Local delivery of mRNA-encoded cytokines promotes antitumor immunity and tumor eradication across multiple preclinical tumor models.

Local immunotherapy ideally stimulates immune responses against tumors while avoiding toxicities associated with systemic administration. Current strategies for tumor-targeted, gene-based delivery, however, are limited by adverse effects such as off-targeting or antivector immunity. We investigated the intratumoral administration of saline-formulated messenger (m)RNA encoding four cytokines that were identified as mediators of tumor regression across different tumor models: interleukin-12 (IL-12) single chain, interferon-α (IFN-α), granulocyte-macrophage colony-stimulating factor, and IL-15 sushi. Effective antitumor activity of these cytokines relied on multiple immune cell populations and was accompanied by intratumoral IFN-γ induction, systemic antigen-specific T cell expansion, increased granzyme B+ T cell infiltration, and formation of immune memory. Antitumor activity extended beyond the treated lesions and inhibited growth of distant tumors and disseminated tumors. Combining the mRNAs with immunomodulatory antibodies enhanced antitumor responses in both injected and uninjected tumors, thus improving survival and tumor regression. Consequently, clinical testing of this cytokine-encoding mRNA mixture is now underway.

HMGB1 promotes CXCL12-dependent egress of murine B cells from Peyer's patches in homeostasis.

High mobility group box-1 protein (HMGB1) is an alarmin that, once released, promotes inflammatory responses, alone and as a complex with the chemokine CXCL12. Here, we report that the HMGB1-CXCL12 complex plays an essential role also in homeostasis by controlling the migration of B lymphocytes. We show that extracellular HMGB1 is critical for the CXCL12-dependent egress of B cells from the Peyer's patches (PP). This promigratory function of the complex was restricted to the PPs, since HMGB1 was not required for B-cell migratory processes in other locations. Accordingly, we detected higher constitutive levels of the HMGB1-CXCL12 complex in PPs than in other lymphoid organs. HMGB1-CXCL12 in vivo inhibition was associated with a reduced basal IgA production in the gut. Collectively, our results demonstrate a role for the HMGB1-CXCL12 complex in orchestrating B-cell trafficking in homeostasis, and provide a novel target to control lymphocyte migration in mucosal immunity.

Harnessing the immune system to fight cancer with Toll-like receptor and RIG-I-like receptor agonists.

Cancer immunotherapy has come of age with the advent of immune checkpoint inhibitors. In this article we review how agonists for receptors of the innate immune system, the Toll-like receptors and the RIG-I-like receptors, impact anticancer immune responses. Treatment with these agonists enhances the activity of anticancer effector cells, such as cytotoxic T cells and NK cells, and at the same time blocks the activity of immunosuppressive cell types such as regulatory T cells and myeloid-derived suppressor cells. These compounds also impact the recruitment of immune cells to the tumor. The phenomena of pattern-recognition receptor tolerance and reprogramming and their implications for immunotherapy are discussed. Finally, novel delivery systems that target the immune-stimulating drugs to the tumor or the tumor-draining lymph nodes to enhance their efficacy and safety are presented.

Hook length of the bacterial flagellum is optimized for maximal stability of the flagellar bundle.

Most bacteria swim in liquid environments by rotating one or several flagella. The long external filament of the flagellum is connected to a membrane-embedded basal body by a flexible universal joint, the hook, which allows the transmission of motor torque to the filament. The length of the hook is controlled on a nanometer scale by a sophisticated molecular ruler mechanism. However, why its length is stringently controlled has remained elusive. We engineered and studied a diverse set of hook-length variants of Salmonella enterica. Measurements of plate-assay motility, single-cell swimming speed, and directional persistence in quasi-2D and population-averaged swimming speed and body angular velocity in 3D revealed that the motility performance is optimal around the wild-type hook length. We conclude that too-short hooks may be too stiff to function as a junction and too-long hooks may buckle and create instability in the flagellar bundle. Accordingly, peritrichously flagellated bacteria move most efficiently as the distance travelled per body rotation is maximal and body wobbling is minimized. Thus, our results suggest that the molecular ruler mechanism evolved to control flagellar hook growth to the optimal length consistent with efficient bundle formation. The hook-length control mechanism is therefore a prime example of how bacteria evolved elegant but robust mechanisms to maximize their fitness under specific environmental constraints.

Engineered hybrid spider silk particles as delivery system for peptide vaccines.

