Spatially organized multicellular immune hubs in human colorectal cancer.

Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd individuals. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors. To discover hubs of interacting malignant and immune cells, we identified expression programs in different cell types that co-varied across tumors from affected individuals and used spatial profiling to localize coordinated programs. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage and an MMRd-enriched immune hub within the tumor, with activated T cells together with malignant and myeloid cells expressing T cell-attracting chemokines. By identifying interacting cellular programs, we reveal the logic underlying spatially organized immune-malignant cell networks.

Meta-research: How many diagnostic or prognostic models published in radiological journals are evaluated externally?

OBJECTIVES: Prognostic and diagnostic models must work in their intended clinical setting, proven via "external evaluation", preferably by authors uninvolved with model development. By systematic review, we determined the proportion of models published in high-impact radiological journals that are evaluated subsequently. METHODS: We hand-searched three radiological journals for multivariable diagnostic/prognostic models 2013-2015 inclusive, developed using regression. We assessed completeness of data presentation to allow subsequent external evaluation. We then searched literature to August 2022 to identify external evaluations of these index models. RESULTS: We identified 98 index studies (73 prognostic; 25 diagnostic) describing 145 models. Only 15 (15%) index studies presented an evaluation (two external). No model was updated. Only 20 (20%) studies presented a model equation. Just 7 (15%) studies developing Cox models presented a risk table, and just 4 (9%) presented the baseline hazard. Two (4%) studies developing non-Cox models presented the intercept. Just 20 (20%) articles presented a Kaplan-Meier curve of the final model. The 98 index studies attracted 4224 citations (including 559 self-citations), median 28 per study. We identified just six (6%) subsequent external evaluations of an index model, five of which were external evaluations by researchers uninvolved with model development, and from a different institution. CONCLUSIONS: Very few prognostic or diagnostic models published in radiological literature are evaluated externally, suggesting wasted research effort and resources. Authors' published models should present data sufficient to allow external evaluation by others. To achieve clinical utility, researchers should concentrate on model evaluation and updating rather than continual redevelopment. CLINICAL RELEVANCE STATEMENT: The large majority of prognostic and diagnostic models published in high-impact radiological journals are never evaluated. It would be more efficient for researchers to evaluate existing models rather than practice continual redevelopment. KEY POINTS: • Systematic review of highly cited radiological literature identified few diagnostic or prognostic models that were evaluated subsequently by researchers uninvolved with the original model. • Published radiological models frequently omit important information necessary for others to perform an external evaluation: Only 20% of studies presented a model equation or nomogram. • A large proportion of research citing published models focuses on redevelopment and ignores evaluation and updating, which would be a more efficient use of research resources.

Morphologic and molecular analysis of liver injury after SARS-CoV-2 vaccination reveals distinct characteristics.

BACKGROUND & AIMS: Liver injury after COVID-19 vaccination is very rare and shows clinical and histomorphological similarities with autoimmune hepatitis (AIH). Little is known about the pathophysiology of COVID-19 vaccine-induced liver injury (VILI) and its relationship to AIH. Therefore, we compared VILI with AIH. METHODS: Formalin-fixed and paraffin-embedded liver biopsy samples from patients with VILI (n = 6) and from patients with an initial diagnosis of AIH (n = 9) were included. Both cohorts were compared by histomorphological evaluation, whole-transcriptome and spatial transcriptome sequencing, multiplex immunofluorescence, and immune repertoire sequencing. RESULTS: Histomorphology was similar in both cohorts but showed more pronounced centrilobular necrosis in VILI. Gene expression profiling showed that mitochondrial metabolism and oxidative stress-related pathways were more and interferon response pathways were less enriched in VILI. Multiplex analysis revealed that inflammation in VILI was dominated by CD8+ effector T cells, similar to drug-induced autoimmune-like hepatitis. In contrast, AIH showed a dominance of CD4+ effector T cells and CD79a+ B and plasma cells. T-cell receptor (TCR) and B-cell receptor sequencing showed that T and B cell clones were more dominant in VILI than in AIH. In addition, many T cell clones detected in the liver were also found in the blood. Interestingly, analysis of TCR beta chain and Ig heavy chain variable-joining gene usage further showed that TRBV6-1, TRBV5-1, TRBV7-6, and IgHV1-24 genes are used differently in VILI than in AIH. CONCLUSIONS: Our analyses support that SARS-CoV-2 VILI is related to AIH but also shows distinct differences from AIH in histomorphology, pathway activation, cellular immune infiltrates, and TCR usage. Therefore, VILI may be a separate entity, which is distinct from AIH and more closely related to drug-induced autoimmune-like hepatitis. IMPACT AND IMPLICATIONS: Little is known about the pathophysiology of COVID-19 vaccine-induced liver injury (VILI). Our analysis shows that COVID-19 VILI shares some similarities with autoimmune hepatitis, but also has distinct differences such as increased activation of metabolic pathways, a more prominent CD8+ T cell infiltrate, and an oligoclonal T and B cell response. Our findings suggest that VILI is a distinct disease entity. Therefore, there is a good chance that many patients with COVID-19 VILI will recover completely and will not develop long-term autoimmune hepatitis.

