Innate Type Research Articles

Exercise intensity and training alter the innate immune cell type and chromosomal origins of circulating cell-free DNA in humans

Exercising regularly promotes health, but these benefits are complicated by acute inflammation induced by exercise. A potential source of inflammation is cell-free DNA (cfDNA), yet the cellular origins, molecular causes, and immune system interactions of exercise-induced cfDNA are unclear. To study these, 10 healthy individuals were randomized to a 12-wk exercise program of either high-intensity tactical training (HITT) or traditional moderate-intensity training (TRAD). Blood plasma was collected pre- and postexercise at weeks 0 and 12 and after 4 wk of detraining upon program completion. Whole-genome enzymatic methylation sequencing (EM-seq) with cell-type proportion deconvolution was applied to cfDNA obtained from the 50 plasma samples and paired to concentration measurements for 90 circulating cytokines. Acute exercise increased the release of cfDNA from neutrophils, dendritic cells (DCs), and macrophages proportional to exercise intensity. Exercise training reduced cfDNA released in HITT participants but not TRAD and from DCs and macrophages but not neutrophils. For most participants, training lowered mitochondrial cfDNA at rest, even after detraining. Using a sequencing analysis approach we developed, we concluded that rapid ETosis, a process of cell death where cells release DNA extracellular traps, was the likely source of cfDNA, demonstrated by enrichment of nuclear DNA. Further, several cytokines were induced by acute exercise, such as IL-6, IL-10, and IL-16, and training attenuated the induction of only IL-6 and IL-17F. Cytokine levels were not associated with cfDNA induction, suggesting that these cytokines are not the main cause of exercise-induced cfDNA. Overall, exercise intensity and training modulated cfDNA release and cytokine responses, contributing to the anti-inflammatory effects of regular exercise.

Tissue-resident natural killer cells derived from conventional natural killer cells are regulated by progesterone in the uterus.

The murine uterus contains three subsets of innate lymphoid cells (ILCs). Innate lymphoid cell type 1 (ILC1) and conventional natural killer (cNK) cells seed the uterus before puberty. Tissue-resident NK (trNK) cells emerge at puberty and vary in number during the estrous cycle. Here, we addressed the origin of uterine trNK cells and the influence of ovarian hormones on their local activation and differentiation in vivo. We used parabiosed mice in combination with intravascular fluorescent antibody labeling and flow cytometry to distinguish tissue-resident from circulating immune cells. Additionally, we used C57BL/6J ovariectomized (OVX) and non-OVX mice supplemented with ovarian hormones to assess their effects on uterine trNK cell function. Strikingly, mice OVX at three weeks of age and analyzed as adults lacked uterine trNK cells unless progesterone was administered. Our parabiosis studies confirmed that the progesterone-responsive trNK cells are derived from peripheral cNK cells. Moreover, medroxyprogesterone 17- acetate-induced expansion of cNK-derived trNK cells was abolished by a progesterone receptor antagonist. These data reveal a novel, uterine-specific differentiation pathway of trNK cells that is tightly regulated by progesterone.

KSRP Deficiency Attenuates the Course of Pulmonary Aspergillosis and Is Associated with the Elevated Pathogen-Killing Activity of Innate Myeloid Immune Cells.

The mRNA-binding protein KSRP (KH-type splicing regulatory protein) is known to modulate immune cell functions post-transcriptionally, e.g., by reducing the mRNA stability of cytokines. It is known that KSRP binds the AU-rich motifs (ARE) that are often located in the 3'-untranslated part of mRNA species, encoding dynamically regulated proteins as, for example, cytokines. Innate myeloid immune cells, such as polymorphonuclear neutrophils (PMNs) and macrophages (MACs), eliminate pathogens by multiple mechanisms, including phagocytosis and the secretion of chemo- and cytokines. Here, we investigated the role of KSRP in the phenotype and functions of both innate immune cell types in the mouse model of invasive pulmonary aspergillosis (IPA). Here, KSRP-/- mice showed lower levels of Aspergillus fumigatus conidia (AFC) and an increase in the frequencies of PMNs and MACs in the lungs. Our results showed that PMNs and MACs from KSRP-/- mice exhibited an enhanced phagocytic uptake of AFC, accompanied by increased ROS production in PMNs upon stimulation. A comparison of RNA sequencing data revealed that 64 genes related to inflammatory and immune responses were shared between PMNs and MACs. The majority of genes upregulated in PMNs were involved in metabolic processes, cell cycles, and DNA repair. Similarly, KSRP-deficient PMNs displayed reduced levels of apoptosis. In conclusion, our results indicate that KSRP serves as a critical negative regulator of PMN and MAC anti-pathogen activity.