The generation of strong T-cell immunity is one of the main challenges for the development of successful vaccines against cancer and major infectious diseases. Here we have engineered spider silk particles as delivery system for a peptide-based vaccination that leads to effective priming of cytotoxic T-cells. The recombinant spider silk protein eADF4(C16) was fused to the antigenic peptide from ovalbumin, either without linker or with a cathepsin cleavable peptide linker. Particles prepared from the hybrid proteins were taken up by dendritic cells, which are essential for T-cell priming, and successfully activated cytotoxic T-cells, without signs of immunotoxicity or unspecific immunostimulatory activity. Upon subcutaneous injection in mice, the particles were taken up by dendritic cells and accumulated in the lymph nodes, where immune responses are generated. Particles from hybrid proteins containing a cathepsin-cleavable linker induced a strong antigen-specific proliferation of cytotoxic T-cells in vivo, even in the absence of a vaccine adjuvant. We thus demonstrate the efficacy of a new vaccine strategy using a protein-based all-in-one vaccination system, where spider silk particles serve as carriers with an incorporated peptide antigen. Our study further suggests that engineered spider silk-based vaccines are extremely stable, easy to manufacture, and readily customizable.

Infrared absorption spectrometer for the determination of temperature and species profiles in an entrained flow gasifier.

An absorption spectrometer utilizing a tunable distributed feedback diode laser at 2.3 µm and an interband cascade laser at 3.1 µm has been developed to measure temperature and concentrations of CO, CH4, C2H2, and H2O under gasification conditions. A wavelength division multiplexing approach using a single ZrF4-fiber was used to measure both wavelength regions simultaneously. The performance of the spectrometer has been tested in laminar flat flames and a heated cell and then applied for measurements at an atmospheric entrained flow gasifier (REGA). A water-cooled optical probe was used to provide optical access at two measurement positions. By moving the burner, axial profiles of temperature and species concentration could be obtained. These profiles were compared with numerical simulations and can be used to validate the simulation.

Reprogramming of TLR7 signaling enhances antitumor NK and cytotoxic T cell responses.

Toll-like receptor (TLR) 7 agonists are effective in topical application for the immunotherapy of skin cancers, but their performance for the systemic treatment of solid tumors is limited by the development of TLR tolerance. In this study, we describe a novel strategy to overcome TLR tolerance and enhance TLR7-dependent antitumor immune responses through reprogramming of TLR signaling pathways. The sensitivity of TLR7 signaling in dendritic cells (DC) was increased by prior stimulation with the dsRNA poly(I:C) that mimics virally induced immune activation. Timing of the stimulations was important, as sequential stimulation with poly(I:C) and the TLR7 agonist R848 interspaced by 24 h induced higher MAPK and NFkB signaling in DC than the simultaneous application of the same ligands. DC activated by sequential poly(I:C)/R848 stimulation efficiently induced Th1 differentiation and primed NK-cell and cytotoxic T-cell responses. We have developed a treatment regimen taking advantage of TLR7 reprogram-ming that cured over 80% of large immunogenic tumors in mice by the action of NK cells and cytotoxic T cells. These results have direct implications for the use of these clinically established ligands in the immunotherapy of cancer.

TLR7-based cancer immunotherapy decreases intratumoral myeloid-derived suppressor cells and blocks their immunosuppressive function.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells with the capacity to inhibit immunological responses. During cancer progression, MDSC are recruited to the tumor sites and secondary lymphoid organs, leading to the suppression of the antitumor function of NK and T cells. Here, we show that the TLR7/8 agonist resiquimod (R848) has a direct effect on MDSC populations in tumor-bearing mice. Systemic application of R848 led to a rapid reduction in both intratumoral and circulating MDSC. The subpopulation of monocytic MDSC (m-MDSC) was the most affected by R848 treatment with an up to 5-fold decrease in the tumor. We found that TLR7 stimulation in tumor-bearing mice led to a maturation and differentiation of MDSC with upregulation of the surface molecules CD11c, F4/80, MHC-I, and MHC-II. MDSC treated with R848 lost their immunosuppressive function and acquired instead an antigen-presenting phenotype with the capability to induce specific T-cell proliferation. Importantly, we found that MDSC co-injected s.c. with CT26 tumor cells lost their ability to support tumor growth after pretreatment with R848. Our results demonstrate that treatment of tumor-bearing mice with a TLR7/8 agonist acts directly on MDSC to induce their maturation and leads them to acquire a non-suppressive status. Considering the obstacles posed by MDSC for cancer immunotherapy, targeting these cells by a TLR7/8 agonist may improve immune responses against cancer.

