Viral vector-mediated reprogramming of the fibroblastic tumor stroma sustains curative melanoma treatment.

The tumor microenvironment (TME) is a complex amalgam of tumor cells, immune cells, endothelial cells and fibroblastic stromal cells (FSC). Cancer-associated fibroblasts are generally seen as tumor-promoting entity. However, it is conceivable that particular FSC populations within the TME contribute to immune-mediated tumor control. Here, we show that intratumoral treatment of mice with a recombinant lymphocytic choriomeningitis virus-based vaccine vector expressing a melanocyte differentiation antigen resulted in T cell-dependent long-term control of melanomas. Using single-cell RNA-seq analysis, we demonstrate that viral vector-mediated transduction reprogrammed and activated a Cxcl13-expressing FSC subset that show a pronounced immunostimulatory signature and increased expression of the inflammatory cytokine IL-33. Ablation of Il33 gene expression in Cxcl13-Cre-positive FSCs reduces the functionality of intratumoral T cells and unleashes tumor growth. Thus, reprogramming of FSCs by a self-antigen-expressing viral vector in the TME is critical for curative melanoma treatment by locally sustaining the activity of tumor-specific T cells.

Type I interferon signaling in fibroblastic reticular cells prevents exhaustive activation of antiviral CD8+ T cells.

Fibroblastic reticular cells (FRCs) are stromal cells that actively promote the induction of immune responses by coordinating the interaction of innate and adaptive immune cells. However, whether and to which extent immune cell activation is determined by lymph node FRC reprogramming during acute viral infection has remained unexplored. Here, we genetically ablated expression of the type I interferon-α receptor (Ifnar) in Ccl19-Cre+ cells and found that sensing of type I interferon imprints an antiviral state in FRCs and thereby preserves myeloid cell composition in lymph nodes of naive mice. During localized lymphocytic choriomeningitis virus infection, IFNAR signaling precipitated profound phenotypic adaptation of all FRC subsets enhancing antigen presentation, chemokine-driven immune cell recruitment, and immune regulation. The IFNAR-dependent shift of all FRC subsets toward an immunostimulatory state reduced exhaustive CD8+ T cell activation. In sum, these results unveil intricate circuits underlying type I IFN sensing in lymph node FRCs that enable protective antiviral immunity.

Remodeling of light and dark zone follicular dendritic cells governs germinal center responses.

Efficient generation of germinal center (GC) responses requires directed movement of B cells between distinct microenvironments underpinned by specialized B cell-interacting reticular cells (BRCs). How BRCs are reprogrammed to cater to the developing GC remains unclear, and studying this process is largely hindered by incomplete resolution of the cellular composition of the B cell follicle. Here we used genetic targeting of Cxcl13-expressing cells to define the molecular identity of the BRC landscape. Single-cell transcriptomic analysis revealed that BRC subset specification was predetermined in the primary B cell follicle. Further topological remodeling of light and dark zone follicular dendritic cells required CXCL12-dependent crosstalk with B cells and dictated GC output by retaining B cells in the follicle and steering their interaction with follicular helper T cells. Together, our results reveal that poised BRC-defined microenvironments establish a feed-forward system that determines the efficacy of the GC reaction.

Expression of AXL receptor tyrosine kinase relates to monocyte dysfunction and severity of cirrhosis.

Infectious complications in patients with cirrhosis frequently initiate episodes of decompensation and substantially contribute to the high mortality. Mechanisms of the underlying immuneparesis remain underexplored. TAM receptors (TYRO3/AXL/MERTK) are important inhibitors of innate immune responses. To understand the pathophysiology of immuneparesis in cirrhosis, we detailed TAM receptor expression in relation to monocyte function and disease severity prior to the onset of acute decompensation. TNF-α/IL-6 responses to lipopolysaccharide were attenuated in monocytes from patients with cirrhosis (n = 96) compared with controls (n = 27) and decreased in parallel with disease severity. Concurrently, an AXL-expressing (AXL+) monocyte population expanded. AXL+ cells (CD14+CD16highHLA-DRhigh) were characterised by attenuated TNF-α/IL-6 responses and T cell activation but enhanced efferocytosis and preserved phagocytosis of Escherichia coli Their expansion correlated with disease severity, complications, infection, and 1-yr mortality. AXL+ monocytes were generated in response to microbial products and efferocytosis in vitro. AXL kinase inhibition and down-regulation reversed attenuated monocyte inflammatory responses in cirrhosis ex vivo. AXL may thus serve as prognostic marker and deserves evaluation as immunotherapeutic target in cirrhosis.

