Journal: Nature Communications

Article Title: ATLAS-seq: a microfluidic single-cell TCR screen for antigen-reactive TCRs

doi: 10.1038/s41467-024-54675-3

Figure Lengend Snippet: A Stem-loop structure of IFNγ-specific aptamer beacon. The 5’ end of the aptamer is decorated with the Cy3 fluorophore. The 3’ end of the aptamer is decorated with the TAO quencher, iSP18 linker and cholesterol. B MART1 or NY-ESO-1 peptide-loaded aAPCs were added to 5 × 10 5 cells/well DMF5 Jurkat cells or control (CTRL) Jurkat cells decorated with the IFNγ-specific aptamer beacons (1nmol/10 6 cells) at 2:1 ratio in 96-well plate. Top: Representative images of control or DMF5 Jurkat cells treated with MART1 aAPCs on day 2. Bottom: Average cellular fluorescence intensities of the aptamer beacons were measured to detect IFNγ secretion due to MART1 antigen-specific DMF5 Jurkat T cell activation. mean ± s.d. of n = 3 independent biological replicates. Arb. Units: arbitrary units. C Control (CTRL) Jurkat and DMF5 Jurkat cells were decorated with IFNγ-specific aptamer beacons (1nmol/10 6 cells) and co-encapsulated in droplets with MART1 peptide-loaded aAPCs or empty aAPCs for 2 days at 37 °C, 5% CO 2 . Fluorescence intensities of aptamer beacons were measured by flow cytometry to analyze the IFNγ secretion from MART1 antigen-specific activated single Jurkat cells in droplets. Aptamer beacon-decorated control Jurkat cells: with empty aAPCs (green); with MART1 peptide-loaded aAPCs (orange); with aptamer cDNA (red). Aptamer beacon-decorated DMF5 Jurkat cells: with empty aAPCs (gray); with MART1 peptide-loaded aAPCs (dark blue); with aptamer cDNA (light blue). Aptamer cDNA: DNA oligo with a complementary sequence to the aptamer stem region. D ATLAS-seq workflow. A single aptamer-labeled T cell is co-encapsulated with antigen peptide-loaded aAPC beads within uniformly-sized water-in-oil microdroplets using a cross junction channel in a microfluidic chip. Droplets are incubated off-chip to accumulate IFNγ secreted from the encapsulated single T cell. Secreted IFNγ will switch on the Cy3 fluorescence signal of the aptamer beacon on the T cell surface. After breaking the emulsion, recovered T cells are analyzed and sorted by flow cytometry based on the intensity of Cy3 fluorescence signal. CD8 + /Cy3 high T cells are used for preparing both single-cell TCR-seq and single-cell RNA-seq libraries using 10X Genomics Chromium Controller. Source data are provided as a Source Data file.

Article Snippet: DMF5 expressing IFNG KO Jurkat cell generation: 2 × 10 7 TU/mL MART1-Specific TCR Lentivirus (Clone DMF5, BPS Bioscience, 78679-1) were used to transduce human IFNG knockout Jurkat cell line (abcam, ab273746) with TransDux MAX transduction reagent (System Biosciences, LV860A-1) following the suspension cell transduction protocol provided by the manufacturer ( https://www.systembio.com/wp/wp-content/uploads/2020/10/Manual_LV860A-1-1.pdf ).

Techniques: Control, Fluorescence, Activation Assay, Flow Cytometry, Sequencing, Labeling, Incubation, Emulsion, RNA Sequencing Assay

