Antigen-presenting Dendritic Cells Research Articles

A novel in vitro model reveals distinctive modulatory roles of Plasmodium falciparum and Plasmodium vivax on na\xefve cell-mediated immunity

BackgroundTo date, human peripheral blood mononuclear cells (PBMCs) have been used mainly in immune stimulation assays and the interpretation of data can be influenced by the previous immunological history of donors and cross reactivity with other infectious agents. Resolving these limitations requires an alternative in vitro model to uncover the primary response profiles.MethodsA novel in vitro model of mononuclear cells (MNCs) generated from haematopoietic stem cells (HSCs) was developed and these cells were then co-cultured with various antigens from Plasmodium falciparum and Plasmodium vivax to investigate the response of naïve immune cells to malaria antigens by flow cytometry.ResultsIn vitro stimulation of naïve lymphocytes showed that CD4+ and CD8+ T lymphocytes were significantly reduced (P < 0.01) by exposure to lysates of infected erythrocytes or intact erythrocytes infected with P. falciparum. The depletion was associated with the expression of CD95 (Fas receptor) on the surface of T lymphocytes. Maturation of T lymphocytes was affected differently, showing elevated CD3+CD4+CD8+ and CD3+CD4−CD8− T lymphocytes after stimulation with cell lysates of P. falciparum and P. vivax, respectively. In addition, antigen presenting monocytes and dendritic cells derived from haematopoietic stem cells showed impaired HLA-DR expression as a consequence of exposure to different species of malaria parasites.ConclusionThese results suggest that naïve mononuclear cells differentiated in vitro from HSCs could provide a valid model for the assessment of immunity. P. falciparum and P. vivax malaria parasites could modulate various populations of immune cells starting from newly differentiated mononuclear cells.

Laminarin promotes anti-cancer immunity by the maturation of dendritic cells.

This research evaluates the effects of laminarin on the maturation of dendritic cells and on the in vivo activation of anti-cancer immunity. In vivo treatment of C56BL/6 mice with laminarin increased the expression levels of co-stimulatory molecules and the production of pro-inflammatory cytokines in spleen dendritic cells. Laminarin enhanced ovalbumin antigen presentation in spleen dendritic cells and promoted the proliferation of OT-I and OT-II T cells. Laminarin also induced the maturation of dendritic cells in tumor-draining lymph nodes and protected interferon-γ and tumor necrosis factor-α and proliferation of OT-I and OT-II T cells in tumors. The combination treatment of laminarin and ovalbumin inhibited B16-ovallbumin melanoma tumor growth and its liver metastasis by antigen-specific immune activation, including cytotoxic T lymphocyte activation and interferon-γ production. Thus, these data demonstrated the potential of laminarin as a new and useful immune stimulatory molecule for use in cancer immunotherapy.

Cathepsin E Deficiency Ameliorates Graft-versus-Host Disease and Modifies Dendritic Cell Motility.

Microbial products influence immunity after allogeneic hematopoietic stem cell transplantation (allo-SCT). In this context, the role of cathepsin E (Ctse), an aspartate protease known to cleave bacterial peptides for antigen presentation in dendritic cells (DCs), has not been studied. During experimental acute graft-versus-host disease (GVHD), we found infiltration by Ctse-positive immune cells leading to higher Ctse RNA- and protein levels in target organs. In Ctse-deficient allo-SCT recipients, we found ameliorated GVHD, improved survival, and lower numbers of tissue-infiltrating DCs. Donor T cell proliferation was not different in Ctse-deficient vs. wild-type allo-SCT recipients in MHC-matched and MHC-mismatched models. Furthermore, Ctse-deficient DCs had an intact ability to induce allogeneic T cell proliferation, suggesting that its role in antigen presentation may not be the main mechanism how Ctse impacts GVHD. We found that Ctse deficiency significantly decreases DC motility in vivo, reduces adhesion to extracellular matrix (ECM), and diminishes invasion through ECM. We conclude that Ctse has a previously unrecognized role in regulating DC motility that possibly contributes to reduced DC counts and ameliorated inflammation in GVHD target organs of Ctse-deficient allo-SCT recipients. However, our data do not provide definite proof that the observed effect of Ctse−/− deficiency is exclusively mediated by DCs. A contribution of Ctse−/−-mediated functions in other recipient cell types, e.g., macrophages, cannot be excluded.

