Activation Of Adaptive Immunity Research Articles

Repetitive sulfur dioxide exposure in mice models post-deployment respiratory syndrome.

Soldiers deployed to Iraq and Afghanistan have a higher prevalence of respiratory symptoms than nondeployed military personnel and some have been shown to have a constellation of findings on lung biopsy termed post-deployment respiratory syndrome (PDRS). Since many of the subjects in this cohort reported exposure to sulfur dioxide (SO2), we developed a model of repetitive exposure to SO2 in mice that phenocopies many aspects of PDRS, including adaptive immune activation, airway wall remodeling, and pulmonary vascular (PV) disease. Although abnormalities in small airways were not sufficient to alter lung mechanics, PV remodeling resulted in the development of pulmonary hypertension and reduced exercise tolerance in SO2-exposed mice. SO2 exposure led to increased formation of isolevuglandins (isoLGs) adducts and superoxide dismutase 2 (SOD2) acetylation in endothelial cells, which were attenuated by treatment with the isoLG scavenger 2-hydroxybenzylamine acetate (2-HOBA). In addition, 2-HOBA treatment or Siruin-3 overexpression in a transgenic mouse model prevented vascular remodeling following SO2 exposure. In summary, our results indicate that repetitive SO2 exposure recapitulates many aspects of PDRS and that oxidative stress appears to mediate PV remodeling in this model. Together, these findings provide new insights regarding the critical mechanisms underlying PDRS.NEW & NOTEWORTHY We developed a mice model of "post-deployment respiratory syndrome" (PDRS), a condition in Veterans with unexplained exertional dyspnea. Our model successfully recapitulates many of the pathological and physiological features of the syndrome, revealing involvement of the ROS-isoLGs-Sirt3-SOD2 pathway in pulmonary vasculature pathology. Our study provides additional knowledge about effects and long-term consequences of sulfur dioxide exposure on the respiratory system, serving as a valuable tool for future PDRS research.

Gene-edited pigs: a translational model for human food allergy against alpha-Gal and anaphylaxis.

The prevalence of food allergy is rising and is estimated to approach 10%. Red meat allergy is the first known food allergy elicited by immunoglobulin E (IgE) antibodies recognizing a carbohydrate. Due to the loss of function of the alpha-1,3-galactosyltransferase (GGTA1) gene in humans, the disaccharide galactose-α-1,3-galactose (α-Gal) cannot be synthesized and therefore became immunogenic. IgE sensitization is elicited through the skin by repetitive tick bites transmitting α-Gal. The underlying mechanisms regarding innate and adaptive immune cell activation, including the B-cell isotype switch to IgE, are poorly understood, requiring further research and physiologically relevant animal models. Here, we describe a new animal model of red meat allergy using percutaneous α-Gal sensitization of gene-edited GGTA1-deficient pigs. Total and α-Gal-specific IgG, IgG1, IgG2, IgG4, and IgE levels were tracked. Further key factors associated with allergic skin inflammation, type 2 immunity, and allergy development were measured in PBMCs and skin samples. Significant increases in α-Gal-specific IgG1 and IgE levels indicated successful sensitization to the allergen α-Gal. Intracutaneous sensitizations with α-Gal recruited lymphocytes to the skin, including elevated numbers of T helper 2 (Th2) cells. Finally, α-Gal-sensitized pigs not only recognized α-Gal as non-self-antigen following α-Gal exposure through the skin but also developed anaphylaxis upon antigen challenge. Based on the similarities between the porcine and human skin, this new large animal model for α-Gal allergy should help to unveil the consecutive steps of cutaneous sensitization and aid the development of prophylactic and treatment interventions.

Harnessing Pyroptosis for Cancer Immunotherapy.

Cancer immunotherapy is a novel pillar of cancer treatment that harnesses the immune system to fight tumors and generally results in robust antitumor immunity. Although immunotherapy has achieved remarkable clinical success for some patients, many patients do not respond, underscoring the need to develop new strategies to promote antitumor immunity. Pyroptosis is an immunostimulatory type of regulated cell death that activates the innate immune system. A hallmark of pyroptosis is the release of intracellular contents such as cytokines, alarmins, and chemokines that can stimulate adaptive immune activation. Recent studies suggest that pyroptosis promotes antitumor immunity. Here, we review the mechanisms by which pyroptosis can be induced and highlight new strategies to induce pyroptosis in cancer cells for antitumor defense. We discuss how pyroptosis modulates the tumor microenvironment to stimulate adaptive immunity and promote antitumor immunity. We also suggest research areas to focus on for continued development of pyroptosis as an anticancer treatment. Pyroptosis-based anticancer therapies offer a promising new avenue for treating immunologically 'cold' tumors.

