Individual Dendritic Cells Research Articles

Proximity-dependent labeling identifies dendritic cells that drive the tumor-specific CD4+ T cell response.

Dendritic cells (DCs) are uniquely capable of transporting tumor antigens to tumor-draining lymph nodes (tdLNs) and interact with effector T cells in the tumor microenvironment (TME) itself, mediating both natural antitumor immunity and the response to checkpoint blockade immunotherapy. Using LIPSTIC (Labeling Immune Partnerships by SorTagging Intercellular Contacts)-based single-cell transcriptomics, we identified individual DCs capable of presenting antigen to CD4+ T cells in both the tdLN and TME. Our findings revealed that DCs with similar hyperactivated transcriptional phenotypes interact with helper T cells both in tumors and in the tdLN and that checkpoint blockade drugs enhance these interactions. These findings show that a relatively small fraction of DCs is responsible for most of the antigen presentation in the tdLN and TME to both CD4+ and CD8+ tumor-specific T cells and that classical checkpoint blockade enhances CD40-driven DC activation at both sites.

Increased Proliferative Drive of Lymphocytes in Sickle Cell Disease

Introduction Patients with sickle cell disease (SCD) have higher levels of inflammation which plays a prominent role in disease pathogenesis. It is well known that individuals with SCD have a higher erythropoietic drive given the ~20 day life span of their red blood cells. We asked whether this increased proliferative drive also occurred in white blood cells (WBC). To characterize WBC turnover we focused on the longest-lived CD4+ and CD8+ T cells. Methods Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient from red cell waste products from consented individuals with sickle cell disease and from leukapheresis products of healthy donors. In ten healthy donors and ten donors with SCD, CD4 and CD8 T cells were purified using EasySep Cell Isolation, labeled with eFluor 670 proliferation dye, and cultured in RPMI with 30% FBS for seven days. Samples were collected daily and stained with naïve and memory T cell markers. CountBright beads were added to obtain absolute cell counts and eFluor 780 live/dead dye was used to capture daily snapshots of cell death. Flow cytometric analysis was performed to track absolute cell numbers, cell division and indirectly monitor cell death. B cells, NK, macrophages, and dendritic cells were similarly tracked. To further probe T cell turnover, T cell receptor (TCR) sequencing was performed on twelve separate aliquots from purified CD4+ T cells at two different timepoints in healthy donors and individuals with SCD. Turnover of clonal sequences was analyzed using the Morisita index. Results CD4+ and CD8+ lymphocytes from individuals with SCD experienced more death in culture by day 7 compared to healthy donor controls. In addition, T lymphocytes from donors with SCD were observed to undergo 15-fold more proliferation compared to healthy donors (p=0.0002, unpaired t-test). Immunophenotyping performed on the CD4 and CD8 T lymphocytes showed a propensity for memory cells to undergo proliferation. We also found individuals with SCD had higher turnover in TCR sequences in two timepoints compared to healthy donors (p=0.05, Chi-square). Notably, we also saw increased proliferation of B cells, NK cells, macrophages, and dendritic cells in individuals with SCD. Conclusion Our data suggest that T lymphocytes and other PBMCs undergo faster turnover in SCD than in healthy donors, suggesting a stronger hematopoietic drive. Our findings may have important implications for gene therapy since an increased proliferative drive may contribute to an increased risk of developing leukemia in SCD.

Activation, Amplification, and Ablation as Dynamic Mechanisms of Dendritic Cell Maturation.

T cell responses to cognate antigens crucially depend on the specific functionality of dendritic cells (DCs) activated in a process referred to as maturation. Maturation was initially described as alterations of the functional status of DCs in direct response to multiple extrinsic innate signals derived from foreign organisms. More recent studies, conducted mainly in mice, revealed an intricate network of intrinsic signals dependent on cytokines and various immunomodulatory pathways facilitating communication between individual DCs and other cells for the orchestration of specific maturation outcomes. These signals selectively amplify the initial activation of DCs mediated by innate factors and dynamically shape DC functionalities by ablating DCs with specific functions. Here, we discuss the effects of the initial activation of DCs that crucially includes the production of cytokine intermediaries to collectively achieve amplification of the maturation process and further precise sculpting of the functional landscapes among DCs. By emphasizing the interconnectedness of the intracellular and intercellular mechanisms, we reveal activation, amplification, and ablation as the mechanistically integrated components of the DC maturation process.

