Journal: Cell reports

Article Title: Consumption of fish oil high-fat diet induces murine hair loss via epidermal fatty acid binding protein in skin macrophages

doi: 10.1016/j.celrep.2022.111804

Figure Lengend Snippet: (A) Representative hematoxylin and eosin staining of skin tissues from mice fed the LFD, fish oil HFD, or cocoa butter diet at different magnifications (red arrows: infiltrating inflammatory cells). Scale bars, 100 μm. (B and C) Flow cytometric analysis of immune cell phenotype in the dermis of mice on different diets for 3 months. A cell population with strong autofluorescence (red trapezoid gate) was specifically accumulated in the dermis of mice fed the fish oil HFD (B). Multichannel signals of the autofluorescent cells were analyzed using a BD Fortessa flow cytometer (C). (D–F) Autofluorescent cells in the dermis of fish oil HFD-fed mice were purified using a BD FACSAria II flow sorter and stained with a panel of metal-tagged CyTOF antibodies. Uniform manifold approximation and projection (UMAP) was used to visualize and identify immune cell populations in unsorted dermal cells (D) and sorted autofluorescent dermal cells (E). Individual surface marker signatures in the CyTOF panel are shown in (F). (G and H) Representative IHC images of F4/80 + macrophages (brown staining, G) and CD8 + T cells (brown staining, H) in the skin of mice fed the LFD, fish oil HFD, or cocoa butter HFD. Scale bars, 100 μm. See also . These in vitro experiments were repeated with at least three biological replicates.

Article Snippet: Rat monoclonal anti-mouse CD8a - 168Er , Fluidigm , Cat#3168003B; RRID:AB_2811241.

Techniques: Staining, Flow Cytometry, Purification, Marker, In Vitro

Journal: Cell reports

Article Title: Consumption of fish oil high-fat diet induces murine hair loss via epidermal fatty acid binding protein in skin macrophages

doi: 10.1016/j.celrep.2022.111804

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rat monoclonal anti-mouse CD8a - 168Er , Fluidigm , Cat#3168003B; RRID:AB_2811241.

Techniques: Purification, Recombinant, Activation Assay, SYBR Green Assay, Reverse Transcription, Detection Assay, Enzyme-linked Immunosorbent Assay, Selection, Software

Journal: Journal of Clinical Investigation

Article Title: OX40+ plasmacytoid dendritic cells in the tumor microenvironment promote antitumor immunity

doi: 10.1172/jci131992

Figure Lengend Snippet: Figure 1. OX40 expression on pDCs in the TME of HNSCC. (A) OX40 expression in the TME (measured by flow cytometry) of HNSCC patients on differ- ent immune cell subsets — pDCs (n = 89), cDCs (n = 53), CD8+ T cells (n = 16), CD4+ T cells (n = 17), CD4+ Th1 T cells (n = 12), and CD4+ Treg cells (n = 14). T cell subsets were gated from live CD45+CD3+ cells. Th1 cells were defined as CD4+Tbet+ T cells and Treg cells were defined as CD4+Foxp3+ cells. (B) Gating strategy for FACS analysis and sorting of OX40+ and OX40lo/– pDCs from patient specimens. After selecting for singlets and live cells, pDCs were gated from HLA-DRhiLineage– cells, followed by CD11c–CD123+ cells. pDCs were further confirmed by expression of CD303 (BDCA-2). OX40 expression on pDCs was determined using internal negative controls. (C) Immunofluorescence of pDCs in the TME demonstrating OX40 and CD123 coexpression. n = 4, with 4 patient repeats. Original magnification, ×63. Scale bar: 5 μm. Red, OX40; green, CD123; blue, DAPI. (D) OX40 expression on pDCs from different anatomic sites: PBMC (n = 17), dLN– (n = 50) or dLN+ (n = 59), and primary tumor (n = 53). (E) Correlation (Pearson, with a line of best fit) between OX40 and ICOSL expression on matched patient TME pDCs (n = 28). One-way ANOVA followed by Tukey’s post hoc test (A and D). **P < 0.01; ***P < 0.001; ****P < 0.0001. Bar graph data are mean ± SEM; middle line of box-and-whisker plot indicates the median, box limits indicate the first and third quartiles, and whiskers indicate “extreme” for all data points. Representative flow plots are shown (A, D, and E).

