Induction of self-tolerance in T cells but not B cells of transgenic mice expressing little self antigen.

Self-tolerance to a transgene-encoded protein, hen egg lysozyme, was examined in the T and B cell repertoires of a series of lines of transgenic mice that expressed different serum concentrations of soluble lysozyme. T cells were tolerant in all lines in which lysozyme was expressed irrespective of the antigen concentration, whereas B cell tolerance did not occur when the serum lysozyme concentration was less than 1.5 nanograms per milliliter (0.1 nM). Induction of elevated transgene expression could restore B cell tolerance. These findings support the hypothesis that autoimmune disease may in some instances arise through a bypass of T cell tolerance.

Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice.

Immunological tolerance has been demonstrated in double-transgenic mice expressing the genes for a neo-self antigen, hen egg lysozyme, and a high affinity anti-lysozyme antibody. The majority of anti-lysozyme B-cells did not undergo clonal deletion, but were no longer able to secrete anti-lysozyme antibody and displayed markedly reduced levels of surface IgM while continuing to express high levels of surface IgD. These findings indicate that self tolerance may result from mechanisms other than clonal deletion, and are consistent with the hypothesis that IgD may have a unique role in B-cell tolerance.

Suppressor T cell memory. Induction and recall of HGG-specific memory suppressor T cells and their role in regulation of antibody production.

Regulation of the antibody response to the hapten, dinitrophenyl (DNP), was studied using human gammaglobulin (HGG) as the carrier in an adoptive transfer system. CBA mice immunized at least 4 weeks previously to HGG or DNP served as the source of T helper cells and hapten-primed B cells, respectively. The addition of spleen cells from donors recently primed to HGG in immunogenic form (aHGG) suppressed the collaborative anti-DNP PFC response as effectively as cells from tolerant donors. The suppressive effect was antigen-specific and was mediated by a T cell with the same phenotype (Ly-1-, Ly-23+, Ia+) and induction kinetics as those previously identified in HGG-tolerant animals. During the primary response to HGG an early burst of helper activity was detected initially, followed by a wave of suppression which peaked at day 7 and subsequently waned, allowing adoptive helper function to reappear during the third week. When previously immunized animals were boosted with soluble HGG after primary suppression had waned, the sequential appearance of helper and suppressor activity was accelerated, and the secondary suppressive effect was more profound and of longer duration than that observed during a primary response. Although helper activity was not apparent during the peak of secondary suppression, helper T cells (Th) had not been functionally deleted since treatment of the donor spleen cells with anti-Ia and complement to deplete suppressor T cells (Ts) before adoptive transfer resulted in significant augmentation of the anti-DNP response. The secondary suppressive effect was formally shown to be mediated by activated Ts effector cells bearing the same surface markers as the cells responsible for primary suppression. The results suggest that the Ts population, like other lymphocyte subsets, contain memory cells capable of rapid reexpression of effector function upon secondary exposure to antigen. These cells are considered to be of a major importance in maintenance of immune homeostasis.

Suppressor T cell memory. II. The role of memory suppressor T cells in tolerance to human gamma globulin.

