Foxn1 regulates key target genes essential for T cell development in postnatal thymic epithelial cells.

Thymic epithelial cell differentiation, growth and function depend on the expression of the transcription factor Foxn1; however, its target genes have never been physically identified. Using static and inducible genetic model systems and chromatin studies, we developed a genome-wide map of direct Foxn1 target genes for postnatal thymic epithelia and defined the Foxn1 binding motif. We determined the function of Foxn1 in these cells and found that, in addition to the transcriptional control of genes involved in the attraction and lineage commitment of T cell precursors, Foxn1 regulates the expression of genes involved in antigen processing and thymocyte selection. Thus, critical events in thymic lympho-stromal cross-talk and T cell selection are indispensably choreographed by Foxn1.

Functional antibodies exhibit light chain coherence.

The vertebrate adaptive immune system modifies the genome of individual B cells to encode antibodies that bind particular antigens1. In most mammals, antibodies are composed of heavy and light chains that are generated sequentially by recombination of V, D (for heavy chains), J and C gene segments. Each chain contains three complementarity-determining regions (CDR1-CDR3), which contribute to antigen specificity. Certain heavy and light chains are preferred for particular antigens2-22. Here we consider pairs of B cells that share the same heavy chain V gene and CDRH3 amino acid sequence and were isolated from different donors, also known as public clonotypes23,24. We show that for naive antibodies (those not yet adapted to antigens), the probability that they use the same light chain V gene is around 10%, whereas for memory (functional) antibodies, it is around 80%, even if only one cell per clonotype is used. This property of functional antibodies is a phenomenon that we call light chain coherence. We also observe this phenomenon when similar heavy chains recur within a donor. Thus, although naive antibodies seem to recur by chance, the recurrence of functional antibodies reveals surprising constraint and determinism in the processes of V(D)J recombination and immune selection. For most functional antibodies, the heavy chain determines the light chain.

Cbl Ubiquitin Ligases Control B Cell Exit from the Germinal-Center Reaction.

Selective expansion of high-affinity antigen-specific B cells in germinal centers (GCs) is a key event in antibody affinity maturation. GC B cells with improved affinity can either continue affinity-driven selection or exit the GC to differentiate into plasma cells (PCs) or memory B cells. Here we found that deleting E3 ubiquitin ligases Cbl and Cbl-b (Cbls) in GC B cells resulted in the early exit of high-affinity antigen-specific B cells from the GC reaction and thus impaired clonal expansion. Cbls were highly expressed in GC light zone (LZ) B cells, where they promoted the ubiquitination and degradation of Irf4, a transcription factor facilitating PC fate choice. Strong CD40 and BCR stimulation triggered the Cbl degradation, resulting in increased Irf4 expression and exit from GC affinity selection. Thus, a regulatory cascade that is centered on the Cbl ubiquitin ligases ensures affinity-driven clonal expansion by connecting BCR affinity signals with differentiation programs.

Salmonella Infection Drives Promiscuous B Cell Activation Followed by Extrafollicular Affinity Maturation.

The B cell response to Salmonella typhimurium (STm) occurs massively at extrafollicular sites, without notable germinal centers (GCs). Little is known in terms of its specificity. To expand the knowledge of antigen targets, we screened plasmablast (PB)-derived monoclonal antibodies (mAbs) for Salmonella specificity, using ELISA, flow cytometry, and antigen microarray. Only a small fraction (0.5%-2%) of the response appeared to be Salmonella-specific. Yet, infection of mice with limited B cell receptor (BCR) repertoires impaired the response, suggesting that BCR specificity was important. We showed, using laser microdissection, that somatic hypermutation (SHM) occurred efficiently at extrafollicular sites leading to affinity maturation that in turn led to detectable STm Ag-binding. These results suggest a revised vision of how clonal selection and affinity maturation operate in response to Salmonella. Clonal selection initially is promiscuous, activating cells with virtually undetectable affinity, yet SHM and selection occur during the extrafollicular response yielding higher affinity, detectable antibodies.

Thymic B Cells Are Licensed to Present Self Antigens for Central T Cell Tolerance Induction.

