Lymphoreticular cells isolated by centrifugal elutriation from a mammary adenocaricinoma. i. characterization of an in situ lymphocyte suppressor population by surface markers and functional reactivity

The procedure of centrifugal elutriation was evaluated as a means of purifying large numbers of in situ lymphocytes from enzymatically disaggregated mouse mammary tumors. The eluate obtained at a flow rate of 3.0 ml/min was optimal for high levels of lymphocyte recovery with low levels of contaminating tumor cells, polymorphonuclear leukocytes, and macrophages. The majority of the tumor infiltrating lymphocytes (90%) expressed the Thy 1.2 antigen, while less than 5% possessed surface immunoglobulin. Further analysis of the T lymphocyte population was accomplished by flow cytometric analysis on in situ lymphocytes stained with fluorescein-conjugated monoclonal anti-Lyt 2 antibodies. The results of such studies reveal an increase in the levels of Lyt 2⁺ lymphocytes within the in situ population. To determine whether these Lyt 2⁺ cells were functionally active as suppressor cells, the ISL were mixed with spleen cells from tumor bearers and then tested for their ability to respond to mitogen and TAA-induced blast transformation of tumor-bearer spleen cells. Removal of macrophages from ISL by Sephadex G-10 columns did not alter the suppression. Treatment with monoclonal anti-Lyt I antibody and complement did not affect the inhibition observed. However, treatment of ISL with anti-Lyt 2⁺ monoclonal antibody and complement resulted in the elimination of the suppressor cell activity. We concluded that within the tumor-infiltrating lymphoreticular cells there is a population of Thy 1.2⁺ Lyt 2.2⁺ lymphocytes responsible for the suppression of mitogen and tumor-antigen-induced blastogenesis.