Journal: iScience

Article Title: TRAIL-dependent apoptosis of peritoneal mesothelial cells by NK cells promotes ovarian cancer invasion

doi: 10.1016/j.isci.2023.108401

Figure Lengend Snippet: Ascites blocks NK cell degranulation but not TRAIL-dependent HPMC apoptosis (A and B) TAL were pre-cultured in RPMI/5% AB media or 100% ascites pool (+/− α-CD3 Ab stimulation) prior to co-culture with HPMC. (A) Apoptosis of HPMC is shown as percentage of annexin V+ cells after gating on CD45 − cells (n = 7 patients). (B) Degranulating NK cells in response to HPMC were measured via flow cytometry and depicted as percentage of CD335+/CD107a+ cells (n = 6 patients). (C and D) TAL were pretreated with α-TRAIL blocking Ab prior to HPMC co-culture. An irrelevant mouse IgG was included as control. Experiments were conducted with TAL cultured in RPMI/5% AB media (n = 11 patients) (C) and 100% ascites pool (n = 5 patients) (D). The amount of Annexin V+ HPMC was determined as described previously. (E) Schematic representation of co-culture experiments applying T cell CM for NK cell activation. CM were collected from CD3 + , CD3+/CD4+, and CD3+/CD8+ T cell subsets cultured in media +/− α-CD3 Ab stimulation. Purified NK cells were then stimulated with T cell CM prior to HPMC co-cultures. (F) The amount of apoptotic HPMC induced by NK cells activated with CM of CD3 + T cells was compared with CM of CD3+/CD4+ and CD3+/CD8+ T cells (n = 4 matched pairs of different patients). (G) Analysis of TRAIL signaling was performed by applying an α-TRAIL blocking Ab to NK cells stimulated with CD3 + T cell CM (α-CD3 Ab activated) prior to the co-culture with HPMC (n = 5 patients). An irrelevant mouse IgG was included as control. The mean is shown by horizontal bars or boxes; vertical error bars represent the standard deviation in A–D, F, and G. ∗ FDR < 0.05; ∗∗ FDR < 0.01; ∗∗∗ FDR < 0.001; determined by paired t test and Benjamini-Hochberg adjustment (ns, not significant).

Article Snippet: To further divide the CD3 + T cell fraction into CD3+/CD4+ T helper cells and CD3+/CD8+ cytotoxic T-cells, the preselected CD14 − lymphocytes were stained with APC-labelled anti-human CD8 antibody (Miltenyi Biotec), incubated with anti-APC microbeads and isolated via MACS.

Techniques: Cell Culture, Co-Culture Assay, Flow Cytometry, Blocking Assay, Control, Activation Assay, Purification, Standard Deviation

Journal: iScience

Article Title: TRAIL-dependent apoptosis of peritoneal mesothelial cells by NK cells promotes ovarian cancer invasion

doi: 10.1016/j.isci.2023.108401

Figure Lengend Snippet:

Article Snippet: To further divide the CD3 + T cell fraction into CD3+/CD4+ T helper cells and CD3+/CD8+ cytotoxic T-cells, the preselected CD14 − lymphocytes were stained with APC-labelled anti-human CD8 antibody (Miltenyi Biotec), incubated with anti-APC microbeads and isolated via MACS.

Techniques: Control, Recombinant, Polymer, 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: Journal of experimental & clinical cancer research : CR

Article Title: Regnase-1 downregulation promotes pancreatic cancer through myeloid-derived suppressor cell-mediated evasion of anticancer immunity.

doi: 10.1186/s13046-023-02831-w

Figure Lengend Snippet: Fig. 6 Suppression of CTLs by CD11b+ MDSCs is responsible for the acceleration of tumor progression by Regnase-1 downregulation. A-D Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1 KO murine pancreatic cancer cells. Representative macro images of pancreatic tumors (A). Relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (B) (N = 6 per group). Representative images of HE (C, left panel) and CD8a immunostaining (C, right panel) and the number of CD8-positive cells (C, right) (N = 6 per group). Dot plots of CD3+CD8+ cells evaluated by flow cytometry (D, left) and the proportion of CD8 + cells among CD45+ cells (D, right) (N = 3 per group). E–H Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1-KO murine pancreatic cancer cells with or without depletion of CD8+ cells upon anti-CD8a antibody or IgG treatment. Experimental schematic (E). Dot plots of CD3+ and CD8.+ cells in WT or Regnase-1-KO syngeneic tumors upon anti-CD8a antibody or IgG treatment evaluated by flow cytometry (F). Tumor weights (G) (N = 6 per group). The relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (H) (N = 6 per group). Student’s t test was used to evaluate differences between 2 groups. One-way ANOVA with Tukey’s post hoc test was used to compare differences among 4 groups. *P < 0.05, scale bars: 100 μm (insets)

Article Snippet: BE0061, a fully neutralizing mAb recognizing CD8a, and control IgG were obtained from Bioxcell.

