The induction of hapten-specific immunologic tolerance and immunity in B lymphocytes. IV. Induction of TNP-specific unresponsiveness in vitro with serum from tolerant mice
Fidler, J.M.
Journal of Immunology 121(4): 1558-1565
1978
ISSN/ISBN: 0022-1767 PMID: 308969 Document Number: 138371
The serum from mice rendered specifically tolerant (TolS) to the trinitrophenyl (TNP) hapten by the injection of trinitrobenzenesulfonic acid (TNBS) was tested in vitro for its ability to induce unresponsiveness in untreated spleen cells. Conjugated with TNP at a concentration of .apprxeq. 4 .times. 10-4 M, TolS induced complete TNP-specific tolerance in cultures of spleen cells challenged with TNP-lipopolysaccharide (TNP-LPS) and 4-hydroxy-3-iodo-5-nitrophenylacetic acid conjugated polymerized flagellin (NIP-POL). By using normal mouse serum (NMS) as a control, TNP-specific tolerance in the in vitro antibody response was induced by cultivation of spleen cells with amounts of TolS of 10-4 to 10-2 ml (equivalent to 4 .times. 10-8 to 4 .times. 10-6 M TNP) in 1 ml cultures of 10 .times. 106 viable spleen cells. Thymic-derived (T) cells were not required for the induction of the unresponsiveness in TNP-specific bone marrow-derived (bursal equivalent, B) lymphocytes. TolS induced tolerance in cultures challenged with T cell independent (TI) or T cell dependent (TD) forms of the TNP hapten. TolS was nonimmunogenic under the in vitro culture conditions used, and did not affect control responses. Although it was most effective when harvested 4 or fewer days after TNBS injection of donor mice, TolS exerted a tolerogenic effect if obtained 2 wk after such treatment. TolS was active across histocompatibility and immunoglobulin allotype barriers and exerted its tolerogenic effect in the presence of concentrations of antigen optimal for the induction of immunity. Complete tolerance was induced by pretreatment of spleen cells with TolS and extensive washing before the addition of immunogen. Following dose response kinetics, the time required and the level of unresponsiveness induced during preincubation with TolS were directly related to the dose of tolerogen utilized. The inability of TNP-lysine to act as a tolerogen and the nondialysable nature of TolS suggested that the active component of this serum is a macromolecule and not merely TNBS. Fractionation of TolS by (NH4)2SO4 resulted in virtually complete recovery of the tolerogenic activity in the globulin (nonalbumin) fraction. Although haptenated serum .gamma.-globulins would be present in this fraction, other constituents with potential tolerogenic capacity may be present (i.e., various serum proteins or cell surface membrane fragments that are trinitrophenylated). TNBS-induced tolerance in vivo may be mediated by a component of TolS that is active as a tolerogen in vitro.