Early-life programming of mesenteric lymph node stromal cell identity by the lymphotoxin pathway regulates adult mucosal immunity.

Redundant mechanisms support immunoglobulin A (IgA) responses to intestinal antigens. These include multiple priming sites [mesenteric lymph nodes (MLNs), Peyer's patches, and isolated lymphoid follicles] and various cytokines that promote class switch to IgA, even in the absence of T cells. Despite these backup mechanisms, vaccination against enteric pathogens such as rotavirus has limited success in some populations. Genetic and environmental signals experienced during early life are known to influence mucosal immunity, yet the mechanisms for how these exposures operate remain unclear. Here, we used rotavirus infection to follow antigen-specific IgA responses through time and in different gut compartments. Using genetic and pharmacological approaches, we tested the role of the lymphotoxin (LT) pathway-known to support IgA responses-at different developmental stages. We found that LT-ß receptor (LTßR) signaling in early life programs intestinal IgA responses in adulthood by affecting antibody class switch recombination to IgA and subsequent generation of IgA antibody-secreting cells within an intact MLN. In addition, early-life LTßR signaling dictates the phenotype and function of MLN stromal cells to support IgA responses in the adult. Collectively, our studies uncover new mechanistic insights into how early-life LTßR signaling affects mucosal immune responses during adulthood.

Microbiota-derived peptide mimics drive lethal inflammatory cardiomyopathy.

Myocarditis can develop into inflammatory cardiomyopathy through chronic stimulation of myosin heavy chain 6-specific T helper (TH)1 and TH17 cells. However, mechanisms governing the cardiotoxicity programming of heart-specific T cells have remained elusive. Using a mouse model of spontaneous autoimmune myocarditis, we show that progression of myocarditis to lethal heart disease depends on cardiac myosin-specific TH17 cells imprinted in the intestine by a commensal Bacteroides species peptide mimic. Both the successful prevention of lethal disease in mice by antibiotic therapy and the significantly elevated Bacteroides-specific CD4+ T cell and B cell responses observed in human myocarditis patients suggest that mimic peptides from commensal bacteria can promote inflammatory cardiomyopathy in genetically susceptible individuals. The ability to restrain cardiotoxic T cells through manipulation of the microbiome thereby transforms inflammatory cardiomyopathy into a targetable disease.

Divergent memory responses driven by adenoviral vectors are impacted by epitope competition.

Adenoviral vectors induce robust epitope-specific CD8+ T cell responses. Within the repertoire of responses generated both conventional memory evolution and the phenomenon of memory inflation are seen. The rules governing which epitopes inflate are not fully known, but may include a role for both antigen processing and competition. To investigate this, we looked at memory generated from vectors targeting the Gp33-41 (KAVYNFATC/K9C) epitope from the gp of lymphocytic choriomeningitis virus (LCMV) in mice. This well-described epitope has both the Gp33-41 and Gp34-41 epitopes embedded within it. Vaccination with a full-length gp or a minigene Ad-Gp33/K9C vector-induced conventional memory responses against the immunodominant Gp33/K9C epitope but a strong inflationary response against the Gp34/A8C epitope. These responses showed sustained in vivo function, with complete protection against LCMV infectious challenge. Given the unexpected competition between epitopes seen in the minigene model, we further tested epitope competition using the full-length Ad-LacZ (ß-galactosidase) model. Generation of an Ad-LacZ vector with a single amino acid disruption of the inflationary ß-gal96-103 /D8V epitope transformed the ß-gal497-504 /I8V epitope from conventional to inflationary memory. This work collectively demonstrates the importance of epitope competition within adenoviral vector inserts and is of relevance to future studies using adenoviral vectored immunogens.