Techniques: Immunostaining, Flow Cytometry

Journal: Cell reports

Article Title: The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8 + T cell differentiation and function

doi: 10.1016/j.celrep.2022.111963

Figure Lengend Snippet: (A and B) Flow cytometry analysis of Ahr expression (GFP) in CD8 + T cells of Ahr +/+ and Ahr dCAIR/+ mice was performed. Histogram plot of GFP( Ahr ) in CD8 + T cells isolated from spleen (Sp), peripheral lymph node (pLN), mesenteric lymph node (mLN), small intestine lamina propria (LPL), or intraepithelial lymphocytes (IEL) (A). Quantification of ΔgMFI in Ahr dCAIR/+ compared with GFP-negative Ahr +/+ mice (B). (C and D) Flow cytometry quantification of ΔgMFI of Ahr -GFP in different CD8 + T cell populations isolated from LPL including CD44 − CD62L + (T naive ), CD127 − KLRG1 − (T EE ), CD127 − KLRG1 + (T SLE ), CD127 + KLRG1 − (T MPE ) (C), and CD44 + CD62L + (T CM ), CD44 + CD62L − (T EM ), and CD69 + CD103 + (T RM ) (D). Data are shown as mean ± SEM (n = 3 mice per group). Data are representative of two independent experiments. See also .

Article Snippet: Anti-human CD8 – APC-Cyanine7 (clone SK1) , TONBO biosciences , Cat# 25-0087-T025; RRID: AB_2848136.

Techniques: Flow Cytometry, Expressing, Isolation

Journal: Cell reports

Article Title: The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8 + T cell differentiation and function

doi: 10.1016/j.celrep.2022.111963

Figure Lengend Snippet: (A–E and H) RNA-seq analysis of IEL resident CD8 + T cells isolated from Ahr f/f and Ahr f/f Cd8 cre mice and statistics (q values shown) were calculated via DESeq2 differential expression analysis, and FPKM values were quantified using RSEM. MA plot highlighting genes upregulated (red) and downregulated (blue) in Ahr f/f Cd8 cre compared with Ahr f/f (A). Gene set enrichment analysis of resident and circulating core gene signatures (B). Heatmap depicting (fold change >1.5) genes enriched in respective signatures (C). FPKM expression values for transcriptional regulators (D) and secreted factors associated with cell function (E). (F and G) Flow cytometry quantification of granzyme B protein levels of IEL resident CD8 + T cells isolated from Ahr f/f and Ahr f/f Cd8 cre mice. The percentages (F) and total cell number (n = 5 mice per group) (G) are shown. Data are representative of two independent experiments. (H) FPKM expression values for proliferation, cell cycle, and apoptosis genes. (I–K) Naive CD8 + T cells were isolated from Ahr +/+ and Ahr − / − mice and then subjected to in vitro T RM -like differentiation culture conditions. RNA was isolated for qRT-PCR expression analysis of Ahrr (I). Flow cytometry quantification of CD69 + CD103 + in vitro T RM -like cells (J) and CD44 + CD62L + in vitro T CM -like population frequency (K). Data are shown as mean ± SEM (n = 3 mice per group). Data are representative of three independent experiments. See also , , and .

Article Snippet: Anti-human CD8 – APC-Cyanine7 (clone SK1) , TONBO biosciences , Cat# 25-0087-T025; RRID: AB_2848136.

Techniques: RNA Sequencing, Isolation, Quantitative Proteomics, Expressing, Cell Function Assay, Flow Cytometry, In Vitro, Quantitative RT-PCR

Journal: Cell reports

Article Title: The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8 + T cell differentiation and function

doi: 10.1016/j.celrep.2022.111963

Figure Lengend Snippet: (A and B) ChIP-seq of Ahr was performed using in vitro T RM -like CD8 + T cells. Analysis of Ahr binding was performed, and pie chart of peak annotation (A) as well as top seven enriched transcription factor motifs (B) are shown. (C and D) Binding and Expression Target Analysis (BETA) was performed to integrate RNA-seq and ChIP-seq data. Visualization of transcription factor activating/repressive function prediction (C) and rank product volcano plot depicting top direct target candidates (D). (E and F) Ahr − / − in vitro T RM -like CD8 + T cells transduced with retroviral constructs encoding MIG-EV (empty vector), MIG-Ahr, MIG-Y9A, or MIG-DbHLH. The cells were treated with DMSO or FICZ on day 3. On day 5, CD69 and CD103 expression was analyzed by flow cytometry (E), and RNA was isolated for qRT-PCR expression analysis of Ahr direct target gene Ahrr (F). Data are shown as mean ± SEM (n = 3 technical replicates per group). Data are representative of two independent experiments. (G) Flow cytometry analysis of CD69 and CD103 expression in Ahr − / − in vitro T RM -like CD8 + T cells transduced with retroviral constructs encoding MIG-EV, MIG-Ahr, hCD2-EV, or hCD2-Blimp1. Data are representative of two independent experiments. See also .

