Two-stage cooperative T cell receptor-peptide major histocompatibility complex-CD8 trimolecular interactions amplify antigen discrimination.

The T cell receptor (TCR) and CD8 bind peptide-major histocompatibility complex (pMHC) glycoproteins to initiate adaptive immune responses, yet the trimolecular binding kinetics at the T cell membrane is unknown. By using a micropipette adhesion frequency assay, we show that this kinetics has two stages. The first consists of TCR-dominant binding to agonist pMHC. This triggers a second stage consisting of a step increase in adhesion after a one second delay. The second-stage binding requires Src family kinase activity to initiate CD8 binding to the same pMHC engaged by the TCR. This induced trimeric-cooperative interaction enhances adhesion synergistically to favor potent ligands, which further amplifies discrimination. Our data reveal a TCR-CD8 positive-feedback loop involved in initial signaling steps that is sensitive to a single pMHC is rapid, reversible, synergistic, and peptide discriminative.

Progression of relapsing-remitting demyelinating disease does not require increased TCR affinity or epitope spread.

In this study, we investigate the basis of T cell recognition of myelin that governs the progression from acute symptoms into disease remission, relapse, and chronic progression in a secondary progressive model of demyelinating disease. Until now, the frequency and affinity of myelin-reactive CD4 T cells that elicit relapsing-remitting disease have not been quantified. The micropipette adhesion frequency assay was used to obtain a sensitive and physiologically relevant two-dimensional measurement of frequency and TCR affinity for myelin, as the inherent low affinity does not allow the use of specific peptide:MHC-II tetramers for this purpose. We found the highest affinity and frequency of polyclonal myelin oligodendrocyte glycoprotein-reactive cells infiltrate the CNS during acute disease, whereas affinities during remission, relapse, and chronic disease are not significantly different from each other. Frequency analysis revealed that the vast majority of CNS-infiltrating CD4 T cells are myelin oligodendrocyte glycoprotein reactive at all time points, demonstrating epitope spread is not a predominant factor for disease progression. Furthermore, time points at which mice were symptomatic were characterized by an infiltration of Th17 cells in the CNS, whereas symptom remission showed an enrichment of cells producing IFN-γ. Also, the ratio of regulatory T cells to Foxp3(-) CD4 T cells was significantly higher in the CNS at remission than during acute disease. The results of this study indicate that a high frequency of T cells specific for a single myelin Ag, rather than increased TCR affinity or epitope spread, governs the transition from acute symptoms through remission, relapse, and chronic disease states.

The kinetics of two-dimensional TCR and pMHC interactions determine T-cell responsiveness.

The T-cell receptor (TCR) interacts with peptide-major histocompatibility complexes (pMHC) to discriminate pathogens from self-antigens and trigger adaptive immune responses. Direct physical contact is required between the T cell and the antigen-presenting cell for cross-junctional binding where the TCR and pMHC are anchored on two-dimensional (2D) membranes of the apposing cells. Despite their 2D nature, TCR-pMHC binding kinetics have only been analysed three-dimensionally (3D) with a varying degree of correlation with the T-cell responsiveness. Here we use two mechanical assays to show high 2D affinities between a TCR and its antigenic pMHC driven by rapid on-rates. Compared to their 3D counterparts, 2D affinities and on-rates of the TCR for a panel of pMHC ligands possess far broader dynamic ranges that match that of their corresponding T-cell responses. The best 3D predictor of response is the off-rate, with agonist pMHC dissociating the slowest. In contrast, 2D off-rates are up to 8,300-fold faster, with the agonist pMHC dissociating the fastest. Our 2D data suggest rapid antigen sampling by T cells and serial engagement of a few agonist pMHCs by TCRs in a large self pMHC background. Thus, the cellular environment amplifies the intrinsic TCR-pMHC binding to generate broad affinities and rapid kinetics that determine T-cell responsiveness.

Signal transducer and activator of transcription (STAT) 3 inhibition delays the onset of lupus nephritis in MRL/lpr mice.

