Human Antigen-presenting Cells Research Articles

The modulation of cathepsin activities by HIV protease inhibitors alters the processing and cross-presentation of pathogens

Abstract Immune recognition by T cells relies on the presentation of pathogen-derived peptides by infected cells but the persistence of chronic infections calls for new approaches to modulate immune recognition. Pathogen uptake in phagosomes initiates infection of macrophages and leads to the priming of T cell responses by dendritic cells. The original degradation of antigens is performed by pH-dependent endolysosomal cathepsins. We show that HIV protease inhibitors (PIs) prescribed to HIV-infected persons variably modulate cathepsin hydrolytic activities in human antigen presenting cells (APCs), dendritic cells and macrophages, and CD4 T cells, three cell subsets infected by HIV. Two HIV PIs acted in two complementary ways on cathepsin activities: They directly enhanced procathepsin maturation and increased cathepsin hydrolytic activities. They indirectly altered regulators of cathepsin functions by inhibiting Akt kinase activities, reducing NADPH oxidase 2 (NOX2) activation, lowering phagolysosomal ROS production and pH, which altogether led to enhanced cathepsin activities. HIV PIs modified endolysosomal degradation and epitope production of proteins from HIV and other pathogens in a sequence-dependent manner. They altered cross-presentation of antigens by dendritic cells and epitope-specific T cell-mediated killing by up to 2.5-fold. HIV PI-induced modulation of antigen processing partly changed the self MHC-peptidome displayed by primary human cells. This first identification of prescription drugs modifying the regulation of cathepsin activities and the MHC-peptidome may provide an alternate therapeutic approach to modulate immune recognition in immune disease beyond HIV.

A novel Flt3-deficient HIS mouse model with selective enhancement of human DC development.

Humanized mice harboring human immune systems (HIS) represent a platform to study immune responses against pathogens and to screen vaccine candidates and novel immunotherapeutics. Innate and adaptive immune responses are suboptimal in HIS mice, possibly due to poor reconstitution of human antigen-presenting cells, including dendritic cells (DCs). DC homeostasis is regulated by cytokine availability, and Flt3-ligand (Flt3L) is one factor that conditions this process. Mouse myelopoiesis is essentially normal in most current HIS models. As such, developing mouse myeloid cells may limit human DC reconstitution by reducing available Flt3L and by cellular competition for specific "niches." To address these issues, we created a novel HIS model that compromises host myeloid cell development via deficiency in the receptor tyrosine kinase Flk2/Flt3. In Balb/c Rag2(-/-) Il2rg(-/-) Flt3(-/-) (BRGF) recipients, human conventional DCs and plasmacytoid DCs develop from hCD34(+) precursors and can be specifically boosted with exogenous Flt3L. Human DCs that develop in this context normally respond to TLR stimulation, and improved human DC homeostasis is associated with increased numbers of human NK and T cells. This new HIS-DC model should provide a means to dissect human DC differentiation and represents a novel platform to screen immune adjuvants and DC targeting therapies.

A novel approach for reliable detection of cathepsin S activities in mouse antigen presenting cells

Cathepsin S (CTSS) is a eukaryotic protease mostly expressed in professional antigen presenting cells (APCs). Since CTSS activity regulation plays a role in the pathogenesis of various autoimmune diseases like multiple sclerosis, atherosclerosis, Sjögren's syndrome and psoriasis as well as in cancer progression, there is an ongoing interest in the reliable detection of cathepsin S activity. Various applications have been invented for specific detection of this enzyme. However, most of them have only been shown to be suitable for human samples, do not deliver quantitative results or the experimental procedure requires technical equipment that is not commonly available in a standard laboratory. We have tested a fluorogen substrate, Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH2, that has been described to specifically detect CTSS activities in human APCs for its potential use for mouse samples. We have modified the protocol and thereby offer a cheap, easy, reproducible and quick activity assay to detect CTSS activities in mouse APCs. Since most of basic research on CTSS is performed in mice, this method closes a gap and offers a possibility for reliable and quantitative CTSS activity detection that can be performed in almost every laboratory.

