1. Calcium Ionophore-Treated Myeloid Cells Acquire Many Dendritic Cell Characteristics Independent of Prior Differentiation State, Transformation Status, or Sensitivity to Biologic Agents
by Gary K. Koski, Gretchen N. Schwartz, David E. Weng, Ronald E. Gress, Friederike H.C. Engels, Maria Tsokos, Brian J. Czerniecki, and Peter A. Cohen
Blood
Volume 94(4):
August 15, 1999
©1999 by American Society of Hematology

2. Time-course analysis of A23187-inducible surface Ag expression.
Time-course analysis of A23187-inducible surface Ag expression. HL-60 cells (5 × 105) were cultured in a final volume of 2 mL with or without an optimal dose (180 ng/mL) of A23187 as described in Materials and Methods. Cells were harvested at 20, 48, and 96 hours post addition of A23187 and stained with PE-conjugated mouse-antihuman CD83, B7.1, B7.2, ICAM-1, CD40, CD1a, CD33, or IgG subclass-matched control Ab, and analyzed by FACS as described in Materials and Methods. Results displayed are at 20 hours without A23187 treatment (UNTREATED), 20 hours with A23187 treatment (20H TREATMENT), 48 hours with A23187 treatment (48H TREATMENT), and 96 hours with A23187 treatment (96H TREATMENT). Cultures not treated with A23187 showed an identical immunophenotypic profile at 20, 48, and 96 hours (not shown). Histograms display staining intensity of viable (PI-excluding) cells, comparing control Ab (light lines) to specific Ab (heavy lines).
Gary K. Koski et al. Blood 1999;94:
©1999 by American Society of Hematology

3. A23187-treated HL-60 cells display progressive acquisition of dendritic processes.
A23187-treated HL-60 cells display progressive acquisition of dendritic processes. HL-60 cells (1 × 106) were cultured in a final volume of 2 mL with or without an optimal dose of A23187 (180 ng/mL) for up to 96 hours. Cells were harvested at 24 or 96 hours, resuspended in fresh medium, transferred to polylysine-coated glass chamber slides, and incubated an additional 20 minutes at 37°C. Cells were either immediately examined microscopically (600×) and photographed using DIC (Nomarski) optics (A, B, and C), or were fixed in 100% ethanol, stained in Wright’s solution, and then photographed (D and E). Photomicrographs A, B, and C were taken at consecutive 30-second intervals and depict living HL-60 cells treated with A23187 for 20 hours. Note motility of dendritic processes. Photomicrographs D and E depict fixed, stained, HL-60 cells previously left untreated (D) or treated with ionophore for 96 hours (E).
Gary K. Koski et al. Blood 1999;94:
©1999 by American Society of Hematology

4. Effects of rhIFN-γ and rhGM-CSF on A23187-induced HL-60 surface Ag expression.
Effects of rhIFN-γ and rhGM-CSF on A23187-induced HL-60 surface Ag expression. HL-60 cells (5 × 105) were cultured in a final volume of 2 mL with or without an optimal dose (180 ng/mL) of CI A23187, rhIFN-γ (1000 U/mL), or rhGM-CSF (20 ng/mL). Cells were harvested 72 hours later, stained with PE-conjugated mouse antihuman B7.2, HLA-DR, HLA-ABC, CD40, CD1a, CD83, or IgG subclass-matched control Ab, and analyzed by FACS as described in Materials and Methods. Cell viabilities (percent) at time of analysis (72 hours of treatment) are indicated below histograms for each treatment group. Histograms display staining intensity of viable (PI-excluding) cells, comparing control Ab (light lines) to specific Ab (heavy lines).
Gary K. Koski et al. Blood 1999;94:
©1999 by American Society of Hematology

5. Treatment with CI A23187 or A23187 with adjunct cytokines enhances T-cell allosensitizing capacity of HL-60 cells.
Treatment with CI A23187 or A23187 with adjunct cytokines enhances T-cell allosensitizing capacity of HL-60 cells. HL-60 cells (5 × 105/well) were cultured in 24-well plates in 2 mL CM (Untreated) or CM supplemented with rhGM-CSF (20 ng/mL, GM-CSF), rhIFN-γ (1000 U/mL, IFN-γ) or combined rhGM-CSF plus rhIFN-γ, each of these conditions with or without CI A23187 (180 ng/mL). HL-60 cells were harvested 72 hours later, washed three times in CM, γ-irradiated (30 Gy), and cocultured at various HL-60:T-cell ratios with freshly prepared allogeneic human T lymphocytes (1 × 105/well) in triplicate 96-well tissue culture plate wells in 200 μL final volume CM. Cells were maintained in culture for 96 hours, pulsed with 1 μCi [3H]-TdR, harvested 18 hours later, and [3H]-TdR incorporation assessed by liquid scintillation spectrometry. Ordinate displays [3H]-TdR incorporation as thousands of CPM per well during the final 18-hour culture period; abscissa displays ratio of APC:T cells. Not shown: irradiated HL-60 cells alone, regardless of treatment, generated < 750 cpm when seeded at 20,000 cells per well; T cells alone generated < 250 cpm. Bars indicate SEM from triplicate wells.
Gary K. Koski et al. Blood 1999;94:
©1999 by American Society of Hematology

