Defective Dendritic Cells Research Articles

Correction of age-associated defects in dendritic cells enables CD4+ T cells to eradicate tumors

Defective host defenses later in life are associated with changes in immune cell activities, suggesting that age-specific considerations are needed in immunotherapy approaches. In this study, we found that PD-1 and CTLA4-based cancer immunotherapies are unable to eradicate tumors in elderly mice. This defect in anti-tumor activity correlated with two known age-associated immune defects: diminished abundance of systemic naive CD8+ Tcells and weak migratory activities of dendritic cells (DCs). We identified a vaccine adjuvant, referred to as a DC hyperactivator, which corrects DC migratory defects in the elderly. Vaccines containing tumor antigens and DC hyperactivators induced T helper type 1 (TH1) CD4+ Tcells with cytolytic activity that drive anti-tumor immunity in elderly mice. When administered early in life, DC hyperactivators were the only adjuvant identified that elicited anti-tumor CD4+ Tcells that persisted into old age. These results raise the possibility of correcting age-associated immune defects through DC manipulation.

Abstract 3001: Investigating the quantity and quality of dendritic cells in human glioblastoma tissues and in orthotopic murine xenograft and syngraft models of glioblastoma

Abstract WHO grade 4 glioblastoma multiforme (GBM) has a very poor prognosis even after standard treatment with surgery, radiotherapy, and chemotherapy. Tumor recurrence is virtually inevitable when it is fatal. Chimeric Antigen Receptor (CAR)-T cell therapy is a promising new modality of therapy for GBM patients. Even though second-generation CAR-T cells may be considered to operate autonomously because co-stimulation is inherent to CAR design, the tumor microenvironment of GBM is particularly hostile to immune cells and their normal functions. In our recently published work (Gargett et al. J Immunother Cancer 2022; 10(9): e005187), CAR-T cells specific for the glioblastoma antigen, GD2, had improved antitumor activity if the CAR-T cells co-expressed IL-15. Although IL-15 co-expression prolonged control of intracranial tumor xenografts, tumors escaped immune control in most cases. Our early data suggest that a relative lack of intratumoral dendritic cells (DCs) may be a contributory factor. DCs are central to the initiation and maintenance of adaptive immune responses. Hence, we were interested to survey and characterize the DC content of glioblastoma tissues obtained either from human surgical samples or mice bearing orthotopic xenografts and syngrafts. We have used multi-colour flow cytometry to enumerate and evaluate the maturation status of subsets of DCs together with other types of immune cells among single cell suspensions of isolated glioblastoma tissues. Our preliminary findings are: Total DCs (mean +/- SD), which were defined as lineage-negative and HLA-DR (+), were significantly reduced among total viable cells of glioblastoma tissues (0.845% +/- 0.569) and peripheral blood mononuclear cells (PBMCs) (0.755% +/- 0.601) from GBM patients (n=12) compared to PBMCs (1.77% +/- 0.611) from healthy donors (n=12).No significant differences were detected in the percentages of total DCs among splenocytes of nontumor-bearing C57BL/6 (B6) mice (n=6) compared to B6 bearing othotopic syngrafts of the GL261 (n=3) or C2TA (n=3) glioblastoma cell lines. We will present more detailed analyses from human glioblastoma samples. In addition, we will present a more extensive characterization of DCs from the orthotopic murine models of glioblastoma: we will compare DCs and other types of immune cells in tumor and in the primary and secondary lymphoid tissues of both tumor-bearing and nontumor-bearing mice. We expect that the final data will fill a current gap in knowledge and indicate possible therapeutic strategies that might correct for the quantitative and qualitative defects in dendritic cells in glioblastoma. We hypothesize that adequate correction of these defects in vivo may augment the anti-glioblastoma activity of CAR-T cell therapies. Citation Format: Michael P. Brown, Tessa Gargett, Bryan J. Gardam. Investigating the quantity and quality of dendritic cells in human glioblastoma tissues and in orthotopic murine xenograft and syngraft models of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3001.

