pmel-1 tcrtg t cell mouse  (Jackson Laboratory)

Journal: STAR Protocols

Article Title: CRISPitope: A generic platform to model target antigens for adoptive T cell transfer therapy in mouse tumor models

doi: 10.1016/j.xpro.2021.101038

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Article Snippet: Mouse: Pmel-1 TCRtg T cell mouse (B6.Cg-Thy1a/Cy Tg(TcraTcrb)8Rest/J) (sex: male or female; age range: 8–16 weeks) , The Jackson Laboratory , Cat-No: 005023.

Techniques: Purification, Virus, Recombinant, Saline, Binding Assay, Red Blood Cell Lysis, Staining, Transfection, DNA Purification, Plasmid Preparation, DNA Extraction, Sequencing, Software, Spectrophotometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD204 suppresses large heat shock protein-facilitated priming of tumor antigen gp100-specific T cells and chaperone vaccine activity against mouse melanoma

doi: 10.4049/jimmunol.1100703

Figure Lengend Snippet: A. SRA/CD204 directly interacts with exogenous hsp110. WT BMDCs were incubated with His-hsp110 at 4 °C for 30 min, and then extensively washed with PBS. Cell lysates were immunoprecipitated with antibodies for SRA/CD204 (top) or His-tag (bottom). The immunocomplexes were subjected to immunoblotting analyses using antibodies for His-tag (1:4000, top) or SRA/CD204 (1:3000, bottom). IgG serves as a negative control. B. BMDCs were incubated with His-hsp110 or His-gp100 protein. SRA/CD204 was immunoprecipitated and analyzed for its association with hsp110 or gp100 using His-tag antibodies. C. Competition assays. DCs were incubated with biotinylated-His-hsp110 (40 μg/ml) in the presence of 3× or 9× excess of His-gp100 or His-Hsp110. Immunocomplexes pulled down by SRA/CD204 antibodies were analyzed by immunoblot using Avidin-HRP or anti-SRA antibodies (left). Alternatively, DCs were incubated with His-hsp110 in the presence of BSA or autologous hsp110-purified from mouse spleen, followed by immunoblotting analysis of His-hsp110 association with immunoprecipitated SRA/CD204 (right). D and E. SRA/CD204 absence results in reduced hsp110 binding and internalization. WT and SRA−/− BMDCs were incubated with His-hsp110 for 30 min at either 4 °C (D) or 37 °C (E). Cells were washed and cultured at 37 °C for the times indicated. Total cell lysates were prepared and analyzed for the presence of His-hsp110 using His-tag antibodies. Immunoblots were quantified by densitometry analysis using ImageJ. The data are presented as fold of change in protein expression for each sample compared with WT DCs at 0 min. The ratio of His-hsp110 toβ-actin in WT DCs at 0 min is set to 1. *p< 0.01, representative results from three independent experiments with similar results are shown.

Article Snippet: SRA/CD204 knockout mice (SRA −/− ) and pmel transgenic mice carrying T-cell receptor transgene specific for the mouse homologue (pmel-17) of human gp100 ( 29 ) were purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Incubation, Immunoprecipitation, Western Blot, Negative Control, Avidin-Biotin Assay, Purification, Binding Assay, Cell Culture, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD204 suppresses large heat shock protein-facilitated priming of tumor antigen gp100-specific T cells and chaperone vaccine activity against mouse melanoma

doi: 10.4049/jimmunol.1100703

Figure Lengend Snippet: A. Increased proliferation of gp100-specific T cells when co-cultured with chaperone vaccine-loaded SRA−/− DCs. DCs were pulsed with hsp110-gp100 complexes for 5 h and then cultured with naïve CD8+ T cells from pmel transgenic mice. Cell proliferation was assayed by incorporation of 3H-TdR. B. Increased stimulation of pmel cells by chaperone vaccine-loaded SRA−/− DCs. Following DC-pmel cell co-culture, IL-2 levels in supernatant were measured by ELISA assays. C. DCs were incubated with BSA-gp100 or hsp110-gp100 complexes, and used to stimulate pmel cells. IL-2 levels in the media were assayed by ELISA. D. DCs were pulsed with hsp110-gp100 complexes or grp170-gp100 complexes, and assayed for their ability to stimulate gp100-specific T cells. E and F. SRA/CD204 silencing enhances large HSP-mediated T cell stimulation. BMDCs were infected with lentiviruses encoding scramble shRNA or SRA/CD204 shRNA. 2 days later, cells were incubated with hsp110-gp100 (E) or grp170-gp100 (F), and co-cultured with pmel cells at different ratios. Pmel cell proliferation was measured using 3H-TdR incorporation assays. ShRNA-mediated downregulation of SRA/CD204 expression in DCs was confirmed using immunoblotting (E). (Values are means ± S.D., *p< 0.005, SRA shRNA vs scramble). Results from three independent experiments are shown. G. Increased IFN-γ production by pmel cells stimulated with SRA/CD204-silenced DCs. SRA/CD204-silenced DCs were incubated with hsp110-gp100 or grp170-gp100 complexes, and then co-cultured with pmel cells. The levels of IL-2 in the supernatant were determined using ELISA assays. *p< 0.005, representative results from two independent experiments are shown.

