TLR9 plus STING Agonist Adjuvant Combination Induces Potent Neopeptide T Cell Immunity and Improves Immune Checkpoint Blockade Efficacy in a Tumor Model.

Immune checkpoint blockade (ICB) immunotherapies have emerged as promising strategies for the treatment of cancer; however, there remains a need to improve their efficacy. Determinants of ICB efficacy are the frequency of tumor mutations, the associated neoantigens, and the T cell response against them. Therefore, it is expected that neoantigen vaccinations that boost the antitumor T cell response would improve ICB therapy efficacy. The aim of this study was to develop a highly immunogenic vaccine using pattern recognition receptor agonists in combination with synthetic long peptides to induce potent neoantigen-specific T cell responses. We determined that the combination of the TLR9 agonist K-type CpG oligodeoxynucleotides (K3 CpG) with the STING agonist c-di-AMP (K3/c-di-AMP combination) significantly increased dendritic cell activation. We found that immunizing mice with 20-mer of either an OVA peptide, low-affinity OVA peptides, or neopeptides identified from mouse melanoma or lung mesothelioma, together with K3/c-di-AMP, induced potent Ag-specific T cell responses. The combined K3/c-di-AMP adjuvant formulation induced 10 times higher T cell responses against neopeptides than the TLR3 agonist polyinosinic:polycytidylic acid, a derivative of which is the leading adjuvant in clinical trials of neoantigen peptide vaccines. Moreover, we demonstrated that our K3/c-di-AMP vaccine formulation with 20-mer OVA peptide was capable of controlling tumor growth and improving survival in B16-F10-OVA tumor-bearing C57BL/6 mice and synergized with anti-PD-1 treatment. Together, our findings demonstrate that the K3/c-di-AMP vaccine formulation induces potent T cell immunity against synthetic long peptides and is a promising candidate to improve neoantigen vaccine platform.

Overcoming immune checkpoint blockade resistance in solid tumors with intermittent ITK inhibition.

Cytotoxic CD8 + T cell (CTL) exhaustion is driven by chronic antigen stimulation. Reversing CTL exhaustion with immune checkpoint blockade (ICB) has provided clinical benefits in different types of cancer. We, therefore, investigated whether modulating chronic antigen stimulation and T-cell receptor (TCR) signaling with an IL2-inducible T-cell kinase (ITK) inhibitor, could confer ICB responsiveness to ICB resistant solid tumors. In vivo intermittent treatment of 3 ICB-resistant solid tumor (melanoma, mesothelioma or pancreatic cancer) with ITK inhibitor significantly improved ICB therapy. ITK inhibition directly reinvigorate exhausted CTL in vitro as it enhanced cytokine production, decreased inhibitory receptor expression, and downregulated the transcription factor TOX. Our study demonstrates that intermittent ITK inhibition can be used to directly ameliorate CTL exhaustion and enhance immunotherapies even in solid tumors that are ICB resistant.

Ibrutinib directly reduces CD8+T cell exhaustion independent of BTK.

Introduction: Cytotoxic CD8+ T cell (CTL) exhaustion is a dysfunctional state of T cells triggered by persistent antigen stimulation, with the characteristics of increased inhibitory receptors, impaired cytokine production and a distinct transcriptional profile. Evidence from immune checkpoint blockade therapy supports that reversing T cell exhaustion is a promising strategy in cancer treatment. Ibrutinib, is a potent inhibitor of BTK, which has been approved for the treatment of chronic lymphocytic leukemia. Previous studies have reported improved function of T cells in ibrutinib long-term treated patients but the mechanism remains unclear. We investigated whether ibrutinib directly acts on CD8+ T cells and reinvigorates exhausted CTLs. Methods: We used an established in vitro CTL exhaustion system to examine whether ibrutinib can directly ameliorate T cell exhaustion. Changes in inhibitory receptors, transcription factors, cytokine production and killing capacity of ibrutinib-treated exhausted CTLs were detected by flow cytometry. RNA-seq was performed to study transcriptional changes in these cells. Btk deficient mice were used to confirm that the effect of ibrutinib was independent of BTK expression. Results: We found that ibrutinib reduced exhaustion-related features of CTLs in an in vitro CTL exhaustion system. These changes included decreased inhibitory receptor expression, enhanced cytokine production, and downregulation of the transcription factor TOX with upregulation of TCF1. RNA-seq further confirmed that ibrutinib directly reduced the exhaustion-related transcriptional profile of these cells. Importantly, using btk deficient mice we showed the effect of ibrutinib was independent of BTK expression, and therefore mediated by one of its other targets. Discussion: Our study demonstrates that ibrutinib directly ameliorates CTL exhaustion, and provides evidence for its synergistic use with cancer immunotherapy.

