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: EMBO Reports

Article Title: Identification of a novel population of highly cytotoxic c‐Met‐expressing CD8 + T lymphocytes

doi: 10.15252/embr.201744075

Figure Lengend Snippet: Day 5 c‐Met+ or c‐Met− CD45.2+ Pmel‐1 CTLs treated with either PBS or HGF (30 ng/ml for 24 h) were transferred i.v. into congenic WT CD45.2+ recipients. Equal numbers of hgp10025–33‐pulsed CFSE‐labeled target CD45.1+ and unpulsed brilliant violet (BV)‐labeled CD45.1+ splenocytes were injected i.v. into the recipient CD45.2+ mice 5 h after the adoptive transfer of Pmel‐1 CTLs. Recipient CD45.2+ mice were sacrificed 24 h following transfer of target cells and control splenocytes, and the percentage of remaining target cells was quantified by FACS analysis.

Article Snippet: Thy1.1 + Pmel‐1 T‐cell receptor (TCR) transgenic mice on a C57BL/6 CD45.2 background, whose TCR recognizes an H‐2D b ‐restricted epitope corresponding to amino acids 25–33 of murine and human gp100 (hgp100 25–33 ), a self‐tumor antigen, were obtained from the Jackson Laboratory.

Techniques: Labeling, Injection, Adoptive Transfer Assay, Control

Journal: EMBO Reports

Article Title: Identification of a novel population of highly cytotoxic c‐Met‐expressing CD8 + T lymphocytes

doi: 10.15252/embr.201744075

Figure Lengend Snippet: For tumor inoculation, mice were injected i.v. with 1 × 105 mouse melanoma B16‐F10 cells. One day after B16‐F10 injection, day 5 c‐Met+ or c‐Met− Pmel‐1 CTLs treated with either PBS or HGF (30 ng/ml for 24 h) were adoptively transferred into recipients. Mice were sacrificed after 14 days.

Article Snippet: Thy1.1 + Pmel‐1 T‐cell receptor (TCR) transgenic mice on a C57BL/6 CD45.2 background, whose TCR recognizes an H‐2D b ‐restricted epitope corresponding to amino acids 25–33 of murine and human gp100 (hgp100 25–33 ), a self‐tumor antigen, were obtained from the Jackson Laboratory.

Techniques: 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: Nature Communications

Article Title: Bempegaldesleukin selectively depletes intratumoral Tregs and potentiates T cell-mediated cancer therapy

doi: 10.1038/s41467-020-14471-1

Figure Lengend Snippet: a – d C57BL/6 mice bearing 7-day-old, s.c. B16.F10 tumors received pmel-1 T-cell and gp100 peptide vaccination followed by either aldesleukin or NKTR-214. a Experimental scheme. b Tumor size in individual mice. c Pmel-1 CD8 + Teff, and d CD4 + CD25 hi Foxp3 + Tregs in blood through time. Data are represented as mean ± SEM ( n = 5, ** P < 0.01, unpaired t test).

Article Snippet: Each mouse was vaccinated s.c. on each flank with 50 μg of heteroclitic mouse gp100 25–33 (hgp100) peptide KVPRNQDWL (Peptides International, Louisville, KY) formulated in l -Tyrosine microparticles along with 50 μg s.c. of anti-CD40 antibody (clone FGK4.5/FGK45, BioXcell, New Hampshire) and 50 mg per mouse Imiquimod cream 5% (Fougera, Melville, NY).

Techniques:

Journal: Nature Communications

Article Title: Bempegaldesleukin selectively depletes intratumoral Tregs and potentiates T cell-mediated cancer therapy

doi: 10.1038/s41467-020-14471-1

Figure Lengend Snippet: a – e C57BL/6 mice bearing 7-day-old, s.c. B16.F10 tumors received pmel-1 T cells and vaccination followed by aldesleukin or NKTR-214. Splenic Thy1.1 + pmel-1 CD8 + Teff, CD4 + Teff, and Tregs were analyzed by flow cytometry on days 3, 5, 7, and 10 post treatment. a Experimental scheme. b Representative plots showing the level of CD8 + Teff (upper panel) and Tregs (lower panel) in tumor through time. c Absolute numbers of Thy1.1 + CD8 + Teff, Tregs, and CD4 + Teff in tumor (upper panel) and spleen (lower panel) analyzed on day 7 post treatment. Histograms showing d CD8/Treg ratio in tumor (upper panel) and spleen (lower panel). e Ki67 and f Annexin-V expression on tumor-infiltrating pmel-1 CD8 + Teff and Tregs on day 7 after treatment. Data represented as mean ± SEM ( n = 4–5, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns nonsignificant, unpaired t test).

Article Snippet: Each mouse was vaccinated s.c. on each flank with 50 μg of heteroclitic mouse gp100 25–33 (hgp100) peptide KVPRNQDWL (Peptides International, Louisville, KY) formulated in l -Tyrosine microparticles along with 50 μg s.c. of anti-CD40 antibody (clone FGK4.5/FGK45, BioXcell, New Hampshire) and 50 mg per mouse Imiquimod cream 5% (Fougera, Melville, NY).

Techniques: Flow Cytometry, Expressing

Journal: Nature Communications

Article Title: Bempegaldesleukin selectively depletes intratumoral Tregs and potentiates T cell-mediated cancer therapy

doi: 10.1038/s41467-020-14471-1

Figure Lengend Snippet: a Mice bearing 4-day established s.c. CT26 tumors received either no treatment denoted by a minus sign (−) or NKTR-214 plus AH-1 vaccine, denoted by the plus sign (+); Treg levels in the spleen and tumor are shown on day 7 post treatment ( n = 5, mean ± SEM, ** P < 0.01, unpaired t test). b Mice bearing 7-day established s.c. B16.F10 tumors received pmel-1 T cells, and were either left untreated (−) or received NKTR-214 plus gp100 vaccine (+); Treg levels in the spleen and tumor on day 7 post treatment, are shown ( n = 5, mean ± SEM, ** P < 0.01, ns nonsignificant; unpaired t test). c Level of Tregs in blood ( n = 13, mean ± SEM) and in tumor ( n = 7, mean ± SEM) pre treatment (−) and 3 weeks post NKTR-214 treatment (+) in melanoma and renal cell carcinoma patients (* P < 0.05, *** P < 0.001; Wilcoxon-matched paired t test).

Article Snippet: Each mouse was vaccinated s.c. on each flank with 50 μg of heteroclitic mouse gp100 25–33 (hgp100) peptide KVPRNQDWL (Peptides International, Louisville, KY) formulated in l -Tyrosine microparticles along with 50 μg s.c. of anti-CD40 antibody (clone FGK4.5/FGK45, BioXcell, New Hampshire) and 50 mg per mouse Imiquimod cream 5% (Fougera, Melville, NY).

Techniques:

Journal: Nature Communications

Article Title: Bempegaldesleukin selectively depletes intratumoral Tregs and potentiates T cell-mediated cancer therapy

doi: 10.1038/s41467-020-14471-1

Figure Lengend Snippet: a C57BL/6 mice bearing 7-day-old, s.c. B16.F10 tumors received pmel-1 T cells followed by treatment with NKTR-214 plus gp100 vaccination. Absolute numbers of pmel-1 Teff and Tregs in tumor and spleen on indicated days are plotted. Correlation between Teff and Treg numbers through time in each tissue was calculated, and Pearson’s and Spearman correlation coefficients are shown. b Level of IFN-γ and TNF-α protein in tumors of untreated or vaccine-NKTR-214-treated mice on day 7. Data represented as mean ± SEM ( n = 5, **** P < 0.0001, unpaired t test). c Gene expression profiling of IFN-γ and TNF-α performed via NanoString in tumor biopsies from melanoma and renal cell carcinoma patients ( n = 8), pre (−) and week 3 post NKTR-214 treatment (+). RNA counts are represented as mean ± SEM (* P < 0.05 ratio, paired t test).

