Intratumoral administration of unconjugated Accum™ impairs the growth of pre-established solid lymphoma tumors.

The Accum™ technology was initially designed to enhance the bioaccumulation of a given molecule in target cells. It does so by triggering endosomal membrane damages allowing endocytosed products to enter the cytosol, escaping the harsh environmental cues of the endosomal lumen. In an attempt to minimize manufacturing hurdles associated with Accum™ conjugation, we tested whether free Accum™ admixed with antigens could lead to outcomes similar to those obtained with conjugated products. Surprisingly, unconjugated Accum™ was found to promote cell death in vitro, an observation further confirmed on various murine tumor cell lines (EL4, CT-26, B16, and 4 T1). At the molecular level, unconjugated Accum™ triggers the production of reactive oxygen species and elicits immunogenic cell death while retaining its innate ability to cause endosomal damages. When administered as a monotherapy to animals with pre-established EL4 T-cell lymphoma, Accum™ controlled tumor growth in a dose-dependent manner, and its therapeutic effect relies on CD4 and CD8 T cells. Although unconjugated Accum™ synergizes with various immune checkpoint inhibitors (anti-CTLA4, anti-PD-1, or anti-CD47) at controlling tumor growth, its therapeutic potency could not be further enhanced when combined with all three tested immune checkpoint inhibitors at once due to its dependency on a specific dosing regimen. In sum, we report in this study an unprecedented new function for unconjugated Accum™ as a novel anticancer molecule. These results could pave the path for a new line of investigation aimed at exploring the pro-killing properties of additional Accum™ variants as a mean to develop second-generation anticancer therapeutics.

Perioperative arginine prevents metastases by accelerating natural killer cell recovery after surgery.

Profound natural killer (NK) cell suppression after cancer surgery is a main driver of metastases and recurrence, for which there is no clinically approved intervention available. Surgical stress is known to cause systemic postoperative changes that negatively modulate NK cell function including the expansion of surgery-induced myeloid-derived suppressor cells (Sx-MDSCs) and a marked reduction in arginine bioavailability. In this study, we determine that Sx-MDSCs regulate systemic arginine levels in the postoperative period and that restoring arginine imbalance after surgery by dietary intake alone was sufficient to significantly reduce surgery-induced metastases in our preclinical murine models. Importantly, the effects of perioperative arginine were dependent upon NK cells. Although perioperative arginine did not prevent immediate NK cell immunoparalysis after surgery, it did accelerate their return to preoperative cytotoxicity, interferon gamma secretion, and activating receptor expression. Finally, in a cohort of patients with colorectal cancer, postoperative arginine levels were shown to correlate with their Sx-MDSC levels. Therefore, this study lends further support for the use of perioperative arginine supplementation by improving NK cell recovery after surgery.

Lipid accumulation impairs natural killer cell cytotoxicity and tumor control in the postoperative period.

BACKGROUND: Natural killer (NK) cell dysfunction following cancer surgery has been shown to promote metastases. Recent studies demonstrate an emerging role for lipids in the modulation of NK cell innate responses. However, the mechanisms involved in lipid modulation of NK cell postoperative anti-tumor function are unknown. This current study will determine whether the lipid accumulation via scavenger receptors on NK cells is responsible for the increase in postoperative metastasis. METHODS: Lipid content in mouse and human NK cells was evaluated by flow cytometry. NK cell scavenger receptor (SR) expression was measured by microarray analysis, validated by qRT-PCR and flow cytometry. NK cell ex vivo and in vivo tumor killing was measured by chromium-release and adoptive transfer assays, respectively. The mediating role of surgery-expanded granulocytic myeloid derived suppressor cells (gMDSC) in SR induction on NK cells was evaluated using co-culture assays. RESULTS: NK cells in surgery-treated mice demonstrated increased lipid accumulation, which occurred via up-regulation of MSR1, CD36 and CD68. NK cells with high lipid content had diminished ability to lyse tumor targets ex vivo. Adoptive transfer of lipid-laden NK cells into NK cell-deficient mice were unable to protect against a lung tumor challenge. Granulocytic MDSC from surgery-treated mice increased SR expression on NK cells. Colorectal cancer surgical patients showed increased NK cell lipid content, higher CD36 expression, decreased granzyme B and perforin production in addition to reduced cytotoxicity in the postoperative period. CONCLUSIONS: Postoperative lipid accumulation promotes the formation of metastases by impairing NK cell function in both preclinical surgical models and human surgical colorectal cancer patient samples. Understanding and targeting the mechanisms underlying lipid accumulation in innate immune NK cells can improve prognosis in cancer surgical patients.

