Both semi-invariant natural killer T (NKT) and mucosal associated invariant T (MAIT) cells are enriched at the lung mucosa but their roles in respiratory diseases are not fully understood. Pulmonary tularemia, caused by Francisella tularensis (Ft) is associated with most severe form of the disease with significantly enhanced mortality. We have found a pro-inflammatory role for NKT cells in tularemia-like disease caused by intranasal (i.n.) LVS infection of C57BL/6 (B6) mice. By contrast, NKT cell deficient CD1d-/- mice were protected from tularemia-like disease caused by i.n. LVS infection. Protected CD1d-/- mice showed increased iBALT structures in the lungs which were absent in NKT-cell sufficient B6 mice. While IL-17 is known to promote iBALT formation, Tregs are known to suppress them. We have found significantly enhanced IL-17 producing MAIT17 cells and reduced Treg numbers in lungs of CD1d-/- mice when compared to B6 mice 7 days post inoculation (dpi) with LVS. In a preliminary experiment we found that adoptive transfer of LVS-activated MAIT cells sorted from the lungs of infected CD1d-/- mice protected Rag2-/- recipients from developing tularemia-like disease while splenic MAIT cells did not. Lung MAIT cells are enriched in MAIT17 in comparison to splenic MAIT cells, which are enriched in interferon (IFN)-g producing MAIT1 cells. Thus, enhanced protection in CD1d-/- mice compared to B6 mice is due to MAIT17-cell-associated qualitative and quantitative differences. Supported by VA merit awards BX001444 & BX001610 and BX000915; NIH grants AI137082, AI139046 and HL136664. GDO and NUC were supported by T32GM007347 and NUC by F30HL159941. Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Early immune dynamics during the initiation of fatal tularemia caused by Francisella tularensis infection remain unknown. Unto that end, we generated a transcriptomic map at single-cell resolution of the innate- like lymphocyte responses to F. tularensis live vaccine strain (LVS) infection of mice. We found that both interferon-g (IFN-g)-producing type 1 and interleukin-17 (IL-17)-producing type 3 innate-like lymphocytes expanded in the infected lungs. Natural killer (NK) and NKT cells drove the type 1 response, whereas mucosal-associated invariant T (MAIT) and gd T cells drove the type 3 response. Furthermore, tularemia- like disease resistant NKT cell-deficient, Cd1d-'- mice accumulated more MAIT1 cells, MAIT17 cells, and cells with a hybrid phenotype between MAIT1 and MAIT17 cells than wild-type mice. Critically, adoptive transfer of LVS-activated MAIT cells from Cd1d-'- mice, which were enriched in MAIT17 cells, was sufficient to protect LVS-susceptible, immunodeficient RAG2-/- mice from severe LVS infection-inflicted pathology. Collectively, our findings position MAIT cells as potential mediators of IL-17-dependent protection from pulmonary tularemia-like disease.

