Cbl-b mitigates the responsiveness of naive CD8+ T cells that experience extensive tonic T cell receptor signaling.

Naive T cells experience tonic T cell receptor (TCR) signaling in response to self-antigens presented by major histocompatibility complex (MHC) in secondary lymphoid organs. We investigated how relatively weak or strong tonic TCR signals influence naive CD8+ T cell responses to stimulation with foreign antigens. The heterogeneous expression of Nur77-GFP, a transgenic reporter of tonic TCR signaling, in naive CD8+ T cells suggests variable intensities or durations of tonic TCR signaling. Although the expression of genes associated with acutely stimulated T cells was increased in Nur77-GFPHI cells, these cells were hyporesponsive to agonist TCR stimulation compared with Nur77-GFPLO cells. This hyporesponsiveness manifested as diminished activation marker expression and decreased secretion of IFN-γ and IL-2. The protein abundance of the ubiquitin ligase Cbl-b, a negative regulator of TCR signaling, was greater in Nur77-GFPHI cells than in Nur77-GFPLO cells, and Cbl-b deficiency partially restored the responsiveness of Nur77-GFPHI cells. Our data suggest that the cumulative effects of previously experienced tonic TCR signaling recalibrate naive CD8+ T cell responsiveness. These changes include gene expression changes and negative regulation partially dependent on Cbl-b. This cell-intrinsic negative feedback loop may enable the immune system to restrain naive CD8+ T cells with higher self-reactivity.

What's the Catch? The Significance of Catch Bonds in T Cell Activation.

One of the main goals in T cell biology has been to investigate how TCR recognition of peptide:MHC (pMHC) determines T cell phenotype and fate. Ag recognition is required to facilitate survival, expansion, and effector function of T cells. Historically, TCR affinity for pMHC has been used as a predictor for T cell fate and responsiveness, but there have now been several examples of nonfunctional high-affinity clones and low-affinity highly functional clones. Recently, more attention has been paid to the TCR being a mechanoreceptor where the key biophysical determinant is TCR bond lifetime under force. As outlined in this review, the fundamental parameters between the TCR and pMHC that control Ag recognition and T cell triggering are affinity, bond lifetime, and the amount of force at which the peak lifetime occurs.

Immunogen-Specific Strengths and Limitations of the Activation-Induced Marker Assay for Assessing Murine Antigen-Specific CD4+ T Cell Responses.

The activation-induced marker (AIM) assay is a cytokine-independent technique to identify Ag-specific T cells based on the upregulated expression of activation markers after Ag restimulation. The method offers an alternative to intracellular cytokine staining in immunological studies, in which limited cytokine production makes the cell subsets of interest difficult to detect. Studies of lymphocytes in human and nonhuman primates have used the AIM assay to detect Ag-specific CD4+ and CD8+ T cells. However, there is a lack of validation of the strengths and limitations of the assay in murine (Mus musculus) models of infection and vaccination. In this study, we analyzed immune responses of TCR-transgenic CD4+ T cells, including lymphocytic choriomeningitis virus-specific SMARTA, OVA-specific OT-II, and diabetogenic BDC2.5-transgenic T cells, and measured the ability of the AIM assay to effectively identify these cells to upregulate AIM markers OX40 and CD25 following culture with cognate Ag. Our findings indicate that the AIM assay is effective for identifying the relative frequency of protein immunization-induced effector and memory CD4+ T cells, whereas the AIM assay had reduced ability to identify specific cells induced by viral infection, particularly during chronic lymphocytic choriomeningitis virus infection. Evaluation of polyclonal CD4+ T cell responses to acute viral infection demonstrated that the AIM assay can detect a proportion of both high- and low-affinity cells. Together, our findings indicate that the AIM assay can be an effective tool for relative quantification of murine Ag-specific CD4+ T cells to protein vaccination, while demonstrating its limitations during conditions of acute and chronic infection.

A single-amino acid substitution in the adaptor LAT accelerates TCR proofreading kinetics and alters T-cell selection, maintenance and function.

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 (LATG135D) disrupts ligand discrimination in vivo. The enhanced self-reactivity of LATG135D T cells triggers excessive thymic negative selection and promotes T-cell anergy. During Listeria infection, LATG135D 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 LATG135D show features associated with autoimmunity and immunopathology. Our data reveal the importance of kinetic proofreading in balancing tolerance and immunity.

