Survival Of Dendritic Cells Research Articles

Effect of physical exercise on the proliferation and inactivation capacity of biological immune cells

Objective: Exploring the effects of sport on the proliferation and inactivation of biological immune cells. Method: Sixty college students enrolled in a university between 1 March 2023 and 1 June 2023 were selected for the experiment. A total of 30 subjects were screened based on exclusion and inclusion criteria. The 30 subjects were evaluated and categorized into three experimental groups: Low-intensity physical activity, high-intensity physical activity and no physical activity. The three groups were tested for a period of 10 weeks, and the changes in cell survival rate, proliferation and inactivation capacity of macrophages, t-lymphocytes and dendritic cells were analyzed in the three groups. Result: There was a statistically significant difference (P < 0.05) in the pre- and post-experimental comparison of the different immune cells in the low-intensity physical exercise group. Only dendritic cell differentiation and survival rate were statistically significantly different in the pre- and post-experiment comparisons of high-intensity physical exercise (P < 0.05). Conclusion: Low-intensity physical exercise can significantly increase the proliferation and inactivation capacity of immune cells, which has a significant effect on the immune system of the organism.

PH and Redox Dual-Responsive Nanoparticle with Enhanced Dendritic Cell Maturation for Cancer Therapy.

Type I interferons (IFNs) are essential for activating dendritic cells (DCs) and presenting tumor-associated antigens to T cells. IFNs are primarily produced from DCs among immune cells. A combination of chemotherapy and metalloimmunotherapy induces IFN production by activating the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. However, chemotherapeutic agents deplete DC populations, suppressing immunostimulatory activities, despite their potent anticancer activities. Furthermore, an optimal ratio between chemotherapeutic agents and metal for activating DCs at the highest level has not been reported, and evidence for ensuring DC survival is lacking. In this study, we hypothesized that there is an optimal ratio to yield the highest DC maturation and anticancer activity with minimal DC depletion. To demonstrate it, we have designed a pH and redox dual-responsive nanoparticle, MnO2@BSA@DOX (MD), to prevent DCs from depleting and activate the cGAS-STING pathway both in cancer cells and DCs, inducing considerable levels of IFNs and maturation. MD consists of a core-layer structure, a manganese dioxide (MnO2) core, and a cross-linked layer with bovine serum albumin (BSA) and doxorubicin (DOX), with a specific ratio of DOX to manganese. MD exhibits structure-based selectivity between cancer cells and DCs by targeting the extracellular pH of the tumor microenvironment and intracellular redox reactions in cancer cells. Among various formulations, the 1:1 ratio shows the highest maturation with no significant depletion. Moreover, it induces distinct cytotoxicity in cancer cells through apoptosis and cGAS-STING activation, leading to increased calreticulin expression and enhanced DC phagocytosis. Consequently, it results in superior tumor suppression and prolonged survival with the high accumulation of MD in the tumor and no observed systemic toxicities, highlighting its potential as a therapeutic agent in cancer treatments.

Endoplasmic Reticulum Stress Response Mediator IRE-1α Promotes Host Dendritic Cells in Graft-versus-Host Disease Development.

Allogeneic hematopoietic cell transplantation is an effective treatment for hematologic malignancies, but the complications such as graft-versus-host disease (GVHD) can limit its benefit. The conditioning regimens before transplant, including chemotherapy or irradiation, can trigger endoplasmic reticulum stress. IRE-1α is a major endoplasmic reticulum stress mediator that can further activate both spliced XBP-1 (XBP-1s) and regulated IRE-1-dependent decay (RIDD). IRE-1α-XBP-1s signaling controls dendritic cell (DC) differentiation and Ag presentation, crucial in GVHD progression. In this study, we used DC-specific XBP-1-deficient mice as donors or recipients and observed that XBP-1s was crucial for host DCs in the induction of GVHD but dispensable for the graft-versus-leukemia response. To specifically target IRE-1α in the host, we treated recipient mice with the IRE-1α inhibitor B-I09 for 3 d prior to bone marrow transplantation, which significantly suppressed GVHD development while maintaining the graft-versus-leukemia effect. XBP-1-deficient or BI09-treated recipients showed reduced DC survival after irradiation and bone marrow transplantation. Inhibition of IRE-1α also led to a reduction in DC alloreactivity, subsequently decreasing the proliferation and activation of allogeneic T cells. With further study using RIDD-deficient DCs, we observed that RIDD was also required for optimal DC activation. Taken together, XBP-1s and RIDD both promote host DC survival and alloreactivity that contribute to GVHD development.

