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: PLoS ONE

Article Title: A Comparative Study of N -glycolylneuraminic Acid (Neu5Gc) and Cytotoxic T Cell (CT) Carbohydrate Expression in Normal and Dystrophin-Deficient Dog and Human Skeletal Muscle

doi: 10.1371/journal.pone.0088226

Figure Lengend Snippet: GRMD muscle (cranial sartorius) was triple stained for Neu5Gc (green), Pax7 (A), CD11b (B), CD4 (C), CD8 (D) (all red), and DAPI (blue). Arrows indicated co-staining of Neu5Gc and Pax7 (in A), CD11b (in B) or CD4 (in C). Bar is 100 µm (A) and 50 µm (B–D) for all panels.

Article Snippet: Co-staining antibodies used in dog were mouse anti-chicken Pax7 (Developmental Studies Hybridoma Bank, clone P3U1), mouse anti-dog CD4 (AbD Serotec, MCA1998S), rat anti-dog CD8 (AbD Serotec, MCA1039GA), mouse anti-dog CD11b (AbD Serotec, MCA1777S), mouse anti-dog CD21 (AbD Serotec, MCA1781R), mouse anti-human β spectrin (Novus, NB300-574) or mouse anti-rat embryonic myosin (NovaCastra, NCL-MHCd).

Techniques: Staining

Journal: PLoS ONE

Article Title: A Comparative Study of N -glycolylneuraminic Acid (Neu5Gc) and Cytotoxic T Cell (CT) Carbohydrate Expression in Normal and Dystrophin-Deficient Dog and Human Skeletal Muscle

doi: 10.1371/journal.pone.0088226

Figure Lengend Snippet: (A) Cranial sartorius muscle sections from GR and GRMD dogs were stained with markers for satellite cells (Pax7), T lymphocytes (CD4 or CD8) or macrophages (CD11b) and quantified for numbers of cells stained per 40X visual field. (B) The percentage of cells co-stained for Neu5Gc and CD4, CD8, CD11b or Pax7 in GRMD muscles was quantified. Errors are SEM. ***P<0.001, for each GR vs. GRMD comparison in A.

Article Snippet: Co-staining antibodies used in dog were mouse anti-chicken Pax7 (Developmental Studies Hybridoma Bank, clone P3U1), mouse anti-dog CD4 (AbD Serotec, MCA1998S), rat anti-dog CD8 (AbD Serotec, MCA1039GA), mouse anti-dog CD11b (AbD Serotec, MCA1777S), mouse anti-dog CD21 (AbD Serotec, MCA1781R), mouse anti-human β spectrin (Novus, NB300-574) or mouse anti-rat embryonic myosin (NovaCastra, NCL-MHCd).

Techniques: Staining, Muscles, Comparison

Journal: Kidney international

Article Title: Role of thymic- and graft-dependent mechanisms in tolerance induction to rat kidney transplant by donor PBMC infusion.

doi: 10.1038/sj.ki.5002202

Figure Lengend Snippet: Figure 3 | Immunofluorescence staining for CD4 þFoxp3 þ T cells in rat kidney allografts. Representative microphotographs of staining for CD4 (phycoerythrin-conjugated anti-rat CD4mAb, digitally converted in green to increase visibility) and Foxp3 (shown in red, anti-rat Foxp3mAb and Cy5-conjugated goat anti-rabbit as secondary antibody) on kidney tissue from BN-PBMC-infused rats killed (a) 5 days and (b) 60 days post-transplant and from an untreated rat killed 5 days post-transplant (c) Original magnification 400. Interstitial CD4 þ T cell infiltrates are shown. In kidney grafts from BN-PBMC rats (both at 5 and 60 days post-transplant), several CD4 þ T cells coexpress Foxp3 (indicated by arrows), whereas in the rejected kidney graft from untreated rats very few Foxp3 þ cells are present.

Article Snippet: Cells were stained with mouse anti-rat CD4, mouse anti-rat CD8 (OX-8), mouse anti-rat CD25, and mouse anti-rat MHCII (OX-6) mAbs (Serotec) followed by fluorescein isothiocyanate–goat antimouse immunoglobulin (Pharmingen, San Diego, CA, USA) as secondary antibody.

Techniques: Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: Effects of Inhibiting Dipeptidyl Peptidase-4 (DPP4) in Cows with Subclinical Ketosis

doi: 10.1371/journal.pone.0136078

Figure Lengend Snippet: Effects of dipeptidyl peptidase IV (DPP4) inhibition via BI 14332 to relative numbers of CD4 + and CD8 + T-lymphocytes (LSmean ± SE).

Article Snippet: Samples were double stained with monoclonal antibodies for CD4 + (mouse anti bovine CD4:FITC) and CD8 + (mouse anti bovine CD8:RPE) or the corresponding isotype controls (mouse IgG2a negative control: RPE and mouse IgG2b negative control: FITC; all AbD Serotec).

Techniques: Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Pathological Analysis of Ocular Lesions in a Murine Model of Sjögren’s Syndrome

doi: 10.3390/ijms18061209

Figure Lengend Snippet: Immune cell infiltration of target organs of the SS model mice. CD4, CD8, CD19, and F4/80 expressing immune cells were observed by confocal microscopy. ( a ) CD4 + T cells, ( b ) CD8 + T cells, ( c ) CD19 + B cells, and ( d ) F4/80 + macrophages in the lacrimal glands of the SS model mice were detected by Alexa-546-conjugated antibodies. Nuclei were stained with 4′,6-diamdino-2-phenylindole dihydrochloride (DAPI). The results are representative of five mice. Scale bar: 50 μm; ( e ) Flow cytometry analysis using lacrimal gland tissues from the SS model mice. CD4 and CD8 T cell subsets ware shown, and the result is representative of 5 mice; and ( f ) Cell numbers of CD4 + , CD8 + T cells, CD19 + B cells, and F4/8 + macrophages were calculated by flow cytometery analysis. Data are means ± SD of 5 mice.

Article Snippet: Lymphocytes from lacrimal gland tissues were stained with antibodies against PE-Cy7-conjugated anti-mouse CD4 mAb (TONBO Biosciences, San Diego, CA, USA), fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD8 mAb (eBioscience, San Diego, CA, USA), PE-Cy5.5-conjugated anti-mouse CD19 mAb (TONBO Biosciences), and APC-Cy7-conjugated anti-mouse F4/80 mAb (BioLegend, San Diego, CA, USA).

Techniques: Expressing, Confocal Microscopy, Staining, Flow Cytometry