human gp100 25–33 (hgp100) peptide  (GenScript corporation)

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

Journal: Cellular and Molecular Immunology

Article Title: Chronic activation of 4-1BB signaling induces granuloma development in tumor-draining lymph nodes that is detrimental to subsequent CD8 + T cell responses

doi: 10.1038/s41423-020-00533-3

Figure Lengend Snippet: Normal trafficking of pmel-1 CD8 + T cells following 4-1BB triggering in vivo. C57BL/6 mice were injected intravenously with CFSE-labeled pmel-1 Thy1.1 + CD8 + T cells ( a , c , d ) or CellTrace Violet-labeled S1PR1-GFP × pmel-1 Thy1.1 + CD8 + T cells ( b ), immunized with hgp100 peptide in IFA, and further stimulated with rat IgG or anti-4-1BB mAb at days 0 and 2. Inguinal LN cells of the mice that received pmel-1 Thy1.1 + CD8 + T cells were stained with the indicated mAbs at day 5, and gated Thy1.1 + cells are plotted CFSE vs. PD-1, LAG3, KLRG-1, CD62L or CCR7 ( a ). Inguinal LN cells of the mice that received S1PR1-GFP × pmel-1 Thy1.1 + CD8 + T cells were stained with anti-Thy1.1-PE on day 5, and gated Thy1.1 + cells were plotted CellTrace Violet vs. S1PR1-GFP ( b ). Percentage and absolute number of pmel-1 Thy1.1 + CD8 + T cells in inguinal LNs of the mice that received pmel-1 Thy1.1 + CD8 + T cells at day 5 ( c ). Division rates of pmel-1 Thy1.1 + CD8 + T cells of rat IgG- or anti-4-1BB-treated mice ( d ). e – g CFSE-labeled naive pmel-1 Thy1.1 + CD8 + T cells were adoptively transferred to B6 mice 7 days after the B16-F10 challenge and simultaneously immunized with hgp100 peptide in IFA. Rat IgG or anti-4-1BB mAb was intraperitoneally injected into the mice on days 7 and 9. The TDLNs and tumor tissues were collected from the mice 4 or 7 days after peptide immunization, counted, and stained with fluorochrome-conjugated anti-Thy1.1, anti-CD8, and anti-CD45 mAbs. Gated CD8 + T cells were plotted as Thy1.1 vs. CFSE ( f ). The percentage and absolute number of Thy1.1 + CD8 + T cells in the TDLNs ( g ). The percentage of transferred Thy1.1 + and endogenous Thy1.1 − CD8 + T cells in the tumor tissues ( h ). Data are from three ( a – d ) and two ( e – h ) independent experiments with 5–6 mice per experiment. Student’s t test was performed in b and shown as the means ± SDs (* p < 0.05; ** p < 0.01; *** p < 0.005)

Article Snippet: Human gp100 25–33 (hgp100, KVPRNQDWL) peptides were synthesized by Peptron (Daejeon, Korea).

Techniques: In Vivo, Injection, Labeling, Staining

Journal: Cellular and Molecular Immunology

Article Title: Chronic activation of 4-1BB signaling induces granuloma development in tumor-draining lymph nodes that is detrimental to subsequent CD8 + T cell responses