Combinatorial depletions of G-protein coupled receptor kinases in immune cells identify pleiotropic and cell type-specific functions

G-protein coupled receptor kinases (GRKs) participate in the regulation of chemokine receptors by mediating receptor desensitization. They can be recruited to agonist-activated G-protein coupled receptors (GPCRs) and phosphorylate their intracellular parts, which eventually blocks signal propagation and often induces receptor internalization. However, there is growing evidence that GRKs can also control cellular functions beyond GPCR regulation. Immune cells commonly express two to four members of the GRK family (GRK2, GRK3, GRK5, GRK6) simultaneously, but we have very limited knowledge about their interplay in primary immune cells. In particular, we are missing comprehensive studies comparing the role of this GRK interplay for (a) multiple GPCRs within one leukocyte type, and (b) one specific GPCR between several immune cell subsets. To address this issue, we generated mouse models of single, combinatorial and complete GRK knockouts in four primary immune cell types (neutrophils, T cells, B cells and dendritic cells) and systematically addressed the functional consequences on GPCR-controlled cell migration and tissue localization. Our study shows that combinatorial depletions of GRKs have pleiotropic and cell-type specific effects in leukocytes, many of which could not be predicted. Neutrophils lacking all four GRK family members show increased chemotactic migration responses to a wide range of GPCR ligands, whereas combinatorial GRK depletions in other immune cell types lead to pro- and anti-migratory responses. Combined depletion of GRK2 and GRK6 in T cells and B cells shows distinct functional outcomes for (a) one GPCR type in different cell types, and (b) different GPCRs in one cell type. These GPCR-type and cell-type specific effects reflect in altered lymphocyte chemotaxis in vitro and localization in vivo. Lastly, we provide evidence that complete GRK deficiency impairs dendritic cell homeostasis, which unexpectedly results from defective dendritic cell differentiation and maturation in vitro and in vivo. Together, our findings demonstrate the complexity of GRK functions in immune cells, which go beyond GPCR desensitization in specific leukocyte types. Furthermore, they highlight the need for studying GRK functions in primary immune cells to address their specific roles in each leukocyte subset.

Experimental Models of Mouse and Human Hematopoietic Stem Cell Transplantation.

Experimental hematopoietic stem cell transplantation (HSCT) is an invaluable tool in determining the function and characteristics of hematopoietic stem cells (HSC) from experimental mouse and human donor groups. These groups could include, but are not limited to, genetically altered populations (gene knockout/knockin models), ex vivo manipulated cell populations, or in vivo modulated cell populations. The basic fundamentals of this process involve taking cells from a mouse/human donor source and putting them into another mouse (recipient) after preconditioning of the recipient with either total body irradiation (TBI) for mouse donor cells or into sublethally irradiated immune-deficient mice for human donor cells. Then, at pre-determined time pointspost-transplant, sampling a small amount of peripheral blood (PB) and at the termination of the evalaution, bone marrow (BM) to determine donor contribution and function by phenotypic analysis. Exploiting the congenic mouse strains of C57BL/6 (CD45.1- CD45.2+), BoyJ (CD45.1+ CD45.2-), and their F1-crossed hybrid C57BL/6×BoyJ (CD45.1+ CD45.2+), we are able to quantify donor, competitor, and recipient mouse cell contributions to the engraftment state. Human donor cell engraftment (e.g., from the cord blood [CB], mobilized PB, or BM) is assessed by human cell phenotyping in sublethally irradiated immune-deficient mouse recipients (e.g., NOD scid gamma mice that are deficient in B cells, T cells, and natural killer cells and have defective dendritic cells and macrophages). Engraftment of cells from primary mouse recipients into secondary mice allows for an estimation of the self-renewal capacity of the original donor HSC. This chapter outlines concepts, methods, and techniques for mouse and human cell models of HSCT and for assessment of donor cells collected and processed in hypoxia versus ambient air.

