Journal: Vaccine

Article Title: Immunogenic potential of DNA vaccine candidate, ZyCoV-D against SARS-CoV-2 in animal models

doi: 10.1016/j.vaccine.2021.05.098

Figure Lengend Snippet: Detection of IFN-γ responses in BALB/c mice post-administration of DNA vaccine. BALB/c mice were immunized with 25 and 100 μg of DNA vaccine. IFN-γ responses were analyzed in the animals on days 14, 28 and 42. T cell responses were measured by IFN-γ ELISpot in splenocytes stimulated for 24 h with overlapping peptide pools spanning the SARS-CoV-2 spike region.

Article Snippet: The 96-well Mouse IFN-γ ELISPOT kit (CTL, USA) plates pre-coated with purified anti-mouse IFN-γ capture antibody were taken out blocked with RPMI-1640 + 10% FBS + 1X Antibiotic-Antimycotic for 1 Hr in CO 2 incubator.

Techniques: Enzyme-linked Immunospot

Journal: BMC Cancer

Article Title: HLA class II-restricted T cell epitopes in public neoantigens of ESR1 and PIK3CA in breast cancer

doi: 10.1186/s12885-025-13992-6

Figure Lengend Snippet: Summary of the ELISPOT assay of healthy donors. A Representative data of the ELISPOT assay for ES#2_E380Q, PIK#5_E545A, PIK#8_H1047L, and PIK#9_H1047Y, measured by IFN-γ (red spots) and IL-2 (blue spots). The numbers denote the spot count for IFN-γ (red). NC, negative control. TNTC: too numerous to count. B , C The ELISPOT profiles for DRB4*01:03 positive donors ( B ) and DP5 positive donors ( C ). The data for peptide pairs that showed penetrance ≥ 0.4 and g15 ratio > 2.0 are presented. HLA alleles of each donor are displayed on the top. Positive ELISPOT responses (IFN-γ) are highlighted in red. Numbers indicate the positive responses/total number of experiments (range of spot counts). The penetrance represents the number of positive donors/numbers of total donors (in red letters). The peptides with %Rank < 10 (NetMHCIIpan-4.1) is highlighted in yellow, and those with a g15 ratio > 2.0 (MHC-density assay) is highlighted in light blue. NA: not analyzed (shadowed in gray). TNTC: too numerous to count

Article Snippet: The ELISPOT assay was conducted using the human interferon-gamma (IFN-γ)/IL-2 double-color ELISPOT kit (Cellular Technology Limited, Cleveland, OH, USA), following the manufacturer’s instructions.

Techniques: Enzyme-linked Immunospot, Negative Control

Journal: NPJ Vaccines

Article Title: Shigella virulence protein VirG is a broadly protective antigen and vaccine candidate

doi: 10.1038/s41541-023-00797-6

Figure Lengend Snippet: a , b Spleen and bone marrow were obtained from mice immunized IM with 20 µg of VirGα (IM) or IN with S. flexneri 2a (sl Sf2a-IN) that survived S. flexneri 2a lethal challenge. Single-cell suspensions from individual mice in each group were pooled, and VirGα-specific and total IgG and IgA ASC were measured by ELISpot in quadruplicates. Bars represent mean values per 10 6 cells ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 by t test.

Article Snippet: The frequencies of total and VirGα-specific IgG- and IgA-secreting cells were determined using a mouse IgA/IgG double-color ELISpot assay kit [Catalogue # mIgGIgA-DCE-1M/2, ImmunoSpot® ELISpot Kit, Cellular Technology Limited (CTL), Cleveland, OH] according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunospot

Journal: Oncotarget

Article Title: The novel complex combination of alum, CpG ODN and HH2 as adjuvant in cancer vaccine effectively suppresses tumor growth in vivo

doi: 10.18632/oncotarget.17504

Figure Lengend Snippet: ( A ) The 4-h NK assay against YAC-1 targets was determined at various effector:target ratios. Error bars represent mean + SEM. ** p < 0.01, *** p < 0.001. ( B ) The cytolytic activity of splenocytes against NY-ESO-1 + B16 cells was measured using a 4 h 51 Cr-release assay at various E:T ratios. Error bars represent mean + SEM. *** p < 0.001. ( C ) The representative graphs of spot-forming cells for IFN-γ and IL-4 at one week post-immunization in the ELISpot assay. ( D ) The average number of IL-4/IFN-γ secreting splenocytes was calculated (n = 3, three independent experiments). Error bars represent mean + SEM. * p < 0.05, ** p < 0. 01. ( E ) T cell analysis in splenocytes after vaccination. Intracellular staining of IFN-γ in CD4 + and CD8 + T cells was analyzed by FACS.

Article Snippet: The mouse IFN-γ/IL-4 Dual-Color ELISpot kit (R&D Systems, Minneapolis, USA) was used for ELISpot assay.

