Strength of tonic T cell receptor signaling instructs T follicular helper cell-fate decisions.

T follicular helper (TFH) cells are critical in adaptive immune responses to pathogens and vaccines; however, what drives the initiation of their developmental program remains unclear. Studies suggest that a T cell antigen receptor (TCR)-dependent mechanism may be responsible for the earliest TFH cell-fate decision, but a critical aspect of the TCR has been overlooked: tonic TCR signaling. We hypothesized that tonic signaling influences early TFH cell development. Here, two murine TCR-transgenic CD4+ T cells, LLO56 and LLO118, which recognize the same antigenic peptide presented on major histocompatibility complex molecules but experience disparate strengths of tonic signaling, revealed low tonic signaling promotes TFH cell differentiation. Polyclonal T cells paralleled these findings, with naive Nur77 expression distinguishing TFH cell potential. Two mouse lines were also generated to both increase and decrease tonic signaling strength, directly establishing an inverse relationship between tonic signaling strength and TFH cell development. Our findings elucidate a central role for tonic TCR signaling in early TFH cell-lineage decisions.

A RORγt+ cell instructs gut microbiota-specific Treg cell differentiation.

The mutualistic relationship of gut-resident microbiota and the host immune system promotes homeostasis that ensures maintenance of the microbial community and of a largely non-aggressive immune cell compartment1,2. The consequences of disturbing this balance include proximal inflammatory conditions, such as Crohn's disease, and systemic illnesses. This equilibrium is achieved in part through the induction of both effector and suppressor arms of the adaptive immune system. Helicobacter species induce T regulatory (Treg) and T follicular helper (TFH) cells under homeostatic conditions, but induce inflammatory T helper 17 (TH17) cells when induced Treg (iTreg) cells are compromised3,4. How Helicobacter and other gut bacteria direct T cells to adopt distinct functions remains poorly understood. Here we investigated the cells and molecular components required for iTreg cell differentiation. We found that antigen presentation by cells expressing RORγt, rather than by classical dendritic cells, was required and sufficient for induction of Treg cells. These RORγt+ cells-probably type 3 innate lymphoid cells and/or Janus cells5-require the antigen-presentation machinery, the chemokine receptor CCR7 and the TGFß activator αv integrin. In the absence of any of these factors, there was expansion of pathogenic TH17 cells instead of iTreg cells, induced by CCR7-independent antigen-presenting cells. Thus, intestinal commensal microbes and their products target multiple antigen-presenting cells with pre-determined features suited to directing appropriate T cell differentiation programmes, rather than a common antigen-presenting cell that they endow with appropriate functions.

Intrinsic CD4+ T cell sensitivity and response to a pathogen are set and sustained by avidity for thymic and peripheral complexes of self peptide and MHC.

Interactions of T cell antigen receptors (TCRs) with complexes of self peptide and major histocompatibility complex (MHC) are crucial to T cell development, but their role in peripheral T cell responses remains unclear. Specific and nonspecific stimulation of LLO56 and LLO118 T cells, which transgenically express a TCR specific for the same Listeria monocytogenes epitope, elicited distinct interleukin 2 (IL-2) and phosphorylated kinase Erk responses, the strength of which was set in the thymus and maintained in the periphery in proportion to the avidity of the binding of the TCR to the self peptide-MHC complex. Deprivation of self peptide-MHC substantially compromised the population expansion of LLO56 T cells in response to L. monocytogenes in vivo. Despite their very different self-reactivity, LLO56 T cells and LLO118 T cells bound cognate peptide-MHC with an identical affinity, which challenges associations made between these parameters. Our findings highlight a crucial role for selecting ligands encountered during thymic 'education' in determining the intrinsic functionality of CD4+ T cells.

c-Myc-induced transcription factor AP4 is required for host protection mediated by CD8+ T cells.