Protein Nanoclustering as Funcional Unit of Immune Cells

Organization by compartmentalization is a general property of natural systems that efficiently facilitates and orchestrates biological events in space and time. In the last decade, compartmentalization of the plasma membrane of living cells has emerged as a dominant feature present at different spatiotemporal scales and regulating key functions of immune cells. I will discuss two important glycoproteins expressed on the surface of various human antigen-presenting cells (APCs), those spatiotemporal organization regulate their function: DC-SIGN and CD1d. DC-SIGN is a pathogen recognition receptor expressed on dendritic cells playing a decisive role in stimulation of different immune responses. Using a combination of multi-color single particle tracking and super-resolution microscopy approaches we investigated the effect of DC-SIGN molecular structure on the formation DC-SIGN nanoclusters and their dynamic interaction with other molecular components of the cell membrane, including the glycocalyx matrix, lipid nanodomains and clathrin coated pits. We find that DC-SIGN compartmentalizes at different spatiotemporal scales and that this hierarchical organization is crucial to facilitate its pathogen binding capabilities and clathrin-dependent endocytosis. In the second example, I will focus on CD1d, a non-classical MHC protein, involved in the presentation of lipid antigens to T cells. Using a combination of advanced biophysical techniques we found that CD1d molecules organize in nanoclusters on the membrane of APCs. We further discover that the actin cytoskeleton prevents enhanced CD1d nanoclustering by hindering physical encountering between CD1d diffusing nanoclusters, reducing basal activation of invariant natural killer T (iNKT) cells. Our results indicate that regulation of CD1d nanoclustering through the actin cytoskeleton constitutes a novel mechanism to fine-tune peripheral iNKT cell autoreactivity.

Uptake of Chitosan-Based Viscoelastic Hydrogel Particles by Antigen Presenting Cells and Activation of Innate Immune Responses

The chitin-derived biopolymer chitosan has been shown to possess immunostimulatory properties in several systems and has rendered interest as a candidate adjuvant in vaccine formulations. The mechanisms underlying this effect are not completely understood at the cellular and molecular level, but activation of inflammasome and caspase-1 in antigen presenting cells (APC) was recently suggested to be vital for immune activation. In this study, chitosan-based viscoelastic hydrogel particles of two sizes, 10 μm and 200 μm, were evaluated in regard to cellular uptake and activation of APCs. Macrophages derived from the human cell line THP-1 were shown by flow cytometry and confocal laser scanning microscopy to take up FITC-labelled chitosan particles of both sizes via an active process that could be inhibited by cytochalasin D. To investigate if the viscoelastic chitosan particles cause inflammasome activation, NFκB and IL-1β was measured in THP-1 derived macrophages after 24 h incubation with the chitosan particles with or without priming of the cells with LPS. We found that chitosan particles of both sizes stimulated upregulation of NFκB and IL-1β in the absence of LPS. Finally the dependence of this effect on inflammasome-mediated activation of caspase-1 was assessed. Active caspase-1 was not detected in THP-1- derived macrophages stimulated with chitosan-based viscoelastic hydrogel particles, neither alone nor in combination with LPS. In conclusion, we show that viscoelastic chitosan particles in the size range of 10-200 μm, are taken up by human APCs. Moreover, our study suggests that the chitosan particles stimulate NFκB upregulation and IL-1β secretion through inflammasome activation via a caspase-1 independent pathway.

Phenotypic and Functional Properties of Human Steady State CD14+ and CD1a+ Antigen Presenting Cells and Epidermal Langerhans Cells.

