The expression and the regulatory role of OX40 and 4-1BB heterodimer in activated human T cells by Bruce Y. Ma, Sebastian A. Mikolajczak, Ali Danesh, Karoline A. Hosiawa, Cheryl M. Cameron, Akifumi Takaori-Kondo, Takashi Uchiyama, David J. Kelvin, and Atsuo Ochi Blood Volume 106(6):2002-2010 September 15, 2005 ©2005 by American Society of Hematology

Induction of OX40+4-1BB+ cells in CD3- and CD28-stimulated PBL T cells. Induction of OX40+4-1BB+ cells in CD3- and CD28-stimulated PBL T cells. In the left panel, total PBL was stimulated with anti-CD3 Ab (OKT3; 1 μg/mL) and anti-CD28 Ab (1 μg/mL) for 3 days before 3-color flow cytometry specific for OX40, 4-1BB, and CD4 or CD8. Right panels show the data for PBL cells without mitogenic stimulation. R3 quadrants in the dot plot indicate cells expressing OX40 gated for CD4+ or CD8+ T cells. Histograms indicate cells positive for 4-1BB gated in quadrant R3. The proportion of OX40+4-1BB+ subsets gated for stimulated CD4+ or CD8+ T cells is indicated in the top portion of the histograms. Bruce Y. Ma et al. Blood 2005;106:2002-2010 ©2005 by American Society of Hematology

Confocal study of OX40 and 4-1BB on activated T cells and transfectant cells. Confocal study of OX40 and 4-1BB on activated T cells and transfectant cells. (A) OX40 and 4-1BB colocalize on activated T cells. Mitogen-stimulated total T cells were stained for OX40 (green), 4-1BB (red), or CD27 (red) and analyzed by confocal microscopy. In the top 3 panels, stimulated PBLs were stained with OX40 and 4-1BB. In the right end panel, green and red fluorescence overlapped and cells exhibiting colocalization of OX40 and 4-1BB are indicated by arrows. In Ai-ii, cells positive for OX40 and 4-1BB staining (i) and cells positive for OX40 and CD27 (negative control) staining (ii) were analyzed at higher magnification for colocalization. (B) OX40 and 4-1BB colocalize in gene-transfected HEK293 cells. HEK293 cells were gene-transfected with OX40-GFP and 4-1BB-RFP or CD40L-RFP for 16 hours before confocal microscopy. In the right end panels, green and red fluorescence were overlapped to show colocalization of receptors. (C) OX40 and 4-1BB coendocytose following cross-linking to 4-1BB. HEK293 cells were gene-transfected with OX40-GFP and 4-1BB–RFP for 16 hours. Cells were treated for 30 minutes with anti–4-1BB Ab (1 μg/mL) at 4°C, 37°C, or at 37°C in the presence of sucrose (0.45 M), which prevented clathrin-dependent endocytosis.27 In the right end panels, green and red fluorescences were overlapped to emphasize the coendocytosis of receptors. Bruce Y. Ma et al. Blood 2005;106:2002-2010 ©2005 by American Society of Hematology