Apoptosis of Dendritic Cells and Autoimmune Disease.

Dendritic cells (DCs), the most efficient antigen-presenting cells (APCs), bridge the innate and adaptive immune systems. As such, the turn-over of DCs is critical during autoimmune responses, and the dysregulation of DC apoptosis could cause severe immune destruction in the host. For example, reduction of immunogenic DCs by increased apoptosis could lead to immune tolerance to pathogen infection that might allow exposure of nuclear autoantigens, whereas reduced apoptosis could result in long-term lymphocyte activation to break the immune tolerance for the development of autoimmune disease. Thus, keeping a balance between survival and apoptosis of DCs is crucial to maintain immune homeostasis. In this review, we summarize the recent development on the factors inducing DC apoptosis and their underlying mechanisms to provide insights into the immunopathogenesis of some autoimmune diseases, which could lead to effective therapeutic interventions in the clinics.

L-methionine and the L-type Ca2+ channel agonist BAY K 8644 collaboratively contribute to the reduction of depressive-like behavior in mice.

The L-type Ca2+ channel (LTCC, also known as Cav1,2) is involved in the regulation of key neuronal functions, such as dendritic information integration, cell survival, and neuronal gene expression. Clinical studies have shown an association between L-type calcium channels and the onset of depression, although the precise mechanisms remain unclear. The development of depression results from a combination of environmental and genetic factors. DNA methylation, a significant epigenetic modification, plays a regulatory role in the pathogenesis of psychiatric disorders such as posttraumatic stress disorder (PTSD), depression, and autism. In our study, we observed reduced Dnmt3a expression levels in the hippocampal DG region of mice with LPS-induced depression compared to control mice. The antidepressant Venlafaxine was able to increase Dnmt3a expression levels. Conversely, Bay K 8644, an agonist of the L-type Ca2+ channel, partially ameliorated depression-like behaviors but did not elevate Dnmt3a expression levels. Furthermore, when we manipulated DNA methylation levels during Bay K 8644 intervention in depression-like models, we found that enhancing the expression of Dnmt3a could improve LPS-induced depression/anxiety-like behaviors, while inhibiting DNA methylation exacerbated anxiety-like behaviors, the combined use of BAY K 8644 and L-methionine can better improve depressive-like behavior. These findings indicate that DNA methylation plays a role in the regulation of depression-like behaviors by the L-type Ca2+ channel, and further research is needed to elucidate the interactions between DNA methylation and L-type Ca2+ channels.

An Organotypic Human Lymph Node Model Reveals the Importance of Fibroblastic Reticular Cells for Dendritic Cell Function.

