mouse monoclonal cd8a  (fluidigm)

Journal: eLife

Article Title: PIM kinase control of CD8 T cell protein synthesis and cell trafficking

doi: 10.7554/eLife.98622

Figure Lengend Snippet: Estimated copies per cell of PIM1 and PIM2 protein from quantitative proteomics analysis of ( A ) OT1 CD8 T cells stimulated with SIINFEKL peptide for indicated times from published dataset or ( B ) naive ex vivo and 24 hr αCD3/αCD28 (TCR) activated WT CD8 T cells (see ( G, H )) for further details. ( C ) Fragments per kilobase million (FPKM) of Pim1 , Pim2, and Pim3 mRNA from published bulk RNAseq analysis of naive and 24 hr gp33-41 peptide stimulated P14 CD8 T cells. Lymph node cell suspensions from C57BL/6 (WT) and Pim1 KO/ Pim2 KO (Pim dKO) mice were activated for 24 hr with αCD3/αCD28 (both 0.5 µg/mL) and CD4 and CD8 T cell ( D ) FSC-A SSC-A profiles, ( E ) expression of surface activation markers (CD25, CD44, CD71) or CD8 T cell intracellular IFNγ were measured by flow cytometry. ( F ) Lymph node single-cell suspensions from WT and Pim dKO mice were labelled with CellTrace Violet (CTV), activated with αCD3/αCD28 (both 0.5 µg/mL) and CD4 and CD8 T cell CTV proliferation profiles were measured at indicated time points. ( G, H ) Lymph node cell suspensions from WT and Pim dKO mice were stimulated for 24 hr with αCD3/αCD28 (both 0.5 µg/mL) and activated CD4 and CD8 T cells were sorted for analysis by quantitative proteomics. Data was analysed using proteomic ruler method to estimate protein copy number per cell. An interactive version of the proteomics expression data is available for exploration on the Immunological Proteome Resource website: immpres.co.uk ( G ) Total protein content (µg/million cells) (one-way ANOVA), ( H ) Volcano plots of p-value (-log 10 ) versus fold-change (log 2 ) in protein copy number between Pim dKO and WT. Horizontal dotted line represents multi-test correction cut-off of q=0.05, vertical dotted line shows 1.5-fold change. Phosphoribosyl Pyrophosphate synthase 1 like 1 (Prps1l1), was found to be higher in Pim dKO CD8 T cells, but was a low confidence quantification (based on only two unique peptides) with no known function in T cells. Lymph node single-cell suspensions from WT and Pim dKO mice were labelled with CellTrace Violet (CTV) and ( I ) cells were cultured in IL-7 (5 ng/mL) +/- rapamycin (20 nM) and CD8 T cell numbers measured over time or ( J ) cells were activated with αCD3/αCD28 (both 0.5 µg/mL) +/- rapamycin (20 nM) and CD8 T cell mean division number was calculated over time (two-way ANOVA). Symbols in bar charts represent biological replicates, symbols in ( I ) represent the mean. Error bars show mean ± S.D. Flow cytometry dot plots and histograms are representative of ( D, E ) n=3, except for IFNγ staining which is n=2, ( F ) n=5, or show pooled data from ( I ) n=3–4 and ( J ) n=5 biological replicates, with data collected over at least two independent experiments. Quantitative proteomics was performed on biological triplicates. Figure 1—source data 1. Raw values plotted in .

Article Snippet: Antibody , anti-mouse CD8a (Rat, monoclonal, 53–6.7) , Thermo Fisher Scientific/eBioscience , Cat # 47-0081-82, RRID: AB_1272185 , cell surface stain 1:200, APC eF780.

Techniques: Quantitative Proteomics, Ex Vivo, Expressing, Activation Assay, Flow Cytometry, Cell Culture, Staining

Journal: eLife

Article Title: PIM kinase control of CD8 T cell protein synthesis and cell trafficking

doi: 10.7554/eLife.98622

Figure Lengend Snippet: Comparison of Pim dKO and age/sex matched WT control mice for ( A ) proportion of splenocytes that were CD4+ and CD8+ (one-way ANOVA), ( B ) total number of CD4 and CD8 T cells in spleens (one-way ANOVA), and ( C ) total number of splenocytes (student t-test). Symbols in bar charts represent biological replicates: error bars show mean ± S.D. ( A–C ) n=8 collected over four independent experiments. ** q<0.01, ***q<0.001. Figure 1—figure supplement 1—source data 1. Raw values plotted in .

