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: 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

Journal: The Journal of Experimental Medicine

Article Title: AIM2 regulates anti-tumor immunity and is a viable therapeutic target for melanoma

doi: 10.1084/jem.20200962

Figure Lengend Snippet: Vaccination with AIM2-deficient DC improves the efficacy of ACT through activation of STING–type I IFN signaling. (A) IFN-β or CXCL10 in the supernatants of indicated BMDCs stimulated with 0, 0.1, or 1 µg/ml B16F10 DNA for 4 h (IFN-β) or 10 h (CXCL10; n = 3). (B) Immunoblotting for pTBK1, TBK1, pIRF3, IRF3, and vinculin in the lysates of indicated BMDCs stimulated with 0, 0.1, or 1 µg/ml B16F10 DNA for 4 h. (C–G) B16F10-bearing WT mice (B16F10 mice) were treated with ACT alone or ACT + 1.0 × 10 6 WT, Aim2 −/− , or Aim2 −/− Sting −/− DC-gp100. On day 20 after PMELs (1.0 × 10 6 cells) transfer, tissues were harvested. (C) The therapy regimen scheme. (D) Tumor growth over time (left; n = 9). Sample photo of B16F10 tumor on day 20 after PMELs transfer (right). Scale bar, 10 mm. (E and F) Flow cytometry analysis of TILs ( n = 9). (E) The numbers of PMELs, CD8 + T cells, and CD4 + T cells among 10 4 live singlet cells, percentage of FoxP3 + cells in CD4 + T cells, and PMEL/T reg cell ratio. (F) Percentages of IFN-γ + and TNF-α + cells in PMELs. (G) IFN-β protein levels within the tumor, TdLN, and spleen ( n = 7). (H and I) B16F10 DNA–stimulated WT or Aim2 −/− DC-gp100 was cocultured with CFSE-labeled PMELs for 72 h ( n = 5). (H) Histograms of PMELs CFSE dilution. (I) Proliferation index of PMELs and amount of IFN-γ + in the supernatants. (J and K) B16F10 mice were treated with ACT using 1.0 × 10 6 CFSE-labeled PMELs + 1.0 × 10 6 WT or Aim2 −/− DC-gp100. On day 3 after PMELs transfer, spleens were harvested. (J) The therapeutic regimen. (K) Histograms of PMELs CFSE dilution, proliferation index of PMELs, and numbers of PMELs among 10 4 live singlet cells in the spleen ( n = 6 or 7). Data are shown as mean ± SEM and are pooled from three (A and D–G) or two (I and K) experiments or are representative of at least two independent experiments (B, H, and K). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; two-way ANOVA with Tukey’s multiple-comparisons test (D), one-way ANOVA with Dunnett’s (A and I) or Tukey’s (E, F, and K) multiple-comparisons test, or Mann–Whitney test (G and I).

Article Snippet: For the generation of peptide-pulsed DC vaccine (DC-gp100), nonadherent cells were pulsed for 3 h at 37°C in 5% CO 2 with 10 μM of the human gp100 25–33 (hgp100) peptide (GenScript) in Opti-MEM medium (Gibco) and washed three times with PBS before their use.

Techniques: Activation Assay, Western Blot, Flow Cytometry, Labeling, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: AIM2 regulates anti-tumor immunity and is a viable therapeutic target for melanoma

doi: 10.1084/jem.20200962

Figure Lengend Snippet: The effect of AIM2-deficient DC vaccine with ACT on tumor, TdLN, and spleen in the B16F10 model. (A) Quantitative RT-PCR analysis of Ifnb , Ifna , Cxcl10 , and Cxcl9 mRNA expression in indicated BMDCs stimulated with 0, 0.1, or 1 µg/ml B16F10 DNA for 4 h ( n = 3), presented in AU, relative to Actb (encoding β-actin) expression. (B) Experimental scheme for analyzing DC vaccine infiltration in the tumor, TdLN, and spleen. B16F10-bearing CD45.1 congenic B6 mice were treated with ACT using 1.0 × 10 6 PMELs (CD45.2) + 1.0 × 10 6 WT or Aim2 −/− DC-gp100 (CD45.2), and tissues were harvested 1.5 d after PMELs transfer. (C) The absolute numbers of transferred DCs present in the tumor, TdLN, and spleen ( n = 8). (D and E) Flow cytometry analysis of the percentage of FoxP3 − cells in total CD4 + T cells, numbers of MACs, DCs, CD103 + DCs, and CD11b + DCs among 10 4 live singlet cells in the tumor (D), numbers of PMELs, CD8 + T cells, CD4 + T cells among 10 4 live singlet cells, and percentages of FoxP3 + cells in CD4 + T cells in the TdLN and spleen (E) of B16F10 mice treated with ACT + WT, Aim2 −/− , or Aim2 −/− Sting −/− DC-gp100 ( n = 9). (F and G) Flow cytometry staining of CD11b and CD103 (F) and the mean fluorescence intensity (MFI) of MHC class I (MHC-I), CD86, or CD80 (G) on freshly generated WT and Aim2 −/− BMDCs ( n = 8). Data are shown as mean ± SEM and are pooled from three (A and C–E) or two (G) independent experiments or are representative of two independent experiments (F). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-way ANOVA with Dunnett’s (A) or Tukey’s (D and E) multiple-comparisons test or Mann–Whitney test (C and G). FMO, fluorescence minus one control.

