(a) Lymphocytes were isolated as described 1 hr after phosphate-buffered saline (PBS) (gray bars) or CD3Ab (1 g, 1452C11) injection (black bars) and intracellular expression levels of interferon (IFN)- were determined by intracellular staining in total T cells or T cells gated for TCR-+ NK11C CD11ahigh (TCR, T-cell receptor; NK, natural killer)

(a) Lymphocytes were isolated as described 1 hr after phosphate-buffered saline (PBS) (gray bars) or CD3Ab (1 g, 1452C11) injection (black bars) and intracellular expression levels of interferon (IFN)- were determined by intracellular staining in total T cells or T cells gated for TCR-+ NK11C CD11ahigh (TCR, T-cell receptor; NK, natural killer). percentage of splenic TCR-+ cells were stimulated and became CD69+. Activation of TCR-+ cells in the liver was at least as efficient as with the spleen, but almost all T cells and all scavenger liver sinusoidal endothelial cells bound CD3Ab. In contrast to CD69 up-regulation, only CD4+ natural killer T (NKT) cells and CD11ahigh CD8+ KIT T cells were activated by CD3Ab and indicated interferon (IFN)-. Again, IFN- launch from NKT/T cells was at least as efficient in the liver as with the spleen. Taken together, our results support the notion SKLB-23bb that the combination of considerable hepatic vascularization and very high scavenger activity allows the liver to fulfill its metabolic jobs and to promote activation SKLB-23bb of the large but widely distributed hepatic human population of NKT/T cells. is definitely lacking. We tackled this query by injecting mice with CD3Ab, which allowed us to study quick T-cell activation in peripheral organs that was dependent on Fc receptor (FcR) cross-linking through APCs. We provide evidence that blood-borne CD3Ab induces quick activation and cytokine manifestation of both NKT and memory space CD11a+ CD44high T cells in the liver and spleen. NKT- and T-cell activation is dependent on cross-linking of CD3Ab through FcR-bearing APCs and is most designated in the liver. The biodistribution and binding of monoclonal antibodies (mAb) to T cells and APCs differ in the liver, spleen, lymph node and thymus, reflecting fundamental variations in practical microanatomy amongst these organs. Materials and methods Mice Mice received humane care and were managed under specific pathogen-free conditions at the animal facilities of the ZMBH (Zentrum fr Molekulare Biologie Heidelberg, Heidelberg, Germany) or Hanover Medical School (Hanover, Germany) SKLB-23bb relating to German recommendations for animal care. Experiments were performed relating to animal experimental ethics committee recommendations. H-2Kb-restricted SIINFEKL-specific TCR-transgenic (OT-I),15 FcRC/C,16 FcRIIC/C,17 FcRIIIC/C,18 and RAG2C/C19 mice have been explained previously. Antibodies and reagents Ovalbumin and saponin were purchased from Sigma (Deisenhofen, Germany). Antibodies for interleukin (IL)-2 enzyme-linked immunosorbent assay (ELISA) (clones JES6-1A12 and JES6-5H4) were from BD Biosciences (Heidelberg, Germany) and the assay was performed according to the manufacturer’s instructions. CD3 (145-2C11), CD4 (GK15), CD8 (53-67), CD11a (2D7), CD11b (M1/70), CD11c (HL3), CD19 (1D3), CD44 (IM7), CD62L (MEL-14), CD69 (H12F3), IFN- (XMG12), NK11 (PK136), TCR- (H57-597), TNF- (MP6-XT22), V2 (B201) and V5 (MR9-4) mAbs, conjugated to different fluorochromes or biotin, and strepavidin conjugates (SA-PerCP-Cy55 and SA-PE-Cy5) were also purchased from BD Biosciences. ME-9F1 mAb was kindly provided by A. Hamann (Deutsches Rheumaforschungszentrum, Berlin, Germany). The CD1d-restricted NKT cell hybridoma DN3A4-1220 was kindly provided by M. Kronenberg ( La Jolla Institute of Allergy and Immunology, San Diego, CA). Cell isolation Isolation of LSECs from murine livers was carried out as explained previously.3 Briefly, murine livers were perfused with 005% collagenase A (Boehringer, Mannheim, Germany) inside a calcium-free phosphate buffer by cannulating the vena porta having a 21-gauge venflon. Following mechanical dissection, the liver was incubated for 30 min inside a rotatory waterbath (240 r.p.m. at 37) in Gey’s balanced salt remedy comprising 005% collagenase A. LSECs were separated from parenchymal cells by denseness gradient centrifugation on a metrizamide (Sigma) gradient (1089 g/cm3) followed by two washing steps to remove cell debris. Further separation was achieved by counterflow centrifugal SKLB-23bb elutriation using a J2-MC centrifuge (Beckman, Mnchen, Germany) equipped with a JE-6B rotor and a standard elutriation chamber (both Beckman). Rotor rate was kept constant at 750 for 20 min), mononuclear cells were harvested from your interface and extensively washed. Peripheral blood lymphocytes were purified by gradient centrifugation (800 for 20 min) using a Lymphoprep remedy (Nycomed Pharmac, Unterschleissheim, Germany) and splenic lymphocytes were obtained by mechanical dissection of the spleen. Immunofluorescent staining.