Role of accessory cells in the in vitro growth of aggregate-derived murine T-cell colonies

C3H/HeJ lymph node cells (LNC) were seeded in 35-mm petri dishes containing 0.8% methylcellulose, 10% fetal calf serum, 2-mercapthoethanol, and supernatant from PHA or Con A-stimulated spleen cells. After 3–4 days incubation at 37 °C, colonies containing >50 cells appeared. The cells from individual colonies stained with a fluorescent anti-Thy-1 antiserum, and colony formation was prevented by treating the LNC with radiation or anti-T-cell serum + complement before culturing. When fewer than 1?2 × 10⁶ LNC were seeded, the number of colonies formed decreased exponentially; this observation suggested colony formation might require cell-cell interaction. Formation of cellular aggregates could be seen as early as 4–20 hr after plating. Colony formation of 2?5 × 10⁵ LNC was promoted by adding irradiated or anti-T serum + complement-treated LNC, and colony formation was inhibited by carbonyl iron treatment to remove adherent cells. Cell separation by velocity sedimentation showed colony promoting activity was associated with cells sedimenting at 4 mm/hr and also >6 mm/hr. These are properties similar to those of accessory cells that are required for immune responses in vitro and in vivo. Colony formation was also increased in LNC from tumor allograft immune mice, and in the uterine lymph nodes from mice bearing an allogeneic fetus. T-Cell colonies produced by direct plating of LNC in this system arise from proliferation of cellular aggregates, and are primarily a measure of accessory cell activity.