Journal: Frontiers in Immunology

Article Title: A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens

doi: 10.3389/fimmu.2024.1444886

Figure Lengend Snippet: In Vitro Validation of a Genetically Engineered T-Cell Activation Model. (A) Schematic representation of the genetically modified Jurkat T-cell line designed to report T-cell activation via NFAT-driven eGFP expression. These cells are equipped with a TCR specific for HLA-A*02-restricted MART1, CD8 co-receptor, PD-1 receptor, and a dCas9-KRAB-MeCP2 transcriptional repression system. When co-cultured with APCs/melanoma cells expressing MART1, eGFP expression indicates robust T-cell activation. In certain melanoma cell lines, robust expression of surface PD-L1 can be achieved by pre-treatment for 24 hours with IFN-γ. (B) Validation of the NFAT-driven eGFP reporter functionality in the T-cell model using flow cytometry. Negligible T-cell activation is observed in the absence of antigen-presenting or tumor cells (quiescent state), while treatment with PMA and ionomycin results in near-total activation. (C) Comparative analysis of T-cell activation following co-culture with different antigen-presenting cells (APCs) pulsed with MART1 peptides and melanoma cell lines. 2D3s were co-cultured at a 1:2 tumor cell/APC to 2D3 TCR/dCas9 ratio for 20-24 hours. The 2D3 TCR/dCas9 T-cell line exhibits moderate activation with peptide-pulsed U266-B1 cells and pronounced activation with peptide-pulsed T2 cells. Negative controls with non-peptide-pulsed APCs confirm assay specificity. As for melanoma cell lines, additional examination of checkpoint blockade with nivolumab and IFN-γ treatment effects on co-culture-mediated T-cell activation demonstrates distinct immune evasion mechanisms. All data shown represent the mean values of triplicate measurements, with error bars indicating the standard deviation.

Article Snippet: For PD-L1 and MART1 protein level evaluation, mouse monoclonal anti-PD-L1 antibody (CellSignaling, Cat. #29122T) and rabbit monoclonal anti-MART1 antibody (Cell Signaling, Cat. #64718) were used at dilutions of 1:1000.

Techniques: In Vitro, Biomarker Discovery, Activation Assay, Genetically Modified, Expressing, Cell Culture, Flow Cytometry, Co-Culture Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens

doi: 10.3389/fimmu.2024.1444886

Figure Lengend Snippet: Immune Modulation Landscape and Mechanistic Insights from Melanoma Cell Lines. (A) Western blot analysis demonstrating MART1 protein levels in SK-MEL-5 dCas9 and MALME-3M dCas9 melanoma cell lines with and without IFN-γ treatment, revealing higher MART1 expression in SK-MEL-5 cells. (B) Heatmap comparison of gene expression profiles, illustrating downregulation of MHC class I pathway genes in untreated SK-MEL-5 dCas9 cells versus MALME-3M dCas9 . Post IFN-γ treatment, a heatmap indicates upregulation of MHC class I-associated proteins in both cell lines. (C) Post IFN-γ treatment, MALME-3M dCas9 cells show an upregulation of the PD-L1 pathway, pointing to a PD-1/PD-L1-mediated reduction in T-cell activation. In contrast, SK-MEL-5 dCas9 cells exhibit negligible PD-L1 pathway upregulation, suggesting a different mechanism for immune modulation. The color intensity in the heatmap corresponds to the Z-score of normalized counts across rows.

Article Snippet: For PD-L1 and MART1 protein level evaluation, mouse monoclonal anti-PD-L1 antibody (CellSignaling, Cat. #29122T) and rabbit monoclonal anti-MART1 antibody (Cell Signaling, Cat. #64718) were used at dilutions of 1:1000.

Techniques: Western Blot, Expressing, Comparison, Gene Expression, Activation Assay

Journal: Frontiers in Immunology

Article Title: A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens

doi: 10.3389/fimmu.2024.1444886

Figure Lengend Snippet: Cytotoxic response of melanoma cell lines to bulk PBL T cell co-culture over 48 hours. Cytotoxic effects of MART1-primed and mock-primed bulk PBL T cells on two melanoma cell lines, MALME-3M dCas9/eGFP and SK-MEL-5 dCas9/eGFP . The plot shows the normalized eGFP fluorescence intensity, indicative of cell viability, across a 48-hour period.