Although the transcription factor c-Myc is essential for the establishment of a metabolically active and proliferative state in T cells after priming, its expression is transient. It remains unknown how T cell activation is maintained after c-Myc expression is downregulated. Here we identified AP4 as the transcription factor that was induced by c-Myc and sustained activation of antigen-specific CD8+ T cells. Despite normal priming, AP4-deficient CD8+ T cells failed to continue transcription of a broad range of c-Myc-dependent targets. Mice lacking AP4 specifically in CD8+ T cells showed enhanced susceptibility to infection with West Nile virus. Genome-wide analysis suggested that many activation-induced genes encoding molecules involved in metabolism were shared targets of c-Myc and AP4. Thus, AP4 maintains c-Myc-initiated cellular activation programs in CD8+ T cells to control microbial infection.

The TCR Takes Some Immune Responsibility.

In this issue of Immunity, Van Braeckel-Budimmir et al. (2017) reveal that the pathogenic response of mice to a Plasmodium berghei infection is dominated by a Vß8.1 T cell response. Mice lacking Vß8.1 T cells fail to mount a pathogenic response, thus showing that the TCR locus can be an Immune response (Ir) gene.

How the TCR balances sensitivity and specificity for the recognition of self and pathogens.

The T cell repertoire is generated during thymic development in preparation for the response to antigens from pathogens. The T cell repertoire is shaped by positive selection, which requires recognition by the T cell antigen receptor (TCR) of complexes of self peptide and major histocompatibility complex proteins (self-pMHC) with low affinity, and negative selection, which eliminates T cells with TCRs that recognize self-pMHC with high affinity. This generates a repertoire with low affinity for self-pMHC but high affinity for foreign antigens. The TCR must successfully engage both of these ligands for development, homeostasis and immune responses. This review discusses mechanisms underlying the interaction of the TCR with peptide-major histocompatibility complex ligands of varying affinity and highlights signaling mechanisms that enable the TCR to generate different responses to very distinct ligands.

A voltage-gated sodium channel is essential for the positive selection of CD4(+) T cells.

The sustained entry of Ca(2+) into CD4(+)CD8(+) double-positive thymocytes is required for positive selection. Here we identified a voltage-gated Na(+) channel (VGSC) that was essential for positive selection of CD4(+) T cells. Pharmacological inhibition of VGSC activity inhibited the sustained Ca(2+) influx induced by positively selecting ligands and the in vitro positive selection of CD4(+) but not CD8(+) T cells. In vivo short hairpin RNA (shRNA)-mediated knockdown of the gene encoding a regulatory ß-subunit of a VGSC specifically inhibited the positive selection of CD4(+) T cells. Ectopic expression of VGSC in peripheral AND CD4(+) T cells bestowed the ability to respond to a positively selecting ligand, which directly demonstrated that VGSC expression was responsible for the enhanced sensitivity. Thus, active VGSCs in thymocytes provide a mechanism by which a weak positive selection signal can induce the sustained Ca(2+) signals required for CD4(+) T cell development.

Polysaccharide Capsules Equip the Human Symbiont Bacteroides thetaiotaomicron to Modulate Immune Responses to a Dominant Antigen in the Intestine.

Bacteria express multiple diverse capsular polysaccharides (CPSs) for protection against environmental and host factors, including the host immune system. Using a mouse TCR transgenic CD4+ T cell, BθOM, that is specific for B. thetaiotaomicron and a complete set of single CPS-expressing B. thetaiotaomicron strains, we ask whether CPSs can modify the immune responses to specific bacterial Ags. Acapsular B. thetaiotaomicron, which lacks all B. thetaiotaomicron CPSs, stimulated BθOM T cells more strongly than wild-type B. thetaiotaomicron Despite similar levels of BθOM Ag expression, many single CPS-expressing B. thetaiotaomicron strains were antistimulatory and weakly activated BθOM T cells, but a few strains were prostimulatory and strongly activated BθOM T cells just as well or better than an acapsular strain. B. thetaiotaomicron strains that expressed an antistimulatory CPS blocked Ag delivery to the immune system, which could be rescued by Fc receptor-dependent Ab opsonization. All single CPS-expressing B. thetaiotaomicron strains stimulated the innate immune system to skew toward M1 macrophages and release inflammatory cytokines in an MyD88-dependent manner, with antistimulatory CPS activating the innate immune system in a weaker manner than prostimulatory CPS. The expression of antistimulatory versus prostimulatory CPSs on outer membrane vesicles also regulated immune responses. Moreover, antistimulatory and prostimulatory single CPS-expressing B. thetaiotaomicron strains regulated the activation of Ag-specific and polyclonal T cells as well as clearance of dominant Ag in vivo. These studies establish that the immune responses to specific bacterial Ags can be modulated by a diverse set of CPSs.

