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Abstract Cancer resistance to immune checkpoint inhibitors motivated investigations into leveraging the immunostimulatory properties of radiotherapy to overcome immune evasion and to improve treatment response. However, clinical benefits of radiotherapy-immunotherapy combinations have been modest. Routine concomitant tumor-draining lymph node irradiation (DLN IR) might be the culprit. As crucial sites for generating anti-tumor immunity, DLNs are indispensable for the in situ vaccination effect of radiotherapy. Simultaneously, DLN sparing is often not feasible due to metastatic spread. Using murine models of metastatic disease in female mice, here we demonstrate that delayed (adjuvant), but not neoadjuvant, DLN IR overcomes the detrimental effect of concomitant DLN IR on the efficacy of radio-immunotherapy. Moreover, we identify IR-induced disruption of the CCR7-CCL19/CCL21 homing axis as a key mechanism for the detrimental effect of DLN IR. Our study proposes delayed DLN IR as a strategy to maximize the efficacy of radio-immunotherapy across different tumor types and disease stages.

Summary: Eosinophils are involved in host protection against multicellular organisms. However, their recruitment to the mesenteric lymph node (mLN) during type 2 immunity is understudied. Our results demonstrate that eosinophil association with lymphoid stromal niches constructed by fibroblastic reticular cells (FRCs) and lymphatic endothelial cells is diminished in mice selectively lacking interleukin (IL)-4Rα or lymphotoxin-β (LTβ) expression on B cells. Furthermore, eosinophil survival, activation, and enhanced Il1rl1 receptor expression are driven by stromal cell and B cell dialogue. The ligation of lymphotoxin-β receptor (LTβR) on FRCs improves eosinophil survival and significantly augments IL-33 expression and eosinophil homing to the mLN, thus confirming the significance of lymphotoxin signaling for granulocyte recruitment. Eosinophil-deficient ΔdblGATA-1 mice show diminished mLN expansion, reduced interfollicular region (IFR) alarmin expression, and delayed helminth clearance, elucidating their importance in type 2 immunity. These findings provide insight into dialogue between stromal cells and B cells, which govern mLN eosinophilia, and the relevance of these mechanisms during type 2 immunity.

Fibroblastic reticular cells (FRCs) support localisation of immune cells in secondary lymphoid tissues but less is known about the lamina propria. Here the authors use scRNA-seq and intestinal infection to characterise FRCs in the intestinal lamina propria and show specialised niches that foster innate lymphoid cells during homeostasis and infection.

Acute graft versus host disease (aGvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT) inflicted by alloreactive T cells primed in secondary lymphoid organs (SLOs) and subsequent damage to aGvHD target tissues. In recent years, Treg transfer and/or expansion has emerged as a promising therapy to modulate aGvHD. However, cellular niches essential for fostering Tregs to prevent aGvHD have not been explored. Here, we tested whether and to what extent MHC class II (MHCII) expressed on Ccl19+ fibroblastic reticular cells (FRCs) shape the donor CD4+ T cell response during aGvHD. Animals lacking MHCII expression on Ccl19-Cre–expressing FRCs (MHCIIΔCcl19) showed aberrant CD4+ T cell activation in the effector phase, resulting in exacerbated aGvHD that was associated with significantly reduced expansion of Foxp3+ Tregs and invariant NK T (iNKT) cells. Skewed Treg maintenance in MHCIIΔCcl19 mice resulted in loss of protection from aGvHD provided by adoptively transferred donor Tregs. In contrast, although FRCs upregulated costimulatory surface receptors, and although they degraded and processed exogenous antigens after myeloablative irradiation, FRCs were dispensable to activate alloreactive CD4+ T cells in 2 mouse models of aGvHD. In summary, these data reveal an immunoprotective, MHCII-mediated function of FRC niches in secondary lymphoid organs (SLOs) after allo-HCT and highlight a framework of cellular and molecular interactions that regulate CD4+ T cell alloimmunity.

Lymphocytic choriomeningitis virus (LCMV)-based viral vectors have been shown to induce potent antitumor immune responses. Here the authors show that a LCMV-based vaccine vector remodels the tumor-associated fibroblastic stroma, sustaining CD8+ T cell activation and reducing tumor growth in a preclinical model of melanoma.