Sepsis increases perioperative metastases in a murine model.

BACKGROUND: Cancer surgery can promote tumour metastases and worsen prognosis, however, the effect of perioperative complications on metastatic disease remains unclear. In this study we sought to evaluate the effect of common perioperative complications including perioperative blood loss, hypothermia, and sepsis on tumour metastases in a murine model. METHODS: Prior to surgery, pulmonary metastases were established by intravenous challenge of CT26LacZ colon cancer cells in BALB/c mice. Surgical stress was generated through partial hepatectomy (PH) or left nephrectomy (LN). Sepsis was induced by puncturing the cecum to express stool into the abdomen. Hemorrhagic shock was induced by removal of 30% of total blood volume (i.e. stage 3 hemorrhage) via the saphenous vein. Hypothermia was induced by removing the heating apparatus during surgery and lowering core body temperatures to 30 °C. Lung tumour burden was quantified 3 days following surgery. RESULTS: Surgically stressed mice subjected to stage 3 hemorrhage or hypothermia did not show an additional increase in lung tumour burden. In contrast, surgically stressed mice subjected to intraoperative sepsis demonstrated an additional 2-fold increase in the number of tumour metastases. Furthermore, natural killer (NK) cell function, as assessed by YAC-1 tumour cell lysis, was significantly attenuated in surgically stressed mice subjected to intraoperative sepsis. Both NK cell-mediated cytotoxic function and lung tumour burden were improved with perioperative administration of polyI:C, which is a toll-like receptor (TLR)-3 ligand. CONCLUSIONS: Perioperative sepsis alone, but not hemorrhage or hypothermia, enhances the prometastatic effect of surgery in murine models of cancer. Understanding the cellular mechanisms underlying perioperative immune suppression will facilitate the development of immunomodulation strategies that can attenuate metastatic disease.

Correction to: Sepsis increases perioperative metastases in a murine model.

It has been highlighted that the original manuscript [1] contains a typesetting error in Fig. 1 and the Fig. 1c panel gas been inadvertently duplicated in panel Fig. 1d. This does not affect the results and conclusions of the article. The correct version of Fig. 1 is included with this Correction. The original article has been updated.

Ly49Q positively regulates type I IFN production by plasmacytoid dendritic cells in an immunoreceptor tyrosine-based inhibitory motif-dependent manner.

Plasmacytoid dendritic cells (pDC) are the major producers of type I IFN during the initial immune response to viral infection. Ly49Q, a C-type lectin-like receptor specific for MHC-I, possesses a cytoplasmic ITIM and is highly expressed on murine pDC. Using Ly49Q-deficient mice, we show that, regardless of strain background, this receptor is required for maximum IFN-α production by pDC. Furthermore, Ly49Q expression on pDC, but not myeloid dendritic cells, is necessary for optimal IL-12 secretion, MHC-II expression, activation of CD4(+) T cell proliferation, and nuclear translocation of the master IFN-α regulator IFN regulatory factor 7 in response to TLR9 agonists. In contrast, the absence of Ly49Q did not affect plasmacytoid dendritic cell-triggering receptor expressed on myeloid cells expression or pDC viability. Genetic complementation revealed that IFN-α production by pDC is dependent on an intact tyrosine residue in the Ly49Q cytoplasmic ITIM. However, pharmacological inhibitors and phosphatase-deficient mice indicate that Src homology 2 domain-containing phosphatase 1 (SHP)-1, SHP-2, and SHIP phosphatase activity is dispensable for this function. Finally, we observed that Ly49Q itself is downregulated on pDC in response to CpG exposure in an ITIM-independent manner. In conclusion, Ly49Q enhances TLR9-mediated signaling events, leading to IFN regulatory factor 7 nuclear translocation and expression of IFN-I genes in an ITIM-dependent manner that can proceed without the involvement of SHP-1, SHP-2, and SHIP.

