Experiments have been done to establish whether the radiation-resistant or A cell has a specific function in the initiation of an immune response in mice to sheep erythrocytes (SRBC). All previous demonstrations using accessory (A) cells have involved in vitro assays and are possibly explainable as tissue culture artifacts. If A cells are essential, it should be possible to demonstrate their requirement in vivo. Therefore we first established such conditions. Two methods were found for creating an A-cell deficiency in vivo: (a) A cells disappear gradually from the spleens of irradiated mice, presumably by migration since A-cell function was shown not to be decreased by irradiation. If 3 days elapse between irradiation and transplantation of mixtures of bone marrow and thymus cells (which provide B and T but few A cells), the usual synergistic response does not occur. Addition of large numbers of freshly irradiated spleen cells to the mixture of bone marrow and thymus completely restores the immune response. (b) Injection of 1010 horse erythrocytes into mice suppresses A-cell activity in these mice 24 hr later; a much reduced response to SRBC is obtained when they are given at this time. The response can be partially restored if irradiated spleen cells are given with the SRBC. This observation formed the basis for a quantitative in vivo assay for A cells in which the magnitude of restoration by various suspensions of irradiated cells was used to estimate the A-cell activity of that suspension. A quantitative in vitro assay for A cells was also developed. It was essential for this assay that the total cell number, B-cell number, and T-cell number be kept constant and that only the number of A cells be allowed to vary. Only under these conditions was the response a linear function of the number of A cells added.

If the in vivo and in vitro assays are detecting the same class of radiation-resistant cells, the physical properties of the cells active in each assay should be identical. Spleen cells were separated on the basis of both density and sedimentation velocity. Fractions from both separation methods were tested for their content of A cells using both the in vivo and in vitro assays. The density and sedimentation profiles of A cells were similar in both assays.

The demonstration that a radiation-resistant cell is required in vivo and that this cell has properties identical to the radiation-resistant cell required in vitro indicates that this cell (the A cell) is directly involved in the initiation of an immune response to erythrocyte antigens.

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