Antigen Stimulation of 3H Thymidine Incorporation by Subpopulations of Human Peripheral Blood Cells

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Abstract
Using human peripheral blood lymphocytes at various stages of purification we have re-examined the question of whether foreign proteins and hapten-protein conjugates to which the cell donor has no known previous immunization can stimulate in vitro 3H thymidine incorporation. Responses were examined in dextran-sedimented mixed leukocytes, purified lymphocytes isolated by isopycnic centrifugation in a Ficoll-Hypaque gradient (FH cells), and FH cells that had been further purified by passage through a short nylon wool column (nylon purified cells). The dextran sedimented, FH, and nylon purified cells were cultured in autologous serum with various antigens for 5 days and the increase in 3H thymidine incorporation was measured. In mixed leukocytes and FH cells all of the antigens examined (bovine serum albumin, bovine γ-globulin, Candida albicans, histoplasmin, trinitrophenyl-bovine serum albumin, dinitrophenyl-bovine serum albumin, and fluorescein-bovine γ-globulin) produced a significant increase in 3H thymidine uptake. Appropriate controls indicated that the response could not be ascribed to endotoxin. Serum protein and fungal antigens stimulated nylon purified cells as well or better than the leukocyte or FH populations. Contrariwise, the three hapten-protein conjugates inhibited 3H thymidine incorporation by nylon-purified cells. Although there are several alternate possibilities, we favor the interpretation that the in vitro response to serum protein and fungal antigens represents a secondary immune phenomenon in which few if any monocytes are required, whereas stimulation by hapten-protein conjugates involves a primary immune (monocyte dependent) response. However, the possibility that the hapten-protein stimulation is a secondary response involving cells sensitized to cross-reacting antigens is by no means excluded. The inhibition of nylon-purified cells by hapten-protein conjugates may conceivably represent an in vitro correlate of tolerance.

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