Objectives To correlate immune responses following a two-dose regimen of mRNA anti-SARS-CoV-2 vaccines in patients with rheumatoid arthritis (RA) to the development of a potent neutralising antiviral activity.Methods The RECOVER study was a prospective, monocentric study including patients with RA and healthy controls (HCs). Assessments were performed before, and 3, 6, 12 and 24 weeks, after the first vaccine dose, respectively, and included IgG, IgA and IgM responses (against receptor binding domain, S1, S2, N), IFN-γ ELISpots as well as neutralisation assays.Results In patients with RA, IgG responses developed slower with lower peak titres compared with HC. Potent neutralising activity assessed by a SARS-CoV-2 pseudovirus neutralisation assay after 12 weeks was observed in all 21 HCs, and in 60.3% of 73 patients with RA. A significant correlation between peak anti-S IgG levels 2 weeks after the second vaccine dose and potent neutralising activity against SARS-CoV-2 was observed at weeks 12 and 24. The analysis of IgG, IgA and IgM isotype responses to different viral proteins demonstrated a delay in IgG but not in IgA and IgM responses. T cell responses were comparable in HC and patients with RA but declined earlier in patients with RA.Conclusion In patients with RA, vaccine-induced IgG antibody levels were diminished, while IgA and IgM responses persisted, indicating a delayed isotype switch. Anti-S IgG levels 2 weeks after the second vaccine dose correlate with the development of a potent neutralising activity after 12 and 24 weeks and may allow to identify patients who might benefit from additional vaccine doses or prophylactic regimen.

In lymphopenic environments, secondary lymphoid organs regulate the size of B and T cell compartments by supporting the homeostatic proliferation of mature lymphocytes. The molecular mechanisms underlying these responses and their functional consequences remain incompletely understood. To evaluate homeostasis of the mature B cell pool during lymphopenia, we turned to an adoptive transfer model of purified follicular B cells into Rag2–/– mouse recipients. Highly purified follicular B cells transdifferentiated into marginal zone–like B cells when transferred into Rag2–/– lymphopenic hosts but not into wild-type hosts. In lymphopenic spleens, transferred B cells gradually lost their follicular phenotype and acquired characteristics of marginal zone B cells, as judged by cell surface phenotype, expression of integrins and chemokine receptors, positioning close to the marginal sinus, and an ability to rapidly generate functional plasma cells. Initiation of follicular to marginal zone B cell transdifferentiation preceded proliferation. Furthermore, the transdifferentiation process was dependent on Notch2 receptors in B cells and expression of Delta-like 1 Notch ligands by splenic Ccl19-Cre+ fibroblastic stromal cells. Gene expression analysis showed rapid induction of Notch-regulated transcripts followed by upregulated Myc expression and acquisition of broad transcriptional features of marginal zone B cells. Thus, naive mature B cells are endowed with plastic transdifferentiation potential in response to increased stromal Notch ligand availability during lymphopenia.

Cancer vaccination aims at inducing an adaptive immune response against tumor-derived antigens. In this study, we utilize recombinant human adenovirus serotype 5 (rAd5) and recombinant lymphocytic choriomeningitis virus (rLCMV)-based vectors expressing the melanocyte differentiation antigen gp100. In contrast to single or homologous vaccination, a heterologous prime boost vaccination starting with a rAd5-gp100 prime immunization followed by a rLCMV-gp100 boost injection induces a high magnitude of polyfunctional gp100-specific CD8+ T cells. Our data indicate that an optimal T cell induction is dependent on the order and interval of the vaccinations. A prophylactic prime boost vaccination with rAd5- and rLCMV-gp100 protects mice from a B16.F10 melanoma challenge. In the therapeutic setting, combination of the vaccination with low-dose cyclophosphamide showed a synergistic effect and significantly delayed tumor growth. Our findings suggest that heterologous viral vector prime boost immunizations can mediate tumor control in a mouse melanoma model.

Morphogens such as chemokines form gradients to direct graded responses and modulate cell behaviors. Here the authors show, using imaging and computer simulation, that the chemokine CXCL13 originated from follicular reticular cells in the lymph nodes forms both soluble and immobilized gradients to regulate B cell recruitment and migration.

Fibroblastic reticular cells (FRC) are important for lymph node (LN) structure and function. Here the authors show that the YAP/TAZ complex downstream of Hippo signalling regulates FRC commitment and maturation, with YAP/TAZ deficiency impairing FRC differentiation, while hyperactivation of YAZ/TAZ inducing myofibroblastic FRCs and LN fibrosis.

The white pulp of spleen is an important immune structure dynamically modulated during development and immune responses. Here the authors define, using multi-color lineage tracing and single-cell transcriptome analysis, the subset distribution and differentiation trajectory of fibroblastic reticular cells to serve structural insights for splenic white pulps.

