Dendritic Cell Populations Research Articles

A multiscale mechanistic model of human dendritic cells for in-silico investigation of immune responses and novel therapeutics discovery.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) with the unique ability to mediate inflammatory responses of the immune system. Given the critical role of DCs in shaping immunity, they present an attractive avenue as a therapeutic target to program the immune system and reverse immune disease disorders. To ensure appropriate immune response, DCs utilize intricate and complex molecular and cellular interactions that converge into a seamless phenotype. Computational models open novel frontiers in research by integrating large-scale interaction to interrogate the influence of complex biological behavior across scales. The ability to model large biological networks will likely pave the way to understanding any complex system in more approachable ways. We developed a logical and predictive model of DC function that integrates the heterogeneity of DCs population, APC function, and cell-cell interaction, spanning molecular to population levels. Our logical model consists of 281 components that connect environmental stimuli with various layers of the cell compartments, including the plasma membrane, cytoplasm, and nucleus to represent the dynamic processes within and outside the DC, such as signaling pathways and cell-cell interactions. We also provided three sample use cases to apply the model in the context of studying cell dynamics and disease environments. First, we characterized the DC response to Sars-CoV-2 and influenza co-infection by in-silico experiments and analyzed the activity level of 107 molecules that play a role in this co-infection. The second example presents simulations to predict the crosstalk between DCs and T cells in a cancer microenvironment. Finally, for the third example, we used the Kyoto Encyclopedia of Genes and Genomes enrichment analysis against the model's components to identify 45 diseases and 24 molecular pathways that the DC model can address. This study presents a resource to decode the complex dynamics underlying DC-derived APC communication and provides a platform for researchers to perform in-silico experiments on human DC for vaccine design, drug discovery, and immunotherapies.

Abstract PD4-05: PD4-05 Integrated multi-cohort profiling identifies CCL19+ dendritic cells to potentiate immunotherapy efficacy in triple-negative breast cancer

Abstract Background: Immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis have emerged as constituting a new pillar for breast cancer; however, they benefit only a subset of patients, and predictive biomarkers are urgently needed. While dendritic cells (DCs) are essential for the orchestration of antitumor immune response, the clinical value of DCs in breast cancer immunotherapy remains unclear. An improved understanding of the functional diversity of DC subsets, as well as their relationship with ICI response, would eventually improve the efficacy of ICIs in breast cancer patients. Objective: We sought to comprehensively characterize DC heterogeneity within the tumor microenvironment, identify distinct DC subsets associated with the response to ICIs, and define clinically applicable predictive biomarkers for ICI therapy. Methods: We performed a comprehensive single-cell RNA sequencing (scRNA-seq) analysis of 53 breast tumors receiving ICIs or chemotherapy in two retrospective studies (NCT03197389 and GSE169246) to identify specific DC populations associated with ICI responsiveness. Next, we harnessed multiplex immunohistochemistry (n = 186), multiparametric flow cytometry of fresh tissues (n = 87), large consecutive transcriptomic datasets (The Cancer Genome Atlas [TCGA] and Fudan University Shanghai Cancer Center [FUSCC]), and in vivo experiments to illustrate the molecular portraits and clinical relevance of the identified DC subsets. Additionally, except for the two scRNA-seq datasets, we leveraged another four independent cohorts (NCT03805399, NCT04613674, NCT04129996, GSE124821) and assessed the clinical utility of the established clinically applicable biomarkers derived from prior analyses to predict response following treatment with ICIs or chemotherapy. Results: By obtaining single-cell transcriptome data of 1,955 high-quality DCs, we identified five distinct DC subpopulations (CLEC9A+, CD1A+, CLEC10A+, CCL19+, and LILRA4+). We then illustrated that a specific CCL19+ DC population, but not whole DCs or other subsets, was associated with ICI responsiveness, particularly in triple-negative breast cancer (TNBC). Mechanistically, a potent CD8+ T-cell response was unleashed, rendering tumors susceptible to PD-1 blockade in vivo and confirming their putative immunomodulatory capacity. This phenomenon was only observed in CCL19+ DCs but not in CCL19- DCs, indicating CCL19 as a specific marker and could reflect the infiltration and functional phenotype of CCL19+ DCs in breast tumors. Finally, by integrating six independent ICI therapy cohorts, we demonstrated that baseline CCL19 in both tumor and blood could predict better response and survival in TNBC patients receiving ICIs, but not so for chemotherapy. Conclusions: Our results provide important insights into the relationship between the clinical outcome of ICIs and DC heterogeneity at single-cell resolution, support the development of therapies modulating CCL19+ DCs to trigger antitumor CD8+ T-cell immunity, and suggest baseline CCL19 levels as a noninvasive predictive biomarker for patients receiving ICIs, that are potentially applicable in the clinic. In light of these results, we suggest CCL19+ DC modulation as a possible precision immunotherapy approach for cancer patients. Citation Format: Song-Yang Wu, Si-Wei Zhang, Ding Ma, Xi Jin, Yi-Zhou Jiang, Zhi-Ming Shao. PD4-05 Integrated multi-cohort profiling identifies CCL19+ dendritic cells to potentiate immunotherapy efficacy in triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD4-05.