Abstract Breast cancer is the most common cancer diagnosis worldwide, and an unfortunately high number of women die every year due to a lack of effective therapeutic strategies for metastatic breast cancer. Patients with brain metastases in particular have a significantly poorer prognosis than women with metastasis to other sites (e.g., lung, liver, bone). Previous studies by our group identified a ligand-receptor complex in the tumor microenvironment (TME) that promotes metastasis of breast cancer to the brain. This interaction between platelet-derived growth factor-BB (PDGFB), produced by cancerous breast epithelial cells, and its receptor, platelet-derived growth factor receptor beta (PDGFRβ), expressed by mesenchymal cells, is also prognostic of breast cancer metastasis to the brain in patients. The mechanism of how the PDGFB-PDGFRβ pathway mediates brain metastasis is still unclear and is the focus of our current work. To further analyze the biological effects of PDGFB, our group evaluated gene expression changes between primary human breast tumors expressing high PDGFB versus those expressing low PDGFB and found significantly more immune signaling in the low-expressing tumors. These results led us to hypothesize that the PDGFB-PDGFRβ pathway creates an immunosuppressive microenvironment, allowing for increased metastasis of cancerous cells. To test this, flow cytometry was completed on the brains, spleens, lungs, and livers of control mice and mutant mice expressing stromal-specific hyperactive PDGFRβ following intracardiac injection of PDGFB-expressing mammary tumor cells. No significant changes in immune cell populations were observed in non-tumor-bearing mice. However, in the brains of tumor-bearing mutant mice, there are significant changes to the myeloid lineage. Specifically, analysis at day 10 post-injection (prior to detectable metastatic lesions) revealed diminished CD45+CD11b+ populations. Segregating by CD45hi/CD45lo confirmed a profound decrease in CD45loCD11b+F4/80+ microglia, likely a tumor-suppressive population. Looking at day 16 post-injection (detectable metastatic lesions), there is a shift towards an increase in CD45+ CD11b+GR1+ infiltrating myeloid-derived suppressor cells (MDSCs) in experimental mice, likely a tumor-promoting population. Minimal changes were observed in the lungs and livers at any time point. Confirmatory immunofluorescence staining showed a decrease in tumor-infiltrating microglia (Iba1+) and a significant increase in tumor-infiltrating peripheral macrophages (F4/80+), mirroring the flow data. This experiment was then repeated to test the contribution of the ligand (PDGFB), where a significant increase of infiltrating microglia (Iba1+) was observed upon tumor cell knockdown of PDGFB. Together, these results suggest that the PDGFB-PDGFRβ pathway creates an immunosuppressive environment in the brain TME at least in part through reorchestration of the myeloid populations. Citation Format: Alexis A Mossing, Johnathon G Schiebel, Rebecca L Packard, Amrendra Kumar, Sarah A Steck, Nathaniel S Grabinski, Katie A Thies, Bri Wasik, Tasneem Ariswala, Gary Tozbikian, Johnathan Godbout, Steven T Sizemore, Paul R Lockman, Anna E Vilgelm, Gina M Sizemore. Stromal PDGFRβ hyperactivation modulates the myeloid lineage in the brain metastatic microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A085.

BACKGROUND:Evidence suggests that COVID-19 predisposes to cardiovascular diseases (CVDs). While monocytes/macrophages play a central role in the immunopathogenesis of atherosclerosis, less is known about their immunopathogenic mechanisms that lead to CVDs during COVID-19. Natural killer (NK) cells, which play an intermediary role during pathologies like atherosclerosis, are dysregulated during COVID-19. Here, we sought to investigate altered immune cells and their associations with CVD risk during severe COVID-19.METHODS:We measured plasma biomarkers of CVDs and determined phenotypes of circulating immune subsets using spectral flow cytometry. We compared these between patients with severe COVID-19 (severe, n=31), those who recovered from severe COVID-19 (recovered, n=29), and SARS-CoV-2-uninfected controls (controls, n=17). In vivo observations were supported using in vitro assays to highlight possible mechanistic links between dysregulated immune subsets and biomarkers during and after COVID-19. We performed multidimensional analyses of published single-cell transcriptome data of monocytes and NK cells during severe COVID-19 to substantiate in vivo findings.RESULTS:During severe COVID-19, we observed alterations in cardiometabolic biomarkers including oxidized-low-density lipoprotein, which showed decreased levels in severe and recovered groups. Severe patients exhibited dysregulated monocyte subsets, including increased frequencies of proinflammatory intermediate monocytes (also observed in the recovered) and decreased nonclassical monocytes. All identified NK-cell subsets in the severe COVID-19 group displayed increased expression of activation and tissue-resident markers, such as CD69 (cluster of differentiation 69). We observed significant correlations between altered immune subsets and plasma oxidized-low-density lipoprotein levels. In vitro assays revealed increased uptake of oxidized-low-density lipoprotein into monocyte-derived macrophages in the presence of NK cells activated by plasma of patients with severe COVID-19. Transcriptome analyses confirmed enriched proinflammatory responses and lipid dysregulation associated with epigenetic modifications in monocytes and NK cells during severe COVID-19.CONCLUSIONS:Our study provides new insights into the involvement of monocytes and NK cells in the increased CVD risk observed during and after COVID-19.