Optimized tetramer analysis reveals Ly49 promiscuity for MHC ligands.

Murine Ly49 receptors, which are expressed mainly on NK and NKT cells, interact with MHC class I (MHC-I) molecules with varying specificity. Differing reports of Ly49/MHC binding affinities may be affected by multiple factors, including cis versus trans competition and species origin of the MHC-I L chain (ß2-microglobulin). To determine the contribution of each of these factors, Ly49G, Ly49I, Ly49O, Ly49V, and Ly49Q receptors from the 129 mouse strain were expressed individually on human 293T cells or the mouse cell lines MHC-I-deficient C1498, H-2(b)-expressing MC57G, and H-2(k)-expressing L929. The capacity to bind to H-2D(b)- and H-2K(b)-soluble MHC-I tetramers containing either human or murine ß2-microglobulin L chains was tested for all five Ly49 receptors in all four cell lines. We found that most of these five inhibitory Ly49 receptors show binding for one or both self-MHC-I molecules in soluble tetramer binding assays when three conditions are fulfilled: 1) lack of competing cis interactions, 2) tetramer L chain is of mouse origin, and 3) Ly49 is expressed in mouse and not human cell lines. Furthermore, Ly49Q, the single known MHC-I receptor on plasmacytoid dendritic cells, was shown to bind H-2D(b) in addition to H-2K(b) when the above conditions were met, suggesting that Ly49Q functions as a pan-MHC-Ia receptor on plasmacytoid dendritic cells. In this study, we have optimized the parameters for soluble tetramer binding analyses to enhance future Ly49 ligand identification and to better evaluate specific contributions by different Ly49/MHC-I pairs to NK cell education and function.

Maraba MG1 virus enhances natural killer cell function via conventional dendritic cells to reduce postoperative metastatic disease.

This study characterizes the ability of novel oncolytic rhabdoviruses (Maraba MG1) to boost natural killer (NK) cell activity. Our results demonstrate that MG1 activates NK cells via direct infection and maturation of conventional dendritic cells. Using NK depletion and conventional dendritic cells ablation studies in vivo, we established that both are required for MG1 efficacy. We further explored the efficacy of attenuated MG1 (nonreplicating MG1-UV(2min) and single-cycle replicating MG1-Gless) and demonstrated that these viruses activate conventional dendritic cells, although to a lesser extent than live MG1. This translates to equivalent abilities to remove tumor metastases only at the highest viral doses of attenuated MG1. In tandem, we characterized the antitumor ability of NK cells following preoperative administration of live and attenuated MG1. Our results demonstrates that a similar level of NK activation and reduction in postoperative tumor metastases was achieved with equivalent high viral doses concluding that viral replication is important, but not necessary for NK activation. Biochemical characterization of a panel of UV-inactivated MG1 (2-120 minutes) revealed that intact viral particle and target cell recognition are essential for NK cell-mediated antitumor responses. These findings provide mechanistic insight and preclinical rationale for safe perioperative virotherapy to effectively reduce metastatic disease following cancer surgery.

Impaired natural killer cell self-education and "missing-self" responses in Ly49-deficient mice.

Ly49-mediated recognition of MHC-I molecules on host cells is considered vital for natural killer (NK)-cell regulation and education; however, gene-deficient animal models are lacking because of the difficulty in deleting this large multigene family. Here, we describe NK gene complex knockdown (NKC(KD)) mice that lack expression of Ly49 and related MHC-I receptors on most NK cells. NKC(KD) NK cells exhibit defective killing of MHC-I-deficient, but otherwise normal, target cells, resulting in defective rejection by NKC(KD) mice of transplants from various types of MHC-I-deficient mice. Self-MHC-I immunosurveillance by NK cells in NKC(KD) mice can be rescued by self-MHC-I-specific Ly49 transgenes. Although NKC(KD) mice display defective recognition of MHC-I-deficient tumor cells, resulting in decreased in vivo tumor cell clearance, NKG2D- or antibody-dependent cell-mediated cytotoxicity-induced tumor cell cytotoxicity and cytokine production induced by activation receptors was efficient in Ly49-deficient NK cells, suggesting MHC-I education of NK cells is a single facet regulating their total potential. These results provide direct genetic evidence that Ly49 expression is necessary for NK-cell education to self-MHC-I molecules and that the absence of these receptors leads to loss of MHC-I-dependent "missing-self" immunosurveillance by NK cells.