Abstract Background Long-term survival of stage IV melanoma patients has improved significantly with the development of immune checkpoint inhibitors (CIs). Reliable biomarkers to predict response and clinical outcome are needed. Methods We investigated the role of melanoma-associated antibodies as predictive markers for CI therapy in two independent cohorts. In cohort 1, a prospective study, we measured specific antibodies before treatment, after one week and after six to nine weeks of treatment. Cohort 2 consisted of serum samples prior to CI therapy initiation. ELISA assays were performed to quantify specific IgG directed against melanocyte differentiation antigens tyrosinase-related proteins 1 and 2 (TRP1/TYRP1 and TRP2/TYRP2), glycoprotein 100 (gp100), MelanA/MART1, and the cancer-testis antigen NY-ESO-1. Response was defined as either complete or partial remission on CT scan according to RECIST 1.1. Results In cohort 1, baseline levels of these antibodies were higher in the responder group, although statistical significance was only reached for NY-ESO-1 (p = 0.007). In cohort 2, significantly higher antibody baseline levels for MelanA/MART1 (p = 0.003) and gp100 (p = 0.029) were found. After pooling the results from both cohorts, higher levels of MelanA/MART1 (p = 0.013), TRP1/TYRP1 (p = 0.048), TRP2/TYRP2 (p = 0.047) and NY-ESO-1 (p = 0.005) specific antibodies at baseline were independently associated with response. Conclusions Melanoma-associated antibodies may be candidate biomarkers for response and survival in metastatic melanoma patients being treated with CIs. These markers may be used to complement patient assessment, in combination with PD-L1 status, tumor-infiltrating lymphocytes and tumor mutational burden, with the aim to predict outcome of CI treatment in patients with metastatic melanoma. Trial registration Ethikkommission Ostschweiz, EKOS 16/079 https://ongoingprojects.swissethics.ch/runningProjects_list.php?q=%28BASECID~contains~2016-00998%29&orderby=dBASECID.

Squamous cell carcinoma of the tonsil is one of the most frequent cancers of the oropharynx. The escalating rate of tonsil cancer during the last decades is associated with the increase of high risk-human papilloma virus (HR-HPV) infections. While the microbiome in oropharyngeal malignant diseases has been characterized to some extent, the microbial colonization of HR-HPV-associated tonsil cancer remains largely unknown. Using 16S rRNA gene amplicon sequencing, we have characterized the microbiome of human palatine tonsil crypts in patients suffering from HR-HPV-associated tonsil cancer in comparison to a control cohort of adult sleep apnea patients. We found an increased abundance of the phyla Firmicutes and Actinobacteria in tumor patients, whereas the abundance of Spirochetes and Synergistetes was significantly higher in the control cohort. Furthermore, the accumulation of several genera such as Veillonella, Streptococcus and Prevotella_7 in tonsillar crypts was associated with tonsil cancer. In contrast, Fusobacterium, Prevotella and Treponema_2 were enriched in sleep apnea patients. Machine learning-based bacterial species analysis indicated that a particular bacterial composition in tonsillar crypts is tumor-predictive. Species-specific PCR-based validation in extended patient cohorts confirmed that differential abundance of Filifactor alocis and Prevotella melaninogenica is a distinct trait of tonsil cancer. This study shows that tonsil cancer patients harbor a characteristic microbiome in the crypt environment that differs from the microbiome of sleep apnea patients on all phylogenetic levels. Moreover, our analysis indicates that profiling of microbial communities in distinct tonsillar niches provides microbiome-based avenues for the diagnosis of tonsil cancer.

Immune checkpoint inhibitors (ICIs) have improved the survival of patients with non-small cell lung cancer (NSCLC) by reinvigorating tumor-specific T cell responses. However, the specificity of such T cells and the human leukocyte antigen (HLA)-associated epitopes recognized, remain elusive. In this study, we identified NSCLC T cell epitopes of recently described NSCLC-associated antigens, termed keratinocyte differentiation antigens. Epitopes of these antigens were presented by HLA-A 03:01 and HLA-C 04:01 and were associated with responses to ICI therapy. Patients with CD8+ T cell responses to these epitopes had improved overall and progression-free survival. T cells specific for such epitopes could eliminate HLA class I-matched NSCLC cells ex vivo and were enriched in patient lung tumors. The identification of novel lung cancer HLA-associated epitopes that correlate with improved ICI-dependent treatment outcomes suggests that keratinocyte-specific proteins are important tumor-associated antigens in NSCLC. These findings improve our understanding of the mechanisms of ICI therapy and may help support the development of vaccination strategies to improve ICI-based treatment of these tumors.

The growth of lymph nodes in response to infection requires lymphangiogenesis. Dubey et al. show that the mesenteric lymph node lymphangiogenesis upon helminth infection depends on the signaling loop between the B and fibroblastic reticular cells (FRCs), whereby the FRCs respond to lymphotoxin secreted by B cells by releasing B cell activating factor.

