In vitro detection of Mycoplasma fermentans binding to B-lymphocytes in fresh peripheral blood using flow cytometry

Previous reports have shown, using fluorescent probes conjugated to the organism, that Mycoplasma fermentans fuses with about 12% of peripheral blood lymphocytes. However, no lymphocyte subset was specified. To elucidate the specific subset of lymphocytes involved, we developed a three-color flow cytometric assay to detect M. fermentans binding to fresh peripheral blood cells. In our assay, two strains of M. fermentans were grown in SP4 glucose broth, mixed with fresh whole blood samples (n > 20), and incubated at 37 degrees C. The blood samples were then stained with a polyclonal antibody to M. fermentans, a monoclonal antibody to B-lymphocytes (CD19), and a monoclonal antibody to T-lymphocytes (CD3). Using three-color flow cytometry, we obtained data confirming binding of M. fermentans to 10%-15% of peripheral blood lymphocytes with minimal granulocyte or monocyte staining detected. Flow cytometric analysis showed that early binding appears predominantly directed towards B-lymphocytes (86.7 +/- 9.0%), and that this binding could not be blocked by antibodies directed towards common B lymphocyte cell surface antigens. M. fermentans binding to B-lymphocytes occurred within 5 min of in vitro inoculation, reached a maximum within 30-60 min (94-97%), and thereafter plateaued. The binding was concentration dependent over a three log dilution using 10(3) color changing units as standard. Binding to T-lymphocytes was minimal (<5% positive). B lineage tumor cells or peripheral blood B cells obtained from HIV infected individuals demonstrated reduced binding of M. fermentans. This assay provides a good method to study the cellular interactions of mycoplasma and may help to elucidate pathogenic mechanisms of mycoplasma infections.