Journal: Cell reports

Article Title: GOT1 regulates CD8 + effector and memory T cell generation

doi: 10.1016/j.celrep.2022.111987

Figure Lengend Snippet: (A) Flow cytometry comparison of CD62L expression (left) and real-time PCR analysis of Sell mRNA (right) between WT and GOT1 −/− CTLs. (B) WT and GOT1 −/− CTLs with different congenic markers were mixed at 1:1 ratio and co-adoptively transferred into WT recipients. Flow cytometry analysis of percentages of WT and GOT1 −/− CTLs before adoptive transfer (left) or from the spleen and lymph nodes 48-h after adoptive transfer (right). (C–G) GOT1 −/− CD8 + T cells show enhanced memory generation in an adoptive transfer model. (C) Experimental scheme. Naive WT and GOT1 −/− OT-I CD8 + T cells were adoptively transferred into WT recipients separately and infected with LMOVA. (D) Percentages of antigen specific Thy1.1 + cells of CD8 + T cells from spleen on day 7. (E) Percentages of KLRG1 + CD127 − and KLRG1 − CD127 + cells of Thy1.1 + T cells from spleen on day 7. (F) Percentages of IL-2 + cells of Thy1.1 + T cells post OVAI peptide stimulation ex vivo from spleen on day 7. (G) Percentages of antigen specific Thy1.1 + cells of CD8 + T cells from blood on day 70 (left) and from spleen 5 days post Vaccinia-OVA (VacOVA) rechallenge (right). (H–J) GOT1 −/− CD8 + T cells show enhanced memory generation in a co-adoptive transfer model. (H) Experimental scheme. WT and GOT1 −/− OT-I CD8 + T cells with different congenic markers were mixed at 1:1 ratio, co-transferred into WT recipients, and infected with LMOVA. (I) Flow cytometry analysis of percentages of WT and GOT1 −/− OT-I CD8 + T cells before transfer (left) and from blood at indicated time points after adoptive transfer (right). (J) Percentages of WT and GOT1 −/− OT-I CD8 + T cells from spleen and lymph nodes on day 60. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant. Unpaired t test (A), paired t test (B, J), Mann-Whitney t test (D, E, F, and G), two-way ANOVA with Sidak’s multiple comparisons test (I). Data are representative of three independent experiments (A–B, D–G, and I–J). Data are represented as mean ± SD.

Article Snippet: OVAI peptide (SIINFEKL) , AnaSpec , Cat# AS-62572-5.

Techniques: Flow Cytometry, Comparison, Expressing, Real-time Polymerase Chain Reaction, Adoptive Transfer Assay, Infection, Ex Vivo, MANN-WHITNEY

Journal: Cell reports

Article Title: GOT1 regulates CD8 + effector and memory T cell generation

doi: 10.1016/j.celrep.2022.111987

Figure Lengend Snippet:

Article Snippet: OVAI peptide (SIINFEKL) , AnaSpec , Cat# AS-62572-5.

Techniques: Blocking Assay, Virus, Recombinant, Cell Isolation, Cell Culture, Transgenic Assay, Software, Real-time Polymerase Chain Reaction

Journal: Genes & Development

Article Title: Dual functions of angiopoietin-like protein 2 signaling in tumor progression and anti-tumor immunity