Human salivary protein-derived peptides specific-salivary SIgA antibodies enhanced by nasal double DNA adjuvant in mice play an essential role in preventing Porphyromonas gingivalis colonization: an in-vitro study

BackgroundWe previously showed that fimbriae-bore from Poryphyromonas gingivalis (Pg), one of the putative periodontopathogenic bacteria specifically bound to a peptide domain (stat23, prp21) shared on statherin or acidic proline-rich protein 1 (PRP1) molecule of human salivary proteins (HSPs). Here, we investigated whether the nasal administration of DNA plasmid expressing Flt3 ligand (pFL) and CpG oligodeoxynucleotide 1826 as double DNA adjuvant (dDA) with stat23 and prpr21 induces antigen (Ag)-specific salivary secretory IgA (SIgA) antibodies (Abs) in mice. Further, we examined that stat23- and prpr21-specific salivary SIgA Abs induced by dDA have an impact on Pg-binding to human whole saliva-coated hydroxyapatite beads (wsHAPs).Material and methodsC57BL/6N mice were nasally immunized with dDA plus sta23 or/and prp21 peptide as Ag four times at weekly intervals. Saliva was collected one week after the final immunization and was subjected to Ag-specific ELISA. To examine the functional applicability of Ag-specific SIgA Abs, SIgA-enriched saliva samples were subjected to Pg binding inhibition assay to wsHAPs.ResultsSignificantly elevated levels of salivary SIgA Ab to stat23 or prp21 were seen in mice given nasal stat23 or prp21 with dDA compared to those in mice given Ag alone. Of interest, mice nasally given the mixture of stat23 and prp21 as double Ags plus dDA, resulted in both stat23- and prp21-specific salivary SIgA Ab responses, which are mediated through significantly increased numbers of CD11c+ dendritic cell populations and markedly elevated Th1 and Th2 cytokines production by CD4+ T cells in the mucosal inductive and effector tissues. The SIgA Ab-enriched saliva showed significantly reduced numbers of live Pg cells binding to wsHAPs as compared with those in mice given double Ags without dDA or naïve mice. Additionally, saliva from IgA-deficient mice given nasal double Ags plus dDA indicated no decrease of live Pg binding to wsHAPs.ConclusionThese findings show that HSP-derived peptides-specific salivary SIgA Abs induced by nasal administration of stat23 and prp21 peptides plus dDA, play an essential role in preventing Pg attachment and colonization on the surface of teeth, suggesting a potency that the SIgA may interrupt and mask fimbriae-binding domains in HSPs on the teeth.

Abstract WP239: Intestinal Alkaline Phosphatase: A Novel Regulator Of The Gut-brain Axis In Acute Ischemic Stroke

While strokes are typically associated with the brain, it is widely known that stroke outcomes are influenced by communication between the brain and peripheral organs. The gastrointestinal (GI) tract comprises a critical organ system that is affected during and after stroke, and the interaction between the GI tract and the brain in stroke is attributed to the modulation of the gut-brain axis. Intestinal alkaline phosphatase (IAP; gene: Akp3 ) is a brush border enzyme localized in the intestinal epithelium that regulates intestinal homeostasis by modulating inflammation and intestinal integrity. We hypothesized that IAP regulates the early post-stroke changes within the gut-brain axis, and that loss of IAP disrupts the gut-brain axis to worsen post-stroke outcomes and disrupt immune responses. We utilized 4-6 month-old male and female mice with a genomic deletion of Akp3 and their wild type (WT) littermate controls. Following the induction of photothrombotic stroke (PTS), stroke-injured mice and their sham controls were for evaluated for bacterial load, intestinal permeability, intestinal motility, neurological deficits, cerebral blood flow, and immune responses over the next 24 hours. Statistical analysis of Clark’s neurological score, laser speckle flowmetry, and 2,3,5-Triphenyltetrazolium chloride (TTC) staining at day 1 post stroke showed a main effect of stroke but no differences between genotypes. While no differences in intestinal motility were observed due to stroke or genotype, gut permeability to 3kD-FITC dextran quantified at 3.5 hours post-stroke was higher in Akp3-/- mice compared to WT mice. Further analysis of bacterial burden showed that anaerobic bacteria in the ileum were diminished in Akp3-/- mice post-stroke compared to WT counterparts. Quantification of immune cell populations in cervical lymph nodes and spleen showed that Akp3-/- mice displayed baseline decreases in monocyte and dendritic cell populations that were either maintained or further decreased at 24-hours post-stroke, suggesting that Akp3-/- mice are unable to mount an appropriate innate immune response in acute stroke. Taken together, the current results support the hypothesis that IAP shapes early outcomes and modulates the gut-brain axis in ischemic stroke.