Article Snippet: Anti-human CD8 – APC-Cyanine7 (clone SK1) , TONBO biosciences , Cat# 25-0087-T025; RRID: AB_2848136.

Techniques: ChIP-sequencing, In Vitro, Binding Assay, Expressing, RNA Sequencing, Transduction, Retroviral, Construct, Plasmid Preparation, Flow Cytometry, Isolation, Quantitative RT-PCR

Journal: Cell reports

Article Title: The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8 + T cell differentiation and function

doi: 10.1016/j.celrep.2022.111963

Figure Lengend Snippet: (A–F) Analysis of antigen-specific (OTI) CD8 + T cell response in the IEL during oral L . m .-OVA infection was performed as depicted in the schematic (A). Flow cytometry quantification of the percentage of Ahr +/+ vs. Ahr − / − OTI cells on day 9, 20, and 34 post infection (B). Flow cytometry quantification of memory precursor populations based on expression of CD127 and KLRG1 (C) as well as CD69 and CD103 (D) in Ahr +/+ vs. Ahr − / − OTI cells present in the IEL on day 9 post infection. Flow cytometry analysis of CD45.1 and GzmB gated on OTI cells depicting CD45 . 1 + Ahr +/+ (CD45.1/.2) and CD45 . 1 − Ahr − / − (CD45.2/.2) OTI cells production of granzyme B on day 34 post infection (E). Quantification of granzyme B + OTI T cells in the mice of indicated genotypes (F). (G–J) L . m .-OVA re-infection was performed and Ahr +/+ vs. Ahr − / − OTI IEL resident CD8 + T cells analyzed on day 3 post re-infection. Flow cytometry analysis of CD45.1 and CD45.2 depicting percentage of Ahr +/+ (CD45.1/.2) and Ahr − / − (CD45.2/.2) OTI IEL resident CD8 + T cells as well as (G) quantification of percentage are shown (H). Flow cytometry analysis (I) and quantification (J) of granzyme B production in analyzed cells. Data are compiled from two independent experiments and shown as mean ± SEM (n = 3–6 replicates per group). See also .

Article Snippet: Anti-human CD8 – APC-Cyanine7 (clone SK1) , TONBO biosciences , Cat# 25-0087-T025; RRID: AB_2848136.

Techniques: Infection, Flow Cytometry, Expressing

Journal: Cell reports

Article Title: The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8 + T cell differentiation and function

doi: 10.1016/j.celrep.2022.111963

Figure Lengend Snippet: (A–D) scRNA-seq analysis of Ahr +/+ (n = 2) vs. Ahr − / − (n = 2) TIL CD8 + T cells was performed. UMAP dimensionality reduction and cluster visualization (left) as well as pie chart frequency depiction (right) color-coded to represent cluster ID (A). Pseudotime visualization (B) and quantification (C). Differential gene expression depicted via color intensity as average expression and circle size as percent expressed in CD8 + T cells (D). (E–H) Ahr f/f and Ahr f/f Cd8 cre mice were inoculated subcutaneously with B16F10 mouse melanoma and tumor size monitored (E). At endpoint, tumor weight was quantified (F), and TILs were isolated for flow cytometry analysis. Pie chart visualization depicting polyfunctionality of TIL CD8 + T cells in mice with indicated genotypes (G). Polyfunctionality score quantification (triple-positive plus double-positive minus triple-negative divided by total cells) of TIL CD8 + T cells isolated from Ahr f/f and Ahr f/f Cd8 cre tumor-bearing mice (H). Data are compiled from two independent experiments and are shown as mean ± SEM (n = 6 to 7 replicates per group). See also .

Article Snippet: Anti-human CD8 – APC-Cyanine7 (clone SK1) , TONBO biosciences , Cat# 25-0087-T025; RRID: AB_2848136.