Shared and unique patterns of autonomous human endogenous retrovirus loci transcriptomes in CD14 + monocytes from individuals with physical trauma or infection with COVID-19

Since previous studies have suggested that the RNAs of human endogenous retrovirus (HERV) might be involved in regulating innate immunity, it is important to investigate the HERV transcriptome patterns in innate immune cell types such as CD14 + monocytes. Using single cell RNA-seq datasets from resting or stimulated PBMCs mapped to 3,220 known discrete autonomous proviral HERV loci, we found individual-specific variation in HERV transcriptomes between HERV loci in CD14 + monocytes. Analysis of paired datasets from the same individual that were cultured in vitro with LPS or without (i.e. control) revealed 36 HERV loci in CD14 + monocytes that were detected only after activation. To extend our analysis to in vivo activated CD14 + monocytes, we used two scRNA-seq datasets from studies that had demonstrated activation of circulating CD14 + monocytes in patients with physical trauma or patients hospitalized with COVID-19 infections. For direct comparison between the trauma and COVID-19 datasets, we first analyzed 1.625 billion sequence reads from a composite pangenome control of 21 normal individuals. Comparison of the sequence read depth of HERV loci in the trauma or COVID-19 samples to the pangenome control revealed that 39 loci in the COVID-19 and 11 HERV loci in the trauma samples were significantly different (Mann-Whitney U test), with 9 HERV loci shared between the COVID-19 and trauma datasets. The capacity to compare HERV loci transcriptome patterns in innate immune cells, like CD14 + monocytes, across different pathological conditions will lead to greater understanding of the physiological role of HERV expression in health and disease.

Peripheral innate immunophenotype in neurodegenerative disease: blood-based profiles and links to survival.

The innate immune system plays an integral role in the progression of many neurodegenerative diseases. In addition to central innate immune cells (e.g., microglia), peripheral innate immune cells (e.g., blood monocytes, natural killer cells, and dendritic cells) may also differ in these conditions. However, the characterization of peripheral innate immune cell types across different neurodegenerative diseases remains incomplete. This study aimed to characterize peripheral innate immune profiles using flow cytometry for immunophenotyping of peripheral blood mononuclear cells in n = 148 people with Alzheimer's disease (AD), frontotemporal dementia (FTD), corticobasal syndrome (CBS), progressive supranuclear palsy (PSP), Lewy body dementia(LBD) as compared to n = 37 healthy controls. To compare groups, we used multivariate dissimilarity analysis and principal component analysis across 19 innate immune cell types. We identified pro-inflammatory profiles that significantly differ between patients with all-cause dementia and healthy controls, with some significant differences between patient groups. Regression analysis confirmed that time to death following the blood test correlated with the individuals' immune profile weighting, positively to TREM2+ and non-classical monocytes and negatively to classical monocytes. Taken together, these results describe transdiagnostic peripheral immune profiles and highlight the link between prognosis and the monocyte cellular subdivision and function (as measured by surface protein expression). The results suggest that blood-derived innate immune profiles can inform sub-populations of cells relevant for specific neurodegenerative diseases that are significantly linked to accelerated disease progression and worse survival outcomes across diagnoses. Blood-based innate immune profiles may contribute to enhanced precision medicine approaches in dementia, helping to identify and monitor therapeutic targets and stratify patients for candidate immunotherapies.

Analysis of lipid uptake, storage, and fatty acid oxidation by group 2 innate lymphoid cells.

