Three classes of immunologically reactive cells, differing only slightly in size from each other, are required for the production of hemolysin-forming cells in culture. The three classes of cells can be detected in the normal mouse spleen by the combined use of rosette formation, velocity sedimentation, and irradiation.

One class of cells (peak sedimentation velocity, 3.2 mm per hr) forms rosettes. The capacity of these cells to participate in immune responses to foreign erythrocytes is inhibited by relatively low doses of irradiation. These cells may be the immediate precursors of hemolysin-forming cells.

A second class of cells (peak sedimentation velocity, 3.6 mm per hr) facilitates the production of hemolysin-forming cells by small numbers of normal spleen cells. Their facilitative activity is resistant to a relatively large dose of radiation. They do not form rosettes.

The requirement of a third class of cells was deduced from the results of mixing experiments. Neither rosette-forming cells nor spleen cells largely depleted of rosette-forming cells could give rise to hemolysin-forming cells when cultured either alone or in the presence of large numbers of heavily irradiated cells. However, when rosette-forming cells, cells depleted of rosette-forming cells, and heavily irradiated cells were mixed together, hemolysin-forming cells were produced. The peak responses were found in fractions sedimenting at 4 mm per hr. Thus, it is suggested that these fractions contain a third class of cells. This class of cells does not form rosettes, but its function is inhibited by relatively low doses of radiation.

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