ViReMaShiny: an interactive application for analysis of viral recombination data.

MOTIVATION: Recombination is an essential driver of virus evolution and adaption, giving rise to new chimeric viruses, structural variants, sub-genomic RNAs and defective RNAs. Next-generation sequencing (NGS) of virus samples, either from experimental or clinical settings, has revealed a complex distribution of recombination events that contributes to intrahost diversity. We and others have previously developed alignment tools to discover and map these diverse recombination events in NGS data. However, there is no standard for data visualization to contextualize events of interest, and downstream analysis often requires bespoke coding. RESULTS: We present ViReMaShiny, a web-based application built using the R Shiny framework to allow interactive exploration and point-and-click visualization of viral recombination data provided in BED format generated by computational pipelines such as ViReMa (Viral-Recombination-Mapper). AVAILABILITY AND IMPLEMENTATION: The application is hosted at https://routhlab.shinyapps.io/ViReMaShiny/ with associated documentation at https://jayeung12.github.io/. Code is available at https://github.com/routhlab/ViReMaShiny. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Next-Generation Sequencing and Image-Guided Tissue Sampling: A Primer for Interventional Radiologists.

The discovery of increasing numbers of actionable molecular and gene targets for cancer treatment has driven the demand for tissue sampling for next-generation sequencing (NGS). Requirements for sequencing can be very specific, and inadequate sampling leads to delays in management and decision making. It is important that interventional radiologists are aware of NGS technologies and their common applications and be cognizant of the factors that contribute to successful sample sequencing. This review summarizes the fundamentals of cancer tissue collection and processing for NGS. It elaborates on sequencing technologies and their applications with the aim of providing readers with a working understanding that can enhance their clinical practice. It then describes imaging, tumor, biopsy, and sample collection factors that improve the chances of NGS success. Finally, it discusses future practice, highlighting the problem of undersampling in both clinical and research settings and the opportunities within interventional radiology to address this.

Thrombus Distribution in Vaccine-induced Immune Thrombotic Thrombocytopenia after ChAdOx1 nCov-19 Vaccination.

Vaccination strategies have been at the forefront of controlling the COVID-19 pandemic. An association between vaccine-induced immune thrombotic thrombocytopenia (VITT) and one of these vaccines, the ChAdOx1 nCov-19 vaccine, is now recognized. The purpose of this study was to investigate the frequency and location of thrombosis in each vascular system using CT, MRI, and US to identify additional sites of thrombus in a United Kingdom-wide sample of patients with confirmed VITT. Thirty-two radiology centers identified through the national collaborative Radiology Academic Network for Trainees were invited from the United Kingdom; seven of these contributed to this study. All patients with confirmed VITT ¬between February 3 and May 12, 2021, who met the inclusion criteria were included. The location and extent of thrombi were evaluated using CT, MRI, and US. A total of 40 patients (median age, 41 years [IQR, 32-52]; 22 [55%] men) with confirmed vaccine-induced immune thrombotic thrombocytopenia after administration of their first ChAdOx1 nCov-19 vaccine were included. Thirty-two patients (80%) developed symptoms within the first 14 days, and eight (20%) developed symptoms within 14-28 days. Twenty-nine patients (72%) experienced neurologic symptoms and were confirmed to have cerebral venous sinus thrombosis, 12 (30%) had clinical deterioration and repeat imaging demonstrated extension of their primary thrombus, and eight (20%) died. Twenty-five of 30 patients (83%) who underwent additional imaging had occult thrombosis. In conclusion, patients with VITT are likely to have multiple sites of thrombosis, with the most frequent being cerebral venous sinus thrombosis in combination with pulmonary embolism and portomesenteric venous thrombosis. Whole-body imaging with contrast-enhanced CT can be used to identify occult thrombosis.

