Journal: bioRxiv

Article Title: CRISPR activation and interference screens in primary human T cells decode cytokine regulation

doi: 10.1101/2021.05.11.443701

Figure Lengend Snippet: (A) Schematic of CRISPRa screens. (B) sgRNA log 2 fold changes for genes of interest in IL-2 (left) and IFN-γ (right) screens. Bars represent the mean log 2 fold change for each sgRNA across two donors. Density plots above represent the distribution of all sgRNAs. (C-D) Scatter plots of median sgRNA log 2 fold change (high/low sorting bins) for each gene, comparing screens in two donors, for IL-2 (C) and IFN-γ screens (D). (E) Comparison of gene log 2 fold change (median sgRNA, mean of two donors) in IL-2 and IFN-γ screens. Sub-panel Venn-diagram shows the number of screen unique and shared hits.

Article Snippet: Unless otherwise noted, thawed T cells were cultured in X-VIVO 15 (Lonza Bioscience cat 04-418Q) supplemented with 5% FCS, 55mM 2-mercaptoethanol, 4mM N-Acetyl L-Cysteine, and 500 IU/ml recombinant human IL-2 (Amerisource Bergen cat 10101641).

Techniques:

Journal: bioRxiv

Article Title: CRISPR activation and interference screens in primary human T cells decode cytokine regulation

doi: 10.1101/2021.05.11.443701

Figure Lengend Snippet: (A) Timeline of stimulation, golgi plug with Brefeldin A, and fixation/staining. (B) Representative flow cytometry plots showing CD4 staining compared to IL-2 and IFN-γ staining. (C) Gating strategy used for sorting screens with representative FACS plots. (D-E) Pairwise Pearson correlation matrices for Calabrese Set A (D) and Calabrese Set B (E) library samples. Correlations are calculated using sgRNA log 2 fold change from the original plasmid pool. (F-G) Volcano plots for IL-2 (F) and IFN-γ (G) screens. Points represent each gene’s median log 2 fold change followed by averaging two donors. Dashed lines represent cut-offs for hit calling, with positive and negative hits colored in red or blue, respectively.

Article Snippet: Unless otherwise noted, thawed T cells were cultured in X-VIVO 15 (Lonza Bioscience cat 04-418Q) supplemented with 5% FCS, 55mM 2-mercaptoethanol, 4mM N-Acetyl L-Cysteine, and 500 IU/ml recombinant human IL-2 (Amerisource Bergen cat 10101641).

Techniques: Staining, Flow Cytometry, Plasmid Preparation

Journal: bioRxiv

Article Title: CRISPR activation and interference screens in primary human T cells decode cytokine regulation

doi: 10.1101/2021.05.11.443701

Figure Lengend Snippet: (A) Comparison of gene log 2 fold change (median sgRNA) in IL-2 and IFN-γ CRISPRi screens. (B) Comparison IL-2 CRISPRi and CRISPRa screens with genes belonging to the T cell receptor signaling pathway (KEGG pathways) indicated in colors other than grey. Genes meeting hit criteria are labeled. CD3 surface complex genes are indicated by black border and bolded labels. Scales represent z-score of log 2 fold change, with positive regulators of IL-2 production having positive z-scores. (C) Unique and common positive and negative regulators identified across CRISPRa and CRISPRi screens. For CRISPRa, positive regulators are defined as having a positive log 2 fold change (high/low bins), and for CRISPRi a negative log 2 fold change (high/low). (D) Comparison IFN-γ CRISPRi and CRISPRa screens with manually selected NF-κB pathway regulators labeled. All other genes are shown in grey. (E) Map of NF-κB pathway regulators labeled in (D). (F) Map of screen hits with previous evidence of defined function in T cell stimulation and costimulation signal transduction pathways. Tiles represent proteins encoded by indicated genes, with the caveat that due to space constraints, subcellular localization is inaccurate, as many of the components shown in the cytoplasm occur at the plasma membrane. Tiles are colored according to log2 fold change z-score as shown in the sub-panel, with examples of different hits. Large arrows at the top represent stimulation/co-stimulation sources by Immunocult. (G) Map of top screen hits with previously undescribed or poorly described function in T cells in the same format as (F).

