Journal: PLoS Pathogens

Article Title: β-catenin regulates HIV latency and modulates HIV reactivation

doi: 10.1371/journal.ppat.1010354

Figure Lengend Snippet: (A) 3–4 human donors were used to establish a primary T CM model of HIV latency using HIV strain NL4-3 (left) or REJO (right). Latently infected cells were stimulated with αCD3/αCD8 activation beads alone (black) or in the presence of 2 μM Bio (blue). The percentage of CD4 negative HIV p24 positive cells is shown. (B) T CM cells latently infected with HIV REJO were stimulated with the indicated drugs in combination with 2 μM Bio (blue). Reactivation as measured by the percentage of CD4 negative HIV p24 positive cells was normalized to reactivation with the drug in the absence of Bio. Mean and SEM of 3 donors is shown. Significance was determined using paired t -tests for all panels, * p<0.05, ** p<0.01.

Article Snippet: CD8+ depleted PBMCs were isolated using the RosetteSep Human CD8 depletion cocktail (StemCell) and suspended in RPMI supplemented with 30 U/ml IL-2 and antiretrovirals raltegravir (1 μM) and nelfinavir (0.5 μM).

Techniques: Infection, Activation Assay

Journal: PLoS Pathogens

Article Title: β-catenin regulates HIV latency and modulates HIV reactivation

doi: 10.1371/journal.ppat.1010354

Figure Lengend Snippet: (A) CD8-depleted PBMCs from n = 5 HIV positive donors on suppressive cART therapy were treated for 48 hours with 50 nM β-catenin inhibitor ADV, αCD3/αCD28 T-cell activating beads alone or combined with 50 nM ADV. Extracellular (released virions, right) HIV RNA copies were quantified. Absolute RNA copy numbers in vehicle control and ADV treated cultures are shown, with lines connecting samples from the same donor. Symbols corresponding to donors in are used for panels a-g. (B) Fold change of RNA copies in ADV treated cultures over vehicle control are shown from released virus. (C) Fold change of RNA copies in ADV co-treated cultures over αCD3/αCD28 single treatment. (D) As in (A), cells were treated with αCD3/αCD28 beads alone or combined with 2 μM 6Bio. HIV RNA quantities are shown with lines connecting cultures from the same donor. (E) Fold change of HIV RNA copies in 6Bio co-treated cells over αCD3/αCD28 single treatment. (f-g) Downstream target of β-catenin, Bcl-xL, was quantified by flow cytometry in cells treated with ADV, αCD3/αCD28, or αCD3/αCD28 with ADV/6Bio, to confirm the modulation of β-catenin by these drugs. Fold change in mean fluorescence intensity of Bcl-xL is shown compared to vehicle control or αCD3/αCD28 treated cells, for single or dual treated cells, respectively. (H) Viability and T cell activation markers in cells following drug treatments were quantified by flow cytometry. The proportion of CD3+ CD4+ T cells expressing Ki67, CD69, CD38/HLA-DR, or LIVE/DEAD stain are plotted for cells treated with the indicated treatments. Significance was determined using paired t -tests for all panels, * p<0.05, ** p<0.01.

Article Snippet: CD8+ depleted PBMCs were isolated using the RosetteSep Human CD8 depletion cocktail (StemCell) and suspended in RPMI supplemented with 30 U/ml IL-2 and antiretrovirals raltegravir (1 μM) and nelfinavir (0.5 μM).

Techniques: Control, Virus, Flow Cytometry, Fluorescence, Activation Assay, Expressing, Staining

Journal: PLoS Pathogens

Article Title: β-catenin regulates HIV latency and modulates HIV reactivation

doi: 10.1371/journal.ppat.1010354

Figure Lengend Snippet: Schematic demonstrating multiple potential mechanisms by which β-catenin may modulate HIV transcription and latency, based on integrated findings from previous studies and data presented here. (1) β-catenin and TCF-4 form a complex with nuclear matrix-associated protein SMAR1 at the HIV LTR just upstream of the transcriptional start site and Sp-1, NFκB, and AP-1 binding sites. This complex pulls the HIV LTR towards the nuclear matrix, occluding access of RNA polymerase . (2) β-catenin positively regulates levels of TCF-4, which has been shown to block binding and transcriptional regulation of NFκB at the HIV LTR . (3) β-catenin further regulates c-Myc levels, which recruit HDAC enzymes, resulting in the viral promoter being more densely packed in chromatin . (4) β-catenin also mediates self-renewal and cell proliferation of memory T cells through CBP, which may contribute to perpetuating the reservoir of latently infected cells , (5) A source of β-catenin signaling are CD8+ T cells, which secrete Wnt proteins resulting in stimulation of the Wnt/β-catenin pathway in CD4+ T cells, which culminates in accumulation of β-catenin in the cytoplasm and translocation to the nucleus . This may explain the observed role of CD8+ T cells in maintaining HIV latency. Notably, other cells may serve as a source of Wnt proteins and β-catenin pathway modulating factors.