doi: 10.1038/s41423-020-00533-3

Figure Lengend Snippet: Proliferation and trafficking of pmel-1 CD8 + T cells and tumor growth in mice pretreated with anti-4-1BB mAb. a – f B16-bearing C57BL/6 mice were treated with rat IgG or anti-4-1BB mAb at days 5 and 10 and received CFSE-labeled naive Thy1.1 + CD8 + T cells at day 14. TLDNs and tumor tissues were analyzed at days 15 and 21. a Schematic diagram of the experiment. b The inguinal TDLNs were collected from the mice 1 or 7 days after CD8 + T cell transfer, stained with fluorochrome-conjugated anti-CD8 and anti-Thy1.1 mAb, and further stained with 7-AAD. Single-cell suspensions of tumor tissues were stained with fluorochrome-conjugated anti-CD45, anti-CD8, and anti-Thy1.1 mAbs. All samples were subsequently analyzed by FACSCalibur (BD Bioscience). c Total cell numbers at day 21. d Percentages of dividing and nondividing pmel-1 Thy1.1 + CD8 + T cells in TDLNs at day 21. e Absolute numbers of dividing and nondividing pmel-1 Thy1.1 + CD8 + T cells in TDLNs at day 21. f Percentages of Thy1.1 − CD8 + TILs in CD45 + cells from tumor tissues at day 21. g – h C57BL/6 mice were immunized with 20 μg OVA in IFA and injected with anti-4-1BB mAb at days 3, 6, and 9. On day 20, the untreated (UnTx) or the OVA + mAb-treated (Ab-Tx) mice were injected subcutaneously with MC38 tumor cells and further received rat IgG or anti-4-1BB mAb every 5 days from day 3 after the tumor challenge. Tumor growth rates were monitored every 3–4 days. Data are from two ( b – f ) or three ( h ) independent experiments with five mice per experiment. Student’s t test was performed in c – f and shown as the means ± SDs (* p < 0.05; ** p < 0.01)

Article Snippet: Human gp100 25–33 (hgp100, KVPRNQDWL) peptides were synthesized by Peptron (Daejeon, Korea).

Techniques: Labeling, Staining, Injection

Journal: Nature Metabolism

Article Title: Inosine is an alternative carbon source for CD8 + -T-cell function under glucose restriction

doi: 10.1038/s42255-020-0219-4

Figure Lengend Snippet: Naive CD8 + T cells from C57BL/6 mice were activated by plate-bound anti-CD3 and anti-CD28 antibodies in complete medium for 24 h, and then the cells were switched to the indicated conditioned media (either glucose free, containing glucose or containing inosine) and were cultured for 48 h. a , b , Cell proliferation ( a ) and cell death ( b ) were determined by carboxyfluorescein succinimidyl ester (CFSE) dilution and 7-aminoactinomycin D (7-AAD) uptake, respectively. Data are presented as mean ± s.d. ( n = 6). *** P = 4 × 10 –7 for glucose free versus inosine. Data are representative of three independent experiments. M denotes million. c , Mouse T eff cells were cultured in glucose-containing medium in the presence or absence of inosine for 72 h, and cell survival was determined by 7-AAD uptake. Data are presented as mean ± s.d. ( n = 4). Data are representative of three independent experiments. d , Splenocytes from Pmel transgenic mice were isolated and cultured with 1 μM human gp100 and 30 U ml –1 recombinant human IL-2 in complete medium for 4 d, and were switched to the indicated conditioned media for 72 h. B16 melanoma cells were co-cultured with activated Pmel T cells, and the percentage of tumour-cell lysis was determined by calcein release with the Spectramax M2 microplate reader. Data are presented as mean ± s.d. ( n = 3). ** P = 0.0025 and 0.0053 for effector to target (E:T) ratio 10:1 and 5:1 for glucose free versus inosine, respectively. e , Naive CD8 + T cells from C57BL/6 mice were activated by plate-bound anti-CD3 and anti-CD28 antibodies and were differentiated in the indicated conditioned media for 4 d. The indicated proteins were quantified by intracellular staining following phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation. Data are presented as mean ± s.d. ( n = 4). *** P = 0.0003, 0.00005, 0.0006 for granzyme B, TNF-α and IFN-γ for glucose free versus inosine, respectively. Data were analysed by unpaired two-sided t -test ( b , d , e ). Sample size ( n ) represents biologically independent samples ( b–e ).

Article Snippet: For CD8 + -T-cell activation and culture, naive CD8 T cells were activated by plate-bound antibodies and incubated with recombinant mouse IL-2 (100 U ml –1 ) and IL-12 (5 ng ml –1 ) for 5 d. For Pmel + CD8 + -T-cell activation and culture, splenocytes from Pmel transgenic mice were isolated and cultured with 1 μM human gp100 (hgp100) (GenScript) and 30 U ml –1 recombinant human IL-2 (PeproTech) in complete medium for 5 d , .

