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Pathogens and vaccines that produce persisting antigens can generate expanded pools of effector memory CD8+ T cells, described as memory inflation. While properties of inflating memory CD8+ T cells have been characterized, the specific cell types and tissue factors responsible for their maintenance remain elusive. Here, we show that clinically applied adenovirus vectors preferentially target fibroblastic stromal cells in cultured human tissues. Moreover, we used cell-type-specific antigen targeting to define critical cells and molecules that sustain long-term antigen presentation and T cell activity after adenovirus vector immunization in mice. While antigen targeting to myeloid cells was insufficient to activate antigen-specific CD8+ T cells, genetic activation of antigen expression in Ccl19-cre-expressing fibroblastic stromal cells induced inflating CD8+ T cells. Local ablation of vector-targeted cells revealed that lung fibroblasts support the protective function and metabolic fitness of inflating memory CD8+ T cells in an interleukin (IL)-33-dependent manner. Collectively, these data define a critical fibroblastic niche that underpins robust protective immunity operating in a clinically important vaccine platform.

Innate lymphoid cells (ILCs) govern immune cell homeostasis in the intestine and protect the host against microbial pathogens. Various cell-intrinsic pathways have been identified that determine ILC development and differentiation. However, the cellular components that regulate ILC sustenance and function in the intestinal lamina propria are less known. Using single-cell transcriptomic analysis of lamina propria fibroblasts, we identify fibroblastic reticular cells (FRCs) that underpin cryptopatches (CPs) and isolated lymphoid follicles (ILFs). Genetic ablation of lymphotoxin-β receptor expression inCcl19-expressing FRCs blocks the maturation of CPs into mature ILFs. Interactome analysis shows the major niche factors and processes underlying FRC-ILC crosstalk. In vivo validation confirms that a sustained lymphotoxin-driven feedforward loop of FRC activation including IL-7 generation is critical for the maintenance of functional ILC populations. In sum, our study indicates critical fibroblastic niches within the intestinal lamina propria that control ILC homeostasis and functionality and thereby secure protective gut immunity.

Pathogens and vaccines that produce persisting antigens can generate expanded pools of effector memory CD8

Innate lymphoid cells (ILC) in the small intestine govern immune homeostasis and protect the host against gut pathogens. While distinct cell-intrinsic signals have been identified that determine ILC development and differentiation, it has remained unclear which cell population regulates ILC sustenance. Using unbiased transcriptomic analysis of intestinal fibroblasts, we have identified a specialized Ccl19-expressing fibroblastic reticular cell (FRC) population that underpins solitary intestinal lymphoid tissue (SILT) structures including cryptopatches and isolated lymphoid follicles. Conditional ablation of lymphotoxin-β receptor (LTβR) signalling in SILT FRC impeded the maturation of isolated lymphoid follicles and blocked ILC maintenance resulting in the elevated susceptibility to bacterial infection. Moreover, specific Ltbr ablation in FRC during adulthood revealed that sustained LTβR-dependent FRC-ILC interaction is required to maintain SILT structures and ILC populations. Taken together, our study unveils a critical intestinal FRC niche that secures protective gut immunity.

Interleukin-33 (IL-33) is a nuclear cytokine of the interleukin-1 family and is released upon cell damage thereby acting as an alarmin. IL-33 plays an essential role in promoting host-protective immune responses against helminth parasites at different mucosal surfaces including the intestine. However, early events after gastrointestinal nematode infection are poorly understood and the cellular source of IL-33 in the gut remains ill-defined. Here we show that Cxcl13-Cre-positive fibroblastic stromal cells (FSCs) of the lamina propria are the main source of IL-33 in the small intestine. Within the first days of nematode infection, lamina propria FSCs showed strongly increased IL-33 expression. Ablation of IL-33 in Cxcl-13-Cre-positive FSCs resulted in an increased infestation with the gastrointestinal nematode Heligmosomoides polygyrus bakeri. We found that myeloid cells were recruited to the site of worm invasion early after infection and that the upregulation of a distinct set of inflammatory cytokines was dependent on FSC-derived IL-33. Collectively, these data unveil that IL-33-mediated crosstalk between stromal and myeloid cells is a critical event during the initial immune response against gastrointestinal nematodes.

Fibroblastic reticular cells influence the function of lymphocytes in secondary lymphoid organs. Ludewig and colleagues demonstrate that they also specifically restrain the activation of group 1 innate lymphoid cells in the presence of microbial stimulation to prevent immunopathology. Supplementary information The online version of this article (doi:10.1038/ni.3566) contains supplementary material, which is available to authorized users., Fibroblastic reticular cells (FRCs) of secondary lymphoid organs form distinct niches for interaction with hematopoietic cells. We found here that production of the cytokine IL-15 by FRCs was essential for the maintenance of group 1 innate lymphoid cells (ILCs) in Peyer's patches and mesenteric lymph nodes. Moreover, FRC-specific ablation of the innate immunological sensing adaptor MyD88 unleashed IL-15 production by FRCs during infection with an enteropathogenic virus, which led to hyperactivation of group 1 ILCs and substantially altered the differentiation of helper T cells. Accelerated clearance of virus by group 1 ILCs precipitated severe intestinal inflammatory disease with commensal dysbiosis, loss of intestinal barrier function and diminished resistance to colonization. In sum, FRCs act as an 'on-demand' immunological 'rheostat' by restraining activation of group 1 ILCs and thereby preventing immunopathological damage in the intestine. Supplementary information The online version of this article (doi:10.1038/ni.3566) contains supplementary material, which is available to authorized users.