Article Snippet: Unless otherwise noted, thawed T cells were cultured in X-VIVO 15 (Lonza Bioscience cat 04-418Q) supplemented with 5% FCS, 55mM 2-mercaptoethanol, 4mM N-Acetyl L-Cysteine, and 500 IU/ml recombinant human IL-2 (Amerisource Bergen cat 10101641).

Techniques: Labeling, Cell Stimulation, Transduction

Journal: bioRxiv

Article Title: CRISPR activation and interference screens in primary human T cells decode cytokine regulation

doi: 10.1101/2021.05.11.443701

Figure Lengend Snippet: (A) Lentiviral constructs used for dCas9-KRAB and sgRNA delivery. (B) Representative histograms of indicated cell surface protein knockdown after delivery of sgRNAs targeting their transcriptional start site or a non-targeting control. (C-D) Pairwise Pearson correlation matrices for Dolcetto Set A (C) and Dolcetto Set B (D) library samples. Correlations are calculated using sgRNA log 2 fold change from the original plasmid pool. (E-F) Scatter plots of median sgRNA log2 fold change (high/low sorting bins) for each gene, comparing screens in two donors, for IL-2 (E) and IFN-γ screens (F). Positive regulator hits are colored in red and negative regulator hits in blue. (G) sgRNA log 2 fold changes for genes of interest in IL-2 (left) and IFN-gamma (right) screens. Bars represent the mean log 2 fold change for each sgRNA across two donors. Density plots above represent the distribution of all sgRNAs. (H) Venn-diagram shows the number of screen unique and shared hits.

Article Snippet: Unless otherwise noted, thawed T cells were cultured in X-VIVO 15 (Lonza Bioscience cat 04-418Q) supplemented with 5% FCS, 55mM 2-mercaptoethanol, 4mM N-Acetyl L-Cysteine, and 500 IU/ml recombinant human IL-2 (Amerisource Bergen cat 10101641).

Techniques: Construct, Plasmid Preparation

Journal: bioRxiv

Article Title: CRISPR activation and interference screens in primary human T cells decode cytokine regulation

doi: 10.1101/2021.05.11.443701

Figure Lengend Snippet: (A) Comparison IFN-γ CRISPRi and CRISPRa screens with genes belonging to the T cell receptor signaling pathway (KEGG pathways) indicated in colors other than grey. Genes meeting hit criteria are labeled. CD3 surface complex genes are indicated by black border and bolded labels. Scales represent z-score of log 2 fold change, with positive regulators of IFN-γ production having positive z-scores. (B) Significantly enriched KEGG pathways from CRISPRa/CRISPRi screen log 2 fold change gene ranks. Points are scaled according to −log 10 FDR adjusted P-value. (C) Discordant hits across CRISPRi and CRISPRa hits, where perturbations identified as positive regulators are colored and red, and negative regulators in blue. Discordant hits between IL-2 and IFN-γ screens include EOMES and CBFB , encoding transcription factors known to have key roles in the differentiation of T cell subsets. The proximal kinase responsible for CD3ζ phosphorylation, LCK, and its activating phosphatase, CD45 (encoded by PTPRC ), are discordant between CRISPRi and CRISPRa screens, suggesting appropriately balanced expression in this module is critical for optimal TCR signal transduction. (D) Comparison IL-2 CRISPRi and CRISPRa screens with genes of interest labeled.

Article Snippet: Unless otherwise noted, thawed T cells were cultured in X-VIVO 15 (Lonza Bioscience cat 04-418Q) supplemented with 5% FCS, 55mM 2-mercaptoethanol, 4mM N-Acetyl L-Cysteine, and 500 IU/ml recombinant human IL-2 (Amerisource Bergen cat 10101641).

