In response to infection, naïve CD4
T cells differentiate into two subpopulations: T follicular helper (T
) cells, which support B cell antibody production, and non-T
cells, which enhance innate ...immune cell functions. Interleukin-2 (IL-2), the major cytokine produced by naïve T cells, plays an important role in the developmental divergence of these populations. However, the relationship between IL-2 production and fate determination remains unclear. Using reporter mice, we found that differential production of IL-2 by naïve CD4
T cells defined precursors fated for different immune functions. IL-2 producers, which were fated to become T
cells, delivered IL-2 to nonproducers destined to become non-T
cells. Because IL-2 production was limited to cells receiving the strongest T cell receptor (TCR) signals, a direct link between TCR-signal strength, IL-2 production, and T cell fate determination has been established.
A robust primary immune response has been correlated with the precursor number of antigen-specific T cells, as identified using peptide MHCII tetramers. However, these tetramers identify only the ...highest-affinity T cells. Here we show the entire CD4+ T-cell repertoire, inclusive of low-affinity T cells missed by tetramers, using a T-cell receptor (TCR) signalling reporter and micropipette assay to quantify naive precursors and expanded populations. In vivo limiting dilution assays reveal hundreds more precursor T cells than previously thought, with higher-affinity tetramer-positive T cells, comprising only 5-30% of the total antigen-specific naive repertoire. Lower-affinity T cells maintain their predominance as the primary immune response progresses, with no enhancement of survival of T cells with high-affinity TCRs. These findings demonstrate that affinity for antigen does not control CD4+ T-cell entry into the primary immune response, as a diverse range in affinity is maintained from precursor through peak of T-cell expansion.
TCR affinity for peptide MHC dictates the functional efficiency of T cells and their propensity to differentiate into effectors and form memory. However, in the context of chronic infections, it is ...unclear what the overall profile of TCR affinity for Ag is and if it differs from acute infections. Using the comprehensive affinity analysis provided by the two-dimensional micropipette adhesion frequency assay and the common indirect affinity evaluation methods of MHC class II tetramer and functional avidity, we tracked IA
GP
-specific cells in the mouse model of acute (Armstrong) and chronic (clone 13) lymphocytic choriomeningitis virus infection. In each response, we show CD4 T cell population affinity peaks at the effector phase and declines with memory. Of interest, the range and average relative two-dimensional affinity was equivalent between acute and chronic infection, indicating chronic Ag exposure did not skew TCR affinity. In contrast, functional and tetramer avidity measurements revealed divergent results and lacked a consistent correlation with TCR affinity. Our findings highlight that the immune system maintains a diverse range in TCR affinity even under the pressures of chronic Ag stimulation.
The LCMV GP33 CD8 epitope has long been one of the most widely used antigens in viral immunology. Of note, almost all of the
analyses of CD8 T cell responses to this epitope make use of an altered ...peptide ligand (APL) in which the cysteine from the original 9-mer peptide (KAVYNFATC) is substituted by a methionine at position 41 (KAVYNFATM). In addition, it is possible that the antigen processed during natural LCMV infection is an 11-mer peptide (KAVYNFATCGI) rather than the widely used 9-mer. Although previous affinity measurements using purified proteins for these antigen variants revealed minimal differences, we applied highly sensitive two dimensional (2D) biophysical based techniques to further dissect TCR interaction with these closely related GP33 variants. The kinetic analyses of affinity provided by the 2D micropipette adhesion frequency assay (2D-MP) and bond lifetime under force analyzed using a biomembrane force probe (BFP) revealed significant differences between 41M, 41C and the 11-mer 41CGI antigen. We found a hierarchy in 2D affinity as 41M peptide displayed augmented TCR 2D affinity compared to 41C and 41CGI. These differences were also maintained in the presence of CD8 coreceptor and when analysis of total TCR:pMHC and CD8:pMHC bonds were considered. Moreover, the three ligands displayed dramatic differences in the bond lifetimes generated under force, in particular the 41CGI variant with the lowest 2D affinity demonstrated a 15-fold synergistic contribution of the CD8 coreceptor to overall bond lifetime. Our analyses emphasize the sensitivity of single cell and single bond 2D kinetic measurements in distinguishing between related agonist peptides.
Antigen receptor diversity underpins adaptive immunity by providing the ground for clonal selection of lymphocytes with the appropriate antigen reactivity. Current models attribute T cell clonal ...selection during the immune response to T-cell receptor (TCR) affinity for either foreign or self peptides. Here, we report that clonal selection of CD4(+) T cells is also extrinsically regulated by B cells. In response to viral infection, the antigen-specific TCR repertoire is progressively diversified by staggered clonotypic expansion, according to functional avidity, which correlates with self-reactivity. Clonal expansion of lower-avidity T-cell clonotypes depends on availability of MHC II-expressing B cells, in turn influenced by B-cell activation. B cells clonotypically diversify the CD4(+) T-cell response also to vaccination or tumour challenge, revealing a common effect.
T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen ...recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.