6. Cultured CD34pos bone marrow cells acquire the capacity to form dendritic processes in response to treatment with A23187 or rhCD40L.
Cultured CD34pos bone marrow cells acquire the capacity to form dendritic processes in response to treatment with A23187 or rhCD40L. Cultures of CD34pos bone marrow cells initially expanded for 6 days in the presence of rh-c-kit ligand, rhGM-CSF, and rhTNF- to induce numerical expansion. Cells were then centrifuged and replated in identical fresh medium except for the absence of rh-c-kit ligand, with individual wells also receiving CI A23187 or rhCD40L. Twenty hours after replating, cells were harvested and centrifuged, resuspended in fresh medium and transferrred onto polylysine-coated glass chamber slides, incubated for an additional 20 minutes at 37oC, and photomicrographed (600×) using DIC (Nomarski) optics. Photomicrographs are representative of multiple CI and CD40L doses tested in two separate experiments: untreated cells (A); cells treated for 20 hours with 375 ng/mL CI A23187 (B); cells treated for 20 hours with 10 μg/mL rhCD40L (C). Note dendritic processes exhibited on cells in panels B and C that are largely absent in A. Similar results were observed after CI treatment at 180 ng or 750 ng/mL or rhCD40L at 2.5 or 40 μg/mL (not shown).
Gary K. Koski et al. Blood 1999;94:
©1999 by American Society of Hematology

7. Alterations in immunophenotype of cultured human CD34pos bone marrow cells and peripheral blood monocytes in the presence of CI A23187 or rhCD40L.
Alterations in immunophenotype of cultured human CD34pos bone marrow cells and peripheral blood monocytes in the presence of CI A23187 or rhCD40L. Cultures of CD34posbone marrow cells previously expanded for 6 days in the presence of cytokines (see Fig 5) were replated and cultured for an additional 20 hours in the same medium without rh-c-kit ligand with various doses of CI A23187 or rhCD40L added. In synchronous experiments, elutriated monocytes were washed and plated overnight as described in Materials and Methods, then cultured for an additional 20 hours with no additive or CI A23187 or rhCD40L. After the 20-hour treatment bone marrow progenitors and monocytes were harvested and stained with PE-conjugated antibodies against human B7.1, B7.2, and CD83 and analyzed by FACS (see Materials and Methods). Representative displayed data depict untreated cells, cells treated with an optimal dose of CI (375 ng/mL for CD34pos cells and 225 ng/mL for monocytes), or cells treated with 10 μg/mL rhCD40L. Similar results were observed in either cultured CD34pos progenitors or peripheral blood monocytes treated with 2.5 or 40 μg/mL doses of rhCD40L (not shown).
Gary K. Koski et al. Blood 1999;94:
©1999 by American Society of Hematology

8. Dose-response analyses of immunophenotypic modulations of cultured CD34pos bone marrow cells during CI A23187 treatment.
Dose-response analyses of immunophenotypic modulations of cultured CD34pos bone marrow cells during CI A23187 treatment. Six-day cultured CD34pos myeloid cells enriched from normal volunteer bone marrow were cultured in medium containing rhGM-CSF and rhTNF- with graded doses of CI A23187 (0 to 750 ng/mL). Cells were harvested and analyzed by flow cytometry with added propidium iodide (PI) as described in Fig 6 and Methods. Abscissa displays ng/mL of A23187 present during CI treatment. Two or three separate dose-response experiments were performed and plotted for each cell surface marker analyzed (B7.1, B7.2, CD83, ICAM-1, HLA-DR, CD1a, and CD14). Also portrayed is the viable cell recovery relative to control for three dose-response experiments (0 ng/mL group = 100%). Cell surface Ag expression data are portrayed as Specific Mean Fluorescence quotients (mean fluorescent intensity of cells stained with specific Ab/mean fluorescent intensity of cells stained with subclass-matched control Ab); a minimum of 5000 cells with background-level PI staining was assayed per determination.
Gary K. Koski et al. Blood 1999;94:
©1999 by American Society of Hematology

9. Treatment with CI A23187 enhances T-cell allosensitizing capacity of cultured CD34pos bone marrow cells.
Treatment with CI A23187 enhances T-cell allosensitizing capacity of cultured CD34pos bone marrow cells. The latter were treated and harvested as in Fig 6, then cocultured at various APC:T lymphocyte ratios with fixed numbers of freshly prepared allogeneic human T lymphocytes as in Fig 4. Ordinate displays CPM/105 input T cells during the last 18 hours in culture. Groups are “Untreated” (continued culture in rhGM-CSF and rhTNF-), “CD40L” (10 μg/mL rhCD40L) and “A23187” (375 ng/mL). Abscissa displays ratio of APC: T cells. Not shown: CD34pos cells alone, regardless of treatment, generated < 250 cpm when seeded at 20,000 cells per well; T cells alone generated < 250 cpm. Bars indicate SEM from triplicate wells.
Gary K. Koski et al. Blood 1999;94:
©1999 by American Society of Hematology