Immune Crosstalk Research Articles

Single-cell RNA-sequencing analysis of immune and mesenchymal cell crosstalk in the developing enthesis

Autoimmunity underlies many painful disorders, such as enthesopathies, which localize to the enthesis. From infiltration of the synovium and axial skeleton by B cells, to disturbances in the ratio of M1/M2 enthesis macrophages, to CD8 + T cell mediated inflammation, autoimmune dysregulation is becoming increasingly well characterized in enthesopathies. Tissue resident B cells, macrophages, neutrophils, and T cells have also been localized in healthy human entheses. However, the potential developmental origins, presence, and role of immune cells (ICs) in enthesis development is not known. Here, we use single-cell RNA-sequencing analysis to describe IC subtypes present in the enthesis before, during, and after mineralization, and to infer regulatory interactions between ICs and mesenchymal cells (MCs). We report the presence of nine phenotypically distinct IC subtypes, including B cells, macrophages, neutrophils, and T cells. We find that specific IC subtypes may promote MC-proliferation and differentiation, and that MCs may regulate IC phenotype and autoimmunity. Our findings suggest that bidirectional regulatory interactions between ICs and MCs may be important to enthesis mineralization, and suggest that progenitor MCs have a unique ability to limit autoimmunity during development.

Therapeutic Immunomodulation of Tumor-Lymphatic Crosstalk via Intratumoral Immunotherapy.

Intra- and peritumoral lymphatics and tumor-draining lymph nodes play major roles in mediating the adaptive immune response to cancer immunotherapy. Despite this, current paradigms of clinical cancer management seldom seek to therapeutically modulate tumor-lymphatic immune crosstalk. This review explores recent developments that set the stage for how this regulatory axis can be therapeutically manipulated, with a particular emphasis on tumor-localized immunomodulation. Building on this idea, the nature of tumor-lymphatic immune crosstalk and relevant immunotherapeutic targets and pathways are reviewed, with a focus on their translational potential. Engineered drug delivery systems that enhance intratumoral immunotherapy by improving drug delivery to both the tumor and lymph nodes are also highlighted.

A compartmentalized microfluidic platform to investigate immune cells cross-talk in rheumatoid arthritis

The dysregulation of the immune system plays a crucial role in the pathogenesis of manyfold diseases, among which we find rheumatoid arthritis (RA), an autoimmune disease characterized by chronic inflammation in synovial joints, leading to pain and disability. Immune cells such as pro-inflammatory macrophages and T helper 1 (Th1) cells drive the inflammatory cascade. Thus, including immune system inin vitromodels is pivotal to recapitulate and better understand the complex interactions between these immune cell subsets and their secreted mediators. Here, a compartmentalized microfluidic platform is presented, for precise confinement of circulating immune cells in organs-on-chip. The integration of innovative normally-closed sieving valves allows, through minimal waste of biological material, to co-culture different immune cell types (e.g. macrophages and Th1). Moreover, the platform allows to stimulate cell subsets separately, and to assess their cross-talk at desired time points. Functional validation of the platform demonstrates its ability to create stable chemotactic gradients, allowing for induction and evaluation of Th1 cells migration. In a proof-of-concept study, the platform allowed to assess Th1 T cells migration towards pro-inflammatory macrophages, thus replicating a characteristic interaction among immune cells triggered during RA onset. These results thus support the suitability of the platform to study immune cells cross-talk and migration phenomena, being potentially applicable to a manyfold immune cell mechanisms, both involved in RA progression and in different immune-mediated pathologies.

Sleep and Immune System Crosstalk: Implications for Inflammatory Homeostasis and Disease Pathogenesis.

Sleep and immune function are interconnected aspects of health that mutually impact each other in disease development and inflammatory homeostasis. Different aspects of immunology are regulated by different sleep characteristics, impacting on specific aspects of immune function including cytokine production and T-cell activity. Ongoing disruptions of sleep have been linked to heightened inflammation and are suspected in the pathogenesis and disease course of a range of life-style-related illnesses, including diabetes and neurodegenerative diseases. This review provides a comprehensive overview of knowledge on the interaction of sleep with the immune system, its modulation of inflammatory balance, and the pathogenesis of many diseases. It emphasizes how sleep deficiency compromises immune function by means of a systemic, low-grade inflammatory response, while adequate sleep promotes intense immune responses and thus enables efficient pathogen clearance and the maintenance of immune memory. The mutual influence of sleep on the immune system underlines its critical involvement in health preservation and the course of disease. Sleep plays an indispensable role in immune health, mediating the efficiency of immune responses and the course of the regulation of inflammation. Chronic sleep deprivation can result in a low-grade inflammation that substantially contributes to the onset and exacerbation of metabolic and neurodegenerative disorders. The intimate linkage between sleep and immune function can be one strategic approach to therapy, improving health outcomes by leveraging this sleep-immune connection.

