Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD10 , REAL318 , 50 , 130-118-368 , FITC , Miltenyi Biotec.

Techniques: Imaging

Journal: Cancer research

Article Title: The alarmin HMGN1 contributes to anti-tumor immunity and is a potent immunoadjuvant

doi: 10.1158/0008-5472.CAN-13-2042

Figure Lengend Snippet: HMGN1 knockout (Hmgn1−/−) and littermate-matched wild-type (Hmgn1+/+) mice (7~8 week-old, female, 10/group) were subcutaneously injected with 2×105 EG7 tumor cells into the right flank and the growth of tumors was monitored. On day 28, mouse splenocytes were analyzed by flow cytometry after staining with anti-CD3-FITC, OVA-Tetramer-PE, anti-CD4-APC, and anti-CD8- PerCP-Cy5.5. A, Tumor growth, *p < 0.001 by ANOVA. B, Dot-plot showing OVA-specific CD8 cells of one mouse spleen. C, Percentage of OVA-specific CD8 cells of Hmgn1+/+ and Hmgn1−/− groups. Shown are the results of one experiment representative of two.

Article Snippet: The recombinant plasmids encoding HMGN1 (Clone Id LIFESEQ1228711) and gp100 (OriGene SC122763) were purchased from Open Biosystems and OriGene Technologies, Inc., respectively.

Techniques: Knock-Out, Injection, Flow Cytometry, Staining

Journal: Cancer research

Article Title: The alarmin HMGN1 contributes to anti-tumor immunity and is a potent immunoadjuvant

doi: 10.1158/0008-5472.CAN-13-2042

Figure Lengend Snippet: A, Mouse BM-derived DCs (5×105/ml) treated without or with recombinant HMGN1 (1 or 5 µg/ml) at 37°C for 48 h in humidified air containing 5% CO2 were immunostained for analysis of surface expression of CD80, CD86, and I-A/E. Shown are the overlay histograms (shaded area = sham treated DCs; solid line = HMGN1-treated DCs) of one experiment representative of three. B, Supernatants of mouse BM-derived DCs (5×105/ml) incubated in the absence (sham) or presence of recombinant human HMGN1 (5 µg/ml) at 37°C for 48 h in humidified air containing 5% CO2 were assayed for the production of indicated cytokines. Shown is the average (mean±SD) of three experiments. *p < 0.05 by Student’s t test.

Article Snippet: The recombinant plasmids encoding HMGN1 (Clone Id LIFESEQ1228711) and gp100 (OriGene SC122763) were purchased from Open Biosystems and OriGene Technologies, Inc., respectively.

Techniques: Derivative Assay, Recombinant, Expressing, Incubation

Journal: Cancer research

Article Title: The alarmin HMGN1 contributes to anti-tumor immunity and is a potent immunoadjuvant

doi: 10.1158/0008-5472.CAN-13-2042

Figure Lengend Snippet: A, Schematic illustration of pcDNA3.1-hHMGN1-hygromycin. EG7 cells were transfected with pcDNA3.1-hygromycin or pcDNA3.1-hHMGN1-hygromycin and selected in the presence of hygromycin for generating control and human HMGN1-expressing EG7 clones. B, in vitro proliferation of control and HMGN1-expressing EG7 tumor cells. Control and HMGN1-expressing EG7 cells were plated in triplicate in a 96-well plate at the indicated number and incubated overnight. Subsequently, the culture was pulsed with 0.5 µCi/well of 3H-TdR for 4 h, harvested, and measured for the incorporation of 3H-TdR. Shown is the average incorporation of 3H-TdR (mean ± SD) of triplicate wells. C, C57BL/6 mice (8-week old, female) were divided into 2 groups (n = 10) and were subcutaneously injected with 5×105 control or HMGN1-expressing EG7 into the right flank. Tumor growth was monitored twice a week for up to 4 weeks. Shown are the results of one experiment representative of three. *p < 0.05 by Repeated Measures ANOVA. D, Two groups of C57BL/6 mice (8-week old, female, n = 10) were subcutaneously injected with 5×105 control or HMGN1-expressing EG7 into the right flank. Mouse morbidity was monitored for 60 days and compared after graphing with Prism software. *p < 0.05 by Mantel-Cox test. E, Five groups of C57BL/6 mice (8-week old, female, n = 10) were subcutaneously injected with 5×105 control (PBS group) or HMGN1-expressing EG7 (groups with Ab treatment) into the right flank on day 1. For depleting CD4 or CD8 T cells, mice were intraperitoneally injected with anti-CD4, anti-CD8, or a combination of anti-CD4 and anti-CD8α antibodies on day-4, -3, -2, 3, 10, 17, and 24 at 150 µg per mouse. Tumor volume was monitored twice a week. Shown are the results of one experiment representative of two. *p < 0.05 by Repeated Measures ANOVA.