Cutaneous antigen presenting cells (APCs) are critical for the induction and regulation of skin immune responses. The human skin contains phenotypically and functionally distinct APCs subsets that are present at two separated locations. While CD1ahigh LCs form a dense network in the epidermis, the CD14+ and CD1a+ APCs reside in the dermal compartment. A better understanding of the biology of human skin APC subsets is necessary for the improvement of vaccine strategies that use the skin as administration route. In particular, progress in the characterization of uptake and activatory receptors will certainly improve APC-targeting strategies in vaccination. Here we performed a detailed analysis of the expression and function of glycan-binding and pattern-recognition receptors in skin APC subsets. The results demonstrate that under steady state conditions human CD1a+ dermal dendritic cells (DCs) were phenotypically most mature as measured by the expression of CD83 and CD86, whereas the CD14+ cells showed a higher expression of the CLRs DC-SIGN, mannose receptor and DCIR and had potent antigen uptake capacity. Furthermore, steady state LCs showed superior antigen cross-presentation as compared to the dermal APC subsets. Our results also demonstrate that the TLR3 ligand polyribosinic-polyribocytidylic acid (pI:C) was the most potent stimulator of cytokine production by both LCs and dDCs. These studies warrant further exploration of human CD1a+ dDCs and LCs as target cells for cancer vaccination to induce anti-tumor immune responses.

Multiparametric Characterization of Human T-Cell Immune Synapses by InFlow Microscopy.

Immune cells need to communicate with each other via direct cell contact formation. The contact zone has similar functions as a neuronal synapse and is therefore named immune synapse. Supramolecular activation clusters consisting of a variety of surface receptors and cytoplasmic proteins are formed within the immune synapse, which are pivotal for T-cell activation. Thus, a malfunction of immune synapse formation has detrimental effects on the healthiness of the individual.Classical confocal microscopy to analyze the supramolecular cluster formation and maturation of the immune synapse between primary human T-cells and antigen-presenting cells is time consuming and the number of cells that can be analyzed is limited. Therefore, we have established an InFlow microscopy approach for the analysis of immune synapses. InFlow microscopy is a hybrid method combining fluorescence microscopy and flow cytometry. Our InFlow microscopy method allows quantifying protein distribution in immune synapses of several hundred or even thousand cell couples in one sample. Importantly, comparisons of different samples with a strong statistical power are possible with InFlow microcopy.

ID: 240: HCMV infected human monocyte-derived macrophages and DC produce type I IFN in a cGAS-dependent manner

The majority of the human population is latently infected with human cytomegalovirus (HCMV). In the immunocompromised host HCMV reactivation can lead to serious symptoms and vertical infection can cause severe disabilities in the infant. We addressed the susceptibility of primary human antigen presenting cells to HCMV infection and their capacity to mount type I interferon (IFN-I) responses. HCMV treated plasmacytoid dendritic cells (pDC) did not efficiently support viral gene expression, while abundant to intermediate viral gene expression was detected in M-CSF macrophages (M-CSF M Φ ) , monocyte-derived DC (moDC), and GM-CSF macrophages (GM-CSF M Φ ) in descending order. Even though HCMV encodes multiple evasion mechanisms, several of which exploit the interferon system, the magnitude of IFN-I responses mounted by the different monocyte-derived subsets correlated with the degree of infection. In contrast, pDC that were largely resistant to HCMV infection showed the highest IFN-I expression. These data implied that pDC sensed HCMV in a different manner than monocyte-derived cells. Since microbial dsDNA can activate cyclic GMP/AMP synthase (cGAS) to produce cyclic GMP-AMP dinucleotides (cGAMP), which trigger the stimulator of interferon genes (STING) that in turn induces IFN-I, we analyzed cGAS-dependent IFN-I responses in pDC, moDC, GM-CSF M Φ , and M-CSF M Φ . We found that upon HCMV infection monocyte-derived macrophages and DC, but not pDC, showed enhanced cGAMP production as a measure of cGAS activity. Moreover, monocyte-derived cells devoid of cGAS showed impaired IFN-I responses. Thus, HCMV stimulated primary monocyte-derived macrophages and DC, but not pDC, showed cGAS dependent cGAMP formation that triggered IFN-I.

