human gp100 25–33 peptide  (GenScript corporation)

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

Journal: Oncoimmunology

Article Title: Intracavitary adoptive transfer of IL-12 mRNA-engineered tumor-specific CD8 + T cells eradicates peritoneal metastases in mouse models

doi: 10.1080/2162402X.2022.2147317

Figure Lengend Snippet: IL-12 mRNA-armoring of T cells enhances the anti-tumor effect of OT-I in other tumor models and of the low affinity TCR tumor-specific T cells. a . Survival follow-up of mice ( n = 8/group) that were IP injected with PBS, OT-I-LUC or OT-I-IL-12 in IP-bearing PANC02-OVA mice is shown. Treatment days are indicated by the dashed lines. b-d . Mice were inoculated with 2.5 × 10 5 B16-F10 intraperitoneally b . Mice were treated with 2.5 × 10 6 PMEL-1 T cells on days 6 and 9, and their survival was monitored ( n = 8). c . 19 h after IP injection of PBS, PMEL-1-LUC or PMEL-1-IL-12, the concentration of 8 cytokines was measured in the peritoneal lavage fluid using a ProcartaPlex multiplex immunoassay ( n = 5/group). d . Mice were IP injected either with saline solution or with two doses of OT-I-IL-12. Adoptive transfer therapy was combined either with a Rat IgG2a isotype control or with antibody against PD-1 (RMP1-14 clone) or CD137 (3H3 clone) on days 6-9-12-15. Data are given as mean ± SD. Statistical significance was determined with one-way Anova with Tukey’s multiple comparison test for panel C. Survival differences between groups in panels a, b , and d were analyzed using log-rank tests (Mantel-Cox). (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: PMEL-1 T cells were resuspended at a concentration of 1.5 × 10 6 cells/ml in complete RPMI medium supplemented with 100 ng/ml of human gp100 peptide 25-33 (GenScript), for 48 h in the incubator.

Techniques: Injection, Concentration Assay, Multiplex Assay, Saline, Adoptive Transfer Assay, Control, Comparison

Journal: Cells

Article Title: Pro- and Anti-Tumoral Factors Involved in Total Body Irradiation and Interleukin-2 Conditioning in Adoptive T Cell Therapy of Melanoma-Bearing Rag1 Knock-Out Mice

doi: 10.3390/cells11233894

Figure Lengend Snippet: Anti-melanoma effect of total body irradiation and interleukin-2 treatment in adoptive T cell therapy. ( A ) Schematic drawing of the experiment. Rag1 knock-out mice were subcutaneously inoculated with B16-F10 melanoma and treated with activated Pmel-1 as a form of adoptive T cell therapy. Pmel-1 stimulated for 2 days was administered into the mice on day 5. On day 3, some mice were exposed to 4 Gy total body irradiation (TBI). The interleukin-2 (IL-2) treatment group was injected daily (intraperitoneally) with 10,000 IU IL-2 on day 5 to day 7. ( B ) Tumor growth rate measured for 100 days. Each symbol and error bar indicate the mean and standard error of the mean (s.e.m.) of the tumor size in the same group. ( C ) Tumor growth rate of each mouse is indicated. ( D ) Kaplan–Meier curves showing the survival rate of the mice. ( E ) Representative images of the surviving mice in the TBI + Pmel-1 and TBI + Pmel-1 + IL-2 groups on day 80. Arrows indicate the tumor inoculation sites. UnTx (untreated) group, n = 7 mice; Pmel-1 and TBI + Pmel-1 groups, n = 5 mice per group; Pmel-1 + IL-2 and TBI + Pmel-1 + IL-2 groups, n = 6 mice per group. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The cells were stimulated with 5 μg/mL human gp100 25–33 peptide (KVPRNQDWL; Peptron, Daejeon, Korea) in RPMI1640 media containing 10% FBS, antibiotics, and 5% of CD8-depleted splenocytes.

