The spleen is the lymphoid organ that induces immune responses toward blood-borne pathogens. Specialized macrophages in the splenic marginal zone are strategically positioned to phagocytose pathogens and cell debris, but are not known to play a role in the activation of T-cell responses. Here we demonstrate that splenic marginal metallophilic macrophages (MMM) are essential for crosspresentation of blood-borne antigens by splenic dendritic cells (DCs). Our data demonstrate that antigens targeted to MMM as well as blood-borne adenoviruses are efficiently captured by MMM and exclusively transferred to splenic CD8 + DCs for cross-presentation and for the activation of cytotoxic T lymphocytes. Depletion of macrophages in the marginal zone prevents cytotoxic T-lymphocyte activation by CD8 + DCs after antibody targeting or adenovirus infection. Moreover, we show that tumor antigen targeting to MMM is very effective as antitumor immunotherapy. Our studies point to an important role for splenic MMM in the initial steps of CD8 + T-cell immunity by capturing and concentrating blood-borne antigens and the transfer to cross-presenting DCs which can be used to design vaccination strategies to induce antitumor cytotoxic T-cell immunity.antigen presentation | infection T he spleen is essential for the induction of immune responses toward blood-borne antigens and has a unique architecture. Arterial blood flow terminates in marginal sinuses situated in the marginal zone (MZ) that surrounds the white pulp containing Bcell follicles and T-cell zones. Marginal sinuses are lined by reticular cells and contain marginal zone B cells and two types of macrophages (Mφ) (1, 2). Marginal metallophilic macrophages (MMM), characterized by the expression of sialic acid-binding Iglike lectin-1 (Siglec-1, Sialoadhesin, CD169) (3, 4), are located as a tight network in the inner part of the MZ near the white pulp, whereas marginal zone macrophages (MZM), which specifically express the C-type lectin SIGN-R1, can be found in the outer MZ toward the red pulp (5). Both MZM and a subset of MMM express the type I scavenger receptor MARCO (6). Although MMM and MZM efficiently take up particulate antigens (Ag) present in the blood (7-9), they are hitherto considered not to be important for the generation of T-cell responses (8-10).In contrast to Mφ, dendritic cells (DCs) are specialized Agpresenting cells that have a dominant role in initiating primary Tcell responses. Murine splenic DCs can be divided into two different subsets based on the expression of phenotypic markers: localization and function (11). CD8 + DCs express the C-type lectin DEC205 and are found in the T-cell zone and the outer marginal zone (12). They are specialized in cross-presentation of Ag and in the activation and tolerization of cytotoxic T cells (CTLs) (13-16). Furthermore, they are important for the generation of antitumor specific immune responses and the elimination of tumors in vivo (17). In contrast, CD8− DCs are specialized in the activation of CD4 + T cells. CD8 − DCs are...

After ingestion, oral antigens distribute systemically and provoke T cell stimulation outside the gastrointestinal tract. Within the liver, scavenger liver sinusoidal endothelial cells (LSEC) eliminate blood-borne antigens and induce T cell tolerance. Here we investigated whether LSEC contribute to oral tolerance. Oral antigens were efficiently cross-presented on H-2K b by LSEC to naive CD8 T cells. Cross-presentation efficiency in LSEC but not dendritic cells was increased by antigen-exposure to heat or low pH. Mechanistically, cross-presentation in LSEC requires endosomal maturation, involves hsc73 and proteasomal degradation. H-2K b -restricted cross-presentation of oral antigens by LSEC in vivo induced CD8 T cell priming and led to development of CD8 T cell tolerance in two independent experimental systems. Adoptive transfer of LSEC from mice fed with antigen (ovalbumin) into RAG2 -/-knockout mice, previously reconstituted with naive ovalbumin-specific CD8 T cells, prevented development of specific cytotoxicity and expression of IFN-c in CD8 T cells. Using a new transgenic mouse line expressing H-2K b only on endothelial cells, we have demonstrated that oral antigen administration leads to tolerance in H-2K b -restricted CD8 T cells. Collectively, our data demonstrate a participation of the liver, in particular scavenger LSEC, in development of CD8 T cell tolerance towards oral antigens.