P024 Effects of anti-TNF and anti-interleukins 12 and 23 drugs on circulating dendritic cells migratory capacity in inflammatory bowel disease

Abstract Background Inflammatory bowel disease (IBD) is an idiopathic and chronic disorder that includes ulcerative colitis (UC) and Crohn’s disease (CD). Both diseases are different but show an uncontrolled intestinal immune response that generates tissue inflammation. Dendritic cells (DC) are phagocytic professional antigen presenting cells that link the innate and adaptive immune systems. Indeed, they are the only cell type able to stimulate naïve T cells generating an antigen-specific immune response. In humans, DC can be divided into plasmacytoid (pDC) and c conventional type 1 and 2 (cDC1, cDC2). Although cDC migration towards the GI mucosa is enhanced in IBD, the mechanisms underlying these migrations and whether biological drugs can modulate them are currently unknown. This study aimed to analyze DC migratory capacity towards the GI mucosa, and whether biological drugs with different mechanisms of action can modulate this process in health and IBD. Methods This is a cross-sectional observational postmarketing study where the modulatory effect of different biological treatments on samples from patients with an endoscopic diagnosis of IBD and healthy controls was studied ex vivo. Peripheral blood mononuclear cells (PBMC) were obtained from 15 patients per group and were cultured in presence of golimumab or ustekinumab. Following culture, surface markers expression was determined, assessing the differential effect of biological drugs (if any) over the DC subsets in the different groups. After that, the migratory capacity of circulating DC towards CCL2, CCL25, and MadCam1 from the different study groups was determined. Finally, we evaluated the DC migratory capacities in presence of biological drugs or with culture media alone. Results The cCD2 subset showed higher expression levels of β7 and CCR5 compared to cCD1 (Figure 1). Golimumab and ustekinumab did not modify the expression of the homing markers in any studied subset (Figure 2). Even though no conclusive results were obtained for the biological drugs used in the study on the expression of homing markers, ustekinumab decreased the migratory capacity of cDC2 from CD patients towards a medium supplemented with CCL25 (Figure 3). Conclusion cDC2 showed more migratory profile markers than other DC populations. Golimumab and ustekinumab did not modify the expression of the homing markers in any DC subset in any group. Nevertheless, ustekinumab decreased the migratory capacity of cDC2 from CD patients. This fact indicates that the behavior of this population is different in CD and UC and that the migration capacity of this subset is not dependent on homing markers.

Features of peritoneal dendritic cells in the development of endometriosis

BackgroundEmerging evidence of immunological dysfunction have been described in endometriosis. Dendritic cells (DCs), one of the main antigen-presenting cells, are specialized in the initiation and modulation of the adaptive immune response. Emerging studies demonstrated both endometrial and circulating differences in DCs populations in women with endometriosis. However, the role and mechanism of peritoneal DCs in endometriosis is still unclear. The present study was undertaken to explore the features of peritoneal DCs in the pathogenesis of endometriosis. This study is beneficial to further clarify the cause of endometriosis and provide a new insight into the medical treatment for endometriosis.MethodsThe study included 12 women with endometriosis and 11 women without endometriosis. The C57BL6 mouse model of endometriosis was established by intraperitoneal injection of endometrial segments. The peritoneal DCs of endometriosis patients and mouse models were analyzed by fluorescence associated cell sorting (FACS) examination.ResultsIncreased cell density of peritoneal DCs were observed in endometriosis patients. Moreover, the proportion of mature DCs (mDCs, CD80highCD1alow cells) in the peritoneal DCs was lower whereas the proportion of immature DCs (iDCs, CD80lowCD1ahigh cells) was increased in endometriosis patients. Similarly, the cell density of peritoneal DCs in murine models increased immediately after the injection of endometrial tissues and reached the highest level at 14 days. In addition, the proportion of mDCs (CD11chighCD80high cells) in the peritoneal DCs decreased immediately after the injection of endometrial tissues and then increased with the time until 42 days, but still lower than the control group. In contrast, the proportion of iDCs (CD11chighCD80low cells) in the peritoneal DCs showed the opposite dynamic changes. However, after treated with LPS, the mDCs proportion was significantly increased, leading to lower volume and weight of the endometriosis lesions.ConclusionsIncreased level of peritoneal DCs facilitated the pathogenesis of endometriosis lesions, especially in the early stage of the disease. Furthermore, peritoneal DCs maturation played an important role in the development of endometriosis.

Blockade of EP4 by ASP7657 Modulates Myeloid Cell Differentiation In Vivo and Enhances the Antitumor Effect of Radiotherapy.

The tumor microenvironment (TME) is thought to influence the antitumor efficacy of immuno-oncology agents through various products of both tumor and stromal cells. One immune-suppressive factor is prostaglandin E2 (PGE2), a lipid mediator whose biosynthesis is regulated by ubiquitously expressed cyclooxygenase- (COX-) 1 and inducible COX-2. By activating its receptors, PGE2 induces immune suppression to modulate differentiation of myeloid cells into myeloid-derived suppressor cells (MDSCs) rather than dendritic cells (DCs). Pharmacological blockade of prostaglandin E receptor 4 (EP4) causes a decrease in MDSCs, reprogramming of macrophage polarization, and increase in tumor-infiltrated T cells, leading to enhancement of antitumor immunity in preclinical models. Here, we report the effects of the highly potent EP4 antagonist ASP7657 on the DC population in tumor and antitumor immune activation in an immunocompetent mouse tumor model. Oral administration of ASP7657 inhibited tumor growth, which was accompanied by an increase in intratumor DC and CD8+ T cell populations and a decrease in the M-MDSC population in a CT26 immunocompetent mouse model. The antitumor activity of ASP7657 was dependent on CD8+ T cells and enhanced when combined with an antiprogrammed cell death-1 (PD-1) antibody. Notably, ASP7657 also significantly enhanced the antitumor efficacy of radiotherapy in an anti-PD-1 antibody refractory model. These results indicate that the therapeutic potential of ASP7657 arises via upregulation of DCs and subsequent CD8+ T cell activation in addition to suppression of MDSCs in mouse models and that combining EP4 antagonists with radiotherapy or an anti-PD-1 antibody can improve antitumor efficacy.

