The MRC OX-62 antigen: a useful marker in the purification of rat veiled cells with the biochemical properties of an integrin.

Abstract

SummaryA mouse immunoglobulin G1 monoclonal antibody (mAb), MRC OX-62 (OX-62), was raised against density gradient-enriched rat veiled (dendritic) cells obtained from lymph. In suspensions of lymphoid cells, the OX-62 mAb only labeled cells with the characteristics of veiled cells. The OX-62 mAb was used with a magnetic cell sorter to enrich or deplete veiled cells, and the enriched veiled cells were potent stimulators in the primary allogeneic mixed leukocyte reaction. Immunohistochemical staining of tissue sections showed that the OX-62 mAb did not label all classical dendritic cells and was not restricted to this cell type. In lymphoid tissues, the labeling correlated with dendritic cells, but in skin, major histocompatibility complex class II + cells were OX-62-, while another CD3 + cell with dendritic morphology was strongly OX-62 + . It seems that the OX-62 mAb may be restricted to dendritic cells and probably to T/$ T cells. The OX-62 mAb will be of use in delineating minor subsets of cells with dendritic morphdogy in various tissues. Sodium dodecyl sulfate-polyacrylamide gel dectrophoresis analysis of veiled ceil-enriched populations immunoprecipitated with the OX-62 mAb gave bands with the biochemical characteristics of an integrin. The OX-62 mAb recognized the c~-like subunit. Dendritic cells are classified as a heterogeneous group of cells with dendritic morphology found in lymphoid and nonlymphoid tissues, and as veiled cells in lymph (1). Dendritic cells originate from bone marrow but the lineage is unresolved (2). Attention focused on dendritic cells after the isolation of the spleen dendritic cell and the observation that these cells are the most potent accessory cells identified at inducing primary T cell responses in vitro (3). The immunoregnlatory role of dendritic ceils in vivo is implicated by their presence in the T-dependent areas of secondary lymphoid organs (4, 5). Identification of dendritic ceils is based on properties of the isolated spleen dendritic cell, which indude dendritic morphology (6), constitutive expression of MHC class II (7), low phagocytic ability in vitro (8), and potent accessory function in the primary allogeneic MLR (3). The need for mAbs that are specific for dendritic calls is evident (9), but few useful mAbs are available (10-17), and anti-MHC class II mAbs are of limited value. One major problem in generating dendritic cell mAbs is that it is difficult to obtain large numbers of dendritic cells for immunization and there are no call lines equivalent to normal dendritic cells (18)(19)(20). In this paper the production of a mouse IgG1 mAb (OX-62) useful for the purification of rat veiled ceils and recognizing an antigen with the biochemical properties of an integrin is described. Materials and MethodsAnimals. BALB/c (H-2 d) and DBA/2 (H-2 d) inbred mice were obtained from the Sir William Dunn School of Pathology (Oxford). F1 hybrids between these two strains were bred at the MRC Cellular Immunology Unit (Oxford). PVG (RT1 ~ and AO (R.T1 u) specific pathogen-free inb...

A new protocol for the propagation of dendritic cells from rat bone marrow using recombinant GM-CSF, and their quantification using the mAb OX-62

“…Furthermore, their stimulatory activity in MLR strongly correlates with the intensity of expression of these surface antigens. This is not surprising, because OX-62 identifies the α subunit of an integrin-like molecule (Brenan and Puklavec, 1992), up regulation of which would be expected under the influence of GM-CSF. Similarly, the increased expression of class II on mature DC is not unexpected, as DC are well known to be the most potent of Ag presenting cells (Steinman, 1991;Steinman and Cohn, 1973).…”

“…The recent availability of a specific mAb for the rat DC (OX-62) (Brenan and Puklavec, 1992) makes it possible to reliably monitor the development of BM-derived DC in culture. Although OX-62 does not stain all DC (Brenan and Puklavec, 1992), it does allow us to standardise culture techniques for the propagation of DC from the rat, an animal used widely for investigating allograft-recipient interactions.…”

“…Although OX-62 does not stain all DC (Brenan and Puklavec, 1992), it does allow us to standardise culture techniques for the propagation of DC from the rat, an animal used widely for investigating allograft-recipient interactions. Therefore in this paper, putative DC (confirmed to be OX-19 − and therefore not γ/δ T cells) are double labelled with OX-62 and the MHC class II marker, OX-6, allowing an accurate (although conservative) determination of cell phenotype and number.…”

“…Phenotypic analysis of these cells was enhanced by the use of OX-62, the new murine monoclonal antibody directed against an integrin-like rat DC surface marker (Brenan and Puklavec, 1992). This method utilises several of the previously described properties of rat DC precursors to maximise the yield of these cells in culture, name1y their lack of adherence to plastic flasks (Klinkert et al, 1980(Klinkert et al, , 1982, FcR status (Klinkert et al, 1980(Klinkert et al, , 1982 and their partial dependence on murine rGM-CSF (MacPherson, 1989;MacPherson et al, 1989).…”

“…In addition to these measures, tissue culture flasks were coated with gelatin to augment DC growth (in a fashion analogous to the use of type 1 collagen to promote the maturation of liver-derived DC (Lu et al, 1994)). Using this approach, bulk cultures of DC can readily be generated from rat BM, as documented by their morphology, functional ability in mixed lymphocyte reactions (MLR) and their expression of MHC class II (Banuls et al, 1993;Bowers and Berkowitz, 1986;Hart and Fabre, 1981) and the DC-specific surface marker, OX62 (Brenan and Puklavec, 1992).…”