Techniques: Gene Expression, Expressing, Isolation, Flow Cytometry

Journal: Cell reports

Article Title: The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8 + T cell differentiation and function

doi: 10.1016/j.celrep.2022.111963

Figure Lengend Snippet: (A–C) Human peripheral blood (PBMC) vs. IEL CD8 + T cells were analyzed via flow cytometry. Staining of CD45RA and CD45RO (top), CD103 and CD69 (bottom) (A), as well as T-BET and AHR (B) in tissue fractions as indicated in the figure. Quantification of AHR protein levels (gMFI) in human PBMC, LPL, and IEL CD8 + T cells (C). Data are compiled from three independent experiments and shown as mean ± SEM (n = 4 replicates per group). (D–G) Human naive CD8 + T cells were isolated from PBMCs and then subjected to in vitro T RM -like differentiation culture conditions. The cells were given differentiation cytokines and treated with DMSO (control), FICZ, or CH223191 on day 2. The assay was collected on day 4, and flow cytometry quantification of CD103 expression was performed (D). Data are compiled from three independent experiments and are shown as mean ± SEM (n = 4 replicates per group). RNA was isolated for qRT-PCR expression analysis of AHR (E), AHR direct target gene AHRR (F), and GZMB (G). Data are shown as mean ± SEM (n = 3 technical replicates per group). Data are representative of three independent experiments.

Article Snippet: Anti-human CD8 – APC-Cyanine7 (clone SK1) , TONBO biosciences , Cat# 25-0087-T025; RRID: AB_2848136.

Techniques: Flow Cytometry, Staining, Isolation, In Vitro, Control, Expressing, Quantitative RT-PCR

Journal: Cell reports

Article Title: The aryl hydrocarbon receptor cell intrinsically promotes resident memory CD8 + T cell differentiation and function

doi: 10.1016/j.celrep.2022.111963

Figure Lengend Snippet:

Article Snippet: Anti-human CD8 – APC-Cyanine7 (clone SK1) , TONBO biosciences , Cat# 25-0087-T025; RRID: AB_2848136.

Techniques: Virus, Isolation, Recombinant, SYBR Green Assay, DNA Library Preparation, Reverse Transcription, Staining, Transgenic Assay, Plasmid Preparation, Software

Journal: PLoS ONE

Article Title: The novel multi-cytokine inhibitor TO-207 specifically inhibits pro-inflammatory cytokine secretion in monocytes without affecting the killing ability of CAR T cells

doi: 10.1371/journal.pone.0231896

Figure Lengend Snippet: A) Effects of PSL and TO-207 on the cytotoxicity of CAR T cells. K562/CD19/fLucEGFP cells (3 × 10 3 ) were co-cultured with CD4 + or CD8 + CAR T cells (1.5 × 10 4 ), and treated with different concentrations of PSL and TO-207. Viable target cells were quantified using the luciferase assay after 72 h of co-culture. Values were normalized to viable K562/CD19/fLucEGFP cells cultured alone. The error bars represent SDs from three independent experiments. B) Effects of PSL and TO-207 on CAR T cell degranulation. K562/CD19 cells (5 × 10 5 ) and CAR T cells (5 × 10 5 ) were co-cultured in 500 μl T-cell expansion medium in the absence or presence of PSL or TO-207. Following 68 h of co-culture, monensin (2 μM) and APC–anti-human CD107a antibody were added. Cells were incubated for an additional 4 h, and membrane expression of CD107a was determined by flow cytometry. Representative data of four independent experiments are shown. C) Mean fluorescence intensity (MFI) of samples in Fig 5B. The error bars represent SEs from four independent experiments. D) Sustained cytotoxicity of CAR T cells following TO-207 treatment. K562/CD19/fLucEGFP cells (1 × 10 4 ), CAR T cells (5 × 10 4 ), and CD14 + cells (5 × 10 4 ) were co-cultured in the absence or presence of PSL or TO-207 for 72 h, and viable target cells were quantified by the luciferase assay. Values were normalized to the well containing K562/CD19/fLucEGFP cells alone. The error bars represent SDs from three independent experiments. The linear dose-response relationship was assessed using log-transformed dose values (to the base 10) in a mixed model, in which the zero dose was replaced by the log (minimal dose) - 1. P < 0.05 was considered statistically significant. n.s.: not significant.

Article Snippet: Flow cytometry was performed using allophycocyanin (APC)-conjugated IgG 1 (BioLegend), APC–anti-human CD19 antibody (BioLegend), APC–anti human CD107a (BioLegend), APC–Cy7–anti-human CD3 antibody (BioLegend), V500–anti-human CD4 antibody (BD Biosciences, San Jose, CA, USA), and PerCP–Cy5.5–anti-human CD8 antibody (TONBO Biosciences, San Diego, CA, USA).

Techniques: Cell Culture, Luciferase, Co-Culture Assay, Incubation, Membrane, Expressing, Flow Cytometry, Fluorescence, Transformation Assay