Dendritic cells (DCs) accumulate in the lung of patients affected by idiopathic pulmonary fibrosis (IPF). We measured the frequencies of circulating conventional CD1c + and CD141+ cells (namely, cDC2 and cDC1) and of plasmacytoid CD303+ DCs in a cohort of 60 therapy naive IPF patients by flow cytometry. Peripheral levels of reactive oxygen species (ROS) and of pro-inflammatory and Th1/Th2 polarizing cytokines were also analyzed. All blood DC subtypes were significantly reduced in IPF patients in comparison to age- and sex-matched controls, while ROS and interleukin (IL-6) levels were augmented. IL-6 expression increased along with disease severity, according to the gender-age-physiology index, and correlated with the frequency of cDC2. IL-6 and cDC2 were not influenced by anti-fibrotic therapies but were associated with a reduced survival, the latter being an independent predictive biomarker of worse prognosis. Deciphering the role of DCs in IPF might provide information on disease pathogenesis and clinical behavior.

Cancer vaccines can amplify existing antitumor responses or prime naïve T cells to elicit effector T-cell functions in patients through immunization. Antigen-specific CD8+ T cells are crucial for the rejection of established tumors. We constructed XCL1-GPC3 fusion molecules as a liver cancer vaccine by linking the XCL1 chemokine to glypican-3 (GPC3), which is overexpressed in hepatocellular carcinoma (HCC). Cells expressing XCL1-GPC3 chemoattracted murine XCR1+CD8α+DCs and human XCR1+CD141+DCs in vitro and promoted their IL-12 production. After subcutaneous mXcl1-GPC3 plasmid injection, mXCL1-GPC3 was mainly detected in CD8α+DCs of mouse draining lymph nodes. XCL1-GPC3-targeted DCs enhanced antigen-specific CD8+ T cell-proliferation and induced the de novo generation of GPC3-specific CD8+ T cells, which abolished GPC3-expressing tumor cells in mouse and human systems. We immunized a murine autochthonous liver cancer model, with a hepatitis B background, with the mXcl1-GPC3 plasmid starting six weeks, when malignant hepatocyte clusters formed, or 14 weeks, when liver tumor nodules developed, after diethylnitrosamine administration. mXcl1-GPC3 immunized mice displayed significantly inhibited tumor formation and growth compared to GPC3-immunized mice. After mXcl1-GPC3 immunization, mouse livers showed elevated production of IFNγ, granzyme B, IL-18, CCL5, CXCL19, Xcl1, and increased infiltration of GPC3-specific CD8+ T cells, activated NK cells and NKT cells. The antitumor effects of these immune cells were further enhanced by the administration of anti-PD1. Anti-HCC effects induced by hXCL1-GPC3 were confirmed in HCC-PDX model from three patients. Thus, XCL1-GPC3 might be a promising cancer vaccine to compensate for the deficiency of the checkpoint blockades in HCC immunotherapy.

Background: Despite efficacy of allergen-specific immunotherapy (AIT), the role of trained immunity and tolerance in this process has not been elucidated.

Objective: Here, we performed a comprehensive, longitudinal analysis of systemic innate immune cell repertoire in the course of AIT.

Methods: Allergic patients received standard preseasonal subcutaneous AIT with allergoids to birch and/or grass. Healthy controls were monitored without any intervention. Flow cytometry of ILC, NK, monocyte, and DC subsets was performed at baseline, 3 months (birch season), 6 months (grass seasons), and 12 months after the therapy in patients or in similar seasonal time points in controls. Additional analyses were performed in the 3^rd-year birch and grass season.

Results: We observed a durable decrease of ILC2 and an increase of ILC1 after AIT with dynamic changes in their composition. We found that an expansion of CD127⁺CD25⁺⁺ clusters caused observed shifts in ILC1 heterogeneity. In addition, we observed development of CD127⁺CD25⁺⁺c-Kit⁺ ILC3 clusters. Moreover, we found an increase in intermediate monocytes, in parallel to the reduction of non-classical monocytes during first year after AIT. Classical and intermediate monocytes presented significant heterogeneity in allergic patients, but AIT reduced HLA-DR⁺⁺ clusters. Finally, an increase in pDCs and CD141⁺ mDCs was observed in allergic individuals, while CD1c⁺ mDCs were reduced during 1^st year of AIT.

Conclusion: AIT induces changes in the composition and heterogeneity of circulating innate immune cells and brings them to the level observed in healthy individuals. Monitoring of ILC, monocytes, and DCs during AIT might serve as a novel biomarker strategy.

Keywords: DCs; ILC; NK cells; allergen immunotherapy; antigen presenting cells; innate immune cells; monocytes.