Mycoplasma hyorhinis-Contaminated Cell Lines Activate Primary Innate Immune Cells via a Protease-Sensitive Factor.

Mycoplasma are a frequent and occult contaminant of cell cultures, whereby these prokaryotic organisms can modify many aspects of cell physiology, rendering experiments that are conducted with such contaminated cells problematic. Chronic Mycoplasma contamination in human monocytic cells lines has been associated with suppressed Toll-like receptor (TLR) function. In contrast, we show here that components derived from a Mycoplasma hyorhinis-infected cell line can activate innate immunity in non-infected primary immune cells. Release of pro-inflammatory cytokines such as IL-6 by dendritic cells in response to Mycoplasma hyorhinis-infected cell components was critically dependent on the adapter protein MyD88 but only partially on TLR2. Unlike canonical TLR2 signaling that is triggered in response to the detection of Mycoplasma infection, innate immune activation by components of Mycoplasma-infected cells was inhibited by chloroquine treatment and sensitive to protease treatment. We further show that in plasmacytoid dendritic cells, soluble factors from Mycoplasma hyorhinis-infected cells induce the production of large amounts of IFN-α. We conclude that Mycoplasma hyorhinis-infected cell lines release protein factors that can potently activate co-cultured innate immune cells via a previously unrecognized mechanism, thus limiting the validity of such co-culture experiments.

TLR and RLR Signaling Are Reprogrammed in Opposite Directions after Detection of Viral Infection.

Innate immune recognition of RNA is key for the initiation of immunity in response to viral infection. Although the factors controlling the detection of viral RNA by innate immune receptors in host cells are increasingly well understood, little is known about the dynamic changes in signaling after the initial triggering of these receptors. In this study, we report that preconditioning with the synthetic dsRNA polyinosinic-polycytidylic acid [poly(I:C)], a mimetic of viral RNA, rapidly reprograms murine APCs by simultaneously augmenting sensitivity of endosomal TLRs and inhibiting activation of RIG-I-like receptors (RLRs) in an IFN-ß-dependent manner. These changes in receptor sensitivity were also seen in vivo after treatment of mice with poly(I:C). Mechanistically, the increased sensitivity of the TLR pathway was associated with elevated MAPK and NF-κB activity. The RLR response was inhibited downstream of TANK-binding kinase-1, resulting in decreased IFN regulatory factor 3 phosphorylation. Reprogramming of pattern-recognition receptor signaling also occurred after viral infection, because infection of host cells with Sendai virus or their exposure to supernatant from virus-infected cells induced the same changes in TLR and RLR sensitivity as poly(I:C). Thus, innate recognition of viral infection critically modifies responses to pattern-recognition receptor stimulation. These dynamic adaptations to infection may reinforce antiviral immunity and at the same time serve to limit pathological inflammation.

Virus-associated activation of innate immunity induces rapid disruption of Peyer's patches in mice.

Early in the course of infection, detection of pathogen-associated molecular patterns by innate immune receptors can shape the subsequent adaptive immune response. Here we investigate the influence of virus-associated innate immune activation on lymphocyte distribution in secondary lymphoid organs. We show for the first time that virus infection of mice induces rapid disruption of the Peyer's patches but not of other secondary lymphoid organs. The observed effect was not dependent on an active infectious process, but due to innate immune activation and could be mimicked by virus-associated molecular patterns such as the synthetic double-stranded RNA poly(I:C). Profound histomorphologic changes in Peyer's patches were associated with depletion of organ cellularity, most prominent among the B-cell subset. We demonstrate that the disruption is entirely dependent on type I interferon (IFN). At the cellular level, we show that virus-associated immune activation by IFN-α blocks B-cell trafficking to the Peyer's patches by downregulating expression of the homing molecule α4ß7-integrin. In summary, our data identify a mechanism that results in type I IFN-dependent rapid but reversible disruption of intestinal lymphoid organs during systemic viral immune activation. We propose that such rerouted lymphocyte trafficking may impact the development of B-cell immunity to systemic viral pathogens.

HLA-DR15-derived self-peptides are involved in increased autologous T cell proliferation in multiple sclerosis.

