Journal: Cell reports

Article Title: Calcineurin is an adaptor required for assembly of the TCR signaling complex

doi: 10.1016/j.celrep.2024.114568

Figure Lengend Snippet:

Article Snippet: Human CD4 + T cells were isolated from buffy coats of healthy volunteers (NIH Blood Bank) using the Human CD4 cell recovery column kit (Cedarlane), according to the manufacturer’s instructions.

Techniques: Purification, Recombinant, Protease Inhibitor, Cell Recovery, In Situ, Plasmid Preparation, Control, Software, Luminex

Journal: medRxiv

Article Title: Cholesterol-lowering Decreased mTOR Complex 2 Signaling and Enhanced Antitumor Immunity

doi: 10.1101/2021.01.29.21250381

Figure Lengend Snippet: A. Mice were treated with ezetimibe for 14 days prior to subcutaneous injection of syngeneic tumor cell lines. B . B16 melanoma cells were injected into B6 mice (8-9 mice/group) C . RM1 prostate adenocarcinoma cells were injected into B6 mice (9-10 mice/group). D . RENCA renal cell carcinoma cells were injected into Balb/c mice (5-6 mice/group). In each experiment, one group received ezetimibe and depleting αCD8 antibody two days after s.c. tumor injection into the flank. P-values were obtained by using the repeated measures ANOVA (A-D). E . To determine if immunity is lymphocyte-dependent, mice were treated with ezetimibe for 14 days to lower cholesterol and then vaccinated with DCs pulsed with B16 tumor lysate. Lymphocytes were harvested from these mice and cultured in vitro with tumor lysate pulsed DC and IL-2 for 3 days, and adoptively transferred into recipient mice, which were challenged with i.v. B16 cells. Lung tumors were examined 25 days later. F . Representative lungs are shown from recipient mice, labeled with the donor mouse treatment. G . Lung weights (top) and CD8+ T cell IFN- γ response to ex vivo restimulation (bottom) are shown. Graphs are labeled with the treatment provided to the donor mice. H . In a similar experiment, B6 mice were treated with DCs pulsed with RM1 tumor lysate. Lymphocytes were transferred into recipient mice that were challenged with i.v. RM1 cells. I . Representative H&E sections of lungs from each treatment group are shown. J . Lung weights (top) and tumor number in the lungs (bottom) are shown. Each group contained 3 mice. Each experiment was conducted at least twice to confirm results (E-J).

Article Snippet: CD8+ cells were isolated using the Mouse CD8 Cell Recovery Column Kit from Cedarlane Laboratories Ltd (ON, Canada).

Techniques: Injection, Cell Culture, In Vitro, Labeling, Ex Vivo

Journal: medRxiv

Article Title: Cholesterol-lowering Decreased mTOR Complex 2 Signaling and Enhanced Antitumor Immunity

doi: 10.1101/2021.01.29.21250381

Figure Lengend Snippet: A . Serum cholesterol levels were measured in mice treated with ezetimibe (n=6). Splenocytes from ezetimibe-treated mice showed decreased AKT/mTOR signaling, with pronounced decrease in phosphorylation of AKT at Ser473. Representative blots from duplicate experiments are shown. C . pAKT (Ser473) in CD8+ lymphocytes from patients before and after cholesterol lowering (CL) with statin+ezetimibe was measured by flow cytometry (n=4). D . Human pAKT (Ser473) in CD8+ lymphocytes in these patients was significantly reduced by CL. E . RM1 prostate adenocarcinoma cells were injected into B6 mice (8-9 mice/group) and ezetimibe treatments were started 3 days later. Tumor growth was monitored and p-value was obtained by using the repeated measures ANOVA. F-I . Lymphocytes were collected from mice treated with ezetimibe or saline for 21 days and analyzed for the indicated markers by flow cytometry. G . Splenocytes were first gated for CD8 prior to flow cytometry analysis for the indicated markers.

Article Snippet: CD8+ cells were isolated using the Mouse CD8 Cell Recovery Column Kit from Cedarlane Laboratories Ltd (ON, Canada).

Techniques: Phospho-proteomics, Flow Cytometry, Injection, Saline

Journal: medRxiv

Article Title: Cholesterol-lowering Decreased mTOR Complex 2 Signaling and Enhanced Antitumor Immunity

doi: 10.1101/2021.01.29.21250381

Figure Lengend Snippet: A . Pmel-1 lymphocytes were adoptive transferred into congenic mice followed by ezetimibe treatment. Mice were injected with DCs pulsed with gp100 peptide on day 0 and day 20 (DC vaccination). Representative results are shown for triplicate experiments with 4-6 mice per experimental group. B-C . Lymphocytes were harvest on day 10 and analyzed for the indicated markers by flow cytometry. B . Donor Pmel-1 lymphocytes (Thy1.1+) were increased in ezetimibe-treated animals following DC vaccination and all CD8+ lymphocytes displayed a memory phenotype with increase in CD62L and CD44 expressing. C . CPT1A staining intensity was assessed in CD8+Thy1.1+ and CD8+Thy1.1-cells. D-F . Mice were restimulated with DC vaccination on day 20 and lymphocytes were harvested 3 days later. D . Ezetimibe treatment was associated with a greater recall proliferation of Pmel-1 CD8+ lymphocytes, which displayed a memory phenotype with increase in CD62L and CD44 expression. E-F . PD1 and Granzyme B expressions were assessd in CD8+Thy1.1+ and CD8+Thy1.1-cells.

