Coreceptor scanning by the T cell receptor provides a mechanism for T cell tolerance.

In the thymus, high-affinity, self-reactive thymocytes are eliminated from the pool of developing T cells, generating central tolerance. Here, we investigate how developing T cells measure self-antigen affinity. We show that very few CD4 or CD8 coreceptor molecules are coupled with the signal-initiating kinase, Lck. To initiate signaling, an antigen-engaged T cell receptor (TCR) scans multiple coreceptor molecules to find one that is coupled to Lck; this is the first and rate-limiting step in a kinetic proofreading chain of events that eventually leads to TCR triggering and negative selection. MHCII-restricted TCRs require a shorter antigen dwell time (0.2 s) to initiate negative selection compared to MHCI-restricted TCRs (0.9 s) because more CD4 coreceptors are Lck-loaded compared to CD8. We generated a model (Lck come&stay/signal duration) that accurately predicts the observed differences in antigen dwell-time thresholds used by MHCI- and MHCII-restricted thymocytes to initiate negative selection and generate self-tolerance.

Affinity for self antigen selects Treg cells with distinct functional properties.

The manner in which regulatory T cells (Treg cells) control lymphocyte homeostasis is not fully understood. We identified two Treg cell populations with differing degrees of self-reactivity and distinct regulatory functions. We found that GITR(hi)PD-1(hi)CD25(hi) (Triple(hi)) Treg cells were highly self-reactive and controlled lympho-proliferation in peripheral lymph nodes. GITR(lo)PD-1(lo)CD25(lo) (Triple(lo)) Treg cells were less self-reactive and limited the development of colitis by promoting the conversion of CD4(+) Tconv cells into induced Treg cells (iTreg cells). Although Foxp3-deficient (Scurfy) mice lacked Treg cells, they contained Triple(hi)-like and Triple(lo)-like CD4(+) T cells zsuper> T cells infiltrated the skin, whereas Scurfy Triple(lo)CD4(+) T cells induced colitis and wasting disease. These findings indicate that the affinity of the T cell antigen receptor for self antigen drives the differentiation of Treg cells into distinct subsets with non-overlapping regulatory activities.

The transcription factor T-bet is induced by IL-15 and thymic agonist selection and controls CD8αα(+) intraepithelial lymphocyte development.

CD8αα(+) intraepithelial lymphocytes (IELs) are instrumental in maintaining the epithelial barrier in the intestine. Similar to natural killer cells and other innate lymphoid cells, CD8αα(+) IELs constitutively express the T-box transcription factor T-bet. However, the precise role of T-bet for the differentiation or function of IELs is unknown. Here we show that mice genetically deficient for T-bet lacked both TCRαß(+) and TCRγδ(+) CD8αα(+) IELs and thus are more susceptible to chemically induced colitis. Although T-bet was induced in thymic IEL precursors (IELPs) as a result of agonist selection and interleukin-15 (IL-15) receptor signaling, it was dispensable for the generation of IELPs. Subsequently, T-bet was required for the IL-15-dependent activation, differentiation, and expansion of IELPs in the periphery. Our study reveals a function of T-bet as a central transcriptional regulator linking agonist selection and IL-15 signaling with the emergence of CD8αα(+) IELs.

T cell affinity regulates asymmetric division, effector cell differentiation, and tissue pathology.

The strength of interactions between T cell receptors and the peptide-major histocompatibility complex (pMHC) directly modulates T cell fitness, clonal expansion, and acquisition of effector properties. Here we show that asymmetric T cell division is an important mechanistic link between increased signal strength, effector differentiation, and the ability to induce tissue pathology. Recognition of pMHC above a threshold affinity drove responding T cells into asymmetric cell division. The ensuing proximal daughters underwent extensive division and differentiated into short-lived effector cells expressing the integrin VLA-4, allowing the activated T cell to infiltrate and mediate destruction of peripheral target tissues. In contrast, T cells activated by below-threshold antigens underwent symmetric division, leading to abortive clonal expansion and failure to fully differentiate into tissue-infiltrating effector cells. Antigen affinity and asymmetric division are important factors that regulate fate specification in CD8(+) T cells and predict the potential of a self-reactive T cell to mediate tissue pathology.

Opposing effects of actin signaling and LFA-1 on establishing the affinity threshold for inducing effector T-cell responses in mice.

