatic stress (PTS) symptoms (including symptom clusters) and subjective and objective measures of functional impairment in the education domain. University students completed online self-report measures of educational impairment, PTS symptoms, intelligence and childhood trauma. We accessed participants' average grades at the end of the semester in which they participated. After controlling for IQ and childhood trauma, increased PTS symptoms were associated with both higher subjective educational impairment and lower Grade Point Average; this relationship was strongest for subjective global ratings of educational impairment, compared to educational impairment assessed according to specific examples. Our results suggest conceptual overlap between symptoms and impairment, and point to the benefit of using both objective and subjective modes of assessing impairment.

JAK inhibition in a patient with a STAT1 gain-of-function variant reveals STAT1 dysregulation as a common feature of aplastic anemia.

BACKGROUND: Idiopathic aplastic anemia is a potentially lethal disease, characterized by T cell-mediated autoimmune attack of bone marrow hematopoietic stem cells. Standard of care therapies (stem cell transplantation or immunosuppression) are effective but associated with a risk of serious toxicities. METHODS: An 18-year-old man presented with aplastic anemia in the context of a germline gain-of-function variant in STAT1. Treatment with the JAK1 inhibitor itacitinib resulted in a rapid resolution of aplastic anemia and a sustained recovery of hematopoiesis. Peripheral blood and bone marrow samples were compared before and after JAK1 inhibitor therapy. FINDINGS: Following therapy, samples showed a decrease in the plasma concentration of interferon-γ, a decrease in PD1-positive exhausted CD8+ T cell population, and a decrease in an interferon responsive myeloid population. Single-cell analysis of chromatin accessibility showed decreased accessibility of STAT1 across CD4+ and CD8+ T cells, as well as CD14+ monocytes. To query whether other cases of aplastic anemia share a similar STAT1-mediated pathophysiology, we examined a cohort of 9 patients with idiopathic aplastic anemia. Bone marrow from six of nine patients also displayed abnormal STAT1 hyper-activation. CONCLUSIONS: These findings raise the possibility that STAT1 hyperactivition defines a subset of idiopathic aplastic anemia patients for whom JAK inhibition may be an efficacious therapy. FUNDING: Funding was provided by the Massachusetts General Hospital Department of Medicine Pathways Program and NIH T32 AI007387. A trial registration is at https://clinicaltrials.gov/ct2/show/NCT03906318.

Multimodal profiling of lung granulomas in macaques reveals cellular correlates of tuberculosis control.

Mycobacterium tuberculosis lung infection results in a complex multicellular structure: the granuloma. In some granulomas, immune activity promotes bacterial clearance, but in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal positron emission tomography and computed tomography imaging, single-cell RNA sequencing, and measures of bacterial clearance. Bacterial persistence occurred in granulomas enriched for mast, endothelial, fibroblast, and plasma cells, signaling amongst themselves via type 2 immunity and wound-healing pathways. Granulomas that drove bacterial control were characterized by cellular ecosystems enriched for type 1-type 17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks involving diverse cell populations. Granulomas that arose later in infection displayed functional characteristics of restrictive granulomas and were more capable of killing Mtb. Our results define the complex multicellular ecosystems underlying (lack of) granuloma resolution and highlight host immune targets that can be leveraged to develop new vaccine and therapeutic strategies for TB.

Orgasm Frequency (Total Sexual Outlet) in a National American Sample.

There has been renewed interest in the conceptualization and diagnosis of conditions marked by excessive sexuality. Researchers and clinicians have often utilized orgasm frequency (e.g., total sexual outlet) as an indicator of hypersexuality. Indeed, some have proposed seven or more (7+) orgasms by any means in a typical week as indicating hypersexuality. Most studies utilizing this criterion, however, have examined clinical or judicial samples of men, as opposed to general population samples. The purpose of the current study was to provide representative population data of total sexual outlet (TSO) for people varying in age, relationship status, and sex, while also examining the impact of the phrasing of the questions (i.e., time frame). A total of 1029 participants were recruited online via a Qualtrics panel, consisting of 442 males and 587 females, from diverse regions across the USA. Results indicated that between 10.3 and 16.7% of the sample met the 7+ criterion for hypersexuality, with considerable variation by age, relationship status, sex, and less variation by wording of the question. Results are discussed in terms of the applicability of the 7+ cut-off for identifying elevated TSO. Results from this survey could be useful to researchers and clinicians looking for comparison data for their research and clinical assessment results.

Design for a cohort-randomized trial of an acceptance and commitment therapy-enhanced weight management and fitness program for Navy personnel.

