Journal: Frontiers in Immunology

Article Title: Targeting Cbx3 /HP1γ Induces LEF-1 and IL-21R to Promote Tumor-Infiltrating CD8 T-Cell Persistence

doi: 10.3389/fimmu.2021.738958

Figure Lengend Snippet: Cbx3 /HP1γ deficiency modulates LEF-1 and IL-21R expression in CD8 + effector T cells. (A) Mouse Lef1 locus. -2 and 12: positions of first and last primer pairs, respectively. (B) Read-density tracks of Cbx3 /HP1γ peaks across Lef1 identified by ChIP-Seq using chromatin from wt day 5 activated/differentiated CD8 + T cells (pooled from 3 mice); y axis: number of reads per million mapped per 25-bp window; representative of 2 independent ChIP-Seq runs. (C) LEF-1 expression was evaluated by Western immunoblot using total protein lysates from activated/differentiated control and Cbx3 /HP1γ-deficient CD8 + T cells; 3/28+IL2: CD8 + T cells activated/differentiated for 5 days with plate-bound anti-CD3 and anti-CD28 plus IL-2; C: control (CD8α-Cre or wt); fl/+: Cbx3 fl/+ and fl/fl: Cbx3 fl/fl (CD8 + T-cell-restricted deletion of Cbx3/ HP1γ using the CD8α-Cre mouse); n = 3; representative of 3 experiments. (D) Mouse Il21r locus. -4 and 4: positions of first and last pair of primers, respectively. (E) Read-density tracks of Cbx3 /HP1γ peaks across Il21r were determined by ChIP-Seq as in (B) . (F) IL-21R expression levels on activated/differentiated CD8 + T cells by flow; numbers: percent cells; representative of 3 experiments; n = 3. (G) IL-21R mean fluorescence intensity (MFI) was evaluated from (F) . (H) Relative expression of Il21r in day 5 activated/differentiated CD8 + T cells was assessed by RT-qPCR and normalized to Gapdh ; Graphpad unpaired student t-test: **p ≤ 0.01; representative of 3 experiments. (I) Number of CD8 + IL-21R + T cells in cultures was calculated from (F) ; Graphpad unpaired student t-test: *p ≤ 0.05, **p ≤ 0.01.

Article Snippet: The following Western blot and ChIP-tested antibodies were purchased from Cell Signaling: tri-methyl-histone H3 (Lys9) (D4W1U) rabbit mAb #13969, phospho-Rpb1 CTD (Ser2) (E1Z3G) rabbit mAb #13499, phospho-Rpb1 CTD (Ser5) (D9N5I) rabbit mAb #13523, LEF-1 (C12A5) rabbit mAb #2230, cleaved caspase-3 (Asp175) antibody #9661 and phospho-HP1γ (Ser83) Ab #2600.

Techniques: Expressing, ChIP-sequencing, Western Blot, Control, Fluorescence, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Targeting Cbx3 /HP1γ Induces LEF-1 and IL-21R to Promote Tumor-Infiltrating CD8 T-Cell Persistence

doi: 10.3389/fimmu.2021.738958

Figure Lengend Snippet: Cbx3 /HP1γ regulates Lef1 and Il21r transcription initiation and chromatin remodeling. (A, B) Levels of Pol II S5 bound to Lef1 (A) and Il21r (B) were quantified by ChIP-qPCR using chromatin from day 5 activated/differentiated CD8 + T cells. TSS: transcription start site; numbers on X axis: positions of primers along the two loci; 150 bp products amplified by Lef1 or Il21r primers. (C, D) Levels of Pol II S2 bound to Lef1 (C) and Il21r (D) were quantified as in (A, B) . (E, F) H3K9me3 deposition on Lef1 (E) and Il21r (F) was quantified as in (A, B) . Graphpad unpaired student t-test: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; representative of 4 independent ChIPs using pooled chromatin from 3 mice.

Article Snippet: The following Western blot and ChIP-tested antibodies were purchased from Cell Signaling: tri-methyl-histone H3 (Lys9) (D4W1U) rabbit mAb #13969, phospho-Rpb1 CTD (Ser2) (E1Z3G) rabbit mAb #13499, phospho-Rpb1 CTD (Ser5) (D9N5I) rabbit mAb #13523, LEF-1 (C12A5) rabbit mAb #2230, cleaved caspase-3 (Asp175) antibody #9661 and phospho-HP1γ (Ser83) Ab #2600.