Article Snippet: Antibody , anti-mouse CD8a (Rat, monoclonal, 53–6.7) , Thermo Fisher Scientific/eBioscience , Cat # 47-0081-82, RRID: AB_1272185 , cell surface stain 1:200, APC eF780.

Techniques: Comparison, Control

Journal: eLife

Article Title: PIM kinase control of CD8 T cell protein synthesis and cell trafficking

doi: 10.7554/eLife.98622

Figure Lengend Snippet: ( A ) OT1 lymph node cell suspensions were SIINFEKL peptide activated for 36 hr, washed then cultured with no cytokine, IL-15 (20 ng/mL) or IL-2 (20 ng/mL) for 4 or 24 hr. Western blots of PIM1 (two isoforms of 44 and 34 kDa, non-specific band indicated by *), PIM2 (three isoforms of 40, 37, and 34 kDa) or pSTAT5 Y694 expression. ( B ) Schematic of cytokine driven memory and effector CD8 T cell expansion and differentiation cultures. Lymph node or spleen cell suspensions were activated for 2 days with TCR stimulus + cytokine, washed, then split daily into fresh media + cytokine. ( C ) WT (Ly5.1) and Pim dKO LN suspensions were mixed at a 50:50 ratio for T cells and cultured as outlined in ( B ) with TCR stimulus αCD3/αCD28 (both 0.5 µg/mL) + cytokine IL-15 (20 ng/mL), and CD8 T cell number was measured daily. ( D ) WT and Pim dKO T cells were expanded with IL-15 in separate cultures as per ( B, C ) and % live cells (PI-ve) were assessed on days 4 and 6 (two-way ANOVA). ( E–J ) WT and Pim dKO CD8 T cells were activated with TCR stimulus αCD3/αCD28 (both 0.5 µg/mL) + cytokine IL-15 (20 ng/mL), expanded with IL-15 as per ( B ), with an additional CD4 T cell magnetic depletion step on day 3 of culture. CD8 T cells were harvested on day 6 for parallel RNAseq and proteomic analysis. An interactive version of the proteomics expression data is available for exploration on the Immunological Proteome Resource website: immpres.co.uk ( E ) Fold-change in mRNA expression between Pim dKO and WT versus average mRNA expression (TPM). mRNA expression (Transcripts per million, TPM) of ( F ) secondary lymphoid homing receptors Sell, Ccr7, S1pr1 and ( G ) key transcription factors involved in CD8 T cell memory differentiation and maintenance Tcf7, Klf2, Foxo1, Foxo3, Id3. ( H ) WT vs Pim dKO protein copy numbers, differentially expression proteins (FC >1.5, q<0.05) are highlighted in red ( I ) Protein copy numbers per cell for key mitochondrial proteins DRP1, OPA1, and CPT1A. ( J ) WT vs Pim dKO protein copy numbers, mitochondrial proteins (as defined in MitoCarta 3.0) are highlight in pink. Symbols in bar charts represent biological replicates, symbols in ( C, E, H, J ) represent the mean. Error bars show mean ± S.D. Data are representative of ( A ) n=3 or show pooled data from ( C ) n=4, and ( D ) n=5 biological replicates with data collected over at least two independent experiments. Quantitative proteomics and RNAseq was performed on biological triplicates. ** q≤0.01, fold-change (FC) shown on bar graphs when q<0.05. Figure 2—source data 1. PDF files containing labelled and uncropped images for western blots displayed in . Figure 2—source data 2. Original files for western blot images displayed in . Figure 2—source data 3. Raw values plotted in .

Article Snippet: Antibody , anti-mouse CD8a (Rat, monoclonal, 53–6.7) , Thermo Fisher Scientific/eBioscience , Cat # 47-0081-82, RRID: AB_1272185 , cell surface stain 1:200, APC eF780.

