Modulation of SF3B1 in the pre-mRNA spliceosome induces a RIG-I-dependent type I IFN response.

Nucleic acid-sensing pathways play critical roles in innate immune activation through the production of type I interferon (IFN-I) and proinflammatory cytokines. These factors are required for effective antitumor immune responses. Pharmacological modulators of the pre-mRNA spliceosome splicing factor 3b subunit 1 (SF3B1) are under clinical investigation as cancer cytotoxic agents. However, potential roles of these agents in aberrant RNA generation and subsequent RNA-sensing pathway activation have not been studied. In this study, we observed that SF3B1 pharmacological modulation using pladienolide B (Plad B) induces production of aberrant RNA species and robust IFN-I responses via engagement of the dsRNA sensor retinoic acid-inducible gene I (RIG-I) and downstream interferon regulatory factor 3. We found that Plad B synergized with canonical RIG-I agonism to induce the IFN-I response. In addition, Plad B induced NF-κB responses and secretion of proinflammatory cytokines and chemokines. Finally, we showed that cancer cells bearing the hotspot SF3B1K700E mutation, which leads to global aberrant splicing, had enhanced IFN-I response to canonical RIG-I agonism. Together, these results demonstrate that pharmacological modulation of SF3B1 in cancer cells can induce an enhanced IFN-I response dependent on RIG-I expression. The study suggests that spliceosome modulation may not only induce direct cancer cell cytotoxicity but also initiate an innate immune response via activation of RNA-sensing pathways.

Antagonism of the STING Pathway via Activation of the AIM2 Inflammasome by Intracellular DNA.

Recent evidence has indicated that innate immune sensing of cytosolic DNA in dendritic cells via the host STING pathway is a major mechanism leading to spontaneous T cell responses against tumors. However, the impact of the other major pathway triggered by intracellular DNA, the absent in melanoma 2 (AIM2) inflammasome, on the functional output from the stimulator of IFN genes (STING) pathway is poorly understood. We found that dendritic cells and macrophages deficient in AIM2, apoptosis-associated specklike protein, or caspase-1 produced markedly higher IFN-ß in response to DNA. Biochemical analyses showed enhanced generation of cyclic GMP-AMP, STING aggregation, and TANK-binding kinase 1 and IFN regulatory factor 3 phosphorylation in inflammasome-deficient cells. Induction of pyroptosis by the AIM2 inflammasome was a major component of this effect, and inhibition of caspase-1 reduced cell death, augmenting phosphorylation of TANK-binding kinase 1/IFN regulatory factor 3 and production of IFN-ß. Our data suggest that in vitro activation of the AIM2 inflammasome in murine macrophages and dendritic cells leads to reduced activation of the STING pathway, in part through promoting caspase-1-dependent cell death.

Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity.

Spontaneous tumor-initiated T cell priming is dependent on IFN-ß production by tumor-resident dendritic cells. On the basis of recent observations indicating that IFN-ß expression was dependent upon activation of the host STING pathway, we hypothesized that direct engagement of STING through intratumoral (IT) administration of specific agonists would result in effective anti-tumor therapy. After proof-of-principle studies using the mouse STING agonist DMXAA showed a potent therapeutic effect, we generated synthetic cyclic dinucleotide (CDN) derivatives that activated all human STING alleles as well as murine STING. IT injection of STING agonists induced profound regression of established tumors in mice and generated substantial systemic immune responses capable of rejecting distant metastases and providing long-lived immunologic memory. Synthetic CDNs have high translational potential as a cancer therapeutic.

The STING pathway and the T cell-inflamed tumor microenvironment.

A major subset of patients with advanced solid tumors show a spontaneous T cell-inflamed tumor microenvironment, which has prognostic import and is associated with clinical response to immunotherapies. As such, understanding the mechanisms governing the generation of spontaneous T cell responses in only a subset of patients is critical for advancing immunotherapeutic approaches further. Here, we discuss the characteristics of T cell-inflamed versus non-inflamed tumors, including a type I interferon (IFN) signature associated with T cell priming against tumor antigens. We review recent findings that have pointed towards the STING (stimulator of interferon genes) pathway of cytosolic DNA sensing as an important innate immune sensing mechanism driving type I IFN production in the tumor context. Knowledge of this pathway is guiding the further development of novel immunotherapeutic strategies.

Innate immune recognition of cancer.

