rosetteseptm human cd8 + t cell enrichment cocktail  (STEMCELL Technologies Inc)

Journal: bioRxiv

Article Title: Synaptic synergy of T cell receptor and interleukin 2 receptor in CD8 + T cells

doi: 10.1101/2024.08.13.607831

Figure Lengend Snippet: (A) UMAP dimensionality reduction embedding of blasted (grey), memory (green) and naive (red) CD8 + T cells coloured by orthogonally generated clusters labelled by manual cell type annotation. (B) UMAP embedding of scRNAseq dataset of CD8 + T cell populations coloured by expression profiles of IL2RA (left), IL2RB (middle) and IL2RG (right). (C) Dot plots depicting mean expression (visualized by colour) and fraction of cells (visualized by the size of the dot) expressing common-gamma chain interleukin receptor genes. (d-f) RA14 CD8 + T cells were exposed to Ag-specific SLBs in the presence of 50 U/ml of IL2 to form ISs and fixed after 10 min. Samples were then permeabilized and immunostained against the IL2 receptor subunits. Panels show representative TIRF images of the different distribution phenotypes (Homogeneous, Central and Annular) observed for the IL2RA (d), IL2RB (e) and IL2RG (f) for each state of CD8 + T cells (Blast, Memory and Naïve). Frequency for each phenotype is represented as parts of a whole for each T cell state. Data is the mean of three independent donors with >50 cells/donor. (G) Representative TIRF images of blast (top), memory (middle) and naïve (bottom) RA14 CD8 + T cells forming ISs with Ag- specific SLBs in the presence of 50 U/ml of IL2 and fixed after 10 min. Samples were then permeabilized and immunostained for the IL2 receptor subunits. Histograms (right) show the intensity line profile for the different proteins. (H) Percentage of the total IL2RG intensity that is within the area covered by IL2RB (left plot) or within the intersection between the IL2RB and IL2RA (right plot). See Fig. S3 for a more detailed explanation. Dots shows the distribution of the values for >100 cells, from three independent donors. Histograms show the mean +/- SE. **pv<0.01, ***pv<0.001, ****pv<0.0001; Kruskal-Wallis test. (I) Representative z-stack Airyscan reconstruction of a RA14 CD8 + blast forming a IS on an Ag-specific SLB in the presence of 50 U/ml of IL2. Sample was fixed after 10 min of SLB exposure, permeabilized and immunostained against the IL2 receptor subunits.

Article Snippet: CD8+ T cells were purified using the negative selection kits RosetteSep™ Human CD8+ T Cell Enrichment Cocktail (StemCell Technologies).

Techniques: Generated, Expressing

Journal: bioRxiv

Article Title: Synaptic synergy of T cell receptor and interleukin 2 receptor in CD8 + T cells

doi: 10.1101/2024.08.13.607831

Figure Lengend Snippet: (A) Heatmap showing the normalized expression of proteins identified in EVs from resting and activated CD8 + T cells (naïve, memory and total/pooled CD8 + differentiation states). All members of the common-gamma chain cytokine receptors and interleukins were interrogated, together with other activation markers and EVs markers. The heatmap shows all species detected. For activation, cells were incubated with anti-CD3/CD28 Dynabeads for 48 hours. (B) Nano-FCM analysis showing the scatter plot for IL2RA (y-axis) and IL2RB (x-axis) positive EVs from resting and activated pooled CD8 + T cell. Marked in red are the double positives (IL2RA and IL2RB), blue exclusively represents the IL2RA positive EVs (negative for IL2RB), green exclusively represents IL2RB positive EVs (negative for IL2RA), and the double negatives are marked in yellow. Similarly, (C) shows the scatter plot for IL2RG (y-axis) and IL2RB (x-axis) positive EVs from resting and activated CD8 + T cell. Marked in red are the double positives (IL2RG and IL2RB), blue exclusively represents the IL2RG positive EVs (negative for IL2RB), green exclusively represents IL2RB positive EVs (negative for IL2RA), and the double negatives are marked in yellow. (D) Naïve CD8 + T cells were exposed to EVs purified from activated CD8 + T cells. After 48 hours with or without EVs incubation, Naïve CD8 + T cells were treated or not with 50 U/ml of IL2 and analysed by phospho-flow cytometry to quantify total pSTAT5 levels. Right plot shows the median intensity for pSTAT5.

Article Snippet: CD8+ T cells were purified using the negative selection kits RosetteSep™ Human CD8+ T Cell Enrichment Cocktail (StemCell Technologies).

