Migration Of Dendritic Cells Research Articles

Applications of Intravital Imaging in Cancer Immunotherapy.

Currently, immunotherapy is one of the most effective treatment strategies for cancer. However, the efficacy of any specific anti-tumor immunotherapy can vary based on the dynamic characteristics of immune cells, such as their rate of migration and cell-to-cell interactions. Therefore, understanding the dynamics among cells involved in the immune response can inform the optimization and improvement of existing immunotherapy strategies. In vivo imaging technologies use optical microscopy techniques to visualize the movement and behavior of cells in vivo, including cells involved in the immune response, thereby showing great potential for application in the field of cancer immunotherapy. In this review, we briefly introduce the technical aspects required for in vivo imaging, such as fluorescent protein labeling, the construction of transgenic mice, and various window chamber models. Then, we discuss the elucidation of new phenomena and mechanisms relating to tumor immunotherapy that has been made possible by the application of in vivo imaging technology. Specifically, in vivo imaging has supported the characterization of the movement of T cells during immune checkpoint inhibitor therapy and the kinetic analysis of dendritic cell migration in tumor vaccine therapy. Finally, we provide a perspective on the challenges and future research directions for the use of in vivo imaging technology in cancer immunotherapy.

Site-specific regulation of Th2 differentiation within lymph node microenvironments.

T helper 2 (Th2) responses protect against pathogens while also driving allergic inflammation, yet how large-scale Th2 responses are generated in tissue context remains unclear. Here, we used quantitative imaging to investigate early Th2 differentiation within lymph nodes (LNs) following cutaneous allergen administration. Contrary to current models, we observed extensive activation and "macro-clustering" of early Th2 cells with migratory type-2 dendritic cells (cDC2s), generating specialized Th2-promoting microenvironments. Macro-clustering was integrin-mediated and promoted localized cytokine exchange among T cells to reinforce differentiation, which contrasted the behavior during Th1 responses. Unexpectedly, formation of Th2 macro-clusters was dependent on the site of skin sensitization. Differences between sites were driven by divergent activation states of migratory cDC2 from different dermal tissues, with enhanced costimulatory molecule expression by cDC2 in Th2-generating LNs promoting prolonged T cell activation, macro-clustering, and cytokine sensing. Thus, the generation of dedicated Th2 priming microenvironments through enhanced costimulatory molecule signaling initiates Th2 responses in vivo and occurs in a skin site-specific manner.

A CFSE-Based Assay to Monitor Skin Dendritic Cell Migration to Draining Lymph Nodes in Infected Mice

A CFSE-Based Assay to Monitor Skin Dendritic Cell Migration to Draining Lymph Nodes in Infected Mice

A CFSE-Based Assay to Monitor Skin Dendritic Cell Migration to Draining Lymph Nodes in Infected Mice

A CFSE-Based Assay to Monitor Skin Dendritic Cell Migration to Draining Lymph Nodes in Infected Mice

CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells.

Dendritic cell (DC) migration from peripheral tissues via afferent lymphatic vessels to draining lymph nodes (dLNs) is important for the organism's immune regulation and immune protection. Several lymphatic endothelial cell (LEC)-expressed adhesion molecules have thus far been found to support transmigration and movement within the lymphatic vasculature. In this study, we investigated the contribution of CD112, an adhesion molecule that we recently found to be highly expressed in murine LECs, to this process. Performing in vitro assays in the murine system, we found that transmigration of bone marrow-derived dendritic cells (BM-DCs) across or adhesion to murine LEC monolayers was reduced when CD112 was absent on LECs, DCs, or both cell types, suggesting the involvement of homophilic CD112-CD112 interactions. While CD112 was highly expressed in murine dermal LECs, CD112 levels were low in endogenous murine dermal DCs and BM-DCs. This might explain why we observed no defect in the in vivo lymphatic migration of adoptively transferred BM-DCs or endogenous DCs from the skin to dLNs. Compared to murine DCs, human monocyte-derived DCs expressed higher CD112 levels, and their migration across human CD112-expressing LECs was significantly reduced upon CD112 blockade. CD112 expression was also readily detected in endogenous human dermal DCs and LECs by flow cytometry and immunofluorescence. Upon incubating human skin punch biopsies in the presence of CD112-blocking antibodies, DC emigration from the tissue into the culture medium was significantly reduced, indicating impaired lymphatic migration. Overall, our data reveal a contribution of CD112 to human DC migration.

