Journal: The Journal of Clinical Investigation

Article Title: S1PR4 ablation reduces tumor growth and improves chemotherapy via CD8 + T cell expansion

doi: 10.1172/JCI136928

Figure Lengend Snippet: (A) Representative t-distributed stochastic neighbor embedding (tSNE) plots show differences in immune cell infiltrates at the endpoint. (B–D) Relative amounts of immune cell populations (B), FoxP3+ Tregs (C), and CD8+ T cells (D) in PyMT tumors of WT (n = 17) and KO (n = 18) mice analyzed by FACS. (E and F) Sections from PyMT tumors were stained for CD8+ cytotoxic T cells. (E) Quantification of CD8+ T cells as a percentage of total cells (WT: n = 10, KO: n = 9) and (F) representative sections stained for CD8 (brown) and DAPI (blue; nuclei). Scale bars: 200 μm; magnified areas: 50 μm. (G) Relative numbers of Trm (CD103+), exhausted (PD-1+), and effector CD8+ T cells (CD49a–CD103–) in tumors (n = 10) determined by FACS. (H) Relative numbers of gMDSCs (CD11b+Ly6GhiLy6Clo) and mMDSCs (CD11b+Ly6GloLy6Chi) in PyMT tumors (WT, n = 5; KO, n = 6) expressing arginase 1 (Arg1) determined by FACS. (I) Relative numbers of proliferating T cells upon coculture with WT (n = 18) and S1PR4-KO (n = 10) MDSCs in different ratios determined by FACS. (J) Chemokine levels in WT (n = 15) and S1PR4-KO PyMT (n = 14) tumors determined by LEGENDplex. (K) Splenocytes of WT mice in the upper well of a modified Boyden chamber were allowed to migrate toward extracellular fluid from WT and S1PR4-KO PyMT tumors (n = 10). Migrated cell populations were analyzed by FACS. Heat-inactivated FCS served as control. Means ± SEM; 2-tailed Student’s t test (D, E, G, and J), 2-way ANOVA with Holm-Šidák correction (K); *P < 0.05, **P < 0.01.

Article Snippet: CD8 + T cells were isolated from murine spleens using a Mouse CD8 + T Cell Isolation Kit (STEMCELL Technologies) and cultured in T cell medium (RPMI 1640, supplemented with 5 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 10% heat-inactivated FCS, 1% nonessential and essential amino acids, 1% sodium pyruvate, and 1% HEPES).

Techniques: Staining, Expressing, Modification

Journal: The Journal of Clinical Investigation

Article Title: S1PR4 ablation reduces tumor growth and improves chemotherapy via CD8 + T cell expansion

doi: 10.1172/JCI136928

Figure Lengend Snippet: (A) Representative images of WT and S1PR4-KO AOM/DSS-treated colons at day 84 stained with H&E. Scale bars: 1 mm. (B) Number of tumors per mouse for WT and S1PR4-KO mice (n = 9) at day 84 after treatment with AOM/DSS. (C) Representative tSNE plots showing the composition of the epithelial layer from WT and S1PR4-KO colons at day 84. (D and E) Relative amounts of CD8+ IEL (D) and CD8+ Trm IELs (E) within the epithelial layer of WT and S1PR4-KO mice at day 0 (n = 6), day 8 (n = 8), day 15 (n = 4), and day 84 (n = 9) analyzed by flow cytometry. Means ± SEM; 2-tailed Student’s t test; *P < 0.05, **P < 0.01.

Article Snippet: CD8 + T cells were isolated from murine spleens using a Mouse CD8 + T Cell Isolation Kit (STEMCELL Technologies) and cultured in T cell medium (RPMI 1640, supplemented with 5 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 10% heat-inactivated FCS, 1% nonessential and essential amino acids, 1% sodium pyruvate, and 1% HEPES).

Techniques: Staining, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: S1PR4 ablation reduces tumor growth and improves chemotherapy via CD8 + T cell expansion