Techniques: Labeling, Expressing, Transduction

Journal: bioRxiv

Article Title: CRISPR activation and interference screens in primary human T cells decode cytokine regulation

doi: 10.1101/2021.05.11.443701

Figure Lengend Snippet: (A) Schematic of arrayed experiments. (B) Comparison of IL-2 and IFN-γ CRISPRa screens, with genes targeted by the arrayed sgRNA panel indicated, as well as their screen hit categorization. Paralogs of arrayed panel genes that were also highly ranked hits are additionally indicated. (C) Intracellular cytokine staining flow cytometry for indicated cytokines in control (NO-TARGET_1 sgRNA) or VAV1 (VAV1_1 sgRNA) CRISPRa T cells after 10 hours of stimulation. (D) Intracellular cytokine staining of full arrayed sgRNA panel, showing percent of cells gated positive for indicated cytokines in CD4 + or CD8 + T cells. Points represent the mean value of 4 donors, with and without stimulation. Dashed vertical lines represent the mean no-target control sgRNA control value with stimulation. *q<0.05, **q<0.01, Mann-Whitney U-test followed by q-value multiple comparison correction. Full data in fig. S8B. Medium stimulation dose shown for IL-2 and IFN-γ, low dose shown for TNF-ɑ. (E) Intracellular cytokine staining arrayed panel binned by indicated gene categories, with sgRNAs targeting IL2 and IFNG removed. Points represent a single sgRNA and donor measurement. *p<0.05, **p<0.01, ***p<0.001, Mann-Whitney U-test. (F) Secreted cytokine staining arrayed panel grouped by indicated gene categories, with sgRNAs targeting IL2 and IFNG genes removed. Points represent a single gene and donor measurement. *p<0.05, **p<0.01, ***p<0.001, Mann-Whitney U-test. (G) Principal component analysis of secreted cytokine measurements from indicated sgRNAs. (H) Heatmap of selected secreted cytokine measurements grouped by indicated biological category. Values represent the median of 4 donors, followed by z-score scaling for each cytokine.

Article Snippet: Unless otherwise noted, thawed T cells were cultured in X-VIVO 15 (Lonza Bioscience cat 04-418Q) supplemented with 5% FCS, 55mM 2-mercaptoethanol, 4mM N-Acetyl L-Cysteine, and 500 IU/ml recombinant human IL-2 (Amerisource Bergen cat 10101641).

Techniques: Staining, Flow Cytometry, MANN-WHITNEY

Journal: bioRxiv

Article Title: CRISPR activation and interference screens in primary human T cells decode cytokine regulation

doi: 10.1101/2021.05.11.443701

Figure Lengend Snippet: (A) Schematic of gating strategy. Note, IFN-γ and TNF-ɑ are not shown, but follow the same strategy as IL-2. (B) Complete data of summary shown in . Points represent the percentage of cells gated as positive for expressing a given cytokine in the indicated donors, across multiple stimulation doses. Low, medium, and high doses represent 3.125, 6.25, and 12.5μl/ml of anti-CD2/CD28/CD2 Immunocult, respectively.

Article Snippet: Unless otherwise noted, thawed T cells were cultured in X-VIVO 15 (Lonza Bioscience cat 04-418Q) supplemented with 5% FCS, 55mM 2-mercaptoethanol, 4mM N-Acetyl L-Cysteine, and 500 IU/ml recombinant human IL-2 (Amerisource Bergen cat 10101641).

Techniques: Expressing

Journal: bioRxiv

Article Title: CRISPR activation and interference screens in primary human T cells decode cytokine regulation

doi: 10.1101/2021.05.11.443701

Figure Lengend Snippet: (A) Cytokine measurements in non-targeting control sgRNA samples after stimulation (2 individual sgRNAs). (B) Measurements of IL-2, IFN-γ, and TNF-ɑ in samples with indicated sgRNAs. (C) Unsupervised clustering of full cytokine panel measurements across different sgRNAs. Each tile represents the median value of 4 donors, z-score scaled across each cytokine. (D) Th1 and Th2 category grouped cytokine measurements across different sgRNAs. Th1 group includes of IFN-γ IL-2, TNF-ɑ, and TNF-β. Th2 group includes IL-4, IL-5, and IL-13. Each point represents a donor, cytokine measurement, z-score scaled for each cytokine.