Article Snippet: For the generation of peptide-pulsed DC vaccine (DC-gp100), nonadherent cells were pulsed for 3 h at 37°C in 5% CO 2 with 10 μM of the human gp100 25–33 (hgp100) peptide (GenScript) in Opti-MEM medium (Gibco) and washed three times with PBS before their use.

Techniques: Quantitative RT-PCR, Expressing, Flow Cytometry, Staining, Fluorescence, Generated, MANN-WHITNEY, Control

Journal: The Journal of Experimental Medicine

Article Title: AIM2 regulates anti-tumor immunity and is a viable therapeutic target for melanoma

doi: 10.1084/jem.20200962

Figure Lengend Snippet: Enhanced anti-melanoma immunity of vaccination with AIM 2 -deficient DCs is dependent on the recognition of tumor-derived DNA and independent of prolonged cell survival of vaccinated DCs. (A–C) B16F10 mice were treated with ACT + WT or Aim2 −/− DC-gp100 and intratumoral (i.t.) administration of DNase I or PBS. On day 20 after PMEL transfer, tissues were harvested. (A) Therapy regimen scheme. (B) Tumor growth over time (left; n = 9). Sample photo of B16F10 tumor on day 20 after PMELs transfer (right). Scale bar, 10 mm. (C) Flow cytometry analysis of the numbers of PMELs, CD8 + T cells, and CD4 + T cells among 10 4 live singlet cells, percentage of FoxP3 + cells in CD4 + T cells, and PMEL/T reg cell ratio in the tumor ( n = 9). (D) Experimental scheme for analyzing DC vaccine infiltration in the tumor, TdLN, and spleen. B16F10-bearing CD45.1 congenic B6 mice were treated with ACT using 1.0 × 10 6 PMELs (CD45.2) + 1.0 × 10 6 WT or Aim2 −/− DC-gp100 (CD45.2), and tissues were harvested on day 10 ( n = 7) and day 20 ( n = 8) after PMELs transfer. (E) Representative contour plot for CD45.2 + Thy1.1 − CD11c + MHC-II + DC-gp100 (DC vaccine) present at the tumor, TdLN, and spleen on day 20 after PMELs transfer. (F) The absolute number of vaccinated DCs present in the tumor, TdLN, and spleen on days 10 ( n = 7) and 20 ( n = 8) after PMELs transfer. Data are shown as mean ± SEM and are pooled from four (B and C) or three (F) independent experiments or are representative of three independent experiments (E). *, P < 0.05; **, P < 0.01; ***, P < 0.001; two-way ANOVA with Tukey’s multiple-comparisons test (B), one-way ANOVA with Tukey’s multiple-comparisons test (C), or Mann–Whitney test (F).

Article Snippet: For the generation of peptide-pulsed DC vaccine (DC-gp100), nonadherent cells were pulsed for 3 h at 37°C in 5% CO 2 with 10 μM of the human gp100 25–33 (hgp100) peptide (GenScript) in Opti-MEM medium (Gibco) and washed three times with PBS before their use.

Techniques: Derivative Assay, Flow Cytometry, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: AIM2 regulates anti-tumor immunity and is a viable therapeutic target for melanoma