Article Snippet: CD8+ depleted PBMCs were isolated using the RosetteSep Human CD8 depletion cocktail (StemCell) and suspended in RPMI supplemented with 30 U/ml IL-2 and antiretrovirals raltegravir (1 μM) and nelfinavir (0.5 μM).

Techniques: Binding Assay, Blocking Assay, Infection, Translocation Assay

Journal: Frontiers in Immunology

Article Title: Targeting Nuclear LSD1 to Reprogram Cancer Cells and Reinvigorate Exhausted T Cells via a Novel LSD1-EOMES Switch

doi: 10.3389/fimmu.2020.01228

Figure Lengend Snippet: LSD1 inhibition re-invigorates CD8 + T cell subsets in mouse models. (A) Schematic of the metastatic breast cancer mouse model. Tumor samples from 3 individual animals/group were collected on day 15, RNA was extracted, and gene expression measured using the nanostring pancancer immune profiling panel. (B) Bar chart of -log10 transformed p -values across cell types by nCounter advanced analysis. Characteristic genes of various immune cell populations measure each population's abundance within the tumor sample. Immune pathway scores against treatment conditions defined using the first principal component of each geneset's data and each sample's gene expression profile condensed into a small set of pathway scores including innate, adaptive, humoral, and inflammation pathways. Total number of induced and inhibited genes for immune-related pathways analyzed by nanostring. Statistically significantly differentially expressed genes are defined by 2-fold linear changes with p < 0.05 compared to control samples. Yellow bar plot represents phenelzine, red bar plot represents anti-PD-1. (C) 4T1 tumor-bearing mice were treated with vehicle control, phenelzine (40 mg/kg), or EPI-111 (20 mg/kg). Fifteen days post-treatment, the primary tumors were measured and harvested and collected for nanostring analysis. NanoString global significance scores for RNA isolated from whole tumors. Heatmap displaying each sample's directed global significance scores. Directed global significance statistics measure the extent to which a geneset's genes are up- or down-regulated with the variable. Red denotes genesets whose genes exhibit extensive over-expression with the covariate, blue denotes genesets with extensive under-expression. NanoString analysis was performed in triplicate. (D) Jurkat T cells were treated with phenelzine or GSK for 10 h followed by inhibition withdrawal and resting for different time points. After resting for 24 h, cells were re-inhibited with phenelzine or GSK followed by repeat inhibition (PMA/CaI for 2 h), withdrawal, and resting. IFN-γ expression levels were measured by RT-PCR and normalized to GAPDH. Data represent the fold changes in expression of stimulated samples compared to control non-stimulated samples. Expression values are the average of the RT-PCR (technical) replicates, and error bars indicate min-max. (E) T cells isolated from 4T1 tumors were stimulated with PMA/ionomycin for 4 h in the presence of brefeldin A, stained for IFN-γ, and analyzed by flow cytometry (* p < 0.05, n = 3). Data overlies total CD8 + T cell infiltration into the primary tumor ( n = 5 mice per group). Treatment groups are control, abraxane (30 mg/kg), anti-PD1 (10 mg/kg), phenelzine (40 mg/kg), anti-PD1 and phenelzine, or triple therapy. (F) 4T1 breast cancer treatment regimen. Treatment groups from left to right: Control, Abraxane (30 mg/kg), phenelzine (40 mg/kg), EPI-111 (1 mg/kg), EPI-111 (4 mg/kg), and EPI-111 (20 mg/kg), with 5 mice per group. (G) Cells were taken for FACS analysis from primary tumors from a 4T1 TNBC immunotherapy-resistant mouse model treated as in 3E and labeled with primary antibodies against CD45 and CD3 positive cells. Graphs plot the % cell population for each group ( n = 5 mice per group). Mann-Whitney test, **** p < 0.0001, *** p = 0.0002, ** p = 0.0021, * p = 0.033, ns > 0.05. (H) FFPE sections were taken from primary tumors from a 4T1 TNBC immunotherapy-resistant mouse model treated as in 2D and labeled with primary antibodies against CD8, LSD1, TIGIT, LAG3, and TIM3. The CD8 + T cell population positive for these markers was analyzed using the ASI digital pathology system to enumerate % total cell population. Graphs plot the % cell population for either CD8 + T cell infiltration or CD8 + LSD1 + TIGIT + LAG3 + TIM3 + T cells ( N > 500 cells per group, 5 mice per group). Mann-Whitney test, **** p < 0.0001, *** p = 0.0002, ** p = 0.0021, * p = 0.033, ns > 0.05. Treatment groups are control, abraxane (30 mg/kg), phenelzine (40 mg/kg), EPI-111 (1 mg/kg), EPI-111 (4 mg/kg), and EPI-111 (20 mg/kg), with 5 mice per group. (I) 4T1 breast cancer treatment regimen. Treatment groups from left to right. Control, abraxane (30 mg/kg), phenelzine (40 mg/kg), EPI-111 (4 mg/kg), and anti-PD1 (10 mg/kg). (J) FFPE sections were taken from primary tumors from a 4T1 TNBC immunotherapy-resistant mouse model treated as in 2D and labeled with primary antibodies against CD8. The CD8 + T cell population within the FFPE section was analyzed using the ASI digital pathology system to enumerate % total cell population. Graphs plot the % cell population for either CD8 + T cell infiltration ( N > 500 cells per group, n = 5 mice per group). Mann-Whitney test, **** p < 0.0001, *** p = 0.0002, ** p = 0.0021, * p = 0.033, ns > 0.05. (K) FFPE sections were taken from primary tumors from a 4T1 TNBC immunotherapy-resistant mouse model treated as in 3J and labeled with primary antibodies against CD8 and IFN-γ. The CD8 + T cell population positive for both of these markers was analyzed using the ASI digital pathology system to enumerate % total double-positive cell population. Graphs plot the % cell population for CD8 + IFN-g + T cells ( n > 500 cells per group; n = 5 mice per group). Mann-Whitney test, **** p < 0.0001, *** p = 0.0002, ** p = 0.0021, * p = 0.033, ns > 0.05.