Thymic antigen-presenting cells (APCs) such as dendritic cells and medullary thymic epithelial cells (mTECs) use distinct strategies of self-antigen expression and presentation to mediate central tolerance. The thymus also harbors B cells; whether they also display unique tolerogenic features and how they genealogically relate to peripheral B cells is unclear. Here, we found that Aire is expressed in thymic but not peripheral B cells. Aire expression in thymic B cells coincided with major histocompatibility class II (MHCII) and CD80 upregulation and immunoglobulin class-switching. These features were recapitulated upon immigration of naive peripheral B cells into the thymus, whereby this intrathymic licensing required CD40 signaling in the context of cognate interactions with autoreactive CD4(+) thymocytes. Moreover, a licensing-dependent neo-antigen selectively upregulated in immigrating B cells mediated negative selection through direct presentation. Thus, autoreactivity within the nascent T cell repertoire fuels a feed forward loop that endows thymic B cells with tolerogenic features.

The carboxypeptidase ACE shapes the MHC class I peptide repertoire.

The surface presentation of peptides by major histocompatibility complex (MHC) class I molecules is critical to CD8(+) T cell-mediated adaptive immune responses. Aminopeptidases have been linked to the editing of peptides for MHC class I loading, but carboxy-terminal editing is thought to be due to proteasome cleavage. By analysis of wild-type mice and mice genetically deficient in or overexpressing the dipeptidase angiotensin-converting enzyme (ACE), we have now identified ACE as having a physiological role in the processing of peptides for MHC class I. ACE edited the carboxyl terminus of proteasome-produced MHC class I peptides. The lack of ACE exposed new antigens but also abrogated some self antigens. ACE had substantial effects on the surface expression of MHC class I in a haplotype-dependent manner. We propose a revised model of peptide processing for MHC class I by introducing carboxypeptidase activity into the process.

Eomes broadens the scope of CD8 T-cell memory by inhibiting apoptosis in cells of low affinity.

The memory CD8 T-cell pool must select for clones that bind immunodominant epitopes with high affinity to efficiently counter reinfection. At the same time, it must retain a level of clonal diversity to allow recognition of pathogens with mutated epitopes. How the level of diversity within the memory pool is controlled is unclear, especially in the context of a selective drive for antigen affinity. We find that preservation of clones that bind the activating antigen with low affinity depends on expression of the transcription factor Eomes in the first days after antigen encounter. Eomes is induced at low activating signal strength and directly drives transcription of the prosurvival protein Bcl-2. At higher signal intensity, T-bet is induced which suppresses Bcl-2 and causes a relative survival advantage for cells of low affinity. Clones activated with high-affinity antigen form memory largely independent of Eomes and have a proliferative advantage over clones that bind the same antigen with low affinity. This causes high-affinity clones to prevail in the memory pool, despite their relative survival deficit. Genetic or therapeutic targeting of the Eomes/Bcl-2 axis reduces the clonal diversity of the memory pool, which diminishes its ability to respond to pathogens carrying mutations in immunodominant epitopes. Thus, we demonstrate on a molecular level how sufficient diversity of the memory pool is established in an environment of affinity-based selection.

Germinal center responses to complex antigens.

Germinal centers (GCs) are the primary sites of antibody affinity maturation, sites where B-cell antigen-receptor (BCR) genes rapidly acquire mutations and are selected for increasing affinity for antigen. This process of hypermutation and affinity-driven selection results in the clonal expansion of B cells expressing mutated BCRs and acts to hone the antibody repertoire for greater avidity and specificity. Remarkably, whereas the process of affinity maturation has been confirmed in a number of laboratories, models for how affinity maturation in GCs operates are largely from studies of genetically restricted B-cell populations competing for a single hapten epitope. Much less is known about GC responses to complex antigens, which involve both inter- and intraclonal competition for many epitopes. In this review, we (i) compare current methods for analysis of the GC B-cell repertoire, (ii) describe recent studies of GC population dynamics in response to complex antigens, discussing how the observed repertoire changes support or depart from the standard model of clonal selection, and (iii) speculate on the nature and potential importance of the large fraction of GC B cells that do not appear to interact with native antigen.

Manipulation of the glycan-specific natural antibody repertoire for immunotherapy.

