Fig 1.
An endogenous MLV env is an immune stimulation-responsive gene in B cells and is expressed by primary lymphocytes and EL4 thymoma cells.
(A) Heatmap of endogenous MLV expression in resting and CD3/CD28-stimulated CD4+ T cells assessed by RNA-seq. (B) Genomic location of Xmv45 and RNA-seq read mapping in unstimulated and LPS-stimulated B cells and in unstimulated CD4+ T cells. (C) Splenocytes of C57BL/6J, 129S8 or BALB/c mice were in vitro stimulated with LPS for 24 or 48 hours and the expression of Xmv45 (left) and AI506816a (right) was assessed by qRT-PCR. Expression levels following LPS stimulation were compared to basal expression levels in unstimulated C57BL/6J splenocytes. (D) Flow cytometric analysis of MLV envelope on the surface of SSChi B cells from C57BL/6J, 129S8 or BALB/c mice following in vitro stimulation with LPS for 48 hours compared with unstimulated, but stained B cells and with stimulated, but 83A25-unstained B cells. Two mice per strain are shown. (E) Heatmap of endogenous MLV expression in resting and CD3/CD28-stimulated EL4 cells assessed by RNA-seq. Two biological replicates are shown. (F) MLV envelope localisation in non-polarised and polarised EL4 cells. EL4 cells were labelled with anti-CD45, anti-CD43 for 30 minutes and 83A25 for 15 minutes at 37°C, counterstained with Hoechst and imaged by IS. Scale bar = 7 μm.
Fig 2.
Endogenous MLV envelope is internalised by EL4 cells and primary lymphocytes.
(A) IS images of EL4 cells incubated with 83A25 antibody for specified periods of time and counterstained with anti-CD45 and Hoechst (left). Percentage of cells with internalised envelope-antibody complexes (right) over time from four independent experiments. A minimum of 5000 cells were analysed in each experiment at each time point. Scale bar = 7 μm. (B) Splenocytes were imaged by IS in the naïve state or were activated with LPS or CD3/CD28 Dynabeads for 48 hours. Prior to imaging, cells were incubated with 83A25 antibody for specified periods of time. At the end of incubation with 83A25, cells were labelled with anti-CD45, anti-CD3, anti-CD19 and Hoechst. Three-hour time point images are shown (top). Percentage of activated cells with internalised env-antibody complexes at specified time points (bottom left). Co-localisation of 83A25 with CD19 in B cells and CD3 in T cells (bottom right) was quantified using the Bright Detail Similarity feature in IDEAS and compared to Hoechst, a non-colocalising probe. A minimum of 10000 cells were analysed at each time point. Scale bar = 7 μm. (C) Antibody binding to MLV envelope reduces the number of envelope molecules present on the cell surface. E.G7-OVA cells were incubated with either 83A25, 522, isotype control antibody or a combination of these for defined periods of time and counterstained with anti-CD45 and Hoechst. Images (left) and fluorophore signal intensities (right) were recorded by IS. Data representative of three independent experiments with a minimum of 10000 cells analysed per treatment group. Scale bar = 7 μm. (D) Internalised envelope-antibody complexes localise to acidic endosomal/lysosomal compartments. IS images of E.G7-OVA cells incubated with pHrodo-conjugated 83A25 for three hours and stained with LysoTracker and Hoechst dyes (left). Co-localisation of 83A25 with LysoTracker was quantified using the Bright Detail Similarity feature in IDEAS and compared to Hoechst, a non-colocalising probe (right). Scale bar = 7 μm.
Fig 3.
Stimulation of EL4 cells with 83A25 induces cell clustering and transcriptional changes.