The HLA-DR15 haplotype confers the largest part of the genetic risk to develop multiple sclerosis, a prototypic CD4+ T cell-mediated autoimmune disease. The mechanisms how certain HLA-class II molecules functionally contribute to autoimmune diseases are still poorly understood, but probably involve shaping an autoimmune-prone T cell repertoire during central tolerance in the thymus and subsequently maintaining or even expanding it in the peripheral immune system. Self-peptides that are presented by disease-associated HLA-class II molecules most likely play important roles during both processes. Here, we examined the functional involvement of the HLA-DR15 haplotype in autologous proliferation in multiple sclerosis and the contribution of HLA-DR15 haplotype-derived self-peptides in an in vitro system. We observe increased autologous T cell proliferation in patients with multiple sclerosis in relation to the multiple sclerosis risk-associated HLA-DR15 haplotype. Assuming that the spectrum of self-peptides that is presented by the two HLA-DR15 allelic products is important for sustaining autologous proliferation we performed peptide elution and identification experiments from the multiple sclerosis-associated DR15 molecules and a systematic analysis of a DR15 haplotype-derived self-peptide library. We identify HLA-derived self-peptides as potential mediators of altered autologous proliferation. Our data provide novel insights about perturbed T cell repertoire dynamics and the functional involvement of the major genetic risk factor, the HLA-DR15 haplotype, in multiple sclerosis.

TLR activation excludes circulating naive CD8+ T cells from gut-associated lymphoid organs in mice.

The trafficking of effector T cells is tightly regulated by the expression of site-specific sets of homing molecules. In contrast, naive T cells are generally assumed to express a uniform pattern of homing molecules and to follow a random distribution within the blood and secondary lymphoid organs. In this study, we demonstrate that systemic infection fundamentally modifies the trafficking of circulating naive CD8(+) T cells. We show that on naive CD8(+) T cells, the constitutive expression of the integrin α4ß7 that effects their entry into GALT is downregulated following infection of mice with Salmonella typhimurium. We further show that this downregulation is dependent on TLR signaling, and that the TLR-activated naive CD8(+) T cells are blocked from entering GALT. This contrasts strongly with Ag-experienced effector T cells, for which TLR costimulation in the GALT potently upregulates α4ß7 and enhances trafficking to intestinal tissues. Thus, TLR activation leads to opposite effects on migration of naive and effector CD8(+) T cells. Our data identify a mechanism that excludes noncognate CD8(+) T cells from selected immune compartments during TLR-induced systemic inflammation.

Systemic cancer immunotherapy with Toll-like receptor 7 agonists: Timing is everything.

Toll-like receptor (TLR) 7 agonists represent a promising strategy for the immunotherapy of cancer. We have recently investigated the influence of TLR tolerance on the efficacy of systemic tumor treatment with TLR7 ligands. We propose that considering the kinetics of receptor sensitivity highly improves the outcome of cancer immunotherapy.

Specific antibody-receptor interactions trigger InlAB-independent uptake of Listeria monocytogenes into tumor cell lines.

BACKGROUND: Specific cell targeting is an important, yet unsolved problem in bacteria-based therapeutic applications, like tumor or gene therapy. Here, we describe the construction of a novel, internalin A and B (InlAB)-deficient Listeria monocytogenes strain (Lm-spa+), which expresses protein A of Staphylococcus aureus (SPA) and anchors SPA in the correct orientation on the bacterial cell surface. RESULTS: This listerial strain efficiently binds antibodies allowing specific interaction of the bacterium with the target recognized by the antibody. Binding of Trastuzumab (Herceptin®) or Cetuximab (Erbitux®) to Lm-spa+, two clinically approved monoclonal antibodies directed against HER2/neu and EGFR/HER1, respectively, triggers InlAB-independent internalization into non-phagocytic cancer cell lines overexpressing the respective receptors. Internalization, subsequent escape into the host cell cytosol and intracellular replication of these bacteria are as efficient as of the corresponding InlAB-positive, SPA-negative parental strain. This specific antibody/receptor-mediated internalization of Lm-spa+ is shown in the murine 4T1 tumor cell line, the isogenic 4T1-HER2 cell line as well as the human cancer cell lines SK-BR-3 and SK-OV-3. Importantly, this targeting approach is applicable in a xenograft mouse tumor model after crosslinking the antibody to SPA on the listerial cell surface. CONCLUSIONS: Binding of receptor-specific antibodies to SPA-expressing L. monocytogenes may represent a promising approach to target L. monocytogenes to host cells expressing specific receptors triggering internalization.

