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RNA vaccines traditionally consist of messenger RNA synthesized by in vitro transcription using a bacteriophage RNA polymerase and template DNA that encodes the antigen(s) of interest. Once administered and internalized by host cells, the mRNA transcripts are translated directly in the cytoplasm and then the resulting antigens are presented to antigen presenting cells to stimulate an immune response. Alternatively, dendritic cells can be loaded with either tumor associated antigen mRNA or total tumor RNA and delivered to the host to elicit a specific immune response. In this review, we will explain why RNA vaccines represent an attractive platform for cancer immunotherapy, discuss modifications to RNA structure that have been developed to optimize mRNA vaccine stability and translational efficiency, and describe strategies for nonviral delivery of mRNA vaccines, highlighting key preclinical and clinical data related to cancer immunotherapy.

Flow cytometry use has significantly increased in clinical laboratories and has significantly helped improve the diagnosis of leukemias, lymphomas, and follow-up of minimal residual disease. Mastering this technique enables the performance of multiparametric single-cell analysis and increases the odds of identifying abnormal populations. As in many fields, there is a need to improve the quality of the data generated for accuracy, reproducibility, and trueness. The implementation of solutions reducing variability is achievable and needed, as the flow cytometry workflow involves many manual steps and items susceptible to operator bias and human error. Standardization of flow cytometry assays is sought and already implemented in many clinical hematology laboratories. However, the clinical community would highly benefit from further efforts in that direction to increase the comparability of findings across laboratories. This review covers the strengths and weaknesses of flow cytometry and focuses on the standardization approaches developed, including recent advances in the field.

Activating intra-tumor innate immunity might enhance tumor immune surveillance. Virotherapy is proposed to achieve tumor cell killing, while indirectly activating innate immunity. Here, we report that recombinant poliovirus therapy primarily mediates antitumor immunotherapy via direct infection of non-malignant tumor microenvironment (TME) cells, independent of malignant cell lysis. Relative to other innate immune agonists, virotherapy provokes selective, TBK1-IRF3 driven innate inflammation that is associated with sustained type-I/III interferon (IFN) release. Despite priming equivalent antitumor T cell quantities, MDA5-orchestrated TBK1-IRF3 signaling, but not NFκB-polarized TLR activation, culminates in polyfunctional and Th1-differentiated antitumor T cell phenotypes. Recombinant type-I IFN increases tumor-localized T cell function, but does not mediate durable antitumor immunotherapy without concomitant pattern recognition receptor (PRR) signaling. Thus, virus-induced MDA5-TBK1-IRF3 signaling in the TME provides PRR-contextualized IFN responses that elicit functional antitumor T cell immunity. TBK1-IRF3 innate signal transduction stimulates eventual function and differentiation of tumor-infiltrating T cells. Innate intratumoral immunity might be important in enhancing oncolytic tumor immunotherapy. Here, the authors show that recombinant poliovirus signalling through TBK-IRF3 enhances tumor-infiltrating T cell activity and multi-cytokine function.

Immunotherapies are rapidly being integrated into standard of care (SOC) therapy in conjunction with surgery, chemotherapy, and radiotherapy for many cancers and a large number of clinical studies continue to explore immunotherapy alone and as part of combination therapies in patients with cancer. It is evident that clinical effectiveness of immunotherapy is limited to a subset of patients and improving immunotherapy related outcomes remains a major scientific and clinical effort. Understanding the immune cell subset phenotype and activation/functional status (cellular immunome) prior to and post therapy is therefore critical to develop biomarkers that (1) will predict if a patient will respond to immunotherapy and (2) are a result of immunotherapy. In this study, we investigated local (tumor) and peripheral (blood) cellular immunome of patients with melanoma, breast cancer, and brain cancer using a rapid and reliable standardized, multiparameter flow cytometry assay. We used this approach to monitor changes in the peripheral cellular immunome in women with breast cancer undergoing SOC therapy. Our analysis is unique because it is conducted using matched fresh tumor tissue and blood from patients in real-time, within 2-3 h of sample acquisition, and provides insight into the innate and adaptive immune cell profile in blood and tumor. Specific to blood, this approach involves no manipulation and evaluates all immune subsets such as T cells, B cells, natural killer (NK) cells, monocytes, dendritic cells (DCs), neutrophils, eosinophils, and basophils using 0.5 ml of blood. Analysis of the corresponding tumor provides much needed insight into the phenotype and activation status of immune cells, especially T and B cells, in the tumor microenvironment vs. the periphery. This analysis will be used to assess baseline and therapy-mediated changes in local and peripheral cellular immunome in patients with glioblastoma, breast cancer, and melanoma in planned immunotherapy clinical studies.

