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Peripheral T cell tolerance is challenging to induce in partially lymphopenic hosts and this is relevant for clinical situations involving transplant tolerance. While the shortage of regulatory cells is thought to be one reason for this, T cell-intrinsic tolerance processes such as anergy are also poorly triggered in such hosts. In order to understand the latter, we used a T cell deficient mouse model system where adoptively transferred autoreactive T cells are significantly tolerized in a cell intrinsic fashion, without differentiation to regulatory T cells. Intriguingly these T cells often retain sufficient effector functions to trigger autoimmune pathology. Here we find that the high population density of the autoreactive T cells that accumulated in such a host limits the progression of the cell-intrinsic tolerance process in T cells. Accordingly, reducing the cell density during a second transfer allowed T cells to further tune down their responsiveness to antigenic stimulation. The retuning of T cells was reflected by a loss of the T cell’s abilities to proliferate, produces cytokines or help B cells. We further suggest, based on altering the levels of chronic antigen using miniosmotic pumps, that the effects of cell-density on T cell re-tuning may reflect the effective changes in the antigen dose perceived by individual T cells. This could proportionally elicit more negative feedback downstream of the TCR. Consistent with this, the retuned T cells showed signaling defects both proximal and distal to the TCR. Therefore, similar to the immunogenic activation of T cells, cell-intrinsic T cell tolerance may also involve a quantitative and progressive process of tuning down its antigen-responsiveness. The progress of such tuning seems to be stabilized at multiple intermediate stages by factors such as cell density, rather than just absolute antigen levels.

The molecular mechanisms that underpin thymocyte selection remain incompletely defined. Groups led by Love, Gascoigne and Schwartz independently identify Themis, a signaling protein essential for the positive selection of thymocytes. T cell antigen receptor (TCR) signaling in CD4+CD8+ double-positive thymocytes determines cell survival and lineage commitment, but the genetic and molecular basis of this process is poorly defined. To address this issue, we used ethylnitrosourea mutagenesis to identify a previously unknown T lineage–specific gene, Themis, which is critical for the completion of positive selection. Themis contains a tandem repeat of a unique globular domain (called 'CABIT' here) that includes a cysteine motif that defines a family of five uncharacterized vertebrate proteins with orthologs in most animal species. Themis-deficient thymocytes showed no substantial impairment in early TCR signaling but did show altered expression of genes involved in the cell cycle and survival before and during positive selection. Our data suggest a unique function for Themis in sustaining positive selection.

SummaryAfter an immune response, the expanded population of antigen-specific CD4+ T cells contract to steady state levels. We have found that the contraction is neither cell-autonomous nor mediated by competition for generic trophic factors, but regulated by relatively rare subsets of neighboring CD4+ T cells not necessarily of a conventional regulatory T cell lineage. These regulators, referred to as deletors, specifically limit the frequency of particular antigen-specific T cells even though they are not reactive to the same agonist as their targets. Instead, an isolated deletor could outcompete the target for recognition of a shared, nonstimulatory endogenous peptide-MHC ligand. This mechanism was sufficient to prevent even agonist-driven autoimmune disease in a lymphopenic environment. Such a targeted regulation of homeostasis within narrow colonies of T cells with related TCR specificities for subthreshold ligands might help to prevent the loss of unrelated TCRs during multiple responses, preserving the valuable diversity of the repertoire.

The behavior of self-reactive T cells in the peripheral immune system has often been studied by following the fate of adoptively transferred antigen-specific T cells in antigen expressing mice. In most cases, after a period of expansion, such cells undergo a slow clonal deletion, accompanied by the onset of anergy and/or suppression in the remaining cells. Here, we demonstrate that at initial frequencies approaching those found in normal repertoires, it is possible to completely avoid deletion and still maintain peripheral tolerance. At starting numbers of

Most gene promoters have multiple binding sequences for many transcription factors, but the contribution of each of these factors to chromatin remodeling is still unclear. Although we previously found a dynamic change in the arrangement of nucleosome arrays at the Il2 promoter during T cell activation, its timing preceded that of a decrease in nucleosome occupancy at the promoter. In this article, we show that the initial nucleosome rearrangement was temporally correlated with the binding of NFAT1 and AP-1 (Fos/Jun), whereas the second step occurred in parallel with the recruitment of other transcription factors and RNA polymerase II. Pharmacologic inhibitors for activation of NFAT1 or induction of Fos blocked the initial phase in the sequential changes. This step was not affected, however, by inhibition of c-Jun phosphorylation, which instead blocked the binding of the late transcription factors, the recruitment of CREB-binding protein, and the acetylation of histone H3 at lysine 27. Thus, the sequential recruitment of transcription factors appears to facilitate two separate steps in chromatin remodeling at the Il2 locus.

