A plaque technique which provides a sensitive and quantitative determination of allohemolysin (isohemolysin) production by lymphoid cells of mice and Syrian hamsters is described. Much ongoing attention has been given to minimizing sources of spurious plaques while improving the reproducibility of the technique. Primary intravenous immunization with allogeneic blood cells led to the early appearance of plaque-forming spleen cells responding to both strong and weak histocompatibility antigens in inbred strains of mice. The strongest histoincompatibility (A/J → BL/6), characterized by H-2 plus non-H-2 differences and a median skin allograft survival time of 8.2 ± 0.5 days, gave a peak average plaque production of 29.6 per 10 x 106 viable spleen cells after 6 days. At the other extreme with an average allograft survival time of 91 days, C3H(H-1a) → C3H.K(H-1b) showed a feeble production of plaque-forming cells with a peak response of 3.7 per 10 x 106 viable spleen cells at 9 days. Histocompatibility antigens determined by genes at the H-1, H-Y, and H-X loci were demonstrated to be erythrocyte antigens as well. Peak plaque production occurred earlier and reached higher levels across the stronger histocompatibility barriers (H-2 plus non-H-2 or H-2 alone) in temporal accord with the rapidity of skin allograft rejection. Weak H-Y or H-1 differences leading to prolonged allograft survival were associated with moderate, but not commensurate delays in the initial occurrence and recruitment of plaque-forming cells. With one exception (C57BL/10 → B10.A), the average plaque counts found at the peak of the primary responses were inversely proportional to the median survival times of skin allografts in the same strain combinations. Nevertheless, in light of the disparity between the times of allograft rejection and the time course of appearance of plaque-forming cells with the "weaker" congenic combinations, the early 19S antibodies do not appear to be directly involved in allograft rejection.
There appears to be no limitation in the weakness of histocompatibility antigens capable of evoking antibodies detectible by plaque assay. The extent to which molecular species of mouse antibodies other than 19S hemolysins are induced and detected under the conditions employed remains to be determined. The responses in strain combinations involving stronger histocompatibility barriers were characterized by exponential increases in plaque-forming cells, for 2 to 4 days until maximal levels were reached. Repeated immunization in several trials led to substantial increases over the maximum primary response in the number of demonstrable plaque-forming cells. Further preliminary results are reported concerning alloimmune plaque production in three strains of Syrian hamsters exhibiting intermediate to weak histocompatibility differences.