human gp100 peptide (kvprnqdwl)  (Thermo Fisher)

Journal: Science Advances

Article Title: IFN-γ–dependent tumor-antigen cross-presentation by lymphatic endothelial cells promotes their killing by T cells and inhibits metastasis

doi: 10.1126/sciadv.abl5162

Figure Lengend Snippet: ( A ) Lyve-1 staining of ndLN (non dLN) sections from IFN-γR WT and IFN-γR Δprox-1 mice 4 weeks after tamoxifen treatment. ( B to F ) Tamoxifen-treated IFN-γR WT and IFN-γR Δprox-1 mice were injected or not with 0.5 × 10 6 in vitro activated OT-1 cells 8 days after B16F10-OVA + VC + inoculation. (B) Tumor growth. Data are representative of three experiments. (C) Tumor LEC density and (D) frequency of cleaved casp-3 + tumor LEC 5 days after T cell injection. Data represent two pooled experiments, N = 6 to 10 mice per group. (E) Black color intensity analysis of inguinal, axillary, and brachial TdLN from indicated mice 6 days after OT-1 transfer. Pictures show representative images, and histograms show the quantification of the intensity for individual LNs. Data are pooled from three experiments, N = 16 mice per group. (F) Gp100 staining of inguinal, axillary, and brachial TdLNs from indicated mice 9 days after OT-1 transfer. Pictures show representative images, and histograms show the quantification of Gp100 + cells in LNs. Data are from six mice per group. (A, C, and D) Mann-Whitney statistical test; (B) two-way ANOVA test; (E) one-way ANOVA test. Error bars show means ± SEM.* P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: B16F10-OVA + VEGF-C hi tumor-bearing mice were injected subcutaneously at day 5 with human Gp100 peptide (KVPRNQDWL) (100 μg per mouse; Thermo Fisher Scientific) and CpG-B 1668 (30 μg per mouse; Invivogen).

Techniques: Staining, Injection, In Vitro, MANN-WHITNEY

Journal: Science Advances

Article Title: IFN-γ–dependent tumor-antigen cross-presentation by lymphatic endothelial cells promotes their killing by T cells and inhibits metastasis

doi: 10.1126/sciadv.abl5162

Figure Lengend Snippet: ( A and B ) C57BL/6 and β 2 m KO were injected or not with 0.5 × 10 6 OT-1 cells 8 days after B16F10-OVA + VC + inoculation. (A) Tumor growth and (B) frequencies of cleaved casp-3 + tumor LECs 4 days after OT-1 transfer. Data are from one experiment, N = 6 to 7 mice per group. ( C to F ) Tamoxifen-treated β 2 m WT and β 2 m Δprox-1 mice were injected with 0.5 × 10 6 OT-1 cells 9 days after B16F10-OVA + VC + inoculation. (C) Tumor growth and (D) frequencies of cleaved casp-3 + tumor LECs 5 days after OT-1 transfer. Data are from one experiment, N = 6 to 7 mice per group. (E) The percentage of metastasis-positive and metastasis-negative (free of metastasis) LNs according to black color. Pictures show representative images. Scale bars, 0.5 cm. (F) Immunofluorescent Gp100 staining of inguinal, axillary, and brachial TdLNs (iLN, aLN, and bLN, respectively) 5 days after OT-1 transfer. Histograms represent the quantification of Gp100 + cells in LNs. (A and C) Two-way ANOVA test; (B, D, and E) Mann-Whitney statistical test. Error bars show means ± SEM. * P < 0.05, ** P < 0.01.

Article Snippet: B16F10-OVA + VEGF-C hi tumor-bearing mice were injected subcutaneously at day 5 with human Gp100 peptide (KVPRNQDWL) (100 μg per mouse; Thermo Fisher Scientific) and CpG-B 1668 (30 μg per mouse; Invivogen).

Techniques: Injection, Staining, MANN-WHITNEY

Journal: Journal for Immunotherapy of Cancer

Article Title: Immunologically programming the tumor microenvironment induces the pattern recognition receptor NLRC4-dependent antitumor immunity

doi: 10.1136/jitc-2020-001595

Figure Lengend Snippet: Programming tumor microenvironment with Flagrp170 induces a potent antitumor immunity. (A, B) C3H/HeN mice (n=5) bearing SCCVII tumors (4~5 mm in diameter) were treated intratumoral with an empty adenovirus (ie, null) or an adenovirus encoding Flagrp170 every other day for a total of five doses. Tumor growth (A) and animal survival (B) were followed. (C) Transcription of ifng and il12a genes in tumor tissues (n=3) following treatments was assayed by quantitative PCR. (D, E) Systemic T cell activation by Flagrp170-mediated immune programming of tumor environment. Splenocytes from treated mice (n=3) were stimulated with SCCVII tumor cell lysates at a ratio of 3:1 for 96 hours. IFN-γ level in the culture media was examined using ELISA (D) and the frequency of IFN-γ-producing CD8 + or CD4 + T cells were determined using intracellular cytokine staining (E). (F–H) Comparable antitumor potency of human and mouse versions of Flagrp170. Mice bearing B16 tumors of 4–5 mm sizes (n=5) received treatment with mouse version of Flagrp170 (mFlagrp170) or its human counterpart, that is, hFlagrp170 (F). Immune activation in the tumor tissues (n=3) was evaluated by analyzing the transcription of ifng and il12a (G). Splenocytes (left) or lymph node cells (right) from mice treated with or without mFlagrp170/hFlagrp170 (n=3) were stimulated with MHC I-restricted gp100 25-33 peptide, followed by assessment of IFN-γ production using ELISA (H). Data represent three different experiments with similar results. *p<0.05, **p<0.01, ***p<0.001, NS, not significant, using two-way repeated measures analysis of variance test (A, F), log-rank test (B) and Student’s t-test (C, D, G, H). IFN, interferon; MHC I, major histocompatibility complex class I; SCC, squamous cellcarcinoma.