Natural immunoglobulin derived from innate-like B lymphocytes plays important roles in the suppression of inflammatory responses and represents a promising therapeutic target in a growing number of allergic and autoimmune diseases. These antibodies are commonly autoreactive and incorporate evolutionarily conserved specificities, including certain glycan-specific antibodies. Despite this conservation, exposure to bacterial polysaccharides during innate-like B lymphocyte development, through either natural exposure or immunization, induces significant changes in clonal representation within the glycan-reactive B cell pool. Glycan-reactive natural antibodies (NAbs) have been reported to play protective and pathogenic roles in autoimmune and inflammatory diseases. An understanding of the composition and functions of a healthy glycan-reactive NAb repertoire is therefore paramount. A more thorough understanding of NAb repertoire development holds promise for the design of both biological diagnostics and therapies. In this article, we review the development and functions of NAbs and examine three glycan specificities, represented in the innate-like B cell pool, to illustrate the complex roles environmental antigens play in NAb repertoire development. We also discuss the implications of increased clonal plasticity of the innate-like B cell repertoire during neonatal and perinatal periods, and the prospect of targeting B cell development with interventional therapies and correct defects in this important arm of the adaptive immune system.

Update on Aire and thymic negative selection.

Twenty years ago, the autoimmune regulator (Aire) gene was associated with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, and was cloned and sequenced. Its importance goes beyond its abstract link with human autoimmune disease. Aire identification opened new perspectives to better understand the molecular basis of central tolerance and self-non-self distinction, the main properties of the immune system. Since 1997, a growing number of immunologists and molecular geneticists have made important discoveries about the function of Aire, which is essentially a pleiotropic gene. Aire is one of the functional markers in medullary thymic epithelial cells (mTECs), controlling their differentiation and expression of peripheral tissue antigens (PTAs), mTEC-thymocyte adhesion and the expression of microRNAs, among other functions. With Aire, the immunological tolerance became even more apparent from the molecular genetics point of view. Currently, mTECs represent the most unusual cells because they express almost the entire functional genome but still maintain their identity. Due to the enormous diversity of PTAs, this uncommon gene expression pattern was termed promiscuous gene expression, the interpretation of which is essentially immunological - i.e. it is related to self-representation in the thymus. Therefore, this knowledge is strongly linked to the negative selection of autoreactive thymocytes. In this update, we focus on the most relevant results of Aire as a transcriptional and post-transcriptional controller of PTAs in mTECs, its mechanism of action, and its influence on the negative selection of autoreactive thymocytes as the bases of the induction of central tolerance and prevention of autoimmune diseases.

Shaping the repertoire of tumor-infiltrating effector and regulatory T cells.

Many tumors express antigens that can be specifically or selectively recognized by T lymphocytes, suggesting that T-cell-mediated immunity may be harnessed for the immunotherapy of cancer. However, since tumors originate from normal cells and evolve within the context of self-tissues, the immune mechanisms that prevent the autoimmune attack of normal tissues function in parallel to restrict anti-tumor immunity. In particular, the purging of autoreactive T cells and the development of immune-suppressive regulatory T cells (Tregs) are thought to be major barriers impeding anti-tumor immune responses. Here, we discuss current understanding regarding the antigens recognized by tumor-infiltrating T-cell populations, the mechanisms that shape the repertoire of these cells, and the role of the transcription factor autoimmune regulator (Aire) in these processes. Further elucidation of these principles is likely to be critical for optimizing emerging cancer immunotherapies, and for the rational design of novel therapies exhibiting robust anti-tumor activity with limited toxicity.

Targeted loss of SHP1 in murine thymocytes dampens TCR signaling late in selection.

SHP1 is a tyrosine phosphatase critical to proximal regulation of TCR signaling. Here, analysis of CD4-Cre SHP1(fl/fl) conditional knockout thymocytes using CD53, TCRß, CD69, CD4, and CD8α expression demonstrates the importance of SHP1 in the survival of post selection (CD53(+) ), single-positive thymocytes. Using Ca(2+) flux to assess the intensity of TCR signaling demonstrated that SHP1 dampens the signal strength of these same mature, postselection thymocytes. Consistent with its dampening effect, TCR signal strength was also probed functionally using peptides that can mediate selection of the OT-I TCR, to reveal increased negative selection mediated by lower-affinity ligand in the absence of SHP1. Our data show that SHP1 is required for the survival of mature thymocytes and the generation of the functional T-cell repertoire, as its absence leads to a reduction in the numbers of CD4(+) and CD8(+) naïve T cells in the peripheral lymphoid compartments.

VprBP Is Required for Efficient Editing and Selection of Igκ+ B Cells, but Is Dispensable for Igλ+ and Marginal Zone B Cell Maturation and Selection.