Dendritic cells (DCs) use effective mechanisms to combat antigens and to bring about adaptive immune responses through their ability to stimulate näive T cells. At present, four major cell types are categorised as DCs: Classical or conventional (cDCs), Plasmacytoid (pDCs), Langerhans cells (LCs), and monocyte-derived DCs (Mo-DCs). It was suggested that pDCs, CD1c+ DCs and CD141+ DCs in humans are equivalent to mouse pDCs, CD11b+ DCs and CD8α+ DCs, respectively. Human CD141+ DCs compared to mouse CD8α+ DCs have remarkable functional and transcriptomic similarities. Characteristic markers, transcription factors, toll-like receptors, T helpers (Th) polarisation, cytokines, etc. of DCs are discussed in this review. Major histocompatibility complex (MHC) I and II antigen presentation, cross-presentation and Th polarisation are defined, and the dual role of DCs in the tumour is discussed. Human DCs are the main immune cells that orchestrate the immune response in the tumour microenvironment.

Background: Dendritic cells (DCs) are the most efficient antigen-presenting cells, hence initiating a potent and cancer-specific immune response. This ability (mainly using monocyte-derived DCs) has been exploited in vaccination strategies for decades with limited clinical efficacy. Another alternative would be the use of conventional DCs (cDCs) of which at least three subsets circulate in human blood: cDC1s (CD141^bright), cDC2s (CD1c⁺) and plasmacytoid DCs. Despite their paucity, technical advances may allow for their selection and clinical use. However, many assumptions concerning the DC subset biology depend on observations from mouse models, hindering their translational potential. In this study, we characterise human DCs in patients with ovarian cancer (OvC) or prostate cancer (PrC).

Patients and methods: Whole blood samples from patients with OvC or PrC and healthy donors (HDs) were evaluated by flow cytometry for the phenotypic and functional characterisation of DC subsets.

Results: In both patient groups, the frequency of total CD141⁺ DCs was lower than that in HDs, but the cDC1 subset was only reduced in patients with OvC. CD141⁺ DCs showed a reduced response to the TLR3 agonist poly (I:C) in both groups of patients. An inverse correlation between the frequency of cDC1s and CA125, the OvC tumour burden marker, was observed. Consistently, high expression of CLEC9A in OvC tissue (The Cancer Genome Atlas data set) indicated a better overall survival.

Conclusions: cDC1s are reduced in patients with OvC, and CD141⁺ DCs are quantitatively and qualitatively impaired in patients with OvC or PrC. CD141⁺ DC activation may predict functional impairment. The loss of cDC1s may be a bad prognostic factor for patients with OvC.

Keywords: CD141; Cross-presenting DC; Ovarian cancer; Prostate cancer; Vaccines; cDC1.

Type 1 conventional dendritic cells (cDC1s) possess efficient antigen presentation and cross-presentation activity, as well as potent T cell priming ability. Tissue-resident cDC1s (CD103⁺ cDC1s in mice, CD141⁺ cDC1s in humans) are linked with improved tumor control, yet the efficacy of immunotherapy using this population is understudied.

We generated murine CD103⁺ cDC1s in vitro and examined their expression of cDC1-related factors, antigen cross-presentation activity, and accumulation in tumor-draining lymph nodes (TdLNs). The antitumor efficacy of the in vitro-generated CD103⁺ cDC1s was studied in murine melanoma and osteosarcoma models. We evaluated tumor responses on vaccination with CD103⁺ cDC1s, compared these to vaccination with monocyte-derived DCs (MoDCs), tested CD103⁺ cDC1 vaccination with checkpoint blockade, and examined the antimetastatic activity of CD103⁺ cDC1s.

In vitro-generated CD103⁺ cDC1s produced cDC1-associated factors such as interleukin-12p70 and CXCL10, and demonstrated antigen cross-presentation activity on stimulation with the toll-like receptor 3 agonist polyinosinic:polycytidylic acid (poly I:C). In vitro-generated CD103⁺ cDC1s also migrated to TdLNs following poly I:C treatment and intratumoral delivery. Vaccination with poly I:C-activated and tumor antigen-loaded CD103⁺ cDC1s enhanced tumor infiltration of tumor antigen-specific and interferon-γ⁺ CD8⁺ T cells, and suppressed melanoma and osteosarcoma growth. CD103⁺ cDC1s showed superior antitumor efficacy compared with MoDC vaccination, and led to complete regression of 100% of osteosarcoma tumors in combination with CTLA-4 antibody-mediated checkpoint blockade. In vitro-generated CD103⁺ cDC1s effectively protected mice from pulmonary melanoma and osteosarcoma metastases.

Our data indicate an in vitro-generated CD103⁺ cDC1 vaccine elicits systemic and long-lasting tumor-specific T cell-mediated cytotoxicity, which restrains primary and metastatic tumor growth. The CD103⁺ cDC1 vaccine was superior to MoDCs and enhanced response to immune checkpoint blockade. These results indicate the potential for new immunotherapies based on use of cDC1s alone or in combination with checkpoint blockade.

