CD80+CD205+ splenic dendritic cells are specialized to induce Foxp3+ regulatory T cells

Foxp3+CD25+CD4+ regulatory T cells (Treg) mediate immunological self-tolerance and suppress immune responses. A subset of dendritic cells (DCs) in the intestine is specialized to induce Treg in a TGF-β- and retinoic acid-dependent manner to allow for oral tolerance. In this study we compare two major DC subsets from mouse spleen. We find that CD8+ DEC-205/CD205+ DCs, but not the major fraction of CD8- DC inhibitory receptor-2 (DCIR2)+ DCs, induce functional Foxp3+ Treg from Foxp3- precursors in the presence of low doses of Ag but without added TGF-β. CD8 +CD205+ DCs preferentially express TGF-β, and the induction of Treg by these DCs in vitro is blocked by neutralizing Ab to TGF-β. In contrast, CD8+DCIR2+ DCs better induce Foxp3+ Treg when exogenous TGF-β is supplied. In vivo, CD8 +CD205+ DCs likewise preferentially induce Treg from adoptively transferred, Ag-specific DO11.10 RAG-/- Foxp3 -CD4+ T cells, whereas the CD8-DCIR2 + DCs better stimulate natural Foxp3+ Treg. These results indicate that a subset of DCs in spleen, a systemic lymphoid organ, is specialized to differentiate peripheral Foxp3+ Treg, in part through the endogenous formation of TGF-β. Targeting of Ag to these DCs might be useful for inducing Ag-specific Foxp3+ Treg for treatment of autoimmune diseases, transplant rejection, and allergy

Regulation des Zelloberflächenmoleküls CD83 durch das Epstein-Barr Virus und Analyse seiner Funktion