Changes in immune cell populations following KappaMab, lenalidomide and low-dose dexamethasone treatment in multiple myeloma.

Lenalidomide (LEN) is used to treat multiple myeloma (MM) and shows in vitro synergy with KappaMab (KM), a chimeric antibody specific for Kappa Myeloma antigen, an antigen exclusively expressed on the surface of kappa-restricted MM cells. Lenalidomide, dexamethasone (DEX) and KM control MM via multiple immunomodulatory mechanisms; however, there are several additional effects of the drug combination on immune cells. Lenalidomide can increase T cell and NKT cell cytotoxicity and dendritic cell (DC) activation in vitro. We investigated the immune cell populations in bone marrow of patients treated with KM, LEN and low-dose DEX in kappa-restricted relapsed/refractory MM ex vivo and assessed association of those changes with patient outcome. A cohort (n = 40) of patients with kappa-restricted relapsed/refractory MM, treated with KM, LEN and low-dose DEX, was analysed using a mass cytometry panel that allowed identification of immune cell subsets. Clustering analyses were used to determine significant changes in immune cell populations at time periods after treatment. We found changes in five DC and 17 T-cell populations throughout treatment. We showed an increase in activated conventional DC populations, a decrease in immature/precursor DC populations, a decrease in activated CD4 T cells and an increase in effector-memory CD4 T cells and effector CD8 T cells, indicating an activated immune response. These data characterise the effects of LEN, DEX, and KM treatment on non-target immune cells in MM. Treatment may support destruction of MM cells by both direct action and indirect mechanisms via immune cells.

A novel in vitro model of the small intestinal epithelium in co-culture with 'gut-like' dendritic cells.

Cross-talk between dendritic cells (DCs) and the intestinal epithelium is important in the decision to mount a protective immune response to a pathogen or to regulate potentially damaging responses to food antigens and the microbiota. Failures in this decision-making process contribute to the development of intestinal inflammation, making the molecular signals that pass between DCs and intestinal epithelial cells potential therapeutic targets. Until now, in vitro models with sufficient complexity to understand these interactions have been lacking. Here, we outline the development of a co-culture model of in vitro differentiated 'gut-like' DCs with small intestinal organoids (enteroids). Sequential exposure of murine bone marrow progenitors to Flt3L, granulocyte macrophage colony-stimulating factor (GM-CSF) and all-trans-retinoic acid (RA) resulted in the generation of a distinct population of conventional DCs expressing CD11b+SIRPα+CD103+/- (cDC2) exhibiting retinaldehyde dehydrogenase (RALDH) activity. These 'gut-like' DCs extended transepithelial dendrites across the intact epithelium of enteroids. 'Gut-like' DC in co-culture with enteroids can be utilized to define how epithelial cells and cDCs communicate in the intestine under a variety of different physiological conditions, including exposure to different nutrients, natural products, components of the microbiota, or pathogens. Surprisingly, we found that co-culture with enteroids resulted in a loss of RALDH activity in 'gut-like' DCs. Continued provision of GM-CSF and RA during co-culture was required to oppose putative negative signals from the enteroid epithelium. Our data contribute to a growing understanding of how intestinal cDCs assess environmental conditions to ensure appropriate activation of the immune response.

Immunoexpression of CD1a and Histopathology in Lesional and Non Lesional Skin in Psoriasis: A Cross-sectional Study

Introduction: Psoriasis is a long-lasting autoimmune disease mediated by T-lymphocytes and dendritic cells. Langerhans cells are a unique population of dendritic cells found in the epidermis, where they can be identified by Cluster of Differentiation 1a (CD1a) positivity. They play an important role in the pathogenesis of psoriasis. Aim: To evaluate the utility of immunoexpression of CD1a in the early diagnosis of psoriasis and to study the histopathology of skin biopsies from clinically diagnosed cases of psoriasis. Materials and Methods: A cross-sectional study was conducted from September 2017 to August 2019 at Gandhi Hospital, Secunderabad, Telangana, India. Skin biopsies were taken from 50 patients with psoriasis from the Outpatient Department (OPD). Histopathological features in early psoriasis were studied, and CD1a immunoexpression in lesional, perilesional, and distant skin was analysed. The data were analysed using Student’s t-test with the Statistical Package for the Social Sciences (SPSS) software. Results: Half of the cases were found in two age groups: 51-60 years (15 patients, 30%) and 11-20 years (10 patients, 20%). The majority of the cases were males (36 patients, 72%). Most of the biopsies were taken from the lower limb (24 patients, 48%), followed by the back (13 patients, 26%). In this study, the average number of CD1a positive Langerhans cells was highest (54.92±5.26) in perilesional skin compared to lesional skin (30±3.96), which was statistically significant (p-value=0.04). Conclusion: Based on the observations in this study, a strong positive reaction of CD1a in perilesional skin can be used to diagnose psoriasis in the early clinical stages, before full-blown clinical psoriatic plaques have appeared. Early diagnosis may prompt physicians to initiate treatment early

In Vitro Generation of Murine Dendritic Cells from Hoxb8-Immortalized Hematopoietic Progenitors.