Strong Basal/Tonic TCR Signals Are Associated with Negative Regulation of Naive CD4+ T Cells.

T cells experience varying intensities of tonic or basal TCR signaling in response to self-peptides presented by MHC (self-pMHC) in vivo. We analyzed four subpopulations of mouse naive CD4+ cells that express different levels of Nur77-GFP and Ly6C, surrogate markers that positively and inversely correlate with the strength of tonic TCR signaling, respectively. Adoptive transfer studies suggest that relatively weak or strong Nur77-GFP intensity in thymocytes tends to be maintained in mature T cells. Two-dimensional affinity measurements were lowest for Nur77-GFPloLy6C+ cells and highest for Nur77-GFPhiLy6C- cells, highlighting a positive correlation between apparent TCR affinity and tonic TCR signal strength. Despite experiencing the strongest tonic TCR signaling, Nur77-GFPhiLy6C- cells were least responsive to multiple concentrations of a cognate or suboptimal pMHC. Gene expression analyses suggest that Nur77-GFPhiLy6C- cells induce a gene expression program that has similarities with that of acutely stimulated T cells. However, strong tonic TCR signaling also correlates with increased expression of genes with inhibitory functions, including coinhibitory receptors. Similarly, assay for transposase-accessible chromatin with sequencing analyses suggested that increased tonic TCR signal strength correlated with increased chromatin accessibility associated with genes that have positive and inhibitory roles in T cell activation. Strikingly, Nur77-GFPhiLy6C- cells exhibited differential accessibility within regions of Cd200r1 and Tox that were similar in location to differentially accessible regions previously identified in exhausted CD8+ T cells. We propose that constitutive strong tonic TCR signaling triggers adaptations detectable at both the transcriptional and epigenetic levels, ultimately contributing to the tuning of T cell responsiveness.

Tuning T cell receptor sensitivity through catch bond engineering.

Adoptive cell therapy using engineered T cell receptors (TCRs) is a promising approach for targeting cancer antigens, but tumor-reactive TCRs are often weakly responsive to their target ligands, peptide-major histocompatibility complexes (pMHCs). Affinity-matured TCRs can enhance the efficacy of TCR-T cell therapy but can also cross-react with off-target antigens, resulting in organ immunopathology. We developed an alternative strategy to isolate TCR mutants that exhibited high activation signals coupled with low-affinity pMHC binding through the acquisition of catch bonds. Engineered analogs of a tumor antigen MAGE-A3-specific TCR maintained physiological affinities while exhibiting enhanced target killing potency and undetectable cross-reactivity, compared with a high-affinity clinically tested TCR that exhibited lethal cross-reactivity with a cardiac antigen. Catch bond engineering is a biophysically based strategy to tune high-sensitivity TCRs for T cell therapy with reduced potential for adverse cross-reactivity.

The magnitude of LFA-1/ICAM-1 forces fine-tune TCR-triggered T cell activation.

T cells defend against cancer and viral infections by rapidly scanning the surface of target cells seeking specific peptide antigens. This key process in adaptive immunity is sparked upon T cell receptor (TCR) binding of antigens within cell-cell junctions stabilized by integrin (LFA-1)/intercellular adhesion molecule-1 (ICAM-1) complexes. A long-standing question in this area is whether the forces transmitted through the LFA-1/ICAM-1 complex tune T cell signaling. Here, we use spectrally encoded DNA tension probes to reveal the first maps of LFA-1 and TCR forces generated by the T cell cytoskeleton upon antigen recognition. DNA probes that control the magnitude of LFA-1 force show that F>12 pN potentiates antigen-dependent T cell activation by enhancing T cell-substrate engagement. LFA-1/ICAM-1 mechanical events with F>12 pN also enhance the discriminatory power of the TCR when presented with near cognate antigens. Overall, our results show that T cells integrate multiple channels of mechanical information through different ligand-receptor pairs to tune function.

Mechanobiology of T Cell Activation: To Catch a Bond.