Phase-variable bacteria simultaneously express multiple capsules.

Capsular polysaccharides (CPSs) protect bacteria from host and environmental factors. Many bacteria can express different CPSs and these CPSs are phase variable. For example, Bacteroides thetaiotaomicron (B. theta) is a prominent member of the human gut microbiome and expresses eight different capsular polysaccharides. Bacteria, including B. theta, have been shown to change their CPSs to adapt to various niches such as immune, bacteriophage, and antibiotic perturbations. However, there are limited tools to study CPSs and fundamental questions regarding phase variance, including if gut bacteria can express more than one capsule at the same time, remain unanswered. To better understand the roles of different CPSs, we generated a B. theta CPS1-specific antibody and a flow cytometry assay to detect CPS expression in individual bacteria in the gut microbiota. Using these novel tools, we report for the first time that bacteria can simultaneously express multiple CPSs. We also observed that nutrients such as glucose and salts had no effect on CPS expression. The ability to express multiple CPSs at the same time may provide bacteria with an adaptive advantage to thrive amid changing host and environmental conditions, especially in the intestine.

An endogenous peptide positively selects and augments the activation and survival of peripheral CD4+ T cells.

Although CD4(+) and CD8(+) T cells differ in the strength of their positively selecting signal, endogenous positively selecting ligands have been identified only for major histocompatibility complex (MHC) class I-restricted T cell antigen receptors (TCRs). Here we screened for ligands able to positively select MHC class II-restricted TCRs using thymocytes from four I-E(k)-restricted TCR-transgenic mice and a large panel of self peptides. One peptide, gp250, induced positive selection of AND CD4(+) T cells, had no homology with the AND TCR agonist ligand and was recognized with a high degree of specificity. The gp250 peptide acted as a coagonist to initiate the activation and enhance the survival of peripheral AND CD4(+) T cells. Thus, positively selecting ligands are critical in thymocyte development and in the activation and maintenance of peripheral T cells.

Tuning T Cell Signaling Sensitivity Alters the Behavior of CD4+ T Cells during an Immune Response.

Intricate processes in the thymus and periphery help curb the development and activation of autoreactive T cells. The subtle signals that govern these processes are an area of great interest, but tuning TCR sensitivity for the purpose of affecting T cell behavior remains technically challenging. Previously, our laboratory described the derivation of two TCR-transgenic CD4 T cell mouse lines, LLO56 and LLO118, which recognize the same cognate Listeria epitope with the same affinity. Despite the similarity of the two TCRs, LLO56 cells respond poorly in a primary infection whereas LLO118 cells respond robustly. Phenotypic examination of both lines revealed a substantial difference in their surface of expression of CD5, which serves as a dependable readout of the self-reactivity of a cell. We hypothesized that the increased interaction with self by the CD5-high LLO56 was mediated through TCR signaling, and was involved in the characteristic weak primary response of LLO56 to infection. To explore this issue, we generated an inducible knock-in mouse expressing the self-sensitizing voltage-gated sodium channel Scn5a. Overexpression of Scn5a in peripheral T cells via the CD4-Cre promoter resulted in increased TCR-proximal signaling. Further, Scn5a-expressing LLO118 cells, after transfer into BL6 recipient mice, displayed an impaired response during infection relative to wild-type LLO118 cells. In this way, we were able to demonstrate that tuning of TCR sensitivity to self can be used to alter in vivo immune responses. Overall, these studies highlight the critical relationship between TCR-self-pMHC interaction and an immune response to infection.