Techniques: Irradiation, Knock-Out, Injection

Journal: Cells

Article Title: Pro- and Anti-Tumoral Factors Involved in Total Body Irradiation and Interleukin-2 Conditioning in Adoptive T Cell Therapy of Melanoma-Bearing Rag1 Knock-Out Mice

doi: 10.3390/cells11233894

Figure Lengend Snippet: Analysis of adoptively transferred T cells in lymphoid tissues. ( A ) Schematic drawing of the experiment. Adoptive T cell transfer, total body irradiation (TBI), and interleukin-2 (IL-2) treatment were conducted as in A. On day 14, the inguinal tumor-draining lymph nodes (TdLNs) and spleen were collected and analyzed. ( B ) Total count of the collected cells in the TdLNs and spleen. ( C ) Gating strategy of flow cytometry analysis. Among the acquired cell data, viable cells were gated in the FSC-A/FVS700 plot. Singlets were further gated in the FSC-A/FSC-H and SSC-A/SSC-H plots prior to the analyses of relevant markers. ( D ) Representative flow cytometry images showing Pmel-1 in the TdLNs and spleen. ( E ) Calculated frequency (left) and cell count (right) of Pmel-1 in lymphoid tissues. Pmel-1 and Pmel-1 + IL-2 groups, n = 3 mice per group; TBI + Pmel-1 and TBI + Pmel-1 + IL-2 groups, n = 5 mice per group. Each bar indicates the mean and standard deviation (s.d.) of each group. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The cells were stimulated with 5 μg/mL human gp100 25–33 peptide (KVPRNQDWL; Peptron, Daejeon, Korea) in RPMI1640 media containing 10% FBS, antibiotics, and 5% of CD8-depleted splenocytes.

Techniques: Irradiation, Flow Cytometry, Cell Counting, Standard Deviation

Journal: Cells

Article Title: Pro- and Anti-Tumoral Factors Involved in Total Body Irradiation and Interleukin-2 Conditioning in Adoptive T Cell Therapy of Melanoma-Bearing Rag1 Knock-Out Mice

doi: 10.3390/cells11233894

Figure Lengend Snippet: Analysis of adoptively transferred T cells in tumor tissues. ( A ) Schematic drawing of the experiment. Adoptive T cell transfer, total body irradiation (TBI), and interleukin-2 (IL-2) treatment were conducted as in A. On day 14, the inoculated tumor tissue was dissociated into a single cell suspension and analyzed using flow cytometry. ( B ) Gating strategy of flow cytometry analysis. ( C ) Representative flow cytometry images showing tumor-infiltrating Pmel-1. Among the acquired cell data, viable cells were gated in the FSC-A/FVS700 plot. Singlets were further gated in the SSC-A/SSC-H and FSC-A/FSC-H plots prior to the analyses of relevant markers. ( D ) Determined frequency of Pmel-1 in tumor tissue. ( E ) Representative flow cytometry images showing PD-1 expression on Pmel-1. ( F ) Determined frequency of PD-1 + cells within the Pmel-1 population. Pmel-1 and Pmel-1 + IL-2 groups, n = 3 mice per group; TBI + Pmel-1 and TBI + Pmel-1 + IL-2 groups, n = 4 mice per group. Each bar indicates the mean and standard deviation (s.d.) of each group. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The cells were stimulated with 5 μg/mL human gp100 25–33 peptide (KVPRNQDWL; Peptron, Daejeon, Korea) in RPMI1640 media containing 10% FBS, antibiotics, and 5% of CD8-depleted splenocytes.

Techniques: Irradiation, Suspension, Flow Cytometry, Expressing, Standard Deviation

Journal: Cells

Article Title: Pro- and Anti-Tumoral Factors Involved in Total Body Irradiation and Interleukin-2 Conditioning in Adoptive T Cell Therapy of Melanoma-Bearing Rag1 Knock-Out Mice

