Central human B cell tolerance manifests with a distinctive cell phenotype and is enforced via CXCR4 signaling in hu-mice.

Central B cell tolerance, the process restricting the development of many newly generated autoreactive B cells, has been intensely investigated in mouse cells while studies in humans have been hampered by the inability to phenotypically distinguish autoreactive and nonautoreactive immature B cell clones and the difficulty in accessing fresh human bone marrow samples. Using a human immune system mouse model in which all human Igκ+ B cells undergo central tolerance, we discovered that human autoreactive immature B cells exhibit a distinctive phenotype that includes lower activation of ERK and differential expression of CD69, CD81, CXCR4, and other glycoproteins. Human B cells exhibiting these characteristics were observed in fresh human bone marrow tissue biopsy specimens, although differences in marker expression were smaller than in the humanized mouse model. Furthermore, the expression of these markers was slightly altered in autoreactive B cells of humanized mice engrafted with some human immune systems genetically predisposed to autoimmunity. Finally, by treating mice and human immune system mice with a pharmacologic antagonist, we show that signaling by CXCR4 is necessary to prevent both human and mouse autoreactive B cell clones from egressing the bone marrow, indicating that CXCR4 functionally contributes to central B cell tolerance.

Is T Cell Negative Selection a Learning Algorithm?

Our immune system can destroy most cells in our body, an ability that needs to be tightly controlled. To prevent autoimmunity, the thymic medulla exposes developing T cells to normal "self" peptides and prevents any responders from entering the bloodstream. However, a substantial number of self-reactive T cells nevertheless reaches the periphery, implying that T cells do not encounter all self peptides during this negative selection process. It is unclear if T cells can still discriminate foreign peptides from self peptides they haven't encountered during negative selection. We use an "artificial immune system"-a machine learning model of the T cell repertoire-to investigate how negative selection could alter the recognition of self peptides that are absent from the thymus. Our model reveals a surprising new role for T cell cross-reactivity in this context: moderate T cell cross-reactivity should skew the post-selection repertoire towards peptides that differ systematically from self. Moreover, even some self-like foreign peptides can be distinguished provided that the peptides presented in the thymus are not too similar to each other. Thus, our model predicts that negative selection on a well-chosen subset of self peptides would generate a repertoire that tolerates even "unseen" self peptides better than foreign peptides. This effect would resemble a "generalization" process as it is found in learning systems. We discuss potential experimental approaches to test our theory.

An AIREless Breath: Pneumonitis Caused by Impaired Central Immune Tolerance.

Autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a monogenic disorder caused by biallelic mutations in the AIRE gene, has historically been defined by the development of chronic mucocutaneous candidiasis together with autoimmune endocrinopathies, primarily hypoparathyroidism and adrenal insufficiency. Recent work has drawn attention to the development of life-threatening non-endocrine manifestations such as autoimmune pneumonitis, which has previously been poorly recognized and under-reported. In this review, we present the clinical, radiographic, autoantibody, and pulmonary function abnormalities associated with APECED pneumonitis, we highlight the cellular and molecular basis of the autoimmune attack in the AIRE-deficient lung, and we provide a diagnostic and a therapeutic roadmap for patients with APECED pneumonitis. Beyond APECED, we discuss the relevance and potential broader applicability of these findings to other interstitial lung diseases seen in secondary AIRE deficiency states such as thymoma and RAG deficiency or in common polygenic autoimmune disorders such as idiopathic Sjögren's syndrome.

Dormant pathogenic CD4+ T cells are prevalent in the peripheral repertoire of healthy mice.

Thymic central tolerance eliminates most immature T cells with autoreactive T cell receptors (TCR) that recognize self MHC/peptide complexes. Regardless, an unknown number of autoreactive CD4+Foxp3- T cells escape negative selection and in the periphery require continuous suppression by CD4+Foxp3+ regulatory cells (Tregs). Here, we compare immune repertoires of Treg-deficient and Treg-sufficient mice to find Tregs continuously constraining one-third of mature CD4+Foxp3- cells from converting to pathogenic effectors in healthy mice. These dormant pathogenic clones frequently express TCRs activatable by ubiquitous autoantigens presented by class II MHCs on conventional dendritic cells, including self-peptides that select them in the thymus. Our data thus suggest that identification of most potentially autoreactive CD4+ T cells in the peripheral repertoire is critical to harness or redirect these cells for therapeutic advantage.

