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European journal of immunology (2022). doi:10.1002/eji.202249816, Published by Wiley-VCH, Weinheim

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Recent studies have provided evidence for an increasing number of phenotypically distinct conventional DC (cDC) subsets that on one hand exhibit a certain functional plasticity, but on the other hand are characterized by their tissue- and context-dependent functional specialization. Here, we describe a selection of assays for the functional characterization of mouse and human cDC. The first two protocols illustrate analysis of cDC endocytosis and metabolism, followed by guidelines for transcriptomic and proteomic characterization of cDC populations. Then, a larger group of assays describes the characterization of cDC migration in vitro, ex vivo, and in vivo. The final guidelines measure cDC inflammasome and antigen (cross)-presentation activity. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Within this article, detailed protocols are presented that allow for the generation of single cell suspensions from human lymphohematopoietic tissues including blood, spleen, thymus, and tonsils with a focus on the subsequent analysis of DC via flow cytometry, as well as flow cytometric cell sorting of primary human DC. Further, prepared single cell suspensions as well as cell sorter-purified DC can be subjected to other applications including cellular enrichment procedures, RNA sequencing, functional assays, and many more. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various nonlymphoid tissues. DC are sentinels of the immune system present in almost every mammalian organ. Since they represent a rare cell population, DC need to be extracted from organs with protocols that are specifically developed for each tissue. This article provides detailed protocols for the preparation of single-cell suspensions from various mouse nonlymphoid tissues, including skin, intestine, lung, kidney, mammary glands, oral mucosa and transplantable tumors. Furthermore, our guidelines include comprehensive protocols for multiplex flow cytometry analysis of DC subsets and feature top tricks for their proper discrimination from other myeloid cells. With this collection, we provide guidelines for in-depth analysis of DC subsets that will advance our understanding of their respective roles in healthy and diseased tissues. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all coauthors, making it an essential resource for basic and clinical DC immunologists.

Dendritic cells (DCs) together with regulatory T cells (Tregs) are essential mediators of immune homeostasis. Disruption of function or frequency of either cell type can lead to fatal autoimmunity. We previously described that mice constitutively lacking DCs (∆DC) develop autoimmunity characterized by reduced body weight, autoantibodies, and pronounced intestinal inflammation. In this study, we show that lack of DCs leads to an altered gene expression profile in peripheral but not thymic Tregs with increased expression of inhibitory receptors. The suppressive function of Tregs from ΔDC mice was impaired in T cell cocultures. In a model of transfer colitis, Tregs from ∆DC mice were only functional in the presence of DCs in recipient mice. Lack of MHC class II on DCs also resulted in upregulation of inhibitory receptors on Tregs, reduced body weight, and elevated serum IgA levels. Further analysis of the IgA response revealed an expansion of IgA+ germinal center B cells and plasma cells in mesenteric lymph nodes and more IgA-coated commensal bacteria in feces of ∆DC mice. Thus, we show a critical role for DCs to establish intestinal homeostasis by regulating Treg function for prevention of spontaneous inflammation.

According to the World Health Organization, cancer is the second most important cause of death in Europe. Due to its manifold manifestations, it is not possible to treat all patients according to a uniform scheme. However, all solid tumors have one thing in common: independent of the tumor’s molecular subgroup and the treatment protocol, the immune status of the tumor, especially the amount of tumor infiltrating lymphocytes (TILs), is important for the patient’s clinical outcome – the higher the number of TILs, the better the outcome. For this reason it seems desirable to increase the number of TILs. One way to accumulate T cells in the tumor area is to make them magnetizable and attract them with an external magnetic field. Magnetization can be achieved by superparamagnetic iron oxide nanoparticles (SPIONs) which can be bound to the cells’ surface or internalized into the cells. For this study, SPIONs with different coatings were synthesized and incubated with immortalized mouse T lymphocytes. SPIONs only stabilized with lauric acid (LA) coated in situ or afterwards showed high toxicity. Addition of an albumin layer increased the biocompatibility but reduced cellular uptake. To increase the cellular uptake the albumin coated particles were aminated, leading to both higher uptake and toxicity, dependent on the degree of amination. In the presence of an externally applied magnetic field, T cells loaded with selected types and amounts of SPIONs were guidable. With this promising pilot study we already can demonstrate that it is possible to attract SPION bearing T cells by an external magnet. To sum up, biocompatibility and uptake of SPIONs by T cells are opposing events. Thus, for the functionalization of T cells with SPIONs the balance between uptake and toxicity must be evaluated carefully.

