quantikine elisa mouse ifnγ immunoassay  (R&D Systems)

Journal: Cancer Discovery

Article Title: Temporal Genomic Analysis of Homogeneous Tumor Models Reveals Key Regulators of Immune Evasion in Melanoma

doi: 10.1158/2159-8290.CD-23-1422

Figure Lengend Snippet: SCCs with the same clonality and >80% genetic similarity show opposite growth phenotypes. A, Phylogenetic tree representing data from the UVB-irradiated B2905 cell line. The tree depicts the results from mutation-based clustering analysis, which was used to define the distinct subclones present within the UVB cell line. The phylogenetic relationship between subclones is shown; each of the 40 UVB-derived SCCs was mapped onto the subclonal branch with the highest genetic similarity. Each of the 40 SCCs is depicted as a ball of 100 tumor cells, with the color coding reflecting the percentage frequency of each branch in each SCC sample. Top, Left boxes show the UVB sample (median and mean variant VAF) as a ball of 100 tumor cells, color-coded to match the subclonal branches. B, Growth curve of nonrejected (top) and rejected (bottom) SCC-derived tumors in vivo in immunocompetent mice. n = 5. C, VAF distribution of parental UVB-irradiated B2905 cells (red), rejected SCC31 (purple), and SCC40 (pink) cells and nonrejected SCC32 (green), SCC35 (blue), and SCC37 (light-blue) cells in the log 2 space. VAF > 0.25 (log 2 = −2) is considered clonal. D, Growth curve of rejected SCC-derived tumors in vivo in NSG immunodeficient mice. n = 3. E, PCA plot based on the TMM-normalized log CPM gene expression of the rejected (green) and nonrejected (pink) clones across in vitro (day 0; open circles) and in vivo (days 6, 10, 16, and 20; closed circles) time points after removing outlier samples (“Methods”). Top two PCs are shown, with the percentage of explained total variance labeled on the corresponding axis. F, T cell function scores and ( G ) T cell infiltration scores, both computed with the TIDE algorithm in the rejected (green) and nonrejected (pink) clones across different time points ( x -axis). The scores between the rejected and nonrejected clones were compared with a linear model at each time point (“Methods”), and the corresponding Benjamini–Hochberg adjusted P values are shown. H, GSVA enrichment scores of the IFNγ pathway (“Methods”) in the rejected (green) and nonrejected (pink) clones across different time points ( x -axis). Enrichment for the IFNγ pathway genes was tested with gene set enrichment analysis (“Methods”), and the corresponding Benjamini–Hochberg adjusted P values are shown.

Article Snippet: To measure IFNγ secretion by T cells co-cultured with the tumor-derived macrophages in the media of the T cell suppression assay, we used Quantikine ELISA Mouse IFNγ Immunoassay (R&D Systems, cat. #MIF00-1) according to the manufacturer’s instructions.

Techniques: Irradiation, Mutagenesis, Derivative Assay, Variant Assay, In Vivo, Gene Expression, Clone Assay, In Vitro, Labeling, Cell Function Assay

Journal: Cancer Discovery

Article Title: Temporal Genomic Analysis of Homogeneous Tumor Models Reveals Key Regulators of Immune Evasion in Melanoma