The observation that a subset of cancer patients show evidence for spontaneous CD8+ T cell priming against tumor-associated antigens has generated renewed interest in the innate immune pathways that might serve as a bridge to an adaptive immune response to tumors. Manipulation of this endogenous T cell response with therapeutic intent-for example, using blocking antibodies inhibiting PD-1/PD-L1 (programmed death-1/programmed death ligand 1) interactions-is showing impressive clinical results. As such, understanding the innate immune mechanisms that enable this T cell response has important clinical relevance. Defined innate immune interactions in the cancer context include recognition by innate cell populations (NK cells, NKT cells, and γδ T cells) and also by dendritic cells and macrophages in response to damage-associated molecular patterns (DAMPs). Recent evidence has indicated that the major DAMP driving host antitumor immune responses is tumor-derived DNA, sensed by the stimulator of interferon gene (STING) pathway and driving type I IFN production. A deeper knowledge of the clinically relevant innate immune pathways involved in the recognition of tumors is leading toward new therapeutic strategies for cancer treatment.

STING-dependent cytosolic DNA sensing mediates innate immune recognition of immunogenic tumors.

Spontaneous T cell responses against tumors occur frequently and have prognostic value in patients. The mechanism of innate immune sensing of immunogenic tumors leading to adaptive T cell responses remains undefined, although type I interferons (IFNs) are implicated in this process. We found that spontaneous CD8(+) T cell priming against tumors was defective in mice lacking stimulator of interferon genes complex (STING), but not other innate signaling pathways, suggesting involvement of a cytosolic DNA sensing pathway. In vitro, IFN-? production and dendritic cell activation were triggered by tumor-cell-derived DNA, via cyclic-GMP-AMP synthase (cGAS), STING, and interferon regulatory factor 3 (IRF3). In the tumor microenvironment in vivo, tumor cell DNA was detected within host antigen-presenting cells, which correlated with STING pathway activation and IFN-? production. Our results demonstrate that a major mechanism for innate immune sensing of cancer occurs via the host STING pathway, with major implications for cancer immunotherapy.

Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis.

Regulatory T cells (Treg cells) have a crucial role in the immune system by preventing autoimmunity, limiting immunopathology, and maintaining immune homeostasis. However, they also represent a major barrier to effective anti-tumour immunity and sterilizing immunity to chronic viral infections. The transcription factor Foxp3 has a major role in the development and programming of Treg cells. The relative stability of Treg cells at inflammatory disease sites has been a highly contentious subject. There is considerable interest in identifying pathways that control the stability of Treg cells as many immune-mediated diseases are characterized by either exacerbated or limited Treg-cell function. Here we show that the immune-cell-expressed ligand semaphorin-4a (Sema4a) and the Treg-cell-expressed receptor neuropilin-1 (Nrp1) interact both in vitro, to potentiate Treg-cell function and survival, and in vivo, at inflammatory sites. Using mice with a Treg-cell-restricted deletion of Nrp1, we show that Nrp1 is dispensable for suppression of autoimmunity and maintenance of immune homeostasis, but is required by Treg cells to limit anti-tumour immune responses and to cure established inflammatory colitis. Sema4a ligation of Nrp1 restrained Akt phosphorylation cellularly and at the immunologic synapse by phosphatase and tensin homologue (PTEN), which increased nuclear localization of the transcription factor Foxo3a. The Nrp1-induced transcriptome promoted Treg-cell stability by enhancing quiescence and survival factors while inhibiting programs that promote differentiation. Importantly, this Nrp1-dependent molecular program is evident in intra-tumoral Treg cells. Our data support a model in which Treg-cell stability can be subverted in certain inflammatory sites, but is maintained by a Sema4a-Nrp1 axis, highlighting this pathway as a potential therapeutic target that could limit Treg-cell-mediated tumour-induced tolerance without inducing autoimmunity.

Cancer immunotherapy strategies based on overcoming barriers within the tumor microenvironment.

For tumor antigen-specific T cells to effectively control the growth of cancer cells in vivo, they must gain access to, and function within, the tumor microenvironment. While tumor antigen-based vaccines and T cell adoptive transfer strategies can result in clinical benefit in a subset of patients, most of the patients do not respond clinically. Even for tumor-infiltrating lymphocyte (TIL)-based adoptive transfer for patients with metastatic melanoma, which can provide tumor shrinkage in around 50% of treated individuals, many patients are not eligible, in part because there are not sufficient TIL present in the resected tumor. Thus, the denominator is in fact larger, and it has been suggested that absence of TIL may be a marker for poor efficacy of immunotherapies in general. While qualitative and/or quantitative features of the T cells are important considerations for efficacy, a major component of primary resistance likely can be attributed to the tumor microenvironment. Data are accumulating suggesting that two major categories of immune resistance within the tumor microenvironment may exist: failure of T cell trafficking due to low levels of inflammation and lack of chemokines for migration, and dominant suppression through immune inhibitory mechanisms. New therapeutic interventions are being guided by these observations, and preliminary clinical success is validating this working model.