The detection of microorganisms and danger signals by pattern recognition receptors on dendritic cells (DCs) and the consequent formation of inflammasomes are pivotal for initiating protective immune responses. Although the activation of inflammasomes leading to secretion of the cytokine IL-1β is typically accompanied by pyroptosis (an inflammatory form of lytic programmed cell death), some cells can survive and exist in a state of hyperactivation. Here, we found that the conventional type 2 DC (cDC2) subset is the major human DC subset that is transcriptionally and functionally poised for inflammasome formation and response without pyroptosis. When cDC2 were stimulated with ligands that relatively weakly activated the inflammasome, the cells did not enter pyroptosis but instead secreted IL-12 family cytokines and IL-1β. These cytokines induced prominent T helper type 1 (T

The detection of microorganisms and danger signals by pattern recognition receptors on dendritic cells (DCs) and the consequent formation of inflammasomes are pivotal for initiating protective immune responses. Although the activation of inflammasomes leading to secretion of the cytokine IL-1β is typically accompanied by pyroptosis (an inflammatory form of lytic programmed cell death), some cells can survive and exist in a state of hyperactivation. Here, we found that the conventional type 2 DC (cDC2) subset is the major human DC subset that is transcriptionally and functionally poised for inflammasome formation and response without pyroptosis. When cDC2 were stimulated with ligands that relatively weakly activated the inflammasome, the cells did not enter pyroptosis but instead secreted IL-12 family cytokines and IL-1β. These cytokines induced prominent T helper type 1 (TH1) and TH17 responses that were superior to those seen in response to Toll-like receptor (TLR) stimulation alone or to stronger, classical inflammasome ligands. These findings not only define the human cDC2 subpopulation as a prime target for the treatment of inflammasome-dependent inflammatory diseases but may also inform new approaches for adjuvant and vaccine development.

Allograft-specific regulatory T cells (Trₑg cells) are crucial for long-term graft acceptance after transplantation. Although adoptive Trₑg cell transfer has been proposed, major challenges include graft-specificity and stability. Thus, there is an unmet need for the direct induction of graft-specific Trₑg cells. We hypothesized a synergism of the immunotolerogenic effects of rapamycin (mTOR inhibition) and plerixafor (CXCR4 antagonist) for Trₑg cell induction. Thus, we performed fully-mismatched heart transplantations and found combination treatment to result in prolonged allograft survival. Moreover, fibrosis and myocyte lesions were reduced. Although less CD3⁺ T cell infiltrated, higher Trₑg cell numbers were observed. Noteworthy, this was accompanied by a plerixafor-dependent plasmacytoid dendritic cells-(pDCs)-mobilization. Furthermore, in vivo pDC-depletion abrogated the plerixafor-mediated Trₑg cell number increase and reduced allograft survival. Our pharmacological approach allowed to increase Trₑg cell numbers due to pDC-mediated immune regulation. Therefore pDCs can be an attractive immunotherapeutic target in addition to plerixafor treatment. CA extern

Signal peptide peptidase–like 2a (SPPL2a) is an aspartyl intramembrane protease essential for degradation of the invariant chain CD74. In humans, absence of SPPL2a leads to Mendelian susceptibility to mycobacterial disease, which is attributed to a loss of the dendritic cell (DC) subset conventional DC2. In this study, we confirm depletion of conventional DC2 in lymphatic tissues of SPPL2a−/− mice and demonstrate dependence on CD74 using SPPL2a−/− CD74−/− mice. Upon contact with mycobacteria, SPPL2a−/− bone marrow–derived DCs show enhanced secretion of IL-1β, whereas production of IL-10 and IFN-β is reduced. These effects correlated with modulated responses upon selective stimulation of the pattern recognition receptors TLR4 and Dectin-1. In SPPL2a−/− bone marrow–derived DCs, Dectin-1 is redistributed to endosomal compartments. Thus, SPPL2a deficiency alters pattern recognition receptor pathways in a CD74-dependent way, shifting the balance from anti- to proinflammatory cytokines in antimycobacterial responses. We propose that in addition to the DC reduction, this altered DC functionality contributes to Mendelian susceptibility to mycobacterial disease upon SPPL2a deficiency.

