Limiting dilution analysis of human peripheral blood mononuclear leukocytes that react to human amnion cells and protect these against viral challenge

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Abstract
Human peripheral blood mononuclear leukocytes (PBL) cocultured with WISH human amnion cells rapidly produced α-type interferon (IFN-α) and in an IFN-dependent manner protected the WISH cells against the cytopathic effects caused by vesicular stomatitis virus. When the number of PBL per WISH cell monolayer culture was diluted out using multiple WISH microtiter plate cultures for each PBL dilution, the protection of the WISH cultures appeared as an all-or-none phenomenon. Thus, at one small mean number of PBL per culture, some cultures were protected against the virus, while others were not. Limiting dilution analysis, with plot of the logarithm of the fraction of nonprotected WISH cell cultures at each PBL dilution against the mean number of PBL per culture, yielded straight lines with an intercept on the ordinate close to 1. This indicated a single-hit event due to the function of a single type of limiting “protecting unit”. The frequency of this limiting unit in the PBL population could reproducibly be determined for an individual blood donor. For a limited number of different blood donors it ranged from 1/400 to 1/3000. These minimal frequency estimates were considerably increased (40-300%) after 3 h preincubation of PBL with IFN-α. Two major alternatives were considered with regard to the nature of the limiting protecting units. They were either IFN-producing cells operating alone, or they were inducer-type cells that possibly used IFN as a mediator to activate protecting cells present in excess in the coculture system. The second possibility was favored, and the relation of the protecting cell type to the natural killer cell system is discussed herein.

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