Techniques: ChIP-qPCR, Amplification

Journal: Frontiers in Immunology

Article Title: Targeting Cbx3 /HP1γ Induces LEF-1 and IL-21R to Promote Tumor-Infiltrating CD8 T-Cell Persistence

doi: 10.3389/fimmu.2021.738958

Figure Lengend Snippet: Cbx3 /HP1γ-deficient CD8 + effector T cells can persist and cause tumor rejection. (A) Ascites from control (CD8α-Cre or wt), Cbx3 Tg , Cbx3 fl/+ and Cbx3 fl/fl mice (X axis) were visualized on day 120 after IP injection of mouse ID8 ovarian tumor cells; Cbx3 Tg : Cbx3/ HP1γ T-cell-restricted expression driven by the human Cd2 promoter; Cbx3 fl/+ and Cbx3 fl/fl : CD8 + T-cell-restricted deletion of Cbx3/ HP1γ using the CD8α-Cre mouse. (B) Ovarian ascites of mice in (A) were measured; bars: group median; Graphpad unpaired student t-test: **p≤0.01, ***p≤0.001; each symbol = one mouse; n = 5-8; representative of 2 experiments. (C) Survival curves of ovarian tumor-bearing mice were determined; Graphpad log-rank (Mantel-Cox) test; n = 5-8. (D) B16 tumor cells were injected subcutaneously (sc) and growth was assessed starting on day 14 after tumor-cell injection then every 2 days through day 20; Graphpad two-way ANOVA: **p ≤ 0.01, ****p ≤ 0.0001; n = 5; representative of 3 experiments. (E) Survival curves of B16 melanoma tumor-bearing mice; Graphpad log-rank (Mantel-Cox) test; n = 5. (F) NB-9464 tumor cells were injected sc, NBL tumor volume was determined every 2 days starting on day 22 through day 28; Graphpad two-way ANOVA: ****p ≤ 0.0001; n = 5; representative of 2 experiments. (G) Survival curves of NBL tumor-bearing mice; Graphpad log-rank (Mantel-Cox) test, n = 5. (H, J, L) Frequencies of CD8 + NKG2D + T cells in ovarian ascites, B16 and NBL tumors from control (CD8α-Cre or wt), Cbx3 Tg , Cbx3 fl/+ and Cbx3 fl/fl mice; data were extracted from flow analysis ( Figure S2C, E, G ); bars: group median; Graphpad unpaired student t-test: **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; each symbol = one mouse; n = 5-8; representative of 2-3 experiments. (I, K, M) Frequencies of CD4 + FOXP3 + Tregs in ovarian ascites, B16 and NBL tumors ( Figure S2D, F, H ); bars: group median; Graphpad unpaired student t-test: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; each symbol = one mouse; n = 5-8; representative of 2-3 experiments. (N, O) Apoptotic cells (brown) in B16 melanoma and NBL tumor sections were identified by TUNEL staining; representative of 4 tumors from each mouse strain.

Article Snippet: The following Western blot and ChIP-tested antibodies were purchased from Cell Signaling: tri-methyl-histone H3 (Lys9) (D4W1U) rabbit mAb #13969, phospho-Rpb1 CTD (Ser2) (E1Z3G) rabbit mAb #13499, phospho-Rpb1 CTD (Ser5) (D9N5I) rabbit mAb #13523, LEF-1 (C12A5) rabbit mAb #2230, cleaved caspase-3 (Asp175) antibody #9661 and phospho-HP1γ (Ser83) Ab #2600.

Techniques: Control, Injection, Expressing, TUNEL Assay, Staining

Journal: Frontiers in Immunology

Article Title: Targeting Cbx3 /HP1γ Induces LEF-1 and IL-21R to Promote Tumor-Infiltrating CD8 T-Cell Persistence

doi: 10.3389/fimmu.2021.738958

Figure Lengend Snippet: Cbx3 /HP1γ-deficient CD8 + T cells remodel the tumor chemokine/receptor landscape. (A, B) Relative expression of chemokines and receptors mediating CD8 + T cells trafficking into B16 tumors was determined by RT-qPCR and normalized to Gapdh . (C, D) Relative expression of chemokines and receptors mediating CD8 + T cells trafficking into NBL tumors was evaluated by RT-qPCR and normalized to Gapdh . (E, F) Relative expression of chemokines and receptors inducing CD4 + Tregs homing into tumors was normalized to Gapdh . (G, H) Relative expression of chemokines and receptors inducing CD4 + Tregs homing into NBL tumors was assessed. X axis: mouse strains; Graphpad unpaired student t-test: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; ns, not significant; representative of 4 experiments; n = 4.