Abstract B034: Characterization of MHC-I-mediated immune evasion in triple-negative breast cancer using human transcriptomic datasets and immunocompetent mouse models

Abstract In Triple-Negative Breast Cancer (TNBC) patients, adaptive immune system activity in the tumor microenvironment is associated with improved prognosis. However, tumors can evolve to evade immune detection, likely leading to worse patient outcomes. Multi-omic data of paired primary and metastatic breast tumors demonstrate that TNBC metastases frequently lose expression of genes involved in major histocompatibility complex class I (MHC-I) antigen presentation, a key pathway for adaptive immune activation (PMID: 36585450). Therefore, understanding and addressing MHC-I-mediated immune evasion may have profound clinical benefits for TNBC patients. We hypothesize that breast tumor MHC-I loss results in a unique immune-evasive phenotype, which we are characterizing using human clinical datasets and immunocompetent mouse models of breast cancer. First, using large transcriptomic datasets (i.e., SCAN-B and TCGA), we are characterizing molecular and clinical features of MHC-I-low human breast tumors using expression of the MHC-I gene human leukocyte antigen A (HLA-A) as a continuous variable. We found HLA-A expression varies significantly between PAM50 molecular and clinical subtypes of breast cancer and has differing impacts on patient survival based on clinical subtype. In TNBCs, low HLA-A expression is associated with worse outcomes, while in HR+/HER2- tumors, low HLA-A predicts improved outcomes. We also identified genes highly correlated with HLA-A regardless of molecular subtype. This list was functionally rich, related to antigen processing machinery, adaptive immune-mediated cytotoxicity, and T cell exhaustion features. To complement human genomic data, we are empirically testing the role of MHC-I in syngeneic mouse mammary tumor models by individually knocking out murine MHC-I genes–H2-K, H2-D, and B2m–through CRISPR/Cas9-mediated genome editing. We have generated and validated knockouts (KOs) of H2-K and B2m, as well as empty vector control clones from the “immune-hot” and immune checkpoint inhibitor (ICI) responsive mammary tumor cell lines KPB25L-UV and T11-APOBEC. RNA sequencing of KPB25L-UV H2-K and B2m KO versus wildtype cell lines in vitro identified lists of differentially expressed genes, many of which are related to proteolytic and peptide antigen processing. Next, these CRISPR-modified cell lines were injected into the mammary fat pad of syngeneic immunocompetent mice, and the resulting tumors were harvested for RNA sequencing. Differentially expressed genes in KPB25L-UV H2-K KO versus wildtype tumors were related to increased MHC Class II antigen presentation and dendritic cell activity. Preliminary data suggests that mice bearing KPB25L-UV H2-K KO tumors have shortened survival compared to H2-K wildtype tumors. Experiments are ongoing to further characterize the growth dynamics, metastatic potential, single-cell transcriptomic profiles, and ICI sensitivities of these KO tumors in vivo. Our long-term goal is to identify therapeutic means to overcome this common immune-evasive phenotype to improve TNBC patient outcomes. Citation Format: Constandina E O'Connell, Brooke M Felsheim, Aranzazu Fernandez-Martinez, Kevin R Mott, Charles M Perou. Characterization of MHC-I-mediated immune evasion in triple-negative breast cancer using human transcriptomic datasets and immunocompetent mouse models [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 B034.

Circadian immunity from bench to bedside: a practical guide.

The immune system is built to counteract unpredictable threats, yet it relies on predictable cycles of activity to function properly. Daily rhythms in immune function are an expanding area of study, and many originate from a genetically based timekeeping mechanism known as the circadian clock. The challenge is how to harness these biological rhythms to improve medical interventions. Here, we review recent literature documenting how circadian clocks organize fundamental innate and adaptive immune activities, the immunologic consequences of circadian rhythm and sleep disruption, and persisting knowledge gaps in the field. We then consider the evidence linking circadian rhythms to vaccination, an important clinical realization of immune function. Finally, we discuss practical steps to translate circadian immunity to the patient's bedside.

Utilization of a Histoplasma capsulatum zinc reporter reveals the complexities of fungal sensing of metal deprivation.