Human lymph node (HuLN) models have emerged with invaluable potential for immunological research and therapeutic application given their fundamental role in human health and disease. While fibroblastic reticular cells (FRCs) are instrumental to HuLN functioning, their inclusion and recognition of importance for organotypic in vitro lymphoid models remain limited. Here, we established an in vitro three-dimensional (3D) model in a collagen-fibrin hydrogel with primary FRCs and a dendritic cell (DC) cell line(MUTZ-3 DC). To study and characterise the cellular interactions seen in this 3D FRC-DC organotypic model compared to the native HuLN; flow cytometry, immunohistochemistry, immunofluorescence and cytokine/chemokine analysis were performed. FRCs were pivotal for survival, proliferation and localisation of MUTZ-3 DCs. Additionally, we found that CD1a expression was absent on MUTZ-3 DCs that developed in the presence of FRCs during cytokine-induced MUTZ-3 DC differentiation, which was also seen with primary monocyte-derived DCs (moDCs). This phenotype resembled HuLN-resident DCs, which we detected in primary HuLNs, and these CD1a- MUTZ-3 DCs induced T cell proliferation within a mixed leukocyte reaction (MLR), indicating a functional DC status. FRCs expressed podoplanin (PDPN), CD90 (Thy-1), CD146 (MCAM) and Gremlin-1, thereby resembling the DC supporting stromal cell subset identified in HuLNs. This 3D FRC-DC organotypic model highlights the influence and importance of FRCs for DC functioning in a more realistic HuLN microenvironment. As such, this work provides a starting point for the development of an in vitro HuLN.

Clusterin protects mature dendritic cells from reactive oxygen species mediated cell death

ABSTRACT Dendritic cells (DCs) play a key role in the induction of the adaptive immune response. They capture antigens in peripheral tissues and prime naïve T lymphocytes, triggering the adaptive immune response. In the course of inflammatory processes DCs face stressful conditions including hypoxia, low pH and high concentrations of reactive oxygen species (ROS), among others. How DCs survive under these adverse conditions remain poorly understood. Clusterin is a protein highly expressed by tumors and usually associated with bad prognosis. It promotes cancer cell survival by different mechanisms such as apoptosis inhibition and promotion of autophagy. Here, we show that, upon maturation, human monocyte-derived DCs (MoDCs) up-regulate clusterin expression. Clusterin protects MoDCs from ROS-mediated toxicity, enhancing DC survival and promoting their ability to induce T cell activation. In line with these results, we found that clusterin is expressed by a population of mature LAMP3+ DCs, called mregDCs, but not by immature DCs in human cancer. The expression of clusterin by intratumoral DCs was shown to be associated with a transcriptomic profile indicative of cellular response to stress. These results uncover an important role for clusterin in DC physiology.

Jiangqi Pingxiao formula regulates dendritic cell apoptosis in an autophagy-dependent manner through the AMPK/mTOR pathway in a murine model of OVA-induced asthma

Ethnopharmacological relevanceAllergic asthma is a recurring respiratory condition that typically manifests during childhood or adolescence. It is characterized by a dominant type II immune response triggered by the identification and capturing of inhaled allergens by dendritic cells (DCs). Jiangqi Pingxiao Formula (JQPXF), a prescription medicine used for the treatment of pediatric asthma, has been clinically proven to be both safe and effective. However, its mechanism of action in the treatment of asthma has not been fully been fully elucidated. Recent research suggests that several natural compounds have the potential to target dendritic cells (DCs) and alleviate ovalbumin (OVA)-induced asthma, which may also be found within JQPXF. Aim of the studyThis study aimed to elucidate the effect of JQPXF on OVA-induced asthma model and its molecular mechanism targeting DCs. Materials and methodsThe main constituents of JQPXF were analyzed by ultra performance liquid chromatography (UPLC). An asthma model was established by OVA. Hematoxylin-eosin staining and measurement of respiratory function was used to evaluate the treatment effect of JQPXF on asthmatic mice. Cytokine (IL-5, IL-13 and IgE) concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was employed to evaluate inflammatory cell infiltration (T helper 2 cells and DCs) in vivo and DC survival in vivo and vitro. Western blot and immunofluorescence were used to verify the molecular mechanisms. ResultsThe results suggest that JQPXF can ameliorate pathological conditions and improve lung function in asthmatic mice, as well as the Th2 cells. Treatment with JQPXF significantly reduced the number of DCs and increased the number of Propidium iodide+ (PI) DCs. Furthermore, JQPXF upregulated protein levels of the pro-apoptotic factors Cleaved-caspase-3 and Bax, while downregulating the anti-apoptotic factor Bcl-2. Simultaneously, JQPXF increased autophagy levels by facilitating p62 degradation and promoting translation from LC3B I to LC3B II of DCs in vitro, as well as reducing the integrated optical density (IOD) of p62 within the CD11c-positive area in the lung. 3-Methyladenine (3-MA) was used to block autophagic flux and the apoptotic effect of JQPXF on DCs was abolished in vitro, with the number of DCs decreased by JQPXF being reversed in vivo. We further investigated the upstream key regulator of autophagy, the AMPK/mTOR pathway, and found that JQPXF increased AMPK phosphorylation while decreasing mTOR phosphorylation levels. Additionally, we employed Compound C (CC) as an AMPK inhibitor to inhibit this signaling pathway, and our findings revealed that both autophagic flux and apoptotic levels in DCs were abolished in vitro. ConclusionsIn summary, we have demonstrated that JQPXF could alleviate type II inflammation in an asthmatic model by promoting the apoptosis of DCs through an autophagy-dependent mechanism, achieved by regulating the AMPK/mTOR signaling pathway.