Traumatic brain injury alters dendritic cell differentiation and distribution in lymphoid and non-lymphoid organs

BackgroundPathophysiological consequences of traumatic brain injury (TBI) mediated secondary injury remain incompletely understood. In particular, the impact of TBI on the differentiation and maintenance of dendritic cells (DCs), which are regarded as the most professional antigen presenting cells of the immune system, remains completely unknown. Here, we report that DC-differentiation, maintenance and functions are altered on day 3 and day 7 after TBI.MethodsLong bones, spleen, peripheral lymph nodes (pLNs), mesenteric lymph nodes (mLNs), liver, lungs, skin and blood were collected from mice with either moderate-level cortical impact (CCI) or sham on day 1, day 3 or day 7 after TBI. Bone marrow cells were isolated from the tibias and femurs of hind limb through flushing. Tissues were digested with Collagenase-D and DNase I. Skin biopsies were digested in the presence of liberase + DNase I. Single cell suspensions were made, red blood cells were lysed with Ammonium chloride (Stem Cell Technology) and subsequently filtered using a 70 μM nylon mesh. DC subsets of the tissues and DC progenitors of the BM were identified through 10-color flow cytometry-based immunophenotyping studies. Intracellular reactive oxygen species (ROS) were identified through H2DCFDA staining.ResultsOur studies identify that; (1) frequencies and absolute numbers of DCs in the spleen and BM are altered on day 3 and day 7 after TBI; (2) surface expression of key molecules involved in antigen presentation of DCs were affected on day 3 and day 7 after TBI; (3) distribution and functions of tissue-specific DC subsets of both circulatory and lymphatic systems were imbalanced following TBI; (4) early differentiation program of DCs, especially the commitment of hematopoietic stem cells to common DC progenitors (CDPs), were deregulated after TBI; and (5) intracellular ROS levels were reduced in DC progenitors and differentiated DCs on day 3 and day 7 after TBI.ConclusionsOur data demonstrate, for the first time, that TBI affects the distribution pattern of DCs and induces an imbalance among DC subsets in both lymphoid and non-lymphoid organs. In addition, the current study demonstrates that TBI results in reduced levels of ROS in DCs on day 3 and day 7 after TBI, which may explain altered DC differentiation paradigm following TBI. A deeper understanding on the molecular mechanisms that contribute to DC defects following TBI would be essential and beneficial in treating infections in patients with acute central nervous system (CNS) injuries, such as TBI, stroke and spinal cord injury.

Interleukin-30 feeds breast cancer stem cells via CXCL10 and IL23 autocrine loops and shapes immune contexture and host outcome

BackgroundBreast cancer (BC) progression to metastatic disease is the leading cause of death in women worldwide. Metastasis is driven by cancer stem cells (CSCs) and signals from their microenvironment. Interleukin...

Semaphorin 3E deficiency dysregulates dendritic cell functions: In vitro and in vivo evidence.

Regulation of dendritic cell functions is a complex process in which several mediators play diverse roles as a network in a context-dependent manner. The precise mechanisms underlying dendritic cell functions have remained to be addressed. Semaphorins play crucial roles in regulation of various cell functions. We previously revealed that Semaphorin 3E (Sema3E) contributes to regulation of allergen-induced airway pathology partly mediated by controlling recruitment of conventional dendritic cell subsets in vivo, though the underlying mechanism remained elusive. In this study, we investigate the potential regulatory role of Sema3E in dendritic cells. We demonstrated that bone marrow-derived dendritic cells differentiated from Sema3e-/- progenitors have an enhanced migration capacity both at the baseline and in response to CCL21. The enhanced migration ability of Sema3E dendritic cells was associated with an overexpression of the chemokine receptor (CCR7), elevated Rac1 GTPase activity and F-actin polymerization. Using a mouse model of allergic airway sensitization, we observed that genetic deletion of Sema3E leads to a time dependent upregulation of CCR7 on CD11b+ conventional dendritic cells in the lungs and mediastinal lymph nodes. Furthermore, aeroallergen sensitization of Sema3e-/- mice lead to an enhanced expression of PD-L2 and IRF-4 as well as enhanced allergen uptake in pulmonary CD11b+ DC, compared to wild type littermates. Collectively, these data suggest that Sema3E implicates in regulation of dendritic cell functions which could be considered a basis for novel immunotherapeutic strategies for the diseases associated with defective dendritic cells in the future.

Monocyte and dendritic cell defects in COVID-19.

Monocytes, plasmacytoid and conventional dendritic cells are crucial for antiviral immune responses. A new study now compares severe and moderate cases of COVID-19 and links defects in viral sensing, interferon and antigen presentation pathways, associated with upregulated apoptosis and inflammatory pathways, to high COVID-19 severity.

