Novel engineered B lymphocytes targeting islet-specific T cells inhibit the development of type 1 diabetes in non-obese diabetic Scid mice

IntroductionIn this study, we report a novel therapeutic approach using B lymphocytes to attract islet-specific T cells in the non-obese diabetic (NOD) mouse model and prevent the development of autoimmune diabetes. Rather than using the antibody receptor of B cells, this approach utilizes their properties as antigen-presenting cells to T cells.MethodsPurified splenic B cells were treated with lipopolysaccharide, which increases regulatory B (Breg) cell function, then electroporated with mRNA encoding either chimeric MHC-I or MHC-II molecules covalently linked to antigenic peptides. Immunoregulatory functions of these engineered B cells (e-B cells) were tested by in vitro assays and in vivo co-transfer experiments with beta-cell-antigen-specific CD8+ or CD4+ T cells in NOD.Scid mice, respectively.ResultsThe e-B cells expressing chimeric MHC-I-peptide inhibited antigen-specific CD8+ T-cell cytotoxicity in vitro. The e-B cells expressing chimeric MHC-II-peptide induced antigen-specific CD4+ T cells to express the regulatory markers, PD-1, ICOS, CTLA-4, Lag3, and Nrp1. Furthermore, e-B cells encoding the chimeric MHC-I and MHC-II peptide constructs protected NOD.Scid mice from autoimmune diabetes induced by transfer of antigen-specific CD8+ and CD4+ T cells.DiscussionMHC–peptide chimeric e-B cells interacted with pathogenic T cells, and protected the host from autoimmune diabetes, in a mouse model. Thus, we have successfully expressed MHC–peptide constructs in B cells that selectively targeted antigen-specific cells, raising the possibility that this strategy could be used to endow different protective cell types to specifically regulate/remove pathogenic cells

Membrane anchored IL-18 linked to constitutively active TLR4 and CD40 improves human T cell antitumor capacities for adoptive cell therapy

BACKGROUND: Adoptive transfer of tumor-infiltrating lymphocytes (TILs) or blood T cells genetically redirected by an antitumor TCR or CAR induces a strong antitumor response in a proportion of patients with cancer; however, the therapeutic efficacy is often limited by rapid decline in T cell functions. Coadministering supportive cytokines frequently provokes systemic side effects preventing their broad clinical application. We recently showed that cytokines can be anchored to the cell membrane in a functional fashion and that cytokine receptor signaling can synergize with TLR4 and CD40 signaling. Here, we aimed at augmenting T cell activation by simultaneous signaling through the cytokine receptor, toll-like receptor and TNF-type receptor using IL-18, TLR4 and CD40 as prototypes. METHODS: Genes were expressed on electroporation of in vitro-transcribed mRNA in CD4(+) and CD8(+) T cells from healthy donors redirected against melanoma cells with an anti-melanotransferrin CAR and in TILs derived from melanoma patients. Functional assays included the activation of signaling pathways, expression of activation and differentiation markers, cytokine secretion and killing of melanoma target cells. RESULTS: To provide IL-18 costimulation to T cells in-cis while avoiding systemic effects, we genetically anchored IL-18 to the T cell membrane, either alone (memIL-18) or fused with constitutively active (ca)TLR4 and caCD40 signaling domains arranged in tandem, creating a synthetic ‘all-in-one’ memIL-18-TLR4-CD40 receptor. MemIL-18-TLR4-CD40, but not memIL-18, triggered strong NF-κB activation in cells lacking the IL-18 receptor, attesting to functionality of the TLR-CD40 moiety. While the membrane-anchored cytokine was found to act mainly in-cis, some T cell activation in-trans was also observed. The electroporated T cells exhibited spontaneous T-bet upregulation and IFN-γ and TNF-α secretion. Melanoma-induced activation of CAR-T cells and TILs as manifested by cytokine secretion and cytolytic activity was substantially augmented by both constructs, with memIL-18-TLR4-CD40 exerting stronger effects than memIL-18 alone. CONCLUSIONS: Linking membrane anchored IL-18 with caTLR4 and caCD40 signaling in one hybrid transmembrane protein provides simultaneous activation of three T cell costimulatory pathways through one genetically engineered membrane molecule, strongly amplifying T cell functions for adoptive T cell therapy of cancer