Article Snippet: Liquid biopsies were pre-enriched using the RosetteSepTM method to isolate CD8 + T cells by employing the RosetteSepTM Human CD8 enrichment Kit (15063, Stemcell Technologies, Vancouver, Canada) to remove CD45 + cells and red blood cells using density gradient centrifugation with SepMateTM-15 (IVD) density gradient tubes (85420, Stemcell Technologies) and LymphoprepTM density gradient medium (07861, Stemcell Technologies).

Techniques: Inhibition, Gene Expression, Transformation Assay, Control, Isolation, Over Expression, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Flow Cytometry, Labeling, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Targeting Nuclear LSD1 to Reprogram Cancer Cells and Reinvigorate Exhausted T Cells via a Novel LSD1-EOMES Switch

doi: 10.3389/fimmu.2020.01228

Figure Lengend Snippet: nLSD1p inhibition induces immune cell infiltration and re-invigoration and enhances Tm in CD8 + T cells in mouse models and humans. (A) CD8 + T cells from TNBC patients were untreated or treated with phenelzine, GSK, or control in vitro and stained with CCR7 and CD45RA antibodies to categorize them into naïve, T effector memory (Tem), and T effector memory RA (Temra). Cells were also stained with perforin and IFN-γ after stimulation with PMA/CaI for 4 h in the presence of brefeldin A. (B) t-distributed stochastic neighbor embedding (tSNE) analysis was performed on patient-derived TNBC CD8 + T cells either pre or post LSD1 therapy after PMA/ionomycin stimulation. Gated populations were divided into Naïve, Tscm, Tcm, Tem, and Temra phenotypes. (C) tSNE analysis was performed on patient-derived TNBC CD8 + T cells post LSD1 therapy or post 25 weeks LSD1 therapy after PMA/ionomycin stimulation. Gated populations (naïve, Tem, and Temra) of distinct phenotypes were overlaid onto a 2-dimensinal tSNE data space, revealing the differential expression of IFN-γ and perforin in total CD8 + T cells or different CD8 subsets.

Article Snippet: Liquid biopsies were pre-enriched using the RosetteSepTM method to isolate CD8 + T cells by employing the RosetteSepTM Human CD8 enrichment Kit (15063, Stemcell Technologies, Vancouver, Canada) to remove CD45 + cells and red blood cells using density gradient centrifugation with SepMateTM-15 (IVD) density gradient tubes (85420, Stemcell Technologies) and LymphoprepTM density gradient medium (07861, Stemcell Technologies).

Techniques: Inhibition, Control, In Vitro, Staining, Derivative Assay, Quantitative Proteomics

Journal: Frontiers in Immunology

Article Title: Targeting Nuclear LSD1 to Reprogram Cancer Cells and Reinvigorate Exhausted T Cells via a Novel LSD1-EOMES Switch

doi: 10.3389/fimmu.2020.01228

Figure Lengend Snippet: Global transcriptome analysis shows that phenelzine-mediated gene signatures are common to reinvigoration phenotypes in different model systems. (A) Volcano plot of the difference in gene expression before and after phenelzine treatment in TNBC and HER2 − donor CD8 + T cell transcriptomes. Significance was determined in DeSeq2, FDR < 0.1, n = 4. Three hundred fifty genes were downregulated with phenelzine (PHE) treatment and 314 were upregulated. (B) Enrichment of the down (PHE_Down) and up (PHE_UP) PHE signatures in different models of T cell exhaustion, activation, and memory. For x vs. y comparisons, a positive normalized enrichment score (NSE) indicates enrichment in x, a negative score enrichment in y. * p < 0.05 ** p < 0.01 *** p < 0.001. (C) Enrichment plots for GSE72752 and GSE24081 showing individual genes from the two phenelzine signatures distributed across the ranking in expression from chronic to resolver or progressor to controller. (D) Expression profiles of the genes up- and downregulated by phenelzine in the two donors (TNBC and HER2 − ). Averages of the duplicate isolations and treatments are shown ( n = 2). The number of SMAD (S) and HIC2 (H) or EOMES (E) and RUNX1 (R) motifs in nearby enhancers are marked. Upregulated genes with at least 5 SMAD motifs in nearby enhancers or that are negative regulators of the TGF-b pathway are named. Downregulated genes with at least 5 EOMES motifs in nearby enhancers are named (red: direct (bound) EOMES targets upregulated by EOMES overexpression; blue: direct (bound) targets downregulated by EOMES overexpression).

