pMHC Multimers Research Articles

Characterization of naive CD4+ T cells with specificity to self-antigen (137.21)

Abstract During thymic development, T cells with specificity to self-antigens are deleted, thereby ensuring that the peripheral repertoire is devoid of potentially autoreactive clones. However, the ultra-low frequency of naive epitope-specific T cells has made the direct test of this theory technically unfeasible in the past. Using peptide:MHC (pMHC) multimers coupled with magnetic bead enrichment, we have directly studied endogenous polyclonal populations of naive CD4+ T cells with specificity for an I-Ab-restricted model antigen, 2W1S, that is foreign in C57BL/6 mice (B6) or self in mice transgenically expressing the model antigen (Act-2W). Act-2W mice contained a population of 2W1S-specific T cells that was about 25% the size of that in B6 mice, demonstrating that thymic deletion of self-antigen specific T cells is incomplete. Nonetheless, these cells were tolerant to 2W1S, as they did not develop autoimmune responses or respond to large doses of exogenous 2W1S antigen administered in vivo. Antigen receptors expressed by these cells exhibited features of reduced pMHC ligand binding affinity, indicating a preferential loss of high-affinity 2W1S-specific clones during thymic selection. Moreover, a greater proportion of 2W1S-specific T cells expressed FoxP3 in Act-2W mice than in B6 counterparts, suggesting that regulatory T cell development may also function as a mechanism of T cell tolerance to self-antigens. This work was funded by NIH grants PO1-AI35296 and F32-AI063793.

Tricks with tetramers: how to get the most from multimeric peptide-MHC.

The development of fluorochrome-conjugated peptide-major histocompatibility complex (pMHC) multimers in conjunction with continuing advances in flow cytometry has transformed the study of antigen-specific T cells by enabling their visualization, enumeration, phenotypic characterization and isolation from ex vivo samples. Here, we bring together and discuss some of the 'tricks' that can be used to get the most out of pMHC multimers. These include: (1) simple procedures that can substantially enhance the staining intensity of cognate T cells with pMHC multimers; (2) the use of pMHC multimers to stain T cells with very-low-affinity T-cell receptor (TCR)/pMHC interactions, such as those that typically predominate in tumour-specific responses; and (3) the physical grading and clonotypic dissection of antigen-specific T cells based on the affinity of their cognate TCR using mutant pMHC multimers in conjunction with new approaches to the molecular analysis of TCR gene expression. We also examine how soluble pMHC can be used to examine T-cell activation, manipulate T-cell responses and study allogeneic and superantigen interactions with TCRs. Finally, we discuss the problems that arise with pMHC class II (pMHCII) multimers because of the low affinity of TCR/pMHCII interactions and lack of 'coreceptor help'.

Impact of CD8–MHC class I interaction in detection and sorting efficiencies of antigen-specific T cells using MHC class I/peptide multimers: contribution of pMHC valency

Recombinant soluble multimeric forms of MHC class I molecules loaded with antigenic peptides (pMHC) have turned out to be particularly useful to detect and isolate specific T cells. These applications both rely on the oligomerization of pMHC monomers in order to compensate for their inherent low affinity for the TCR. In this study, we have evaluated the precise contribution of CD8-pMHC interaction on the specificity and sorting efficiency of pMHC multimers according to their degree of oligomerization. To this end several wild-type versus mutated pMHC complexes (A*0201, B*0701, B*0801, B*3501) carrying point mutations known to reduce (245V mutants) or to abrogate (227,8KA mutants) CD8-pMHC interaction and showing various degrees of valency have been used. We show that irrespective of the HLA allele tested, 245V mutation strongly improves the binding specificity and immunomagnetic sorting efficiency of pMHC multimers. Our results also indicate that the contribution of CD8 to the binding of pMHC multimers to specific CD8+ T cells is inversely correlated to the degree of pMHC oligomerization. Consequently, efficient staining or specific sorting of high-affinity T cells (i.e. CD8 independent) can only be achieved using 227,8KA pMHC complexes with low-order oligomerization.

MHC multimers: expanding the clinical toolkit

T cell recognition of specific antigen is initiated by the interaction between TCR molecules and peptide–MHC complexes (pMHC) expressed on the surface of antigenpresenting cells. Soluble pMHC molecules can substitute as ligands in this interaction, binding specifically to T cells expressing the appropriate TCR. Because the monomeric interaction of TCR–pMHC is of low avidity [1–6], multimerization of the pMHC complex facilitates the binding reaction, and a large number of studies have now been performed in which pMHC multimers are used to detect and characterize specific T cells in patients and normal individuals. This technology, commonly referred to as “MHC tetramers,” was originally developed for the analysis of class I restricted CD8 T cells, in which the TCR recognizes a MHC class I–antigen complex [7]. Similar technology has been used for class I recognition by NK receptors [8–10], for CD1 recognition by NKT cells [11,12], for MIC recognition by -TCR molecules [13], and for recognition of the peptide–HLA class II complex by CD4 T lymphocytes in humans [14–19] and mice [20,21].