Flt3L, LIF, and IL-10 combination promotes the selective in vitro development of ESAMlow cDC2B from murine bone marrow.

The development of two conventional dendritic cells (DC) subsets (cDC1 and cDC2) and the plasmacytoid DC (pDC) in vivo and in cultures of bone marrow (BM) cells is mediated by the growth factor Flt3L. However, little is known about the factors that direct the development of the individual DC subsets. Here, we describe the selective in vitro generation of murine ESAMlow CD103- XCR1- CD172a+ CD11b+ cDC2 from BM by treatment with a combination of Flt3L, LIF, and IL-10 (collectively named as FL10). FL10 promotes common dendritic cell progenitors (CDP) proliferation in the cultures, similar to Flt3L and CDP sorted and cultured in FL10 generate exclusively cDC2. These cDC2 express the transcription factors Irf4, Klf4, and Notch2, and their growth is reduced using BM from Irf4-/- mice, but the expression of Batf3 and Tcf4 is low. Functionally they respond to TLR3, TLR4, and TLR9 signals by upregulation of the surface maturation markers MHC II, CD80, CD86, and CD40, while they poorly secrete proinflammatory cytokines. Peptide presentation to TCR transgenic OT-II cells induced proliferation and IFN-γ production that was similar to GM-CSF-generated BM-DC and higher than Flt3L-generated DC. Together, our data support that FL10 culture of BM cells selectively promotes CDP-derived ESAMlow cDC2 (cDC2B) development and survival in vitro.

Skin sensitization to fragrance hydroperoxides: interplay between dendritic cells, keratinocytes and free radicals.

Skin sensitization to hydroperoxides (R-OOHs) of the commonly used fragrance terpenes limonene, linalool and citronellol is frequently reported. R-OOHs are believed to initiate the process leading to sensitization and allergic contact dermatitis through mechanisms involving radical intermediates. Thus, radical intermediates, keratinocytes and dendritic cells (DCs) may act in concert to initiate the process. To evaluate individual DC activation profiles by R-OOHs in the context of keratinocytes with regard to frequency, specificity and magnitude of upregulation. We used 2D and 3D cocultures with keratinocytes/reconstructed human epidermis (RHE) and DCs to evaluate cell surface levels of the costimulatory molecules CD86, CD80 and the adhesion molecule CD54 on cocultured DCs. Analysis of radical formation from limonene hydroperoxides in RHE was performed using electron paramagnetic resonance combined with the spin trapping technique. R-OOHs induce donor-dependent DC activation. Major differences were found between the limonene-OOHs. Limonene-1-OOH was stronger with respect to both frequency and magnitude of response. Using a 3D coculture model, no DC activation was detected after topical application of 0·2% limonene-OOHs (20µgcm-2 ), while 1·2% limonene-1-OOH or 2% limonene-2-OOH induced DC activation. Furthermore, we demonstrated differences in the carbon and oxygen radicals formed from the limonene-OOHs using RHE, mimicking what may happen in vivo. We report clear individual differences in DC maturation induced by the most important hydroperoxides. Response rates and magnitude of response both indicate that very small structural alterations in the hydroperoxides are translated into specific DC responses. In addition, we provide more insight into the amounts of hydroperoxides that can activate DCs and induce sensitization.

Morphometric Changes to Corneal Dendritic Cells in Individuals With Mild Cognitive Impairment.

