Competent antigen-presenting cells are generated from the long-term culture of splenocytes with granulocyte-macrophage colony-stimulating factor

Extended Culture of Bone Marrow with Granulocyte Macrophage-Colony Stimulating Factor Generates Immunosuppressive Cells

“…Every 4 wk or so, we compared the capacity to stimulate allogeneic T cells from BALB/c mice in MLR between the 1-wk cultured BM cells and the long-term cultured BM cells. Similarly to our previous study ( 19 ), non-adherent or loosely-attached cells in the 4-wk old BM culture were found less efficient to stimulate allogeneic T cells than those in the 1-wk old BM culture ( Fig. 1A ).…”

“…In our previous study ( 19 ), we found that BM-DCs isolated from 3- or 4-wk old BM cultures were much less efficient to stimulate T cells than BM-DCs from 1-wk old BM culture. To follow up on our previous finding that BM-DCs lose their capacity to induce the proliferation of allogeneic T cells in MLR after the extended culture with GM-CSF ( 19 ), we set out to examine the culture of BM cells from C57BL/6 mice for lengthy periods. To sustain and examine the extended culture of BM cells, non-adherent or loosely-attached cells were removed from tissue culture plates every 1 or 2 wk and only the remaining adherent cells were maintained in culture medium containing GM-CSF.…”

“…Non-adherent or loosely-attached BM cells in the 1-wk culture with GM-CSF are composed of heterogeneous populations including a large majority of CD11c + CD11b + MHC II int/lo macrophages and a relative smaller number of CD11c + CD11b + MHC II hi DCs ( 19 20 21 ). Both of the BM cells cultured with GM-CSF for 1 wk as well as for 4 wk exhibited heterogeneous populations with regard to their surface level of MHC II ( Fig.…”

Two Distinct Subsets Are Identified from the Peritoneal Myeloid Mononuclear Cells Expressing both CD11c and CD115

“…Then, CFSE or CTV labeled T cells were added into round bottom 96-well plates at 2.5×10 4 /well and mixed with isolated APCs at an indicated APC: T cell ratio in the media containing 57.2 μM β-mercaptoethanol (Sigma-Aldrich). After co-culture with APCs for 3–4 days, the proliferation of live T cells was evaluated by the dilution of CFSE or CTV (626). For in vitro T-cell polarization assays, the mixture of APCs and T cells (APC:T cell = 1:10) included additional cytokines, neutralizing Abs, and reagents as follows: Th0 (media alone), Th1 (1 μg/ml LPS) (27), Th2 (10 ng/ml IL-4) (2829), Th17 (3 ng/ml TGF-β, 20 ng/ml IL-6) (30), iTreg (3 ng/ml TGF-β, 1 nM all-trans retinoic acid (ATRA); (3132), type 0 cytotoxic T cell (Tc0) (media alone), Tc1 (1 μg/ml LPS) (33), Tc2 (10 μg/ml anti-IFN-γ, 20 ng/ml IL-4) (33), Tc17 (10 μg/ml anti-IFN-γ, 5 ng/ml TGF- β, 20 ng/ml IL-6) (33).…”

“…Macrophages also promote the resolution of inflammatory process by inducing angiogenesis and tissue repair. Macrophages and DCs are morphologically different; adherent macrophages display large irregular shapes while non-adherent DCs exhibit long probing processes or dendrites (6).…”