Journal: Cell metabolism

Article Title: Increased Tumor Glycolysis Characterizes Immune Resistance to Adoptive T cell Therapy

doi: 10.1016/j.cmet.2018.02.024

Figure Lengend Snippet: A. List of pathways that are significantly modulated by silencing PTEN in melanoma cells. The mRNA expression profiles of PTEN-silenced and control cell lines were established from a melanoma cell line (A375) by microarray analysis. The two PTEN-silenced cell lines expressed shRNA hairpins that target different positions of the PTEN sequence (PTEN-silenced Tu-17 and Tu-60), and the corresponding control cell line (Control Tu) expressed a non-targeting shRNA hairpin. IPA was used to determine the pathways that are directly regulated by PTEN expression. B. Bioenergetic profiles of melanoma cells with or without silenced PTEN are shown. The Seahorse XF cell Mito stress test was used to define the bioenergetic profiles of PTEN-silenced and control cells. The changes in ECAR and OCR for the time indicated during a XF24 extracellular flux analyzer run are plotted. Oligomycin (1 uM) and FCCP (0.3 uM) were sequentially injected to the assay medium. The dash lines indicate the injection time points. *P<0.05. FCCP: carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone. C. A modified dropout screen used to identify genes that promote resistance to T cell-mediated tumor killing is shown. Patient-derived melanoma cells were transduced with a pooled shRNA library, followed by puromycin selection to eliminate non-transduced cells. After a 2-week cell culture to remove the genes that directly modulate cell survival, the cells were selected by autologous TILs with an effector to target (E:T) ratio of 1:1 that caused approximately 30% killing efficiency. The shRNA-targeted genes that promote resistance to T cell-induced tumor killing were underrepresented in the samples with TIL treatment. D. The identified candidate immune regulators involved in enzymatic steps of glycolysis are shown. LogFC: the base 2 log of the fold change of TIL-treated groups vs. control; FDR: false discovery rate. ALDOA and ALDOC: aldolase A and C; ENO3: enolase 3, GPI: glucose-6-phosphate isomerase; PGAM2: phosphoglycerate mutase 2.

Article Snippet: Human A375 Melanoma Cells , ATCC , ATCC CRL-1619; RRID:CVCL_0132.

Techniques: Expressing, Control, Microarray, shRNA, Sequencing, Injection, Modification, Derivative Assay, Transduction, Selection, Cell Culture

Journal: Cell metabolism

Article Title: Increased Tumor Glycolysis Characterizes Immune Resistance to Adoptive T cell Therapy

doi: 10.1016/j.cmet.2018.02.024

Figure Lengend Snippet: KEY RESOURCE TABLE

Article Snippet: Human A375 Melanoma Cells , ATCC , ATCC CRL-1619; RRID:CVCL_0132.

Techniques: Derivative Assay, Recombinant, Plasmid Preparation, Software

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Dasatinib is a potent enhancer for CAR T cell generation by CD3-targeted lentiviral vectors

doi: 10.1016/j.omtm.2022.12.002

Figure Lengend Snippet: Dasatinib enhances CD3-targeted gene transfer into activated PBMCs in a time- and dose-dependent manner Freshly thawed primary human PBMCs were activated for 3 days with αCD3 and αCD28 antibodies in presence of IL-7/15 prior to transduction with CD3-LV. (A) Cells were incubated in 50 nM dasatinib-containing medium for 5 h with increasing particle numbers. Fold increase in transduction compared with transduction in absence of dasatinib is shown for four vector stocks. Each data point is an average value referring to a particular vector stock tested on one to four donors. ∗p < .05, by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. (B) Cells were incubated in 0.39–100 nM dasatinib-containing medium for 7 h in presence of vector particles. Dose-dependent increase in GFP-expression 3 days after transduction with 2.5 × 10 10 CD3-LV particles is shown for four to five donors. (C and D) Cells were incubated without (w/o), 25 nM, 50 nM, or 100 nM dasatinib-containing medium for 3 h, 5 h, or 7 h in presence of vector particles. (C) Representative dot plots of GFP-expression 3 days after transduction. (D) Superimposed symbols for different concentrations connected by line show GFP-expression 3 days after transduction. Data are based on three donors, and values are shown as mean ± standard error of the mean (SEM).

