Summary In order to survey a universe of MHC-presented peptide antigens whose numbers greatly exceed the diversity of the T cell repertoire, T cell receptors (TCRs) are thought to be cross-reactive. However, the nature and extent of TCR cross-reactivity has not been conclusively measured experimentally. We developed a system to identify MHC-presented peptide ligands by combining TCR selection of highly diverse yeast-displayed peptide-MHC libraries with deep sequencing. While we identified hundreds of peptides reactive with each of five different mouse and human TCRs, the selected peptides possessed TCR recognition motifs that bore a close resemblance to their known antigens. This structural conservation of the TCR interaction surface allowed us to exploit deep sequencing information to computationally identify activating microbial and self-ligands for human autoimmune TCRs. The mechanistic basis of TCR cross-reactivity described here enables effective surveillance of diverse self and foreign antigens, but without necessitating degenerate recognition of non-homologous peptides.

Biomaterial scaffolds that enrich and modulate immune cells in situ can form the basis for potent immunotherapies to elicit immunity or reëstablish tolerance. Here, we explore the potential of an injectable, porous hydrogel to induce a regulatory T cell (Treg) response by delivering a peptide antigen to dendritic cells (DCs) in a non-inflammatory context. Two methods are described for delivering the BDC peptide from pore-forming gels in the NOD (non-obese diabetic) mouse model of type 1 diabetes: encapsulation in poly(lactide-co-glycolide) (PLG) microparticles, or direct conjugation to the alginate polymer. While particle-based delivery leads to antigen-specific T cells responses in vivo, PLG particles alter the phenotype of the cells infiltrating the gels. Following gel-based peptide delivery, transient expansion of endogenous antigen-specific T cells is observed in the draining lymph nodes. Antigen-specific T cells accumulate in the gels, and, strikingly, ~60% of the antigen-specific CD4+ T cells in the gels are Tregs. Antigen-specific T cells are also enriched in the pancreatic islets, and administration of peptide-loaded gels does not accelerate diabetes. This work demonstrates that a non-inflammatory biomaterial system can generate antigen-specific Tregs in vivo, which may enable the development of new therapies for the treatment of transplant rejection or autoimmune diseases.

The T cell costimulatory receptor CD28 is required for the full activation of naïve T cells and for the development and maintenance of Foxp3+ regulatory T (Treg) cells. We showed that the cytoplasmic domain of CD28 was bound to the plasma membrane in resting cells and that ligand binding to CD28 resulted in its release. Membrane binding by the CD28 cytoplasmic domain required two clusters of basic amino acid residues, which interacted with the negatively charged inner leaflet of the plasma membrane. These same clusters of basic residues also served as interaction sites for Lck, a Src family kinase critical for CD28 function. This signaling complex was further stabilized by the Lck-mediated phosphorylation of CD28 Tyr207 and the subsequent binding of the Src homology 2 (SH2) domain of Lck to this phosphorylated tyrosine. Mutation of the basic clusters in the CD28 cytoplasmic domain reduced the recruitment to the CD28-Lck complex of protein kinase Cθ (PKCθ), which serves as a key effector kinase in the CD28 signaling pathway. Consequently, mutation of either a basic cluster or Tyr207 impaired CD28 function in mice as shown by the reduced thymic differentiation of FoxP3+ Treg cells. On the basis of these results, we propose a previously un-described model for the initiation of CD28 signaling.

The T cell receptor (TCR):CD3 complex transduces signals that are critical for optimal T cell development and adaptive immunity. In resting T cells, the CD3ε cytoplasmic tail associates with the plasma membrane via a proximal basic-rich stretch (BRS). Here we show that mice lacking a functional CD3ε-BRS exhibited substantial reductions in thymic cellularity and limited CD4−CD8− double negative-3 (DN3) to DN4 thymocyte transition, due to enhanced DN4 TCR signaling resulting in increased cell death and TCR downregulation in all subsequent populations. Furthermore, positive, but not negative, T cell selection was affected in mice lacking a functional CD3ε-BRS, which led to limited peripheral T cell function and substantially reduced responsiveness to influenza infection. Collectively, these results indicate membrane association of the CD3ε signaling domain is required for optimal thymocyte development and peripheral T cell function.