Article Snippet: Liquid biopsies were pre-enriched using the RosetteSepTM method to isolate CD8 + T cells by employing the RosetteSepTM Human CD8 enrichment Kit (15063, Stemcell Technologies, Vancouver, Canada) to remove CD45 + cells and red blood cells using density gradient centrifugation with SepMateTM-15 (IVD) density gradient tubes (85420, Stemcell Technologies) and LymphoprepTM density gradient medium (07861, Stemcell Technologies).

Techniques: Gene Expression, Activation Assay, Expressing, Over Expression

Journal: Frontiers in Immunology

Article Title: Targeting Nuclear LSD1 to Reprogram Cancer Cells and Reinvigorate Exhausted T Cells via a Novel LSD1-EOMES Switch

doi: 10.3389/fimmu.2020.01228

Figure Lengend Snippet: The EOMES:LSD1p nuclear complex is enriched in PD-1 + CD8 + T cells from resistant, high disease burden patients. (A) CD8 + T cells were isolated from healthy donors, melanoma patient cohorts, or metastatic breast cancer patient (ER + /PR + /HER2- or TNBC) liquid biopsies and labeled with primary antibodies targeting T-bet, EOMES, and PD-1. ASI Digital Pathology Analysis was carried out to calculate the percentage population of T-bet Low EOMES Hi PD-1 + T cells. Patient cohorts included n = 5 healthy donors, n = 20 patients in the responder cohort, and n = 15 patients in the resistant cohort, with sampling as in with 4 samples per patient. (B) Primary antibodies against LSD1 and EOMES were used to label PD-1 + CD8 + T cells from healthy donors or melanoma patients with different immunotherapy susceptibility profiles. Samples were processed by ASI Digital Pathology Analysis based on >500 cells (patient cohorts included n = 5 healthy donors, n = 20 patients in the responder cohort, and n = 15 patients in the resistant cohort, with sampling as in with 4 samples per patient). The % EOMES Hi LSD1 + PD-1 + CD8 + T cell population was analyzed for each group as well as the TNFI of LSD1 and EOMES. Representative images for each dataset are shown with scale bar = 10 mm. Graphs represent either the % cell population (positive for EOMES/LSD1) or the TNFI for LSD1 and EOMES measured using digital pathology (ASI) minus background ( n > 500 cells/patient sample for 40 patient samples). The EOMES:LSD1 PCC was determined for at least 40 individual cells ± SEM. Mann-Whitney test, **** p < 0.0001, *** p = 0.0002, ** p = 0.0021, * p = 0.033, ns p > 0.05. (C) Primary antibodies against LSD1 and EOMES were used to label PD-1 + CD8 + T cells from healthy donors or metastatic breast cancer patients (ER + /PR + /HER2 − or TNBC). Samples were processed by ASI digital pathology analysis based on >500 cells. Patient cohorts included n = 5 healthy donors, n = 20 patients in the responder cohort, and n = 15 patients in the resistant cohort, with sampling as in with 4 samples per patient. The % EOMES Hi LSD1 + PD-1 + CD8 + T cell population was analyzed for each group. Graphs represent the mean % positive cell population measured using digital pathology (ASI) minus background ( n > 500 cells/patient sample for 40 patient samples). The EOMES:LSD1 PCC was determined for at least 40 individual cells ± SEM. Mann-Whitney test, **** p < 0.0001, *** p = 0.0002, ** p = 0.0021, * p = 0.033, ns p > 0.05. (D) Proximity ligation assay (DuoLink) for EOMES and LSD1 in CD8 + T cells from melanoma patients with different immunotherapy susceptibility profiles. Representative images shown with scale bar = 10 mm. Graph plotted measures the PLA (ligation intensity measured by high-resolution microscopy) protein interaction. Patient cohorts = 3 patients per group and 4 repeat samples per patient. (E) Melanoma primary tumor baseline tissue biopsies for either responder or resistant melanoma patient cohorts were stained for LSD1, EOMES, and CD8 with DAPI. Representative images for each dataset are shown. Graphs represent either the % population of EOMES + LSD1 + CD8 + T cells or the mean TNFI for LSD1 and EOMES measured using ASI Digital Pathology minus background ( n > 50 cells/patient sample for 20 patient samples). The EOMES:LSD1 PCC was determined for at least 20 individual cells ± SEM. Mann-Whitney test, **** p < 0.0001, *** p = 0.0002, ** p = 0.0021, * p = 0.033, ns p > 0.05. n = 20 patient samples total; n = 10 responder patient samples, n = 10 resistant patient samples (with 4 repeat samples per patient). (F) Metastatic brain cancer lesions from a metastatic breast cancer patient were stained for LSD1, EOMES, and CD8 with DAPI. Representative images for each dataset are shown. Graphs represent the mean TNFI for LSD1 and EOMES measured using ASI Digital Pathology minus background ( n > 50 cells/patient sample for 30 patient samples). The EOMES:LSD1 PCC was determined for at least 20 individual cells ± SEM. Mann-Whitney test, **** p < 0.0001, *** p = 0.0002, ** p = 0.0021, * p = 0.033, ns p > 0.05.