Article Snippet: For PD-L1 and MART1 protein level evaluation, mouse monoclonal anti-PD-L1 antibody (CellSignaling, Cat. #29122T) and rabbit monoclonal anti-MART1 antibody (Cell Signaling, Cat. #64718) were used at dilutions of 1:1000.

Techniques: Co-Culture Assay, Fluorescence

Journal: Frontiers in Immunology

Article Title: A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens

doi: 10.3389/fimmu.2024.1444886

Figure Lengend Snippet: Assessment of dCas9-KRAB-MeCP2 Expression and Gene Targeting Efficacy. (A) Quantitative evaluation of gene knockdown efficiency in MALME-3M, SK-MEL-5, and 2D3 TCR/dCas9 cell lines. Knockdown achieved for all evaluated genes confirmed the precision of CRISPRi-mediated gene silencing. (B) Functional validation of the disrupted PD-1/PD-L1 pathway and MART1 in co-cultures of 2D3 TCR/dCas9 T-cells and MALME-3M melanoma cells. Disruption in either cell type restored T-cell activation, despite significant when IFN-γ was used to induce PD-L1 expression. MART1 knockdown further reduced T-cell activation. All data shown represent the mean values of triplicate measurements, with error bars indicating standard deviation. Statistical significance is denoted by asterisks: *p<0.05 (t-test with Benjamini-Hochberg multiple testing correction).

Article Snippet: For PD-L1 and MART1 protein level evaluation, mouse monoclonal anti-PD-L1 antibody (CellSignaling, Cat. #29122T) and rabbit monoclonal anti-MART1 antibody (Cell Signaling, Cat. #64718) were used at dilutions of 1:1000.

Techniques: Expressing, Knockdown, Functional Assay, Biomarker Discovery, Disruption, Activation Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens

doi: 10.3389/fimmu.2024.1444886

Figure Lengend Snippet: Impact of sgRNA-mediated Knockdown on T-cell Activation in MALME-3M Cells. (A) Knockdown efficiency of sgRNA-mediated gene silencing in MALME-3M dCas9 cells, as measured by quantitative PCR. The bar graph presents the relative normalized expression levels of five targeted genes: MART1, PD-L1, IFNGR2, STAT1, and MYC, in comparison to non-targeting controls (sgNCs). (B) Percentage of eGFP-positive 2D3 TCR/dCas T-cells, indicative of T-cell activation levels following sgRNA-mediated knockdown of MART1, PD-L1, IFNGR2, STAT1, and MYC genes in MALME-3M dCas target cells. Positive controls MART1 and PD-L1 knockdowns, conducted with single sgRNA guides, resulted in expected modulation of T-cell activation affirming the arrayed screen’s ability to discern positive and negative modulators of T-cell activation. For MYC, STAT1, and IFNGR2, two pairs of sgRNAs were used to ensure knockdown efficiency. The genetic perturbation of these genes was consistent with their known roles, promoting T-cell activation and thereby validating the sgRNA design and highlighting this method’s potential in investigating gene function in immune response regulation. We used four diverse negative control sgRNAs (sgNC) in the screening to ensure a robust baseline. All data shown represent the mean values of triplicate measurements, with error bars indicating standard deviation. Statistical significance is denoted by asterisks: *p<0.05 (t-test with Benjamini-Hochberg multiple testing correction), calculated based on all combined sgNC.

Article Snippet: For PD-L1 and MART1 protein level evaluation, mouse monoclonal anti-PD-L1 antibody (CellSignaling, Cat. #29122T) and rabbit monoclonal anti-MART1 antibody (Cell Signaling, Cat. #64718) were used at dilutions of 1:1000.