Histoplasma capsulatum is a dimorphic fungal pathogen acquired via inhalation of soil-resident spores. Upon exposure to mammalian body temperatures, these fungal elements transform into yeasts that reside primarily within phagocytes. Macrophages (MΦ) provide a permissive environment for fungal replication until T cell-dependent immunity is engaged. MΦ activated by granulocyte macrophage colony stimulating factor (GM-CSF) induces metallothioneins (MTs) that bind zinc (Zn) and deprive yeast cells of labile Zn, thereby disabling fungal growth. Prior work demonstrated that the zinc transporter, ZRT2, was important for fungal survival in vivo. Hence, we constructed a yeast cell reporter strain that expresses green fluorescent protein (GFP) under control of the ZRT2 zinc-regulated promoter. This reporter accurately responds to a medium devoid of Zn. ZRT2 expression increased in GM-CSF, but not interferon-γ, stimulated MΦ. To examine the in vivo response, we infected mice with a reporter yeast strain and assessed ZRT2 expression at 0, 3, 7, and 14 days post-infection (dpi). ZRT2 expression minimally increased at 3 dpi and peaked at 7 dpi, corresponding with the onset of adaptive immunity. We discovered that the major MΦ populations that restrict Zn from the fungus are interstitial MΦ and exudate MΦ. Neutralizing GM-CSF blunted the control of infection but unexpectedly increased ZRT2 expression. This increase was dependent on another cytokine that activates MΦ to control H. capsulatum replication, M-CSF. These findings illustrate the reporter's ability to sense Zn in vitro and in vivo and correlate ZRT2 expression with GM-CSF and M-CSF activation of MΦ.IMPORTANCEPhagocytes use an arsenal of defenses to control the replication of Histoplasma yeasts, one of which is the limitation of trace metals. On the other hand, H. capsulatum combats metal restriction by upregulating metal importers such as the Zn importer ZRT2. This transporter contributes to H. capsulatum pathogenesis upon activation of adaptive immunity. We constructed a fluorescent ZRT2 transcriptional reporter to probe H. capsulatum Zn sensing during infection and exposed the role for M-CSF activation of macrophages when GM-CSF is absent. These data highlight the ways in which fungal pathogens sense metal deprivation in vivo and reveal the potential of metal-sensing reporters. The work adds a new dimension to study how intracellular pathogens sense and respond to the changing environments of the host.

Sirpα on tumor-associated myeloid cells restrains antitumor immunity in colorectal cancer independent of its interaction with CD47.

Immunosuppressive myeloid cells hinder immunotherapeutic efficacy in tumors, but the precise mechanisms remain undefined. Here, by performing single-cell RNA sequencing in colorectal cancer tissues, we found tumor-associated macrophages and granulocytic myeloid-derived suppressor cells increased most compared to their counterparts in normal tissue and displayed the highest immune-inhibitory signatures among all immunocytes. These cells exhibited significantly increased expression of immunoreceptor tyrosine-based inhibitory motif-bearing receptors, including SIRPA. Notably, Sirpa-/- mice were more resistant to tumor progression than wild-type mice. Moreover, Sirpα deficiency reprogramed the tumor microenvironment through expansion of TAM_Ccl8hi and gMDSC_H2-Q10hi subsets showing strong antitumor activity. Sirpa-/- macrophages presented strong phagocytosis and antigen presentation to enhance T cell activation and proliferation. Furthermore, Sirpa-/- macrophages facilitated T cell recruitment via Syk/Btk-dependent Ccl8 secretion. Therefore, Sirpα deficiency enhances innate and adaptive immune activation independent of expression of CD47 and Sirpα blockade could be a promising strategy to improve cancer immunotherapy efficacy.

Lipid peroxidation of immune cells in cancer.

Growing evidence indicates that cellular metabolism is a critical determinant of immune cell viability and function in antitumor immunity and lipid metabolism is important for immune cell activation and adaptation to the tumor microenvironment (TME). Lipid peroxidation is a process in which oxidants attack lipid-containing carbon-carbon double bonds and is an important part of lipid metabolism. In the past decades, studies have shown that lipid peroxidation participates in signal transduction to control cell proliferation, differentiation, and cell death, which is essential for cell function execution and human health. More importantly, recent studies have shown that lipid peroxidation affects immune cell function to modulate tumor immunity and antitumor ability. In this review, we briefly overview the effect of lipid peroxidation on the adaptive and innate immune cell activation and function in TME and discuss the effectiveness and sensitivity of the antitumor ability of immune cells by regulating lipid peroxidation.

A Bidirectional Immunological Relationship between Diabetes Mellitus and Tuberculosis: A Narrative Review

Tuberculosis (TB) is one of the most common contagious diseases worldwide. Over the past few decades, researchers have made significant efforts to prevent TB. Recent predictions indicate that the prevalence of Diabetes Mellitus (DM) will reach 552 million by 2023 due to ongoing epidemiological changes in several countries across the globe. India is expected to have 62-80 million people with diabetes by 2030, making it the "Diabetes Capital" of the world. Present review discusses the urgent need to understand the mechanisms and implications of DM on the immune systems of TB patients. The correlation between TB and DM has been shown to be bidirectional. Numerous studies have revealed a higher prevalence of diabetes in TB patients, as their immune systems are compromised. TB patients with DM experience progressive deficits in both innate and adaptive immune cell activity, reducing their ability to suppress Mycobacterium tuberculosis (M. tuberculosis) and increasing their risk of developing TB. Increased inflammation and elevated levels of inflammatory cytokines in circulation are characteristics of TB with DM co-morbidity, which indicate the active involvement of the angiogenesis-inflammation nexus. Therefore, to alleviate the burden of both disorders, it would be beneficial to make informed therapy choices by gaining a better understanding of TB immunology in the context of DM.

Proteogenomic Data Integration Reveals CXCL10 as a Potentially Downstream Causal Mediator for IL-6 Signaling on Atherosclerosis.