Article Snippet: Unless otherwise noted, thawed T cells were cultured in X-VIVO 15 (Lonza Bioscience cat 04-418Q) supplemented with 5% FCS, 55mM 2-mercaptoethanol, 4mM N-Acetyl L-Cysteine, and 500 IU/ml recombinant human IL-2 (Amerisource Bergen cat 10101641).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Safe and effective treatment of spontaneous neoplasms with interleukin 12 electro-chemo-gene therapy

doi: 10.1111/jcmm.12382

Figure Lengend Snippet: Details of the enrolled subjects

Article Snippet: To produce the canine IL12 used in this study, canine IL12 subunits P35 and P40 DNA were synthesized based on Genebank sequences NM_001003293 and NM_001003292 (GenScript USA) and subcloned to control vector pVC1157 under CMV promoter , .

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Safe and effective treatment of spontaneous neoplasms with interleukin 12 electro-chemo-gene therapy

doi: 10.1111/jcmm.12382

Figure Lengend Snippet: ECGT with IL12 pDNA is equally effective with gemcitabine or bleomycin. Analysing all treatment cycles with IL12 pDNA and either bleomycin (IL12 + blm) or gemcitabine (IL12 + gem) in squamous cell carcinoma (A, SCC, n + 9, 10), plasmacytoma (B, PC, n + 2, 3) and sarcoma (C, Sarc, n + 5, 4) reveals that ECGT is equally effective with blm or gem. (D) Comparison of all tumour histotypes shows that sarcomas do not respond to ECGT while PC and SCC are very responsive. (E) ECGT treatments extends survival compared to non-surgical standard treatments. Survival curve comparing ECGT ( n + 16) and non-surgical standard treatments in subjects with complete records at the clinical trial host site. Error bars represent SEM.

Article Snippet: To produce the canine IL12 used in this study, canine IL12 subunits P35 and P40 DNA were synthesized based on Genebank sequences NM_001003293 and NM_001003292 (GenScript USA) and subcloned to control vector pVC1157 under CMV promoter , .

Techniques: Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: Safe and effective treatment of spontaneous neoplasms with interleukin 12 electro-chemo-gene therapy

doi: 10.1111/jcmm.12382

Figure Lengend Snippet: IL12 pDNA can confer an antitumour immune response. (A) In squamous cell carcinoma, ECGT with IL12 pDNA and a chemotherapeutic induces tumour regression equally compared to IL12 pDNA alone plus electroporation, but the tumour growth resumes by day 21 after treatment ( n + 5 for IL12 pDNA alone and n + 20 for ECGT). (B) In acanthomatous ameloblastoma, IL12 pDNA alone plus electroporation appears more effective than ECGT with IL12 pDNA and a chemotherapeutic, although the difference is not significant ( n + 4). (C) In Sarcomas, IL12 pDNA alone plus electroporation ( n + 2) appears to stabilize the tumour volume while ECGT with IL12 pDNA and a chemotherapeutic ( n + 9) cannot inhibit tumour growth; however, the low sample size of IL12 pDNA prohibits statistical analysis. (D) SCC tumours located anywhere except the nasal planum (non-NP SCC, n + 20) respond to ECGT with an average 30% tumour reduction in only 21 days, but nasal planum SCC tumours (NP SCC, n + 8) do not respond to ECGT. (E) A non-treated metastatic lymph node SCC tumour steadily regressed after two treatments in the initial dorsal carpus tumours on days 0 and 7. At 96 days after the treatments in the dorsal carpus tumours, progression of the metastatic nodule was noted. X denotes surgical removal of the metastatic tumour. Error bars represent SEM.

Article Snippet: To produce the canine IL12 used in this study, canine IL12 subunits P35 and P40 DNA were synthesized based on Genebank sequences NM_001003293 and NM_001003292 (GenScript USA) and subcloned to control vector pVC1157 under CMV promoter , .

Techniques: Electroporation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Safe and effective treatment of spontaneous neoplasms with interleukin 12 electro-chemo-gene therapy

doi: 10.1111/jcmm.12382

Figure Lengend Snippet: IL12 pDNA alone or with chemotherapy can equally affect large and small tumours. (A) EP-mediated EGT (IL12 pDNA alone) equally regresses small (<3 cm, n + 4) and large (>3 cm, n + 5) tumours for 14 days, but tumour progression returns by day 21. (B) ECGT (IL12 pDNA plus either bleomycin or gemcitabine) equally regresses small (<3 cm, n + 11) and large (>3 cm, n + 19) tumours. Error bars represent SEM.