Article Snippet: Tissue sections were incubated for 1 hour in blocking solution at 4°C with the following primary antibodies: rat anti–human CD8 (1:200, clone: YTC182.2, Bio-Rad), mouse anti–human CD123 (1:75, clone: 7G3, BD), and rabbit anti–human OX40 (1:80, E9U7O; Cell Signaling Technology).

Techniques: Expressing, Flow Cytometry, Immunofluorescence, Whisker Assay

Journal: Journal of Clinical Investigation

Article Title: OX40+ plasmacytoid dendritic cells in the tumor microenvironment promote antitumor immunity

doi: 10.1172/jci131992

Figure Lengend Snippet: Figure 3. OX40+ pDCs promote antigen-specific CD8+ T cell responses. (A) Illustration of the antigen-specific in vitro coculture model, in which autologous OX40+/OX40lo/– pDCs from the TME/non-TME of HNSCC patients (n = 9) were cocultured with autologous TAA peptide-loaded mDCs and CD8+ T cells for 5 to 6 days, at which point antigen-specific CD8+ T cell responses were measured, (B) including for proliferation (eFluor 450–low) and IFN-γ production as demonstrated in flow plots of a patient’s CD8+ T cells cocultured with OX40+ or OX40lo/– pDCs sorted from their tumors. CD8+ T cell positivity was also measured for (C) Tbet and (D) eFluor 450–low in these coculture experiments. (E) CD8+ T cell positivity for CD69 after coculture with TAA peptide–loaded mDCs without pDCs (control) or with OX40+ or OX40lo/– pDCs from the TME versus non-TME (dLN–) (n = 5). (F) Illustration depicting the Transwell coculture assay in which OX40+ or OX40lo/– pDCs in the top chamber were separated from autologous CD8+ T cells and peptide-loaded mDCs in the bottom chamber. (G) Percentage of proliferating (eFluor 450–low) and GzB+ CD8+ T cells in Transwell versus contact coculture (n = 3). Representative flow plots show GzB production by CD8+ T cells cocultured with E7-loaded mDCs and OX40+ or OX40lo/– pDCs in coculture contact or separated by Transwell. (H) Flow plots comparing antigen presentation capacities of autologous OX40+ and OX40lo/– pDCs with mDCs, based on cytolytic CD8+ T cell responses (no peptide controls for these plots are shown in Supplemental Figure 2B). Shown is GzB production by CD8+ T cells in the presence or absence of OX40+/OX40lo/– pDCs (top) and IL-12p70 production by mDC/pDC subsets (bottom). n = 2; 2 experimen- tal repeats. One-way ANOVA followed by Tukey’s post hoc test (C–E and G). Bar graph data are mean ± SEM; *P < 0.05. NS, not significant. Middle line of box-and-whisker plot indicates the median, box limits indicate the first and third quartiles, and whiskers indicate “extreme” for all data points. Representative flow plots are shown (C–E and G).

Article Snippet: Tissue sections were incubated for 1 hour in blocking solution at 4°C with the following primary antibodies: rat anti–human CD8 (1:200, clone: YTC182.2, Bio-Rad), mouse anti–human CD123 (1:75, clone: 7G3, BD), and rabbit anti–human OX40 (1:80, E9U7O; Cell Signaling Technology).

Techniques: In Vitro, Control, Co-culture Assay, Immunopeptidomics, Whisker Assay

Journal: Journal of Clinical Investigation

Article Title: OX40+ plasmacytoid dendritic cells in the tumor microenvironment promote antitumor immunity