Dendritic cells resist to disulfiram-induced cytotoxicity, but reduced interleukin-12/23(p40) production.

Disulfiram (DSF), a medication for alcoholism, has recently been used as a repurposing drug owing to its anticancer effects. Despite the crucial role of dendritic cells (DCs) in immune homeostasis and cancer therapy, the effects of DSF on the survival and function of DCs have not yet been studied. Therefore, we treated bone marrow-derived DCs with DSF and lipopolysaccharide (LPS) and performed various analyses. DCs are resistant to DSF and less cytotoxic than bone marrow cells and spleen cells. The viability and metabolic activity of DCs hardly decreased after treatment with DSF in the absence or presence of LPS. DSF did not alter the expression of surface markers (MHC II, CD86, CD40, and CD54), antigen uptake capability, or the antigen-presenting ability of LPS-treated DCs. DSF decreased the production of interleukin (IL)-12/23 (p40), but not IL-6 or tumor necrosis factor-α, in LPS-treated DCs. We considered the granulocyte-macrophage colony-stimulating factor (GM-CSF) as a factor to make DCs resistant to DSF-induced cytotoxicity. The resistance of DCs to DSF decreased when GM-CSF was not given or its signaling was inhibited. Also, GM-CSF upregulated the expression of a transcription factor XBP-1 which is essential for DCs' survival. This study demonstrated for the first time that DSF did not alter the function of DCs, had low cytotoxicity, and induced differential cytokine production.

Evaluation of Serum Level of Interleukin 15 in Active Vitiligo Compared to Stable Disease and Healthy Control

Abstract Background Vitiligo is a common acquired disorder of depigmentation. Its pathogenesis entails a T helper (TH) 1-cytotoxic T (CT) lymphocytes mediated autoimmune melanocyte destruction. Interleukin (IL)-15 is one of the IL-2 family of cytokines and shares several actions with IL-2. IL-15 enhances survival, maturation, and functional activity of natural killer, neutrophils, and dendritic cells. Furthermore, it potentiates survival, maturation, and cytotoxicity of memory CT cells. IL-15 has been shown to play a crucial role in the pathogenesis of several autoimmune diseases but was poorly investigated in patients with vitiligo. Aims To investigate the role of IL-15 in the pathogenesis of vitiligo. Patients and Methods In total, 40 Patients with vitiligo and 20 controls matched for age and gender were enrolled in the study. Vitiligo Extent Score (VES) and Vitiligo Disease Activity (VIDA) score were used to assess vitiligo severity and activity, respectively. Serum level of IL-15 was assessed by enzyme-linked immune-sorbent assay. Results Serum IL-15 level, in patients with vitiligo, was significantly higher in comparison with the control group (P =. 001). A significant positive correlation was found between serum IL-15 level and VES score (P =. 001) Conclusion IL-15 level was elevated in the sera of patients with vitiligo. IL-15 may therefore have a significant impact on vitiligo autoimmune pathogenesis, and further identification of its molecular roles may highlight new therapeutic strategies for vitiligo.