Atypical cofilin signaling drives dendritic cell migration through the extracellular matrix via nuclear deformation

To mount an adaptive immune response, dendritic cells must migrate to lymph nodes to present antigens to Tcells. Critical to 3D migration is the nucleus, which is the size-limiting barrier for migration through the extracellular matrix. Here, we show that inflammatory activation of dendritic cells leads to the nucleus becoming spherically deformed and enables dendritic cells to overcome the typical 2- to 3-μm diameter limit for 3D migration through gaps in the extracellular matrix. We show that the nuclear shape change is partially attained through reduced cell adhesion, whereas improved 3D migration is achieved through reprogramming of the actin cytoskeleton. Specifically, our data point to a model whereby the phosphorylation of cofilin-1 at serine 41 drives the assembly of a cofilin-actomyosin ring proximal to the nucleus and enhances migration through 3D collagen gels. In summary, these data describe signaling events through which dendritic cells deform their nucleus and enhance their migratory capacity.

Musashi-2 potentiates colorectal cancer immune infiltration by regulating the post-translational modifications of HMGB1 to promote DCs maturation and migration

Post-translational modifications (PTMs) of the non-histone protein high-mobility group protein B1 (HMGB1) are involved in modulating inflammation and immune responses. Recent studies have implicated that the RNA-binding protein (RBP) Musashi-2 (MSI2) regulates multiple critical biological metabolic and immunoregulatory functions. However, the precise role of MSI2 in regulating PTMs and tumor immunity in colorectal cancer (CRC) remains unclear. Here, we present data indicating that MSI2 potentiates CRC immunopathology in colitis-associated colon cancer (CAC) mouse models, cell lines and clinical specimens, specifically via HMGB1-mediated dendritic cell (DC) maturation and migration, further contributes to the infiltration of CD4+ and CD8+ T cells and inflammatory responses. Under stress conditions, MSI2 can exacerbate the production, nucleocytoplasmic transport and extracellular release of damage-associated molecular patterns (DAMPs)-HMGB1 in CRC cells. Mechanistically, MSI2 mainly enhances the disulfide HMGB1 production and protein translation via direct binding to nucleotides 1403–1409 in the HMGB1 3′ UTR, and interacts with the cytoplasmic acetyltransferase P300 to upregulate its expression, further promoting the acetylation of K29 residue in HMGB1, thus leading to K29-HMGB1 nucleocytoplasmic translocation and extracellular release. Furthermore, blocking HMGB1 activity with glycyrrhizic acid (Gly) attenuates MSI2-mediated immunopathology and immune infiltration in CRC in vitro and in vivo. Collectively, this study suggests that MSI2 may improve the prognosis of CRC patients by reprogramming the tumor immune microenvironment (TIME) through HMGB1-mediated PTMs, which might be a novel therapeutic option for CRC immunotherapy.

Enhanced tumor control activities of anti-mPD-L1 antibody and antigen-presenting cell-like natural killer cell in an allograft model

BackgroundDespite the utilization of immune checkpoint inhibitors (ICIs) in treating numerous types of cancers being approved, their efficacy in tumor control in the clinic is not satisfactory. Since adoptive cell therapy (ACT) can alter the tumor microenvironment, we hypothesized that ACT potentially synergized with ICI in tumor control and examined this hypothesis via a murine allograft model.MethodsFemale C57BL/6 mice were stimulated with interleukin 15 and granulocyte monocyte-colony stimulating factor, followed by collecting their bone marrow cells for murine NKDC cultivation. Then, female C57BL/6 mice, inoculated with lymphoma cancer cell line E.G7-OVA, were administrated with murine NKDC cells, murine anti-program cell death ligand-1 antibody (α-mPD-L1), or both for 28 days. After 28 days of treatment, mice were sacrificed whose inoculated tumors, spleen, sentinel lymph nodes, and peripheral blood were collected to measure tumor size, lymphocyte infiltration, and change of immune cell profile.ResultsCombined treatment of NKDCs with α-mPD-L1 exhibited significantly stronger tumor control efficacy than treatment of NKDCs or α-mPD-L1 alone. NKDCs/α-mPD-L1 combination increased migration of dendritic cells, CD4, CD8 T cells, and activated CD8 T cells to the tumor-bedding site, and promoted endogenous tumor-specific cytotoxic T-cell response.ConclusionThe current study confirmed our hypothesis that combining NKDC ACT with ICI therapy can potentiate tumor control efficacy by manipulating the tumor microenvironment. This study provided a novel circumstance on tumor immunotherapy.

