Activated alpha 2-macroglobulin reverses the immunosuppressive activity in human breast cancer cell-conditioned medium by selectively neutralizing transforming growth factor-beta in the presence of interleukin-2.
The immunosuppressive activity of tumor cells may be mediated by tumor-derived cytokines such as transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10). A human breast cancer cell line derived from malignant ascites (BRC 173) secreted TGF-beta, 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 alpha 2-macroglobulin (alpha 2M), a plasma protein and cytokine carrier that binds isoforms in the TGF-beta family, was tested for its ability to neutralize the immunosuppressive activity in BRC 173 TCS. alpha 2M was converted to its activated conformation by reaction with methylamine (alpha 2M-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 alpha 2M-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-beta-neutralizing antibody was added to the PBL culture medium with IL-2 and TCS, a majority of the lytic activity was restored. alpha 2M-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 alpha 2M-MA to counteract immunosuppressive cytokines in breast cancer TCS was evident in serum-containing and serum-free medium. These studies demonstrate the activated alpha 2M can function as a selective cytokine neutralizer to thereby promote the activation of NK, LAK, and tumor-specific CTL responses.
Harthun
N L
NL
Department of Surgery, University of Virginia Health Sciences Center, Charlottesville, USA.
Weaver
A M
AM
Brinckerhoff
L H
LH
Deacon
D H
DH
Gonias
S L
SL
Slingluff
C L
CL
D001943:Breast Neoplasms / Q000276:immunology; D002478:Cells, Cultured / Q000187:drug effects; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000494:pharmacology; D016895:Culture Media, Serum-Free; D016207:Cytokines / Q000494:pharmacology; D005260:Female; D006801:Humans; D007108:Immune Tolerance / Q000187:drug effects; D016753:Interleukin-10 / Q000032:analysis; D007376:Interleukin-2 / Q000276:immunology*; D015979:Killer Cells, Lymphokine-Activated / Q000187:drug effects; D007694:Killer Cells, Natural / Q000187:drug effects; D009500:Neutralization Tests; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000032:analysis / Q000276:immunology* / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis; D000511:alpha-Macroglobulins / Q000494:pharmacology*
D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D016207:Cytokines; D007376:Interleukin-2; D016212:Transforming Growth Factor beta; D014409:Tumor Necrosis Factor-alpha; D000511:alpha-Macroglobulins; D016753:Interleukin-10