Adaptive tolerance is a hyporesponsive state in which lymphocyte Ag receptor signaling becomes desensitized after prolonged in vivo encounter with Ag. The molecular mechanisms underlying this hyporesponsive state in T cells are not fully understood, although a major signaling block has been shown to be present at the level of ZAP70 phosphorylation of linker for activation of T cells (LAT). In this study, we investigated the ability of adaptively tolerant mouse T cells to form conjugates with Ag-bearing APCs and to translocate signaling molecules into the interface between the T cells and APCs. Compared with naive or preactivated T cells, adaptively tolerant T cells showed no dramatic impairment in their formation of conjugates with APCs. In contrast, there was a large impairment in immunological synapse formation. Adaptively tolerant T cells were defective in their translocation of signaling molecules, such as ZAP70, LAT, and phospholipase C γ1, into the T cell–APC contact sites. Although Ag-induced activation of VAV1 was normal, VAV’s recruitment into the synapse was also impaired. Interestingly, expressions of both IL-2–inducible T cell kinase and growth factor receptor-bound protein 2-related adaptor downstream of SHC were decreased by 60–80% in adaptively tolerant T cells. These decreases, in addition to the impairment in LAT phosphorylation by ZAP70, appear to be the major impediments to the phosphorylation of SLP76 (SRC homology 2 domain-containing leukocyte protein of 76 kDa) and the recruitment of VAV1, which are important for stable immunological synapse formation.

Anergy is a state of long term hyporesponsiveness in T cells that is characterized by an active repression of TCR signaling and IL-2 expression [1]. Several forms of anergy have been described and the last few years have brought to light an increasing number of “anergic factors” involved in the induction and the active maintenance of the state in lymphocytes. The role of mTOR and other related metabolic sensors and regulators has recently emerged as of particular importance in broadening our view of anergy-inducing signals. We will discuss the role of these molecules in regulating the choice between anergy and activation, a decision faced by all T cells undergoing TCR stimulation. We will then explore the relationship between the induction of anergy and the induction of regulatory T cells as well as the potential crosstalk responsible for the phenomenon of infectious tolerance.

Induction of T-cell clonal anergy involves serial activation of transcription factors, including NFAT and Egr2/3. However, downstream effector mechanisms of these transcription factors are not fully understood yet. Here we identify Ndrg1 as an anergy factor induced by Egr2. Ndrg1 is upregulated by anergic signalling and maintained at high levels in resting anergic T cells. Overexpression of Ndrg1 mimics the anergic state and knockout of the gene prevents anergy induction. Interestingly, Ndrg1 is phosphorylated and degraded by CD28 signalling in a proteasome-dependent manner, explaining the costimulation dependence of anergy prevention. Similarly, IL-2 treatment of anergic T cells, under conditions that lead to the reversal of anergy, also induces Ndrg1 phosphorylation and degradation. Finally, older Ndrg1-deficient mice show T-cell hyperresponsiveness and Ndrg1-deficient T cells aggravate inducible autoimmune inflammation. Thus, Ndrg1 contributes to the maintenance of clonal anergy and inhibition of T-cell-mediated inflammation., T cell clonal anergy is mediated by Egr2 but its downstream effectors have not been characterized. Here the authors show that Egr2 activates Ndrg1, which is critical for the anergy state, while anergy-counteracting signals IL-2 or CD-28 promote Ndrg1 phosphorylation and degradation.

Adaptive tolerance is a process by which T cells become desensitized when Ag stimulation persists following an initial immune response in vivo. To examine the biochemical changes in TCR signaling present in this state, we used a mouse model in which Rag2−/− TCR-transgenic CD4+ T cells were transferred into CD3ε−/− recipients expressing their cognate Ag. Compared with naive T cells, adaptively tolerant T cells had normal levels of TCR and slightly increased levels of CD4. Following activation with anti-TCR and anti-CD4 mAbs, the predominant signaling block in the tolerant cells was at the level of Zap70 kinase activity, which was decreased 75% in vitro. Phosphorylations of the Zap70 substrates (linker of activated T cells and phospholipase Cγ1 were also profoundly diminished. This proximal defect impacted mostly on the calcium/NFAT and NF-κB pathways, with only a modest decrease in ERK1/2 phosphorylation. This state was contrasted with T cell clonal anergy in which the RAS/MAPK pathway was preferentially impaired and there was much less inhibition of Zap70 kinase activity. Both hyporesponsive states manifested a block in IκB degradation. These results demonstrate that T cell adaptive tolerance and clonal anergy are distinct biochemical states, possibly providing T cells with two molecular mechanisms to curtail responsiveness in different biological circumstances.