Type I interferon response and innate immune sensing of cancer.

Unexpectedly, many cancers appear to induce a spontaneous adaptive T cell response. The presence of a T cell infiltrate has been linked to favorable clinical outcome in multiple cancer types. However, the innate immune pathways that bridge to an adaptive immune response under sterile conditions are poorly understood. Recent data have indicated that tumors can induce type I interferon (IFN) production by host antigen-presenting cells (APCs), which is required for a spontaneous T cell response in vivo. The innate immune sensing pathways that trigger type I IFN production are being elucidated. Host type I IFNs are also required for optimal therapeutic efficacy with radiation. This recently uncovered role for host type I IFNs for antitumor immunity has important fundamental and clinical implications.

Innate immune sensing of cancer: clues from an identified role for type I IFNs.

A subset of patients with a variety of cancers shows evidence of a natural adaptive immune response against their tumor, as evidenced by spontaneous T-cell infiltration, circulating anti-tumor T cells, or antibody responses. Evidence has indicated that such natural immune responses have positive prognostic import in early stage disease and may be predictive of clinical response to immunotherapeutics in advanced disease. However, these observations raise a new critical fundamental question-what innate immune signals might be generated in the context of non-pathogen-induced cancers that drive productive antigen presentation toward induction of an adaptive immune response? Gene expression profiling in melanoma revealed that tumors having high expression of T-cell markers also show evidence of a type I IFN transcriptional signature. Mechanistic experiments in mice have revealed that a spontaneous CD8(+) T-cell response against transplantable tumors depends on host type I IFN signaling, through a mechanism dependent upon CD8α(+) dendritic cells (DCs). The requirement for type I IFN production by host DCs has suggested a subset of innate immune sensing receptors and signaling pathways that might be involved with initiating this process. Elucidating further these innate immune mechanisms should provide new insights into cancer immunotherapy.

Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape.

Inhibitory receptors on immune cells are pivotal regulators of immune escape in cancer. Among these inhibitory receptors, CTLA-4 (targeted clinically by ipilimumab) serves as a dominant off-switch while other receptors such as PD-1 and LAG-3 seem to serve more subtle rheostat functions. However, the extent of synergy and cooperative interactions between inhibitory pathways in cancer remain largely unexplored. Here, we reveal extensive coexpression of PD-1 and LAG-3 on tumor-infiltrating CD4(+) and CD8(+) T cells in three distinct transplantable tumors. Dual anti-LAG-3/anti-PD-1 antibody treatment cured most mice of established tumors that were largely resistant to single antibody treatment. Despite minimal immunopathologic sequelae in PD-1 and LAG-3 single knockout mice, dual knockout mice abrogated self-tolerance with resultant autoimmune infiltrates in multiple organs, leading to eventual lethality. However, Lag3(-/-)Pdcd1(-/-) mice showed markedly increased survival from and clearance of multiple transplantable tumors. Together, these results define a strong synergy between the PD-1 and LAG-3 inhibitory pathways in tolerance to both self and tumor antigens. In addition, they argue strongly that dual blockade of these molecules represents a promising combinatorial strategy for cancer.

Host type I IFN signals are required for antitumor CD8+ T cell responses through CD8{alpha}+ dendritic cells.

Despite lack of tumor control in many models, spontaneous T cell priming occurs frequently in response to a growing tumor. However, the innate immune mechanisms that promote natural antitumor T cell responses are undefined. In human metastatic melanoma, there was a correlation between a type I interferon (IFN) transcriptional profile and T cell markers in metastatic tumor tissue. In mice, IFN-ß was produced by CD11c(+) cells after tumor implantation, and tumor-induced T cell priming was defective in mice lacking IFN-α/ßR or Stat1. IFN signaling was required in the hematopoietic compartment at the level of host antigen-presenting cells, and selectively for intratumoral accumulation of CD8α(+) dendritic cells, which were demonstrated to be essential using Batf3(-/-) mice. Thus, host type I IFNs are critical for the innate immune recognition of a growing tumor through signaling on CD8α(+) DCs.

Differential subcellular localization of the regulatory T-cell protein LAG-3 and the coreceptor CD4.

