Journal: Science immunology

Article Title: The tumor-intrinsic role of the m 6 A reader YTHDF2 in regulating immune evasion

doi: 10.1126/sciimmunol.adl2171

Figure Lengend Snippet: ( A and B ) The tumor growth of WT and Ythdf2 -KO MC38 tumors ( n = 5) and B16-OVA tumors ( n = 6) in C57BL/6 mice. C57BL/6 mice were subcutaneously injected with either 1 × 10 6 WT and Ythdf2 -KO MC38 cells (A) or 1 × 10 6 WT and Ythdf2 -KO B16-OVA cells (B). Tumor sizes were measured every other day starting from the 12th or 14th day after tumor inoculation. ( C and D ) Overall survival of immunocompetent mice subcutaneously implanted with WT or Ythdf2 -KO B16-OVA tumor cells ( n = 14 for WT and n = 15 for KO) (C) or MC38 tumor cells ( n = 8 for WT and n = 10 for KO) (D). ( E and F ) WT or Ythdf2 -KO B16-OVA tumors were transplanted from Rag1 −/− to C57BL/6 mice ( n = 4). Tumor volume (E) and tumor weight (F) were measured on the 14th day after the tumor was transplanted. ( G ) A total of 1 × 10 6 WT or Ythdf2 -KO B16-OVA tumor cells were subcutaneously implanted in C57BL/6 mice pretreated with anti–immunoglobulin G (IgG), anti-NK1.1, anti-CD4, and anti-CD8 antibodies ( n = 5 or 6). Tumor growth was monitored by measuring tumor sizes every other day starting from the eighth day after tumor inoculation. ( H and I ) WT or Ythdf2 -KO MC38-OVA (H) and B16-OVA (I) tumor growth in Rag1 −/− mice that were adoptively transferred with OT1 CD8 + T cells. The mice were subcutaneously injected with either 1 × 10 6 WT or Ythdf2 -KO tumor cells. Tumor sizes were measured every other day starting from the sixth day after tumor inoculation ( n = 5). ( J ) WT or Ythdf2 -KO B16F10 tumor growth in Rag1 −/− mice that were adoptively transferred with gp100-stimulated pmel-1 splenocytes. The mice were subcutaneously injected with either 1 × 10 6 WT or Ythdf2 -KO tumor cells. Tumor sizes were measured every other day starting from the sixth day after tumor inoculation ( n = 5). Data are represented as means ± SD. Statistical analysis was performed using two-way ANOVA with a mixed-effects model with P values adjusted by a Holm-Šídák method for multiple comparisons (A, B, G, H, I, and J), unpaired two-tailed t test (E and F), or Kaplan-Meier survival analysis and log-rank test (C and D). The data presented represent one of two or three independent experiments. * P < 0.05, ** P < 0.01, and **** P < 0.001.

Article Snippet: Splenocytes isolated from pmel-1 T cell receptor transgenic mice were stimulated with gp100 25–33 (5 μg/ml; MedChemExpress) and mouse recombinant IL-2 (100 IU/ml) in RPMI 1640 complete medium at 37°C for 24 hours.

Techniques: Injection, Two Tailed Test

Journal: The Journal of Clinical Investigation

Article Title: Aptamer-targeted inhibition of mTOR in T cells enhances antitumor immunity

doi: 10.1172/JCI69856

Figure Lengend Snippet: CD90.2+ C57BL/6 mice were transferred with congenic CD90.1+ Pmel-1 cells, activated with gp100 peptide 2 days later, and treated either with rapamycin or with 4-1BB-GFP or 4-1BB-raptor conjugates. (A) On day 30, mice were revaccinated with gp100 peptide, and the proportion of CD8+CD90.1+ Pmel-1 cells in the spleen was determined by flow cytometry (n = 2). (B) On day 30 (day 0 on the x axis), mice were challenged s.c. with 105 B16.F10 melanoma cells, and the survival (time to sacrifice when tumors reached 12 mm in diameter) was determined (5 mice per group) (n = 3).

Article Snippet: Forty-eight hours later, 100 μg of KVPRNQDWL human gp100 ( 25 – 33 ) peptide (AnaSpec) and 10 μg of E . coli LPS were injected i.v., and mice were treated with rapamycin or aptamer-siRNA conjugates as described above.