T cell activation is a critical event in the adaptive immune response, indispensable for cell-mediated and humoral immunity as well as for immune regulation. Recent years have witnessed an emerging trend emphasizing the essential role that physical force and mechanical properties play at the T cell interface. In this review, we integrate current knowledge of T cell antigen recognition and the different models of T cell activation from the perspective of mechanobiology, focusing on the interaction between the T cell receptor (TCR) and the peptide-major histocompatibility complex (pMHC) antigen. We address the shortcomings of TCR affinity alone in explaining T cell functional outcomes and the rising status of force-regulated TCR bond lifetimes, most notably the TCR catch bond. Ultimately, T cell activation and the ensuing physiological responses result from mechanical interaction between TCRs and the pMHC.

Canonical T cell receptor docking on peptide-MHC is essential for T cell signaling.

T cell receptor (TCR) recognition of peptide-major histocompatibility complexes (pMHCs) is characterized by a highly conserved docking polarity. Whether this polarity is driven by recognition or signaling constraints remains unclear. Using "reversed-docking" TCRß-variable (TRBV) 17+ TCRs from the naïve mouse CD8+ T cell repertoire that recognizes the H-2Db-NP366 epitope, we demonstrate that their inability to support T cell activation and in vivo recruitment is a direct consequence of reversed docking polarity and not TCR-pMHCI binding or clustering characteristics. Canonical TCR-pMHCI docking optimally localizes CD8/Lck to the CD3 complex, which is prevented by reversed TCR-pMHCI polarity. The requirement for canonical docking was circumvented by dissociating Lck from CD8. Thus, the consensus TCR-pMHC docking topology is mandated by T cell signaling constraints.

MHC class II tetramers engineered for enhanced binding to CD4 improve detection of antigen-specific T cells.

The ability to identify T cells that recognize specific peptide antigens bound to major histocompatibility complex (MHC) molecules has enabled enumeration and molecular characterization of the lymphocytes responsible for cell-mediated immunity. Fluorophore-labeled peptide:MHC class I (p:MHCI) tetramers are well-established reagents for identifying antigen-specific CD8+ T cells by flow cytometry, but efforts to extend the approach to CD4+ T cells have been less successful, perhaps owing to lower binding strength between CD4 and MHC class II (MHCII) molecules. Here we show that p:MHCII tetramers engineered by directed evolution for enhanced CD4 binding outperform conventional tetramers for the detection of cognate T cells. Using the engineered tetramers, we identified about twice as many antigen-specific CD4+ T cells in mice immunized against multiple peptides than when using traditional tetramers. CD4 affinity-enhanced p:MHCII tetramers, therefore, allow direct sampling of antigen-specific CD4+ T cells that cannot be accessed with conventional p:MHCII tetramer technology. These new reagents could provide a deeper understanding of the T cell repertoire.

Targeting transcriptional coregulator OCA-B/Pou2af1 blocks activated autoreactive T cells in the pancreas and type 1 diabetes.

The transcriptional coregulator OCA-B promotes expression of T cell target genes in cases of repeated antigen exposure, a necessary feature of autoimmunity. We hypothesized that T cell-specific OCA-B deletion and pharmacologic OCA-B inhibition would protect mice from autoimmune diabetes. We developed an Ocab conditional allele and backcrossed it onto a diabetes-prone NOD/ShiLtJ strain background. T cell-specific OCA-B loss protected mice from spontaneous disease. Protection was associated with large reductions in islet CD8+ T cell receptor specificities associated with diabetes pathogenesis. CD4+ clones associated with diabetes were present but associated with anergic phenotypes. The protective effect of OCA-B loss was recapitulated using autoantigen-specific NY8.3 mice but diminished in monoclonal models specific to artificial or neoantigens. Rationally designed membrane-penetrating OCA-B peptide inhibitors normalized glucose levels and reduced T cell infiltration and proinflammatory cytokine expression in newly diabetic NOD mice. Together, the results indicate that OCA-B is a potent autoimmune regulator and a promising target for pharmacologic inhibition.

Localized hydrogel delivery of dendritic cells for attenuation of multiple sclerosis in a murine model.

