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Interferons (IFNs) are a family of proteins that are generated in response to viral infection and induce an antiviral response in many cell types. The COVID-19 pandemic revealed that patients with inborn errors of type-I IFN immunity were more prone to severe infections, but also found that many patients with severe COVID-19 had anti-IFN autoantibodies that led to acquired defects in type-I IFN immunity. These findings revealed the previously unappreciated finding that central immune tolerance to IFN is essential to immune health. Further evidence has also highlighted the importance of IFN within the thymus and its impact on T-cell development. This review will highlight what is known of IFN's role in T-cell development, T-cell central tolerance, and the impact of IFN on the thymus. [ABSTRACT FROM AUTHOR]

• Intestinal IEL are a heterogeneous lymphocyte population. • In adult animals, the majority of CD8αα IEL precursors are self-reactive thymocytes that undergo agonist selection. • Alternative, and likely functionally distinct, precursors exist. The intestinal epithelium is the outermost cellular layer that separates the body from the gut lumen. The integrity of this protective mucosal barrier is crucial and maintained by specialized cells—intraepithelial lymphocytes (IEL). Much research has been conducted on these cells and our overall understanding of them is increasing rapidly. In this review we focus on the TCRαβ+ subset of CD8αα IEL. We discuss recent studies that shed light on the development, ontogeny, maintenance, and functional characteristics of CD8αα IEL, and highlight yet unanswered questions for future studies. [ABSTRACT FROM AUTHOR]

Clonal deletion of T cells specific for self-antigens in the thymus has been widely studied, primarily by approaches that focus on a single receptor (using TCR transgenes) or a single specificity (using peptide-MHC tetramers). However, less is known about clonal deletion at the population level. In this article, we report an assay that measures cleaved caspase 3 to define clonal deletion at the population level. This assay distinguishes clonal deletion from apoptotic events caused by neglect and approximates the anatomic site of deletion using CCR7. This approach showed that 78% of clonal deletion events occur in the cortex in mice. Medullary deletion events were detected at both the semimature and mature stages, although mature events were associated with failed regulatory T cell induction. Using this assay, we showed that bone marrow-derived APC drive approximately half of deletion events at both stages. We also found that both cortical and medullary deletion rely heavily on CD28 costimulation. These findings demonstrate a useful strategy for studying clonal deletion within the polyclonal repertoire. [ABSTRACT FROM AUTHOR]

T cells can sometimes acquire properties of a memory cell without encountering foreign antigen. Miller et al. now show that development of such 'memory phenotype' (MP) CD8+ T cells starts with recognition of self-ligands in the thymus, and finishes in peripheral tissues. These cells respond rapidly during infection, and infiltrate tumors, where they express high amounts of PD-1. [ABSTRACT FROM AUTHOR]

Single-positive thymocytes that successfully complete positive and negative selection must still undergo one final step, generally termed T cell maturation, before they gain functional competency and enter the long-lived T cell pool. Maturation initiates after positive selection in single-positive thymocytes and continues in the periphery in recent thymic emigrants, before these newly produced T cells gain functional competency and are ready to participate in the immune response as peripheral naive T cells. Recent work using genetically altered mice demonstrates that T cell maturation is not a single process, but a series of steps that occur independently and sequentially after positive selection. This review focuses on the changes that occur during T cell maturation, as well as the molecules and pathways that are critical at each step. [ABSTRACT FROM AUTHOR]

Self-reactivity was once seen as a potential characteristic of T cells that was eliminated by clonal selection to protect the host from autoimmune pathology. It is now understood that the T cell repertoire is in fact broadly self-reactive, even self-centered. The strength with which a T cell reacts to self ligands and the environmental context in which this reaction occurs influence almost every aspect of T cell biology, from development to differentiation to effector function. Here we highlight recent advances and discoveries that relate to T cell self-reactivity, with a particular emphasis on T cell antigen receptor (TCR) signaling thresholds. [ABSTRACT FROM AUTHOR]