L-Myc expression by dendritic cells is required for optimal T-cell priming.

The transcription factors c-Myc and N-Myc--encoded by Myc and Mycn, respectively--regulate cellular growth and are required for embryonic development. A third paralogue, Mycl1, is dispensable for normal embryonic development but its biological function has remained unclear. To examine the in vivo function of Mycl1 in mice, we generated an inactivating Mycl1(gfp) allele that also reports Mycl1 expression. We find that Mycl1 is selectively expressed in dendritic cells (DCs) of the immune system and controlled by IRF8, and that during DC development, Mycl1 expression is initiated in the common DC progenitor concurrent with reduction in c-Myc expression. Mature DCs lack expression of c-Myc and N-Myc but maintain L-Myc expression even in the presence of inflammatory signals such as granulocyte-macrophage colony-stimulating factor. All DC subsets develop in Mycl1-deficient mice, but some subsets such as migratory CD103(+) conventional DCs in the lung and liver are greatly reduced at steady state. Importantly, loss of L-Myc by DCs causes a significant decrease in in vivo T-cell priming during infection by Listeria monocytogenes and vesicular stomatitis virus. The replacement of c-Myc by L-Myc in immature DCs may provide for Myc transcriptional activity in the setting of inflammation that is required for optimal T-cell priming.

LysMD3 is a type II membrane protein without an in vivo role in the response to a range of pathogens.

Germline-encoded receptors recognizing common pathogen-associated molecular patterns are a central element of the innate immune system and play an important role in shaping the host response to infection. Many of the innate immune molecules central to these signaling pathways are evolutionarily conserved. LysMD3 is a novel molecule containing a putative peptidoglycan-binding domain that has orthologs in humans, mice, zebrafish, flies, and worms. We found that the lysin motif (LysM) of LysMD3 is likely related to a previously described peptidoglycan-binding LysM found in bacteria. Mouse LysMD3 is a type II integral membrane protein that co-localizes with GM130+ structures, consistent with localization to the Golgi apparatus. We describe here two lines of mLysMD3-deficient mice for in vivo characterization of mLysMD3 function. We found that mLysMD3-deficient mice were born at Mendelian ratios and had no obvious pathological abnormalities. They also exhibited no obvious immune response deficiencies in a number of models of infection and inflammation. mLysMD3-deficient mice exhibited no signs of intestinal dysbiosis by 16S analysis or alterations in intestinal gene expression by RNA sequencing. We conclude that mLysMD3 contains a LysM with cytoplasmic orientation, but we were unable to define a physiological role for the molecule in vivo.

Taking a toll road to better vaccines.

Effective subunit vaccines must elicit strong CD4(+) T cell responses. In this issue of Immunity, Malherbe et al. (2008) find that the ability of adjuvants to stimulate high-avidity T cell responses correlates with Toll-like-receptor engagement.

The balance between T cell receptor signaling and degradation at the center of the immunological synapse is determined by antigen quality.

The role of the center of the immunological synapse (the central supramolecular activation cluster or cSMAC) is controversial. One model suggests that the role of the cSMAC depends on antigen quality and can both enhance signaling and receptor downregulation, whereas a second model proposes that the sole function of the cSMAC is to downregulate signaling. An important distinction between the models is whether signaling occurs in the cSMAC. Here, we demonstrate that at early time points, signaling occurs outside the cSMAC, but occurs in the cSMAC at later time points. Additionally, we show that cSMAC formation enhances the stimulatory potency of weak agonists for the TCR. Combined with previous studies showing that cSMAC formation decreases the signaling by strong agonists, our data support a model proposing that signaling and receptor degradation both occur in the cSMAC and that the balance between signaling and degradation in the synapse is determined by antigen quality.

