I t has recently been shown that activated t h y m u s -d e p e n d e n t (T) lymphocytes, in addition to their well-recognized ability to cooperate with thymusindependent (B) lymphocytes, can exert "suppressor" effects on a n t i b o d y formation in vivo (1-4) and in vitro (5,6). 1 This effect is exerted both on t h y m u s -d e p e n d e n t and t h y m u s -i n d e p e n d e n t a n t i b o d y responses, and appears to be mediated b y soluble factors. We present here evidence for a similar suppressor effect on the nonspecific mitotic response of B cells to lipopolysaccharide endotoxin (LPS). Materials and MethodsAnimals.--Inbred DA rats were thymectomized at 8 wk of age, irradiated 2 wk later with 900 R (Siemens Stabilipan, 250 kv; Siemens Corp., Iselin, N. J.), and reconstituted with 3 X 10 s syngeneic bone marrow cells. Their spleens and lymph nodes (cervical, axillary, inguinal, and popliteal) harvested 7 wk after irradiation provided "T-deprived" cells, which were compared with cells of nontreated animals. Cell Suspensions and Culture Media.--Teased lymph node cells (LNC) were gently squeezedwith sterile slides in ice-cold medium, passed through nylon mesh, and washed twice by centrifugation. Spleen cells, prepared in the same way, were treated with tris(hydroxymethy])aminomethane-ammonium chloride buffer, pH 7.2, to remove red cells and washed three times. Both suspensions were routinely diluted to 4 X 106/ml viable cells (estimated by trypan blue dye exclusion). Ham's F10 medium (Microbiological Associates, Inc., Bethesda, Md.) containing 10% heat-inactivated fetal calf serum

Cytochalasin B, at concentrations between 0.02 and 0.2 ;Lg/ml, was slightly stimulatory to lymph-node cells from normal rats and greatly potentiated their response to phytohemagglutinin and low concentrations of concanavalin A (mitogens for thymus-derived lymphocytes); it also potentiated the response of thymocytes to phytohemagglutinin. The response of lymphnode cells to lipopolysaccharide endotoxin (a mitogen for thymus-independent lymphocytes) was also enhanced, but only at concentrations in the usual inhibitory range, possibly by inhibition of a "suppressor T-cell" response. Sensitized lymphocytes responding to antigen were not stimulated at all, except at a very high cell density, where inhibition of a "suppressor cell" response was also considered likely. At concentrations of 5-10 Mg/ml or higher, cytochalasin 1B inhibited all responses tested.Cytochalasin B is a fungal product (1) widely used as an inhibitor of cell functions that involve contraction of microfilaments (2). Its target within the cell was undetermined (3) until recently; new evidence establishes firmly its ability to interact directly with the actin moiety of actomyosin, producing a loss of viscosity and of ATPase activity (4). Cytochalasin B, in a restricted low-concentration range (<1 Mug/ml), stimulates chemotaxis (5) and phagocytosis ( §), but inhibits these functions at higher concentrations (>5 Mkg/ml). We report here its ability, in a concentration range of 0.02-0.5 ,g/ml, to stimulate lymphocyte DNA synthesis and to potentiate the responses of lymphocytes to mitogens. MATERIALS AND METHODSAnimals and Sensitization. Inbred DA rats of both sexes, 3-5 months of age, were sensitized by injection of ovalbumin (Nutritional Biochemicals Corp., Cleveland, Ohio), 100 ,g in Freund's complete adjuvant in both hind foot pads (6). Their inguinal and iliac nodes, 9 days after sensitization, were used as sources of sensitized lymph-node cells (LNC). As sources of normal LNC, cervical, axillary, inguinal, and popliteal lymph nodes were harvested from untreated rats. (Grand Island Biological Co., Grand Island, N.Y.), 100 units of penicillin per ml, and 100 ,lg of streptomycin per ml, was used throughout.Reagents. Ovalbumin, PHA (phytohemagglutinin P; Difco Laboratories, Detroit, Mich., ¶), Con A (concanavalin A, twice crystallized, Nutritional Biochemicals Corp., Cleveland, Ohio), and lipopolysaccharide B, Escherichia coli 0111:1B4 (Difco Laboratories, Detroit, Mich.) were dissolved in phosphate-buffered saline (pH 7.4) at various concentrations, and 0.1 ml of the solutions were added to appropriate cultures. Cytochalasin B (Imperial Chemical Industries Ltd., Macclesfield, Chesire, England) was dissolved at 3 mg/ml in (CH3)2SO. Just before use, the stock solution was diluted with phosphate-buffered saline to the desired concentrations, and corresponding dilutions of (CH3)2SO were prepared as controls.Estimation RESULTSEffect of Cytochalasin B on Nonstimulated Cells. Normal LNC (2 X 106/ml) were incubated with cytochalasin B at concentrati...