Milica Ng

Evaluation of the bioactivity of influenza vaccine strains in vitro suggests that the introduction of new strains in the 2010 Southern Hemisphere trivalent influenza vaccine is associated with adverse events.

In Australia, during the 2010 Southern Hemisphere (SH) influenza season, there was an unexpected increase in post-marketing adverse event reports of febrile seizures (FS) in children under 5 years of age shortly after vaccination with the CSL trivalent influenza vaccine (CSL 2010 SH TIV) compared to previous CSL TIVs and other licensed 2010 SH TIVs. The present study describes the outcomes of a series of in vitro experiments directed at elucidating the root cause. The scientific investigations found that a subset of paediatric donors displayed elevated cytokine/chemokine responses to the CSL 2010 SH TIV but not to previous CSL TIVs nor other 2010 SH TIVs. The induction of elevated cytokines/chemokines in paediatric whole blood correlated with elevated NF-κB activation in a HEK293 cell reporter assay. The data indicate that the introduction of the B/Brisbane/60/2008 strain within the CSL manufacturing process (such as occurred in the preceding 2009/10 NH season) appears to have raised the pyrogenic potential of the CSL 2009/10 NH TIV but that this was insufficient to elicit FS in children <5 years. The 2010 SH season coincided with the first introduction of the H1N1 A/California/07/2009 in combination with the B/Brisbane/60/2008 strain. Our data demonstrates that the introduction of the H1N1 A/California/07/2009 (and to a much lesser degree, H3N2 A/Wisconsin/15/2009) in combination with B/Brisbane/60/2008 (as expressed through the CSL method of manufacture) combined and likely compounded the bioactivity of the CSL 2010 SH TIV. This was associated with stronger immune responses, which in a proportion of children <5 years were associated with FS. The assays and systems developed during these investigations should greatly assist in determining the bioactivity of new influenza strains, and thus aid with the manufacture of CSL TIVs indicated for use in the paediatric population.

Therapeutic Targeting of the G-CSF Receptor Reduces Neutrophil Trafficking and Joint Inflammation in Antibody-Mediated Inflammatory Arthritis

G-CSF is a hemopoietic growth factor that has a role in steady state granulopoiesis, as well as in mature neutrophil activation and function. G-CSF– and G-CSF receptor–deficient mice are profoundly protected in several models of rheumatoid arthritis, and Ab blockade of G-CSF also protects against disease. To further investigate the actions of blocking G-CSF/G-CSF receptor signaling in inflammatory disease, and as a prelude to human studies of the same approach, we developed a neutralizing mAb to the murine G-CSF receptor, which potently antagonizes binding of murine G-CSF and thereby inhibits STAT3 phosphorylation and G-CSF receptor signaling. Anti–G-CSF receptor rapidly halted the progression of established disease in collagen Ab-induced arthritis in mice. Neutrophil accumulation in joints was inhibited, without rendering animals neutropenic, suggesting an effect of G-CSF receptor blockade on neutrophil homing to inflammatory sites. Consistent with this, neutrophils in the blood and arthritic joints of anti–G-CSF receptor–treated mice showed alterations in cell adhesion receptors, with reduced CXCR2 and increased CD62L expression. Furthermore, blocking neutrophil trafficking with anti–G-CSF receptor suppressed local production of proinflammatory cytokines (IL-1β, IL-6) and chemokines (KC, MCP-1) known to drive tissue damage. Differential gene expression analysis of joint neutrophils showed a switch away from an inflammatory phenotype following anti–G-CSF receptor therapy in collagen Ab-induced arthritis. Importantly, G-CSF receptor blockade did not adversely affect viral clearance during influenza infection in mice. To our knowledge, we describe for the first time the effect of G-CSF receptor blockade in a therapeutic model of inflammatory joint disease and provide support for pursuing this therapeutic approach in treating neutrophil-associated inflammatory diseases.