Summary Understanding the thymic processes that support the generation of functionally competent and self-tolerant lymphocytes requires dissection of the T-cell receptor (TCR) response to ligands of different affinities. In spatially segregated regions of the thymus, with unique expression of proteases and cytokines, TCR affinity guides a number of cell fate decisions. Yet affinity alone does not explain the selection paradox. Increasing evidence suggests that the 'altered peptide' model of the 1980s together with the affinity model might best explain how the thymus supports conventional and regulatory T-cell development. Development of new tools to study the strength of TCR signals perceived by T cells, novel regulatory T-cell transgenic mice, and tetramer enrichment strategies have provided an insight into the nature of TCR signals perceived during thymocyte development. These topics are discussed and support for the prevailing hypotheses is presented. [ABSTRACT FROM AUTHOR]

We have used organ culture of fetal thymic lobes from T cell receptor (TCR) transgenic &brg;2M(-/-) mice to study the role of peptides in positive selection. The TCR used was from a CD8+ T cell specific for ovalbumin 257-264 in the context of Kb. Several peptides with the ability to induce positive selection were identified. These pep-tide-selected thymocytes have the same phenotype as mature CD8+ T cells and can respond to antigen. Those peptides with the ability to induce positive se-lection were all variants of the antigenic peptide and were identified as TCR antagonist peptides for this re-ceptor. One peptide tested, E1, induced positive selec-tion on the &brg;2M[-l-) background but negative selec-tion on the &brg;2M(+l-) background. These results show that the process of positive selection is exquisitely peptide specific and sensitive to extremely low ligand density and support the notion that low efficacy li-gands mediate positive selection. [ABSTRACT FROM AUTHOR]

The Krüppel-like factor family of transcription factors plays an important role in differentiation, function, and homeostasis of many cell types. While their role in lymphocytes is still being determined, it is clear that these factors influence processes as varied as lymphocyte quiescence, trafficking, differentiation, and function. This review will present an overview of how these factors operate and coordinate with each other in lymphocyte regulation. [ABSTRACT FROM AUTHOR]

Producing a healthy immune system capable of defending against pathogens, while avoiding autoimmunity, is dependent on thymic selection. Positive selection yields functional T cells that have the potential to recognize both self and foreign antigens. Therefore, negative selection exists to manage potentially self-reactive cells. Negative selection results from the induction of anergy, receptor editing, clonal diversion (agonist selection), and/or clonal deletion (apoptosis) in self-reactive clones. Clonal deletion has been inherently difficult to study because the cells of interest are undergoing apoptosis and being eliminated quickly. Furthermore, analysis of clonal deletion in humans has proved even more difficult due to availability of samples and lack of reagents. Mouse models have thus been instrumental in achieving our current understanding of central tolerance, and the evolution of elegant model systems has led to an explosion of new data to be assimilated. This review will focus on recent advances in the field of clonal deletion with respect to three aspects: the development of physiological model systems, signaling pathways that lead to apoptosis, and antigen presenting cell types involved in the induction of clonal deletion. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Clonal selection is central to immune function, but it is complemented by ‘receptor selection’, which regulates the immune repertoire not by cell death or proliferation but through the control of antigen receptor gene recombination. Inappropriate receptors, such as those that are autoreactive, underexpressed, or that fail to promote positive selection of thymocytes or B cells, stimulate secondary V-to-J recombinations that destroy and replace receptor genes. These processes play a central role in lymphocyte repertoire development. Recent work on the role of receptor selection in B and T cells has uncovered evidence for and against antigen-induced editing in thymocytes. Many studies suggest that editing plays a central role in B and T lymphocyte repertoire development. Important recent evidence has been uncovered addressing the role of tolerance-induced editing in thymocytes. [Copyright &y& Elsevier]

The fate and functional activity of T lymphocytes depend largely on the precise timing of gene expression and protein production. However, it is clear that post-translational modification of proteins affects their functional properties. Although modifications such as phosphorylation have been intensely studied by immunologists, less attention has been paid to the impact that changes in glycosylation have on protein function. However, there is considerable evidence that glycosylation plays a key role in immune regulation. We will focus here on examples in which differential glycosylation affects the development, survival or reactivity of T cells. [ABSTRACT FROM AUTHOR]

Studies the role of peptides in positive selection using a T cell receptor. Identification of positive-selection inducing peptides; Antigenic peptide causing negative selection; Peptides inducing CD8 development.