doi: 10.1084/jem.20200962

Figure Lengend Snippet: The role of DNA sensing, IFNAR, and CXCL10 in AIM2-deficient DC vaccine with ACT on tumor, TdLN, and spleen in the B16F10 model. (A and B) Flow cytometry analysis of the numbers of PMELs, CD8 + T cells (A), and CD4 + T cells among 10 4 live singlet cells and percentages of FoxP3 + cells in CD4 + T cells (B) in the TdLN and spleen of B16F10 mice treated with ACT + WT or Aim2 −/− DC-gp100 and intratumoral administration of DNase I or PBS ( n = 9). (C–E) Flow cytometry analysis of the percentage of FoxP3 − cells in total CD4 + T cells, numbers of CD103 + and CD11b + DCs among 10 4 live singlet cells in the tumor (C), numbers of PMELs, CD8 + T cells (D), and CD4 + T cells among 10 4 live singlet cells and percentages of FoxP3 + cells in CD4 + T cells (E) in the TdLN and spleen of B16F10 mice treated with ACT + WT, Aim2 −/− , Aim2 −/− Ifnar −/− , or Aim2 −/− Cxcl10 −/− DC-gp100 ( n = 10 or 11). Data are shown as mean ± SEM and are pooled from four (A and B) or three (C–E) independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-way ANOVA with Tukey’s multiple-comparisons test (A–E).

Article Snippet: For the generation of peptide-pulsed DC vaccine (DC-gp100), nonadherent cells were pulsed for 3 h at 37°C in 5% CO 2 with 10 μM of the human gp100 25–33 (hgp100) peptide (GenScript) in Opti-MEM medium (Gibco) and washed three times with PBS before their use.

Techniques: Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: AIM2 regulates anti-tumor immunity and is a viable therapeutic target for melanoma

doi: 10.1084/jem.20200962

Figure Lengend Snippet: AIM2-deficient DC vaccination facilitates tumor antigen–specific CD8 + T cell infiltration into the tumor via IFNAR signaling and CXCL10 production. (A) IFN-β or CXCL10 in the supernatants of indicated BMDCs stimulated with 0, 0.1, or 1 µg/ml B16F10 DNA for 4 (IFN-β) or 10 h (CXCL10; n = 3). (B–D) B16F10 mice were treated with ACT + WT, Aim2 −/− , Aim2 −/− Ifnar −/− , or Aim2 −/− Cxcl10 −/− DC-gp100. On day 20 after PMELs transfer, tissues were harvested ( n = 10 or 11). (B) Tumor growth over time. (C and D) Flow cytometry analysis of TILs. (C) The numbers of PMELs, CD8 + T cells, and CD4 + T cells among 10 4 live singlet cells, percentages of FoxP3 + cells in CD4 + T cells, and PMEL/T reg cell ratio. (D) The percentages of IFN-γ + and TNF-α + in CD8 + T cells. (E–G) Similar analysis as in B–D was performed on B16F10 mice treated by ACT with WT, Aim2 −/− Cxcl10 −/− , or Cxcl10 −/− DC-gp100 ( n = 8 or 9). Data are shown as mean ± SEM and are pooled from three independent experiments (A–G). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; two-way ANOVA with Tukey’s multiple-comparisons test (B and E) or one-way ANOVA with Dunnett’s (A) or Tukey’s (C, D, and F) multiple-comparisons test.

Article Snippet: For the generation of peptide-pulsed DC vaccine (DC-gp100), nonadherent cells were pulsed for 3 h at 37°C in 5% CO 2 with 10 μM of the human gp100 25–33 (hgp100) peptide (GenScript) in Opti-MEM medium (Gibco) and washed three times with PBS before their use.

Techniques: Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: AIM2 regulates anti-tumor immunity and is a viable therapeutic target for melanoma

doi: 10.1084/jem.20200962

Figure Lengend Snippet: Reduced IL-1β and IL-18 production by AIM2-deficient DC vaccination restricts T reg cell infiltration into the tumor. (A) IL-1β, IL-18, IFN-β, and CXCL10 in the supernatants of indicated BMDCs stimulated with 0, 0.1, or 1 µg/ml B16F10 DNA for 4 (IFN-β) or 10 h (IL-1β, IL-18, and CXCL10; n = 3). (B–E) B16F10 mice were treated with ACT + WT, Aim2 −/− , or Il1β −/− DC-gp100. On day 20 after PMELs transfer, tissues were harvested ( n = 12–14). (B) Tumor growth over time. (C–E) Flow cytometry analysis of TILs. The numbers of PMELs, CD8 + T cells (C), and CD4 + T cells among 10 4 live singlet cells, percentage of FoxP3 + cells in CD4 + T cells, PMEL/T reg ratio (D), and the percentages of IFN-γ + and TNF-α + (E) in CD8 + T cells. (F–I) B16F10 mice were treated with ACT + WT, Aim2 −/− , or Il18 −/− DC-gp100. On day 20 after PMELs transfer, tissues were harvested ( n = 8 or 9), and similar analysis as in B–E was performed. Data are shown as mean ± SEM and are pooled from three independent experiments (A–I). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; two-way ANOVA with Tukey’s multiple-comparisons test (B and F) or one-way ANOVA with Dunnett’s (A) or Tukey’s (C–E and G–I) multiple-comparisons test.