Techniques: Cell Culture, Transgenic Assay, Isolation, Recombinant, Lysis, Staining

Journal: Nature Metabolism

Article Title: Inosine is an alternative carbon source for CD8 + -T-cell function under glucose restriction

doi: 10.1038/s42255-020-0219-4

Figure Lengend Snippet: a , Naive CD8+ T cells from C57BL/6 mice were activated by plate-bound anti-CD3 and anti-CD28 antibodies in the indicated conditional media for 3 days. Cell proliferation and cell death of active CD8+ T cells were determined by CFSE dilution and 7AAD uptake, respectively. Data are presented as mean ± SD (n=6). NS, P =0.4668 for Glucose free versus Adenosine. b , B16 melanoma cells were co-cultured with activated Pmel T cell that are generated in complete media followed by 72 h incubation in the indicated conditional media and the percentage of tumor cell lysis was determined by calcein release with the Spectramax M2 microplate reader. Data are presented as mean ± SD (n=4). NS, P =0.4107 and 0.2385 for E:T ratio 10:1 and 5:1 for Glucose free versus Adenosine, respectively. c , Naive CD8+ T cells from C57BL/6 mice were activated by plate bound anti-CD3 and anti-CD28 antibodies and differentiated in the indicated conditional media for 4 days. The indicated proteins were quantified by intracellular staining following PMA and ionomycin stimulation. Data are presented as mean ± SD (n=4). NS, P =0.6305, 0.6906, 0.9495 for Granzyme B, TNF-α, and IFN-γ for Glucose free versus Adenosine, respectively. d , Naive CD8+ T cells from C57BL/6 mice were activated in the indicated conditional media for 3 days. Cell proliferation and cell death of active CD8+ T cells were determined by CFSE dilution and 7AAD uptake, respectively. Data are presented as mean ± SD (n=6). ***, P =2E-10 for Inosine versus Inosine+Adenosine. Data were analyzed by unpaired two-sided t-test ( a – d ). Sample size (n) represents biologically independent samples ( a - d ).

Article Snippet: For CD8 + -T-cell activation and culture, naive CD8 T cells were activated by plate-bound antibodies and incubated with recombinant mouse IL-2 (100 U ml –1 ) and IL-12 (5 ng ml –1 ) for 5 d. For Pmel + CD8 + -T-cell activation and culture, splenocytes from Pmel transgenic mice were isolated and cultured with 1 μM human gp100 (hgp100) (GenScript) and 30 U ml –1 recombinant human IL-2 (PeproTech) in complete medium for 5 d , .

Techniques: Cell Culture, Generated, Incubation, Lysis, Staining

Journal: Nature Metabolism

Article Title: Inosine is an alternative carbon source for CD8 + -T-cell function under glucose restriction

doi: 10.1038/s42255-020-0219-4

Figure Lengend Snippet: a , Diagram of the PNP inhibitor foro blocking the breakdown of inosine into hypoxanthine and R1P. b , PNP protein and messenger RNA expression levels in mouse T cells at the indicated time points following activation were determined by immunoblot (top) and quantitative PCR (bottom), respectively. Data are representative of two independent experiments. c , Bar graph representing mouse T-cell bioenergetic activity in the indicated conditioned media. Activated mouse T cells were incubated without glucose (background), with glucose or with inosine, as well as with glucose or inosine in combination with increased concentrations of foro (0.1 μM, 0.5 μM and 2 μM) for 24 h, followed by Biolog redox dye mix MB incubation, and were measured spectrophotometrically at 590 nm. d , Bar graph representing cell-survival percentages in the indicated condidioned media. Naive CD8 + cells from C57BL/6 mice were activated as previously described in complete medium for 24 h, and then the cells were switched to the indicated conditioned media in combination with 2 μM foro for 72 h. Cell proliferation and cell death were determined by CFSE dilution (top) and 7-AAD uptake (bottom), respectively. *** P = 0.000009 for inosine versus inosine + foro. e , Naive CD8 + T cells from C57BL/6 mice were activated and cultured in the presence of glucose or inosine and were treated with MTX for 72 h. Cell death was determined by 7-AAD uptake. *** P = 5.68 × 10 –7 , 1.23 × 10 –7 , 1.07 × 10 –7 and 3.19 × 10 –10 , from left to right. f , B16 melanoma cells were co-cultured with activated Pmel + T cells generated in the presence of inosine with or without foro, and the percentage of tumour-cell lysis was determined by calcein release. *** P = 0.000008 and 0.000007 for E:T ratios 10:1 and 5:1, respectively. g , Naive CD8 + T cells from C57BL/6 mice were activated as previously described and differentiated in the indicated conditioned media with or without 2 μM foro for 4 d. The indicated proteins were quantified by intracellular staining following PMA and ionomycin stimulation. Data are presented as mean ± s.d. ( n = 3 for b , f ; n = 4 for c – e , g ). ** P = 0.0042, 0.0048 and 0.0056 for granzyme B, TNF-α and IFN-γ for inosine versus inosine + foro. Data were analysed by unpaired two-sided t -test ( d – g ). Sample size ( n ) represents biologically independent samples ( b – g ).