TTK spindle assembly checkpoint kinase is an emerging cancer target. This preclinical study explored the antitumor mechanism of TTK inhibitor OSU13 to define a strategy for clinical development. We observed prominent antitumor activity of OSU13 in melanoma, colon and breast cancer cells, organoids derived from patients with melanoma, and mice bearing colon tumors associated with G2 cell cycle arrest, senescence, and apoptosis. OSU13-treated cells displayed DNA damage and micronuclei that triggered the cytosolic DNA-sensing cGAS/STING pathway. STING was required for the induction of several proteins involved in T cell recruitment and activity. Tumors from OSU13-treated mice showed an increased proportion of T and NK cells and evidence of PD-1/PD-L1 immune checkpoint activation. Combining a lowtoxicity dose of OSU13 with anti-PD-1 checkpoint blockade resulted in prominent STING- and CD8+ T cell-dependent tumor inhibition and improved survival. These findings provide a rationale for utilizing TTK inhibitors in combination with immunotherapy in STING-proficient tumors.

Immune checkpoint blockade (ICB) has revolutionized cancer treatment and led to complete and durable responses, but only for a minority of patients. Resistance to ICB can largely be attributed to insufficient number and/or function of antitumor CD8(+) T cells in the tumor microenvironment. Neoantigen targeted cancer vaccines can activate and expand the antitumor T cell repertoire, but historically, clinical responses have been poor because immunity against peptide antigens is typically weak, resulting in insufficient activation of CD8(+) cytotoxic T cells. Herein, we describe a nanoparticle vaccine platform that can overcome these barriers in several ways. First, the vaccine can be reproducibly formulated using a scalable confined impingement jet mixing method to coload a variety of physicochemically diverse peptide antigens and multiple vaccine adjuvants into pH-responsive, vesicular nanoparticles that are monodisperse and less than 100 nm in diameter. Using this approach, we encapsulated synergistically acting adjuvants, cGAMP and monophosphoryl lipid A (MPLA), into the nanocarrier to induce a robust and tailored innate immune response that increased peptide antigen immunogenicity. We found that incorporating both adjuvants into the nanovaccine synergistically enhanced expression of dendritic cell costimulatory markers, pro-inflammatory cytokine secretion, and peptide antigen cross-presentation. Additionally, the nanoparticle delivery increased lymph node accumulation and uptake of peptide antigen by dendritic cells in the draining lymph node. Consequently, nanoparticle codelivery of peptide antigen, cGAMP, and MPLA enhanced the antigen-specific CD8(+) T cell response and delayed tumor growth in several mouse models. Finally, the nanoparticle platform improved the efficacy of ICB immunotherapy in a murine colon carcinoma model. This work establishes a versatile nanoparticle vaccine platform for codelivery of peptide neoantigens and synergistic adjuvants to enhance responses to cancer vaccines.

This study investigates the impact of nearby structures on the cyclic settlement mechanisms of shallow foundations in liquefiable soils using a numerical model based on Biot’s porous media theory. The model predicts excess pore water pressure and settlement by coupling equilibrium and continuity equations, solved using an implicit time integration scheme. Soil nonlinearity under cyclic loading is represented using generalized plasticity, boundary surfaces, and non-associated models. Three scenarios are simulated to study the effect of spacing between light and heavy foundations and variation in acceleration intensity. Results show that as spacing between foundations increases, lateral displacement and settlement decrease. Excess pore water pressure generation also decreases with increased foundation spacing. Soil just below the foundation exhibits maximum settlement, decreasing with depth. When input acceleration increases from 0.1 g to 0.15 g and 0.2 g, settlement increases by 40%–55% and 90%–110% respectively for both light and heavy foundations, regardless of spacing. Excess pore water pressure also increases sharply with higher acceleration intensity. The findings highlight the importance of considering foundation-soil-foundation interaction effects in liquefaction-prone urban settings and provide insights for designing resilient shallow foundations. The advanced numerical modeling approach offers engineers a more informed way to mitigate liquefaction risk and build safer, more durable structures in earthquake-prone areas.