Article Snippet: Each mouse was vaccinated s.c. on each flank with 50 μg of heteroclitic mouse gp100 25–33 (hgp100) peptide KVPRNQDWL (Peptides International, Louisville, KY) formulated in l -Tyrosine microparticles along with 50 μg s.c. of anti-CD40 antibody (clone FGK4.5/FGK45, BioXcell, New Hampshire) and 50 mg per mouse Imiquimod cream 5% (Fougera, Melville, NY).

Techniques: Gene Expression

Journal: Nature Communications

Article Title: Bempegaldesleukin selectively depletes intratumoral Tregs and potentiates T cell-mediated cancer therapy

doi: 10.1038/s41467-020-14471-1

Figure Lengend Snippet: a – d C57BL/6 mice bearing 7-day-old, s.c. B16.F10 tumors received pmel-1 T cells, and were either left untreated or received gp100 peptide vaccine and/or NKTR-214. Where indicated, mice received neutralizing antibodies against IFN-γ and TNF-α on days 1, 2, 4, and 6. a Tregs in tumor (upper panel) and spleen (lower panel). b Expression of Ki67 and c Annexin V on intratumoral Tregs on day 7 post treatment. d Heat map depicting protein level of indicated cytokines and chemokines in tumor and spleen of mice 7 days after treatment. Color scale denotes levels of cytokines/chemokines in pg/ml. Data represented as mean ± SEM ( n = 5, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns nonsignificant, unpaired t test).

Article Snippet: Each mouse was vaccinated s.c. on each flank with 50 μg of heteroclitic mouse gp100 25–33 (hgp100) peptide KVPRNQDWL (Peptides International, Louisville, KY) formulated in l -Tyrosine microparticles along with 50 μg s.c. of anti-CD40 antibody (clone FGK4.5/FGK45, BioXcell, New Hampshire) and 50 mg per mouse Imiquimod cream 5% (Fougera, Melville, NY).

Techniques: Expressing

Journal: Cells

Article Title: IL7-Fc Enhances the Efficacy of Adoptive T Cell Therapy under Lymphopenic Conditions in a Murine Melanoma Model

doi: 10.3390/cells10082018

Figure Lengend Snippet: Anti-tumor effects of IL7-Fc in non-lymphopenic and lymphopenic conditions. ( A ) C57BL/6 mice were subcutaneously implanted with 2 × 10 5 B16-F10 melanoma cells in their backs. Mice were i.p. injected with the indicated dose of CTX to induce prior lymphodepletion on day 3. Activated pmel-1 Thy1.1 + CD8 + T cells were prepared by isolating CD8 + T cells from Thy1.1 + pmel-1 Tg mice and stimulating them with 5 μg/mL of human gp100 peptide in the presence of 5% CD8-depleted splenocytes for 2 days, and transferred into mice on day 5 via the i.v. route. All mice received 25,000 IU of rhIL-2 daily for 6 days. Human IgG (hIgG) or IL7-Fc was also injected daily into mice for 6 days. ( B ) The growth rate of B16-F10 melanoma. ( C ) Flow cytometric analysis of tumor-draining lymph nodes (TDLNs) and tumor tissues. Single cell suspensions were prepared from inguinal TDLNs and tumor tissues on days 13 and 19, and stained with anti-CD8-PE-Cy5 and anti-Thy1.1-FITC. All samples were further stained with propidium iodide (PI) and subsequently analyzed using FACSCalbur (BD Bioscience). Live cells were gated and plotted as the indicated markers. ( D ) Absolute numbers of TDLN cells were calculated using ADAM-MC2 (Nanoentek, Seoul, Korea). Absolute numbers of Thy1.1 + CD8 + T cells were calculated from TDLN cell numbers and the percentages of Thy1.1 + CD8 + T cells. Percentages of transferred pmel-1 Thy1.1 + CD8 + T cells and endogenous Thy1.1 − CD8 + T cells in tumor tissues were calculated from ( C ). CTX; cyclophosphamide, mpk; mg per Kg. Data are from three ( B ) or two ( C , D ) independent experiments with 5 ( B ) or 3 ( C , D ) mice per experiment. Student’s t -test was performed in D, and the results are expressed as mean ± SD (* p < 0.05; ** p < 0.01; *** p < 0.005).