Article Snippet: SRA/CD204 knockout mice (SRA −/− ) and pmel transgenic mice carrying T-cell receptor transgene specific for the mouse homologue (pmel-17) of human gp100 ( 29 ) were purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Cell Culture, Transgenic Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Incubation, Cell Stimulation, Infection, shRNA, Expressing, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD204 suppresses large heat shock protein-facilitated priming of tumor antigen gp100-specific T cells and chaperone vaccine activity against mouse melanoma

doi: 10.4049/jimmunol.1100703

Figure Lengend Snippet: A. Increased expansion of adoptively transferred pmel cells in SRA−/− mice. Mice received pmel cells on day -1, and were immunized with the hsp110-gp100 complex i.p. on day 0. Spleens (SP) and lymph nodes (LN) were harvested and subjected to FACS analysis following cell staining with CD90.1 and CD8 antibodies. Data are representative of three separate experiments with similar results. B. Increased IFN-γ production by pmel cells in the hsp110-gp100-immunized SRA−/− mice. Following pmel cell transfer and vaccination, gp10025–33–stimulated CD90.1+CD8+ T cell activation was determined by intracellular IFN-γ staining and FACS analysis. Data are representative of three separate experiments in which at least 3 mice in each group were analyzed.

Article Snippet: SRA/CD204 knockout mice (SRA −/− ) and pmel transgenic mice carrying T-cell receptor transgene specific for the mouse homologue (pmel-17) of human gp100 ( 29 ) were purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Staining, Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD204 suppresses large heat shock protein-facilitated priming of tumor antigen gp100-specific T cells and chaperone vaccine activity against mouse melanoma

doi: 10.4049/jimmunol.1100703

Figure Lengend Snippet: A. Increased IFN-γ production by gp100-specific CD8+ T cells in SRA−/− mice. Mice were immunized with the hsp110-gp100 complex. Splenocytes and lymph node cells were stimulated with gp10025–33 and analyzed by intracellular cytokine staining. B. Increased cytolytic activity of effector CD8+ T cells in SRA−/− mice. Splenocytes were stimulated with gp10025–33 in the presence of IL-2 for 5 days, and chromium release assays were carried out using B16-gp100 cells as targets. Data shown as means ± S.D. are representative of two experiments. C. Following immunization with grp170-gp100 complexes, splenocytes were stimulated with gp10025–33 peptide or gp100 protein, and assayed for IFN-γ production using ELISPOT assays. * p< 0.01, the representative results from three independent experiments are shown.

Article Snippet: SRA/CD204 knockout mice (SRA −/− ) and pmel transgenic mice carrying T-cell receptor transgene specific for the mouse homologue (pmel-17) of human gp100 ( 29 ) were purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Staining, Activity Assay, Enzyme-linked Immunospot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD204 suppresses large heat shock protein-facilitated priming of tumor antigen gp100-specific T cells and chaperone vaccine activity against mouse melanoma

doi: 10.4049/jimmunol.1100703

Figure Lengend Snippet: A. Increased tumor protective activity of chaperone vaccine in SRA−/− mice. WT and SRA−/− mice (n=5) were immunized with the hsp110-gp100 complexes and then challenged with 2×105 cells B16-gp100 tumor cells. * p< 0.01, the results are representative of two independent experiments. B. Enhanced therapeutic activity of chaperone vaccine in tumor-bearing SRA−/− mice. Mice (n=5) were established with B16-gp100 tumors on day 0 and treated with the hsp110-gp100 vaccine on days 4, 7 and 10. Values are means ± S.D. * p< 0.01, the results representative of three independent experiments are shown.

Article Snippet: SRA/CD204 knockout mice (SRA −/− ) and pmel transgenic mice carrying T-cell receptor transgene specific for the mouse homologue (pmel-17) of human gp100 ( 29 ) were purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Activity Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CD204 suppresses large heat shock protein-facilitated priming of tumor antigen gp100-specific T cells and chaperone vaccine activity against mouse melanoma

doi: 10.4049/jimmunol.1100703

Figure Lengend Snippet: A. Increased gp100-specific CD8+ T cells in treated SRA−/− mice. After hsp110-gp100 vaccine therapy, tumor-draining lymph node cells were prepared and stimulated with gp10025–33. The frequency of IFN-γ-producing CD8+ T cells was assayed using intracellular cytokine staining and FACS analysis. B. Increased tumor infiltration of CD8+ cells in treated SRA−/− mice. Tumor tissues harvested after chaperone vaccine therapy were digested and prepared as single cell suspensions. Cells were stained with anti-CD8 antibodies and analyzed using FACS. C. Enhanced immune recognition of B16-gp100 tumor cells in treated SRA−/− mice. Splenocytes were co-cultured with irradiated B16 cells for 48 h and IFN-γ levels in the culture media were assayed using ELISA. Values are means ± S.D. * p< 0.01, the results represent two independent experiments. D. Similar antibody response to gp100 antigen in WT and SRA−/− mice. Sera were collected from tumor-bearing mice after chaperone vaccine therapy and analyzed for IgG levels against gp100 using ELISA assays (p> 0.5, WT vs SRA−/−).

Article Snippet: SRA/CD204 knockout mice (SRA −/− ) and pmel transgenic mice carrying T-cell receptor transgene specific for the mouse homologue (pmel-17) of human gp100 ( 29 ) were purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Staining, Cell Culture, Irradiation, Enzyme-linked Immunosorbent Assay