In this issue of Immunity, studies from and show that mice deficient in Tec kinases support development of a population of CD8+ T cells with “innate-immune” properties. The similarity of these cells to NKT and MHC-class-Ib-restricted T cells suggests a common developmental pathway. [Copyright &y& Elsevier]

Resolution of inflammation is pivotal to restoring tissue homeostasis, yet there is limited understanding of how this process is regulated. In this issue of Immunity , Liew et al. (2017) reveal a critical role for invariant natural killer T (iNKT) cells in switching inflammation to tissue repair in an interlukin-4-dependent process. [ABSTRACT FROM AUTHOR]

Examines peptide fine specificity in positive selection of thymocytes. Identification of individual major histocompatibility complex (MHC)-binding peptides; Direct T-cell receptor (TCR)-peptide interaction; Self-tolerance by thymocytes.

The article offers information on several papers on immunology that was published in the year 1980 and 1989. Topics discussed include the generation of muromonab CD-3 (OKT3) monoclonal antibodies (mAb) to the human T cells and CD3, the characterization of monoclonal antibodies to the murine T-cell receptorβ-chain (TCR β-chain), and the immunoglobulin (Ig)-binding properties of streptavidin and protein A.

Graphical abstract Abstract The development of a self-tolerant and effective T cell receptor repertoire is dependent on interactions coordinated by various antigen presenting cells (APC) within the thymus. T cell receptor–self-peptide–MHC interactions are essential for determining T cell fate, however different cytokine and co-stimulatory signals provided by the diverse APCs within the thymus are also critical. Here, we outline the different localization and functional capabilities of thymic APCs. We also discuss how these distinct APCs work collectively to facilitate the establishment of a diverse T cell receptor repertoire that is tolerant to an array of different self-antigens. [ABSTRACT FROM AUTHOR]

A thymus-specific subunit of the proteasome, β5t, was previously described and shown to be essential for positive selection of CD8+ T cells. In this issue of Immunity, suggest that β5t is essential because it alters the specific peptide-MHC complexes displayed by cortical epithelial cells. [Copyright &y& Elsevier]

The article offers information about Kruppellike factor 2 (KLF2) which are a large family of proteins that activate and repress genes and regulate various biological processes. It mentions that KLF2 is required for thymic emigration and lymph node homing. According to research findings, KLF2 coordinates expression of multiple types of cell surface receptors to control T-cell trafficking during an immune response. Also presented is a figure depicting KLF2 controlling T-cell trafficking.

Discusses the function of major histocompatibility complex (MHC) in the positive selection of T lymphocyte antigens. Effect of MHC glycoproteins on peptide-binding specificity; Role in determining antigen presentation and thymocyte development; Disclosure of natural peptide ligands for positive selection.

The activation of thymic B cells is critical for their licensing as antigen presenting cells and resulting ability to mediate T cell central tolerance. The processes leading to licensing are still not fully understood. By comparing thymic B cells to activated Peyer’s patch B cells at steady state, we found that thymic B cell activation starts during the neonatal period and is characterized by TCR/CD40-dependent activation, followed by immunoglobulin class switch recombination (CSR) without forming germinal centers. Transcriptional analysis also demonstrated a strong interferon signature, which was not apparent in the periphery. Thymic B cell activation and CSR were primarily dependent on type III IFN signaling, and loss of type III IFN receptor in thymic B cells resulted in reduced thymocyte regulatory T cell (Treg) development. Finally, from TCR deep sequencing, we estimate that licensed B cells induce development of a substantial fraction of the Treg cell repertoire. Together, these findings reveal the importance of steady-state type III IFN in generating licensed thymic B cells that induce T cell tolerance to activated B cells. [ABSTRACT FROM AUTHOR]

