Tolerance Induction to a Foreign Protein Antigen: Analysing the Role of B Cells in Establishing Peripheral Tolerance

Abstract

Tolerance to self proteins is largely dependent upon the deletion of immature, self-specific T and B cells in the thymus and bone marrow. Although highly efficient, the elimination of these self-reactive lymphocytes is dependent on the expression of their target antigen in these primary lymphoid organs. Many proteins, however, such as hormones, are developmentally regulated and expressed at different stages of life, while other proteins are expressed outside the thymus and marrow. To ensure self-tolerance, other mechanisms must exist to inactivate or prevent the activation of mature, potentially self-reactive lymphocytes and maintain peripheral tolerance. T cell activation requires direct recognition of a specific protein fragment, presented on the surface of an antigen presenting cell (APC), as well as the interaction between various T cell and APC surface molecules. In the absence of the costimulatory signals provided by these ligand-pair interactions and lymphokines, antigen recognition leads to T cell inactivation and tolerance to the protein. Since many autoimmune disorders appear to be based upon the aberrant activation of mature T lymphocytes, it is important to identify and understand the mechanisms of peripheral tolerance. The obvious importance of the APC in initiating the T cell immune response has led our lab to examine one of the many antigen-processing cells, the B lymphocyte. Our studies have shown that B cells are highly efficient APC and can present antigen at very low doses to cultured T cell lines. In addition, we have found that we can induce tolerance, as measured by a reduced antibody response to an immunogenic form of the protein, in naive, normal mice by targeting a foreign protein to their B cells for antigen processing and presentation. Tolerance in the treated mice can be traced to a lesion in the T cell compartment of the animals, thus suggesting that B cells can act as tolerizing APC for peripherally expressed antigens. To further explore this idea and find more direct evidence for the role of B cells in establishing peripheral tolerance, we developed a model system that would more closely resemble in vivo conditions. This thesis tests and provides additional evidence for the hypothesis that B cells are tolerizing antigen presenting cells for peripherally expressed protein antigens. Tolerance to the foreign protein human μ chain, is induced in normal recipient mice by the transfusion of splenocytes from transgenic mice that express the membrane-bound form of μ on their B cells. Tolerance is antigen-specific since the transfused recipients' antibody production to the irrelevant protein chicken IgG is not compromised. Only viable transgenic spleen cells are tolerogenic and even when human μ chain is accessible to other APCs for presentation, tolerance can be induced by the transfusion of live μ transgenic splenoctyes. These data suggested that the transfused μ chain-expressing B cells are the tolerizing APCs which was confirmed by experiments that compared the tolerizing abilities of purified B and T cells from the transgenic mice. Adoptive transfer experiments showed that the recipients' T cell response to human μ was impaired but an analysis of the isotypes produced by tolerized mice did not indicate that either helper T cell subset was specifically compromised. Splenocytes from human μ chain-secreting transgenic B cells also induce tolerance to human μ in nontransgenic mice. Although human μ chain-expressing B cells were not detected in transfused mice, the presence of measurable levels of human IgM in the sera of mice transfused with μ chain-secreting spleen cells suggests that the transfused transgenic B cells persist in their new host. In addition, the tolerizing ability of both resting and activated membrane-bound μ chain B cells was compared. Lipopolysaccharide (LPS)-activated transgenic spleen cells do not tolerize, nor do they prime for antibody to human μ, thus suggesting that the induction of costimulatory molecules on the transgenic B cells inhibits tolerance induction. To more specifically address this, human μ chain-expressing mice were bred to transgenic mice that express the costimulatory molecule, B7-1 (CD80), on their B cells. Double transgenic splenocytes, in which the B cells bear both human μ and B7-1, did not induce tolerance to human μ chain, a result that supports the idea that activated B cells are not tolerogenic. Together the data in this thesis show that resting B cells can process and present a foreign endogenous antigen in a tolerogenic manner to the immune system and suggest a role for the B cell in the maintenance of peripheral tolerance.