Peripheral tolerance is crucial for avoiding activation of self-reactive T cells to tissue-restricted antigens. Sterile tissue injury can break peripheral tolerance, but it is unclear how autoreactive T cells get activated in response to self. An example of a sterile injury is myocardial infarction (MI). We hypothesized that tissue necrosis is an activator of dendritic cells (DCs), which control tolerance to self-antigens. DC subsets of a murine healthy heart consisted of IRF8-dependent conventional (c)DC1, IRF4-dependent cDC2, and monocyte-derived DCs. In steady state, cardiac self-antigen α-myosin was presented in the heart-draining mediastinal lymph node (mLN) by cDC1s, driving the proliferation of antigen-specific CD4+ TCR-M T cells and their differentiation into regulatory cells (Tregs). Following MI, all DC subsets infiltrated the heart, whereas only cDCs migrated to the mLN. Here, cDC2s induced TCR-M proliferation and differentiation into interleukin-(IL)-17/interferon-(IFN)γ-producing effector cells. Thus, cardiac-specific autoreactive T cells get activated by mature DCs following myocardial infarction.

Summary: Fibroblastic reticular cells (FRCs) form a road-like cellular network in lymph nodes (LNs) that provides essential chemotactic, survival, and regulatory signals for immune cells. While the topological characteristics of the FRC network have been elaborated, the network properties of the micro-tubular conduit system generated by FRCs, which drains lymph fluid through a pipeline-like system to distribute small molecules and antigens, has remained unexplored. Here, we quantify the crucial 3D morphometric parameters and determine the topological properties governing the structural organization of the intertwined networks. We find that the conduit system exhibits lesser small-worldness and lower resilience to perturbation compared to the FRC network, while the robust topological organization of both networks is maintained in a lymphotoxin-β-receptor-independent manner. Overall, the high-resolution topological analysis of the “roads-and-pipes” networks highlights essential parameters underlying the functional organization of LN micro-environments and will, hence, advance the development of multi-scale LN models. : Novkovic et al. elaborate the topological organization of the FRC network and conduit system in lymph nodes and demonstrate their distinct small-world topologies, which are maintained in a lymphotoxin-β-receptor-independent manner. The FRC network exhibited higher tolerance and robustness to perturbation than the conduit system did. Keywords: lymph node, fibroblastic reticular cell, stromal cell, topology, network, conduit system, robustness, graph theory

Summary: Adaptive type 2 immune responses against the intestinal helminth Heligmosomoides polygyrus (Hp) require the interaction of follicle-associated CXCR5+ dendritic cells with naive T cells in the draining mesenteric lymph nodes (mLNs). However, the source of CXCL13 responsible for attracting CXCR5+ dendritic cells has remained unclear. Using multiplex imaging combined with deep tissue analysis, we observed new CXCL13+ fibroblastic reticular cells surrounding paracortical and cortical B cell follicles in the mLNs of infected mice. CXCL13+ fibroblasts expressed markers of marginal reticular cells (MRCs), and their expansion required lymphotoxin (LT)-dependent interactions between IL-4Rα-expressing B cells and CCL19+ fibroblasts. Infection-induced follicles did not necessarily contain follicular dendritic cells (FDCs), indicating that CXCL13+ fibroblasts may instead drive their formation. These data reveal a role for lymphotoxin signaling to CCL19+ fibroblasts in the development of CXCL13+ MRC-like cells and adaptive type 2 immunity in response to helminth infection. : MRCs are a specialized, CXCL13-producing, stromal population located beneath the subcapsular sinus of lymph nodes. Dubey et al. demonstrate that intestinal helminth infection drives the remodeling and development of MRCs toward the paracortical region within the draining mLNs and requires the expression of type 2 cytokines and intimate B cell-stromal cell interactions. Keywords: marginal reticular cells, fibroblastic reticular cells, IL-4Rα, helminth infection, Heligmosomoides polygyrus, interfollicular region, CXCL13, mesenteric lymph node, MAdCAM-1, intestinal infection

Vesicular stomatitis virus (VSV) is an insect-transmitted rhabdovirus that is neurovirulent in mice. Upon peripheral VSV infection, CD169+ subcapsular sinus (SCS) macrophages capture VSV in the lymph, support viral replication, and prevent CNS neuroinvasion. To date, the precise mechanisms controlling VSV infection in SCS macrophages remain incompletely understood. Here, we show that Toll-like receptor-7 (TLR7), the main sensing receptor for VSV, is central in controlling lymph-borne VSV infection. Following VSV skin infection, TLR7−/− mice display significantly less VSV titers in the draining lymph nodes (dLN) and viral replication is attenuated in SCS macrophages. In contrast to effects of TLR7 in impeding VSV replication in the dLN, TLR7−/− mice present elevated viral load in the brain and spinal cord highlighting their susceptibility to VSV neuroinvasion. By generating novel TLR7 floxed mice, we interrogate the impact of cell-specific TLR7 function in anti-viral immunity after VSV skin infection. Our data suggests that TLR7 signaling in SCS macrophages supports VSV replication in these cells, increasing LN infection and may account for the delayed onset of VSV-induced neurovirulence observed in TLR7−/− mice. Overall, we identify TLR7 as a novel and essential host factor that critically controls anti-viral immunity to VSV. Furthermore, the novel mouse model generated in our study will be of valuable importance to shed light on cell-intrinsic TLR7 biology in future studies.