Expression of tissue restricted antigens by lymph node stromal cells has been described, and results in T cell tolerance to self antigens. We have identified a model in which presentation of a skin antigen by non-hematopoietic cells induces autoimmunity. We employed K14-OVAp mice expressing an ovalbumin peptide in the epidermis to study CD8 T cell responses to a skin self antigen, and found that a radioresistant APC initiated a robust T cell response and autoimmunity. Langerhans cells (LC), the APC in the epidermis of the skin were the likely APC because they are radioresistant in hematopoietic chimeras. We addressed the same question in K14-mOVA mice, which express a transmembrane ovalbumin. In K14-mOVA mice, CD8 T cells became activated and divided, but did not expand or acquire effector function. In mice expressing both mOVA and OVAp transgenes, robust expansion occurred, suggesting that K14-mOVA antigen does not induce full immune activation or tolerance. Use of inducibly LC deficient K14-OVAp and K14-mOVA mice suggested that LC were dispensable for activation of T cells in both systems. Future experiments will identify the non-hematopoietic APC, and the mechanism behind K14-OVAp autoimmunity. [ABSTRACT FROM AUTHOR]

The T-cell antigen receptor binds to self-MHC molecules with low affinity. Recent reports disagree as to whether this interaction sensitizes or desensitizes the receptor. Here we discuss how these findings might be reconciled. [Copyright &y& Elsevier]

Dual TCRα-expressing T cells outnumber dual TCRβ-expressing cells by ~10:1. As a result, efforts to understand how dual TCR T cells impact immunity have focused on dual TCRα expression; dual TCRβ expression remains understudied. We recently demonstrated, however, that dual TCRβ expression accelerated disease in a TCR transgenic model of autoimmune arthritis through enhanced positive selection efficiency, indicating that dual TCRβ expression, though rare, can impact thymic selection. Here we generated mice hemizygous for TCRα, TCRβ, or both on the C57BL/6 background to investigate the impact bi-allelic TCR chain recombination has on T cell development, repertoire diversity, and autoimmunity. Lack of bi-allelic TCRα or TCRβ recombination reduced αβ thymocyte development efficiency, and the absence of bi-allelic TCRβ recombination promoted γδ T cell development. However, we observed no differences in the numbers of naïve and expanded antigen-specific T cells between TCRα+/-β+/- and wildtype mice, and TCR repertoire analysis revealed only subtle differences in Vβ gene usage. Finally, the absence of dual TCR T cells did not impact induced experimental autoimmune encephalomyelitis pathogenesis. Thus, despite more stringent allelic exclusion of TCRβ relative to TCRα, bi-allelic TCRβ expression can measurably impact thymocyte development and is necessary for maintaining normal αβ/γδ T cell proportions. [ABSTRACT FROM AUTHOR]

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 β5t5i 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 Nur77GFP reporter, we show that the T cells generated in the absence of β5T have higher reactivity to self, generating predominantly CD44hi 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. [ABSTRACT FROM AUTHOR]

On the whole, the healthy adaptive immune system is responsive to foreign antigens and tolerant to self. However, many individual lymphocytes have, and even require, substantial self-reactivity for their particular functions in immunity. In this review, we discuss several populations of lymphocytes that are thought to experience agonist stimulation through the T cell receptor during selection: nTreg cells, iNKT cells, nIELs, and nTh17s. We discuss the nature of this self-reactivity, how it compares with conventional T cells, and why it is important for overall immune health. We also outline molecular pathways unique to each lineage and consider possible commonalities to their development and survival. [ABSTRACT FROM AUTHOR]