Successful adaptive immunity against a broad range of pathogens depends on the diversity of randomly generated T-lymphocyte and B-lymphocyte receptors. A subset of these receptors will be self-reactive and must be regulated to prevent autoimmunity. The process of immunologic tolerance addresses this problem by either purging autoreactive receptors from the system or tuning down their reactivity sufficiently to prevent disease. Immature lymphocytes generate a novel receptor during development in the thymus or bone marrow. Engagement of self antigens by these nascent receptors leads to their purging, either by the apoptotic death of the lymphocyte or by the initiation of receptor editing, a process in which the autoreactive receptor is replaced. If the lymphocytes mature further, the activation threshold of autoreactive cells can be tuned by the co-expression of inhibitory receptors or negative signaling molecules, allowing the persistence of the receptor without an increased risk of autoimmunity. T-cell and B-cell receptors that escape these checkpoints can still be regulated in the peripheral immune system by both purging and tuning mechanisms. A separate set of mechanisms, mediated by various regulatory cells, also operates to tune peripheral receptors in a cell-extrinsic fashion. The combined action of these processes ensures that the organism does not suffer autoimmune pathology, even if autoreactive receptors are generated and maintained in the immune system.

The adaptive immune system allows individual organisms to mount defensive reactions against unanticipated pathogens by developmentally creating a diverse repertoire of clonally distributed receptors capable of recognizing a multitude of antigens and then expanding as effector cell populations those that can recognize molecules from the pathogens. To function properly, the system must deal with the problem of randomly generated receptors that can recognize self components. Most solutions to this self-tolerance problem are cell intrinsic and involve the deletion or inactivation of autoreactive cells. However, an extrinsic form of dominant tolerance has been demonstrated that takes the form of CD4(+) regulatory T cells. This perspective discusses why such a mechanism might have evolved and the problems it presents for self-non-self discrimination.

TGF-β1 is critical for maintaining T cell homeostasis. Smad3 has been implicated in this regulatory process, yet the cellular targets and molecular details remain poorly understood. In this study, we report that TGF-β1 impairs the entry of CD4+ and CD8+ T cells into the cell cycle as well as their progression through subsequent rounds of division, and show that Smad3 is essential for TGF-β1 to inhibit TCR-induced division of only CD4+ and not CD8+ T cells. Both CD8+ and CD4+ T cells from Smad3−/− mice were refractory to TGF-β1-induced inhibition of IL-2 production, thus demonstrating that not all CD8+ T cell responses to TGF-β1 are Smad3 independent. These TGF-β1 effects were all T cell intrinsic, as they were reproduced in purified CD4+ and CD8+ T cells. Finally, we found that Smad3 was critical for the survival of CD8+, but not CD4+ T cells following activation ex vivo. The TCR-induced death of Smad3−/− CD8+ T cells was not dependent upon TNF-α production. Exogenous TGF-β1 partially rescued the CD8+ T cells by signaling through a Smad3-independent pathway. TGF-β1 also enhanced survival of TCR-stimulated CD4+CD44high T cells in a Smad3-independent manner. Collectively, these findings firmly establish for the first time that TGF-β1 discriminately regulates CD4+ and CD8+ T cell expansion by signaling through distinct intracellular pathways.

Epithin, a mouse type II transmembrane serine protease, is processed at Gly149 and released from the membrane. Here, we report the identification of an epithin isoform, epithin(Delta), containing a 66 amino acid deletion from the full-length epithin, which is missing the 4th LDLRA domain and the protease activation sequence. This truncated isoform showed the same characteristic N-terminal processing at Gly149 as the full-length form, however, no protease activity was detected. The N-terminal processed epithin(Delta) short form (Epi(Delta)-S) was not released into the medium under conditions in which the processed epithin short form (Epi-S) is released. This type of epithin shedding was also prevented when serine protease inhibitors were added to cells expressing the full-length form. These results strongly suggest that the serine protease activity is involved in the shedding process. The presence of epithin(Delta) message was detected in multiple tissues and its significance is discussed.

The secretion of IL-2 is a critical and early landmark in the activation program of CD4+ T cells in vitro, but the lack of sensitive assays has limited its application for studying T cell activation in vivo. Using a mouse cytokine capture assay we were able to detect the rapid secretion of IL-2 after an in vivo stimulus by 1–2 h in naive T cells and as early as 30 min in memory T cells. Maximal secretion was achieved within 1–2 h for memory cells or 6–8 h for naive T cells. Surprisingly IL-2 production terminated quickly in vivo and secretion was undetectable by 20–24 h in either cell type. We further demonstrated that this short duration of secretion can be influenced by cellular competition between Ag-specific CD4+ T cells. The consequences of competition were mimicked by reducing the strength of the antigenic stimulus. These data argue that early competition between T cells influences both the eventual frequency of IL-2 producers in the population and also the duration of their secretion, potentially by altering the strength or duration of the stimulus available to each T cell.