Techniques: Activity Assay, Release Assay, Enzyme-linked Immunospot, Cell Analysis, Staining

Journal: bioRxiv

Article Title: mRNA-based influenza vaccine expands breadth of B cell response in humans

doi: 10.1101/2024.10.10.617255

Figure Lengend Snippet: a , HA proteins encoded by mRNA-1010 for the 2022-2023 Northern Hemisphere influenza vaccine strains. Fluarix vaccine included the same strains, except H3N2 was egg-based (A/Darwin/9/2021). b , ELISpot quantification of mean HA-binding IgM-secreting PBs in blood at baseline, 1, and 2 weeks post vaccination in mRNA-1010 (red) and Fluarix (black) participants. Numbers of HA-specific PBs were quantified against the four vaccine HAs and averaged. c , ELISpot quantification of HA-binding IgM-secreting PBs at 1 week post vaccination in mRNA-1010 (red) and Fluarix (black) participants. Horizontal bars represent geometric means. P values determined by Mann-Whitney U test. d , Flow cytometry gating strategy for HA-specific MBCs from PBMCs e , Quantification of HA-specific MBCs as a percentage of CD19+ cells in blood by flow cytometry at baseline, 4, and 17/26 weeks post vaccination in mRNA-1010 (red) and Fluarix (black) participants. Numbers represent geometric mean frequencies of MBCs for each time point. f , Fold change in HA-specific MBCs as a percentage of CD19+ cells in blood by flow cytometry at indicated time points over week 0 for mRNA-1010 (red) and Fluarix (black) participants. Numbers represent mean values. In e and f , P values determined by Mann-Whitney U test. For participants that did not complete a blood collection at week 26 or PBMCs were not available, samples from 17 weeks post vaccination were used for analysis (mRNA-1010, n=3; Fluarix, n=2).

Article Snippet: Secreting cells were detected using three-color FluoroSpot IgA/IgG/IgM ELISpot Kits (Cellular Technology Limited) according to the manufacturer’s instructions.

Techniques: Northern Blot, Enzyme-linked Immunospot, Binding Assay, MANN-WHITNEY, Flow Cytometry

Journal: bioRxiv

Article Title: mRNA-based influenza vaccine expands breadth of B cell response in humans

doi: 10.1101/2024.10.10.617255

Figure Lengend Snippet: a , WU397 study design. We enrolled 29 healthy adults (ages 24-51) who received Fluarix (n=15) or mRNA-1010 (n=14) intramuscularly. Blood was collected before vaccination and at 1, 2, 4, 8, 17, and 26 weeks after vaccination. FNAs of ipsilateral axillary lymph nodes were collected before vaccination and at 2, 8, 17, and 26 weeks after vaccination. b , ELISpot quantification of mean HA-binding IgG- and IgA-secreting PBs in blood at baseline, 1, and 2 weeks after vaccination in mRNA-1010 (red) and Fluarix (black) participants. Numbers of HA-binding PBs were quantified against the four vaccine HAs and averaged. c , ELISpot quantification of HA-binding IgG- and IgA-secreting PBs at 1 week post vaccination in mRNA-1010 (red) and Fluarix (black) participants. Horizontal bars represent geometric means. P values determined by Mann-Whitney U test. d , Plasma IgG titers at baseline, 4, and 17/26 weeks post vaccination (left) and fold change in plasma IgG titers at 4 and 17/26 weeks over baseline (right) against the four vaccine HAs in mRNA-1010 (red) and Fluarix (black) participants. Numbers on left panels represent geometric mean titers for each time point; numbers on right panels represent mean fold changes. P values determined by Mann-Whitney U test. e , HAI titers for the four vaccine virus strains in mRNA-1010 (red) and Fluarix (black) participants at baseline, 4, and 17/26 weeks. Bars represent geometric mean with 95% confidence interval. P values determined by Wilcoxon matched pairs signed rank test. In d-e , for participants that did not complete a blood collection at week 26, samples from 17 weeks post vaccination were used for analysis (mRNA-1010, n=2; Fluarix, n=2).

Article Snippet: Secreting cells were detected using three-color FluoroSpot IgA/IgG/IgM ELISpot Kits (Cellular Technology Limited) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunospot, Binding Assay, MANN-WHITNEY, Clinical Proteomics, Virus

Journal: Frontiers in immunology

Article Title: Evaluation of Immune and Vaccine Competence in Steroid-Sensitive Nephrotic Syndrome Pediatric Patients.

doi: 10.3389/fimmu.2021.602826

Figure Lengend Snippet: FIGURE 2 | Total and antigen-specific IgG-secreting B cells in steroid-sensitive nephrotic syndrome pediatric patients at onset. (A–E) Isolated PBMCs were stimulated for 5 days with CpG plus rhIL-21 and rhIL-4. Following stimulation, (A) total, (B,C) anti-tetanus and (D,E) anti-HBV IgG-secreting B cells were enumerated by ELISPOT in steroid-sensitive nephrotic syndrome pediatric patients at disease onset (SSNS, n=11) and in age-matched controls (CTRL, n=5). Antigen-specific memory B cells were represented as (B,D) absolute count/106 cells and as (C,E) percentage of total IgG-secreting B cells. Each plot represents a different patient. Horizontal lines indicate the means and differences between groups were compared using the unpaired t test.