Article Snippet: Human gp100 25–33 (KVPRNQDWL) peptides were from AnaSpec (Fremont, California, USA).

Techniques: Real-time Polymerase Chain Reaction, Activation Assay, Enzyme-linked Immunosorbent Assay, Staining, Immunopeptidomics

Journal: Journal for Immunotherapy of Cancer

Article Title: Immunologically programming the tumor microenvironment induces the pattern recognition receptor NLRC4-dependent antitumor immunity

doi: 10.1136/jitc-2020-001595

Figure Lengend Snippet: TLR5 is not required for Flagrp170-enhanced dendritic cell (DC) activation in vitro. (A) WT or Tlr5 −/− bone marrow-derived DCs were infected with an empty or Flagrp170-encoding virus at a multiplicity of infection of 300. 24 hours later, protein lysates were prepared and analyzed for NF-κB activation by immunoblotting. (B) The expression of costimulatory molecules CD86 and CD80 was also assessed using flow cytometry. (C) Production of TNF-α, IL-6, and IL-12p40 by DCs were measured using ELISA. (D) Virus-infected WT or Tlr5 −/− bone morrow-derived dendritic cells were pulsed with gp100 25-33 peptide and cocultured with gp100-specific Pmel cells for 3 days. T-cell proliferation was assessed by 3 H-TdR incorporation assays (left) and ELISA of the IL-2 levels in culture medium (right). The experiments were repeated three times with similar results. NS, not significant, using Student’s t-test (C and D). IL, interleukin; TNF, tumor necrosis factor; WT, wild-type.

Article Snippet: Human gp100 25–33 (KVPRNQDWL) peptides were from AnaSpec (Fremont, California, USA).

Techniques: Activation Assay, In Vitro, Derivative Assay, Infection, Virus, Western Blot, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Immunologically programming the tumor microenvironment induces the pattern recognition receptor NLRC4-dependent antitumor immunity

doi: 10.1136/jitc-2020-001595

Figure Lengend Snippet: Toll-like receptor 5 is dispensable for Flagrp170-elicited antitumor immunity in vivo. (A) WT or Tlr5 −/− mice (n=5) with B16 tumors of 4~5 mm sizes were treated with an empty or Flagrp170-encoding adenovirus. Tumor infiltration by T cells (B), NK cells (C), and dendritic cells (DCs) (D) and their activation status were assayed by intracellular cytokine staining and flow cytometry. (E) Transcription of il12a , ifng , gmcsf , and tbet in tumors (n=3) was examined 2 weeks after treatment. Splenocytes from mock or Flagrp170 treated mice (n=3) were stimulated with B16 tumor lysates (F) or gp100 25-33 peptide (G), followed by analyzes of IFN-γ and IL-2 levels in the culture media using ELISA. Data shown are representative of three independent experiments. NS, not significant, using two-way repeated measures analysis of variance test (A) and Student’s t-test (E–G). IFN, interferon; IL, interleukin; WT, wild-type.

Article Snippet: Human gp100 25–33 (KVPRNQDWL) peptides were from AnaSpec (Fremont, California, USA).

Techniques: In Vivo, Activation Assay, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Immunologically programming the tumor microenvironment induces the pattern recognition receptor NLRC4-dependent antitumor immunity

doi: 10.1136/jitc-2020-001595

Figure Lengend Snippet: The NLR family CARD domain-containing protein (NLRC4) inflammasome is required for antitumor efficacy of in situ Flagrp170 therapy. (A) Abrogation of therapeutic activity of Flagrp170 in the absence of NLRC4. B16 tumor-bearing WT and Nlrc4 −/− mice (n=5) were treated with an empty or Flagrp170-encoding virus. Tumor growth was monitored by measuring tumor sizes. Transcription of ifng , tbet , and il1b in tumor tissues (B) and the frequency or activation of tumor-infiltrating T cells (C) were examined (n=3). (D) Splenocytes (top) or lymph node cells (bottom) from mock or Flagrp170 treated mice (n=3) were stimulated with gp100 25-33 peptide for 72 hours, followed by ELISA analysis of IFN-γ production. (E) 48 hours after infection, WT or Nlrc4 −/− dendritic cells (DCs) was examined for their production of IL-1β, IL-1α, and IL-18. (F) Reduced T-cell priming activity of DCs in the absence of NLRC4. Infected WT or Nlrc4 −/− DCs were pulsed with gp100 25-33 peptide and cocultured with Pmel cells for 3 days, followed by ELISA analysis of IFN-γ production. Data shown are representative of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, using two-way repeated measures analysis of variance test (A) and Student’s t-test (B–F). CARD, caspase activation and recruitment domain; IFN, interferon; IL, interleukin; NLR, NOD like receptor; WT, wild-type.

Article Snippet: Human gp100 25–33 (KVPRNQDWL) peptides were from AnaSpec (Fremont, California, USA).

Techniques: In Situ, Activity Assay, Virus, Activation Assay, Enzyme-linked Immunosorbent Assay, Infection

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: 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