Clone 33D1 is a mouse-rat hybridoma that secretes a specific anti-dendritic cell (DC) monoclonal antibody (14). Because the antibody kills DC in the presence of rabbit complement, it can be used to study the functional consequences of selective DC depletion. Previous data on the cell specificity of 33D1 were first extended. By cytotoxicity (rabbit complement) and indirect immunofluorescence (biotin-avidin technique), 33D1 reacted with DC but not with macrophages nor other splenocytes. In contrast, the monoclonal antibody, F4/80 (15), reacted with macrophages but not DC. The functional assay evaluated in this paper was stimulation of the primary mixed leukocyte reaction (MLR). 33D1 antibody itself did not inhibit stimulation by enriched populations of DC. In the presence of complement, 33D1 killed DC and ablated stimulatory function. The effect of 33D1 and complement on MLR stimulation by heterogenous cell mixtures was then evaluated. Removal of DC from unfractionated spleen suspensions reduced stimulatory capacity 75-90 percent, comparable to that produced with specific anti-Ia antibody and complement. Stimulation of both proliferative and cytotoxic responses was reduced. DC depletion had similar effects on MLR generated across full strain differences, or across selected subregions (H2I, H-2K/D) of the major histocompatibility complex. To further compare the functional properties of spleen DC and macrophages, MLR stimulation by adherent and nonadherent fractions of spleen were tested separately. 62 +/- 8 percent of the total stimulatory capacity of spleen was in the plastic adherent population. Activity was ablated greater than 90 percent after elimination of DC. MLR stimulation by 24-h cultures of spleen adherent cells, which contained a three- to sixfold excess of Ia(+) macrophages, was also ablated when DC were removed. Stimulation by nonadherent spleen was more resistant, but was reduced 50-75 percent by 33D1 and complement. The function of spleen cells treated with 33D1 or anti-Ia antibody and complement was restored with a small inoculum of purified DC. The latter corresponded to 0.5 percent of total stimulator cells and were enriched by previously described techniques that did not require the 33D1 antibody. We conclude that the DC, a trace component of mouse spleen, is the principal cell type required for stimulation of the primary MLR. Because other cells are not immunogenic, but do express Ia and H-2 alloantigens, DC likely represent the critical accessory cell required for the induction of lymphocyte responses.

Dendritic cells (DC) are a small subpopulation of lymphoid cells with distinctive cytologic features, surface properties, and functions. This report describes the DC-specific antibody (Ab) secreted by clone 33D1. Rat spleen cells immune to mouse DC were fused to the P3U myeloma. Hybrid culture supernatants were screened simultaneously against DC, a macrophage (M40) cell line, and mitogen-stimulated lymphoblasts. 33Di Ab specifically killed 80-90% of DC from spleen and lymph node, but no other leukocytes, including Ia+ and la-MC (Ia, I-regionassociated antigen). Quantitative Dendritic cells (DC) are morphologically distinct cells discovered in adherent populations from mouse lymphoid organs (1). DC can be highly enriched on the basis oflow buoyant density, adherence to glass or plastic, and lack of Fc fragment receptors (2). DC express abundant I-region-associated antigen (Ia Ag) and lack the characteristic surface markers of B cells, T cells, macrophages (MF), and granulocytes (refs. 3 and 4, reviewed in ref. 5). DC are also functionally distinct. For example, DC are 100-fold more effective than other leukocytes in stimulating allogeneic and syngeneic mixed leukocyte reactions (6, 7) and function as potent accessory cells during the generation of cytotoxic lymphocytes (8) and oxidative mitogenesis (9, 10).A DC-specific antibody (Ab) would be a valuable probe for further in vitro and in vivo studies. Attempts to generate such a reagent by injecting mouse DC into rabbits failed*because the principal reactivity was directed to Ia and H-2 Ag (unpublished data). In this communication, we have used hybridoma technology to generate the DC-specific Ab, clone 33D1. Specificity was monitored by cytotoxicity assays as well as quantitative binding and autoradiographic studies with radiolabeled Ab. Expression of the 33D1 Ag in mouse lymphoid suspensions precisely paralleled the distribution of DC as previously assessed by cytologic (1-3) and functional (6) assays. MATERIALS AND METHODSAnimals. (DBA/2 X BALB/c) FI/Tru, (A x C57BL/6) F1/ Tru, C57BL/6 Tru, (BALB/c X C57BL/6) FI/Tru, and C3H/ J, C3H. OH/J, and Swiss mice were obtained from the Trudeau Institute (Saranac Lake, NY), The Jackson Laboratory, Taconic Farms (Germantown, NY), and The Rockefeller University (New York). Sprague female rats were from Charles River Breeding Laboratories.Antibodies. Rabbit antiserum to rat Ig (Cappel Laboratories, Cochranville, PA) was affinity purified on Sepharose-rat-Ig and digested to F(ab')2 fragments as described (4). Rabbit antiserum to mouse Ig was from Cappel Laboratories. A number of monoclonal antibodies of defined specificity (4) were employed: B21-2 and 2E8 are anti-I-A reagents; B25-1 is anti-H-2D; 2.4G2 identifies the Fcyreceptor; B5-3 is anti-Thy-i; 1.21 J reacts with the "mac-l" Ag on Mb and granulocytes; and 2.D2C and 2.6 react with Ag found on DC as well as other cell types.Cells. Suspensions of spleen, lymph node, and thymus were prepared by teasing and disruption on a stainless steel sieve. Bone marrow ...