(A) EL4 cells cluster following incubation with 83A25. Light microscopy images of EL4 cells incubated with indicated antibodies for 18 hours (left). Scale bar = 200 μm. Quantification of area occupied by the cells as a percentage of total per field of view (right). Pooled data from three independent experiments with at least ten fields of view per experiment. (B) Blocking LFA-1 α subunit (CD11a) prevents EL4 cluster formation. Light microscopy images of EL4 cells incubated with indicated antibodies for 18 hours (left). Scale bar = 200 μm. Quantification of area occupied by the cells as a percentage of total per field of view (right). Pooled data from three independent experiments with at least five fields of view per experiment. (C) EL4 cells were incubated with indicated antibodies for 18 hours and assessed for surface CD107a by flow cytometry. Representative flow cytometric plots (left) and quantitation of CD107a positive cells as a percentage of total (right). Pooled data from three independent experiments. (D) EL4 cells were co-cultured with 293T or 293T.mCAT-1 cells in the absence or presence of 83A25 antibody for 18 hours and assessed for surface CD107a by flow cytometry. Representative flow cytometric plots (left) and quantitation of CD107a positive EL4 cells as a percentage of total (right). Pooled data from two independent experiments. (E) Heatmap of differentially expressed genes assessed by RNA-seq showing a comparison of untreated, isotype control antibody treated, 83A25 antibody treated or CD3/CD28 Dynabeads treated EL4 cells following 1, 3 or 18 hours of stimulation. Two biological replicates for each treatment and time point are shown. (F) Expression of Nhsl2, Tgfb3, Abhd12b and Pmaip1 genes assessed by qRT-PCR in EL4 cells stimulated with 83A25 antibody for 18 hours.
Fig 4.
Internalisation of MLV envelope-antibody complexes is necessary for the initiation of intracellular signalling.
(A) Expression of Nhsl2, Tgfb3 and Il2 genes assessed by qRT-PCR in EL4 cells stimulated with plate-bound or soluble 83A25 antibody for 18 hours. Pooled data from two independent experiments. (B) Expression of Nhsl2, Tgfb3 and Il2 genes assessed by qRT-PCR in EL4 cells stimulated with bead-bound or soluble 83A25 antibody for 18 hours. Pooled data from two independent experiments. (C) Chemical inhibition of signalling in EL4 cells. EL4 cells were either left untreated or pre-treated with indicated inhibitors for 5 minutes prior to addition of the stimulating 83A25 antibody. Cells were incubated for 18 hours and assessed for Nhsl2 and Tgfb3 expression by qRT-PCR. Pooled data from two independent experiments.
Fig 5.
Sorting motif within the cytoplasmic tail of MLV envelope is required for intracellular signalling.
(A) Heatmap of genes differentially expressed by Jurkat and Jurkat.Emv2env cells assessed by RNA-seq. Four biological replicates are shown. (B) Heatmap of genes affected by 83A25 antibody treatment in Jurkat.Emv2 cells assessed by RNA-seq at 3 hours and 18 hours post-stimulation. Four biological replicates are shown. (C) Jurkat cells were transduced with a retrovirus carrying the Emv2 env gene and a gene encoding GFP as a selectable marker, in a bicistronic genome. Three to four days following transduction cells were sorted as GFP negative, GFP intermediate or GFP high (left). Cells transduced with a retrovirus carrying only the GFP encoding gene were sorted for high expression of GFP and were also included. Sorted populations were assessed for EGR1 expression by qRT-PCR (right). (D) Flow cytometric analysis of GFP and Emv2 envelope expression in Jurkat.Emv2env WT, Jurkat.Emv2env ΔCT, Jurkat.Emv2env ΔSM and Jurkat.Emv2env Y656A cells. (E) IS images of Jurkat.Emv2env WT and mutant cells incubated with 83A25 antibody for 15 minutes and counterstained with anti-CD45 and Hoechst showing envelope distribution on the cell surface (top panel, left). IS images of Jurkat.Emv2env WT and mutant cells incubated with 83A25 antibody for 6 hours and counterstained with anti-CD45 and Hoechst showing levels of envelope internalisation (bottom panel, left). Percentage of cells with internalised envelope-antibody complexes in Jurkat.Emv2env WT and mutant cells (right). A minimum of 10000 cells were analysed at each time point. Scale bar = 7 μm. (F) Expression of FLT3, NELL1 and PRKG2 genes assessed by qRT-PCR in Jurkat.Emv2env WT and mutant cells.