A hallmark of many chronic inflammatory and autoimmune diseases is that there is an impaired resolution of inflammation and return to the steady state. The infusion of high doses of pooled serum IgG preparations from thousands of donors [intravenous immunoglobulin (IVIG) therapy] has been shown to induce resolution of inflammation in a variety of chronic inflammatory and autoimmune diseases, suggesting that IgG molecules can instruct the immune system to stop inflammatory processes and initiate the return to the steady state. The aim of this review is to discuss how insights into the mechanism of IVIG activity may help to understand the molecular and cellular pathways underlying resolution of inflammation. We will put a special emphasis on pathways dependent on the IgG FC domain and IgG sialylation, as several recent studies have provided new insights into how this glycosylation-dependent pathway modulates innate and adaptive immune responses through different sets of C-type or I-type lectins.

Dendritic cells (DCs) orchestrate adaptive immune responses. In healthy individuals, DCs are drivers and fine-tuners of T cell responses directed against invading pathogens or cancer cells. In parallel, DCs control autoreactive T cells, thereby maintaining T cell tolerance. Under various disease conditions, a disruption of this delicate balance can lead to chronic infections, tumor evasion, or autoimmunity. While great efforts have been made to unravel the origin and development of this powerful cell type in mice, only little is known about the ontogeny of human DCs. Here, we summarize the current understanding of the developmental path of DCs from hematopoietic stem cells to fully functional DCs in their local tissue environment and provide a template for the identification of DCs across various tissues.

Dendritic cells (DCs) are major regulators of adaptive immunity, as they are not only capable to induce efficient immune responses, but are also crucial to maintain peripheral tolerance and thereby inhibit autoimmune reactions. DCs bridge the innate and the adaptive immune system by presenting peptides of self and foreign antigens as peptide MHC complexes to T cells. These properties render DCs as interesting target cells for immunomodulatory therapies in cancer, but also autoimmune diseases. Several subsets of DCs with special properties and functions have been described. Recent achievements in understanding transcriptional programs on single cell level, together with the generation of new murine models targeting specific DC subsets, advanced our current understanding of DC development and function. Thus, DCs arise from precursor cells in the bone marrow with distinct progenitor cell populations splitting the monocyte populations and macrophage populations from the DC lineage, which upon lineage commitment can be separated into conventional cDC1, cDC2, and plasmacytoid DCs (pDCs). The DC populations harbor intrinsic programs enabling them to react for specific pathogens in dependency on the DC subset, and thereby orchestrate T cell immune responses. Similarities, but also varieties, between human and murine DC subpopulations are challenging, and will require further investigation of human specimens under consideration of the influence of the tissue micromilieu and DC subset localization in the future.

For magnetic control of cells for biomedical applications such as targeting of immune cells to tumors, cells must be magnetizable. For that, cells are incubated with superparamagnetic iron oxide nanoparticles (SPIONs) to take them up and thus become magnetizable. When using adherent cells, non-ingested SPIONs can be easily removed by rinsing of the particles regardless of their colloidal stability in cell culture medium. However, if suspension cells such as T cells are to be loaded with SPIONs, established methods to separate excess nanoparticles from cells are based on physicochemical parameters such as density, size or magnetizability. Thus, colloidal stability of the particles is of great importance, since only colloidally stable SPIONs can be completely separated from the cells due to their physicochemical differences. Aggregates of colloidally meta- or unstable particles cannot, however, be separated from cells due to their overlapping sizes and densities. Thus, development of an alternative method for the separation of nanoparticle aggregates from suspension cells is urgently needed. Here, we present an affinity chromatographic separation method based on immunohistochemical properties of the respective cells. A desthiobiotinylated antibody against a cellular surface antigen (here CD90.2 receptor on EL4 T cells) is immobilized on a streptavidin agarose column optimized for cell purification. Subsequently the column is loaded with the particle/cell suspension so that the cells bind to the column. After removing the particles by washing, the cells can be gently eluted with biotin solution under physiological conditions. This allows >95% of the excess iron concentration to be removed while maintaining cell viability.