Antibodies as biomarker candidates for response and survival to checkpoint inhibitors in melanoma patients.

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 .

Fibroblastic reticular cells initiate immune responses in visceral adipose tissues and secure peritoneal immunity.

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.

Fibroblastic reticular cells regulate intestinal inflammation via IL-15-mediated control of group 1 ILCs.

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.

Dendritic cell-specific delivery of Flt3L by coronavirus vectors secures induction of therapeutic antitumor immunity.

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.

Maturation of lymph node fibroblastic reticular cells from myofibroblastic precursors is critical for antiviral immunity.

The stromal scaffold of the lymph node (LN) paracortex is built by fibroblastic reticular cells (FRCs). Conditional ablation of lymphotoxin-ß receptor (LTßR) expression in LN FRCs and their mesenchymal progenitors in developing LNs revealed that LTßR-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 LTßR-dependent maturation of FRCs guarantees full immunocompetence and hence optimal LN function during infection.

Salmonella Typhi OmpS1 and OmpS2 porins are potent protective immunogens with adjuvant properties.

Salmonella enterica serovar Typhi (S. Typhi) is the causal agent of typhoid fever, a disease that primarily affects developing countries. Various antigens from this bacterium have been reported to be targets of the immune response. Recently, the S. Typhi genome has been shown to encode two porins--OmpS1 and OmpS2--which are expressed at low levels under in vitro culture conditions. In this study, we demonstrate that immunizing mice with either OmpS1 or OmpS2 induced production of specific, long-term antibody titres and conferred protection against S. Typhi challenge; in particular, OmpS1 was more immunogenic and conferred greater protective effects than OmpS2. We also found that OmpS1 is a Toll-like receptor 4 (TLR4) agonist, whereas OmpS2 is a TLR2 and TLR4 agonist. Both porins induced the production of tumour necrosis factor and interleukin-6, and OmpS2 was also able to induce interleukin-10 production. Furthermore, OmpS1 induced the over-expression of MHC II molecules in dendritic cells and OmpS2 induced the over-expression of CD40 molecules in macrophages and dendritic cells. Co-immunization of OmpS1 or OmpS2 with ovalbumin (OVA) increased anti-OVA antibody titres, the duration and isotype diversity of the OVA-specific antibody response, and the proliferation of T lymphocytes. These porins also had adjuvant effects on the antibody response when co-immunized with either the Vi capsular antigen from S. Typhi or inactivated 2009 pandemic influenza A(H1N1) virus [A(H1N1)pdm09]. Taken together, the data indicate that OmpS1 and OmpS2, despite being expressed at low levels under in vitro culture conditions, are potent protective immunogens with intrinsic adjuvant properties.

T helper cell- and CD40-dependent germline IgM prevents chronic virus-induced demyelinating disease.

Generation of antiviral IgM is usually considered as a marker of a short-lived initial antibody response that is replaced by hypermutated and more-efficient IgG. However, once viruses have established a particular niche for their persistence (e.g., within the CNS), the immune system has to specifically mobilize a broad range of antimicrobial effectors to contain the pathogen in the long term. Infection of the CNS with the mouse hepatitis virus (MHV) provides a unique model situation in which the extent of inflammatory CNS disease is determined by the balance between antiviral immune control, viral replication, and immune-mediated damage. We show here that whereas antibody- or B cell-deficient mice failed to contain MHV CNS infection and developed progressive demyelinating disease, germline IgM produced in activation-induced cytidine deaminase-deficient mice (aicda(-/-)) provided long-term protection against the chronic multiple sclerosis-like disease. Furthermore, we found that appropriate B-cell activation within the CNS-draining lymph node and subsequent CXCR3-mediated migration of antiviral IgM-secreting cells to the infected CNS was dependent on CD40-mediated interaction of B cells with T helper cells. These data indicate that the CD40-mediated collaboration of T and B cells is critical to secure neuroprotective IgM responses during viral CNS infection.