A cytotoxic anti-IL-3Rα antibody targets key cells and cytokines implicated in systemic lupus erythematosus

To date, the major target of biologic therapeutics in systemic lupus erythematosus (SLE) has been the B cell, which produces pathogenic autoantibodies. Recently, targeting type I IFN, which is elaborated by plasmacytoid dendritic cells (pDCs) in response to endosomal TLR7 and TLR9 stimulation by SLE immune complexes, has shown promising results. pDCs express high levels of the IL-3Rα chain (CD123), suggesting an alternative potential targeting strategy. We have developed an anti-CD123 monoclonal antibody, CSL362, and show here that it affects key cell types and cytokines that contribute to SLE. CSL362 potently depletes pDCs via antibody-dependent cell-mediated cytotoxicity, markedly reducing TLR7, TLR9, and SLE serum-induced IFN-α production and IFN-α-upregulated gene expression. The antibody also inhibits TLR7- and TLR9-induced plasmablast expansion by reducing IFN-α and IL-6 production. These effects are more pronounced than with IFN-α blockade alone, possibly because pDC depletion reduces production of other IFN subtypes, such as type III, as well as non-IFN proinflammatory cytokines, such as IL-6. In addition, CSL362 depletes basophils and inhibits IL-3 signaling. These effects were confirmed in cells derived from a heterogeneous population of SLE donors, various IFN-dependent autoimmune diseases, and healthy controls. We also demonstrate in vivo activity of CSL362 following its s.c. administration to cynomolgus monkeys. This spectrum of effects provides a preclinical rationale for the therapeutic evaluation of CSL362 in SLE.

Comparison of clustering tools in R for medium-sized 10x Genomics single-cell RNA-sequencing data

Background: The commercially available 10x Genomics protocol to generate droplet-based single-cell RNA-seq (scRNA-seq) data is enjoying growing popularity among researchers. Fundamental to the analysis of such scRNA-seq data is the ability to cluster similar or same cells into non-overlapping groups. Many competing methods have been proposed for this task, but there is currently little guidance with regards to which method to use. Methods: Here we use one gold standard 10x Genomics dataset, generated from the mixture of three cell lines, as well as three silver standard 10x Genomics datasets generated from peripheral blood mononuclear cells to examine not only the accuracy but also robustness of a dozen methods. Results: We found that some methods, including Seurat and Cell Ranger, outperform other methods, although performance seems to be dependent on the complexity of the studied system. Furthermore, we found that solutions produced by different methods have little in common with each other. Conclusions: In light of this, we conclude that the choice of clustering tool crucially determines interpretation of scRNA-seq data generated by 10x Genomics. Hence practitioners and consumers should remain vigilant about the outcome of 10x Genomics scRNA-seq analysis.

Cluster Headache: Comparing Clustering Tools for 10X Single Cell Sequencing Data

The commercially available 10X Genomics protocol to generate droplet-based single cell RNA-seq (scRNA-seq) data is enjoying growing popularity among researchers. Fundamental to the analysis of such scRNA-seq data is the ability to cluster similar or same cells into non-overlapping groups. Many competing methods have been proposed for this task, but there is currently little guidance with regards to which method offers most accuracy. Answering this question is complicated by the fact that 10X Genomics data lack cell labels that would allow a direct performance evaluation. Thus in this review, we focused on comparing clustering solutions of a dozen methods for three datasets on human peripheral mononuclear cells generated with the 10X Genomics technology. While clustering solutions appeared robust, we found that solutions produced by different methods have little in common with each other. They also failed to replicate cell type assignment generated with supervised labeling approaches. Furthermore, we demonstrate that all clustering methods tested clustered cells to a large degree according to the amount of genes coding for ribosomal protein genes in each cell.

100 Depletion of plasmacytoid dendritic cells with JNJ-56022473 minimises induction of an interferon gene signature in response to TLR9 and SLE immune complex stimulation