Dendritic cells (DCs), which are essential for initiating immune responses, are comprised of different subsets. Tetraspanins organize dendritic cell membranes by facilitating protein-protein interactions within the so called tetraspanin web. In this study we analyzed expression of the complete tetraspanin superfamily in primary murine (CD4+, CD8+, pDC) and human DC subsets (CD1c+, CD141+, pDC) at the transcriptome and proteome level. Different RNA and protein expression profiles for the tetraspanin genes across human and murine DC subsets were identified. Although RNA expression levels of CD37 and CD82 were not significantly different between human DC subsets, CD9 RNA was highly expressed in pDCs, while CD9 protein expression was lower. This indicates that relative RNA and protein expression levels are not always in agreement. Both murine CD8α+ DCs and its regarded human counterpart, CD141+ DCs, displayed relatively high protein levels of CD81. CD53 protein was highly expressed on human pDCs in contrast to the relatively low protein expression of most other tetraspanins. This study demonstrates that tetraspanins are differentially expressed by human and murine DC subsets which provides a valuable resource that will aid the understanding of tetraspanin function in DC biology.

BACKGROUND

Myeloid and plasmacytoid dendritic cell (DC) subsets have been recently identified in the human lung based on their differential expression of Blood DC Antigens 1-3 (BDCAs). We investigated the expression of these antigens by isolated human pulmonary CD1a(+) DCs, namely Langerhan's cells.

METHODS

Using an in vitro cell culture system we successfully isolated a population of relatively pure (>70%) CD1a(+) cells from human lung tissue (n=5 subject samples) and stained these with antibodies against the myeloid DC markers BDCA1 (CD1c) and BDCA3 (CD303), the plasmacytoid DC marker BDCA2 (CD141), the Langerhan's cell marker Langerin and the maturation marker CD83.

RESULTS

Among different subject samples, the isolated CD1a(+) cells showed variable expression of Langerin, BDCAs and CD83. Interestingly, in two subject samples, which contained >70% CD83(+) mature CD1a(+) cells, >50% of the cells were positive for all of the BDCAs.

CONCLUSIONS

We conclude that isolated pulmonary CD1a(+) DCs in vitro have the capacity to express both myeloid and plasmacytoid BDCA markers and that rather than subset restriction in pulmonary DCs, a significant degree of flexibility/plasticity can be induced, albeit experimentally.

BACKGROUND

Airway dendritic cells (DC) are critical mediators of lung inflammation in asthma, but the characteristics of DC in the airways of healthy children, and children with asthma, are currently unknown.

OBJECTIVE

We sought to identify changes in DC subset distribution and activation profile in paediatric asthma using flow cytometry to analyse induced sputum samples obtained from healthy and asthmatic children.

METHODS

Lung function and atopic status were determined by spirometry and skin prick testing. Induced sputum samples were analysed using 7-colour flow cytometry to identify airway DC populations (lineage(-) HLA-DR(+) sputum cells expressing either CD11c as conventional DC or CD123 as plasmacytoid DC).

RESULTS

Sputum samples containing lower airway plugs were obtained from 10 healthy children and 8 children with asthma. Lineage(-) HLA-DR(+) DC were successfully identified in all samples, and DC comprised a significantly higher proportion of sputum cells in children with asthma compared with age-matched healthy controls (1.29% vs. 0.67%, P = 0.02). DC expression of the costimulatory marker CD86 was significantly reduced in asthmatic children (73.4% vs. 59.7%, P = 0.04). Sputum DC also included numerous CD1c(+) cells (mean 57% of the total DC population) and low frequencies of cells expressing the subset markers CD141 or CD123, although the proportions of these did not differ between groups.

CONCLUSIONS

Airway DC can be identified and characterized non-invasively using flow cytometry to analyse paediatric sputum samples. Our data reveal that children with steroid-treated asthma exhibit increased frequency of airway DC with reduced expression of the costimulatory marker CD86, suggesting altered trafficking and/or maturation of these cells either due to asthma or steroid therapies.