Mature CD8(+) T cells use a narrow antigen affinity threshold to generate tissue-infiltrating cytotoxic effector T cells and induce autoimmune pathology, but the mechanisms that establish this antigen affinity threshold are poorly understood. Only antigens with affinities above the threshold induce stable contacts with APCs, polarization of a T cell, and asymmetric T-cell division. Previously published data indicate that LFA-1 inside-out signaling might be involved in establishing the antigen affinity threshold. Here, we show that subthreshold antigens weakly activate all major distal TCR signaling pathways. Low-affinity antigens are more dependent on LFA-1 than suprathreshold antigens. Moreover, augmenting the inside-out signaling by hyperactive Rap1 does not increase responses to the subthreshold antigens. Thus, LFA-1 signaling does not contribute to the affinity-based antigen discrimination. However, we found that subthreshold antigens do not induce actin rearrangement toward an APC, mediated by Rho-family GTPases, Cdc42, and Rac. Our data suggest that Rac and Cdc42 contribute to the establishment of the antigen affinity threshold in CD8(+) T cells by enhancing responses to high-affinity antigens, or by reducing the responses to low-affinity antigens.

A minimum number of autoimmune T cells to induce autoimmunity?

While autoimmune T cells are present in most individuals, only a minority of the population suffers from an autoimmune disease. To better appreciate the limits of T cell tolerance, we carried out experiments to determine how many autoimmune T cells are required to initiate an experimental autoimmune disease. Variable numbers of autoimmune OT-I T cells were transferred into RIP-OVA mice, which were injected with antigen-loaded DCs in a single footpad; this restricted T cell priming to a few OT-I T cells that are present in the draining popliteal lymph node. Using selective plane illumination microscopy (SPIM) we counted the number of OT-I T cells present in the popliteal lymph node at the time of priming. Analysis of our data suggests that a single autoimmune T cell cannot induce an experimental autoimmune disease, but a "quorum" of 2-5 autoimmune T cells clearly has this capacity.

Optimal T-cell receptor affinity for inducing autoimmunity.

T-cell receptor affinity for self-antigen has an important role in establishing self-tolerance. Three transgenic mouse strains expressing antigens of variable affinity for the OVA transgenic-I T-cell receptor were generated to address how TCR affinity affects the efficiency of negative selection, the ability to prime an autoimmune response, and the elimination of the relevant target cell. Mice expressing antigens with an affinity just above the negative selection threshold exhibited the highest risk of developing experimental autoimmune diabetes. The data demonstrate that close to the affinity threshold for negative selection, sufficient numbers of self-reactive T cells escape deletion and create an increased risk for the development of autoimmunity.

Negative selection--clearing out the bad apples from the T-cell repertoire.

Dead cells are a prominent feature of the thymic landscape as only 5% of developing thymocytes are exported as mature T cells. The remaining thymocytes die by one of two mechanisms; most thymocytes die because they are not positively selected and do not receive a survival signal, whereas a minority of thymocytes undergo T-cell receptor (TCR)-mediated apoptosis, a process known as negative selection. Negative selection is extremely important for establishing a functional immune system, as it provides an efficient mechanism for ridding the T-cell repertoire of self-reactive and potentially autoimmune lymphocytes. This review discusses several cellular and molecular aspects of negative selection.

What is the role of autoimmunity in type 1 diabetes? A clinical perspective.

Despite tremendous research efforts, type 1 diabetes is one of the few remaining autoimmune diseases without any approved immunological treatment. This observation compels us to reconsider the role of autoimmunity in the pathogenesis of this disease. In this commentary, we will review solely human data in an attempt to appreciate, in an unbiased manner, the importance and relevance of the immunological alterations in patients with type 1 diabetes. The aim of this paper is to generate reflection on this topic, rather than a controversy.

Physical and functional bivalency observed among TCR/CD3 complexes isolated from primary T cells.

Unlike BCR and secreted Ig, TCR expression is not thought to occur in a bivalent form. The conventional monovalent model of TCR/CD3 is supported by published studies of complexes solubilized in the detergent digitonin, in which bivalency was not observed. We revisited the issue of TCR valency by examining complexes isolated from primary αß T cells after solubilization in digitonin. Using immunoprecipitation followed by flow cytometry, we unexpectedly observed TCR/CD3 complexes that contained two TCRs per complex. Standard anti-TCR Abs, being bivalent themselves, tended to bind with double occupancy to bivalent TCRs; this property masked the presence of the second TCR per complex in certain Ab binding assays, which may partially explain why previous data did not reveal these bivalent complexes. We also found that the prevalence of bivalency among fully assembled, mature TCR/CD3 complexes was sufficient to impact the functional performance of immunoprecipitated TCRs in binding antigenic peptide/MHC-Ig fusion proteins. Both TCR positions per bivalent complex required an Ag-specific TCR to effect optimal binding to these soluble ligands. Therefore, we conclude that in primary T cells, TCR/CD3 complexes can be found that are physically and functionally bivalent. The expression of bivalent TCR/CD3 complexes has implications regarding potential mechanisms by which Ag may trigger signaling. It also suggests the possibility that the potential for bivalent expression could represent a general feature of Ag receptors.