Flow cytometric gating for spleen monocyte and DC subsets: differences in autoimmune NOD mice and with acute inflammation

The role of antigen presenting cells (APCs) in the pathogenesis of autoimmune and other inflammatory diseases is now better understood due to advances in multicolor flow cytometry, gene expression analysis of APC populations, and functional correlation of mouse to human APC populations. A simple but informative nomenclature of conventional and plasmacytoid dendritic cell subsets (cDC1, cDC2, pDC) and monocyte-derived populations incorporates these advances, but accurate subset identification is critical. Ambiguous gating schemes and alterations of cell surface markers in inflammatory condition can make comparing results between studies difficult. Both acute inflammation, such as TLR–ligand stimulation, and chronic inflammation as found in mouse models of autoimmunity can alter DC subset gating. Here, we address these issues using in vivo CpG stimulation as an example of acute inflammation and the non-obese diabetic (NOD) mouse as a model of chronic inflammation.We provide a flow cytometric antibody panel and gating scheme that differentiate 2 monocytic and 3DC subsets in the spleen both at steady state and after CpG stimulation. Using this method, we observed differences in the composition of NOD DCs that have been previously reported, and newly identified increases in the number of NOD monocyte-derived DCs. Finally, we established a protocol for DC phosphoflow to measure the phosphorylation state of intracellular proteins, and use it to confirm functional differences in the identified subsets. Therefore, we present optimized methods for distinguishing monocytic and DC populations with and without inflammation and/or autoimmunity associated with NOD mice.

MGL ligand expression is correlated to BRAF mutation and associated with poor survival of stage III colon cancer patients.

Colorectal cancer (CRC) is the third most prevalent cancer type worldwide with a mortality rate of approximately 50%. Elevated cell-surface expression of truncated carbohydrate structures such as Tn antigen (GalNAcα-Ser/Thr) is frequently observed during tumor progression. We have previously demonstrated that the C-type lectin macrophage galactose-type lectin (MGL), expressed by human antigen presenting cells, can distinguish healthy tissue from CRC through its specific recognition of Tn antigen. Both MGL binding and oncogenic BRAF mutations have been implicated in establishing an immunosuppressive microenvironment. Here we aimed to evaluate whether MGL ligand expression has prognostic value and whether this was correlated to BRAF(V600E) mutation status. Using a cohort of 386 colon cancer patients we demonstrate that high MGL binding to stage III tumors is associated with poor disease-free survival, independent of microsatellite instability or adjuvant chemotherapy. In vitro studies using CRC cell lines showed an association between MGL ligand expression and the presence of BRAF(V600E). Administration of specific BRAF(V600E) inhibitors resulted in decreased expression of MGL-binding glycans. Moreover, a positive correlation between induction of BRAF(V600E) and MGL binding to epithelial cells of the gastrointestinal tract was found in vivo using an inducible BRAF(V600E) mouse model. We conclude that the BRAF(V600E) mutation induces MGL ligand expression, thereby providing a direct link between oncogenic transformation and aberrant expression of immunosuppressive glycans. The strong prognostic value of MGL ligands in stage III colon cancer patients, i.e. when tumor cells disseminate to lymph nodes, further supports the putative immune evasive role of MGL ligands in metastatic disease.

In vitro and in vivo imaging of initial B-T-cell interactions in the setting of B-cell based cancer immunotherapy

There has been a growing interest in the use of B cells for cancer vaccines, since they have yielded promising results in preclinical animal models. Contrary to dendritic cells (DCs), we know little about the migration behavior of B cells in vivo. Therefore, we investigated the interactions between CD40-activated B (CD40B) cells and cytotoxic T cells in vitro and the migration behavior of CD40B cells in vivo. Dynamic interactions of human antigen-presenting cells (APCs) and T cells were observed by time-lapse video microscopy. The migratory and chemoattractant potential of CD40B cells was analyzed in vitro and in vivo using flow cytometry, standard transwell migration assays, and imaging of fluorescently labeled murine CD40B cells. Murine CD40B cells show migratory features similar to human CD40B cells. They express important lymph node homing receptors which were functional and induced chemotaxis of T cells in vitro. Striking differences were observed with regard to interactions of human APCs with T cells. CD40B cells differ from DCs by displaying a rapid migratory pattern undergoing highly dynamic, short-lived and sequential interactions with T cells. In vivo, CD40B cells are home to the secondary lymphoid organs where they accumulate in the B cell zone before traveling to the B/T cell boundary. Moreover, intravenous (i.v.) administration of murine CD40B cells induced an antigen-specific cytotoxic T cell response. Taken together, this data show that CD40B cells home secondary lymphoid organs where they physically interact with T cells to induce antigen-specific T cell responses, thus underscoring their potential as cellular adjuvant for cancer immunotherapy.

A new TLR2 agonist promotes cross-presentation by mouse and human antigen presenting cells

Cross-presentation is the process by which professional APCs load peptides from an extracellularly derived protein onto class I MHC molecules to trigger a CD8+ T cell response. The ability to enhance this process is therefore relevant for the development of antitumor and antiviral vaccines. We investigated a new TLR2-based adjuvant, Small Molecule Immune Potentiator (SMIP) 2.1, for its ability to stimulate cross-presentation. Using OVA as model antigen, we demonstrated that a SMIP2.1-adjuvanted vaccine formulation induced a greater CD8+ T cell response, in terms of proliferation, cytokine production and cytolytic activity, than a non-adjuvanted vaccine. Moreover, using an OVA-expressing tumor model, we showed that the CTLs induced by the SMIP2.1 formulated vaccine inhibits tumor growth in vivo. Using a BCR transgenic mouse model we found that B cells could cross-present the OVA antigen when stimulated with SMIP2.1. We also used a flow cytometry assay to detect activation of human CD8+ T cells isolated from human PBMCs of cytomegalovirus-seropositive donors. Stimulation with SMIP2.1 increased the capacity of human APCs, pulsed in vitro with the pp65 CMV protein, to activate CMV-specific CD8+ T cells. Therefore, vaccination with an exogenous antigen formulated with SMIP2.1 is a successful strategy for the induction of a cytotoxic T cell response along with antibody production.

The stimulatory effect of the TLR4-mediated adjuvant glucopyranosyl lipid A is well preserved in old age

Many subunit vaccines require adjuvants to improve their limited immunogenicity. Various adjuvant candidates targeting toll-like receptors (TLRs) are currently under development including the synthetic TLR4 agonist glucopyranosyl lipid A (GLA). GLA has been investigated in the context of influenza vaccine, which is of particular importance for the elderly population. This study investigates the effect of GLA on antigen-presenting cells from young (median age 29years, range 26-33years) and older (median age 72years, range 61-78years) adults. Treatment with GLA efficiently increases the expression of co-stimulatory molecules on human monocyte-derived dendritic cells (DC)as well as on ex vivo myeloid DC. Expression of co-stimulatory molecules is less pronounced on ex vivo monocytes. Production of pro-inflammatory cytokines (IL-6, TNF-α, IL-12) as well as of the anti-inflammatory cytokine IL-10 is induced in monocyte-derived DC. In PBMC cultures myeloid DC and to an even greater extent monocytes produce TNF-α and IL-6 after stimulation with GLA. Production of IL-12 can also be observed in these cultures. There are no age-related differences in the capacity of GLA to induce expression of co-stimulatory molecules or production of cytokines by human antigen-presenting cells. Therefore, TLR4 agonists like GLA are particularly promising candidates as adjuvants of vaccines designed for elderly individuals.