Article Snippet: For T cell activation prior to transduction, plates were coated with 1 μg/mL anti-human CD3 (αCD3; clone OKT3; Miltenyi Biotec), and freshly isolated or thawed PBMCs were cultivated with 3 μg/mL anti-human CD28 (αCD28; clone 15E8; Miltenyi Biotec)-containing medium for 3 days.

Techniques: Transduction, Incubation, Plasmid Preparation, Expressing

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Dasatinib is a potent enhancer for CAR T cell generation by CD3-targeted lentiviral vectors

doi: 10.1016/j.omtm.2022.12.002

Figure Lengend Snippet: Dasatinib prevents T cell activation causing increased availability of the CD3 receptor for CD3-LV binding and transduction (A) PBMCs were cultured in IL-7/15 supplemented medium overnight without further stimulation. The next day, cells were treated with activating antibodies αCD3/αCD28, CD3-LV, VSV-LV, CD8-LV or remained untreated (ut). Cells were incubated in 100 nM dasatinib-containing medium or medium without (w/o) dasatinib for 7 h during vector incubation. Bar diagrams show expression of the activation marker CD25 4 days after transduction. Mean ± SEM of three to four donors is shown, of which one was measured in technical triplicates. ∗p < .05, ∗∗p < .01, by paired t test. (B) Activated PBMCs were incubated in 100 nM dasatinib-containing medium or without dasatinib for 7 h during incubation with CD3-LVs. Timelines show mean fluorescence intensity (MFI) of anti-His staining, normalized to 7 h HuM291-LV, over 72 h after treatment with dasatinib, allowing detection of LVs bound to PBMCs. Filled symbols and lines show the binding of LVs to cells treated with dasatinib (+dasat.), and blank dots connected by dashed lines show binding of LVs to cells in absence of dasatinib. Mean ± SEM of three donors measured in technical duplicates. (C) MFI of the CD3 receptor 3 days after transduction with CD3-LV in presence or absence of dasatinib. Each data point is an average value referring to a particular vector stock tested on one to three donors. Values are shown as mean ± SEM. ∗p < .05, ∗∗p < .01, by repeated measure one-way ANOVA followed by Tukey’s multiple comparison test. (D) Activated PBMCs were incubated in 0–20 nM bafilomycin A1 (BflA1)-containing medium for 5 h during transduction. Fold change in transduction compared with transduction in absence of BflA is shown for three donors as mean ± SEM.

Article Snippet: For T cell activation prior to transduction, plates were coated with 1 μg/mL anti-human CD3 (αCD3; clone OKT3; Miltenyi Biotec), and freshly isolated or thawed PBMCs were cultivated with 3 μg/mL anti-human CD28 (αCD28; clone 15E8; Miltenyi Biotec)-containing medium for 3 days.

Techniques: Activation Assay, Binding Assay, Transduction, Cell Culture, Incubation, Plasmid Preparation, Expressing, Marker, Fluorescence, Staining, Comparison

Journal: Clinical and Translational Medicine

Article Title: LRH‐1/NR5A2 targets mitochondrial dynamics to reprogram type 1 diabetes macrophages and dendritic cells into an immune tolerance phenotype