The transient presence of suppressor T cell (Ts) activity in high-dose tolerance to human gamma globulin (HGG), and its (apparent) absence in low-dose tolerance, have been advanced as strong evidence against the concept that Ts play an important role in maintenance of immunological unresponsiveness. To analyze this question, CBA mice were exposed to high or low doses of deaggregated HGG (dHGG) and later challenged with HGG in immunogenic form (aHGG); their capacity to mount a primary or secondary suppressive response was assessed in an adoptive hapten-carrier system. Primary suppression reached a maximum 7 d after high-dose tolerance induction and gradually waned thereafter, being no longer detectable by day 30-35. Subsequent challenge of tolerant mice with aHGG, however, led to a rapid reactivation of suppression that bore the hallmarks of an anamnestic secondary response, and this effect was still demonstrable 135 d after tolerance induction. It was also shown that a single low dose of dHGG was capable of generating memory for suppression despite the absence of detectable primary suppression, indicating that the latter is not a prerequisite for induction of memory cells. The results were interpreted as indicating that tolerance, like immunity, is a manifestation of specific immunological memory. If tolerance to self-antigens is maintained by a similar mechanism, it would be expected that memory Ts could be induced during the early stages of fetal development. Mice were therefore exposed to tolerogen in utero by injection of their mothers with dHGG at day 7 of gestation, and were assessed at various times after birth for the capacity to exhibit primary or secondary suppression in adoptive transfer. Nonspecific suppression masked any specific effects during the first 5 wk of life. Antigen-specific, primary suppression was demonstrable subsequently until 10-12 wk of age, and if the animals were challenged with aHGG before transfer an anamnestic secondary suppressive response could be elicited up to 6 mo of age. These observations are consistent with the notion that memory Ts may play an important role in the maintenance of self-tolerance.

T cell-dependent suppression of antibody production. I. Characteristics of suppressor T cells following tolerance induction.

Specific immunological tolerance was induced in adult CBA mice by a single injection of deaggregated human IgG (dHGG). Spleen cells taken 7 to 42 days later, produced consistent suppression of a DNP-HGG collaborative antibody response on adoptive transfer into heavily irradiated recipients. Noncentrifuged F(ab')2 fragments of HGG were as effective as dHGG in the production of suppressor cells. Suppression was antigen-specific since HGG-tolerant cells failed to abrogate either a DNP-keyhole limpet hemocyanin collaborative response or antibody production to the noncross-reactive antigen, horse erythrocytes. Pretreatment of the tolerant cell population with anti-Thy-1 serum and complement reversed the suppressive effect. However, purified tolerant T cells obtained by passage through nylon wool or anti-Ig columns were less effective than the original spleen cells in mediating suppression. Analysis of the cell types appearing in the column effluents indicated that the reduction in suppressive activity is best explained by retention of T cells rather than macrophages. Different T cell populations, however, were retained on the two types of columns. In the case of anti-Ig columns, these consisted of Ly-2,3+, Ia+ effector cells, whereas nylon wool columns caused depletion of Ly-1,2,3+ cells which are known to act as amplifiers of suppression. Suppression could not be explained in terms of delay in differentiation of antibody-forming cell precursors since the effect persisted for up to 15 days after transfer of tolerant cells. The demonstration of a reduction in serum anti-DNP and anti-HGG antibodies excluded the possibility of antibody production in sites other than the spleen. A role for anti-carrier antibody-antigen complexes in mediating the effector phase of suppression was rendered unlikely by the finding that the suppressive effect of tolerant cells persisted in the absence of detectable anti-HGG antibody production. Effector T cells mediating suppression in this system were shown to bear the phenotype Ia+, Ly-2,3+ as judged by the effect of pretreatment with appropriate antisera and complement. They were spleen-seeking, but were not detected in the thymus or recirculating lymphocyte pool. Adult thymectomy failed to cause a significant reduction in suppressive activity by tolerant spleen cells indicating that at least a major component of the immediate precursors is not of recent thymic origin.

Influence of antigenic competition on the development of antibody-forming cell clones.

Isoelectric focusing in polyacrylamide gels was used to investigate the anti-sheep red blood cell antibody responses of mice subjected to antigenic competition. A reduction in the number and intensity of antibody bands was found, even in situations where the suppression of IgG antibody titres was minimal, while with large reductions in titre, antibody bands were rarely seen. It thus appeared that the output of individual B-cell clones was severely depressed during competition. It was concluded that inhibition of clonal expansion is an important feature of competition, and that this may reflect a normal regulatory activity which acts to limit cellular proliferation during immune responses. This conclusion was supported by observations on the level of DNA synthesis, following immunization with sheep red cells, in the spleens of normal and suppressed mice.