Techniques: Knockdown, Activation Assay, Real-time Polymerase Chain Reaction, Expressing, Comparison, Negative Control, Standard Deviation

Journal: Frontiers in Immunology

Article Title: A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens

doi: 10.3389/fimmu.2024.1444886

Figure Lengend Snippet: In Vitro Validation of a Genetically Engineered T-Cell Activation Model. (A) Schematic representation of the genetically modified Jurkat T-cell line designed to report T-cell activation via NFAT-driven eGFP expression. These cells are equipped with a TCR specific for HLA-A*02-restricted MART1, CD8 co-receptor, PD-1 receptor, and a dCas9-KRAB-MeCP2 transcriptional repression system. When co-cultured with APCs/melanoma cells expressing MART1, eGFP expression indicates robust T-cell activation. In certain melanoma cell lines, robust expression of surface PD-L1 can be achieved by pre-treatment for 24 hours with IFN-γ. (B) Validation of the NFAT-driven eGFP reporter functionality in the T-cell model using flow cytometry. Negligible T-cell activation is observed in the absence of antigen-presenting or tumor cells (quiescent state), while treatment with PMA and ionomycin results in near-total activation. (C) Comparative analysis of T-cell activation following co-culture with different antigen-presenting cells (APCs) pulsed with MART1 peptides and melanoma cell lines. 2D3s were co-cultured at a 1:2 tumor cell/APC to 2D3 TCR/dCas9 ratio for 20-24 hours. The 2D3 TCR/dCas9 T-cell line exhibits moderate activation with peptide-pulsed U266-B1 cells and pronounced activation with peptide-pulsed T2 cells. Negative controls with non-peptide-pulsed APCs confirm assay specificity. As for melanoma cell lines, additional examination of checkpoint blockade with nivolumab and IFN-γ treatment effects on co-culture-mediated T-cell activation demonstrates distinct immune evasion mechanisms. All data shown represent the mean values of triplicate measurements, with error bars indicating the standard deviation.

Article Snippet: For PD-L1 and MART1 protein level evaluation, mouse monoclonal anti-PD-L1 antibody (CellSignaling, Cat. #29122T) and rabbit monoclonal anti-MART1 antibody (Cell Signaling, Cat. #64718) were used at dilutions of 1:1000.

Techniques: In Vitro, Biomarker Discovery, Activation Assay, Genetically Modified, Expressing, Cell Culture, Flow Cytometry, Co-Culture Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens

doi: 10.3389/fimmu.2024.1444886

Figure Lengend Snippet: Immune Modulation Landscape and Mechanistic Insights from Melanoma Cell Lines. (A) Western blot analysis demonstrating MART1 protein levels in SK-MEL-5 dCas9 and MALME-3M dCas9 melanoma cell lines with and without IFN-γ treatment, revealing higher MART1 expression in SK-MEL-5 cells. (B) Heatmap comparison of gene expression profiles, illustrating downregulation of MHC class I pathway genes in untreated SK-MEL-5 dCas9 cells versus MALME-3M dCas9 . Post IFN-γ treatment, a heatmap indicates upregulation of MHC class I-associated proteins in both cell lines. (C) Post IFN-γ treatment, MALME-3M dCas9 cells show an upregulation of the PD-L1 pathway, pointing to a PD-1/PD-L1-mediated reduction in T-cell activation. In contrast, SK-MEL-5 dCas9 cells exhibit negligible PD-L1 pathway upregulation, suggesting a different mechanism for immune modulation. The color intensity in the heatmap corresponds to the Z-score of normalized counts across rows.

Article Snippet: For PD-L1 and MART1 protein level evaluation, mouse monoclonal anti-PD-L1 antibody (CellSignaling, Cat. #29122T) and rabbit monoclonal anti-MART1 antibody (Cell Signaling, Cat. #64718) were used at dilutions of 1:1000.

Techniques: Western Blot, Expressing, Comparison, Gene Expression, Activation Assay

Journal: Frontiers in Immunology

Article Title: A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens

doi: 10.3389/fimmu.2024.1444886

Figure Lengend Snippet: Cytotoxic response of melanoma cell lines to bulk PBL T cell co-culture over 48 hours. Cytotoxic effects of MART1-primed and mock-primed bulk PBL T cells on two melanoma cell lines, MALME-3M dCas9/eGFP and SK-MEL-5 dCas9/eGFP . The plot shows the normalized eGFP fluorescence intensity, indicative of cell viability, across a 48-hour period.