doi: 10.1172/jci131992

Figure Lengend Snippet: Figure 4. The OX40-OX40L axis is utilized by pDCs in the TME. (A) OX40L expression measured by flow cytometry on OX40+ and OX40lo/– pDCs (n = 7). Correla- tion (Pearson, with line of best fit) of OX40 and OX40L expression intensities (per-cell normalized counts, total weighting) on pDCs measured on an HNSCC TME tissue section. n = 4; 4 patient repeats. (B) Immunofluorescence images from the TME showing a cell’s coexpression of OX40 (red) with CD123 (green), sitting adjacent to CD8-expressing (magenta) cells. Gallery view of Z-stacks (collected at 0.29-μm intervals). Original magnification, ×63. Scale bar: 5 μm. n = 4; 4 patient repeats. (C) Processed multispectral image (steps outlined in Supplemental Figure 2E for same example image) of the TME. Original magnification, ×40. Inset: a cluster CD123+ (green) cells, including one with OX40 expression (orange), next to a CD8+ (magenta) cell. (D) Representative image file written using Phenoptr to calculate touching pairs of phenotyped OX40L+CD68+ macrophages with phenotyped OX40+ pDCs. (E and F) Percentage of phenotyped OX40+/ OX40lo/– pDCs touching other OX40L+ cells, including pDCs, macrophages (Mφ), and “other cells” (Methods), in the TME and non-TME of patients (n = 3). (G) Ratios of CD8+ T cell counts within 30 μm of pDC subsets to total counts of CD123+ pDC subsets (OX40+/OX40lo/–) in the TME and non-TME of patients (n = 5). (H) Intercellular distances of phenotyped pDC subsets to the closest tumor margin (μm). Original magnification, ×40. n = 3; 3 patient repeats. One-way ANOVA followed by Tukey’s post hoc test (F) and unpaired (E), and paired (A, G, and H) t tests. *P < 0.05; **P < 0.01; NS, not significant. Bar graph data are mean ± SEM; middle line of box-and-whisker plot indicates the median, box limits indicate the first and third quartiles, and whiskers indicate “extreme” for all data points.

Article Snippet: Tissue sections were incubated for 1 hour in blocking solution at 4°C with the following primary antibodies: rat anti–human CD8 (1:200, clone: YTC182.2, Bio-Rad), mouse anti–human CD123 (1:75, clone: 7G3, BD), and rabbit anti–human OX40 (1:80, E9U7O; Cell Signaling Technology).

Techniques: Expressing, Flow Cytometry, Immunofluorescence, Whisker Assay

Journal: Journal of Clinical Investigation

Article Title: OX40+ plasmacytoid dendritic cells in the tumor microenvironment promote antitumor immunity

doi: 10.1172/jci131992

Figure Lengend Snippet: Figure 6. OX40+ pDCs correlate to survival in cancer patients and suppress tumor growth. (A) Prospective recurrence-free survival (log-rank, Mantel-Cox test) of HNSCC cohort (n = 80), stratified by median (45%) intratumoral pDC OX40 expression, as measured by flow cytometry. (B) Overall survival (log-rank, Mantel-Cox test) of HNSCC patients (n = 500) from the GDC data portal, stratified first by median pDC gene signature Z scores followed by stratification of mean TNFRSF4 (encodes OX40) mRNA levels. (C) Correlation (Pearson, with line of best fit) of TNFRSF4 log2 mRNA levels (among cases with pDChi gene signatures) with CD8+ T effector scores in HNSCC (n = 172). (D) OX40 expression on intratumoral pDCs from different murine tumor mod- els. n =4; 4 experimental replicates. (E) gp100-specific Pmel-1 CD8+ T cell IFN-γ production by proliferating (eFluor 450–low) CD8+ T cells, measured in the presence or absence of pDCs from the dLNs of B16-F10– and B16CCR7-bearing mice. n = 2; 2 experimental repeats. (F) gp100-specific proliferating (eFluor 450–low) Pmel-1 CD8+ T cells in the presence or absence of B16CCR7 pDCs prestimulated with Resiquimod and OX86. n = 2; 2 experimental repeats. (G) Effect of pDC depletion (anti-PDCA1) in B16-F10– and B16CCR7-bearing mice compared with controls (anti-polyclonal IgG). Data are pooled from at least 2 independent experiments with 3 to 5 mice per group. (H) Quantification (by flow cytometry) of conventional cDCs (CD11c+CD11b–) and CD8a+ cDCs from B16CCR7-bearing mice treated with anti-PDCA1 or anti-polyclonal IgG. Data are pooled from individual experiments and normalized to 5 × 105 live cells. One-way ANOVA followed by Tukey’s post hoc test (D), 2-way ANOVA with Sidak’s test for multiple comparisons (H), and unpaired Student’s t test (G). **P < 0.01; ***P < 0.001. Tumor burden data and bar graph data are mean ± SEM.

Article Snippet: Tissue sections were incubated for 1 hour in blocking solution at 4°C with the following primary antibodies: rat anti–human CD8 (1:200, clone: YTC182.2, Bio-Rad), mouse anti–human CD123 (1:75, clone: 7G3, BD), and rabbit anti–human OX40 (1:80, E9U7O; Cell Signaling Technology).

