Oncodevelopmental
antigens may cause immunologic suppression in the host through release of suppressor molecules from the host's own immunoregulatory cells. This concept has been difficult to study until recently when
carcinoembryonic antigen was shown to induce the release of such molecules from normal circulating human mononuclear cells in vitro. However, the amount of the suppressor moiety generated was too small to adequately characterize, and its presence in vivo, i.e., in the
cancer-bearing host, was unknown. Therefore, we sought to isolate and characterize a similar or identical macromolecule from
ascites having an elevated CEA level in patients with
cancer. A single malignant
ascites, when precipitated at 0 to 35%
ammonium sulfate saturation, was the source of suppressive factor for purposes of isolation and standardization. Suppression was quantitated by reduction of [3H]
thymidine incorporation by
phytohemagglutinin-stimulated normal human peripheral blood mononuclear cells.
Sephadex G-200 chromatography revealed probable aggregation of the factor in isotonic
buffers; aggregation was reduced in the presence of 8 M
urea. Purification was achieved by precipitation with 5%
trichloroacetic acid (TCA). The suppressor factor remained soluble in TCA and demonstrated a 95-fold increase in specific activity. Analytical
sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated a single
protein band of 50,000 daltons.
Ascites from three additional
cancer patients gave identical results. Physicochemical characterization of the suppressor moiety revealed stability at 70 degrees C for 30 min and at pH 2 and pH 10 for 24 hr. Delipidation by
chloroform-
methanol extraction,
proteolytic enzyme digestion, and
protamine sulfate precipitation did not affect activity, suggesting that
lipid, simple
peptides, and
nucleic acids were not crucial. However,
periodate oxidation irreversibly destroyed suppressor activity, suggesting the importance of
carbohydrate to the molecule and offering one explanation for
protease resistance. Similarities in m.w. (50,000 daltons), isoelectric point (pI = 3.4), physical properties (heat and
acid stability and resistance to
proteases), and immunologic activity of this factor with that released from lymphocytes after in vitro exposure to
carcinoembryonic antigen indicates they may be identical. Our results suggest that early aberrant events induced in the immunoregulatory network by
tumor-associated
antigens may be relevant and may lead to better understanding of immunosuppression in the
cancer-bearing host.