The thymic epithelium forms specialized niches to enable thymocyte differentiation. While the common epithelial progenitor of medullary and cortical thymic epithelial cells (mTECs and cTECs) is well defined, early stages of mTEC lineage specification have remained elusive. Here, we utilized in vivo targeting of mTECs to resolve their differentiation pathways and to determine whether mTEC progenitors participate in thymocyte education. We found that mTECs descend from a lineage committed, podoplanin (PDPN)-expressing progenitor located at the cortico-medullary junction. PDPN(+) junctional TECs (jTECs) represent a distinct TEC population that builds the thymic medulla, but only partially supports negative selection and thymocyte differentiation. Moreover, conditional gene targeting revealed that abrogation of alternative NF-κB pathway signaling in the jTEC stage completely blocked mTEC development. Taken together, this study identifies jTECs as lineage-committed mTEC progenitors and shows that NF-κB-dependent progression of jTECs to mTECs is critical to secure central tolerance.

The splenic white pulp is underpinned by poorly characterized stromal cells that demarcate distinct immune cell microenvironments. Here, definition of the embryonic origin and tracing of the differentiation trajectories of fibroblastic reticular cells (FRCs) was enabled by the establishment of FRC-specific fate-mapping in mice. We found that all reticular cell subsets descend from pluripotent progenitors that emerge at embryonic day 19.5 from Sca-1 periarterial progenitors. Commitment of FRC progenitors was concluded during the first week of postnatal life through occupation of niches along developing central arterioles. Single cell transcriptomic analysis facilitated deconvolution of FRC differentiation trajectories and indicated that perivascular reticular cells function both as adult lymphoid organizer cells and mural cell progenitors. Finally, the lymphotoxin-β receptor-independent sustenance of postnatal progenitor stemness unveiled that systemic immune surveillance in the splenic white pulp is governed through subset specification of reticular cells from a pluripotent periarterial progenitor cell.

Immune protection of the body cavities depends on the swift activation of innate and adaptive immune responses in nonclassical secondary lymphoid organs known as fat-associated lymphoid clusters (FALCs). Compared with classical secondary lymphoid organs such as lymph nodes and Peyer's patches, FALCs develop along distinct differentiation trajectories and display a reduced structural complexity. Although it is well established that fibroblastic reticular cells (FRCs) are an integral component of the immune-stimulating infrastructure of classical secondary lymphoid organs, the role of FRCs in FALC-dependent peritoneal immunity remains unclear. Using FRC-specific gene targeting, we found that FRCs play an essential role in FALC-driven immune responses. Specifically, we report that initiation of peritoneal immunity was governed through FRC activation in a myeloid differentiation primary response 88 (MYD88)-dependent manner. FRC-specific ablation of MYD88 blocked recruitment of inflammatory monocytes into FALCs and subsequent CD4+ T cell-dependent B-cell activation and IgG class switching. Moreover, containment of Salmonella infection was compromised in mice lacking MYD88 expression in FRCs, indicating that FRCs in FALCs function as an initial checkpoint in the orchestration of protective immune responses in the peritoneal cavity.

Lymph nodes (LNs) are strategically situated throughout the body at junctures of the blood vascular and lymphatic systems to direct immune responses against antigens draining from peripheral tissues. The current paradigm describes LN development as a programmed process that is governed through the interaction between mesenchymal lymphoid tissue organizer (LTo) cells and hematopoietic lymphoid tissue inducer (LTi) cells. Using cell-type-specific ablation of key molecules involved in lymphoid organogenesis, we found that initiation of LN development is dependent on LTi-cell-mediated activation of lymphatic endothelial cells (LECs) and that engagement of mesenchymal stromal cells is a succeeding event. LEC activation was mediated mainly by signaling through receptor activator of NF-κB (RANK) and the non-canonical NF-κB pathway and was steered by sphingosine-1-phosphate-receptor-dependent retention of LTi cells in the LN anlage. Finally, the finding that pharmacologically enforced interaction between LTi cells and LECs promotes ectopic LN formation underscores the central LTo function of LECs.

Abs play a significant role in protection against the intracellular bacterium Salmonella Typhi. In this article, we investigated how long-term protective IgM responses can be elicited by a S. Typhi outer-membrane protein C– and F–based subunit vaccine (porins). We found that repeated Ag exposure promoted a CD4+ T cell–dependent germinal center reaction that generated mutated IgM-producing B cells and was accompanied by a strong expansion of IFN-γ–secreting T follicular helper cells. Genetic ablation of individual cytokine receptors revealed that both IFN-γ and IL-17 are required for optimal germinal center reactions and production of porin-specific memory IgM+ B cells. However, more profound reduction of porin-specific IgM B cell responses in the absence of IFN-γR signaling indicated that this cytokine plays a dominant role. Importantly, mutated IgM mAbs against porins exhibited bactericidal capacity and efficiently augmented S. Typhi clearance. In conclusion, repeated vaccination with S. Typhi porins programs type I T follicular helper cell responses that contribute to the diversification of B cell memory and promote the generation of protective IgM Abs.

Attenuation of T cell-mediated damage of blood endothelial cells (BECs) in transplanted organs is important to prevent transplant vasculopathy (TV) and chronic rejection. Here, we assessed the importance of minor histocompatibility antigen (mHA) distribution and different coinhibitory molecules for T cell-BEC interaction. A transgenic mHA was directed specifically to BECs using the Tie2 promoter and cellular interactions were assessed in graft-versus-host disease-like and heterotopic heart transplantation settings. We found that cognate CD4+ T-cell help was critical for the activation of BEC-specific CD8+ T cells. However, systemic mHA expression on BECs efficiently attenuated adoptively transferred, BEC-specific CD4+ and CD8+ T cells and hence prevented tissue damage, whereas restriction of mHA expression to heart BECs precipitated the development of TV. Importantly, the lack of the coinhibitory molecules programed death-1 (PD-1) and B and T lymphocyte attenuator fostered the initial activation of BEC-specific CD4+ T cells, but did not affect development of TV. In contrast, TV was significantly augmented in the absence of PD-1 on BEC-specific CD8+ T cells. Taken together, these results indicate that antigen distribution in the vascular bed determines the impact of coinhibition and, as a consequence, critically impinges on T cell-mediated vascular immunopathology.

