Tonsil Stromal-Cell Lines Expressing FDC-Like Properties: Isolation, Characterization, and Interaction with B Lymphocytes

Journal of Immunology Research, Aug 2018

The microenvironment of secondary lymphoid organs consists of two major populations of cells, the lymphoid cells and a population of stromal cells that contribute to both tissue architecture and function. Interactions of both populations are essential for the development and control of humoral immune responses. In this study, stromal-cell preparations were obtained by a multistage process. This involved culturing 300-400-μm slices of human tonsil for 6-8 days at 25°C, trypsin digestion of the residual explant, followed by CD45-positive-cell depletion using magnetic beads, and a final period of culture for 4 days to remove remaining nonadherent cells. Phenotypifig with a panel of monoclonal antibodies revealed that the cells express HLA-DR, CD54 (ICAM-1), CD44, but no CD45 nor a range of other markers for epithelial and endothelial cells. Immunoassays of supernatants from stromal cells revealed that IL-6 was produced constitutively, and its production was increased by treatment with TNF-α and IFN-γ. In contrast IL-1, IL-2, IL-4, IL-7, IL-8, IL-10, IL-12, TNF-α, and IFNγ were not produced. Functional tests showed that these cells express follicular dendritic cell-like properties. Coculturing of tonsilar B cells with stromal cells resulted in enhanced proliferation and also led to increased production of immunoglobulins and IL-6, suggesting crucial signaling between these populations.

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Tonsil Stromal-Cell Lines Expressing FDC-Like Properties: Isolation, Characterization, and Interaction with B Lymphocytes

Journal of Tonsil Stromal-Cell Lines Expressing FDC-Like Properties: Isolation, Characterization, and Interaction with B Lymphocytes GRZEGORZ SKIBINSKI 0 ANNA SKIBINSKA 0 MARTINE DECKERS 0 KEITH JAMES 0 0 Lister Research Laboratories, Department of Surgery, University of Edinburgh, Royal Infirmary , Lauriston Place, Edinburgh EH3 9YW, Scotland , UK The microenvironment of secondary lymphoid organs consists of two major populations of cells, the lymphoid cells and a population of stromal cells that contribute to both tissue architecture and function. Interactions of both populations are essential for the development and control of humoral immune responses. In this study, stromal-cell preparations were obtained by a multistage process. This involved culturing 300-400-/zm slices of human tonsil for 6-8 days at 25C, trypsin digestion of the residual explant, followed by CD45-positive-cell depletion using magnetic beads, and a final period of culture for 4 days to remove remaining nonadherent cells. Phenotypifig with a panel of monoclonal antibodies revealed that the cells express HLA-DR, CD54 (ICAM-1), CD44, but no CD45 nor a range of other markers for epithelial and endothelial cells. Immunoassays of supernatants from stromal cells revealed that IL-6 was produced constitutively, and its production was increased by treatment with TNF-ce and IFN-y. In contrast IL-1, IL-2, IL-4, IL-7, IL-8, IL-10, IL-12, TNF-ce, and IFN y were not produced. Functional tests showed that these cells express follicular dendritic cell-like properties. Coculturing of tonsilar B cells with stromal cells resulted in enhanced proliferation and also led to increased production of immunoglobulins and IL-6, suggesting crucial signaling between these populations. FDC-like cell lines; FDC/B lymphocyte interactions; human tonsil INTRODUCTION During antigen-specific T-cell-dependent immune al., 1990; M611er, 1992). This maturation process is highly controlled and depends on numerous interactions with interdigidating dendritic cells, T cells, responses in secondary lymphoid tissues, antigenand stromal cells, including highly specialized follispecific naive B cells undergo a series of events cular dendritic cells (Kosco and Gray, 1992). The including activation, expansion, somatic mutations, crosstalk between B-cell subsets and their microisotype switch, selection, and differentiation into environment is controlled by membrane/ligand recepeither antibody-secreting or memory cells (Kroese et tor interactions (MacLennan, 1994) and signals from *Corresponding author. the cytokine network (Banchereau and Rousset, 1992). Most of the studies on follicular dendritic cells in vitro have been hampered by the fact that it is very difficult to isolate pure FDC with reasonable yields. Tsunoda et al. (1990) first reported isolation and maintenance of human FDC in long-term culture; these cells, however, did not grow in vitro. Lindhout et al. (1994) obtained EBV-transformed human FDC lines, but their long doubling time makes their cultivation in vitro difficult. Clark et al. (1995) and Kim et al. (1995) described cell lines with FDC-like properties that grew in vitro without exogenous growth factors. In this paper, we followed the idea used by Jenkinson et al. (1992) in the mouse and by Gady et al. (1993) in human systems to obtain thymic stromal elements. We cut tonsil tissue into 300-400-/zm thick slices and then cultured them at 25C in order to deplete them of CD45-positive cells. CD45-negative cells obtained in this way were cultured in vitro. Stromal elements thus obtained were enzymedigested and expanded in vitro. In this study, we show that the stromal-cell lines (SCL) obtained by this procedure exhibit FDC-like properties. Furthermore, we have characterized their phenotype, cytokine secretion, and their interaction with B lymphocytes. Isolation of Human Tonsil Stromal Cells Tonsils were cut into 300-400-#m thick slices and incubated in RPMI containing 1% FBS for 6-8 days at 25C with gentle rocking. During this time, most of the lymphocytes were shed into the medium. At the end of the culture period, the tissue slices were washed and digested with 0.25% trypsin. The cells obtained still contained a variable proportion of CD45-positive cells (5-20%, depending on the experiment); it was therefore further subjected to a depletion step with the aid of magnetic beads. The resulting cell population contained less then 2% of CD45-positive cells and was seeded into culture flasks. After 24 hr incubation, large adherent cells appeared. During the first 3-4 days of culture, the few remaining lymphoid cells degenerated. The remaining cells were adherent and uniform, grew in the absence of exogenous cytokines, and exhibited characteristic fibroblastoidlike morphology (Fig. 1). Confluent cells were split using 0.25% trypsin to detach them from plastic. The results presented here were obtained from tonsils of seven different donors. The cells were passaged u (...truncated)


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Grzegorz Skibinski, Anna Skibinska, Martine Deckers, Keith James. Tonsil Stromal-Cell Lines Expressing FDC-Like Properties: Isolation, Characterization, and Interaction with B Lymphocytes, Journal of Immunology Research, 6, DOI: 10.1155/1998/81637