The human bone marrow (BM) gives rise to all distinct blood cell lineages, including CD1c+ (cDC2) and CD141+ (cDC1) myeloid dendritic cells (DC) and monocytes. These cell subsets are also present in peripheral blood (PB) and lymphoid tissues. However, the difference between the BM and PB compartment in terms of differentiation state and immunological role of DC is not yet known. The BM may represent both a site for development as well as a possible effector site and so far, little is known in this light with respect to different DC subsets. Using genome-wide transcriptional profiling we found clear differences between the BM and PB compartment and a location-dependent clustering for cDC2 and cDC1 was demonstrated. DC subsets from BM clustered together and separate from the corresponding subsets from PB, which similarly formed a cluster. In BM, a common proliferating and immature differentiating state was observed for the two DC subsets, whereas DC from the PB showed a more immune-activated mature profile. In contrast, BM-derived slan+ non-classical monocytes were closely related to their PB counterparts and not to DC subsets, implying a homogenous prolife irrespective of anatomical localization. Additional functional tests confirmed these transcriptional findings. DC-like functions were prominently exhibited by PB DC. They surpassed BM DC in maturation capacity, cytokine production, and induction of CD4+ and CD8+ T cell proliferation. This first study on myeloid DC in healthy human BM offers new information on steady state DC biology and could potentially serve as a starting point for further research on these immune cells in healthy conditions as well as in diseases.

Dendritic cells (DCs) are highly specialized antigen-presenting cells that bridge innate and adaptive immune responses in vertebrates, being key modulators in the initiation of specific responses. Although teleost fish present the main elements of a fully developed adaptive immune system, not many studies have focused on identifying specific DC subsets in teleost species. Previous work from our group identified in rainbow trout (Oncorhynchus mykiss) skin a DC subpopulation co-expressing CD8α and major histocompatibility complex II β on the cell surface. Interestingly, these CD8+ DCs expressed common unique markers of mammalian cross-presenting DCs, a DC subset with an important role in antigen presentation and activation of CD8+ T cytotoxic lymphocytes. In this study, we have identified a similar DC subset in rainbow trout gills that also transcribes molecules uniquely expressed on diverse mammalian cross-presenting DC populations such as CD8, CD103, CD141, Batf3, IFN regulatory protein 8, and toll-like receptor 3. Hence, we have undertaken a broad phenotypic and functional characterization of this new DC subset that includes the confirmation of novel capacities for DCs in teleost, such an IgM-binding capacity and responsiveness to CD40 ligand. Furthermore, our results show that in gills, this DC subset shows some different phenotypic and functional characteristics when compared with their homologs in the skin, suggesting an adaptation of the cells to different mucosal tissues or different maturation status depending on their location. Our findings contribute to increase our knowledge on fish cross-presenting DCs, an important cell population to take into account for the future design of mucosal vaccination strategies.

OBJECTIVE

Anti-TNF-α treatment constitutes a mainstay in the treatment of Crohn's disease (CD), but its mechanisms of action are not fully understood. We aimed to investigate the effects of adalimumab, a human monoclonal TNF-α antibody, on macrophage (MQ) and dendritic cell (DC) subsets in mucosal biopsies and peripheral blood.

METHODS

Intestinal biopsies and blood samples were obtained from 12 different CD patients both before and 4 weeks after the initiation of the induction of adalimumab treatment. Endoscopic disease activity was estimated by the Simple Endoscopic Score for Crohn's Disease. Biopsies were obtained from inflamed and non-inflamed areas. The numbers of lamina propria CD14 (+) DR(int) and CD14 (+) DR(hi) MQs, CD141(+), CD141(-) and CD103(+) DCs subsets, and circulating monocytes and DCs were analyzed using flow cytometry.

RESULTS

At baseline, we observed higher numbers of DR(int) MQs and lower numbers of CD103(+) DCs in inflamed versus non-inflamed mucosa [843 vs. 391/10(5) lamina propria mononuclear cells (LPMCs) (p < 0.05) and 9 vs. 19 × 10(5) LPMCs (p = 0.01), respectively]. After four weeks of adalimumab treatment, the numbers of DR(int) MQs decreased [843 to 379/10(5) LPMCs (p = 0.03)], whereas the numbers of CD103(+) DCs increased [9-20 × 10(5) LPMCs (p = 0.003)] compared with baseline. In peripheral blood, no alterations were observed in monocyte or DC numbers between baseline and week 4.

CONCLUSIONS

In CD, mucosal inflammation is associated with high numbers of DR(int) MQs and low numbers of CD103(+) DCs. This composition of intestinal myeloid subsets is reversed by anti-TNF-α treatment. These results suggest that DR(int) MQs play a pivotal role in CD inflammation.