Focal adhesion kinase-mediated interaction between tumor and immune cells in the tumor microenvironment: implications for cancer-associated therapies and tumor progression.

Focal adhesion kinase (FAK) expression has been linked to tumor growth, immunosuppression, metastasis, angiogenesis, and therapeutic resistance through kinase-dependent and kinase scaffolding functions in the nucleus and cytoplasm. Hence, targeting FAK alone or with other agents has gained attention as a potential therapeutic strategy. Moreover, mounting evidence shows that FAK activity can influence the tumor immune microenvironment crosstalk to support tumor progression. Recently, tumor immune microenvironment interaction orchestrators have shown to be promising therapeutic agents for cancer immunotherapies. Therefore, this review highlights how FAK regulates the tumor immune microenvironment interplay to promote tumor immune evasive mechanisms and their potential for combination therapies with standard cancer treatments.

Focal Adhesion Kinase and Colony Stimulating Factors: Intestinal Homeostasis and Innate Immunity Crosstalk.

Thousands struggle with acute and chronic intestinal injury due to various causes. Epithelial intestinal healing is dependent on phenotypic transitions to a mobile phenotype. Focal adhesion kinase (FAK) is a ubiquitous protein that is essential for cell mobility. This phenotype change is mediated by FAK activation and proves to be a promising target for pharmaceutical intervention. While FAK is crucial for intestinal healing, new evidence connects FAK with innate immunity and the importance it plays in macrophage/monocyte chemotaxis, as well as other intracellular signaling cascades. These cascades play a part in macrophage/monocyte polarization, maturation, and inflammation that is associated with intestinal injury. Colony stimulating factors (CSFs) such as macrophage colony stimulating factor (M-CSF/CSF-1) and granulocyte macrophage colony stimulating factor (GM-CSF/CSF-2) play a critical role in maintaining homeostasis within intestinal mucosa by crosstalk capabilities between macrophages and epithelial cells. The communication between these cells is imperative in orchestrating healing upon injury. Diving deeper into these connections may allow us a greater insight into the role that our immune system plays in healing, as well as a better comprehension of inflammatory diseases of the gut.

Beneficial effects of Dendrobium officinale National Herbal Drink on metabolic immune crosstalk via regulate SCFAs-Th17/Treg

The development of gut-liver axis metabolic immune crosstalk is intimately associated with intestinal barrier disorder, intestinal SCFAs-Th17/Treg immunological imbalance, and disorders of the gut microbiota. Prior research has discovered that Dendrobium officinale National Herbal Drink (NHD), a traditional Chinese medicine drink with enhanced immunity, may enhance the immunological response in animals with impaired immune systems brought on by cyclophosphamide by repairing intestinal barrier function and controlling turbulence in the gut microbiota. However, whether NHD can further improve the gut-liver axis metabolic immune crosstalk and its related mechanisms need to be systematically studied. The purpose of this study is to clarify the function and mechanism of NHD in enhancing the gut-liver axis metabolic immunological crosstalk brought on by excessive alcohol intake. In this work, we set up a mouse model to analyze the metabolic and immunological crosstalk involving the gut-liver axis across 7 weeks of continuous, excessive drinking. At the same time, high and low doses (20,10 ml/kg) of NHD were given by gavage. The effect of NHD on improving the metabolism of gut-liver axis was evaluated by blood lipid, liver lipid deposition, liver function and intestinal pathophysiology. By measuring serum immunological indices, intestinal barrier, and intestinal immune barrier, the impact of NHD on enhancing immune and intestinal barrier function was assessed. Furthermore, immunohistochemistry, immunofluorescence, 16S rRNA, Western blot, q-PCR and other methods were used to detect gut microbiota, SCFAs-GPR41/43 pathway, intestinal Th17/Treg immune cells and PPAR-α-NPC1L1/SREBP1 pathway to elucidate the mechanism by which NHD enhances the gut-liver axis' metabolic immune crosstalk. Our study demonstrated that NHD has the potential to improve the pathophysiological damage caused by gut-liver axis in model mice. NHD also ameliorated the disorder of lipid metabolism. In addition, it regulated the levels of peripheral blood T cell immunity and serum immune factors. And NHD can restore intestinal mechanical and immune barrier damage. NHD has a favorable impact on the quantity of beneficial bacteria, including uncultured_bacterium_g__norank_f__muribaculacea and uncultured_bacterium_g__Turicibacter. Additionally, it raised the model mice's levels of SCFAs (n-butyric acid, isovaleric acid, etc.). This resulted in the promotion of intestinal GPR41/43-ERK1/2 expression and the reshaping of intestinal CD4+T cell Th17/Treg homeostasis. As a consequence, colon IL-22 and IL-10 levels increased, while colon IL-17A levels decreased. Lastly, NHD raised the amount of intestinal IAP/LPS, regulated the development of PPAR-α-NPC1L1/SREBP1 pathway in gut-liver axis, and improve lipid metabolism disorder. Our study found that NHD can improve the gut-liver axis metabolic immune crosstalk in model mice caused by excessive drinking. The mechanism might be connected to how NHD controls gut microbiota disorders in model mice, the activation of intestinal SCFAs-GPR41/43 pathway, the remodeling of Th17/Treg immune homeostasis of intestinal CD4+T cells, the improvement of IAP/LPS abnormality, and further mediating the PPAR-α-NPC1L1/SREBP1 pathway of lipid metabolism in gut-liver axis.