Article Snippet: For the simultaneous detection of IgM and IgG we used the Human IgG/IgM Dual-Color B Cell ELISpot Kit (R&D System).

Techniques: Isolation, Enzyme-linked Immunospot

Journal: Nature immunology

Article Title: YTHDF2 orchestrates tumor-associated macrophage reprogramming and controls antitumor immunity through CD8 + T cells

doi: 10.1038/s41590-022-01398-6

Figure Lengend Snippet: a, b, qPCR (a) (n = 4) and immunoblotting (b) showing the expression of YTHDF2 in splenicCD3−CD19−CD49b−CD11b+ myeloid cells from Ythdf2f/f and Ythdf2cKO mice. c, d, qPCR (c) (n = 4) and immunoblotting (d) showing the expression of YTHDF2 in bone marrow-derived macrophages (BMDMs) from Ythdf2f/f and Ythdf2cKO mice. e-g, Gating strategy (e), percentages (f), and absolute numbers (g) of CD11c+MHC-II+ DCs, CD11b+F4/80+ macrophages, CD11b+Ly6c+ monocytes, and CD11b+Ly6G+ neutrophils in the spleen of Ythdf2f/f and Ythdf2cKO (n = 3). h-k, Percentages (h, j) and absolute numbers (i, k) of tumor-infiltrating CD11b+Gr-1+ MDSCs and CD11b+CD11c+MHC-II+ DCs from Ythdf2f/f and Ythdf2cKO mice on day 14 post-B16-OVA (h, i) or MC38 (j, k) tumor inoculation (n = 5). l, Percentages and representative plots of IFN-γ-producing OT-I CD8+ T cells co-cultured with purified tumor-infiltrated CD11b+Gr-1+ MDSCs from B16-OVA bearing Ythdf2f/f and Ythdf2cKO mice in the presence of SIINFEKEL (n = 3). m, Percentages and representative plots of IFN-γ-producing OT-I CD8+ T cells co-cultured with purified tumor-infiltrated CD11b+CD11c+MHC-II+ DCs from B16-OVA-bearing Ythdf2f/f and Ythdf2cKO mice in the presence of SIINFEKEL (n = 3). n, o, Percentages of IFN-γ-producing OT-I CD8+ T cells co-cultured with purified tumor-infiltrated MDSCs (n) or DCs (o) from B16-OVA-bearing Ythdf2f/f and Ythdf2cKO mice in the presence of ovalbumin protein (n = 3). p, q, Immunoblotting showing the expression of YTHDF2 in BMDCs (p) or splenic DCs (q) from Ythdf2f/f and Ythdf2cKO mice. r, Percentages of tumor-infiltrating CD11b+F4/80+ macrophages after injection with clodronate liposomes or PBS liposomes in B16-OVA-bearing Ythdf2f/f and Ythdf2cKO mice (Ythdf2f/f + PBS liposome, n = 6; Ythdf2cKO + PBS liposome, n = 5; Ythdf2cKO + clodronate liposome, n = 4). s, Schematic diagram showing s.c. co-injection of B16-OVA cells with BMDMs from CD45.2 mice into CD45.1 mice and representative plots of the macrophages in the tumor tissues on day 14 post-B16-OVA tumor inoculation. Data are shown as mean ± SD and were analyzed with an unpaired two-tailed t-test (a-o) or one-way ANOVA with the Holm-Šídák post-test (r). Data in a, b, c, d, f-r are representative of at least two independent experiments. NS, not significant.

Article Snippet: Fourteen days later, 3 × 10 5 lymphocytes were isolated from draining lymph nodes and restimulated with 10 μg ml –1 SIINFEKEL for 48 h. IFNγ production was determined with an IFNγ single-color ELISpot kit (Cellular Technology) according to the manufacturer’s protocol (Cellular Technology).

Techniques: Western Blot, Expressing, Derivative Assay, Cell Culture, Purification, Injection, Liposomes, Two Tailed Test