Patterns of Herpes Simplex Virus 1 Infection in Neural Progenitor Cells.

Herpes simplex virus 1 (HSV-1) can induce damage in brain regions that include the hippocampus and associated limbic structures. These neurogenic niches are important because they are associated with memory formation and are highly enriched with neural progenitor cells (NPCs). The susceptibility and fate of HSV-1-infected NPCs are largely unexplored. We differentiated human induced pluripotent stem cells (hiPSCs) into NPCs to generate two-dimensional (2D) and three-dimensional (3D) culture models to examine the interaction of HSV-1 with NPCs. Here, we show that (i) NPCs can be efficiently infected by HSV-1, but infection does not result in cell death of most NPCs, even at high multiplicities of infection (MOIs); (ii) limited HSV-1 replication and gene expression can be detected in the infected NPCs; (iii) a viral silencing mechanism is established in NPCs exposed to the antivirals (E)-5-(2-bromovinyl)-2'-deoxyuridine (5BVdU) and alpha interferon (IFN-α) and when the antivirals are removed, spontaneous reactivation can occur at low frequency; (iv) HSV-1 impairs the ability of NPCs to migrate in a dose-dependent fashion in the presence of 5BVdU plus IFN-α; and (v) 3D cultures of NPCs are less susceptible to HSV-1 infection than 2D cultures. These results suggest that NPC pools could be sites of HSV-1 reactivation in the central nervous system (CNS). Finally, our results highlight the potential value of hiPSC-derived 3D cultures to model HSV-1-NPC interaction.IMPORTANCE This study employed human induced pluripotent stem cells (hiPSCs) to model the interaction of HSV-1 with NPCs, which reside in the neurogenic niches of the CNS and play a fundamental role in adult neurogenesis. Herein, we provide evidence that in NPCs infected at an MOI as low as 0.001, HSV-1 can establish a latent state, suggesting that (i) a variant of classical HSV-1 latency can be established during earlier stages of neuronal differentiation and (ii) neurogenic niches in the brain may constitute additional sites of viral reactivation. Lytic HSV-1 infections impaired NPC migration, which represents a critical step in neurogenesis. A difference in susceptibility to HSV-1 infection between two-dimensional (2D) and three-dimensional (3D) NPC cultures was observed, highlighting the potential value of 3D cultures for modeling host-pathogen interactions. Together, our results are relevant in light of observations relating HSV-1 infection to postencephalitic cognitive dysfunction.

Label-Free Quantitative Proteomics Distinguishes General and Site-Specific Host Responses to Pseudomonas aeruginosa Infection at the Ocular Surface.

Mass spectrometry-based proteomics enables the unbiased and sensitive profiling of cellular proteomes and extracellular environments. Recent technological and bioinformatic advances permit identifying dual biological systems in a single experiment, supporting investigation of infection from both the host and pathogen perspectives. At the ocular surface, Pseudomonas aeruginosa is commonly associated with biofilm formation and inflammation of the ocular tissues, causing damage to the eye. The interaction between P. aeruginosa and the immune system at the site of infection describes limitations in clearance of infection and enhanced pathogenesis. Here, the extracellular environment (eye wash) of murine ocular surfaces infected with a clinical isolate of P. aeruginosa is profiled and neutrophil marker proteins are detected, indicating neutrophil recruitment to the site of infection. The first potential diagnostic markers of P. aeruginosa-associated keratitis are also identified. In addition, the deepest murine corneal proteome to date is defined and proteins, categories, and networks critical to the host response are detected. Moreover, the first identification of bacterial proteins attached to the ocular surface is reported. The findings are validated through in silico comparisons and enzymatic profiling. Overall, the work provides comprehensive profiling of the host-pathogen interface and uncovers differences between general and site-specific host responses to infection.

