Journal: bioRxiv

Article Title: Different signaling interpretations by PKC eta and theta control T cell function and exhaustion

doi: 10.1101/2024.09.26.615103

Figure Lengend Snippet: (A) MFIs from phospho-flow cytometry of the indicated markers are shown. T EX-PROG or T EX-TERM cells from LCMV Cl 13 infected mice 30 d.p.i. were stimulated with PMA ex vivo for 30 mins (all but c-Fos and c-Jun) or 4 hours (c-Fos and c-Jun). (B) Expression of PKC theta and eta by subset from LCMV Cl 13, 30 d. p.i. (C) Validation of PKC theta ( Prkcq ) and PKC eta ( Prkch ) knockouts by flow cytometry and Western blot. (D) Mice were infected with LCMV clone 13, and congenic P14 T cells were negative selected and activated in vitro via platebound anti-CD3/28. After 24 hours, cells were subjected to Cas9-sgRNA electroporation to delete genes of interest, and after a further 24 hours of recovery, electroporated cells were mixed and co-transferred into infected mice (E-F) The frequencies of each congenic population of electroporated cells at the time of co-transfer (E), and the frequencies of cells of each genotype relative to the total CD8 T cell population at the indicated timepoints post-infection (F) (G-H) The frequencies of indicated markers among transferred PD1+ CD8 T cells, 8 d.p.i (I-K) The frequencies of indicated markers (I-J) and TOX MFI (K) in transferred PD1+ cells, 28-30 d.p.i Scatter plots show mean +/- s.e.m. Statistical significance was determined using Student’s t-test (A, I-K) or one way ANOVA (panels B-H), with significance shown as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Inhibitor screening of kinases downstream of PKC theta or eta : GSK-626616 (MedChem Express) was used to inhibit DYRK and was diluted from a 10 mM stock to a final concentration of 1 μM.

Techniques: Flow Cytometry, Infection, Ex Vivo, Expressing, Western Blot, In Vitro, Electroporation

Journal: bioRxiv

Article Title: Different signaling interpretations by PKC eta and theta control T cell function and exhaustion

doi: 10.1101/2024.09.26.615103

Figure Lengend Snippet: (A) Schematic of in vitro exhaustion experiments. P14 T cells were activated in vitro , expanded, and co-cultured with B16-gp33 cells from days 3-7 post-activation. (B) Representative flow plots showing the effect of chronic PMA treatment on T cells in vitro . (C) Cumulative bar graphs of PMA effects on in vitro exhausted cells. (D) nCounter data highlighting PMA-driven gene expression changes. T EX-TERM associated genes are in red, while T EX-PROG associated genes are in blue. Data are shown as log2 fold change. (E) Cartoon schematic of the regulation of PKC kinase activity. (F) Western blots showing PKC protein levels in cultured primary CD8 + T cells after 4 days of PMA treatment. (G) Effects of KO of Prkcq or Prkch on responsiveness to PMA during in vitro exhaustion. (H) Domain architecture of PKC theta. Numbers represent the amino acids defining each domain and the three phosphorylation sites key for PKC kinase activity. (I) A two-round K-to-R mutagenesis screen to identify lysine residues required for PKC activity-induced degradation after 24 hours of PMA treatment. Lysine residues were first mutated in blocks of four to six at a time (left), and then individual lysine residues were mutated within the 409-451 subset (right) (J-K) P14 T cells were transduced with EV or PKC OE vectors, subjected to in vitro exhaustion, and assessed for their capacity to produce cytokines. Scatter plots show mean +/- s.e.m. Statistical significance was determined using Student’s t-test (panel C) or one way ANOVA (panels G, I-K), with significance shown as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Inhibitor screening of kinases downstream of PKC theta or eta : GSK-626616 (MedChem Express) was used to inhibit DYRK and was diluted from a 10 mM stock to a final concentration of 1 μM.