Tumour-retained activated CCR7+ dendritic cells are heterogeneous and regulate local anti-tumour cytolytic activity

Tumour dendritic cells (DCs) internalise antigen and upregulate CCR7, which directs their migration to tumour-draining lymph nodes (dLN). CCR7 expression is coupled to an activation programme enriched in regulatory molecule expression, including PD-L1. However, the spatio-temporal dynamics of CCR7+ DCs in anti-tumour immune responses remain unclear. Here, we use photoconvertible mice to precisely track DC migration. We report that CCR7+ DCs are the dominant DC population that migrate to the dLN, but a subset remains tumour-resident despite CCR7 expression. These tumour-retained CCR7+ DCs are phenotypically and transcriptionally distinct from their dLN counterparts and heterogeneous. Moreover, they progressively downregulate the expression of antigen presentation and pro-inflammatory transcripts with more prolonged tumour dwell-time. Tumour-residing CCR7+ DCs co-localise with PD-1+CD8+ T cells in human and murine solid tumours, and following anti-PD-L1 treatment, upregulate stimulatory molecules including OX40L, thereby augmenting anti-tumour cytolytic activity. Altogether, these data uncover previously unappreciated heterogeneity in CCR7+ DCs that may underpin a variable capacity to support intratumoural cytotoxic T cells.

Blockade of CD93 in pleural mesothelial cells fuels anti-lung tumor immune responses.

Background: CD93 reportedly facilitates tumor angiogenesis. However, whether CD93 regulates antitumor immunity remains undeciphered. Methods: Lung tumor tissues, malignant pleural effusions (MPEs) were obtained from lung cancer patients. Blood was obtained from healthy volunteers and lung cancer patients with anti-PD-1 therapy. Furthermore, p53fl/flLSL-KrasG12D, Ccr7-/-, Cd93-/- mice and CD11c-DTR mice were generated. Specifically, EM, NTA and western blotting were utilized to identify Tumor extracellular vesicles (TEVs). EV labeling, detection of EV uptake in vitro and in vivo, degradation of EV proteins and RNAs were performed to detect the role of TEVs in tumor progression. Pleural mesothelial cells (pMCs) were isolated to investigate related signaling pathways. Recombinant proteins and antibodies were generated to test which antibody was the most effective one to increase CCL21a in p-pMCs. RNA-Seq, MiRNA array, luciferase reporter assay, endothelial tube formation assay, protein labeling and detection, transfection of siRNAs and the miRNA mimic and inhibitor, chemotaxis assay, immunohistochemical staining, flow cytometry, Real-time PCR, and ELISA experiments were performed. Results: We show that CD93 of pMCs reduced lung tumor migration of dendritic cells by preventing pMCs from secreting CCL21, thereby suppressing systemic anti-lung tumor T-cell responses. TEV-derived miR-5110 promotes CCL21 secretion by downregulating pMC CD93, whereas C1q, increasing in tumor individuals, suppresses CD93-mediated CCL21 secretion. CD93-blocking antibodies (anti-CD93) inhibit lung tumor growth better than VEGF receptor-blocking antibodies because anti-CD93 inhibit tumor angiogenesis and promote CCL21 secretion from pMCs. Anti-CD93 also overcome lung tumor resistance to anti-PD-1 therapy. Furthermore, lung cancer patients with higher serum EV-derived miR-5193 (human miR-5110 homolog) are more sensitive to anti-PD-1 therapy, while patients with higher serum C1q are less sensitive, consistent with their regulatory functions on CD93. Conclusions: Our study identifies a crucial role of CD93 in controlling anti-lung tumor immunity and suggests a promising approach for lung tumor therapy.

Ectromelia Virus Affects the Formation and Spatial Organization of Adhesive Structures in Murine Dendritic Cells In Vitro.