The path to next-generation disease-modifying immunomodulatory combination therapies in Alzheimer's disease.

The cautious optimism following recent anti-amyloid therapeutic trials for Alzheimer's disease (AD) provides a glimmer of hope after years of disappointment. Although these encouraging results represent discernible progress, they also highlight the need to enhance further the still modest clinical efficacy of current disease-modifying immunotherapies. Here, we highlight crucial milestones essential for advancing precision medicine in AD. These include reevaluating the choice of therapeutic targets by considering the key role of both central neuroinflammation and peripheral immunity in disease pathogenesis, refining patient stratification by further defining the inflammatory component within the forthcoming ATN(I) (amyloid, tau and neurodegeneration (and inflammation)) classification of AD biomarkers and defining more accurate clinical outcomes and prognostic biomarkers that better reflect disease heterogeneity. Next-generation immunotherapies will need to go beyond the current antibody-only approach by simultaneously targeting pathological proteins together with innate neuroinflammation and/or peripheral-central immune crosstalk. Such innovative immunomodulatory combination therapy approaches should be evaluated in appropriately redesigned clinical therapeutic trials, which must carefully integrate the neuroimmune component.

Bone voyage: immune crosstalk sets sail.

Bone voyage: immune crosstalk sets sail.

The mechanisms of white matter injury and immune system crosstalk in promoting the progression of Parkinson's disease: a narrative review.

Parkinson's disease (PD) is neurodegenerative disease in middle-aged and elderly people with some pathological mechanisms including immune disorder, neuroinflammation, white matter injury and abnormal aggregation of alpha-synuclein, etc. New research suggests that white matter injury may be important in the development of PD, but how inflammation, the immune system, and white matter damage interact to harm dopamine neurons is not yet understood. Therefore, it is particularly important to delve into the crosstalk between immune cells in the central and peripheral nervous system based on the study of white matter damage in PD. This crosstalk could not only exacerbate the pathological process of PD but may also reveal new therapeutic targets. By understanding how immune cells penetrate through the blood-brain barrier and activate inflammatory responses within the central nervous system, we can better grasp the impact of structural destruction of white matter in PD and explore how this process can be modulated to mitigate or combat disease progression. Microglia, astrocytes, oligodendrocytes and peripheral immune cells (especially T cells) play a central role in its pathological process where these immune cells produce and respond to pro-inflammatory cytokines such as tumor necrosis factor (TNF-α), interleukin-1β(IL-1β) and interleukin-6(IL-6), and white matter injury causes microglia to become pro-inflammatory and release inflammatory mediators, which attract more immune cells to the damaged area, increasing the inflammatory response. Moreover, white matter damage also causes dysfunction of blood-brain barrier, allows peripheral immune cells and inflammatory factors to invade the brain further, and enhances microglia activation forming a vicious circle that intensifies neuroinflammation. And these factors collectively promote the neuroinflammatory environment and neurodegeneration changes of PD. Overall, these findings not only deepen our understanding of the complexity of PD, but also provide new targets for the development of therapeutic strategies focused on inflammation and immune regulation mechanisms. In summary, this review provided the theoretical basis for clarifying the pathogenesis of PD, summarized the association between white matter damage and the immune cells in the central and peripheral nervous systems, and then emphasized their potential specific mechanisms of achieving crosstalk with further aggravating the pathological process of PD.