PurposeThere has been increasing interest in identifying non-invasive, imaging biomarkers for neurodegenerative disorders of the central nervous system (CNS). The aim of this proof-of-concept study was to investigate whether corneal sensory nerve and dendritic cell (DC) parameters, captured using in vivo confocal microscopy (IVCM), are altered in individuals with mild cognitive impairment (MCI) and Alzheimer’s disease (AD).MethodsFifteen participants were recruited from the Australian Imaging Biomarkers and Lifestyle (AIBL) study in Melbourne, VIC, Australia. The cohort consisted of cognitively normal (CN) individuals (n = 5), and those with MCI (n = 5) and AD (n = 5). Participants underwent a slit lamp examination of the anterior segment, followed by corneal imaging using laser-scanning in vivo confocal microscopy (IVCM) of the central and inferior whorl regions. Corneal DC density, field area, perimeter, circularity index, aspect ratio, and roundness were quantified using Image J. Quantitative data were derived for corneal nerve parameters, including nerve fiber length (CNFL), fiber density (CNFD), branch density (CNBD), and diameter.ResultsCorneal DC field area and perimeter were greater in individuals with MCI, relative to CN controls, in both the central and inferior whorl regions (p < 0.05 for all comparisons). In addition, corneal DCs in the whorl region of MCI eyes had lower circularity and roundness indices and a higher aspect ratio relative to CNs (p < 0.05 for all comparisons). DC density was similar across participant groups in both corneal regions. There was a trend toward lower quantitative parameters for corneal nerve architecture in the AD and MCI groups compared with CN participants, however, the inter-group differences did not reach statistical significance. Central corneal nerve diameters were similar between groups.ConclusionThis study is the first to report morphological differences in corneal DCs in humans with MCI. These differences were evident in both the central and mid-peripheral cornea, and in the absence of significant nerve abnormalities or a difference in DC density. These findings justify future large-scale studies to assess the utility of corneal IVCM and DC analysis for identifying early stage pathology in neurodegenerative disorders of the CNS.

Glyco-Dendrimers as Intradermal Anti-Tumor Vaccine Targeting Multiple Skin DC Subsets.

The human skin is an attractive anti-tumor vaccination site due to the vast network of dendritic cell (DC) subsets that carry antigens to the draining lymph nodes and stimulate tumor specific CD4+ and CD8+ T cells in. Specific vaccine delivery to skin DC can be accomplished by targeting glycan coated antigens to C-type lectin receptors (CLRs) such as DC-SIGN expressed by human dermal DCs and Langerin expressed by Langerhans cells (LCs), which facilitate endocytosis and processing for antigen presentation and T cell activation. Although there are multiple human skin DC subsets, targeting individual DC subsets and receptors has been a focus in the past. However, the simultaneous targeting of multiple human skin DC subsets that mobilize the majority of the skin antigen presenting cells (APC) is preferred to accomplish more robust and efficient T cell stimulation. Dual CLR targeting using a single tumor vaccine has been difficult, as we previously showed Langerin to favor binding and uptake of monovalent glyco-peptides whereas DC-SIGN favors binding of larger multivalent glyco-particles such as glyco-liposomes.Methods: We used branched polyamidoamine (PAMAM) dendrimers as scaffold for melanoma specific gp100 synthetic long peptides and the common DC-SIGN and Langerin ligand Lewis Y (LeY), to create multivalent glyco-dendrimers with varying molecular weights for investigating dual DC-SIGN and Langerin targeting. Using DC-SIGN+ monocyte derived DC (moDC) and Langerin+ primary LC we investigated glyco-dendrimer CLR targeting properties and subsequent gp100 specific CD8+ T cell activation in vitro. In situ targeting ability to human dermal DC and LC through intradermal injection in a human skin explant model was elucidated.Results: Dual DC-SIGN and Langerin binding was achieved using glyco-dendrimers of approximately 100kD, thereby fulfilling our criteria to simultaneously target LCs and CD1a+ and CD14+ dermal DC in situ. Both DC-SIGN and Langerin targeting by glyco-dendrimers resulted in enhanced internalization and gp100 specific CD8+ T cell activation.Conclusion: We designed the first glyco-vaccine with dual CLR targeting properties, thereby reaching multiple human skin DC subsets in situ for improved anti-tumor CD8+ T cell responses.

Class II MHC-independent suppressive adhesion of dendritic cells by regulatory T cells in vivo.