Ectromelia virus (ECTV) is a causative agent of mousepox. It provides a suitable model for studying the immunobiology of orthopoxviruses, including their interaction with the host cell cytoskeleton. As professional antigen-presenting cells, dendritic cells (DCs) control the pericellular environment, capture antigens, and present them to T lymphocytes after migration to secondary lymphoid organs. Migration of immature DCs is possible due to the presence of specialized adhesion structures, such as podosomes or focal adhesions (FAs). Since assembly and disassembly of adhesive structures are highly associated with DCs' immunoregulatory and migratory functions, we evaluated how ECTV infection targets podosomes and FAs' organization and formation in natural-host bone marrow-derived DCs (BMDC). We found that ECTV induces a rapid dissolution of podosomes at the early stages of infection, accompanied by the development of larger and wider FAs than in uninfected control cells. At later stages of infection, FAs were predominantly observed in long cellular extensions, formed extensively by infected cells. Dissolution of podosomes in ECTV-infected BMDCs was not associated with maturation and increased 2D cell migration in a wound healing assay; however, accelerated transwell migration of ECTV-infected cells towards supernatants derived from LPS-conditioned BMDCs was observed. We suggest that ECTV-induced changes in the spatial organization of adhesive structures in DCs may alter the adhesiveness/migration of DCs during some conditions, e.g., inflammation.

Circadian clocks drive rhythmic antitumor immune responses mediated by migratory dendritic cells

Circadian clocks drive rhythmic antitumor immune responses mediated by migratory dendritic cells

A mechanistic marker-based screening tool to predict clinical immunogenicity of biologics

BackgroundThe efficacy and safety of therapeutic proteins are undermined by immunogenicity driven by anti-drug antibodies. Immunogenicity risk assessment is critically necessary during drug development, but current methods lack predictive power and mechanistic insight into antigen uptake and processing leading to immune response. A key mechanistic step in T-cell-dependent immune responses is the migration of mature dendritic cells to T-cell areas of lymphoid compartments, and this phenomenon is most pronounced in the immune response toward subcutaneously delivered proteins.MethodsThe migratory potential of monocyte-derived dendritic cells is proposed to be a mechanistic marker for immunogenicity screening. Following exposure to therapeutic protein in vitro, dendritic cells are analyzed for changes in activation markers (CD40 and IL-12) in combination with levels of the chemokine receptor CXCR4 to represent migratory potential. Then a transwell assay captures the intensity of dendritic cell migration in the presence of a gradient of therapeutic protein and chemokine ligands.ResultsHere, we show that an increased ability of the therapeutic protein to induce dendritic cell migration along a gradient of chemokine CCL21 and CXCL12 predicts higher immunogenic potential. Expression of the chemokine receptor CXCR4 on human monocyte-derived dendritic cells, in combination with activation markers CD40 and IL-12, strongly correlates with clinical anti-drug antibody incidence.ConclusionsMechanistic understanding of processes driving immunogenicity led to the development of a predictive tool for immunogenicity risk assessment of therapeutic proteins. These predictive markers could be adapted for immunogenicity screening of other biological modalities.

Abstract A053: Notch completes CD4 T cell help in CD8 T cells and ensures timely departure

Abstract Efficient activation, expansion and differentiation of CD8 T cells depend on help from CD4+ T cells. Such help is especially critical for CD8 T cell responses against tumor antigens. Priming of CD8 T cells in lymph nodes (LN) involves two steps that are geographically and temporally separate. Naïve CD8 T cells are first preactivated by a migratory dendritic cell (DC) carrying antigen from tissues. Preactivated CD8 T cells must then find and interact with lymph node-resident XCR1+ DC that have been licensed for optimal CD8 T cell activation via CD4 T cell-mediated activation of CD40. The efferent signals provided by licensed CD8+XCR1+ DC to install the CD4 T cell help program in CD8 T cells are incompletely known. Moreover, it is not clear whether and, if so, how premature lymph node exit by preactivated CD8 T cells before receipt of help is avoided. Here, we show that completion of CD4 help delivery is coupled to the ability to leave lymph nodes through activation of Notch receptors on CD8 T cells. Expression of Notch receptors is induced on naïve CD8 T cells during the first step of priming, while CD40 ligation elicits expression of Notch ligands on XCR1+ lymph node resident DCs. Using a DNA vaccination model that allows induction of CD8 T cell responses in the presence or absence of CD4 T cell help, we show that the Notch1/2 receptors are necessary in CD8+ T cells for CD4+ T cell help-dependent proliferation and acquisition of effector capability. Correspondingly, RNA sequencing revealed that more than half of the genetic CD4 T cell help program in CD8 T cells depends on Notch. Strikingly, activated Notch1/2 deficient and unhelped CD8 T cells remain largely stuck in lymph nodes owing to a lack of expression of the lymph node exit receptor S1PR5. Control of expression of S1PR5 by Notch therefore ensures that preactivated CD8 T cells remain in lymph nodes until completion of their activation process by receiving the CD4 T cell help signal. Citation Format: Adrien Remi Gabriel Laurent, Julien Karrich, Nikolina Babala, Derk Amsen. Notch completes CD4 T cell help in CD8 T cells and ensures timely departure [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A053.