Group 2 Innate Lymphoid Cells (ILC2) are critical drivers of both innate and adaptive type 2 immune responses, known to orchestrate processes involved in tissue restoration and wound healing. In addition, ILC2 have been implicated in chronic inflammatory barrier disorders in type 2 immunopathologies such as allergic rhinitis and asthma. ILC2 in the context of allergen-driven airway inflammation have recently been shown to influence local and systemic metabolism, as well as being rich in lipid-storing organelles called lipid droplets. However, mechanisms of ILC2 lipid anabolism and catabolism remain largely unknown and the impact of these metabolic processes in regulating ILC2 phenotypes and effector functions has not been extensively characterized. ILC2 phenotypes and effector functions are shaped by their metabolic status, and determining the metabolic requirements of ILC2 is critical in understanding their role in type 2 immune responses and their associated pathophysiology. We detail here a novel experimental method of implementing flow cytometry for large scale analysis of fatty acid uptake, storage of neutral lipids, and fatty acid oxidation in primary murine ILC2 with complementary morphological analysis of lipid storage using confocal microscopy. By combining flow cytometry and confocal microscopy, we can identify the metabolic lipid requirements for ILC2 functions as well as characterize the phenotype of lipid storage in ILC2. Linking lipid metabolism pathways to ILC2 phenotypes and effector functions is critical for the assessment of novel pharmaceutical strategies to regulate ILC2 functions in type 2 immunopathologies.

Expression of Epithelial Alarmin Receptor on Innate Lymphoid Cells Type 2 in Eosinophilic Chronic Obstructive Pulmonary Disease.

Studies have shown that eosinophilic COPD (eCOPD) is a distinct phenotype of the disease. It is well established that innate lymphoid cells are involved in the development of eosinophilic inflammation. Interleukin(IL)-25, thymic stromal lymphopoietin (TSLP) and IL-33 are a group of cytokines produced by epithelium in response to danger signals, e.g., cigarette smoke, and potent activators of ILC2s. In the present study, we examined circulating and sputum ILC2 numbers and expression of intracellular IL-5 as well as receptors for TSLP, IL-33 and IL-25 by ILC2s in non-atopic COPD patients with and without (neCOPD) airway eosinophilic inflammation and healthy smokers. In addition, we examined the association between ILC2s and clinical indicators of COPD burden (i.e., symptom intensity and risk of exacerbations). ILC2s were enumerated in peripheral blood and induced sputum by means of flow cytometry. We noted significantly greater numbers of airway IL-5+ILC2s and TSLPR+ILC2s in eCOPD compared with neCOPD (p < 0.05 and p < 0.01, respectively) and HSs (p < 0.001 for both). In addition, we showed that IL-5+ILC2s, IL-17RB+ILC2s and ST2+ILC2s are significantly increased in the sputum of eCOPD patients compared with HSs. In all COPD patients, sputum ILC2s positively correlated with sputum eosinophil percentage (r = 0.48, p = 0.002). We did not find any significant correlations between sputum ILC2s and dyspnea intensity as measured by the modified Medical Research Council scale (mMRC) and symptom intensity measured by the COPD Assessment Test (CAT). These results suggest the involvement of epithelial alarmin-activated ILC2s in the pathobiology of eosinophilic COPD.

Regulation of Bacteroides acidifaciens by the aryl hydrocarbon receptor in IL-22-producing immune cells has sex-dependent consequential impact on colitis.

Colitis is an inflammatory bowel disease (IBD) characterized by immune cell dysregulation and alterations in the gut microbiome. In our previous report, we showed a natural product in cruciferous vegetables and ligand of the aryl hydrocarbon receptor (AhR), indole-3-carbinol (I3C), was able to reduce colitis-induced disease severity and microbial dysbiosis in an interleukin-22 (IL-22) dependent manner. In the current study, we performed single-cell RNA sequencing (scRNAseq) from colonocytes during colitis induction and supplementation with I3C and show how this treatment alters expression of genes involved in IL-22 signaling. To further define the role of IL-22 signaling in I3C-mediated protection during colitis and disease-associated microbial dysbiosis, we generated mice with AhR deficiency in RAR-related orphan receptor c (Rorc)-expressing cells (AhR ΔRorc ) which depletes this receptor in immune cells involved in production of IL-22. Colitis was induced in wildtype (WT), AhR ΔRorc , and littermate (LM) mice with or without I3C treatment. Results showed AhR ΔRorc mice lost the efficacy effects of I3C treatment which correlated with a loss of ability to increase IL-22 by innate lymphoid type 3 (ILC3s), not T helper 22 (Th22) cells. 16S rRNA microbiome profiling studies showed AhR ΔRorc mice were unable to regulate disease-associated increases in Bacteroides, which differed between males and females. Lastly, inoculation with a specific disease-associated Bacteroides species, Bacteroides acidifaciens (B. acidifaciens), was shown to exacerbate colitis in females, but not males. Collectively, this report highlights the cell and sex-specific role of AhR in regulating microbes that can impact colitis disease.