Techniques: In Vitro, Cell Culture, Activation Assay, Expressing, Activity Assay, Western Blot, Mutagenesis, Transduction

Journal: bioRxiv

Article Title: Different signaling interpretations by PKC eta and theta control T cell function and exhaustion

doi: 10.1101/2024.09.26.615103

Figure Lengend Snippet: (A) Timeline of adoptive transfer experiments of P14 T cells into mice infected with LCMV Cl 13. Overexpression of PKC theta variants or an EV control were introduced using a retroviral (RV) vector. (B-C) MFI of PKC theta among all transferred GFP+ (B) and GFP+ T TERM cells (C). (D-F) Numbers of total GFP+ cells of the indicated genotypes, in total (D), or in SLAMF6 + TIM3 - (E) or SLAMF6 - TIM3 + (F) subsets. (G) Frequency GFP+ P14 T cells expressing CXCR6 in LCMV Cl 13. (H-I) Normalized MFI of the indicated markers in LCMV Cl 13. (J) Timeline of adoptive transfer experiments in B16gp33 tumor-bearing mice. (K-L) Tumor volumes (K) and masses at endpoint (L) of tumors by P14 genotype. (M) IFNγ producing functionality of P14 T cells infiltrating B16gp33 tumors by genotype. Scatter plots show mean +/- s.e.m. Statistical significance was determined using one way ANOVA where applicable, with significance shown as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Inhibitor screening of kinases downstream of PKC theta or eta : GSK-626616 (MedChem Express) was used to inhibit DYRK and was diluted from a 10 mM stock to a final concentration of 1 μM.

Techniques: Adoptive Transfer Assay, Infection, Over Expression, Control, Retroviral, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: Different signaling interpretations by PKC eta and theta control T cell function and exhaustion

doi: 10.1101/2024.09.26.615103

Figure Lengend Snippet: (A) Schematic of preparation of primary T cells for global phosphoproteomics. (B) Cartoon highlighting and simplifying major known pathways downstream of PKC. (C) Select, top-ranking PKC theta-specific or PKC eta-specific phosphopeptides identified from the anti-CD3 stimulation condition. Phosphorylated residues are highlighted in bold and followed with an asterisk (*). The gene name of the protein from which the peptide originates is shown to the left of each peptide. (D) GO terms associated with the top 100 PKC theta- vs eta-specific downstream phosphoproteins. (E) Volcano plot of Kinase Library analysis of kinases active downstream of PKC theta or eta after anti-CD3 stimulation. Re-stimulated sgPrkch or sgPrkcq samples were normalized to unstimulated controls of the matching genetic background, and the normalized phosphoproteomes were subsequently compared to one another. The kinases active in each condition (plotted) are inferred by identifying enrichment of ideal peptide motifs for each kinase in the entire proteomics dataset. (F) Phospho-flow cytometry of the indicated markers within in vitro activated CD8 T cells, stimulated for 30 minutes with PMA, in the indicated genetic backgrounds. (G) Fraction of top 100 PKC eta-specific peptides that score highly as CK1 family targets according to Kinase Library peptide motifs. (H) Schematic of in vitro exhaustion experiments used to screen inhibitors of kinases downstream of PKC theta and eta. (I) Screening effects of inhibitors of kinase families putatively downstream of the PKCs for markers of T EX-PROG differentiation. Scatter plots show mean +/- s.e.m. Statistical significance was determined using one way ANOVA where applicable, with significance shown as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Inhibitor screening of kinases downstream of PKC theta or eta : GSK-626616 (MedChem Express) was used to inhibit DYRK and was diluted from a 10 mM stock to a final concentration of 1 μM.

Techniques: Flow Cytometry, In Vitro

Journal: bioRxiv

Article Title: Different signaling interpretations by PKC eta and theta control T cell function and exhaustion

doi: 10.1101/2024.09.26.615103

Figure Lengend Snippet: (A) Cartoon of PKC theta signaling through the CARMA1-BCL10-MALT1 complex. (B) Western blot showing Regnase-1 levels after 30 minutes of PMA or vehicle treatment. T cells were collected after in vitro activation and 5 days of expansion. (C) Cartoon schematic of PKC eta proximity labeling strategy and conditions via TurboID. The TurboID-PKC eta fusion protein was transduced into primary T cells, and the expanded transductants were treated and collected 6-7 days post-activation. (D) Venn diagram of raw TurboID statistics. (E) Scatterplot of comparing the log2 fold enrichment of candidate PKC eta interacting proteins in anti-CD3-stimulated vs unstimulated conditions. Dashed lines and colors indicate protein subsets based on enrichment thresholds. Enrichment value was determined by dividing the peak area of candidate proteins by the mean peak area of negative control conditions. Only proteins with p adj < 0.01 are plotted. (F) Changes between the subcellular localizations and cellular functions of top candidate PKC eta interactors between stimulated and unstimulated conditions.