CD40–CD40L cross-talk drives fascin expression in dendritic cells for efficient antigen presentation to CD4+ T cells

Fascin is an actin-bundling protein that, among immune cells, is restricted to expression in dendritic cells (DCs). Previous reports have suggested that fascin plays an important role in governing DC antigen presentation to CD4+ T cells. However, no report has clearly linked the receptor-ligand engagement that can direct downstream regulation of fascin expression. In this study, bone marrow-derived DCs from wild-type versus CD40-knockout C57BL/6 mice were used to elucidate the mechanisms of fascin expression and activity upon CD40-CD40 ligand (CD40L) engagement. These investigations now show that CD40 engagement governs fascin expression in DCs to promote CD4+ T-cell cytokine production. Absence of CD40 signaling resulted in diminished fascin expression in DCs and was associated with impaired CD4+ T-cell responses. Furthermore, the study found that loss of CD40-CD40L engagement resulted in reduced DC-T-cell contacts. Rescue by ectopic fascin expression in CD40-deficient DCs was able to re-establish sustained contacts with T cells and restore cytokine production. Taken together, these results show that cross-talk through CD40-CD40L signaling drives elevated fascin expression in DCs to support acquisition of full T-cell responses.

Myeloid Cells in Asthma.

Asthma is a heterogeneous chronic inflammatory disorder of the airways, and not surprisingly, many myeloid cells play a crucial role in pathogenesis. Antigen-presenting dendritic cells are the first to recognize the allergens, pollutants, and viruses that are implicated in asthma pathogenesis, and subsequently initiate the adaptive immune response by migrating to lymph nodes. Eosinophils are the hallmark of type 2 inflammation, releasing toxic compounds in the airways and contributing to airway remodeling. Mast cells and basophils control both the early- and late-phase allergic response and contribute to alterations in smooth muscle reactivity. Finally, relatively little is known about neutrophils and macrophages in this disease. Although many of these myeloid cells respond well to treatment with inhaled steroids, there is now an increasing armamentarium of targeted biologicals that can specifically eliminate only one myeloid cell population, like eosinophils. It is only with those new tools that we will be able to fully understand the role of myeloid cells in chronic asthma in humans.

Crosstalk Among UV-Induced Inflammatory Mediators, DNA Damage and Epigenetic Regulators Facilitates Suppression of the Immune System.

The suppression of the immune system by overexposure to ultraviolet (UV) radiation has been implicated in the initiation and progression of photocarcinogenesis. Numerous changes occur in the skin on UVB exposure, including the generation of inflammatory mediators, DNA damage, epigenetic modifications, and migration and functional alterations in the antigen-presenting dendritic cells. Although each of these alterations can elicit a cascade of events that have the potential to modulate immune sensitivity alone, there is emerging evidence that there is considerable crosstalk between these cascades. The development of an understanding of UV-induced changes in the skin that culminate in UV-induced immunosuppression, which has been implicated in the risk of nonmelanoma skin cancer, as a network of events has implications for the development of more effective chemopreventive strategies. In the current review article, we discuss the evidence of interactions between the various molecular targets and signaling mechanisms associated with UV-induced immunosuppression.