The deubiquitinating enzyme OTUD7b protects dendritic cells from TNF-induced apoptosis by stabilizing the E3 ligase TRAF2

The cytokine tumor necrosis factor (TNF) critically regulates the intertwined cell death and pro-inflammatory signaling pathways of dendritic cells (DCs) via ubiquitin modification of central effector molecules, but the intrinsic molecular switches deciding on either pathway are incompletely defined. Here, we uncover that the ovarian tumor deubiquitinating enzyme 7b (OTUD7b) prevents TNF-induced apoptosis of DCs in infection, resulting in efficient priming of pathogen-specific CD8+ T cells. Mechanistically, OTUD7b stabilizes the E3 ligase TNF-receptor-associated factor 2 (TRAF2) in human and murine DCs by counteracting its K48-ubiquitination and proteasomal degradation. TRAF2 in turn facilitates K63-linked polyubiquitination of RIPK1, which mediates activation of NF-κB and MAP kinases, IL-12 production, and expression of anti-apoptotic cFLIP and Bcl-xL. We show that mice with DC-specific OTUD7b-deficiency displayed DC apoptosis and a failure to induce CD8+ T cell-mediated brain pathology, experimental cerebral malaria, in a murine malaria infection model. Together, our data identify the deubiquitinating enzyme OTUD7b as a central molecular switch deciding on survival of human and murine DCs and provides a rationale to manipulate DC responses by targeting their ubiquitin network downstream of the TNF receptor pathway.

Bio-adhesive Macroporous Hydrogels for In Situ Recruitment and Modulation of Dendritic Cells.

Biomaterials that enable in situ recruitment and modulation of immune cells have demonstrated tremendous promise for developing potent cancer immunotherapy such as therapeutic cancer vaccine. One challenge related to biomaterial scaffold-based cancer vaccines is the development of macroporous materials that are biocompatible and stable, enable controlled release of chemokines to actively recruit a large number of dendritic cells (DCs), contain macropores that are large enough to home the recruited DCs, and support the survival and proliferation of DCs. Bio-adhesive macroporous gelatin hydrogels were synthesized and characterized for mechanical properties, porous structure, and adhesion towards tissues. The recruitment of immune cells including DCs to chemokine-loaded bioadhesive macroporous gels was analyzed. The ability of gels loaded with granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor extracellular vesicles (EVs) to elicit tumor-specific CD8+ T cell responses was also analyzed. Here we develop a bioadhesive macroporous hydrogel that can strongly adhere to tissues, contain macropores that are large enough to home immune cells, are mechanically tough, and enable controlled release of chemokines to recruit and modulate immune cells in situ. The macroporous hydrogel is composed of a double crosslinked network of gelatin and polyacrylic acid, and the macropores are introduced via cryo-polymerization. By incorporating GM-CSF and tumor EVs into the macroporous hydrogel, a high number of DCs can be recruited in situ to process and present EV-encased antigens. These tumor antigen-presenting DCs can then traffic to lymphatic tissues to prime antigen-specific CD8+ T cells. This bioadhesive macroporous hydrogel system provides a new platform for in situ recruitment and modulation of DCs and the development of enhanced immunotherapies including tumor EV vaccines. We also envision the promise of this material system for drug delivery, tissue regeneration, long-term immunosuppression, and many other applications. The online version contains supplementary material available at 10.1007/s12195-023-00770-2.