doi: 10.3390/cells11233894

Figure Lengend Snippet: Analysis of the immune cell population in the spleen after adoptive T cell therapy. Adoptive T cell transfer, total body irradiation (TBI), and interleukin-2 (IL-2) treatment were conducted as in A. On day 14, the spleen was collected and subjected to multi-parameter flow cytometry analysis. ( A ) Gating strategy of the analysis. Among the acquired cell data, viable cells were gated in the FSC-A/FVS700 plot. Singlets were further gated in the SSC-A/SSC-H and FSC-A/FSC-H plots prior to the analyses of relevant markers. First, Pmel-1 (CD8b + /Thy1.1 + ) and natural killer cells (NK; NK1.1 + ) were defined among the singlets. Myeloid cells (CD11c + and/or CD11b + ) were divided into macrophages (F4/80 + ), neutrophils (Ly6G + ), and other cells (double negative) in the Ly6G/F4/80 plot. After dendritic cells (DCs; CD11c + /MHC-II + ) were excluded from the double negative subset, monocytes (Ly6C + /CD11b + ) were defined. Types 1 and 2 macrophages (M1 and M2) were gated in the CD206/MHC-II plot. ( B ) Frequency of diverse immune cell subsets in the spleen. The frequency of Pmel-1, NKs, neutrophils, macrophages, DCs, and monocytes among lineage marker-positive cells (Lin + ; positive for Thy1.1, NK1.1, CD11b, and/or CD11c) was calculated. For M1 and M2, the ratio is indicated. ( C ) Representative flow cytometry images showing conventional types 1 and 2 DCs (cDC1 and cDC2) within the DC population. ( D ) Calculated ratio between cDC1 and cDC2. Pmel-1 + IL-2 and TBI + Pmel-1 groups, n = 3 mice per group; TBI + Pmel-1 + IL-2 group, n = 4 mice per group. Each bar indicates the mean and standard deviation (s.d.) of each group. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The cells were stimulated with 5 μg/mL human gp100 25–33 peptide (KVPRNQDWL; Peptron, Daejeon, Korea) in RPMI1640 media containing 10% FBS, antibiotics, and 5% of CD8-depleted splenocytes.

Techniques: Irradiation, Flow Cytometry, Marker, Standard Deviation

Journal: Cells

Article Title: Pro- and Anti-Tumoral Factors Involved in Total Body Irradiation and Interleukin-2 Conditioning in Adoptive T Cell Therapy of Melanoma-Bearing Rag1 Knock-Out Mice

doi: 10.3390/cells11233894

Figure Lengend Snippet: Analysis of the immune cell population in the tumor after adoptive T cell therapy. Adoptive T cell transfer, total body irradiation (TBI), and interleukin-2 (IL-2) treatment were conducted as in A. On day 14, tumor tissue was collected and subjected to multi-parameter flow cytometry analysis. ( A ) Gating strategy of the analysis. Among the acquired cell data, viable cells were gated in the FSC-A/FVS700 plot. Singlets were further gated in the SSC-A/SSC-H and FSC-A/FSC-H plots prior to the analyses of relevant markers. First, Pmel-1 (CD8b + /Thy1.1 + ) and natural killer cells (NK; NK1.1 + ) were defined among the singlets. Dendritic cells (DCs; CD11c+/MHC-II+) were defined among CD11c- and/or CD11b-expressing myeloid cells. Monocytic myeloid-derived suppressor cells (Mo-MDSCs) and polymorphonuclear MDSC (PMN-MDSC) were gated in the Ly6G/Ly6C plot. ( B ) Frequency of diverse immune cell subsets in the tumor. The frequency of Pmel-1, NKs, DCs, monocytic myeloid-derived suppressor cells (Mo-MDSCs), and polymorphonuclear MDSC (PMN-MDSC) among lineage marker-positive cells (Lin + ; positive for Thy1.1, NK1.1, CD11b, and/or CD11c) was calculated. For Pmel-1, the PD-1-positive proportion is additionally indicated. ( C) Representative flow cytometry images showing monocyte-derived DCs (MoDCs) and conventional types 1 and 2 DCs (cDC1 and cDC2) within the DC population. ( D ) The frequency of cDC1, cDC2, and MoDC is indicated. Pmel-1 + IL-2 and TBI + Pmel-1 groups, n = 3 mice per group; TBI + Pmel-1 + IL-2 group, n = 4 mice per group. Each bar indicates the mean and standard deviation (s.d.) of each group. * p < 0.05; ** p < 0.01.

Article Snippet: The cells were stimulated with 5 μg/mL human gp100 25–33 peptide (KVPRNQDWL; Peptron, Daejeon, Korea) in RPMI1640 media containing 10% FBS, antibiotics, and 5% of CD8-depleted splenocytes.

Techniques: Irradiation, Flow Cytometry, Expressing, Derivative Assay, Marker, Standard Deviation