The transcription factor Krüppel-like factor 2 (KLF2) controls the emigration of conventional T cells from the thymus through its regulation of the cell surface receptor S1P1. Prior to KLF2 expression, developing T cells require a positive selection signal through the TCR. However, following positive selection there are time, spatial, and maturational events that occur before KLF2 is finally upregulated and emigration occurs. We are interested in determining the signals that upregulate KLF2 and allow thymocytes to emigrate into circulation and whether they are linked to functional maturation. In endothelial cells KLF2 expression has been shown to be dependent on the mitogen-activated protein kinase ERK5. Furthermore, it has been reported that IL-7 signaling leads to the phosphorylation of ERK5. Thus, we hypothesized that IL-7R signaling through ERK5 could drive the expression of KLF2. In this study, we provide evidence that this hypothesis is incorrect. We also found that CD8 lineage specification occurred normally in the absence of IL-7R signaling, in contrast to a recently proposed model. We showed that both CD4 and CD8 T cells complete maturation and express KLF2 independently of ERK5 and IL-7. [ABSTRACT FROM AUTHOR]

A prominent population of innate CD8+ T cells develops in the thymus of several gene-deficient mouse strains, including Itk, KLF2, CBP and Id3. These cells have the phenotype and function of memory CD8+ T cells, without previous exposure to antigen. Surprisingly, the cytokine IL-4 plays a key role in their development. As this developmental mechanism was discovered, it came to light that innate CD8+ T cells exist also in normal mice and in humans. In this review, we discuss how these cells develop, compare and contrast them to other CD8 memory cells, and discuss their potential physiological relevance. [Copyright &y& Elsevier]

The transcription factor Krüppel-like factor 2 (KLF2) is critical for normal trafficking of T lymphocytes, but its role in B cells is unclear. We report that B cell-specific KLF2 deficiency leads to de- creased expression of the trafficking molecules CD62L and β7- integrin, yet expression of sphingosine-1 phosphate receptor 1 (which is a critical target of KLF2 in T cells) was, unexpectedly, minimally altered. Unexpectedly, Klf2 deletion led to a drastic reduction in the B1 B-cell pool and a substantial increase in transitional and marginal zone B-cell numbers. In addition, we observed that KLF2-deficient B cells showed increased apoptosis and impaired proliferation after B-cell receptor cross-linking. Gene expression analysis indicated that KLF2-deficient follicular B cells display numerous characteristics shared by normal marginal zone B cells, including reduced expression of several signaling molecules that may contribute to defective activation of these cells. Hence, our data indicate that KLF2 plays a critical role in dictating normal subset differentiation and functional reactivity of mature B cells. [ABSTRACT FROM AUTHOR]

It is unknown how the contrasting events of positive and negative selection can lead to the distinct biological outcomes of life or death. An increasing body of evidence suggests that the duration of extracellular signal-regulated kinase (ERK) signaling plays a role in thymocyte selection. However, it remains unclear what the kinetics of ERK activation are during positive selection in vivo. In this study, we examined the magnitude and duration of ERK signaling in intact murine thymic tissues cultured under conditions of negative or positive selection. We found that negative selection induced a rapid and robust ERK activation that is associated with death, whereas positive selection stimulated a lower intensity and brief ERK activation that quickly declined and then gradually increased and was sustained over several days. The expression pattern of Egr-1 (early growth response-i), a downstream ERK effector, correlates with the biphasic kinetics of ERK during positive selection. Id3 (inhibitor of differentiation/DNA binding 3) also exhibits biphasic kinetics but appeared to be independent of ERK signaling. Furthermore, inhibitors of T cell receptor ligation and ERK activation block maturation of CDS single-positive thymocytes even when added after 24 h. These results demonstrate that the in vivo duration of ERK signaling must be sustained to support positive selection. [ABSTRACT FROM AUTHOR]