Transforming growth factor-β1 is essential to maintain T cell homeostasis, as illustrated by multiorgan inflammation in mice deficient in TGF-β1 signaling. Despite the physiological importance, the mechanisms that TGF-β1 uses to regulate T cell expansion remain poorly understood. TGF-β1 signals through transmembrane receptor serine/threonine kinases to activate multiple intracellular effector molecules, including the cytosolic signaling transducers of the Smad protein family. We used Smad3−/− mice to investigate a role for Smad3 in IL-2 production and proliferation in T cells. Targeted disruption of Smad3 abrogated TGF-β1-mediated inhibition of anti-CD3 plus anti-CD28-induced steady state IL-2 mRNA and IL-2 protein production. CFSE labeling demonstrated that TGF-β1 inhibited entry of wild-type anti-CD3 plus anti-CD28-stimulated cells into cycle cell, and this inhibition was greatly attenuated in Smad3−/− T cells. In contrast, disruption of Smad3 did not affect TGF-β1-mediated inhibition of IL-2-induced proliferation. These results demonstrate that TGF-β1 signals through Smad3-dependent and -independent pathways to inhibit T cell proliferation. The inability of TGF-β1 to inhibit TCR-induced proliferation of Smad3−/− T cells suggests that IL-2 is not the primary stimulus driving expansion of anti-CD3 plus anti-CD28-stimulated T cells. Thus, we establish that TGF-β1 signals through multiple pathways to suppress T cell proliferation.

Motivation: We present a statistical framework for the analysis of high-dimensional microarray data, where the goal is to compare intensities among several groups based on as few as a single sample from each group. In this setting, it is of interest to compare gene expression among several phenotypes to define candidate genes that simultaneously characterize several criteria, simultaneously, among the comparison groups. We motivate the approach by a comparative microarray experiment in which clones of a cell were singly exposed to several distinct but related conditions. The experiment was conducted to elucidate genes involved in pathways leading to T cell clonal anergy. Results: By integrating inference principles within a bioinformatics setting, we introduce a two-stage approach to select candidate genes that characterize several criteria. The method is unified in its non-parametric approach to inference and description. For inference, we construct a testable hypothesis based on the criteria of interest in a high-dimensional space, while preserving the dependence among genes. Upon rejecting the null, we estimate the cardinality of a set of individual candidate genes (or gene pairs) that depict the events of interest. With this estimate, we then select individual genes (or gene pairs) based upon a two-dimensional ranking that examines relations within and between genes, among comparison groups, using singular value decomposition in combination with inner product concepts. Availability: The functions developed for obtaining results from our approach are available upon request. A detailed documentation of the methods and the experiment may be obtained from http://www.cancerbiostats.onc.jhmi.edu/Kowalski.htm

The quantitative adaptation of receptor thresholds allows cells to tailor their responses to changes in ambient ligand concentration in many biological systems. Such a cell-intrinsic calibration of T cell receptor (TCR) sensitivity could be involved in regulating responses to autoantigens, but this has never been demonstrated for peripheral T cells. We examined the ability of monoclonal naive T cells to modulate their responsiveness differentially after exposure to fourfold different levels of persistent antigen stimulation in vivo. T cells expanded and entered a tolerant state with different kinetics in response to the two levels of stimulation, but eventually adjusted to a similar slow rate of turnover. In vivo restimulation revealed a greater impairment in the proliferative ability of T cells resident in a higher antigen presentation environment. We also observed subtle differences in TCR signaling and in vitro cytokine production consistent with differential adaptation. Unexpectedly, the system failed to similarly compensate to the persistent stimulus in vivo at the level of CD69 expression and actin polymerization. This greater responsiveness of T cells residing in a host with a lower level of antigen presentation allows us to demonstrate for the first time an intrinsic tuning process in mature T lymphocytes, albeit one more complex than current theories predict.

A new era of cellular immunology is clearly at hand. It is now possible, with a little bit of effort, to isolate monoclonal populations of T cells specific for any given antigen. The implications o f this technological advance are enormous in terms of applications to basic research and clinical medicine. In this article the two basic approaches that have been used to clone T lymphocytes are outlined, the pros and cons of each technique discussed and examples are given of recent experiments which have exploited this technology to gain new insights into T-cell specificity.

This Research Topic covers all of the major lectures and symposia addresses delivered by invited speakers at the 2013 International Congress in Immunology (ICI) at Milan, Italy, August 22-27, 2013.

We have proposed a model in which the prevention of anergy by costimulation is the result of IL-2-induced G1 to S phase cell cycle progression. Here we demonstrate that the reversal of anergy by exogenous IL-2 also occursduring this window of the cell cycle. Recently, it has been proposed that the cell cycle inhibitor p27 K i p 1 is an anergic factor. In contrast, our data demonstrate that during the induction, maintenance and rechallenge phases of anergy, p27 K i p 1 levels do not correlate with the anergic phenotype. Although p27 K i p 1 levels were down-regulated by IL-2 during the G1 to S phase transition, the amount of IL-2 required to produce this effect was far lower than that required to prevent the induction of anergy. Furthermore, T cell lines from p27 K i p 1 knockout mice were anergized as well as T cells from mice that were heterozygous for p27 K i p 1 . Interestingly, the forced overexpression of p27 K i p 1 was able to decrease IL-2 promoter-induced transcription, suggesting that the cell cycle machinery may be involved in T cell activation; however, physiological levels of p27 K i p 1 did not prevent IL-2 transcription. Overall, our data serve to disassociate the ability of IL-2 to down-regulate p27 K i p 1 and its ability to prevent or reverse anergy.