Journal: Nature immunology

Article Title: YTHDF2 orchestrates tumor-associated macrophage reprogramming and controls antitumor immunity through CD8 + T cells

doi: 10.1038/s41590-022-01398-6

Figure Lengend Snippet: a, b, Median fluorescence intensity (MFI, a) and representative histogram (b) of pHrodo red zymosan bioparticles uptake by anti-tumoral BMDMs from Ythdf2f/f and Ythdf2cKO mice (n = 3). c, B16-OVA tumor growth on day 18 post-B16-OVA tumor inoculation in Rag1−/− mice s.c. transplanted with BMDMs from Ythdf2f/f and Ythdf2cKO mice (n = 3). d, B16-OVA tumor growth on day 13 post-B16-OVA tumor inoculation in Rag1−/− mice that were i.v. injected with CD3+ T cells and s.c. transplanted with BMDMs from Ythdf2f/f and Ythdf2cKO mice (n = 5). e-h, Percentages (e, g) and absolute numbers (f, h) of tumor-infiltrating CD4+ T cells, CD8+ T cells, and CD3−NK1.1+ NK cells from Ythdf2f/f and Ythdf2cKO mice on day 14 post-B16-OVA tumor cell implantation (e, f) or MC38 (g, h) tumor inoculation (n = 5). i, j, Percentages and representative plots of tumor-infiltrating CD4+ IFN-γ+ TH1 cells, CD4+ IL-17A+ TH17 cells, CD4+ Foxp3+ Treg cells, and granzyme B-producing NK cells from Ythdf2f/f and Ythdf2cKO mice on day 14 post B16-OVA (i) or MC38 (j) tumor inoculation (n = 4). Data are shown as mean ± SD and were analyzed with an unpaired two-tailed t-test (a, e-j) or two-way ANOVA with mixed-effects model and adjusted by the Holm-Šídák post-test (c, d). Data in a-j are representative of at least two independent experiments. NS, not significant.

Article Snippet: Fourteen days later, 3 × 10 5 lymphocytes were isolated from draining lymph nodes and restimulated with 10 μg ml –1 SIINFEKEL for 48 h. IFNγ production was determined with an IFNγ single-color ELISpot kit (Cellular Technology) according to the manufacturer’s protocol (Cellular Technology).

Techniques: Fluorescence, Injection, Two Tailed Test

Journal: Nature immunology

Article Title: YTHDF2 orchestrates tumor-associated macrophage reprogramming and controls antitumor immunity through CD8 + T cells

doi: 10.1038/s41590-022-01398-6

Figure Lengend Snippet: a, Top six enriched pathways in Ythdf2cKO antitumoral macrophages compared to the Ythdf2f/f macrophages from GSEA. b, Enrichment of the IFNγ response in antitumoral macrophages from Ythdf2cKO mice compared to those from Ythdf2f/f mice; NES, normalized enrichment score; FDR, false discovery rate. c, Volcano plot showing the DEGs between antitumoral macrophages from Ythdf2f/f mice and those from Ythdf2cKO mice. DEGs (differentially expressed genes) with an absolute log-transformed fold change of >0.25 and adjusted P value of <0.05 (determined by two-sided Wilcoxon rank-sum test and adjusted using the Bonferroni correction) were defined as significant. d, Immunoblotting assay of STAT1 phosphorylation (pSTAT1) in IFNγ-treated BMDMs from Ythdf2f/f and Ythdf2cKO mice. e, qPCR of Il15 and Cxcl9 mRNA expression in antitumoral macrophages transfected with Stat1 siRNA or control siRNA (n = 3). f, B16-OVA tumor growth on day 15 after tumor inoculation in immunocompetent C57BL/6 mice transplanted with Stat1-knockdown (siRNAStat1) or control (siRNACtr) BMDMs from Ythdf2f/f or Ythdf2cKO mice (n = 5). g, B16-OVA tumor growth on day 15 after tumor inoculation in immunocompetent C57BL/6 mice transplanted with Stat1-knockout (gRNAStat1) or control (gRNACtr) BMDMs from Ythdf2f/f or Ythdf2cKO mice (n = 5); gRNA, guide RNA. h, B16-OVA tumor growth on day 15 after tumor inoculation in Ythdf2f/f and Ythdf2cKO mice treated with IgG or IFNγ antibody (Ythdf2f/f + IgG, n = 5; Ythdf2cKO + IFNγ antibody, n = 5; Ythdf2cKO + IgG, n = 4). i, B16-OVA tumor growth on day 13 after tumor inoculation in immunocompetent C57BL/6 mice transplanted with Ifngr1-knockout BMDMs (gRNAIfngr1) from Ythdf2cKO mice (n = 5) or control (gRNACtr) BMDMs from Ythdf2f/f or Ythdf2cKO mice (n = 5). Data are shown as mean ± s.d. and were analyzed by one-way ANOVA with a Holm–Šídák post test (e) or two-way ANOVA with mixed-effects model followed by a Holm–Šídák post test (f–i). Data in d–i are representative of at least two independent experiments.

Article Snippet: Fourteen days later, 3 × 10 5 lymphocytes were isolated from draining lymph nodes and restimulated with 10 μg ml –1 SIINFEKEL for 48 h. IFNγ production was determined with an IFNγ single-color ELISpot kit (Cellular Technology) according to the manufacturer’s protocol (Cellular Technology).