doi: 10.1158/2159-8290.CD-23-1422

Figure Lengend Snippet: Opal and CODEX multiplexed tissue imaging data validate increased M2-like macrophage infiltration and T cell exhaustion in the nonrejected SCCs. A, Representative Opal Multiplex IHC stains for F4/80 (red), CD204 (yellow), and CD206 (green) in tumors derived from SCC31, SCC40, SCC32, SCC35, and SCC37 on day 10 after inoculation. Four areas from each tumor and three tumors from each SCC were examined. Scale bars, 20 μm. DAPI staining is not shown. B, Representative CODEX multiplexed tissue images for CD3 (green), CD8 (red), TIM3 (magenta), and LAG3 (cyan blue) in tumors derived from SCC31, SCC40, SCC32, SCC35, and SCC37 on day 6 after inoculation. Five areas from each tumor and three tumors from each SCC were examined. Scale bars, 20 μm. C, Quantification of the percentage of F4/80 + CD204 + CD206 + cells described in A . Data are mean ± SEM. The rejected and nonrejected groups were compared using the Wilcoxon rank-sum test; W = 456; P = 2.55 × 10 −8 . D, Quantification of the percentage of CD3 + CD8 + cells described in B . Data are mean ± SEM. P = 0.00055; t = −5.27; degrees of freedom (df) = 9, unpaired t test. E, Quantification of the percentage of TIM3 + LAG3 + /CD3 + CD8 + cells described in B . Data are mean ± SEM. P = 0.02522; t = 2.6798; df = 9, unpaired t test. F, Boxplot quantifying the percentage of total cells for each CD8 cluster identified in CyTOF analysis. G, Boxplot quantifying the percentage of total cells for each macrophage cluster identified in CyTOF analysis. H, CFSE-based T cell proliferation assay after co-culture for 48 hours with nonrejected SCC35 tumor-derived macrophages isolated 10 days after inoculation. Representative histogram plot and quantification of CFSE intensity ( n = 5). CD8 + /CD4 + T cells isolated from healthy mouse spleens cultured without macrophages served as the controls. CD8: Kruskal–Wallis χ 2 test, χ 2 = 12.5; P = 0.001930454. Pairwise comparison by the Wilcoxon test with Bonferroni correction is shown in the figure. *, P value < 0.05. CD4: Kruskal–Wallis χ 2 test, χ 2 = 12.5; P = 0.00193. Pairwise comparison by the Wilcoxon test with Bonferroni correction is shown in the figure. *, P value < 0.05. ELISA measurement of IFNγ in culture media 48 hours after co-culture of T cells with or without tumor-derived macrophages. n = 6. Data are mean ± SEM. Kruskal–Wallis χ 2 test, χ 2 = 15.726; P = 0.0003847. Pairwise comparison by the Wilcoxon test with Bonferroni correction is shown. ***, P value < 0.01. MFI, mean fluorescence intensity.

Article Snippet: To measure IFNγ secretion by T cells co-cultured with the tumor-derived macrophages in the media of the T cell suppression assay, we used Quantikine ELISA Mouse IFNγ Immunoassay (R&D Systems, cat. #MIF00-1) according to the manufacturer’s instructions.

Techniques: Imaging, Multiplex Assay, Derivative Assay, Staining, Proliferation Assay, Co-Culture Assay, Isolation, Cell Culture, Comparison, Enzyme-linked Immunosorbent Assay, Fluorescence

Journal: Clinical Cancer Research

Article Title: Targeting Dendritic Cell Dysfunction to Circumvent Anti-PD1 Resistance in Head and Neck Cancer

doi: 10.1158/1078-0432.CCR-23-3477

Figure Lengend Snippet: aPD1-resistant model shows reduced priming due to cDC1 dysfunction in tumor-draining lymph nodes (DLN). A, In vivo tumor growth of MOC1P-ova after treatment with aPD1 (250 μg IP on days 3, 6, and 9) or FTY720 (10 μg IP daily from one day before inoculation). ( n = 4 tumors for each group). and C, Flow-cytometric analysis of MOC1P-ova/MOC1esc1-ova DLN on day 10 after inoculation ( n = 4 for each group, representative data of two independent experiments). D, Representation of experiment in . E, Tumor DLN from orthotopically inoculated MOC1P-ova/esc1-ova–bearing mice were harvested on day 10 and stimulated with SIINFEKL peptide for 48 hours to assess IFNγ production by ELISA ( n = 4 for each group, representative data of two independent experiments). F, Flow-cytometric analysis of costimulatory markers on Xcr1 + DC in DLN of MOC1P-ova/MOC1esc1-ova harvested 10 days after tumor inoculation ( n = 4 for each group, representative data of two independent experiments). G, Representation of experiment in . H, Xcr1 + DC magnetically isolated from DLN of MOC1P-ova/MOC1esc1-ova were cocultured with CD8 + OT1 T cells to test priming ability evaluated by IFNγ ELISA ( n = 5–6, representative data of two independent experiments). Data are plotted as mean ± SEM in and individual data with mean ± SD in all other panels. Data were analyzed using two-way ANOVA with multiple comparison for and Mann–Whitney U test to generate two-tailed P values in , C , , F , and . ( and G were generated by using BioRender under granted license.) *, P < 0.05; **, P < 0.01; ns, not significant.