Regulatory T (T reg) cells are essential for peripheral homeostasis and known to target and suppress dendritic cells (DCs). One important mechanism is through prolonged interaction between antigen-specific T reg cells and DCs that down-regulates the co-stimulatory capacity of DCs. However, the dynamics and TCR specificities of such T reg cell-DC interaction and its relevance to the suppressive outcomes for individual DCs have not been clarified. To gain insights into the underlying cellular events in vivo, we analyzed individual T reg cell-DC interaction events in lymph nodes by intravital microscopy. Our results show that, upon exposure to interleukin-2, T reg cells formed prolonged adhesive contact with DCs, independent of antigen or MHC recognition, which significantly suppressed the contemporaneous interaction of the same DCs with antigen-specific conventional T cells and impaired T cell priming. Therefore, T reg cells may function in part as feedback regulators in inflammatory milieu, by suppressing local DCs and interrupting immune activation in a contact-dependent and class II MHC-independent manner.

Functional Impairment of Murine Dendritic Cell Subsets following Infection with Infective Larval Stage 3 of Brugia malayi.

Filarial parasites cause functional impairment of host dendritic cells (DCs). However, the effects of early infection on individual DC subsets are not known. In this study, we infected BALB/c mice with infective stage 3 larvae of the lymphatic filarial parasite Brugia malayi (Bm-L3) and studied the effect on fluorescence-activated cell sorter (FACS)-sorted DC subsets. While myeloid DCs (mDCs) accumulated by day 3 postinfection (p.i.), lymphoid DCs (LDCs) and CD8+ plasmacytoid DCs (pDCs) peaked at day 7 p.i. in the spleens and mesenteric lymph nodes (mLNs) of infected mice. Increased tumor necrosis factor alpha (TNF-α) but reduced interleukin 12 (IL-12) and Toll-like receptor 4 (TLR4), -6, and -9 and reciprocal secretion of IL-4 and IL-10 were also observed across all DC subsets. Interestingly, Bm-L3 increased the expression of CD80 and CD86 across all DC subsets but decreased that of major histocompatibility complex class II (MHC-II) on mDCs and pDCs, resulting in their impaired antigen uptake and presentation capacities, but maximally attenuated the T-cell proliferation capacity of only mDCs. Furthermore, Bm-L3 increased phosphorylated p38 (p-p38), but not p-ERK, in mDCs and LDCs but downregulated them in pDCs, along with differential modulation of protein tyrosine phosphatases SHP-1, TCPTP, PTEN, and PTP1B across all DC subsets. Taken together, we report hitherto undocumented effects of early Bm-L3 infection on purified host DC subsets that lead to their functional impairment and attenuated host T-cell response.

Conventional Dendritic Cells Confer Protection against Mouse Cytomegalovirus Infection via TLR9 and MyD88 Signaling

Cytomegalovirus (CMV) is an opportunistic virus severely infecting immunocompromised individuals. In mice, endosomal Toll-like receptor 9 (TLR9) anddownstream myeloid differentiation factor 88 (MyD88) are central to activating innate immune responses against mouse CMV (MCMV). In this respect, the cell-specific contribution of these pathways in initiating anti-MCMV immunity remains unclear. Using transgenic mice, we demonstrate that TLR9/MyD88 signaling selectively in CD11c+ dendritic cells (DCs) strongly enhances MCMV clearance by boosting natural killer (NK) cell CD69 expression and IFN-γ production. In addition, we show that in the absence of plasmacytoid DCs (pDCs), conventional DCs (cDCs) promote robust NK cell effector function and MCMV clearance in a TLR9/MyD88-dependent manner. Simultaneously, cDC-derived IL-15 regulates NK cell degranulation by TLR9/MyD88-independent mechanisms. Overall, we compartmentalize the cellular contribution of TLR9 and MyD88 signaling in individual DC subsets and evaluate the mechanism by which cDCs control MCMV immunity.

Characterization of Dendritic Cell Subsets Through Gene Expression Analysis.