doi: 10.1101/gad.329417.119

Figure Lengend Snippet: ANGPTL2 stimulates dendritic cell-mediated immune responses. ( A ) In vitro cross-presentation of OVA proteins by LPS-stimulated or LPS-unstimulated bone-marrow-derived dendritic cells (BMDCs) derived from WT or Angptl2 −/− mice using the CD8 + T-cell hybridoma B3Z. β-galactosidase activity in B3Z cells incubated with unstimulated BMDCs untreated with OVA proteins was set at 1. Data are means ± SD; n = 4 per group. (ns) Not significant ( P > 0.05), one-way ANOVA test followed by Tukey's multiple comparison test. ( B ) Comparison of levels of Il12p40 transcripts in LPS-stimulated or LPS-unstimulated BMDCs derived from WT or Angptl2 −/− mice. Levels in unstimulated WT BMDCs were set to 1. Data are means ± SD; n = 3 per group. (ns) Not significant ( P > 0.05), one-way ANOVA test followed by Tukey's multiple comparison test. ( C ) In vitro cross-presentation of OVA proteins by WT BMDCs stimulated with or without recombinant ANGPTL2 (rANGPTL2) protein using B3Z cells. β-galactosidase activity in B3Z cells incubated with unstimulated BMDCs untreated with OVA proteins was set to 1. Data are means ± SD; n = 4 per group. (ns) Not significant ( P > 0.05); (***) P < 0.001, one-way ANOVA test followed by Tukey's multiple comparison test. ( D ) Comparison of levels of Il12p40 transcripts in WT BMDCs treated with or without rANGPTL2. Levels in ANGPTL2-untreated BMDCs were set to 1. Data are means ± SD; n = 3 per group. (***) P < 0.001, unpaired t -test. ( E ) Frequency of CD11c + MHC class II (MHC II) hi cells in WT BMDCs treated with or without rANGPTL2, as assessed by FACS analysis. Frequency is calculated as a percentage of total CD11c + cells. Data are means ± SD; n = 3 per group. (***) P < 0.001, unpaired t -test. ( F ) Relative expression levels (median fluorescence intensity, MFI) of costimulatory molecules (CD86, CD80, and CD40) in WT BMDCs treated with or without rANGPTL2, as assessed by FACS analysis. Levels in ANGPTL2-untreated BMDCs were set to 1. Data are means ± SD; n = 3 per group. (***) P < 0.001, unpaired t -test. ( G ) Schematic illustrating the experimental design of dendritic cell immunization of WT male mice. rA2, recombinant ANGPTL2 protein; SINFEKL, H-2K b -restricted OVA class I epitope. ( H ) Comparison of tumor volumes in C57BL/6 male mice bearing B16-OVA cells transferred with untreated dendritic cells (DC) or ANGPTL2-treated (DC + rA2 + SIINFEKL) or untreated (DC + SIINFEKL) DCs pulsed with SIINFEKL. Data are means ± SEM; n = 6 tumors per group. (ns) Not significant ( P > 0.05); (**) P < 0.01, two-way ANOVA test comparing each group with the PBS group. ( I ) Kaplan–Meier tumor-free survival curves of mice bearing B16-OVA cells transferred with untreated dendritic cells (DC) or ANGPTL2-treated (DC + rA2 + SIINFEKL) or untreated (DC + SIINFEKL) DCs pulsed with SIINFEKL ( n = 5 mice for PBS and DC groups or n = 4 mice for DC + SIINFEKL and DC + rA2 + SIINFEKL groups). (ns) Not significant ( P > 0.05); (*) P < 0.05, log-rank test comparing each group with the PBS group.

Article Snippet: WT BMDCs were stimulated with 5 μg/mL recombinant mouse ANGPTL2 protein for 4 h and then pulsed with OVA peptide SIINFEKL (MBL) for 6 h more.

Techniques: In Vitro, Derivative Assay, Activity Assay, Incubation, Comparison, Recombinant, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Oxygen levels at the time of activation determine T cell persistence and immunotherapeutic efficacy

doi: 10.1101/2022.11.25.517976

Figure Lengend Snippet: (A) Heat map illustrating expression of markers of differentiation as median fluorescence intensity (MFI) determined by flow cytometry 72h after activation of naive CD8 + T cells in 21%, 5% or 1% O 2 . Viridis was used as colour scale and rows represent averaged z-scores; n=4-29. (B) Number of CD8 + T cells following activation in 21%, 5% or 1% O 2 shown in cells/mL and determined with automated cell counter. Exponential growth curve fit (represented by thick lines) was used to calculate doubling time in hours of T cells in each oxygen tension; n=4. (C) HIF-1α protein expression in nuclear extracts from CD8 + T cells activated in 21% (grey), 5% (light blue) or 1% (dark blue) O 2 . Representative immunoblots (left) and HIF-1α protein signal normalized to lamin B1 (right); n=1. (D) Representation of inhibition of negative regulators of HIFα with hypoxia and chemical inhibition of PHD with FG-4592. (E) HIF-1α protein expression in nuclear extracts from OT-I CD8 + T cells activated for 72h. Conditions analysed: wild-type (WT) cells activated in 21%, 5% or 1% O 2 ; WT cells activated in 21% O 2 and treated with 50 μM FG-4592. Representative immunoblot (top) and HIF-1α levels relative to 21% after normalization to HDAC or Histone 3 loading controls (bottom); n=3. (F) Analysis of cell number, proliferation, and expression of differentiation markers in the experimental con0ditions described in E. Cell number determined with automated cell counter and cell proliferation with CTV staining. Expression of differentiation markers was measured after stimulation with SIINFEKL and brefeldin for 3 hours and is shown as a percentage of live CD8 + cells. Histograms are representative flow cytometry plots for each parameter and are pre-gated on live, singlet, CD8 + events. The dotted line defines in violin plot graphs the median of 21%-grown cells and in histograms the gate for each marker; n=8-30. (G) In vitro cytotoxicity assay using OT-I CD8 + T cells shown in F. OT-I cells were co-cultured with B16–OVA tumour cells at different effector:target (E:T) ratios and cytotoxicity was assessed after 14-18 hours. (H) Cytotoxicity assay obtained according to G performed at 1%O 2 and shown in dose-response curves (plotted with 95% confidence intervals as shaded areas) determined with non-linear regression ([agonist] vs normalised response). Asterisk represents significantly lower EC 50 values as compared to 21%; n=4-7. (I) Correlation between log 2 fold change (FC) of cell counts on day 3 (obtained in F) and EC 50 values (obtained in H) relative to 21% O 2 . All results were pooled from at least two independent experiments and are shown as median ± interquartile range (IQR). Each data point represents an independent animal. * P<0.05, ** P<0.01, *** P<0.001; Holm-Šídák’s multiple comparisons test relative to 21% (E-H) or # P<0.05, ### P<0.001; paired T test (F). Full data and statistical analysis from can be found in Source data 1.