Background and aims Systemic Lupus Erythematosus (SLE) is associated with an increased IFN gene signature detectable in the peripheral blood. Plasmacytoid dendritic cells (pDC) are potent producers of IFNα in response to TLR9 and TLR7-agonists. pDCs which express high levels of CD123 (IL-3Rα) can be depleted by JNJ-56022473 (JNJ-473), a novel Fc-engineered neutralising and depleting therapeutic antibody targeting CD123. Methods We investigated the effects of pDC depletion with JNJ-473 on IFNα production and gene expression within SLE patient PBMC (n=8) stimulated with TLR-agonists, SLE-immune complexes (IC, SLE IgG with necrotic cell lysates (NCL)) and sera from SLE patients with NCL. Results Stimulation with CpGc, SLE-IC or SLE sera was able to induce high levels of IFNα, which was greatly decreased by pDC depletion with JNJ-473. SLE-IC and SLE sera stimulation also induced the differential expression of hundreds of genes and could induce similar genes to TLR9-agonism. pDC depletion with JNJ-473 prevented the upregulation of TLR9-induced genes. JNJ-473 conferred minimal effects on the induction of genes in response to the TLR4-agonist LPS. Furthermore, a distinct 11-gene IFN signature was induced by CpGc and SLE-IC that was significantly reduced by treatment with JNJ-473, suggesting that the depletion of pDCs with JNJ-473 could have distinct and specific effects on the detectable IFN signature in many SLE patients. Conclusions Depletion of pDCs with JNJ-473 is able to dramatically decrease IFNα production and IFN gene signature induced by TLR9-agonists and SLE-IC.

302 Serum cytokine analysis and transcriptional blood profiling reveal an association between il-3 and ifn in human sle

Background and aims IFNα, produced by plasmacytoid dendritic cells (pDCs) is a major contributor to SLE pathogenesis. IL-3 promotes pDC survival, but its role in SLE has not been well characterised. This study investigated serum IL-3 and IFNα levels, and a whole blood ‘IL-3 gene signature’ in human SLE. Methods Serum cytokine levels were measured by ELISA in n=42 SLE donors from The Royal Melbourne Hospital and n=44 healthy donors (HD). IL-3 upregulated genes were determined by RNASeq of HD whole blood (WB) stimulated in vitro with IL-3 for 6 or 24 hours. WB RNASeq analysis was also undertaken in n=31 SLE donors from the Monash Lupus Clinic and n=28 HDs. Results Serum IL-3 levels correlated with serum IFNα (r=0.612, 95% CI 0.455–0.733, p<0.001). IL-3 stimulation in vitro altered 794 genes (−1≥logFC ≥1, FDR<0.05). Thirty-five of these genes overlapped with differentially expressed genes between SLE and HD. These 35 genes were expressed in 28/31 SLE donors, revealing the presence of an ‘IL-3 gene signature’. There was strong correlation between the IL-3 signature and an IFN signature determined by heirarchical clustering of the five hundred most variable genes in SLE donors (r=0.939, 95% CI 0.898–0.964, p<0.0001). Conclusions We have previously reported a novel anti-IL-3Rα mAb (CSL362/JNJ-473), which depletes pDCs and reduces IFNα production, as well as neutralising IL-3 signalling (Oon S, JCI Insight, 2016). An association between IL-3 and IFNα was found in this study, raising the possibility that CSL362 may be especially useful for lupus patients with a dual IL-3/IFN gene signature.

Easy and efficient ensemble gene set testing with EGSEA

Gene set enrichment analysis is a popular approach for prioritising the biological processes perturbed in genomic datasets. The Bioconductor project hosts over 80 software packages capable of gene set analysis. Most of these packages search for enriched signatures amongst differentially regulated genes to reveal higher level biological themes that may be missed when focusing only on evidence from individual genes. With so many different methods on offer, choosing the best algorithm and visualization approach can be challenging. The EGSEA package solves this problem by combining results from up to 12 prominent gene set testing algorithms to obtain a consensus ranking of biologically relevant results.This workflow demonstrates how EGSEA can extend limma-based differential expression analyses for RNA-seq and microarray data using experiments that profile 3 distinct cell populations important for studying the origins of breast cancer. Following data normalization and set-up of an appropriate linear model for differential expression analysis, EGSEA builds gene signature specific indexes that link a wide range of mouse or human gene set collections obtained from MSigDB, GeneSetDB and KEGG to the gene expression data being investigated. EGSEA is then configured and the ensemble enrichment analysis run, returning an object that can be queried using several S4 methods for ranking gene sets and visualizing results via heatmaps, KEGG pathway views, GO graphs, scatter plots and bar plots. Finally, an HTML report that combines these displays can fast-track the sharing of results with collaborators, and thus expedite downstream biological validation. EGSEA is simple to use and can be easily integrated with existing gene expression analysis pipelines for both human and mouse data.

