fab3791p  (R&D Systems)

Journal: PLoS Pathogens

Article Title: EBV Promotes Human CD8 + NKT Cell Development

doi: 10.1371/journal.ppat.1000915

Figure Lengend Snippet: ( A ) The experimental and analysis scheme for detecting co-receptor-expressing NKT cells and T cells in various reaggregated fetal thymic organ cultures (RTOCs). After 14-days of culture, the various cell types were identified by flow cytometry as in . ( B – D ) Frequency of total NKT cells and total T cells ( B ), co-receptor-expressing NKT cells ( C ) and T cells ( D ), in the different RTOCs. The protocols for the establishment of the RTOCs were described in the leftmost panels in each sub-figure: DP thymocytes were reaggregated with either total thymic stromal cells (DC-included), purified thymic dendritic cells, or BM-derived dendritic cells. The stimuli were added as indicated. Nil, no stimulus; EBV-epitopes, HLA-A2-restricted, derived from the lytic cycle protein BMLF1 and HLA-DRB1-restricted, derived from nuclear antigen EBNA1 (10 µg/ml each); EBV, infectious EBV (10 7 pfu); Solvent, 0.005% polysorbate 20; α-GalCer (0.1 µg/ml). The RTOCs were harvested, and assessed by flow cytometry. Only the data for CD4 + and CD8 + NKT cells, and CD4 SP and CD8 SP T cells were shown. VL, very low level (below detectable level). Data were mean ± s.d. (n = 10). *, p <0.001, EBV-challenged RTOCs vs. non-challenged RTOCs.

Article Snippet: For analysis of co-receptor-expressing NKT cells, single cell suspensions were stained with mAbs to human CD4 and CD8α (R&D Systems, clone 11830 and 37006, isotype mouse IgG 2a and IgG 2b ), unless otherwise noted.

Techniques: Expressing, Flow Cytometry, Purification, Derivative Assay, Solvent

Journal: PLoS Pathogens

Article Title: EBV Promotes Human CD8 + NKT Cell Development

doi: 10.1371/journal.ppat.1000915

Figure Lengend Snippet: ( A ) The experimental and analysis scheme for detecting co-receptor-expressing NKT cells and T cells in various human fetal thymic organ cultures. After 14-day-culture, the various cell types were identified by flow cytometry as in . ( B – D ) Frequency of total NKT cells and total T cells ( B ), co-receptor-expressing NKT cells ( C ) and T cells ( D ) in the different FTOCs. The protocols for the establishment of different FTOCs were described in the leftmost panels in each sub-figure. The various stimuli and blockers were added as indicated. Nil, no stimulus; EBV-epitopes, HLA-A2-restricted, derived from the lytic cycle protein BMLF1 and HLA-DRB1-restricted, derived from nuclear antigen EBNA1 (1 µg/ml each); EBV, infectious EBV (10 7 pfu); Solvent, 0.005% polysorbate 20; α-GalCer (0.1 µg/ml). IL-7 (10 ng/ml); IL-15 (10 ng/ml), Abs, either mouse anti-human IL-7 monoclonal Ab plus mouse anti-human IL-7Rα monoclonal Ab, or mouse anti-human IL-15 monoclonal Ab plus mouse anti-human IL-15Rα monoclonal Ab, respectively (5 µg/ml each); The FTOCs were harvested and assessed by flow cytometry. Only the data for CD4 + and CD8 + NKT cells and CD4 SP and CD8 SP T cells were shown. VL, very low level (below detectable level). Data were mean ± s.d. (n = 10). ** p <0.05. *, p <0.001, EBV-challenged FTOCs vs. non-challenged FTOCs; IL-7-challenged FTOCs vs. non-challenged FTOCs.

Article Snippet: For analysis of co-receptor-expressing NKT cells, single cell suspensions were stained with mAbs to human CD4 and CD8α (R&D Systems, clone 11830 and 37006, isotype mouse IgG 2a and IgG 2b ), unless otherwise noted.

Techniques: Expressing, Flow Cytometry, Derivative Assay, Solvent

Journal: PLoS Pathogens

Article Title: EBV Promotes Human CD8 + NKT Cell Development

doi: 10.1371/journal.ppat.1000915

Figure Lengend Snippet: PBMC were from healthy latent EBV-infected subjects [EBV + (La)], IM patients at year 1 post-onset [EBV + (IMy)], EBV-associated HL patients [EBV + (HL)] and EBV-negative normal control subjects (NS). Thymic cell suspension were from EBV-exposed (EBV + ), un-exposed (EBV − ) or HTLV-1-exposed (HTLV-1 + ) hu-thy/liv-SCID chimeras, or from EBV-exposed (EBV + ) or un-exposed (EBV − ) RTOC and FTOC. For detection of intracellular expression of perforin, cells were stimulated with α-GalCer (1 µg/ml) for 24 hrs, intracellular stained, and assessed by flow cytometry using the experimental strategy shown in the upper panel of each subfigure. The NKT cells were gated by either CD1d tetramers vs. anti-αβTCR mAb (middel panels) or anti-Vα24 mAb vs. 6B11 mAb (bottom panels). Perforin positive cells (%) in gated CD4 + ( A ) and CD8 + ( B ) NKT cells were shon. Solvent for α-GalCer was 0.005% polysorbate 20 (not shown). Nil, negative stimulation control. ND, no determination. Data were mean ± s.d. (n = 8). **, p<0.05; *, p<0.001. CD8 + in ( B ) vs. counterpart CD4 + NKT cells in ( A ).

Article Snippet: For analysis of co-receptor-expressing NKT cells, single cell suspensions were stained with mAbs to human CD4 and CD8α (R&D Systems, clone 11830 and 37006, isotype mouse IgG 2a and IgG 2b ), unless otherwise noted.

Techniques: Infection, Control, Suspension, Expressing, Staining, Flow Cytometry, Solvent

Journal: PLoS Pathogens

Article Title: EBV Promotes Human CD8 + NKT Cell Development

doi: 10.1371/journal.ppat.1000915

Figure Lengend Snippet: ( A ) The experimental and analysis scheme for detecting total and co-receptor CD8α- and CD8β-expressing NKT cells. ( B ) and ( C ) NKT cells in PBMCs from healthy latent EBV-infected subjects [EBV + (La)], IM patients at year 1 post-onset [EBV + (IMy)], EBV-associated HL patients [EBV + (HL)] and EBV-negative normal control subjects (NS) were assessed by flow cytometry using the gate of either CD1d tetramers vs. anti-αβTCR mAb ( B ) or anti-Vα24 mAb vs. 6B11 mAb ( C ). Further dot plot analysis of CD8α vs. CD8β in gated NKT cells was shown. ( D ) and ( E ), NKT cells in thymus (Thy) and liver (Liv) from hu-thy/liv-SCID chimeras challenged i.t. with EBV (EBV + ) or unchallenged (EBV − ) were assessed by flow cytometry using the gate of either CD1d tetramers vs. anti-αβTCR mAb ( D ) or anti-Vα24 mAb vs. 6B11 mAb ( E ). Further dot plot analysis of CD8α vs. CD8β in gated NKT cells was shown. Data were representatives of 5 similar experiments in each group.

Article Snippet: For analysis of co-receptor-expressing NKT cells, single cell suspensions were stained with mAbs to human CD4 and CD8α (R&D Systems, clone 11830 and 37006, isotype mouse IgG 2a and IgG 2b ), unless otherwise noted.

Techniques: Expressing, Infection, Control, Flow Cytometry