Immune-mediated disorders such as autoimmune diseases, graft-versus-host disease or allergies, are a major contemporary burden. Despite significant advances in the field of immunological tolerance, immunosuppressive drugs are still the current hallmark for treatment of transplanted recipients or patients with autoimmune disorders. While these drugs ameliorate the lives of the patients, benefits are unfortunately counterbalanced by the need for life-long treatment and non-negligible adverse side effects. The development of a therapy affecting the immune system in an antigen-specific manner is thus required for these and many other situations. Pursuing this aim, our laboratory has recently developed a novel approach to induce antigen-specific T cell tolerance through targeting antigens to erythrocytes in situ. Apoptotic cells are known to be processed tolerogenically in vivo. The concept of our therapeutic approach is based on the premise that as aged erythrocytes are cleared through an apoptotic-like mechanism, the surface-bound antigen payload will be cleared tolerogenically along with the eryptotic debris. While great strides have been made in showing the potential of the approach in inducing antigen-specific immune tolerance, little is known about the mechanisms driving tolerance. The aim of this thesis is to further understand the mechanisms behind induction of tolerance by erythrocyte-targeted antigens. Aspects such as memory of tolerance, dependence on T cell receptor (TCR) affinity for the peptide major histocompatibility complex (pMHC) and molecular pathways involved in this process will be addressed. Using ovalbumin (OVA) as a model protein antigen, we demonstrated long-term induction of immune tolerance after treatment with erythrocyte-targeted antigens, and requirement of CD25+FOXP3+ regulatory T cells for maintenance of the tolerogenic state. Signaling through programmed death-1/programmed death ligand-1 (PD-1/PD-L1), but not through cytotoxic T lymphocyte antigen 4 (CTLA4), was shown to be required for antigen-specific T cell deletion, anergy and expression of regulatory markers. The recent evidences of a direct implication of low affinity T cells as active contributor of self-destruction in autoimmunity raise questions regarding the ability of peripheral tolerance to deal with T cells of lower affinities. To explore the impact of TCR-pMHC affinity on induction of peripheral tolerance by erythrocyte-targeted antigens, erythrocyte-targeted altered peptide ligands with gradated affinity for the TCR of OTI CD8+ T cells were used. We demonstrated that tolerance induction is dependent on TCR strength as well as activation state of low affinity T cells. Finally, RNA sequencing was performed to determine how genes are regulated in CD8+ T cells and which pathways are activated after treatment with erythrocyte-targeted antigens. Comparison with gene sets characteristic of T cells undergoing anergy or deletional tolerance revealed the similarity between response induced by the natural event of peripheral tolerance and tolerance induced by erythrocyte-targeted antigens. Together, these results pave the way for the continued exploration of erythrocyte-targeting for prophylactic and therapeutic modulation of the immune system.