Article Snippet: For CD8 + -T-cell activation and culture, naive CD8 T cells were activated by plate-bound antibodies and incubated with recombinant mouse IL-2 (100 U ml –1 ) and IL-12 (5 ng ml –1 ) for 5 d. For Pmel + CD8 + -T-cell activation and culture, splenocytes from Pmel transgenic mice were isolated and cultured with 1 μM human gp100 (hgp100) (GenScript) and 30 U ml –1 recombinant human IL-2 (PeproTech) in complete medium for 5 d , .

Techniques: Blocking Assay, RNA Expression, Activation Assay, Western Blot, Real-time Polymerase Chain Reaction, Activity Assay, Incubation, Cell Culture, Generated, Lysis, Staining

Journal: Nature Metabolism

Article Title: Inosine is an alternative carbon source for CD8 + -T-cell function under glucose restriction

doi: 10.1038/s42255-020-0219-4

Figure Lengend Snippet: a , A murine melanoma xenograft model was established in C57BL/6 mice by subcutaneous inoculation of B16-F10 tumour cells. The indicated experimental mice were treated with IgG control (200 μg, intraperitoneal (i.p.) injection twice per week), inosine (300 mg per kg (body weight), oral gavage daily), anti-PDL1 antibody (200 μg, i.p. twice per week) or anti-PDL1 antibody (200 μg, i.p. twice per week) + inosine (300 mg per kg (body weight), oral gavage daily). Tumour size and mouse survival were monitored. Data represent mean ± s.d. ( n = 10). *** P < 0.0001 for anti-PDL1 versus anti-PDL1 + inosine, by two-way ANOVA (left). ** P = 0.0018 for anti-PDL1 versus anti-PDL1 + inosine (right), by one-sided Mantel–Cox test. b , C57BL/6 mice were injected (subcutaneously) with B16-F10 melanoma cells and were sublethally irradiated (500 cGy) at day 6 after tumour-cell inoculation. One day later, mice were i.v. injected with activated Pmel CD8 + T cells (4 × 10 6 cells per mouse). Mice were administered inosine (300 mg per kg (body weight) per day by oral gavage from day 8). Tumour size and mouse survival were monitored. Data are presented as mean ± s.d. ( n = 10). *** P = 0.0091 for Pmel versus Pmel + inosine, by two-way ANOVA (left). ** P < 0.0001 for Pmel versus Pmel + inosine with one-sided Mantel–Cox test (right). c , A human neuroblastoma xenograft model was established in NSG mice by subcutaneous inoculation of LAN-1 neuroblastoma cells. The indicated experimental mice were treated, from day 6 when tumours reached 100–150 mm 3 , with PBS (i.v. as control), inosine (300 mg per kg (body weight) by oral gavage daily), GD2-CAR T cells (8 × 10 6 cells per mouse, i.v.) and GD2-CAR T cells (8 × 10 6 cells per mouse, i.v.) + inosine (300 mg per kg (body weight) by oral gavage daily). Tumour growth and mouse survival were monitored. Data are presented as mean ± s.e.m. ( n = 20 for control and inosine, 19 for CAR T and 16 for CAR T + inosine group). *** P < 0.0001 for CAR T versus CAR T + inosine with two-way ANOVA (left). * P = 0.0111 for CAR T versus CAR T + inosine with one-sided Mantel–Cox test (right). Data are representative of two independent experiments ( a – c ). Sample size ( n ) represents biologically independent animals ( a , b ) or tumours ( c ).