Article Snippet: CD8+ cells were isolated using the Mouse CD8 Cell Recovery Column Kit from Cedarlane Laboratories Ltd (ON, Canada).

Techniques: Injection, Flow Cytometry, Expressing, Staining

Journal: medRxiv

Article Title: Cholesterol-lowering Decreased mTOR Complex 2 Signaling and Enhanced Antitumor Immunity

doi: 10.1101/2021.01.29.21250381

Figure Lengend Snippet: A . CD4+ and CD8+ lymphocytes from Rictor KO mice (left 2 panels) or wild type (WT; right 2 panels) were assessed for Rictor expression. B . CD8+ Lymphocytes from Rictor KO mice were assessed for pAKT (Ser473) by flow cytometry. β CD was added to cell culture to bind and reduce cholesterol in the medium. C . CD8+ lymphocytes were assessed for pS6k (Thr389). D . CD8+ lymphocytes were also stimulated with αCD3 and αCD28 to assess activation as measured by TNFα and IFN γ using flow cytometry. Summarized data (B-C) are for triplicate experiments. E . B16 tumors were also grown subcutaneously in mice with lymphocytes-specific Rictor KO and control ( Rictor flox ), with or without daily ezetimibe treatment. Tumors and spleens were harvested on day 18. Tumor growth was monitored and p-value was obtained by using the repeated measures ANOVA. F . Tumors were photographed. G . Splenocytes were stained with αCD8 and gp100 tetramer. I . Splenocytes were cultured ex vivo with gp100 peptide stimulation for 5 days. Lymphocytes were stained with αCD8, gp100 tetramer, and αIFN γ before flow cytometry analysis.

Article Snippet: CD8+ cells were isolated using the Mouse CD8 Cell Recovery Column Kit from Cedarlane Laboratories Ltd (ON, Canada).

Techniques: Expressing, Flow Cytometry, Cell Culture, Activation Assay, Control, Staining, Ex Vivo

Journal: medRxiv

Article Title: Cholesterol-lowering Decreased mTOR Complex 2 Signaling and Enhanced Antitumor Immunity

doi: 10.1101/2021.01.29.21250381

Figure Lengend Snippet: A . RM1 tumors were grown subcutaneously in mice with lymphocyte-specific Rictor KO (n=9) and control ( Rictor flox , n=6). Tumor growth was monitored and on day 18, tumors were removed and photographed. Tumor growth was monitored and p-value was obtained by using the repeated measures ANOVA. B-E . Lymphocytes were collected from mice and analyzed for the indicated markers by flow cytometry. G . Splenocytes were first gated for CD8+ prior to flow cytometry analysis for the indicated markers.

Article Snippet: CD8+ cells were isolated using the Mouse CD8 Cell Recovery Column Kit from Cedarlane Laboratories Ltd (ON, Canada).

Techniques: Control, Flow Cytometry

Journal: medRxiv

Article Title: Cholesterol-lowering Decreased mTOR Complex 2 Signaling and Enhanced Antitumor Immunity

doi: 10.1101/2021.01.29.21250381

Figure Lengend Snippet: A . CL with ezetimibe increased tumor infiltration of CD8+ lymphocytes in mice (p=0.03). Mouse experiments were performed in duplicate and representative result is shown. B . In patient tumor tissue, CL with a statin and ezetimibe increased tumor CD8+ lymphocytes and M1 macrophages as determined by RNAseq. This analysis was performed for 2 patients and a representative result is shown. C . In similarly treated patients, αCD8 immunofluorescent staining are shown using matched pre-treatment prostate biopsy and post-treatment prostatectomy tissue. For the summarized data, each column shows mean values from 6 matched pre-treatment biopsies and post-treatment prostatectomies. D . Using gene expressions from 1,321 tumors in the PCTA database, scatter plot and regression line were generated to show the relationship between genes involved in cholesterol homeostasis (biosynthesis, metabolism, efflux and import) and genes specific for CD8+ T cells. The correlation coefficient was computed by Spearman’s method. E . Using the RM1 lung metastases from the experiment described in , representation H&E section and Ki67 staining are shown. All scale bars represent 100μm (C,E).