Abstract TMP96: Neuroinflammatory Mechanism of Vascular Cognitive Impairment

Introduction: The mechanisms of vascular cognitive impairment (VCI) are poorly understood. We previously demonstrated increased expression and activity of soluble epoxide hydrolase (sEH) in microvascular endothelium of human brain tissue from deceased patients with pre-mortem VCI. sEH is a key enzyme in the inactivation of endogenous lipid signaling mediators, epoxyeicosatrienoic acids (EETs), known to have vasodilator and anti-inflammatory properties. Hypothesis: Upregulation of endothelial sEH impairs cognition, by mechanisms involving reduced cerebral blood flow (CBF) and enhanced inflammation. Methods: Mice constitutively expressing human sEH in endothelium (Tie2-hsEH) and wild-type (WT) underwent unilateral common carotid artery occlusion (UCCAO) or sham surgery, then analyzed 4 months later for cognitive performance using the Morris water maze (MWM), CBF by perfusion MRI, immune cell profiling in brain, blood and spleen using fluorescence-activated cell sorting (FACS) and immunohistochemistry (IHC). Additionally, immune profiling was performed on WT mice 28 days following surgery, with and without pretreatment with immune modulator DRa1-hMOG-35-55 (HLA-DR a1 moiety linked to human MOG-35-55 peptide). Results: Tie2-hsEH mice showed impaired spatial memory retention in the MWM. No differences were observed in CBF between genotypes. IHC demonstrated a higher density of CD45-positive cells in Tie2-hsEH brain compared to WT. FACS analysis revealed a higher frequency of CD11b + CD45 hi cells in Tie2-hsEH vs. WT mice. Furthermore, these cells express higher levels of CD74, the receptor for macrophage migration inhibitory factor (MIF). Similarly, following UCCAO, the frequency of antigen-presenting dendritic cells and CD74 expression on CD11b + CD45 hi cells were increased in brain, but reduced in spleen and blood compared to naïve mice; both changes were reversed by treatment with DRa1-MOG-35-55, resulting in a decreased frequency and activation state of these cells. Conclusions: Our data suggest that infiltration of monocyte-derived macrophages may contribute to cognitive impairment in VCI. Our findings suggest that CD74 expression could serve as a biomarker for VCI, and that DRa1-MOG-35-55 may be effective in treating VCI.

Splenic Immune Response Is Down-Regulated in C57BL/6J Mice Fed Eicosapentaenoic Acid and Docosahexaenoic Acid Enriched High Fat Diet.

Dietary n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are associated with reduction of inflammation, although the mechanisms are poorly understood, especially how the spleen, as a secondary lymphoid organ, is involved. To investigate the effects of EPA and DHA on spleen gene expression, male C57BL/6J mice were fed high fat diets (HFD) differing in fatty acid composition, either based on corn oil (HFD-CO), or CO enriched with 2 g/100 g EPA and DHA (HFD-ED), for eight weeks. Spleen tissue was analyzed using transcriptomics and for fatty acids profiling. Biological processes (BPs) related to the immune response, including T-cell receptor signaling pathway, T-cell differentiation and co-stimulation, myeloid dendritic cell differentiation, antigen presentation and processing, and the toll like receptor pathway were downregulated by HFD-ED compared with control and HFD-CO. These findings were supported by the down-regulation of NF-κB in HFD-ED compared with HFD-CO fed mice. Lower phospholipid arachidonic acid levels in HFD-ED compared with HFD-CO, and control mice suggest attenuation of pathways via prostaglandins and leukotrienes. The HFD-ED also upregulated BPs related to erythropoiesis and hematopoiesis compared with control and HFD-CO fed mice. Our findings suggest that EPA and DHA down-regulate the splenic immune response induced by HFD-CO, supporting earlier work that the spleen is a target organ for the anti-inflammatory effects of these n-3 fatty acids.

Characterization of αX I-Domain Binding to Receptors for Advanced Glycation End Products (RAGE).

