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The precise steps of iNKT subset differentiation in the thymus and periphery have been controversial. We demonstrate here that the small proportion of thymic iNKT and mucosal associated invariant T cells that express CCR7 represent a multi-potent progenitor pool that gives rise to effector subsets within the thymus. Using intra-thymic labeling, we also showed that CCR7+ iNKT cells emigrate from the thymus in a Klf2 dependent manner, and undergo further maturation after reaching the periphery. Ccr7 deficiency impaired differentiation of iNKT effector subsets and localization to the medulla. Parabiosis and intra-thymic transfer showed that thymic NKT1 and NKT17 were resident—they were not derived from and did not contribute to the peripheral pool. Finally, each thymic iNKT effector subset produces distinct factors that influence T cell development. Our findings demonstrate how the thymus is both a source of iNKT progenitors and a unique site of tissue dependent effector cell differentiation.

Mature invariant natural killer T cells ( i NKT cells) are an early innate source of cytokines. Hogquist and colleagues classify three lineages of i NKT cells by their distinct transcription factors and cytokine profiles. Invariant natural killer T cells ( i NKT cells) can produce copious amounts of interleukin 4 (IL-4) early during infection. However, indirect evidence suggests they may produce this immunomodulatory cytokine in the steady state. Through intracellular staining for transcription factors, we have defined three subsets of i NKT cells (NKT1, NKT2 and NKT17) that produced distinct cytokines; these represented diverse lineages and not developmental stages, as previously thought. These subsets exhibited substantial interstrain variation in numbers. In several mouse strains, including BALB/c, NKT2 cells were abundant and were stimulated by self ligands to produce IL-4. In those strains, steady-state IL-4 conditioned CD8 + T cells to become 'memory-like', increased serum concentrations of immunoglobulin E (IgE) and caused dendritic cells to produce chemokines. Thus, i NKT cell–derived IL-4 altered immunological properties under normal steady-state conditions.

In contrast to peptide-recognizing T cells, invariant natural killer T (iNKT) cells express a semi-invariant T cell receptor that specifically recognizes self- or foreign-lipids presented by CD1d molecules. There are three major functionally distinct effector states for iNKT cells. Owning to these innate-like effector states, iNKT cells have been implicated in early protective immunity against pathogens. Yet, growing evidence suggests that iNKT cells play a role in tissue homeostasis as well. In this review, we discuss current knowledge about the underlying mechanisms that regulate the effector states of iNKT subsets, with a highlight on the roles of a variety of transcription factors and describe how each subset influences different facets of thymus homeostasis.

TCRβ + CD8αα + intraepithelial lymphocytes arise from CD4 − CD8 − CD5 + thymic cells, but the exact precursor source has been not been established. Hogquist and colleagues identify two distinct thymic populations that both give rise mainly to gut-homing intraepithelial lymphocytes. TCRαβ + CD4 − CD8α + CD8β − intestinal intraepithelial lymphocytes (CD8αα IELs) are an abundant population of thymus-derived T cells that protect the gut barrier surface. We sought to better define the thymic IEL precursor (IELp) through analysis of its maturation, localization and emigration. We defined two precursor populations among TCRβ + CD4 − CD8 − thymocytes by dependence on the kinase TAK1 and rigorous lineage-exclusion criteria. Those IELp populations included a nascent PD-1 + population and a T-bet + population that accumulated with age. Both gave rise to intestinal CD8αα IELs after adoptive transfer. The PD-1 + IELp population included more strongly self-reactive clones and was largely restricted by classical major histocompatibility complex (MHC) molecules. Those cells localized to the cortex and efficiently emigrated in a manner dependent on the receptor S1PR1. The T-bet + IELp population localized to the medulla, included cells restricted by non-classical MHC molecules and expressed the receptor NK1.1, the integrin CD103 and the chemokine receptor CXCR3. The two IELp populations further differed in their use of the T cell antigen receptor (TCR) α-chain variable region (V α ) and β-chain variable region (V β ). These data provide a foundation for understanding the biology of CD8αα IELs.