#2027 Activation of histone deacetylase-1 in fibroblasts and pericytes induces renal interstitial fibrosis after ischemia-reperfusion injury

Abstract Background and Aims Following an acute kidney ischemic event the chromatin remodelling enzyme, histone deacetylase-1 (HDAC1), is activated in many cell types of the kidney including fibroblasts and pericytes. During kidney injury, the fibroblasts/pericytes differentiate into the myofibroblasts and produce extracellular matrix that can lead to a permanent decline in kidney function. Pharmacological inhibition of HDACs can attenuate ischemia-reperfusion-injury (IRI) mediated interstitial fibrosis. The hypothesis of this study was that fibroblast/pericyte HDAC1 activation promotes myofibroblast differentiation and interstitial fibrosis. Method Tamoxifen inducible, fibroblast/pericyte HDAC1 knockout (KO) mice (HDAC1Fl/Fl; Col1a2-CreEr) and littermate controls (HDAC1Fl/Fl) were used. Male and female mice were given tamoxifen by ip at 8-10 weeks of age and IRI or sham surgery completed after a 2-week tamoxifen washout period. A mild, 18 min, bilateral, warm IRI model was used, and samples collected over 4 weeks. Glomerular filtration rate (GFR) was assessed using transdermal FITC-Sinistrin clearance. In vitro experiments with kidney fibroblast cells (NRK49F) overexpressing empty vector or HDAC1 were used for RNA-sequencing to determine the HDAC1-dependent myofibroblast transcriptome. Results We confirmed that HDAC1 was knockdown in myofibroblast cells from KO mice with co-localization of HDAC1 and PDGFRβ or α-smooth muscle actin in the kidneys of IRI mice. Twenty-four hours post-surgery, all IRI mice exhibited 2-3 times increase in serum creatinine and a ∼50% reduction in GFR compared to sham mice (control IRI = 513 ± 129 μl /min/100 g body mass, control sham = 1010 ± 218, n = 4-5, p = 0.02; KO IRI = 529 ± 116, KO sham = 1187 ± 59 μl/min/100 g body mass, n = 4-5, p = 0.004). Subsequent GFR measurements over the next 4 weeks showed a gradual recovery of renal function of IRI mice starting at week 2 and fully restored to sham values by week 4. However, the male control IRI mice had significant focal interstitial fibrosis 2- and 4-weeks post injury (4-week data: sham = 0.3 ± 0.09 % of cortical area, IRI = 1.2 ± 0.2%, n = 6/group, p = 0.0011) but this was attenuated in the KO male IRI mice (sham = 0.4 ± 0.07% n = 4, IRI = 0.5 ± 0.1%, n = 7, p = 0.93). The female mice, regardless of genotype, had very little kidney damage or interstitial fibrosis at 4 weeks. Transcriptomes of NRK49F cells overexpressing HDAC1 had an upregulation of 55 genes related to pro-inflammatory cytokines and tumour necrosis factor signalling and downregulation of 367 genes related to cell junctions, cytoskeleton proteins, and cell adhesion compared to the empty vector control cells. Fibroblast proliferation rates were not statistically different among the groups, suggesting HDAC1 does not affect myofibroblast proliferation but rather pro-inflammatory gene expression. Conclusion In male mice, fibroblast/pericyte activation of HDAC1 leads to the production of pro-inflammatory cytokines that we predict will lead to immune cell infiltration and crosstalk that leads to the excessive production of interstitial fibrosis following IRI.

Resident and recruited macrophages differentially contribute to cardiac healing after myocardial ischemia.

Cardiac macrophages are heterogenous in phenotype and functions, which has been associated with differences in their ontogeny. Despite extensive research, our understanding of the precise role of different subsets of macrophages in ischemia/reperfusion (I/R) injury remains incomplete. We here investigated macrophage lineages and ablated tissue macrophages in homeostasis and after I/R injury in a CSF1R-dependent manner. Genomic deletion of a fms-intronic regulatory element (FIRE) in the Csf1r locus resulted in specific absence of resident homeostatic and antigen-presenting macrophages, without affecting the recruitment of monocyte-derived macrophages to the infarcted heart. Specific absence of homeostatic, monocyte-independent macrophages altered the immune cell crosstalk in response to injury and induced proinflammatory neutrophil polarization, resulting in impaired cardiac remodeling without influencing infarct size. In contrast, continuous CSF1R inhibition led to depletion of both resident and recruited macrophage populations. This augmented adverse remodeling after I/R and led to an increased infarct size and deterioration of cardiac function. In summary, resident macrophages orchestrate inflammatory responses improving cardiac remodeling, while recruited macrophages determine infarct size after I/R injury. These findings attribute distinct beneficial effects to different macrophage populations in the context of myocardial infarction.

Abstract PO4-28-10: Single-Cell Transcriptomic Assessment of Stromal and Immune Crosstalk on Breast Cancer Invasion Using 3D Tumor-On-Chip Model

