I-Ag7 ß56/57 polymorphisms regulate non-cognate negative selection to CD4+ T cell orchestrators of type 1 diabetes.

Genetic susceptibility to type 1 diabetes is associated with homozygous expression of major histocompatibility complex class II alleles that carry specific beta chain polymorphisms. Why heterozygous expression of these major histocompatibility complex class II alleles does not confer a similar predisposition is unresolved. Using a nonobese diabetic mouse model, here we show that heterozygous expression of the type 1 diabetes-protective allele I-Ag7 ß56P/57D induces negative selection to the I-Ag7-restricted T cell repertoire, including beta-islet-specific CD4+ T cells. Surprisingly, negative selection occurs despite I-Ag7 ß56P/57D having a reduced ability to present beta-islet antigens to CD4+ T cells. Peripheral manifestations of non-cognate negative selection include a near complete loss of beta-islet-specific CXCR6+ CD4+ T cells, an inability to cross-prime islet-specific glucose-6-phosphatase catalytic subunit-related protein and insulin-specific CD8+ T cells and disease arrest at the insulitis stage. These data reveal that negative selection on non-cognate self-antigens in the thymus can promote T cell tolerance and protection from autoimmunity.

A temporal thymic selection switch and ligand binding kinetics constrain neonatal Foxp3+ Treg cell development.

The neonatal thymus generates Foxp3+ regulatory T (tTreg) cells that are critical in controlling immune homeostasis and preventing multiorgan autoimmunity. The role of antigen specificity on neonatal tTreg cell selection is unresolved. Here we identify 17 self-peptides recognized by neonatal tTreg cells, and reveal ligand specificity patterns that include self-antigens presented in an age- and inflammation-dependent manner. Fate-mapping studies of neonatal peptidyl arginine deiminase type IV (Padi4)-specific thymocytes reveal disparate fate choices. Neonatal thymocytes expressing T cell receptors that engage IAb-Padi4 with moderate dwell times within a conventional docking orientation are exported as tTreg cells. In contrast, Padi4-specific T cell receptors with short dwell times are expressed on CD4+ T cells, while long dwell times induce negative selection. Temporally, Padi4-specific thymocytes are subject to a developmental stage-specific change in negative selection, which precludes tTreg cell development. Thus, a temporal switch in negative selection and ligand binding kinetics constrains the neonatal tTreg selection window.

Resolving the Instructions for αß T Cell Development.

In this issue of Immunity, Chopp et. al. use single-cell transcriptomics and epigenomics in mice and human samples to delineate developmental trajectories of αß T cell subsets and refine the kinetic selection model of CD4+ and CD8+ T cell lineage commitment.

Coreceptor scanning by the T cell receptor provides a mechanism for T cell tolerance.

In the thymus, high-affinity, self-reactive thymocytes are eliminated from the pool of developing T cells, generating central tolerance. Here, we investigate how developing T cells measure self-antigen affinity. We show that very few CD4 or CD8 coreceptor molecules are coupled with the signal-initiating kinase, Lck. To initiate signaling, an antigen-engaged T cell receptor (TCR) scans multiple coreceptor molecules to find one that is coupled to Lck; this is the first and rate-limiting step in a kinetic proofreading chain of events that eventually leads to TCR triggering and negative selection. MHCII-restricted TCRs require a shorter antigen dwell time (0.2 s) to initiate negative selection compared to MHCI-restricted TCRs (0.9 s) because more CD4 coreceptors are Lck-loaded compared to CD8. We generated a model (Lck come&stay/signal duration) that accurately predicts the observed differences in antigen dwell-time thresholds used by MHCI- and MHCII-restricted thymocytes to initiate negative selection and generate self-tolerance.

Hydrophobic CDR3 residues promote the development of self-reactive T cells.

Studies of individual T cell antigen receptors (TCRs) have shed some light on structural features that underlie self-reactivity. However, the general rules that can be used to predict whether TCRs are self-reactive have not been fully elucidated. Here we found that the interfacial hydrophobicity of amino acids at positions 6 and 7 of the complementarity-determining region CDR3ß robustly promoted the development of self-reactive TCRs. This property was found irrespective of the member of the ß-chain variable region (Vß) family present in the TCR or the length of the CDR3ß. An index based on these findings distinguished Vß2(+), Vß6(+) and Vß8.2(+) regulatory T cells from conventional T cells and also distinguished CD4(+) T cells selected by the major histocompatibility complex (MHC) class II molecule I-A(g7) (associated with the development of type 1 diabetes in NOD mice) from those selected by a non-autoimmunity-promoting MHC class II molecule I-A(b). Our results provide a means for distinguishing normal T cell repertoires versus autoimmunity-prone T cell repertoires.