Techniques: Cell Culture, Western Blot, Expressing, Quantitative Proteomics

Journal: eLife

Article Title: PIM kinase control of CD8 T cell protein synthesis and cell trafficking

doi: 10.7554/eLife.98622

Figure Lengend Snippet: ( A ) Estimated copies per cell of PIM1 and PIM2 protein from published quantitative proteomics analysis ; of CD8 T cells expanded in IL-2 or IL-15 as outlined in . ( B–D, F, G ) WT (Ly5.1) and Pim dKO lymph node or spleen single-cell suspensions were mixed at a 50:50 ratio of T cells, activated for 2 days with αCD3/αCD28 (both 0.5 µg/mL) and IL-2 (20 ng/mL), washed then split into fresh medium containing IL-2 (20 ng/mL) daily (as per ). Some of the mixed cell suspensions were also cultured in IL-7 (5 ng/mL) to sustain a naive T cell reference. ( B ) WT and Pim dKO CTL were treated 1 hr +/- Jak1/3 inhibitor Tofacitinib (100 nM; negative control) before pSTAT5 Y694 expression was measured on day 3 and 6 of culture, ( C ) surface CD25 expression was measured on days 3 and 6 of culture, ( D ) CD8 T cell number vs time was calculated, ( F ) CD8 T cell FSC-A, SSC-A and surface activation markers (CD44, CD71) were measured on days 3 and 6 of culture ( G ) expression of adhesion molecule CD62L was measured daily. ( E ) WT and Pim dKO T cells were activated and expanded with IL-2 as per ( B-D ) and ( F, G ) except in separate cultures and % live cells (PI-ve) was assessed on days 4 and 6 (two-way ANOVA). Symbols in bar charts represent biological replicates, symbols in ( D ) represent the mean. Error bars show mean ± S.D. Data are representative of ( B, G ) n=4, ( C, F ) n=6 or show pooled data from ( D ) n=4, ( E ) n=6 biological replicates with data collected over at least two independent experiments. Figure 3—source data 1. Raw values plotted in .

Article Snippet: Antibody , anti-mouse CD8a (Rat, monoclonal, 53–6.7) , Thermo Fisher Scientific/eBioscience , Cat # 47-0081-82, RRID: AB_1272185 , cell surface stain 1:200, APC eF780.

Techniques: Quantitative Proteomics, Cell Culture, Negative Control, Expressing, Activation Assay

Journal: eLife

Article Title: PIM kinase control of CD8 T cell protein synthesis and cell trafficking

doi: 10.7554/eLife.98622

Figure Lengend Snippet: WT and Pim dKO CD8 T cells were activated for 2 days with αCD3/αCD28 (both 0.5 µg/mL) and IL-2 (20 ng/mL), washed then split into fresh medium containing IL-2 (20 ng/mL) daily (as per ), with an additional CD4 T cell magnetic depletion step on day 3 of culture. CD8 T cells were harvested on day 6 of culture for high-resolution mass spectrometry. An interactive version of the proteomics expression data is available for exploration on the Immunological Proteome Resource website: immpres.co.uk ( A ) Estimated total protein content per cell (student t-test). ( B ) Volcano plots of Pim dKO vs WT protein copy numbers, differentially expressed proteins (FC >1.5, q<0.05) are highlighted in red. Estimated protein copy number per cell of ( C ) transcription factor TBX21 and TCF1 ( D ) glucose transporters SLC2A1 and SLC2A3. ( E ) Volcano plots of Pim dKO vs WT protein copy numbers. Proteins with KEGG term = ‘terpenoid backbone biosynthesis’, ‘biosynthesis of unsaturated fatty acids’ or ‘steroid biosynthesis’ are highlighted with proteins with FC >1.5, q<0.05 shown in red and proteins with FC <1.5 and/or q>0.05 shown in pink. ( F ) Heatmap of protein copy numbers for granzymes, perforin, and effector cytokines. Estimated protein copies for ( G ) major cytolytic Granzymes A and B and ( H ) IFNγ. ( I ) Granzyme B and IFNγ expression was measured by flow cytometry in day 6 IL-2 expanded WT and Pim dKO CTL. Symbols in bar charts show biological replicates. Error bars show mean ± S.D. Data are representative of ( I ) n=3–4, with data collected over at least two independent experiments. Quantitative proteomics was performed on biological triplicates. * indicates q<0.05, fold-change (FC) shown on graph when q<0.05. Figure 4—source data 1. Raw values plotted in .