T-cell immunity in the liver is tightly regulated to prevent chronic liver inflammation in response to antigens and toxins derived from food and intestinal bacterial flora. Since the main sites of T cell activation in response to foreign components entering solid tissues are the draining lymph nodes (LN), we aimed to study whether Antigen-Presenting Cell (APC) subsets in human liver lymph-draining LN show features that may contribute to the immunologically tolerant liver environment. Healthy liver LN, iliac LN, spleen and liver perfusates were obtained from multi-organ donors, while diseased liver LN were collected from explanted patient livers. Inguinal LN were obtained from kidney transplant recipients. Mononuclear cells were isolated from fresh tissues, and immunophenotypic and functional characteristics of APC subsets were studied using flowcytometry and in ex vivo cultures. Healthy liver-draining LN contained significantly lower relative numbers of CD1c⁺ conventional dendritic cells (cDC2), plasmacytoid DC (PDC), and CD14⁺CD163⁺DC-SIGN⁺ macrophages (MF) compared to inguinal LN. Compared to spleen, both types of LN contained low relative numbers of CD141^hi cDC1. Both cDC subsets in liver LN showed a more activated/mature immunophenotype than those in inguinal LN, iliacal LN, spleen and liver tissue. Despite their more mature status, cDC2 isolated from hepatic LN displayed similar cytokine production capacity (IL-10, IL-12, and IL-6) and allogeneic T cell stimulatory capacity as their counterparts from spleen. Liver LN from patients with inflammatory liver diseases showed a further reduction of cDC1, but had increased relative numbers of PDC and MF. In steady state conditions human liver LN contain relatively low numbers of cDC2, PDC, and macrophages, and relative numbers of cDC1 in liver LN decline during liver inflammation. The paucity of cDC in liver LN may contribute to immune tolerance in the liver environment.

OBJECTIVE

To study whether in-vivo recruitment of dendritic cells in response to antigen administration in the skin is altered during HIV-1 infection.

METHODS

Skin punch biopsies were collected from HIV-1-positive as well as seronegative individuals at 48 h after intradermal injection of inactivated antigens of mumps virus, Candida albicans, or purified protein derivate (PPD) from Mycobacterium tuberculosis.

METHODS

Cryosections were analyzed by in-situ staining and computerized imaging.

RESULTS

Control skin biopsies showed that there was no difference in the number of skin-resident dendritic cells between seronegative and HIV-1-positive individuals. Antigen injection resulted in substantial infiltration of dendritic cells compared to the frequencies found in donor-matched control skin. In HIV-1-positive individuals, CD123(+)/CD303(+) plasmacytoid dendritic cells and CD11c myeloid dendritic cells, including the CD141(+) cross-presenting subset, were recruited at lower levels compared to healthy controls in response to PPD and mumps but not C. albicans. The level of dendritic cell recruitment correlated with the frequencies of T cells infiltrating the respective antigen sites. Ki67(+) cycling T cells at the injection sites were much more frequent in response to each of the antigens in the HIV-1-positive individuals, including those with AIDS, compared to healthy controls.

CONCLUSIONS

Multiple dendritic cell subsets infiltrate the dermis in response to antigen exposure. There was no obvious depletion or deficiency in mobilization of dendritic cells in response to antigen skin tests during chronic HIV-1 infection. Instead, the levels of antigen-specific memory T cells that accumulate at the antigen site may determine the level of dendritic cell infiltration.

Blood represents the most accessible source of human dendritic cells (DCs). We present here a method to isolate three DC subtypes, as identified until now, from peripheral blood: plasmacytoid dendritic cells (pDCs), CD141(+) myeloid DCs, and CD1c(+) myeloid DCs. The method is based on the sequential depletion of non-DCs. First, depletion of granulocytes, erythrocytes, and platelets is obtained by blood centrifugation over a Ficoll gradient. Then, antibodies recognizing non-DCs, combined with magnetic beads, allow enrichment of DCs from peripheral blood mononuclear cells (PBMCs). Finally, enriched DCs are purified and separated into the different subtypes by immunolabeling and fluorescence-activated cell sorting (FACS) using DC-specific surface markers.DC studies might contribute to the comprehension of human immune processes in physiological and pathological conditions. Human blood DCs targeting might be a useful tool to ameliorate inflammatory diseases and improve vaccination strategies.

OBJECTIVE

The pathogenesis of cardiac allograft vasculopathy after heart transplant remains controversial. Histologically, cardiac allograft vasculopathy is characterized by intimal hyperplasia of the coronary arteries induced by infiltrating cells. The origin of these infiltrating cells in cardiac allograft vasculopathy is unclear. Endothelial progenitor cells are reportedly involved in cardiac allograft vasculopathy; however, the role of CD14(+) monocyte-derived progenitor cells in cardiac allograft vasculopathy pathogenesis remains unknown.

METHODS

Monocyte-derived progenitor cells were isolated from blood mononuclear cell fractions obtained from 25 patients with cardiac allograft vasculopathy and 25 patients without cardiac allograft vasculopathy.