Techniques: Transformation Assay, Western Blot, Phospho-proteomics, Expressing, Transfection, Control, Knockdown, Knock-Out

Journal: Nature immunology

Article Title: YTHDF2 orchestrates tumor-associated macrophage reprogramming and controls antitumor immunity through CD8 + T cells

doi: 10.1038/s41590-022-01398-6

Figure Lengend Snippet: a, Violin plot showing the Tlr9 expression across 15 cell clusters from scRNA-seq data in this study. b, Sequence of the CpG-linked mouse Ythdf2 siRNA conjugate (CpG–siRNAYthdf2). c, d, Representative histograms showing the expression of Cy3-CpG-siRNAYthdf2 in wild-type BMDMs incubated with various doses of Cy3-CpG-siRNAYthdf2 (c) or with 100 nM Cy3-labeled CpG-Ythdf2 siRNA for the indicated times (d). e, Representative histograms showing the uptake of Cy3-CpG-siRNAYthdf2 by CD11b+F4/80+ TAMs isolated from tumor tissues of B16-OVA-bearing wild-type mice that received intratumoral (i.t.) injection of CpG-siRNAYthdf2. f, qPCR showing the mRNA expression of mRNA Ythdf2 in CD11b+F4/80+ TAMs sorted from tumor tissues of B16-OVA-bearing mice i.t. injected with CpG-siRNAYthdf2 or CpG-siRNACtrl (n = 3). g, h, Representative dot plots and percentages of CD11b+F4/80+iNOS+ anti-tumoral macrophages (g) or h, CD11b+F4/80+Arg1+ pro-tumoral macrophages (h) in tumor tissues from B16-OVA-bearing mice that received i.t. injection of CpG-siRNAYthdf2 or CpG-siRNACtrl (n = 5). i, qPCR showing the mRNA expression of Ythdf2 in CD11b+F4/80+ TAMs isolated from lung tissues of B16-F10-bearing mice that received i.v. injected of CpG-siRNAYthdf2 or CpG-siRNACtrl (n = 3). j, k, Metastatic nodules (j) and percentages of tumor-infiltrating IFN-γ producing CD8+ T cells (k) in the lung of B16F10-bearing mice that received i.v. injection of CpG-siRNAYthdf2, CpG-siRNACtrl, CpG, or PBS (n = 5). l, B16-OVA tumor growth on day 13 post-tumor inoculation in Batf3−/− mice treated with CpG-siRNAYthdf2 or CpG-siRNACtrl (n = 5). m, n, Violin plots showing the TLR9 expression across cell clusters in scRNA-seq datasets of patients with glioblastoma (m) or kidney cancer (n). o, p, Representative histograms showing expression levels of PD-L1 in B16-OVA cells (o) or BMDMs (p) after treatment with IFN-γ for 24 h. Median fluorescence intensity (MFI) is shown. Data are shown as mean ± SD and were analyzed with an unpaired two-tailed t-test (f-i) or one-way ANOVA with the Holm-Šídák post-test (j, k) or two-way ANOVA with the mixed-effects model and adjusted by Holm-Šídák post-test (l). Data in c-l, o, and p are representative of at least two independent experiments.

Article Snippet: Fourteen days later, 3 × 10 5 lymphocytes were isolated from draining lymph nodes and restimulated with 10 μg ml –1 SIINFEKEL for 48 h. IFNγ production was determined with an IFNγ single-color ELISpot kit (Cellular Technology) according to the manufacturer’s protocol (Cellular Technology).

Techniques: Expressing, In Vivo, Sequencing, Incubation, Labeling, Isolation, Injection, Fluorescence, Two Tailed Test

Journal: Nature immunology

Article Title: YTHDF2 orchestrates tumor-associated macrophage reprogramming and controls antitumor immunity through CD8 + T cells

doi: 10.1038/s41590-022-01398-6

Figure Lengend Snippet: a, HOMER motif discovery tool showing the m6A motif in BMDMs from both Ythdf2f/f and Ythdf2cKO mice. b, Density distribution of m6A peaks across the length of mRNA. c, Pie chart depicting the fraction of m6A peaks in four transcript segments. d, Volcano plot showing the differentially expressed genes in anti-tumoral macrophages from Ythdf2cKO mice compared with those from Ythdf2f/f mice from RNA-seq. DEGs with absolute log-transformed fold change > 0.25 and adjusted P < 0.05 (determined by two-sided Wilcoxon rank sum test and adjusted using Bonferroni correction) were defined as significant. e, GSEA results showing the top six pathways enriched in Ythdf2cKO anti-tumoral macrophages compared to Ythdf2f/f anti-tumoral macrophages. The P values were calculated by a permutation test. f, GSEA results showing enrichment of IFN-γ response sets in Ythdf2cKO anti-tumoral macrophages compared to Ythdf2f/f anti-tumoral macrophages. g, Heatmaps showing the relative expression of representative genes in Ifng, Tnf, Ifna, Irf7, Tlr4, and Stat1 signaling pathways in BMDMs from Ythdf2f/f and Ythdf2cKO mice from RNA-seq data (n = 2). Transcript methylation is depicted by filled (m6A-modified) or unfilled (non-m6A-modified) circles. h, Scatterplot showing the overlap of high confidence YTHDF2-binding peaks between replicate 1 and replicate 2 in YTHDF2 RIP-seq. i, Density distribution of YTHDF2-binding peaks across the length of mRNA. j, Pie chart depicting the fraction of YTHDF2-binding peaks in four transcript segments.