Mouse dendritic cells (DCs) are routinely generated based on cells isolated form the bone marrow (BM) and cultured in the presence of growth factors that support DC development, such as FMS-like tyrosine kinase 3 ligand (FLT3L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Guo et al., J Immunol Methods 432:24-29, 2016). In response to these growth factors, DC progenitors expand and differentiate, while other cell types die during the in vitro culture period, ultimately leading to relatively homogenous DC populations. An alternative method, which is discussed in detail in this chapter, relies on conditional immortalization of progenitor cells with DC potential in vitro using an estrogen-regulated form of Hoxb8 (ERHBD-Hoxb8). Such progenitors are established by retroviral transduction of largely unseparated BM cells with a retroviral vector expressing ERHBD-Hoxb8. Treatment of ERHBD-Hoxb8-expressing progenitors with estrogen results in Hoxb8 activation, which blocks cell differentiation and allows for expansion of homogenous progenitor cell populations in the presence of FLT3L. These cells, referred to as Hoxb8-FL cells, retain lineage potential for lymphocyte and myeloid lineages, including the DC lineage. Upon removal of estrogen (inactivation of Hoxb8), Hoxb8-FL cells differentiate into highly homogenous DC populations in the presence of GM-CSF or FLT3L akin to their endogenous counterparts. Given their unlimited proliferative capacity and amenability for genetic manipulation, for example, by CRISPR/Cas9, these cells provide a large number of options to investigate DC biology. Here, I am describing the method to establish Hoxb8-FL cells from mouse BM, as well as procedures for DC generation and gene deletion using lentivirally delivered CRISPR/Cas9.

Polyphenol-rich <italic>Sargassum horneri</italic> alleviates atopic dermatitis-like skin lesions in NC/Nga mice by suppressing Th2-mediated cytokine IL-13

Atopic dermatitis (AD) is one of major skin inflammatory diseases characterized by excessive Th2-mediated immune responses. Recent evidence provides that interlukin-13 (IL-13) plays the role of a key Th2 cytokine that drives the inflammation underlining AD. Due to adverse effects of commercially available synthetic drugs, the need for treatments based on natural products is gaining much attention. Sargassum horneri is an edible brown algae known for beneficial bioactivities including anti-inflammation. We investigated if polyphenol-rich S. horneri extracts (SHE) could suppress AD-like skin lesions in NC/Nga mice and if that involved inhibition of the infiltration of Th2-mediated cytokine IL-13. We observed markedly increased infiltration of IL-13 positive cells in AD-like skin lesions of mice but SHE treatments decreased it. Also, the dermal expression of IL-13 was sufficient to cause inflammatory responses in mice skin resembling human AD. SHE suppressed the dermal infiltration of inflammatory cells where IL-13 plays a crucial role in skin tissues and in the recruitment of inflammatory cells. Furthermore, it was confirmed that SHE reduced T cell, dendritic cell, and macrophage populations in spleen. Moreover, SHE decreased the collagen deposition in skin and ear dermis resulting in reduced fibrosis that occurs in AD due to excessive collagen. Taken together, our results reveal that SHE suppressed the infiltration of inflammatory cells into skin dermis by decreasing the infiltration of IL-13 positive cells. Therefore, SHE could be taken as a useful therapeutic agent to alleviate AD.

Polyelectrolyte Coating of Ferumoxytol Differentially Impacts the Labeling of Inflammatory and Steady-State Dendritic Cell Subtypes.

Engineered magnetic nanoparticles (MNPs) are emerging as advanced tools for medical applications. The coating of MNPs using polyelectrolytes (PEs) is a versatile means to tailor MNP properties and is used to optimize MNP functionality. Dendritic cells (DCs) are critical regulators of adaptive immune responses. Functionally distinct DC subsets exist, either under steady-state or inflammatory conditions, which are explored for the specific treatment of various diseases, such as cancer, autoimmunity, and transplant rejection. Here, the impact of the PE coating of ferumoxytol for uptake into both inflammatory and steady-state DCs and the cellular responses to MNP labeling is addressed. Labeling efficiency by uncoated and PE-coated ferumoxytol is highly variable in different DC subsets, and PE coating significantly improves the labeling of steady-state DCs. Uncoated ferumoxytol results in increased cytotoxicity of steady-state DCs after labeling, which is abolished by the PE coating, while no increased cell death is observed in inflammatory DCs. Furthermore, uncoated and PE-coated ferumoxytol appear immunologically inert in inflammatory DCs, but they induce activation of steady-state DCs. These results show that the PE coating of MNPs can be applied to endow particles with desired properties for enhanced uptake and cell type-specific responses in distinct target DC populations.