Techniques: Expressing, Flow Cytometry

Journal: Journal of Virology

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8αβ

doi: 10.1128/jvi.79.17.11422-11433.2005

Figure Lengend Snippet: FIG. 1. Flow cytometric evaluation of Nef-mediated CD8 down-regulation in retrovirally transduced cells. (A) Bivariate dot plots (CD8- allophycocyanin, CD8-phycoerythrin versus EGFP) of flow cytometric measurement of Nef (control) and Nef (NA-7 allele) transduced peripheral blood mononuclear cells, gated on CD8 cells, at day 3 after transduction. (B) Bivariate dot plots of flow cytometric measurement (CD8-allophycocyanin, CD8-phycoerythrin versus EGFP) of Nef NA-7 wild-type and NA-7 LLAA transduced SupT1 cells (left) and SupT1 cells overexpressing CD8 (right), at day 2 after transduction. (C) The solid and open histograms show the CD8 expression profile of SupT1 CD8 cells and SupT1 CD8 cells (CD8-transduced population), respectively, gated as shown in the inset. (D) Daudi CD8 cells and Daudi CD8 cells were transduced with control, HIV-1 (NL4-3, LAI and NA-7), SIV (mac239), and HIV-2 (Rod) Nef. Percent down-regulation is shown with white bars for CD8 in Daudi CD8, with gray bars for CD8 in Daudi CD8, and with black bars for CD8 in Daudi CD8. All percentages were calculated, as described in Materials and Methods, using the ranges E and E, as indicated in A.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8 primary antibody (5F2 [15], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Control, Transduction, Expressing

Journal: Journal of Virology

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8αβ

doi: 10.1128/jvi.79.17.11422-11433.2005

Figure Lengend Snippet: FIG. 3. Flow cytometric analysis of Nef-mediated receptor down- regulation and internalization in retrovirally transduced SupT1 cells. SupT1 cells were transduced with an inducible NA-7.ER construct. At time zero, 4-hydroxytamoxifen (1 M) was added to the culture me- dium. (A) Percent down-regulation was calculated, as described in the Materials and Methods. CD4-allophycocyanin (solid line) and CD8- phycoerythrin (dashed line) were measured as a function of time. (B) The figure shows the percentage of CD4, CD8, and CD28 molecules internalized by HIV-1 NA-7.ER, calculated as described in Materials and Methods. In each graph, Nef-positive (EGFP expressing Nef, solid line) and Nef-negative (EGFP not expressing Nef, dashed line) cells are depicted. The EGFP ranges used for calculation are indicated in Fig. 1A.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8 primary antibody (5F2 [15], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Transduction, Construct, Expressing

Journal: Journal of Virology

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8αβ

doi: 10.1128/jvi.79.17.11422-11433.2005

Figure Lengend Snippet: FIG. 4. Mutations in the CD8 -chain and their effect on endocytosis and down-regulation. Daudi cells were cotransduced with CD8, wild-type or mutant CD8, and control or wild-type Nef. (A and C) Alignment of amino acid sequences of wild-type (210*) and mutant CD8 -chain cytoplasmic tails. An asterisk indicates a stop codon. (A) The bar chart shows the percent down-regulation of (mutant) CD8 by HIV-1 Nef alleles NA-7, LAI, and NL4-3. In both B and D the percentage of (mutant) CD8 molecules internalized by wild-type HIV-1 NA-7 is shown, including in both the same data for a Nef-negative construct as a control (wild-type Nef ). (C) The bar chart represents the percentage of (mutant) CD8 down-regulation after transduction with either control virus or wild-type Nef NL4-3. Each bar represents a (mutant) CD8 -chain, as indicated by the changed amino acid sequence compared with CD8. Percent down-regulation and internalization were calculated as described in Materials and Methods, using the ranges indicated in Fig. 1A. In A, B, and D, mean values are shown and standard deviations are calculated from the data generated from three independent experiments. In B the results for NL4-3 are representative of the results with HIV-1 alleles NA-7 and LAI.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8 primary antibody (5F2 [15], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Mutagenesis, Control, Construct, Transduction, Virus, Sequencing, Generated

