naive cd8a t cell isolation kit  (R&D Systems)

Journal: bioRxiv

Article Title: A SREBF2-dependent gene program drives an immunotolerant dendritic cell population during cancer progression

doi: 10.1101/2023.04.26.538456

Figure Lengend Snippet: ( A to E ) T cell proliferation assays with CFSE-labeled OTI T cells co-cultured with antigen-pulsed DCs. ( A ) Co-culture of OTI CD8 + T cells with either CD63 - or CD63 + DCs isolated from the TDLN of mice bearing OVA-expressing BRAF V600E PTEN -/- melanomas (5:1 T cells: DC) (Left) Schematic of experiment (Right) Flow cytometry plots and quantification ( n = 4). ( B ) Co-culture of OTI CD8 + T cells with either CD63 - or CD63 + DCs pulsed with SIINFEKL peptide prior to co-culture. ( C ) Co-culture of OTI CD8 + T cells with OVA-pulsed DCs and increasing numbers of CD63 + DCs (Left) Schematic of experiment (Right) Flow cytometry plots and quantification ( n = 4). ( D ) Co-culture of OTI CD8 + T cells with SIINFEKL-pulsed DCs and increasing numbers of CD63 + DCs ( n = 5). ( E ) Co-culture of OTI CD8 + T cells with SIINFEKL-pulsed DCs (1 x 10 4 ) in the bottom chamber of a 0.4 μm transwell. Ratios of CD63 + :CD63 - DCs ranging from 1 x 10 4 :0 to 0:1 x 10 4 in upper chamber while maintaining the same total cell number across conditions. ( F ) Intracellular staining of IL-4 in CD4 + T cells following co-culture with CD63 + or CD63 - DCs (T cell:DC ratio, 5:1) ( n = 4). ( G ) MHCII-OVA 323-329 tetramer staining of CD4 + T cells following co-culture with CD63 + or CD63 - DCs isolated from OVA-expressing melanoma-bearing mice (T cell:DC ratio, 5:1) ( n = 5). ( H ) Flow cytometry plots (left) and quantification (right) of Foxp3-expressing Tregs after co-culture of CD63 - or CD63 + DCs (H-2 d ) with naïve CD4 + T cells isolated from Foxp3-GFP mice (H-2 b ) (T cell:DC ratio, 5:1) ( n = 10). ( I ) Flow cytometry plots (left) and quantification (right) demonstrating the effect of the IDO1 inhibitor, epacadostat, on CD63 + DC-induced Treg differentiation ( n = 5). All two-group comparisons were analyzed using unpaired t tests. ( C to E and I ) Statistical analysis was performed by two-way ANOVA followed by Sidak’s multiple comparisons test. All data presented as a mean ± SEM. All data is representative of 2-3 independent experiments. * P < 0.05, ** P < 0.005. *** P < 0.0005.

Article Snippet: Naïve CD8+ T cells were isolated from OTI mice by magnetic selection (R&D Systems, Cat# MAGM207) labeled with cell trace CFSE (ThermoFisher Scientific, Cat# C34554) and co-cultured with the sorted DCs at a 5:1 ratio for 72 hours.

Techniques: Labeling, Cell Culture, Co-Culture Assay, Isolation, Expressing, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: A SREBF2-dependent gene program drives an immunotolerant dendritic cell population during cancer progression

doi: 10.1101/2023.04.26.538456

Figure Lengend Snippet: ( A ) Flow cytometry analysis following CD63 + or CD63 - DC co-culture with FoxP3-GFP naïve CD4 + T cells pre-treated with Fatostatin or vehicle control. (Left) Flow cytometry dot plots (Right) Quantification of flow cytometry plots ( n = 3). ( B ) Flow cytometry CFSE proliferation assay following OVA-pulsed CD63 + or CD63 - DCs co-cultured with naïve OTI CD8 + T cells ± fatostatin, simvastatin, or GGTI-2147. (Left) Flow cytometry histograms. (Right) Quantification of flow cytometry plots ( n = 5). ( C ) (Left) Tumor growth curves of primary melanoma tissues in autochthonous BRAF V600E PTEN -/- transgenic melanoma mice treated with fatostatin (30mg/kg) or vehicle control. (Right) Growth curves showing individual replicates ( n = 9). ( D to E ) Flow cytometry quantification of immune cell populations within TDLN or tumor tissues following fatostatin versus vehicle control treatment of an autochthonous BRAF V600E PTEN -/- transgenic melanoma model. ( D ) Quantification of DC subtypes in the (Left) TDLN and (Right) Tumor. ( E ) Quantification of (Left) Foxp3 + Tregs and (Right) CD8 + T cells in the TDLN and Tumor. ( F ) Extracellular flux analysis measuring oxygen consumption rate (OCR) in CD11c-SREBF2 -/- control littermate SREBF2 fl/fl hosts. ( n = 5). ( G ) Flow cytometry quantification of FAOblue staining in BMDCs isolated from CD11c -Cre , Srebf2 -/- mice compared to littermates lacking Cre expression (SREBF2 fl/fl ). Data normalized to cDC1 FAOblue MFI ( n = 4). ( H ) Flow cytometry CFSE proliferation assay following OVA-pulsed BMDCs from CD11c-SREBF2 -/- mice and their SREBF2 fl/fl littermate controls co-cultured with naïve OTI CD8 + T cells. (Right) Quantification of flow cytometry plots ( n = 8). ( I ) (Top) Tumor growth curves of BRAF V600E PTEN -/- melanomas in CD11c-SREBF2 -/- mice and their SREBF2 fl/fl littermate controls. (Bottom) Growth curves showing individual replicates ( n = 12). ( J and K ) Flow cytometry quantification of tumor and TDLN infiltrating immune cell populations in CD11c-SREBF2 -/- mice and their SREBF2 fl/fl littermate controls. ( J ) Quantification of DC subtypes in the (Above) TDLN and (Below) Tumor. ( K ) Quantification of tumor-infiltrating CD4 + T cells, Foxp3 + CD4 + Tregs, KLRG1 + -activated Tregs and CD8 + T cells ( n = 11 for SREBF2 fl/fl controls, n = 9 for CD11c-SREBF2 fl/fl ). ( l ) CD11c-SREBF2 -/- mice and their SREBF2 fl/fl littermate controls were implanted with YUMM1.1g BRAF V600E PTEN -/- CDKN2A -/- melanoma cells and allowed to grow for 28 Days. Lungs were sectioned and H&E and S100β IHC was used to quantify micro-metastases. All two-group comparisons were analyzed using unpaired t tests. ( A and B ) Statistical analysis was performed by two-way ANOVA followed by Sidak’s multiple comparisons test. Data is shown as mean ± SEM with individual data points. All data is representative of 2-3 independent experiments.* P < 0.05, ** P < 0.005, *** P < 0.0005.

Article Snippet: Naïve CD8+ T cells were isolated from OTI mice by magnetic selection (R&D Systems, Cat# MAGM207) labeled with cell trace CFSE (ThermoFisher Scientific, Cat# C34554) and co-cultured with the sorted DCs at a 5:1 ratio for 72 hours.

Techniques: Flow Cytometry, Co-Culture Assay, Control, Proliferation Assay, Cell Culture, Transgenic Assay, Staining, Isolation, Expressing