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.3

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.3

A Landscape of Dendritic Cells Development, Activation and Migration in Mouse Model of Sickle Cell Anemia

Dendritic cells (DCs) are crucial players of innate immunity in initiating, polarizing, and regulating the adaptive immune response. They become activated upon encountering either pathogen- or damage-associated molecular patterns (PAMPs or DAMPs), which are often released due to tissue necrosis or intravascular hemolysis, common events in individuals with sickle cell disease (SCD), one of the most common hemoglobinophaties that affects millions of people worldwide. Upon activation, DCs migrate to peripheral lymph nodes, where they present antigens to T lymphocyte together with co-stimulatory or inhibitory molecules. In addition, they can polarize T cell response by releasing pro- or anti-inflammatory cytokines. We previously showed that SCD patients have higher circulating total and inflammatory DCs, whereas the ratio of type 1 conventional DCs (cDC1) is lower than control individuals. These changes correlated with number of reticulocytes and with the skewed T response towards Th17. Alterations in the development and activation of DC subpopulations, as well as in their function can impair the immune response or tolerance, which may account for the dysfunction in T lymphocyte responses observed in SCD patients. In order to investigate the development, activation and migration of DCs during SCD, we characterized these cells by flow cytometry in lymphoid organs of Townes mice, which have human S hemoglobin (HbS). Our data show that Townes mice (SS) have lower number of total DCs in bone marrow (p=0.057; N=8 each), however, they have higher number of these cells in the spleen (p<0.0001; N=8 each) and lymph nodes (p<0.0021; N=8 each) when compared with the littermate WT mice, suggesting migration of DCs to these peripheral organs. Evaluation of the ratios of DCs subsets shows that the frequency of cDC1 (CD11c +CD8a +CD11b -) among total DCs is higher in the three organs of SS mice compared with WT mice (p=0.004 in spleen; p=0.0015 in bone marrow; and p=0.01 in lymph nodes; N=8 each group). In contrast, the percentage of type 2 conventional DCs, cDC2 (CD11c +CD8a -CD11b +) is similar or lower in SS mice than WT mice (p=0.0005 in spleen; N=8 each). In addition, the frequency of plasmacytoid dendritic cell, pDC (CD11c +B220 +SiglecH +) is higher in lymph nodes (p=0.0006; N=8 each) and lower in bone marrow (p=0.0017; N=8 each) of SS mice when compared with WT mice, indicating that the migration of DCs to peripheral organs is due to cDC1 and pDC. Regarding their maturation and activation phenotype, cDC1 and cDC2 from SS mice have a more immature or inhibitory phenotype than WT mice, as showed by MHC-II (cDC1: p=0.01 in spleen; p=0.0004 in lymph nodes; N=8 each) and CD86 expression (cDC1: p=0.013 in spleen; p=0.0003 in bone marrow; and p= 0.03 in lymph nodes; cDC2: p=0.0067 in bone marrow and p=0.038 in lymph nodes; N=8 each). On the other hand, pDCs from the three organs of SS mice showed a more mature profile than WT mice, as demonstrated by an increase in Sca-1 expression (p=0.0024 in spleen; p=0.037 in bone marrow; and p=0.04 in lymph nodes; N=8 each), indicating that cDCs are being inhibited whereas pDCs are activated during SCD even in steady state. Aimed to understand whether at least part of the alterations on DCs is due to changes in their development, we quantified monocyte and DC progenitor, MDP (Lin -(CD3/CD19/CD56/CD14) CD135 +CD11c -CD115 +CD117 +) and common DC progenitor, CDP (Lin -CD135 +CD11c -CD115 +CD117 -) in bone barrow. Our data show that the ratio of MDP, the earlier progenitor, is increased (p=0.0009; N=8 each), whereas CDP, the progenitor committed to DCs, is diminished (p=0.014; N=8 each) in SS mice when compared with WT mice, indicating a change in the DC compartment in SS mice since their developmental stage. When we performed an in vitro differentiation of DC from bone marrow cells in the presence of flt3l, we did not observe statistical difference in either development and apoptosis of DCs, or in the percentages of DC subsets between SS and WT mice, indicating that the alteration in DC development in SS mice may be due to environmental conditions instead of CDP or DC intrinsic factor. Our findings so far improve our understanding of DCs dynamics in SCD, including the development, activation and migration of the different subsets that may have an important impact on adaptive immune response disfunction in SCD, which may account for the chronic inflammatory state and the susceptibility to infections.