Human Primary Immune Cells Exhibit Distinct Mechanical Properties that Are Modified by Inflammation

T lymphocytes are key modulators of the immune response. Their activation requires cell-cell interaction with different myeloid cell populations of the immune system called antigen-presenting cells (APCs). Although T lymphocytes have recently been shown to respond to mechanical cues, in particular to the stiffness of their environment, little is known about the rigidity of APCs. In this study, single-cell microplate assays were performed to measure the viscoelastic moduli of different human myeloid primary APCs, i.e., monocytes (Ms, storage modulus of 520 +90/−80 Pa), dendritic cells (DCs, 440 +110/−90 Pa), and macrophages (MPHs, 900 +110/−100 Pa). Inflammatory conditions modulated these properties, with storage moduli ranging from 190 Pa to 1450 Pa. The effect of inflammation on the mechanical properties was independent of the induction of expression of commonly used APC maturation markers, making myeloid APC rigidity an additional feature of inflammation. In addition, the rigidity of human T lymphocytes was lower than that of all myeloid cells tested and among the lowest reported (Young’s modulus of 85 ± 5 Pa). Finally, the viscoelastic properties of myeloid cells were dependent on both their filamentous actin content and myosin IIA activity, although the relative contribution of these parameters varied within cell types. These results indicate that T lymphocytes face different cell rigidities when interacting with myeloid APCs in vivo and that this mechanical landscape changes under inflammation.

Characterization of Peroxidases Expressed in Human Antigen Presenting Cells and Analysis of the Covalent Binding of Nitroso Sulfamethoxazole to Myeloperoxidase.

Drug hypersensitivity remains a major concern, as it causes high morbidity and mortality. Understanding the mechanistic basis of drug hypersensitivity is complicated by the multiple risk factors implicated. This study utilized sulfamethoxazole (SMX) as a model drug to (1) relate SMX metabolism in antigen presenting cells (APCs) to the activation of T-cells and (2) characterize covalent adducts of SMX and myeloperoxidase, which might represent antigenic determinants for T-cells. The SMX metabolite nitroso-SMX (SMX-NO) was found to bind irreversibly to APCs. Time- and concentration-dependent drug-protein adducts were also detected when APCs were cultured with SMX. Metabolic activation of SMX was significantly reduced by the oxygenase/peroxidase inhibitor methimazole. Similarly, SMX-NO-specific T-cells were activated by APCs pulsed with SMX, and the response was inhibited by pretreatment with methimazole or glutaraldehyde, which blocks antigen processing. Western blotting, real-time polymerase chain reaction (RT-PCR), and mass spectrometry analyses suggested the presence of low concentrations of myeloperoxidase in APCs. RT-PCR revealed mRNA expression for flavin-containing monooxygenases (FMO1-5), thyroid peroxidase, and lactoperoxidase, but the corresponding proteins were not detected. Mass spectrometric characterization of SMX-NO-modified myeloperoxidase revealed the formation of N-hydroxysulfinamide adducts on Cys309 and Cys398. These data show that SMX's metabolism in APCs generates antigenic determinants for T-cells. Peptides derived from SMX-NO-modified myeloperoxidase may represent one form of functional antigen.

Convergent chemo-enzymatic synthesis of mannosylated glycopeptides; targeting of putative vaccine candidates to antigen presenting cells.

The combination of solid phase peptide synthesis and endo-β-N-acetylglucosaminidase (ENGase) catalysed glycosylation is a powerful convergent synthetic method allowing access to glycopeptides bearing full-length N-glycan structures. Mannose-terminated N-glycan oligosaccharides, produced by either total or semi-synthesis, were converted into oxazoline donor substrates. A peptide from the human cytomegalovirus (CMV) tegument protein pp65 that incorporates a well-characterised T cell epitope, containing N-acetylglucosamine at specific Asn residues, was accessed by solid phase peptide synthesis, and used as an acceptor substrate. High-yielding enzymatic glycosylation afforded glycopeptides bearing defined homogeneous high-mannose N-glycan structures. These high-mannose containing glycopeptides were tested for enhanced targeting to human antigen presenting cells (APCs), putatively mediated via the mannose receptor, and for processing by the APCs for presentation to human CD8+ T cells specific for a 9-mer epitope within the peptide. Binding assays showed increased binding of glycopeptides to APCs compared to the non-glycosylated control. Glycopeptides bearing high-mannose N-glycan structures at a single site outside the T cell epitope were processed and presented by the APCs to allow activation of a T cell clone. However, the addition of a second glycan within the T cell epitope resulted in ablation of T cell activation. We conclude that chemo-enzymatic synthesis of mannosylated glycopeptides enhances uptake by human APCs while preserving the immunogenicity of peptide epitopes within the glycopeptides, provided those epitopes are not themselves glycosylated.