Article Snippet: For PD-L1 and MART1 protein level evaluation, mouse monoclonal anti-PD-L1 antibody (CellSignaling, Cat. #29122T) and rabbit monoclonal anti-MART1 antibody (Cell Signaling, Cat. #64718) were used at dilutions of 1:1000.

Techniques: Co-Culture Assay, Fluorescence

Journal: Frontiers in Immunology

Article Title: A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens

doi: 10.3389/fimmu.2024.1444886

Figure Lengend Snippet: Assessment of dCas9-KRAB-MeCP2 Expression and Gene Targeting Efficacy. (A) Quantitative evaluation of gene knockdown efficiency in MALME-3M, SK-MEL-5, and 2D3 TCR/dCas9 cell lines. Knockdown achieved for all evaluated genes confirmed the precision of CRISPRi-mediated gene silencing. (B) Functional validation of the disrupted PD-1/PD-L1 pathway and MART1 in co-cultures of 2D3 TCR/dCas9 T-cells and MALME-3M melanoma cells. Disruption in either cell type restored T-cell activation, despite significant when IFN-γ was used to induce PD-L1 expression. MART1 knockdown further reduced T-cell activation. All data shown represent the mean values of triplicate measurements, with error bars indicating standard deviation. Statistical significance is denoted by asterisks: *p<0.05 (t-test with Benjamini-Hochberg multiple testing correction).

Article Snippet: For PD-L1 and MART1 protein level evaluation, mouse monoclonal anti-PD-L1 antibody (CellSignaling, Cat. #29122T) and rabbit monoclonal anti-MART1 antibody (Cell Signaling, Cat. #64718) were used at dilutions of 1:1000.

Techniques: Expressing, Knockdown, Functional Assay, Biomarker Discovery, Disruption, Activation Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens

doi: 10.3389/fimmu.2024.1444886

Figure Lengend Snippet: Impact of sgRNA-mediated Knockdown on T-cell Activation in MALME-3M Cells. (A) Knockdown efficiency of sgRNA-mediated gene silencing in MALME-3M dCas9 cells, as measured by quantitative PCR. The bar graph presents the relative normalized expression levels of five targeted genes: MART1, PD-L1, IFNGR2, STAT1, and MYC, in comparison to non-targeting controls (sgNCs). (B) Percentage of eGFP-positive 2D3 TCR/dCas T-cells, indicative of T-cell activation levels following sgRNA-mediated knockdown of MART1, PD-L1, IFNGR2, STAT1, and MYC genes in MALME-3M dCas target cells. Positive controls MART1 and PD-L1 knockdowns, conducted with single sgRNA guides, resulted in expected modulation of T-cell activation affirming the arrayed screen’s ability to discern positive and negative modulators of T-cell activation. For MYC, STAT1, and IFNGR2, two pairs of sgRNAs were used to ensure knockdown efficiency. The genetic perturbation of these genes was consistent with their known roles, promoting T-cell activation and thereby validating the sgRNA design and highlighting this method’s potential in investigating gene function in immune response regulation. We used four diverse negative control sgRNAs (sgNC) in the screening to ensure a robust baseline. All data shown represent the mean values of triplicate measurements, with error bars indicating standard deviation. Statistical significance is denoted by asterisks: *p<0.05 (t-test with Benjamini-Hochberg multiple testing correction), calculated based on all combined sgNC.

Article Snippet: For PD-L1 and MART1 protein level evaluation, mouse monoclonal anti-PD-L1 antibody (CellSignaling, Cat. #29122T) and rabbit monoclonal anti-MART1 antibody (Cell Signaling, Cat. #64718) were used at dilutions of 1:1000.

Techniques: Knockdown, Activation Assay, Real-time Polymerase Chain Reaction, Expressing, Comparison, Negative Control, Standard Deviation