Journal: The Journal of Clinical Investigation

Article Title: Pharmacologic LDH inhibition redirects intratumoral glucose uptake and improves antitumor immunity in solid tumor models

doi: 10.1172/JCI177606

Figure Lengend Snippet: ( A ) Schematic depicting tumor-killing assay with LDHi in which B16-YFP cells were treated with 20 μM LDHi or vehicle 24 hours apart and T cells were added 24 hours after the first LDHi treatment. ( B ) Quantified media glucose from killing assay coculture. ( C ) Flow cytometry quantification of 2-NBDG (MFI) in B16-YFP and CD8 + Pmel-1 T cells from killing assay cocultures 48 hours after last treatment. ( D – F ) ( D ) Quantified YFP + tumor cells and ( E ) representative in vitro killing assay images of YFP + tumor cells after 48 hours of coincubation with Pmel-1 CD8 + T cells as in A . ( F ) Corresponding quantified YFP + tumor cells and percentages of tumor killing in the same conditions as above alongside vehicle supplemented with 10 mM glucose. ( G ) Quantification of killing of OVA 257-264 –pulsed live B16-YFP tumor cells by OVA-primed CD8 + T cells from OT1 transgenic mice upon 48 hours of coculture in the presence of LDHi (as indicated in A ). E:T = 2:1, cocultured over 48 hours. ( H ) Schematic depicting in vitro Treg suppression assay with MACS column–sorted Tregs (CD4 + CD25 + Regulatory T Cell Isolation Kit, mouse) cocultured with αCD3/αCD28-activated CTV-labeled syngeneic CD8 + T cells for 48 hours with the addition of conditioned media from B16 cells treated with 20 μM LDHi or vehicle or fresh media containing 10 mM glucose. ( I ) Percentage of suppression was calculated as percentage reduction in CD8 + T cell proliferation with respect to CD8 + T cells cultured alone in the same treatment and glucose conditions. Data show 1 representative experiment of 3 independent experiments ( n = 3–4 technical replicates). All statistics produced by 2-way ANOVA with Bonferroni’s multiple-comparisons test implemented in GraphPad Prism. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Data are represented as mean ± SEM.

Article Snippet: Splenocytes were primed with OVA (SIINFEKL 257-264, AnaSpec, AS-60193-1) or human gp100 peptide (25-33, AnaSpec, AS-62589) in RPMI media supplemented with 10% FCS and 50 mM BME as previously described ( ).

Techniques: Flow Cytometry, In Vitro, Transgenic Assay, Suppression Assay, Cell Isolation, Labeling, Cell Culture, Produced

Journal: bioRxiv

Article Title: WASP facilitates tumor mechanosensitivity in T lymphocytes

doi: 10.1101/2023.10.02.560434

Figure Lengend Snippet: (A) Confocal images showing a s ide view of F-actin in the B16-pmel-1 cell synapses. Pmel-1 cells transfected with either GFP-WASP (WT) or GFP-WASPΔC (WASPΔC) were allowed to form synapses with B16 cells for 5min. The graphs show F-actin foci and phospho-CD3ζ intensity at B16-pmel-1 synapses. Each point in the scatter plot represents the values obtained from individual cells; the bars show Mean± SEM. This experiment was repeated thrice with similar results. p-values were obtained using Mann-Whitney two-tailed test; p***<0.0001, and **<0.005. (B) Cytotoxic activity of WT or WASPΔC expressing pmels. The data points in the graph represent Mean ± SEM from four replicates. (C) Images showing actin foci and pCasL levels in synapses formed between B16 cells and WT or WASP-/- pmel-1 CTLs, either expressing empty control vector (‘WT or WASP-/-’) or WASP construct. The graph shows the percentage of cells with discernable actin foci in the synapse. (D) Cytotoxic activity of WASP overexpressing pmels compared to the WT or WASP-/- pmels. The experiment was conducted as described in (B). Scale bar in the images, 5µm.

Article Snippet: For both synapse and cytotoxicity assays, B16 cells were incubated with 100ng/ml of IFNγ overnight, pulsed with 10µM GP100 peptide (25-33; EGSRNQDWL; Anaspec AS-64752) for 1h, washed and utilized for the experiments.

Techniques: Transfection, MANN-WHITNEY, Two Tailed Test, Activity Assay, Expressing, Plasmid Preparation, Construct

Journal: bioRxiv

Article Title: WASP facilitates tumor mechanosensitivity in T lymphocytes

doi: 10.1101/2023.10.02.560434

Figure Lengend Snippet: Surface expression of LFA-1 (A), CD69 (B), or cytokine production (C-D) was assessed after activation of CD8+T cells with indicated reagents for 24h. (E) The activation-induced proliferation of CD8+T cells was determined using a CFSE dilution assay. All these experiments were repeated at least three times with similar results. The bar graphs represent mean ± SEM. p-value *<0.05; ***<0.005, as determined by paired two-way t-test. (F, G) C57BL/6 mice lacking WASP in their immune cells show significantly higher growth of tumors. WT or WASP-/- animals were subcutaneously injected with 0.5 million B16 cells. Tumor growth was measured using the calipers method at the indicated time points. Each data point represents data from at least six animals, and the experiment was repeated four times. (H) Adoptive transfer of WT or WASP-/- pmel-1 CTLs (left graph) or OTI CTLs in animals bearing B16 (left panels) or B16F10-ova (right panels) tumors respectively. Tumor growth was measured and plotted as described in (I-J). This experiment was repeated thrice with similar results.

Article Snippet: For both synapse and cytotoxicity assays, B16 cells were incubated with 100ng/ml of IFNγ overnight, pulsed with 10µM GP100 peptide (25-33; EGSRNQDWL; Anaspec AS-64752) for 1h, washed and utilized for the experiments.

Techniques: Expressing, Activation Assay, Dilution Assay, Injection, Adoptive Transfer Assay