doi: 10.1172/JCI136928

Figure Lengend Snippet: (A) WT and S1PR4-KO PyMT mice were treated with 5 mg/kg DXR once a week without (B–E) or with CD8-depleting antibodies (F and G). (B) Tumor size kinetics in DXR-treated PyMT mice (n = 10). (C) Relative numbers of lymphocyte subsets in PyMT tumors of WT (n = 10) and S1PR4-KO (n = 9) mice after 5 weeks of DXR treatment. (D) Representative FACS plots showing percentage of CD8+ T cells in PyMT tumors. (E) Relative numbers of Trm (CD103+), exhausted (PD-1+), and effector CD8+ T cells (CD49a–CD103–) in DXR-treated WT (n = 11) and KO (n = 12) PyMT tumors determined by FACS. (F) Tumor size kinetics in DXR and anti-CD8 antibody (n = 6) or IgG control antibody (WT, n = 11; S1PR4-KO, n = 8) treated PyMT mice. (G) Representative FACS plots showing percentage of CD8+ and CD8/4 DN T cells in IgG- or anti–CD8-treated WT PyMT tumors. Means ± SEM; 2-way ANOVA with Holm-Šidák correction (B, C, and F), 2-tailed Student’s t test (E); *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: CD8 + T cells were isolated from murine spleens using a Mouse CD8 + T Cell Isolation Kit (STEMCELL Technologies) and cultured in T cell medium (RPMI 1640, supplemented with 5 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 10% heat-inactivated FCS, 1% nonessential and essential amino acids, 1% sodium pyruvate, and 1% HEPES).

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: S1PR4 ablation reduces tumor growth and improves chemotherapy via CD8 + T cell expansion

doi: 10.1172/JCI136928

Figure Lengend Snippet: (A–D) WT and S1PR4-KO PyMT mice were treated with either anti–PD-1 or IgG isotype control antibodies (n = 17 each) on days 0, 6, 12, and 18 after the first tumor reached 0.6 cm in diameter. Tumors were harvested at day 27. (A) Experimental outline. (B) Relative numbers of CD8+PD-1+ and total CD8+ T cells infiltrated into PyMT tumors (n = 17) 4 weeks after treatment began. (C and D) Tumor size kinetics of treated WT (C) and S1PR4-KO (n = 16) PyMT mice (D). (E) Ifna and Ifnb mRNA expression in WT (n = 8) and S1PR4-KO (n = 9) PyMT tumors determined by quantitative PCR (qPCR). (F) Relative numbers of exhausted CD8+PD-1+ T cells in PyMT tumors of IFNAR1 WT S1PR4 WT (IWSW, n = 9), IFNAR1 WT S1PR4-KO (IWSK, n = 10), IFNAR1 KO S1PR4 WT (IKSW, n = 5), and IFNAR1 KO S1PR4-KO (IKSK, n = 4) after 1 tumor reached a size of approximately 1.5 cm. Means ± SEM; 2-way ANOVA with Holm-Šidák correction (B and D), 1-way ANOVA with Holm-Šidák correction (F); *P < 0.05, **P < 0.01.

Article Snippet: CD8 + T cells were isolated from murine spleens using a Mouse CD8 + T Cell Isolation Kit (STEMCELL Technologies) and cultured in T cell medium (RPMI 1640, supplemented with 5 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 10% heat-inactivated FCS, 1% nonessential and essential amino acids, 1% sodium pyruvate, and 1% HEPES).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: The Journal of Clinical Investigation

Article Title: S1PR4 ablation reduces tumor growth and improves chemotherapy via CD8 + T cell expansion

doi: 10.1172/JCI136928

Figure Lengend Snippet: (A) Unsupervised hierarchical cluster analysis of FACS-sorted WT and S1PR4-KO PyMT CD8+ T cell gene expression profiles (top 300 differentially expressed genes). (B and C) Representative gene set enrichment plots of gene sets enriched in KO PyMT CD8+ T cells. (D) Absolute cell number of splenic WT and S1PR4-KO CD8+ T cells after activation at days 0, 2, and 4. One representative experiment of 3 independent biological replicates is shown, which was repeated 5 times with similar outcomes. (E) Ki67 expression of WT and S1PR4-KO CD8+ T cells (n = 3) at day 2 determined by qPCR. (F) Relative numbers of viable WT and S1PR4-KO CD8+ T cells determined by annexin V/7-AAD staining at day 2 and day 13 for this individual experiment. One representative experiment with 5 independent biological replicates is shown, which was repeated 3 times with similar outcomes. (G) Absolute cell number of WT and S1PR4-KO CD103+ CD8+ Trm T cells at day 13. One representative experiment with 5 independent biological replicates, which was repeated 3 times with similar outcomes, is shown. Means ± SEM; 2-tailed Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: CD8 + T cells were isolated from murine spleens using a Mouse CD8 + T Cell Isolation Kit (STEMCELL Technologies) and cultured in T cell medium (RPMI 1640, supplemented with 5 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 10% heat-inactivated FCS, 1% nonessential and essential amino acids, 1% sodium pyruvate, and 1% HEPES).