Translational control of the innate immune response through IRF-7.

Transcriptional activation of cytokines, such as type-I interferons (interferon (IFN)-alpha and IFN-beta), constitutes the first line of antiviral defence. Here we show that translational control is critical for induction of type-I IFN production. In mouse embryonic fibroblasts lacking the translational repressors 4E-BP1 and 4E-BP2, the threshold for eliciting type-I IFN production is lowered. Consequently, replication of encephalomyocarditis virus, vesicular stomatitis virus, influenza virus and Sindbis virus is markedly suppressed. Furthermore, mice with both 4E- and 4E-BP2 genes (also known as Eif4ebp1 and Eif4ebp2, respectively) knocked out are resistant to vesicular stomatitis virus infection, and this correlates with an enhanced type-I IFN production in plasmacytoid dendritic cells and the expression of IFN-regulated genes in the lungs. The enhanced type-I IFN response in 4E-BP1-/- 4E-BP2-/- double knockout mouse embryonic fibroblasts is caused by upregulation of interferon regulatory factor 7 (Irf7) messenger RNA translation. These findings highlight the role of 4E-BPs as negative regulators of type-I IFN production, via translational repression of Irf7 mRNA.

Surgical stress promotes the development of cancer metastases by a coagulation-dependent mechanism involving natural killer cells in a murine model.

OBJECTIVE: To determine whether the postoperative hypercoagulable state is responsible for the increase in metastases observed after surgery. BACKGROUND: Surgery precipitates a hypercoagulable state and increases the formation of cancer metastases in animal models. Coagulation promotes metastases by facilitating the formation of microthrombi around tumor cell emboli (TCE), thereby inhibiting natural killer (NK) cell-mediated destruction. METHODS: Mice underwent surgery preceded by tumor cell inoculation to establish pulmonary metastases in the presence or absence of various perioperative anticoagulants. Pulmonary TCE were quantified and characterized using fluorescently labeled fibrinogen and platelets. The role of NK cells was evaluated by repeating these experiments after antibody depletion in a genetically deficient strain and by adoptively transferring NK cells into NK-deficient mice. RESULTS: Surgery resulted in a consistent and significant increase in metastases while a number of different anticoagulants and platelet depletion attenuated this effect. Impaired clearance of TCE from the lungs associated with an increase in peritumoral fibrin and platelet clot formation was observed in surgically stressed mice, but not in control mice or mice that received perioperative anticoagulation. The increase in TCE survival conferred by surgery and inhibited by perioperative anticoagulation was eliminated by the immunological or genetic depletion of NK cells. Adoptive transfer experiment confirms that surgery impairs NK cell function. CONCLUSIONS: Surgery promotes the formation of fibrin and platelet clots around TCE, thereby impairing NK cell-mediated tumor cell clearance, whereas perioperative anticoagulation attenuates this effect. Therapeutic interventions aimed at reducing peritumoral clot formation and enhancing NK cell function in the perioperative period will have important clinical implications in attenuating metastatic disease after cancer surgery.

ORFV: a novel oncolytic and immune stimulating parapoxvirus therapeutic.

Replicating viruses for the treatment of cancer have a number of advantages over traditional therapeutic modalities. They are highly targeted, self-amplifying, and have the added potential to act as both gene-therapy delivery vehicles and oncolytic agents. Parapoxvirus ovis or Orf virus (ORFV) is the prototypic species of the Parapoxvirus genus, causing a benign disease in its natural ungulate host. ORFV possesses a number of unique properties that make it an ideal viral backbone for the development of a cancer therapeutic: it is safe in humans, has the ability to cause repeat infections even in the presence of antibody, and it induces a potent T(h)-1-dominated immune response. Here, we show that live replicating ORFV induces an antitumor immune response in multiple syngeneic mouse models of cancer that is mediated largely by the potent activation of both cytokine-secreting, and tumoricidal natural killer (NK) cells. We have also highlighted the clinical potential of the virus by demonstration of human cancer cell oncolysis including efficacy in an A549 xenograft model of cancer.

