In the past few years, there has been a flurry of discoveries and advancements in our understanding of how the thymus prepares T cells to exist at peace in normal healthy tissue: that is, to be self-tolerant. In the thymus, one of the main mechanisms of T-cell central tolerance is clonal deletion, although the selection of regulatory T cells is also important and is gaining enormous interest. In this Review, we discuss the emerging consensus about which models of clonal deletion are most physiological, and we review recent data that define the molecular mechanisms of central tolerance.

Sequential rearrangement of the T cell receptor for antigen (TCR) β and α chains is a hallmark of thymocyte development. This temporal control is lost in TCR transgenics because the α chain is expressed prematurely at the CD4−CD8− double negative (DN) stage. To test the importance of this, we expressed the HYα chain at the physiological CD4+CD8+ double positive (DP) stage. The reduced DP and increased DN cellularity typically seen in TCR transgenics was not observed when the α chain was expressed at the appropriate stage. Surprisingly, antigen-driven selection events were also altered. In male mice, thymocyte deletion now occurred at the single positive or medullary stage. In addition, no expansion of CD8αα intestinal intraepithelial lymphocytes (IELs) was observed, despite the fact that HY transgenics have been used to model IEL development. Collectively, these data establish the importance of proper timing of TCR expression in thymic development and selection and emphasize the need to use models that most accurately reflect the physiologic process.

The thymic medulla is generally held to be a specialized environment for negative selection. However, many self-reactive thymocytes fi rst encounter ubiquitous self-antigens in the cortex. Cortical epithelial cells are vital for positive selection, but whether such cells can also promote negative selection is controversial. We used the HY cd4 model, where T cell receptor for antigen (TCR) expression is appropriately timed and a ubiquitous self-antigen drives clonal deletion in male mice. We demonstrated unambiguously that this deletion event occurs in the thymic cortex. However, the kinetics in vivo indicated that apoptosis was activated asynchronously relative to TCR activation. We found that radioresistant antigen-presenting cells and, specifi cally, cortical epithelial cells do not effi ciently induce apoptosis, although they do cause TCR activation. Rather, thymocytes undergoing clonal deletion were preferentially associated with rare CD11c + cortical dendritic cells, and elimination of such cells impaired deletion.

Lymphopenia driven T cell activation is associated with autoimmunity. That lymphopenia does not always lead to autoimmunity suggests that control mechanisms may exist. We assessed the importance of the co-inhibitory receptor programmed death-1 (PD-1) in the control of lymphopenia-driven autoimmunity in newly generated T cells vs. established peripheral T cells and in thymic selection. PD-1 was not required for negative selection in the thymus or for maintenance of self tolerance following transfer of established PD-1⁻/⁻ peripheral T cells to a lymphopenic host. In contrast, PD-1 was essential for systemic self tolerance in newly generated T cells under lymphopenic conditions, as PD-1⁻/⁻ recent thymic emigrants (RTE), generated after transfer of PD-1⁻/⁻ hematopoietic stem cell (HSC) precursors or thymocytes into lymphopenic adult Rag⁻/⁻ recipients, induced a rapidly lethal multi-organ inflammatory disease. Disease could be blocked by using lymph node deficient recipients, indicating that lymphopenia driven PD-1⁻/⁻ T cell activation required access to sufficient lymph node stroma. These data suggested that PD-1⁻/⁻ mice themselves might be substantially protected from autoimmunity because their T cell repertoire is first generated early in life, a period naturally deficient in lymph node stroma. Consistent with this idea, neonatal Rag⁻/⁻ recipients of PD-1⁻/⁻ HSC were resistant to disease. Thus, a critical role of PD-1 resides in the control of RTE in lymphopenia. The data suggest that PD-1 and a paucity of lymphoid stroma cooperate to control autoimmunity in newly generated T cells. Clinical therapies for autoimmune disease employing lymphoablation and hematopoietic stem cell transplantation will need to take into account functional polymorphisms in the PD-1 pathway, if the treatment is to ameliorate rather than exacerbate autoimmunity.

Material

Calnexin is a molecular chaperone and a component of the quality control of the secretory pathway. We have generated calnexin gene-deficient mice (cnx ؊/؊ ) and showed that calnexin deficiency leads to myelinopathy. Calnexin-deficient mice were viable with no discernible effects on other systems, including immune function, and instead they demonstrated dysmyelination as documented by reduced conductive velocity of nerve fibers and electron microscopy analysis of sciatic nerve and spinal cord. Myelin of the peripheral and central nervous systems of cnx ؊/؊ mice was disorganized and decompacted. There were no abnormalities in neuronal growth, no loss of neuronal fibers, and no change in fictive locomotor pattern in the absence of calnexin. This work reveals a previously unrecognized and important function of calnexin in myelination and provides new insights into the mechanisms responsible for myelin diseases.The endoplasmic reticulum (ER) 5 is the first compartment in the secretory pathway responsible for protein synthesis, posttranslational modification, and correct folding. The resident molecular chaperones ensure that only correctly folded proteins leave the ER. Calnexin is a type I ER membrane protein, a major component in assuring the quality control of the secretory pathway, and together with calreticulin and the oxidoreductase ERp57, it promotes the correct folding of newly synthesized glycoproteins (2). Calnexin and calreticulin bind monoglucosylated carbohydrate on newly synthesized glycoproteins, whereas ERp57 catalyzes rearrangements of disulfide bonds within the calnexin/calreticulin substrate proteins (2). Despite its ubiquitous expression, the absence of calnexin has a different effect in different organisms. Calnexin deficiency is lethal in Schizosaccharomyces pombe but not in Saccharomyces cerevisiae (3), Dictyostelium (4, 5), or Caenorhabditis elegans (6, 7). The loss of calnexin affects phagocytosis in Dictyostelium (4, 5) and promotes necrotic cell death in C. elegans (7). It has been reported that deletion of the calnexin gene in a mouse results in early postnatal death (1), and thus the molecular consequences of calnexin deficiency could not be studied.Here we show that calnexin deficiency in the mouse did not result in early postnatal death (1). These animals developed myelinopathy with no discernible effects on other systems, including immune function. The phenotype was linked to slow nerve conduction velocities in the absence of calnexin with evidence of peripheral axon dysmyelination. The dysmyelinating phenotype described here underscores the emerging importance of calnexin and ER-associated pathways as contributors to these severe neurological disorders.

During late stages of thymic development, T cells must chose between different fates, dictated by their TCR specificity. Typically, this is thought of as a choice between three alternatives (being positive selection for useful T cells vs negative selection or neglect for harmful or useless T cells). However, there is growing evidence for a fourth alternative, in which T cells are positively selected by agonist ligands, which would normally be expected to induce T cell deletion. In this review, we will discuss where and when agonist selection is induced and whether this should be considered as a novel form of thymic selection or as an alternative differentiation state for Ag-exposed T cells.

The nuclear orphan receptors NR4A1, NR4A2, and NR4A3 are immediate early genes that are induced by various signals. They act as transcription factors and their activity is not regulated by ligand binding and are thus regulated via their expression levels. Their expression is transiently induced in T cells by triggering of the T cell receptor following antigen recognition during both thymic differentiation and peripheral T cell responses. In this review, we will discuss how NR4A family members impact different aspects of the life of a T cell from thymic differentiation to peripheral response against infections and cancer.