Techniques: Expressing, Activation Assay, Incubation, Purification, Flow Cytometry

Journal: bioRxiv

Article Title: Synaptic synergy of T cell receptor and interleukin 2 receptor in CD8 + T cells

doi: 10.1101/2024.08.13.607831

Figure Lengend Snippet: (A) Histograms of phospho-flow cytometry data showing total levels of pSTAT5 after 10 min incubation at 37 ° C in the presence (+IL2) or absence (No IL2) of 50 U/ml of IL2 treatment; for naïve (yellow), memory (magenta) and blast (blue) CD8 + T cells. Bar plots (right) show the mean +/- SE of the pSTAT5 median fluorescence intensity (MFI) from three independent donors. (B) Representative z-stack Airyscan reconstruction of RA14 CD8 + blasts forming a IS on an HLA-A2-p65 peptide bearing SLB in the presence or absence of 50 U/ml of IL2. Sample was fixed after 10 min of SLB exposure, permeabilized and stained for F-actin and immunostained against pSTAT5. (C) Representative TIRF images of Naïve (top), Memory (middle) and Blast (bottom) CD8 + T cells forming an IS with a SLB and fixed after the indicated time-point. 0 min represents cells fixed after 15 min of contact with a SLB without UCHT1 (no IS formation). pSTAT5, magenta; pZAP70, green. White pixels show overlap between pSTAT5 and pZAP70. (D-F) Plots show the mean intensity for pSTAT5 (magenta) and pZAP70 (green) normalized to the 0 min time-point (left Y axis) and the Pearson’s Correlation Coefficient (PCC, grey) between pSTAT5 and pZAP70 signals (right Y axis) over time for naïve (d), memory (e) and blast (f) CD8 + T cells. Data is the mean +/- SE of 25-50 cells. (G-K) Naïve (g-h), memory (i-j) or blast (k-l) RA14 CD8 + T cells were exposed to Ag-specific SLBs in the presence or absence of 50 U/ml of IL2 to visualize IS formation. Cells were fixed after 10 min of bilayer exposure and immunostained for pSTAT5 (magenta), pZAP70 (yellow), IL2RG (cyan), TCR (green). (g, i and k) Representative TIRF images of ISs. Merge is the overlap between channels. Histograms on the right show the intensity profile across the yellow line shown in the merge images. (h, j and l) show the dot plots with the mean intensity for pSTAT5 and pZAP70 normalized to the condition without IL2. Data is the mean +/- SE, n=90-150 from 3 independent donors (50 cells/donor for naïve and blast and 30 cells/donor for memory). ns, not significant, *pv<0.05, **pv<0.01; Mann-Whitney test.

Article Snippet: CD8+ T cells were purified using the negative selection kits RosetteSep™ Human CD8+ T Cell Enrichment Cocktail (StemCell Technologies).

Techniques: Flow Cytometry, Incubation, Fluorescence, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Synaptic synergy of T cell receptor and interleukin 2 receptor in CD8 + T cells

doi: 10.1101/2024.08.13.607831

Figure Lengend Snippet: (A) Cartoon representing the different structural variants of IST used in this work. Two HLA-A*02 complexes with the cognate Ag (CMV pp65) or a non-specific control Ag (MART-1) are connected, or not, to four units of engineered IL2 (Cue IL2), with abrogated binding to IL2RA and reduced binding to IL2RB. (B) RA14 CD8 + blasts were exposed to SLBs containing the different structural variants of ISTs linked to the D-peptide at SLB-saturating conditions without any additional adhesion or co-stimulatory molecules and imaged by interference reflection microscopy (IRM) and bright field after 15 min. (C) Percentage of cells forming a contact with the SLB (detected by IRM) from all cells exposed to the SLB (bright field) in (b). Data is the mean +/- SE of 4 independent donors; *pv<0.05, **pv<0.01, one-way ANOVA test. (D) Quantification of the contact area between the SLB and the T cell (detected by IRM) from cells in (b). Data is the mean +/- SE of >60 established contacts. *pv<0.05; Mann-Whitney test. (E) Time-lapse of a RA14 CD8 + blast T cell forming a contact with a SLB containing fluorescently tagged ISTs with the CMV peptide (IST, green; IRM, grey). (F) RA14 CD8 + blast T cells were exposed to SLBs containing CMV-specific or MART1-specific ISTs (green) linked to the D-peptide at 3nM and ICAM1 (magenta) and imaged after 15 min of exposure.

Article Snippet: CD8+ T cells were purified using the negative selection kits RosetteSep™ Human CD8+ T Cell Enrichment Cocktail (StemCell Technologies).