Pulling RANK on Cancer: Blocking Aire-Mediated Central Tolerance to Enhance Immunotherapy.

A major breakthrough in cancer treatment occurred with the development of strategies that overcome T-cell tolerance toward tumor cells. These approaches enhance antitumor immunity by overcoming mechanisms that are normally in place to prevent autoimmunity but simultaneously prevent rejection of tumor cells. Although tolerance mechanisms that restrict antitumor immunity take place both in the thymus and periphery, only immunotherapies that target peripheral tolerance mechanisms occurring outside of the thymus are currently available. We review here recent gains in our understanding of how thymic tolerance mediated by the autoimmune regulator (Aire) impedes antitumor immunity. It is now clear that transient depletion of Aire-expressing cells in the thymus can be achieved with RANKL blockade. Finally, we discuss key findings that support the repurposing of anti-RANKL as a cancer immunotherapy with a unique mechanism of action.

Live-cell imaging reveals the relative contributions of antigen-presenting cell subsets to thymic central tolerance.

Both medullary thymic epithelial cells (mTEC) and dendritic cells (DC) present tissue-restricted antigens (TRA) to thymocytes to induce central tolerance, but the relative contributions of these antigen-presenting cell (APC) subsets remain unresolved. Here we developed a two-photon microscopy approach to observe thymocytes interacting with intact APCs presenting TRAs. We find that mTECs and DCs cooperate extensively to induce tolerance, with their relative contributions regulated by the cellular form of the TRA and the class of major histocompatibility complex (MHC) on which antigen is presented. Even when TRA expression is restricted to mTECs, DCs still present self-antigens at least as frequently as mTECs. Notably, the DC subset cDC2 efficiently acquires secreted mTEC-derived TRAs for cross-presentation on MHC-I. By directly imaging interactions between thymocytes and APCs, while monitoring intracellular signaling, this study reveals that distinct DC subsets and AIRE+ mTECs contribute substantially to presentation of diverse self-antigens for establishing central tolerance.

Active PI3K abrogates central tolerance in high-avidity autoreactive B cells.

Autoreactive B cells that bind self-antigen with high avidity in the bone marrow undergo mechanisms of central tolerance that prevent their entry into the peripheral B cell population. These mechanisms are breached in many autoimmune patients, increasing their risk of B cell-mediated autoimmune diseases. Resolving the molecular pathways that can break central B cell tolerance could therefore provide avenues to diminish autoimmunity. Here, we show that B cell-intrinsic expression of a constitutively active form of PI3K-P110α by high-avidity autoreactive B cells of mice completely abrogates central B cell tolerance and further promotes these cells to escape from the bone marrow, differentiate in peripheral tissue, and undergo activation in response to self-antigen. Upon stimulation with T cell help factors, these B cells secrete antibodies in vitro but remain unable to secrete autoantibodies in vivo. Overall, our data demonstrate that activation of the PI3K pathway leads high-avidity autoreactive B cells to breach central, but not late, stages of peripheral tolerance.

Central Tolerance Mechanisms to TSHR in Graves' Disease: Contributions to Understand the Genetic Association.

In the last 3 years, the association of thyrotropin receptor gene (TSHR) variations to Graves' disease (GD) has been confirmed. It is now well established that a 30 Kb region of intron 1 of the TSHR gene is linked to GD predisposition. Elucidating the mechanism(s) by which these polymorphisms confer susceptibility is difficult but would constitute an important advance in endocrine autoimmunity in general. Two hypotheses, both postulating TSHR gene regulatory mechanisms, are discussed. One postulates differential level of expression in the thymus, involving central tolerance. The other postulates a shift in TSHR differential splicing leading to the production of soluble proteins that will have easy access to antigen presenting cells, so it is focused in peripheral tolerance. A combination of the 2 hypothesis is feasible, especially under the light of recent evidence that have identified epigenetic factors acting on TSHR intron 1.

IL-4 and IL-13 Guide Early Thymic Progenitors To Mature toward Dendritic Cells.