Abstract Introduction: Our understanding of cancer has evolved over the last decades, with treatments increasingly targeting stromal cells in the tumor microenvironment (TME). The TME comprises transformed cancer cells and various non-cancerous cell types such as fibroblasts, macrophages, and endothelial cells. Despite this understanding, disrupting the tumor-stromal interactions are not targeted mainstream, as the stromal cells in the TME are genetically stable and can induce both beneficial and adverse effect on tumorigenesis. Thus, ex vivo tumor models that can faithfully recapitulate these critical tumor-stroma interactions are required to mechanistically understand the multifaceted reaction occurring within the TME. In this study, we established an innovative and multi-cellular TME on-a-chip model and assessed the synergistic effect of patient-derived Cancer Associated Fibroblast (CAF’s) and macrophages (M𝟇) on breast cancer progression. Materials and Methods: The influence of stromal components on cancer progression was assessed using the microfluidic model that allows spatial organization of various cell types. Tri-culture invasion model was established using Sum159 breast cancer cells, patient-derived CAFs, and THP-1 derived naïve (M𝟇) macrophages. Sum159 mixed with collagen and Matrigel forms the tumor region, while CAF’s and M𝟇 embedded in collagen form the stromal region. Functional and real-time assessments such as migration, morphometric analysis, and proliferation studies were conducted to understand the stromal influence on cancer cells. Gene expression analysis on an array of macrophage activation genes was performed to identify the polarized state of the macrophages after cellular interactions. Finally, Single Cell-RNA sequencing was carried out to unveil the drivers of stromal-immune crosstalk on invasion. Results and Discussion: Using the TME on-a-chip model, we compared cancer cell invasion across 4 different culture conditions namely monoculture (Sum only), co-culture (Sum+CAF, Sum+ M𝟇) and tri-culture (Sum+CAF+ M𝟇). Cancer cell migration was significantly increased in the triculture condition compared to others and stromal components also enhanced cancer cell proliferation. Hierarchical clustering of migration and morphological changes demonstrated that the co-culture conditions grouped together while CAF+ M𝟇 exerted the highest influence on cell migration and clustered separately. To understand the bi-directional communication between tumor and stroma, we assessed the polarized states of naïve macrophages using qPCR. Gene expression analysis showed that tumor-educated macrophages showed characteristics of M1 and M2 phenotypes and communicated reciprocally with tumor cells. Finally, to investigate the molecular mechanics of complex cellular interactions, we performed sc-RNA sequencing on cells extracted from the device and 2D cells (controls). Unsupervised clustering identified 11 different clusters, categorizing cells as Sum159 (tumor cells), CAFs (Stromal), and M𝟇 (Immune) based on cell-specific markers. Notably, we observed a noticeable shift in the clusters of Sum159 cells between 2D and 3D samples, emphasizing the difference between these two cultures. Conclusion: Using the TME on-a-chip, we confirmed the critical influence of microenvironmental components on tumor progression through functional assessment. Gene-expression analysis revealed bi-directional communication between noncancerous stromal components and tumor cells highlighting the importance of microenvironmental crosstalk. Furthermore, sc-RNA sequencing revealed the differences between 2D and 3D cultures. Ongoing work aims to perform transcriptomic and bioinformatic analysis on the sc-RNA sequencing data to gain a deeper understanding of the multifaceted communication within our platform. Citation Format: Kalpana Ravi, Lydia Sakala, Jin Park, Joshua LaBaer, Mehdi Nikkhah. Single-Cell Transcriptomic Assessment of Stromal and Immune Crosstalk on Breast Cancer Invasion Using 3D Tumor-On-Chip Model [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-10.

Abstract PO2-03-11: A novel peripheral CD4+ T cell immune network associated with response to PD-1 inhibitor in early-stage triple negative breast cancer

