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 th e organism, that Mycoplasma fermentans fuses with about 12% of periphe ral 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. fermenta ns were grown in SP4 glucose broth, mixed with fresh whole blood sampl es (n > 20), and incubated at 37 degrees C. The blood samples were the n stained with a polyclonal antibody to M. fermentans, a monoclonal an tibody to B-lymphocytes (CD19), and a monoclonal antibody to T-lymphoc ytes (CD3), Using three-color now cytometry, we obtained data confirmi ng binding of M. fermentans to 10%-15% of peripheral blood lymphocytes with minimal granulocyte or monocyte staining detected, Flow cytometr ic analysis showed that early binding appears predominantly directed t owards B-lymphocytes (86.7 +/- 9.0%), and that this binding could not be blocked by antibodies directed towards common B lymphocyte cell. su rface 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 depende nt over a three log dilution using 10(3) color changing units as stand ard. Binding to T-lymphocytes was minimal (< 5% positive), B lineage t umor cells or peripheral blood B cells obtained from HIV infected indi viduals demonstrated reduced binding of M. fermentans. This assay prov ides a good method to study the cellular interactions of mycoplasma an d may help to elucidate pathogenic mechanisms of mycoplasma infections . (C) 1997 WLley-Liss, Inc.