RESULTS

Both patients with cardiac allograft vasculopathy and those without cardiac allograft vasculopathy had CD45(+), CD34(+), CD14(+), CD141(-), CD31(-) monocyte-derived progenitor cells that differentiated into mesenchymal lineages. Monocyte-derived progenitor cells formed significantly higher numbers of colonies in patients with cardiac allograft vasculopathy than in those without cardiac allograft vasculopathy; this correlated with posttransplant follow-up time. Importantly, monocyte-derived progenitor cells from patients with cardiac allograft vasculopathy expressed significantly more α smooth muscle actin and proliferated at a higher rate than did monocyte-derived progenitor cells of patients without cardiac allograft vasculopathy. In vitro experiments suggested a paracrine control mechanism in proliferation of monocyte-derived progenitor cells in cardiac allograft vasculopathy.

CONCLUSIONS

These results indicate that monocyte-derived progenitor cells are associated with cardiac allograft vasculopathy, have the ability to transdifferentiate into smooth muscle cells, and thus may contribute to intimal hyperplasia of coronary arteries in cardiac allograft vasculopathy. Targeting monocyte-derived progenitor cell recruitment could be beneficial in cardiac allograft vasculopathy treatment.

One strategy in cancer immunotherapy is to capitalize on the key immunoregulatory and antigen presenting capabilities of dendritic cells (DCs). This approach is dependent on efficient delivery of tumor specific antigens to DCs, which subsequently induce an anti-tumor T-cell mediated immune response. Human adenovirus serotype 5 (HAdV5) has been used in human studies for gene delivery, but has limited infection in DCs, which lack the proper receptors. Addition of the porcine fiber knob (PK) from porcine adenovirus type 4 to HAdV5 allows the virus to deliver genetic material via binding to glycosylated surface proteins and bypasses the coxsackie-and-adenovirus receptor required by wild-type HAdV5. In this study we explored the potential therapeutic applications of an adenovirus with PK-based tropism against cancers expressing mesothelin. Infectivity and gene transfer assays were used to compare Ad5-PK to wild-type HAdV5. Mouse models were used to demonstrate peptide specificity and T-cell responses. We show that the PK modification highly augmented infection of DCs, including the CD141+ DC subset, a key subset for activation of naïve CD8+ T-cells. We also show that Ad5-PK increases DC infectivity and tumor specific antigen expression. Finally, vaccination of mice with the Ad5-PK vector resulted in enhanced T-cell-mediated interferon gamma (IFN-γ) release in response to both mesothelin peptide and a tumor line expressing mesothelin. Ad5-PK is a promising tool for cancer immunotherapy as it improves infectivity, gene transfer, protein expression, and subsequent T-cell activation in DCs compared to wild-type HAdV5 viruses.

Allergen-specific immunotherapy (AIT) induces tolerance and shifts the Th2 response towards a regulatory T-cell profile. The underlying mechanisms are not fully understood, but dendritic cells (DC) play a vital role as key regulators of T-cell responses. DCs interact with allergens via Fc receptors (FcRs) and via certain C-type lectin receptors (CLRs), including CD209/DC-SIGN, CD206/MR and Dectin-2/CLEC6A. In this study, the effect of AIT on the frequencies as well as the FcR and CLR expression profiles of human DC subsets was assessed. PBMC was isolated from peripheral blood from seven allergic donors before and after 8 weeks and 1 year of subcutaneous AIT, as well as from six non-allergic individuals. Cells were stained with antibodies against DC subset-specific markers and a panel of FcRs and CLRs and analyzed by flow cytometry. After 1 year of AIT, the frequency of CD123+ DCs was increased and a larger proportion expressed FcεRI. Furthermore, the expression of CD206 and Dectin-2 was reduced on CD141+ DCs after 1 year of treatment and CD206 as well as Dectin-1 was additionally down regulated in CD1c+ DCs. Interestingly, levels of DNGR1/CLEC9A on CD141+ DCs were increased by AIT, reaching levels similar to cells isolated from non-allergic controls. The modifications in phenotype and occurrence of specific DC subsets observed during AIT suggest an altered capacity of DC subsets to interact with allergens, which can be part of the mechanisms by which AIT induces allergen tolerance.

Hepatitis C virus (HCV) induces interferon (IFN) stimulated genes in the liver despite of distinct innate immune evasion mechanisms, suggesting that beyond HCV infected cells other cell types contribute to innate immune activation. Upon coculture with HCV replicating cells, human CD141+ myeloid dendritic cells (DC) produce type III IFN, whereas plasmacytoid dendritic cells (pDC) mount type I IFN responses. Due to limitations in the genetic manipulation of primary human DCs, we explored HCV mediated stimulation of murine DC subsets. Coculture of HCV RNA transfected human or murine hepatoma cells with murine bone marrow-derived DC cultures revealed that only Flt3-L DC cultures, but not GM-CSF DC cultures responded with IFN production. Cells transfected with full length or subgenomic viral RNA stimulated IFN release indicating that infectious virus particle formation is not essential in this process. Use of differentiated DC from mice with genetic lesions in innate immune signalling showed that IFN secretion by HCV-stimulated murine DC was independent of MyD88 and CARDIF, but dependent on TRIF and IFNAR signalling. Separating Flt3-L DC cultures into pDC and conventional CD11b-like and CD8α-like DC revealed that the CD8α-like DC, homologous to the human CD141+ DC, release interferon upon stimulation by HCV replicating cells. In contrast, the other cell types and in particular the pDC did not. Injection of human HCV subgenomic replicon cells into IFN-β reporter mice confirmed the interferon induction upon HCV replication in vivo. These results indicate that HCV-replicating cells stimulate IFN secretion from murine CD8α-like DC independent of infectious virus production. Thus, this work defines basic principles of viral recognition by murine DC populations. Moreover, this model should be useful to explore the interaction between dendritic cells during HCV replication and to define how viral signatures are delivered to and recognized by immune cells to trigger IFN release.