Article Snippet: Antibody , anti-mouse CD8a (Rat, monoclonal, 53–6.7) , Thermo Fisher Scientific/eBioscience , Cat # 47-0081-82, RRID: AB_1272185 , cell surface stain 1:200, APC eF780.

Techniques: Mass Spectrometry, Expressing, Flow Cytometry, Quantitative Proteomics

Journal: eLife

Article Title: PIM kinase control of CD8 T cell protein synthesis and cell trafficking

doi: 10.7554/eLife.98622

Figure Lengend Snippet: RNAseq analysis was performed in day 6 IL-2 expanded WT and Pim dKO CD8 T cells which were collected in parallel with proteomics analysis described in . ( A ) Volcano plot of RNAseq data, differentially expressed mRNA (FC >1.5, q<0.05) are highlighted in red. ( B ) Volcano plot of RNAseq data, Granzymes C-K, perforin, Pdcd4 and Sell are highlighted in red. ( C ) Heatmap of mRNA expression (TPM) for granzymes, perforin and effector cytokines. Bar chart of mRNA expression (TPM) of ( D ) Granzymes A and B ( E ) Glucose transporters Slc2a1 and Slc2a3. ( F, G ) Fold change of PimdKO/WT protein from proteomics analysis described in vs mRNA ( F ) highlighting in red proteins that are differentially expressed (FC >1.5, q<0.05) where mRNA is not substantially different (FC <1.2) and ( G ) highlighting in red protein and mRNA that are both differentially expressed (FC >1.5, q<0.05). ( H ) Estimated cytosolic ribosome content per cell (left), % ribosome of total cellular protein content (right). ( I ) Estimated protein copy number per cell of translation repressor PDCD4. ( J ) PDCD4 expression measured by flow cytometry on day 3 and 6 in IL-2 expanded WT vs Pim dKO CD8 T cells. ( K ) Estimated protein copy number per cell of EIF4A1. ( L ) Adjusted ratio of PDCD4: EIF4A1 (assuming 1 PDCD4 binds 2 x EIF4A1) in WT and Pim dKO proteomes. ( M ) Protein synthesis measured by OPP incorporation in day 6 IL-2-expanded WT CTL treated for 24 hours with pan PIM kinase inhibitors PIM447 (5 µM) or AZD1208 (10 µM). 30-min cycloheximide (100 µg/mL) treatment gives no protein synthesis background control. Symbols in bar charts show biological replicates: error bars show mean ± S.D. Data are representative of ( M ) n=2 biological replicates collected over two independent experiments, ( J ) n=2 biological replicates. Quantitative proteomics and RNAseq were performed on biological triplicates. * indicates q<0.05, fold-change (FC) shown on graph when q<0.05. Figure 5—source data 1. Raw values plotted in .

Article Snippet: Antibody , anti-mouse CD8a (Rat, monoclonal, 53–6.7) , Thermo Fisher Scientific/eBioscience , Cat # 47-0081-82, RRID: AB_1272185 , cell surface stain 1:200, APC eF780.

Techniques: Expressing, Flow Cytometry, Control, Quantitative Proteomics

Journal: eLife

Article Title: PIM kinase control of CD8 T cell protein synthesis and cell trafficking

doi: 10.7554/eLife.98622

Figure Lengend Snippet: PCA plots from parallel ( A ) RNAseq and ( B ) proteomics analysis from Day 6 IL-2 and IL-15 expanded WT and Pim dKO CD8 T cells (as described in , and ).

Article Snippet: Antibody , anti-mouse CD8a (Rat, monoclonal, 53–6.7) , Thermo Fisher Scientific/eBioscience , Cat # 47-0081-82, RRID: AB_1272185 , cell surface stain 1:200, APC eF780.

Techniques:

Journal: eLife

Article Title: JAK inhibition decreases the autoimmune burden in Down syndrome

doi: 10.7554/eLife.99323

Figure Lengend Snippet:

Article Snippet: Antibody , Mouse monoclonal anti-human CD8a (clone RPA-T8) , Fluidigm , Cat # 3162015; RRID: AB_2661802 , Lot 0171813, 1:100.

Techniques: Isolation, Biomarker Discovery, Staining, Antibody Labeling, Software, Sequencing