Role of T-Cells in the Mechanism of Reactivity of the Microplate Leukocyte Adherence Inhibition Assay1

To characterize the role of mononuclear cells in the microplate leukocyte adherence inhibition assay, enriched popula tions of T-cells, B-cells, macrophages, and a nylon wool-ad herent fraction consisting of B-cells and macrophages were prepared by a two-stage adherence procedure from spleen cells of normal C57BL/6J mice and mice bearing progressively growing murine colon adenocarcinoma 38 (MCA-38) tumors. These studies indicate that the reactive cell undergoing specific antigen-induced adherence inhibition was present in the mac rophage fraction. This cell was programmed to undergo spe cific antigen-induced adherence inhibition by an MCA-38-sen-sitized B-cell. An enriched population of MCA-38-sensitized T-cells but not normal T-cells abolished the in vitro leukocyte adherence inhibition response of MCA-38-reactive cells. Pre treatment of the MCA-38-sensitized T-cell fraction with anti-Thy 1:2 serum and complement but not anti-immunoglobulin and complement or carbonyl iron to selectively deplete mac rophages abolished the regulatory effect of the MCA-38-sen-sitized T-cell fraction. MCA-38-sensitized T-cells could prevent MCA-38-sensitized B-cells from programming normal macro phages in vitro but could not abolish the leukocyte adherence inhibition response of presensitized macrophages. Temporal studies of suppressor T-cell activity were per formed throughout different phases of progressive tumor growth. Suppressor T-cells could be detected as early as 4 days post-MCA-38 tumor cell inoculation and persisted throughout MCA-38 progressive tumor growth. Titration stud ies of suppressor T-cells revealed the need for 4 times the number of MCA-38-sensitized T-cells from small tumor bearers (Day 4) in comparison to large tumor bearers (Day 20) to achieve the same degree of suppression. Thus, in the MCA-38 system, sensitized T-cells exert a negative regulatory influence by preventing B-cell programming of normal macrophages. This negative regulatory influence increased in magnitude with the progressive growth of the MCA-38 tumor. normal mice and from MCA-38 tumor-bearing mice harvested at 4, 8, 12,16, and 20 days post-MCA-38 tumor cell inoculation were fraction ated on nylon wool columns into T-cell and nylon wool-adherent frac tions. MCA-38-sensitized T-cells were admixed with normal T-cells, as previously described, prior to recombination with MCA-38 nylon wool-adherent cells, in order to determine the minimum number of MCA-38-sensitized T-cells needed to abolish the in vitro LAI response. Preliminary studies indicated that a specific antitumor response could be detected between Days 8 and 16 with the MCA-38-sensitized nylon wool-adherent fraction. Before Day 8, this specific antitumor response could not be detected; by Day 20, this specific antitumor response disappeared. Thus, studies on the development of suppressor T-cell activity before Day 8 and after Day 16 were performed with MCA-38-sensitized nylon wool-adherent cells harvested between Days 8 and 12 after MCA-38 tumor cell inoculation.