SummaryThe stromal scaffold of the lymph node (LN) paracortex is built by fibroblastic reticular cells (FRCs). Conditional ablation of lymphotoxin-β receptor (LTβR) expression in LN FRCs and their mesenchymal progenitors in developing LNs revealed that LTβR-signaling in these cells was not essential for the formation of LNs. Although T cell zone reticular cells had lost podoplanin expression, they still formed a functional conduit system and showed enhanced expression of myofibroblastic markers. However, essential immune functions of FRCs, including homeostatic chemokine and interleukin-7 expression, were impaired. These changes in T cell zone reticular cell function were associated with increased susceptibility to viral infection. Thus, myofibroblasic FRC precursors are able to generate the basic T cell zone infrastructure, whereas LTβR-dependent maturation of FRCs guarantees full immunocompetence and hence optimal LN function during infection.

It is not known how naive B cells compute divergent chemoattractant signals of the T-cell area and B-cell follicles during in vivo migration. Here, we used two-photon microscopy of peripheral lymph nodes (PLNs) to analyze the prototype G-protein-coupled receptors (GPCRs) CXCR4, CXCR5, and CCR7 during B-cell migration, as well as the integrin LFA-1 for stromal guidance. CXCR4 and CCR7 did not influence parenchymal B-cell motility and distribution, despite their role during B-cell arrest in venules. In contrast, CXCR5 played a nonredundant role in B-cell motility in follicles and in the T-cell area. B-cell migration in the T-cell area followed a random guided walk model, arguing against directed migration in vivo. LFA-1, but not α4 integrins, contributed to B-cell motility in PLNs. However, stromal network guidance was LFA-1 independent, uncoupling integrin-dependent migration from stromal attachment. Finally, we observed that despite a 20-fold reduction of chemokine expression in virus-challenged PLNs, CXCR5 remained essential for B-cell screening of antigen-presenting cells. Our data provide an overview of the contribution of prototype GPCRs and integrins during naive B-cell migration and shed light on the local chemokine availability that these cells compute.

Fibroblastic reticular cells (FRCs) form the cellular scaffold of lymph nodes (LNs) and establish distinct microenvironmental niches to provide key molecules that drive innate and adaptive immune responses and control immune regulatory processes. Here, we have used a graph theory-based systems biology approach to determine topological properties and robustness of the LN FRC network in mice. We found that the FRC network exhibits an imprinted small-world topology that is fully regenerated within 4 wk after complete FRC ablation. Moreover, in silico perturbation analysis and in vivo validation revealed that LNs can tolerate a loss of approximately 50% of their FRCs without substantial impairment of immune cell recruitment, intranodal T cell migration, and dendritic cell-mediated activation of antiviral CD8+ T cells. Overall, our study reveals the high topological robustness of the FRC network and the critical role of the network integrity for the activation of adaptive immune responses., Topological complex network analysis reveals an underlying robust "small-world" organization of fibroblastic reticular cells and its critical role in the maintenance of lymph node functionality., Author Summary Fibroblastic reticular cells (FRCs) in lymph nodes are organized in a highly connected cellular network that not only acts as a scaffold for lymphocyte migration but also provides key factors for induction and maintenance of immune responses. By utilizing high-resolution microscopy coupled with computational approaches to complex network analysis, we determined the topological properties and robustness of the FRC network. The underlying structure of the FRC network has been identified as a small-world network analogous to many other biological networks. Moreover, we demonstrate that this distinct structural organization is an imprinted trait of the FRC network, which is capable of fully regenerating after complete FRC ablation. In silico perturbation analysis of the FRC network confirmed that lymph nodes are able to tolerate FRC loss of approximately 50%. In vivo experiments corroborated these findings by demonstrating substantial impairment of immune cell recruitment, migration, and dendritic cell-mediated activation of antiviral CD8+ T cells, after critical loss of FRCs. In conclusion, the present study reveals the extraordinary topological robustness of the FRC network, crucial for establishing effective immunity.

Lymph nodes (LNs) are highly confined environments with a cell-dense three-dimensional meshwork, in which lymphocyte migration is regulated by intracellular contractile proteins. However, the molecular cues directing intranodal cell migration remain poorly characterized. Here we demonstrate that lysophosphatidic acid (LPA) produced by LN fibroblastic reticular cells (FRCs) acts locally to LPA2 to induce T-cell motility. In vivo, either specific ablation of LPA-producing ectoenzyme autotaxin in FRCs or LPA2 deficiency in T cells markedly decreased intranodal T cell motility, and FRC-derived LPA critically affected the LPA2-dependent T-cell motility. In vitro, LPA activated the small GTPase RhoA in T cells and limited T-cell adhesion to the underlying substrate via LPA2. The LPA-LPA2 axis also enhanced T-cell migration through narrow pores in a three-dimensional environment, in a ROCK-myosin II-dependent manner. These results strongly suggest that FRC-derived LPA serves as a cell-extrinsic factor that optimizes T-cell movement through the densely packed LN reticular network. DOI: http://dx.doi.org/10.7554/eLife.10561.001, eLife digest Small organs called lymph nodes are found throughout the body and help to filter out harmful particles and cells. Lymph nodes are packed with different types of immune cells, such as the T-cells that play a number of roles in detecting and destroying bacteria, viruses and other disease-causing microbes. Within the lymph node, T-cells crawl along a meshwork made up of cells called fibroblastic reticular cells. The T-cells appear to move in random patterns, but the signals that drive this movement remain ill-defined. Now, Takeda et al. reveal that a lipid called lysophosphatidic acid (LPA), which is produced by the fibroblastic reticular cells, is responsible for regulating how T-cells move around inside the lymph nodes. T-cells are able to detect LPA via certain receptor proteins on their surface. Takeda et al. engineered mice that were either unable to produce a particular LPA receptor on their T-cells, or that produced less LPA than normal. The T-cells of these mice moved around less than T-cells in normal mice. Further experiments revealed that LPA signaling also affects the signaling pathway that alters how well the T-cells stick to nearby surfaces. This suggests that LPA helps to optimize T-cell movement to allow the cells to navigate the small spaces found between the fibroblastic reticular cells. In the future, targeting the processes involved in LPA signaling could help to develop new treatments for disorders of the immune system. DOI: http://dx.doi.org/10.7554/eLife.10561.002

