Delineation of the dendritic cell lineage by generating large numbers of Birbeck granule-positive Langerhans cells from human peripheral blood progenitor cells in vitro

Abstract

It is well established by in vivo and in vitro studies that dendritic cells (DCs) originate from hematopoietic progenitor cells. However, the presumed intermediate of Birbeck granule (BG)+ Langerhans cells (LCs) has not been detected in cultures derived from bone marrow or peripheral blood progenitor cells (PBPCs), thus contrasting with the data obtained with cord blood. We show here that large numbers of BG+ LCs can be generated from human CD34+ PBPCs in vitro, when granulocyte-macrophage colony-stimulating factor and interleukin-4, potent promotors of LC/DC differentiation, are combined with a cocktail of early acting hematopoietic growth factors. LCs were found to emerge from CD33+CD11b+CD14-progenitor cells that they share with the monocytic lineage. During culture, these cells exhibited a sequence of dramatic morphologic changes, starting with a major increase in granularity followed by an increase in size herein exceeding that of all peripheral blood cells. At the same time, CD1a and major histocompatibility complex class II expression were upregulated and virtually all CD1a++ cells were BG+ by electron microscopy. With prolonged culture, CD1a was downregulated on a major population of cells, paralleled by a loss of BG and an increase of CD4, CD25, and CD80 expression that may correspond to the maturation of epidermal LC in vitro. However, these cells were consistently CD5- and did not exhibit changes in the CD45-isoform expression during culture. The availability of large numbers of these highly purified BG+ LCs and mature DCs allows for specific analysis of these subpopulations and provides a source of potent antigen-presenting cells from individual patients for vaccination protocols against infectious or tumor-associated antigens.

Conversion of In Vitro Cultured Human Monocytes into Effective Presenters of an HER2/neu‐Encoded CTL Peptide Epitope

“…Large round cells with ruffled uropodia and adherent cells with dendritic appearance were seen. In agreement with previously published data [11,16], FCM analysis indicated that viable cultured cells were distributed into two discrete regions corresponding to large cells with high granularity, and smaller cells with low granularity (data not shown). The large cell region contained as low as 30% and as high as 95% of the cultured cells, but consistently constituted approximately 4% of the total input population.…”

“…Since the time they were described [1], dendritic cells (DC) [2] have been under intense investigation because they are thought to induce primary CTL responses [3][4][5][6] and when pulsed with relevant CTL peptide epitopes, regression and elimination of established tumour burden [7][8][9]. A variety of procedures which employ gradients, magnetic beads and/or depletion by immunoglobulin and complement, have been developed in order to obtain purified DC from human peripheral blood, cord blood, or bone marrow CD34 + haematopoietic progenitors [10][11][12][13][14][15]. Human monocytes, following in vitro culture in the presence of GM-CSF and IL-4, also display adhesion molecules and co-stimulatory proteins characteristic of purified dendritic cells [16][17][18][19].…”

Generation of multinuclear tartrate‐resistant acid phosphatase positive osteoclasts in liquid culture of purified human peripheral blood CD34+ progenitors

“…However, the self-renewing ability and the multipotentiality of PBPC have been tested in vitro exclusively along those myeloid differentiation pathways through which circulating mature blood cells are generated (namely erythropoiesis, granulo/monocytopoiesis and thrombocytopoiesis). Recently, Mackensen et al (1995) showed that human PBPC generate large number of Birbeck granulepositive Langerhans cells when they are in vitro expanded in liquid culture, in the presence of either tumour necrosis factor or interleukin-4. The above-mentioned culture technique provides a useful model to study in vitro the biological properties of these elements and to generate large numbers of antigen presenting cells for vaccination protocols.…”

Developmental biology of the dendritic cell system

“…Generation of myeloid DCs in vitro from blood or bone marrow requires the inclusion of granulocyte-macrophage colony-stimulating factor (GM-CSF) in culture (6). Other cytokines supporting myeloid DC production include tumor necrosis factora (TNFa ), interleukin-4 (IL-4), stem cell factor (SCF) and FLT-3 ligand (7). They are high producers of proin ammatory and immunoregulatory cytokines upon stimulation (8).…”