Article Snippet: Inhibitor screening of kinases downstream of PKC theta or eta : GSK-626616 (MedChem Express) was used to inhibit DYRK and was diluted from a 10 mM stock to a final concentration of 1 μM.

Techniques: Western Blot, In Vitro, Activation Assay, Labeling, Negative Control

Journal: bioRxiv

Article Title: Different signaling interpretations by PKC eta and theta control T cell function and exhaustion

doi: 10.1101/2024.09.26.615103

Figure Lengend Snippet: (A) Timeline of casein kinase I gene deletions in LCMV Cl 13 adoptive transfer experiments. (B) Frequency of the indicated genotype transferred P14 T cells among all CD8 + T cells .(C-E) Representative data (C), frequencies (C-D), and MFIs (E) of markers of differentiation and functionality of P14 T cells of the indicated genotypes. (F) Timeline of Csnk1g2 deletion in B16gp33 adoptive transfer experiments. (G) Masses of B16gp33 tumors at sacrifice timepoint by indicated genotype. (H) MFI of PD-1 in TTERM P14 T cells infiltrating B16gp33 tumors. (I) (Left) UMAP of integrated single cell RNA-seq datasets of CD8 + T cells isolated from human cancer biopsies (GSE146771, GSE99254, GSE98638), with cell subsets indicated by color. (Middle, right) Violin plots of PRKCQ and PRKCH expression by T cell subset. (J) Schematic of in vitro exhaustion experiments used to test the effects of CK1 inhibition and perturbations of PKC signaling on human CAR-T cells. (K) TNF production of in vitro exhausted human CAR-T cells by the indicated genotypes and treatments. Scatter plots show mean +/- s.e.m. Statistical significance was determined using Student’s t-test (panels C-E, G-H) or one way ANOVA (panels B, K), with significance shown as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Inhibitor screening of kinases downstream of PKC theta or eta : GSK-626616 (MedChem Express) was used to inhibit DYRK and was diluted from a 10 mM stock to a final concentration of 1 μM.

Techniques: Adoptive Transfer Assay, RNA Sequencing Assay, Isolation, Expressing, In Vitro, Inhibition

Journal: Molecular and cellular neurosciences

Article Title: Protein kinase C mediates peroxynitrite toxicity to oligodendrocytes.

doi: 10.1016/j.mcn.2011.06.006

Figure Lengend Snippet: Fig. 2. The pan PKC inhibitor Bis-1 blocked PKC activation and mature OL toxicity induced by SIN-1 and ZnCl2. A, Mature OLs were exposed to a peroxynitrite donor SIN-1 (0.5 mM) or ZnCl2 (100 μM) in the absence or presence of Bis-1 for 30 min, and then lysed for assaying the PKC activity. **, pb0.01 and ***, pb0.001 were obtained when the SIN-1 and the zinc treated groups were compared with the control group. ##, pb0.01 was obtained when the Bis-1 treated groups were compared with SIN-1 alone and ZnCl2 alone groups. A representative experiment of three that were performed is shown. B, OLs were exposed to SIN-1 (0.5 mM) in the absence or presence of Bis-1 for 2 h, and the toxicity was assessed at 24 h. Bis-1 dose-dependently protected against SIN-1 induced toxicity. *, pb0.05 and **, pb0.01 were obtained when the Bis-1 treated SIN-1 groups were compared with the SIN-1 alone group. A representative experiment of six that were performed is shown. C, OLs were exposed to ZnCl2 (100 μM) in the absence or presence of Bis-1 for 2 h, and the toxicity was assessed at 24 h. Bis-1 dose-dependently protected against ZnCl2 induced toxicity. *, pb0.05 and ***, pb0.001 were obtained when the Bis-1 treated ZnCl2 groups were compared with the zinc alone group. A representative experiment of six that were performed is shown.

