antibody cocktail to the three melanoma cell markers mart1, tyrosinase, and gp100  (Novus Biologicals)

Journal: bioRxiv

Article Title: Patient-derived xenografts and single-cell sequencing identifies three subtypes of tumor-reactive lymphocytes in uveal melanoma metastases

doi: 10.1101/2023.05.16.540908

Figure Lengend Snippet: a) IHC staining of HMB45, SOX10, S100, MELAN-A (MART1) and PRAME expression in one PDX (See also for additional PDX models), as well as Sanger sequencing to verify mutation of GNA11 at Q209L. Color reactions were with magenta. b) Tumor growth curves of established PDX models. c-d) TIL therapy experiments in PDX models. UM1 and UM22 were transplanted subcutaneously into NOG or hIL2-NOG mice. When tumors were palpable, 20 million autologous TILs were injected in the tail vein. Tumor growth was monitored by a caliper. Tumor sizes are average size ± SEM. e) Images of tumor spheroids created (scale bar represents 100uM). UM23 was unable to form spheres. f-g) Activation (41BB + ) and degranulation (CD107a + ) status for spheres and TIL co-cultures, (f) and (g) respectively, using n = 2 or 3 replicates.

Article Snippet: MART1 specific yTILs were identified as previously described and sorted using FACSAria III (BD Biosciences). yTILs were expanded using a standard REP (Rapid Expansion Protocol), constituting irradiated (401lGy) allogeneic feeder cells, CD3 antibody (clone: OKT3) (301lng/ml) (Miltenyi), medium (AIM-V, Invitrogen) supplemented with 10% HS and 60001lIU/ml IL2 and monitored for 14 days with media changes .

Techniques: Immunohistochemistry, Expressing, Sequencing, Mutagenesis, Injection, Activation Assay

Journal: bioRxiv

Article Title: Patient-derived xenografts and single-cell sequencing identifies three subtypes of tumor-reactive lymphocytes in uveal melanoma metastases

doi: 10.1101/2023.05.16.540908

Figure Lengend Snippet: a) Proportions of cells from each sample that match clonotypes found in experimentally identified 4-1BB + and MART1-reactive T cells and which are present in a given biopsy CD8 + T cell cluster. The difference between matching and non-matching cells in each cluster is shown on the right, highlighting subsets that are enriched among the reactive T cells. b) Biopsy CD8 T cells with clonotypes matching identified reactive T cells, highlighted in the UMAP representation. c) As in (b), but highlighting cells with clonotypes shared between biopsies and TILs. d) Proportions of biopsy CD8 + T cells in each cluster matching either clonotypes from TIL cultures or experimentally identified reactive T cells. Statistical differences in frequency were determined using binomial tests between the frequency of the latter in each cluster relative to the background frequency of all TIL clonotypes present in the same cluster. Frequencies were calculated as number of cells from a given category in a given cluster divided by the total number of cells from that category, where category refers to either TILs or 4-1BB /MART1-reactive cells. p -values were adjusted for multiple testing using Bonferroni correction. e) Distributions of cells matching the two different categories of experimentally identified reactive cells among biopsy CD8 + T cells clusters. f) Cells from scRNA-seq of TIL cultures that have clonotypes matching experimentally identified reactive cells. g) Shared clonotypes among all biopsy and TIL samples. h) GLIPH2 clusters of significantly similar and HLA-restricted clonotypes , mapped to known antigens in public databases using TCRMatch , based on CDR3β sequences. i) Biopsy CD8 + T cells with clonotypes in either high or low confidence GLIPH2 clusters that match MART1 motifs in public databases.

Article Snippet: MART1 specific yTILs were identified as previously described and sorted using FACSAria III (BD Biosciences). yTILs were expanded using a standard REP (Rapid Expansion Protocol), constituting irradiated (401lGy) allogeneic feeder cells, CD3 antibody (clone: OKT3) (301lng/ml) (Miltenyi), medium (AIM-V, Invitrogen) supplemented with 10% HS and 60001lIU/ml IL2 and monitored for 14 days with media changes .