Article Snippet: Purified OT-I CD8 + T cells were activated with 0.1-1 μg/mL of the OVA-derived peptide SIINFEKL (ProImmune) or with anti-mouse CD3/CD28 dynabeads.

Techniques: Expressing, Fluorescence, Flow Cytometry, Activation Assay, Western Blot, Inhibition, Staining, Marker, In Vitro, Cytotoxicity Assay, Cell Culture

Journal: bioRxiv

Article Title: Oxygen levels at the time of activation determine T cell persistence and immunotherapeutic efficacy

doi: 10.1101/2022.11.25.517976

Figure Lengend Snippet: ( A ) Human CD8 + T cells were activated in 1 %O 2 for 1, 3 or 7 days. Cells continuously grown in 21% were used as control (CT). ( B ) Number of CD8 + T cells after 7 days of increasing time of exposure to 1%O 2 as shown in A. Cell counts assessed by flow cytometry using counting beads and presented as log 2 FC relative to 21% cultures; n=14-29. (C) Expression of differentiation markers shown as log2 FC in MFI relative to 21% CT (horizontal grey line) following conditioning to 1% O 2 as described in A; n=8-38. (D and E) Seahorse analysis of activated human CD8 + T cells exposed for 1 day to 1%O 2 as shown in A. ECAR determined after injection of glucose (Glc), Oligomycin (O) and 2-DG (D). OCR determined after injection of Oligomycin (O), FCCP (F) and rotenone+antimycin A (R+A) (E); n=12. (F) Signal of mitotracker DeepRed and TMRM in 7 day-cultured cells exposed to 1%O 2 for 1 day as shown in A. Left: log 2 FC relative to 21% controls; Right: histograms representative for cells activated in 21% O 2 or in 1% O 2 for 1 day (blue). (G) OT-I T cells were activated for 24 hours in 1%O 2 with SIINFEKL and expanded for 6 days in 21% O 2 before single-cell cytosolic Ca 2+ was measured by microscopy using the radiometric dye Fura-2AM (F 340nm /F 380nm ) in presence of 1 mM Ca 2+ at day 7. Single-cell time-lapse imaging was performed at 5 s intervals with baseline fluorescence measured for 4 min, followed by another 6 min reading after addition of SIINFEKL. Number of cells analysed (21% grey, n=249; 1%1day blue, n=128) are derived from multiple coverslips and 2 OT-I donor spleens; mean ± SEM. (H) TCR signalling. (I) Protein analysis 24h after activation of human CD8 + T cells with anti-CD3/CD28 dynabeads in 21% O 2 , 1% O 2 or 21% O 2 + 50μM FG-4592 (FG). Non-activated cells (N) were used as negative control. Histone 3, Lamin B, HDAC or total protein stain (TPS) were used as loading control for nuclear fraction proteins and vinculin was used as loading control for the cytoplasmic fraction. Left: representative immunoblots; right: log 2 FC in protein expression of cells activated in 1% (top) or with FG (bottom) relative to 21% controls; n=3-12. (J) Flow cytometry analysis of the proportion of cells positive for phospho-S6 Ribosomal Protein (Ser235/236, pS6) for the same experimental conditions described in H; n=10-15. (K) IL-2 ELISA performed on supernatant of cells activated for 1 day as described in I; n=6-12. (L) Flow cytometry analysis of Blimp1, phopho STAT5 (Y694, pSTAT5) and CD25 in cells activated in 1% O 2 for 24 hours. Left: MFI log 2 fold change relative to 21% O 2 . Right: representative histograms for naive (light grey) and cells activated in 21% (dark grey) and 1% O 2 (dark blue). All results were pooled from at least two independent experiments and are shown as median ± IQR (except G). Apart from D and E, each data point represents an independent donor; * P<0.05, ** P<0.01, *** P<0.001; Wilcoxon matched-pairs signed rank test (B-C and L), Šídák’s multiple comparisons test relative to 21% (D-E, J-K) and unpaired (G) and paired (F and I) T tests. Full data and statistical analysis from can be found in Source data 4.

Article Snippet: Purified OT-I CD8 + T cells were activated with 0.1-1 μg/mL of the OVA-derived peptide SIINFEKL (ProImmune) or with anti-mouse CD3/CD28 dynabeads.

Techniques: Flow Cytometry, Expressing, Injection, Cell Culture, Microscopy, Imaging, Fluorescence, Derivative Assay, Activation Assay, Negative Control, Staining, Western Blot, Enzyme-linked Immunosorbent Assay