Ubiquitination of MHC Class II by March-I Regulates Dendritic Cell Fitness.

The expression and turnover of Ag-specific peptide-MHC class II (pMHC-II) on the surface of dendritic cells (DCs) is essential for their ability to efficiently activate CD4 T cells. Ubiquitination of pMHC-II by the E3 ubiquitin ligase March-I regulates surface expression and survival of pMHC-II in DCs. We now show that despite their high levels of surface pMHC-II, MHC class II (MHC-II) ubiquitination-deficient mouse DCs are functionally defective; they are poor stimulators of naive CD4 T cells and secrete IL-12 in response to LPS stimulation poorly. MHC-II ubiquitination-mutant DC defects are cell intrinsic, and single-cell RNA sequencing demonstrates that these DCs have an altered gene expression signature as compared with wild-type DCs. Curiously, these functional and gene transcription defects are reversed by activating the DCs with LPS. These results show that dysregulation of MHC-II turnover suppresses DC development and function.

Expansion of dysfunctional CD56-CD16+ NK cells in chronic hepatitis B patients.

Natural killer (NK) cells are primary innate effector cells that play an important role in the control of human viral infections. During chronic viral infection, NK cells undergo significant changes in phenotype, function and subset distribution, including the appearance of CD56-CD16+ (CD56-) NK cells, previously identified in chronic human immunodeficiency virus (HIV) and hepatitis C virus infection. However, the presence of CD56- NK cells in the pathogenesis of chronic hepatitis B (CHB) remains unknown. Phenotype and function of CD56- NK cells from patients with CHB (n=28) were assessed using flow cytometry and in vitro stimulation with HBV antigen. CHB patients had a higher frequency of CD56- NK cells compared to healthy controls in peripheral blood (6.2% vs 1.4%, P<.0001). Compared to CD56+ NK cells, CD56- NK cells had increased expression of inhibitory receptors, and reduced expression of activating receptors, as measured by MFI and qPCR. CD56- NK cells were less responsive to target cell and cytokine stimulation compared to their CD56+ counterparts. In addition, CD56- NK cells demonstrated defective dendritic cells (DCs) interactions resulting in reduced DCs maturation, lower expression of NK CD69 and impaired capacity of NK cells to eliminate immature DCs in co-culture studies. Finally, frequency of CD56- NK cells was positively correlated with serum HBV DNA levels. Chronic HBV infection induces the expansion of highly dysfunctional of CD56- NK cells that likely contribute to inefficient innate and adaptive antiviral immune response in chronic HBV infection.

Dendritic Cell-Mediated Th2 Immunity and Immune Disorders.

Dendritic cells (DCs) are the professional antigen-presenting cells that recognize and present antigens to naïve T cells to induce antigen-specific adaptive immunity. Among the T-cell subsets, T helper type 2 (Th2) cells produce the humoral immune responses required for protection against helminthic disease by activating B cells. DCs induce a Th2 immune response at a certain immune environment. Basophil, eosinophil, mast cells, and type 2 innate lymphoid cells also induce Th2 immunity. However, in the case of DCs, controversy remains regarding which subsets of DCs induce Th2 immunity, which genes in DCs are directly or indirectly involved in inducing Th2 immunity, and the detailed mechanisms underlying induction, regulation, or maintenance of the DC-mediated Th2 immunity against allergic environments and parasite infection. A recent study has shown that a genetic defect in DCs causes an enhanced Th2 immunity leading to severe atopic dermatitis. We summarize the Th2 immune-inducing DC subsets, the genetic and environmental factors involved in DC-mediated Th2 immunity, and current therapeutic approaches for Th2-mediated immune disorders. This review is to provide an improved understanding of DC-mediated Th2 immunity and Th1/Th2 immune balancing, leading to control over their adverse consequences.

Cell-free miR-17-5p as a diagnostic biomarker for gastric cancer inhibits dendritic cell maturation.