Dissecting TCR-MHC/peptide complex interactions with A2/peptide multimers incorporating tumor antigen peptide variants: crucial role of interaction kinetics on functional outcomes.

Soluble peptide/MHC-class-I (pMHC) multimers have recently emerged as unique reagents for the study of specific interactions between the pMHC complex and the TCR. Here, we assessed the relative binding efficiency of a panel of multimers incorporating single-alanine-substituted variants of the tumor-antigen-derived peptide MAGE-A10(254-262) to specific CTL clones displaying different functional avidity. For each individual clone, the efficiency of binding of multimers incorporating MAGE-A10 peptide variants was, in most cases, in good although not linear correlation with the avidity of recognition of the corresponding variant. In addition, we observed two types of discrepancies between efficiency of recognition and multimer binding. First, for some peptide variants, efficient multimer binding was detected in the absence of measurable effector functions. Some of these peptide variants displayed antagonist activity. Second, when comparing different clones we found clear discrepancies between the dose of peptide required to obtain half-maximal lysis in CTL assays and the binding efficiency of the corresponding multimers. These discrepancies, however, were resolved when the differential stability of the TCR/pMHC complexes was determined. For individual clones, decreased recognition correlated with increased TCR/pMHC off-rate. TCR/pMHC complexes formed by antagonist ligands displayed off-rates faster than those of TCR/pMHC complexes formed with weak agonists. In addition, when comparing different clones, the efficiency of multimer staining correlated better with relative multimer off-rates than with half-maximal lysis values. Altogether, the data presented here reconcile and extend our previous results on the impact of the kinetics of interaction of TCR with pMHC complexes on multimer binding and underline the crucial role of TCR/pMHC off-rates for the functional outcome of such interactions.

T Cell Activity Correlates with Oligomeric Peptide-Major Histocompatibility Complex Binding on T Cell Surface

Recognition of virally infected cells by CD8+ T cells requires differentiation between self and nonself peptide-class I major histocompatibility complexes (pMHC). Recognition of foreign pMHC by host T cells is a major factor in the rejection of transplanted organs from the same species (allotransplant) or different species (xenotransplant). AHIII12.2 is a murine T cell clone that recognizes the xenogeneic (human) class I MHC HLA-A2.1 molecule (A2) and the syngeneic murine class I MHC H-2 D(b) molecule (D(b)). Recognition of both A2 and D(b) are peptide-dependent, and the sequences of the peptides recognized have been determined. Alterations in the antigenic peptides bound to A2 cause large changes in AHIII12.2 T cell responsiveness. Crystal structures of three representative peptides (agonist, null, and antagonist) bound to A2 partially explain the changes in AHIII12.2 responsiveness. Using class I pMHC octamers, a strong correlation is seen between T cell activity and the affinity of pMHC complexes for the T cell receptor. However, contrary to previous studies, we see similar half-lives for the pMHC multimers bound to the AHIII12.2 cell surface.

The Human CD8 Coreceptor Effects Cytotoxic T Cell Activation and Antigen Sensitivity Primarily by Mediating Complete Phosphorylation of the T Cell Receptor ζ Chain

Recognition of antigen by cytotoxic T lymphocytes (CTL) is determined by interaction of both the T cell receptor and its CD8 coreceptor with peptide-major histocompatibility complex (pMHC) class I molecules. We examine the relative roles of these receptors in the activation of human CTL using mutations in MHC class I designed to diminish or abrogate the CD8/pMHC interaction. We use surface plasmon resonance to determine that point mutation of the alpha3 loop of HLA A2 abrogates the CD8/pMHC interaction without affecting the affinity of the T cell receptor/pMHC interaction. Antigen-presenting cells expressing HLA A2 which does not bind to CD8 fail to activate CTL at any peptide concentration. Comparison of CTL activation by targets expressing HLA A2 with normal, abrogated, or diminished CD8/pMHC interaction show that the CD8/pMHC interaction enhances sensitivity to antigen. We determine that the biochemical basis for coreceptor dependence is the activation of the 23-kDa phosphoform of the CD3zeta chain. In addition, we produce mutant MHC class I multimers that specifically stain but do not activate CTL. These reagents may prove useful in circumventing undesirable activation-related perturbation of intracellular processes when pMHC multimers are used to phenotype antigen-specific CD8+ lymphocytes.