Epithin was originally identified as a mouse type II membrane serine protease. Its human orthologue membrane type-serine protease 1 (MT-SP1)/matriptase has been reported to be localized on the plasma membrane. In addition, soluble forms of matriptase were isolated from human breast milk and breast cancer cell-conditioned medium. In this paper, we report a processing mechanism that appears to be required for the release of epithin. CHO-K1 or COS7 cells transfected with single full-length epithin cDNA generated two different-sized proteins in cell lysates, 110 and 92 kDa. The 92-kDa epithin was found to be an N-terminally truncated form of the 110-kDa epithin, and it was the only form detected in the culture medium. The 92-kDa epithin was also found on the cell surface, where it was anchored by the N-terminal fragment. The results of in vivo cell labeling experiments indicate that the 110-kDa epithin is rapidly processed to the 92-kDa epithin. Using site-directed mutagenesis experiments, we identified Gly(149) of the GSVIA sequence in epithin as required for the processing and release of the protein. These results suggest that N-terminal processing of epithin at Gly(149) is a necessary prerequisite step for release of the protein.

The regulation of gene expression in thymic epithelial cells is critical for T cell development. The mouse thymic epithelial gene Tscot encodes a protein with weak homology to bacterial 12 transmembrane co-transporters. Using competitive reverse transcription-polymerase chain reaction (RT-PCR), we show that low level Tscot expression is detectable in several other tissues. Tscot was mapped to chromosome 4 and was also detected in other mammalian species by Southern blotting. The human cDNA clone showed 77% amino acid identity with the mouse sequence. The highest conservation was in the TM regions and in a small segment of the central cytoplasmic loop. Genomic clones spanning 17 164 bases of the Tscot gene revealed four exons with nine of the TM domains encoded in the first exon. The major transcriptional start site in mouse was identified by a primer extension analysis and confirmed by RT-PCR. Comparison of 1.7 kb of the human and mouse promoters identified six conserved possible regulatory elements, one containing a potential binding site for an interferon α inducible factor. Finally, as a functional test, 3 kb of the murine promoter was used to create a transgenic mouse that expresses enhanced green fluorescent protein message strongly in the thymus, weakly in the kidney and undetectably in the spleen, liver and heart.

The gene LamC2 encoding the γ2 chain of laminin 5, an epithelial cell-specific extracellular matrix protein, was identified in a PCR-based subtracted cDNA library from mouse thymic stromal cells. The mRNA existed in two alternative forms (5.1 and 2.4 kb). The full-length message was highly expressed in SCID thymus and in a nurse cell line, but not in other thymic epithelial cell lines, while the short form was more widely expressed. In situ hybridization and immunohistochemical staining revealed laminin 5 expression mostly in the subcapsular region of the adult thymus. Addition to fetal thymic organ cultures of a cell adhesion-blocking mAb to the α3 chain of laminin 5 interrupted T cell development. There was a 40% reduction in the total yield of thymocytes, and the most profound decrease (75–90%) was seen in the CD25+CD44+ and CD25+CD44−subsets of the CD4−CD8− double negative fraction. Most of the surviving double negative thymocytes expressed Sca-1, and there were significant increases in the number of cells with CD69 expression and in the fraction of annexin V-stained cells. None of these changes were observed with a nonblocking anti-laminin α3 chain mAb. These results suggest that the interaction between double negative thymoctyes and laminin 5 made by subcapsular epithelial cells is required for the survival and differentiation of mouse thymocytes.

Resistance toToxoplasma gondiihas been shown to be mediated by gamma interferon (IFN-γ) produced by NK, CD4+, and CD8+T cells. While studies of SCID mice have implicated NK cells as the source of the cytokine in acute infection, several lines of evidence suggest that IFN-γ production by CD4+T lymphocytes also plays an important role in controlling early parasite growth. To evaluate whether this function is due to nonspecific as opposed to T-cell receptor (TCR)-dependent stimulation by the parasite, we have examined the resistance toT. gondiiinfection of pigeon cytochromectransgenic (PCC-Tg) Rag-2−/−mice in which all CD4+T lymphocytes are unreactive with the protozoan. When inoculated with the ME49 strain, PCC-Tg animals exhibited only temporary control of acute infection and succumbed by day 17. Intracellular cytokine staining by flow cytometry revealed that, in contrast to infected nontransgenic controls, infected PCC-Tg animals failed to develop IFN-γ-producing CD4+T cells. Moreover, the CD4+lymphocytes from these mice showed no evidence of activation as judged by lack of upregulated expression of CD44 or CD69. Nevertheless, when acutely infected transgenic mice were primed by PCC injection, the lymphokine responses measured after in vitro antigen restimulation displayed a strong Th1 bias which was shown to be dependent on endogenous interleukin 12 (IL-12). The above findings argue that, whileT. gondii-induced IL-12 cannot trigger IFN-γ production by CD4+T cells in the absence of TCR ligation, the pathogen is able to nonspecifically promote Th1 responses against nonparasite antigens, an effect that may explain the immunostimulatory properties ofT. gondiiinfection.