EBV is a γ-herpes virus which is able to infect human resting B-cells and to transform them into permanently growing lymphoblastoid cell lines (LCLs). EBNA2 (Epstein-Barr virus nuclear antigen 2) is one of the first viral proteins expressed after in vitro infection and interacts with different cellular proteins like RBP-Jκ and PU.1. The EBNA2 protein acts as a transcriptional activator of the viral Latent Membrane Proteins 1 and 2 (LMP1 and LMP2) and the viral nuclear genes EBNA1, EBNA3A, -3B, -3C, EBNA-LP. Additionally EBNA2 is also able to transactivate cellular genes like CD21, CD23 or c-myc. To study the different EBNA2 target genes and the function of EBNA2 a LCL was established (ER/EB2-5 cells, Kempkes et al., 1995) harboring an estrogen-inducible EBNA2. In the presence of estrogen the ER/EBNA2 fusion protein (estrogen receptor binding domain) is located in the nucleus were EBNA2 can transactivate its target genes, whereas in the absence of estrogen the ER/EBNA2 fusion protein is kept in the cytoplasm and therefore inactive. The cells proliferate in the presence of estrogen and they arrest in the absence resulting in a phenotype similar to resting B-lymphocytes. By using the ER/EB2-5 cell line I could clearly show that the cell surface molecule CD83, belonging to the immunoglobuline superfamily (Zhou et al., 1992), is upregulated after the activation of EBNA2. By using a derivative ER/EB2-5 cell line that constitutively expressed LMP1 I could show that CD83 is still expressed even in the absence of functional EBNA2 suggesting that LMP1, the viral target gene of EBNA2, is responsible for the induction of CD83. Therefore I analysed the activation of the CD83 promoter by LMP1. LMP1 is a transmembrane protein with a short intracellular N-terminus, 6 hydrophobic transmembrane domains and a long intracellular C-terminus, containing C-terminal activator regions CTAR1, 2 and 3. The different CTAR regions are responsible for activating genes via NF-κB, ATF, AP1 and STAT signaling pathways. For the activation of its target genes LMP1 uses the same signaling molecules (TRAF, TRADD) as family members of the TNF-R family (CD40, TNF-R1, TNF-R2). The CD83 promoter was activated by LMP1 as shown by promoter luciferase reporter assays in 293-T cells. The induction was not observed in the absence of a NF-κB binding site in a CD83 promoter mutant. Furthermore LMP1 mutants which are mutated in the binding regions for TRAF2 (CTAR1) or TRADD (CTAR2) are not able to transactivate the CD83 promoter. By co-transfection of LMP1 and dominant/negative IκB the CD83 promoter could not be activated because of inactivation of NF-κB. These experiments clearly demonstrate that the CD83 promoter is transactivated by LMP1 via NF-κB. Additionally to the regulation of CD83 I was also interested in the functional role of CD83. Until now only little is known about the function of CD83. CD83 seems to have a specific role in the decision to single positive CD4+ T-cells in the thymus (Fujimoto et al., 2002). I have tested a possible co-stimulatory function of CD83 to CD4+ T-cells by retroviral expression of CD83 in non-professional antigen presenting cells (RCC). Indeed CD83 expression increased the CD4+ response in comparison to CD80 or GFP retroviral infected RCC cells. In mixed lymphocyte reactions this co-stimulatory effect could not be clearly demonstrated although a soluble CD83-Ig showed a small inhibitory influence. The identification of a CD83 ligand molecule could give new insights into the function of CD83. Therefore a CD83-Ig fusion protein as well as a CD83-tetramer construct were generated and used to screen for a potential ligand of CD83. First results showed that the CD83-Ig fusion protein and the CD83-tetramer construct bound to CD4+ and to CD8+ T-cells of isolated PBMCs as well as to activated T-cells in a culture of mixed T-cell populations.Das Epstein-Barr Virus (EBV) infiziert humane ruhende B-Zellen und transformiert diese in vitro zu kontinuierlich wachsenden lymphoblastoiden Zellinien (LCLs). EBNA-2 ist eines der ersten viralen Proteine, die nach Infektion exprimiert werden. Es interagiert mit zellulären Transkriptionsfaktoren wie PU.1 oder RBP-Jκ und induziert dadurch sowohl zelluläre als auch virale Gene wie LMP-1, LMP-2, EBNA-3A, -3B und -3C. Da EBNA-2 in der LCL ER/EB2-5 als Fusionsprodukt mit der Hormonbindungsdomäne des Östrogenrezeptors exprimiert wird, kann die Aktivität von EBNA-2 durch Zugabe und Entzug von Östrogen in diesen Zellen reguliert werden. Nach Entzug von Östrogen werden die Zellen arretiert, was in einem Phänotyp ähnlich dem ruhender B-Zellen resultiert. Die Wiederzugabe von Östrogen induziert die Proliferation und Aktivierung der ER/EB2-5 Zellen mit Expression von Aktivierungsmarkern und Adhäsionsmolekülen wie CD21, CD23, CD39 und CD54. In dieser Arbeit wurde der Frage nachgegangen, ob die Expression des Oberflächenmoleküls CD83, eines Markers für die Reifung dendritischer Zellen und die Aktivierung von Lymphozyten, durch EBV beeinflußt wird. CD83 wird in ER/EB2-5 Zellen nur in Gegenwart von aktivem EBNA-2 (+Östrogen) auf der Oberfläche exprimiert. Es zeigte sich, daß die CD83-Expression sowohl auf RNA- als auch auf Proteinebene reguliert ist. Weiterhin wurde gefunden, daß CD83 nicht direkt über EBNA-2, sondern über das virale latente Membranprotein LMP-1 induziert wird. In Promotor-Reporter-Assays konnte durch Co-Transfektion von verschiedenen CD83-Promotor Luziferase-Konstrukten mit Expressionsplasmiden für LMP-1, LMP-1 Mutanten und den an der LMP-1 Signalübertragung beteiligten Proteine gezeigt werden, daß der CD83-Promotor durch LMP-1 über NF-κB reguliert wird. Der CD83-Promotor wird auch durch Mitglieder der TNF-Rezeptor Familie (CD40, TNF-R1, TNF-R2) in sehr ähnlicher Weise wie durch LMP-1 aktiviert. Über die Funktion von CD83 ist bislang sehr wenig bekannt. Aufgrund seiner Immunglobulin-ähnlichen Domäne und seines Expressionsmusters wird CD83 eine wichtige Rolle in der Antigenpräsentation und/oder den zellulären Interaktionen, die der Lymphozytenaktivierung folgen, zugesprochen. Nach retroviraler Infektion mit CD83 wurde in der nicht professionellen Antigen-präsentierenden Zellinie RCC p18.S bei der Untersuchung einer möglichen co-stimulatorischen Funktion von CD83 eine leichte Aktivierung eines spezifischen CD4+ T-Zellklons beobachtet. In einer gemischten Lymphozytenkultur konnte eine co-stimulatorische Aktivität von CD83 nicht direkt demonstriert werden, obwohl ein lösliches CD83-Ig Konstrukt eine schwache inhibitorische Wirkung zeigte. Ein Hinweis auf die Existenz von löslichen Formen von CD83 wurden auf RNA-Niveau durch Analyse alternativer Spleißprodukte gefunden. Ein weiterer wichtiger Schwerpunkt war die Identifikation einer Zellpopulation, die den Liganden von CD83 exprimiert. Mit Hilfe eines CD83-Ig Fusionsproteins und eines CD83-Tetramer-Konstrukts konnten CD83-Ligand positive Zellen sowohl auf CD4+ als auch auf CD8+ T-Zellen isolierter PBMCs sowie auf dem T-Zellklon KS festgestellt werden. Weiterhin ließ sich eine kleine Population aktivierter Granulozyten mit dem CD83-Tetramer färben. Diese Ergebnisse bieten gute Voraussetzungen für die Identifikation eines Liganden und zur Aufklärung der Funktion von CD83