Dendritic cells (DCs) are immune sentinels of the body and play a key role in the orchestration of the communication between the innate and the adaptive immune systems. DCs can polarize innate and adaptive immunity toward a variety of functions, sometimes with opposite roles in the overall control of immune responses (e.g., tolerance or immunosuppression versus immunity) or in the balance between various defense mechanisms promoting the control of different types of pathogens (e.g., antiviral versus antibacterial versus anti-worm immunity). These multiple DC functions result both from the plasticity of individual DC to exert different activities and from the existence of various DC subsets specialized in distinct functions. Functional genomics represents a powerful, unbiased, approach to better characterize these two levels of DC plasticity and to decipher its molecular regulation. Indeed, more and more experimental immunologists are generating high-throughput data in order to better characterize different states of DC based, for example, on their belonging to a specific subpopulation and/or on their exposure to specific stimuli and/or on their ability to exert a specific function. However, the interpretation of this wealth of data is severely hampered by the bottleneck of their bioinformatics analysis. Indeed, most experimental immunologists lack advanced computational or bioinformatics expertise and do not know how to translate raw gene expression data into potential biological meaning. Moreover, subcontracting such analyses is generally disappointing or financially not sustainable, since companies generally propose canonical analysis pipelines that are often unadapted for the structure of the data to analyze or for the precise type of questions asked. Hence, there is an important need of democratization of the bioinformatics analyses of gene expression profiling studies, in order to accelerate interpretation of the results by the researchers at the origin of the research project, of the data and who know best the underlying biology. This chapter will focus on the analysis of DC subset transcriptomes as measured by microarrays. We will show that simple bioinformatics procedures, applied one after the other in the framework of a pipeline, can lead to the characterization of DC subsets. We will develop two tutorials based on the reanalysis of public gene expression data. The first tutorial aims at illustrating a strategy for establishing the identity of DC subsets studied in a novel context, here their in vitro generation in cultures of human CD34(+) hematopoietic progenitors. The second tutorial aims at illustrating how to perform a posteriori bioinformatics analyses in order to evaluate the risk of contamination or of improper identification of DC subsets during preparation of biological samples, such that this information is taken into account in the final interpretation of the data and can eventually help to redesign the sampling strategy.

Skin dendritic cells induce follicular helper T cells and protective humoral immune responses

The contribution of individual subsets of dendritic cells (DCs) to generation of adaptive immunity is central to understanding immune homeostasis and protective immune responses. We sought to define functions for steady-state skin DCs. We present an approach in which we restrict antigen presentation to individual DC subsets in the skin and monitor the effects on endogenous antigen-specific CD4(+) T- and B-cell responses. Presentation of foreign antigen by Langerhans cells (LC) in the absence of exogenous adjuvant led to a large expansion of T follicular helper (TFH) cells. This was accompanied by B-cell activation, germinal center formation, and protective antibody responses against influenza. The expansion of TFH cells and antibody responses could be elicited by both systemic and topical skin immunization. TFH cell induction was not restricted to LCs and occurred in response to antigen presentation by CD103(+) dermal DCs. CD103(+) DCs, despite inducing similar TFH responses as LCs, were less efficient in induction of germinal center B cells and humoral immune responses. We also found that skin DCs are sufficient to expand CXCR5(+) TFH cells through an IL-6- and IFN-α/β receptor-independent mechanism, but B cells were required for sustained Bcl-6(+) expression. These data demonstrate that a major unappreciated function of skin DCs is their promotion of TFH cells and humoral immune responses that potentially represent an efficient approach for vaccination.

Differential T-Helper Cell Polarization after Allergen-Specific Stimulation of Autologous Dendritic Cells in Polysensitized Allergic Patients