Gemcitabine promotes autophagy and lysosomal function through ERK- and TFEB-dependent mechanisms.

Gemcitabine is a first-line treatment agent for pancreatic ductal adenocarcinoma (PDAC). Contributing to its cytotoxicity, this chemotherapeutic agent is primarily a DNA replication inhibitor that also induces DNA damage. However, its therapeutic effects are limited owing to chemoresistance. Evidence in the literature points to a role for autophagy in restricting the efficacy of gemcitabine. Autophagy is a catabolic process in which intracellular components are delivered to degradative organelles lysosomes. Interfering with this process sensitizes PDAC cells to gemcitabine. It is consequently inferred that autophagy and lysosomal function need to be tightly regulated to maintain homeostasis and provide resistance to environmental stress, such as those imposed by chemotherapeutic drugs. However, the mechanism(s) through which gemcitabine promotes autophagy remains elusive, and the impact of gemcitabine on lysosomal function remains largely unexplored. Therefore, we applied complementary approaches to define the mechanisms triggered by gemcitabine that support autophagy and lysosome function. We found that gemcitabine elicited ERK-dependent autophagy in PDAC cells, but did not stimulate ERK activity or autophagy in non-tumoral human pancreatic epithelial cells. Gemcitabine also promoted transcription factor EB (TFEB)-dependent lysosomal function in PDAC cells. Indeed, treating PDAC cells with gemcitabine caused expansion of the lysosomal network, as revealed by Lysosome associated membrane protein-1 (LAMP1) and LysoTracker staining. More specific approaches have shown that gemcitabine promotes the activity of cathepsin B (CTSB), a cysteine protease playing an active role in lysosomal degradation. We showed that lysosomal function induced by gemcitabine depends on TFEB, the master regulator of autophagy and lysosomal biogenesis. Interfering with TFEB function considerably limited the clonogenic growth of PDAC cells and hindered the capacity of TFEB-depleted PDAC cells to develop orthotopic tumors.

Perioperative oncolytic virotherapy to counteract surgery-induced immunosuppression and improve outcomes in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a high fatality cancer with one of the worst prognoses in solid tumors. Most patients present with late stage, metastatic disease and are not eligible for potentially curative surgery. Despite complete resection, the majority of surgical patients will recur within the first two years following surgery. Postoperative immunosuppression has been described in different digestive cancers. While the underlying mechanism is not fully understood, there is compelling evidence to link surgery with disease progression and cancer metastasis in the postoperative period. However, the idea of surgery-induced immunosuppression as a facilitator of recurrence and metastatic spread has not been explored in the context of pancreatic cancer. By surveying the existing literature on surgical stress in mostly digestive cancers, we propose a novel practice-changing paradigm: alleviate surgery-induced immunosuppression and improve oncological outcome in PDAC surgical patients by administering oncolytic virotherapy in the perioperative period.

Heterologous prime-boost cellular vaccination induces potent antitumor immunity against triple negative breast cancer.