CpG-rich oligonucleotides (CpG-ODN) bind to Toll-like receptor 9 (TLR9) and are used as powerful adjuvants for vaccination. Here we report that CpG-ODN not only act as immune stimulatory agents but can also induce strong immune suppression depending on the anatomical location of application. In agreement with the adjuvant effect, subcutaneous application of antigen plus CpG-ODN resulted in antigen-specific T cell activation in local lymph nodes. In contrast, systemic application of CpG-ODN resulted in suppression of T cell expansion and CTL activity in the spleen. The suppressive effect was mediated by indoleamine 2,3-dioxygenase (IDO) as indicated by the observation that CpG-ODN induced IDO in the spleen and that T cell suppression could be abrogated by 1-methyl-tryptophan (1-MT), an inhibitor of IDO. No expression of IDO was observed in lymph nodes after injection of CpG-ODN, explaining why suppression was restricted to the spleen. Studies with a set of knockout mice demonstrated that the CpG-ODN-induced immune suppression is dependent on TLR9 stimulation and independent of type I and type II interferons. The present study shows that for the use of CpG-ODN as an adjuvant in vaccines, the route of application is crucial and needs to be considered. In addition, the results indicate that down-modulation of immune responses by CpG-ODN may be possible in certain pathological conditions. See accompanying commentary: http://dx

Suspension cultures, in which human mesenchymal stem cells are cultivated on microcarriers in scalable single‐use stirred bioreactor types, have been shown to be a promising alternative to planar flask cultures. However, stirred single‐use bioreactors were originally developed for production processes with robust, permanent cell lines. Human mesenchymal stem cells are adherent primary cells and thus expanding them in such bioreactor systems imposes more stringent requirements on bioreactor systems. For low‐serum conditions (5%) and different types of stirred single‐use bioreactors, a suspension criteria‐based approach for expanding human adipose tissue‐derived mesenchymal stem cells (hASCs) from milliliter to pilot scale was successfully developed. For process scale‐up, experimental and numerical investigations were performed to (i) predict optimum impeller speeds, (ii) determine the main engineering parameters (local shear stress, turbulent dissipation rate, Kolmogorov microscale), and (iii) verify suspension criteria NS1 and NS1u for rapid process transfer from 100 mL to 2 L and 35 L cultures. Using optimized medium‐microcarrier combinations as well as NS1 and NS1u as scale‐up factors, total hASC quantities between 3 × 107 (100 mL scale) and 1 × 1010 (35 L scale) were obtained. The cell quantities obtained are the highest reported to date for scalable single‐use bioreactors under low‐serum conditions.

Early during Gram-negative sepsis, excessive release of pro-inflammatory cytokines can cause septic shock that is often followed by a state of immune paralysis characterized by the failure to mount adaptive immunity towards secondary microbial infections. Especially, the early mechanisms responsible for such immune hypo-responsiveness are unclear. Here, we show that TLR4 is the key immune sensing receptor to initiate paralysis of T-cell immunity after bacterial sepsis. Downstream of TLR4, signalling through TRIF but not MyD88 impaired the development of specific T-cell immunity against secondary infections. We identified type I interferon (IFN) released from splenic macrophages as the critical factor causing Tcell immune paralysis. Early during sepsis, type I IFN acted selectively on dendritic cells (DCs) by impairing antigen presentation and secretion of pro-inflammatory cytokines. Our results reveal a novel immune regulatory role for type I IFN in the initiation of septic immune paralysis, which is distinct from its well-known immune stimulatory effects. Moreover, we identify potential molecular targets for therapeutic intervention to overcome impairment of T-cell immunity after sepsis.