Myocarditis is a potentially lethal inflammatory heart disease of children and young adults that frequently leads to dilated cardiomyopathy (DCM). Since diagnostic procedures and efficient therapies are lacking, it is important to characterize the critical immune effector pathways underlying the initial cardiac inflammation and the transition from myocarditis to DCM. We describe here a T-cell receptor (TCR) transgenic mouse model with spontaneously developing autoimmune myocarditis that progresses to lethal DCM. Cardiac magnetic resonance imaging revealed early inflammation-associated changes in the ventricle wall including transient thickening of the left ventricle wall. Furthermore, we found that IFN-γ was a major effector cytokine driving the initial inflammatory process and that the cooperation of IFN-γ and IL-17A was essential for the development of the progressive disease. This novel TCR transgenic mouse model permits the identification of the central pathophysiological and immunological processes involved in the transition from autoimmune myocarditis to DCM.

Nonhematopoietic stromal cells of secondary lymphoid organs form important scaffold and fluid transport structures, such as lymph node (LN) trabeculae, lymph vessels, and conduits. Furthermore, through the production of chemokines and cytokines, these cells generate a particular microenvironment that determines lymphocyte positioning and supports lymphocyte homeostasis. IL-7 is an important stromal cell-derived cytokine that has been considered to be derived mainly from T-cell zone fibroblastic reticular cells. We show here that lymphatic endothelial cells (LECs) are a prominent source of IL-7 both in human and murine LNs. Using bacterial artificial chromosome transgenic IL-7–Cre mice, we found that fibroblastic reticular cells and LECs strongly up-regulated IL-7 expression during LN remodeling after viral infection and LN reconstruction after avascular transplantation. Furthermore, IL-7–producing stromal cells contributed to de novo formation of LyveI-positive lymphatic structures connecting reconstructed LNs with the surrounding tissue. Importantly, diphtheria toxin–mediated depletion of IL-7–producing stromal cells completely abolished LN reconstruction. Taken together, this study identifies LN LECs as a major source of IL-7 and shows that IL-7–producing stromal cells are critical for reconstruction and remodeling of the distinct LN microenvironment.

Adaptive immune responses are characterized by substantial restructuring of secondary lymphoid organs. The molecular and cellular factors responsible for virus-induced lymphoid remodeling are not well known to date. Here we applied optical projection tomography, a mesoscopic imaging technique, for a global analysis of the entire 3-dimensional structure of mouse peripheral lymph nodes (PLNs), focusing on B-cell areas and high endothelial venule (HEV) networks. Structural homeostasis of PLNs was characterized by a strict correlation between total PLN volume, B-cell volume, B-cell follicle number, and HEV length. After infection with lymphocytic choriomeningitis virus, we observed a substantial, lymphotoxin (LT) β-receptor–dependent reorganization of the PLN microarchitecture, in which an initial B-cell influx was followed by 3-fold increases in PLN volume and HEV network length on day 8 after infection. Adoptive transfer experiments revealed that virus-induced PLN and HEV network remodeling required LTα1β2-expressing B cells, whereas the inhibition of vascular endothelial growth factor-A signaling pathways had no significant effect on PLN expansion. In summary, lymphocytic choriomeningitis virus-induced PLN growth depends on a vascular endothelial growth factor-A–independent, LT- and B cell–dependent morphogenic pathway, as revealed by an in-depth mesoscopic analysis of the global PLN structure.

The generation of lymphoid microenvironments in early life depends on the interaction of lymphoid tissue-inducer cells with stromal lymphoid tissue-organizer cells. Whether this cellular interface stays operational in adult secondary lymphoid organs has remained elusive. We show here that during acute infection with lymphocytic choriomeningitis virus, antiviral cytotoxic T cells destroyed infected T cell zone stromal cells, which led to profound disruption of secondary lymphoid organ integrity. Furthermore, the ability of the host to respond to secondary antigens was lost. Restoration of the lymphoid microanatomy was dependent on the proliferative accumulation of lymphoid tissue-inducer cells in secondary lymphoid organs during the acute phase of infection and lymphotoxin alpha(1)beta(2) signaling. Thus, crosstalk between lymphoid tissue-inducer cells and stromal cells is reactivated in adults to maintain secondary lymphoid organ integrity and thereby contributes to the preservation of immunocompetence.

Objective— The purpose of this study was to examine the relative contribution of different immunopathological mechanisms during murine cytomegalovirus (MCMV)-mediated acceleration of atheroma formation in apolipoprotein E–deficient (apoE −/− ) mice. Methods and Results— To distinguish between the effects of systemic activation and cognate immune reactivity against a pathogen-derived persisting antigen in the vasculature, we used hypercholesterolemic transgenic mice constitutively expressing the β-galactosidase (β-gal) transgene in the cardiovascular system (apoE −/− ×SM-LacZ). After infection with β-gal–recombinant MCMV-LacZ, apoE −/− , and apoE −/− ×SM-LacZ mice mounted comparable cellular immune responses against the virus. β-gal–specific CD8 + T cells expanded rapidly and remained detectable for at least 100 days in both mouse strains. However, compared with apoE −/− mice, apoE −/− ×SM-LacZ mice developed drastically accelerated atherosclerosis. Moreover, atherosclerotic lesions in MCMV-LacZ–infected apoE −/− ×SM-LacZ but not apoE −/− mice were associated with pronounced inflammatory infiltrates. Conclusions— Taken together, our data indicate that chronic immune reactivity against pathogen-derived antigens persisting in the vasculature significantly exacerbates atherogenesis.