Heterocomplex formation between OX40 and 4-1BB in gene-transfectant cells and T-cell blasts. Heterocomplex formation between OX40 and 4-1BB in gene-transfectant cells and T-cell blasts. (A) OX40 and 4-1BB coimmunoprecipitate from cell extracts derived from HEK293 transfectants. HEK293 cells were transfected with OX40-HA (full-length HA-tagged OX40) and 4-1BB-FLAG (full-length FLAG-tagged 4-1BB) as indicated on the top of the figure. One percent NP-40 cell extracts were immunoprecipitated with anti-FLAG (for 4-1BB immunoprecipitation) or anti-HA (for OX40 immunoprecipitation), then analyzed by HA-specific or FLAG-specific Western blotting as indicated. Lanes e-f show total cell lysates of OX40-HA–transfected HEK293 or nontransfected HEK293. Positions of OX40-HA and 4-1BB–FLAG are indicated by arrows on the right. (B) OX40 and 4-1BB coprecipitate by OX40L-GST or 4-1BB–GST from cell extracts of gene-transfectant cells. HEK293 cells were transfected with OX40-HA and 4-1BB–FLAG as indicated on the top of the figure. One percent NP-40 cell extracts were subjected to OX40-GST–or 4-1BB-GST–specific precipitation then analyzed by HA-specific Western blotting (WB). Lane a, precipitated by GST beads; lane b, precipitated by 4-1BBL–GST; lane c, precipitated by OX40L-GST; lane d, precipitated by 4-1BBL–GST; lane e, precipitated by OX40L-GST; and lane f, precipitated by GST. Position of OX40-HA is indicated by the arrow on the right. Bruce Y. Ma et al. Blood 2005;106:2002-2010 ©2005 by American Society of Hematology

Detection of OX40 and 4-1BB heterodimer by SDS-PAGE under a nonreduced condition. Detection of OX40 and 4-1BB heterodimer by SDS-PAGE under a nonreduced condition. (A) HEK293 cells were transfected with OX40-HA and 4-1BB–FLAG as indicated on the top of figure. One percent NP-40 cell extracts were characterized by HA- or FLAG-specific Western blotting under reduced or nonreduced conditions. In subpanels i-ii, monomer of OX40 (∼48 kDa) is indicated by filled arrows. In subpanels ii and iv, presumed dimer of OX40 (∼96 kDa) and dimer of 4-1BB (∼60 kDa) are indicated by open arrows. Blots for presumed heterodimer (∼78 kDa) and the dimer of heterodimer (∼160 kDa) were observed in both HA-specific and FLAG-specific Western blots and indicated at right side in subpanels ii and iv. (B) The OX40-HA and 4-1BB–FLAG double-transfectant HEK cell extracts were immunoprecipitated with Abs specific for HA, FLAG, OX40, 4-1BB, or isotype control (indicated below panels). Immunoprecipitates were characterized by OX40, 4-1BB, HA-tag–or FLAG-tag–specific Western blotting under nonreduced conditions. Blots for presumed heterodimer (∼78 kDa) were observed in both OX40-HA–specific and 4-1BB-FLAG–specific Western blotting and indicated at the right side of each panel. Bruce Y. Ma et al. Blood 2005;106:2002-2010 ©2005 by American Society of Hematology

Presence of OX40/4-1BB complex in cell lysate of PBL T-cell blasts. Presence of OX40/4-1BB complex in cell lysate of PBL T-cell blasts. Total T cells were stimulated with anti-CD3 Ab (OKT3; 1 μg/mL) and anti-CD28 Ab (1 μg/mL) for 3 days. One percent NP-40 cell extracts were immunoprecipitated with anti-OX40 Ab or anti-4-1BB Ab, then analyzed by OX40-specific or 4-1BB-specific Western blotting in a reduced condition. Immunoprecipitation Abs or control Ab is indicated on the top of each panel. H indicates the position of immunoglobulin heavy chain; L, position of immunoglobulin light chain. The arrow in the top panel indicates the position of OX40 and the arrow in the bottom panel indicates the position of 4-1BB. Bruce Y. Ma et al. Blood 2005;106:2002-2010 ©2005 by American Society of Hematology

The association of OX40/4-1BB heterodimer with TRAF proteins. The association of OX40/4-1BB heterodimer with TRAF proteins. HEK293 cells were transfected with HA-tagged TRAF1, 2, 3, 4, or 5 for 16 hours as indicated in the top of each panel. Cells were concomitantly transfected with OX40-HA and 4-1BB–FLAG or both as indicated on the left of each panel. One percent NP-40 cell extracts were immunoprecipitated with anti-OX40 Ab in panel A and immunoprecipitated with anti–4-1BB Ab in panels B-C. Immunoprecipitates were analyzed in a reduced condition by SDS-PAGE and Western blotting specific for HA or FLAG. In panel A, positions of TRAF proteins are indicated by open arrows. Bruce Y. Ma et al. Blood 2005;106:2002-2010 ©2005 by American Society of Hematology