B cell development past the pro-B cell stage in mice requires the Cul4-Roc1-DDB1 E3 ubiquitin ligase substrate recognition subunit VprBP. Enforced Bcl2 expression overcomes defects in distal VH-DJH and secondary Vκ-Jκ rearrangement associated with VprBP insufficiency in B cells and substantially rescues maturation of marginal zone and Igλ(+) B cells, but not Igκ(+) B cells. In this background, expression of a site-directed Igκ L chain transgene increases Igκ(+) B cell frequency, suggesting VprBP does not regulate L chain expression from a productively rearranged Igk allele. In site-directed anti-dsDNA H chain transgenic mice, loss of VprBP function in B cells impairs selection of Igκ editor L chains typically arising through secondary Igk rearrangement, but not selection of Igλ editor L chains. Both H and L chain site-directed transgenic mice show increased B cell anergy when VprBP is inactivated in B cells. Taken together, these data argue that VprBP is required for the efficient receptor editing and selection of Igκ(+) B cells, but is largely dispensable for Igλ(+) B cell development and selection, and that VprBP is necessary to rescue autoreactive B cells from anergy induction.

mir-181a-1/b-1 Modulates Tolerance through Opposing Activities in Selection and Peripheral T Cell Function.

Understanding the consequences of tuning TCR signaling on selection, peripheral T cell function, and tolerance in the context of native TCR repertoires may provide insight into the physiological control of tolerance. In this study, we show that genetic ablation of a natural tuner of TCR signaling, mir-181a-1/b-1, in double-positive thymocytes dampened TCR and Erk signaling and increased the threshold of positive selection. Whereas mir-181a-1/b-1 deletion in mice resulted in an increase in the intrinsic reactivity of naive T cells to self-antigens, it did not cause spontaneous autoimmunity. Loss of mir-181a-1/b-1 dampened the induction of experimental autoimmune encephalomyelitis and reduced basal TCR signaling in peripheral T cells and their migration from lymph nodes to pathogenic sites. Taken together, these results demonstrate that tolerance can be modulated by microRNA gene products through the control of opposing activities in T cell selection and peripheral T cell function.

Distinct phases in the positive selection of CD8+ T cells distinguished by intrathymic migration and T-cell receptor signaling patterns.

Positive selection of CD8 T cells in the thymus is thought to be a multistep process lasting 3-4 d; however, the discrete steps involved are poorly understood. Here, we examine phenotypic changes, calcium signaling, and intrathymic migration in a synchronized cohort of MHC class I-specific thymocytes undergoing positive selection in situ. Transient elevations in intracellular calcium concentration ([Ca(2+)]i) and migratory pauses occurred throughout the first 24 h of positive selection, becoming progressively briefer and accompanied by a gradual shift in basal [Ca(2+)]i over time. Changes in chemokine-receptor expression and relocalization from the cortex to medulla occurred between 12 and 24 h after the initial encounter with positive-selecting ligands, a time frame at which the majority of thymocytes retain CD4 and CD8 expression and still require T-cell receptor (TCR) signaling to efficiently complete positive selection. Our results identify distinct phases in the positive selection of MHC class I-specific thymocytes that are distinguished by their TCR-signaling pattern and intrathymic location and provide a framework for understanding the multistep process of positive selection in the thymus.

Promiscuous Foxp3-cre activity reveals a differential requirement for CD28 in Foxp3⁺ and Foxp3⁻ T cells.

Costimulatory signals by CD28 are critical for thymic regulatory T-cell (Treg) development. To determine the functional relevance of CD28 for peripheral Treg post thymic selection, we crossed the widely used Forkhead box protein 3 (Foxp3)-CreYFP mice to mice bearing a conditional Cd28 allele. Treg-specific CD28 deficiency provoked a severe autoimmune syndrome as a result of a strong disadvantage in competitive fitness and proliferation of CD28-deficient Tregs. By contrast, Treg survival and lineage integrity were not affected by the lack of CD28. This data demonstrate that, even after the initial induction requirement, Treg maintain a higher dependency on CD28 signalling than conventional T cells for homeostasis. In addition, we found the Foxp3-CreYFP allele to be a hypomorph, with reduced Foxp3 protein levels. Furthermore, we report here the stochastic activity of the Foxp3-CreYFP allele in non-Tregs, sufficient to recombine some conditional alleles (including Cd28) but not others (including R26-RFP). This hypomorphism and 'leaky' expression of the Foxp3-CreYFP allele should be considered when analysing the conditionally mutated Treg.