Genetic and experimental studies support a causal involvement of IL-6 (interleukin-6) signaling in atheroprogression. Although trials targeting IL-6 signaling are underway, any benefits must be balanced against an impaired host immune response. Dissecting the mechanisms that mediate the effects of IL-6 signaling on atherosclerosis could offer insights about novel drug targets with more specific effects. Leveraging data from 522 681 individuals, we constructed a genetic instrument of 26 variants in the gene encoding the IL-6R (IL-6 receptor) that proxied for pharmacological IL-6R inhibition. Using Mendelian randomization, we assessed its effects on 3281 plasma proteins quantified with an aptamer-based assay in the INTERVAL cohort (n=3301). Using mediation Mendelian randomization, we explored proteomic mediators of the effects of genetically proxied IL-6 signaling on coronary artery disease, large artery atherosclerotic stroke, and peripheral artery disease. For significant mediators, we tested associations of their circulating levels with incident cardiovascular events in a population-based study (n=1704) and explored the histological, transcriptomic, and cellular phenotypes correlated with their expression levels in samples from human atherosclerotic lesions. We found significant effects of genetically proxied IL-6 signaling on 70 circulating proteins involved in cytokine production/regulation and immune cell recruitment/differentiation, which correlated with the proteomic effects of pharmacological IL-6R inhibition in a clinical trial. Among the 70 significant proteins, genetically proxied circulating levels of CXCL10 (C-X-C motif chemokine ligand 10) were associated with risk of coronary artery disease, large artery atherosclerotic stroke, and peripheral artery disease, with up to 67% of the effects of genetically downregulated IL-6 signaling on these end points mediated by decreases in CXCL10. Higher midlife circulating CXCL10 levels were associated with a larger number of cardiovascular events over 20 years, whereas higher CXCL10 expression in human atherosclerotic lesions correlated with a larger lipid core and a transcriptomic profile reflecting immune cell infiltration, adaptive immune system activation, and cytokine signaling. Integrating multiomics data, we found a proteomic signature of IL-6 signaling activation and mediators of its effects on cardiovascular disease. Our analyses suggest the interferon-γ-inducible chemokine CXCL10 to be a potentially causal mediator for atherosclerosis in 3 vascular compartments and, as such, could serve as a promising drug target for atheroprotection.

Immunogenic ferroptosis protects against the development of tumour growth

Immunotherapy is a proven and effective anti-tumor strategy, which can be used alongside surgery, radiation therapy, and chemotherapy [1]. Immunogenic cell death (ICD) was identified as a critical factor determining the effectiveness of cancer treatment [2]. The concept of ICD combines the capacity to destroy cancer cells effectively, with the activation of a cancer cell-specific immune response, leading to potent and long-lasting anti-cancer immunity. ICD-inducing agents activate a perilous pathway that triggers the release of ICD mediators called damage-associated molecular patterns (DAMPs). DAMPs encompass a group of naturally occurring molecules that gain immunostimulatory qualities upon exposure to the outer cell membrane or when liberated into the extracellular matrix in a specific spatiotemporal fashion. ATP, the nuclear protein HMGB1, calreticulin (CRT), and type I interferons (IFNs) are among the identified factors [8]. The concept of ICD was initially described for cancer cells undergoing apoptosis, but it was expanded to encompass additional forms of cell death, such as necroptosis, pyrroptosis, ferroptosis, nontosis, etc. [11]. Ferroptosis is a regulated iron-dependent type of cell death that is characterized by the buildup of reactive oxygen species in the cell. In this study, the immunogenicity of ferroptotic cancer cells in vitro was assessed and their potential as an alternative approach to cancer immunotherapy was tested. Glioma GL261 and fibrosarcoma MCA205 cells were induced with one of the well-known inducers of ferroptosis, RSL3 (RAS-Selective Lethal 3). After 24 hours of RSL3 stimulation, 80% of GL261 cells and 90% of MCA205 cells showed positivity to Annexin V/Sytox Blue, indicating they were in the late stage of ferroptosis. Similarly, after 3 hours of RSL3 stimulation, 50% of GL261 cells and 45% of MCA205 cells were double positive with Annexin V/Sytox Blue indicating their late-stage ferroptotic state. We evaluated the immunogenic features of early and late ferroptotic cells in vitro, specifically at 3 or 24 hours after RSL3 stimulation. To achieve this, we compared the phenotype of dendritic cells (BMDCs) exposed to late ferroptotic cells with BMDCs exposed to viable cancer cells. Furthermore, immunogenic apoptosis was induced with MTX as a positive control and LPS as a secondary positive control. Late ferroptotic MCA 205 cells surprisingly did not induce phenotypic BMDC maturation, as indicated by the lack of surface activation of costimulatory molecules CD86, CD80, and MHCII. In contrast, a less pronounced phenotypic response compared to MCA205 cells was induced by early ferroptotic glioma GL261 cells. Nonetheless, a decrease in the ability to activate dendritic cells was observed for late ferroptotic glioma cells as well. The study used the standard tumor prophylactic vaccination model on immunocompetent C57BL/6 J mice to assess the adaptive immune system activation by early ferroptotic cancer cells. Mice were immunized with early or late ferroptosis MCA205 cells. As a negative control, we used PBS or cells that underwent spontaneous necrosis. The mice that were immunized were later confronted with viable MCA205 tumor cells. Protection against tumor growth at the site of infection was deemed indicative of successful activation of the adaptive immune system. Remarkably, mice that received immunization with late ferroptotic MCA205 cells, induced with RSL3 for 24 hours, exhibited conspicuous tumor growth at the infection site, signifying that late ferroptotic cells are not immunogenic in vivo, as per our preliminary observations in vitro.