TCR signaling requirements for activating T cells and for generating memory.

Over the last two decades the molecular and cellular mechanisms underlying T cell activation, expansion, differentiation, and memory formation have been intensively investigated. These studies revealed that the generation of memory T cells is critically impacted by a number of factors, including the magnitude of the inflammatory response and cytokine production, the type of dendritic cell [DC] that presents the pathogen derived antigen, their maturation status, and the concomitant provision of costimulation. Nevertheless, the primary stimulus leading to T cell activation is generated through the T cell receptor [TCR] following its engagement with a peptide MHC ligand [pMHC]. The purpose of this review is to highlight classical and recent findings on how antigen recognition, the degree of TCR stimulation, and intracellular signal transduction pathways impact the formation of effector and memory T cells.

Thymic selection threshold defined by compartmentalization of Ras/MAPK signalling.

A healthy individual can mount an immune response to exogenous pathogens while avoiding an autoimmune attack on normal tissues. The ability to distinguish between self and non-self is called 'immunological tolerance' and, for T lymphocytes, involves the generation of a diverse pool of functional T cells through positive selection and the removal of overtly self-reactive thymocytes by negative selection during T-cell ontogeny. To elucidate how thymocytes arrive at these cell fate decisions, here we have identified ligands that define an extremely narrow gap spanning the threshold that distinguishes positive from negative selection. We show that, at the selection threshold, a small increase in ligand affinity for the T-cell antigen receptor leads to a marked change in the activation and subcellular localization of Ras and mitogen-activated protein kinase (MAPK) signalling intermediates and the induction of negative selection. The ability to compartmentalize signalling molecules differentially in the cell endows the thymocyte with the ability to convert a small change in analogue input (affinity) into a digital output (positive versus negative selection) and provides the basis for establishing central tolerance.

Effects of self-rated health on sick leave, disability pension, hospital admissions and mortality. A population-based longitudinal study of nearly 15,000 observations among Swedish women and men.

BACKGROUND: Simple global self-ratings of health (SRH) have become increasingly used in national and international public health monitoring, and in recent decades recommended as a standard part of health surveys. Monitoring developments in population health requires identification and use of health measures, valid in relation to targets for population health. The aim of the present study was to investigate associations between SRH and sick leave, disability pension, hospital admissions, and mortality, adjusted for effects of significant covariates, in a large population-based cohort. METHODS: The analyses were based on screening data from eight population-based cohorts in southern and central Sweden, and on official register data regarding sick-leave, disability pension, hospital admissions, and death, with little or no data loss. Sampling was performed 1973-2003. The study population consisted of 11,880 women and men, age 25-99 years, providing 14,470 observations. Information on SRH, socio-demographic data, lifestyle variables and somatic and psychological symptoms were obtained from questionnaires. RESULTS: There was a significant negative association between SRH and sick leave (Beta -13.2, p<0.0001, and -9.5, p<0.01, in women and men, respectively), disability pension (Hazard ratio 0.77, p<0.0001 and 0.76, p<0.0001, in women and men, respectively), and mortality, adjusted for covariates. SRH was also significantly associated with hospital admissions in men (Hazard ratio 0.87, p<0.0001), but not in women (Hazard ratio 0.96, p0.20). Associations between SRH on the one hand, and sick leave, disability pension, hospital admission, and mortality, on the other, were robust during the follow-up period. CONCLUSIONS: SRH had strong predictive validity in relation to use of social insurance facilities and health care services, and to mortality. Associations were strong and robust during follow-up.

T cell receptor engagement by peptide-MHC ligands induces a conformational change in the CD3 complex of thymocytes.

The T cell receptor (TCR) can recognize a variety of cognate peptide/major histocompatibility complex (pMHC) ligands and translate their affinity into distinct cellular responses. To achieve this, the nonsignaling alphabeta heterodimer communicates ligand recognition to the CD3 signaling subunits by an unknown mechanism. In thymocytes, we found that both positive- and negative-selecting pMHC ligands expose a cryptic epitope in the CD3 complex upon TCR engagement. This conformational change is induced in vivo and requires the expression of cognate MHC. We conclude that TCR engagement with a cognate pMHC ligand induces a conformational change in the CD3 complex of thymocytes and propose that this marks an initial event during thymic selection that signals the recognition of self-antigen.