BCG infection dose guides dendritic cell migration and T cell priming in the draining lymph node.

In contrast to delayed-type hypersensitivity (DTH) and other hallmark reactions of cell-mediated immunity that correlate with vaccine-mediated protection against Mycobacterium tuberculosis, the contribution of vaccine dose on responses that emerge early after infection in the skin with Bacille Calmette-Guérin (BCG) is not well understood. We used a mouse model of BCG skin infection to study the effect of BCG dose on the relocation of skin Dendritic cells (DCs) to draining lymph node (DLN). Mycobacterium antigen 85B-specific CD4+ P25 T cell-receptor transgenic (P25 TCRTg) cells were used to probe priming to BCG in DLN. DC migration and T cell priming were studied across BCG inocula that varied up to 100-fold (104 to 106 Colony-forming units-CFUs). In line with earlier results in guinea pigs, DTH reaction in our model correlated with BCG dose. Importantly, priming of P25 TCRTg cells in DLN also escalated in a dose-dependent manner, peaking at day 6 after infection. Similar dose-escalation effects were seen for DC migration from infected skin and the accompanying transport of BCG to the DLN. BCG-triggered upregulation of co-stimulatory molecules on migratory DCs was restricted to the first 24 hour after infection and was independent of BCG dose over a 10-fold range (105 to 106 CFUs). The dose seemed to be a determinant of the number of total skin DCs that move to the DLN. In summary, our results support the use of higher BCG doses to detect robust DC migration and T cell priming.

Phenotypic features of dendritic cells when using different modes of their stimulated maturation

Monocyte-derived dendritic cells (DCs) can be used for cell immunotherapy of cancer. In most cases, mature DCs, loaded with tumor-associated antigens, are used for immune therapy. The functionality of DCs for immunotherapy substantially depends on their immunophenotype and secretory profile, which are established after DCs maturation. The purpose of this research was to explore the phenotype of DCs after using various approaches for stimulation of their maturation.Maturation of DCs was stimulated by pro-inflammatory cytokines and their mixtures, or by ligands to the TLRs of DCs. DCs were stimulated by the following means: TNF; poly I:C; LPS; cytokine cocktail (TNF + IL-1 + IL-6 + PGE2); the cocktail mixed with poly I:C; and melanoma cells lysate. Forty-eight hours after stimulation, the expression of DCs’ receptors involved into their interaction with T cells, was evaluated by flow cytometry. Moreover, the secretion of IL-12 (activator of T cell response) and IL-10 (inhibitor of T cell response) was estimated by ELISA technique.We have shown that, following stimulation with cytokine cocktail, the DCs exhibit highest expression of receptors, which are necessary for interaction with T cells and for activation of T cell mediated immune response, i.e., antigen-presenting receptors (HLA-DR), co-stimulatory receptors (CD83, CD40, CD86), and receptors controlling the migration of DCs to lymph nodes (CCR7). Moreover, the cocktail-stimulated DCs intensively secrete both IL-12 and IL-10. The stimulatory effect of TNF and poly I:C proved to be moderate: the expression of most receptors was significantly lower than after using the cocktail; no significant differences from control (in absence of induced maturation) in IL-12 secretion were detected. LPS and melanoma cell lysate did not affect both expression of receptors and secretory profile of DCs. Addition of poly I:C to the cytokine cocktail did not affect the receptor expression, but significantly increased the secretion of both proinflammatory IL-12 and anti-inflammatory IL-10.The results of experiments demonstrate that the mixture of cytokine cocktail and poly I:C seems to be the most effective tool for stimulation of DCs maturation. However, further experiments are required to compare the functionality of DCs when using different tools for induced DC maturation.