Force-Regulated In Situ TCR-Peptide-Bound MHC Class II Kinetics Determine Functions of CD4+ T Cells.

We have recently shown that two-dimensional (2D) and force-regulated kinetics of TCR-peptide-bound MHC class I (pMHC-I) interactions predict responses of CD8(+) T cells. To test whether these findings are applicable to CD4(+) T cells, we analyzed the in situ 3.L2 TCR-pMHC-II interactions for a well-characterized panel of altered peptide ligands on the T cell surface using the adhesion frequency assay with a micropipette and the thermal fluctuation and force-clamp assays with a biomembrane force probe. We found that the 2D effective TCR-pMHC-II affinity and off-rate correlate with, but better predict the T cell response than, the corresponding measurements with the surface plasmon resonance in three dimensions. The 2D affinity of the CD4 for MHC-II was very low, approaching the detection limit, making it one to two orders of magnitude lower than the affinity of CD8 for MHC-I. In addition, the signal-dependent cooperation between TCR and coreceptor for pMHC binding previously observed for CD8 was not observed for CD4. Interestingly, force elicited TCR-pMHC-II catch-slip bonds for agonists but slip-only bonds for antagonists, thereby amplifying the power of discrimination between altered peptide ligands. These results show that the force-regulated 2D binding kinetics of the 3.L2 TCR for pMHC-II determine functions of CD4(+) T cells.

The ability to rearrange dual TCRs enhances positive selection, leading to increased Allo- and Autoreactive T cell repertoires.

Thymic selection is designed to ensure TCR reactivity to foreign Ags presented by self-MHC while minimizing reactivity to self-Ags. We hypothesized that the repertoire of T cells with unwanted specificities such as alloreactivity or autoreactivity are a consequence of simultaneous rearrangement of both TCRα loci. We hypothesized that this process helps maximize production of thymocytes capable of successfully completing thymic selection, but results in secondary TCRs that escape stringent selection. In T cells expressing two TCRs, one TCR can mediate positive selection and mask secondary TCR from negative selection. Examination of mice heterozygous for TRAC (TCRα(+/-)), capable of only one functional TCRα rearrangement, demonstrated a defect in generating mature T cells attributable to decreased positive selection. Elimination of secondary TCRs did not broadly alter the peripheral T cell compartment, though deep sequencing of TCRα repertoires of dual TCR T cells and TCRα(+/-) T cells demonstrated unique TCRs in the presence of secondary rearrangements. The functional impact of secondary TCRs on the naive peripheral repertoire was evidenced by reduced frequencies of T cells responding to autoantigen and alloantigen peptide-MHC tetramers in TCRα(+/-) mice. T cell populations with secondary TCRs had significantly increased ability to respond to altered peptide ligands related to their allogeneic ligand as compared with TCRα(+/-) cells, suggesting increased breadth in peptide recognition may be a mechanism for their reactivity. Our results imply that the role of secondary TCRs in forming the T cell repertoire is perhaps more significant than what has been assumed.

Self-peptides in TCR repertoire selection and peripheral T cell function.

The vertebrate antigen receptors are anticipatory in their antigen recognition and display a vast diversity. Antigen receptors are assembled through V(D)J recombination, in which one of each Variable, (Diverse), and Joining gene segment are randomly utilized and recombined. Both gene rearrangement and mutational insertion are generated through randomness; therefore, the process of antigen receptors generation requires a rigorous testing system to select every receptor which is useful to recognize foreign antigens, but which would cause no harm to self cells. In the case of T cell receptors (TCR), such a quality control responsibility rests in thymic positive and negative selection. In this review, we focus on the critical involvement of self-peptides in the generation of a T cell repertoire, discuss the role of T cell thymic development in shaping the specificity of TCR repertoire, and directing function fitness of mature T cells in periphery. Here, we consider thymic positive selection to be not merely a one-time maturing experience for an individual T cell, but a life-long imprinting which influences the function of each individual T cell in periphery.