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The immunosuppressive activity of tumor cells may be mediated by tumor-derived cytokines such as transforming growth factor-β (TGF-β) and interleukin-10 (EL-10). A human breast cancer celt line derived from malignant ascites (BRC 173) secreted TGF-β, but not IL-10, into tissue culture supernatant (TCS). BRC 173 TCS suppressed natural killer (NK) and lymphokine-activated killer (LAK) cell activity and also blocked the generation of HLA-A*0201-restricted tumor-reactive cytotoxic T-lymphocyte (CTL) lines in vitro. Human α2-macroglobulin (α2M), a plasma protein and cytokine carrier that binds isoforms in the TGF-β family, was tested for its ability to neutralize the immunosuppressive activity in BRC 173 TCS. α2M was converted to its activated conformation by reaction with methylamine (α2M-MA) and then incubated with normal human peripheral blood lymphocytes (PBL) in the presence of IL-2 and BRC 173 TCS. Lysis of NK targets (K562) and LAK cell targets (DM6 melanoma) by the PBL was examined after 6 days of culture. PBL cultured in IL-2, without TCS or α2M-MA, were lytic for both target cells. BRC 173 TCS substantially suppressed the lytic activity of the PBL in the presence of IL-2. When TGF-β-neutralizing antibody was added to the PBL culture medium with IL-2 and TCS, a majority of the lytic activity was restored. α2M-MA (280 nM) neutralized almost all of the immunosuppressive activity in the TCS, restoring 80–100% of the lytic activity without any apparent effect on the activity of IL-2. The ability of α2M-MA to counteract immunosuppressive cytokines in breast cancer TCS was evident in serum-containing and serum-free medium. These studies demonstrate that activated α2M can function as a selective cytokine neutralizer to thereby promote the activation of NK, LAK, and tumor-specific CTL responses.