502 The T cell-specific surface protein 41BB (CD137) is induced upon activation, interacts with a specific ligand (41BBL) expressed by APCs and has been shown to transduce costimulatory signals even in the absence of CD28/B7 costimulation. Recent evidence suggests that this costimulatory pathway may be particularly important for CD8+ T cell activation. We have investigated the effects of blocking 41BB/41BBL interaction in human mixed lymphocyte cultures (MLR) using a soluble fusion protein - h41BBIg. The extracellular domain of human 41BB was amplified by RT-PCR. This sequence was spliced to a sequence encoding the Fc portion of human IgG1 and the resulting cDNA was expressed in CHO cells and protein purified from culture supernatant. The fusion protein reacted specifically with human IgGFc and human 41BB-specifc antibodies by ELISA. A panel of six MLRs was carried out with freshly isolated human Peripheral Blood Mononuclear Cells (PBMC) from three donors. Responder and irradiated stimulator cells were mixed at 1:1 ratio. Additions were: control human IgG1 (50μg/ml); h41BBIg (50μg/ml); hCTLA4Ig (25μg/ml) + IgG1 (50μg/ml), or hCTLA4Ig (25μg/ml) + h41BBIg (50μg/ml). Proliferation was assessed by thymidine incorporation between 96 and 112 hours and assessment of cell numbers, viability, activation and subset analysis was carried out by flow cytometry after 5 days of culture. Compared to control reactions h41BBIg resulted in a mean reduction in proliferation of 37% (range 20-49%), hCTLA4Ig resulted in a mean reduction of 43% (range 27-64%), while the two fusion proteins together resulted in a 52% (range 28-71%) mean reduction. Viable activated CD8+ T cells were reduced in number in the presence of h41BBIg by a mean of 48% (range 16-78%) while viable activated CD4+ T cell numbers were reduced by only 27% (0-57%). In contrast, the effects of hCTLA4Ig on T cell subset numbers were more marked for CD4+ than CD8+ with a 55% (range 35-67%) mean reduction vs. 41% (16-64%) mean reduction, respectively. The addition of the two fusion proteins had additive effects on both subsets [CD4+ - mean reduction 59% (range 30-76%); CD8+ - mean reduction 67% (range 50-80%)]. We conclude that blockade of 41BB/41BBL interactions significantly reduces human alloreactivity in vitro, exerts additive effects to blockade of CD28/B7 and merits investigation as an immunosuppressive strategy.