Dendritic cells (DCs) are major regulators of adaptive immunity, as they are not only capable to induce efficient immune responses, but are also crucial to maintain peripheral tolerance and thereby inhibit autoimmune reactions. DCs bridge the innate and the adaptive immune system by presenting peptides of self and foreign antigens as peptide MHC complexes to T cells. These properties render DCs as interesting target cells for immunomodulatory therapies in cancer, but also autoimmune diseases. Several subsets of DCs with special properties and functions have been described. Recent achievements in understanding transcriptional programs on single cell level, together with the generation of new murine models targeting specific DC subsets, advanced our current understanding of DC development and function. Thus, DCs arise from precursor cells in the bone marrow with distinct progenitor cell populations splitting the monocyte populations and macrophage populations from the DC lineage, which upon lineage commitment can be separated into conventional cDC1, cDC2, and plasmacytoid DCs (pDCs). The DC populations harbor intrinsic programs enabling them to react for specific pathogens in dependency on the DC subset, and thereby orchestrate T cell immune responses. Similarities, but also varieties, between human and murine DC subpopulations are challenging, and will require further investigation of human specimens under consideration of the influence of the tissue micromilieu and DC subset localization in the future.

Background Mast cells (MCs) are best known as key effector cells of allergic reactions, but they also play an important role in host defense against pathogens. Despite increasing evidence for a critical effect of MCs on adaptive immunity, the underlying mechanisms are poorly understood. Objective Here we monitored MC intercellular communication with dendritic cells (DCs), MC activation, and degranulation and tracked the fate of exocytosed mast cell granules (MCGs) during skin inflammation. Methods Using a strategy to stain intracellular MCGs in vivo, we tracked the MCG fate after skin inflammation–induced MC degranulation. Furthermore, exogenous MCGs were applied to MC-deficient mice by means of intradermal injection. MCG effects on DC functionality and adaptive immune responses in vivo were assessed by combining intravital multiphoton microscopy with flow cytometry and functional assays. Results We demonstrate that dermal DCs engulf the intact granules exocytosed by MCs on skin inflammation. Subsequently, the engulfed MCGs are actively shuttled to skin-draining lymph nodes and finally degraded inside DCs within the lymphoid tissue. Most importantly, MCG uptake promotes DC maturation and migration to skin-draining lymph nodes, partially through MC-derived TNF, and boosts their T-cell priming efficiency. Surprisingly, exogenous MCGs alone are sufficient to induce a prominent DC activation and T-cell response. Conclusion Our study highlights a unique feature of peripheral MCs to affect lymphoid tissue–borne adaptive immunity over distance by modifying DC functionality through delivery of granule-stored mediators.

Dendritic cells (DCs) are major players for the induction of immune responses. Apart from plasmacytoid DCs (pDCs), human DCs can be categorized into two types of conventional DCs: CD141+ DCs (cDC1) and CD1c+ DCs (cDC2). Defining uniquely expressed surface markers on human immune cells is not only important for the identification of DC subpopulations but also a prerequisite for harnessing the DC subset-specific potential in immunomodulatory approaches, such as antibody-mediated antigen targeting. Although others identified CLEC9A as a specific endocytic receptor for CD141+ DCs, such a receptor for CD1c+ DCs has not been discovered, yet. By performing transcriptomic and flow cytometric analyses on human DC subpopulations from different lymphohematopoietic tissues, we identified CLEC10A (CD301, macrophage galactose-type C-type lectin) as a specific marker for human CD1c+ DCs. We further demonstrate that CLEC10A rapidly internalizes into human CD1c+ DCs upon binding of a monoclonal antibody directed against CLEC10A. The binding of a CLEC10A-specific bivalent ligand (the MUC-1 peptide glycosylated with N-acetylgalactosamine) is limited to CD1c+ DCs and enhances the cytokine secretion (namely TNFα, IL-8, and IL-10) induced by TLR 7/8 stimulation. Thus, CLEC10A represents not only a candidate to better define CD1c+ DCs-due to its high endocytic potential-CLEC10A also exhibits an interesting candidate receptor for future antigen-targeting approaches.