Recently we reported that IL-4 and IL-13 signaling in murine early thymic progenitors (ETPs) expressing the heteroreceptor (HR) comprising IL-4 receptor α (IL-4Rα) and IL-13 receptor α 1 (IL-13Rα1) activate STAT6 and inhibit ETP maturation potential toward T cells. In this study, we asked whether IL-4 and IL-13 signaling through the HR mobilizes other STAT molecules to shape ETP fate decision. The findings indicate that HR+ ETPs undergoing cytokine signaling display increased STAT1, but not STAT3, phosphorylation in addition to STAT6 activation. In parallel, the ETPs had a STAT1-dependent heightened expression of IRF-8, a transcription factor essential for development of CD8α+ dendritic cells (DCs). Interestingly, STAT1 phosphorylation and IRF-8 upregulation, which are independent of STAT6 activation, guided ETP maturation toward myeloid cells with a CD8α+ DC phenotype. Furthermore, these CD8α+ DCs display a thymic resident phenotype, as they did not express SIRPα, a molecule presumed to be involved in cell migration. These findings suggest that IL-4 and IL-13 cytokine-induced HR signaling provides a double-edged sword that simultaneously blocks T cell lineage potential but advances myeloid maturation that could impact T cell selection and central tolerance.

Activation of the MEK-ERK Pathway Is Necessary but Not Sufficient for Breaking Central B Cell Tolerance.

Newly generated bone marrow B cells are positively selected into the peripheral lymphoid tissue only when they express a B cell receptor (BCR) that is nonautoreactive or one that binds self-antigen with only minimal avidity. This positive selection process, moreover, is critically contingent on the ligand-independent tonic signals transduced by the BCR. We have previously shown that when autoreactive B cells express an active form of the rat sarcoma (RAS) oncogene, they upregulate the receptor for the B cell activating factor (BAFFR) and undergo differentiation in vitro and positive selection into the spleen in vivo, overcoming central tolerance. Based on the in vitro use of pharmacologic inhibitors, we further showed that this cell differentiation process is critically dependent on the activation of the mitogen-activated protein kinase kinase pathway MEK (MAPKK)-extracellular signal-regulated kinase (ERK), which is downstream of RAS. Here, we next investigated if activation of ERK is not only necessary but also sufficient to break central B cell tolerance and induce differentiation of autoreactive B cells in vitro and in vivo. Our results demonstrate that activation of ERK is critical for upregulating BAFFR and overcoming suboptimal levels of tonic BCR signals or low amounts of antigen-induced BCR signals during in vitro B cell differentiation. However, direct activation of ERK does not lead high avidity autoreactive B cells to increase BAFFR levels and undergo positive selection and differentiation in vivo. B cell-specific MEK-ERK activation in mice is also unable to lead to autoantibody secretion, and this in spite of a general increase of serum immunoglobulin levels. These findings indicate that additional pathways downstream of RAS are required for high avidity autoreactive B cells to break central and/or peripheral tolerance.

Age-Associated Decline in Thymic B Cell Expression of Aire and Aire-Dependent Self-Antigens.

Although autoimmune disorders are a significant source of morbidity and mortality in older individuals, the mechanisms governing age-associated increases in susceptibility remain incompletely understood. Central T cell tolerance is mediated through presentation of self-antigens by cells constituting the thymic microenvironment, including epithelial cells, dendritic cells, and B cells. Medullary thymic epithelial cells (mTECs) and B cells express distinct cohorts of self-antigens, including tissue-restricted self-antigens (TRAs), such that developing T cells are tolerized to antigens from peripheral tissues. We find that expression of the TRA transcriptional regulator Aire, as well as Aire-dependent genes, declines with age in thymic B cells in mice and humans and that cell-intrinsic and cell-extrinsic mechanisms contribute to the diminished capacity of peripheral B cells to express Aire within the thymus. Our findings indicate that aging may diminish the ability of thymic B cells to tolerize T cells, revealing a potential mechanistic link between aging and autoimmunity.

Rapid chromatin repression by Aire provides precise control of immune tolerance.

Aire mediates the expression of tissue-specific antigens in thymic epithelial cells to promote tolerance against self-reactive T lymphocytes. However, the mechanism that allows expression of tissue-specific genes at levels that prevent harm is unknown. Here we show that Brg1 generates accessibility at tissue-specific loci to impose central tolerance. We found that Aire has an intrinsic repressive function that restricts chromatin accessibility and opposes Brg1 across the genome. Aire exerted this repressive influence within minutes after recruitment to chromatin and restrained the amplitude of active transcription. Disease-causing mutations that impair Aire-induced activation also impair the protein's repressive function, which indicates dual roles for Aire. Together, Brg1 and Aire fine-tune the expression of tissue-specific genes at levels that prevent toxicity yet promote immune tolerance.