Amyloid-beta1-42 induced glutamatergic receptor and transporter expression changes in the mouse hippocampus.

Alzheimer's disease (AD) is the leading type of dementia worldwide. With an increasing burden of an aging population coupled with the lack of any foreseeable cure, AD warrants the current intense research effort on the toxic effects of an increased concentration of beta-amyloid (Aß) in the brain. Glutamate is the main excitatory brain neurotransmitter and it plays an essential role in the function and health of neurons and neuronal excitability. While previous studies have shown alterations in expression of glutamatergic signaling components in AD, the underlying mechanisms of these changes are not well understood. This is the first comprehensive anatomical study to characterize the subregion- and cell layer-specific long-term effect of Aß1-42 on the expression of specific glutamate receptors and transporters in the mouse hippocampus, using immunohistochemistry with confocal microscopy. Outcomes are examined 30 days after Aß1-42 stereotactic injection in aged male C57BL/6 mice. We report significant decreases in density of the glutamate receptor subunit GluA1 and the vesicular glutamate transporter (VGluT) 1 in the conus ammonis 1 region of the hippocampus in the Aß1-42 injected mice compared with artificial cerebrospinal fluid injected and naïve controls, notably in the stratum oriens and stratum radiatum. GluA1 subunit density also decreased within the dentate gyrus dorsal stratum moleculare in Aß1-42 injected mice compared with artificial cerebrospinal fluid injected controls. These changes are consistent with findings previously reported in the human AD hippocampus. By contrast, glutamate receptor subunits GluA2, GluN1, GluN2A, and VGluT2 showed no changes in expression. These findings indicate that Aß1-42 induces brain region and layer specific expression changes of the glutamatergic receptors and transporters, suggesting complex and spatial vulnerability of this pathway during development of AD neuropathology. Read the Editorial Highlight for this article on page 7. Cover Image for this issue: https://doi.org/10.1111/jnc.14763.

The Association Between Common Clinical Characteristics and Postoperative Morbidity and Overall Survival in Patients with Glioblastoma.

BACKGROUND: The impact of noncancerous factors on the morbidity and mortality of glioblastoma multiforme (GBM) has not been well studied. Using a large surgical cohort, we examined the association between multiple clinical characteristics and postoperative morbidities and survival in patients with GBM. MATERIALS AND METHODS: The study included 404 consecutive GBM patients who underwent initial tumor resection at MD Anderson Cancer Center between January 1, 2010, and December 31, 2014. Data about clinical characteristics, treatments, and postoperative complications were collected. The associations between clinical parameters and postoperative complications and survival were analyzed. RESULTS: Charlson Comorbidity Index was positively related to a higher incidence of postoperative total (odds ratio [OR] = 1.20; p = .002) and neurological (OR = 1.18; p = .011) complications. Preoperative systolic blood pressure (SBp) over 140 mmHg was associated with a higher incidence of postoperative intracranial hemorrhage (OR = 4.42; p = .039) and longer hospital stay (OR = 2.48; p = .015). Greater postoperative fluctuation of SBp (OR = 1.14; p = .025) and blood glucose (mmol/L; OR = 1.48; p = .023) were related to a higher incidence of neurological complications, whereas higher postoperative blood glucose (OR = 0.64; p < .001) was related to a lower incidence. Long-term lower SBp (<124 mmHg; hazard ratio [HR] = 1.47; p = .010) and higher blood glucose (HR = 1.12; p < .001) were associated with shorter survival. Long-term serum albumin level (g/dL; HR = 0.32; p < .001) was positively associated with survival. CONCLUSION: Short-term SBp and blood glucose levels and fluctuations are associated with postoperative complications in GBM patients. Their long-term optimization may impact survival of these patients. Future clinical trials are needed to confirm the benefit of optimizing medical comorbidities on GBM patients' outcomes. IMPLICATIONS FOR PRACTICE: Glioblastoma multiforme (GBM) is one of the most feared cancer diagnoses because of its limited survival and treatment. This study revealed significant associations of noncancerous factors on the morbidity and mortality of GBM. The complexity of medical comorbidities, as well as short-term postoperative levels and fluctuations of blood pressure and blood glucose, was associated with postoperative complications, but not overall survival. However, long-term levels of these common clinical parameters were significantly associated with survival. Optimization of medical conditions may be critical for reducing the morbidity and mortality of GBM patients. Future clinical trials are needed to validate the observed associations in an independent cohort.