PurposeGastric cancer (GC) patients display aberrant miRNA expression and defective dendritic cell function. However, the role of cancer cell-derived oncomiR in GC detection and dendritic cell (DC) maturation remains largely elusive.MethodsCandidate miRNAs were selected by deep sequencing (8 GC plasma samples vs 8 control plasma samples; 8 GC tissues vs 8 adjacent normal gastric tissues) and confirmed by PCR with 164 plasma samples and 72 formalin-fixed paraffin-embedded GC tissue samples. Their diagnostic performance was evaluated by receiver operating characteristic curve. Cy3 fluorescence signals in DCs, exposed to conditioned medium obtained from BGC-823 cells pre-transfected with Cy3-miR-17-5p, were determined by flow cytometry and visualized by confocal microscopy. Functional and phenotypical alterations of DCs were assayed when DCs were transfected with miR-17-5p in vitro.ResultsDeep sequencing and RT-PCR confirmed that five shared miRNAs were upregulated in plasma and tissue samples of GC patients. Cell-free miR-17-5p was superior to others in GC detection with an area under the curve of 0.82, and correlated with lymphatic metastasis and poor overall survival. GC cell-shuttled miR-17-5p can be delivered to immature DCs, and they significantly inhibited LPS-stimulated phenotypic maturation by diminishing the expression of maturation markers (MHC II, CD80 and CD86 molecules). In line with those alterations in the phenotypic markers, functional experiments demonstrated that miR-17-5p triggered an inhibitory effect on DCs endocytic activity and decreased tumor necrosis factor-α and IL-12 secretion, while enhancing IL-10 production. Mixed lymphocyte reaction showed that miR-17-5p inhibited the T cell stimulating effect of DCs and favored regulatory T cells expansion.ConclusionGC cell-derived miR-17-5p is a potential biomarker for GC detection. Taken up by DCs, miR-17-5p weakened antitumor immune responses via inhibiting the maturation of dendritic cells.

Abstract IA10: PMN-MDSC and neutrophils: Tale of two cells in cancer

Abstract Myeloid cells are a critical component of the tumor microenvironment. In cancer, the myeloid compartment is dramatically affected, which is now considered as one of the major immunological hallmarks of cancer. Accumulation of immunosuppressive macrophages (MΦ), defective dendritic cells (DC) function and expansion of pathologically activated immune suppressive immature myeloid cells – myeloid-derived suppressor cells (MDSC) are major changes in the myeloid compartment in cancer. The total population of MDSC consists of three groups of cells: the most abundant (&amp;gt;75%) immature, pathologically activated neutrophils (PMN-MDSC); the less abundant population of pathologically activated monocytes – (M-MDSC). Tumor associated macrophages (TAM) and DCs can persist in tissues for a long time, whereas PMN-MDSC have short lifespan (&amp;lt; 48 hours) and are constantly replaced from bone marrow (BM). The role of MDSC in mouse tumor models is well established. In most of the studies PMN-MDSC were cells that expanded the most. In recent years, the clinical role of MDSC has emerged. Results showed positive correlation of MDSC in peripheral blood with cancer stage and tumor burden. In a meta-analysis, elevated MDSC in the circulation was found to be an independent indicator of poor outcomes in patients with solid tumors and predicting response to chemotherapy therapy, radiotherapy failure, and immunotherapy. It is widely accepted that the population of myeloid cells in cancer is heterogenic. It consists of cells able to exert antitumor and pro-tumorigenic activity. The nature of this heterogeneity is highly controversial. Some data suggested that PMN-MDSC in tumors may undergo polarization in situ, similar to that observed in TAM. Based on available data on the very short lifespan of MDSC in animal models and cancer patients as well as on functional data, we have postulated that at any given moment, myeloid cells in tissues are comprised of classically activated neutrophils (PMN) and monocytes, with pro-inflammatory and antitumor activity, and pathologically activated MDSC with potent immune suppressive and pro-tumorigenic activity. The balance between these cells defines the tumor microenvironment as immunosuppressive. This balance depends on the stage of cancer. It is equally plausible that in tissues, MDSC are present not in a binary state but in a range of functional states that are characterized by different markers and different functional activities. We will discuss evidence supporting this hypothesis. This question has a direct implication for cancer therapy. It is not clinically feasible to deplete the entire population of PMN and monocytes in cancer patients. However, if a specific group of functionally defined M-MDSC and PMN-MDSC is determined, then much more precise methods of therapeutic targeting could be developed. Potential approaches to such targeting are currently being investigated. Citation Format: Dmitry I. Gabrilovich. PMN-MDSC and neutrophils: Tale of two cells in cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr IA10.