Article Snippet: The PCR product was run on 1.5% agarose gel and visualized under UV light. shRNA transfection Mature OLs were infected with PKCθ shRNA lentiviral particles (sc36247v, Santa Cruz Biotechnology) in the presence of polybrene (2 μg/ml) for 24 h. Polybrene was used to enhance the binding of lentiviral particles to cell membranes.

Techniques: Activation Assay, Activity Assay, Control

Journal: Molecular and cellular neurosciences

Article Title: Protein kinase C mediates peroxynitrite toxicity to oligodendrocytes.

doi: 10.1016/j.mcn.2011.06.006

Figure Lengend Snippet: Fig. 4. Expression of various PKC isoforms in mature OLs. A, RNA samples isolated from OLs were analyzed by reverse transcriptase PCR using the primer pairs (see Table 1) specific for rat PKC α, β, γ, η, ε, δ, θ, λ and ζ. The products were run on a 1% agarose gel impregnated with ethidium bromide. The bands were visualized under UV light. A representative experiment of three that were performed is shown. B, Mature OLs were treated with PMA (100 nM) for 24 h and then lysed to detect the expression of various PKC isoforms. The isoform specific antibodies were used to compare the expression of each PKC specific isoform with and without PMA prolonged treatment. β-actin was used as a loading control for lysates from control (CON) and PMA pretreated cells. A representative experiment of three that were performed is shown.

Article Snippet: The PCR product was run on 1.5% agarose gel and visualized under UV light. shRNA transfection Mature OLs were infected with PKCθ shRNA lentiviral particles (sc36247v, Santa Cruz Biotechnology) in the presence of polybrene (2 μg/ml) for 24 h. Polybrene was used to enhance the binding of lentiviral particles to cell membranes.

Techniques: Expressing, Isolation, Reverse Transcription, Agarose Gel Electrophoresis, Control

Journal: Molecular and cellular neurosciences

Article Title: Protein kinase C mediates peroxynitrite toxicity to oligodendrocytes.

doi: 10.1016/j.mcn.2011.06.006

Figure Lengend Snippet: Fig. 8. Downregulation of PKCθ attenuated OL toxicity induced by zinc. A, OLs were treated with ZnCl2 in the presence of Bis-1, Go6976 (Go) or rottlerin (Ro) for 2 h, and then lysed for western blot. ZnCl2 caused a significant increase of PKCθ phosphorylation, which was completely blocked by rottlerin, but not by Go6976. Bis-1 also slightly attenuated the phosphorylation of PKCθ. There was no phosphorylation of PKCδ following zinc treatment. A representative experiment of three that were performed is shown. B, OLs were infected with Lentiviral PKCθ shRNA particles for 4 days and then lysed for detection of the expression of PKCθ. Transfection with PKCθ shRNA caused a dramatic reduction of the expression of PKCθ, when compared to the GFP shRNA transfected cells. Prolonged treatment with PMA also completely blocked the expression of PKCθ. A representative experiment of three that were performed is shown. C–D, OLs were infected with Lentiviral PKCθ shRNA particles for 4 days and then treated with ZnCl2 (C) or SIN-1 (D) for 2 h. The toxicity was assessed at an additional 24 h. Transfection with GFP shRNA had no effect on zinc or SIN-1 induced toxicity. However, transfection with PKCθ shRNA significantly attenuated cell death induced by ZnCl2 or SIN-1. *, pb0.05 and **, pb0.01 were obtained when PKCθ shRNA transfected groups were compared with GFP shRNA transfected and the control groups following zinc or SIN-1 treatment. A representative experiment of three that were performed is shown.

Article Snippet: The PCR product was run on 1.5% agarose gel and visualized under UV light. shRNA transfection Mature OLs were infected with PKCθ shRNA lentiviral particles (sc36247v, Santa Cruz Biotechnology) in the presence of polybrene (2 μg/ml) for 24 h. Polybrene was used to enhance the binding of lentiviral particles to cell membranes.

Techniques: Western Blot, Phospho-proteomics, Infection, shRNA, Expressing, Transfection, Control