The Extracellular Domain of CD83 Inhibits Dendritic Cell–mediated T Cell Stimulation and Binds to a Ligand on Dendritic Cells

CD83 is an immunoglobulin (Ig) superfamily member that is upregulated during the maturation of dendritic cells (DCs). It has been widely used as a marker for mature DCs, but its function is still unknown. To approach its potential functional role, we have expressed the extracellular Ig domain of human CD83 (hCD83ext) as a soluble protein. Using this tool we could show that immature as well as mature DCs bind to CD83. Since CD83 binds a ligand also expressed on immature DCs, which do not express CD83, indicates that binding is not a homophilic interaction. In addition we demonstrate that hCD83ext interferes with DC maturation downmodulating the expression of CD80 and CD83, while no phenotypical effects were observed on T cells. Finally, we show that hCD83ext inhibits DC-dependent allogeneic and peptide-specific T cell proliferation in a concentration dependent manner in vitro. This is the first report regarding functional aspects of CD83 and the binding of CD83 to DCs

A subset of dendritic cells induces CD4+ T cells to produce IFN-γ by an IL-12–independent but CD70-dependent mechanism in vivo

Interferon (IFN)-γ, a cytokine critical for resistance to infection and tumors, is produced by CD4+ helper T lymphocytes after stimulation by cultured dendritic cells (DCs) that secrete a cofactor, interleukin (IL)-12. We have identified a major IL-12–independent pathway whereby DCs induce IFN-γ–secreting T helper (Th)1 CD4+ T cells in vivo. This pathway requires the membrane-associated tumor necrosis family member CD70 and was identified by targeting the LACK antigen from Leishmania major within an antibody to CD205 (DEC-205), an uptake receptor on a subset of DCs. Another major DC subset, targeted with 33D1 anti-DCIR2 antibody, also induced IFN-γ in vivo but required IL-12, not CD70. Isolated CD205+ DCs expressed cell surface CD70 when presenting antigen to T cell receptor transgenic T cells, and this distinction was independent of maturation stimuli. CD70 was also essential for CD205+ DC function in vivo. Detection of the IL-12–independent IFN-γ pathway was obscured with nontargeted LACK, which was presented by both DC subsets. This in situ analysis points to CD70 as a decision maker for Th1 differentiation by CD205+ DCs, even in Th2-prone BALB/c animals and potentially in vaccine design. The results indicate that two DC subsets have innate propensities to differentially affect the Th1/Th2 balance in vivo and by distinct mechanisms

Harnessing the Complete Repertoire of Conventional Dendritic Cell Functions for Cancer Immunotherapy

The onset of checkpoint inhibition revolutionized the treatment of cancer. However, studies from the last decade suggested that the sole enhancement of T cell functionality might not suffice to fight malignancies in all individuals. Dendritic cells (DCs) are not only part of the innate immune system, but also generals of adaptive immunity and they orchestrate the de novo induction of tolerogenic and immunogenic T cell responses. Thus, combinatorial approaches addressing DCs and T cells in parallel represent an attractive strategy to achieve higher response rates across patients. However, this requires profound knowledge about the dynamic interplay of DCs, T cells, other immune and tumor cells. Here, we summarize the DC subsets present in mice and men and highlight conserved and divergent characteristics between different subsets and species. Thereby, we supply a resource of the molecular players involved in key functional features of DCs ranging from their sentinel function, the translation of the sensed environment at the DC:T cell interface to the resulting specialized T cell effector modules, as well as the influence of the tumor microenvironment on the DC function. As of today, mostly monocyte derived dendritic cells (moDCs) are used in autologous cell therapies after tumor antigen loading. While showing encouraging results in a fraction of patients, the overall clinical response rate is still not optimal. By disentangling the general aspects of DC biology, we provide rationales for the design of next generation DC vaccines enabling to exploit and manipulate the described pathways for the purpose of cancer immunotherapy in vivo. Finally, we discuss how DC-based vaccines might synergize with checkpoint inhibition in the treatment of malignant diseases