The β2 integrins are cell surface transmembrane proteins regulating leukocyte functions, such as adhesion and migration. Two members of β2 integrin, αMβ2 and αXβ2, share the leukocyte distribution profile and integrin αXβ2 is involved in antigen presentation in dendritic cells and transendothelial migration of monocytes and macrophages to atherosclerotic lesions. Receptor for advanced glycation end products (RAGE), a member of cell adhesion molecules, plays an important role in chronic inflammation and atherosclerosis. Although RAGE and αXβ2 play an important role in inflammatory response and the pathogenesis of atherosclerosis, the nature of their interaction and structure involved in the binding remain poorly defined. In this study, using I-domain as a ligand binding motif of αXβ2, we characterize the binding nature and the interacting moieties of αX I-domain and RAGE. Their binding requires divalent cations (Mg2+ and Mn2+) and shows an affinity on the sub-micro molar level: the dissociation constant of αX I-domains binding to RAGE being 0.49 μM. Furthermore, the αX I-domains recognize the V-domain, but not the C1 and C2-domains of RAGE. The acidic amino acid substitutions on the ligand binding site of αX I-domain significantly reduce the I-domain binding activity to soluble RAGE and the alanine substitutions of basic amino acids on the flat surface of the V-domain prevent the V-domain binding to αX I-domain. In conclusion, the main mechanism of αX I-domain binding to RAGE is a charge interaction, in which the acidic moieties of αX I-domains, including E244, and D249, recognize the basic residues on the RAGE V-domain encompassing K39, K43, K44, R104, and K107.

Langerhans Cell Sarcoma: A Case Report Demonstrating Morphological and Immunophenotypical Variability within a Single Lesion

Langerhans cells are antigen presenting dendritic cells and tumours arising from these are rare. The tumours arising from these dendritic cells are divided into two categories according to a WHO classification: Langerhans cell histiocytosis and Langerhans cell sarcoma. It is the degree of atypia and clinical aggressiveness that distinguishes the two subtypes. Langerhans cell sarcoma (LCS) is a neoplastic proliferation of Langerhans cells which can occur in skin, bone marrow, lymph nodes, spleen, liver, and lung. LCS can present with multiple cutaneous and systemic lesions. We present a case of Langerhans cell sarcoma with a unique morphological appearance and variable immunohistochemical profile within a single cutaneous lesion. LCS is a rare malignancy and its diagnosis is based on morphology, immunophenotypical examination, and ultrastructural analysis by electron microscopy. Our case highlights a unique morphological description of LCS wherein the pleomorphic neoplastic cells show epidermotropism and are surrounded by a variable amount of inflammatory infiltrate within a single cutaneous lesion. A single cutaneous lesion of Langerhans cell sarcoma with variable immunohistochemical profile has not been described so far.

Mining the Complex Family of Protein Tyrosine Phosphatases for Checkpoint Regulators in Immunity.

The family of protein tyrosine phosphatases (PTPs) includes 107 genes in humans that are diverse in their structures and expression profiles. The majority are present in immune cells and play various roles in either inhibiting or promoting the duration and amplitude of signaling cascades. Several PTPs, including TC-PTP (PTPN2) and SHP-1 (PTPN6), have been recognized as being crucial for maintaining proper immune response and self-tolerance, and have gained recognition as true immune system checkpoint modulators. This chapter details the most recent literature on PTPs and immunity by examining their known functions in regulating signaling from either established checkpoint inhibitors or by their intrinsic properties, as modulators of the immune response. Notably, we review PTP regulatory properties in macrophages, antigen-presenting dendritic cells, and T cells. Overall, we present the PTP gene family as a remarkable source of novel checkpoint inhibitors wherein lies a great number of new targets for immunotherapies.

Gastric cancer-derived heat shock protein-gp96 peptide complex enhances dendritic cell activation.