Regulation of the Target Protein (Transgene) Expression in the Adenovirus Vector Using Agonists of Toll-Like Receptors

Replication-defective adenoviral vectors are effective molecular tools for both gene therapy and gene vaccination. Using such vectors one can deliver and express target genes in different epithelial, liver, hematopoietic and immune system cells of animal and human origin. The success of gene therapy and gene vaccination depends on the production intensity of the target protein encoded by the transgene. In this work, we studied influence of Toll-like receptors (TLR) agonists on transduction and expression efficacy of adenoviral vectors in animal and human antigen-presenting cells. We found that agonists of TLR2, 4, 5, 7, 8 and 9 significantly enhance a production of the target protein in cells transduced with adenoviral vector having the target gene insert. The enhancement was observed in dendritic cells and macrophages expressing cytoplasmic (GFP), membrane (HA) or secretory (SEAP) proteins encoded by the respective rAd-vectors. Experiments in mice showed that enhancement of the transgene expression can be achieved in the organism of animals using a pharmaceutical-grade TLR4-agonist. In contrast to other TLR-agonists, the agonist of TLR3 substantially suppressed the expression of transgene in cells transduced with adenoviral vectors having insert of GFP or SEAP target genes. We propose that the enhancement of transgene expression is linked to the activation of MyD88→ NF-kB, while the inhibition of transgene expression depends on TRIF→ IRF signaling pathways. Both of these pathways jointly exploited by TLR4-agonists lead to the enhancement of transgene expression due to the dominant role of the MyD88→ NF-kB signaling.

Intratumoral Inhibition of C5a Using Anti-C5a Monoclonal Antibody Inhibits Lymphoma Progression

Background: The complement system can have both pro-inflammatory and immunosuppressive effects. Our preliminary studies demonstrated that C5a can limit antigen-specific responses, in part, by enhancing the induction of immunosuppressive T regulatory (Treg) cells by primary human antigen-presenting cells and malignant B cells. The current study explored the effect of intratumoral C5a on B lymphoma progression and intratumoral T cells using the A20 murine lymphoma mouse model.Methods:(A) Co-injection approach: Female Balb/C mice were subcutaneously injected with A20 B lymphoma cells mixed with either 20 μg of rat anti-mouse C5a neutralizing monoclonal antibody (mAb) or matching isotype control mAb. Tumor growth was followed and the phenotype of tumor-infiltrating lymphocytes analyzed by flow cytometry.(B) Therapeutic approach: Established subcutaneous A20 tumors (7-9 mm diameter) were treated intratumorally with 20 µg of anti-mouse C5a neutralizing mAb (considered as day 1). A second dose (20 µg) of mAb was given intratumorally on day 5. Control mice were treated similarly with matching isotype control mAb. Tumor growth was followed.Data was analyzed using standard t-test (for tumor growth and immune responses) and Log-rank (Mantel-Cox) test (for comparing survival).Results:(A) Co-injection approach: Co-injection of anti-C5a mAb at the time of tumor cell inoculation significantly delayed A20 tumor growth (Fig. 1; p<0.05) and led to prolonged survival of anti-C5a-treated tumor-bearing mice when compared to the control mice (p=0.001).Tumors from mice that were co-injected with anti-C5a mAb showed significantly higher infiltration of activated CD8+ T cells producing IFNg than the control mice (Fig. 2; p=0.034). Anti-C5a-treated mice also had a lower percentage of circulating Tregs (p=0.023) and splenic Tregs (p=0.0001) compared to control mice.(B) Therapeutic approach: Treatment of pre-established subcutaneous A20 tumors with anti-mouse C5a mAb slowed tumor growth significantly when compared to the control mice (Fig. 3; **p<0.01; *p<0.05). Tumor-bearing mice treated intratumorally with anti-C5a also showed significant improvement in the survival when compared to tumor-bearing mice treated with isotype control Ab (p=0.008).Conclusions:Intratumoral anti-C5a mAb therapy inhibits progression of B lymphoma and improves survival, likely by reducing immunosuppression. We are currently exploring the mechanisms behind C5a-promoted tumor immunosuppression. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