doi: 10.1002/ctm2.70134

Figure Lengend Snippet: LRH‐1/NR5A2 agonism alters CD4 + and CD8 + T‐cell subpopulations in T1D individuals. PBMCs were purified from individuals with T1D and exposed to 10 µM BL001 every 24 h for a total duration of 48 h with a final dose given 30 min prior to analysis. Flow cytometry immunophenotyping of (A) CD4 + CD25 + and (B) Tregs; CD4 + CD25 + FoxP3 + cell subpopulations. The initial gating defining the CD4 population was always set at 10 000 cells. n = 8 independent individuals with T1D. Relative transcript levels of (C) FoxP3, (D) CTLA4 and (E) GATA3. Data were normalised to the housekeeping gene RSP9. n = 5 T1D independent donors. Flow cytometry immunophenotyping of (F) Th1; CD4 + CD196 − CD183 + CD194 − and (G) Th2; CD4 + CD196 − CD183 − CD194 + . The initial gating defining the CD4 population was always set at 10000 cells. n = 8 independent individuals with T1D. Relative (H) transcript and (I) secreted levels of IFNγ as well as of (J) IL‐4. Transcript levels of IFNγ were normalised to the housekeeping gene RSP9. n = 4–6 T1D independent donors. (K) Th17/22; CD4 + CD196 + CD183 − CD194 + immunophenotyping and (L) IL17 transcript levels normalised to the housekeeping gene RSP9. n = 6–8 T1D independent donors. (M) CD8 + immunophenotyping. n = 8 independent individuals with T1D. (O) CD4 + cells were isolated from PBMCs and treated with BL001 as described above. Cells were then analysed by flow cytometry for the cell surface markers CD25 + FoxP3 + . Relative (P) LRH‐1/NR5A2 and (Q) FoxP3 transcript levels in either siScrambled (siSc) or siNR5A2‐treated PBMCs. Data were normalised to the housekeeping gene RSP9. n = 3 T1D independent donors. CD14 − PBMCs were labelled with CFSE and stimulated/expanded using antiCD3/CD28 before the addition of CD4 + T‐cells (at a 1:2 ratio, respectively, from the same donor). Proliferation of (R) CD4 + /CFSE + and (S) CD8 + /CFSE + subpopulations was assessed by flow cytometry 4 days post co‐culturing. n = 6 T1D independent donors. Each donor is colour coded. Data are presented as means ± SEM. Paired Student t ‐test * p < 0.05 and ** p < 0.01 as compared with untreated cells.

Article Snippet: T cell activation was induced using a cocktail of CD3 and CD28 antibodies (T Cell TransAct; Miltenyi Biotech).

Techniques: Purification, Flow Cytometry, Isolation

Journal: The American journal of pathology

Article Title: The Neuropeptide Alpha-Melanocyte-Stimulating Hormone Is Critical for Corneal Endothelial Cell Protection and Graft Survival after Transplantation.

doi: 10.1016/j.ajpath.2021.10.016

Figure Lengend Snippet: Figure 1 Corneal endothelial cells (CEnC) express high levels of the receptor for a-MSH. A: Immunostaining for a-MSH receptor, melanocortin receptor 1 (MC1R), on murine (mCEnC) and human (hCEnC) CEnC lines and primary CEnCs. Blue: DAPI, purple: MC1R. B and C: Representative Western blot bands of MC1R in murine and human CEnC lines (B) and in primary murine corneal endothelium lysates (C). SIT lysate: small intestine tissue lysate used as positive control for mouse MC1R (mMC1R). A-375: human skin epithelial cell lysate used as positive control for human MC1R (hMC1R). Scale bars Z 50 mm.

Article Snippet: The mouse small intestine tissue (SIT) lysate (No. PK-AB718-1408; PromoCell, Heidelberg, Germany) and A375 human skin epithelial cell lysate (No. sc-3811; Santa Cruz Biotechnology, Dallas, TX) were used as positive controls for mouse and human MC1R, respectively.

Techniques: Immunostaining, Western Blot, Positive Control

Journal: Molecular Therapy Oncolytics

Article Title: Molecular characterization of type I IFN-induced cytotoxicity in bladder cancer cells reveals biomarkers of resistance

doi: 10.1016/j.omto.2021.11.006

Figure Lengend Snippet: IFNα does not alter proliferation in bladder cancer cell lines Proliferation was measured by EdU (a nucleoside analog of thymidine) incorporation following 24 h of exposure to palbociclib or rhIFNα2b. EdU incorporation was calculated by dividing the number of EdU-positive nuclei by the total number of nuclei. The graph depicts EdU incorporation in treated cells divided by EdU incorporation in untreated cells multiplied by 100 for each cell line. Results are presented as mean ± SD from three independent experiments performed in quadruplicate. EdU, 5-ethynyl-2′-deoxyuridine; h, hour; IFN, interferon; SD, standard deviation.

Article Snippet: The following other reagents were used in this study: rhIFNα2b (Stem Cell Technologies, 78,077), palbociclib (Cayman, 16,273), rhTRAIL (R&D 375-TL).

Techniques: Standard Deviation