Article Snippet: Quantikine ELISA Mouse IFNγ Immunoassays (R&D Systems) were performed for IFNγ quantification, and mouse CCL5 ELISA Kit (KE10017, Proteintech) was used for CCL5 quantification according to the manufacturer's instructions.

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Isolation, Comparison, MANN-WHITNEY, Two Tailed Test, Generated

Journal: Clinical Cancer Research

Article Title: Targeting Dendritic Cell Dysfunction to Circumvent Anti-PD1 Resistance in Head and Neck Cancer

doi: 10.1158/1078-0432.CCR-23-3477

Figure Lengend Snippet: Xcr1+ DC vaccine is sufficient to induce antigen-reactive T cells, alter the tumor microenvironment and attenuate tumor growth in aPD1-resistant mouse model. A, DC generation, Xcr1 + DC isolation, and Xcr1 + DC intratumoral vaccine. Vaccination was performed with 1 million Xcr1 + DC. B, CD8 + T cells were isolated from intratumoral DC-vaccinated MOC1esc1 mouse DLN or spleen on day 14 after inoculation, stimulated with peptides and evaluated for reactivity by IFNγ ELISA. PC; positive control (PMA + ionomycin), NC; negative control (no peptides), or p15e or mYipf1 peptide (0.1 μmol/L) stimulation. ( n = 8, representative data of two independent experiments). C and D, Flow-cytometric analysis of MOC1esc1 tumors ( C ) and DLN treated with intratumoral PBS or DC vaccine on days 1/4/7 after inoculation and harvested on day 14 after tumor inoculation. ( n = 8, representative data of two independent experiments, gating strategies shown in Supplementary Fig. S2F and S2G). E, Tumor growth of aPD1-resistant MOC1esc1 model treated with intratumoral PBS (on days 1/4/7), intraperitoneal aPD1 (250 μg on days 3/6/9), intratumoral DC vaccine (1 million Xcr1+ DC on days 1/4/7), or the combination. n = 8 per group. F, DLN and spleens of MOC1esc1-bearing mice treated as in (separate experiment) were harvested on day 13 after inoculation and cocultured with indicated peptides to test reactivity evaluated by IFNγ ELISPOT. PC; positive control (PMA + ionomycin), NC; negative control (no peptides), or p15e + mYipf1 peptide (0.1 μmol/L) stimulation. ( n = 8 per group). G, Quantification of spots analyzed in experiment . ( n = 2). Individual data with mean ± SD are plotted in – . Data are plotted as mean ± SEM in . Data were analyzed using the Mann–Whitney U test to generate two-tailed P values in – . Two-way ANOVA with multiple comparison was used for growth curve analysis in . ( was generated by using BioRender under granted license.) *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Article Snippet: Quantikine ELISA Mouse IFNγ Immunoassays (R&D Systems) were performed for IFNγ quantification, and mouse CCL5 ELISA Kit (KE10017, Proteintech) was used for CCL5 quantification according to the manufacturer's instructions.

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Positive Control, Negative Control, Enzyme-linked Immunospot, MANN-WHITNEY, Two Tailed Test, Comparison, Generated

Journal: Clinical Cancer Research

Article Title: Targeting Dendritic Cell Dysfunction to Circumvent Anti-PD1 Resistance in Head and Neck Cancer