Background: Dendritic cells (DCs) play an important role in the induction and regulation of adaptive immune responses by polarizing T-helper (Th) cells. In allergic disease this response is dominated by Th2 cells. It is still unclear whether the activation of Th cells by DCs in atopic individuals is allergen specific. Methods: Monocyte-derived DCs (MoDCs) obtained from polysensitized patients were stimulated with purified Bet v 1, Phl p 5 and Act d 10, and the surface marker expression was analysed. Proliferation and cytokine profiles of autologous naïve CD4+ T cells co-cultured with allergen-pulsed MoDCs were assessed. Results: The addition of either Bet v 1 or Phl p 5 did not further increase the expression of surface markers from matured MoDCs in all study groups. In co-cultures, autologous naïve CD4+ T cells proliferated when DCs obtained from individuals allergic to birch and grass pollen were stimulated with Bet v 1 and Phl p 5, respectively. In the co-culture supernatants, significantly increased levels of IL-5 and IL-13 were detected. This effect correlated with the sensitization background and was absent when applying an unspecific allergen, Act d 10. The levels of IL-10 in supernatants of MoDCs and the levels of IL-10 and IFN-γ in supernatants of T cells remained unchanged upon stimulation with allergens. Conclusions: In this study we observed that allergen-specific stimulation of MoDCs induces T-cell proliferation and upregulation of Th2-type cytokines. Interestingly, this Th2 polarization was only observed in cells stimulated with the allergen to which the patients were sensitized.

Prothymosin α and a prothymosin α-derived peptide enhance TH1-type immune responses against defined HER-2/neu epitopes

BackgroundActive cancer immunotherapies are beginning to yield clinical benefit, especially those using peptide-pulsed dendritic cells (DCs). Different adjuvants, including Toll-like receptor (TLR) agonists, commonly co-administered to cancer patients as part of a DC-based vaccine, are being widely tested in the clinical setting. However, endogenous DCs in tumor-bearing individuals are often dysfunctional, suggesting that ex vivo educated DCs might be superior inducers of anti-tumor immune responses. We have previously shown that prothymosin alpha (proTα) and its immunoreactive decapeptide proTα(100–109) induce the maturation of human DCs in vitro. The aim of this study was to investigate whether proTα- or proTα(100–109)-matured DCs are functionally competent and to provide preliminary evidence for the mode of action of these agents.ResultsMonocyte-derived DCs matured in vitro with proTα or proTα(100–109) express co-stimulatory molecules and secrete pro-inflammatory cytokines. ProTα- and proTα(100–109)-matured DCs pulsed with HER-2/neu peptides induce TH1-type immune responses, prime autologous naïve CD8-positive (+) T cells to lyse targets expressing the HER-2/neu epitopes and to express a polyfunctional profile, and stimulate CD4+ T cell proliferation in an HER-2/neu peptide-dependent manner. DC maturation induced by proTα and proTα(100–109) is likely mediated via TLR-4, as shown by assessing TLR-4 surface expression and the levels of the intracellular adaptor molecules TIRAP, MyD88 and TRIF.ConclusionsOur results suggest that proTα and proTα(100–109) induce both the maturation and the T cell stimulatory capacity of DCs. Although further studies are needed, evidence for a possible proTα and proTα(100–109) interaction with TLR-4 is provided. The initial hypothesis that proTα and the proTα-derived immunoactive decapeptide act as “alarmins”, provides a rationale for their eventual use as adjuvants in DC-based anti-cancer immunotherapy.

Altered phenotype and function of dendritic cells in individuals with chronic periodontitis

ObjectiveTo investigate the effects of periodontal bacterial lysates on maturation and function of mature monocyte-derived dendritic cells (m-MDDCs) derived from individuals with chronic periodontitis (CP) or healthy periodontal tissue (HP). Designm-MDDCs derived from peripheral blood monocytes, cultured for 7 days in presence of interleukin (IL)-4 and granulocyte-macrophage colony stimulating factor (GM-CSF), were stimulated with lysates of Streptococcus sanguinis, Prevotella intermedia, Porphyromonas gingivalis, or Treponema denticola on day 4, and were then phenotyped. IL-10, IL-12 and IFN-gamma concentration in the supernatant of cultures were measured. ResultsExpression of HLA-DR was lower in bacterial-unstimulated mature m-MDDC from CP compared to HP (p=0.04), while expression of CD1a and CD123 were higher in CP. The expression pattern of HLA-DR, CD11c, CD123, and CD1a did not change on bacterial stimulation, regardless of the bacteria. Stimulation with P. intermedia upregulated CD80 and CD86 in CP cells (p≤0.05). Production of IL-12p70 by bacterial-unstimulated m-MDDCs was 5.8-fold greater in CP compared to HP. Bacterial stimulation further increased IL-12p70 production while decreasing IL-10. Significantly more IFN-gamma was produced in co-cultures of CP m-MDDCs than with HP m-MDDCs when cells were stimulated with P. intermedia (p=0.009). ConclusionsBacterial-unstimulated m-MDDC from CP exhibited a more immature phenotype but a cytokine profile biased towards proinflammatory response; this pattern was maintained/exacerbated after bacterial stimulation. P. intermedia upregulated co-stimulatory molecules and IFN-gamma expression in CP m-MDDC. These events might contribute to periodontitis pathogenesis.