Affinity for self antigen selects Treg cells with distinct functional properties.

The manner in which regulatory T cells (Treg cells) control lymphocyte homeostasis is not fully understood. We identified two Treg cell populations with differing degrees of self-reactivity and distinct regulatory functions. We found that GITR(hi)PD-1(hi)CD25(hi) (Triple(hi)) Treg cells were highly self-reactive and controlled lympho-proliferation in peripheral lymph nodes. GITR(lo)PD-1(lo)CD25(lo) (Triple(lo)) Treg cells were less self-reactive and limited the development of colitis by promoting the conversion of CD4(+) Tconv cells into induced Treg cells (iTreg cells). Although Foxp3-deficient (Scurfy) mice lacked Treg cells, they contained Triple(hi)-like and Triple(lo)-like CD4(+) T cells zsuper> T cells infiltrated the skin, whereas Scurfy Triple(lo)CD4(+) T cells induced colitis and wasting disease. These findings indicate that the affinity of the T cell antigen receptor for self antigen drives the differentiation of Treg cells into distinct subsets with non-overlapping regulatory activities.

Single naive CD4+ T cells from a diverse repertoire produce different effector cell types during infection.

A naive CD4(+) T cell population specific for a microbial peptide:major histocompatibility complex II ligand (p:MHCII) typically consists of about 100 cells, each with a different T cell receptor (TCR). Following infection, this population produces a consistent ratio of effector cells that activate microbicidal functions of macrophages or help B cells make antibodies. We studied the mechanism that underlies this division of labor by tracking the progeny of single naive T cells. Different naive cells produced distinct ratios of macrophage and B cell helpers but yielded the characteristic ratio when averaged together. The effector cell pattern produced by a given naive cell correlated with the TCR-p:MHCII dwell time or the amount of p:MHCII. Thus, the consistent production of effector cell subsets by a polyclonal population of naive cells results from averaging the diverse behaviors of individual clones, which are instructed in part by the strength of TCR signaling.

How persistent infection overcomes peripheral tolerance mechanisms to cause T cell-mediated autoimmune disease.

T cells help orchestrate immune responses to pathogens, and their aberrant regulation can trigger autoimmunity. Recent studies highlight that a threshold number of T cells (a quorum) must be activated in a tissue to mount a functional immune response. These collective effects allow the T cell repertoire to respond to pathogens while suppressing autoimmunity due to circulating autoreactive T cells. Our computational studies show that increasing numbers of pathogenic peptides targeted by T cells during persistent or severe viral infections increase the probability of activating T cells that are weakly reactive to self-antigens (molecular mimicry). These T cells are easily re-activated by the self-antigens and contribute to exceeding the quorum threshold required to mount autoimmune responses. Rare peptides that activate many T cells are sampled more readily during severe/persistent infections than in acute infections, which amplifies these effects. Experiments in mice to test predictions from these mechanistic insights are suggested.

CD4 Effector TCR Avidity for Peptide on APC Determines the Level of Memory Generated.

Initial TCR affinity for peptide Ag is known to impact the generation of memory; however, its contributions later, when effectors must again recognize Ag at 5-8 d postinfection to become memory, is unclear. We examined whether the effector TCR affinity for peptide at this "effector checkpoint" dictates the extent of memory and degree of protection against rechallenge. We made an influenza A virus nucleoprotein (NP)-specific TCR transgenic mouse strain, FluNP, and generated NP-peptide variants that are presented by MHC class II to bind to the FluNP TCR over a broad range of avidity. To evaluate the impact of avidity in vivo, we primed naive donor FluNP in influenza A virus-infected host mice, purified donor effectors at the checkpoint, and cotransferred them with the range of peptides pulsed on activated APCs into second uninfected hosts. Higher-avidity peptides yielded higher numbers of FluNP memory cells in spleen and most dramatically in lung and draining lymph nodes and induced better protection against lethal influenza infection. Avidity determined memory cell number, not cytokine profile, and already impacted donor cell number within several days of transfer. We previously found that autocrine IL-2 production at the checkpoint prevents default effector apoptosis and supports memory formation. Here, we find that peptide avidity determines the level of IL-2 produced by these effectors and that IL-2Rα expression by the APCs enhances memory formation, suggesting that transpresentation of IL-2 by APCs further amplifies IL-2 availability. Secondary memory generation was also avidity dependent. We propose that this regulatory pathway selects CD4 effectors of highest affinity to progress to memory.