A functional immune system requires the selection of T lymphocytes expressing receptors that are major histocompatibility complex restricted but tolerant to self-antigens. This selection occurs predominantly in the thymus, where lymphocyte precursors first assemble a surface receptor. In this review we summarize the current state of the field regarding the natural ligands and molecular factors required for positive and negative selection and discuss a model for how these disparate outcomes can be signaled via the same receptor. We also discuss emerging data on the selection of regulatory T cells. Such cells require a high-affinity interaction with self-antigens, yet differentiate into regulatory cells instead of being eliminated. [ABSTRACT FROM AUTHOR]

A central tenet of T cell development postulates that if a developing thymocyte encounters self-antigen, it is induced to die via apoptosis, thereby protecting the organism from autoreactive T cells. We created transgenic mice that expressed a peptide antigen in the cortical epithelial cells of the thymus. This did not, however, result in deletion of specific T cells. Instead, antigen presentation by epithelial cells caused T cell receptor (TCR) internalization and increased gene rearrangement at the endogenous TCRα locus, or receptor editing. This editing mechanism in immature T cells parallels that which occurs in immature B cells, and has important implications for understanding positive and negative selection signaling in the thymus, and the limits of self-tolerance. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Programmed death-1 (PD-1) inhibits T and B cell function upon ligand binding. PD-1 blockade revolutionized cancer treatment, and although numerous patients respond, some develop autoimmune-like symptoms or overt autoimmunity characterized by autoantibody production. PD-1 inhibition accelerates autoimmunity in mice, but its role in regulating germinal centers (GC) is controversial. To address the role of PD-1 in the GC reaction in type 1 diabetes, we used tetramers to phenotype insulin-specific CD4+ T and B cells in NOD mice. PD-1 or PD-L1 deficiency, and PD-1 but not PD-L2 blockade, unleashed insulin-specific T follicular helper CD4+ T cells and enhanced their survival. This was concomitant with an increase in GC B cells and augmented insulin autoantibody production. The effect of PD-1 blockade on the GC was reduced when mice were treated with a mAb targeting the insulin peptide:MHC class II complex. This work provides an explanation for autoimmune side effects following PD-1 pathway inhibition and suggests that targeting the self-peptide:MHC class II complex might limit autoimmunity arising from checkpoint blockade. [ABSTRACT FROM AUTHOR]

Peripheral invariant NKT cells (iNKT) and CD8+ tissue-resident memory T cells (TRM) express high levels of the extracellular ATP receptor P2RX7 in mice. High extracellular ATP concentrations or NAD-mediated P2RX7 ribosylation by the enzyme ARTC2.2 can induce P2RX7 pore formation and cell death. Because both ATP and NAD are released during tissue preparation for analysis, cell death through these pathways may compromise the analysis of iNKT and CD8+ TRM. Indeed, ARTC2.2 blockade enhanced recovery of viable liver iNKT and TRM. The expression of ARTC2.2 and P2RX7 on distinct iNKT subsets and TRM is unclear, however, as is the impact of recovery from other nonlymphoid sites. In this study, we performed a comprehensive analysis of ARTC2.2 and P2RX7 expression in iNKT and CD8+ T cells in diverse tissues, at steady-state and after viral infection. NKT1 cells and CD8+ TRM express high levels of both ARTC2.2 and P2RX7 compared with NKT2, NKT17, and CD8+ circulating memory subsets. Using nanobody-mediated ARTC2.2 antagonism, we showed that ARTC2.2 blockade enhanced NKT1 and TRM recovery from nonlymphoid tissues during cell preparation. Moreover, blockade of this pathway was essential to preserve functionality, viability, and proliferation of both populations. We also showed that short-term direct P2RX7 blockade enhanced recovery of TRM, although to a lesser degree. In summary, our data show that short-term in vivo blockade of the ARTC2.2/ P2RX7 axis permits much improved flow cytometry-based phenotyping and enumeration of murine iNKT and TRM from nonlymphoid tissues, and it represents a crucial step for functional studies of these populations. [ABSTRACT FROM AUTHOR]