Nitric Oxide (NO) regulation of Dendritic cells (DC) metabolism and immune effector functions

Abstract Dendritic cell (DC) activation is characterized by a sustained commitment to glycolysis, a requirement for survival in DC subsets expressing inducible nitric oxide synthase (iNOS, encoded by Nos2 gene). Nitric oxide (NO) can mediate both host-protective and host-cytotoxic effects by interfering with pathogen replication and/or by inhibiting the host mitochondrial respiration, resulting in metabolic rewiring in DCs. These phenomena primarily have been studied in DCs from the classic laboratory inbred mouse strain C57BL/6J (B6), where DCs experience a loss of mitochondrial function due to NO accumulation. To assess the conservation of NO-driven metabolic regulation in DCs, we compared B6 mice to the wild-derived genetically divergent PWD/PhJ (PWD) strain. We show preserved mitochondrial respiration and enhanced post-activation survival in LPS-stimulated PWD DCs due to attenuated NO production. To genetically map PWD’s NO phenotype, we used a congenic mouse strain (B6.PWD-Chr11.2; 11.2) that carries a PWD-derived portion of chromosome 11, including Nos2, on a B6 background. The 11.2 DCs show comparable iNOS expression with lower NO production levels than B6 DCs. We demonstrate that activated 11.2 DCs maintain mitochondrial respiration and TCA cycle carbon flux, compared with B6 DCs. However, reduced NO production by the PWD Nos2 allele results in impaired cellular control of Listeria monocytogenes replication. These studies establish a natural genetic model for restrained endogenous NO production and suggest a useful model to investigate the contribution/regulation of NO in DC metabolism and immune function. This work was supported by the National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (P30GM118228, 1R21AI135385-01A, T32AI055402-15, and R21 AI145306), the NIH, National Institute of Neurological Disorders and Stroke (R01 NS097596), and the National Multiple Sclerosis Society (RG-1901-33309).

Gain‑of‑function of IDO in DCs inhibits Tcell immunity by metabolically regulating surface molecules and cytokines.

Both tolerogenicity and immunogenicity of dendritic cells (DCs) are regulated by their intracellular metabolism. As a rate-limiting enzyme of tryptophan (Trp) metabolism, indoleamine 2,3-dioxygenase (IDO) is involved in regulating the functions of numerous cell types, including DCs, a subset of which has a high capacity for producing IDO to control over-activated inflammation. To identify the mechanisms of IDO in DCs, stable DC lines with both gain- and reduction-of-function of IDO were established using a recombinant DNA technique. Although the IDO variation did not affect DC survival and migration, it altered Trp metabolism and other features of DCs analyzed by high-performance liquid chromatography and flow cytometry. On the surface of the DCs, IDO inhibited co-stimulatory CD86 but promoted co-inhibitory programmed cell death ligand 1 expression, and suppressed the antigen uptake, which ultimately led to the compromised ability of DCs to activate T cells. Furthermore, IDO also suppressed IL-12 secretion but enhanced that of IL-10 in DCs, which eventually induced T cells into tolerogenic phenotypes by inhibiting the differentiation of Th1 but promoting that of regulatory T cells. Collectively, the findings of the present study demonstrated that IDO is a key molecule for tolerogenic DC induction by metabolically regulating surface molecule and cytokine expression. This conclusion may lead to the targeted development of therapeutic drugs for autoimmune diseases.

Normal tissue homeostasis and impairment of selective inflammatory responses in dendritic cells deficient for ATF6α.