Evaluation of pre-processing methods for tear fluid proteomics using proximity extension assays.

Tear fluid forms a potential source for biomarker identification, and can be minimal invasively collected via Schirmer strips. The lack of knowledge on the processing of Schirmer strips however complicates the analysis and between-study comparisons. We studied two different pre-processing methods, specifically the use of punches of the strip versus elution of the strip in a buffer. Tear fluid filled Schirmer strips were collected from 5 healthy participants, and divided into two halves over the length of the strip. In either part, punches or eluates were obtained from 4 different locations, from the first part touching the eye (head) to the end, to assess the protein distribution along the strips. The levels of 92 inflammatory proteins were measured in the punches/eluates using proximity extension assays. The punch method yielded higher protein detectability compared to the elution method (76% vs 66%; p ≤ 0.001). Protein expression level was found to be slightly higher in the head of the strip, however, 3 out of 5 punches from the head failed quality control. Protein expression levels over the remaining parts of the strips were similar. Our study showed beneficial use of punches of any part of the strip except the head in future biomarker research.

Trained Immunity in Primary Sjögren's Syndrome: Linking Type I Interferons to a Pro-Atherogenic Phenotype.

Background: Trained immunity - or innate immune memory - can be described as the long-term reprogramming of innate immune cells towards a hyperresponsive state which involves intracellular metabolic changes. Trained immunity has been linked to atherosclerosis. A subgroup of patients with primary Sjögren's syndrome (pSS) exhibits systemic type I interferon (IFN) pathway activation, indicating innate immune hyperactivation. Here, we studied the link between type I IFNs and trained immunity in an in vitro monocytic cell model and peripheral blood mononuclear cells (PBMCs) from pSS patients. Methods: The training stimuli heat killed Candida albicans, muramyl dipeptide, IFNß, and patient serum were added to THP-1 cells for 24 hours, after which the cells were washed, rested for 48 hours and subsequently re-stimulated with LPS, Pam3Cys, poly I:C, IFNß or oxLDL for 4-24 hours. PBMCs from pSS patients and healthy controls were stimulated with LPS, Pam3Cys, poly I:C or IFNß for 0.5-24 hours. Results: Training with IFNß induced elevated production of pro-atherogenic cytokines IL-6, TNFα and CCL2, differential cholesterol- and glycolysis-related gene expression, and increased glucose consumption and oxLDL uptake upon re-stimulation. Type I IFN production was increased in Candida albicans- and IFNß-trained cells after LPS re-stimulation, but was reduced after poly I:C re-stimulation. Training with muramyl dipeptide and IFNß, but not Candida albicans, affected the IFN-stimulated gene expression response to IFNß re-stimulation. PBMCs from pSS patients consumed more glucose compared with healthy control PBMCs and tended to produce more TNFα and type I IFNs upon LPS stimulation, but less type I IFNs upon poly I:C stimulation. Conclusions: Type I IFN is a trainer inducing a trained immunity phenotype with pro-atherogenic properties in monocytes. Conversely, trained immunity also affects the production of type I IFNs and transcriptional response to type I IFN receptor re-stimulation. The phenotype of pSS PBMCs is consistent with trained immunity. This connection between type I IFN, trained immunity and cholesterol metabolism may have important implications for pSS and the pathogenesis of (subclinical) atherosclerosis in these patients.