Sensors of the NLR family generally activate innate immunity. Ting et al ., however, demonstrate that the little-known NLRC3 negatively regulates Toll-like receptor signaling by altering ubiquitination of the signaling adaptor TRAF6. Several members of the NLR family of sensors activate innate immunity. In contrast, we found here that NLRC3 inhibited Toll-like receptor (TLR)-dependent activation of the transcription factor NF-κB by interacting with the TLR signaling adaptor TRAF6 to attenuate Lys63 (K63)-linked ubiquitination of TRAF6 and activation of NF-κB. We used bioinformatics to predict interactions between NLR and TRAF proteins, including interactions of TRAF with NLRC3. In vivo , macrophage expression of Nlrc3 mRNA was diminished by the administration of lipopolysaccharide (LPS) but was restored when cellular activation subsided. To assess biologic relevance, we generated Nlrc3 −/− mice. LPS-treated Nlrc3 −/− macrophages had more K63-ubiquitinated TRAF6, nuclear NF-κB and proinflammatory cytokines. Finally, LPS-treated Nlrc3 −/− mice had more signs of inflammation. Thus, signaling via NLRC3 and TLR constitutes a negative feedback loop. Furthermore, prevalent NLR-TRAF interactions suggest the formation of a 'TRAFasome' complex.

BackgroundAlthough sentinel lymph node (SLN) biopsy is a standard procedure used to identify patients at risk for melanoma recurrence, it fails to risk-stratify certain patients accurately. Because processes in SLNs regulate anti-tumor immune responses, the authors hypothesized that SLN gene expression may be used for risk stratification.MethodsThe Nanostring nCounter PanCancer Immune Profiling Panel was used to quantify expression of 730 immune-related genes in 60 SLN specimens (31 positive [pSLNs], 29 negative [nSLNs]) from a retrospective melanoma cohort. A multivariate prediction model for recurrence-free survival (RFS) was created by applying stepwise variable selection to Cox regression models. Risk scores calculated on the basis of the model were used to stratify patients into low- and high-risk groups. The predictive power of the model was assessed using the Kaplan–Meier and log-rank tests.ResultsDuring a median follow-up period of 6.3 years, 20 patients (33.3%) experienced recurrence (pSLN, 45.2% [14/31] vs nSLN, 20.7% [6/29]; p = 0.0445). A fitted Cox regression model incorporating 12 genes accurately predicted RFS (C-index, 0.9919). Improved RFS was associated with increased expression of TIGIT (p = 0.0326), an immune checkpoint, and decreased expression of CXCL16 (p = 0.0273), a cytokine important in promoting dendritic and T cell interactions. Independent of SLN status, the model in this study was able to stratify patients into cohorts at high and low risk for recurrence (p < 0.001, log-rank).ConclusionsExpression profiles of the SLN gene are associated with melanoma recurrence and may be able to identify patients as high or low risk regardless of SLN status, potentially enhancing patient selection for adjuvant therapy.

Development of effective, yet safe, antithrombotic agents has been challenging because such agents increase the propensity of patients to bleed. Recently, naturally occurring polyphosphates such as extracellular DNA, RNA, and inorganic polyphosphates have been shown to activate blood coagulation. In this report, we evaluate the anticoagulant and antithrombotic activity of nucleic acid-binding polymers in vitro and in vivo. Such polymers bind to DNA, RNA, and inorganic polyphosphate molecules with high affinity and inhibit RNA- and polyphosphate-induced clotting and the activation of the intrinsic pathway of coagulation in vitro. Moreover, [NH ₂(CH ₂) ₂NH ₂]∶(G = 3);dendri PAMAM(NH ₂) ₃₂ (PAMAM G-3) prevents thrombosis following carotid artery injury and pulmonary thromboembolism in mice without significantly increasing blood loss from surgically challenged animals. These studies indicate that nucleic acid-binding polymers are able to scavenge effectively prothrombotic nucleic acids and other polyphosphates in vivo and represent a new and potentially safer class of antithrombotic agents.

