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

Abstract

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.

Engineering and Characterization of a Stabilized α1/α2 Module of the Class I Major Histocompatibility Complex Product Ld

“…In addition, class I molecules that lack ␣3/␤2m might be of some use in regulating the activity of T cells by either stimulating the highest avidity, CD8-independent T cells or by eliciting inhibitory signals through the TCR complex in the absence of CD8/lck signaling (58). In this regard, a recent study compared QL9⅐L d monomers and multimers in the 2C system to examine activation requirements (59). It will be of interest to examine how the QL9⅐L d -m31 ligand, which lacks the CD8-binding epitope, will function in eliciting T-cell activity.…”

Conditional MHC class I ligands and peptide exchange technology for the human MHC gene products HLA-A1, -A3, -A11, and -B7

“…Fluorescently labeled MHC multimers are commonly used for the detection of antigen-specific T cells by flow cytometry. In addition, there is an increasing interest in the development of high-throughput assay systems, such as MHC microarrays or combinatorial coding schemes, to visualize pathogen-specific or other disease-associated immune responses in a more comprehensive manner (2,3,21). A major obstacle in the development of these high-throughput approaches for the dissection of antigen-specific CD8 ϩ T cell immunity has been the fact that for each specific peptide-MHC class I complex, a separate production run is required (1,22), limiting the practical use of MHC multimer-based T cell detection to a few T cell specificities.…”

Bi-specific MHC Heterodimers for Characterization of Cross-reactive T Cells*

“…b Complexes-Extracellular domains of the murine MHC class I H-2K b heavy chain carrying a C121R mutation and a non-native C-terminal cysteine introduced at position 282 (17), and full-length human light chain ␤ 2 -microglobulin, were expressed separately as inclusion bodies in Escherichia coli and were folded in vitro by dilution in the presence of excess peptide, as previously described for human class I MHCs (1). Synthetic peptides purified by reverse phase-HPLC were purchased from 21st Century Biochemicals.…”

“…One such reagent would be a bi-specific, MHC heterodimer carrying two different MHC-peptide complexes. MHC homodimers, prepared as fusion proteins using immunoglobulin Fc domains (18) or by cysteine-mediated coupling (19), have already been developed for use as staining reagents, and can be used similarly to MHC (homo)tetramers, although with somewhat lowered binding avidity (17). If MHC heterodimers were available, they would be expected to exhibit specific binding only to T cells carrying receptors able to bind to both component MHC-peptide complexes, as the affinity of monomeric MHCpeptide engagement by T cell receptors (ϳ10 M (28)) generally is too low to survive typical washing protocols.…”

“…In this technique, biotin-labeled recombinant MHC molecules loaded with specific antigenic peptides are oligomerized using fluorescent streptavidin to form highly specific reagents for analysis of antigen-specific T cells in mixed populations using flow cytometry. The use of oligomeric species is necessary because the MHC-TCR interaction is characterized by low affinity and rapid dissociation kinetics (16), which precludes use of labeled MHC monomers as specific staining reagents (17). MHC tetramers conventionally are used in T cell staining protocols, mostly because of the ease of introducing biotin labels into recombinant proteins and the availability of a wide variety of fluorescent streptavidin conjugates (15).…”