THU0311 IL3R-Alpha as A Novel Therapeutic Target in Systemic Lupus Erythematosus

Background Despite currently available therapies, a significant morbidity, and mortality remains in SLE. Major therapeutic targets have been the auto-antibody producing B cell, and more recently, type I IFN, which is produced by pDCs activated via TLR7 and TLR9 stimulation by SLE immune complexes. Alternate ways of reducing type I IFN, such as by modulating pDCs, are now emerging (1, 2). We have developed a novel mAb targeting the IL-3Rα, CSL362, which both depletes IL-3Rα expressing cells, and neutralizes signaling through IL-3. Objectives Our objective was to explore the potential utility of CSL362 as a therapeutic in SLE, via its effects on multiple pathogenic cell types and cytokines. Methods In vitro studies were undertaken in a heterogenous population of SLE donors, and healthy and autoimmune disease controls, and major findings confirmed in vivo in cynomolgus macaques. The effect of CSL362 on peripheral blood mononuclear cell types was evaluated by flow cytometry, on cytokine production by ELISA or Luminex assays, and on gene expression by qRT-PCR. Results CSL362 potently and specifically depleted CD123hi pDCs and basophils (average depletion 86.07%±2.07 (SEM), p<0.05, n=72 for pDCs, and 59.39%±3.57, p<0.05, n=71 for basophils). This occurred mainly through antibody-dependent cell-mediated cytotoxicity, although neutralization of IL-3 alone with the Fab fragment of CSL362 resulted in pDC, but not basophil, depletion at higher doses. Through pDC depletion, TLR7-, TLR9- and SLE serum- stimulated IFNα production, and IFN-inducible gene expression was markedly reduced (average reduction in TLR9-stimulated IFNα production 99.36%±0.39, p<0.05, n=29). Additionally, TLR7- and TLR9-induced plasmablast expansion was inhibited by CSL362. This depended upon depletion of pDCs by CSL362, which decreased not only IFNα, but IL-6, production that was required for plasmablast expansion. Importantly, we found that IFN-inducible gene expression and plasmablast expansion were more effectively inhibited by CSL362 than by IFNα blocking mAbs. This may be because pDC depletion by CSL362 reduced production of cytokines other than IFNα, such as IL-6, and interferon types other than type I. Indeed, CSL362 reduced production of type III IFN, a cytokine which has been postulated as a cause of ongoing disease activity despite type I IFN blockade (3). Administration of a single subcutaneous dose of CSL362 to cynomolgus macaques confirmed pDC and basophil depletion, correlating with a decrease in IFN-inducible gene expression, that lasted several weeks post dose, with no major adverse events observed. Conclusions The unique ability of CSL362 to affect the major pathogenic targets of type I IFN and the B cell, and newer targets such as the basophil (4), IL-3 (5) and type III IFN, presents a strong rationale for its therapeutic evaluation in SLE. References Pellerin A et al, EMBO Mol Med, 2015;7:464–476 Zhan Y et al, Arthritis Rheumatol, 2015;67:797–808 Amezcua-Guerra LM et al, Rheumatology (Oxford), 2015;54:203–205 Charles N et al, Nat Med, 2010;16:701–707 Renner K et al, Kidney Int, 2015;88:1088–1098 Disclosure of Interest S. Oon Grant/research support from: CSL Limited, H. Huynh Employee of: CSL Limited, T. Y. Tai Employee of: CSL Limited, M. Ng Employee of: CSL Limited, K. Monaghan Shareholder of: CSL Limited, Employee of: CSL Limited, M. Biondo Employee of: CSL Limited, G. Vairo Shareholder of: CSL Limited, Employee of: CSL Limited, E. Maraskovsky Shareholder of: CSL Limited, Employee of: CSL Limited, A. Nash Shareholder of: CSL Limited, Employee of: CSL Limited, N. Wilson Shareholder of: CSL Limited, Employee of: CSL Limited, I. Wicks Grant/research support from: CSL Limited