AIMTo investigate the role of heat shock protein (HSP)-glycoprotein (gp)96 in dendritic cells (DCs) and lymphocytes induction in gastric cancer (GC).METHODSHuman GC cell lines KATOIII, MKN-28 and SGC-7901 were infected with adenovirus gp96 at a multiplicity of infection of 100. gp96-GC antigen peptide complexes were purified. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, lactate dehydrogenase (LDH) release assay and enzyme-linked immunosorbent assay were used to determine allo-reactive T cell stimulation, natural killer (NK) cell activity and expression of cytokines (such as interleukin (IL)-10, IL-12, interferon (IFN)-γ and tumor necrosis factor (TNF)-α), respectively. Effect of cytotoxic T lymphocyte (CTL) on DCs incubated with HSP-gp96 was also evaluated by LDH release. All assays were performed in triplicate and the average values were reported. Comparison between groups was conducted using Student’s t test.RESULTST cells incubated with HSP-gp96 exhibited a marked increase in proliferation in a dose-dependent manner (P < 0.05). NK cell activity after gp96-GC peptide complex treatment was significantly higher than that after antigen peptide treatment (P < 0.05). The activity of CTLs incubated with DCs from three GC cells lines was obviously higher than that stimulated by GC antigen at ratios of 50: 1, 25: 1, 10: 1, and 5: 1 (P < 0.05). Furthermore, the secretion of TNF-α, IL-10, IL-12 (P70) and IFN-γ markedly increased after incubation with HSP-gp96 (P < 0.05).CONCLUSIONHSP-gp96 promotes T cell response, enhances DC antigen presentation and induces cytokine secretion, as well. HSP-gp96 has potential as immunotherapy for elimination of residual GC cells.

Dendritic cells and isolevuglandins in immunity, inflammation, and hypertension.

Hypertension is the major risk factor for morbidity and mortality from myocardial infarction, stroke, heart failure, and chronic kidney disease. Despite its importance, the pathogenesis of essential hypertension is poorly understood. During the past several years, it has become evident that T cells contribute to hypertension. Activated T cells accumulate in the perivascular space and the kidney and release cytokines that promote vascular dysfunction and end-organ damage. Although dendritic cells play a pivotal role in initiating adaptive immune responses, T cells have taken center stage in studies implicating the immune system in the genesis of hypertension. The mechanisms by which T cells are activated and the antigens involved are poorly understood. We recently showed that hypertension is associated with increased dendritic cell production of the TH17 polarizing cytokines, IL-6, IL-1β, and IL-23. This occurs in part by increased superoxide production via NADPH oxidase and protein modification by highly reactive isolevuglandins (IsoLGs). IsoLGs are produced via the isoprostane pathway of free radical-mediated lipid peroxidation and, when adducted to proteins, have the potential to act as neoantigens. In this review, we discuss recent advances in our understanding of the role of antigen-presenting dendritic cells in the pathophysiology of hypertension and highlight potential neoantigens that may contribute to this disease.

CD16+ Dendritic Cells Are a Unique Myeloid Antigen Presenting Cell Population

Dendritic cells (DC) are phenotypically identified in human blood as HLA-DR+ cells, which lack major cell surface lineage markers. We demonstrated that myeloid antigen presenting cells, including monocytes and DC display a continuum of CD14 and CD16 expression (10th Human Leucocyte Differentiation Antigen Workshop). The robustness of DC and monocyte identification, particularly when identifying cell subsets with little or no surface CD14 is limited by subjective gating strategies for determining rare cell populations. Application of Poisson counting statistics established that rare cell types such as "CD14- CD16+ DC" are often overlooked in analyses powered to detect the much larger populations of "classical" and "non-classical" monocytes.We used fluorescent and mass cytometry, in conjunction with unsupervised high dimensional clustering, to show that the continuum of CD14 expression separates CD14lo CD16+ non-classical monocytes and CD14- CD16+ DC. We have defined the CD14-CD16+ DC using a broad panel of cell surface markers and established a CD14-CD16+ DC phenotypic signature that is distinct from both classical and non-classical monocytes in healthy donor blood. The CD14-CD16+ DC differ in both size to CD14lo CD16+ monocytes and functional antigen uptake, with slower kinetics of soluble antigen uptake into lysozymes. Their proteasome processing and presentation of influenza matrix protein by MHC I was comparable to other primary blood monocytes and DC antigen presenting cell populations. The CD14-CD16+ DC had limited capacity for further in vitro differentiation. The recovery of CD14-CD16+ DC after autologous and allogeneic myeloablative hematopoietic cell transplants (HCT) followed similar kinetics to other monocytic and DC populations. CD14lo CD16+ monocytes expressed CCR5 as did other myeloid DC but CD14-CD16+ DC lacked CCR5, although interferon induced CCR5. The early differentiation and induction of CCR5 on circulating CD16+ DC after allogeneic HCT predicted for the onset of acute graft versus host disease.These data demonstrate that "CD14- CD16+ DC" represents a distinct clinically relevant human white blood cell population, whose ontogeny and function are under further investigation. DisclosuresFromm:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Papadimitrious:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Hsu:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Larsen:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Gibson:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Bradstock:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Kupresanin:DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Clark:DendroCyte BioTech Ltd: Equity Ownership, Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd. Hart:DendroCyte BioTech Ltd: Equity Ownership, Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd.