Article Snippet: Mouse gp100 25–33 (mgp100, EGSRNQDWL) and human gp100 25–33 (hgp100, KVPRNQDWL) peptides were synthesized by Peptron (Daejeon, Korea).

Techniques: Injection, Staining

Journal: Cells

Article Title: IL7-Fc Enhances the Efficacy of Adoptive T Cell Therapy under Lymphopenic Conditions in a Murine Melanoma Model

doi: 10.3390/cells10082018

Figure Lengend Snippet: Anti-tumor effects of IL7-Fc with IL-2 in lymphopenia-induced B16-F10 melanoma-bearing B6 mice. ( A ) B16-F10 melanoma-bearing C57BL/6 mice were i.p. injected with 300 mpk of CTX on day 3 followed by 2 × 10 6 activated pmel-1 Thy1.1 + CD8 + T cells, and further treated with PBS, rhIL-2, hIL7-Fc, or rhIL-2 plus hIL7-Fc as described above. ( B ) The growth rate of B16-F10 melanoma cells. ( C ) Inguinal TDLN, popliteal non-TDLN, and spleen (SP) were collected from each group of mice on day 13 and single cell suspensions were stained with anti-CD8-PE-Cy5 and anti-Thy1.1-FITC. All samples were further stained with propidium iodide (PI) and subsequently analyzed using FACSCalbur (BD Bioscience). Live cells were gated and plotted as the indicated markers. ( D ) Absolute numbers of live cells in non-DLN, TDLN, and spleen. Percentages and absolute numbers of CD8 + T cells, the transferred pmel-1 Thy1.1 + CD8 + T cells, and the endogenous Thy1.1 − CD8 + T cells in non-TDLN, TDLN, and spleen on day 13. ( E ) TDLN cells were stained with anti-CD44-PE, anti-CD62L-FITC, and anti-Thy1.1-PE-Cy5 along with anti-CD4-APC or anti-CD8-APC. Gated Thy1.1-negative CD4 + or CD8 + cells were plotted as CD44 vs. CD62L. Data are from three independent experiments with 5 ( B ) or 3 ( C , D ) mice per experiment. Student’s t -test was performed in D, and the results are expressed as mean ± SD (* p < 0.05; ** p < 0.01).

Article Snippet: Mouse gp100 25–33 (mgp100, EGSRNQDWL) and human gp100 25–33 (hgp100, KVPRNQDWL) peptides were synthesized by Peptron (Daejeon, Korea).

Techniques: Injection, Staining

Journal: Cells

Article Title: IL7-Fc Enhances the Efficacy of Adoptive T Cell Therapy under Lymphopenic Conditions in a Murine Melanoma Model

doi: 10.3390/cells10082018

Figure Lengend Snippet: Effects of IL7-Fc on myeloid and lymphoid cells in bone marrow and spleen. ( A ) B16-F10 melanoma-bearing C57BL/6 mice were sequentially injected with CTX, activated pmel-1 Thy1.1 + CD8 + T cells, and further treated with PBS, rhIL-2, hIL7-Fc, or rhIL-2 plus hIL7-Fc as described above. ( B ) BMs and spleens were collected from mice on day 13 and single cell suspensions were stained with anti-CD11b-PE-Cy5 and anti-B220-FITC. All samples were further stained with propidium iodide (PI) and subsequently analyzed using FACSCalbur (BD Bioscience). Live cells were gated and plotted as the indicated markers. ( C ) Absolute numbers of spleen cells were counted, and the percentages and absolute numbers of CD11b + monocytes and B220 + B cells were calculated. ( D ) Absolute numbers of BM cells were counted, and the percentages and absolute numbers of CD11b + monocytes and B220 + B cells were calculated. Data are from two independent experiments with 3 mice per experiment. Student’s t -test was performed in ( C , D ), and the results are expressed as mean ± SD (* p < 0.05; ** p < 0.01).