FGL2 promotes tumor progression in the CNS by suppressing CD103+ dendritic cell differentiation

Few studies implicate immunoregulatory gene expression in tumor cells in arbitrating brain tumor progression. Here we show that fibrinogen-like protein 2 (FGL2) is highly expressed in glioma stem cells and primary glioblastoma (GBM) cells. FGL2 knockout in tumor cells did not affect tumor-cell proliferation in vitro or tumor progression in immunodeficient mice but completely impaired GBM progression in immune-competent mice. This impairment was reversed in mice with a defect in dendritic cells (DCs) or CD103+ DC differentiation in the brain and in tumor-draining lymph nodes. The presence of FGL2 in tumor cells inhibited granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced CD103+ DC differentiation by suppressing NF-κB, STAT1/5, and p38 activation. These findings are relevant to GBM patients because a low level of FGL2 expression with concurrent high GM-CSF expression is associated with higher CD8B expression and longer survival. These data provide a rationale for therapeutic inhibition of FGL2 in brain tumors.

Defective Dendritic Cell Cytotoxic Activity of High-Grade Glioma Patients' Results from the Low Expression of Membrane TNFα and Can Be Corrected In Vitro by Treatment with Recombinant IL-2 or Exogenic Double-Stranded DNA.

Besides initiation of tumor-specific T cell immunity, dendritic cells (DCs) are endowed with tumoricidal activity. Previously, we showed that monocyte-derived DCs of high-grade glioma patients generated in the presence of interferon alpha (IFNα) (IFN-DCs) have impaired cytotoxic activity against tumor necrosis factor alpha (TNFα)-sensitive HEp-2 tumor cells. Herein, we demonstrate that decreased transmembrane TNFα (tmTNFα) expression, but not soluble TNFα (sTNFα) production by high-grade glioma patient IFN-DCs, determines the defective tumoricidal activity against TNFα-sensitive HEp-2 cells. Blocking TNFα-converting enzyme or stimulation of patient IFN-DCs with rIL-2 or dsDNA enhances tmTNFα expression on IFN-DCs and significantly increases their cytotoxicity. Decreased tmTNFα expression on patient IFN-DCs is not caused by downregulation of pNFκB. Neither rIL-2 nor dsDNA upregulates tmTNFα expression on patient IFN-DCs via an increase of pNFκB. The current study shows an important role of tmTNFα as mediator of IFN-DC tumoricidal activity and as molecular target for the restoration of defective DC killer activity in high-grade glioma patients.

NH36 and F3 Antigen-Primed Dendritic Cells Show Preserved Migrating Capabilities and CCR7 Expression and F3 Is Effective in Immunotherapy of Visceral Leishmaniasis.

Physical contact between dendritic cells (DCs) and T cell lymphocytes is necessary to trigger the immune cell response. CCL19 and CCL21 chemokines bind to the CCR7 receptor of mature DCs, and of T cells and regulate DCs migration to the white pulp (wp) of the spleen, where they encounter lymphocytes. In visceral leishmaniasis (VL), cellular immunosuppression is mediated by impaired DC migration due to the decreased chemokine secretion by endothelium and to the reduced DCs CCR7 expression. The Leishmania (L.) donovani nucleoside hydrolase NH36 and its C-terminal domain, the F3 peptide are prominent antigens in the generation of preventive immunity to VL. We assessed whether these vaccines could prevent the migrating defect of DCs by restoring the expression of CCR7 receptors. C57Bl6 mice were vaccinated with NH36 and F3 and challenged with L. (L.) infantum chagasi. The F3 vaccine induced a 100% of survival and a long-lasting immune protection with an earlier CD4+Th1 response, with secretion of higher IFN-γ and TNF-α/IL-10 ratios, and higher frequencies of CD4+ T cells secreting IL-2+, TNF-α+, or IFN-γ+, or a combination of two or the three cytokines (IL-2+TNF-α+IFN-γ+). The CD8+ T cell response was promoted earlier by the NH36-vaccine, and later by the F3-vaccine. Maximal number of F3-primed DCs migrated in vitro in response to CCL19 and showed a high expression of CCR7 receptors (26.06%). Anti-CCR7 antibody treatment inhibited DCs migration in vitro (90%) and increased parasite load in vivo. When transferred into 28-day-infected mice, only 8% of DCs from infected, 59% of DCs from NH36-vaccinated, and 84% of DCs from F3-vaccinated mice migrated to the wp. Consequently, immunotherapy of infected mice with F3-primed DCs only, promoted increases in corporal weight and reductions of spleen and liver parasite loads and relative weights. Our findings indicate that vaccination with F3-vaccine preserves the maturation, migration properties and CCR7 expression of DCs, which are essential processes for the generation of cell-mediated immunity. The F3 vaccine is more potent in reversing the migration defect that occurs in VL and, therefore, more efficient in immunotherapy of VL.