The transcription factor STAT3 is overexpressed and hyperactivated in T cells from SLE patients. STAT3 plays a central role in T cell differentiation into Th17 and T follicular helper cells, two subsets that orchestrate autoimmune responses in SLE. Moreover, STAT3 is important in chemokine-mediated T cell migration. To better understand its role in SLE, we inhibited STAT3 in lupus-prone mice using the small molecule Stattic. Stattic-treated mice exhibited delayed onset of proteinuria (3 weeks later than controls), and had lower levels of anti-dsDNA antibodies and inflammatory cytokines. Inhibitor treatment reduced lymphadenopathy, resulted in a 3-fold decrease in total T cell number, and a 4-fold decrease in the numbers of T follicular helper cells. In vitro experiments showed that Stattic-treated T cells exhibited decreased proliferation and a decrease in ability to migrate to CXCL12. We propose that STAT3 inhibition represents a therapeutic target in SLE, particularly lupus nephritis.

Destabilization of peptide:MHC interaction induces IL-2 resistant anergy in diabetogenic T cells.

Autoreactive T cells are responsible for inducing several autoimmune diseases, including type 1 diabetes. We have developed a strategy to induce unresponsiveness in these cells by destabilizing the peptide:MHC ligand recognized by the T cell receptor. By introducing amino acid substitutions into the immunogenic peptide at residues that bind to the MHC, the half life of the peptide:MHC complex is severely reduced, thereby resulting in abortive T cell activation and anergy. By treating a monoclonal diabetogenic T cell population with an MHC variant peptide, the cells are rendered unresponsive to the wild type ligand, as measured by both proliferation and IL-2 production. Stimulation of T cells with MHC variant peptides results in minimal Erk1/2 phosphorylation or cell division. Variant peptide stimulation effectively initiates a signaling program dominated by sustained tyrosine phosphatase activity, including elevated SHP-1 activity. These negative signaling events result in an anergic phenotype in which the T cells are not competent to signal through the IL-2 receptor, as evidenced by a lack of phospho-Stat5 upregulation and proliferation, despite high expression of the IL-2 receptor. This unique negative signaling profile provides a novel means to shut down the anti-self response.

A unique unresponsive CD4+ T cell phenotype post TCR antagonism.

The functional outcomes of the T cell's interaction with the peptide:MHC complex can be dramatically altered by the introduction of a single amino acid substitution. Previous studies have described the varied effects of these altered peptide ligands (APL) on T cell responses. These outcomes of T cell interaction with an APL include the induction of clonal unresponsiveness (anergy) and inhibition of T cell responses (antagonism). The phenotype of peptide-induced anergy, i.e. low proliferation and low IL-2 production, has been extensively described, and a number of groups have demonstrated antagonism. However, the response of T cells to an agonist ligand after encountering an antagonistic stimulus has not been previously characterized. Here, we show that T cells post-antagonism fail to proliferate but produce large quantities of IL-2 upon stimulation with their wild type ligand. This unique phenotype is not due to differences in IL-2 receptor expression or rates of apoptosis, and cannot be overcome by the addition of recombinant IL-2. The response of CD4 T cells to agonist stimulation after encountering an antagonist is a novel phenotype, and is distinct from previously described forms of anergy.

TCR antagonism by peptide requires high TCR expression.

Current models of T cell activation focus on the kinetics of TCR-ligand interactions as the central parameter governing T cell responsiveness. However, these kinetic parameters do not adequately predict all T cell behavior, particularly the response to antagonist ligands. Recent studies have demonstrated that TCR number is a critical parameter influencing the responses of CD4(+) T cells to weak agonist ligands, and receptor density represents an important means of regulating tissue responsiveness in other receptor ligand systems. To systematically address the impact of TCR expression on CD8(+) T cell responses, mAbs to the TCR alpha-chain and T cells expressing two TCR species were used as two different methods to manipulate the number of available TCRs on P14 and OT-I transgenic T cells. Both methods of TCR reduction demonstrated that the efficacy of antagonist peptides was significantly reduced on T cells bearing low numbers of available receptors. In addition, the ability of weak agonists to induce proliferation was critically dependent on the availability of high numbers of TCRs. Therefore, in this report we show that TCR density is a major determinant of CD8(+) T cell reactivity to weak agonist and antagonist ligands but not agonist ligands.

Insights into T cell recognition of antigen: significance of two-dimensional kinetic parameters.