BackgroundIn melanoma patients, microscopic tumor in the sentinel lymph-node biopsy (SLN) increases the risk of distant metastases, but the transition from tumor in the SLN to metastatic disease remains poorly understood.MethodsFluorescent staining for CD3, CD20, CD11c, and DNA was performed on SLN tissue and matching primary tumors. Regions of interest (ROI) were then chosen geometrically (e.g., tumor) or by fluorescent cell subset markers (e.g., CD11c). Each ROI was further analyzed using NanoString Digital Spatial Profiling high-resolution multiplex profiling. Digital counts for 59-panel immune-related proteins were collected and normalized to account for system variation and ROI area. ResultsTumor regions of SLNs had variable infiltration of CD3 cells among patients. The patient with overall survival (OS) > 8 years had the most CD11c- and CD3-expressing cells infiltrating the SLN tumor region. All patients had CD11c (dendritic cell, DC) infiltration into the SLN tumor region. Selecting ROI by specific cell subtype, we compared protein expression of CD11c cells between tumor and non-tumor/normal tissue SLN regions. Known markers of DC activation such as CD86, HLA-DR, and OX40L were lowest on CD11c cells within SLN tumor for the patient with OS < 1 year and highest on the patient with OS > 8 years.ConclusionWe demonstrate the feasibility of profiling the protein expression of CD11c cells within the SLN tumor. Identifying early regulators of melanoma control when the disease is microscopically detected in the SLN is beneficial and requires follow-up studies in a larger cohort of patients.

Nucleic acid-containing debris released from dead and dying cells can be recognized as damage-associated molecular patterns (DAMPs) or pattern-associated molecular patterns (PAMPs) by the innate immune system. Inappropriate activation of the innate immune response can engender pathological inflammation and autoimmune disease. To combat such diseases, major efforts have been made to therapeutically target the pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs) that recognize such DAMPs and PAMPs, or the downstream effector molecules they engender, to limit inflammation. Unfortunately, such strategies can limit the ability of the immune system to combat infection. Previously, we demonstrated that nucleic acid-binding polymers can act as molecular scavengers and limit the ability of artificial nucleic acid ligands to activate PRRs. Herein, we demonstrate that nucleic acid scavengers (NASs) can limit pathological inflammation and nucleic acid-associated autoimmunity in lupus-prone mice. Moreover, we observe that such NASs do not limit an animal’s ability to combat viral infection, but rather their administration improves survival when animals are challenged with lethal doses of influenza. These results indicate that molecules that scavenge extracellular nucleic acid debris represent potentially safer agents to control pathological inflammation associated with a wide range of autoimmune and infectious diseases.

Plexins are a family of genes (A,B,C, and D) that are expressed in many organ systems. Plexins expressed in the immune system have been implicated in cell movement and cell-cell interaction during the course of an immune response. In this study, the expression pattern of Plexin-B2 and Plexin-D1 in dendritic cells (DCs), which are central in immune activation, was investigated. Plexin-B2 and Plexin-D1 are reciprocally expressed in myeloid and plasmacytoid DC populations. Plasmacytoid DCs have high Plexin-B2 but low Plexin-D1, while the opposite is true of myeloid DCs. Expression of Plexin-B2 and Plexin-D1 is modulated upon activation of DCs by TLR ligands, TNFα, and anti-CD40, again in a reciprocal fashion. Semaphorin3E, a ligand for Plexin-D1 and Plexin-B2, is expressed by T cells, and interestingly, is dramatically higher on Th2 cells and on DCs. The expression of Plexins and their ligands on DCs and T cells suggest functional relevance. To explore this, we utilized chimeric mice lacking Plxnb2 or Plxnd1. Absence of Plexin-B2 and Plexin-D1 on DCs did not affect the ability of these cells to upregulate costimulatory molecules or the ability of these cells to activate antigen specific T cells. Additionally, Plexin-B2 and Plexin-D1 were dispensable for chemokine-directed in-vitro migration of DCs towards key DC chemokines, CXCL12 and CCL19. However, the absence of either Plexin-B2 or Plexin-D1 on DCs leads to constitutive expression of IL-12/IL-23p40. This is the first report to show an association between Plexin-B2 and Plexin-D1 with the negative regulation of IL-12/IL-23p40 in DCs. This work also shows the presence of Plexin-B2 and Plexin-D1 on mouse DC subpopulations, and indicates that these two proteins play a role in IL-12/IL-23p40 production that is likely to impact the immune response.