Jennifer D. Stone

Soluble peptide–MHC monomers cause activation of CD8+ T cells through transfer of the peptide to T cell MHC molecules

T cell receptor (TCR)-mediated activation of CD4+ T cells is known to require multivalent engagement of the TCR by, for example, oligomeric peptide–MHC complexes. In contrast, for CD8+ T cells, there is evidence for TCR-mediated activation by univalent engagement of the TCR. We have here compared oligomeric and monomeric Ld and Kb peptide–MHC complexes and free peptide as stimulators of CD8+ T cells expressing the 2C TCR. We found that the monomers are indeed effective in activating naïve and effector CD8+ T cells, but through an unexpected mechanism that involves transfer of peptide from soluble monomers to T cell endogenous MHC (Kb) molecules. The result is that T cells, acting as antigen-presenting cells, are able to activate other naïve T cells.

T-cell activation by soluble MHC oligomers can be described by a two-parameter binding model.

T-cell activation is essential for initiation and control of immune system function. T cells are activated by interaction of cell-surface antigen receptors with major histocompatibility complex (MHC) proteins on the surface of other cells. Studies using soluble oligomers of MHC-peptide complexes and other types of receptor cross-linking agents have supported an activation mechanism that involves T cell receptor clustering. Receptor clustering induced by incubation of T cells with MHC-peptide oligomers leads to the induction of T-cell activation processes, including downregulation of engaged receptors and upregulation of the cell-surface proteins CD69 and CD25. Dose-response curves for these T-cell activation markers are bell-shaped, with different maxima and midpoints, depending on the valency of the soluble oligomer used. In this study, we have analyzed the activation behavior using a mathematical model that describes the binding of multivalent ligands to cell-surface receptors. We show that a simple equilibrium binding model accurately describes the activation data for CD4(+) T cells treated with MHC-peptide oligomers of varying valency. The model can be used to predict activation and binding behavior for T cells and MHC oligomers with different properties.

CD8 T Cells, Like CD4 T Cells, Are Triggered by Multivalent Engagement of TCRs by MHC-Peptide Ligands but Not by Monovalent Engagement

T cell activation is initiated by recognition of antigenic peptide presented in complex with MHC molecules on the surface of APCs. The mechanism by which this recognition occurs is still unclear, and many models exist in the literature. CD4 T cells have been shown to respond to soluble oligomers of activating class II MHC-peptide complexes, but not to soluble monomers. In determining the reactivity of CD8 T cells to soluble activating class I MHC-peptide complexes, a complicating phenomenon had been observed whereby peptide from soluble complexes was loaded onto cell surface MHCs on the T cells and re-presented to other T cells, clouding the true valency requirement for activation. This study uses soluble allogeneic class I MHC-peptide monomers and oligomers to stimulate murine CD8 T cells without the possible complication of peptide re-presentation. The results show that MHC class I monomers bind to, but do not activate, CD8 T cells whether the cells are in solution or adhered to a surface. Monomeric MHC class I binding can antagonize the stimulation triggered by soluble oligomers, a phenomenon also observed for CD4 T cells. Dimeric engagement is necessary and sufficient to stimulate downstream activation processes including TCR down-regulation, Zap70 phosphorylation, and CD25 and CD69 up-regulation, even in T cells that do not express the MHC coreceptor CD8. Thus, the valency dependence of the response of CD8 T cells to soluble MHC-peptide reagents is the same as previously observed for CD4 T cells.

Exploration of the P6/P7 region of the peptide-binding site of the human class II Major Histocompatability Complex Protein HLA-DR1

Crystal structures of the class II major histocompatibilty complex (MHC) protein, HLA-DR1, generally show a tight fit between MHC and bound peptide except in the P6/P7 region of the peptide-binding site. In this region, there is a shallow water-filled pocket underneath the peptide and between the pockets that accommodate the P6 and P7 side chains. We investigated the properties of this pocket with the idea of engineering substitutions into the corresponding region of peptide antigens to increase their binding affinity for HLA-DR1. We investigated d-amino acids and N-alkyl modifications at both the P6 and P7 positions of the peptide and found that binding of peptides to HLA-DR1 could be increased by incorporating an N-methyl substitution at position 7 of the peptide. The crystal structure of HLA-DR1 bound to a peptide containing a P7 N-methyl alanine was determined. The N-methyl group orients in the P6/P7 pocket, displacing one of the waters usually bound in this pocket. The structure shows that the substitution does not alter the conformation of the bound peptide, which adopts the usual polyproline type II helix. An antigenic peptide carrying the N-methyl modification is taken up by antigen-presenting cells and loaded onto endogenous class II MHC molecules for presentation, and the resultant MHC-peptide complexes activate antigen-specific T-cells. These results suggest a possible strategy for increasing the affinity of weakly immunogenic peptides that might be applicable to the development of vaccines and diagnostic reagents.

Engineering High-Affinity T Cell Receptor/ Cytokine Fusions for Therapeutic Targeting

Jennifer D. Stone1, Yiyuan Yin2, Min Mo2, K. Scott Weber3, David L. Donermeyer3, Paul M. Allen3,Roy A. Mariuzza2,4 and David M. Kranz 1 1Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, 2Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, 3Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, 4Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, USA