Article Snippet: Mouse gp100 25–33 (mgp100, EGSRNQDWL) and human gp100 25–33 (hgp100, KVPRNQDWL) peptides were synthesized by Peptron (Daejeon, Korea).

Techniques: Injection, Staining

Journal: Cells

Article Title: IL7-Fc Enhances the Efficacy of Adoptive T Cell Therapy under Lymphopenic Conditions in a Murine Melanoma Model

doi: 10.3390/cells10082018

Figure Lengend Snippet: Effects of IL7-Fc on homeostatic and Ag-dependent proliferation of pmel-1 CD8 + T cells in non-lymphopenic and lymphopenic conditions. ( A ) WT or RAG1 −/− C57BL/6 mice were injected i.v. with 5 × 10 5 of CFSE-labeled pmel-1 Thy1.1 + CD8 + T cells and daily injected i.p. with 1 μg of hIgG or IL7-Fc for 3 days. After seven days, a single cell suspension of inguinal LN cells was stained with anti-CD8-PE and anti-Thy1.1-PE-Cy5. CFSE dilution rates of the gated Thy1.1 + CD8 + cells were assessed by flow cytometry ( A ). ( B ) Total numbers of live LN cells, percentages of Thy1.1 + CD8 + T cells, and absolute numbers of Thy1.1 + CD8 + T cells were determined by flow cytometry and cell counting. Proliferation index was calculated using Flow Jo software. ( C , D ) WT or RAG1 −/− C57BL/6 mice were administered 5 × 10 6 of CFSE-labeled pmel-1 Thy1.1 + CD8 + T cells, immunized s.c. with mgp100 peptide emulsified in IFA, and injected daily with hIgG or IL7-Fc for 3 days. After four days, the CFSE dilution rates of the Thy1.1 + CD8 + cells in inguinal LNs were assessed by flow cytometry ( C ). ( D ) Total numbers of live LN cells, percentages of Thy1.1 + CD8 + T cells, absolute numbers of Thy1.1 + CD8 + T cells, and proliferation index were calculated as described above. Data are from two independent experiments with 3 mice per experiment. Student’s t -test was performed in ( B , D ), and the results are expressed as mean ± SD (* p < 0.05; ** p < 0.01; *** p < 0.005).

Article Snippet: Mouse gp100 25–33 (mgp100, EGSRNQDWL) and human gp100 25–33 (hgp100, KVPRNQDWL) peptides were synthesized by Peptron (Daejeon, Korea).

Techniques: Injection, Labeling, Suspension, Staining, Flow Cytometry, Cell Counting, Software

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Article Title: Cloning and Characterization of the Genes Encoding the Murine Homologues of the Human Melanoma Antigens MART1 and gp100

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Figure Lengend Snippet: Northern blot analyses of the expression of mouse MART1 and gp100 mRNA. Fifteen micrograms of total RNAs from different murine melanoma tumor lines, nonmelanoma lumor lines, and normal tissues were loaded in each lane. Mouse MART1 mRNA (A) and gp100 mRNA (B) were detected using the full-length murine MART1 and gp100 cDNA as the probes, respectively. Ethidium bromide staining of RNA as shown in (C) was used as a control. The positions of mMART1 RNA, mgp100 RNA, AND 28S and 18S RNA were marked.

Article Snippet: The recombinant vaccinia and fowlpox viruses encoding human MART1 or gp100 (named as rVV-hMART1, rVV-hgp100, rFPV-hMART1, and rFPV-hgp100, respectively)were generated in a similar fashion and provided by Therion Biologics (Cambridge, MA, U.S.A.).The control vaccinia and fowlpox viruses containing LacZ (rVV-LacZ and rFPV-LacZ, respectively) were described previously ( 16 ).

Techniques: Northern Blot, Expressing, Staining, Control

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Article Title: Cloning and Characterization of the Genes Encoding the Murine Homologues of the Human Melanoma Antigens MART1 and gp100

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Figure Lengend Snippet: Western blot analyses of mouse MART1 and gp100 proteins. Equal quantities of protein (10 μg) from each cell line were subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblotting with MART1-specific monoclonal anibody (mAb)2D12 (A) or gp100-specific mAb α PEP13 (B). The 16-kDa mouse MART1 and the 85-kDa mouse gp100 proteins are marked. The position and size of molecular weight markers are indicated.