CD4 binds to MHC class II molecules and enhances T-cell activation. The CD4-related transmembrane protein LAG-3 (lymphocyte activation gene-3, CD223) binds to the same ligand but inhibits T-cell proliferation. We have previously shown that LAG-3 cell surface expression is tightly regulated by extracellular cleavage in order to regulate its potent inhibitory activity. Given this observation and the contrasting functions of CD4 and LAG-3, we investigated the cell distribution, location and transport of these related cell surface molecules. As expected, the vast majority of CD4 is expressed at the cell surface with minimal intracellular localization, as determined by flow cytometry, immunoblotting and confocal microscopy. In contrast, nearly half the cellular content of LAG-3 is retained in intracellular compartments. This significant intracellular storage of LAG-3 appears to facilitate its rapid translocation to the cell surface following T-cell activation, which was much faster for LAG-3 than CD4. Increased vesicular pH inhibited translocation of both CD4 and LAG-3 to the plasma membrane. While some colocalization of the microtubule organizing center, early/recycling endosomes and secretory lysosomes was observed with CD4, significantly greater colocalization was observed with LAG-3. Analysis of CD4:LAG-3 chimeras suggested that multiple domains may contribute to intracellular retention of LAG-3. Thus, in contrast with CD4, the substantial intracellular storage of LAG-3 and its close association with the microtubule organizing center and recycling endosomes may facilitate its rapid translocation to the cell surface during T-cell activation and help to mitigate T-cell activation.

Tonic ubiquitylation controls T-cell receptor:CD3 complex expression during T-cell development.

Expression of the T-cell receptor (TCR):CD3 complex is tightly regulated during T-cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3varepsilon proline-rich sequence, Lck, c-Cbl, and SLAP, which collectively trigger the dynamin-dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3zeta-monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T-cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T-cell development.

ATP release by infected bovine monocytes increases the intracellular survival of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis is the etiologic agent of Johne's disease, a chronic intestinal infection in ruminants. Adenosine 5'-Triphosphate (ATP) has been reported to induce killing of several Mycobacterium species in human and murine macrophages. We investigated whether ATP secreted from M. avium subsp. paratuberculosis-infected bovine monocytes affects intracellular survival of the bacilli. Bovine monocytes constitutively secreted ATP during an 8-day incubation period in vitro; however, M. avium subsp. paratuberculosis infection did not enhance ATP release. Removal of extracellular ATP by the addition of apyrase increased the viability of infected monocytes, but surprisingly decreased the number of viable intracellular bacilli. In contrast to previous reports, addition of extracellular ATP (1mM) increased intracellular survival of M. avium subsp. paratuberculosis in bovine monocytes. Neither apyrase nor ATP altered production of reactive oxygen intermediates (ROI) or reactive nitrogen intermediates (RNI) by bovine monocytes. These results suggest that ATP release from infected bovine monocytes improves, rather than decreases, the intracellular survival of M. avium subsp. paratuberculosis.

Control of viral immunoinflammatory lesions by manipulating CD200:CD200 receptor interaction.

Previous investigators have demonstrated that the CD200:Fc that engages CD200 receptors (CD200R) shows promise as an immunosuppressive and anti-inflammatory reagent. In this report, we evaluate the use of CD200:Fc to control a viral induced immunoinflammatory reactions caused by ocular infection with herpes simplex virus (HSV). Our results show that HSV infection causes invasion of the cornea by CD200R(+) cells most of which were CD11b(+) cells. Systemic administration of CD200:Fc, starting at 5 days post infection (p.i.), resulted in diminished incidence and severity of lesions compared to controls. Splenocytes isolated from treated animals showed reduced IL-12 and IFN-gamma responses when stimulated in vitro and ex vivo. Treated animals also had increased frequencies of Foxp3(+) regulatory T cells in both the cornea and draining lymph nodes perhaps contributing also to the control of the corneal immunopathology. Treatment of animals in the chronic phase was minimally effective. Our data are the first to demonstrate the use of CD200R stimulation to control lesion severity in a viral induced inflammatory disease.

Tactics of Mycobacterium avium subsp. paratuberculosis for intracellular survival in mononuclear phagocytes.

Johne's disease is a condition that refers to chronic granulomatous enteritis in ruminants. It is believed that survival and replication of Mycobacterium (M.) paratuberculosis in mononuclear phagocytes plays an important role in the pathogenesis of Johne's disease. However, it is not clear how M. paratuberculosis survives for long time periods in mononuclear phagocytes, nor is it clear which factors trigger multiplication of these bacilli and result in the development of Johne's disease. Investigating the intracellular fate of M. paratuberculosis is challenging because of its very slow growth (more than two months to form visible colonies on media). Existing animal models also have limitations. Despite those obstacles, there has been progress in understanding the intracellular survival tactics of M. paratuberculosis and the host response against them. In this review, we compare known aspects of the intracellular survival tactics of M. paratuberculosis with those of other mycobacterial species, and consider possible mycobactericidal mechanisms of mononuclear phagocytes.