This study provides a detailed spatiotemporal interaction analysis between B cells, Th cells, and dendritic cells (DC) during the generation of protective antiviral B cell immunity. Following vesicular stomatitis virus (VSV) infection, conditional ablation of CD11c-positive DC at the time-point of infection did not impair extrafollicular plasma cell generation and Ig class switching. In contrast, the generation of Th and B cell responses following immunization with recombinant VSV-glycoprotein was DC-dependent. Furthermore, we show that the CCR7-dependent interplay of the three cell-types is crucial for virus-neutralizing B cell responses in the presence of limiting amounts of Ag. An immediate event following VSV infection was the CCR7-mediated interaction of VSV-specific B and Th cells at the T cell-B cell zone border that facilitated plasma cell differentiation and Th cell activation. Taken together, these experiments provide evidence for a direct, CCR7-orchestrated and largely DC-independent mutual activation of Th cells and Ag-specific B cells that is most likely a critical step during early immune responses against cytopathic viruses.

The thymus is an anatomically compartmentalized primary lymphoid organ that fosters the production of self‐tolerant T cells. The thymic cortex provides a specialized microenvironment in which cortical thymic epithelial cells (cTECs) support the positive selection and further differentiation of self‐MHC‐restricted thymocytes. Following their migration into the medulla, positively selected thymocytes are further screened for self‐reactivity, which involves both negative selection and Foxp3+ regulatory T cell generation via interactions with medullary thymic epithelial cells (mTECs). Given the importance of both cortical and medullary microenvironments for T cell development, studies that address the developmental origins of cTECs and mTECs are important in understanding the processes that shape the developing T cell receptor repertoire, and reduce the frequency of self‐reactive T cells that initiate autoimmune disease. In this issue of the European Journal of Immunology, Onder et al. [Eur. J. Immunol. 2015. 45: 2218‐2231] identified a subset of podoplanin+ mTECs in mice that reside at the corticomedullary junction (CMJ), show that their development is important to establish self‐tolerance, and require the presence of self‐reactive T cells. Collectively, their findings highlight the CMJ as a potential repository for precursors of the mTEC lineage, and provide a better understanding of thymus medulla formation.

The development of preventive or therapeutic recombinant vaccines and the generation of serodiagnostic assays for infectious diseases depend essentially on the availability of molecularly defined antigens. A major bottleneck for the identification of suitable target antigens for many pathogens is the isolation of sufficient amounts of material for subsequent genomic or proteomic screening. Applying a highly efficient expression cloning strategy to the human pathogens vaccinia virus (VV) and Chlamydia pneumoniae (CP), we demonstrate that sub-nanogram amounts of isolated nucleic acids can be utilized to determine comprehensive sets of immunodominant antigens. Remarkably, the approach not only confirmed the immunogenicity of previously reported antigens but also disclosed novel vaccine candidates conserved in orthopoxviruses, including antigenic envelope proteins and immunodominant CTL epitopes. Moreover, as illustrated for CP infection, we show that a panel of novel antigens can be readily selected from the initially discovered pool to build up pathogen-specific seroassays. The established approach is rapid, making it an attractive procedure for the comprehensive dissection of immunomes of known human pathogens and newly emerging infectious agents.

Due to their unique ability to initiate immune responses against invading pathogens, dendritic cells (DC) are considered the most potent antigen-presenting cells (APC). DC are specialized in antigen transport to secondary lymphoid organs and subsequent stimulation of primary and secondary T-cell responses. However, peptides presented by DC are not only of foreign origin but may include peptides derived from self-proteins, a process that can eventually lead to the development of autoimmune disease. However, a variety of mechanisms impact on the immunogenicity of DC in order to prevent autoimmune responses. Hence, targeted modulation of DC function may open new treatment options aiming at alleviating DC-driven autoimmune responses.

Prostate cancer (PCa) like other tumors expresses antigens that may serve as target for specific immunotherapy. Special antigen-presenting cells (e. g., dendritic cells) are capable of generating tumor-specific immunity. Cytotoxic T-cells (killer cells) are very effective against antigens and, consequently, against the respective tissue or tumor. Cancer testis antigens (CTA) are expressed in various human cancers but, aside from the testicles, not in normal tissue. Therefore, they are suitable for a specific tumor immunotherapy. We looked at different CTA (LAGE-1, PRAME, MAGE-C2, NY-ESO-1, SSX-2 and PAGE4) and their occurrence in prostatic cancer. Expression of CTA in various PCa cell lines and PCa material from patients was very heterogeneous. Only PAGE4 was expressed in primary PCa and in LnCaP cells as well as in hormone-dependent and hormone-refractory PCa probes. We conclude that PAGE4 should be further evaluated as a potential target for immunotherapy of PCa.

A previously unknown coronavirus (CoV) is the aetiological agent causing severe acute respiratory syndrome (SARS), for which an effective antiviral treatment is urgently needed. To enable the rapid and biosafe identification of coronavirus replicase inhibitors, we have generated a non-cytopathic, selectable replicon RNA (based on human CoV 229E) that can be stably maintained in eukaryotic cells. Most importantly, the replicon RNA mediates reporter gene expression as a marker for coronavirus replication. We have used a replicon RNA-containing cell line to test the inhibitory effect of several compounds that are currently being assessed for SARS treatment. Amongst those, interferon-α displayed the strongest inhibitory activity. Our results demonstrate that coronavirus replicon cell lines provide a versatile and safe assay for the identification of coronavirus replicase inhibitors. Once this technology is adapted to SARS-CoV replicon RNAs, it will allow high throughput screening for SARS-CoV replicase inhibitors without the need to grow infectious SARS-CoV.