Article Snippet: The following Western blot and ChIP-tested antibodies were purchased from Cell Signaling: tri-methyl-histone H3 (Lys9) (D4W1U) rabbit mAb #13969, phospho-Rpb1 CTD (Ser2) (E1Z3G) rabbit mAb #13499, phospho-Rpb1 CTD (Ser5) (D9N5I) rabbit mAb #13523, LEF-1 (C12A5) rabbit mAb #2230, cleaved caspase-3 (Asp175) antibody #9661 and phospho-HP1γ (Ser83) Ab #2600.

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Targeting Cbx3 /HP1γ Induces LEF-1 and IL-21R to Promote Tumor-Infiltrating CD8 T-Cell Persistence

doi: 10.3389/fimmu.2021.738958

Figure Lengend Snippet: LEF-1 and IL-21R are indispensable for halting tumor growth. (A) Western blot of LEF-1 expression was done using total protein lysates of day 5 activated/differentiated CD8 + T cells; C: Cbx3 /HP1γ-deficient; Lef1 fl/+ and Lef1 fl/fl : Cbx3 - Lef1 -deficient; representative of 4 experiments. (B) IL-21R expression was measured by flow; Cbx3 fl/fl : Cbx3 /HP1γ-deficient; Il21r +/- and Il21r -/- : Cbx3 - Il21r -deficient; representative of 4 experiments. (C) Ovarian ascites were measured on day 125 after tumor injection; bars: group median; Graphpad unpaired student t-test: ***p ≤ 0.001, ****p ≤ 0.0001; each symbol = one mouse; n = 3-5; Cbx3 fl/fl Lef1 fl/fl : Cbx3 - Lef1 -deficient; representative of 2 experiments. (D) B16 melanoma tumor burden was determined on day 18 after tumor injection; bars: group median; Graphpad unpaired student t-test: *p ≤ 0.05, ****p ≤ 0.0001; each symbol = mouse; for each genotype n = 4-7; Lef1 fl/fl : Lef1 ablation alone using CD8α-Cre mice; representative of 3 experiments. (E) On day 10 after injection of tumor cells, tumor bearing B6SJ/L mice (CD45.1 + ) were treated with in vitro -activated/differentiated CD8 + T cells (CD45.2 + ) from control, Cbx3 fl/fl , Cbx3 fl/fl , Il21r -/- ( Cbx3 - Il21r -deficient) or Il-21r -/- (germline deleted) mice; **p ≤ 0.01, ***p ≤ 0.001; n = 3-4 recipients; representative of 2 experiments. (F) NBL tumor burden was determined starting on day 25 after injection of tumor cells then every 2 days until day 33; Graphpad two-way ANOVA: ***p≤0.001; n = 2-5; representative of 2 experiments. (G) On day 14 after NBL injection, tumor bearing B6SJ/L mice were treated with in vitro -activated/differentiated CD8 + T cells from control, Cbx3 fl/fl or Cbx3 fl/fl , Il21r -/- ( Cbx3 - Il21r -deficient) mice; ****p ≤ 0.0001, n = 3-4 recipients; representative of 2 experiments.

Article Snippet: The following Western blot and ChIP-tested antibodies were purchased from Cell Signaling: tri-methyl-histone H3 (Lys9) (D4W1U) rabbit mAb #13969, phospho-Rpb1 CTD (Ser2) (E1Z3G) rabbit mAb #13499, phospho-Rpb1 CTD (Ser5) (D9N5I) rabbit mAb #13523, LEF-1 (C12A5) rabbit mAb #2230, cleaved caspase-3 (Asp175) antibody #9661 and phospho-HP1γ (Ser83) Ab #2600.

Techniques: Western Blot, Expressing, Injection, In Vitro, Control

Journal: Frontiers in Immunology

Article Title: Targeting Cbx3 /HP1γ Induces LEF-1 and IL-21R to Promote Tumor-Infiltrating CD8 T-Cell Persistence

doi: 10.3389/fimmu.2021.738958

Figure Lengend Snippet: LEF-1 and IL-21R are required for CD8 + T cells to maintain their effector capacity. (A, B) Prf 1 and Gzmb relative expression in B16 melanoma tumors was determined by RT-qPCR; Graphpad unpaired student t-test: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; n = 3 tumors; representative of 3 experiments. (C–E) Prf1 , Gzmb and Ifng relative expression in NBL tumors was determined by RT-qPCR; Graphpad unpaired student t-test: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; n = 3 tumors; representative of 3 experiments. (F–H) Prf1 , Gzmb and Ifng relative expression in day 5 in vitro -activated/differentiated CD8 + T cells from Cbx3 /HP1γ-deficient and compound mutant mice was quantified by RT-qPCR; Graphpad unpaired student t-test: **p ≤ 0.01, ***p ≤ 0.001; representative of 3 experiments.

