cd3 coated culture plate  (Biogems International)

Journal: The Journal of Experimental Medicine

Article Title: Suppressive effects of the obese tumor microenvironment on CD8 T cell infiltration and effector function

doi: 10.1084/jem.20210042

Figure Lengend Snippet: CD8 T cell activation in dLNs is not impaired in obese mice. (A and B) C57BL/6 mice were fed an SFD or HFD, and MC38 (A) or B16-F10 (B) tumor cells were injected s.c. Results depict flow cytometric analysis of CD8 + T cells in dLNs of tumor-bearing mice. Data are shown as individual mice and mean ± SEM; unpaired Student’s t test. **, P < 0.01 for three independent experiments for each tumor model. (C and D) CD8 T cells isolated from SFD-fed ( n = 3; pooled) or HFD-fed ( n = 3; pooled) mice were activated with anti-CD3/anti-CD28 and analyzed by Seahorse flux assay (C) or flow cytometry (D). Oxphos, oxidative phosphorylation. (E) CD8 T cells isolated from dLNs or spleens from SFD-fed (black; n = 3 technical replicates from 7 pooled mice) or HFD-fed (red; n = 3 technical replicates from 7 pooled mice) mice were activated with anti-CD3/anti-CD28 (+) or unstimulated (−) and analyzed by flow cytometry. Representative data from at least two independent experiments.

Article Snippet: For activation assays, isolated CD8 T cells, total LN cells, or total spleen cells were cultured in anti-CD3–coated (1 µg/ml; Sigma-Aldrich) cell culture plates in the presence of IL-2 (5 ng/ml) and anti-CD28 (3 µg/ml; Sigma-Aldrich) for 48 h, unless otherwise stated.

Techniques: Activation Assay, Injection, Isolation, Flux Assay, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Suppressive effects of the obese tumor microenvironment on CD8 T cell infiltration and effector function

doi: 10.1084/jem.20210042

Figure Lengend Snippet: Obesity-induced functional defects in CD8 T cells are associated with impaired amino acid metabolism. (A–D) C57BL/6 mice were fed an HFD or SFD, and B16-F10 (A–C; n = 5 SFD, n = 7 HFD) and MC38 (D; n = 7 per group) tumors were injected. LipidTOX uptake by CD8 + T cells from tumors was analyzed by flow cytometry. Graphs depict MFI (A and D), correlation of LipidTOX versus tumor volume (B), and correlation of LipidTOX versus IFN-γ expression by CD8 + T cells (C). (E–I) C57BL/6 mice were fed an HFD ( n = 8) or SFD ( n = 8) for 6 wk, and MC38 tumors were injected s.c. On day 12 after tumor inoculation, tumors were dissected and stained for flow cytometry. (E and F) Representative graph, and quantification of kynurenine uptake by CD8 + T cells in tumors. Cells were treated with the system L blocker BCH or not treated with kynurenine (fluorescence minus one control [FMO]) as negative controls. (G and H) MFI or frequency of kynurenine, CD98, and pS6 in CD8 + T cells. (I) Correlation between kynurenine uptake and expression of intracellular molecules in CD8 T cells. Black dots indicate SFD-fed mice, and red dots indicate HFD-fed mice. (J–L) Serum taken from male SFD-fed ( n = 10) or HFD-fed ( n = 10) mice was analyzed by mass spectrometry for metabolite composition. Data are represented as fold change over SFD. Pooled data from two experiments. (M) CD8 T cells isolated from naive spleens were activated with anti-CD3/anti-CD28 in the presence or absence of L-glutamine, and expression of Ki-67, IFN-γ, GzmB, and pS6 was analyzed by flow cytometry. Experiment was performed twice ( n = 3 technical replicates). (A, D, F–H, and L) Unpaired Student’s t test. (B, C, and I) Simple linear regression. (M) One-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: For activation assays, isolated CD8 T cells, total LN cells, or total spleen cells were cultured in anti-CD3–coated (1 µg/ml; Sigma-Aldrich) cell culture plates in the presence of IL-2 (5 ng/ml) and anti-CD28 (3 µg/ml; Sigma-Aldrich) for 48 h, unless otherwise stated.

Techniques: Functional Assay, Injection, Flow Cytometry, Expressing, Staining, Fluorescence, Mass Spectrometry, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Suppressive effects of the obese tumor microenvironment on CD8 T cell infiltration and effector function

doi: 10.1084/jem.20210042

Figure Lengend Snippet: Obesity is associated with impaired immune infiltration in humans with endometrial cancer. Immune profiles of endometrial cancer tumors and IF in a blinded normal and obese patient study ( n = 24). (A) Exemplary images of CD3, CD8, and PD-L1 immunohistochemical staining in IF of two stage 1a, grade 2 endometrial cancer patients (BMI 26.5 kg/m 2 and BMI 46 kg/m 2 , respectively). (B) Correlation of CD3, CD8, and PD-L1 scores with patient BMI. Correlation was computed using nonparametric Spearman correlation.

Article Snippet: For activation assays, isolated CD8 T cells, total LN cells, or total spleen cells were cultured in anti-CD3–coated (1 µg/ml; Sigma-Aldrich) cell culture plates in the presence of IL-2 (5 ng/ml) and anti-CD28 (3 µg/ml; Sigma-Aldrich) for 48 h, unless otherwise stated.

Techniques: Immunohistochemical staining, Staining

Journal: The Journal of Experimental Medicine

Article Title: Suppressive effects of the obese tumor microenvironment on CD8 T cell infiltration and effector function

doi: 10.1084/jem.20210042

Figure Lengend Snippet: Weight loss restores T cell infiltration in endometrial cancer. (A and B) Patients with endometrial cancer ( n = 9) were analyzed for weight loss (A) and cancer disease progression (B) after VSG. (C and D) Immune profile of tissue samples taken from a patient with endometrial cancer over the course of their treatment. The patient received VSG in April 2018, and samples were taken before and after VSG. (C) H&E (top panel) and IHC images of tumor tissue (T; blue) and IF (pink) stained for CD3, CD8, and PD-L1. Numbers in images represent the number of CD3 + or CD8 + T cells per mm 2 tumor or PD-L1 combined score. (D) CD3 and CD8 infiltration and PD-L1 scores over the course of weight loss. Significance was calculated using one-way ANOVA (A). ***, P < 0.001; ****, P < 0.0001.

Article Snippet: For activation assays, isolated CD8 T cells, total LN cells, or total spleen cells were cultured in anti-CD3–coated (1 µg/ml; Sigma-Aldrich) cell culture plates in the presence of IL-2 (5 ng/ml) and anti-CD28 (3 µg/ml; Sigma-Aldrich) for 48 h, unless otherwise stated.

Techniques: Staining

Journal: The Journal of Experimental Medicine

Article Title: Suppressive effects of the obese tumor microenvironment on CD8 T cell infiltration and effector function

doi: 10.1084/jem.20210042

Figure Lengend Snippet: FACS antibodies

Article Snippet: For activation assays, isolated CD8 T cells, total LN cells, or total spleen cells were cultured in anti-CD3–coated (1 µg/ml; Sigma-Aldrich) cell culture plates in the presence of IL-2 (5 ng/ml) and anti-CD28 (3 µg/ml; Sigma-Aldrich) for 48 h, unless otherwise stated.

Techniques: