Journal: Human reproduction (Oxford, England)

Article Title: A monoclonal antibody, HCL-2, raised against human luteal cells reacts with apolipoprotein-B and detects the uptake of low density lipoprotein by luteinizing granulosa cells.

doi: 10.1093/humrep/13.4.936

Figure Lengend Snippet: Figure 1. HCL-2 antigen expression in corpus luteum on day 7 detected by indirect immunofluorescence staining. (A) Haematoxylin and eosin staining. (B) Staining with HCL-2 mAb. (C) Staining with anti-apolipoprotein-B mAb (MAB012). (D) Negative control stained with anti-TNP mAb. HCL-2 antigen and apolipoprotein-B were expressed on the granular-shaped structure near the nuclei in the cytoplasm of both large (LL) and small (SL) luteal cells. Both proteins were also detected along the cell membrane, showing the similar expression profiles. Original magnification 3120. Bar 5 100 µm.

Article Snippet: The immunoglobulin isotype was determined using an isotyping kit for mouse monoclonal antibodies (Serotec Ltd, Oxford, UK).

Techniques: Expressing, Staining, Negative Control, Membrane

Journal: Human reproduction (Oxford, England)

Article Title: A monoclonal antibody, HCL-2, raised against human luteal cells reacts with apolipoprotein-B and detects the uptake of low density lipoprotein by luteinizing granulosa cells.

doi: 10.1093/humrep/13.4.936

Figure Lengend Snippet: Figure 5. Detection of HCL-2 antigen on human luteinizing granulosa cells cultured in a medium with or without low density lipoprotein (LDL) by indirect immunofluorescence staining. (A–D) Culture with LDL. (E–H) Culture without LDL. (A, C, E and G) Phase-contrast pictures. (B and F) HCL-2 antigen. (D and H) Negative controls (anti-TNP mAb). HCL-2 antigen was clearly detected in the cytoplasm of human granulosa cells (arrows) cultured in the medium containing LDL for 3 days (B), whereas it was hardly detected in those cultured without LDL (F). Original magnification 3240. Bar 5 50 µm.

Article Snippet: The immunoglobulin isotype was determined using an isotyping kit for mouse monoclonal antibodies (Serotec Ltd, Oxford, UK).

Techniques: Cell Culture, Staining

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

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 2. Characteristics of the HHR thymus and differentiation status of thymocytes in the thymus. (A) Graphs show the weights and cellularities of SDR and HHR thymi at 4–5 wk of age. Three SDRs and HHRs were used for each analysis. Data are shown as mean 6 SD. *p , 0.05. (B) Histological sections of thymi from 4-wk-old SDRs and HHRs were stained with H&E. Scale bars represent 1 mm. Representative results of two SDRs and HHRs are shown. (C) The differentiation status of thymocytes was analyzed with a flow cytometer. Representative results of three SDRs and HHRs are shown. An arrow indicates a population of DP thymocytes with decreased CD4 levels. Averaged values from three independent flow cytometric analyses for the proportions of DP, CD4-SP, CD8-SP, and DN thymocytes are shown in the lower graph. Data are shown as mean 6 SD. (D) Expression levels of Cd4 and Cd8 genes in the thymus were analyzed by real-time RT-PCR. Three SDRs and HHRs were used. The level of each gene is shown relative to the value for the SDR, which is set at 1. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Staining, Cytometry, Expressing, Quantitative RT-PCR

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

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 3. nTreg numbers in the HHR thymus. (A) Expression levels of Cd25 and Foxp3 genes in CD4-SP thymocytes were analyzed by real-time RT- PCR. Three SDRs and HHRs were used. The level of each gene is shown relative to the value for the SDR, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (B) Upper, The proportion of CD4+CD25+ cells in the thymus was determined by flow cytometric analysis. Representative results of three SDRs and HHRs are shown. Averaged values from three independent analyses for the proportion of CD4+CD25+ cells are shown in the graph. Data are shown as mean 6 SD. Lower, The proportion of Foxp3+ cells in the CD4+CD25+ cell fraction was determined by flow cytometric analysis. Representative results of three SDRs and HHRs are shown. Averaged values from three independent analyses for the proportion of Foxp3+ cells in the CD4+CD25+ cell fraction are shown in the graph. Data are shown as mean 6 SD. *p , 0.05. (C) Upper, Estimated values for the proportion of CD4+CD25+Foxp3+ nTregs in the thymus were calculated using the values of the proportion of CD4+CD25+ cells in the thymus and those of Foxp3+ cells in the CD4+CD25+ cell fraction obtained from flow cytometric analysis and are shown in the graph. Data are shown as mean 6 SD. *p , 0.05. Lower, Estimated values for the absolute number of CD4+CD25+Foxp3+ nTregs in the thymus were calculated using the values of the proportion of CD4+CD25+ cells in the thymus and those of total thymus cell number (Fig. 2A) and are shown in the graph. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Expressing, Quantitative RT-PCR

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

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 5. Loss of Ly49s3 gene expression in HHR thymic cDCs. (A) Left, Genome-wide microarray CGH analysis, performed with genomic DNA from SDR and HHR livers, shows the deletion of four Ly49 family genes—Ly49s4, Ly49i4, Ly49s3, and Ly49i3—in chromosome 4 at the q42 region (shaded area). Data shown are representative of two independent analyses. Right, Genomic PCR with DNA from SDR and HHR livers was performed to confirm the deletion of DNA in this region. As an internal standard, the Ccr4 gene, located at chromosome 8q32, was used. (B) Left, RT-PCR analysis of the expression of the Ly49s3 gene was performed with total RNA from SDR and HHR thymi. As an internal standard, the Gapdh gene was used. Middle, RT- PCR analysis of Ly49s3 gene expression in DP, CD4-SP, and CD8-SP thymocytes of the SDR thymus was performed with total RNA from the cells. Note that it was not possible to isolate pure DN thymocytes by positive and/or negative selection using CD4 and CD8a microbeads because the remaining cells after the selection of DP, CD4-SP, and CD8-SP cells are a mixture of DN thymocytes and all of the other types of cells. Right, RT-PCR analysis of Ly49s3 gene expression in cDCs of SDR and HHR thymi was performed with total RNA from the cells.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Gene Expression, Genome Wide, Microarray, Reverse Transcription Polymerase Chain Reaction, Expressing, Selection

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

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 6. Expression of the nTreg marker and MHC class II genes in the mixed-cell culture. (A) A total of 1 3 106 CD4-SP thymocytes isolated from the SDR thymus were cultured with 2.5 3 105 cDCs from the SDR thymus, and 1 3 106 CD4-SP thymocytes from the HHR thymus were cultured with 2.5 3 105 cDCs from the HHR thymus. At 3 d later, total RNA was extracted and expression levels of nTreg marker genes Foxp3, Cd25, Ctla4, and Pd-1 and MHC class II genes Rt1-Ba and Rt1-Bb were determined by real-time RT-PCR. Three independent experiments were performed for each mixed-cell culture. The level of each gene is shown relative to the value for the mixed culture of cells from the SDR thymus, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (B) To confirm the expression levels of Foxp3 and CD25, an additional mixed-cell culture experiment was performed, and the proportion of Foxp3+ or CD25+ cells was determined by flow cytometric analysis. (C) As control experiments, 1 3 106 CD4-SP thymocytes were cultured alone for 3 d and the same real-time RT-PCR analyses as above were performed. Three independent experiments were performed for each culture. The level of each gene is shown relative to the value for SDR cells, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (D) Effects of cDCs on nTreg marker gene expression. The expression levels of nTreg marker genes in mixed-cell (A) and control cultures (C) are shown in the same graph relative to the value for the SDR control culture, which is set at 1. Statistical analysis was performed between the induction ratios of gene expression. Data are shown as mean 6 SD. *p , 0.05. (E) A total of 1 3 106 CD4+CD82CD252 thymocytes isolated from SDR and HHR thymi were cultured with 2.5 3 105 cDCs isolated from SDR and HHR thymi, respectively. At 3 d later, total RNA was extracted and expression levels of nTreg marker genes Foxp3, Cd25, Ctla4, and Pd-1 were determined by real-time RT-PCR. Three independent experiments were performed for each mixed-cell culture. The level of each gene is shown relative to the value for the mixed culture of cells from the SDR thymus, which is set at 1. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Expressing, Marker, Cell Culture, Isolation, Quantitative RT-PCR, Control, Gene Expression

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

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 7. Expression of the nTreg marker and MHC class II genes in the mixed-cell culture using Ly49s3-expressing HHR thymic cDCs. (A) The FLAG- tagged Ly49s3 structure is schematically represented. R: arginine residue. (B) Left, 293T cells were transfected with recombinant vectors before being packaged into the virus, and cell lysates were subjected to Western blot analysis with the anti-FLAG M2 Ab. Right, The proteins on the membrane were stained with fast green. (C) Left, cDCs from the HHR thymus were transduced with the lentiviral vector of FLAG-tagged Ly49s3, and the expression of the fusion protein on the surface of cDCs was confirmed by fluorescence microscopic observations with the anti-FLAG M2 Ab. Right, Phase contrast appearance of the identical cells shown in the left photographs. Note dendrites on the surface of the cells. The cells were suspended in buffer and all the photos were taken. Original magnification 3800. (D) A total of 2.5 3 105 cDCs isolated from the HHR thymus were transduced with the lentiviral vector of FLAG-tagged Ly49s3 (Ly) or the mock vector (Mo) and then mixed with 1 3 106 CD4-SP thymocytes isolated from the HHR thymus. At 3 d later, total RNAwas extracted and the expression levels of nTreg marker genes Foxp3, Cd25, Ctla4, and Pd-1 and MHC class II genes Rt1-Ba and Rt1-Bb were determined by real-time RT-PCR. Three independent experiments were performed for each culture. The level of each gene is shown relative to the value for the mixed culture, using cDCs transduced with the mock vector, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (E) A total of 2.5 3 105 cDCs from the HHR thymus were transduced with the lentiviral vector of FLAG-tagged Ly49s3 and then mixed with 1 3 106 CD4-SP thymocytes from the HHR thymus. Next 5 mg of anti-rat MHC class I Ab or normal IgG was added to the culture. At 3 d later, total RNA was extracted, and the expression levels of nTreg marker genes and MHC class II genes were determined by real-time RT-PCR. Three independent experiments were performed for each culture. The level of each gene is shown relative to the value for the mixed culture in the presence of normal IgG, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (F) Summary of the experiments performed using the lentiviral vector of the Ly49s3 gene and anti-MHC class I Ab. The level of each gene is shown relative to the value for mock-transduced cells, which is set at 1. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Expressing, Marker, Cell Culture, Residue, Transfection, Recombinant, Virus, Western Blot, Membrane, Staining, Transduction, Plasmid Preparation, Isolation, Quantitative RT-PCR

Journal: Nature

Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack

doi: 10.1038/s41586-022-04772-4

Figure Lengend Snippet: a, Design of the MICA/B vaccine. pAbs, polyclonal serum IgG. b, MICB-specific serum antibody (Ab) titres quantified by flow cytometry (n = 4 mice per group) in MICB-transgenic mice immunized with Ctrl-vax (blue) or MICB-vax (red). MFI, median fluorescence intensity. c, MICB-specific CD4+ T cell responses following immunization with MICB-vax or Ctrl-vax; CFSE dilution of splenocytes stimulated with MICB or control protein (ovalbumin (OVA)); shown are representative flow cytometry plots (left) and quantification for three mice per group (right). d, Cell-surface levels of MICB on B16F10 (MICB) tumours from mice immunized with MICB-vax or Ctrl-vax (n = 4 mice per group); staining of tumour cells with isotype-control monoclonal antibody (grey) or anti-MICA/B monoclonal antibody (specific for the α1–α2 domains, monoclonal antibody not blocked by vaccine-induced antibodies). e, Therapeutic efficacy of MICB-vax (red) or Ctrl-vax (blue) in mice with established B16F10 (MICB) tumours immunized at the indicated time points (n = 7 mice per group). Vax, vaccination. *P = 0.0137, ****P <0.0001; NS (not significant), P > 0.999. f, g, Vaccine efficacy in two models of spontaneous metastasis. Mice were immunized with Ctrl-vax (blue) or MICB-vax (red) following surgical removal of primary tumours using the B16-B6 melanoma (f; 10 mice per group) or 4T1 breast cancer (g; 13 mice per group) models. Shown are the size of primary tumours at the time of surgery (left), representative images of lung metastases (middle) and quantification of the total number of lung surface metastases (right). D, day; s.c., subcutaneous. h, i, Immunogenicity of the rhesus MICA/B α3 domain vaccine in the rhesus macaque model. h, Timeline of vaccination; blood was drawn 24 h before indicated immunization or boost. i, Serum titres of antibody to rhesus MICA/B for animal ID 9312. Representative data are shown from at least three (b) or two (c–g) independent experiments. Data from a single experiment with technical replicates for each time point are shown in i. Statistical significance was assessed by two-tailed unpaired Student’s t test (b), two-way ANOVA with Sidak’s multiple-comparison test (c), one-way ANOVA with Tukey’s multiple-comparison test (d), two-way ANOVA with Bonferroni’s post hoc test (left) and the log-rank (Mantel–Cox) test (right) (e), two-tailed Mann–Whitney test (f, g) and two-way ANOVA with Tukey’s multiple-comparison test (i). Data are depicted as the mean ± s.e.m. (b–g) or mean±s.d. (i).

Article Snippet: The following isotype-control antibodies were used: rat IgG2b (clone LTF-2, BioXcell; control for anti-CD4), rat IgG1 (clone TNP6A7, BioXcell; control for anti-CD8β), mouse IgG2a (clone C1.18.4, BioXcell; control for anti-NK1.1) and rat IgG2a (clone 2A3, BioXcell; control for anti-CSF1R).

Techniques: Flow Cytometry, Transgenic Assay, Fluorescence, Control, Staining, Drug discovery, Immunopeptidomics, Two Tailed Test, Comparison, MANN-WHITNEY

Journal: Nature

Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack

doi: 10.1038/s41586-022-04772-4

Figure Lengend Snippet: a, Characterization of the MICA/B α3 immunogen used in the primate study. The α3 domains of rhesus macaque MICA and MICB were expressed as a fusion protein with ferritin to generate nanoparticles that displayed both α3 domains on the surface. Nanoparticles formed by this fusion protein were conjugated using click chemistry to CpG ODN 2935 as the adjuvant and characterized by HPLC gel filtration chromatography. Shown are HPLC traces of the protein following conjugation to the CpG oligonucleotide (red: 280 nm trace for detection of protein; blue: 350 nm trace for detection of bis-aryl hydrazone bond). b, SDS-PAGE analysis of purified macaque MICA/B α3–ferritin protein under reducing (+) and non-reducing (−) conditions following CpG conjugation and final purification using a HPLC gel filtration column. c–e, Characterization of serum antibody responses to rhesus macaque MICA (left) and MICB (right) proteins (full-length extracellular domains without ferritin fusion partner) at different steps in the immunization process. Antibody responses were investigated in three animals (RBQ12, RVf10 and RQq15) at multiple timepoints (pre-immunization; three weeks following initial immunization and boosts 1-3, as illustrated in Fig. 1h) using a fluorescence-based ELISA (RFU, relative fluorescence units) at multiple serum dilutions (1:102 to 1:104). f–m, Binding of purified polyclonal serum IgG to cell surface MICA (left) and MICB (right) using HEK293T transfectants that displayed rhesus macaque MICA (Mamu-A*01) or MICB (Mamu-B*01) proteins. Preimmune sera were used as a control (grey) for sera obtained following immunization (red). Representative histograms (left in f, g) and graphical summaries of flow cytometry data (right in f, g, h–m) are shown for the four immunized macaques (9312, RBQ12, RVf10 and RQq15). Representative data from two experiments (a–b). Data from a single macaque immunization experiment with technical replicates for each macaque analyzed (c–m). Two-way ANOVA with Tukey’s multiple comparison test (c-e). Data depict mean+/− SD.

Article Snippet: The following isotype-control antibodies were used: rat IgG2b (clone LTF-2, BioXcell; control for anti-CD4), rat IgG1 (clone TNP6A7, BioXcell; control for anti-CD8β), mouse IgG2a (clone C1.18.4, BioXcell; control for anti-NK1.1) and rat IgG2a (clone 2A3, BioXcell; control for anti-CSF1R).

Techniques: Adjuvant, Filtration, Chromatography, Conjugation Assay, SDS Page, Purification, Fluorescence, Enzyme-linked Immunosorbent Assay, Binding Assay, Control, Flow Cytometry, Comparison

Journal: Nature

Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack

doi: 10.1038/s41586-022-04772-4

Figure Lengend Snippet: a–b, Representative histograms (a) and quantification (b) of PD-1, CTLA-4, Tim-3, Tigit and Lag3 expression by tumor-infiltrating CD8 T-cells from Ctrl-vax (blue) or MICB-vax (red) mice (n = 9 mice/group). c–d, Comparison of T-cell and NK cell populations in B16F10 (MICB) tumors following treatment with a MICA/B mAb or the MICB vaccine. In the vaccine arm, mice received Ctrl-vax (C-vax) (n = 9 mice) or MICB-vax (M-vax) (n = 10 mice) on days 0 and 14, while mice in the mAb treatment group (n = 8 mice/ group) received two buffer injections. Mice were implanted with B16F10 (MICB-dox) tumor cells on day 21. MICB expression was induced on tumor cells by doxycycline treatment starting on day 28, and mice in the mAb treatment group received either mouse IgG2a isotype control mAb (iso) or MICA/B mAb (mAb) treatment every 48 h starting on day 28. Tumor-infiltrating immune cells were analyzed in all groups on day 35. Total numbers of tumor-infiltrating CD4+ T-cells, CD8+ T-cells and NK cells were quantified by flow cytometry (c), and intracellular staining was performed for IFNγ (d) in all four treatment groups. e–g, scRNA-seq analysis of changes in tumor-infiltrating immune cells induced by the vaccine. CD45+ immune cells in B16F10 (MICB-dox) tumors were investigated by scRNA-seq under four experimental conditions: the MICB-vax (+dox) experimental group and the three control groups, Ctrl-vax (−dox), Ctrl-vax (+dox) and MICB-vax (−dox). Doxycycline was administered to mice for seven days prior to scRNA-seq analysis to induce MICB expression on tumor cells in two of these groups (+dox). For each of the four groups, CD45+ immune cells were pooled from five mice to reduce variation from individual tumors. e, UMAP projection of CD45+ immune cells combined from all experimental groups. Major immune cell populations are annotated based on differentially expressed genes. f, Comparison of immune subpopulations across all clusters for the experimental MICB-vax (+dox) group (red) versus the three combined control groups (blue). g, Distribution of CD45+ cells across individual clusters (color-coded as in e) for the experimental MICB-vax (+dox) group (MICB) and the three combined control groups (Ctrl). Representative data of two experimental repeats (a–b). Data from a single experiment (c–d). ScRNA-seq data from a single experiment with sorted CD45+ cells pooled from 5 mice/group (e–g). Two-tailed Mann Whitney test (b); one-way ANOVA with Tukey’s multiple comparison test (c–d). Data depict mean+/− SEM.

Article Snippet: The following isotype-control antibodies were used: rat IgG2b (clone LTF-2, BioXcell; control for anti-CD4), rat IgG1 (clone TNP6A7, BioXcell; control for anti-CD8β), mouse IgG2a (clone C1.18.4, BioXcell; control for anti-NK1.1) and rat IgG2a (clone 2A3, BioXcell; control for anti-CSF1R).

Techniques: Expressing, Comparison, Control, Flow Cytometry, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack

doi: 10.1038/s41586-022-04772-4

Figure Lengend Snippet: a, b, Comparison of vaccine efficacy against B16F10 (MICB) WT tumours and tumours with resistance mutations in the B2m (a) or Ifngr1 (b) gene. Mice received MICB-vax or Ctrl-vax and were then challenged with tumours of the indicated genotype (n = 7 mice per group). KO, knockout. c, Effect of CD4+ T cell and NK cell depletion on immunity to B2m-knockout tumours. Mice were immunized with MICB-vax or Ctrl-vax; treatment with depleting or isotype-control monoclonal antibody was started 2 d before injection of B2m-knockout B16F10 (MICB) tumour cells (n = 7 mice per group). d, Contribution of vaccine-induced anti-MICB antibodies to NK cell-mediated cytotoxicity against B2m-knockout B16F10 (MICB) tumour cells. CFSE-labelled B2m-knockout B16F10 (MICB) tumour cells were pre-incubated with 10 μg per well of purified serum IgG from mice immunized with MICB-vax or Ctrl-vax before the addition of NK cells at different effector to target (E:T) ratios as indicated. The percentage of dead target cells was assessed by flow cytometry. e, Effect of CD4+ T cell depletion on vaccine-induced NK cell infiltration into tumours. Flow cytometry quantification of total NK cell numbers is shown in WT (left) and B2m-knockout (right) tumours for the following treatment groups: Ctrl-vax+isotype-control monoclonal antibody (blue), Ctrl-vax + anti-CD4 (orange), MICB-vax+isotype-control monoclonal antibody (red) and MICB-vax + anti-CD4 (green) (n = 7 mice per group). Representative data from two independent experiments are shown in a–e. Statistical significance was assessed by two-way ANOVA with Bonferroni’s post hoc test (left) and log-rank (Mantel–Cox) test (right) (a–c), two-way ANOVA with Sidak’s multiple-comparison test (d) and one-way ANOVA with Tukey’s multiple-comparison test (e). Data are depicted as the mean±s.e.m.

Article Snippet: The following isotype-control antibodies were used: rat IgG2b (clone LTF-2, BioXcell; control for anti-CD4), rat IgG1 (clone TNP6A7, BioXcell; control for anti-CD8β), mouse IgG2a (clone C1.18.4, BioXcell; control for anti-NK1.1) and rat IgG2a (clone 2A3, BioXcell; control for anti-CSF1R).

Techniques: Comparison, Knock-Out, Control, Injection, Incubation, Purification, Flow Cytometry

Journal: Nature

Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack

doi: 10.1038/s41586-022-04772-4

Figure Lengend Snippet: a, Effect of CD4+ T cells on migratory DC populations in the tdLNs of mice immunized with MICB-vax versus Ctrl-vax. Total migratory DCs as well as cDC1 and cDC2 cells were quantified 2 d after induction of MICB expression in tumour cells by doxycycline treatment (n = 7 mice per group, except n = 6 for Ctrl-vax without anti-CD4). b, Migratory DC subsets within the tdLN of MICB-vax-immunized mice treated following immunization (days 28 + 30) with isotype-control, CD4-depleting or CD40L-blocking monoclonal antibody (n = 7 mice per group). c, Quantification of DC populations within the tumours of mice immunized with Ctrl-vax (blue) or MICB-vax (red) on day 7 following induction of MICB expression with doxycycline (n = 7 mice per group). d, Effect of cDC1 depletion on MICB vaccine-induced T cell and NK cell accumulation within tumours in Xcr1DTR mice. Mice were treated with DT or left untreated starting on day 26 following immunization with Ctrl-vax or MICB-vax (days 0 + 14) and B16F10 (MICB-dox) tumour implantation (day 21). Immune cells were analysed in tumours 7 d after induction of MICB expression on tumours with doxycycline (day 37) (n = 7 mice per group). e, Contribution of vaccine-induced anti-MICB antibodies to DC-mediated cross-presentation of tumour antigens to CD8+ T cells. Bone marrow-derived DCs (BMDCs) were pre-incubated with B2m-knockout B16F10 (MICB-OVA) tumour cells in the presence of affinity-purified serum IgG from mice immunized with Ctrl-vax or MICB-vax at the indicated concentrations. DCs were co-cultured with CFSE-labelled OT-1 CD8+ T cells with T cell proliferation as the readout. The role of activating Fc receptor (FcR) was assessed using BMDCs from Fcer1g−/− mice (orange) or pre-incubation of BMDCs with FcR-blocking antibody (yellow) before tumour cell addition. Representative data from two independent experiments are shown in a–e. Statistical significance was assessed by one-way ANOVA with Tukey’s multiple-comparison test (a, b, d), two-tailed Mann–Whitney test (c) and two-way ANOVA with Tukey’s multiple-comparison test (e). Data are depicted as the mean ± s.e.m. (a–d) or mean ± s.d. (e).

Article Snippet: The following isotype-control antibodies were used: rat IgG2b (clone LTF-2, BioXcell; control for anti-CD4), rat IgG1 (clone TNP6A7, BioXcell; control for anti-CD8β), mouse IgG2a (clone C1.18.4, BioXcell; control for anti-NK1.1) and rat IgG2a (clone 2A3, BioXcell; control for anti-CSF1R).

Techniques: Expressing, Control, Blocking Assay, Derivative Assay, Incubation, Knock-Out, Affinity Purification, Cell Culture, Comparison, Two Tailed Test, MANN-WHITNEY

Journal:

Article Title: Induction of Specific Cytotoxic Lymphocytes in Mice Vaccinated with Brucella abortus RB51

doi: 10.1128/IAI.69.9.5502-5508.2001

Figure Lengend Snippet: Different roles of CD4+ and CD8+ T cells in the cytotoxic lysis and release of IFN-γ. The two T-cell populations were isolated by the MACS magnetic kit and showed more than 92% purity by flow cytometry. The individual T-cell populations were cocultured at various concentrations with either strain RB51-infected or normal J774.A1 cells. The cytotoxic activity (A) and the amount of IFN-γ released into the supernatants (B) were measured. The data are means for triplicate estimations, and standard deviations did not exceed 20% of the means. Cocultures of CD4+ T cells with noninfected target macrophages (solid squares) and RB51-pulsed target macrophages (solid triangles) and CD8+ T cells with noninfected target cells (solid diamonds) and RB51-pulsed target cells (solid circles) were tested.

Article Snippet: Briefly, live T cells isolated by Histopaque column purification were incubated with MACS magnetic MicroBeads to which monoclonal antibodies against CD4 molecule (clone GK1.5; isotype, rat IgG2b) or CD8 molecule (clone 53-6.7; isotype, rat IgG2a) had been coupled (Miltenyi Biotec, Auburn, Calif.) at the concentration of 10 μl of MicroBeads per 10 7 total cells for 15 min in a refrigerator at 4°C (see Fig. ).

Techniques: Lysis, Isolation, Flow Cytometry, Infection, Activity Assay

Journal:

Article Title: Induction of Specific Cytotoxic Lymphocytes in Mice Vaccinated with Brucella abortus RB51

doi: 10.1128/IAI.69.9.5502-5508.2001

Figure Lengend Snippet: Specific phenotype analysis of effector cells by flow cytometry a

Article Snippet: Briefly, live T cells isolated by Histopaque column purification were incubated with MACS magnetic MicroBeads to which monoclonal antibodies against CD4 molecule (clone GK1.5; isotype, rat IgG2b) or CD8 molecule (clone 53-6.7; isotype, rat IgG2a) had been coupled (Miltenyi Biotec, Auburn, Calif.) at the concentration of 10 μl of MicroBeads per 10 7 total cells for 15 min in a refrigerator at 4°C (see Fig. ).

Techniques: Flow Cytometry