Article Snippet: Liquid biopsies were pre-enriched using the RosetteSepTM method to isolate CD8 + T cells by employing the RosetteSepTM Human CD8 enrichment Kit (15063, Stemcell Technologies, Vancouver, Canada) to remove CD45 + cells and red blood cells using density gradient centrifugation with SepMateTM-15 (IVD) density gradient tubes (85420, Stemcell Technologies) and LymphoprepTM density gradient medium (07861, Stemcell Technologies).

Techniques: Isolation, Labeling, Sampling, MANN-WHITNEY, Proximity Ligation Assay, Ligation, Microscopy, Staining

Journal: Frontiers in Immunology

Article Title: Targeting Nuclear LSD1 to Reprogram Cancer Cells and Reinvigorate Exhausted T Cells via a Novel LSD1-EOMES Switch

doi: 10.3389/fimmu.2020.01228

Figure Lengend Snippet: An EOMES methylation/acetylation switch regulated by LSD1 in CD8 + T cells indicates immunotherapy responsiveness. (A) Jurkat T cells transfected with either VO, LSD1 wildtype (LSD1-WT), or LSD1 NLS mutant plasmids (LSD1-NLSmut) were probed with primary antibodies targeting EOMES and screened by IF. Graphs represent the TNFI and Fn/c for n = 20 or more cells ( n = 3 experiments). Mean NFI and Fn/c are shown. Mann-Whitney test, **** p < 0.0001, *** p = 0.0002, ** p = 0.0021, ns >0.05. (B) Schematic of EOMES plasmids and 641k motif. (C) Jurkat T cells transfected with either VO, EOMES-WT, EOMES-Mut1, or EOMES-Mut2 plasmids and probed with primary antibodies targeting EOMES and screened by IF. Graphs represent the TNFI and Fn/c for n = 20 or more cells ( n = 3 experiments). Representative images for each dataset are shown; scale bar indicates 10 μM. Mean NFI and Fn/c are shown. Mann-Whitney test, **** p < 0.0001, *** p = 0.0002, ** p = 0.0021, ns >0.05. (D) Nuclear extracts from jurkat T cells transfected with LSD1 WT or mutant plasmids and subjected to half-way CHIP using LSD1 pull down or a no antibody control. Samples were subject to immunoblot analysis and probed with a primary rabbit antibody to human EOMES; representative bands are shown. EOMES band intensity was plotted using ImageJ software minus background for n = 3 with mean ± SEM. Group 1, EOMES WT; Group 2, EOMES-Mut2; Group 3, LSD1 WT; Group 4, LSD1 NLS mutant. (E) EOMES plasmids and Jurkat T cells transfected with either VO, EOMES-WT, EOMES-Mut1, or EOMES-Mut2 plasmids and probed with antibodies targeting Ki67, IFN-γ, and TNF-α. Graphs show the percentage of cells calculated from ≥ 500 cells in 3 experiments ( n = 3) and the mean TFI of markers ( n ≥ 20 cells). Representative images for each dataset are shown; scale bar indicates 10 μM. Mean TFI and the population % are shown. Mann-Whitney test, **** p < 0.0001, *** p = 0.0002, ** p = 0.0021, ns >0.05. (F) EOMES protein structure showing the NLS and DNA-binding domain and the custom antibodies raised against specific PTMs. (G) Primary melanoma baseline biopsies classified as responder or resistant and processed for 3D