Exosomes in melanoma: a role in tumor progression, metastasis and impaired immune system activity.

Exosomes (Exo) are small vesicles produced by melanoma cells and the accessory cells of the tumor microenvironment. They emerge via both classical and direct pathways and actively participate in tumor colonisation of distant tissues. The proteins, nucleic acids, cytokines and growth factors engulfed by Exo are transferred to recipient cells, where they drive numerous functions required for the tumor escape from immune system control and tumor progression. By positively or negatively modulating immune cell properties, Exo provoke immune suppression and, in turn, defective dendritic cell (DC) functions. Together, these effects limit the cytotoxicity of T-cells and expand both T-regulatory and myeloid-derived suppressor populations. They also hinder perforin and granzyme production by natural killer cells. Finally, Exo also control the organotropism of melanoma cells. The distinct phenotypic properties of Exo can be exploited both for diagnostic purposes and in the early identification of melanoma patients likely to respond to immunotherapy. The potential therapeutic application of Exo derived from DCs has been demonstrated in vaccination trials, which showed an increase in anti-melanoma activity with respect to circulating tumor cells. However, additional studies are required before Exo can be effectively used in diagnostic and therapeutic applications in melanoma.

Manifold Roles of CCR7 and Its Ligands in the Induction and Maintenance of Bronchus-Associated Lymphoid Tissue

The processes underlying the development and maintenance of tertiary lymphoid organs are incompletely understood. Using a Ccr7 knockout/knockin approach, we show that spontaneous bronchus-associated lymphoid tissue (BALT) formation can be caused by CCR7-mediated migration defects ofdendritic cells (DCs) in the lung. Plt/plt mice that lack the CCR7 ligands CCL19 and CCL21-serine do not form BALT spontaneously because lung-expressed CCL21-leucine presumably suffices to maintain steady-state DC egress. However, plt/plt mice are highly susceptible to modified vaccinia virus infection, showing enhanced recruitment of immune cells as well as alterations in CCR7-ligand-mediated lymphocyte egress from the lungs, leading to dramatically enhanced BALT. Furthermore, we identify two independent BALT homing routes for blood-derived lymphocytes. One is HEV mediated and depends on CCR7 and L-selectin, while the second route is via the lung parenchyma and is independent of these molecules. Together, these data provide insights into CCR7/CCR7-ligand-orchestrated aspects in BALT formation.

Abstract 31: The INSPIRE trial: A randomized trial of neoadjuvant and adjuvant therapy with the IRX-2 regimen in patients with newly diagnosed stage II, III, or IVa squamous cell carcinoma of the oral cavity