In silico investigation into dendritic cell regulation of CD8Treg mediated killing of Th1 cells in murine experimental autoimmune encephalomyelitis

BackgroundExperimental autoimmune encephalomyelitis has been used extensively as an animal model of T cell mediated autoimmunity. A down-regulatory pathway through which encephalitogenic CD4Th1 cells are killed by CD8 regulatory T cells (Treg) has recently been proposed. With the CD8Treg cells being primed by dendritic cells, regulation of recovery may be occuring around these antigen presenting cells. CD4Treg cells provide critical help within this process, by licensing dendritic cells to prime CD8Treg cells, however the spatial and temporal aspects of this help in the CTL response is currently unclear.ResultsWe have previously developed a simulator of experimental autoimmune encephalomyelitis (ARTIMMUS). We use ARTIMMUS to perform novel in silico experimentation regarding the priming of CD8Treg cells by dendritic cells, and the resulting CD8Treg mediated killing of encephalitogenic CD4Th1 cells. Simulations using dendritic cells that present antigenic peptides in a mutually exclusive manner (either MBP or TCR-derived, but not both) suggest that there is no significant reliance on dendritic cells that can prime both encephalitogenic CD4Th1 and Treg cells. Further, in silico experimentation suggests that dynamics of CD8Treg priming are significantly influenced through their spatial competition with CD4Treg cells and through the timing of Qa-1 expression by dendritic cells.ConclusionThere is no requirement for the encephalitogenic CD4Th1 cells and cytotoxic CD8Treg cells to be primed by the same dendritic cells. We conjecture that no significant portion of CD4Th1 regulation by Qa-1 restricted CD8Treg cells occurs around individual dendritic cells, and as such, that CD8Treg mediated killing of CD4Th1 cells occurring around dendritic cells is not critical for recovery from the murine autoimmune disease. Furthermore, the timing of the CD4Treg licensing of dendritic cells and the spatial competition between CD4Treg and CD8Treg cells around the dendritic cell is critical for the size of the cytotoxic T lymphocyte response, because dendritic cells have a limited lifespan. If treatments can be found to either speed up the licensing process, or increase the spatial competitiveness of CD8Treg cells, the magnitude of the cytotoxic T lymphocyte response can be increased.

Transcriptional Profiling of Human Dendritic Cell Populations and Models - Unique Profiles of In Vitro Dendritic Cells and Implications on Functionality and Applicability

BackgroundDendritic cells (DCs) comprise heterogeneous populations of cells, which act as central orchestrators of the immune response. Applicability of primary DCs is restricted due to their scarcity and therefore DC models are commonly employed in DC-based immunotherapy strategies and in vitro tests assessing DC function. However, the interrelationship between the individual in vitro DC models and their relative resemblance to specific primary DC populations remain elusive.ObjectiveTo describe and assess functionality and applicability of the available in vitro DC models by using a genome-wide transcriptional approach.MethodsTranscriptional profiling was performed with four commonly used in vitro DC models (MUTZ-3-DCs, monocyte-derived DCs, CD34-derived DCs and Langerhans cells (LCs)) and nine primary DC populations (dermal DCs, LCs, blood and tonsillar CD123+, CD1c+ and CD141+ DCs, and blood CD16+ DCs).ResultsPrincipal Component Analysis showed that transcriptional profiles of each in vitro DC model most closely resembled CD1c+ and CD141+ tonsillar myeloid DCs (mDCs) among primary DC populations. Thus, additional differentiation factors may be required to generate model DCs that more closely resemble other primary DC populations. Also, no model DC stood out in terms of primary DC resemblance. Nevertheless, hierarchical clustering showed clusters of differentially expressed genes among individual DC models as well as primary DC populations. Furthermore, model DCs were shown to differentially express immunologically relevant transcripts and transcriptional signatures identified for each model DC included several immune-associated transcripts.ConclusionThe unique transcriptional profiles of in vitro DC models suggest distinct functionality in immune applications. The presented results will aid in the selection of an appropriate DC model for in vitro assays and assist development of DC-based immunotherapy.