Patients with CD3G mutations reveal a role for human CD3γ in Treg diversity and suppressive function.

Integrity of the T-cell receptor/CD3 complex is crucial for positive and negative selection of T cells in the thymus and for effector and regulatory functions of peripheral T lymphocytes. In humans, CD3D, CD3E, and CD3Z gene defects are a cause of severe immune deficiency and present early in life with increased susceptibility to infections. By contrast, CD3G mutations lead to milder phenotypes, mainly characterized by autoimmunity. However, the role of CD3γ in establishing and maintaining immune tolerance has not been elucidated. In this manuscript, we aimed to investigate abnormalities of T-cell repertoire and function in patients with genetic defects in CD3G associated with autoimmunity. High throughput sequencing was used to study composition and diversity of the T-cell receptor ß (TRB) repertoire in regulatory T cells (Tregs), conventional CD4+ (Tconv), and CD8+ T cells from 6 patients with CD3G mutations and healthy controls. Treg function was assessed by studying its ability to suppress proliferation of Tconv cells. Treg cells of patients with CD3G defects had reduced diversity, increased clonality, and reduced suppressive function. The TRB repertoire of Tconv cells from patients with CD3G deficiency was enriched for hydrophobic amino acids at positions 6 and 7 of the CDR3, a biomarker of self-reactivity. These data demonstrate that the T-cell repertoire of patients with CD3G mutations is characterized by a molecular signature that may contribute to the increased rate of autoimmunity associated with this condition.

A single T cell receptor bound to major histocompatibility complex class I and class II glycoproteins reveals switchable TCR conformers.

Major histocompatibility complex class I (MHCI) and MHCII proteins differ in structure and sequence. To understand how T cell receptors (TCRs) can use the same set of variable regions to bind both proteins, we have presented a comparison of a single TCR bound to both MHCI and MHCII ligands. The TCR adopts similar orientations on both ligands with TCR amino acids thought to be evolutionarily conserved for MHC interaction occupying similar positions on the MHCI and MHCII helices. However, the TCR antigen-binding loops use different conformations when interacting with each ligand. Most importantly, we observed alternate TCR core conformations. When bound to MHCI, but not MHCII, Vα disengages from the Jα ß strand, switching Vα's position relative to Vß. In several other structures, either Vα or Vß undergoes this same modification. Thus, both TCR V-domains can switch among alternate conformations, perhaps extending their ability to react with different MHC-peptide ligands.

A role for differential variable gene pairing in creating T cell receptors specific for unique major histocompatibility ligands.

A limited set of T cell receptor (TCR) variable (V) gene segments are used to create a repertoire of TCRs that recognize all major histocompatibility complex (MHC) ligands within a species. How individual αßTCRs are constructed to specifically recognize a limited set of MHC ligands is unclear. Here we have identified a role for the differential pairing of particular V gene segments in creating TCRs that recognized MHC class II ligands exclusively, or cross-reacted with classical and nonclassical MHC class I ligands. Biophysical and structural experiments indicated that TCR specificity for MHC ligands is not driven by germline-encoded pairwise interactions.Rather, identical TCRß chains can have altered peptide-MHC (pMHC) binding modes when paired with different TCRα chains. The ability of TCR chain pairing to modify how V region residues interact with pMHC helps to explain how the same V genes are used to create TCRs specific for unique MHC ligands.

Indoctrinating T cells to attack pathogens through homeschooling.

Adaptive immunity is predicated on the ability of the T cell repertoire to have pre-existing specificity for the universe of potential pathogens. Recent findings suggest that T cell receptor (TCR)-self-major histocompatibility protein (pMHC) interactions limit autoimmune responses while enhancing T cell response to foreign antigens. We review these findings here, placing them in context of the current understanding of how TCR-self-pMHC interactions regulate T cell activation thresholds, and suggest that TCR-self-pMHC interactions increase the efficiency of the T cell repertoire by giving a competitive advantage to peptide cross-reactive T cells. We propose that self-reactivity and peptide cross-reactivity are controlled by particular CDR3 sequence motifs, which would allow thymic selection to contribute to solving the feat of broad pathogen specificity by exporting T cells that are pre-screened by positive and negative selection for the ability to be 'moderately' peptide cross-reactive.

Identifying environmental antigens that activate myelin-specific T cells.