The cellular microenvironment and neoplastic population in mycosis fungoides skin lesions: a clinicopathological correlation.

The cellular microenvironment has been proven to play a crucial role in solid tumours and seems to be important in haematologic malignancies, however, it has not been adequately investigated in primary cutaneous Tcell lymphomas. The aim of this study was to register the composition of the cellular microenvironment in mycosis fungoides skin lesions and correlate the composing parameters with the clinical data and follow-up results. The presence of eosinophilic polymorphonuclear leukocytes, Blymphocytes, CD68+ macrophages, and CD1a+ epidermal Langerhans and antigen-presenting dermal dendritic cells, as well as their relation to clinicopathological parameters, were studied in 16 mycosis fungoides cases of different disease stages. The presence and nature of the participating Tcell populations was also investigated. CD8+ tumour infiltrating Tcells and CD56+ cells were found among neoplastic CD4+ Tcells in the lesions. Generally, eosinophils and Blymphocytes were absent or in low numbers, regardless of clinical presentation, contrary to tumourous lesions. Macrophages and CD1a+ cells were constantly present, even in early-stage mycosis fungoides. The reduced presence of the CD1a+ population was associated with resistance to therapy (x2; p=0.012). There is a striking difference in cellular microenvironment composition between early and advanced mycosis fungoides lesions.

Abstract A121: Analysis of tumor microenvironment identifies pathways predicting response to checkpoint control inhibitors: A case study comparing the immune microenvironment of uveal melanoma vs skin cutaneous melanoma