Extracellular ATP is cytotoxic to mononuclear phagocytes but does not induce killing of intracellular Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis is the etiologic agent of Johne's disease, a chronic granulomatous enteritis in ruminants. ATP has been reported to induce cell death of macrophages and killing of Mycobacterium species in human and murine macrophages. In this study we investigated the short-term effect of ATP on the viability of M. avium subsp. paratuberculosis-infected bovine mononuclear phagocytes and the bacilli within them. Addition of 5 mM ATP to M. avium subsp. paratuberculosis-infected bovine monocytes resulted in 50% cytotoxicity of bovine monocytes at 24 h. Addition of 2'(3')-O-(4-benzoylbenzoyl) ATP triethylammonium salt (Bz-ATP), which is a longer-lived ATP homologue and purinergic receptor agonist, significantly increased the uptake of YO-PRO, which is a marker for membrane pore activation by P2X receptors. Addition of Bz-ATP also stimulated lactate dehydrogenase release and caspase-3 activity in infected bovine monocytes. Neither ATP nor Bz-ATP reduced the survival of M. avium subsp. paratuberculosis in bovine mononuclear phagocytes. Likewise, addition of ATP or Bz-ATP was cytotoxic to murine macrophage cell lines (RAW 264.7 and J774A.1 cells) but did not affect the intracellular survival of M. avium subsp. paratuberculosis, nor were the numbers of viable Mycobacterium avium subsp. avium or Mycobacterium bovis BCG cells altered in bovine mononuclear phagocytes or J774A.1 cells following ATP or Bz-ATP treatment. These data suggest that extracellular ATP does not induce the killing of intracellular M. avium subsp. paratuberculosis in bovine mononuclear phagocytes.

Life and death in bovine monocytes: the fate of Mycobacterium avium subsp. paratuberculosis.

We previously reported that the number of acid-fast bacilli within Mycobacterium paratuberculosis-infected bovine monocytes increased steadily during an 8-day incubation period in vitro, despite a decrease in the number of viable bacilli as estimated by a radiometric method. In this study, we used differential live/dead staining of bacilli from infected monocytes to show that the percentage of viable bacilli decreased during an 8-day incubation period. We observed poor phagosome-lysosome fusion in monocytes that had ingested viable M. paratuberculosis (30% phagosome-lysosome fusion), while monocytes that ingested heat killed M. paratuberculosis exhibited 94% phagosome-lysosome fusion at 24h after infection. Treatment with the selective Ca(2+)/CaM and PI3 kinase inhibitors (i.e. KN62 and Wortmannin) in combination increased the survival of M. paratuberculosis in bovine monocytes without significantly altering phagosome-lysosome fusion. Scanning electron microscopy suggested that M. paratuberculosis-infected monocytes were less differentiated (smaller and less spreading) than uninfected monocytes at 4 and 8 days of infection. Overall, these data suggest that both multiplication and killing of intracellular M. paratuberculosis occur concomitantly in bovine monocytes. Monocytes in turn may be adversely affected by the bacilli, their products, or factors released from infected monocytes.

Bovine monocytes and a macrophage cell line differ in their ability to phagocytose and support the intracellular survival of Mycobacterium avium subsp. paratuberculosis.

Bovine monocytes exhibited a greater ability to phagocytose Mycobacterium avium subsp. paratuberculosis (i.e. greater percentage of infected cells, and more bacilli per infected cell), than did a bovine macrophage cell line (BoMac). Phagocytosis of M. paratuberculosis by monocytes, but not the cell line, was significantly enhanced by the addition of autologous serum. Following ingestion, the numbers of viable M. paratuberculosis cells in monocytes increased during the first 4 days and then declined between day 4 and day 8 after infection, as determined by a radiometric method. In contrast, BoMac cells were not permissive for bacillary multiplication; the numbers of M. paratuberculosis remained largely unchanged in the cell line during the 8 day incubation period. The numbers of microscopically visible acid-fast bacilli increased with time in monocytes but not in the macrophage cell line. These observations suggest that replication and inactivation of bacilli may both occur in monocytes. The differing abilities of bovine monocytes and the macrophage cell line to ingest and restrain the intracellular growth of M. paratuberculosis provide contrasting model systems for investigating how M. paratuberculosis enters and persists within its preferred niche, the mononuclear phagocyte.