IL-2 family cytokines IL-9 and IL-21 differentially regulate innate and adaptive type 2 immunity in asthma

BackgroundAsthma is often accompanied by type 2 immunity rich in IL-4, IL-5 and IL-13 cytokines produced by TH2 lymphocytes or type 2 innate lymphoid cells (ILC2s). Interleukin-2 family cytokines play a key role in the differentiation, homeostasis and effector function of innate and adaptive lymphocytes. ObjectiveIL-9 and IL-21 boost the activation and proliferation of TH2 and ILC2s, but the relative importance and potential synergism between these γc cytokines is currently unknown. MethodsUsing newly generated antibodies, we inhibited IL-9 and IL-21 alone or in combination, in various murine models of asthma. In a translational approach using segmental allergen challenge, we recently described elevated IL-9 levels in human allergic asthmatics in comparison to non-asthmatic controls. Here, we also measured IL-21 in both groups. ResultsIL-9 played a central role in controlling innate IL-33 induced lung inflammation by promoting proliferation and activation of ILC2s, in an IL-21 independent manner. Conversely, chronic house dust mite induced airway inflammation, mainly driven by adaptive immunity, was solely dependent on IL-21, that controlled TH2 activation, eosinophilia, total serum IgE and formation of tertiary lymphoid structures. In a model of innate on adaptive immunity driven by papain allergen, a clear synergy was found between both pathways, since combined anti-IL-9 or anti-IL-21 blockade was superior in reducing key asthma features. In human bronchoalveolar lavage (BAL) samples we measured elevated IL-21 protein within the allergic asthmatic group, compared with the allergic control group. We also found increased IL21R transcripts and predicted IL-21 ligand activity in various disease-associated cell subsets. ConclusionIL-9 and IL-21 play important and non-redundant roles in allergic asthma by boosting ILC2s and TH2 cells, revealing a dual IL-9 and IL-21 targeting strategy as a new and testable approach.

Monocyte Single-Cell Multimodal Profiling in Cardiovascular Disease Risk States.

Monocytes are a critical innate immune system cell type that serves homeostatic and immunoregulatory functions. They have been identified historically by the cell surface expression of CD14 and CD16. However, recent single-cell studies have revealed that they are much more heterogeneous than previously realized. We utilized cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and single-cell RNA sequencing to describe the comprehensive transcriptional and phenotypic landscape of 437 126 monocytes. This high-dimensional multimodal approach identified vast phenotypic diversity and functionally distinct subsets, including IFN-responsive, MHCIIhi (major histocompatibility complex class II), monocyte-platelet aggregates, as well as nonclassical, and several subpopulations of classical monocytes. Using flow cytometry, we validated the existence of MHCII+CD275+ MHCIIhi, CD42b+ monocyte-platelet aggregates, CD16+CD99- nonclassical monocytes, and CD99+ classical monocytes. Each subpopulation exhibited unique characteristics, developmental trajectories, transcriptional regulation, and tissue distribution. In addition, alterations associated with cardiovascular disease risk factors, including race, smoking, and hyperlipidemia were identified. Moreover, the effect of hyperlipidemia was recapitulated in mouse models of elevated cholesterol. This integrative and cross-species comparative analysis provides a new perspective on the comparison of alterations in monocytes in pathological conditions and offers insights into monocyte-driven mechanisms in cardiovascular disease and the potential for monocyte subpopulation targeted therapies.