316 A unique population of dermal conventional dendritic cells enhances TH2 and inhibits TH17 cell polarization in healthy skin

Dendritic cells (DC) play a crucial role in informing the adaptive immune system about infectious, innocuous or self antigens. Two developmentally distinct lineages of conventional DC, termed cDC1 and cDC2, have been described in mice and humans. cDC1 predominantly interact with CD8 T cells, while cDC2 interact with CD4 T cells to promote T helper (Th) and T regulatory cell responses. cDC2 present a high degree of phenotypic and functional heterogeneity in different tissues, suggesting that they adapt to diverse peripheral environments. Using high-dimensional proteomic and transcriptomic single-cell technologies, we could show that cDC2 exhibit unique signatures in different murine tissues at the steady state. Focusing on the developmental requirements of these subsets we could show that CD11blow cDC2, a population uniquely found in skin, required STAT6- and KLF4-dependent IL-13 signaling, while subsets in other tissues were STAT6-independent. IL-13 was not only necessary for the development of CD11blow cDC2, but also sufficient to rescue CD11blow cDC2 development in IL-13 KO mice and to differentiate CD11blow cDC2 in vitro. In naïve murine skin, dermal IL-13 was mostly derived from innate lymphoid cells expressing a resting ICOS+KLRG1-ST2- phenotype. In the absence of IL-13 signaling, dermal cDC2 were stable in number and continued to take up antigen but remained CD11bhi. They furthermore showed defective activation in response to different antigens and a diminished ability to support IL-4+ GATA3+ Th2 cell development, whereas anti-fungal IL-17+ RORyt+ responses were increased. Thus, our work identified that homeostatic IL-13 in healthy skin mediates a DC-driven protective non-inflammatory environment at the steady state, but might also contribute to its pro-allergic conditioning upon dysregulation.

Salvage with Sequential T Cell Redirection for Myeloma Patients Who Have Previously Progressed on T Cell Directed Therapy Is Associated with Significant Cellular Activation of the Immune Microenvironment

Background: With the recent approval of two anti-BCMA CAR-Ts and multiple anti-BCMA and anti-GPRC5D bispecific antibodies (BiAbs) in clinical trials, rational salvage strategies for patients who progress after these therapies is an area of unmet need in multiple myeloma. We have previously reported favorable outcomes for patients receiving sequential T cell directed therapies with depth of response and progression free survival (PFS) (Mouhieddine et al Blood (2021) 138 (Supplement 1: 821). However, the underlying immune mechanisms are not well known and would be essential to design sequential strategies combining different T cell directed therapies. Here we show that sequential T cell mediated therapies promote not only T cell engagement but also immune activation in natural killer (NK) and dendritic cells (DC) cellular subsets in patients with relapsed/refractory multiple myeloma (RRMM). Methods: We identified 15 RRMM patients who had progressed on a T cell directed therapy, either anti-BCMA CAR-T (n=10) or BiAbs (n=5) and then received another BiAb as a subsequent line of therapy. 70% of patients who progressed on anti-BCMA CAR-T (7/10) and 20% who progressed on BiAbs had interim therapies before receiving subsequent BiAbs therapy. The median time between the first T cell directed therapy and subsequent therapy was 5 months (range 0- 26 months). We profiled immune cell activation in peripheral blood (PB) by high dimensional flow cytometry and Olink Proteomics. Clinical data such as patient demographics, disease characteristics and post-clinical trial outcomes were collected retrospectively until July of 2022. Data and sample collection for this study was approved by the institutional review board (IRB) and follows the Declaration of Helsinki and International Conference on Harmonization Guidelines for Good Clinical Practice (IRB: GCO#: 11-1433 & GCO #: 18-00456). Results: We compared baseline immune cell populations of 15 RRMM patients in the PB post relapse on T cell mediated therapy and during subsequent BiAb treatment (Figure 1a). 93% of patients (14/15) received BiAbs therapy with a different target than their first T cell directed regimen. 73% of patients (11/15) received sequential BiAbs therapy in combination with an anti-CD38 monoclonal antibody. Patients had a median age of 63 years (range 43-89) at time of progression after the first T cell directed therapy. MM patients were heavily pre-treated with a median of 6 prior lines of therapy (range 3-16). All patients were triple-class refractory while 66% were penta-drug refractory. Immune profiling at time of progression on the first T cell directed therapy, was associated with increased expression of exhaustion markers on CD4+ and CD8+ T cells. Treatment with a second BiAb was associated with significant re-activation of endogenous T cells. Specifically, CD8+ T cells demonstrated increased expression of hallmark surface activation markers such as HLA-DR and CD69+ (p<0.05, Figure 1b), and this increase was consistent in CD4+ T cells as well (p<0.01, Figure 1b). Alongside T cell engagement we saw an increase in surface activation markers CD69+ and HLA-DR on NK and DC populations respectively (p<0.01). This indicates that the application of the second T cell directed therapy not only reactivates T cell compartment but potentiates widespread immune cell activation. Plasma cytokine analysis from these patients revealed elevated CXCL10, CXCL9, and TNFa during bispecific therapy; reinforcing the activation state of the patients during treatment. Although the CD4:CD8 ratios remain conserved, we observed a significant differentiation of CD8+ T cells through sequential therapy, with a reduction of naïve CD8+ T cells (p=0.022) and increase of effector memory CD8+ T cells (p<0.01). Conclusion: Our data suggests that patients who progressed on T cell directed therapy and went on to receive T cell mediated BiAbs, not only respond clinically, but also have hallmark expression of activation markers. T cells can be reactivated post relapse from T cell mediated therapies, indicating that T cell targeted treatments can be applied successfully in sequential order with favorable patient outcomes. We are currently performing immune profiling on more PB and bone marrow samples to further confirm these findings in a larger patient population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Expansion of a CD8+ Temra Population and Activating T-Cell Interactions Characterize the Graft Versus Leukemia Response in Relapsed AML