Abstract A novel peripheral CD4+ T cell immune network associated with response to PD-1 inhibitor in early-stage triple negative breast cancer Background: Immunotherapy has showed benefits in early-stage triple-negative breast cancer (eTNBC). However, there is an urgent need to identify novel biomarkers that can guide stratification for immunotherapy in eTNBC. Anti-tumor immunity is a systemic response that encompasses the mobilization of dendritic cells, lymphocytes, and myeloid cells involving the tumor, blood, and lymph nodes. The activation of anti-tumor immune responses via immunotherapy is expected to reflect in the subset change of circulation. Combined with advantage of dynamic monitoring by liquid biopsy, we detected peripheral blood in the eTNBC patients under immunotherapy to explore potential immune therapeutic predictive subsets. Methods: Single-cell (sc) RNA sequencing was utilized to determine the transcriptomics of immune cells in the peripheral blood. Meanwhile scTCR sequencing was used to trace the dynamic changes of clonetypes under multi-therapy. Nanostring analysis of primary tumor and in vivo models were used to validate the mechanism of immune crosstalk. Prediction index was built based on the proportion of immune subsets at each time point and change of tumor size in each patient. Results: 12 eTNBC patients who participated in neoadjuvant chemotherapy in combination with PD-1 inhibitor (Toripalimab) clinical trial were included in this study. The clinical trial consists of two stages. All eligible patients received four cycles of EC (cyclophosphamide plus epirubicin) regimen and followed four cycles of nab-paclitaxel plus PD-1 inhibitor. Among the 12 patients, 5 patients achieved pCR, while the remaining 7 patients were non-pCR. Peripheral blood samples were collected at baseline, after chemotherapy induction, and on PD-1 inhibitor treatment for scRNA- and scTCR-sequencing. Through prediction index, a novel predictive pattern based on ratio of memory T cells (central memory T and resident memory T) to effective T cells in the peripheral was vividly defined, by which the patients could be classified into C-type (memory T cells dominated) and E-type (effective T cells dominated). The C-type indicated better efficacy of immunotherapy combination therapy, while the E-type suggested better efficacy of chemotherapy alone. Meanwhile, a novel peripheral CD4+Trm (resident-memory-like) subset that possessed both strong differentiation potential and memory function was higher in pCR group at baseline. TCR-tracing analysis revealed that the new clones induced by chemotherapy mainly belong to the CD4+ Trm and CD8+ CD161+ Tcm (central memory) subsets, and the upregulation of CD8+ CD161+ Tcm during therapy was a dynamic predictor for immunotherapy benefits. Besides, most of CD4+Trm transitioned into CD4+Th1-like cells as an activated status, which considered CD4+Trm as a group of reservoirs with polyclonality and differentiation potential for tumor-killing. Meanwhile, the increase in CD5+ cDC2 (type 2 conventional dendritic cell) after chemotherapy was associated with enhanced antigen-presenting function and cell communication and co-stimulation with CD4+ Trm cells which helped the activated transition from CD4+Trm into CD4+Th1-like cells in lymph nodes. Moreover, CD4+Th1-like cells increased secretion of Th1 cytokines thus leading to promote CD8+CD161+ Tcm upregulation and tumor-infiltration after immunotherapy. Conclusions: A novel peripheral pattern based on subsets of T cells as C-type and E-type was established as potential predictors for immunotherapy stratification in eTNBC patients. Meanwhile, a novel peripheral CD4+Trm immune network was identified, in which CD5+cDC2 activated the transition of CD4+Trm into CD4+Th1-like subset leading to activation and tumor-infiltration of CD8+CD161+Tcm. All the above provided potential therapeutic targets for enhancing immunotherapy. Citation Format: Zehao Wang, Zhibo Shao, Bingqiu Xiu, Jiong Wu. A novel peripheral CD4+ T cell immune network associated with response to PD-1 inhibitor in early-stage triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-03-11.

The IBD Risk gene, PTPN2, Is Required for Commensal-associated Th17 Responses to Protect Against Pathobiont Colonization

Background/Aim: Inflammatory bowel disease (IBD) involves multiple factors including genetic susceptibilities, intestinal permeability, microbiome dysbiosis, and expansion of intestinal pathobionts such as adherent-invasive E. coli (AIEC). Mice lacking the IBD risk gene, Ptpn2, exhibit expansion of murine adherent-invasive Escherichia coli ( mAIEC strain, UCR-PP2). Internal transcribed spacer analysis of luminal and mucosal-associated bacteria also indicated a decrease in the protective commensal bacteria, segmented filamentous bacteria (SFB). SFB adherence to intestinal epithelial cells (IEC) induces Th17 cell secretion of IL-17 and IL-22 to promote mucosal defense. The goal of this study was to determine if constitutive whole-body, or IEC-specific Ptpn2 loss in mice modulates SFB abundance, adherence, and downstream production of protective Th17 cytokine in mice. Methods: All mice were housed in specific pathogen free (SPF) vivarium. Distal ileum (2-3 cm) was collected from whole body Ptpn2-wild-type (WT), heterozygous (Het) and Ptpn2 constitutive knockout (KO) mice [Balb/c]; or tamoxifen (TMX)-induced IEC specific Ptpn2 knockout mice ( Ptpn2ΔIEC) and TMX-treated control littermates ( Ptpn2fl/fl) [C57Bl/6]. Tissues were fixed and observed using the Hitachi TM4000 Scanning Electron Microscope (SEM) to visualize SFB. qPCR was used to quantify relative abundance of SFB in the Ileum tissue obtained from Ptpn2ΔIEC and Ptpn2fl/fl mice. Confirmed SFB-free C57Bl/6 mice (10 weeks old; JAX labs; Stock# 000664) were infected for four consecutive days by 109 mAIEC bacteria in PBS. Cytokine protein levels were quantified by Luminex Multiplex Array of whole tissue lysates from cecum, proximal and distal colon regions from 18-21day old WT, HET or KO mice. Immune cells were isolated from the cecum, colon, lamina propria, the mesenteric lymph nodes and the spleen of WT, HET and KO mice and analyzed by flow cytometry for IL-22+, and IL-17+ T cells. Results: SFB adherence to IECs in Ptpn2 constitutive KO mice was visibly decreased by SEM compared to WT and Ptpn2 Het littermates. SEM showed a dramatic decrease in SFB abundance in Ptpn2ΔIEC mice compared to control littermates ( Ptpn2fl/fl). This was confirmed by PCR analysis showing a significant decrease in SFB in Ptpn2ΔIEC mice (p<0.05; n=8). The absence of SFB permitted mAIEC to colonize and cause mild disease in SFB-free mice (JAX; p<0.01; n=8). SFB loss in whole-body Ptpn2-KO mice correlated with a decrease in IL-17+ CD4+ cells in the colon compared to Ptpn2-Het (p<0.001; n=7) and Ptpn2-WT littermates (p<0.05; n=5). Ptpn2-KO mice exhibited higher IL-22 levels in the distal colon (p<0.01; n=3), and increased IL-22+CD4+ T-cells in the cecum, mesenteric lymph nodes and spleen compared to Ptpn2-WT and Het littermates (p<0.001). Conclusion: Loss of the IBD risk gene Ptpn2 in mice, either constitutively or in IECs, reduces the abundance of SFB a commensal inducer of Th17 responses. Consistent with reduced SFB, IL-17+CD4+ T cells were reduced in whole-body Ptpn2-KO mice indicating a loss of SFB-induced Th17 maturation. Despite reduced IL-17, we observed increased IL-22 expression in Ptpn2-KO mice suggesting that loss of PTPN2 provokes a compensatory SFB/IL-17-independent increase in IL-22 production. These findings identify how loss of PTPN2 activity disrupts homeostatic commensal-mucosal immune crosstalk to facilitate IBD-relevant pathobiont colonization. This study was supported by grants NIH-2R01-DK091281, 1R01AI153314-01, R21AI152017, 1R01DK130373, and R01AI165490 (D.F.M.) from the National Institutes of Health (NIH). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Biofabrication Directions in Recapitulating the Immune System-on-a-Chip.