Endogenous nucleic acids and their receptors may be involved in the initiation of systemic autoimmune diseases including rheumatoid arthritis (RA). As the role of the DNA sensing Toll‐like receptor (TLR) 9 in RA is unclear, we aimed to investigate its involvement in the pathogenesis of autoimmune arthritis using three different experimental models of RA. The data obtained revealed involvement of TLR9 in the T cell‐dependent phase of inflammatory arthritis. In rats with pristane‐induced arthritis (PIA), TLR9 inhibition before disease onset reduced arthritis significantly and almost completely abolished bone erosion. Accordingly, serum levels of IL‐6, α‐1‐acid‐glycoprotein and rheumatoid factor were reduced. Moreover, in TLR9−/− mice, streptococcal cell wall (SCW)‐induced arthritis was reduced in the T cell‐dependent phase, whereas T cell‐independent serum‐transfer arthritis was not affected. Remarkably, while TLR7 expression did not change during in vitro osteoclastogenesis, TLR9 expression was higher in precursor cells than in mature osteoclasts and partial inhibition of osteoclastogenesis was achieved only by the TLR9 antagonist. These results demonstrate a pivotal role for TLR9 in the T cell‐dependent phases of inflammatory arthritis and additionally suggest some role during osteoclastogenesis. Hence, endogenous DNA seems to be crucially involved in the pathophysiology of inflammatory autoimmune arthritis.

Despite recent advances in activating immune cells to target tumors, the presence of some immune cells, such as tumor-associated macrophages (TAMs) or tumor-associated neutrophils (TANs), may promote rather than inhibit tumor growth. However, it remains unclear how antibody-dependent tumor immunotherapies, such as cytotoxic or checkpoint control antibodies, affect different TAM or TAN populations, which abundantly express activating Fcγ receptors. In this study, we show that the tissue environment determines which cellular effector pathways are responsible for antibody-dependent tumor immunotherapy. Although TAMs derived from Ly6Chigh monocytes recruited by the CCL2-CCR2 axis were critical for tumor immunotherapy of skin tumors, the destruction of lung tumors was CCL2-independent and required the presence of colony-stimulating factor 2-dependent tissue-resident macrophages. Our findings suggest that TAMs may have a dual role not only in promoting tumor growth in certain tissue environments on the one hand but also in contributing to tumor cell destruction during antibody-mediated immunotherapy on the other hand.

Lehmann et al. targeted antigens to Fcγ receptors expressed on various antigen-presenting cells. Induced CD4+ and CD8+ T cell responses were solely dependent on CD11b+ and CD8+ DC subsets, respectively, but independent of receptor intrinsic ITAM or ITIM signaling domains., Dendritic cells (DCs) are efficient antigen-presenting cells equipped with various cell surface receptors for the direct or indirect recognition of pathogenic microorganisms. Interestingly, not much is known about the specific expression pattern and function of the individual activating and inhibitory Fcγ receptors (FcγRs) on splenic DC subsets in vivo and how they contribute to the initiation of T cell responses. By targeting antigens to select activating and the inhibitory FcγR in vivo, we show that antigen uptake under steady-state conditions results in a short-term expansion of antigen-specific T cells, whereas under inflammatory conditions especially, the activating FcγRIV is able to induce superior CD4+ and CD8+ T cell responses. Of note, this effect was independent of FcγR intrinsic activating signaling pathways. Moreover, despite the expression of FcγRIV on both conventional splenic DC subsets, the induction of CD8+ T cell responses was largely dependent on CD11c+CD8+ DCs, whereas CD11c+CD8− DCs were critical for priming CD4+ T cell responses.

Dendritic cells (DCs) are central modulators of immune responses and, therefore, interesting target cells for the induction of antitumor immune responses. Ag delivery to select DC subpopulations via targeting Abs to DC inhibitory receptor 2 (DCIR2, clone 33D1) or to DEC205 was shown to direct Ags specifically to CD11c+CD8− or CD11c+CD8+ DCs, respectively, in vivo. In contrast to the increasing knowledge about the induction of immune responses by efficiently cross-presenting CD11c+CD8+ DCs, little is known about the functional role of Ag-presenting CD11c+CD8− DCs with regard to the initiation of protective immune responses. In this study, we demonstrate that Ag targeting to the CD11c+CD8− DC subpopulation in the presence of stimulating anti-CD40 Ab and TLR3 ligand polyinosinic-polycytidylic acid induces protective responses against rapidly growing tumor cells in naive animals under preventive and therapeutic treatment regimens in vivo. Of note, this immunization protocol induced a mixed Th1/Th2-driven immune response, irrespective of which DC subpopulation initially presented the Ag. Our results provide important information about the role of CD11c+CD8− DCs, which have been considered to be less efficient at cross-presenting Ags, in the induction of protective antitumor immune responses.