Article Snippet: CD8+ cells were isolated using the Mouse CD8 Cell Recovery Column Kit from Cedarlane Laboratories Ltd (ON, Canada).

Techniques: Staining, Generated

Journal:

Article Title: A role for surface lymphotoxin in experimental autoimmune encephalomyelitis independent of LIGHT

doi: 10.1172/JCI200318648

Figure Lengend Snippet: CD4+ T cells from LTβR-Ig–treated rats are hyporesponsive ex vivo. (a) Pooled CD4+ T cells were isolated at D10 from rats in the EAE model (n = 5), stimulated in vitro with irradiated splenocytes, and indicated concentrations of MBP-peptide and proliferation measured by 3H-thymidine incorporation. Cells were derived from rats treated in vivo with control huIgG (diamonds), LTβR-Ig (circles), or from naive, untreated rats as controls (squares). (b) Supernatants from cultures in a (0 and 2.5 μg/ml MBP-peptide, white and gray bars, respectively) were collected at 72 hours and measured by ELISA for IFN-γ content. The experiment was repeated three times with similar results and also performed at day 7 with similar results.

Article Snippet: CD4 + T cells were purified by negative selection from dissociated LN cells by passing them through a rat CD4 cell-recovery column, according to the manufacturer’s specifications (Cedarlane Laboratories Inc.).

Techniques: Ex Vivo, Isolation, In Vitro, Irradiation, Derivative Assay, In Vivo, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: A role for surface lymphotoxin in experimental autoimmune encephalomyelitis independent of LIGHT

doi: 10.1172/JCI200318648

Figure Lengend Snippet: Lymphocyte infiltrates are detected in the spinal cords of day-13 EAE rats in both groups; however, the positioning of infiltrates in LTβR-Ig–treated rats is altered. FACS analysis of lymphocytes obtained from the spinal cords is shown in a. The percentage of CD4+ (b) and CD8+ (c) for all cells in the lymphoid size gate was compared between treatment groups, where control rats were naive and untreated. Mean and standard deviation of five separate animals per group is shown. Spinal cords were also harvested, fixed, tangential paraffin sections stained with H&E, and visualized at ×50 (d, f, h) or ×400 (e, g, i) with a Leica microscope. Unimmunized naive rats (d and e) were compared with control huIgG–treated rats (f and g) or LTβR-Ig–treated rats (h and i). The experiments were performed twice with similar results.

Article Snippet: CD4 + T cells were purified by negative selection from dissociated LN cells by passing them through a rat CD4 cell-recovery column, according to the manufacturer’s specifications (Cedarlane Laboratories Inc.).

Techniques: Standard Deviation, Staining, Microscopy

Journal:

Article Title: A role for surface lymphotoxin in experimental autoimmune encephalomyelitis independent of LIGHT

doi: 10.1172/JCI200318648

Figure Lengend Snippet: KJ126+ DO11.10 T cells expand normally in vivo in LTβR-Ig–treated mice, but ex vivo recall responses are impaired. (a) DO11.10 T cell expansion in vivo at days 2, 5, and 7 after immunization is represented as percentage of CD4+ T cells that are KJ126+. DO11.10 T cells were transferred to control huIgG-treated (gray bars) or LTβR-Ig–treated (black bars) BALB/c recipients, and the mice were then immunized with OVA323–339 plus CFA (gray and black bars) or not immunized (white bars). (b) Proliferation of DO11.10 T cells was assessed in vivo by prelabeling KJ126+ CD4+ T cells with CFSE and transferring these cells to recipients that were not immunized (top panel) or immunized with OVA323–339 plus CFA and treated with huIgG (middle panel) or LTβR-Ig (bottom panel) as in a. A representative FACS plot 5 days after immunization is shown here, and in each experiment five immunized animals (both treatment groups) and three unimmunized animals were evaluated separately. (c) Proliferation of CD4+ T cells from control huIgG-treated mice (diamonds) was compared with that of LTβR-Ig–treated mice at day 7 (circles). CD4+ T cells from nonimmunized mice were used as controls (squares). These experiments were performed three times with similar results. (d) Proliferation of CD4+ KJ126+ T cells was assessed using CFSE labeling at day 7. CD4+ KJ126+ T cells from control huIgG-treated mice stimulated with OVA323–339 (gray line) were compared with that of LTβR-Ig–treated mice (black line) and unstimulated LN cells (dotted line). These data are representative of three separate mice.

Article Snippet: CD4 + T cells were purified by negative selection from dissociated LN cells by passing them through a rat CD4 cell-recovery column, according to the manufacturer’s specifications (Cedarlane Laboratories Inc.).

Techniques: In Vivo, Ex Vivo, Transferring, Labeling