high-resolution digital pathology with primary antibodies to CD8, EOMES-Ac, or EOMES-Me2 with DAPI. Representative images for each dataset are shown ( n ≥ 90 cells/sample). The % population of each target is plotted along with scale bar = 15 mm. n = 20 patient samples total; n = 10 responder patient samples, n = 10 resistant patient samples. (H) CD8 + T cells were isolated from healthy donors (HD) and immunotherapy resistant or responsive melanoma patients and screened by IF microscopy for (H) EOMES-641k-Ac or (I) EOMES-641k-Me2 or (J) EOMES-373k-Me2 and LSD1. For each LSD1:EOMES pair, the PCC was determined ( n = 10 patients, ≥20 cells/patient). Both the nuclear and cytoplasmic fluorescence intensity of each marker are plotted with significant differences indicated as well as the Fn/c; Mann-Whitney test, **** p < 0.0001, ** p = 0.0021, ns>0.05. IF microscopy patient cohorts included n = 5 healthy donors, n = 15 patients in the responder cohort, and n = 15 patients in the resistant cohort, with sampling as in with 4 samples per patient. (I) CD8 + T cells were isolated from healthy donors (HD), immunotherapy resistant or responsive melanoma patients and screened by IF microscopy for EOMES-641k-Me2 and LSD1. For each LSD1:EOMES-641k-Me2 pair, the PCC was determined ( n = 10 patients per group, ≥20 cells/patient). Both the nuclear and cytoplasmic fluorescence intensities of each marker are plotted with significant differences indicated as well as the Fn/c; Mann-Whitney test, **** p < 0.0001, ** p = 0.0021, * p < 0.05, ns>0.05. IF microscopy patient cohorts included n = 5 healthy donors, n = 15 patients in the responder cohort, and n = 15 patients in the resistant cohort, with sampling as in with 4 samples per patient. (J) CD8 + T cells were isolated from healthy donors (HD) and immunotherapy resistant or responsive melanoma patients and screened by IF microscopy for EOMES-373k-Me2 and LSD1. For each LSD1:EOMES-373k-Me2 pair, the PCC was determined ( n = 10 patients per group, ≥20 cells/patient). Both the nuclear and cytoplasmic fluorescence intensities of each marker are plotted with significant differences indicated as well as the Fn/c; Mann-Whitney test, **** p < 0.0001, ** p = 0.0021, * p < 0.05, ns>0.05. IF microscopy patient cohorts included n = 5 healthy donors, n = 15 patients in the responder cohort, and n = 15 patients in the resistant cohort, with sampling as in with 4 samples per patient. (K) IF microscopy was performed on 4T1 syngeneic metastatic cancer model-derived CD8 + T cells fixed and probed with antibodies targeting EOMES-641k-Ac and CD8 or EOMES-641k-Me2 and CD8 with DAPI. >10,000 cells/group were scanned to profile the % positive population of infiltrating CD8 + T cells in the primary tumor microenvironment for each EOMES marker ( n = 5 mice per group). Significant differences between groups are indicated as per the Mann-Whitney test, **** p < 0.0001, ** p = 0.0021, ns>0.05.