Article Snippet: For CD8 + -T-cell activation and culture, naive CD8 T cells were activated by plate-bound antibodies and incubated with recombinant mouse IL-2 (100 U ml –1 ) and IL-12 (5 ng ml –1 ) for 5 d. For Pmel + CD8 + -T-cell activation and culture, splenocytes from Pmel transgenic mice were isolated and cultured with 1 μM human gp100 (hgp100) (GenScript) and 30 U ml –1 recombinant human IL-2 (PeproTech) in complete medium for 5 d , .

Techniques: Control, Injection, Irradiation

Journal: Frontiers in Immunology

Article Title: IRE1α Activation in Bone Marrow-Derived Dendritic Cells Modulates Innate Recognition of Melanoma Cells and Favors CD8 + T Cell Priming

doi: 10.3389/fimmu.2018.03050

Figure Lengend Snippet: Inhibition of IRE1α endonuclease function reduces the cross-presentation of a melanoma-endogenous antigen in vitro . (A) FL-DCs were preincubated with 50 μM 4μ8C or DMSO for 6 h and pulsed with 100 μg/ml MEL for the last 5 h of culture. Alternatively, cells were pulsed with 2.5 μM human gp100 peptide for the last 20 min of culture. Cells were counted, fixed and 5 × 10 4 FL-DCs were cocultured with 5 × 10 4 pmel-1 CD8 + T cells. Pmel-1 CD8 + T cell activation was quantified by expression of CD69 on day 1 through flow cytometry. Data in graph shows seven independent experiments. (B) FL-DCs were treated as in (A) but were not fixed and 2 × 10 4 FL-DCs were cultured with 5 × 10 4 CFSE-labeled pmel-1 CD8 + T cells. Proliferation was quantified on day 3 by flow cytometry. Data in graph shows three independent experiments. (C) GM-CSF BMDCs were treated and cocultured as in (A) . Data in graph shows six independent experiments. (D) GM-CSF BMDCs were treated and cocultured as in (B) . Data in graph shows four independent experiments. (E) FL-DCs were treated as in (B) but were cultured with 5 × 10 4 CellTrace Violet-labeled CTV = CD4 + T cells isolated from Trp1 mice. Proliferation was measured on day 5 by flow cytometry. Data in graph shows two independent experiments of (A) . Each symbol in the graphs represents data derived from one independent experiment. For all error bars represent mean ± SEM. * p < 0.05, ** p < 0.01 (paired Student's t -test).

Article Snippet: Human gp100 peptide (hgp100 25−33 , KVPRNQDWL) and Mouse TRP-1 peptide (TRP-1 106−130 , SGHNCGTCRPGWRGAACNQKILTVR) were purchased from Genetel Laboratories LLC.

Techniques: Inhibition, In Vitro, Activation Assay, Expressing, Flow Cytometry, Cell Culture, Labeling, Isolation, Derivative Assay

Journal: Oncoimmunology

Article Title: The nitric oxide radical scavenger carboxy-PTIO reduces the immunosuppressive activity of myeloid-derived suppressor cells and potentiates the antitumor activity of adoptive cytotoxic T lymphocyte immunotherapy

doi: 10.1080/2162402X.2015.1019195

Figure Lengend Snippet: Adoptively-transferred CTLs are impaired in the tumor. (A) C57BL/6 mice were injected with B16 melanoma cells (1 × 106), and 9 d later (designated as day 0), tumor-bearing mice (n = 5) received in vitro-activated pmel-1 splenocytes (1 × 107) as CTLs. Tumor volumes were measured every other day. (B) Mice (n = 3) were killed on days 3, 5, 7, and 17 after CTL transfer and infiltration of CTLs into the tumor was analyzed by flow cytometry. The frequency of CTLs in the tumor was determined by quantifying eFluor450−CD45+CD8+CD90.1+ cells. (C) The absolute number of CTLs was calculated as described in the Materials and Methods section and adjusted by the tumor weight (cells/g). (D) IFNγ production by CTLs with or without stimulation with 1 μg/mL hgp100 peptide for 4 h was analyzed on days 3, 5, 7, and 17 after CTL transfer by intracellular staining (n = 3 per group). The experiments were performed independently at least three times with similar results.