Article Snippet: The recombinant vaccinia and fowlpox viruses encoding human MART1 or gp100 (named as rVV-hMART1, rVV-hgp100, rFPV-hMART1, and rFPV-hgp100, respectively)were generated in a similar fashion and provided by Therion Biologics (Cambridge, MA, U.S.A.).The control vaccinia and fowlpox viruses containing LacZ (rVV-LacZ and rFPV-LacZ, respectively) were described previously ( 16 ).

Techniques: Western Blot, Polyacrylamide Gel Electrophoresis, Molecular Weight

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Article Title: Cloning and Characterization of the Genes Encoding the Murine Homologues of the Human Melanoma Antigens MART1 and gp100

doi:

Figure Lengend Snippet: Comparison of amino acid sequences of HLA-A2 restricted epitopes between human (h) and mouse (m) MART1 or gplO0 (identical amino acids are indicated by dashes)

Article Snippet: The recombinant vaccinia and fowlpox viruses encoding human MART1 or gp100 (named as rVV-hMART1, rVV-hgp100, rFPV-hMART1, and rFPV-hgp100, respectively)were generated in a similar fashion and provided by Therion Biologics (Cambridge, MA, U.S.A.).The control vaccinia and fowlpox viruses containing LacZ (rVV-LacZ and rFPV-LacZ, respectively) were described previously ( 16 ).

Techniques: Comparison, Sequencing

Journal:

Article Title: Cloning and Characterization of the Genes Encoding the Murine Homologues of the Human Melanoma Antigens MART1 and gp100

doi:

Figure Lengend Snippet: Recognition of mouse gp100 by human gp100-reactuve tumor-infiltrating lymphocytes (TILs)

Article Snippet: The recombinant vaccinia and fowlpox viruses encoding human MART1 or gp100 (named as rVV-hMART1, rVV-hgp100, rFPV-hMART1, and rFPV-hgp100, respectively)were generated in a similar fashion and provided by Therion Biologics (Cambridge, MA, U.S.A.).The control vaccinia and fowlpox viruses containing LacZ (rVV-LacZ and rFPV-LacZ, respectively) were described previously ( 16 ).

Techniques: Transfection

Journal:

Article Title: Cloning and Characterization of the Genes Encoding the Murine Homologues of the Human Melanoma Antigens MART1 and gp100

doi:

Figure Lengend Snippet: Protection of C57BL/6 mice against challenge with B16 melanoma using different immunization strategies

Article Snippet: The recombinant vaccinia and fowlpox viruses encoding human MART1 or gp100 (named as rVV-hMART1, rVV-hgp100, rFPV-hMART1, and rFPV-hgp100, respectively)were generated in a similar fashion and provided by Therion Biologics (Cambridge, MA, U.S.A.).The control vaccinia and fowlpox viruses containing LacZ (rVV-LacZ and rFPV-LacZ, respectively) were described previously ( 16 ).

Techniques:

Journal:

Article Title: Cloning and Characterization of the Genes Encoding the Murine Homologues of the Human Melanoma Antigens MART1 and gp100

doi:

Figure Lengend Snippet: Specific interferon-γ (IFNγ) secretion by murine T-cell lines induced by adenovirus type 2-human gp100 (Ad2-hgp100) immunization a

Article Snippet: The recombinant vaccinia and fowlpox viruses encoding human MART1 or gp100 (named as rVV-hMART1, rVV-hgp100, rFPV-hMART1, and rFPV-hgp100, respectively)were generated in a similar fashion and provided by Therion Biologics (Cambridge, MA, U.S.A.).The control vaccinia and fowlpox viruses containing LacZ (rVV-LacZ and rFPV-LacZ, respectively) were described previously ( 16 ).

Techniques: In Vitro, Infection