Techniques:

Journal: bioRxiv

Article Title: Patient-derived xenografts and single-cell sequencing identifies three subtypes of tumor-reactive lymphocytes in uveal melanoma metastases

doi: 10.1101/2023.05.16.540908

Figure Lengend Snippet: a) Biopsy CD8 + T cells with clonotypes that match scRNA-sequenced TILs, as in but shown separately for each sample. b) TIL cells with clonotypes that match any corresponding biopsy. c) Proportions within each CD8 + T cell cluster that match either 4-1BB or MART1-selected TILs from UM1, UM9 and UM46, respectively. d) Overlap among TCRβ chains from the reactive cells sorted out from these three samples. e) Shared TCRβ chains among the subset of cells from biopsies and TIL cultures that also have a match to any TCRβ chain among experimentally identified reactive TILs. f-g) Matches to any CDR3β-antigen pair in public databases, as inferred by TRCMatch, for CD8 + T cells from biopsies and TILs. Samples from liver and subcutaneous metastases shown in (f) and (g), respectively. h) As in (f-g), for experimentally identified reactive TILs. i) Cells from biopsy CD8 + T cells that were included in any high-confidence GLIPH2 cluster.

Article Snippet: MART1 specific yTILs were identified as previously described and sorted using FACSAria III (BD Biosciences). yTILs were expanded using a standard REP (Rapid Expansion Protocol), constituting irradiated (401lGy) allogeneic feeder cells, CD3 antibody (clone: OKT3) (301lng/ml) (Miltenyi), medium (AIM-V, Invitrogen) supplemented with 10% HS and 60001lIU/ml IL2 and monitored for 14 days with media changes .

Techniques:

Journal: bioRxiv

Article Title: Patient-derived xenografts and single-cell sequencing identifies three subtypes of tumor-reactive lymphocytes in uveal melanoma metastases

doi: 10.1101/2023.05.16.540908

Figure Lengend Snippet: a) Bulk unsorted TILs or MART1 selected TILs were injected into hIL2-NOG mice carrying liver tumors from patient UM22. b) Flow Cytometry analysis of single cell suspension liver metastasis, comparing treatment of UM22 TILs and UM22 MART1-specific TILs for CD3 + , CD3 + CD8 + CD69 + or CD3 + CD8 + CD137 + . c) IHC with diaminobenzidine (DAB) showing tumor (SOX10) and TILs (CD3) within a liver metastasis, d-e) corresponding analysis of 4-1BB TILs in a section (d) and in image analysis comparing both treatments (e). Statistical tests in b and e were unpaired two-tailed t-tests, assuming equal variance. *: p < 0.05; **: p < 0.01. f) Samples of tumor and TILs from the liver and spleen, respectively, were sequenced with scRNA-seq. n = 3 biological replicates were performed for each group of liver samples, and n = 2 for spleen samples (out of which one spleen sample for MART1-selected TILs was the pooled material of two independent mice). Sequencing reads mapping to human and mouse were separated with XenoCell , after which cells were clustered and annotated as described in Methods . g) Subclustering of CD8 + T cells identified three overall clusters, one of represented a mixed profile of the other two, but with marked cell cycle activity. h) Contributions of the different experimental conditions to each CD8 + T cell cluster. i) Markers distinguishing CD8 + T cell clusters, identified using the FindAllMarkers function of Seurat (the union of the top 25 genes per condition, ranked by log 2 fold change). Expression per experimental condition is shown below. j) All TCRβ chains identified in each experimental condition. Subsets found in TIL culture scRNA-seq data are highlighted, as are any matches to antigens in public databases. k) Differentially expressed genes between bulk TIL mixtures or MART1-selected TILs present in the livers of mice. A pseudo-bulk approach was used, summing read counts across all cells within a given replicate, and statistical testing performed with DESeq2 . Genes with q < 0.05 after Benjamini-Hochberg correction were considered significant. l-m) Expression of KRT86, DUSP4 and LAYN in biopsy CD8 + T cells (l) and the phenotypic clusters they are members of (m).

Article Snippet: MART1 specific yTILs were identified as previously described and sorted using FACSAria III (BD Biosciences). yTILs were expanded using a standard REP (Rapid Expansion Protocol), constituting irradiated (401lGy) allogeneic feeder cells, CD3 antibody (clone: OKT3) (301lng/ml) (Miltenyi), medium (AIM-V, Invitrogen) supplemented with 10% HS and 60001lIU/ml IL2 and monitored for 14 days with media changes .

Techniques: Injection, Flow Cytometry, Suspension, Two Tailed Test, Sequencing, Activity Assay, Expressing

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