doi: 10.1158/1078-0432.CCR-23-3477

Figure Lengend Snippet: CCL5 recruits cDC1 and restores aPD1 responsiveness. A, Comparative tumor growth of MOC1esc1_Ctrl and MOC1esc1_CCL5 cells in C57BL/6 WT mice. Tumor weight measured on day 16 after tumor inoculation ( n = 6 for E1_Ctrl, n = 8 for E1_CCL5, representative data of two independent experiments). B and C, Flow-cytometric analysis of MOC1esc1_Ctrl and MOC1esc1_CCL5 tumors harvested on day 16 after tumor inoculation ( n = 6 for E1_Ctrl, n = 8 for E1_CCL5, representative data of two independent experiments, gating strategies shown in Supplementary Fig. S2F and S2G). D, Flow-cytometric analysis of MOC1esc1_Ctrl and MOC1esc1_CCL5 DLN harvested on day 16 after tumor inoculation ( n = 20 for Ctrl, n = 22 for CCL5, pooled data from three independent experiments, gating strategies shown in Supplementary Fig. S2G). E, Tumor growth experiment of MOC1esc1, MOC1esc1_Ctrl, and MOC1esc1_CCL5 cells (1×10 6 cells/mouse) treated with aPD1 (250 μg/mouse) on days 3, 6, and 9 (black arrows). Left shows mean ± SEM, and right shows individual tumor sizes ( n = 4 for E1 and E1_Ctrl, n = 6 for E1_CCL5, representative data of two independent experiments). F, CD8 + T cells isolated from aPD1-treated MOC1esc1_Ctrl or MOC1esc1_CCL5 DLN were stimulated with indicated peptides for 48 hours and evaluated by IFNγ ELISA. PC; positive control (PMA + ionomycin), NC; negative control (no peptides, n = 2) or p15e, mYipf1 peptide stimulation (0.1 μmol/L). Data are plotted as mean ± SEM in and and individual data with mean ± SD are plotted in – and F . Two-way ANOVA with multiple comparison was used for growth curve analysis in and . Data were analyzed using the Mann–Whitney U test to generate two-tailed P values in – . *, P < 0.05; **, P < 0.01; ns, not significant.

Article Snippet: Quantikine ELISA Mouse IFNγ Immunoassays (R&D Systems) were performed for IFNγ quantification, and mouse CCL5 ELISA Kit (KE10017, Proteintech) was used for CCL5 quantification according to the manufacturer's instructions.

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Positive Control, Negative Control, Comparison, MANN-WHITNEY, Two Tailed Test

Journal: PLoS Pathogens

Article Title: CCR5 Is Essential for NK Cell Trafficking and Host Survival following Toxoplasma gondii Infection

doi: 10.1371/journal.ppat.0020049

Figure Lengend Snippet: (A and B) Proliferation. Magnetically purified positively selected CD4 + (A) and CD8 + (B) T cells (>95% pure) isolated from the spleens of C57BL/6x129 on day 7 PI. Cells were stimulated either with ConA (2.5 μg/ml) or Toxoplasma lysate antigen (15 μg/ml). After 72 h incubation, proliferation was measured by 3 H thymidine incorporation. Data are represented as mean cpm ± standard deviation and are representative of two experiments. (C and D) IFNγ secretion. 10 6 purified CD4 + (C) and CD8 + (D) T cells from 7 d infected C57BL/6x129 mice were cultured in presence of 15 μg/ml of Toxoplasma lysate antigen and irradiated feeder cells (5 × 10 5 cells/well) in 24-well plates. After 72 h of incubation the supernatants were collected, centrifuged, and assayed for IFNγ production by ELISA. (E) Intracellular IFNγ production. Female CCR5 −/− (5–8 wk old) and wild-type mice were infected perorally with T. gondii cysts and splenocytes were harvested at day 7 PI, pooled (three mice per group), and cultured in vitro with phorbol 12-myristate 13-acetate, ionomycin, and monensin for 4 h. The cultured cells were stained for CD4 or CD8 before intracellular staining for IFNγ. Data are presented as percentage (mean ± standard deviation) of CD4 + or CD8 + T cells positive for IFNγ and are pooled from two experiments.

Article Snippet: Supernatants were analyzed for IFNγ by ELISA using mouse IFNγ immunoassay kit (R&D Systems, Minneapolis, Minnesota, United States).

Techniques: Purification, Isolation, Incubation, Standard Deviation, Infection, Cell Culture, Irradiation, Enzyme-linked Immunosorbent Assay, In Vitro, Staining