Despite accounting for 10-30% of the T cell population in mice and humans, the role of dual TCR-expressing T cells in immunity remains poorly understood. It has been hypothesized that dual TCR T cells pose an autoimmune hazard by allowing self-reactive TCRs to escape thymic selection. We revisited this hypothesis using the NOD murine model of type 1 diabetes. We bred NOD mice hemizygous at both TCRα and b (TCRα+/- β+/-) loci, rendering them incapable of producing dual TCR T cells. We found that the lack of dual TCRα expression skewed the insulin-specific thymocyte population toward greater regulatory T (Treg) cell commitment, resulting in a more tolerogenic Treg to conventional T cell ratio and protection from diabetes. These data support a novel hypothesis by which dual TCR expression can promote autoimmunity by limiting agonist selection of self-reactive thymocytes into the Treg cell lineage. [ABSTRACT FROM AUTHOR]

Invariant NKT cells differentiate into three predominant effector lineages in the steady state. To understand these lineages, we sorted undifferentiated invariant NK T progenitor cells and each effector population and analyzed their transcriptional profiles by RNAseq. Bioinformatic comparisons were made to effector subsets among other lymphocytes, specifically Th cells, innate lymphoid cells (ILC), and γδ T cells. Myc-associated signature genes were enriched in NKT progenitors, like in other hematopoietic progenitors. Only NKT1 cells, but not NKT2 and NKT17 cells, had transcriptome similarity to NK cells and were also similar to other IFN-γ-producing lineages such as Th1, ILC1, and intraepithelial γδ T cells. NKT2 and NKT17 cells were similar to their analogous subsets of γδ T cells and ILCs, but surprisingly, not to Th2 and Th17 cells. We identified a set of genes common to each effector lineage regardless of Ag receptor specificity, suggesting the use of conserved regulatory cores for effector function. [ABSTRACT FROM AUTHOR]

Acute infectious mononucleosis (IM) is associated with altered expression of inflammatory cytokines and disturbed T-cell homeostasis, however, the precise mechanism of this immune dysregulation remains unresolved. In the current study we demonstrated a significant loss of circulating myeloid and plasmacytoid dendritic cells (DCs) during acute IM, a loss correlated with the severity of clinical symptoms. In vitro exposure of blood DCs to acute IM plasma resulted in loss of plasmacytoid DCs, and further studies with individual cytokines showed that exposure to interleukin 10 could replicate this effect. Our data provide important mechanistic insight into dysregulated immune homeostasis during acute IM. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary Three subsets of invariant natural killer T (iNKT) cells have been identified, NKT1, NKT2, and NKT17, which produce distinct cytokines when stimulated, but little is known about their localization. Here, we have defined the anatomic localization and systemic distribution of these subsets and measured their cytokine production. Thymic NKT2 cells that produced interleukin-4 (IL-4) at steady state were located in the medulla and conditioned medullary thymocytes. NKT2 cells were abundant in the mesenteric lymph node (LN) of BALB/c mice and produced IL-4 in the T cell zone that conditioned other lymphocytes. Intravenous injection of α-galactosylceramide activated NKT1 cells with vascular access, but not LN or thymic NKT cells, resulting in systemic interferon-γ and IL-4 production, while oral α-galactosylceramide activated NKT2 cells in the mesenteric LN, resulting in local IL-4 release. These findings indicate that the localization of iNKT cells governs their cytokine response both at steady state and upon activation. [ABSTRACT FROM AUTHOR]

Here we present evidence for previously unappreciated B-cell immune dysregulation during acute Epstein-Barr virus (EBV)-associated infectious mononucleosis (IM). Longitudinal analyses revealed that patients with acute IM have undetectable EBVspecific neutralizing antibodies and gp350-specific B-cell responses, which were associated with a significant reduction in memory B cells and no evidence of circulating antibody-secreting cells. These observations correlate with dysregulation of tumor necrosis factor family members BAFF and APRIL and increased expression of FAS on circulating B cells. [ABSTRACT FROM AUTHOR]