Initial validation of equilibrium contrast imaging for extracellular volume quantification using a three-dimensional engineered tissue model.

BACKGROUND: To test the principles underpinning equilibrium contrast imaging estimation of tissue extracellular volume (ECV) fraction, using a three-dimensional (3D) engineered tissue model with known cellular and extracellular volumes. METHODS: Six 3D tissue models (tumoroids) consisting of cell cultures within a collagen containing hydrogel were constructed after culture centrifugation and direct measurement of the cell component volume. Measured tumoroid ECV ranged from 0.89 to 1. ECV was calculated after measuring the T1 relaxation time at 3 Tesla using inversion recovery relaxometry (TI 100-1500 ms) within the tumoroids and surrounding medium before and 375 min after spiking the medium with Gadolinium (to achieve a concentration of 1.4 mM/L). Linear regression model prediction of directly measured ECV (ECVm ) by EQ-MRI measured ECV (ECVeq ); and Bland-Altman agreement between measures was assessed. RESULTS: The fractional cellular volume measured by EQ-MRI (ECVeq ) within the tumoroids ranged from 0.821 to 0.963. ECVeq was a good predictor of ECVm (R2 = 0.77, P = 0.02). The regression line Y-axis intercept (when X = 0) was 0.045 ± 0.019 with a slope of 1.28 ± 0.35. Bland-Altman comparison demonstrated 95% limits of agreement between -0.002 and 0.114 with a bias (SD) of 0.056 (0.03). CONCLUSION: This study supports the principles of ECV estimation using equilibrium contrast MRI, but future development of this model may allow validation over a wider, more physiological ECV range and a greater understanding of the effect of tissue extracellular protein burden on ECV.

Coordination chemistry and FLP reactivity of 1,1- and 1,2-bis-boranes.

Coordination chemistry and frustrated Lewis pair (FLP) chemistry have been most commonly studied using monodentate Lewis acids. In this paper, we examine the corresponding reactions employing the 1,1- and 1,2-bis-boranes, PhCH2CH(B(C6F5)2)21 and Me3SiCH(B(C6F5)2)CH2B(C6F5)22, respectively. Coordination of isocyanide to these species results in the formation of the products RCH(B(C6F5)2CNtBu)CH2(B(C6F5)2CNtBu) (R = Ph 3, Me3Si 4). The rearrangement of 1 to give the 1,2-bis-borane adduct 3 was probed and attributed to a donor-induced retrohydroboration and subsequent hydroboration. The analogous reaction of 1 is evident in efforts to use the Gutman-Beckett method to assess its Lewis acidity. However, in combination with tBu3P, bis-boranes 1 and 2 form FLPs and react with H2 to give [tBu3PH][PhCH2CH(B(C6F5)2)2(µ-H)] 5a and [tBu3PH][Me3SiCH(B(C6F5)2)CH2(B(C6F5)2)(µ-H)] 6, respectively. Reactions of 1 and 2 with various donors and PhCCH were shown to give deprotonation and addition products, depending on the nature of the base. However, in the case of 1, products resulting from retrohydroboration, and subsequent hydroboration are evident. Several of these alkyne products are crystallographically characterized.