A novel approach to tracking antigen-experienced CD4 T cells into functional compartments via tandem deep and shallow TCR clonotyping.

Extensive diversity in the human repertoire of TCRs for Ag is both a cornerstone of effective adaptive immunity that enables host protection against a multiplicity of pathogens and a weakness that gives rise to potential pathological self-reactivity. The complexity arising from diversity makes detection and tracking of single Ag-specific CD4 T cells (ASTs) involved in these immune responses challenging. We report a tandem, multistep process to quantify rare TCRß-chain variable sequences of ASTs in large polyclonal populations. The approach combines deep high-throughput sequencing (HTS) within functional CD4 T cell compartments, such as naive/memory cells, with shallow, multiple identifier-based HTS of ASTs identified by activation marker upregulation after short-term Ag stimulation in vitro. We find that clonotypes recognizing HLA class II-restricted epitopes of both pathogen-derived Ags and self-Ags are oligoclonal and typically private. Clonotype tracking within an individual reveals private AST clonotypes resident in the memory population, as would be expected, representing clonal expansions (identical nucleotide sequence; "ultraprivate"). Other AST clonotypes share CDR3ß amino acid sequences through convergent recombination and are found in memory populations of multiple individuals. Tandem HTS-based clonotyping will facilitate studying AST dynamics, epitope spreading, and repertoire changes that arise postvaccination and following Ag-specific immunotherapies for cancer and autoimmune disease.

MicroRNAs in autoimmune rheumatic diseases.

The etiology of autoimmune diseases remains largely unknown. In recent years, besides genetic factors, several studies proposed that the epigenome may hold the key to a better understanding of autoimmunity initiation and perpetuation. More specifically epigenetic regulatory mechanisms comprise DNA methylation, a variety of histone modifications, and microRNA (miRNA) activity, all of which act upon gene and protein expression levels. In particular it is well known that epigenetic mechanisms are important for controlling the pattern of gene expression during development, the cell cycle, and the response to biological or environmental changes. In the present review a description of the most frequent epigenetic deregulations, in particular the role of miRNAs, in rheumatic autoimmune disorders will be investigated.

Factors that influence the thymic selection of CD8αα intraepithelial lymphocytes.

Thymocytes bearing αß T cell receptors (TCRαß) with high affinity for self-peptide-MHC complexes undergo negative selection or are diverted to alternate T cell lineages, a process termed agonist selection. Among thymocytes bearing TCRs restricted to MHC class I, agonist selection can lead to the development of precursors that can home to the gut and give rise to CD8αα-expressing intraepithelial lymphocytes (CD8αα IELs). The factors that influence the choice between negative selection versus CD8αα IEL development remain largely unknown. Using a synchronized thymic tissue slice model that supports both negative selection and CD8αα IEL development, we show that the affinity threshold for CD8αα IEL development is higher than for negative selection. We also investigate the impact of peptide presenting cells and cytokines, and the migration patterns associated with these alternative cell fates. Our data highlight the roles of TCR affinity and the thymic microenvironments on T cell fate.

Role, function and regulation of the thymocyte selection-associated high mobility group box protein in CD8+ T cell exhaustion.

Thymocyte selection-associated high mobility group box protein (TOX), a member of the high-motility group box (HMG) protein superfamily, is an evolutionarily conserved DNA-binding protein. It functions as a transcription factor that modulates transcriptional programs by binding to DNA in a structure-dependent manner. It has been well established that TOX is required for the development of CD4+ T cells, natural killer (NK) cells and innate lymphoid cells (ILCs), as well as the autoimmunity mediated by CD8+ T cells. Recently, emerging evidence supports an essential role for TOX in the induction of T cell exhaustion in the setting of tumor or chronic viral infection by mediating transcriptional and epigenetic changes, which are cardinal hallmarks of exhausted T cells. Moreover, TOX plays a key role in the persistence of antigen-specific T cells and in the mitigation of tissue damage caused by immunopathology over the course of tumorigenesis and chronic infection. Additionally, TOX contributes to the high level of programmed cell death protein 1 (PD-1) on the cell surface by participating in the process of endocytic recycling of PD-1. In this review, we summarize the most recent information about the role of TOX in the process of T cell exhaustion, which enriches our understanding of the molecular mechanisms of CD8+ T cell exhaustion upon chronic antigen stimulation and reveals promising therapeutic targets for persisting infection and cancer.