The Tolerant Cell: Direct Evidence for Receptor Blockade by Tolerogen

Abstract

Abstract Autoradiographic studies were done with spleen lymphoid cells of normal and tolerant mice to investigate three questions: 1) Does the specificity of lymphocyte receptors change during tolerance? 2) Are the antigen-binding cells (ABC) eliminated in tolerant animals? 3) Does the tolerogen and/or antigen stay on the surface of the ABC in tolerant animals? BDF1 mice were made tolerant by dinitrophenyl isogeneic IgG (DNP-MGG, the tolerogen) and the state of tolerance was tested by challenging the animals with dinitrophenyl-keyhole limpet hemocyanine (DNP-KLH, the antigen). It was found that the lymphocyte receptor of the ABC was specific for the hapten DNP in normal and tolerant animals. Thus, we have no evidence that the hapten-specific B cell receptor changes its specificity during tolerance and recognizes antigenic determinants present on the tolerogen but not the antigen. The number of splenic lymphocytes with receptors free to bind either the antigen or the tolerogen was the same in normal animals and in animals tolerant for 1 week, whereas it was markedly decreased in animals tolerant for 4 weeks. For determining whether the tolerogen and/or the antigen stayed on the cell surface receptor of the ABC in tolerant animals, indirect autoradiography with labeled anti-DNP antibody was done. Purified antibody to DNP was labeled with 125I and reacted with splenic lymphocytes. We found that animals tolerant for 1 week had significantly more cells which bound anti-DNP than either normal animals or animals which received DNP-KLH. Animals tolerant for 4 weeks had even larger numbers of cells which bound anti-DNP-125I than the mice tolerant for 1 week. Blocking studies showed that the binding of anti-DNP in tolerant animals was specific for DNP on cell surfaces. These experiments demonstrated the functional divergence in the tolerogenic and immunogenic forms of the same antigenic determinant (DNP). The tolerogen remains on the cell surface of the ABC although the antigen does not. They also show that the DNP clone of cells is not deleted in tolerance. This provides direct evidence for the receptor blockade mechanism of tolerance induction and the existence of the tolerant cell (i.e., ABC with receptor “paralyzed” by tolerogen). We suggest that the existence of this tolerant cell may account for the mechanism of acquired and self tolerance.