pcdh ef1a effly siinfekl t2a mcherry  (Addgene inc)

Journal: bioRxiv

Article Title: Intratumoral amino acid insufficiency limits CD8 + T-cell effector function

doi: 10.1101/2025.04.28.651077

Figure Lengend Snippet: Gene set enrichment plot of KRIGE_AMINO_ACID_DEPRIVATION in CD8 + CD44 + PD-1 + T-cells from B16-F10 melanomas, E0771 breast tumors, or LCMV-Cl13 infected mice compared with CD8 + CD44 + T-cells from LCMV-Armstrong infected mice. b, Experimental design to measure the impact of chronic TCR stimulation and amino acid deprivation on T-cell protein synthesis. c, Protein synthesis rates in acutely or chronically stimulated CD8 + T-cells cultured in media containing or lacking amino acids for 24 hours as indicated. d, Experimental design to assess cytokine release and cytotoxic capacity of amino acid-deprived T-cells in vitro . e, IFN-γ and TNF-α secretion of chronically stimulated T-cells cultured in the indicated amino acid conditions for 24 hours following re-stimulation with PMA and ionomycin. f, In vitro killing of B16-Luc cells pulsed with indicated concentration of SIINFEKL by acutely or chronically stimulated T-cells cultured in 100% or 2% amino acids for 24 hours. g, Comparison of chronic TCR stimulation-induced amino acid consumption (shown on y-axis with positive values reflecting increased consumption by chronically vs. acutely stimulated T-cells) with environmental amino acid availability (shown on x-axis with positive values reflecting increased abundance in TIF vs. plasma). Amino acid consumption is measured by subtracting metabolite abundances in cell culture supernatant from a media only control and normalizing by cell number in each sample. h, Diagram depicting an optimized reporter to detect glutamine insufficiency in vivo (HTT -1 -mGreenLantern-IRES-mCherry). i, HTT -1 -mGL fluorescence intensity of adoptively transferred TCR-Vα2 + mCherry + OT-I T-cells in spleen, tdLN, and tumor from B16-OVA tumor-bearing mice. j, mGreenLantern fluorescence in intratumoral TCR-Vα2 + mCherry + OT-I T-cells, normalized to PD-1-low T-cells. P values were calculated by one-way ANOVA with Tukey’s multiple comparisons test ( c ), Dunnett’s multiple comparisons test ( e ), or two-tailed Student’s t-test within groups ( f,j ). Data are presented as the mean ± s.d. of n = 3 biologically independent samples or n = 5 or 6 ( i,j ) independent mice from a representative experiment.

Article Snippet: E0771-OVA cells were generated by introducing PresentER-SIINFEKL (mCherry), a gift from Dr. David Scheinberg (Addgene plasmid # 102945), into E0771 cells.

Techniques: Infection, Cell Culture, In Vitro, Concentration Assay, Comparison, Clinical Proteomics, Control, In Vivo, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Intratumoral amino acid insufficiency limits CD8 + T-cell effector function

doi: 10.1101/2025.04.28.651077

Figure Lengend Snippet: Schematic depicting the role of glutamine in supporting amino acid homeostasis and transport. b, Experimental design to test whether SLC6A15 overexpression in CD8 + T-cells improves anti-tumor immunity. c, Growth of subcutaneous E0771-OVA tumors following adoptive cell transfer (ACT) of OT-I T-cells expressing either vector control (Ctrl) or SLC6A15 (6A15). d, Percentages of PD-1 + Tim-3 + cells within CD44 + CD8 + T-cell population expressing either empty vector (Ctrl) or SLC6A15 (6A15) cultured in either 100% or 2% amino acid conditions in the presence of persistent TCR stimulation in vitro . e, OPP-AF647 MFI in Control or SLC6A15 -overexpressing, chronically stimulated T-cells cultured in 100% or 2.5% amino acids for 24h. f, Cytokine release by Control or SLC6A15 -overexpressing, chronically stimulated T-cells cultured in 100%, 2%, or 0% amino acids for 24 hours following re-stimulation with PMA and ionomycin. g, In vitro killing of B16-Luc cells pulsed with indicated concentrations of SIINFEKL by control or SLC6A15-overexpressing acutely or chronically stimulated T-cells cultured in 100% or 2% amino acids for 24 hours. P values were calculated by one-way ANOVA with Tukey’s multiple comparisons test ( d,e,f ), or multiple two-tailed Student’s t-test ( c,g ). Data are presented as the mean ± s.d. of n = 3 biologically independent samples or n = 7 independent mice from a representative experiment.

Article Snippet: E0771-OVA cells were generated by introducing PresentER-SIINFEKL (mCherry), a gift from Dr. David Scheinberg (Addgene plasmid # 102945), into E0771 cells.

Techniques: Over Expression, Expressing, Plasmid Preparation, Control, Cell Culture, In Vitro, Two Tailed Test