Journal: Journal of Virology

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8αβ

doi: 10.1128/jvi.79.17.11422-11433.2005

Figure Lengend Snippet: FIG. 5. Chimeric constructs. Daudi cells were retrovirally trans- duced with the CD8(EC-TM)-CD8(IC) chimera (cyt tail) or a CD8(EC-TM)-CD8(IC) chimera (cyt tail), using bicistronic con- structs with NGFR as the reporter. Bivariate dot plots are gated on NGFR-positive cells, at day 2 after transduction of these cells with control virus, HIV-1 Nef (NA-7 allele), and SIV Nef (mac239), using bicistronic constructs with EGFP as the reporter. CD8-phycoerythrin versus EGFP expression is shown.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8 primary antibody (5F2 [15], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Construct, Transduction, Control, Virus, Expressing

Journal: Journal of Virology

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8αβ

doi: 10.1128/jvi.79.17.11422-11433.2005

Figure Lengend Snippet: FIG. 6. Confocal images of 293T cells. Nef.EGFP was detected by direct fluorescence (green, left panels) and CD8.HA or CD8 by monoclonal antibodies as indicated in Materials and Methods (red, middle panels). Nuclei were visualized by DAPI staining (blue). Right panels show the merged images from Nef.EGFP and CD8. Areas of colocalization of Nef.EGFP/CD8 are shown in yellow. As indicated, the upper panels show cells expressing wild-type LAI, the middle panels show the LLAA mutant, and alower panels show the PPAA mutant. Scale bars represent 5 m.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8 primary antibody (5F2 [15], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Bioprocessing, Staining, Expressing, Mutagenesis

Journal: Journal of Virology

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8αβ

doi: 10.1128/jvi.79.17.11422-11433.2005

Figure Lengend Snippet: FIG. 7. Blocking Nef-mediated internalization and down-regulation by ikarugamycin and RNA interference. (A) The figures show the percentage of CD8 molecules internalized in Daudi CD8 cells by HIV-1 NA-7.ER. Cells were incubated for 2 h with (IKA) or without (IKA ) ikarugamycin (2 M) prior to the internalization experiment. At time zero, 4-hydroxytamoxifen (1 M) was added to NA-7.ER- transduced Daudi cells. (B) Western blot, performed as indicated in Materials and Methods, shows protein expression levels of AP-2 2 subunit, clathrin heavy chain (Chc), and dynamin 2 (Dyn-2, arrowhead) in control and RNAi-transduced SupT1 cells, with equal amounts of protein loaded. (C) Bivariate dot plots of flow cytometric measurement of SupT1 cells transduced with AP-2i and HIV-1 Nef (LAI). CD8 (phycoerythrin) versus NGFR (allophycocyanin) expression is shown, gated on EGFP-negative and EGFP-positive cells. (D) The bar charts represent the effect of AP-2, clathrin heavy chain (Chc), and dynamin 2 RNAi on HIV-1 Nef (LAI) and SIV (mac239) Nef-induced CD4 (left panel) and CD8 (right panel) down-regulation in transduced SupT1 cells, gated on EGFP-negative and EGFP-positive cells. Percent down-regulation was calculated, as described in Materials and Methods, using the ranges N and N, as indicated in C. Mean values and standard deviations are shown, calculated from data generated from three independent experiments.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8 primary antibody (5F2 [15], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Blocking Assay, Incubation, Western Blot, Expressing, Control, Transduction, Generated

Journal: Cell Transplantation

Article Title: A Nerve Conduit Containing a Vascular Bundle and Implanted with Bone Marrow Stromal Cells and Decellularized Allogenic Nerve Matrix

doi: 10.3727/096368916X692951

Figure Lengend Snippet: Immunogenicity of the grafts. Longitudinal sections of the distal part of the bridging materials were investigated in the auto group (A, D), the VD group (B, E), and the allo group (C, F) at 2 (A–C) and 4 (D–F) weeks using immunohistochemistry for CD8. The number of CD8+ cells was determined per field, and quantification is shown (G). Scale bars: 100 mm. ∗p < 0.01. VD group, the group with implantation of the sural vessels and a decellularized allogenic nerve matrix.

Article Snippet: The sections were incubated at 4°C for 24 h with mouse monoclonal antibodies against CD8a (surface marker on cytotoxic T cells) (1:10; AbD Serotec) as primary antibodies.

