Various strains of bacteria were previously shown to bind to different lymphocyte subpopulations, i.e., to B cells and to four T cell subpopulations (T1, T2, T3, T4, our classification). We prepared bacterial monolayers and used them to separate lymphocyte subpopulations according to their binding properties. Bacteria were coupled to glutaraldehyde-treated gelatin layers and were subsequently fixed with formaldehyde. Suspensions of peripheral blood lymphocytes (PBL) were centrifuged against the bacterial monolayers and the cells that did not attach were resuspended with the help of a rocking platform. The percentage of PBL that remained attached was similar to the expected values according to the percentage of PBL-binding bacteria in a “rosette formation” assay. The cells that did not adhere to Escherichia coli-0 monolayers, i.e., practically all PBL, responded to the same extent as the unseparated PBL to four mitogens: phytohemagglutinin, concanavalin A, pokeweed mitogen, and streptolysin 0-associated mitogen. The lymphocytes that did not adhere to E. coli-2 monolayers, namely the T3T4 cells, responded poorly to all four mitogens. Also, the response to various doses of concanavalin A of T3T4 cells was much lower than that of purified T cells. The poor response of T3T4 cells was not due to the reduction in viability or to monocyte depletion. Moreover, the T3T4 cells responded in one-way mixed lymphocyte reactions as well as the unseparated PBL. We concluded that T3T4 lymphocytes could be separated by adherence to bacterial monolayers and that they may be functionally different from the other lymphocyte subpopulations identified by binding of bacteria.

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