Introduction: Triple negative breast cancer (TNBC) is the most aggressive and hard-to-treat subtype of breast cancer, affecting 10-20% of all women diagnosed with breast cancer. Surgery, chemotherapy and hormone/Her2 targeted therapies are the cornerstones of treatment for breast cancer, but women with TNBC do not benefit from these treatments. Although the prognosis is dismal, immunotherapies hold significant promise in TNBC, even in wide spread disease because TNBC is infiltrated with more immune cells. This preclinical study is proposing to optimize an oncolytic virus-infected cell vaccine (ICV) based on a prime-boost vaccination strategy to address this unmet clinical need. Methods: We used various classes of immunomodulators to improve the immunogenicity of whole tumor cells in the prime vaccine, followed by their infection with oncolytic Vesicular Stomatitis Virus (VSVd51) to deliver the boost vaccine. For in vivo studies, we compared the efficacy of a homologous prime-boost vaccination regimen to a heterologous strategy by treating 4T1 tumor bearing BALB/c mice and further by conducting re-challenge studies to evaluate immune memory responses in surviving mice. Due to the aggressive nature of 4T1 tumor spread (akin to stage IV TNBC in human patients), we also compared early surgical resection of primary tumors versus later surgical resection combined with vaccination. Results: In vitro results demonstrated that immunogenic cell death (ICD) markers and pro-inflammatory cytokines were released at the highest levels following treatment of mouse 4T1 TNBC cells with oxaliplatin chemotherapy and influenza vaccine. These ICD inducers also contributed towards higher dendritic cell recruitment and activation. With the top ICD inducers at hand, we observed that treatment of TNBC-bearing mice with the influenza virus-modified prime vaccine followed by VSVd51 infected boost vaccine resulted in the best survival. Furthermore, higher frequencies of both effector and central memory T cells along with a complete absence of recurrent tumors were observed in re-challenged mice. Importantly, early surgical resection combined with prime-boost vaccination led to improved overall survival in mice. Conclusion: Taken together, this novel cancer vaccination strategy following early surgical resection could be a promising therapeutic avenue for TNBC patients.

Synthetic virology approaches to improve the safety and efficacy of oncolytic virus therapies.

The large coding potential of vaccinia virus (VV) vectors is a defining feature. However, limited regulatory switches are available to control viral replication as well as timing and dosing of transgene expression in order to facilitate safe and efficacious payload delivery. Herein, we adapt drug-controlled gene switches to enable control of virally encoded transgene expression, including systems controlled by the FDA-approved rapamycin and doxycycline. Using ribosome profiling to characterize viral promoter strength, we rationally design fusions of the operator element of different drug-inducible systems with VV promoters to produce synthetic promoters yielding robust inducible expression with undetectable baseline levels. We also generate chimeric synthetic promoters facilitating additional regulatory layers for VV-encoded synthetic transgene networks. The switches are applied to enable inducible expression of fusogenic proteins, dose-controlled delivery of toxic cytokines, and chemical regulation of VV replication. This toolbox enables the precise modulation of transgene circuitry in VV-vectored oncolytic virus design.

Fatty acid transport protein inhibition sensitizes breast and ovarian cancers to oncolytic virus therapy via lipid modulation of the tumor microenvironment.

Introduction: Adipocytes in the tumour microenvironment are highly dynamic cells that have an established role in tumour progression, but their impact on anti-cancer therapy resistance is becoming increasingly difficult to overlook. Methods: We investigated the role of adipose tissue and adipocytes in response to oncolytic virus (OV) therapy in adipose-rich tumours such as breast and ovarian neoplasms. Results: We show that secreted products in adipocyte-conditioned medium significantly impairs productive virus infection and OV-driven cell death. This effect was not due to the direct neutralization of virions or inhibition of OV entry into host cells. Instead, further investigation of adipocyte secreted factors demonstrated that adipocyte-mediated OV resistance is primarily a lipid-driven phenomenon. When lipid moieties are depleted from the adipocyte-conditioned medium, cancer cells are re-sensitized to OV-mediated destruction. We further demonstrated that blocking fatty acid uptake by cancer cells, in a combinatorial strategy with virotherapy, has clinical translational potential to overcome adipocyte-mediated OV resistance. Discussion: Our findings indicate that while adipocyte secreted factors can impede OV infection, the impairment of OV treatment efficacy can be overcome by modulating lipid flux in the tumour milieu.

Effect of Ly49 haplotype variance on NK cell function and education.