Graft-versus-leukemia (GvL) activity underlies the curative potential of allogeneic hematopoietic stem cell transplantation. Its effectiveness is well-exemplified by donor lymphocyte infusion (DLI), an established adoptive cellular therapy for treatment of relapsed disease. Whereas ~80% of chronic myelogenous leukemia (CML) patients achieve durable remissions to DLI, responses for acute myelogenous leukemia (AML) are only 15-20%. We hypothesized that systematic characterization of immune cell composition and cellular interactions within the marrow tumor microenvironment in relation to DLI would illuminate the molecular basis of the disease-specific susceptibility to GvL. Given the heterogeneous nature of AML, we suspected AML response to comprise more complex leukemic-immune cell interactions than in CML. To these ends, we generated 5’ single-cell RNA- and TCR-sequencing (scTCRseq) and cell surface marker profiling (scCITE-seq) on 24 bone marrow biopsy samples collected before and after DLI from 9 patients with relapsed AML (5 responders [R], 4 nonresponders [NR]). We merged this dataset with our previously reported CML dataset (14 patients, 8 R, 6 NR [PMID: 34758319]) and batch corrected these data with scVI to obtain a total of 414,000 high-quality single-cell transcriptomes for analysis. Evaluation of the immune cell subset proportions over time revealed 10 populations with distinct dynamics in AML responders to DLI but not CML, while 3 other populations showed similar dynamics across diseases. Both CML and AML DLI response were associated with pre-treatment enrichment of an NK cell population highly expressing XCL1, XCL2, CCL3, CCL4, CCL4L2, and CCL5, consistent with a tumor-infiltrating expression profile. Unique to CML (i.e. not observed in AML), in addition to post-DLI enrichment of the TPEX population previously identified, we found a γδ-like T cell population and a classical dendritic cell population exhibiting post-DLI enrichment associated with response to DLI. In AML, response to DLI was most consistently associated with expansion of a CD8+ T cell population (6.03% of total pre-DLI cells vs 18.31% post-DLI for R [p=0.016] compared to 8.68% pre-DLI vs 9.55% post-DLI for NR [p=0.454]) and a naïve B cell population (4.02% pre-DLI vs 11.68% post-DLI for R [p=0.002] compared to 5.42% pre-DLI vs 5.17% post-DLI for NR [p=0.471]). The CD8+ T cell population was characterized by high expression of cytotoxicity genes (e.g. GZMA, GZMB, GZMH, GZMM, PRF1, KLRG1, TOX, EOMES, TBX21) and was most consistent with a T effector memory re-expressing RA (TEMRA) profile, supported by high CD45RA expression by scCITE-seq. This population also highly expressed ZNF683, which encodes the transcription factor Hobit, known to regulate cytotoxicity in CD8+ T cells. Differential gene expression between AML R and NR TEMRA cells revealed enriched expression of ZNF683, GZMH, NKG7, CCL5, and CXCR4 in R and the exhaustion marker TIGIT and the senescence marker KLRG1 in NR. scTCR-seq of the TEMRA subset demonstrated clonal expansion and differentiation into a more proliferative expression profile post-DLI. Among AML cases, 2-20% of the post-DLI repertoire of responders originated from the DLI product while no TCR from the DLI product was detected in the single NR patient available for analysis. We applied predictive interaction modeling among all 50 cell type pairs using CellPhoneDB (PMID: 32103204), which utilizes a publicly available curated database of receptor-ligand interactions. Predicted interactions unique to R between CD8+ TEMRA and AML leukemia cells included CD226-PVR, CD6-ALCAM, and CD46-JAG1, which have been associated with T cell activation. Interactions unique to NR between CD8+ TEMRA and AML leukemia included the inhibitory interactions TIGIT-NECTIN and PTPRC-MRC1. These interactions were not observed in CML patients in either R or NR between CD8+ TEMRA and CML cells, suggesting disease-specific response and resistance mechanisms. Here we show that response to DLI in AML is associated with largely distinct expansion of immune cell populations compared to CML, including not only T cells but an orchestration of B, NK, and myeloid cell subsets. AML responders were uniquely characterized by expansion of a TEMRA subset marked by high expression of the pro-cytotoxic gene ZNF683. Further investigation is needed to evaluate the role of this population in mediating GvL response.