In Vitro and in Vivo Imaging of Initial T-B Cell Interactions in the Setting of B Cell-Based Cancer Immunotherapy

In recent years, there has been a growing interest in the use of B cells for cellular immunotherapy, since B cell-based cancer vaccines have yielded promising results in preclinical animal models. Contrary to dendritic cells (DCs), we know little about the migration behavior of B cells in vivo. Therefore, we investigated the interactions between CD40-activated (CD40) B cells and cytotoxic T cells in vitro and the migration behavior of CD40B cells in vivo.The dynamic interactions of human antigen-presenting cells and antigen-specific T cells were observed by time-lapse videomicroscopy. The migratory and chemoattractant potential of CD40B cells was analyzed by flow cytometry and standard transwell migration assays. GFP+ CD40B cells or CD40B cells isolated from Luciferase+mice were used for subsequent in vivo studies.Murine CD40B cells show similar migratory and chemotactic characteristics compared to human CD40B cells. Upon CD40-activation, B cells upregulate the important molecules involved in lymh node homing (CD62L, CCR7/CDCR4), which are functional and induce chemotaxis of T cells in vitro. Striking differences were observed for interactions of human CD40B cells or DCs with T cells. Antigen-loaded CD40B cells differ from immature and mature DCs by displaying a rapid migratory pattern undergoing highly dynamic, short-lived (7.5 min) and sequential interactions with cognate T cells. In vivo, CD40B cells migrate to the spleen and the lymph nodes, where they enrich in the B cell zone before traveling to B cell/ T cell boundary close to the T cell zone.CD40B cell interactions with T cells are dynamic and short-lived and thereby differ from DCs. Taken together, the migration behavior of CD40B cells and their interaction with T cells underline their potential as cellular adjuvant for cancer immunotherapy. DisclosuresNo relevant conflicts of interest to declare.

Malassezia yeasts activate the NLRP3 inflammasome in antigen-presenting cells via Syk-kinase signalling.

Although being a normal part of the skin flora, yeasts of the genus Malassezia are associated with several common dermatologic conditions including pityriasis versicolour, seborrhoeic dermatitis (SD), folliculitis, atopic eczema/dermatitis (AE/AD) and dandruff. While Malassezia spp. are aetiological agents of pityriasis versicolour, a causal role of Malassezia spp. in AE/AD and SD remains to be established. Previous reports have shown that fungi such as Candida albicans and Aspergillus fumigatus are able to efficiently activate the NLRP3 inflammasome leading to robust secretion of the pro-inflammatory cytokine IL-1β. To date, innate immune responses to Malassezia spp. are not well characterized. Here, we show that different Malassezia species could induce NLRP3 inflammasome activation and subsequent IL-1β secretion in human antigen-presenting cells. In contrast, keratinocytes were not able to secrete IL-1β when exposed to Malassezia spp. Moreover, we demonstrate that IL-1β secretion in antigen-presenting cells was dependent on Syk-kinase signalling. Our results identify Malassezia spp. as potential strong inducers of pro-inflammatory responses when taken up by antigen-presenting cells and identify C-type lectin receptors and the NLRP3 inflammasome as crucial actors in this process.