Overweight/obesity and inadequate fitness in active duty personnel impact the wellbeing of service members and have significant costs for military readiness and budget. ShipShape (SS), the Navy's weight management program, was designed to promote nutritional, behavioral, and exercise education to service members. Although SS is an evidence-based program, about half of those who complete the program pass the Body Composition Assessment (BCA), one part of the Navy's comprehensive Physical Fitness Assessment (PFA). SS may not fully address underlying behavioral, psychological, and emotional barriers that influence poor eating and exercise habits. A novel solution to improve outcomes is to incorporate acceptance and commitment therapy (ACT) to promote mindful awareness of present moment experiences, improve psychological flexibility, and support commitment to behavior change. This paper describes a cohort-randomized controlled trial of ACT-enhanced SS (ACT + SS) compared to the standard SS-only program. Active duty service members referred to the SS program are randomized to receive 8-weekly ACT + SS or SS-only group interventions. Our aims are to: 1) determine the effectiveness of ACT + SS compared to SS-only; 2) examine psychological flexibility as a mechanism underlying intervention response; and 3) explore potential moderators of intervention response. The primary outcome is weight, one of the key components of the BCA; secondary outcomes include Body Mass Index (BMI), body fat %, self-reported BCA results, physical activity, problematic eating, and quality of life. We have designed a cohort-randomized trial with interventions that are pragmatically implemented in a real-life military setting, and outcomes that are immediately relevant to service members and leadership.

Identification of rfk-1, a Meiotic Driver Undergoing RNA Editing in Neurospora.

Sk-2 is a meiotic drive element that was discovered in wild populations of Neurospora fungi over 40 years ago. While early studies quickly determined that Sk-2 transmits itself through sexual reproduction in a biased manner via spore killing, the genetic factors responsible for this phenomenon have remained mostly unknown. Here, we identify and characterize rfk-1, a gene required for Sk-2-based spore killing. The rfk-1 gene contains four exons, three introns, and two stop codons, the first of which undergoes RNA editing to a tryptophan codon during sexual development. Translation of an unedited rfk-1 transcript in vegetative tissue is expected to produce a 102-amino acid protein, whereas translation of an edited rfk-1 transcript in sexual tissue is expected to produce a protein with 130 amino acids. These findings indicate that unedited and edited rfk-1 transcripts exist and that these transcripts could have different roles with respect to the mechanism of meiotic drive by spore killing. Regardless of RNA editing, spore killing only succeeds if rfk-1 transcripts avoid silencing caused by a genome defense process called meiotic silencing by unpaired DNA (MSUD). We show that rfk-1's MSUD avoidance mechanism is linked to the genomic landscape surrounding the rfk-1 gene, which is located near the Sk-2 border on the right arm of chromosome III. In addition to demonstrating that the location of rfk-1 is critical to spore-killing success, our results add to accumulating evidence that MSUD helps protect Neurospora genomes from complex meiotic drive elements.

Uncovering complex molecular networks in host-pathogen interactions using systems biology.

Interactions between pathogens and their hosts can induce complex changes in both host and pathogen states to privilege pathogen survival or host clearance of the pathogen. To determine the consequences of specific host-pathogen interactions, a variety of techniques in microbiology, cell biology, and immunology are available to researchers. Systems biology that enables unbiased measurements of transcriptomes, proteomes, and other biomolecules has become increasingly common in the study of host-pathogen interactions. These approaches can be used to generate novel hypotheses or to characterize the effects of particular perturbations across an entire biomolecular network. With proper experimental design and complementary data analysis tools, high-throughput omics techniques can provide novel insights into the mechanisms that underlie processes from phagocytosis to pathogen immune evasion. Here, we provide an overview of the suite of biochemical approaches for high-throughput analyses of host-pathogen interactions, analytical frameworks for understanding the resulting datasets, and a vision for the future of this exciting field.

Are Navy Weight Management Programs Ensuring Sailor Physical Readiness? An Analysis at Naval Medical Center San Diego.

INTRODUCTION: The obesity epidemic in the USA includes active duty service members in the military and effects physical readiness. At the Naval Medical Center San Diego command, the Health & Wellness Department is charged with administering the Weight Management Programs (WMP) for sailors in the San Diego area to ensure military physical readiness requirements. The optimal allocation of personnel and resources to manage these programs is paramount for mission success. We analyzed the cost and effectiveness of the WMPs for the active duty population stationed at Naval Medical Center San Diego (NMCSD) with the intent of offering potential recommendations for program optimization. METHODS: As an approved quality improvement program, the cost and effectiveness of the WMP, namely Fitness Enhancement Program (FEP) and ShipShape (SS), for the active duty population stationed at NMCSD were analyzed from 2013 to 2014 by utilizing various official sources. Data analysis included reviewing historical data for trends and developing a budgetary analysis to include Direct Labor Hour rates and opportunity costs. Interviews were conducted with key staff and participants in the WMPs to determine essential aspects and potential beneficial changes to the programs. Overall results were evaluated to identify potential opportunities for program expansion and improvement. RESULTS: Data analysis revealed that the FEP is producing a 78% success rate, with approximately 30% of the enrolled personnel actively participating. Concurrently, the SS program at NMCSD is producing a 71% success rate with 90% course completion rate. This success rate is significantly higher than the national SS average of 34%. Furthermore, our cost analysis revealed that the SS program a significantly higher return on investment. Interviews conducted of key staff and participants yielded several commonalities regarding key factors involved with WMPs success or needed improvements. CONCLUSIONS: To improve the WMPs at NMCSD, the findings in this report support the following recommendations: (1) maximize the SS program, (2) increase utilization of FEP, and (3) increase the participation and training of Assistant Command Fitness Leaders. WMPs navy-wide may benefit from incorporating similar program improvements to increase physical readiness of service members and, therefore, support command mission success.