Plasmodium vivax malaria remains a major public health problem. The requirements for acquisition of protective immunity to the species are not clear. Dendritic cells (DC) are essential for immune cell priming but also perform immune regulatory functions, along with regulatory T cells (Treg). An important function of DC involves activation of the kynurenine pathway via indoleamine 2,3-dioxygenase (IDO). Using a controlled human experimental infection study with blood-stage P. vivax, we characterized plasmacytoid DC (pDC) and myeloid DC (mDC) subset maturation, CD4+ CD25+ CD127lo Treg activation, and IDO activity. Blood samples were collected from six healthy adults preinoculation, at peak parasitemia (day 14; ∼31,400 parasites/ml), and 24 and 48 h after antimalarial treatment. CD1c+ and CD141+ mDC and pDC numbers markedly declined at peak parasitemia, while CD16+ mDC numbers appeared less affected. HLA-DR expression was selectively reduced on CD1c+ mDC, increased on CD16+ mDC, and was unaltered on pDC. Plasma IFN-γ increased significantly and was correlated with an increased kynurenine/tryptophan (KT) ratio, a measure of IDO activity. At peak parasitemia, Treg presented an activated CD4+ CD25+ CD127lo CD45RA- phenotype and upregulated TNFR2 expression. In a mixed-effects model, the KT ratio was positively associated with an increase in activated Treg. Our data demonstrate that a primary P. vivax infection exerts immune modulatory effects by impairing HLA-DR expression on CD1c+ mDC while activating CD16+ mDC. Induction of the kynurenine pathway and increased Treg activation, together with skewed mDC maturation, suggest P. vivax promotes an immunosuppressive environment, likely impairing the development of a protective host immune response.

Background: The conventional type 1 dendritic cell subset (cDC1) is indispensable for tumor immune responses and the efficacy of immune checkpoint inhibitor (ICI) therapies in animal models but little is known about the role of the human CD141⁺ DC cDC1 equivalent in patients with melanoma.

Methods: We developed a flow cytometry assay to quantify and characterize human blood DC subsets in healthy donors and patients with stage 3 and stage 4 metastatic melanoma. To examine whether harnessing CD141⁺ DCs could improve responses to ICIs in human melanoma, we developed a humanized mouse model by engrafting immunodeficient NSG-SGM3 mice with human CD34⁺ hematopoietic stem cells (HSCs) from umbilical cord blood followed by transplantation of a human melanoma cell line and treatment with anti-programmed cell death protein-1 (anti-PD-1).

Results: Blood CD141⁺ DC numbers were significantly reduced in patients with stage 4 melanoma compared with healthy controls. Moreover, CD141⁺ DCs in patients with melanoma were selectively impaired in their ability to upregulate CD83 expression after stimulation with toll-like receptor 3 (TLR3) and TLR7/8 agonists ex vivo. Although DC numbers did not correlate with responses to anti-PD-1 and/or anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) ICIs, their numbers and capacity to upregulate CD83 declined further during treatment in non-responding patients. Treatment with anti-PD-1 was ineffective at controlling tumor growth in humanized mice but efficacy was enhanced by indirectly expanding and activating DCs in vivo with fms-like tyrosine kinase-3 ligand (Flt3L) and a TLR3 agonist. Moreover, intratumoral injections of CD141⁺ DCs resulted in reduced tumor growth when combined with anti-PD-1 treatment.

Conclusions: These data illustrate quantitative and qualitative impairments in circulating CD141⁺ DCs in patients with advanced melanoma and that increasing CD141⁺ DC number and function is an attractive strategy to enhance immunogenicity and response rates to ICIs.

Keywords: Melanoma; dendritic cells; immunogenicity; immunotherapy; vaccine.