Transneuronal Dpr12/DIP-delta interactions facilitate compartmentalized dopaminergic innervation of Drosophila mushroom body axons

The mechanisms controlling wiring of neuronal networks are not completely understood. The stereotypic architecture of the Drosophila mushroom body (MB) offers a unique system to study circuit assembly. The adult medial MB gamma-lobe is comprised of a long bundle of axons that wire with specific modulatory and output neurons in a tiled manner, defining five distinct zones. We found that the immunoglobulin superfamily protein Dpr12 is cell-autonomously required in gamma-neurons for their developmental regrowth into the distal gamma 4/5 zones, where both Dpr12 and its interacting protein, DIP-delta, are enriched. DIP-delta functions in a subset of dopaminergic neurons that wire with gamma-neurons within the gamma 4/5 zone. During metamorphosis, these dopaminergic projections arrive to the gamma 4/5 zone prior to gamma-axons, suggesting that gamma-axons extend through a prepatterned region. Thus, Dpr12/DIP-delta transneuronal interaction is required for gamma 4/5 zone formation. Our study sheds light onto molecular and cellular mechanisms underlying circuit formation within subcellular resolution

Epidermal Growth-Factor – Induced Transcript Isoform Variation Drives Mammary Cell Migration

<div><p>Signal-induced transcript isoform variation (TIV) includes alternative promoter usage as well as alternative splicing and alternative polyadenylation of mRNA. To assess the phenotypic relevance of signal-induced TIV, we employed exon arrays and breast epithelial cells, which migrate in response to the epidermal growth factor (EGF). We show that EGF rapidly – within one hour – induces widespread TIV in a significant fraction of the transcriptome. Importantly, TIV characterizes many genes that display no differential expression upon stimulus. In addition, similar EGF-dependent changes are shared by a panel of mammary cell lines. A functional screen, which utilized isoform-specific siRNA oligonucleotides, indicated that several isoforms play essential, non-redundant roles in EGF-induced mammary cell migration. Taken together, our findings highlight the importance of TIV in the rapid evolvement of a phenotypic response to extracellular signals.</p></div

EGF-induced alternative promoter usage.

<p>(<b>A</b>) <i><u>Upper panel:</u></i> Organization of <i>LAMA3</i> transcripts as defined by the UCSC gene model. Vertical boxes represent exons; the arrow (100 kb) indicates the direction of transcription. Positions of exonic PS (blue) that passed all filtering steps are indicated. The upmost isoform was not expressed in MCF10A cells (neither of two isoform-specific PS interrogating the alternative last exon passed the required signal intensity threshold). <i><u>Lower panels:</u></i> FC (log2 scale) of each PS with respect to pre-stimulus values is shown for each time point. Only PS with ‘present’ calls in all replicates are depicted at each time point. Bars represent standard errors of FC from three biological replicates. Note strong induction of the 3′ portion (the dashed green line marks the median FC of this region). In contrast, the longer isoforms (median FC shown by the dashed red line) are slightly downregulated. (<b>B</b>) The heat map displays the FC ratios of the short to long isoforms of the top 40 genes identified as undergoing EGF-induced alternative usage of well-annotated promoters (FDR<1%) resulting in alternative first exons; if the relative abundance of the short isoforms compared to the long isoforms increases after EGF stimulation the FC ratio becomes ‘high’ (see for instance <i>LAMA3</i>) (<b>C</b>) For qPCR validation of TIV events primer pairs specific to transcript isoforms were used for each gene, along with pairs spanning regions common to all transcripts. Error bars represent standard deviations from three technical replicates. The experiment was repeated twice.</p

Validation of hits of the siRNA screen.