Reduced induction of NF-κB activation by OX40/4-1BB double-transfectant cells stimulated with specific Abs. Reduced induction of NF-κB activation by OX40/4-1BB double-transfectant cells stimulated with specific Abs. OX40-HA and 4-1BB–FLAG double-transfectant or nontransfectant HEK cells were stimulated by anti-OX40 (1 μg/mL), anti–4-1BB (1 μg/mL), or control Ab (1 μg/mL) as indicated. □, nonstimulated cells; ▪, cells stimulated by control or specific Abs. The value of NF-κB reporter gene only transfected cells (the top group of cells indicated as “None” in the left) was set as 100 to calculate relative luciferase activity. Each column indicates an average and SD of triplicate samples. Bruce Y. Ma et al. Blood 2005;106:2002-2010 ©2005 by American Society of Hematology

TNF-α–induced apoptosis of 4-1BB/OX40-transfected cells. TNF-α–induced apoptosis of 4-1BB/OX40-transfected cells. HEK293 cells were transfected with OX40-HA or 4-1BB–FLAG or both or control pcDNA3 as indicated on the left. Sixteen hours later cells were stimulated with TNF-α in the presence and absence of Abs against OX40 or 4-1BB. Fifteen hours later, cells were stained with FITC–annexin V to determine the proportion of cells undergoing apoptosis (indicated as percent in the panels of flow cytometry results). Alternatively, cell extracts were analyzed by SDS-PAGE for PARP-specific Western blotting that detects native PARP (115 kDa, shown as the top bands in panels on right) and bottom bands (89 kDa) indicated by arrows resulting from the caspase cleavages. Bruce Y. Ma et al. Blood 2005;106:2002-2010 ©2005 by American Society of Hematology

Cell growth inhibition of CD25+CD4+ T cells and CD8+ T cells by soluble OX40L and 4-1BBL. Cell growth inhibition of CD25+CD4+ T cells and CD8+ T cells by soluble OX40L and 4-1BBL. (A) CD4+ T cells were stimulated with anti-CD3 Ab (1 μg/mL) and anti-CD28 Ab (1 μg/mL) for 4 days. CD25+ cells were positively purified by anti-CD25 Ab and cell separation kit (StemCell Technologies). Enriched (∼90%) CD25+CD4+ cells were cultured with anti-CD3 Ab (1 μg/mL), anti-CD28 Ab (1 μg/mL), and IL-2 (50 U/mL). Cells were then stimulated with OX40L-GST (5 μg/mL) or 4-1BBL–GST (5 μg/mL) or both, as indicated. When cells were stimulated by one ligand only, additional GST (5 μg/mL) was added. After 3 days of stimulation [3H]thymidine incorporation assay (16 hours of pulsing) was performed. Data indicate the average and the SD of triplicate samples. Two results obtained from independent experiments are shown. (B) CD8+ T cells were stimulated with anti-CD3 Ab (1 μg/mL) and anti-CD28 Ab (1 μg/mL) for 4 days. These preactivated cells were cultured with anti-CD3 Ab (1 μg/mL) and IL-2 (50 U/mL). Cells were simultaneously then stimulated with OX40L-GST (5 μg/mL) or 4-1BBL-GST (5 μg/mL) or both, as indicated. When cells were stimulated by one ligand only, additional GST (5 μg/mL) was added. After 3 days of stimulation, [3H]thymidine incorporation assay (16 hours of pulsing) was performed. Data indicate the average and the SD of triplicate samples. The result represents 3 experiments with similar results. Bruce Y. Ma et al. Blood 2005;106:2002-2010 ©2005 by American Society of Hematology