Techniques: 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 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

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

Article Title: A Prime-Pull-Amplify Vaccination Strategy to Maximize Induction of Circulating and Genital-Resident Intraepithelial CD8 + Memory T Cells

doi: 10.4049/jimmunol.1800219

Figure Lengend Snippet: In situ TCR engagement promotes both recruitment of cognate and non-cognate activated CD8+ T cells. (A-D) CTV-labelled activated CD45.1+OT-I were pretreated or not with pertussis toxin and transferred to naïve C57BL/6 mice one day prior to ivag immunization with HPV PsV expressing OVA or a luciferase as a control. (A-B, E-F) CTV-labelled activated CD45.1+OT-I and Pmel-1 CD8+ T cells were co transferred to naïve C57BL/6 mice one day prior to ivag immunization with HPV PsV expressing OVA or a luciferase as a control. (B) In co-transfer condition, OT-I and Pmel-1 CD8+ T cells were distinguished by CD45.1 and H-2Kb/OVA257–264 tetramer staining. (C, E) On day 4 after immunization, absolute numbers and percent of OT-I and Pmel-1 CD8+ T cells were assessed in the cervicovaginal and dLN tissues, respectively. The mean absolute number and percentage of transferred OT-I CD8+ T cells per organs is shown as horizontal bar ± SD, each symbol represents an individual mouse. (D, F) Representative FACS plot of CD103 expression and CTV labeling on transferred OT-I CD8+ T from cervicovaginal and dLN tissues. Data are representative of two independent experiments (n=4–5 mice per group). P values (***P≤0.001, ****P≤0.0001) were determined by one-way ANOVA with post-hoc Tukey analysis.

Article Snippet: Spleen cells were incubated for three days at 37°C in RPMI medium containing recombinant human IL-2 (100IU/ml, TECIN) and OVA 254-262 (20ng/ml, Genscript) or gp100 25-33 peptide (100ng/ml, Genscript).

Techniques: In Situ, Expressing, Luciferase, Control, Staining, Labeling

Journal: The Journal of Clinical Investigation

Article Title: Pharmacologic LDH inhibition redirects intratumoral glucose uptake and improves antitumor immunity in solid tumor models

doi: 10.1172/JCI177606

Figure Lengend Snippet: ( A ) Schematic depicting tumor-killing assay with LDHi in which B16-YFP cells were treated with 20 μM LDHi or vehicle 24 hours apart and T cells were added 24 hours after the first LDHi treatment. ( B ) Quantified media glucose from killing assay coculture. ( C ) Flow cytometry quantification of 2-NBDG (MFI) in B16-YFP and CD8 + Pmel-1 T cells from killing assay cocultures 48 hours after last treatment. ( D – F ) ( D ) Quantified YFP + tumor cells and ( E ) representative in vitro killing assay images of YFP + tumor cells after 48 hours of coincubation with Pmel-1 CD8 + T cells as in A . ( F ) Corresponding quantified YFP + tumor cells and percentages of tumor killing in the same conditions as above alongside vehicle supplemented with 10 mM glucose. ( G ) Quantification of killing of OVA 257-264 –pulsed live B16-YFP tumor cells by OVA-primed CD8 + T cells from OT1 transgenic mice upon 48 hours of coculture in the presence of LDHi (as indicated in A ). E:T = 2:1, cocultured over 48 hours. ( H ) Schematic depicting in vitro Treg suppression assay with MACS column–sorted Tregs (CD4 + CD25 + Regulatory T Cell Isolation Kit, mouse) cocultured with αCD3/αCD28-activated CTV-labeled syngeneic CD8 + T cells for 48 hours with the addition of conditioned media from B16 cells treated with 20 μM LDHi or vehicle or fresh media containing 10 mM glucose. ( I ) Percentage of suppression was calculated as percentage reduction in CD8 + T cell proliferation with respect to CD8 + T cells cultured alone in the same treatment and glucose conditions. Data show 1 representative experiment of 3 independent experiments ( n = 3–4 technical replicates). All statistics produced by 2-way ANOVA with Bonferroni’s multiple-comparisons test implemented in GraphPad Prism. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Data are represented as mean ± SEM.

Article Snippet: Splenocytes were primed with OVA (SIINFEKL 257-264, AnaSpec, AS-60193-1) or human gp100 peptide (25-33, AnaSpec, AS-62589) in RPMI media supplemented with 10% FCS and 50 mM BME as previously described ( ).

Techniques: Flow Cytometry, In Vitro, Transgenic Assay, Suppression Assay, Cell Isolation, Labeling, Cell Culture, Produced