Techniques: Control, Binding Assay, Microscopy, MANN-WHITNEY

Journal: bioRxiv

Article Title: Synaptic synergy of T cell receptor and interleukin 2 receptor in CD8 + T cells

doi: 10.1101/2024.08.13.607831

Figure Lengend Snippet: (A) Representative TIRF images of RA14 CD8 + blasts forming IS with SLBs containing the indicated IST with ICAM1, CD80 and CD58. Cells were fixed after 15 min of SLB exposure and stained for the IL2 receptor subunits (IL2RA, magenta; IL2RB cyan; IL2RG yellow) and the TCR (CD3, green). (B) Quantification of the mean grey values for each protein (IL2RA, IL2RB, IL2RG and TCR) at the IS. Values were normalized to the negative control MART1 (noIL2). Data is the mean +/- SE, n>100 cells from 3 independent donors; **pv<0.01, ****pv<0.0001; Kruskal-Wallis test. (C-H) Quantification of the Pearson’s correlation coefficient (PCC) between the TCR and the IL2RA (c), IL2RB (d) and the IL2RG (e) and between the IL2 receptor subunits IL2RA and IL2RB (f), IL2RA and IL2RG (g) and the IL2RB and IL2RG (h). Data is the mean +/- SE, n>100 cells from 3 independent donors; *pv<0.05, **pv<0.01, ****pv<0.0001; Kruskal-Wallis test. Left images show the zoomed in area for the square inset indicated in (a).

Article Snippet: CD8+ T cells were purified using the negative selection kits RosetteSep™ Human CD8+ T Cell Enrichment Cocktail (StemCell Technologies).

Techniques: Staining, Negative Control

Journal: bioRxiv

Article Title: Synaptic synergy of T cell receptor and interleukin 2 receptor in CD8 + T cells

doi: 10.1101/2024.08.13.607831

Figure Lengend Snippet: RA14 CD8 + Naive (A-D) , Memory (E-H) and Blasts (I-L) T cells forming ISs with SLBs containing the indicated variant of IST AF488 (yellow), ICAM1, CD80 and CD58. Cells were fixed after 15 min of SLB exposure and stained for pSTAT5 (magenta) and pZAP70 (cyan). Middle plots show the quantification of the mean grey values for pSTAT5 and pZAP70 at the IS for Naïve (b, c), Memory (f, g) and Blast (j, k). Values were normalized to the negative control MART1(noIL2). Right plots show the Pearson’s correlation coefficient (PCC) between pSTAT5 and pZAP70 for Naïve (d), Memory (h) and Blast (l). Images in (d, h and l) show the zoomed in area for the square insets indicated in (a, e and i), respectively. Data is the mean +/- SE, n=200 cells from 3 independent donors; **pv<0.01, ****pv<0.0001; Kruskal-Wallis test. CMV, cognate antigen; MART1, negative control antigen.

Article Snippet: CD8+ T cells were purified using the negative selection kits RosetteSep™ Human CD8+ T Cell Enrichment Cocktail (StemCell Technologies).

Techniques: Variant Assay, Staining, Negative Control

Journal: bioRxiv

Article Title: Synaptic synergy of T cell receptor and interleukin 2 receptor in CD8 + T cells

doi: 10.1101/2024.08.13.607831

Figure Lengend Snippet: Blast CD8 + T cells were electroporated to express the RA14 TCR and reintroduced to the PBMC sample from the same donor. After 2h incubation in a small volume at 37 °C with or without the CMV IL2 IST (1 µM), the cell mix was fixed and blocked (TrueStain, Biolegend) before adding the antibody mix for ImageStream analysis. (A) Gating strategy. Images in focus were used to identify the cell doublets (yellow population, left dot plot). (B) Doublets were further gated to identify possible CD8 + T-T cell conjugates (Dot plots, high values for CD3 and CD8 markers, cyan population). Histograms show the intensity for CMV IL2 IST for the cyan population (possible CD8 + T-T cell conjugates). (C) Examples of CD8 + T-T cell conjugates identified. Note the accumulation of IST at the contact between the two T cells. (D) Examples of CD8 + T cell multifocal clusters. Note the accumulation of IST at the contact between any two T cells. (E) Doublets were further gated to identify possible CD8 + T cell conjugates with CD14 + monocytes (Dot plots, doublets positive for CD8 + and CD14 + markers, magenta population). Histograms show the intensity for CMV IL2 IST for the magenta population (possible CD8 + T cell – CD14 + monocytes conjugates). (F) Examples of CD8 + T cell – CD14 + monocytes conjugates identified. Note the engulfment of ISTs by the CD14 + cell together with CD3. Data is representative from two independent donors. The rest of the channels imaged are shown in Fig. S8.

Article Snippet: CD8+ T cells were purified using the negative selection kits RosetteSep™ Human CD8+ T Cell Enrichment Cocktail (StemCell Technologies).

Techniques: Incubation