T cells specific for post-translational modifications escape intrathymic tolerance induction.

Establishing effective central tolerance requires the promiscuous expression of tissue-restricted antigens by medullary thymic epithelial cells. However, whether central tolerance also extends to post-translationally modified proteins is not clear. Here we show a mouse model of autoimmunity in which disease development is dependent on post-translational modification (PTM) of the tissue-restricted self-antigen collagen type II. T cells specific for the non-modified antigen undergo efficient central tolerance. By contrast, PTM-reactive T cells escape thymic selection, though the PTM variant constitutes the dominant form in the periphery. This finding implies that the PTM protein is absent in the thymus, or present at concentrations insufficient to induce negative selection of developing thymocytes and explains the lower level of tolerance induction against the PTM antigen. As the majority of self-antigens are post-translationally modified, these data raise the possibility that T cells specific for other self-antigens naturally subjected to PTM may escape central tolerance induction by a similar mechanism.

Redefining thymus medulla specialization for central tolerance.

During αßT cell development, the thymus medulla represents an essential microenvironment for T cell tolerance. This functional specialization is attributed to its typical organized topology consisting of a branching structure that contains medullary thymic epithelial cell (mTEC) networks to support negative selection and Foxp3+ T-regulatory cell (T-reg) development. Here, by performing TEC-specific deletion of the thymus medulla regulator lymphotoxin ß receptor (LTßR), we show that thymic tolerance mechanisms operate independently of LTßR-mediated mTEC development and organization. Consistent with this, mTECs continue to express Fezf2 and Aire, regulators of intrathymic self-antigens, and support T-reg development despite loss of LTßR-mediated medulla organogenesis. Moreover, we demonstrate that LTßR controls thymic tolerance by regulating the frequency and makeup of intrathymic dendritic cells (DCs) required for effective thymocyte negative selection. In all, our study demonstrates that thymus medulla specialization for thymic tolerance segregates from medulla organogenesis and instead involves LTßR-mediated regulation of the thymic DC pool.

EBI2 contributes to the induction of thymic central tolerance in mice by promoting rapid motility of medullary thymocytes.

Maturing thymocytes enter the thymic medulla, where they encounter numerous self-antigens presented by antigen presenting cells (APCs). Those thymocytes that are strongly self-reactive undergo either negative selection or diversion into the regulatory T-cell lineage. Although the majority of the proteome is expressed in the medulla, many self-antigens are expressed by only a minor fraction of medullary APCs; thus, thymocytes must efficiently enter the medulla and scan APCs to ensure central tolerance. Chemokine receptors promote lymphocyte migration, organization within tissues, and interactions with APCs in lymphoid organs. The chemokine receptor EBI2 governs localization of T cells, B cells, and dendritic cells (DCs) during immune responses in secondary lymphoid organs. However, the role of EBI2 in thymocyte development has not been elucidated. Here, we demonstrate that EBI2 is expressed by murine CD4+ single positive (CD4SP) thymocytes and thymic DCs. EBI2 deficiency alters the TCR repertoire, but does not grossly impact thymocyte cellularity or subset distribution. EBI2 deficiency also impairs negative selection of OT-II TCR transgenic thymocytes responding to an endogenous self-antigen. Two-photon imaging revealed that EBI2 deficiency results in reduced migration and impaired medullary accumulation of CD4SP thymocytes. These data identify a role for EBI2 in promoting efficient thymic central tolerance.

The multiple pathways to autoimmunity.

Efforts to understand autoimmunity have been pursued relentlessly for several decades. It has become apparent that the immune system evolved multiple mechanisms for controlling self-reactivity, and defects in one or more of these mechanisms can lead to a breakdown of tolerance. Among the multitude of lesions associated with disease, the most common seem to affect peripheral tolerance rather than central tolerance. The initial trigger for both systemic autoimmune disorders and organ-specific autoimmune disorders probably involves the recognition of self or foreign molecules, especially nucleic acids, by innate sensors. Such recognition, in turn, triggers inflammatory responses and the engagement of previously quiescent autoreactive T cells and B cells. Here we summarize the most prominent autoimmune pathways and identify key issues that require resolution for full understanding of pathogenic autoimmunity.

Essential role of CCL21 in establishment of central self-tolerance in T cells.