The class I MHC-specific receptors expressed by murine NK cells exhibit remarkable variation. Specific activating killer Ig-related receptor/Ly49 have major effects on autoimmune and infectious disease induction and outcome in humans and mice. However, these studies are greatly affected by individual background genetics. Furthermore, the educational impact of variable inhibitory KIR/Ly49 gene numbers on NK cell development and the subsequent ability to survey for MHC class I (MHC-I) expression remain unknown. To address these questions, Ly49 congenic mice were generated that maintain a 129-derived Ly49 gene cluster on a C57BL/6 genetic background (B6.Ly49(129) mice), and the in vitro and in vivo NK cell function of these mice was compared with their inbred parental 129S1 and C57BL/6 counterparts. Notably, target cell recognition directed by activating Ly49 receptors was profoundly affected by allelic variation in B6.Ly49(129) congenic cells versus C57BL/6 NK cells. Furthermore, when assessing NK cell function based on education and subsequent recognition of the C57BL/6 MHC-I haplotype by inhibitory Ly49 receptors, B6.Ly49(129) congenic mice exhibited robust NK cell activity, demonstrating efficient NK cell education by the 129S1 Ly49 cluster during development. The responsiveness of NK cells expressing 129S1 Ly49 was shown to be mediated by subsets expressing one or more self-MHC receptors, including Ly49I, Ly49O, Ly49V, and NKG2A. These findings demonstrate that the genetically segregating and diverse MHC-I and Ly49 loci in mice exhibit independent and epistatic effects on NK cell education that can be uncoupled during the intercrossing of inbred strains.

Remodeling the tumor immune microenvironment with oncolytic viruses expressing miRNAs.

MiRNAs (miRNA, miR) play important functions in the tumor microenvironment (TME) by silencing gene expression through RNA interference. They are involved in regulating both tumor progression and tumor suppression. The pathways involved in miRNA processing and the miRNAs themselves are dysregulated in cancer. Consequently, they have become attractive therapeutic targets as underscored by the plethora of miRNA-based therapies currently in pre-clinical and clinical studies. It has been shown that miRNAs can be used to improve oncolytic viruses (OVs) and enable superior viral oncolysis, tumor suppression and immune modulation. In these cases, miRNAs are empirically selected to improve viral oncolysis, which translates into decreased tumor growth in multiple murine models. While this infectious process is critical to OV therapy, optimal immunomodulation is crucial for the establishment of a targeted and durable effect, resulting in cancer eradication. Through numerous mechanisms, OVs elicit a strong antitumor immune response that can also be further improved by miRNAs. They are known to regulate components of the immune TME and promote effector functions, antigen presentation, phenotypical polarization, and varying levels of immunosuppression. Reciprocally, OVs have the power to overcome the limitations encountered in canonical miRNA-based therapies. They deliver therapeutic payloads directly into the TME and facilitate their amplification through selective tumoral tropism and abundant viral replication. This way, off-target effects can be minimized. This review will explore the ways in which miRNAs can synergistically enhance OV immunotherapy to provide the basis for future therapeutics based on this versatile combination platform.

Time-course full profiling of circulating miRNAs in neurologically deceased organ donors: a proof of concept study to understand the onset of the cytokine storm.

Neurologically deceased organ donors (NDDs) generally display an immune response involving an intense production of pro-inflammatory cytokines referred to as the cytokine storm. The sudden surge of inflammatory mediators in circulation promotes tissue and organ damages and ultimately leads to poor transplant outcome. As microRNAs (miRNAs) are frequently proposed as key regulators of inflammation and are relatively stable in circulation, changes in their profiles could play a role in the onset of the cytokine storm in NDDs. In this proof-of-concept study, we sought to investigate differentially abundant circulating miRNAs in a temporal manner between neurological death and organ recovery and to assess the association between specific miRNAs and levels of inflammatory cytokines in blood. Plasma samples from five NDDs were obtained at multiple time points between organ donation consent and organ recovery. Using a time-course analysis and miRNA sequencing, we identified 32 plasma miRNAs fluctuating between consent and organ recovery (false discovery rate; q-value < 0.1). Eleven miRNAs relatively abundant (>100 reads) and detected in all samples were selected for further biological pathway analysis (miR-486-3p, miR-103a-3p, miR-106b-3p, miR-182-5p, miR-101-3p, miR-10a-5p, miR-125a-5p, miR-146b-5p, miR-26a-5p, miR-423-5p, miR-92b-3p). These miRNAs targeted genes such as c-JUN (TNF signalling pathway) and eEF2 (AMPK pathway), suggesting a potential role in regulation of inflammation. Our results contribute to a better understanding of the miRNAs dynamic after neurological death in organ donors and could potentially be used to predict the related early cytokine storm.Trial registration: ClinicalTrials.gov ID NCT03786991. Registered December 2018.