Article Snippet: Fourteen days later, 3 × 10 5 lymphocytes were isolated from draining lymph nodes and restimulated with 10 μg ml –1 SIINFEKEL for 48 h. IFNγ production was determined with an IFNγ single-color ELISpot kit (Cellular Technology) according to the manufacturer’s protocol (Cellular Technology).

Techniques: Binding Assay, RNA Sequencing, Transformation Assay, Expressing, Protein-Protein interactions, Methylation, Modification

Journal: Nature immunology

Article Title: YTHDF2 orchestrates tumor-associated macrophage reprogramming and controls antitumor immunity through CD8 + T cells

doi: 10.1038/s41590-022-01398-6

Figure Lengend Snippet: a,b, Representative plots and percentages of tumor-infiltrating IFNγ-producing CD8+ T cells from Ythdf2f/f and Ythdf2cKOmice on day 14 after B16-OVA (a) or MC38 (b) tumor inoculation (n = 5). c,d, Representative plots and percentages of tumor-infiltrating effector memory (TEM), central memory (TCM) and naive (TN) CD8+ T cells from Ythdf2f/f and Ythdf2cKO mice on day 14 after B16-OVA (c) or MC38 (d) tumor inoculation (n = 5). e,f, Representative plots and percentages of SIINFEKL-specific CD8+ T cells (e) or KSPWFTTL-specific CD8+ T cells (f) from Ythdf2f/f and Ythdf2cKO mice on day 14 after B16-OVA (e) or MC38 (f) tumor inoculation (n = 5). g, IFNγ-producing CD8+ T cells in the draining lymph nodes from Ythdf2f/f and Ythdf2cKO mice on day 14 after B16-OVA tumor inoculation. Cytokine levels are expressed as the number of spot-forming cells out of 3 × 105 draining lymph nodes cells (n = 6). h, B16-OVA tumor growth on day 18 after tumor inoculation in Ythdf2f/f and Ythdf2cKO mice treated with IgG or CD8 antibody (Ab; Ythdf2f/f + IgG, n = 6; Ythdf2cKO + IgG, n = 6; Ythdf2f/f + CD8 Ab, n = 5; Ythdf2cKO + CD8 Ab, n = 5). i, B16-OVA tumor growth on day 21 after tumor inoculation in Rag1−/− mice s.c. implanted with BMDMs from Ythdf2f/f and Ythdf2cKO mice together with i.v. injection of CD8+ T cells from OT-I mice (Ythdf2f/f BMDMs + OT-I, n = 4; Ythdf2cKO BMDMs + OT-I, n = 5). Data are shown as mean ± s.d. and were analyzed by two-way ANOVA with a mixed-effects model and adjusted by Holm–Šídák post test (h and i) or unpaired two-tailed t-test (a–g). Data in a–i are representative of at least two independent experiments.

Article Snippet: Fourteen days later, 3 × 10 5 lymphocytes were isolated from draining lymph nodes and restimulated with 10 μg ml –1 SIINFEKEL for 48 h. IFNγ production was determined with an IFNγ single-color ELISpot kit (Cellular Technology) according to the manufacturer’s protocol (Cellular Technology).

Techniques: Injection, Two Tailed Test

Journal: Nature immunology

Article Title: YTHDF2 orchestrates tumor-associated macrophage reprogramming and controls antitumor immunity through CD8 + T cells