Article Snippet: The recombinant plasmids encoding HMGN1 (Clone Id LIFESEQ1228711) and gp100 (OriGene SC122763) were purchased from Open Biosystems and OriGene Technologies, Inc., respectively.

Techniques: Transfection, Control, Expressing, Clone Assay, In Vitro, Incubation, Injection, Software

Journal: Cancer research

Article Title: The alarmin HMGN1 contributes to anti-tumor immunity and is a potent immunoadjuvant

doi: 10.1158/0008-5472.CAN-13-2042

Figure Lengend Snippet: A, Schematic illustration of a series of eukaryotic expressing plasmids (DNA vaccines) harboring the genes of HMGN1, gp100, or HMGN1-gp100 fusion. The eukaryotic expressing vector used in the DNA vaccines is pcDNA3.1/myc-His B (Invitrogen), which provides a myc epitope and a polyhistidine-tag at the C-terminus. The signal peptide of insulin was introduced in front of target genes to ensure secretion. The Kozak sequence was optimized. In the fusion gene HMGN1-gp100, a flexible linker (Gly4Ser)3 was inserted between HMGN1 and gp100. B, HEK293 cells (106/flask) were transfected with 10 µg of pcDNA3.1, pcDNA3.1-HMGN1, pcDNA3.1-gp100, or pcDNA3.1-HMGN1-gp100 plasmid using Lipofectamine™ 2000. Six hours after the transfection, the culture medium was replaced with DMEM containing 1% FBS and after an additional 24 hour-incubation, supernatants were analyzed by Western Blot. Purified human HMGN1 (1 µg) was used as a positive control. Left panel: lanes loaded with the supernatant of HEK293 cells transfected with pcDNA3.1-HMGN1 or purified HMGN1 were probed with α-polyHis (left two lanes) or α-HMGN1 (right two lanes). Right panel: lanes loaded with the supernatants of HEK293 cells transfected with indicated plasmids were probed with α-HMGN1. C, Transfection and subsequent analysis were similar as in B. Supernatants of HEK293 cells transfected with indicated plasmids were analyzed Western blot using anti-HMGN1 (left panel) or anti-gp100 (right panel) antibodies.

Article Snippet: The recombinant plasmids encoding HMGN1 (Clone Id LIFESEQ1228711) and gp100 (OriGene SC122763) were purchased from Open Biosystems and OriGene Technologies, Inc., respectively.

Techniques: Expressing, Vaccines, Plasmid Preparation, Sequencing, Transfection, Incubation, Western Blot, Purification, Positive Control

Journal: Cancer Immunology Research

Article Title: IDO Vaccine Ablates Immune-Suppressive Myeloid Populations and Enhances Antitumor Effects Independent of Tumor Cell IDO Status