Neurotensin-specific corticothalamic circuit regulates innate response conflict

Animals must simultaneously select and balance multiple action contingencies in ambiguous situations: for instance, evading danger during feeding. This has rarely been examined in the context of information selection; despite corticothalamic pathways that mediate sensory attention being relatively well characterized, neural mechanisms filtering conflicting actions remain unclear. Here, we develop a new loom/feed test to observe conflict between naturally induced fear and feeding and identify a novel anterior cingulate cortex (ACC) output to the ventral anterior and ventral lateral thalamus (VA/VL) that adjusts selectivity between these innate actions. Using micro-endoscopy and fiber photometry, we reveal that activity in corticofugal outputs was lowered during unbalanced/singularly occupied periods, as were the resulting decreased thalamic initiation-related signals for less-favored actions, suggesting that the integration of ACC-thalamic firing may directly regulate the output of behavior choices. Accordingly, the optoinhibition of ACC-VA/VL circuits induced high bias toward feeding at the expense of defense. To identify upstream "commander" cortical cells gating this output, we established dual-order tracing (DOT)-translating ribosome affinity purification (TRAP)-a scheme to label upstream neurons with transcriptome analysis-and found a novel population of neurotensin-positive interneurons (ACCNts). The photoexcitation of ACCNts cells indeed caused similarly hyper-selective behaviors. Collectively, this new "corticofugal action filter" scheme suggests that communication in multi-step cingulate circuits may critically influence the summation of motor signals in thalamic outputs, regulating bias between innate action types.

Assessing the implications of sentinel lymph node removal in cervical cancer: an immunogenetic perspective – a SENTICOL ancillary study

BackgroundCervical cancer’s lymphatic spread primarily begins from the sentinel lymph nodes (SLNs), underlining their pivotal role in disease metastasis. However, these nodes’ immune gene expression profiles and immunoregulation mechanisms have...

Maresin-1 promotes neuroprotection and prevents disease progression in experimental models of multiple sclerosis through metabolic reprogramming and shaping innate and adaptive disease-associated cell types.

Multiple sclerosis (MS) is one of the most common inflammatory neurodegenerative diseases in young adults and causes neurological abnormalities and disability. We studied the effect of maresin 1 (MaR1) on the progression of disease in a relapsing-remitting form of experimental allergic encephalomyelitis (RR-EAE). Treatment with MaR1 in RR-EAE accelerated inflammation resolution, protected against neurological deficits, and delayed disease progression by decreasing immune cell infiltration (CD4+IL17+ and CD4+IFNγ+) into the CNS. Furthermore, the administration of MaR1 increased the production of IL-10, predominantly in macrophages and CD4+ cells. However, neutralizing IL-10 with an anti-IL-10 antibody abolished the protective effect of MaR1 on RR-EAE, suggesting that IL-10 plays a role in mediating the protective effect of MaR1 on EAE. Metabolism is rapidly becoming recognized as an important factor influencing the effector function of many immune cells. Using cutting-edge metabolic assays, our study revealed that compared with vehicle treatment, MaR1 treatment effectively restored the metabolic dysregulation observed in CD4+ cells, macrophages, and microglia in the treated group. Furthermore, MaR1 treatment reversed defective efferocytosis in EAE mice, which was potentially facilitated by the induction of metabolic alterations in macrophages and microglia. MaR1 treatment also protected myelin in the EAE group and regulated the metabolism of O4+ oligodendrocytes by restoring metabolic dysregulation through improved mitochondrial function and decreased glycolysis. Overall, in a preclinical MS animal model, MaR1 treatment produced anti-inflammatory and neuroprotective effects. It also triggered metabolic reprogramming in disease-associated cell types, accelerated efferocytosis, and preserved myelination. These data support that MaR1 has potential as a novel treatment agent for MS and other autoimmune diseases.

Insulin and leptin oscillations license food-entrained browning and metabolic flexibility

Timed feeding drives adipose browning, although the integrative mechanisms for the same remain unclear. Here, we show that twice-a-night (TAN) feeding generates biphasic oscillations of circulating insulin and leptin, representing their entrainment by timed feeding. Insulin and leptin surges lead to marked cellular, functional, and metabolic remodeling of subcutaneous white adipose tissue (sWAT), resulting in increased energy expenditure. Single-cell RNA-sequencing (scRNA-seq) analyses and flow cytometry demonstrate a role for insulin and leptin surges in innate lymphoid type 2 (ILC2) cell recruitment and sWAT browning, since sWAT depot denervation or loss of leptin or insulin receptor signaling or ILC2 recruitment each dampens TAN feeding-induced sWAT remodeling and energy expenditure. Consistently, recreating insulin and leptin oscillations via once-a-day timed co-injections is sufficient to favorably remodel innervated sWAT. Innervation is necessary for sWAT remodeling, since denervation of sWAT, but not brown adipose tissue (BAT), blocks TAN-induced sWAT remodeling and resolution of inflammation. In sum, reorganization of nutrient-sensitive pathways remodels sWAT and drives the metabolic benefits of timed feeding.