The initiation of adaptive immunity relies on the performance of dendritic cells (DCs), which are specialized leukocytes with professional antigen presenting capabilities. As such, the molecular mechanisms safeguarding DC homeostasis are matter of intense research. Sensors of the unfolded protein response (UPR) of the endoplasmic reticulum, a three-pronged signaling pathway that maintains the fidelity of the cellular proteome, have emerged as regulators of DC biology. The archetypical example is the IRE1/XBP1s axis, which supports DC development and survival of the conventional type 1 DC (cDC1) subtype. However, the role of additional UPR sensors in DC biology, such as the ATF6α branch, has not been clearly elucidated. Even though Xbp1 is transcriptionally induced by ATF6α under ER stress, it is unclear if cDCs also co-opt the ATF6α branch in tissues. Here, we examine the role of ATF6α in cDC homeostasis in vivo and upon innate stimulation in vitro. In steady state, animals lacking ATF6α in CD11c+ cells (Itgax Cre x Atf6 fl/fl mice) display normal cDC frequencies in spleen, intestine, liver, and lung. Also, ATF6α deficient cDCs express normal levels of Xbp1 mRNA and additional UPR components. However, a reduction of lung monocytes is observed in Itgax Cre x Atf6 fl/fl conditional deficient animals suggesting that ATF6α may play a role in the biology of monocyte subsets. Notably, in settings of DC activation, ATF6α contributes to the production of IL-12 and IL-6 to inflammatory stimuli. Thus, although ATF6α may be dispensable for tissue cDC homeostasis in steady state, the transcription factor plays a role in the acquisition of selective immunogenic features by activated DCs.

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine.

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been used in tumor vaccines to activate innate immunity and enhance antitumor responses. This protocol demonstrates a therapeutic model using cell-based tumor vaccine consisting of Flt3L-expressing B16-F10 melanoma cells along with phenotypic and functional analysis of immune cells in the tumor microenvironment (TME). Procedures for cultured tumor cell preparation, tumor implantation, cell irradiation, tumor size measurement, intratumoral immune cell isolation, and flow cytometry analysis are described. The overall goal of this protocol is to provide a preclinical solid tumor immunotherapy model, and a research platform to study the relationship between tumor cells and infiltrating immune cells. The immunotherapy protocol described here can be combined with other therapeutic modalities, such as immune checkpoint blockade (anti-CTLA-4, anti-PD-1, anti-PD-L1 antibodies) or chemotherapy in order to improve the cancer therapeutic effect of melanoma.

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been used in tumor vaccines to activate innate immunity and enhance antitumor responses. This protocol demonstrates a therapeutic model using cell-based tumor vaccine consisting of Flt3L-expressing B16-F10 melanoma cells along with phenotypic and functional analysis of immune cells in the tumor microenvironment (TME). Procedures for cultured tumor cell preparation, tumor implantation, cell irradiation, tumor size measurement, intratumoral immune cell isolation, and flow cytometry analysis are described. The overall goal of this protocol is to provide a preclinical solid tumor immunotherapy model, and a research platform to study the relationship between tumor cells and infiltrating immune cells. The immunotherapy protocol described here can be combined with other therapeutic modalities, such as immune checkpoint blockade (anti-CTLA-4, anti-PD-1, anti-PD-L1 antibodies) or chemotherapy in order to improve the cancer therapeutic effect of melanoma.

Glutamine synthetase expression rescues human dendritic cell survival in a glutamine-deprived environment.

Glutamine deficiency is a well-known feature of the tumor environment. Here we analyzed the impact of glutamine deprivation on human myeloid cell survival and function. Different types of myeloid cells were cultured in the absence or presence of glutamine and/or with L-methionine-S-sulfoximine (MSO), an irreversible glutamine synthetase (GS) inhibitor. GS expression was analyzed on mRNA and protein level. GS activity and the conversion of glutamate to glutamine by myeloid cells was followed by 13C tracing analyses. The absence of extracellular glutamine only slightly affected postmitotic human monocyte to dendritic cell (DC) differentiation, function and survival. Similar results were obtained for monocyte-derived macrophages. In contrast, proliferation of the monocytic leukemia cell line THP-1 was significantly suppressed. While macrophages exhibited high constitutive GS expression, glutamine deprivation induced GS in DC and THP-1. Accordingly, proliferation of THP-1 was rescued by addition of the GS substrate glutamate and 13C tracing analyses revealed conversion of glutamate to glutamine. Supplementation with the GS inhibitor MSO reduced the survival of DC and macrophages and counteracted the proliferation rescue of THP-1 by glutamate. Our results show that GS supports myeloid cell survival in a glutamine poor environment. Notably, in addition to suppressing proliferation and survival of tumor cells, the blockade of GS also targets immune cells such as DCs and macrophages.

