Murine Langerhans cells cultured under serum-free conditions mature into potent stimulators of primary immune responses in vitro and in vivo.

The ability of Ag-pulsed dendritic cells (DC) to induce primary immune responses has led them to be used for vaccination purposes. However, irrelevant Ags (e.g., FCS) can also be taken up by DC during their isolation and culture and then presented in vivo. To circumvent this, we have established a serum-free (SF) culture system. Murine epidermal cell (EC) suspensions were prepared with and without FCS and cultured for 3 days either in SF or FCS-containing medium. In spite of the lower Langerhans cell (LC) yields under SF conditions, both SF- and FCS-cultured LC (SF-cLC, FCS-cLC) underwent a similar maturation process, as evidenced by a similar increase in the cell surface expression of MHC class II and of costimulatory molecules. The further observation that SF-EC cultures elaborated comparable amounts of granulocyte-macrophage (GM)-CSF as FCS-cultured EC, but were relatively impaired in their IL-1alpha and TNF-alpha production, supports the role of GM-CSF in LC maturation and, less so, in LC survival. Functionally, freshly isolated SF-LC compared with FCS-LC in their Ag-processing capacity. Three-day-cultured SF-LC were as potent stimulators of polyclonal T cell responses and of the primary allogeneic MLR as FCS-cLC, but were relatively poor activators of naive, syngeneic CD4+ T cells. In vivo, hapten-modified SF-cLC induced a contact hypersensitivity response similar in magnitude and kinetics to that evoked by FCS-cLC. Our data show that, in the absence of serum and exogenous cytokines, LC mature into potent activators of T cell responses and could thus be a valuable cellular source for DC-based immunotherapy.

[Modification of a panoptic method of staining isolated cells]. / Modifikácia panoptickej farbiacej metódy pre izolované bunky.

A simple and rapid method of isolated cell staining was achieved by modification of the Pappenheim's method. It is based on the following steps: (1) dilution of commonly used stock solutions May-Grünwald (1:1), Giemsa-Romanowsky (1.25% v/v), (2) reverse sequence of the used staining solutions in comparison with the original method, (3) shorter time of procedure (5 min/slide), (4) utilization of distilled water of pH 5.1-5.6 for dilution of solutions and rinsing of slides, (5) it is not necessary to add serum to cell suspensions. The presented histological method is suitable for cytomorphological examination of cells. It can be applied in routine hematological laboratories not equipped with cytospins. (Fig. 5, Ref. 6.).