Recent clinical studies indicate that the number of microbial infections (the "pathogen bur- den") critically determines the development and progression of atherosclerotic disease. Viruses or bacteria with a specific tropism for cells of the vascular wall may contribute to the initial vascular injury via direct cytopathic effects or via the induc- tion of genuine autoimmune responses. Immuno- pathological processes such as molecular mimicry, epitope spreading, or bystander activation of self- reactive lymphocytes most likely fuel the chronic inflammatory process in the vascular wall. Recog- nition of atherogenesis as a pathogen-driven, im- munopathological process makes this disease ame- nable to new treatment strategies such as vaccina- tion or immunomodulation. J. Leukoc. Biol. 76: 000-000; 2004.

ICln is an ion channel identified by expression cloning using a cDNA library from Madin-Darby canine kidney cells. In all organisms tested so far, only one transcript for the ICln protein could be identified. Here we show that two splice variants of the ICln ion channel can be found in Caenorhabditis elegans. Moreover, we show that these two splice variants of the ICln channel protein, which we termed IClnN1 and IClnN2, can be functionally reconstituted and tested in an artificial lipid bilayer. In these experiments, the IClnN1-induced currents showed no voltage-dependent inactivation, whereas the IClnN2-induced currents fully inactivated at positive potentials. The molecular entity responsible for the voltage-dependent inactivation of IClnN2 is a cluster of positively charged amino acids encoded by exon 2a, which is absent in IClnN1. Our experiments suggest a mechanism of channel inactivation that is similar to the “ball and chain” model proposed for the Shaker potassium channel,i.e. a cluster of positively charged amino acids hinders ion permeation through the channel by a molecular and voltage-dependent interaction at the inner vestibulum of the pore. This hypothesis is supported by the finding that synthetic peptides with the same amino acid sequence as the positive cluster can transform the IClnN1-induced current to the current observed after reconstitution of IClnN2. Furthermore, we show that the nematode ICln gene is embedded in an operon harboring two additional genes, which we termed Nx and Ny. Co-reconstitution of Nx and IClnN2 and functional analysis of the related currents revealed a functional interaction between the two proteins, as evidenced by the fact that the IClnN2-induced current in the presence of Nx was no longer voltage-sensitive. The experiments described indicate that the genome organization in nematodes allows an effective approach for the identification of functional partner proteins of ion channels.

1. It was postulated that swelling dependent chloride channels are involved in the proton secretion of parietal cells. Since omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 are structurally related to phenol derivatives known to block swelling dependent chloride channels, we set out to test, whether these substances--which are known to block the H,K-ATPase--could also lead to an inhibition of swelling-dependent chloride channels. Swelling-dependent chloride channels--characterized in many different cell types--show highly conserved biophysical and pharmacological features, therefore we investigated the effect of omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 on swelling-dependent chloride channels elicited in fibroblasts, after the reduction of the extracellular osmolarity. 2. Omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 are able to block swelling-dependent chloride channels (IClswell). 3. Lansoprazole and its protonated metabolite AG2000 act on at least two different sites of the IClswell protein: on an extracellular site which seems to be in a functional proximity to the nucleotide binding site, and on an intracellular site which allows the formation of disulfide-bridges. 4. The inhibition of the proton pump and the simultaneous blocking of chloride channels by omeprazole, lansoprazole and its acid activated sulphenamide form AG2000, as described here could be an effective mode to restrict proton secretion in parietal cells.

Endothelial cell ablation of the lymphotoxin-β receptor results in failure to develop peripheral lymph nodes and normal high endothelial venues, which impairs lymphocyte homing., The development of lymph nodes (LNs) and formation of LN stromal cell microenvironments is dependent on lymphotoxin-β receptor (LTβR) signaling. In particular, the LTβR-dependent crosstalk between mesenchymal lymphoid tissue organizer and hematopoietic lymphoid tissue inducer cells has been regarded as critical for these processes. Here, we assessed whether endothelial cell (EC)–restricted LTβR signaling impacts on LN development and the vascular LN microenvironment. Using EC-specific ablation of LTβR in mice, we found that conditionally LTβR-deficient animals failed to develop a significant proportion of their peripheral LNs. However, remnant LNs showed impaired formation of high endothelial venules (HEVs). Venules had lost their cuboidal shape, showed reduced segment length and branching points, and reduced adhesion molecule and constitutive chemokine expression. Due to the altered EC–lymphocyte interaction, homing of lymphocytes to peripheral LNs was significantly impaired. Thus, this study identifies ECs as an important LTβR-dependent lymphoid tissue organizer cell population and indicates that continuous triggering of the LTβR on LN ECs is critical for lymphocyte homeostasis.

Abs confer protection from secondary infection with Legionella pneumophila, the causative agent of a severe form of pneumonia known as Legionnaires’ disease. In this study, we demonstrate that Ab-mediated protection is effective across L. pneumophila serogroups, suggesting that Abs specific for conserved protein Ags are sufficient to mediate this protective effect. We used two independent methods to identify immunogenic L. pneumophila protein Ags, namely, the screening of a λ phage library representing the complete L. pneumophila genome and two-dimensional gel electrophoresis combined with Western blot analysis and protein spot identification by mass spectrometry. A total of 30 novel L. pneumophila B cell Ags were identified, the majority of which are located in or associated with the bacterial membrane, where they are accessible for Abs and, therefore, likely to be relevant for Ab-mediated protection against L. pneumophila. Selected B cell Ags were recombinantly expressed and tested in a vaccination protocol. Mice immunized with either single-protein Ags or an Ag combination showed reduced bacterial titers in bronchoalveolar lavage and lung after L. pneumophila challenge. To determine the clinical relevance of these findings, we tested Legionnaires’ disease patient sera for reactivity with the identified L. pneumophila Ags. The recognized Ags were indeed conserved across host species, because Abs specific for all three selected Ags could be detected in patient sera, rendering the identified protein Ags potential vaccine candidates.