Techniques: Immunopeptidomics, Immunohistochemistry

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 3. Fn facilitates anti-PD-1 efficacy by systematically activating CD8+ TILs in germ-free immunohumanized mice bearing subcutane- ous MSS CRC xenografts and SPF mice bearing orthotopic MSS CRC allografts (A) Model design for germ-free, CD34+-humanized mice bearing subcutaneous HT29 xenografts and experimental groups (n = 6/group). Mice received Fn, Streptococcus mutans UA159 (UA159), or PBS by gavage 3 times a week. Related results can be found in B–F. (B) Engraftment of human immune cells in CD34+-humanized mice were evaluated 14 weeks post implantation (n = 6/group). (C) Representative tumor morphology, weight, and volume (n = 6/group). Red circles indicate the location of subcutaneous tumors. (D) Ki-67, PCNA, and TUNEL staining of HT29 xenografts (n = 6/group). Tumor tissues were stained using anti-Ki-67, anti-PCNA, or TUNEL kit. For Ki-67 and PCNA IHC staining, blue: hematoxylin+ cells, brown: Ki-67+ or PCNA+ cells. For TUNEL staining, green: methyl green+ cells, brown: apoptotic cells. (E) CD8 and PD-1 co-immunofluorescence staining (n = 4/group) and PD-1 IHC in HT29 xenografts (n = 6/group). Tumor tissues were stained using DAPI, Alexa Fluor 647 anti-human CD8, anti-mouse PD-1, and Alexa Fluor 488 anti-Rabbit IgG H&L antibodies for immunofluorescence staining, and anti-PD-1 for IHC. For immunofluorescence staining, red: CD8+ cells, green: PD-1+ cells, blue: DAPI+ cells, yellow: PD-1+ CD8+ cells. For PD-1 IHC staining, blue: hematoxylin+ cells, brown: PD-1+ cells. (F) Flow cytometry analyses of infiltration of CD8+ T cells in HT29 xenografts, and PD-1, IFN-g, TNF-a, and GZMB expression in CD8+ TILs (n = 6/group). (G) Model design for SPF BALB/c mice bearing orthotopic CT26 allografts and experimental groups. Mice were gavaged with Fn conditioned medium (CM), UA159 CM (UACM), or broth Ctrl daily, with anti-PD-1 mAb (aPD-1) or IgG twice a week (n = 8–12/group). Red circles indicate the location of orthotopic tumors. Related results can be found in (H) and (I). (H) Representative tumor morphology, weight, and volume of CT26 allografts (n = 8–12/group).

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: TUNEL Assay, Staining, Immunohistochemistry, Flow Cytometry, Expressing

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 4. Fn decreases PD-1 expression in CD8+ T cell through its small-molecule metabolites (A) Effects of Fn CM fractions on decreasing PD-1 expression in CD8+ T cell after co-culture for 18 h, as determined by flow cytometry (n = 6/group). Fn CM PK: Fn CM treated with proteinase K; >100 kDa: Fn CM > 100 kDa fraction; 10–100 kDa: Fn CM 10–100 kDa fraction; 3–10 kDa: Fn CM 3–10 kDa fraction; <3 kDa: Fn CM < 3 kDa fraction. (B) Workflow to define candidate metabolites from Fn culture medium that suppress PD-1 expression in CD8+ T and promote CD8+ T cell activation in vitro. (C) Orthogonal partial least squares discriminant analysis (OPLS-DA) score plot based on non-targeted metabolomic analysis of Fn CM < 3 kDa vs. control (left) and Fn gavage vs. PBS gavage germ-free mouse feces (right) (n = 6/group). (D) Venn diagram of enriched metabolites in Fn CM < 3 kDa vs. control CM, and Fn gavage vs. PBS gavage in germ-free mouse feces, and metabolomic heatmap of metabolites enriched after Fn gavage vs. PBS gavage germ-free mouse feces as determined by untargeted metabolomics (n = 6/group). (E) Targeted metabolomic analysis of short-chain fatty acids (SFCAs) in Fn- and PBS-gavaged germ-free mouse feces (left). Butyric acid quantification in CM and feces from germ-free mice (right) (n = 6/group). (F) Effect of sodium butyrate (NaB) on the expression of PD-1 and GZMB in splenic CD8+ T cells from tumor-bearing mice (n = 6/group).