Pulmonary Toxicity of Boron Nitride Nanomaterials Is Aspect Ratio Dependent.

Boron nitride (BN) nanomaterials have drawn a lot of interest in the material science community. However, extensive research is still needed to thoroughly analyze their safety profiles. Herein, we investigated the pulmonary impact and clearance of two-dimensional hexagonal boron nitride (h-BN) nanosheets and boron nitride nanotubes (BNNTs) in mice. Animals were exposed by single oropharyngeal aspiration to h-BN or BNNTs. On days 1, 7, and 28, bronchoalveolar lavage (BAL) fluids and lungs were collected. On one hand, adverse effects on lungs were evaluated using various approaches (e.g., immune response, histopathology, tissue remodeling, and genotoxicity). On the other hand, material deposition and clearance from the lungs were assessed. Two-dimensional h-BN did not cause any significant immune response or lung damage, although the presence of materials was confirmed by Raman spectroscopy. In addition, the low aspect ratio h-BN nanosheets were internalized rapidly by phagocytic cells present in alveoli, resulting in efficient clearance from the lungs. In contrast, high aspect ratio BNNTs caused a strong and long-lasting inflammatory response, characterized by sustained inflammation up to 28 days after exposure and the activation of both innate and adaptive immunity. Moreover, the presence of granulomatous structures and an indication of ongoing fibrosis as well as DNA damage in the lung parenchyma were evidenced with these materials. Concurrently, BNNTs were identified in lung sections for up to 28 days, suggesting long-term biopersistence, as previously demonstrated for other high aspect ratio nanomaterials with poor lung clearance such as multiwalled carbon nanotubes (MWCNTs). Overall, we reveal the safer toxicological profile of BN-based two-dimensional nanosheets in comparison to their nanotube counterparts. We also report strong similarities between BNNTs and MWCNTs in lung response, emphasizing their high aspect ratio as a major driver of their toxicity.

Humoral and T cell responses to SARS-CoV-2 reveal insights into immunity during the early pandemic period in Pakistan

BackgroundProtection against SARS-CoV-2 is mediated by humoral and T cell responses. Pakistan faced relatively low morbidity and mortality from COVID-19 through the pandemic. To examine the role of prior immunity in the population, we studied IgG antibody response levels, virus neutralizing activity and T cell reactivity to Spike protein in a healthy control group (HG) as compared with COVID-19 cases and individuals from the pre-pandemic period (PP).MethodsHG and COVID-19 participants were recruited between October 2020 and May 2021. Pre-pandemic sera was collected before 2018. IgG antibodies against Spike and its Receptor Binding Domain (RBD) were determined by ELISA. Virus neutralization activity was determined using a PCR-based micro-neutralization assay. T cell – IFN-γ activation was assessed by ELISpot.ResultsOverall, the magnitude of anti-Spike IgG antibody levels as well as seropositivity was greatest in COVID-19 cases (90%) as compared with HG (39.8%) and PP (12.2%). During the study period, Pakistan experienced three COVID-19 waves. We observed that IgG seropositivity to Spike in HG increased from 10.3 to 83.5% during the study, whilst seropositivity to RBD increased from 7.5 to 33.3%. IgG antibodies to Spike and RBD were correlated positively in all three study groups. Virus neutralizing activity was identified in sera of COVID-19, HG and PP. Spike reactive T cells were present in COVID-19, HG and PP groups. Individuals with reactive T cells included those with and without IgG antibodies to Spike.ConclusionsAntibody and T cell responses to Spike protein in individuals from the pre-pandemic period suggest prior immunity against SARS-CoV-2, most likely from cross-reactive responses. The rising seroprevalence observed in healthy individuals through the pandemic without known COVID-19 may be due to the activation of adaptive immunity from cross-reactive memory B and T cells. This may explain the more favourable COVID-19 outcomes observed in this population.

Soluble TNFR1 has greater reproducibility than IL-6 for the assessment of chronic inflammation in older adults: the case for a new inflammatory marker in aging.