Article Snippet: The H-2D b -restricted peptide human gp100 (hgp100 25–33, KVPRNQDWL) was purchased from GenScript Japan (Tokyo, Japan) at a purity of >90%, with a free amino terminal and carboxyl terminal.

Techniques: Injection, In Vitro, Flow Cytometry, Staining

Journal: Oncoimmunology

Article Title: The nitric oxide radical scavenger carboxy-PTIO reduces the immunosuppressive activity of myeloid-derived suppressor cells and potentiates the antitumor activity of adoptive cytotoxic T lymphocyte immunotherapy

doi: 10.1080/2162402X.2015.1019195

Figure Lengend Snippet: MDSCs inhibit the proliferation of antigen-specific CTLs via NO production. C57BL/6 mice were treated as described in Fig. 1. Tumor-infiltrating cells were prepared from pooled B16 tumors (n = 12) 3 d after CTL transfer and CD11b+Gr1+ cells were positively selected using anti-CD11b magnetic beads. CFSE-labeled pmel-1 CTL were stimulated with hgp100 peptide in the presence or absence of CD11b+Gr1+ cells at the indicated ratio. The proliferation of pmel-1 cells was evaluated by flow cytometry. This was studied in the presence of carboxy-PTIO or L-NMMA. Numbers on the images show the percentage of gated cells (mean ±SD). All experiments shown were performed independently at least three times with similar results.

Article Snippet: The H-2D b -restricted peptide human gp100 (hgp100 25–33, KVPRNQDWL) was purchased from GenScript Japan (Tokyo, Japan) at a purity of >90%, with a free amino terminal and carboxyl terminal.

Techniques: Magnetic Beads, Labeling, Flow Cytometry

Journal: Oncoimmunology

Article Title: The nitric oxide radical scavenger carboxy-PTIO reduces the immunosuppressive activity of myeloid-derived suppressor cells and potentiates the antitumor activity of adoptive cytotoxic T lymphocyte immunotherapy

doi: 10.1080/2162402X.2015.1019195

Figure Lengend Snippet: NO Scavenger C-PTIO restores CTL function. (A) Mice were treated as described in the legend to Fig. 4. TILs were harvested from tumors on days 3 or 7. CTLs in the tumor were analyzed by flow cytometry (left). The absolute number of CTLs was calculated (right). (B) BrdU incorporation by CTLs in the tumor and draining lymph node (DLN) on days 3 and 7 was analyzed by flow cytometry as described in the Materials and Methods section. (C) IFNγ production by CTLs on days 3 and 7 with or without 1 μg/mL hgp100 peptide. (D) The absolute number of IFNγ+ CTLs is shown. (E) CD107a expression on CTLs on days 3 and 7 with or without 1 μg/mL hgp100 peptide. (F) The absolute number of CD107a+ CTLs is shown.

Article Snippet: The H-2D b -restricted peptide human gp100 (hgp100 25–33, KVPRNQDWL) was purchased from GenScript Japan (Tokyo, Japan) at a purity of >90%, with a free amino terminal and carboxyl terminal.

