Histological analysis of CD11c-DTR/GFP mice after in vivo depletion of dendritic cells

Abstract

To investigate the dependence of individual immunological processes on DC, a transgenic mouse system (CD11c-DTR/GFP mice) has been developed that allows conditional depletion of CD11c+ DC in vivo through administration of diphtheria toxin. We have performed careful histological analysis of CD11c-DTR/GFP mice at different time points after diphtheria toxin injection and confirmed the transient depletion of CD11c+ cells from lymph nodes and spleen. Unexpectedly, the injection of diphtheria toxin completely depleted marginal zone and metallophilic M(Phi) from the spleen and their sinusoidal counterparts from the lymph nodes. This finding limits the use of CD11c-DTR/GFP mice for the analysis of the role of DC to models and read outs that are proven to be independent of marginal zone and sinusoidal M(Phi).

Fig. 1

Injection of Diphtheria Toxin depletes dendritic cells and marginal zone MΦ from CD11c-DTR mice. Spleen sections of CD11c-DTR mice were stained with Abs of the indicated specificity at indicated time points after i.p. injection of 100 ng DT. Ab staining resulted in a red precipitate. GFP, indicating transgene (DTR)-expressing cells; CD11c, indicating DC; MOMA-1, indicating metallophilic macrophages; ERTR-9, indicating marginal zone macrophages. Original magnification: ×100 for all panels.

Fig. 2

Injection of Diphtheria Toxin results in a long lasting depletion of marginal zone macrophages in CD11c-DTR/GFP mice. Spleen sections of CD11c-DTR mice were stained with Abs of the indicated specificity at indicated time points after i.p. injection of 100 ng DT. Ab staing resulted in a red precipitate. MOMA-1, indicating metallophilic macrophages; ERTR-9, indicating marginal zone macrophages; F4/80, indicating red pulp macrophages. Original magnification: ×100 for all panels.

Fig. 3

Injection of Diphtheria Toxin does not affect red pulp macrophages in CD11c-DTR/GFP mice. Spleen sections of CD11c-DTR mice were stained with Abs of the indicated specificity at indicated time points after i.p. injection of 100 ng DT. Ab staining resulted in a red precipitate. F4/80, indicating red pulp macrophages; CD11b, indicating pan-macrophages. Original magnification: ×100 for all panels.

Fig. 4

Injection of Diphtheria Toxin depletes transgene-expressing DC and marginal zone macrophages from H-2D^b−/−+ CD11c-DTR/GFP → H-2D^b−/− mixed bone marrow chimeras. Bone marrow chimeras were injected i.v. with 10 mg clodronate liposomes to deplete marginal zone macrophages 8 weeks after transplantation. Six weeks thereafter, mice were injected with 100 ng diphtheria toxin or were untreated, and spleens were isolated 18 h later. Cryosections were stained with anti-H-2D^b FITC plus anti-MOMA-1 biotin/streptavidin TRITC or anti-CD11c biotin/streptavidin TRITC and were analysed for immunofluorescence. Double positive (FITC/TRITC) cells appear orange, whereas H-2D^b negative cells appear red. Original magnification was ×200.