This study establishes that dendritic cells (DC) are the critical accessory cells for the development of anti-trinitrophenol (TNP) cytotoxic T lymphocytes (CTL) in vitro. We developed a model in which nylon wool-nonadherent spleen cells were used both as the responding and stimulating cells, the latter having been TNP-modified and x-irradiated. Thy-1-bearing CTL developed in C57BL/6, B6D2F1, and CBA mice only when small numbers of DC were added. Maximal responses in 5-d cultures were achieved with 0.5-1 DC/100 responding T cells. The DC did not have to be TNP modified directly. Anti-Ia and complement inactivated accessory cells, whereas similar treatment of the responders had no effect. DC exposed to ultraviolet radiation were ineffective, but x-irradiated DC were fully active. Culture media from DC, or from DC-nylon wool-passed spleen T cell cocultures that contained abundant CTL, would not substitute for viable DC. Enriched preparations of macrophages (M phi) were obtained from blood, peritoneal cavity, and spleens of BCG-immune and unprimed mice. M phi added at doses of 0.2-4% were weak or inactive as accessory cells. The level of Ia antigens on test M phi populations was quantitated and visualized by binding of a radioiodinated monoclonal anti-I-Ab,d antibody, clone B-21. M phi that bore substantial amounts of Ia from all organs were weak accessory cells. Addition of M phi to DC-T cell cocultures produced inhibitory effects, usually at a dose of 2% M phi. In contrast, 0.5% Ia-bearing M phi from BCG-immune boosted mice inhibited > 80% of the DC-mediated CTL response. Addition of indomethacin reversed M phi inhibition, and 10(-9) M prostaglandin E2 in turn blocked the indomethacin effect. Indomethacin also restored a low level of accessory cell function in immune-boosted adherent peritoneal cells, but not in preparations of monocytes and spleen M phi. Small numbers of DC were identified in preparations of immune-boosted peritoneal cells and may have accounted for the observed accessory activity. We conclude that the development of anti-TNP CTL is an immune response in which (a) DC are the critical accessory cells; (b) Ia-bearing M phi are weak or inactive; and (c) M phi can inhibit DC-mediated response by an indomethacin-sensitive mechanism.

Our laboratory has focused on the structure and function of the murine dendritic cell (DC; 1). 1 This lymphoid element has a characteristic structure and surface topography and represents a minor population of the cells (1% or less) released from dissociated spleen and lymph node. DC can be purified, and by cytologic criteria they are >90% pure. Purified DC exhibited unique functional properties. They are the most potent stimulators of the allogeneic and syngeneic mixed leukocyte reactions (2, 3) and act as accessory cells in proliferative and cytotoxic T cell responses (4-7).Cell surface markers have contributed to the characterization of spleen DC. Initially it was noted that DC lacked surface immunoglobulin (Ig), brain antigen (Ag), Thy-1 Ag, and Fc and C3 receptors (8). These markers set DC apart from macrophages (Mth) and lymphocytes, particularly when combined with such other differences as distinctive morphology, steroid and radiosensitivity, rapid turnover, weak endocytic activity, and distribution in situ (9). The lack of Fc receptors was useful in purifying DC from DC-Mth mixtures (10). Purified DC all expressed Ia Ag, and did not acquire the surface markers of Mq~ or lymphocytes when cultured in vitro for several days. Further analysis of the cell surface should facilitate the identification of DC in complex cell mixtures, help outline its lineage, and explain its functional capacities.We have therefore performed a detailed study comparing the surface of DC with other bone marrow-derived elements. Monoclonal antibodies and lactoperoxidasemediated surface iodination provided sensitive quantitative and biochemical information. The data show that spleen DC constitutively express high levels of Ia and H-2D alloantigens. DC carry the leukocyte common antigen, but can be distinguished from other leukocytes by several surface markers detected with monoclonal antibodies. Our findings indicate that spleen DC are part of a distinct, Ia-rich, leukocyte differentiation pathway.