Functionalization Of T Lymphocytes With Citrate-Coated Superparamagnetic Iron Oxide Nanoparticles For Magnetically Controlled Immune Therapy

Purpose: Immune activation with T cell tumor infiltration is beneficial for the prognosis of patients suffering from solid cancer. Depending on their immune status, solid tumors can be immunologically classified into three groups: “hot” tumors are infiltrated with T lymphocytes, “cold” tumors are not infiltrated and “immune excluded” tumors are only infiltrated in the peripheral tumor tissue. Checkpoint inhibitors provide new therapeutic options for “hot” tumors by triggering the immune response of T cells. In order to enable this for cold tumors as well, T cells must be enriched in the tumor. Therefore, we use the principle of magnetic targeting to guide T cells loaded with citrate-coated superparamagnetic iron oxide nanoparticles (SPIONCitrate) to the tumor by an externally applied magnetic field. Methods: SPIONCitrate were produced by alkaline coprecipitation of iron(II) and iron(III) chloride and in situ coating with sodium citrate. The concentration-dependent cytocompatibility of the particles was determined by flow cytometry and blood stability assays. Atomic emission spectroscopy was used for the quantification of the particle uptake into T lymphocytes. The attractability of the loaded cells was observed by live-cell imaging in the presence of an externally applied magnetic field. Results: SPIONCitrate displayed good cytocompatibility to T cells and did not show any sign of aggregation in blood. Finally, SPIONCitrate-loaded T cells were strongly attracted by a small external magnet. Conclusion: T cells can be “magnetized” by incorporation of SPIONCitrate for magnetic targeting. The production of the particle-cell hybrid system is straightforward, as the loading process only requires basic laboratory devices and the loading efficiency is sufficient for cells being magnetically controllable. For these reasons, SPIONCitrate are potential suitable candidates for magnetic T cell targeting

Mechanical Regulation of Epithelial Tissue Homeostasis

Despite recent efforts to understand homeostasis in epithelial tissues, there are many unknowns surrounding this steady state. It is considered to be regulated by mechanoresponse, but unlike for single cells, this remains heavily debated for tissues. Here, we show that changes in matrix stiffness induce a non-equilibrium transition from tubular to squamous Madin-Darby Canine Kidney II tissues. Nonetheless, despite different cell morphologies and densities, all homeostatic tissues display equivalent topologies, which, hence, must be actively targeted and regulated. On the contrary, the mechanoresponse induces dramatic changes in the large-scale organization of the colonies. On stiff gels, this yields an unreported cooperative state of motile cells displaying higher densities than in the arrested homeostatic state. This suggests a more complex relation between cell density and motility than previously anticipated. Our results unequivocally relate the mechanosensitive properties of individual cells to the evolving macroscopic structures, an effect that could be important for understanding the emergent pathologies of living tissues

Monocytes Elicit a Neutrophil-Independent Th1/Th17 Response Upon Immunization With a Mincle-Dependent Glycolipid Adjuvant

Successful subunit vaccination with recombinant proteins requires adjuvants. The glycolipid trehalose-dibehenate (TDB), a synthetic analog of the mycobacterial cord factor, potently induces Th1 and Th17 immune responses and is a candidate adjuvant for human immunization. TDB binds to the C-type lectin receptor Mincle and triggers Syk-Card9-dependent APC activation. In addition, interleukin (IL)-1 receptor/MyD88-dependent signaling is required for TDB adjuvanticity. The role of different innate immune cell types in adjuvant-stimulated Th1/Th17 responses is not well characterized. We investigated cell recruitment to the site of injection (SOI) and to the draining lymph nodes (dLNs) after immunization with the TDB containing adjuvant CAF01 in a protein-based vaccine. Recruitment of monocytes and neutrophils to the SOI and the dramatic increase in lymph node cellularity was partially dependent on both Mincle and MyD88. Despite their large numbers at the SOI, neutrophils were dispensable for the induction of Th1/Th17 responses. In contrast, CCR2-dependent monocyte recruitment was essential for the induction of Th1/Th17 cells. Transport of adjuvant to the dLN did not require Mincle, MyD88, or CCR2. Together, adjuvanticity conferred by monocytes can be separated at the cellular level from potential tissue damage by neutrophils