Summary: Secondary lymphoid tissues provide specialized niches for the initiation of adaptive immune responses and undergo a remarkable expansion in response to inflammatory stimuli. Although the formation of B cell follicles was previously thought to be restricted to the postnatal period, we observed that the draining mesenteric lymph nodes (mLN) of helminth-infected mice form an extensive number of new, centrally located, B cell follicles in response to IL-4Rα-dependent inflammation. IL-4Rα signaling promoted LTα1β2 (lymphotoxin) expression by B cells, which then interacted with CCL19 positive stromal cells to promote lymphoid enlargement and the formation of germinal center containing B cell follicles. Importantly, de novo follicle formation functioned to promote both total and parasite-specific antibody production. These data reveal a role for type 2 inflammation in promoting stromal cell remodeling and de novo follicle formation by promoting B cell-stromal cell crosstalk. : Intestinal helminth infection drives type 2 immune responses in the draining mLN. Dubey et al. demonstrate in mice that type 2 inflammation drives lymphoid remodeling resulting from lymphotoxin-dependent crosstalk between B cells and FRCs. Such crosstalk promotes de novo follicle formation and supports the production of parasite-specific antibodies.

Autoimmune myocarditis often leads to dilated cardiomyopathy (DCM). Although T cell reactivity to cardiac self-antigen is common in the disease, it is unknown which antigen presenting cell (APC) triggers autoimmunity. Experimental autoimmune myocarditis (EAM) was induced by immunizing mice with α-myosin loaded bone marrow APCs cultured in GM-CSF. APCs found in such cultures include conventional type 2 CD11b+ cDCs (GM-cDC2s) and monocyte-derived cells (GM-MCs). However, only α-myosin loaded GM-cDC2s could induce EAM. We also studied antigen presenting capacity of endogenous type 1 CD24+ cDCs (cDC1s), cDC2s, and MCs for α-myosin-specific TCR-transgenic TCR-M CD4+ T cells. After EAM induction, all cardiac APCs significantly increased and cDCs migrated to the heart-draining mediastinal lymph node (LN). Primarily cDC2s presented α-myosin to TCR-M cells and induced Th1/Th17 differentiation. Loss of IRF4 in Irf4fl/fl.Cd11cCre mice reduced MHCII expression on GM-cDC2s in vitro and cDC2 migration in vivo. However, partly defective cDC2 functions in Irf4fl/fl.Cd11cCre mice did not suppress EAM. MCs were the largest APC subset in the inflamed heart and produced pro-inflammatory cytokines. Targeting APC populations could be exploited in the design of new therapies for cardiac autoimmunity.

The spatiotemporal regulation of immune responses in the lymph node (LN) depends on its sophisticated tissue architecture, consisting of several subcompartments supported by distinct fibroblastic stromal cells (FSCs). However, the intricate details of stromal structures and associated FSC subsets are not fully understood. Using several gene reporter mice, we sought to discover unrecognized stromal structures and FSCs in the LN. The four previously identified FSC subsets in the cortex are clearly distinguished by the expression pattern of reporters including PDGFRβ, CCL21-ser, and CXCL12. Herein, we identified a unique FSC subset expressing both CCL21-ser and CXCL12 in the deep cortex periphery (DCP) that is characterized by preferential B cell localization. This subset was clearly different from CXCL12highLepRhigh FSCs in the medullary cord, which harbors plasma cells. B cell localization in the DCP was controlled chiefly by CCL21-ser and, to a lesser extent, CXCL12. Moreover, the optimal development of the DCP as well as medulla requires B cells. Together, our findings suggest the presence of a unique microenvironment in the cortex-medulla boundary and offer an advanced view of the multi-layered stromal framework constructed by distinct FSC subsets in the LN.

