Functional comparison of spleen dendritic cells and dendritic cells cultured in vitro from bone marrow precursors

Abstract

We have compared dendritic cells (DC) isolated from mouse spleen, or generated in vitro from bone marrow (BM) precursors cultured in granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), for the ability to process and present soluble antigen and stimulate major histocompatibility complex (MHC) Class II-restricted T cells. DC from spleen or BM cultures were equally able to stimulate the in vitro proliferation of allogeneic T cells or of antigen-specific T-cell receptor (TCR)-transgenic T cells. Both DC populations also induced comparable levels of IL-2 secretion by a T-cell hybridoma. Therefore, splenic and BM-derived DC express comparable levels of (Antigen + MHC Class II) ligands and/or costimulatory molecules and have comparable ability to stimulate T-cell responses. When presentation of a native protein antigen, rather than peptide, was evaluated, BM-derived DC were at least 50 times better than splenic DC at stimulating the proliferation of TCR-transgenic T cells. The antigen processing ability of the two populations was similar only when splenic DC were used immediately ex vivo. Therefore, unlike spleen DC, BM-derived DC maintain the capacity to process protein antigen for MHC Class II presentation during in vitro culture. Due to these characteristics, BM-derived DC may represent a useful tool in immunotherapy studies, as they combine high T-cell stimulatory properties with the capacity to process and present native antigen.

Chitosan hydrogel vaccine generates protective CD8 T cell memory against mouse melanoma

“…Bone marrow harvested from the leg bones of naive C57BL/6 mice was cultured for 7 days in RPMI (Invitrogen, Carlsbad, CA, USA) containing 5% FCS (PAA Laboratories, Pasching, Austria), 1% Pen/Strep (Invitrogen), 0.1% 2ME (Invitrogen) and 20 ng ml -1 GM-CSF (MyBiosource, San Diego, CA, USA) as described. 45 For OVA vaccinations, cells were pulsed with OVA protein overnight on day 5 at a final concentration of 200 μg ml -1 followed by 1 μg ml -1 of lipopolysaccharide (Sigma) overnight on day 6. For TRP2 180-188 vaccination, cells were cultured with 1 ug ml -1 lipopolysaccharide overnight on day 6 and pulsed for 3 h with TRP2 180-188 peptide (Mimotopes, Clayton, Vic Australia) on day 7.…”

The phenotype of type 1 and type 2 CD8+ T cells activated in vitro is affected by culture conditions and correlates with effector activity

“…Bone marrow cells from C57BL/6 J mice were cultured in medium containing 20 ng/ml IL-4 and 20 ng/ml granulocyte-macrophage colony-stimulating factor as described. 10 DC were harvested on day 7, and 1 · 10 4 cells were cultured in 24-well plates with 1 · 10 6 purified CD8 + T cells prepared as described above, and 0Á01 lm LCMV 33)41 . Cultures were supplemented with 20 U/ml rhIL-2, 10 ng/ml rhIL-6 and, for Tc2 cultures, 10 ng/ml rmIL-4.…”

Rat bone marrow‐derived dendritic cells, but not ex vivo dendritic cells, secrete nitric oxide and can inhibit T‐cell proliferation

“…Relatively few studies have attempted to compare DC generated in vitro with those obtained ex vivo and, where this has been attempted, the comparisons have been limited. 12,22 In the experiments described in the present report we have generated DC from rat bone marrow using granulocyte±macrophage colony-stimulating factor (GM±CSF) and interleukin (IL)-4 [bone marrow-derived DC (BMDC)], and have compared their phenotypic and functional properties with DC obtained ex vivo from spleen (SDC) and pseudo-afferent intestinal lymph (LDC).…”