Introduction: Few published in vitro studies have shown antitumor drug action or possible synergistic effects of fluoroquinolones. To assess the potential role of combination therapy, cytotoxic effects of ciprofloxacin and epirubicin alone and in combination were determined. Material and Methods: Human urothelial cancer cell lines HT1197 and HT1376 were exposed in vitro for 1 h to different concentrations of epirubicin (0.02–2 mg/ml) and for 72 h to ciprofloxacin (0.004–0.8 mg/ml). Cytotoxicity was determined using the microculture tetrazolium assay and flow cytometry. Synergistic cytotoxic effects were determined by calculating combination indices. Results: Median effect concentrations of epirubicin for HT1376 and HT1197 cells were as low as 124 and 117 µg/ml, respectively. Ciprofloxacin-treated cells exhibited profound cytotoxic effects at concentrations of 50–100 µg/ml, which is far below the intravesical concentration reached by standard oral application. In addition, a pronounced synergistic effect was found when the two treatments were combined. Conclusions: This study provides evidence that ciprofloxacin and epirubicin exhibit synergistic cytotoxic effects in vitro. After confirmatory animal experiments, future clinical studies of adjuvant chemotherapy after transurethral bladder resection may include treatment arms with combinations of fluoroquinolones based on the observed synergistic effects to reduce both side effects and costs.

Stromal cells provide the structural foundation of secondary lymphoid organs (SLOs), and regulate leukocyte access and cell migration within the different compartments of spleen and lymph nodes. Furthermore, several stromal cell subsets have been implied in shaping of T cell responses through direct presentation of antigen. Despite significant gain of knowledge on the biology of different SLO-resident stromal cell subsets, their molecular and functional characterization has remained incomplete. To address this need, we have generated a bacterial artificial chromosome (BAC)-transgenic mouse model that utilizes the podoplanin (pdpn) promoter to express the Cre-recombinase exclusively in stromal cells of SLOs. The characterization of the pdpn:Cre mouse revealed transgene expression in distinct subsets of fibroblastic reticular cells (FRCs) and lymphatic endothelial cells (LECs) in lymph nodes (LNs). Furthermore, the transgene facilitated the identification of a novel splenic perivascular stromal cell subpopulation that forms web-like structures around central arterioles. Assessment of the in vivo antigen presenting function of the genetically tagged stromal cells in pdpn:Cre mice revealed activation of both MHC I and II-restricted TCR transgenic T cells. Taken together, stromal pdpn:Cre-recombinase expression is well-suited to dissect antigen presentation by FRCs and LECs in LNs and by the novel splenic perivascular stromal cell population.

Secondary lymphoid organs (SLOs) are tissues that facilitate the induction of adaptive immune responses. These organs capture pathogens to limit their spread throughout the body, bring antigen-presenting cells into productive contact with their cognate lymphocytes and provide niches for the differentiation of immune effector cells. Therefore, the microanatomy of SLOs defines the ability of an organism to respond to pathogens. SLO microarchitecture is, at the same time, extremely adaptable to environmental changes. In this Review, we discuss recent insights into the function and plasticity of the SLO microenvironment with regards to antimicrobial immune defence.

Endothelial cells (ECs) presenting minor histocompatibility antigen (mhAg) are major target cells for alloreactive effector CD8+ T cells during chronic transplant rejection and graft-versus-host disease (GVHD). The contribution of ECs to T-cell activation, however, is still a controversial issue. In this study, we have assessed the antigen-presenting capacity of ECs in vivo using a transgenic mouse model with beta-galactosidase (β-gal) expression confined to the vascular endothelium (Tie2-LacZ mice). In a GVHD-like setting with adoptive transfer of β-gal–specific T-cell receptor–transgenic T cells, β-gal expression by ECs was not sufficient to either activate or tolerize CD8+ T cells. Likewise, transplantation of fully vascularized heart or liver grafts from Tie2-LacZ mice into nontransgenic recipients did not suffice to activate β-gal–specific CD8+ T cells, indicating that CD8+ T-cell responses against mhAg cannot be initiated by ECs. Moreover, we could show that spontaneous activation of β-gal–specific CD8+ T cells in Tie2-LacZ mice was exclusively dependent on CD11c+ dendritic cells (DCs), demonstrating that mhAgs presented by ECs remain immunologically ignored unless presentation by DCs is granted.

Dendritic cell (DC)-based immunotherapy has been hampered by the lack of suitable methods for antigen delivery. Here, we use biodegradable poly(D,L-lactide-co-glycolide) microspheres (PLGA-MS) as carriers of peptides and proteins for antigen delivery to human monocyte-derived DC (MoDC). Compared to soluble proteins, MHC classes I and II-restricted presentation of PLGA-MS-encapsulated proteins and peptides by MoDC was markedly prolonged and proteins were presented 50-fold more efficiently on class I molecules. The vaccination of mice with DC loaded with PLGA-MS-encapsulated proteins raised strong and persisting cytotoxic T cell responses. In conclusion, antigen delivery via PLGA-MS markedly enhanced the duration of antigen presentation by human MoDC and the potency of DC-based vaccination.