doi: 10.1038/s41590-022-01398-6

Figure Lengend Snippet: a, Rose diagram showing the proportions of 15 cell types in Ythdf2f/f and Ythdf2cKO mice. b, Violin plots showing the enrichment of signatures of early activated (TEA), cytokine/effector (TC/E), memory (TM) and memory precursor (TMP) CD8+ T cells in scRNA-seq data from CD45+ tumor-infiltrating immune cells sorted from Ythdf2f/f and Ythdf2cKO mice on day 14 after B16-OVA tumor inoculation. The horizontal line indicates the median value, and the box represents the first and third quartiles. P values were calculated by two-sided Wilcoxon rank-sum test. c, Dot plots showing mean strength of selected ligand–receptor pairs of antitumoral and CD8+ Teff cells. Dot size indicates the mean strength level, and color shows the P values calculated by the permutation test. d, Empirical cumulative distribution function (ECDF) plot of enrichment score for the MHC class I gene signature in antitumoral macrophages from Ythdf2f/f and Ythdf2cKO mice in scRNA-seq. Data were analyzed by two-sided unpaired Wilcoxon test. e, Dot plots showing expression features of selected genes from the MHC class I presentation signature in antitumoral macrophages from Ythdf2f/f and Ythdf2cKO mice in scRNA-seq. Dot size indicates the percentage of cells expressing the indicated gene, and color intensity represents the relative gene expression level. f, Representative histograms and percentages of H-2Kb SIINFEKL-positive antitumoral or protumoral macrophages in tumor tissues from Ythdf2f/f and Ythdf2cKO mice on day 14 after B16-OVA tumor inoculation (n = 3). g–j, Representative plots and percentages of IFNγ-producing OT-I CD8+ T cells cocultured with the SIINFEKEL peptide (g and i) or the OVA protein (h and j)-loaded antitumoral BMDMs (g, n = 3; h, n = 4) or CD11b+F4/80+ TAMs isolated from B16-OVA tumor-bearing mice on day 14 after B16-OVA tumor inoculation (i, n = 3; j, n = 3). k, qPCR showing the expression of Ifng4 and Ifnb1 from CD11b+F4/80+ macrophages isolated from B16-OVA tumor-bearing Ythdf2f/f and Ythdf2cKO mice on day 14 after B16-OVA tumor inoculation (n = 3). Data are representative of at least three independent experiments, are shown as mean ± s.d. and were analyzed by unpaired two-tailed t-test (f–k).

Article Snippet: Fourteen days later, 3 × 10 5 lymphocytes were isolated from draining lymph nodes and restimulated with 10 μg ml –1 SIINFEKEL for 48 h. IFNγ production was determined with an IFNγ single-color ELISpot kit (Cellular Technology) according to the manufacturer’s protocol (Cellular Technology).

Techniques: Expressing, Gene Expression, Isolation, Two Tailed Test

Journal: Nature immunology

Article Title: YTHDF2 orchestrates tumor-associated macrophage reprogramming and controls antitumor immunity through CD8 + T cells

doi: 10.1038/s41590-022-01398-6

Figure Lengend Snippet: a, Ingenuity pathway analysis showing the top four pathways enriched in Ythdf2cKO anti-tumoral macrophages compared to Ythdf2f/f anti-tumoral macrophages. P values were determined by hypergeometric test and adjusted for multiple testing using the Benjamini–Hochberg method. b, qPCR showing the expression of IFN-γ response genes in BMDMs from Ythdf2f/f and Ythdf2cKO mice that were treated with IFN-γ (100 ng/ml) for 6 h (n = 3). c, qPCR showing the expression of Stat1 in CD11b+F4/80+ TAMs isolated from tumor tissues of B16-OVA tumor-bearing Ythdf2f/f and Ythdf2cKO on day 14 post B16-OVA tumor inoculation (n = 4). d, Immunoblotting showing the expression of phosphor (p)-STAT1 and STAT1 in CD11b+F4/80+ TAMs isolated from tumor tissues of B16-OVA tumor-bearing Ythdf2f/f and Ythdf2cKO mice on day 14 post-B16-OVA tumor inoculation. e, Immunoblotting showing the expression of STAT1 in BMDMs transfected with Stat1 siRNA or control siRNA. f, Immunoblotting showing the expression of STAT1 in BMDMs nucleofected with Stat1 gRNA or control gRNA. g, Representative histogram of the expression of IFNGR1 in BMDMs nucleofected with Ifngr1 gRNA or control gRNA. h, B16-OVA tumor growth on day 18 post-tumor inoculation in Rag1−/− mice s.c. implanted with BMDMs from Ythdf2f/f and Ythdf2cKO mice together with i.v. injection of CD8+ T cells from IFN-γ−/− mice (n = 4). Data are shown as mean ± SD and were analyzed with an unpaired two-tailed t-test (b, c) or two-way ANOVA with mixed-effects model and adjusted by the Holm-Šídák post-test (h). The numbers below a lane indicate relative expression levels of target proteins normalized to the expression of β-actin. Data in b-h are representative of at least two independent experiments. NS, not significant.

Article Snippet: Fourteen days later, 3 × 10 5 lymphocytes were isolated from draining lymph nodes and restimulated with 10 μg ml –1 SIINFEKEL for 48 h. IFNγ production was determined with an IFNγ single-color ELISpot kit (Cellular Technology) according to the manufacturer’s protocol (Cellular Technology).

Techniques: Expressing, Isolation, Western Blot, Transfection, Control, Injection, Two Tailed Test