Abstract Introduction: IRX-2 is a primary-cell-derived immune-restorative biologic consisting of a well-defined set of human cytokines that act on multiple immune system cell types to overcome tumor-mediated immunosuppression. The IRX-2 biologic stimulates T cells and natural killer cells and turns immature or defective dendritic cells into mature antigen-presenting cells. The IRX-2 biologic is provided as part of the IRX-2 regimen, which contains cyclophosphamide, indomethacin, and zinc to support an anticancer immune response. In a phase 2a clinical trial in 27 patients with therapy-naïve head and neck squamous cell carcinoma (HNSCC), treatment with the IRX-2 regimen was well tolerated and associated with immunologically mediated antitumor effects. The new multicenter randomized IRX-2 Neoadjuvant Therapy in Head and Neck SCC to Provide Immune Response Enhancement (INSPIRE) trial (NCT02609386) is designed to evaluate the safety and effectiveness of combined neoadjuvant and adjuvant therapy with the IRX-2 regimen in patients with oral cavity HNSCC, a disease known to have a disordered immune function. Study Design: Patients enrolled in INSPIRE must be at least 18 years of age; have previously untreated Stage II, III, or IVa SCC of the oral cavity that is surgically resectable with curative intent; and have a Karnofsky Performance status ≥70%. Up to 200 patients will be randomized 2:1 to either the IRX-2 biologic regimen arm or the IRX-2 regimen control arm (the IRX regimen minus the IRX-2 biologic). The neoadjuvant stage of INSPIRE begins 21 days before resection, when patients in both treatment arms will receive the IRX-2 regimen each day until resection. This regimen consists of the immunomodulatory chemotherapeutic agent cyclophosphamide, provided as a low-dose intravenous infusion, as well as two oral drugs, a zinc-containing multivitamin and indomethacin, a nonselective COX-1/COX-2 inhibitor. During this period, patients in the IRX-2 biologic regimen arm will also receive 10 days of the IRX-2 biologic subcutaneously injected into their bilateral sternocleidomastoid insertion regions. After resection, patients receive standard adjuvant radiation or chemoradiation therapy followed by adjuvant IRX-2 booster regimens at 3, 6, 9, and 12 months. During each 10-day booster period, patients in the IRX-2 biologic regimen arm also receive, for 5 consecutive days, the IRX-2 biologic subcutaneously injected into their bilateral deltoid regions. Study Endpoints: INSPIRE is now enrolling patients at 12 selected institutions in the United States. The primary endpoint of INSPIRE is event-free survival (EFS). Secondary endpoints include overall survival (OS) and safety as assessed by the incidence and severity of adverse events. Exploratory endpoints include changes in tumor size and histologic differences between pre- and post-treatment specimens in lymphocytic infiltration, assessed by both cell-surface marker expression (Perkin Elmer multiplex IHC) and immune cell gene signatures (NanoString). Conclusion: With enrollment of up to 200 patients, the randomized INSPIRE trial provides the opportunity to assess the ability of the IRX-2 regimen to improve EFS and OS by inhibiting tumor-mediated immunosuppression. Analysis of the exploratory endpoints will generate data to better describe the mechanism of action of the IRX-2 regimen and provide insight into the differences between inflammatory and non-inflammatory responses to SCCs of the oral cavity. Citation Format: Gregory T. Wolf, Mihir Patel, Audrey Erman, Jason G. Newman, Greg Krempl, Jorge Nieva, R. Bryan Bell, Michael Kaplan, Dennis Kraus, Jeffrey Moyer, Aru Panwar, Joseph Valentino. The INSPIRE trial: A randomized trial of neoadjuvant and adjuvant therapy with the IRX-2 regimen in patients with newly diagnosed stage II, III, or IVa squamous cell carcinoma of the oral cavity [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 31.

A Review on Defects of Dendritic Cells in Common Variable Immunodeficiency.

Common variable immunodeficiency (CVID) is the most important primary disorder that is associated with clinical complications including recurrent infections, malignancy and autoimmune diseases. The genetic cause of CVID is mostly unknown and only a few genetic causes are identified. The various options are proposed for determining the etiology of CVID patients, such as T- and B-cell defects, Toll-like receptors (TLRs) impairments, altered cytokine production as well as blemished dendritic cells (DCs). The patients with CVID show a reduction in number and frequency of DCs in blood, an altered expression of cell surface molecules, and defective activation through toll-like receptors (TLRs). Also loss of IFNα has a critical role in B-cell impairments of CVID patients. The aim of this review is to collect under one umbrella, all the recent knowledge about DCs defects of CVID patients. This review covers basic information about physiology of DCs followed by reports of DCs situation in CVID. According to the results of researches assessing DCs frequency and function in CVID, the roll of DCs in the pathogenesis of CVID cannot be ruled out. The article is expected to encourage the researchers to do comprehensive researches about complex connections between DCs and other immune cells in CVID.