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: Expressing, Co-Culture Assay, Cytometry, Activation Assay, In Vitro, Control

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 5. Fn boosts response to anti-PD-1 therapy through its metabolite butyric acid (A) Workflow for the construction of FN0271 mutant Fn strain deficient in butyric acid biosynthesis. PCR products of FN0271 were analyzed by agarose gel electrophoresis to confirm the insertion mutant of Fn (left). GC-MS was conducted to detect butyric acid levels in Fn CM and FN0271 mutant (Mut) Fn CM (right). (B) Effect of Fn CM, Mut Fn CM, and NaB on PD-1 and GZMB expression in splenic CD8+ T cells (n = 6/group). (C) Evaluation of T cell proliferation by carboxyfluorescein succinimidyl ester (CFSE) assay and flow cytometry (n = 6/group). (D) Workflow to assess Fn CM and Mut Fn CM on anti-PD-1 mAb efficacy in vitro using CD8+ TILs from CT26 tumor-bearing mice. (E) Effect of Fn CM and Mut Fn CM in combination with anti-PD-1 mAb (aPD-1) on PD-1, GZMB, and IFN-g expression in CD8+ TILs (n = 6/group). All results are presented as mean ± SD. Each data point in bar plots represents one subject. Statistical significance was determined by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. See also Figure S6.

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: Mutagenesis, Agarose Gel Electrophoresis, Gas Chromatography-Mass Spectrometry, Expressing, CFSE Assay, Cytometry, In Vitro

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 6. Fn boosts anti-PD-1 mAb tumor-killing effects through its metabolite butyric acid in autologous co-culture system of PBMCs with tumor organoids from patients with MSS CRC (A) Schematic diagram for the evaluation of Fn CM and metabolites on anti-PD-1 therapy-mediated killing of CRC organoids with autologous peripheral blood mononuclear cell (PBMC)-derived CD8+ T cells. (B) Representative images of anti-PD-1 (aPD-1) therapy combined with Fn CM, FN0271 Mut Fn CM, or NaB on tumor killing efficiency. MSS CRC organoids (red) were labeled with CellTrace Far Red and apoptotic cells were labeled with green caspase-3/7 probe (n = 6/group). Black circle: organoid, small dot: T cell, red: CellTrace far red+ cells, green: caspase-3/7+ cells. (C) Apoptosis in CRC organoids, as determined by flow cytometry (n = 6/group). (D) Flow cytometry of PD-1, GZMB, and IFN-g in autologous CD8+ T cells (n = 3/group). All results are presented as mean ± SD. Each data point in bar plots represents one subject. Statistical significance was determined by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. See also Figure S7 and Table S2.

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: Co-Culture Assay, Derivative Assay, Labeling, Cytometry, Flow Cytometry

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 7. Fn-derived butyrate facilitates anti-PD-1 therapy in MSS CRC in a CD8+ T cell-dependent manner (A) CT26 subcutaneous allografts tumor-bearing BALB/c mice received NaB in drinking water, together with anti-PD-1 mAb (aPD-1), anti-CD8a mAb, or IgG twice a week (n = 6–10/group). Related results can be found in B–H. (B) Representative tumor gross morphology, tumor weight, and tumor volume. NaB potentiated with anti-PD-1 to suppress tumor growth, an effect abolished by the depletion of CD8+ T cells by anti-CD8a (n = 6–10/group). Red circles indicate the location of subcutaneous tumors. (C) Tumoral butyrate among different groups (n = 6/group). (D) TUNEL, (E) Ki-67, and PCNA IHC in CT26 allografts to analyze cell apoptosis and proliferation (n = 6/group). Tumor tissues were stained using anti-Ki-67, anti- PCNA, or TUNEL kit. For TUNEL staining, green: methyl green+ cells, brown: apoptotic cells. For Ki-67 and PCNA IHC staining, blue: hematoxylin+ cells, brown: Ki-67+ or PCNA+ cells. (F) Infiltration of CD8+ TILs in CT26 allografts, as determined by flow cytometry and (G) IHC (n = 6/group). Tumor tissues were stained using anti-CD8. Blue: hematoxylin+ cells, brown: CD8+ cells.

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: Derivative Assay, TUNEL Assay, Staining, Immunohistochemistry, Cytometry

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 8. Fn metabolite butyric acid regulates CD8+ T cell through an epigenetic mechanism leading to upregulation of TBX21 (T-bet) (A) CD8+ T cells treated with Fn CM, FN0271 Mut Fn CM, and control CM were analyzed by RNA sequencing. Principal component analysis (PCA) and heatmap analysis of differentially expressed genes, and validation by qPCR (n = 3/group). qPCR data were normalized using Actb as an internal control.

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: Control, RNA Sequencing, Biomarker Discovery