Recent experiments have suggested that the IL-2 locus is monoallelically expressed. We tested this hypothesis using TCR-transgenic mice carrying one inactivated IL-2 allele. The frequency in single-cell assays of IL-2-producing cells following optimal stimulation by antigen and antigen-presenting cells was equivalent to that from wild-type mice, but the amount of IL-2 produced per cell was twofold less. Similar observations were made by intracellular staining for IL-2, although stimulation in bulk culture was less optimal, showing only a 1.7-fold difference. Importantly, the frequency of responding cells from the heterozygotes was less than from the wild-type mice if the IL-2 assay was performed after only 24 - 30 h of activation, suggesting that the targeted allele could compete with the normal allele early after stimulation and give the misimpression that the heterozygotes had fewer IL-2-producing cells. These data strongly argue that the IL-2 locus can be expressed biallelically under optimum stimulation conditions.

Anergic T cells display a marked decrease in their ability to produce IL-2 even in the presence of optimal TCR and costimulatory signals. Using IL-2 enhancer/promoter-driven reporter constructs, we have previously identified a region that appears to be a target for cis transcriptional repression in anergy. This region of the promoter, which shares partial homology with a consensus AP-1-binding sequence, is located about −180 bp from the transcriptional start site. In the present study, we demonstrate that cAMP response element-binding protein/cAMP response element modulator (CREB/CREM), activating transcription factor-2/c-Jun, and Jun-Jun/Oct complexes bind to this site. However, the induction of anergy by prolonged stimulation through the TCR led to an increase in binding of only the CREB/CREM complex. Furthermore, the level of binding of this complex appeared to be up-regulated in both resting and restimulated anergic T cells. Finally, an IL-2 promoter-driven reporter construct that contained a mutation that specifically reduced the binding of the CREB/CREM complex displayed a decreased ability to be affected by anergy, while a construct that contained a mutation that decreased the binding of the Jun-Jun/Oct complex was still susceptible to anergy. These findings suggest that the −180 region of the IL-2 promoter is the target of a CREB/CREM transcriptional inhibitor that contributes to the repression of IL-2 production in T cell anergy.

To investigate the role that translation plays in the stabilization of the IL-2 mRNA, we inhibited protein synthesis in both cis and trans. To block translation in trans, we utilized the inhibitors puromycin (PUR) and cycloheximide (CHX), which differentially effect polysome structure. We found that CHX enhances the stability of IL-2 mRNA in cells stimulated with anti-TCR Ab alone, but it inhibits CD28-induced message stabilization in costimulated cells. In contrast, PUR had a minimal effect on IL-2 mRNA stability in either the presence or absence of costimulation. The differential effects of these two inhibitors suggest that: 1) CHX is unlikely to stabilize the IL-2 mRNA by inhibiting the expression of a labile RNase; 2) CD28-mediated IL-2 mRNA stabilization does not require translation; and 3) IL-2 mRNA decay is not coupled to translation. To block translation in cis, we generated sequence-tagged IL-2 genomic reporters that contain a premature termination codon (PTC). In both the presence and absence of costimulation, these PTC-containing mRNAs exhibit drastically diminished stability. Interestingly, the addition of CHX but not PUR completely restored CD28-mediated stabilization, suggesting that CHX can block the enhanced decay induced by a PTC. Finally, CHX was able to superinduce IL-2 mRNA levels in anti-TCR Ab-stimulated cells but not in CD28-costimulated cells, suggesting that CHX may also act by other mechanisms.

Using sequence-tagged genomic reporter constructs, we investigated the contribution of IL-2 sequences to CD28-mediated regulation of mRNA stability. We find that CD28 signaling acts transiently to stabilize the IL-2 mRNA following T cell activation. Such stabilization requires sequences within both exon 2 and the coding region of exon 4. Unexpectedly, CD28 signaling at later times enhances the decay of the IL-2 mRNA. This CD28-dependent decay of IL-2 mRNA requires sequences localized between exon 3 and the stop codon. Our findings demonstrate that the coding region of the IL-2 mRNA contains previously undefined CD28-responsive sequence elements that are critical for the regulation of mRNA stability.