The thymus is responsible for generating a diverse yet self-tolerant pool of T cells 1 . Although the thymic medulla consists mostly of developing and mature AIRE + epithelial cells, recent evidence has suggested that there is far greater heterogeneity among medullary thymic epithelial cells than was previously thought 2 . Here we describe in detail an epithelial subset that is remarkably similar to peripheral tuft cells that are found at mucosal barriers 3 . Similar to the periphery, thymic tuft cells express the canonical taste transduction pathway and IL-25. However, they are unique in their spatial association with cornified aggregates, ability to present antigens and expression of a broad diversity of taste receptors. Some thymic tuft cells pass through an Aire -expressing stage and depend on a known AIRE-binding partner, HIPK2, for their development. Notably, the taste chemosensory protein TRPM5 is required for their thymic function through which they support the development and polarization of thymic invariant natural killer T cells and act to establish a medullary microenvironment that is enriched in the type 2 cytokine, IL-4. These findings indicate that there is a compartmentalized medullary environment in which differentiation of a minor and highly specialized epithelial subset has a non-redundant role in shaping thymic function. A comprehensive analysis of the thymic medulla identifies a tuft-cell-like thymic epithelial cell population that is necessary for shaping thymic function.

Regulatory T (Treg) cells control self-tolerance, inflammatory responses and tissue homeostasis. In mature Treg cells, continued expression of FOXP3 maintains lineage identity, while T cell receptor (TCR) signaling and interleukin-2 (IL-2)/STAT5 activation support the suppressive effector function of Treg cells, but how these regulators synergize to control Treg cell homeostasis and function remains unclear. Here we show that TCR-activated posttranslational modification by O-linked N-Acetylglucosamine (O-GlcNAc) stabilizes FOXP3 and activates STAT5, thus integrating these critical signaling pathways. O-GlcNAc-deficient Treg cells develop normally but display modestly reduced FOXP3 expression, strongly impaired lineage stability and effector function, and ultimately fatal autoimmunity in mice. Moreover, deficiency in protein O-GlcNAcylation attenuates IL-2/STAT5 signaling, while overexpression of a constitutively active form of STAT5 partially ameliorates Treg cell dysfunction and systemic inflammation in O-GlcNAc deficient mice. Collectively, our data demonstrate that protein O-GlcNAcylation is essential for lineage stability and effector function in Treg cells. The transcription factor Foxp3 and Stat5 modulate lineage stability and function of regulatory T (Treg) cells to promote immune homeostasis. Here the authors show that O-GlcNAcylation of Foxp3 and Stat5, mediated by O-GlcNAc transferase (OGT), is essential for Treg-mediate immune balance, with Treg-specific deficiency of OGT leading to severe autoimmunity.

Epstein-Barr virus (EBV) is a human herpesvirus that causes acute infectious mononucleosis and is associated with cancer and autoimmune disease. While many studies have been performed examining acute disease in adults following primary infection, little is known about the virological and immunological events during EBV's lengthy 6 week incubation period owing to the challenge of collecting samples from this stage of infection. We conducted a prospective study in college students with special emphasis on frequent screening to capture blood and oral wash samples during the incubation period. Here we describe the viral dissemination and immune response in the 6 weeks prior to onset of acute infectious mononucleosis symptoms. While virus is presumed to be present in the oral cavity from time of transmission, we did not detect viral genomes in the oral wash until one week before symptom onset, at which time viral genomes were present in high copy numbers, suggesting loss of initial viral replication control. In contrast, using a sensitive nested PCR method, we detected viral genomes at low levels in blood about 3 weeks before symptoms. However, high levels of EBV in the blood were only observed close to symptom onset-coincident with or just after increased viral detection in the oral cavity. These data imply that B cells are the major reservoir of virus in the oral cavity prior to infectious mononucleosis. The early presence of viral genomes in the blood, even at low levels, correlated with a striking decrease in the number of circulating plasmacytoid dendritic cells well before symptom onset, which remained depressed throughout convalescence. On the other hand, natural killer cells expanded only after symptom onset. Likewise, CD4+ Foxp3+ regulatory T cells decreased two fold, but only after symptom onset. We observed no substantial virus specific CD8 T cell expansion during the incubation period, although polyclonal CD8 activation was detected in concert with viral genomes increasing in the blood and oral cavity, possibly due to a systemic type I interferon response. This study provides the first description of events during the incubation period of natural EBV infection in humans and definitive data upon which to formulate theories of viral control and disease pathogenesis.