The transcription factor STAT6 plays a key role in mediating signaling downstream of the receptors for IL-4 and IL-13. In B cells, STAT6 is required for class switch recombination to IgE and for germinal center formation during type 2 immune responses directed against allergens or helminths. In this study, we compared the transcriptomes and proteomes of primary mouse B cells from wild-type and STAT6-deficient mice cultured for 4 d in the presence or absence of IL-4. Microarray analysis revealed that 214 mRNAs were upregulated and 149 were downregulated >3-fold by IL-4 in a STAT6-dependent manner. Across all samples, ∼5000 proteins were identified by label-free quantitative liquid chromatography/mass spectrometry. A total of 149 proteins was found to be differentially expressed >3-fold between IL-4–stimulated wild-type and STAT6−/− B cells (75 upregulated and 74 downregulated). Comparative analysis of the proteome and transcriptome revealed that expression of these proteins was mainly regulated at the transcriptional level, which argues against a major role for posttranscriptional mechanisms that modulate the STAT6-dependent proteome. Nine proteins were selected for confirmation by flow cytometry or Western blot. We show that CD30, CD79b, SLP-76, DEC205, IL-5Rα, STAT5, and Thy1 are induced by IL-4 in a STAT6-dependent manner. In contrast, Syk and Fc receptor–like 1 were downregulated. This dataset provides a framework for further functional analysis of newly identified IL-4–regulated proteins in B cells that may contribute to germinal center formation and IgE switching in type 2 immunity.

The invariant chain (CD74) mediates assembly and targeting of MHC class II (MHCII) complexes. In endosomes, CD74 undergoes sequential degradation by different proteases, including cathepsin S (CatS) and the intramembrane protease signal peptide peptidase-like 2a (SPPL2a). In their absence, CD74 N-terminal fragments (NTFs) accumulate. In SPPL2a−/− B cells, such an NTF impairs endosomal trafficking and BCR signal transduction. In mice, this leads to a loss of splenic B cells beyond the transitional stage 1. To gain insight into CD74 determinants and the role of MHCII, we compared B cells from CatS−/−, SPPL2a−/−, and SPPL2a-MHCII double-deficient mice. We assessed differentiation of B cells in bone marrow and spleen and analyzed their endosomal morphology, BCR expression, and signal transduction. We demonstrate that MHCII is dispensable for the B cell phenotype of SPPL2a−/− mice, further supporting a CD74-intrinsic effect. Despite significant vacuolization of endosomal compartments similar to SPPL2a−/− B cells, CatS−/− traditional stage 1 B cells show unimpaired degradation of endocytic cargo, have intact BCR signaling, and do not exhibit any relevant defects in maturation. This could indicate that CD74 NTF–induced structural changes of endosomes are not directly involved in these processes. We further found that the block of CD74 degradation in CatS−/− B cells is incomplete, so that NTF levels are significantly lower than in SPPL2a−/− B cells. This suggests a dose dependency and threshold for the CD74 NTF–associated impairment of B cell signaling and maturation. In addition, different functional properties of the longer, MHCII-bound CD74 NTF could contribute to the milder phenotype of CatS−/− B cells.

International audience; Dendritic cells (DCs) are the most potent professional antigen presenting cells and are therefore indispensable for the control of immunity. The technique of antibody mediated antigen targeting to DC subsets has been the basis of intense research for more than a decade. Many murine studies have utilized this approach of antigen delivery to various kinds of endocytic receptors of DCs both in vitro and in vivo. Today, it is widely accepted that different DC subsets are important for the induction of select immune responses. Nevertheless, many questions still remain to be answered, such as the actual influence of the targeted receptor on the initiation of the immune response to the delivered antigen. Further efforts to better understand the induction of antigen-specific immune responses will support the transfer of this knowledge into novel treatment strategies for human diseases. In this review, we will discuss the state-of-the-art aspects of the basic principles of antibody mediated antigen targeting approaches. A table will also provide a broad overview of the latest studies using antigen targeting including addressed DC subset, targeted receptors, outcome, and applied coupling techniques.