The control of dendritic cell (DC) migration is pivotal for the initiation of cellular immune responses. In this study, we demonstrate that the migration of human monocyte-derived (Mo)DCs as well as of ex vivo peripheral blood DCs toward CCL21, CXCL12, and C5a is stringently dependent on the presence of the proinflammatory mediator PGE2, although DCs expressed CXCR4 and C5aR on their surface and DC maturation was accompanied by CCR7 up-regulation independently of PGE2. The necessity of exogenous PGE2 for DC migration is not due to the suppression of PGE2 synthesis by IL-4, which is used for MoDC differentiation, because maturation-induced endogenous production of PGE2 cannot promote DC migration. Surprisingly, PGE2 was absolutely required at early time points of maturation to enable MoDC chemotaxis, whereas PGE2 addition during terminal maturation events was ineffective. In contrast to mouse DCs, which exclusively rely on EP4 receptor triggering for migration, human MoDCs require a signal mediated by EP2 or EP4 either alone or in combination. Our results provide clear evidence that PGE2 is a general and mandatory factor for the development of a migratory phenotype of human MoDCs as well as for peripheral blood myeloid DCs.

In order to provide a rapid and safe assay for the identification and evaluation of coronavirus replicase inhibitors, we have generated a non-cytopathic, selectable replicon RNA (based on human coronavirus 229E [HCoV-229E]) that can be stably maintained in eukaryotic cells. Stable, replicon RNA-containing cell lines that express green fluorescent protein (GFP) as a marker for coronavirus replication have been used to test the inhibitory effect of several compounds that are currently being assessed for SARS treatment or have been predicted to target replicative proteins. Amongst those, interferonalpha and cinanserin displayed the strongest inhibitory activities. Interestingly, cinanserin is a well-characterized serotonin antagonist that has already undergone preliminary clinical testing in humans in the 1960s. Cinanserin has been identified as candidate inhibitor of the SARS-CoV 3C-like proteinase by virtual screening of a database containing structural information of more than 8,000 existing drugs using a docking approach for potential binding to the SARS-CoV 3C-like proteinase. Subsequently, binding of cinanserin to bacterially expressed SARS-CoV 3C-like proteinase and inhibition of its enzymatic activity was demonstrated experimentally. Antiviral activity of cinaserin was further evaluated in two tissue culture–based assays. First, we have used our safe replicon assay and could demonstrate a strong inhibitory activity of cinanserin. Second, we could demonstrate a strong inhibition (up to 4 log reduction of virus RNA and infectious particles) of SARS-CoV and HCoV-229E replication in tissue culture.

BACKGROUND Dendritic cell (DC)-based vaccination has been investigated as immunotherapy for several types of cancer. A potential drawback to vaccination with autologous monocyte-derived DCs (MoDCs) could be that MoDCs from patients are functionally impaired. In case of androgen-independent prostate cancer (CaP), the cancer itself, diverse prior therapies, and the hormone manipulation may affect the immune system. METHODS MoDCs from patients suffering from androgen-independent CaP were generated according to a clinically applicable protocol to evaluate the phenotype, maturation capacity, migration, and T-cell stimulation of these cells compared with those generated from tumor-free donors. RESULTS MoDCs generated from CaP patients could be fully matured and efficiently migrated towards the chemokine CCL21. They had a strong potency to activate allogeneic CD4+ and CD8+ T-cells and to present antigens to specific CTL. CONCLUSIONS Our data suggest that MoDCs from patients with androgen-independent CaP are functionally intact and hence qualify as cellular vaccines for immunotherapy of advanced stage CaP. © 2005 Wiley-Liss, Inc.

The 3C-like proteinase (3CLpro) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is one of the most promising targets for anti-SARS-CoV drugs due to its crucial role in the viral life cycle. In this study, a database containing structural information of more than 8,000 existing drugs was virtually screened by a docking approach to identify potential binding molecules of SARS-CoV 3CLpro. As a target for screening, both a homology model and the crystallographic structure of the binding pocket of the enzyme were used. Cinanserin (SQ 10,643), a well-characterized serotonin antagonist that has undergone preliminary clinical testing in humans in the 1960s, showed a high score in the screening and was chosen for further experimental evaluation. Binding of both cinanserin and its hydrochloride to bacterially expressed 3CLproof SARS-CoV and the related human coronavirus 229E (HCoV-229E) was demonstrated by surface plasmon resonance technology. The catalytic activity of both enzymes was inhibited with 50% inhibitory concentration (IC50) values of 5 μM, as tested with a fluorogenic substrate. The antiviral activity of cinanserin was further evaluated in tissue culture assays, namely, a replicon system based on HCoV-229E and quantitative test assays with infectious SARS-CoV and HCoV-229E. All assays revealed a strong inhibition of coronavirus replication at nontoxic drug concentrations. The level of virus RNA and infectious particles was reduced by up to 4 log units, with IC50values ranging from 19 to 34 μM. These findings demonstrate that the old drug cinanserin is an inhibitor of SARS-CoV replication, acting most likely via inhibition of the 3CL proteinase.

Characterization of autoantigen-specific CD4+ T cells at the single cell level is crucial for understanding the immunopathological mechanisms underlying autoimmune diseases. Cardiac myosin heavy chain (myhca) is the major autoantigen associated with autoimmune myocarditis both in humans and in experimental autoimmune myocarditis (EAM) in mice. In the current study, we evaluated two methods for the enumeration and phenotypic characterization of myhca-specific CD4+ T cells during the course of EAM. Both enzyme-linked immunospot (ELISPOT) and cytokine flow cytometry (CFC) assays were suitable for the detection and characterization of myhca-specific Th cells during acute myocardial inflammation and the late healing phase of the disease. Cytokine production of myhca-specific Th cells was restricted to interferon-γ (IFNγ). Only trace amounts of the Th2 cytokines IL-4 and IL-5 could be detected. Concomitant surface marker analysis in the CFC assay revealed the prototypical effector phenotype of myhca-specific Th1 cells during the acute phase of the disease. Taken together, the combination of both methods appears to be most appropriate for a comprehensive ex vivo single cell analysis of Th cells in heart-specific autoimmune disorders.