All efficient cell separation procedure and specific anti-macrophage serum were used to investigate the requirement of macrophages in the in vitro allograft response of mouse lymphoid cells. The efficiency of the macrophage-depletion procedure used and the undiminished capacity of the purified lymphocytes to respond were verified by also testing the antibody responses to sheep red cells (SRC) and dinitrophenylated polymeric flagellin (DNP POL) as well as the proliferative response to allogeneic cells. It was found that the generation of cytotoxic lymphocytes were diminished after macrophage depletion by surface adherence. The combination of anti-macrophage serum and column purification resulted in the total abolition of cytotoxic activity. The cell-mediated immune response was restored completely by addition of peritoneal macrophages, with as few as 1 macrophage to 600 lymphocytes permitting a significant restoration. Macrophages were not involved in the cytotoxic effector phase, but were essential in immune induction. A subcellular H-2 alloantigen preparation was only immunogenic in the presence of macrophages, indicating that a mere reduction in the size of the antigen from cell-bound alloantigens to membrane fragments was not the sole function of macrophages. The results suggest that macrophages collaborate with T cells in the initiation of an allograft response in vitro.

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