In multiple sclerosis (MS), abnormally activated immune cells responsive to myelin proteins result in widespread damage throughout the central nervous system (CNS) and ultimately irreversible disability. Immunomodulation by delivering dendritic cells (DCs) utilizes a potent and rapid MS disease progression driver therapeutically. Here, we investigated delivering DCs for disease severity attenuation using an experimental autoimmune encephalomyelitis preclinical MS model. DCs treated with interleukin-10 (IL-10) (DC10s) were transplanted using in situ gelling poly(ethylene glycol)-based hydrogel for target site localization. DC delivery increased hydrogel longevity and altered the injection site recruited, endogenous immune cell profile within 2 days postinjection. Furthermore, hydrogel-mediated DC transplantation efficacy depended on the injection-site. DCs delivered to the neck local to MS-associated CNS-draining cervical lymph nodes attenuated paralysis, compared to untreated controls, while delivery to the flank did not alter paralysis severity. This study demonstrates that local delivery of DC10s modulates immune cell recruitment and attenuates disease progression in a preclinical model of MS.

An Engineered T Cell Receptor Variant Realizes the Limits of Functional Binding Modes.

T cell receptors (TCRs) orchestrate cellular immunity by recognizing peptides presented by a range of major histocompatibility complex (MHC) proteins. Naturally occurring TCRs bind the composite peptide/MHC surface, recognizing peptides that are structurally and chemically compatible with the TCR binding site. Here we describe a molecularly evolved TCR variant that binds the human class I MHC protein HLA-A2 independent of the bound peptide, achieved by a drastic perturbation of the TCR binding geometry that places the molecule far from the peptide binding groove. This unique geometry is unsupportive of normal T cell signaling. A substantial divergence between affinity measurements in solution and in two dimensions between proximal cell membranes leads us to attribute the lack of signaling to steric hindrance that limits binding in the confines of a cell-cell interface. Our results provide an example of how receptor binding geometry can impact T cell function and provide further support for the view that germline-encoded residues in TCR binding loops evolved to drive productive TCR recognition and signaling.

Relationship of 2D Affinity to T Cell Functional Outcomes.

T cells are critical for a functioning adaptive immune response and a strong correlation exists between T cell responses and T cell receptor (TCR): peptide-loaded MHC (pMHC) binding. Studies that utilize pMHC tetramer, multimers, and assays of three-dimensional (3D) affinity have provided advancements in our understanding of T cell responses across different diseases. However, these technologies focus on higher affinity and avidity T cells while missing the lower affinity responders. Lower affinity TCRs in expanded polyclonal populations almost always constitute a significant proportion of the response with cells mediating different effector functions associated with variation in the proportion of high and low affinity T cells. Since lower affinity T cells expand and are functional, a fully inclusive view of T cell responses is required to accurately interpret the role of affinity for adaptive T cell immunity. For example, low affinity T cells are capable of inducing autoimmune disease and T cells with an intermediate affinity have been shown to exhibit an optimal anti-tumor response. Here, we focus on how affinity of the TCR may relate to T cell phenotype and provide examples where 2D affinity influences functional outcomes.

IL-21 from high-affinity CD4 T cells drives differentiation of brain-resident CD8 T cells during persistent viral infection.

Development of tissue-resident memory (TRM) CD8 T cells depends on CD4 T cells. In polyomavirus central nervous system infection, brain CXCR5hi PD-1hi CD4 T cells produce interleukin-21 (IL-21), and CD8 T cells lacking IL-21 receptors (IL21R-/-) fail to become bTRM IL-21+ CD4 T cells exhibit elevated T cell receptor (TCR) affinity and higher TCR density. IL21R-/- brain CD8 T cells do not express CD103, depend on vascular CD8 T cells for maintenance, are antigen recall defective, and lack TRM core signature genes. CD4 T cell-deficient and IL21R-/- brain CD8 T cells show similar deficiencies in expression of genes for oxidative metabolism, and intrathecal delivery of IL-21 to CD4 T cell-depleted mice restores expression of electron transport genes in CD8 T cells to wild-type levels. Thus, high-affinity CXCR5hi PD-1hi CD4 T cells in the brain produce IL-21, which drives CD8 bTRM differentiation in response to a persistent viral infection.

CD45RB Status of CD8+ T Cell Memory Defines T Cell Receptor Affinity and Persistence.