Human epicardial adipose tissue contains innate and adaptive lymphoid cells and a higher proportion of innate type 2 lymphoid cells compared to other adipose tissues

ImportanceEpicardial adipose tissue (EAT) is a biologically active organ surrounding myocardium and coronary arteries that has been associated with coronary artery disease (CAD) and atrial fibrillation. Previous work has shown that EAT exhibits beige features. ObjectiveOur objective was to determine whether the stromal vascular fraction of the human EAT contains innate or adaptive lymphoid cells compared to thoracic subcutaneous (thSAT), visceral abdominal (VAT) and subcutaneous abdominal (abSAT). ParticipantsNew pangenomic microarray analysis was performed on previous transcriptomic dataset using significance analysis of microarray and ingenuity pathway analysis (n=41) to identify specific immune signature and its link with browning genes. EAT, thSAT, VAT and abSAT samples from explanted patients with severe cardiomyopathies and multi-organ donor patients (n=17) were used for flow cytometry (FC) immunophenotyping assay. Patients were on average 55±16 years-old; 47% had hypertension and 6% CAD. Phenotypic adaptive and innate immune profiles were performed using a TBNK panel and a specific ILC1-2-3 panel including CD127, CD117, CRTH2 (CD294) and activation markers such as CD25 and CD69. ResultsTranscriptomic analysis showed a significant positive correlation between the TH2 immune pathway (IL-4, IL-5, IL-13, IL-25, IL-33) and browning genes (UCP-1, PRDM16, TMEM26, CITED1, TBX1) in EAT versus thSAT (R=0.82, P<0.0001). Regarding adaptive immune cells, a preponderance of CD8T cells, a contingent of CD4T cells, and a few B cells were observed in all ATs (P<0.0001). In innate lymphoid cells (ILCs), an increase was observed in visceral ATs (i.e. EAT; VAT 35±8ILCs/g of tissue) compared to their subcutaneous counterpart (i.e. thSAT+abSAT: 8±3 ILCs/g of AT, P=0.002), with a difference in the proportion of the 3 subtypes of ILCs (ILC1>ILC3>ILC2). In addition, we observed an increase in EAT-ILC2 compared to other ATs and almost all these EAT-ILC2 expressed CD69 and/or CD25 activation markers (99.75±0.16%; P<0.0001). We also observed more NKs in EAT and VAT (1520±71 cells/g of AT) than in SATs (562±17 cells/g of AT); P=0.01. ConclusionThis is the first study to provide a comparison between innate and adaptive lymphoid cells in human epicardial versus abdominal or thoracic adipose tissues. Further studies are ongoing to decipher whether these cells could be involved in EAT beiging. Trial RegistrationCODECOH No. DC-2021-4518 The French agency of biomedicine PFS21-005.

Administration of intratumoral GD2-directed interleukin-2 immunocytokine and local radiation therapy to activate immune rejection of spontaneous canine melanoma.

Canine malignant melanoma provides a clinically relevant, large animal parallel patient population to study the GD2-reactive hu14.18-IL-2 immunocytokine as it is similar to human melanoma and expresses GD2. The objectives of this study were to evaluate safety, radiation fractionation, and identify informative biomarkers of an in-situ tumor vaccine involving local radiation therapy plus intratumoral-immunocytokine in melanoma tumor-bearing dogs. Twelve dogs (six dogs/arm) with locally advanced or metastatic melanoma were randomized to receive a single 8 Gy fraction (arm A) or three 8 Gy fractions over 1 week (arm B) to the primary site and regional lymph nodes (when clinically involved) with the single or last fraction 5 days before intratumoral-immunocytokine at 12 mg/m 2 on 3 consecutive days. Serial tumor biopsies were obtained. All 12 dogs completed protocol treatment, and none experienced significant or unexpected adverse events. Evidence of antitumor activity includes one dog with a complete response at day 60, one dog with a partial response at day 60, and four dogs with mixed responses. Histology of serial biopsies shows a variably timed increase in intratumoral lymphocytic inflammation in some dogs. Canine NanoString analyses of serial biopsies identified changes in gene signatures of innate and adaptive cell types versus baseline. There were no significant differences in NanoString results between arm A and arm B. We conclude that intratumoral-immunocytokine in combination with local radiation therapy in canine melanoma is well tolerated and has antitumor activity with the potential to inform clinical development in melanoma patients.

