In vivo administration of trinitrobenzenesulfonic acid (TNBS) induces a state of immunologic tolerance to the trinitrophenyl (TNP) hapten in the bone marrow-derived (bursal equivalent, B) lymphocytes of mice. An in vitro culture system utilized to generate an antibody forming cell (AFC) response was characterized and used to evaluate the unresponsive state. The tolerance induced by TNBS injection was TNP-specific and dose-dependent, and was present in response to both the thymic-derived (T) cell independent (TI) antigen TNP-lipopolysaccharide (TNP-LPS) and a T cell dependent (TD) form of TNP (TNP-sheep erythrocytes, TNP-SRC). Unresponsiveness persisted for over 8 weeks in response to either antigen.
Experiments were conducted to determine the mechanism of TNBS-induced immunologic tolerance with emphasis upon differentiation between B cell deletion and blockade of surface immunoglobulin (sIg) receptors by tolerogen. Tolerant and normal spleen cells responded independently to TNP-LPS when co-cultured, indicating that the observed unresponsiveness in TNBS-treated spleen cells was not due to suppression of TNP-specific B lymphocytes by other cells in the preparation. Used at polyclonal B cell-activating concentrations, TNP-LPS did not reverse the tolerant state. Additionally, unconjugated LPS in either serum-containing or serum-free cultures did not break tolerance. Because polyclonal B cell activators do not act in triggering through sIg receptors, the failure of a polyclonal stimulator to reverse the tolerant state suggests that blockade of TNP-specific surface receptors is not the mechanism of tolerance. Tolerant spleen cells did not reduce the number of TNP-specific plaque forming cells (PFC) produced upon stimulation of normal cells by a wide range of LPS doses, supporting the conclusion that suppressive cells or factors are absent. Antigen-free incubation of tolerant spleen cells for 24 hr and washing at 4°C did not abrogate the unresponsive state when followed by challenge with either TNP-LPS or TNP-SRC. Washing at elevated temperatures after pre-culturing in order to avoid possible reassociation of sIg-bound TNP conjugates was equally unsuccessful in reversing tolerance. Thus, antigen-free precultivation where unblocking or turnover and resynthesis of sIg receptors should have taken place yielded no evidence supporting receptor blockade as the basis of TNBS-induced tolerance. Treatment with the proteolytic enzyme pronase, in order to strip potential tolerogen from the cell surface, failed to reverse the unresponsive state. Incubation with anti-TNP or anti-Ig antibody under conditions designed to allow capping and removal of surface-bound trinitrophenylated moieties or sIg-bound tolerogen also revealed a stable tolerant state.
These results and evidence presented previously support the conclusion that TNBS induces tolerance in vivo through a deletion of TNP-specific B lymphocytes.