The identity of CD45 isoforms on the T cell surface changes following the activation of naive T cells and impacts intracellular signaling. In this study, we find that the anti-viral memory CD8+ T pool is unexpectedly comprised of both CD45RBhi and CD45RBlo populations. Relative to CD45RBlo memory T cells, CD45RBhi memory T cells have lower affinity and display greater clonal diversity, as well as a persistent CD27hi phenotype. The CD45RBhi memory population displays a homeostatic survival advantage in vivo relative to CD45RBlo memory, and long-lived high-affinity cells that persisted long term convert from CD45RBlo to CD45RBhi. Human CD45RO+ memory is comprised of both CD45RBhi and CD45RBlo populations with distinct phenotypes, and antigen-specific memory to two viruses is predominantly CD45RBhi. These data demonstrate that CD45RB status is distinct from the conventional central/effector T cell memory classification and has potential utility for monitoring and characterizing pathogen-specific CD8+ T cell responses.

A Hybrid Insulin Epitope Maintains High 2D Affinity for Diabetogenic T Cells in the Periphery.

ß-Cell antigen recognition by autoreactive T cells is essential in type 1 diabetes (T1D) pathogenesis. Recently, insulin hybrid peptides (HIPs) were identified as strong agonists for CD4 diabetogenic T cells. Here, using BDC2.5 transgenic and NOD mice, we investigated T-cell recognition of the HIP2.5 epitope, which is a fusion of insulin C-peptide and chromogranin A (ChgA) fragments, and compared it with the WE14 and ChgA29 -42 epitopes. We measured in situ two-dimensional affinity on individual live T cells from thymus, spleen, pancreatic lymph nodes, and islets before and after diabetes. Although preselection BDC2.5 thymocytes possess higher affinity than splenic BDC2.5 T cells for all three epitopes, peripheral splenic T cells maintained high affinity only to the HIP2.5 epitope. In polyclonal NOD mice, a high frequency (∼40%) of HIP2.5-specific islet T cells were identified at both prediabetic and diabetic stages comprising two distinct high- and low-affinity populations that differed in affinity by 100-fold. This high frequency of high- and low-affinity HIP2.5 T cells in the islets potentially represents a major risk factor in diabetes pathogenesis.

A Critical Insulin TCR Contact Residue Selects High-Affinity and Pathogenic Insulin-Specific T Cells.

Type 1 diabetes is an autoimmune-mediated disease that culminates in the targeted destruction of insulin-producing ß-cells. CD4 responses in NOD mice are dominated by insulin epitope B:9-23 (InsB9-23) specificity, and mutation of the key T-cell receptor (TCR) contact residue within the epitope prevents diabetes development. However, it is not clear how insulin self-antigen controls the selection of autoimmune and regulatory T cells (Tregs). Here we demonstrate that mutation of insulin epitope results in escape of highly pathogenic T cells. We observe an increase in antigen reactivity, clonality, and pathogenicity of insulin-specific T cells that develop in the absence of cognate antigen. Using a single TCR system, we demonstrate that Treg development is greatly diminished in mice with the Y16A mutant epitope. Collectively, these results suggest that the tyrosine residue at position 16 is necessary to constrain TCR reactivity for InsB9-23 by both limiting the development of pathogenic T cells and supporting the selection of Tregs.

Understanding TCR affinity, antigen specificity, and cross-reactivity to improve TCR gene-modified T cells for cancer immunotherapy.

Adoptive cell transfer (ACT) using T cell receptor (TCR) gene-modified T cells is an exciting and rapidly evolving field. Numerous preclinical and clinical studies have demonstrated various levels of feasibility, safety, and efficacy using TCR-engineered T cells to treat cancer and viral infections. Although evidence suggests their use can be effective, to what extent and how to improve these therapeutics are still matters of investigation. As TCR affinity has been generally accepted as the central role in defining T cell specificity and sensitivity, selection for and generation of high affinity TCRs has remained a fundamental approach to design more potent T cells. However, traditional methods for affinity-enhancement by random mutagenesis can induce undesirable cross-reactivity causing on- and off-target adverse events, generate exhausted effectors by overstimulation, and ignore other kinetic and cellular parameters that have been shown to impact antigen specificity. In this Focussed Research Review, we comment on the preclinical and clinical potential of TCR gene-modified T cells, summarize our contributions challenging the role TCR affinity plays in antigen recognition, and explore how structure-guided design can be used to manipulate antigen specificity and TCR cross-reactivity to improve the safety and efficacy of TCR gene-modified T cells used in ACT.