Article Snippet: The following Western blot and ChIP-tested antibodies were purchased from Cell Signaling: tri-methyl-histone H3 (Lys9) (D4W1U) rabbit mAb #13969, phospho-Rpb1 CTD (Ser2) (E1Z3G) rabbit mAb #13499, phospho-Rpb1 CTD (Ser5) (D9N5I) rabbit mAb #13523, LEF-1 (C12A5) rabbit mAb #2230, cleaved caspase-3 (Asp175) antibody #9661 and phospho-HP1γ (Ser83) Ab #2600.

Techniques: Expressing, Quantitative RT-PCR, In Vitro, Mutagenesis

Journal: Nature Communications

Article Title: Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells

doi: 10.1038/s41467-020-15174-3

Figure Lengend Snippet: a Fibroblasts (F) and endothelial cells were obtained from otherwise healthy young and aged individuals. Young untreated cells ( n = 3 distinct individuals for both fibroblasts and endothelial cells, dark blue), aged untreated cells ( n = 8 individuals for fibroblast, n = 7 individuals for endothelial cells, red), and aged treated cells ( n = 8 for fibroblast, n = 7 for endothelial cells, light blue) were analyzed for a panel of 11 different hallmarks of aging. Most of the assays were performed by high-throughput imaging on 500–1000 cells per sample to allow population-wide studies with single-cell resolution (Supplementary Figs. – ). 100 cells per sample (i.e., individuals) were randomly selected and pooled per treatment group to do a statistical comparison across the three groups (young fibroblasts n = 300; aged fibroblasts n = 800; aged treated fibroblasts n = 800; young endothelial cells n = 300; aged endothelial cells n = 700; aged treated endothelial cells n = 700). Pairwise statistical analysis was done by one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001. b Quantification of single-nucleus levels of trimethylated H3K9, a repressive mark of gene expression. Both cell types show significant elevation of the mark towards the youthful distribution. c Quantification of single-nucleus levels of heterochromatin marker HP1γ by immunocytochemistry showing a trend toward youth upon treatment. d Quantification of the inner nuclear membrane polypeptide LAP2α, a regulator of nuclear lamina by regulating the binding of lamin B1 and chromatin. This again shows a trend toward youth after cells are treated. e Results of live cells imaging with florescent marker of autophagosome formation in single cells. f Cleavage of fluorescent-tagged chymotrypsin-like substrate elevated in treated and young fibroblasts and endothelial cells corresponding to increased proteasome 20S core particle activity. g Individual cell mitochondria membrane potential measurements also showing more active mitochondria as a result of transient reprogramming. Quantification of pro-inflammatory factors secreted by the cells in each cohort. h Individual cell mitochondria ROS measurements also showing less accumulated ROS as a result of transient reprogramming. i Inflammatory cytokine profiling in endothelial cells, with a significant elevation and depression specifically in aged and treated endothelial cells, respectively. In b – h data are represented as box–whisker plots with median, and bars represent whiskers with distribution variability 10th–90th percentile. In f – j data are represented as mean values and bars represent SD.

Article Snippet: Rabbit::H3K9me3 (Abcam #ab8898 1:4000), LAP2α (Abcam #ab5162 1:500), SIRT1 (Abcam #ab7343 1:200); Rabbit: Mouse: HP1γ (Millipore Sigma #05-690 1:200), Lamin A/C (Abcam #ab40567 1:200), GFP (Invitrogen, # A11122 , 1:250); Luciferase (Sigma-Aldrich, #L0159, 1:200); Collagen I (Cedarlane Labs, #CL50151AP, 1:200); HSP47 (Abcam, #ab77609, 1:200), Laminin (Abcam, #AB11576, 1:1000), anti-CD31-Alexa Fluor 488 (clone WM59; BioLegend; #303110, 1:75), anti-CD45-Alexa Fluor 488 (clone HI30; Invitrogen; #MHCD4520, 1:75), anti-CD34-FITC (clone 581; BioLegend; #343503, 1:75), anti-CD29-APC (clone TS2/16; BioLegend; #303008, 1:75) and anti-NCAM-Biotin (clone HCD56; BioLegend; #318319, 1:75), anti-CD31-Alexa Fluor 488 (clone WM59; BioLegend; #303110, 1:75), anti-CD45-Alexa Fluor 488 (clone HI30; Invitrogen; #MHCD4520, 1:75), anti-CD34-FITC (clone 581; BioLegend; #343503, 1:75), anti-CD29-APC (clone TS2/16; BioLegend; #303008, 1:75), and anti-NCAM-biotin (clone HCD56; BioLegend; #318319, 1:75).

Techniques: High Throughput Screening Assay, Imaging, Expressing, Marker, Immunocytochemistry, Binding Assay, Activity Assay, Whisker Assay