Abstract The remarkable success of checkpoint control inhibitors in treating a variety of different cancers has necessitated a deeper assessment of the tumor and its microenvironment at the genetic and phenotypic level. Data from recent clinical trials have unequivocally established that the tumor microenvironment significantly impacts the efficacy of immune-oncology drugs. We have taken a gene expression signature-based approach to qualitatively and quantitatively assess the epithelial, stromal and immune content of tumors from RNA-seq data. The immune cell content of the tumors was further stratified to determine the infiltration pattern of nine different immune cell types including CD8+/CD4+ T-cells, Treg cells, NK cells, dendritic cells, B-cells, myeloid-derived suppressor cells (MDSC) and M1/M2 macrophages in the tumors using gene signatures specific to each immune cell type. We applied these signatures singly, or in combination on the TCGA RNA-seq data from 33 cancers. Our analysis supports that there is an underlying molecular symmetry in tumors with higher CD8+ T-cell infiltration across most cancers characterized by the upregulation of a common set of genes that mapped to interferon signaling, antigen presentation and a pro-inflammatory cytokine network pathways. The upregulated genes were strongly correlated with patient survival in skin cutaneous melanoma (SKCM) and other cancers providing a framework for identifying biomarkers of patient response to checkpoint control inhibitors. We present a case study to support the idea that gene expression signatures can address a critical unmet need in the immuno-oncology space, which is to create a framework for treating tumors that carry less mutation burden combined with poor T-cell infiltration. As an example, we analyzed 476 skin cutaneous melanoma (SKCM) and 80 uveal melanoma (UVM) samples from TCGA. The UVM melanoma has ∼10-fold lower median mutational burden compared to SKCM, which correlates with a lower (&amp;lt;10-fold) T-cell neo-epitope content in these tumors. As expected, immune cell infiltration of UVM was significantly lower compared to SKCM and so were the infiltration of different immune cell types, indicating that UVMs are immunologically barren compared to SKCMs. However, we observed interesting differences in the immune cell content of the two tumors. Whereas CD8+ T-cell infiltrated tumors were far fewer in UVM &amp;lt;5% compared to SKCM (∼30%), the immune microenvironment was qualitatively different in these tumors. In UVM CD8 T-cell infiltration was correlated positively with pro-inflammatory antigen presenting M1 macrophages and dendritic cells as well as immune-suppressive MDSCs, Treg cells and M2 macrophages. By contrast, CD8+ T-cell infiltrated SKCM tumors had significantly lower levels of MDSCs and M2 macrophages and were enriched in dendritic cells, M1 macrophages and Treg cells. Significantly, in UVM, the macrophage content was dominated by M2 macrophages (M1:M2, 1:2), whereas in SKCM they were similar. Taken together, our analysis suggests that in UVM, the immunesuppressive tumor microenvironment is contributed by the combined presence of MDSCs and M2 macrophages, whereas in SKCM it is contributed by the infiltration of Treg cells. Based on our findings, we propose that therapies targeting MDSC cells, or those that can shift the balance towards increased M1 macrophage content over M2 are likely to show efficacy in UVM melanoma, which are largely unresponsive to checkpoint inhibitor molecules. Citation Format: Ravi Gupta, Nitin Mandloi, Ashwini Patil, Malini Manoharan, Rekha Sathian, Kiran V. Paul, Amitabha Chaudhuri. Analysis of tumor microenvironment identifies pathways predicting response to checkpoint control inhibitors: A case study comparing the immune microenvironment of uveal melanoma vs skin cutaneous melanoma [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A121.

Enhanced antitumor immunity by targeting dendritic cells with tumor cell lysate-loaded chitosan nanoparticles vaccine.

Whole tumor cell lysates (TCL) have been implemented as tumor antigens for cancer vaccine development, although clinical outcomes of TCL-based antitumor immunotherapy remain unsatisfactory. In order to improve the efficacy of TCL-based vaccines, biomaterials have been employed to enhance antigen delivery and presentation. Here, we have developed chitosan nanoparticles (CTS NPs) with surface mannose (Man) moieties for specific dendritic cells (DCs) targeting (Man-CTS NPs). The Man-CTS NPs were then loaded with TCL generated from B16 melanoma cells (Man-CTS-TCL NPs) for invitro and invivo assessment. Potency of the Man-CTS-TCL NPs as cancer vaccine was also assessed invivo by immunization of mice with Man-CTS-TCL NPs followed by re-challenge with B16 melanoma cell inoculation. We have shown here that Man-CTS-TCL NPs promote bone marrow-derived dendritic cells (BMDCs) maturation and antigen presentation invitro. Invivo evaluation further demonstrated that the Man-CTS-TCL NPs were readily taken up by endogenous DCs within the draining lymph node (DLN) following subcutaneous administration accompanied by increasing in serum IFN-γ and IL-4 levels. Tumor growth was also significantly delayed in mice primed with Man-CTS-TCL NPs vaccine, attributable at least in part to cytotoxic T lymphocytes response. Moreover, Man-CTS-TCL NPs vaccine also exhibited therapeutic effects in mice with melanoma. Thus, we report here the Man-CTS-TCL NPs as effective anti-tumor vaccine for cancer immunotherapy.