Article Snippet: For the generation of peptide-pulsed DC vaccine (DC-gp100), nonadherent cells were pulsed for 3 h at 37°C in 5% CO 2 with 10 μM of the human gp100 25–33 (hgp100) peptide (GenScript) in Opti-MEM medium (Gibco) and washed three times with PBS before their use.

Techniques: Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: AIM2 regulates anti-tumor immunity and is a viable therapeutic target for melanoma

doi: 10.1084/jem.20200962

Figure Lengend Snippet: Effect of IL-1β– and IL-18–deficient DC vaccine, as well as Aim2 siRNA–transfected WT DC vaccine with ACT on tumor, TdLN, and spleen in the B16F10 model. (A–C) Flow cytometry analysis of the percentage of FoxP3 − cells in total CD4 + T cells, numbers of CD103 + and CD11b + DCs among 10 4 live singlet cells in the tumor (A), numbers of PMELs, CD8 + T cells (B), and CD4 + T cells among 10 4 live singlet cells, and percentages of FoxP3 + cells in CD4 + T cells (C) in the TdLN and spleen of B16F10 mice treated with ACT + WT, Aim2 −/− , or Il1β −/− DC-gp100 ( n = 12–14). (D–F) Similar analysis as in A–C was performed on B16F10 mice treated with ACT + WT, Aim2 −/− , or Il-18 −/− DC-gp100 ( n = 9). (G) Flow cytometry analysis of the numbers of PMELs, CD8 + T cells, and CD4 + T cells among 10 4 live singlet cells and percentages of FoxP3 + cells in CD4 + T cells in the TdLN and spleen of B16F10 mice treated with ACT with control- or Aim2 siRNA–transfected DC-gp100 ( n = 9). Data are shown as mean ±SEM and are pooled from three (A–F) or two (G) independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; one-way ANOVA with Tukey’s (A–F) or Dunnett’s (G) multiple-comparisons test.

Article Snippet: For the generation of peptide-pulsed DC vaccine (DC-gp100), nonadherent cells were pulsed for 3 h at 37°C in 5% CO 2 with 10 μM of the human gp100 25–33 (hgp100) peptide (GenScript) in Opti-MEM medium (Gibco) and washed three times with PBS before their use.

Techniques: Transfection, Flow Cytometry, Control

Journal: The Journal of Experimental Medicine

Article Title: AIM2 regulates anti-tumor immunity and is a viable therapeutic target for melanoma

doi: 10.1084/jem.20200962

Figure Lengend Snippet: AIM2-silenced DC vaccine improves the efficacy of ACT against melanoma. (A) Immunoblotting for AIM2 and vinculin in the lysates of mock-, control siRNA–, or Aim2 siRNA– (-1 or -2) transfected WT BMDCs 48 h after transfection. (B) Quantitative RT-PCR analysis of the Aim2 mRNA expression in mock-, control siRNA–, or Aim2 siRNA–transfected WT BMDCs 2, 10, and 22 d after transfection ( n = 6). (C–E) B16F10 mice were treated with ACT + control siRNA– or Aim2 siRNA–transfected WT DC-gp100. On day 20 after PMELs transfer, tissues were harvested. (C) Therapy regimen scheme. (D) Tumor growth over time (left; n = 9). Sample photo of B16F10 tumor on day 20 after PMELs transfer (right). Scale bar, 10 mm. (E) Flow cytometry analysis of the numbers of PMELs, CD8 + , and CD4 + T cells among 10 4 live singlet cells, percentage of FoxP3 + cells in CD4 + T cells, and PMEL/T reg cell ratio in the tumor ( n = 9). Data are shown as mean ± SEM and are representative of three independent experiments (A) or are pooled from two independent experiments (B, D, and E). *, P < 0.05; **, P < 0.01; ***, P < 0.001; two-way ANOVA with Tukey’s multiple-comparisons test (D) or one-way ANOVA with Dunnett’s multiple-comparisons test (B and E).

Article Snippet: For the generation of peptide-pulsed DC vaccine (DC-gp100), nonadherent cells were pulsed for 3 h at 37°C in 5% CO 2 with 10 μM of the human gp100 25–33 (hgp100) peptide (GenScript) in Opti-MEM medium (Gibco) and washed three times with PBS before their use.

Techniques: Western Blot, Control, Transfection, Quantitative RT-PCR, Expressing, Flow Cytometry