Article Snippet: Liquid biopsies were pre-enriched using the RosetteSepTM method to isolate CD8 + T cells by employing the RosetteSepTM Human CD8 enrichment Kit (15063, Stemcell Technologies, Vancouver, Canada) to remove CD45 + cells and red blood cells using density gradient centrifugation with SepMateTM-15 (IVD) density gradient tubes (85420, Stemcell Technologies) and LymphoprepTM density gradient medium (07861, Stemcell Technologies).

Techniques: Methylation, Transfection, Mutagenesis, MANN-WHITNEY, Control, Western Blot, Software, Binding Assay, Isolation, Microscopy, Fluorescence, Marker, Sampling, Derivative Assay

Journal: Cancers

Article Title: Selective Targeting of Protein Kinase C (PKC)-θ Nuclear Translocation Reduces Mesenchymal Gene Signatures and Reinvigorates Dysfunctional CD8 + T Cells in Immunotherapy-Resistant and Metastatic Cancers

doi: 10.3390/cancers14061596

Figure Lengend Snippet: nPKC-θ signatures are enriched in CTCs and metastatic tissues and are associated with poor patient survival in immunotherapy-resistant disease. ( A ) Immunohistofluorescence analysis of PKC-θ expression in CD4 + and CD8 + T cells isolated from healthy donor liquid biopsies. Bar/dot plots show the Fn/c (nuclear to cytoplasmic ratio) of PKC-θ phosphorylated at threonine 568 (PKC-θ-Thr568p). A score below 1 indicates cytoplasmic bias. Data are from three separate patients, n ≥ 20 cells per patient. Representative images are shown for each dataset. PKC-θ-Thr568p (red); CD8/CD4 (purple), and DAPI (cyan) were used to visualize expression and nuclei; scale bar represents 10 µM. ( B ) Immunohistofluorescence analysis of PKC-θ expression in human MCF-7 inducible model (MCF-7-IM) and MDA-MB-231 breast cancer cells. Human MCF-7 epithelial cells (MCF-7epi) were activated with PMA to induce EMT and generate MCF-7 mesenchymal-like (MCF-7mes) breast cancer cells. Bar graphs show the mean nuclear fluorescence intensity (NFI), cytoplasmic fluorescence intensity (CFI), and Fn/c for PKC-θ-Thr568p, n ≥ 20 cells per group. Representative images are shown for each dataset. PKC-θ-Thr568p (green) and DAPI (blue) were used to visualize nuclei. Scale bar represents 10 µM. ( C ) Contrast-enhanced CT scans of tumors in CR and PD metastatic melanoma patients at baseline and 12 weeks after treatment with immunotherapy (nivolumab). Red circles indicate tumor lesions. Tumor lesions are reduced in CR compared with baseline, while PD shows increased tumor burden. ( D ) Dot plot quantification of PKC-θ-Thr568p, CSV, and ABCB5 fluorescence intensity in circulating tumor cells (CTCs) isolated from immunotherapy-responsive (CR, partial response (PR)) or resistant (stable disease (SD), PD) melanoma patients defined using RECIST 1.1 criteria. The Fn/c for PKC-θ-Thr568p, mean CFI for CSV, and mean TFI for ABCB5 were quantified using ASI digital pathology. Representative images are shown for each cohort (six patients were profiled per cohort, n ≥ 20 cells per group); scale bar represents 10 µM. ( E ) Dot plot quantification of PKC-θ-Thr568p, CSV, and ABCB5 fluorescence in FFPE sections of primary melanomas from patients ( n = 18 patients) with CR, SD, or PD. The Fn/c for PKC-θ-Thr568p, mean CFI for CSV, and mean TFI for ABCB5 were quantified by ASI digital pathology ( n ≥ 40 cells per patient sample, four samples per patient). Representative images for each dataset are shown, scale bar represents 30 µM. ( F ) Dot plot quantification of PKC-θ-Thr568p, CSV, and ABCB5 fluorescence intensity in FFPE sections from breast cancer brain metastases ( n = 30 patients) and primary breast cancer biopsies ( n = 15 patients). The Fn/c for PKC-θ-Thr568p, mean CFI for CSV, and mean TFI for ABCB5 were quantified by ASI digital pathology ( n > 40 cells per patient sample). Representative images for each dataset are shown (top); scale bar represents 30 µM. ( G ) Percent change in tumor lesion from baseline for a single patient with metastatic melanoma (Patient D) who was resistant to first-line treatment with pembrolizumab and displayed PD (baseline, 12 weeks) as defined by RECIST 1.1 criteria but subsequently responded to second-line nivolumab and ipilimumab to show a CR (24 weeks, 36 weeks). ( H ) CT scan showing overall tumor burden in Patient D at baseline, SD, and PR (partial response). ( I ) Mesenchymal protein expression (PKC-θ-Thr568p, CSV, and ABCB5) was profiled in CTCs isolated from Patient D at 0 (baseline) and 12-, 24-, and 36-weeks post-immunotherapy. The Fn/c for PKC-θ-Thr568p, mean CFI for CSV, and mean TFI for ABCB5 were determined by ASI Digital Pathology. Representative images for each dataset are shown ( n ≥ 20 cells per group); scale bar represents 10 µM. ( J ) Metastatic melanoma patients ( n = 18 patients) were scored for the Fn/c of PKC-θ from four liquid biopsies over 12 months, with Fn/c categorized as <3 or ≥3 (Fn/c >1 indicates nuclear bias, whereas <1 indicates cytoplasmic bias). These patients were tracked for an additional two years (total 36 months), and their survival data are plotted as Fn/c <3 or ≥3. Statistical significance is denoted by ns (not significant), * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.0005, and **** p ≤ 0.0001.

Article Snippet: CD8 + T cells and CTCs were isolated from blood using the RosetteSepTM human CD8 + enrichment (15063, Stemcell Technologies, Vancouver, BC, Canada) or CD45 + depletion cocktail, LymphoprepTM, and SepMateTM tubes (Stemcell Technologies) according to the manufacturer’s protocol.

Techniques: Immunohistofluorescence, Expressing, Isolation, Fluorescence, Computed Tomography

Journal: Cancers

Article Title: Selective Targeting of Protein Kinase C (PKC)-θ Nuclear Translocation Reduces Mesenchymal Gene Signatures and Reinvigorates Dysfunctional CD8 + T Cells in Immunotherapy-Resistant and Metastatic Cancers