Techniques: Flow Cytometry, BrdU Incorporation Assay, Expressing

Journal: Oncoimmunology

Article Title: The nitric oxide radical scavenger carboxy-PTIO reduces the immunosuppressive activity of myeloid-derived suppressor cells and potentiates the antitumor activity of adoptive cytotoxic T lymphocyte immunotherapy

doi: 10.1080/2162402X.2015.1019195

Figure Lengend Snippet: Adoptively-transferred CTLs are impaired in the tumor. (A) C57BL/6 mice were injected with B16 melanoma cells (1 × 106), and 9 d later (designated as day 0), tumor-bearing mice (n = 5) received in vitro-activated pmel-1 splenocytes (1 × 107) as CTLs. Tumor volumes were measured every other day. (B) Mice (n = 3) were killed on days 3, 5, 7, and 17 after CTL transfer and infiltration of CTLs into the tumor was analyzed by flow cytometry. The frequency of CTLs in the tumor was determined by quantifying eFluor450−CD45+CD8+CD90.1+ cells. (C) The absolute number of CTLs was calculated as described in the Materials and Methods section and adjusted by the tumor weight (cells/g). (D) IFNγ production by CTLs with or without stimulation with 1 μg/mL hgp100 peptide for 4 h was analyzed on days 3, 5, 7, and 17 after CTL transfer by intracellular staining (n = 3 per group). The experiments were performed independently at least three times with similar results.

Article Snippet: The H-2D b -restricted peptide human gp100 (hgp100 25–33, KVPRNQDWL) was purchased from GenScript Japan (Tokyo, Japan) at a purity of >90%, with a free amino terminal and carboxyl terminal.

Techniques: Injection, In Vitro, Flow Cytometry, Staining

Journal: Oncoimmunology

Article Title: The nitric oxide radical scavenger carboxy-PTIO reduces the immunosuppressive activity of myeloid-derived suppressor cells and potentiates the antitumor activity of adoptive cytotoxic T lymphocyte immunotherapy

doi: 10.1080/2162402X.2015.1019195

Figure Lengend Snippet: MDSCs inhibit the proliferation of antigen-specific CTLs via NO production. C57BL/6 mice were treated as described in Fig. 1. Tumor-infiltrating cells were prepared from pooled B16 tumors (n = 12) 3 d after CTL transfer and CD11b+Gr1+ cells were positively selected using anti-CD11b magnetic beads. CFSE-labeled pmel-1 CTL were stimulated with hgp100 peptide in the presence or absence of CD11b+Gr1+ cells at the indicated ratio. The proliferation of pmel-1 cells was evaluated by flow cytometry. This was studied in the presence of carboxy-PTIO or L-NMMA. Numbers on the images show the percentage of gated cells (mean ±SD). All experiments shown were performed independently at least three times with similar results.

Article Snippet: The H-2D b -restricted peptide human gp100 (hgp100 25–33, KVPRNQDWL) was purchased from GenScript Japan (Tokyo, Japan) at a purity of >90%, with a free amino terminal and carboxyl terminal.

Techniques: Magnetic Beads, Labeling, Flow Cytometry

Journal: Oncoimmunology

Article Title: The nitric oxide radical scavenger carboxy-PTIO reduces the immunosuppressive activity of myeloid-derived suppressor cells and potentiates the antitumor activity of adoptive cytotoxic T lymphocyte immunotherapy

doi: 10.1080/2162402X.2015.1019195

Figure Lengend Snippet: NO Scavenger C-PTIO restores CTL function. (A) Mice were treated as described in the legend to Fig. 4. TILs were harvested from tumors on days 3 or 7. CTLs in the tumor were analyzed by flow cytometry (left). The absolute number of CTLs was calculated (right). (B) BrdU incorporation by CTLs in the tumor and draining lymph node (DLN) on days 3 and 7 was analyzed by flow cytometry as described in the Materials and Methods section. (C) IFNγ production by CTLs on days 3 and 7 with or without 1 μg/mL hgp100 peptide. (D) The absolute number of IFNγ+ CTLs is shown. (E) CD107a expression on CTLs on days 3 and 7 with or without 1 μg/mL hgp100 peptide. (F) The absolute number of CD107a+ CTLs is shown.

Article Snippet: The H-2D b -restricted peptide human gp100 (hgp100 25–33, KVPRNQDWL) was purchased from GenScript Japan (Tokyo, Japan) at a purity of >90%, with a free amino terminal and carboxyl terminal.

Techniques: Flow Cytometry, BrdU Incorporation Assay, Expressing