In mice, conventional and plasmacytoid dendritic cells (DCs) derive from separate hematopoietic precursors before they migrate to peripheral tissues. Moreover, two classes of conventional DCs (cDC1 and cDC2 DCs) and one class of plasmacytoid DCs (pDCs) have been shown to be transcriptionally and functionally distinct entities. In humans, these three DC subtypes can be identified using the cell surface markers CD1c (cDC2), CD141 (cDC1), and CD303 (pDCs), albeit it remains elusive whether DC functionality is mainly determined by ontogeny or the tissue microenvironment. By phenotypic and transcriptional profiling of these three DC subtypes in different human tissues derived from a large number of human individuals, we demonstrate that DC subpopulations in organs of the lymphohematopoietic system (spleen, thymus, and blood) are strongly defined by ontogeny rather than by signals from the microenvironment. In contrast, DC subsets derived from human lung or skin differed substantially, strongly arguing that DCs react toward modulatory signals from tissue microenvironments. Collectively, the data obtained in this study may serve as a major resource to guide further studies into human DC biology during homeostasis and inflammation.

Dendritic cells (DCs) are sentinels of the immune system. Under inflammatory conditions DCs undergo various phenotypical changes, including DC migration, upregulation of co-stimulatory and activating molecules, secretion of cytokines, but also the processing of the engulfed antigens. The DC presents short antigenic peptides on the DC surface as peptide MHC class I or MHC class II complexes to CD8 + or CD4 + T cells, respectively. This article will provide a detailed overview on the antigen processing machineries and specialized functions of the increasing variety of DC subsets in mice and men.

Cellular Fcγ receptors are essential for IgG-dependent effector functions in vivo. There is convincing evidence that selective activating Fcγ receptors are responsible for the activity of individual IgG subclasses. Thus, IgG1 activity is absent in FcγRIII-deficient mice, and several studies suggest that the activity of the most potent IgG subclasses, IgG2a and IgG2b, might be dependent on either individual or a combination of activating FcγRs. To study the role of individual activating FcγRs for IgG subclass activity, we generated an FcγRIV-deficient mouse and showed that a variety of IgG2a- and IgG2b-dependent effector functions are impaired in the absence of this activating Fc receptor in models of autoimmunity and antibody-dependent cellular cytotoxicity.

Dendritic cells (DCs) are important antigen presenting cells (APCs) and responsible for the induction of immune responses and preserving peripheral tolerance. We showed that targeting of antigens via C-type lectin receptors to different specialized DC subpopulations induced either CD4+ or CD8+ T cell responses in vivo. Fc receptors are also highly active in endocytosis enabling APCs to take up antigens in form of immune complexes. As they are expressed on various APCs, we aimed to identify responsible APCs for primary and secondary immune responses. Therefore, we assessed their expression in various organs and delivered antigens by specific recombinant antibodies. The targeting of the Fc receptors induced CD4+ and CD8+ T cell responses in a transgenic as well as a naïve system. Moreover, especially antigen delivery to the activating FcγRIV was superior in inducing CD4+ and CD8+ T cell responses at the same time, which could not be observed by classical targeting of the C-type lectin receptors DEC205 or DCIR2. Additionally, targeting of antigens to FcγRIV induced a pronounced CD4 helper response in naïve mice, whereas targeting to DCIR2 was inefficient. As FcγRIV is expressed on both major splenic DC subsets, we used this receptor to verify the subset intrinsic preference of DCs for CD4+ or CD8+ T cell responses. This was clearly shown by the induction of CD4+ T cell responses by splenic CD8− DCs, whereas splenic CD8+ DCs induced a CD8+ T cell response. The induced naïve CD8+ T cell responses were functional relevant, as we could demonstrate efficient dose-dependent killing of target cells in vivo. Therefore, we suggest this strategy as useful tool for the induction of de novo as well as the modulation of immune responses for therapeutic applications.

Mast cells are critical promoters of adaptive immunity in the contact hypersensitivity model, but the mechanism of allergen sensitization is poorly understood. Using Mcpt5-CreTNF(FL/FL) mice, we show here that the absence of TNF exclusively in mast cells impaired the expansion of CD8(+) T cells upon sensitization and the T-cell-driven adaptive immune response to elicitation. T cells primed in the absence of mast cell TNF exhibited a diminished efficiency to transfer sensitization to naive recipients. Specifically, mast cell TNF promotes CD8(+) dendritic cell (DC) maturation and migration to draining lymph nodes. The peripherally released mast cell TNF further critically boosts the CD8(+) T-cell-priming efficiency of CD8(+) DCs, thereby linking mast cell effects on T cells to DC modulation. Collectively, our findings identify the distinct potential of mast cell TNF to amplify CD8(+) DC functionality and CD8(+) T-cell-dominated adaptive immunity, which may be of great importance for immunotherapy and vaccination approaches.