Supplementary Table 1 and Figures 1-7

Membrane ion channels of the calcium homeostasis modulator (CALHM) family promote cell–cell crosstalk at neuronal synapses via ATP release, where ATP acts as a neurotransmitter. CALHM6, the only CALHM highly expressed in immune cells, has been linked to the induction of natural killer (NK) cell anti‐tumour activity. However, its mechanism of action and broader functions in the immune system remain unclear. Here, we generated Calhm6 −/− mice and report that CALHM6 is important for the regulation of the early innate control of Listeria monocytogenes infection in vivo . We find that CALHM6 is upregulated in macrophages by pathogen‐derived signals and that it relocates from the intracellular compartment to the macrophage‐NK cell synapse, facilitating ATP release and controlling the kinetics of NK cell activation. Anti‐inflammatory cytokines terminate CALHM6 expression. CALHM6 forms an ion channel when expressed in the plasma membrane of Xenopus oocytes, where channel opening is controlled by a conserved acidic residue, E119. In mammalian cells, CALHM6 is localised to intracellular compartments. Our results contribute to the understanding of neurotransmitter‐like signal exchange between immune cells that fine‐tunes the timing of innate immune responses.

Objectives: To determine the important Mechanical properties and Elastic modulus of Geopolymer concrete (GPC).Methods: Four mix proportions of GPC using two molarities (12 and 14) of NaOH have been used to achieve M25 grade of GPC.Compressive strength, Tensile strength, Flexural strength and Young's Modulus (E GPC ) tests have been performed as per Codal provisions.Findings: The modulus of elasticity of GPC (E GPC ) for the four mixes has been determined experimentally and the average of four values was noted.With the help of this value,5000 random variables have been generated using Monte Carlo Technique and the average E GPC (for 5000 values) has also been determined.Finally, it has been perceived that the elastic modulus of GPC is nearly equal to that of OPC in contrast to the previous studies.

Supplemental Data and Supplemental Figures

BackgroundCyclin-dependent kinase 4/6 inhibitors (CDK4/6i) combined with endocrine therapy are a mainstay treatment for hormone receptor-positive breast cancer. While their principal mechanism is inhibition of cancer cell proliferation, preclinical and clinical evidence suggests that CDK4/6i can also promote antitumor T-cell responses. However, this pro-immunogenic property is yet to be successfully harnessed in the clinic, as combining CDK4/6i with immune checkpoint blockade (ICB) has not shown a definitive benefit in patients.MethodWe performed an in-depth analysis of the changes in the tumor immune microenvironment and systemic immune modulation associated with CDK4/6i treatment in muring breast cancer models and in patients with breast cancer using high dimensional flow cytometry and RNA sequencing. Gain and loss of function in vivo experiments employing cell transfer and depletion antibody were performed to uncover immune cell populations critical for CDK4/6i-mediated stimulation of antitumor immunity.ResultsWe found that loss of dendritic cells (DCs) within the tumor microenvironment resulting from CDK4/6 inhibition in bone marrow progenitors is a major factor limiting antitumor immunity after CDK4/6i and ICB. Consequently, restoration of DC compartment by adoptively transferring ex vivo differentiated DCs to mice treated with CDK4/6i and ICB therapy enabled robust tumor inhibition. Mechanistically, the addition of DCs promoted the induction of tumor-localized and systemic CD4 T-cell responses in mice receiving CDK4/6i-ICB-DC combination therapy, as characterized by enrichment of programmed cell death protein-1-negative T helper (Th)1 and Th2 cells with an activated phenotype. CD4 T-cell depletion abrogated the antitumor benefit of CDK4/6i-ICB-DC combination, with outgrowing tumors displaying an increased proportion of terminally exhausted CD8 T cells.ConclusionsOur findings suggest that CDK4/6i-mediated DC suppression limits CD4 T-cell responses essential for the sustained activity of CD8 T cells and tumor inhibition. Furthermore, they imply that restoring DC-CD4 T-cell crosstalk via DC transfer enables effective breast cancer immunity in response to CDK4/6i and ICB treatment.

