Activation of Mouse Spleen Cells with Acetylated Concanavalin Coupled to Red Blood Cell Stroma

Abstract

There is currently much interest in how antigen must be presented to the lymphocyte surface to induce blast transformation, and in recent reviews both Moller (1) and Mitchison (2) stressed the importance of multipoint binding. Mitogens also are useful agents for studying events at the lymphocyte membrane: phytohemagglutinin (PHA) bound to red blood cells (RBC) potentiates lymphocyte stimulation (3), and mouse B cells can be stimulated by PHA bound to “Sepharose” particles (4) or concanavalin A (Con A) cross-linked to the bottom of tissue culture petri dishes (5) but not by soluble PHA or by soluble Con A (5). The following experiments show that the acetylation of concanavalin A eliminates its ability to stimulate murine splenic lymphocytes to undergo DNA synthesis. Activity was restored by coupling tritiated acetylated Con A ([³H] AcConA) to RBC stroma.

Materials and Methods. Cell suspensions were made from spleens of SWR inbred mice raised in our laboratory from pairs obtained from Jackson Memorial Laboratory, Bar Harbor, ME. Pooled samples were from all males or all females. Cells were cultured by the method of Adler et al. (6). We prepared Con A by the procedure of Agrawal and Goldstein (7), and the New England Nuclear Corp., Boston, MA acetylated it with [³H] acetic anhydride (8); specific activity of the [³H] AcConA was 10 μCi/mg.

Binding of [³H]AcConA to spleen cells was measured by a procedure described by Stobo, Rosenthal and Paul (10). [³H] AcConA was added to 5 × 10⁶ cells at 4° for 30 min, with and without unmodified Con A. Cells were suspended in 0.5 ml RPMI-1640 containing 0.1% sodium azide and 1% bovine serum albumin (BSA). After 30 min, binding was determined: the cells were washed with RPMI-1640 containing 5% BSA, and lysed with 0.5 ml of 1 N NaOH, and radioactivity was measured in a mixture of Liquifiuor (New England Nuclear Corp.) and BBS-2 (Beckman).