Costimulation (signal 2) has been proposed to inhibit the induction of T cell clonal anergy by either directly antagonizing negative signals arising from TCR engagement (signal 1) or by synergizing with signal 1 to produce IL-2, which in turn leads to proliferation and dilution of negative regulatory factors. To better define the cellular events that lead to the induction of anergy, we used the immunosuppressive agent rapamycin, which blocks T cell proliferation in late G1 phase but does not affect costimulation-dependent IL-2 production. Our data demonstrate that full T cell activation (signal 1 plus 2) in the presence of rapamycin results in profound T cell anergy, despite the fact that these cells produce copious amounts of IL-2. Similar to conventional anergy (induction by signal 1 alone), the rapamycin-induced anergic cells show a decrease in mitogen-activated protein kinase activation, and these cells can be rescued by culture in IL-2. Interestingly, the rapamycin-induced anergic cells display a more profound block in IL-3 and IFN-γ production upon rechallenge. Finally, in contrast to rapamycin, full T cell activation in the presence of hydroxyurea (which inhibits the cell cycle in early S phase) did not result in anergy. These data suggest that it is neither the direct effect of costimulation nor the subsequent T cell proliferation that prevents anergy induction, but rather the biochemical events that occur upon progression through the cell cycle from G1 into S phase.

Recombinant-activating gene 2 (RAG-2−/−) T cell receptor–transgenic mice repeatedly injected with the superantigen staphylococcal enterotoxin A entered a tolerant state in which splenic CD4+ T cells produced little interleukin (IL)-2, interferon γ, or IL-4. This state resulted from a combination of both clonal anergy and cytokine-mediated immunosuppression. The anergy persisted for at least 3 wk and could be distinguished from the suppression by a decrease in IL-2 production per cell, a block in the activation of early response kinases, and a failure to be reversed with anti–transforming growth factor (TGF)-β. Full suppression lasted for only 1 wk and involved both IL-10 and TGF-β, but required additional unknown molecules for optimal effect. These experiments show that complex in vivo interactions of multiple peripheral tolerance mechanisms can now be dissected at both the cellular and molecular levels.

Recent experiments have elucidated two molecular mechanisms that may account for the failure of anergic T cell clones to initiate IL-2 gene transcription following TCR stimulation. First, a block has been identified in the ERK and JNK mitogen-activated protein kinase pathways; the block results from a failure to activate p21ras. It leads to reduced induction of c-Fos and JunB proteins and to a failure to form and phosphorylate the activator protein (AP)-1 heterodimers required for IL-2 gene transcriptional activation. Second, repressor molecules (Nil-2-a and a molecule related to AP-1) have been characterized that dominantly inhibit IL-2 gene transcription.

SummaryDifferences in gut commensal flora can dramatically influence autoimmune responses, but the mechanisms behind this are still unclear. We report, in a Th1-cell-driven murine model of autoimmune arthritis, that specific gut commensals, such as segmented filamentous bacteria, have the ability to modulate the activation threshold of self-reactive T cells. In the local microenvironment of gut-associated lymphoid tissues, inflammatory cytokines elicited by the commensal flora dynamically enhanced the antigen responsiveness of T cells that were otherwise tuned down to a systemic self-antigen. Together with subtle differences in early lineage differentiation, this ultimately led to an enhanced recruitment of pathogenic Th1 cells and the development of a more severe form of autoimmune arthritis. These findings define a key role for the gut commensal flora in sustaining ongoing autoimmune responses through the local fine tuning of T-cell-receptor-proximal activation events in autoreactive T cells.

BACKGROUND The hyperacute rejection mediated by preexisting antibodies is a major impediment to the success of transplants across allogeneic and xenogeneic barriers. We report a new mouse model that allows us to not only monitor the sensitization of B cells mediating the hyperacute response but also validate therapeutic strategies for tolerizing them. MODEL The new model system uses 5C.C7,RAG2 T-cell receptor transgenic T cells and B10.S(9R),CD3[Latin Small Letter Open E] hosts for adoptive transfer experiments. RESULTS AND CONCLUSIONS In the allogeneic hosts, transgenic T cells expanded briefly before being chronically deleted. Once the deletion was initiated, a second graft of donor cells was used to assess a hyperacute response. The rapid rejection of the second cohort correlated with the appearance of donor-specific antibodies in the serum. Interestingly, chronically stimulated T cells were relatively resistant to hyperacute rejection, suggesting an explanation for the slower rejection kinetics of the first cohort even as the second cohort of identical donor cells was being hyperacutely rejected. Finally, we could tolerize the potential for a hyperacute response, by pretreating recipients with a single infusion of naive donor B cells before the first T-cell transfer. This treatment not only abrogated the development of a hyperacute response but also allowed the primary graft to survive in vivo for extended periods.

Ligation of CD28 on CD4 Th1 clones and freshly isolated mixtures of naive and memory CD4 T cells triggered their T cell receptors (TCR) is sufficient to induce the costimulatory signals necessary for interleukin 2 (IL-2) production by these cells. CTLA-4-reactive ligands expressed on antigen-presenting cells (APC) are critical in providing costimulatory signals to these T cell populations. We demonstrate that these activation characteristics apply equally to purified naive CD4 T cells. Because B cell blasts express CTLA-4-reactive ligands and high levels of adhesion and major histocompatibility complex class II molecules, they would be expected to engage both the TCR and CD28 and consequently stimulate IL-2 production by naive CD4 T cells. Using purified populations of cells in limiting dilution cultures, we have carried out a quantitative analysis of the interaction between naive CD4 T cells and either activated B or dendritic cells. We demonstrate that B cell blasts stimulate a high frequency of naive CD4 T cells. Slight differences in TCR signaling efficiency between the two APC types were observed. Even at optimal peptide concentrations, however, the amount of IL-2 made by individual T cells was fourfold lower in response to B cell blasts than to dendritic cells. This relative deficiency of activated B cells was due to their inability to optimally costimulate naive CD4 T cells.