Clinical data demonstrate an increased predisposition to cardiovascular disease (CVD) following severe COVID-19 infection. This may be driven by a dysregulated immune response associated with severe disease. Monocytes and vascular tissue resident macrophages play a critical role in atherosclerosis, the main pathology leading to ischemic CVD. Natural killer (NK) cells are a heterogenous group of cells that are critical during viral pathogenesis and are known to be dysregulated during severe COVID-19 infection. Their role in atherosclerotic cardiovascular disease has recently been described. However, the contribution of their altered phenotypes to atherogenesis following severe COVID-19 infection is unknown. We demonstrate for the first time that during and after severe COVID-19, circulating proinflammatory monocytes and activated NK cells act synergistically to increase uptake of oxidized low-density lipoprotein (Ox-LDL) into vascular tissue with subsequent foam cell generation leading to atherogenesis despite recovery from acute infection. Our data provide new insights, revealing the roles of monocytes/macrophages, and NK cells in COVID-19-related atherogenesis.

The respiratory mucosa is under constant immune surveillance because of its vulnerability to infectious diseases. Infection of the lungs with the live vaccine strain (LVS) derived from Francisella tularensis (Ft) subspecies holarctica models pulmonary tularaemia-like disease in mice. Current evidence suggest many immune cells and cytokines respond to Ft LVS infection, but how these various immune system components are integrated to mount a protective response remains unclear. Hence, in a transcriptomics study at single cell resolution, we characterized the acute immune landscape in the lungs of C57BL/6 mice at day 0 and day 7 post intranasal inoculation with Ft LVS. Defining features of the immune response include a robust type 1 immune response characterized by the accumulation of inflammatory neutrophils and the expansion of innate-like effector lymphocytes, primarily interferon-γ producing NKT1, NK, and effector CD8 +T cells. Increased accumulation of MAIT17 over MAIT1 cells is another feature, which is in line with previous findings linking MAIT cells and IL-17 to LVS immune response. Surprisingly, a significant proportion of MAIT17 cells either maintained or upregulated type 1 inflammatory markers. Similarly, MAIT1 cells maintained or upregulated Type 3 inflammatory markers. Additionally, a highly active MAIT cell subset has increased Nr4a1 expression, which encodes Nur77, suggesting a T cell receptor-mediated activation. By contrast, NKT1 cells poorly upregulated Nr4a1 expression but induced Il18r1 expression suggesting, a cytokine-mediated activation. Consequently, we predict that unconventional NKT & MAIT cells integrate innate cues to control tularaemia-like disease caused by Ft LVS infection in the mouse. VUSM MSTP NIGMS of the National Institutes of Health T32GM007347 (GDO); IO1 BX001444, BX001610, BX00xxx, IK6 BX004595 & RO1 AI137082; IBX000915A (HMA)

Abstract The durability of sand screen completions is essential to longer well life, especially for high rate wells with sand screen erosion concerns. An excessive fluid flow enters the conventional screens near the heel or high permeability/fracture zones, causing premature sand control loss. The high rate screens with a simulation-driven approach address this concern by achieving the annulus-to-tubing flow equalization and reducing the influx spike near the heel or high permeability/fracture zone. The study presents a comprehensive modeling approach including a single-well model workflow for initial production screening along the wellbore with different reservoir conditions, which provides input to the novel multiscale 3D-2D-3D computational fluid dynamics (CFD) modeling technique to design or validate high-rate completions for the specific operating conditions. The principle of operation is based on equalizing the production influx along the screen by achieving the distributed inflow control devices (ICD) effect on the basepipe. The modeling approach was used to compute maximum local velocities in the vicinity of the screen near the heel under 39,000 RB/D of ultra-light oil production in one case and 200 MMscf/D of gas production in another. The design methodology is validated through erosion and sand retention tests performed to verify the screens’ correct slot/gauge size. The high-rate completion case history consists of seven deepwater wells with chemical tracers. The novel design and the modeling methodology are validated by physical erosion tests and verified through field installations.