Hepatocellular carcinoma after direct-acting antivirals for hepatitis C is associated with KIR-HLA types predicting weak NK cell-mediated immunity.

Second-generation direct-acting antivirals (2G DAA) to cure HCV have led to dramatic clinical improvements. HCV-associated hepatocellular carcinoma (HCC), however, remains common. Impaired immune tumor surveillance may play a role in HCC development. Our cohort evaluated the effects of innate immune types and clinical variables on outcomes including HCC. Participants underwent full HLA class I/KIR typing and long-term HCV follow-up. A total of 353 HCV+ participants were followed for a mean of 7 years. Cirrhosis: 25% at baseline, developed in 12% during follow-up. 158 participants received 2G DAA therapy. HCC developed without HCV therapy in 20 subjects, 24 HCC after HCV therapy, and 10 of these after 2G DAA. Two predictors of HCC among 2G DAA-treated patients: cirrhosis (OR, 10.0, p = 0.002) and HLA/KIR profiles predicting weak natural killer (NK) cell-mediated immunity (NK cell complementation groups 6, 9, 11, 12, OR of 5.1, p = 0.02). Without 2G DAA therapy: cirrhosis was the main clinical predictor of HCC (OR, 30.8, p < 0.0001), and weak NK-cell-mediated immunity did not predict HCC. Cirrhosis is the main risk state predisposing to HCC, but weak NK-cell-mediated immunity may predispose to post-2G DAA HCC more than intermediate or strong NK-cell-mediated immunity.

Non-specific effects of inactivated Mycobacterium bovis oral and parenteral treatment in a rabbit scabies model

Tuberculosis BCG vaccination induced non-specific protective effects in humans led to postulate the concept of trained immunity (TRAIM) as an innate type of immune mechanism that triggered by a pathogen, protects against others. Killed vaccines have been considered not to be effective. However, field efficacy of a commercial vaccine against paratuberculosis, as well as of a recently developed M. bovis heat-inactivated vaccine (HIMB) prompted to test whether it could also induce TRAIM. To this, we used a sarcoptic mange rabbit model. Twenty-four weaned rabbits were treated orally or subcutaneously with a suspension of either HIMB (107 UFC) or placebo. Eighty-four days later the animals were challenged with approximately 5000 S. scabiei mites on the left hind limb. Skin lesion extension was measured every 2 weeks until 92 days post-infection (dpi). Two animals were killed at 77 dpi because of extensive skin damage. The rest were euthanized and necropsied and the lesion area and the mite burden per squared cm were estimated. Specific humoral immune responses to S. scabiei and to M. bovis were investigated with the corresponding specific ELISA tests. Subcutaneously and orally HIMB vaccinated animals compared with placebo showed reduced lesion scores (up to 74% and 62%, respectively) and mite counts (−170% and 39%, respectively). This, together with a significant positive correlation (r = 0.6276, p = 0.0031) between tuberculosis-specific antibodies and mite count at 92 dpi supported the hypothesis of non-specific effects of killed mycobacterial vaccination. Further research is needed to better understand this mechanism to maximize cross protection.

Abstract 6733: Multiomics analysis of impact of CD8+ and other cell populations in STING agonist treated ex vivo mesothelioma samples