Studying the effect of double-stranded RNA upon activity of mouse splenocytes using flow cytometry

Ribonucleic acids (RNA), in particular, double-stranded RNAs, due to their ability to modulate innate immune responses, are of undoubted interest in view of their usage as vaccine adjuvants. However, despite the fact that dsRNA preparations have been known for a long time, the issues of cellular interactions and orientation of immune response upon their exposure have not yet been properly studied. The aim of this work was to evaluate the in vitro response of mouse splenocytes to dsRNA exposure in cell cultures, and after drug administration in vivo. The studies were carried out in female Balb/c mice. Activation status of various splenocyte populations after treatment with yeast dsRNA and reference substance (PolyI:PolyC) was assessed by means of flow cytometry by expression of CD69 and CD86 activation markers on CD19+B lymphocytes and CD11c+ dendritic cells (DC). During in vitro studies, the splenocytes were incubated in DMEM medium containing 10% fetal calf serum for 22 hours following addition of the yeast dsRNA preparations, or PolyI:PolyC (2.5 μg/mL) preparation. Single-stranded high-polymer RNA (hpRNA), which is a component of the substance, was used as an additional control at the dose of 16 μg/mL. Our study has shown that the activating effect of dsRNA and PolyI:PolyC on expression of CD86 and CD69 markers upon the cells of the entire pool of splenocytes, B lymphocytes and DC. Highly polymeric RNA increased the total number of CD86+ cells in the population without changing the expression level of these markers upon B lymphocytes and DCs. When performing the in vivo studies, yeast dsRNA substance was administered intravenously into mice at a dose of 2.5 mg/kg, and hpRNA was used at a dose of 16 mg/kg. The number of CD69+ and CD86+ splenocytes was assessed 4 hours after drug administration. The highest stimulating effect of dsRNA was registered with CD69 expression marker: significantly increased numbers of CD69+ cells were registered for B lymphocytes and the entire cell population. The stimulation of CD86 co-receptor expression on B lymphocytes was less pronounced, but statistically significant. The ability of single-stranded and double-stranded RNAs to cause significant increase in CD86+ cell numbers was demonstrated among dendritic cell population. The results of the study made it possible to evaluate the effect of dsRNA on the immune cell function, with respect of their interaction, maturation, and migration. This approach may be useful for developing optimal strategies for selection and screening of new nucleic acid-based adjuvants.

IMMU-18. MIGRATION OF DENDRITIC CELLS THROUGH THE BRAIN-MENINGEAL LYMPHATIC-DRAINING LYMPH NODE NETWORK IN ORTHOTOPIC GLIOMA MODELS

Abstract INTRODUCTION Limited migration of dendritic cell (DC) vaccines to draining lymph nodes (DLN) remains a major limitation to DC efficacy for malignant gliomas. Our prior work demonstrated that increased peripheral DC migration to DLN results in enhanced antitumor efficacy. Given this, we studied DC trafficking in mouse gliomas, the meningeal lymphatic vessel (MLV) system, and the draining cervical lymph nodes (CLN). METHODS To elevate host DC populations, C57BL/6 and VMDk mice were implanted with B16-FMS-like tyrosine kinase 3 ligand (FLT3L) melanoma or with B16-FLT3L supernatant. Mice were implanted intracranially with CT2A, GL261-OVA, and SMA560 syngeneic glioma lines in the right parietal lobe. Antitumor efficacy was measured by tumor weights and median overall survival (mOS). RESULTS DC injected into glioma hemispheres display delayed kinetics of peak migration to CLN compared to control (7 days (d) vs 3d post-injection (pi), respectively). Migration of intraventricular (ivn)-injected DC to CLN is superior at days 1, 3 and 7 pi when glioma is present (p = 0.026). B16-FLT3L supernatant results in a disproportionate systemic expansion in conventional type 1 over type 2 DC (cDC1 &amp;gt; cDC2) compared to media control (cDC1, cDC2 fold change over media for spleen = 27,11; DLN = 6,2; blood = 6,3). Pulsed dosing of FLT3L supernatant results in significantly greater numbers of splenic cDC1 compared to daily FLT3L dosing (p = 0.002). Mice with CT2A and continuous FLT3L secretion in vivo show longer survival (mOS 30d) compared to mice with continuous GM-CSF secretion (mOS 22d) (p = 0.009). Mice with SMA560 and FLT3L secretion have significantly reduced glioma growth compared to tumor alone, GM-CSF-secreting tumors, and mixed FLT3L/GM-CSF-secreting tumors (p = 0.029). CONCLUSION FLT3L signaling in vivo results in cDC1 &amp;gt; cDC2 generation and migration through the CNS with more favorable glioma efficacy. Modeling of DC migration through the glioma-MLV-CLN system with FLT3L stimulation permits testing of strategies to improve DC therapy.

Immunopathogenesis of Acute Pancreatitis

The leading positions in terms of frequency, severity of the course and high mortality belong to infectious forms of acute pancreatitis. However, not all pathophysiological mechanisms of the development of this disorder have been studied. Given that immune responses are an integral part of the pathogenesis of pancreatitis, it is extremely important to study the relationship between the mechanisms of inflammation and activation of the immune response. This review will discuss the role of various populations of innate immune cells, including macrophages, neutrophils, dendritic and mast cells, and regulatory immune cells in the pathogenesis of gland tissue destruction and the relationship between immune responses and systemic inflammatory response syndrome. Targeting innate immune cell populations and metabolite signaling pathways in acute pancreatitis may lead to a broader and ultimately more effective redirection of the treatment program towards disease resolution and improved clinical outcomes.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.