Cortical thymic epithelial cells (cTECs) express a unique thymoproteasome subunit-β5T that plays an essential role in the development of CD8 T cells. In contrast, the immunoproteasome subunit-β5i is expressed in other thymic antigen-presenting cells (APCs). The thymoproteasome may generate peptides that are specialized for positive selection, or it may simply serve to generate peptides that are distinct from other APCs that cause negative selection, thereby promoting an overall larger number of surviving clones to mature and function in the immune system. To distinguish these models, we genetically engineered mice to express distinct peptide repertoires in cTECs vs. other APCs without expressing β5T, by generating β5t ⁵ⁱ knockin mice, in which β5i replaced β5T in cTECs. When such animals were crossed to β5i ⁻/⁻ mice, β5i was exclusively expressed in cTECs, whereas β5 was expressed in other cells. However, this mouse did not support normal positive selection, suggesting that β5T generates peptides that are intrinsically better for positive selection (i.e., β5i could not replace β5T) and not merely because these peptides are distinct from peptides presented by other APCs. Finally, using an Nur77 ᴳFᴾ reporter, we show that the T cells generated in the absence of β5T have higher reactivity to self, generating predominantly CD44 ʰⁱ memory phenotype peripheral CD8 ⁺ T cells. Altogether, our results suggest that the thymoproteasome supports positive selection by generating peptides that are optimized for the selection of weakly self-reactive, naïve T-cell clones.

Interleukin-4 (IL-4) is produced by a unique subset of invariant natural killer T (iNKT) cells (NKT2) in the thymus in the steady state, where it conditions CD8⁺ T cells to become “memory-like” among other effects. However, the signals that cause NKT2 cells to constitutively produce IL-4 remain poorly defined. Using histocytometry, we observed IL-4–producing NKT2 cells localized to the thymic medulla, suggesting that medullary signals might instruct NKT2 cells to produce IL-4. Moreover, NKT2 cells receive and require T cell receptor (TCR) stimulation for continuous IL-4 production in the steady state, since NKT2 cells lost IL-4 production when intrathymically transferred into CD1d-deficient recipients. In bone marrow chimeric recipients, only hematopoietic, not stromal, antigen-presenting cells (APCs), provided such stimulation. Furthermore, using different Cre-recombinase transgenic mouse strains to specifically target CD1d deficiency to various APCs, together with the use of diphtheria toxin receptor (DTR) transgenic mouse strains to deplete various APCs, we found that macrophages were the predominant cell to stimulate NKT2 IL-4 production. Thus, NKT2 cells appear to encounter and require different activating ligands for selection in the cortex and activation in the medulla.

Conventional T cells are selected by peptide-MHC expressed by cortical epithelial cells in the thymus, and not by cortical thymocytes themselves that do not express MHC I or MHC II. Instead, cortical thymocytes express non-peptide presenting MHC molecules like CD1d and MR1, and promote the selection of PLZF + iNKT and MAIT cells, respectively. Here, we report an inducible class-I transactivator mouse that enables the expression of peptide presenting MHC I molecules in different cell types. We show that MHC I expression in DP thymocytes leads to expansion of peptide specific PLZF + innate-like (PIL) T cells. Akin to iNKT cells, PIL T cells differentiate into three functional effector subsets in the thymus, and are dependent on SAP signaling. We demonstrate that PIL and NKT cells compete for a narrow niche, suggesting that the absence of peptide-MHC on DP thymocytes facilitates selection of non-peptide specific lymphocytes. Conventional T cell subsets are selected in the thymus by peptide bearing MHC expressed by cortical epithelial cells, in contrast cortical thymocytes express non-peptide bearing MHC molecules including CD1d and MR1 and select iNKT and MAIT cell populations respectively. Here, the authors generate a novel inducible MHC class-I trasnactivator murine system and suggest the absence of peptide-MHC on thymocytes is involved in the selection of non-peptide specific lymphocytes.