Ever since the implementation of microfluidics in the biomedical field, in vitro models have experienced unprecedented progress that has led to a new generation of highly complex miniaturized cell culture platforms, known as Organs-on-a-Chip (OoC). These devices aim to emulate biologically relevant environments, encompassing perfusion and other mechanical and/or biochemical stimuli, to recapitulate key physiological events. While OoCs excel in simulating diverse organ functions, the integration of the immune organs and immune cells, though recent and challenging, is pivotal for a more comprehensive representation of human physiology. This comprehensive review covers the state of the art in the intricate landscape of immune OoC models, shedding light on the pivotal role of biofabrication technologies in bridging the gap between conceptual design and physiological relevance. The multifaceted aspects of immune cell behavior, crosstalk, and immune responses that are aimed to be replicated within microfluidic environments, emphasizing the need for precise biomimicry are explored. Furthermore, the latest breakthroughs and challenges of biofabrication technologies in immune OoC platforms are described, guiding researchers toward a deeper understanding of immune physiology and the development of more accurate and human predictive models for a.o., immune-related disorders, immune development, immune programming, and immune regulation.

Oncolytic adenovirus in treating malignant ascites: A phase II trial and longitudinal single-cell study

Malignant ascites is a common complication resulting from the peritoneal spread of malignancies, and currently lacks effective treatments. We conducted a phase II trial (NCT04771676) to investigate the efficacy and safety of oncolytic adenovirus H101 and virotherapy-induced immune response in 25 patients with malignant ascites. Oncolytic virotherapy achieved an increased median time to repeat paracentesis of 45 days (95% confidence interval 16.5–73.5 days), compared to the preset control value of 13 days. Therapy was well-tolerated, with pyrexia, fatigue, nausea, and abdominal pain as the most common toxicities. Longitudinal single-cell profiling identified marked oncolysis, early virus replication, and enhanced CD8+ T cells-macrophages immune checkpoint crosstalk, especially in responsive patients. H101 also triggered a proliferative burst of CXCR6+ and GZMK+CD8+ T cells with promoted tumor-specific cytotoxicity. Further establishment of oncolytic virus-induced T cell expansion signature (OiTE) implicated the potential benefits for H101-responsive patients from subsequent anti-PD(L)1 therapy. Patients with up-regulated immune-signaling pathways in tumor cells and a higher proportion of CLEC10A+ DCs and GZMK+CD8+ T cells at baseline showed a superior response to H101 treatment. Our study demonstrates promising clinical responses and tolerability of oncolytic adenovirus in treating malignant ascites and provided insights into the relevant cellular processes following oncolytic virotherapy.

