Determination of the specificity of plasma cells for bacterial antigens in situ.

We developed a method for determining the specificity of plasma cells for bacterial antigens in situ. The method consists of applying rhodamine-labeled whole bacteria to frozen sections of tissue in which inflammation had been induced by injection of the same bacterial strain. After appropriate washings, the tissue sections were briefly treated with a fluorescein-labeled antiserum to endogenous immunoglobulins to detect plasma cells. Plasma cells which could recognize bacterial somatic antigens appeared as clumps of orange-fluorescing bacteria overlying a green-fluorescing cell. The fidelity of the procedure was determined by performing several competition experiments, which showed that pretreatment of the inflamed tissue sections with homologous unlabeled bacteria (i.e., the same bacteria used to induce the tissue inflammation) decreased binding of the labeled bacteria to a significant extent. Pretreatment of the inflamed tissue sections with heterologous unlabeled bacteria, however, did not significantly decrease binding of the labeled bacterium. Additionally, binding of rhodamine-labeled heterologous bacteria to the tissue sections was insignificant. In further experiments, we demonstrated that there was no natural affinity between the bacterial strains employed and lymphocytes taken from the strain of rabbit used and that pretreatment of the sections with anti-immunoglobulin abrogated the binding of the bacteria to which the animal had been specifically sensitized.