Techniques: Expressing, Activation Assay, Staining

Journal: The Journal of Clinical Investigation

Article Title: S1PR4 ablation reduces tumor growth and improves chemotherapy via CD8 + T cell expansion

doi: 10.1172/JCI136928

Figure Lengend Snippet: (A) Venn diagram and the gene list show shared and divergent up- or downregulated genes in PyMT tumor-derived CD8+ T cells and total AOM/DSS-treated colons (day 84) comparing WT and S1PR4-KO mice. Genes selected for in vitro validation are highlighted in green. (B and C) Absolute number of WT and S1PR4-KO CD8+ T cells either untreated (w/o) or treated with (B) 0.5 μM PI3K inhibitor (Ly294002) or (C) 5 μM LTA4H inhibitor (SC 57461A) at day 2. One representative experiment with 5 independent biological replicates is shown, which was repeated 3 times with similar outcomes. (D–I) PyMT tumor spheroids were cocultured with WT and S1PR4-KO CD8+ T cells. One representative experiment with 5 independent biological replicates (each containing means of 6 technical replicates) is shown. (D–F) PyMT spheroid size upon coculture with untreated (black), Ly294002-treated (green), or SC 57461A–treated (red) CD8+ T cells over 6 days (D and E) and at day 6 (F) after initial activation with representative photographs (G–I). Scale bars: 200 μm. (J) Intracellular staining of p-AKT in NTC or PIK3AP1 siRNA-treated WT and S1PR4-KO CD8+ T cells 30 minutes after activation (n = 4). p-AKT expression is shown as MFI. (K) LTB4 concentration in supernatants of WT and S1PR4-KO CD8+ T cells 1 day after activation determined by ELISA (n = 5). (L and M) Absolute number of S1PR4 agonist (Cym 50308) or antagonist (Cym 50358) pretreated CD8+ T cells either untreated (w/o) or treated with 20 μM PGP 6 days (L) or 8 days (M) after activation (n = 5). Means ± SEM; 1-way ANOVA (B–F and J) or 2-way ANOVA (L and M), each with Holm-Šidák correction; *P < 0.05, **P < 0.01.

Article Snippet: CD8 + T cells were isolated from murine spleens using a Mouse CD8 + T Cell Isolation Kit (STEMCELL Technologies) and cultured in T cell medium (RPMI 1640, supplemented with 5 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 10% heat-inactivated FCS, 1% nonessential and essential amino acids, 1% sodium pyruvate, and 1% HEPES).

Techniques: Derivative Assay, In Vitro, Activation Assay, Staining, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Clinical Investigation

Article Title: S1PR4 ablation reduces tumor growth and improves chemotherapy via CD8 + T cell expansion

doi: 10.1172/JCI136928

Figure Lengend Snippet: (A) Human PBMCs were preactivated with 10 ng/mL of LPS and 100 U/mL IFN-γ and prestimulated with or without 200 nM Cym 50358 (S1PR4 antagonist, n = 25) or 200 nM Cym 50308 (S1PR4 agonist, n = 15) for 30 minutes before being cocultured with MCF-7 spheroids for 6 days. The relative number of CD8+ T cells as fold of control is shown. P values were calculated using 1-sample Wilcoxon test. *P < 0.05. (B–G) The METABRIC data set (B–D) and the TCGA colon adenocarcinoma data set (E–G) were used to calculate an in silico S1P ratio, which was correlated with overall patient survival (B and E; Q1, 25% of patients with lowest S1P ratio; Q4, 25% of patients with highest S1P ratio) and CD8A or CD103 expression of human breast (C and D) and colon (F and G) tumors. (H–L) Tissue microarrays of human colon adenocarcinoma (H and I) and human invasive mammary carcinoma (J–L) cores were stained for CD3, CD8, PIK3AP1, LTA4H, and KI67 by PhenOptics. Nuclei were counterstained with DAPI. (H) Representative images show magnified areas of colon adenocarcinoma tissue cores (full cores in Supplemental Figure 5A). Proliferating (KI67+) CD8+PIK3AP1+ T cells are marked by arrows. Scale bars: 50 μm. (I) Correlation matrix of CD8+ T cell subsets in colon adenocarcinoma tissue cores compared with proliferating tumor cells, nodal involvement, stage, and metastasis. Spearman r values are indicated. (J–L) Correlation of CD8+ T cell, PIK3AP1+CD8+ T cell, and LTA4H+CD8+ T cell infiltrates in mammary carcinoma cores with overall patient survival (Q1 indicates 25% lowest abundance; Q4 indicates 25% highest abundance each).

Article Snippet: CD8 + T cells were isolated from murine spleens using a Mouse CD8 + T Cell Isolation Kit (STEMCELL Technologies) and cultured in T cell medium (RPMI 1640, supplemented with 5 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 10% heat-inactivated FCS, 1% nonessential and essential amino acids, 1% sodium pyruvate, and 1% HEPES).

Techniques: In Silico, Expressing, Staining