Stratification of hospitalized COVID-19 patients into clinical severity progression groups by immuno-phenotyping and machine learning.

Quantitative or qualitative differences in immunity may drive clinical severity in COVID-19. Although longitudinal studies to record the course of immunological changes are ample, they do not necessarily predict clinical progression at the time of hospital admission. Here we show, by a machine learning approach using serum pro-inflammatory, anti-inflammatory and anti-viral cytokine and anti-SARS-CoV-2 antibody measurements as input data, that COVID-19 patients cluster into three distinct immune phenotype groups. These immune-types, determined by unsupervised hierarchical clustering that is agnostic to severity, predict clinical course. The identified immune-types do not associate with disease duration at hospital admittance, but rather reflect variations in the nature and kinetics of individual patient's immune response. Thus, our work provides an immune-type based scheme to stratify COVID-19 patients at hospital admittance into high and low risk clinical categories with distinct cytokine and antibody profiles that may guide personalized therapy.

Hyperresponsive cytosolic DNA-sensing pathway in monocytes from primary Sjögren's syndrome.

OBJECTIVES: Cytosolic DNA-sensing pathway stimulation prompts type I IFN (IFN-I) production, but its role in systemic IFN-I pathway activation in primary SS (pSS) is poorly studied. Here we investigate the responsiveness of pSS monocytes and plasmacytoid dendritic cells (pDCs) to stimulator of interferon genes (STING) activation in relation to systemic IFN-I pathway activation and compare this with SLE. METHODS: Expression of DNA-sensing receptors cGAS, IFI16, ZBP-1 and DDX41, signalling molecules STING, TBK1 and IRF3, positive and negative STING regulators, and IFN-I-stimulated genes MxA, IFI44, IFI44L, IFIT1 and IFIT3 was analysed in whole blood, CD14+ monocytes, pDCs, and salivary glands by RT-PCR, monocyte RNA sequencing data, flow cytometry and immunohistochemical staining. Peripheral blood mononuclear cells (PBMCs) from pSS, SLE and healthy controls (HCs) were stimulated with STING agonist 2'3'-cGAMP. STING phosphorylation (pSTING) and intracellular IFNα were evaluated using flow cytometry. RESULTS: STING activation induced a significantly higher proportion of IFNα-producing monocytes, but not pDCs, in both IFN-low and IFN-high pSS compared with HC PBMCs. Additionally, a trend towards more pSTING+ monocytes was observed in pSS and SLE, most pronounced in IFN-high patients. Positive STING regulators TRIM38, TRIM56, USP18 and SENP7 were significantly higher expression in pSS than HC monocytes, while the dual-function STING regulator RNF26 was downregulated in pSS monocytes. STING was expressed in mononuclear infiltrates and ductal epithelium in pSS salivary glands. STING stimulation induced pSTING and IFNα in pSS and SLE pDCs. CONCLUSION: pSS monocytes and pDCs are hyperresponsive to stimulation of the STING pathway, which was not restricted to patients with IFN-I pathway activation.

A non-lethal method for studying scorpion venom gland transcriptomes, with a review of potentially suitable taxa to which it can be applied.

Scorpion venoms are mixtures of proteins, peptides and small molecular compounds with high specificity for ion channels and are therefore considered to be promising candidates in the venoms-to-drugs pipeline. Transcriptomes are important tools for studying the composition and expression of scorpion venom. Unfortunately, studying the venom gland transcriptome traditionally requires sacrificing the animal and therefore is always a single snapshot in time. This paper describes a new way of generating a scorpion venom gland transcriptome without sacrificing the animal, thereby allowing the study of the transcriptome at various time points within a single individual. By comparing these venom-derived transcriptomes to the traditional whole-telson transcriptomes we show that the relative expression levels of the major toxin classes are similar. We further performed a multi-day extraction using our proposed method to show the possibility of doing a multiple time point transcriptome analysis. This allows for the study of patterns of toxin gene activation over time a single individual, and allows assessment of the effects of diet, season and other factors that are known or likely to influence intraindividual venom composition. We discuss the gland characteristics that may allow this method to be successful in scorpions and provide a review of other venomous taxa to which this method may potentially be successfully applied.