Adaptive immune responses develop in secondary lymphoid organs such as lymph nodes (LNs) in a well-coordinated series of interactions between migrating immune cells and resident stromal cells. Although many processes that occur in LNs are well understood from an immunological point of view, our understanding of the fundamental organization and mechanisms that drive these processes is still incomplete. The aim of systems biology approaches is to unravel the complexity of biological systems and describe emergent properties that arise from interactions between individual constituents of the system. The immune system is greater than the sum of its parts, as is the case with any sufficiently complex system. Here, we review recent work and developments of computational LN models with focus on the structure and organization of the stromal cells. We explore various mathematical studies of intranodal T cell motility and migration, their interactions with the LN-resident stromal cells, and computational models of functional chemokine gradient fields and lymph flow dynamics. Lastly, we discuss briefly the importance of hybrid and multi-scale modeling approaches in immunology and the technical challenges involved.

Cytomegalovirus (CMV) infection induces an atypical CD8 T cell response, termed inflationary, that is characterised by accumulation and maintenance of high numbers of effector memory like cells in circulation and peripheral tissues-a feature being successfully harnessed for vaccine purposes. Although stability of this population depends on recurrent antigen encounter, the requirements for prolonged survival in peripheral tissues remain unknown. Here, we reveal that murine CMV-specific inflationary CD8 T cells are maintained in an antigen-independent manner and have a half-life of 12 weeks in the lung tissue. This half-life is drastically longer than the one of phenotypically comparable inflationary effector cells. IL-15 alone, and none of other common γ-cytokines, was crucial for survival of inflationary cells in peripheral organs. IL-15, mainly produced by non-hematopoietic cells in lung tissue and being trans-presented, promoted inflationary T cell survival by increasing expression of Bcl-2. These results indicate that inflationary CD8 T cells are not just simply effector-like cells, rather they share properties of both effector and memory CD8 T cells and they appear to be long-lived cells compared to the effector cells from acute virus infections.

The tumor microenvironment harbors cancer-associated fibroblasts that function as major modulators of cancer progression. Here, we assessed to which extent distinct cancer-associated fibroblast subsets impact mammary carcinoma growth and cancer cell stemness in an orthotopic murine model. We found that fibroblasts expressing the Cre recombinase under the control of the interleukin 7 promoter occupied mainly the tumor margin where they physically interacted with tumor cells. Intratumoral ablation of interleukin 7-expressing fibroblasts impaired breast tumor growth and reduced the clonogenic potential of cancer cells. Moreover, cDNA expression profiling revealed a distinct oncogenic signature of interleukin 7-producing fibroblasts. In particular, Cxcl12 expression was strongly enhanced in interleukin 7-producing fibroblasts and cell type-specific genetic ablation and systemic pharmacological inhibition revealed that the CXCL12/CXCR4 axis impacts breast tumor cell stemness. Elevated expression of CXCL12 and other stem cell factors in primary human breast cancer-associated fibroblasts indicates that certain fibroblast populations support tumor cell stemness and thereby promote breast cancer growth.

Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I). This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU) activity is key to prevent early induction of double-stranded RNA (dsRNA) host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis-within the replicase complex-suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses.

Vaccination against complex pathogens such as typhoidal and non-typhoidal Salmonella requires the concerted action of different immune effector mechanisms. Outer membrane proteins (Omps) of Salmonella Typhi are potent immunogens, which elicit long-lasting and protective immunity. Here, we followed the evolution of S. Typhi OmpC and F-specific T and B cell responses in healthy volunteers after vaccination with the vaccine strain Ty21a. To follow humoral and cellular immune responses, pre- and post-vaccination samples (PBMC, serum and stool) collected from 15 vaccinated and 5 non-vaccinated individuals. Immunoglobulin levels were assessed in peripheral blood by enzyme-linked immunosorbent assay. B cell and T cell activation were analyzed by flow cytometry. We observed a significant increase of circulating antibody-secreting cells and maximal Omp-specific serum IgG titers at day 25 post vaccination, while IgA titers in stool peaked at day 60. Likewise, Omp-specific CD4+ T cells in peripheral blood showed the highest expansion at day 60 post vaccination, concomitant with a significant increase in IFN-γ and TNFα production. These results indicate that S. Typhi Omp-specific B cell responses and polyfunctional CD4+ T cell responses evolve over a period of at least two months after application of the live attenuated vaccine. Moreover, these findings underscore the potential of S. Typhi Omps as subunit vaccine components.ISRCTN18360696.

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.

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.