Journal: Nature immunology

Article Title: YTHDF2 orchestrates tumor-associated macrophage reprogramming and controls antitumor immunity through CD8 + T cells

doi: 10.1038/s41590-022-01398-6

Figure Lengend Snippet: a, Immunoblotting showing the expression of YTHDF2 in CD11b+F4/80+ macrophages isolated from spleen or tumor tissues of B16-OVA tumor-bearing C57BL/6 mice on day 14-post B16-OVA tumor inoculation. b, Immunoblotting showing the expression of YTHDF2 in BMDMs stimulated without or with IL-4, TGF-β, IL-10, or IFN-γ for 24 h. c, Immunoblotting showing the expression of YTHDF2 and phospho-Stat3 in BMDMs stimulated with different doses of IL-10 for 24 h. d, Binding sites for STAT3 in the promoter regions of Ythdf2 (predicted from http://jaspar.genereg.net). e, Luciferase reporter assay showing the luciferase activity of Ythdf2 in BMDMs transfected with STAT3 expressing plasmid or empty vector (n = 3). f, ChIP-qPCR showing the binding of STAT3 to the Ythdf2 promoter in IL-10 (10 ng/ml)–treated BMDMs (n = 3). g, Immunoblotting showing the expression of YTHDF2 and phospho-Stat3 in BMDMs from Stat3f/fand Stat3cKO mice stimulated without or with IL-10 (10 ng/ml). Data are shown as mean ± SD and were analyzed with an unpaired two-tailed t-test (e, f). The numbers below a lane indicate the expression of YTHDF2 normalized to the expression of β-actin. Data in a, b, c, e, f, and g are representative of at least two independent experiments. NS, not significant.

Article Snippet: Fourteen days later, 3 × 10 5 lymphocytes were isolated from draining lymph nodes and restimulated with 10 μg ml –1 SIINFEKEL for 48 h. IFNγ production was determined with an IFNγ single-color ELISpot kit (Cellular Technology) according to the manufacturer’s protocol (Cellular Technology).

Techniques: Western Blot, Expressing, Isolation, Binding Assay, Luciferase, Reporter Assay, Activity Assay, Transfection, Plasmid Preparation, ChIP-qPCR, Two Tailed Test

Journal: Nature immunology

Article Title: YTHDF2 orchestrates tumor-associated macrophage reprogramming and controls antitumor immunity through CD8 + T cells

doi: 10.1038/s41590-022-01398-6

Figure Lengend Snippet: a, B16-OVA (left; n = 5) or MC38 (right; n = 5) tumor growth in mice i.t. injected with CpG–siRNAYthdf2, CpG–siRNACtrl, CpG or PBS 7, 9, 11 and 13 days after tumor inoculation on day −7. Data on day 8 were used for statistical analysis. b, Percentages of tumor-infiltrating IFNγ-producing CD8+ T cells on day 8 in B16-OVA (n = 5) or MC38 tumor models (PBS, n = 5; CpG, n = 3; CpG–siRNACtrl, n = 3; CpG–siRNAYthdf2, n = 3), as described in a. c, B16-OVA tumor growth in mice treated with CpG–siRNAYthdf2, CpG–siRNACtrl or in combination with anti-PD-L1 (left; CpG–siRNACtrl + IgG, n = 5; CpG–siRNAYthdf2 + IgG, n = 5; CpG–siRNACtrl + PD-L1 antibody, n = 6; siRNAYthdf2 + PD-L1 antibody, n = 6) and percentages of tumor-infiltrating IFNγ-producing CD8+ T cells (right; CpG–siRNACtrl + IgG, n = 5; CpG–siRNAYthdf2 + IgG, n = 3; CpG–siRNACtrl + PD-L1 antibody, n = 5; siRNAYthdf2 + PD-L1 antibody, n = 3). CpG–siRNACtrl and CpG–siRNAYthdf2 were treated as in a, while PD-L1 antibody was injected on days −7 and 0. Data on day 8 were used for statistical analysis. d, Representative plots and percentages of IFNγ and granzyme B expression in Ly95 T cells when cocultured with wild-type (WT) or YTHDF2−/− MDMs preloaded with NY-ESO157-C165 peptide (n = 5); MFI, median fluorescence intensity. e, Hematoxylin and eosin (H&E) staining and immunohistochemical staining of YTHDF2 (yellow) plus CD68 (purple) or CD8 (brown) in tumor tissues from individuals with COAD. Dashed lines outline the edge of tumor (T) zones. Asterisks mark the stroma tissues. A representative specimen of YTHDF2+CD68+ (double positive) with a high (top) or low (bottom) number of cells is shown (f); scale bars, 100 μm. Black arrows mark YTHDF2+CD68+ cells. f, Correlations between YTHDF2+CD68+ cells and CD8+ T cells in the stroma area as in e are shown (n = 18). Data are shown as mean ± s.d. and were analyzed by one-way ANOVA with a Holm–Šídák post test (b, c right and d) or two-way ANOVA with mixed-effects model (a and c left) and were adjusted by a Holm–Šídák post test or Spearman correlation (f). Data in a, b, c and d are representative of at least two independent experiments.

Article Snippet: Fourteen days later, 3 × 10 5 lymphocytes were isolated from draining lymph nodes and restimulated with 10 μg ml –1 SIINFEKEL for 48 h. IFNγ production was determined with an IFNγ single-color ELISpot kit (Cellular Technology) according to the manufacturer’s protocol (Cellular Technology).

Techniques: Injection, Expressing, Fluorescence, Staining, Immunohistochemical staining