Human genetic and environmental factors underlie susceptibility to the T cell-mediated autoimmune disease, multiple sclerosis (MS). How the environment influences the pathogenesis of MS has been difficult to parse. A recent paper in Cell shows that environmental antigens that activate myelin-specific T cells can be identified with unprecedented accuracy.

Crossreactive T Cells spotlight the germline rules for alphabeta T cell-receptor interactions with MHC molecules.

To test whether highly crossreactive alphabeta T cell receptors (TCRs) produced during limited negative selection best illustrate evolutionarily conserved interactions between TCR and major histocompatibility complex (MHC) molecules, we solved the structures of three TCRs bound to the same MHC II peptide (IAb-3K). The TCRs had similar affinities for IAb-3K but varied from noncrossreactive to extremely crossreactive with other peptides and MHCs. Crossreactivity correlated with a shrinking, increasingly hydrophobic TCR-ligand interface, involving fewer TCR amino acids. A few CDR1 and CDR2 amino acids dominated the most crossreactive TCR interface with MHC, including Vbeta8 48Y and 54E and Valpha4 29Y, arranged to impose the familiar diagonal orientation of TCR on MHC. These interactions contribute to MHC binding by other TCRs using related V regions, but not usually so dominantly. These data show that crossreactive TCRs can spotlight the evolutionarily conserved features of TCR-MHC interactions and that these interactions impose the diagonal docking of TCRs on MHC.

Peripheral CD8+ T cell tolerance to self-proteins is regulated proximally at the T cell receptor.

CD8(+) T cell tolerance, although essential for preventing autoimmunity, poses substantial obstacles to eliciting immune responses to tumor antigens, which are generally overexpressed normal proteins. Development of effective strategies to overcome tolerance for clinical applications would benefit from elucidation of the immunologic mechanism(s) regulating T cell tolerance to self. To examine how tolerance is maintained in vivo, we engineered dual-T cell receptor (TCR) transgenic mice in which CD8(+) T cells recognize two distinct antigens: a foreign viral-protein and a tolerizing self-tumor protein. Encounter with peripheral self-antigen rendered dual-TCR T cells tolerant to self, but these cells responded normally through the virus-specific TCR. Moreover, proliferation induced by virus rescued function of tolerized self-tumor-reactive TCR, restoring anti-tumor activity. These studies demonstrate that peripheral CD8(+) T cell tolerance to self-proteins can be regulated at the level of the self-reactive TCR complex rather than by central cellular inactivation and suggest an alternate strategy to enhance adoptive T cell immunotherapy.

Antigen affinity and antigen dose exert distinct influences on CD4 T-cell differentiation.

Cumulative T-cell receptor signal strength and ensuing T-cell responses are affected by both antigen affinity and antigen dose. Here we examined the distinct contributions of these parameters to CD4 T-cell differentiation during infection. We found that high antigen affinity positively correlates with T helper (Th)1 differentiation at both high and low doses of antigen. In contrast, follicular helper T cell (TFH) effectors are generated after priming with high, intermediate, and low affinity ligand. Unexpectedly, memory T cells generated after priming with very low affinity antigen remain impaired in their ability to generate secondary Th1 effectors, despite being recalled with high affinity antigen. These data challenge the view that only strongly stimulated CD4 T cells are capable of differentiating into the TFH and memory T-cell compartments and reveal that differential strength of stimulation during primary T-cell activation imprints unique and long lasting T-cell differentiation programs.

Effect of CDR3 sequences and distal V gene residues in regulating TCR-MHC contacts and ligand specificity.

The mature T cell repertoire has the ability to orchestrate immunity to a wide range of potential pathogen challenges. This ability stems from thymic development producing individual T cell clonotypes that express TCRs with unique patterns of Ag reactivity. The Ag specificity of TCRs is created from the combinatorial pairing of one of a set of germline encoded TCR Vα and Vß gene segments with randomly created CDR3 sequences. How the amalgamation of germline encoded and randomly created TCR sequences results in Ag receptors with unique patterns of ligand specificity is not fully understood. Using cellular, biophysical, and structural analyses, we show that CDR3α residues can modulate the geometry in which TCRs bind peptide-MHC (pMHC), governing whether and how germline encoded TCR Vα and Vß residues interact with MHC. In addition, a CDR1α residue that is positioned distal to the TCR-pMHC binding interface is shown to contribute to the peptide specificity of T cells. These findings demonstrate that the specificity of individual T cell clonotypes arises not only from TCR residues that create direct contacts with the pMHC, but also from a collection of indirect effects that modulate how TCR residues are used to bind pMHC.