doi: 10.3390/cancers14061596

Figure Lengend Snippet: PKC-θ is enriched in the nuclei of dysfunctional CD8+ T cells isolated from stage IV metastatic cancers. ( A ) Quantification of PKC-θ-Thr568p Fn/c in CD8+ T cells or CSV+ CTCs isolated from immunotherapy-responsive (complete response, CR; partial response PR) or resistant (PD, progressive disease) melanoma patients defined using RECIST 1.1 criteria. The Fn/c for PKC-θ-Thr568p was quantified by ASI digital pathology and ImageJ-Fiji ( n ≥ 20 cells per group). ( B ) Quantification of PKC-θ-Thr568p Fn/c in CD8+ T cells or CSV+ CTCs isolated from healthy donors (HD), stage IV metastatic breast cancer patients (Mets), or stage IV breast cancer patients with brain metastases (Brain Mets). The Fn/c for PKC-θ-Thr568p was quantified by ASI digital pathology and ImageJ-Fiji ( n ≥ 40 cells per group). ( C ) The amino acid sequence of ZEB1 indicating the top eight peptides for peptide phosphorylation by PKC-θ. The top peptide sequences are displayed in a table with the mean signal intensity (2 SD above the mean was considered a positive phosphorylation event). ( D ) Top peptides positive for phosphorylation and their overlap with the ZEB1 amino acid sequence as well as the structure of ZEB1, adapted from . ( E ) Duolink ® proximity ligation assay (PLA) for PKC-θ and ZEB1 in CD8+PD1+ T cells isolated from immunotherapy-resistant or responder melanoma patients. Representative images are shown for PKC-θ/ZEB1, scale bar represents 10 µM. Graphs represent the PLA signal intensity of the Duolink ® assay; data represent n ≥ 100 cells/sample. Graphs plot the percentage of PLA signal positive cells out of total cells for (A). Data represent n ≥ 100 cells/sample. ( F ) FFPE sections from primary breast cancers ( n = 6 patients, >500 cells counted per patient) or breast cancer brain metastases ( n = 20 patients, >500 cells counted per patient) were processed for high-resolution microscopy using the BondRX platform. FFPE sections were fixed and immunofluorescence microscopy performed probing with primary antibodies targeting CD8, PKC-θ (T53p), and ZEB1 with DAPI. Plots represent the % population of CD8+ T cells positive for PKC-θ and ZEB1 out of total CD8+ T cells. Example images are shown with 20 µM scale bar. ( G ) CD8+ cells were isolated from melanoma patient liquid biopsies (responder = complete response (CR) or resistant, where primary = primary resistance, secondary = secondary resistance, PD = progression of disease) and stimulated with phorbol 12-myristate 13-acetate (PMA) and calcium ionophore (CI) and pre-clinically screened with either vehicle control or nPKC-i2. Samples were then fixed and immunofluorescence microscopy performed with primary antibodies targeting ZEB1, PKC-θ, and CD8. Representative images for each dataset are shown in . Graph represents the mean TNFI for PKC-θ and ZEB1 measured using ImageJ to select the nucleus minus background ( n > 20 cells/sample). ( H ) Plot profiles for each cohort for ZEB1 and PKC-θ are also depicted (red = ZEB1, green = PKC-θ) with the Pearson correlation coefficient (PCC) used to quantify colocalization between fluorophore-tagged proteins indicated and plotted. −1 = inverse of colocalization; 0 = no colocalization; +1 = perfect colocalization. Statistical significance is denoted by ns (not significant), ** p ≤ 0.005 and **** p ≤ 0.0001.

Article Snippet: CD8 + T cells and CTCs were isolated from blood using the RosetteSepTM human CD8 + enrichment (15063, Stemcell Technologies, Vancouver, BC, Canada) or CD45 + depletion cocktail, LymphoprepTM, and SepMateTM tubes (Stemcell Technologies) according to the manufacturer’s protocol.

Techniques: Isolation, Sequencing, Phospho-proteomics, Proximity Ligation Assay, Microscopy, Immunofluorescence, Control

Journal: Cancers

Article Title: Selective Targeting of Protein Kinase C (PKC)-θ Nuclear Translocation Reduces Mesenchymal Gene Signatures and Reinvigorates Dysfunctional CD8 + T Cells in Immunotherapy-Resistant and Metastatic Cancers

doi: 10.3390/cancers14061596

Figure Lengend Snippet: nPKC-θi2 disrupts the nuclear ZEB1/PKC-θ complex and induces cytokine production in CD8+ T cells. ( A ) Graphs depicting the % inhibition or induction based on protein expression were also plotted for each protein target relative to untreated sample. ( B ) Percent of PKC-θ+/ZEB1+/CD8+ T cells in samples isolated from melanoma patients responsive (PR/CR) or primary/secondary resistant (PD) to immunotherapy. CD8+ T cells were treated with nPKC-θi2 before activation ex vivo with PMA/ionomycin. ( C ) Gene expression of key effector cytokines IL2, IFNG, and TNFA in PBMCs isolated from resistant and responder patients either treated with vehicle control or nPKC-θi2 before activation ex vivo with PMA/ionomycin. ( D ) Protein expression of TNF-α and IFN-γ in CD8+ T cells isolated from primary or secondary resistant or responder patient liquid biopsies and treated with nPKC-θi2. Graphs show the % CD8+ increase in expression of TNF-α or IFN-γ in CD8+ T cells stimulated with PMA/ionomycin in addition to treatment with nPKC-θi2. One-way ANOVA was used to compare groups, where ns (not significant), **** p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05.

Article Snippet: CD8 + T cells and CTCs were isolated from blood using the RosetteSepTM human CD8 + enrichment (15063, Stemcell Technologies, Vancouver, BC, Canada) or CD45 + depletion cocktail, LymphoprepTM, and SepMateTM tubes (Stemcell Technologies) according to the manufacturer’s protocol.

Techniques: Inhibition, Expressing, Isolation, Activation Assay, Ex Vivo, Gene Expression, Control