Article Snippet: To produce the canine IL12 used in this study, canine IL12 subunits P35 and P40 DNA were synthesized based on Genebank sequences NM_001003293 and NM_001003292 (GenScript USA) and subcloned to control vector pVC1157 under CMV promoter , .

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Safe and effective treatment of spontaneous neoplasms with interleukin 12 electro-chemo-gene therapy

doi: 10.1111/jcmm.12382

Figure Lengend Snippet: ECGT with IL12 pDNA and either bleomycin or gemcitabine can eradicate or debulk large squamous cell carcinoma tumours. (A) Squamous cell carcinoma tumours on Day 0 prior to ECGT with IL12 pDNA and bleomycin. The larger tumour is outlined in blue and the samller tumour is outlined in red. (B) Eradicated tumours on Day 70. (C) Tumour volume curves showing the eradication as per cent of tumour volume on Day 0. The coloured lines corresponds to the tumours outlined in A. (D) Maxillary Squamous cell carcinoma tumours on Day 0 prior to ECGT with IL12 pDNA and gemcitabine. (E) Healthy maxillary tissue with no evidence of residual SCC tumour cells as determined by histopathological analysis. (F) Tumour volume curve showing the eradication as per cent of tumour volume on Day 0. CT scan (G) and photograph (H) of an invasive squamous cell carcinoma tumour on Day 0 prior to ECGT with IL12 pDNA and bleomycin. (I) Photograph illustrating quick debulking of SCC on day 7 after only 1 ECGT treatment. (J) Tumour volume curve showing tumour reduction as per cent of tumour volume on Day 0. (K) Tumour volume curve showing tumour reduction as per cent of tumour volume on Day 0 of a subject with an SCC tumour in the retromolar trigone after only 1 ECGT treatment with IL12 pDNA and bleomycin. (L) Average tumour volume reduction in SCC tumours with ECGT with IL12 pDNA and either bleomycin or gemcitabine. Error bars represent SEM ( n + 8).

Article Snippet: To produce the canine IL12 used in this study, canine IL12 subunits P35 and P40 DNA were synthesized based on Genebank sequences NM_001003293 and NM_001003292 (GenScript USA) and subcloned to control vector pVC1157 under CMV promoter , .

Techniques: Computed Tomography

Journal:

Article Title: RhoC is dispensable for embryogenesis and tumor initiation but essential for metastasis

doi: 10.1101/gad.1310805

Figure Lengend Snippet: RhoC is not essential in T- or B-cell development, activation, apoptosis, or migration. (A) Flow cytometric analyses of thymic, lymph node, and splenic total T- and B-lymphocyte populations in wild-type (upper panels) or RhoC-/- (lower panels) mice. Results representative of seven independent experiments are shown. (B) Proliferative responses of T and B lymphocytes to stimuli. Purified T and B cells from wild-type (Wt; black bars) and RhoC-/- (white bars) mice were stimulated for 48 h with anti-CD3 with or without costimulation by anti-CD28 and/or IL-2, or PMA plus ionomycine (Iono) for T cells; or anti-IgM, anti-CD40, anti-IgM plus anti-CD40, LPS, or CpG for B cells. [3H]-thymidine incorporation was then assessed. (C) Thymocyte sensitivity to apoptosis. Triplicate cultures of thymocytes from wild-type (black bars) or RhoC-/- (white bars) mice were treated with dexamethasone (DEX) to induce mitochondria-mediated apoptosis, or with anti-CD95 to induce death-receptor-mediated apoptosis. Cell viability was determined 24 h later by Annexin V FITC/PI staining. (UT) Untreated control. (D) Cell migration. T and B cells and thymocytes (∼106 cells) from wild-type (black bars) or RhoC-/- (white bars) mice were placed in the upper chamber of Transwell motility plates. Data shown are the mean percentages of duplicate sample of cells that migrated to the lower chamber after 24 h. For B, C and D, data are presented as the mean ± SD and are representative of three independent experiments.

Article Snippet: T cells were stimulated in triplicate with soluble anti-CD3 (5 μg/mL, Pharmingen) with or without anti-CD28 (2 μg/mL, Pharmingen), or IL-2 (100 U/mL, Pharmingen).

Techniques: Activation Assay, Migration, Purification, Staining