T cells are triggered when the T-cell receptor (TCR) encounters its antigenic ligand, the peptide-major histocompatibility complex (pMHC), on the surface of antigen presenting cells (APCs). Because T ...cells are highly migratory and antigen recognition occurs at an intermembrane junction where the T cell physically contacts the APC, there are long-standing questions of whether T cells transmit defined forces to their TCR complex and whether chemomechanical coupling influences immune function. Here we develop DNA-based gold nanoparticle tension sensors to provide, to our knowledge, the first pN tension maps of individual TCR-pMHC complexes during T-cell activation. We show that naïve T cells harness cytoskeletal coupling to transmit 12–19 pN of force to their TCRs within seconds of ligand binding and preceding initial calcium signaling. CD8 coreceptor binding and lymphocyte-specific kinase signaling are required for antigen-mediated cell spreading and force generation. Lymphocyte function-associated antigen 1 (LFA-1) mediated adhesion modulates TCR-pMHC tension by intensifying its magnitude to values >19 pN and spatially reorganizes the location of TCR forces to the kinapse, the zone located at the trailing edge of migrating T cells, thus demonstrating chemomechanical crosstalk between TCR and LFA-1 receptor signaling. Finally, T cells display a dampened and poorly specific response to antigen agonists when TCR forces are chemically abolished or physically “filtered” to a level below ∼12 pN using mechanically labile DNA tethers. Therefore, we conclude that T cells tune TCR mechanics with pN resolution to create a checkpoint of agonist quality necessary for specific immune response.
Abstract
T cells have been shown to undergo affinity/avidity maturation through selective expansion of higher affinity/avidity (tetramer positive) cells, resulting in the narrowing of the antigen ...specific repertoire over the course of primary and secondary immune responses. Using a highly sensitive two dimensional micropipette adhesion frequency assay (2D) to measure polyclonal CD4+ T cell affinities, we determined if affinity maturation occurred between primary and secondary peak effectors in lymphocytic choriomenengitis virus (LCMV) infection, and if measured affinities correlated with functional avidity. The 2D assay detected 4-5 fold more antigen specific high and low affinity cells than tetramer in both primary and secondary infection, but both 2D and tetramer indicated a 2-3 fold decrease in the frequency of LCMV specific CD4+ cells in the homologous viral rechallenge response. The range of affinities of the antigen specific repertoire was more restricted during secondary infection contracting to a narrower sixteen fold from the wider hundred fold range of the primary response. By functional avidity, secondary effectors were more sensitive to antigen despite a 3 fold decrease in their measured average affinity. Therefore, in this homologous viral rechallenge model, affinity maturation did not occur between primary and secondary effectors, and affinity did not correlate with functional avidity.
Abstract
Shortly after encountering antigen, T cells are considered transiently unresponsive or refractory to subsequent simulation. To determine whether antigen stimulation also changes the cell ...surface biophysical interaction between TCR and pMHC, we assessed the 2D TCR affinity and bond lifetime under force between this receptor ligand pair. Virus (LCMV) specific SMARTA transgenic CD4+ T cells were stimulated with peptide in vitro and 2D TCR affinity and bond lifetime determined at various time points after stimulation using the 2D micropipette adhesion frequency assay and the biomembrane force probe (BFP) respectively. We found activated T cells downregulated 2D affinity at twelve and twenty-four hours after seeing antigen with affinity recovering to naïve levels by forty-eight hours. Unlike pMHC tetramer staining which was dependent on TCR expression levels, the decrease in affinity was independent of antigen dose and degree of TCR downregulation. TCR:pMHC bond lifetime under force was also reduced early after antigen encounter with a steady recovery observed with time. Our data demonstrate a decrease in bond lifetime and relative 2D affinity of TCR and pMHC result from early T cell activation events followed by a recovery phase with both changes occurring independent of activation induced modulation of TCR expression. Thus, TCR affinity for pMHC is dynamic in the 2D T cell membrane context with cellular events allowing the T cell to fine-tune the ability of TCR to interact with antigen.
Mature T cells must discriminate between brief interactions with self-peptides and prolonged binding to agonists. The kinetic proofreading model posits that certain T-cell antigen receptor signaling ...nodes serve as molecular timers to facilitate such discrimination. However, the physiological significance of this regulatory mechanism and the pathological consequences of disrupting it are unknown. Here we report that accelerating the normally slow phosphorylation of the linker for activation of T cells (LAT) residue Y136 by introducing an adjacent Gly135Asp alteration (LAT
) disrupts ligand discrimination in vivo. The enhanced self-reactivity of LAT
T cells triggers excessive thymic negative selection and promotes T-cell anergy. During Listeria infection, LAT
T cells expand more than wild-type counterparts in response to very weak stimuli but display an imbalance between effector and memory responses. Moreover, despite their enhanced engagement of central and peripheral tolerance mechanisms, mice bearing LAT
show features associated with autoimmunity and immunopathology. Our data reveal the importance of kinetic proofreading in balancing tolerance and immunity.
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