EXTH-68. SYSTEMIC NEUTRALIZATION OF IL6 SENSITIZES IDH-WILDTYPE GLIOBLASTOMA PRECLINICAL MODELS TO IMMUNE CHECKPOINT INHIBITION

Abstract IDH-wildtype glioblastomas are resistant to immune checkpoint inhibition (ICI). Here we integrate spatial and single-cell approaches across paired human glioblastomas before or after ICI with functional studies in immunocompetent intracranial mouse glioblastoma models to reveal mechanisms and therapeutic vulnerabilities underlying glioblastoma resistance to ICI. Spatial proteomic and transcriptomic approaches were used to identify biochemical and gene expression programs underlying ICI response or resistance in paired human glioblastoma samples (n = 7). Intracranial SB28 glioblastoma allografts were implanted in C57BL/6J mice and treated with (1) convection enhanced delivery (CED) of AAV9 gene therapy vectors encoding cytokines that reprogram the tumor immune microenvironment (TIME) in other cancers (Il1b, Ccl4, Apoa1), (2) systemic anti-PD1, (3) local anti-IL6, (4) systemic anti-IL6, or (5) combination systemic treatment with anti-PD1 and anti-IL6. Histological, mass cytometry (CyTOF), and multiplexed cytokine assays were used to assess the glioblastoma TIME in response to treatment over time. Mice were monitored for survival and tumor growth was measured using intracranial bioluminescence. Human glioblastomas with radiographic responses to ICI had lower pre-treatment IL6 levels on spatial profiling than glioblastomas resistant to ICI. Intratumor IL6 was suppressed by all AAV9 cytokine conditions compared to AAV9-GFP in mice. Mouse survival was improved by combination systemic treatment with anti-PD1 and anti-IL6, but was not improved by either antibody alone, or by anti-IL6 CED. CyTOF revealed combination systemic therapy reprogrammed the glioblastoma TIME by increasing activated immune cell types such as MHCII+ monocytes, CD103+ migratory dendritic cells (DCs), CD11b+ conventional DCs, and effector CD8+ T cells, and by decreasing immunosuppressive Tregs. In summary, systemic anti-IL6 in combination with systemic anti-PD1 reprograms the glioblastoma TIME to sensitize preclinical models to ICI. These results provide mechanistic validation of findings from human glioblastomas that respond to ICI and shed light on a therapeutic vulnerability underlying the glioblastoma TIME.

Vedolizumab Efficacy Is Associated With Decreased Intracolonic Dendritic Cells, Not Memory T Cells.

Vedolizumab, an antibody blocking integrin α4β7, is a safe and effective therapy for Crohn's disease and ulcerative colitis. Blocking α4β7 from binding its cognate addressin MAdCAM-1 on intestinal blood vessel endothelial cells prevents T cells from migrating to the gut mucosa in animal models. However, data supporting this mechanism of action in humans is limited. We conducted a cross-sectional case-control study to evaluate the effect of vedolizumab on intestinal immune cell populations while avoiding the confounding effect of resolving inflammation on the cellularity of the colonic mucosa in treatment-responsive patients. Colon biopsies from 65 case subjects receiving vedolizumab were matched with biopsies from 65 control individuals, similar in disease type, medications, anatomic location, and inflammation. Biopsies were analyzed by flow cytometry and full messenger RNA transcriptome sequencing of sorted T cells. No difference was seen between vedolizumab recipients and control individuals in the quantity of any antigen-experienced T lymphocyte subset or in the quality of the transcriptome in any experienced T cell subset. Fewer naïve colonic B and T cells were seen in vedolizumab recipients than control individuals, regardless of response. However, the most striking finding was a marked reduction in CD1c+ (BDCA1+) dendritic cells exclusively in vedolizumab-responsive patients. In blood, these dendritic cells ubiquitously express high levels of α4β7, which is rapidly downregulated upon vedolizumab exposure. The clinical effects of vedolizumab reveal integrin α4β7-dependent dendritic cell migration to the intestinal mucosa to be central to inflammatory bowel disease pathogenesis.