Dendritic cells are the principal stimulators of the primary mixed leukocyte reaction in mice

Abstract

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.

The Toll‐like receptor ligand MALP‐2 stimulates dendritic cell maturation and modulates proteasome composition and activity

“…The strong T cell stimulatory activity of mature DC is commonly demonstrated in primary MLR [28][29][30]. Our results showed that MALP-2 pretreatment of DC results in an enhanced proliferation of allogeneic spleen and CD4 + T cells.…”

Dendritic cells: Understanding immunogenicity

“…The absence of DC ablated much of the MLR stimulating activity from the total suspension of spleen cells [17]. We could not interpret the residual activity in 33D1-depleted spleen cells until 7 years later, when we noted that there were two subsets of DC in mouse spleen, a major 33D1 + and a minor NLDC-145 + (now DEC-205/CD205 + ) subset [30].…”

“…These subsets were denoted as CD8 -and CD8 + by Ken Shortman [31]. Because we could selectively deplete most of the MLR stimulating function by removal of DC from a fresh spleen cell suspension, we concluded that "antigens, to be immunogens, require accessory cells" [17].…”

“…Instead, we and others used the distinct morphology of DC to identify, enrich and begin to characterize these cells in several tissues and species, and to prepare the first monoclonals, i.e. 33D1, now anti-DCIR2 [16][17][18] and N418 anti-CD11c [19,20]. The distinct morphology that we used to guide our preparation of DC has now been observed in living lymph nodes, where each DC continually probes its environment by extending and retracting processes [21].…”

À propos de Ralph M. Steinman et des cellules dendritiques

“…Les preuves expérimentales de l'activité présenta-trice d'antigènes des cellules dendritiques in vivo furent apportées grâce à l'utilisation des modèles de transplantation rénale et pancréatique : la déplétion de ces cellules dendritiques par un anticorps produit dans le laboratoire de R. Steinman [5] réduisait ou prévenait le rejet du greffon [6,7]. Ce rôle central joué par la cellule dendritique dans les mécanismes de rejet était la traduction in vivo de ce que Ralph Steinman avait parallèlement démontré in vitro : la mise en évidence de l'extraordinaire capacité de ces cellules à induire une réponse proliférative des lymphocytes T allogéniques (provenant d'un autre individu différant par ses molécules du CMH) in vitro [2] et de son rôle central dans ce phénomène [8]. Il fallut ensuite quinze ans de recherches assidues dans le domaine pour que la cellule dendritique soit utilisée en routine dans les tests de proliféra-tion in vitro (test de réaction lymphocytaire mixte ou MLR).…”