A Web of Coagulotoxicity: Failure of Antivenom to Neutralize the Destructive (Non-Clotting) Fibrinogenolytic Activity of Loxosceles and Sicarius Spider Venoms.

Envenomations are complex medical emergencies that can have a range of symptoms and sequelae. The only specific, scientifically-validated treatment for envenomation is antivenom administration, which is designed to alleviate venom effects. A paucity of efficacy testing exists for numerous antivenoms worldwide, and understanding venom effects and venom potency can help identify antivenom improvement options. Some spider venoms can produce debilitating injuries or even death, yet have been largely neglected in venom and antivenom studies because of the low venom yields. Coagulation disturbances have been particularly under studied due to difficulties in working with blood and the coagulation cascade. These circumstances have resulted in suboptimal spider bite treatment for medically significant spider genera such as Loxosceles and Sicarius. This study identifies and quantifies the anticoagulant effects produced by venoms of three Loxoscles species (L. reclusa, L. boneti, and L. laeta) and that of Sicarius terrosus. We showed that the venoms of all studied species are able to cleave the fibrinogen Aα-chain with varying degrees of potency, with L. reclusa and S. terrosus venom cleaving the Aα-chain most rapidly. Thromboelastography analysis revealed that only L. reclusa venom is able to reduce clot strength, thereby presumably causing anticoagulant effects in the patient. Using the same thromboelastography assays, antivenom efficacy tests revealed that the commonly used Loxoscles-specific SMase D recombinant based antivenom failed to neutralize the anticoagulant effects produced by Loxosceles venom. This study demonstrates the fibrinogenolytic activity of Loxosceles and Sicarius venom and the neutralization failure of Loxosceles antivenom, thus providing impetus for antivenom improvement.

Transcriptome annotation and characterization of novel toxins in six scorpion species.

BACKGROUND: Venom has evolved in parallel in multiple animals for the purpose of self-defense, prey capture or both. These venoms typically consist of highly complex mixtures of toxins: diverse bioactive peptides and/or proteins each with a specific pharmacological activity. Because of their specificity, they can be used as experimental tools to study cell mechanisms and develop novel medicines and drugs. It is therefore potentially valuable to explore the venoms of various animals to characterize their toxins and identify novel toxin-families. This study focuses on the annotation and exploration of the transcriptomes of six scorpion species from three different families. The transcriptomes were annotated with a custom-built automated pipeline, primarily consisting of Basic Local Alignment Search Tool searches against UniProt databases and filter steps based on transcript coverage. RESULTS: We annotated the transcriptomes of four scorpions from the family Buthidae, one from Iuridae and one from Diplocentridae using our annotation pipeline. We found that the four buthid scorpions primarily produce disulfide-bridged ion-channel targeting toxins, while the non-buthid scorpions have a higher abundance of non-disulfide-bridged toxins. Furthermore, analysis of the "unidentified" transcripts resulted in the discovery of six novel putative toxin families containing a total of 37 novel putative toxins. Additionally, 33 novel toxins in existing toxin-families were found. Lastly, 19 novel putative secreted proteins without toxin-like disulfide bonds were found. CONCLUSIONS: We were able to assign most transcripts to a toxin family and classify the venom composition for all six scorpions. In addition to advancing our fundamental knowledge of scorpion venomics, this study may serve as a starting point for future research by facilitating the identification of the venom composition of scorpions and identifying novel putative toxin families.