The chemokine receptor CCR7 directs T cell relocation into and within lymphoid organs, including the migration of developing thymocytes into the thymic medulla. However, how three functional CCR7 ligands in mouse, CCL19, CCL21Ser, and CCL21Leu, divide their roles in immune organs is unclear. By producing mice specifically deficient in CCL21Ser, we show that CCL21Ser is essential for the accumulation of positively selected thymocytes in the thymic medulla. CCL21Ser-deficient mice were impaired in the medullary deletion of self-reactive thymocytes and developed autoimmune dacryoadenitis. T cell accumulation in the lymph nodes was also defective. These results indicate a nonredundant role of CCL21Ser in the establishment of self-tolerance in T cells in the thymic medulla, and reveal a functional inequality among CCR7 ligands in vivo.

Central immune tolerance depends on crosstalk between the classical and alternative NF-κB pathways in medullary thymic epithelial cells.

Medullary thymic epithelial cells (mTECs) contribute to self-tolerance by expressing and presenting peripheral tissue antigens for negative selection of autoreactive T cells and differentiation of natural regulatory T cells. The molecular control of mTEC development remains incompletely understood. We here demonstrate by TEC-specific gene manipulation in mice that the NF-κB transcription factor subunit RelB, which is activated by the alternative NF-κB pathway, regulates development of mature mTECs in a dose-dependent manner. Mice with conditional deletion of Relb lacked mature mTECs and developed spontaneous autoimmunity. In addition, the NF-κB subunits RelA and c-Rel, which are both activated by classical NF-κB signaling, were jointly required for mTEC differentiation by directly regulating the transcription of Relb. Our data reveal a crosstalk mechanism between classical and alternative NF-κB pathways that tightly controls the development of mature mTECs to ensure self-tolerance.

Self-Antigen-Driven Thymic B Cell Class Switching Promotes T Cell Central Tolerance.

B cells are unique antigen-presenting cells because their antigen presentation machinery is closely tied to the B cell receptor. Autoreactive thymic B cells can efficiently present cognate self-antigens to mediate CD4+ T cell-negative selection. However, the nature of thymocyte-thymic B cell interaction and how this interaction affects the selection of thymic B cell repertoire and, in turn, the T cell repertoire are not well understood. Here we demonstrate that a large percentage of thymic B cells have undergone class switching intrathymically. Thymic B cell class switching requires cognate interaction with specific T cells. Class-switched thymic B cells have a distinct repertoire compared with unswitched thymic B cells or splenic B cells. Particularly, autoreactive B cell specificities preferentially expand in the thymus by undergoing class switching, and these enriched, class-switched autoreactive thymic B cells play an important role in CD4 T cell tolerance.

Medullary thymic epithelial cells and CD8α+ dendritic cells coordinately regulate central tolerance but CD8α+ cells are dispensable for thymic regulatory T cell production.

In the thymus, antigen presenting cells (APCs) namely, medullary thymic epithelial cells (mTECs) and thymic dendritic cells (tDCs) regulate T cell tolerance through elimination of autoreactive T cells and production of thymic T regulatory (tTreg) cells. How the different APCs in the thymus share the burden of tolerazing the emerging T cell repertoire remains unclear. For example, while mutations that inhibit mTEC development or function associate with peripheral autoimmunity, the role of tDCs in organ-specific autoimmunity and tTreg cell production remains controversial. In this report we used mice depleted of mTECs and/or CD8α+ DCs, to examine the contributions of these cell populations in thymic tolerance. We found that while mice depleted of CD8α+ DCs or mTECs were normal or developed liver inflammation respectively, combined depletion of mTECs and CD8α+ DCs resulted in overt peripheral autoimmunity. The autoimmune manifestations in mice depleted of both mTECs and CD8α+ cDCs associated with increased percentages of CD4+ and CD8+ T cells in the thymus. In contrast, while mTEC depletion resulted in reduced percentages of tTreg cells, no additional effect was observed when CD8α+ DCs were also depleted. These results reveal that: 1) mTECs and CD8α+ DCs cooperatively safeguard against peripheral autoimmunity through thymic T cell deletion; 2) CD8α+ DCs are dispensable for tTreg cell production, whereas mTECs play a non-redundant role in this process; 3) mTECs and CD8α+ DCs make unique contributions to tolerance induction that cannot be compensated for by other thymic APCs such as migratory SIRPα+ or plasmacytoid DCs.