Cancer therapies trigger diverse cellular responses, ranging from apoptotic death to acquisition of persistent therapy-refractory states such as senescence. Tipping the balance toward apoptosis could improve treat-ment outcomes regardless of therapeutic agent or malignancy. We find that inhibition of the mitochondrial protein BCL-xL increases the propensity of cancer cells to die after treatment with a broad array of oncology drugs, including mitotic inhibitors and chemotherapy. Functional precision oncology and omics analyses suggest that BCL-xL inhibition redirects the outcome of p53 transcriptional response from senescence to apoptosis, which likely occurs via caspase-dependent down-modulation of p21 and downstream cytostatic proteins. Consequently, addition of a BCL-2/xL inhibitor strongly improves melanoma response to the senes-cence-inducing drug targeting mitotic kinase Aurora kinase A (AURKA) in mice and patient-derived organo-ids. This study shows a crosstalk between the mitochondrial apoptotic pathway and cell cycle regulation that can be targeted to augment therapeutic efficacy in cancers with wild-type p53.

Natural infection as well as vaccination with live or attenuated viruses elicit tissue resident, CD8+ memory T cell (Trm) response. Trm cells so elicited act quickly upon reencounter with the priming agent to protect the host. These Trm cells express a unique molecular signature driven by the master regulators-Runx3 and Hobit. We previously reported that intranasal instillation of a subunit vaccine in a prime boost vaccination regimen installed quick-acting, CD8+ Trm cells in the lungs that protected against lethal vaccinia virus challenge. It remains unexplored whether CD8+ Trm responses so elicited are driven by a similar molecular signature as those elicited by microbes in a real infection or by live, attenuated pathogens in conventional vaccination. We found that distinct molecular signatures distinguished subunit vaccine-elicited lung interstitial CD8+ Trm cells from subunit vaccine-elicited CD8+ effector memory and splenic memory T cells. Nonetheless, the transcriptome signature of subunit vaccine elicited CD8+ Trm resembled those elicited by virus infection or vaccination. Clues to the basis of tissue residence and function of vaccine specific CD8+ Trm cells were found in transcripts that code for chemokines and chemokine receptors, purinergic receptors, and adhesins when compared to CD8+ effector and splenic memory T cells. Our findings inform the utility of protein-based subunit vaccination for installing CD8+ Trm cells in the lungs to protect against respiratory infectious diseases that plague humankind.

Performance of ten varieties of rapeseed and mustard were assessed through cluster frontline demonstration (CFLD) under different agro-climatic condition of Bihar and Jharkhandcontinuously for four years (2017-18 to 2020-21) by the KVKs of Bihar and Jharkhand.Among the agro-climatic zones of Bihar, highest yield was recorded in the demonstratedplot from zone III (B) i.e. South-West alluvial plain in case of variety RH 0749 (15.29 q/ha) and in Jharkhand state from zone I (Central and North astern plateau) with varietyNRCHB 101 (13.31 q/ha). Lowest technology index was observed for variety RajendraSufalam in all the zone (-4.96, 5.53, 7.18 and 22.92 % in zone I, III (B), III (A) & II) ofBihar and NRCHB 101 (23.15% in zone I) in Jharkhand. The range of extension gapwas1.38 to 4.37 and 1.13 to 4.86 q/ha under the agro-climatic zone of Bihar and Jharkhand,respectively. Pooled data shows that in Bihar, maximum net return (Rs. 46986 /ha) wasobtained in variety Rajendra Sufalam in agro-climatic zone III A of Bihar with benefit costratio 3.85:1 whereas under agro-climatic condition of Jharkhand maximum net return (Rs.39598 /ha) was recoded from variety NRCHB 101 with benefit cost ratio 2.80:1