The mice produced an initial and a vigorous secondary humoral response to SRBC (Fig. T cells, exhibited a normal humoral immune response against SRBC after transfer, demonstrating that SRBC-specific B cells were left unaffected by antiC4-1BB mAbs. Keywords: 4-1BB receptor, costimulation, humoral HDM201 immunity, anergy The 4-1BB receptor, CDw137, is a member of the TNFR superfamily 1, which is reportedly expressed on activated T and NK cells in mice 1 2. Several studies have demonstrated that the 4-1BB receptor serves as a potent costimulatory molecule for T cells 3 4 5 6 and, in vivo, for NK cells (our unpublished observations). The natural ligand for the 4-1BB receptor, a molecule known as 4-1BB ligand, is constitutively expressed on resting B cells and macrophages and is costimulatory for anti-Cmediated B cell activation 7. We have previously demonstrated through a combination of in vitro and in vivo studies in the mouse that antiC4-1BB mAbs preferentially activate CD8+ T cells 8 and protect them from superantigen (SAg)-induced apoptotic death 9. AntiC4-1BB mAbCcostimulated CD8+ T cells secreted large quantities of IFN- 8 and TNF- (our unpublished observations) and developed into antigen-specific CTLs 8. In tumor-bearing mice, we found that antiC4-1BBCinduced CTLs eradicated large established tumors even when the tumors were poorly immunogenic and refractive to CD28/CD80-mediated costimulation 10. Given the fact that B cells express the 4-1BB ligand and that CD8+ T cells are known to function as suppressor cells, we examined in vivo the effect of antiC4-1BB mAbs on the generation of humoral immunity to thymus-dependent and thymus-independent (TI) antigens. We made use of three model antigens commonly employed for the study of humoral immunity in mice. Sheep (S)RBCs and human (hu)IgG are thymus-dependent antigens. Trinintrophenol (TNP)CFicoll is a type II TI antigen. The studies described here demonstrate that injection of antiC4-1BB mAbs into mice undergoing immunization to T cellCdependent antigens blocked the development of humoral immunity. In contrast, injection of antiC4-1BB mAbs in mice immunized with TNPCFicoll was without effect, and the mice generated a normal humoral anti-TNP response. AntiC4-1BBCinduced immune suppression is long lasting and independent of circulating antiC4-1BB mAbs. Materials and Methods Animals. 8C12-wk-old female BALB/c, C57BL/6, and C57BL/6 2-microglobulinCdeficient mice were purchased from The Jackson Laboratory. Animals were maintained under a standard protocol with free access to food and water. Antibodies and Fusion Proteins. The generation and characterization of 1D8 and 3E1 antiCmouse 4-1BB mAbs and murine 4-1BBChuIg soluble fusion protein has been previously described 8, and both antibodies are rat IgG2A molecules having identical functional properties. 6E9 is a rat IgG2A antiChuman CD40 ligand mAb that does not react with mouse CD40 ligand and was provided by Dr. Tony Siadak (Bristol-Myers Squibb). Experimental Design. Female BALB/c mice (The Jackson Laboratory) were immunized intravenously with 108 SRBCs (Colorado Serum Co.) on day 0 and challenged 7 wk later in the same manner. In some experiments, mice received multiple HDM201 challenges at varying time points following the same procedure. huIgG (Calbiochem Corp.) was administered in two doses of 50 g each on days 0 and 6 and then challenged at varying time points depending on the nature of the experiment with 10 g of huIgG injected intravenously. Mice were bled at indicated intervals, and total antibody response to solubilized SRBC membrane proteins was measured 11. Humoral immunity to TNPCFicoll (TNP-Ficoll-TNP[20]-AGG-AECM-Ficoll), purchased from Biosearch Technologies, was HDM201 established by injection of 50 g of TNPCFicoll intravenously on day 0 and again on day 14. Antibody responses to TNP were measured by ELISA using TNP-conjugated OVA as the substrate. ELISA. 4-1BB Ig was bound to 96-well plates (Immunolon-2; Dynatech Labs, Inc.) at 0.1 g/ml in PBS overnight at 4C. Wells were washed and blocked by incubation for 1 h with specimen diluent (Genetic Systems, Inc.). Antibodies or antisera were diluted or solubilized in specimen diluent for 1 h at 22C. Wells were washed and incubated with several different reagents, depending on the assay. For routine binding assays and hybridoma supernatant screening, wells were incubated with peroxidase-conjugated goat antiCrat IgG (Calbiochem Tmem44 Corp.). For mAb isotyping, wells were incubated with peroxidase-conjugated isotype-specific mouse antiCrat mAbs (Zymed Labs., Inc.). For pharmacokinetic assay, wells were incubated with biotinylated RG7 (mouse antiCrat chain), washed, and then incubated with streptavidinCHRPO (horseradish peroxidase; Amersham). After final washing, all assays were developed with TMB substrate (3,35,5-tetramethylbenzidine; Kirkegaard &.