Impaired regulatory T-cell function results in a severe chronic autoimmune disease affecting multiple organs in Scurfy mice and humans with the immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. Previous studies have shown that T helper cells but not cytotoxic T cells are critical for the disease pathology. Whether this T-cell subset is responsible directly for tissue inflammation or rather indirectly via the interaction with B cells or myeloid cells is largely unknown. To study this and to identify potential therapeutic targets for this lethal disease we investigated the contribution of B cells to this complex autoimmune phenotype. We show that B cells and the production of autoantibodies plays a major role for skin, liver, lung, and kidney inflammation and therapeutic depletion of B cells resulted in reduced tissue pathology and in prolonged survival. In contrast, the absence of B cells did not impact systemic T-cell activation and hyperreactivity, indicating that autoantibody production by B cells may be a major factor for the autoimmune pathology in mice deficient for regulatory T cells.

Clusterin/Apolipoprotein J is a protein that is upregulated in a broad spectrum of diverse pathological processes. The predominant form is a secreted glycoprotein (sCLU) with cytoprotective and anti-inflammatory properties which shows enhanced expression in vascular smooth muscle cells (VSMC) following aortic injury and in atherosclerotic disease. Recent evidence indicates that during atherosclerosis, Toll-like receptors (TLRs) are activated in vascular cells by endogenous ligands. Here, we analyzed whether CLU expression in VSMC is controlled by TLRs, and stimulated by factors associated with or released by necrotic cells. Activation of TLR3 by the synthetic RNA analogue polyinosinic-polycytidylic acid (poly(I:C)) in CRL2018 VSMC and in mice led to induction of CLU mRNA and protein synthesis, respectively. In TLR3-deficient 10A yolk sac cells, induction of CLU by poly(I:C) challenge depended on the ectopic expression of human TLR3. In mice lacking the TLR3-signaling adaptor protein TRIF (TIR-domain-containing adaptor protein inducing IFN-β) CLU induction by poly(I:C) was abrogated. In addition to poly(I:C) CLU gene expression in CRL2018 cells was induced by purified cellular RNA and RNA present in necrotic cell lysate. Our data indicate that cellular RNA following its release from necrotic cells in atherosclerotic lesions can act as an endogenous TLR3 ligand to induce CLU expression in VSMC and in vivo. Thus, they expand the view on TLR2 and TLR4 as known pro-atherosclerotic effectors toward TLR3. Conclusively, TLR3 activation induces expression of cytoprotective and anti-inflammatory CLU by VSMC and mice, to potentially counteract atherosclerotic pathology.

Clinical studies investigating the impact of natural killer (NK) cells in allogeneic hematopoietic stem cell transplantation settings have yielded promising results. However, NK cells are a functionally and phenotypically heterogeneous population. Therefore, we addressed the functional relevance of specific NK cell subsets distinguished by expression of CD117, CD27 and CD11b surface markers in graft-versus-leukemia (GVL)-reaction and graft-versus-host-disease (GVHD). Our results clearly demonstrate that the subset of c-Kit-CD27-CD11b+ NK cells expressed multiple cytotoxic pathway genes and provided optimal graft-versus-leukemia-effects, while significantly reducing T cell proliferation induced by allogeneic dendritic cells. Furthermore, these NK cells migrated to inflamed intestinal tissues where graft-versus-host-colitis was efficiently mitigated. For the first time, we identified the c-Kit-CD27-CD11b+ NK cell population as the specific effector NK cell subset capable of significantly diminishing GVHD in fully mismatched bone marrow transplantation settings. In conclusion, the subset of c-Kit-CD27-CD11b+ NK cells not only supports GVL, but also plays a unique role in the protection against GVHD by migrating to the peripheral GVHD target organs where they exert efficient immunoregulatory activities. These new insights demonstrate the importance of selecting the optimal NK cell subset for cellular immunotherapy following allogeneic hematopoietic stem cell transplantation.