Experimental models to investigate the function of dendritic cell subsets: challenges and implications

The dendritic cell (DC) lineage is remarkably heterogeneous. It has been postulated that specialized DC subsets have evolved in order to select and support the multitude of possible T cell differentiation pathways. However, defining the function of individual DC subsets has proven remarkably difficult, and DC subset control of key T cell fates such as tolerance, T helper cell commitment and regulatory T cell induction is still not well understood. While the difficulty in assigning unique functions to particular DC subsets may be due to sharing of functions, it may also reflect a lack of appropriate physiological in-vivo models for studying DC function. In this paper we review the limitations associated with many of the current DC models and highlight some of the underlying difficulties involved in studying the function of murine DC subsets.

Reconstitution of 6-Sulfo LacNAc Dendritic Cells After Allogeneic Stem-Cell Transplantation

Infections and acute graft-versus-host disease (GvHD) represent major complications of allogeneic stem-cell transplantation (SCT). Dendritic cells (DCs) display an extraordinary capacity to induce innate and adaptive immune responses. Therefore, they play a crucial role in the elimination of pathogens and in the pathogenesis of acute GvHD. 6-Sulfo LacNAc DCs (slanDCs) are a major subpopulation of human blood DCs with a high proinflammatory capacity. We investigated for the first time the reconstitution of slanDCs in the blood of patients after SCT and the modulation of their frequency by bacterial infection, cytomegalovirus (CMV) reactivation, and acute GvHD. The frequency of slanDCs, CD1c myeloid DCs (mDCs), and plasmacytoid DCs (pDCs) in the peripheral blood was quantified by flow cytometry in 80 patients after SCT. To assess individual DC subsets, we used pregating of the HLADRLin subset and antibodies against slanDCs, blood DC antigen 1 (CD1c mDCs), and blood DC antigen 2 (pDCs). SlanDCs showed the slowest reconstitution in the first month after SCT compared with CD1c mDCs and pDCs. Interestingly, in the second and third months after SCT, their percentage steadily increased, and slanDCs were the most abundant DC subset. In addition, we observed a markedly reduced frequency of slanDCs in the blood of patients with bacterial infection, CMV reactivation, or severe acute GvHD. Furthermore, slanDCs showed the most prominent reduction after steroid treatment of acute GvHD. These results indicate that SCT-associated complications such as bacterial infection, CMV reactivation, and acute GvHD can significantly modulate the frequency of slanDCs.

Spatiotemporally separated antigen uptake by alveolar dendritic cells and airway presentation to T cells in the lung

Asthma pathogenesis is focused around conducting airways. The reasons for this focus have been unclear because it has not been possible to track the sites and timing of antigen uptake or subsequent antigen presentation to effector T cells. In this study, we use two-photon microscopy of the lung parenchyma and note accumulation of CD11b(+) dendritic cells (DCs) around the airway after allergen challenge but very limited access of these airway-adjacent DCs to the contents of the airspace. In contrast, we observed prevalent transepithelial uptake of particulate antigens by alveolar DCs. These distinct sites are temporally linked, as early antigen uptake in alveoli gives rise to DC and antigen retention in the airway-adjacent region. Antigen-specific T cells also accumulate in the airway-adjacent region after allergen challenge and are activated by the accumulated DCs. Thus, we propose that later airway hyperreactivity results from selective retention of allergen-presenting DCs and antigen-specific T cells in airway-adjacent interaction zones, not from variation in the abilities of individual DCs to survey the lung.