Alzheimer's Disease-associated Region-specific Decrease of Vesicular Glutamate Transporter Immunoreactivity inthe Medial Temporal Lobe and Superior Temporal Gyrus.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder for which there are very limited treatment options. Dysfunction of the excitatory neurotransmitter system is thought to play a major role in the pathogenesis of this condition. Vesicular glutamate transporters (VGLUTs) are key to controlling the quantal release of glutamate. Thus, expressional changes in disease can have implications for aberrant neuronal activity, raising the possibility of a therapeutic target. There is no information regarding the expression of VGLUTs in the human medial temporal lobe in AD, one of the earliest and most severely affected brain regions. This study aimed to quantify and compare the layer-specific expression of VGLUT1 and VGLUT2 between control and AD cases in the hippocampus, subiculum, entorhinal cortex, and superior temporal gyrus. Free-floating fluorescent immunohistochemistry was used to label VGLUT1 and VGLUT2 in the hippocampus, subiculum, entorhinal cortex, and superior temporal gyrus. Sections were imaged using laser-scanning confocal microscopy and transporter densitometric analysis was performed. VGLUT1 density was not significantly different in AD tissue, except lower staining density observed in the dentate gyrus stratum moleculare (p = 0.0051). VGLUT2 expression was not altered in the hippocampus and entorhinal cortex of AD cases but was significantly lower in the subiculum (p = 0.015) and superior temporal gyrus (p = 0.0023). This study indicates a regionally specific vulnerability of VGLUT1 and VGLUT2 expression in the medial temporal lobe and superior temporal gyrus in AD. However, the causes and functional consequences of these disturbances need to be further explored to assess VGLUT1 and VGLUT2 as viable therapeutic targets.

A Spatial Multi-Modal Dissection of Host-Microbiome Interactions within the Colitis Tissue Microenvironment.

The intricate and dynamic interactions between the host immune system and its microbiome constituents undergo dynamic shifts in response to perturbations to the intestinal tissue environment. Our ability to study these events on the systems level is significantly limited by in situ approaches capable of generating simultaneous insights from both host and microbial communities. Here, we introduce Microbiome Cartography (MicroCart), a framework for simultaneous in situ probing of host features and its microbiome across multiple spatial modalities. We demonstrate MicroCart by comprehensively investigating the alterations in both gut host and microbiome components in a murine model of colitis by coupling MicroCart with spatial proteomics, transcriptomics, and glycomics platforms. Our findings reveal a global but systematic transformation in tissue immune responses, encompassing tissue-level remodeling in response to host immune and epithelial cell state perturbations, and bacterial population shifts, localized inflammatory responses, and metabolic process alterations during colitis. MicroCart enables a deep investigation of the intricate interplay between the host tissue and its microbiome with spatial multiomics.

Cross-domain information fusion for enhanced cell population delineation in single-cell spatial-omics data.

Cell population delineation and identification is an essential step in single-cell and spatial-omics studies. Spatial-omics technologies can simultaneously measure information from three complementary domains related to this task: expression levels of a panel of molecular biomarkers at single-cell resolution, relative positions of cells, and images of tissue sections, but existing computational methods for performing this task on single-cell spatial-omics datasets often relinquish information from one or more domains. The additional reliance on the availability of "atlas" training or reference datasets limits cell type discovery to well-defined but limited cell population labels, thus posing major challenges for using these methods in practice. Successful integration of all three domains presents an opportunity for uncovering cell populations that are functionally stratified by their spatial contexts at cellular and tissue levels: the key motivation for employing spatial-omics technologies in the first place. In this work, we introduce Cell Spatio- and Neighborhood-informed Annotation and Patterning (CellSNAP), a self-supervised computational method that learns a representation vector for each cell in tissue samples measured by spatial-omics technologies at the single-cell or finer resolution. The learned representation vector fuses information about the corresponding cell across all three aforementioned domains. By applying CellSNAP to datasets spanning both spatial proteomic and spatial transcriptomic modalities, and across different tissue types and disease settings, we show that CellSNAP markedly enhances de novo discovery of biologically relevant cell populations at fine granularity, beyond current approaches, by fully integrating cells' molecular profiles with cellular neighborhood and tissue image information.

MAPS: pathologist-level cell type annotation from tissue images through machine learning.

Highly multiplexed protein imaging is emerging as a potent technique for analyzing protein distribution within cells and tissues in their native context. However, existing cell annotation methods utilizing high-plex spatial proteomics data are resource intensive and necessitate iterative expert input, thereby constraining their scalability and practicality for extensive datasets. We introduce MAPS (Machine learning for Analysis of Proteomics in Spatial biology), a machine learning approach facilitating rapid and precise cell type identification with human-level accuracy from spatial proteomics data. Validated on multiple in-house and publicly available MIBI and CODEX datasets, MAPS outperforms current annotation techniques in terms of speed and accuracy, achieving pathologist-level precision even for typically challenging cell types, including tumor cells of immune origin. By democratizing rapidly deployable and scalable machine learning annotation, MAPS holds significant potential to expedite advances in tissue biology and disease comprehension.