A discrete affinity-driven elevation of ZAP-70 kinase activity initiates negative selection.

CONTEXT: Although ZAP-70 is required for T-cell development, it's unclear how this kinase controls both positive and negative selection. OBJECTIVE AND METHODS: Using OT-I pre-selection thymocytes and a panel of peptide major histocompatibility complex (pMHC) ligands of defined affinity, the recruitment, phosphorylation and activity of ZAP-70 was determined at the interface with antigen-presenting cells (APCs). RESULTS: pMHC ligands promoting negative selection induce a discrete elevation of ZAP-70 recruitment, phosphorylation and enzymatic activity in the thymocyte:APCs interface. DISCUSSION: The quantity of ZAP-70 kinase activity per cell is a key parameter controlling the fate of a developing thymocyte since partial inhibition of ZAP-70 kinase activity converted negative into positive selection. Surprisingly, the amount of ZAP-70 enzymatic activity observed during negative selection is not controlled by differential phosphorylation of the ZAP-70 protein but rather by the total amount of T-cell receptor and co-associated ZAP-70 recruited to the thymocyte:APC interface. CONCLUSIONS: These data provide evidence that a burst of ZAP-70 activity initiates the signaling pathways for negative selection.

Visualizing intermolecular interactions in T cells.

The use of appropriate fluorescent proteins has allowed the use of FRET microscopy for investigation of intermolecular interactions in living cells. This method has the advantage of both being dynamic and of working at the subcellular level, so that the time and place where proteins interact can be visualized. We have used FRET microscopy to analyze the interactions between the T cell antigen receptor and the coreceptors CD4 and CD8. This chapter reviews data on how these coreceptors are recruited to the immunological synapse, and how they interact when the T cell is stimulated by different ligands.

Lck regulates the threshold of activation in primary T cells, while both Lck and Fyn contribute to the magnitude of the extracellular signal-related kinase response.

The src family kinases p56lck (Lck) and p59fyn (Fyn) are the most proximal signaling molecules to be activated downstream of the T-cell receptor. Using an inducible transgenic model, we can regulate the expression of Lck in primary T cells and ask how the signaling cascade and differentiation potential are affected by the absence or the presence of reduced levels of Lck. We show that in naïve T cells, Lck controls the threshold of activation by preferentially regulating multiple signaling pathways that result in the mobilization of Ca2+ through activation of phospholipase C-gamma and protein kinase C as well as activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway. Fyn is also able to stimulate the ERK/MAPK pathway in primary T cells but has little influence on the mobilization of Ca2+. Only Lck efficiently stimulates production of diacylglycerol and therefore RasGRP1 recruitment to the plasma membrane and phosphorylation of Shc, suggesting that Fyn activates ERK via a different upstream signaling route. Finally, we show that signals through Lck are essential for the development of T-cell-effector potential, particularly for effective cytokine transcription.

High-sensitivity detection and quantitative analysis of native protein-protein interactions and multiprotein complexes by flow cytometry.

Most mechanisms of cell development, physiology, and signal transduction are controlled by protein-protein interactions. Immunoprecipitation of multiprotein complexes detected by flow cytometry (IP-FCM) is a means to quantitatively measure these interactions. The high sensitivity of this method makes it useful even when very little biomaterial is available for analysis, as in the case of rare primary cell subsets or patient samples. Detection of the T cell antigen receptor associated with the CD3 multiprotein complex from as few as 300 primary murine T cells is presented as an example. The method is compatible with quantitative flow cytometry techniques, making it possible to estimate the number of coimmunoprecipitated molecules. Both constitutive and inducible protein-protein interactions can be analyzed, as illustrated in related methodology using glutathione S-transferase-fusion protein pull-down experiments. IP-FCM represents a robust, quantitative, biochemical technique to assess native protein-protein interactions, without requiring genetic engineering or large sample sizes.

The T-cell receptor signalosome: a dynamic structure with expanding complexity.

Signal transduction in T cells is a dynamic process involving a large number of membrane and cytosolic proteins. The TCR macromolecular complex (signalosome) is initiated by receptor occupancy and becomes more elaborate over time. This review describes how 'vertical' displacement mechanisms and lateral coalescence of lipid-raft-associated scaffold proteins combine to form distinct signalosomes, which control signal specificity.