Mouse dendritic cells in the steady state: Hypoxia, autophagy, and stem cell factor.

Dendritic cells (DCs) are innate immune cells with a central role in immunity and tolerance. Under steady-state, DCs are scattered in tissues as resting cells. Upon infection or injury, DCs get activated and acquire the full capacity to prime antigen-specific CD4+ and CD8+ T cells, thus bridging innate and adaptive immunity. By secreting different sets of cytokines and chemokines, DCs orchestrate diverse types of immune responses, from a classical proinflammatory to an alternative pro-repair one. DCs are highly heterogeneous, and physiological differences in tissue microenvironments greatly contribute to variations in DC phenotype. Oxygen tension is normally low in some lymphoid areas, including bone marrow (BM) hematopoietic niches; nevertheless, the possible impact of tissue hypoxia on DC physiology has been poorly investigated. We assessed whether DCs are hypoxic in BM and spleen, by staining for hypoxia-inducible-factor-1α subunit (HIF-1α), the master regulator of hypoxia-induced response, and pimonidazole (PIM), a hypoxic marker, and by flow cytometric analysis. Indeed, we observed that mouse DCs have a hypoxic phenotype in spleen and BM, and showed some remarkable differences between DC subsets. Notably, DCs expressing membrane c-kit, the receptor for stem cell factor (SCF), had a higher PIM median fluorescence intensity (MFI) than c-kit- DCs, both in the spleen and in the BM. To determine whether SCF (a.k.a. kit ligand) has a role in DC hypoxia, we evaluated molecular pathways activated by SCF in c-kit+ BM-derived DCscultured in hypoxic conditions. Gene expression microarrays and gene set enrichment analysissupported the hypothesis that SCF had an impact on hypoxia response and inhibited autophagy-related gene sets. Our results suggest that hypoxic response and autophagy, and their modulation by SCF, can play a role in DC homeostasis at the steady state, in agreement with our previous findings on SCF's role in DC survival.

Loss of KCC2 in GABAergic Neurons Causes Seizures and an Imbalance of Cortical Interneurons.

K-Cl transporter KCC2 is an important regulator of neuronal development and neuronal function at maturity. Through its canonical transporter role, KCC2 maintains inhibitory responses mediated by γ-aminobutyric acid (GABA) type A receptors. During development, late onset of KCC2 transporter activity defines the period when depolarizing GABAergic signals promote a wealth of developmental processes. In addition to its transporter function, KCC2 directly interacts with a number of proteins to regulate dendritic spine formation, cell survival, synaptic plasticity, neuronal excitability, and other processes. Either overexpression or loss of KCC2 can lead to abnormal circuit formation, seizures, or even perinatal death. GABA has been reported to be especially important for driving migration and development of cortical interneurons (IN), and we hypothesized that properly timed onset of KCC2 expression is vital to this process. To test this hypothesis, we created a mouse with conditional knockout of KCC2 in Dlx5-lineage neurons (Dlx5 KCC2 cKO), which targets INs and other post-mitotic GABAergic neurons in the forebrain starting during embryonic development. While KCC2 was first expressed in the INs of layer 5 cortex, perinatal IN migrations and laminar localization appeared to be unaffected by the loss of KCC2. Nonetheless, the mice had early seizures, failure to thrive, and premature death in the second and third weeks of life. At this age, we found an underlying change in IN distribution, including an excess number of somatostatin neurons in layer 5 and a decrease in parvalbumin-expressing neurons in layer 2/3 and layer 6. Our research suggests that while KCC2 expression may not be entirely necessary for early IN migration, loss of KCC2 causes an imbalance in cortical interneuron subtypes, seizures, and early death. More work will be needed to define the specific cellular basis for these findings, including whether they are due to abnormal circuit formation versus the sequela of defective IN inhibition.