doi: 10.1158/2326-6066.CIR-21-0457

Figure Lengend Snippet: IDO vaccine enhances immune-mediated antitumor effects of tumor antigen–specific vaccination and prolongs survival in the B16F10 tumor model. Gating strategy and IDO pentamer specificity in Supplementary Fig. S2. A, Schematic of the treatment schedule in the tumor model. On day 10 of tumor growth, B16F10 tumor-bearing mice were given IDO vaccine (IDO Vax) with the gp100 25–33 peptide vaccine, along with PADRE (20 μg/mouse) and QuilA (10 μg/mouse) subcutaneously, every 7 days for a total of three doses. Tumor growth and survival were measured. B, Average tumor volume in mice following treatment (* vs. untreated; green * vs. IDO Vax; red * vs. gp100). C, Percent survival of mice depicted by the Kaplan–Meier plot. D, SK plot showing tumor volume and survival for each mouse at different days. Data are shown as an average of two independent experiments ( n = 10–18 per group). Error bars indicate the SEM. For tumor growth, statistical analysis was performed by unpaired, one-tailed Student t test. Survival in various groups was compared using log-rank (Mantel–Cox) tests. *, P ≤ 0.05; and **, P ≤ 0.01. E–K, C57BL/6J mice ( n = 5–8 per group) were treated as in A , except 3 days after second vaccination, mice were sacrificed, and tumors were harvested for immune response study. The frequency of B16F10 tumor-infiltrating cells was determined. Total ( E ), IFNγ + and GB + ( F ), CD40L + ( G ), Penta-IDO + ( H ), IFNγ + penta-IDO + ( I ), Dextra-gp100 + ( J ), IFNγ + dextra-gp100 + ( K ) CD8 + T cells were measured by flow cytometry. Data are shown from one representative experiment of two independent experiments. Error bars indicate the SEM. Statistical analysis was performed by unpaired, one-tailed Student t test. NS: nonsignificant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Article Snippet: The CTL epitope from human gp100 25–33 (KVPRNQDWL; an altered peptide ligand vaccine for mice (100 μg/mouse; Celtek Bioscience) for the B16F10 tumor model or HPV16 E7 49–57 (RAHYNIVTF) for TC-1 tumor model [9-amino acid (aa) peptide, 100 μg/mouse] mixed with synthetic T-helper epitope PADRE (13 aa peptide, aK-Cha-VAAWTLKAAa, where “a” is D-alanine and Cha is L-cyclohexylalanine, 20 μg/mouse; both from Celtek Bioscience) and QuilA adjuvant (10 μg/mouse; Brenntag) were used as the model vaccine (subcutaneous injections, as described below), which contains both CD8 and CD4 epitopes in all studies ( ).

Techniques: One-tailed Test, Flow Cytometry

Journal: Cancer Immunology Research

Article Title: IDO Vaccine Ablates Immune-Suppressive Myeloid Populations and Enhances Antitumor Effects Independent of Tumor Cell IDO Status

doi: 10.1158/2326-6066.CIR-21-0457

Figure Lengend Snippet: IDO vaccine reduces frequency of Tregs and enhances therapeutic ratio in the TME. C57BL/6J mice were treated as in Fig. 1A and , except 3 days after second vaccination, mice were sacrificed, and tumors were harvested for immune response study. The frequency of B16F10 tumor-infiltrating CD4 + ( A ), FoxP3 + CD4 + ( B ), CD8 + /Tregs ( C ), Dextra-gp100 + CD8 + /Tregs ( D ), and Penta-IDO + CD8 + /Tregs ( E ) was determined by flow cytometry. The frequency of TC-1 tumor-infiltrating CD4 + ( F ), FoxP3 + CD4 + ( G ), CD8 + /Tregs ( H ), Dextra-E7 + CD8 + /Tregs ( I ), and Penta-IDO + CD8 + /Tregs ( J ) were also measured by flow cytometry. Data are shown as an average of two independent experiments ( n = 8–13 per group). Error bars indicate the SEM. Statistical analysis was performed by unpaired, one-tailed Student t test. NS, nonsignificant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Article Snippet: The CTL epitope from human gp100 25–33 (KVPRNQDWL; an altered peptide ligand vaccine for mice (100 μg/mouse; Celtek Bioscience) for the B16F10 tumor model or HPV16 E7 49–57 (RAHYNIVTF) for TC-1 tumor model [9-amino acid (aa) peptide, 100 μg/mouse] mixed with synthetic T-helper epitope PADRE (13 aa peptide, aK-Cha-VAAWTLKAAa, where “a” is D-alanine and Cha is L-cyclohexylalanine, 20 μg/mouse; both from Celtek Bioscience) and QuilA adjuvant (10 μg/mouse; Brenntag) were used as the model vaccine (subcutaneous injections, as described below), which contains both CD8 and CD4 epitopes in all studies ( ).

Techniques: Flow Cytometry, One-tailed Test