Although immunomodulatory drugs (IMiDs) were originally developed as anti-inflammatory drugs, they are effective for multiple myeloma. In order to gain further insights into the immunomodulatory mechanisms of IMiDs for the treatment of inflammatory disorders and myeloma, we investigated the influence of a representative IMiD, lenalidomide, on human primary dendritic cell (DC) subsets: myeloid-derived CD1c⁺ DCs, CD141⁺ DCs, and plasmacytoid DCs. Lenalidomide did not affect the viability or expression of costimulatory molecules, but it potently suppressed the production of the key inflammatory cytokines IL-12 and IL-23, and enhanced the production of the anti-inflammatory cytokine IL-10 by CD1c⁺ DCs. Lenalidomide also suppressed the production of IFN-α by CD141⁺ DCs but not that by plasmacytoid DCs. Lenalidomide likely targets pathways downstream of the nuclear translocation of the transcription factors nuclear factor κB (NF-κB) and IFN regulatory 5 (IRF5) in CD1c⁺ DCs. Consistent with the direct immunomodulatory effects on DCs, lenalidomide decreased the capacity of CD1c⁺ DCs to induce differentiation of naïve CD4⁺ T cells into effector cells producing immune activating and myeloma-promoting cytokines. This study demonstrated that lenalidomide has anti-inflammatory effects via the modulation of cytokine production by human myeloid-derived DCs. Such effects on DCs may allow for beneficial immunomodulation aiding in the treatment of inflammatory disorders and multiple myeloma.

Dengue virus (DENV) is the most prevalent arthropod-borne viral disease in humans. DENV causes a spectrum of illness ranging from mild to potentially severe complications. Dendritic cells (DCs) play a critical role in initiating and regulating highly effective antiviral immune response that include linking innate and adaptive immune responses. This study was conducted to comparatively characterize in detail the relative proportion, phenotypic changes, and maturation profile of subsets of both myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in children with dengue fever (DF), dengue hemorrhagic fever (DHF) and for purposes of control healthy individuals. The mDCs (Lin-CD11c+CD123lo), the pDCs (Lin-CD11c-CD123+) and the double negative (DN) subset (Lin-/HLA-DR+/CD11c-CD123-) were analyzed by polychromatic flow cytometry. The data were first analyzed on blood samples collected from DENV-infected patients at various times post-infection. Results showed that the relative proportion of mDCs were significantly decreased which was associated with an increase in disease severity in samples from DENV-infected patients. While there was no significant difference in the relative proportion of pDCs between healthy and DENV-infected patients, there was a marked increase in the DN subset. Analysis of the kinetics of changes of pDCs showed that there was an increase but only during the early febrile phase. Additionally, samples from patients during acute disease showed marked decreases in the relative proportion of CD141+ and CD16+ mDC subsets that were the major mDC subsets in healthy individuals. In addition, there was a significant decrease in the level of CD33-expressing mDCs in DENV patients. While the pDCs showed an up-regulation of maturation profile during acute DENV infection, the mDCs showed an alteration of maturation status. This study suggests that different relative proportion and phenotypic changes as well as alteration of maturation profile of DC subsets may play a critical role in the dengue pathogenesis and disease outcome.

Dendritic cells (DCs) play a key role in initiating and regulating the immune responses to pathogens, self-antigens, and cancers. Human blood DCs comprise a family of different subsets: plasmacytoid DCs (pDCs) and CD16⁺, CD1c/BDCA1⁺, and BDCA3⁺ (CD141⁺) myeloid DCs and possess different phenotypes and functional characteristics. Lung cancer is the most common cancer, with the highest morbidity and mortality in the world. However, which DC subset plays a leading role in the lung cancer immune responses is unclear. We reanalyzed C-type lectin domain family 9 member A (CLEC9A) and CD141 (THBD) gene expression profiles from the Cancer Genome Atlas (TCGA) database and performed the Kaplan-Meier survival analysis of overall survival for several cancers according to their expression levels. Next, we investigated the capacities of five human blood DC subsets to stimulate T cell proliferation and capture, process and (cross-) present tumor antigen. Human BDCA3⁺ (CD141⁺) DCs have a superior capacity to stimulate allogeneic CD4⁺T cells proliferation and induce superior Th1 response compared with other DC subsets. Interestingly, toll-like receptor (TLR) agonists have little effect on DCs to induce the proliferation of naïve CD4⁺ T cells, but contribute to their differentiation. Importantly, BDCA3⁺ (CD141⁺) DCs possess the most potent ability to cross-present human tumor antigen after their uptake of necrotic lung cancer cells despite their lower antigen uptake. These findings suggest that human BDCA3⁺ (CD141⁺) DCs are critical mediators of cytotoxic T lymphocyte responses against EGFR-positive lung cancer. Therefore, our findings may provide theoretical basis for the development of DC-based antitumor vaccines.

Keywords: EGFR; TLR; antitumor vaccine; cytotoxic T lymphocyte; dendritic cell; lung cancer.