Abstract Background: Collective evidence highlights that agonism of the host STING pathway plays a critical role in eliciting robust and durable anti-tumor immunity. Dazostinag (TAK-676) is a novel systemically delivered STING agonist that ignites the innate immune system and mobilizes adaptive immunity. Preclinical studies with dazostinag demonstrated a strong, persistent CD8+ T cell anti-tumor response driven by innate immune derived type I IFN and pro-inflammatory cytokines. The role of CD8+ T cells in response to dazostinag was further supported by a previously demonstrated correlation between response and CD8+ T cell frequency in a patient-derived organotypic tumor spheroids (PDOTS) model of mesothelioma. Within the current work, we sought to determine the extent to which response to dazostinag in mesothelioma depends on CD8+ T cells versus the contribution of other immune cells. Methods: Surgical mesothelioma cases were collected along with clinicopathological variables at Brigham and Women’s Hospital under an IRB-approved protocol. PDOTS were generated as described previously. Ex vivo response was determined by fluorescent life/dead imaging, and orthogonally evaluated by multiplexed cytokine array, immunofluorescence, and flow cytometry; subset of tumors was studied using single cell RNA sequencing (scRNAseq). Results: Dazostinag-driven tumor cell killing was evaluated in 20 mesothelioma PDOTS. 11 samples were characterized as responders (R) with a cut-off of &amp;gt;30% reduction in live cell area; 9 as non-responders (NR). In 12 out of 20 PDOTS, CD8 neutralization effect was assessed: four were TAK-676 NR, and out of 8 R 4 had at least 20% killing abrogated by aCD8. Effect did not depend on the baseline amount of CD8+ T cells in the sample. scRNAseq analysis showed that R and NR T cells have different activation/exhaustion phenotypes and varying levels of interferon stimulated genes (ISG) in response to STING agonism. Other cell populations emerged as candidates for dazostinag resistance: NR cases carried remodeled immunosuppressive myeloid cells or matrix metalloproteinases-expressing fibroblasts. In general, myeloid cell abundance at baseline characterized by flow had a trend in negatively correlating with dazostinag response. Intrinsic expression of type I interferons shown by multiplexed cytokine array was linked to dazostinag sensitivity (R vs. NR IFN-β p=0.028; IFNα2a p=0.016) and can potentially be used as a predictor for response. Conclusion: We evaluated immune modulations in mesothelioma after treatment with a systemic STING agonist dazostinag using human tumor ex vivo culture. Innate interferon type I expression was higher in responding samples. TAK-676 efficacy was partially reduced in absence of effector lymphocytes, however other cells, such as immunosuppressive myeloid cells and fibroblasts, can have a negative impact on dazostinag response in mesothelioma. Citation Format: Elena Ivanova, Sung R. Park, Ari P. Zlota, Nathaniel Spicer, Minh Ha, Sophie Kivlehan, Iliana Gjeci, Simona Innocenti, Lauren Zasadil, Patrick Lizotte, Michael Y. Tolstorukov, Kai Ding, Adnan O. Abu-Yousif, Jeffrey Raizer, Vicky A. Appleman, Raphael Bueno, David A. Barbie, Cloud P. Paweletz. Multiomics analysis of impact of CD8+ and other cell populations in STING agonist treated ex vivo mesothelioma samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6733.

Molecular Profiling of Axial Spondyloarthritis Patients Reveals an Association between Innate and Adaptive Cell Populations and Therapeutic Response to Tumor Necrosis Factor Inhibitors.

This study aims at identifying molecular biomarkers differentiating responders and non-responders to treatment with Tumor Necrosis Factor inhibitors (TNFi) among patients with axial spondyloarthritis (axSpA). Whole blood mRNA and plasma proteins were measured in a cohort of biologic-naïve axSpA patients (n = 35), pre and post (14 weeks) TNFi treatment with adalimumab. Differential expression analysis was used to identify the most enriched pathways and in predictive models to distinguish responses to TNFi. A treatment-associated signature suggests a reduction in inflammatory activity. We found transcripts and proteins robustly differentially expressed between baseline and week 14 in responders. C-reactive protein (CRP) and Haptoglobin (HP) proteins showed strong and early decrease in the plasma of axSpA patients, while a cluster of apolipoproteins (APOD, APOA2, APOA1) showed increased expression at week 14. Responders to TNFi treatment present higher levels of markers of innate immunity at baseline, and lower levels of adaptive immunity markers, particularly B-cells. A logistic regression model incorporating ASDAS-CRP, gender, and AFF3, the top differentially expressed gene at baseline, enabled an accurate prediction of response to adalimumab in our cohort (AUC = 0.97). In conclusion, innate and adaptive immune cell type composition at baseline may be a major contributor to response to adalimumab in axSpA patients. A model including clinical and gene expression variables should also be considered.