Chronic inflammatory pathway activation, commonly referred to as "Inflammaging" or chronic inflammation (CI), is associated with frailty, cognitive and functional decline, and other causes of health span decline in older adults. We investigated the variability of candidate serum measures of CI among community-dwelling older adults selected for mild low-grade inflammation. We focused on serum cytokines known to be highly predictive of adverse health outcomes in older adults (sTNFR1, IL-6) during a short-term (weeks) and medium-term (months) follow-up, as well as immune markers that are less studied in aging but reflect other potentially relevant domains such as adaptive immune activation (sCD25), innate immune activation (sCD14 and sCD163), and the inflammation-metabolism interface (adiponectin/Acrp30) during short-term (weeks) follow up. We found that sTNFR1 was more reproducible than IL-6 over a period of weeks and months short-term and medium-term. The intra-class correlation coefficient (ICC) for sTNFR1 was 0.95 on repeated measures over 6weeks, and 0.79 on repeated measures with mean interval of 14weeks, while the ICC for IL-6 was 0.52 over corresponding short-term and 0.67 over corresponding medium-term follow-up. This suggests that sTNFR1 is a more reliable marker of CI than IL-6. This study provides new insights into the reproducibility of serum markers of CI in older adults. The findings suggest that sTNFR1 may be a better marker of CI than IL-6 in this population. Further studies are needed to confirm these findings and to investigate the clinical utility of sTNFR1 in older adults.

Mayaro virus pathogenesis and immunity in rhesus macaques.

Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that causes debilitating and persistent arthritogenic disease. While MAYV was previously reported to infect non-human primates (NHP), characterization of MAYV pathogenesis is currently lacking. Therefore, in this study we characterized MAYV infection and immunity in rhesus macaques. To inform the selection of a viral strain for NHP experiments, we evaluated five MAYV strains in C57BL/6 mice and showed that MAYV strain BeAr505411 induced robust tissue dissemination and disease. Three male rhesus macaques were subcutaneously challenged with 105 plaque-forming units of this strain into the arms. Peak plasma viremia occurred at 2 days post-infection (dpi). NHPs were taken to necropsy at 10 dpi to assess viral dissemination, which included the muscles and joints, lymphoid tissues, major organs, male reproductive tissues, as well as peripheral and central nervous system tissues. Histological examination demonstrated that MAYV infection was associated with appendicular joint and muscle inflammation as well as presence of perivascular inflammation in a wide variety of tissues. One animal developed a maculopapular rash and two NHP had viral RNA detected in upper torso skin samples, which was associated with the presence of perivascular and perifollicular lymphocytic aggregation. Analysis of longitudinal peripheral blood samples indicated a robust innate and adaptive immune activation, including the presence of anti-MAYV neutralizing antibodies with activity against related Una virus and chikungunya virus. Inflammatory cytokines and monocyte activation also peaked coincident with viremia, which was well supported by our transcriptomic analysis highlighting enrichment of interferon signaling and other antiviral processes at 2 days post MAYV infection. The rhesus macaque model of MAYV infection recapitulates many of the aspects of human infection and is poised to facilitate the evaluation of novel therapies and vaccines targeting this re-emerging virus.

Evaluating TLR4 Gene Expression to Monitor Disease Progression in Iraqi Patients with Rheumatoid Arthritis

Background: Toll-like receptors (TLRs) play a significant role in the activation of adaptive immunity and may have an essential role in the development of rheumatoid arthritis (RA). Objectives: To assess the gene expression of TLR4 in individuals with RA compared to healthy individuals. Methods: From July to December 2022. A total of 100 individuals were encompassed in the study, consisting of 50 individuals diagnosed with RA, of whom 42 were females and 8 were males, with an average age of 45.22 years. Additionally, there were 50 healthy control participants, 40 of whom were females and 10 were males, with an average age of 45.64 years. To assess the TLR4 transcript levels, blood samples were collected from each participant, and RNA extraction was performed. cDNA synthesis was carried out, and real-time PCR was utilized for the analysis. The researchers also assessed the clinicopathological characteristics of the patients. Results: The serum TLR4 gene was significantly overexpressed in RA patients (fold change 2.59) compared to the controls (fold change 1.07). The expression level of the TLR4 gene was correlated with the clinicopathological characteristics of the patients, including erythrocyte sedimentation rates (ESR), RF, anti-CCP antibody, and DAS28. Conclusion: TLR4 was overexpressed in RA patients and was correlated with disease activity. It might be a therapeutic target and may contribute to the pathogenesis of RA.

The cross-talk between macrophages and tumor cells as a target for cancer treatment.