<p>(<b>A</b>) Time lapse microscopy images (one of 5 repeats) of scratch assays using MCF10A cells transfected with isoform-specific siRNAs to <i>PTHLH</i> (left) and quantification of all images (right). Error bars represent the standard deviations of five repeats per condition; asterisks indicate significant differences from siCONTROL (*p<0.05, **p<0.01). (<b>B</b> and <b>C</b>) Results of Real-time Cell Analyzer (RTCA) experiments measuring the effects of isoform-specific oligonucleotides to <i>PTHLH</i> (B) or to <i>LAMA3</i> (C) on cell adhesion (left panels) and single cell migration through filters (right panels). Oligonucleotides targeting transcript regions common to all expressed isoforms of the respective gene were also employed (si gene-level). Cells treated with transfection reagent alone (‘mock’) and cells transfected with scrambled control siRNA oligonucleotides (siCONTROL) are shown as controls. Error bars represent the standard deviations of 3 or more repeats per condition; asterisks indicate significant differences relative to siCONTROL (*p<0.05, **p<0.01). The experiment was repeated thrice.</p

Isoform-specific knockdown and absence of significant proliferation, viability and morphological effects.

<p>(<b>A</b>) MCF10A cells were transfected with siRNA oligonucleotides (20 nM) targeting individual isoforms of <i>LAMA3</i> (left panel) or <i>PTHLH</i> (right panel), siRNA pools targeting all isoforms (‘gene-level’) or with control scrambled siRNA oligonucleotides (sictrl denotes siRNA control, ‘i1,o1’ denotes the first oligonucleotide targeting isoform 1; ‘i2,o2’ the second oligonucleotide targeting isoform 2, etc. and ‘GL’ denotes ‘gene level’). Forty-eight hours later, RNA was extracted and qPCR measurements were performed using primers specific to all expressed individual isoforms, as well as primers amplifying transcript regions common to all expressed isoforms. Measurements were normalized to results obtained with an independent siRNA control replicate. Error bars denote standard errors from three technical replicates and asterisks denote statistically significant (p<0.05) differences relative to siCtrl. Similar results were obtained in three independent repeat experiments. (<b>B</b>) Oligonucleotides for <i>EGFR</i>, which impair migration, and for <i>CSNK1G2</i>, which accelerate migration are shown as additional controls. Cell cycle analysis was performed as described in <i>Methods</i>. Error bars represent the standard errors from the analysis of 24 images taken per from each of four wells per condition. (<b>C</b>) Viability was measured by tetrazolium-based WST-1 assay. MCF10A cells were transfected with the indicated oligonucleotides (siRNA to polo-like kinase, <i>PLK1</i>, served as positive control). Error bars represent standard errors from 3 biological replicates. The experiment was performed thrice. (<b>D</b>) Shown are morphologic effects of isoform-specific oligonucleotides targeting <i>LAMA3</i> or <i>PTHLH</i>. Nine thousand MCF10A cells per well were transfected, starved and stimulated as described for the proliferation assays, followed by DAPI and phalloidin staining, 14 hours after stimulation. Automated image analysis was used to systematically assess changes in cell size and shape by analyzing 24 images taken per condition. No difference between the individual knockdowns was apparent (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080566#pone.0080566.s006" target="_blank">Figure S6B</a>); one representative image for each condition is shown.</p

Functional tests of EGF-induced TIV by an isoform-specific siRNA screen.

<p>(<b>A</b>) For 35 genes, each transcript isoform undergoing EGF-induced TIV was targeted by two individual oligonucleotides and by ‘gene-level’ siRNA oligonucleotide pools knocking down all isoforms. An automated robotic platform was used for performing scratches. Each candidate was screened in eleven biological replicates. (<b>B</b>) Scheme of the automated image analysis used to calculate the average migration distance (AMD) for each well compared to control siRNA oligonucleotides (<b>C</b>) <i><u>Upper panel:</u></i> Each bar represents the measured mean relative AMD (and standard error) for one oligonucleotide. <i><u>Lower panel:</u></i> The FDR q-values, relative to the control siRNA, are shown.</p