Delayed hypersensitivity in vitro: its mediation by cell-free substances formed by lymphoid cell-antigen interaction.

It has long been thought that delayed hypersensitivity is mediated by cells or cell-associated substances. In an attempt to investigate the mechanism of delayed hypersensitivity, in vitro studies using the inhibition of cell migration by specific antigen as an assay have been carried out. In this in vitro system, it has been shown that peritoneal exudate cells taken from guinea pigs exhibiting delayed hypersensitivity and placed in capillary tubes are inhibited from migration by specific antigen.1' 2 Moreover, when mixed populations of normal and sensitive cells were prepared, it was observed that if as few as 2.5 per cent of the cells in a population were from a sensitive animal, the whole population (97.5% normal cells) would be inhibited by antigen. 3 The results of recent experiments suggest that the lymphocyte is the specifically sensitive cell in this system. It was found that lymph node cells (approximately 95% lymphocytes) obtained from sensitive guinea pigs will, when mixed with a population of normal peritoneal exudate cells, cause the whole population to be inhibited by specific antigen.4 It is of note that such sensitive lymphoid cells, when assayed alone in culture, are not themselves inhibited from migrating by antigen. Experiments were initiated in an attempt to determine the manner by which these sensitive lymphoid cells achieved their effect. The results, here reported, demonstrate that, following incubation of sensitive lymphoid cells with specific antigen for 24 hr, a nondialyzable substance is detected in the cell-free supernatants which inhibits the migration of normal peritoneal cells. Materials and Methods. -Sensitization: The antigen-s used were ovalbumin (Worthington) and o-chlorobenzoyl chloride conjugated to bovine gamma globulin (OCBC-BGG) by the method of Benacerraf and Levine,5 kindly supplied by Dr. Y. Borel. Guiniea pigs of the Hartley strain weighing 500-800 gm were sensitized with the appropriate antigen diluted in saline and emulsified in an equal volume of complete adjuvant (Difco TI37Ra). Each animal received a total dose of 100 ,ug of antigen distributed iTito the four footpads, 0.1 ml per footpad. Tissue culture media: The basic tissue culture media used throughouit were mirnimal essential media, Eagle 12-1.25 (Microbiological Associates, Bethesda, Md.) containing 15% normal guinea pig serum and 85 units of penicillin and 85 ,ug of streptomycin/ml. Preparation of lymph node cell suspensions: Twelve to twenty one days after sensitization axillary, inguinal and popliteal lymph nodes were obtained aseptically from guinea pigs which had beeni anesthetized with ether arid exsangulinated by cardiac puncture. The nodes were diced into tissue culture medium, teased gently with mouse-toothed forceps, and the resulting cell sIIspensioni was pipetted into centrifuge tubes. The suspension was allowed to stand for 4 min so that the tissue fragments settled by gravity. The superiiatanit was then removed to a fresh tube. After three such settlings the cell suspensions were essentially free of tissue fragments. The majority of cells were viable as assessed by trypan-blue exclusion, and the preparations containied 90-95% lymphocytes by Wright's stain and phase microscopy. Suspensions were adjusted to a final concentration of 1.8 X 107 cells per ml. Aliquots of these suspensions were made to contain ovalbumin, or OCBC-BGG 100 ,ug/ml. Suspensions not containing antigen were also prepared. In each experiment, suspensions were incuibated in specific antigen and an unrelated antigen.