Macrophages represent an important component of the innate immune system. Under physiological conditions, macrophages, which are essential phagocytes, maintain a proinflammatory response and repair damaged tissue. However, these processes are often impaired upon tumorigenesis, in which tumor-associated macrophages (TAMs) protect and support the growth, proliferation, and invasion of tumor cells and promote suppression of antitumor immunity. TAM abundance is closely associated with poor outcome of cancer, with impediment of chemotherapy effectiveness and ultimately a dismal therapy response and inferior overall survival. Thus, cross-talk between cancer cells and TAMs is an important target for immune checkpoint therapies and metabolic interventions, spurring interest in it as a therapeutic vulnerability for both hematological cancers and solid tumors. Furthermore, targeting of this cross-talk has emerged as a promising strategy for cancer treatment with the antibody against CD47 protein, a critical macrophage checkpoint recognized as the "don't eat me" signal, as well as other metabolism-focused strategies. Therapies targeting CD47 constitute an important milestone in the advancement of anticancer research and have had promising effects on not only phagocytosis activation but also innate and adaptive immune system activation, effectively counteracting tumor cells' evasion of therapy as shown in the context of myeloid cancers. Targeting of CD47 signaling is only one of several possibilities to reverse the immunosuppressive and tumor-protective tumor environment with the aim of enhancing the antitumor response. Several preclinical studies identified signaling pathways that regulate the recruitment, polarization, or metabolism of TAMs. In this review, we summarize the current understanding of the role of macrophages in cancer progression and the mechanisms by which they communicate with tumor cells. Additionally, we dissect various therapeutic strategies developed to target macrophage-tumor cell cross-talk, including modulation of macrophage polarization, blockade of signaling pathways, and disruption of physical interactions between leukemia cells and macrophages. Finally, we highlight the challenges associated with tumor hypoxia and acidosis as barriers to effective cancer therapy and discuss opportunities for future research in this field.

Biomimetic Metal–Organic Framework Combats Biofilm‐Associated Infections via Hyperthermia‐Enhanced Bacterial Metabolic Interference and Autophagy‐Promoted Adaptive Immunity

AbstractRobust bacterial metabolism and the immunosuppression on peripheral immune cells cause biofilm‐associated infections (BAIs) extremely refractory to be eradicated via antibiotics alone. Herein, hierarchical mesoporous UiO‐66 metal–organic framework is decorated with selenite, polypyrrole, and macrophage membrane (MM) to develop a biomimetic nanosphere (USPM). Following the recruitment of USPM to the biofilm microenvironment (BME) via the pathogen‐targeting ability derived from MM. The BME‐responsive USPM can precisely release selenite to penetrate the loosened biofilm in synergy with near‐infrared‐induced mild photothermal therapy (mPTT). Selenite can quickly react with reducing substances to generate hydrogen selenide (H2Se) inside the biofilm. H2Se can competitively inhibit bacterial metabolic processes and disrupt biofilm metabolic homeostasis by cascade amplification effects. Furthermore, H2Se inside the biofilm further sensitizes photothermia to exert a precise local photothermal effect. Outside the biofilm, USPM can simultaneously promote the phagocytosis and autophagy of macrophages to kill and decompose the phagocytosed bacteria. Finally, the well‐decomposed bacterial antigens in macrophages can be presented to antigen‐presenting cells to arouse adaptive immune responses and enhance anti‐biofilm effectiveness further. Such powerful mPTT‐enhanced bacterial metabolic disruption and macrophagic autophagy‐promoted adaptive immune activation suggest an alternative therapeutic strategy to cure refractory BAIs.

EXTH-81. RE-EXPRESSION OF CXCL9 FOLLOWING ADENO-ASSOCIATED VIRUS (AAV) TRANSGENE DELIVERY TRANSFORMS THE IMMUNOLOGICAL LANDSCAPE OF GLIOBLASTOMA, SENSITIZING TUMORS TO PD-1 IMMUNE CHECKPOINT BLOCKADE (ICB)

Abstract BACKGROUND To date, few glioblastoma (GBM) patients have demonstrated clinical benefit from treatment with anti-PD-1 ICB. GBM is notoriously devoid of lymphocytes, signifying that a possible obstacle for effective ICB response may be the limited ability of cytotoxic T-lymphocytes (CTLs) to home to these tumors. CTLs are recruited via long-range signaling mediated by the diffusion of chemokines present in inflammatory environments. Following a comprehensive chemokine screen of clinical tumor specimens, we identified that CXC motif ligand 9 (CXCL9) is notably absent in GBM. To test if GBM reconstitution with CXCL9 could transform these intractable tumors into ICB-responsive, we leveraged AAV gene therapy to generate durable production of this potent lymphotactic signal. HYPOTHESIS AAV mediated re-expression of CXCL9 will improve lymphotaxis in GBM, sensitizing these tumors to PD-1 ICB. METHODS 3-dimensional whole brain fluorescent imaging was performed to characterize the distribution, tropism, and durability of AAV transgene expression in preclinical GBM models. Single-cell RNA sequencing (scRNAseq) and multiparameter flow cytometry was utilized to immunologically profile tumors in response to single and combination treatment. Survival studies with and without lymphocyte depletion were performed to evaluate immunogenic responsiveness of therapy. RESULTS Intra-tumor delivery of AAV6 yields stable transduction of tumor-reactive astrocytes, producing a localized, peri-tumoral pattern of transgene signal distribution. Combination therapy increases tumor infiltration by lymphocytes. scRNAseq analyses reveal widespread innate and adaptive immune activation in response to each CXCL9 and PD-1 ICB treatment. Infiltrating CD8 T-lymphocytes demonstrate inflammatory signatures associated with increased effector function, including antigen presentation, cytotoxicity, and TCR signaling. Combination treatment considerably improved overall survival in two distinct GBM models, with durable responses dependent on CD8 T-lymphocyte infiltration. CONCLUSIONS This work suggests direct conditioning of the tumor microenvironment by AAV-CXCL9 could potentially overcome a key resistance factor in GBM, sensitizing tumors to immune mediated therapies such as PD-1 ICB.