The T cell receptor (TCR) interacts with peptide-major histocompatibility complex (pMHC) to enable T cell development and trigger adaptive immune responses. For this reason, TCR:pMHC interactions have been intensely studied for over two decades. However, the details of how various binding parameters impact T cell activation remain elusive. Most measurements were made using recombinant proteins by surface plasmon resonance, a three-dimensional (3D) technique in which fluid-phase receptors and ligands are removed from their cellular environment. This approach found TCR:pMHC interactions with relatively low affinities and slow off-rates for agonist peptides. Newer generation techniques have analyzed TCR:pMHC interactions in two dimensions (2D), with both proteins anchored in apposing plasma membranes. These approaches reveal in situ TCR:pMHC interaction kinetics that are of high affinity and exhibit rapid on- and off-rates upon interaction with agonist ligands. Importantly, 2D binding parameters correlate better with T cell functional responses to a spectrum of ligands than 3D measures.

Low 2-dimensional CD4 T cell receptor affinity for myelin sets in motion delayed response kinetics.

T cells recognizing self-peptides that mediate autoimmune disease and those that are responsible for efficacious immunity against pathogens may differ in affinity for antigen due to central and peripheral tolerance mechanisms. Here we utilize prototypical self-reactive (myelin) and viral-specific (LCMV) T cells from T cell receptor (TCR) transgenic mice (2D2 and SMARTA, respectively) to explore affinity differences. The T cells responsive to virus possessed >10,000 fold higher 2D affinity as compared to the self-reactive T cells. Despite their dramatically lower affinity for their cognate ligand, 2D2 T cells respond with complete, albeit delayed, activation (proliferation and cytokine production). SMARTA activation occurs rapidly, achieving peak phosphorylation of p38 (1 minute), Erk (30 minutes), and Jun (3 hours) as well as CD69 and CD25 upregulation (3 and 6 hours, respectively), with a corresponding early initiation of proliferation. 2D2 stimulation with MOG results in altered signaling--no phospho-Erk or phospho-p38 accumulation, significantly delayed activation kinetics of Jun (12 hours), and delayed but sustained SHP-1 activity--as well as delayed CD69 and CD25 expression (12-24 hours), and slow initiation of proliferation. This delay was not intrinsic to the 2D2 T cells, as a more potent antigen with >100-fold increased 2D affinity restored rapid response kinetics in line with those identified for the viral antigen. Taken together, these data demonstrate that time can offset low TCR affinity to attain full activation and suggest a mechanism by which low affinity T cells participate in autoimmune disease.

T cell recognition of weak ligands: roles of signaling, receptor number, and affinity.

T cell recognition of antigen is a crucial aspect of the adaptive immune response. One of the most common means of pathogen immune evasion is mutation of T cell epitopes. T cell recognition of such ligands can result in a variety of outcomes including activation, apoptosis and anergy. The ability of a given T cell to respond to a specific peptide-MHC ligand is regulated by a number of factors, including the affinity, on- and off-rates and half-life of the TCR-peptide-MHC interaction. Interaction of T cells with low-potency ligands results in unique signaling patterns and requires engagement with a larger number of T cell receptors than agonist ligands. This review will address these aspects of T cell interaction with weak ligands and the ways in which these ligands have been utilized therapeutically.

Kinetics of MHC-CD8 interaction at the T cell membrane.

CD8 plays an important role in facilitating TCR-MHC interaction, promoting Ag recognition, and initiating T cell activation. MHC-CD8 binding kinetics have been measured in three dimensions by surface plasmon resonance technique using purified molecules. However, CD8 is a membrane-anchored, signaling kinase-linked, and TCR-associated molecule whose function depends on the cell membrane environment. Purified molecules lack their linkage to the membrane, which precludes interactions with other structures of the cell as well as signaling. Furthermore, three-dimensional binding in the fluid phase is biologically and physically distinct from two-dimensional binding across apposing cell membranes. As a first step toward characterizing the molecular interactions between T cells and APCs, we used a micropipette adhesion frequency assay to measure the adhesion kinetics of single mouse T cells interacting with single human RBCs coated with MHC. Using anti-TCR mAb we isolated and characterized the specific two-dimensional MHC-CD8 binding from the trimolecular TCR-MHC-CD8 interaction. The TCR-independent MHC-CD8 interaction has a very low affinity that depends on the MHC alleles, but not on the peptide complexed to the MHC and whether CD8 is an alphaalpha homodimer or an alphabeta heterodimer. Surprisingly, MHC-CD8 binding affinity varies with T cells from different TCR transgenic mice and these affinity differences were abolished by treatment with cholesterol oxidase to disrupt membrane rafts. These data highlight the relevance and importance of two-dimensional analysis of T cells and APCs and indicate that membrane rafts play an important role in modulating the affinity of cell-cell interactions.