Epstein-Barr Virus Orchestrates Spatial Reorganization and Immunomodulation within the Classic Hodgkin Lymphoma Tumor Microenvironment.

Classic Hodgkin Lymphoma (cHL) is a tumor composed of rare malignant Hodgkin and Reed-Sternberg (HRS) cells nested within a T-cell rich inflammatory immune infiltrate. cHL is associated with Epstein-Barr Virus (EBV) in 25% of cases. The specific contributions of EBV to the pathogenesis of cHL remain largely unknown, in part due to technical barriers in dissecting the tumor microenvironment (TME) in high detail. Herein, we applied multiplexed ion beam imaging (MIBI) spatial pro-teomics on 6 EBV-positive and 14 EBV-negative cHL samples. We identify key TME features that distinguish between EBV-positive and EBV-negative cHL, including the relative predominance of memory CD8 T cells and increased T-cell dysfunction as a function of spatial proximity to HRS cells. Building upon a larger multi-institutional cohort of 22 EBV-positive and 24 EBV-negative cHL samples, we orthogonally validated our findings through a spatial multi-omics approach, coupling whole transcriptome capture with antibody-defined cell types for tu-mor and T-cell populations within the cHL TME. We delineate contrasting transcriptomic immunological signatures between EBV-positive and EBV-negative cases that differently impact HRS cell proliferation, tumor-immune interactions, and mecha-nisms of T-cell dysregulation and dysfunction. Our multi-modal framework enabled a comprehensive dissection of EBV-linked reorganization and immune evasion within the cHL TME, and highlighted the need to elucidate the cellular and molecular fac-tors of virus-associated tumors, with potential for targeted therapeutic strategies.

Improvement of mucosal immunity by a live-attenuated SARS-CoV-2 nasal vaccine.

The effectiveness of early COVID-19 vaccines in reducing the severity of the disease has led to a focus on developing next-generation vaccines that can prevent infection and transmission of the virus. One promising approach involves the induction of mucosal immunity through nasal administration and a variety of mucosal vaccine candidates using different platforms are currently in development. Live-attenuated viruses, less pathogenic versions of SARS-CoV-2, have promising features as a mucosal vaccine platform and have the potential to induce hybrid immunity in individuals who have already received mRNA vaccines. This review discusses the potential benefits and considerations for the use of live-attenuated SARS-CoV-2 intranasal vaccines and highlights the authors' work in developing such a vaccine platform.

Mutations in SARS-CoV-2 variant nsp6 enhance type-I interferon antagonism.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve after its emergence. Given its importance in viral infection and vaccine development, mutations in the viral Spike gene have been studied extensively; however, the impact of mutations outside the Spike gene are poorly understood. Here, we report that a triple deletion (ΔSGF or ΔLSG) in nonstructural protein 6 (nsp6) independently acquired in Alpha and Omicron sublineages of SARS-CoV-2 augments nsp6-mediated antagonism of type-I interferon (IFN-I) signaling. Specifically, these triple deletions enhance the ability of mutant nsp6 to suppress phosphorylation of STAT1 and STAT2. A parental SARS-CoV-2 USA-WA1/2020 strain containing the nsp6 ΔSGF deletion (ΔSGF-WA1) shows reduced susceptibility to IFN-I treatment in vitro, outcompetes the parental strain in human primary airway cultures, and increases virulence in mice; however, the ΔSGF-WA1 virus is less virulent than the Alpha variant (which has the nsp6 ΔSGF deletion and additional mutations in other genes). Analyses of host responses from ΔSGF-WA1-infected mice and primary airway cultures reveal activation of pathways indicative of a cytokine storm. These results provide evidence that mutations outside the Spike protein affect virus-host interactions and may alter pathogenesis of SARS-CoV-2 variants in humans.