Microenvironment and activation signals likely imprint heterogeneity in the lymphatic endothelial cell (LEC) population. Particularly LECs of secondary lymphoid organs are exposed to different cell types and immune stimuli. However, our understanding of the nature of LEC activation signals and their cell source within the secondary lymphoid organ in the steady state remains incomplete. Here we show that integrin alpha 2b (ITGA2b), known to be carried by platelets, megakaryocytes and hematopoietic progenitors, is expressed by a lymph node subset of LECs, residing in medullary, cortical and subcapsular sinuses. In the subcapsular sinus, the floor but not the ceiling layer expresses the integrin, being excluded from ACKR4+ LECs but overlapping with MAdCAM-1 expression. ITGA2b expression increases in response to immunization, raising the possibility that heterogeneous ITGA2b levels reflect variation in exposure to activation signals. We show that alterations of the level of receptor activator of NF-κB ligand (RANKL), by overexpression, neutralization or deletion from stromal marginal reticular cells, affected the proportion of ITGA2b+ LECs. Lymph node LECs but not peripheral LECs express RANK. In addition, we found that lymphotoxin-β receptor signaling likewise regulated the proportion of ITGA2b+ LECs. These findings demonstrate that stromal reticular cells activate LECs via RANKL and support the action of hematopoietic cell-derived lymphotoxin.

γδ T cells contribute to first line immune defense, particularly through their ability for rapid production of proinflammatory cytokines. The cytokine profile of γδ T cells is hard-wired already during thymic development. Yet, the molecular pathways underlying this phenomenon are incompletely understood. Here we show that signaling via the NFκB-inducing kinase (NIK) is essential for the formation of a fully functional γδ T cell compartment. In the absence of NIK, development of Vγ5+ dendritic epidermal T cells (DETCs) was halted in the embryonic thymus, and impaired NIK function caused a selective loss of IL-17 expression by γδ T cells. Using a novel conditional mutant of NIK, we could show in vivo that NIK signaling in thymic epithelial cells is essential for the thymic hardwiring of γδ T cell cytokine production.

Heart diseases are an important cause of morbidity and mortality in industrialized countries. Dilated cardiomyopathy (DCM), one of the most common heart diseases, may be the consequence of infectionassociated myocardits. Coxsackievirus B3 (CVB3) can be frequently detected in the inflamed heart muscle. CVB3-induced acute myocarditis is most likely the consequence of direct virus-induced myocyte damage, whereas chronic CVB3 infection-associated heart disease is dominated by its immunopathological sequelae. Bona fide autoimmunity, for example, directed against cardiac myosin, may favor chronic destructive immune damage in the heart muscle and thereby promote the development of DCM. The immunopathogenesis of myocarditis and subsequent DCM induced either by pathogens or autoantigens can be investigated in well-established animal models. In this article, we review recent studies on the role of viruses, with particular emphasis on CVB3, and different immunological effector mechanisms in initiation and progression of myocarditis.

Reversible lysine acetylation plays an important role in the regulation of T cell responses. HDAC1 has been shown to control peripheral T helper cells, however the role of HDAC1 in CD8+ T cell function remains elusive. By using conditional gene targeting approaches, we show that LckCre-mediated deletion of HDAC1 led to reduced numbers of thymocytes as well as peripheral T cells, and to an increased fraction of CD8+CD4- cells within the CD3/TCRβlo population, indicating that HDAC1 is essential for the efficient progression of immature CD8+CD4- cells to the DP stage. Moreover, CD44hi effector CD8+ T cells were enhanced in mice with a T cell-specific deletion of HDAC1 under homeostatic conditions and HDAC1-deficient CD44hi CD8+ T cells produced more IFNγ upon ex vivo PMA/ionomycin stimulation in comparison to wild-type cells. Naïve (CD44l°CD62L+) HDAC1-null CD8+ T cells displayed a normal proliferative response, produced similar amounts of IL-2 and TNFα, slightly enhanced amounts of IFNγ, and their in vivo cytotoxicity was normal in the absence of HDAC1. However, T cell-specific loss of HDAC1 led to a reduced anti-viral CD8+ T cell response upon LCMV infection and impaired expansion of virus-specific CD8+ T cells. Taken together, our data indicate that HDAC1 is required for the efficient generation of thymocytes and peripheral T cells, for proper CD8+ T cell homeostasis and for an efficient in vivo expansion and activation of CD8+ T cells in response to LCMV infection.

Efficacy of antitumor vaccination depends to a large extent on antigen targeting to dendritic cells (DCs). Here, we assessed antitumor immunity induced by attenuated coronavirus vectors which exclusively target DCs in vivo and express either lymphocyte- or DC-activating cytokines in combination with a GFP-tagged model antigen. Tracking of in vivo transduced DCs revealed that vectors encoding for Fms-like tyrosine kinase 3 ligand (Flt3L) exhibited a higher capacity to induce DC maturation compared to vectors delivering IL-2 or IL-15. Moreover, Flt3L vectors more efficiently induced tumor-specific CD8(+) T cells, expanded the epitope repertoire, and provided both prophylactic and therapeutic tumor immunity. In contrast, IL-2- or IL-15-encoding vectors showed a substantially lower efficacy in CD8(+) T cell priming and failed to protect the host once tumors had been established. Thus, specific in vivo targeting of DCs with coronavirus vectors in conjunction with appropriate conditioning of the microenvironment through Flt3L represents an efficient strategy for the generation of therapeutic antitumor immunity.