Modakafusp Alfa Demonstrates Type I Interferon-Mediated Innate and Adaptive Immune Enhancement in a Phase 1/2 Study in Patients with Relapsed and/or Refractory Multiple Myeloma (RRMM)

Modakafusp alfa (moda), a novel immunocytokine, is an innate immunity enhancer comprising two attenuated interferon (IFN) α2b molecules fused to an anti-CD38 IgG4 monoclonal antibody backbone, driving preferential IFNα signalling in CD38-expressing innate and adaptive immune cells as well as myeloma cells. Here, we present pharmacodynamic (PD) data from the first-in-human Phase 1/2 study of moda as a monotherapy in patients with RRMM (iinnovate-1; NCT03215030). A total of 37 patients treated with moda at 1.5 or 3 mg/kg every 4 weeks were included in the analysis. Peripheral blood (PB) and bone marrow (BM) samples were collected during screening or pre-dose on cycle 1 day 1 (C1D1) and at multiple timepoints after treatment. BM and PB samples were evaluated by cytometry time of flight (CyTOF) and bulk RNA sequencing (RNAseq) to assess pharmacodynamic changes in immune cell populations, cell activation, and gene expression. Administration of moda led to enhanced innate immune cell activation and cytotoxic function as demonstrated by increased proportions of CD69+ and granzyme B+ in peripheral NK cells analyzed by CyTOF. This was accompanied by a decrease in the proportion of TIGIT+ NK cells, indicating reduced inhibitory signaling. Furthermore, enhanced proliferation (%Ki67+) of NK cells was observed in both BM and PB. Moda also impacted myeloid cell populations in BM and PB. Dendritic cells (DCs) and monocytes in PB showed increased surface expression of the co-stimulatory molecule CD86, suggesting a potential for enhanced antigen presentation and co-stimulation ability. Increased proliferation (%Ki67) of DCs and monocytes was detected in both PB and BM, accompanied by a decrease in numbers of peripheral DCs which may indicate recruitment or homing to secondary lymphoid organs. Additionally, RNAseq analysis revealed that treatment with moda led to upregulation of CD68 expression in both PB and BM, indicating a proinflammatory M1 macrophage type response. Finally, CyTOF analysis also indicated that moda significantly impacted adaptive immunity. We observed enhanced activation (%CD69, %PD1 and %CD40L) and cytotoxic function (%granzyme B) of peripheral CD8 T cells, and increased CD8 T cell proliferation (%Ki67 CD8) in both PB and BM samples. Enhanced activation of peripheral CD4 T cells (%CD69, %CD40L) was also detected. Consistent with activation of the adaptive immune system, there was a reduction in naïve CD8 T cells accompanied by an increase in CD8 T effector memory cells in PB. Analysis of exhaustion markers (TIGIT, PD-1) in cycle 1 (C1D8, C1D15, and C2D1 predose) showed that moda did not induce CD8 T cell exhaustion in either PB or BM, and there was no change in FoxP3+ Treg cells. Moda-induced innate and adaptive immune cell phenotypic changes were evaluated for correlation with clinical response at timepoints with sufficient number of evaluable samples. While none of the associations were statistically significant after correcting for multiplicity testing (using the false discovery rate method), we observed trends warranting further investigation. There was a greater increase of CD40L+ NK cells and PD1+ CD8 cells in responders vs. non-responders at C1D2. Additionally, at C1D15 responders showed a lower increase in proliferation (%Ki67+) of classical monocytes, NK cells, and myeloid DCs in PBas compared to non-responders. A similar trend was also observed in the BM classical monocytes. Even though the peak of Ki67 expression was observed at C1D8, diminished increases of proliferating cells at C1D15 in responders was a surprising observation that needs further consideration. The PD biomarker data from this first-in-human clinical trial demonstrated that treatment with moda enhances both innate and adaptive immune cell activation in PB and BM. Importantly, moda-mediated immune activation did not result in T cell exhaustion during cycle 1, as evaluated in both PB and BM. We have observed interesting trends of immune phenotypic changes that may correlate with response. Follow-up analyses of the correlation of biomarkers with clinical response are in progress and will be presented. Further clinical trials are underway (iinnovate-2, NCT05556616; iinnovate-3, NCT05590377) to evaluate moda's novel immune activating mechanism in combination with standard of care anti-myeloma therapies.