Systemic cancer therapy with a small molecule agonist of toll-like receptor 7 can be improved by circumventing TLR tolerance.

Topical application of small molecule Toll-like receptor 7 (TLR7) agonists is highly effective for the treatment of skin tumors, whereas their systemic application has been largely unsuccessful for cancer therapy. One reason may be that repeated systemic application of TLR ligands can induce a state of immune unresponsiveness, termed TLR tolerance. We show here that a single injection of the TLR7 agonist R848 in mice induces a short period of increased response to TLR stimulation followed by a state of hyporesponsiveness lasting several days. This state is characterized by inhibited secretion of the key cytokines interleukin (IL)-12p70 and IL-6 as well as by a block in IFN-α production. We show for the first time that at the cellular level, TLR7 tolerance occurs in both plasmacytoid and myeloid dendritic cells, two cell populations that play a critical role in the initiation and amplification of antitumor immune responses. We further show that TLR7 tolerance in plasmacytoid dendritic cells is accompanied by downregulation of the adaptor protein IL-1 receptor-associated kinase 1. On the basis of these findings, we have designed a novel strategy for the treatment of tumors by using cycles of repeated R848 injections separated by treatment-free intervals. We show in CT26 tumor-bearing mice that this protocol circumvents TLR7 tolerance and improves the efficacy of cancer immunotherapy.

Shigella mediated depletion of macrophages in a murine breast cancer model is associated with tumor regression.

A tumor promoting role of macrophages has been described for a transgenic murine breast cancer model. In this model tumor-associated macrophages (TAMs) represent a major component of the leukocytic infiltrate and are associated with tumor progression. Shigella flexneri is a bacterial pathogen known to specificly induce apotosis in macrophages. To evaluate whether Shigella-induced removal of macrophages may be sufficient for achieving tumor regression we have developed an attenuated strain of S. flexneri (M90TDeltaaroA) and infected tumor bearing mice. Two mouse models were employed, xenotransplantation of a murine breast cancer cell line and spontanous breast cancer development in MMTV-HER2 transgenic mice. Quantitative analysis of bacterial tumor targeting demonstrated that attenuated, invasive Shigella flexneri primarily infected TAMs after systemic administration. A single i.v. injection of invasive M90TDeltaaroA resulted in caspase-1 dependent apoptosis of TAMs followed by a 74% reduction in tumors of transgenic MMTV-HER-2 mice 7 days post infection. TAM depletion was sustained and associated with complete tumor regression.These data support TAMs as useful targets for antitumor therapy and highlight attenuated bacterial pathogens as potential tools.

Improvement of the live vaccine strain Salmonella enterica serovar Typhi Ty21a for antigen delivery via the hemolysin secretion system of Escherichia coli.

The attenuated Salmonella enterica serovar Typhi strain Ty21a (Ty21a) is the only attenuated live oral vaccine against typhoid fever. Ty21a is also an attractive carrier for the delivery of heterologous antigens. We have used Ty21a for antigen delivery via the hemolysin (HlyA) secretion system of Escherichia coli, the prototype of the type I secretion system (T1SS). In this study, we identified by genetic complementation that the specific mutation of rpoS correlated with the hemolysin production of strain Ty21a. We furthermore showed that complementation with a plasmid encoding rfaH, which is described to be a downstream target of rpoS, led to increased expression and secretion of hemolysin. Finally, we demonstrated a significant enhancement of antibody responses against the heterologous HlyA antigen of Ty21a after immunization of mice with rfaH complemented S. typhi strain secreting HlyA compared with the same strain without rfaH plasmid.

C-RAF activation promotes BAD poly-ubiquitylation and turn-over by the proteasome.

BAD, a member of the BCL2 family, exhibits an original mode of regulation by phosphorylation. In the present report, we examine the role of the kinase C-RAF in this process. We show that the inducible activation of C-RAF promotes the rapid phosphorylation of BAD on Serine-112 (Ser-75 in the human protein), through a cascade involving the kinases MEK and RSK. Our findings reveal a new aspect of the regulation of BAD protein and its control by the RAF pathway: we find that C-RAF activation promotes BAD poly-ubiquitylation in a phosphorylation-dependent fashion, and increases the turn-over of this protein through proteasomal degradation.