ABSTRACT Efficient vaccination against infectious agents and tumors depends on specific antigen targeting to dendritic cells (DCs). We report here that biosafe coronavirus-based vaccine vectors facilitate delivery of multiple antigens and immunostimulatory cytokines to professional antigen-presenting cells in vitro and in vivo. Vaccine vectors based on heavily attenuated murine coronavirus genomes were generated to express epitopes from the lymphocytic choriomeningitis virus glycoprotein, or human Melan-A, in combination with the immunostimulatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). These vectors selectively targeted DCs in vitro and in vivo resulting in vector-mediated antigen expression and efficient maturation of DCs. Single application of only low vector doses elicited strong and long-lasting cytotoxic T-cell responses, providing protective antiviral and antitumor immunity. Furthermore, human DCs transduced with Melan-A-recombinant human coronavirus 229E efficiently activated tumor-specific CD8+ T cells. Taken together, this novel vaccine platform is well suited to deliver antigens and immunostimulatory cytokines to DCs and to initiate and maintain protective immunity. IMPORTANCE Vaccination against infectious agents has protected many individuals from severe disease. In addition, prophylactic and, most likely, also therapeutic vaccination against tumors will save millions from metastatic disease. This study describes a novel vaccine approach that facilitates delivery of viral or tumor antigens to dendritic cells in vivo. Concomitant immunostimulation via the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) was achieved through delivery by the same viral vector. Single immunization with only low doses of coronavirus-based vaccine vectors was sufficient to elicit (i) vigorous expansion and optimal differentiation of CD8+ T cells, (ii) protective and long-lasting antiviral immunity, and (iii) prophylactic and therapeutic tumor immunity. Moreover, highly efficient antigen delivery to human DCs with recombinant human coronavirus 229E and specific stimulation of human CD8+ T cells revealed that this approach is exceptionally well suited for translation into human vaccine studies.

Plasmacytoid dendritic cell (pDC)-mediated protection against cytopathic virus infection involves various molecular, cellular, tissue-scale, and organism-scale events. In order to better understand such multiscale interactions, we have implemented a systems immunology approach focusing on the analysis of the structure, dynamics and operating principles of virus-host interactions which constrain the initial spread of the pathogen. Using high-resolution experimental data sets coming from the well-described mouse hepatitis virus (MHV) model, we first calibrated basic modules including MHV infection of its primary target cells, i.e. pDCs and macrophages (Mphis). These basic building blocks were used to generate and validate an integrative mathematical model for in vivo infection dynamics. Parameter estimation for the system indicated that on a per capita basis, one infected pDC secretes sufficient type I IFN to protect 10(3) to 10(4) Mphis from cytopathic viral infection. This extremely high protective capacity of pDCs secures the spleen's capability to function as a 'sink' for the virus produced in peripheral organs such as the liver. Furthermore, our results suggest that the pDC population in spleen ensures a robust protection against virus variants which substantially down-modulate IFN secretion. However, the ability of pDCs to protect against severe disease caused by virus variants exhibiting an enhanced liver tropism and higher replication rates appears to be rather limited. Taken together, this systems immunology analysis suggests that antiviral therapy against cytopathic viruses should primarily limit viral replication within peripheral target organs.

Attenuated viral vaccines can be generated by targeting essential pathogenicity factors. We report here the rational design of an attenuated recombinant coronavirus vaccine based on a deletion in the coding sequence of the non-structural protein 1 (nsp1). In cell culture, nsp1 of mouse hepatitis virus (MHV), like its SARS-coronavirus homolog, strongly reduced cellular gene expression. The effect of nsp1 on MHV replication in vitro and in vivo was analyzed using a recombinant MHV encoding a deletion in the nsp1-coding sequence. The recombinant MHV nsp1 mutant grew normally in tissue culture, but was severely attenuated in vivo. Replication and spread of the nsp1 mutant virus was restored almost to wild-type levels in type I interferon (IFN) receptor-deficient mice, indicating that nsp1 interferes efficiently with the type I IFN system. Importantly, replication of nsp1 mutant virus in professional antigen-presenting cells such as conventional dendritic cells and macrophages, and induction of type I IFN in plasmacytoid dendritic cells, was not impaired. Furthermore, even low doses of nsp1 mutant MHV elicited potent cytotoxic T cell responses and protected mice against homologous and heterologous virus challenge. Taken together, the presented attenuation strategy provides a paradigm for the development of highly efficient coronavirus vaccines.