Novel immune cross-talk between inflammatory bowel disease and IgA nephropathy

The mechanisms underlying the complex correlation between immunoglobulin A nephropathy (IgAN) and inflammatory bowel disease (IBD) remain unclear. This study aimed to identify the optimal cross-talk genes, potential pathways, and mutual immune-infiltrating microenvironments between IBD and IgAN to elucidate the linkage between patients with IBD and IgAN. The IgAN and IBD datasets were obtained from the Gene Expression Omnibus (GEO). Three algorithms, CIBERSORTx, ssGSEA, and xCell, were used to evaluate the similarities in the infiltrating microenvironment between the two diseases. Weighted gene co-expression network analysis (WGCNA) was implemented in the IBD dataset to identify the major immune infiltration modules, and the Boruta algorithm, RFE algorithm, and LASSO regression were applied to filter the cross-talk genes. Next, multiple machine learning models were applied to confirm the optimal cross-talk genes. Finally, the relevant findings were validated using histology and immunohistochemistry analysis of IBD mice. Immune infiltration analysis showed no significant differences between IBD and IgAN samples in most immune cells. The three algorithms identified 10 diagnostic genes, MAPK3, NFKB1, FDX1, EPHX2, SYNPO, KDF1, METTL7A, RIDA, HSDL2, and RIPK2; FDX1 and NFKB1 were enhanced in the kidney of IBD mice. Kyoto Encyclopedia of Genes and Genomes analysis showed 15 mutual pathways between the two diseases, with lipid metabolism playing a vital role in the cross-talk. Our findings offer insights into the shared immune mechanisms of IgAN and IBD. These common pathways, diagnostic cross-talk genes, and cell-mediated abnormal immunity may inform further experimental studies.

NFĸB signaling drives myocardial injury via CCR2+ macrophages in a preclinical model of arrhythmogenic cardiomyopathy.

Nuclear factor κ-B (NFκB) is activated in iPSC-cardiac myocytes from patients with arrhythmogenic cardiomyopathy (ACM) under basal conditions, and inhibition of NFκB signaling prevents disease in Dsg2mut/mut mice, a robust mouse model of ACM. Here, we used genetic approaches and single-cell RNA-Seq to define the contributions of immune signaling in cardiac myocytes and macrophages in the natural progression of ACM using Dsg2mut/mut mice. We found that NFκB signaling in cardiac myocytes drives myocardial injury, contractile dysfunction, and arrhythmias in Dsg2mut/mut mice. NFκB signaling in cardiac myocytes mobilizes macrophages expressing C-C motif chemokine receptor-2 (CCR2+ cells) to affected areas within the heart, where they mediate myocardial injury and arrhythmias. Contractile dysfunction in Dsg2mut/mut mice is caused both by loss of heart muscle and negative inotropic effects of inflammation in viable muscle. Single nucleus RNA-Seq and cellular indexing of transcriptomes and epitomes (CITE-Seq) studies revealed marked proinflammatory changes in gene expression and the cellular landscape in hearts of Dsg2mut/mut mice involving cardiac myocytes, fibroblasts, and CCR2+ macrophages. Changes in gene expression in cardiac myocytes and fibroblasts in Dsg2mut/mut mice were dependent on CCR2+ macrophage recruitment to the heart. These results highlight complex mechanisms of immune injury and regulatory crosstalk between cardiac myocytes, inflammatory cells, and fibroblasts in the pathogenesis of ACM.

Ad-derived bone marrow transplant induces proinflammatory immune peripheral mechanisms accompanied by decreased neuroplasticity and reduced gut microbiome diversity affecting AD-like phenotype in the absence of Aβ neuropathology

Immune system dysfunction is increasingly recognized as a significant feature that contributes to Alzheimer’s disease (AD) pathogenesis, reflected by alterations in central and peripheral responses leading to detrimental mechanisms that can contribute to the worsening of the disease. The damaging alterations in the peripheral immune system may disrupt the peripheral-central immune crosstalk, implicating the gut microbiota in this complex interaction. The central hypothesis posits that the immune signature inherently harbored in bone marrow (BM) cells can be transferred through allogeneic transplantation, influencing the recipient’s immune system and modulating peripheral, gut, and brain immune responses.Employing a genetically modified mouse model to develop AD-type pathology we found that recipient wild-type (WT) mice engrafted with AD-derived BM, recapitulated the peripheral immune inflammatory donor phenotype, associated with a significant acceleration of cognitive deterioration in the absence of any overt change in AD-type amyloid neuropathology. Moreover, transcriptomic and phylogenetic 16S microbiome analysis evidence on these animals revealed a significantly impaired expression of genes associated with synaptic plasticity and neurotransmission in the brain and reduced bacteria diversity, respectively, compared to mice engrafted with WT BM.This investigation sheds light on the pivotal role of the peripheral immune system in the brain-gut-periphery axis and its profound potential to shape the trajectory of AD.In summary, this study advances our understanding of the complex interplay among the peripheral immune system, brain functionality, and the gut microbiome, which collectively influence AD onset and progression.