HIV Interferes with Mycobacterium tuberculosis Antigen Presentation in Human Dendritic Cells

HIV coinfection is the most prominent risk factor for progression of Mycobacterium tuberculosis (Mtb) infection into active tuberculosis (TB) disease. The mechanisms behind the increased transition from latent to active TB in coinfected individuals have not been well elucidated at the cellular level. We hypothesized that HIV infection contributes to Mtb pathogenesis by interfering with the dendritic cell (DC)-mediated immune control. Mtb-antigen processing and presentation are key events in the immune response against TB. Human immature DCs coinfected with HIV/Mtb had decreased expression of human leukocyte antigen antigen D related and the costimulatory molecules CD40, CD80, and CD86. In addition, Mtb-infected DCs triggered a significant release of the proinflammatory cytokines IL-6, IL-1β, and tumor necrosis factor-α, whereas coinfected DCs did not. To assess the DC antigen presentation capacity, we measured interferon-γ from co-cultures of DCs and autologous Mtb antigen-specific CD4+ T cells. Interferon-γ release was significantly reduced when purified protein derivative- and Ag85B-specific CD4+ T cells had been activated with coinfected DCs compared to Mtb-infected DCs, and this effect was attributed to Mtb antigen processing rather than peptide-major histocompatibility complex class II loading. Evaluating autophagy as a measure of vesicular processing and maturation further revealed that HIV efficiently blocks initiation of this pathway during coinfection. Overall, our results demonstrate that HIV impairs Mtb antigen presentation in DCs, thereby suppressing an important cell linking innate and adaptive immune response in TB.

Characterization of Antigen-Presenting Macrophages and Dendritic Cells in the Healthy Human Sclera.

The sclera is mainly made of collagen and fibroblasts. The aim of this study was to analyze whether immune cells are present in the healthy human sclera. Ten human anterior episcleral or stromal tissue samples from globe donors were immunohistochemically examined using confocal microscopy. The expression of the macrophage markers CD68, CD163 and CD11b, CD45 (a general leukocyte marker), MHCII (expressed by antigen-presenting cells [APCs]), CD11c (dendritic cell marker), lymphatic endothelium hyaluronan receptor-1 (LYVE1; expressed on lymphatic endothelium and macrophage subsets), chemokine receptor 7 (CCR7, a homing receptor for leukocytes), CXCL12 (expressed by activated leukocytes), CCR2 (a marker for inflammatory monocytes), and glial fibrillary acidic protein (GFAP; expressed by astrocytes) was analyzed and quantified. In the episclera, a high number of cells (≥40 cells/mm2) were immunoreactive for CD68, CD45, MHCII, CCR7, LYVE1, and CD11b. Lower numbers (<20 cells/mm2) were positive for CXCL12, CCR2, and GFAP. The episclera showed a significantly higher number of cells compared to the stroma (P = 0.008). MHCII+ cells could be double positive for CCR7, CD45, CD11c, or CD11b and seldom CXCL12. Macrophages were most likely from the M1 type (CD68+, CD163-). The healthy human sclera contains several macrophage populations, which can function as APCs, with the highest density being present in the episclera. Most cells express macrophage markers and may function as APCs. The presence of these cells might indicate that scleral immune cells are important for maintaining physiological functions in the eye and may potentially contribute to blood vessel homeostasis.

Interactions between canonical Wnt signaling pathway and MAPK pathway regulate differentiation, maturation and function of dendritic cells

Antigen-presenting dendritic cells interpret environmental signals to orchestrate local and systemic immune responses. In this study, the roles of Wnt proteins and their signaling pathway members in the maturation and function of monocyte-derived DCs were investigated. The present study showed higher expression of β-catenin, as well as pGSK-3β in DCs than those in monocytes. Wnt3a, Wnt5a and inhibition of GSK-3β promoted differentiation of DCs, but inhibited maturation of DCs. GSK-3β induced DCs maturation with unconventional phenotypes. Together with β-catenin silence, these treatment lead to reduced secretion of cytokines and chemokines except for IL-10 in comparison with LPS treatment, and significantly promoted proliferation of T cells. Wnt3a and inhibition of GSK-3β increased expression of MAPK signalings (p-ERK, p-p38, p-JNK). However, inhibition of MAPK signalings in turn differently regulated Wnt signaling proteins expression. These data suggest that Wnt pathway regulates DCs differentiation, maturation and function with interaction of MAPK signaling pathways.