To complete their maturation, most immature thymocytes depend on the simultaneous engagement of their antigen receptor [alpha beta T cell receptor (TCR)] and their CD4 or CD8 coreceptors with major histocompatibility complex class II or I ligands, respectively. However, a normal subset of mature alpha beta TCR+ thymocytes did not follow these rules. These thymocytes expressed NK1.1 and a restricted set of alpha beta TCRs that are intrinsically class I-reactive because their positive selection was class I-dependent but CD8-independent. These cells were CD4+ and CD4-8- but never CD8+, because the presence of CD8 caused negative selection. Thus, neither CD4 nor CD8 contributes signals that direct their maturation into the CD4+ and CD4-8- lineages.

T cell anergy is a tolerance mechanism in which the lymphocyte is intrinsically functionally inactivated following an antigen encounter, but remains alive for an extended period of time in a hyporesponsive state. Models of T cell anergy affecting both CD4+and CD8+cells fall into two broad categories. One, clonal anergy, is principally a growth arrest state, whereas the other, adaptive tolerance or in vivo anergy, represents a more generalized inhibition of proliferation and effector functions. The former arises from incomplete T cell activation, is mostly observed in previously activated T cells, is maintained by a block in the Ras/MAP kinase pathway, can be reversed by IL-2 or anti-OX40 signaling, and usually does not result in the inhibition of effector functions. The latter is most often initiated in naïve T cells in vivo by stimulation in an environment deficient in costimulation or high in coinhibition. Adaptive tolerance can be induced in the thymus or in the periphery. The cells proliferate and differentiate to varying degrees and then downregulate both functions in the face of persistent antigen. The state involves an early block in tyrosine kinase activation, which predominantly inhibits calcium mobilization, and an independent mechanism that blocks signaling through the IL-2 receptor. Adaptive tolerance reverses in the absence of antigen. Aspects of both of the anergic states are found in regulatory T cells, possibly preventing them from dominating initial immune responses to foreign antigens and shutting down such responses prematurely.

The delivery of costimulation and the effects of the anergic state impinge on IL-2 production via different molecular mechanisms. The strongest experimental support at this stage suggests that CD28 signaling effects mRNA stability of several lymphokine genes including IL-2. While there may also be transcriptional effects of CD28 signals in human cells, controversy surrounding relevant TCR mimics must be addressed. In the case of clonal anergy, however, transcriptional non-responsiveness is evident when anergic cells are restimulated with TCR and costimulatory signals. This repression affects predominantly AP-1 activity. So far, the nature of the repression has not been identified.

During their differentiation in the mouse thymus, CD4+8- cells undergo several of the sequential changes observed upon normal activation of mature, peripheral CD4+ lymphocytes. Expression of CD69, an early activation marker, is first observed on a minority of cells at the T cell receptor (TCR)lo/med double-positive stage, is maximal (50-90%) on heat-stable antigen (HSA)hi TCRhi double-positive, HSAhi TCRmed CD4+8lo, and HSAhi TCRhi CD4+8- cells, and is downmodulated at the mature HSAlo CD4+8- stage. In contrast, CD44, a late activation marker, is selectively expressed at the HSAlo stage. The set of lymphokines that CD4+8- thymocytes can produce upon stimulation also characteristically expands from mainly interleukin 2 (IL-2) at the HSAhi stage, to IL-2 and very large amounts of IL-4, IL-5, IL-10, and interferon gamma (IFN-gamma) at the HSAlo stage. 1 in 30 HSAlo CD4+8- adult thymocytes secrete IL-4 upon stimulation through their TCR. This frequency is 25% of the frequency of IL-2 producers, about 100-fold above that of peripheral (mainly resting) CD4+ T cells. With time after their generation in organ culture, CD4+8- thymocytes lose their capacity to secrete IL-4, IL-5, and IFN-gamma, but not IL-2. Similarly, the frequency of IL-4, but not of IL-2, producers progressively decreases after emigration to the periphery as judged by direct comparison between thymic and splenic CD4+ cells in newborns, or by following the fate of intrathymically labeled CD4+8- cells in adults after their migration to the spleen. This sequence suggests that thymic selection results from an activation process rather than a simple rescue from death at the double-positive stage, and shows that the functional changes induced after intrathymic activation, although transient, are still evident after export to the periphery.

Stimulation of a normal type I mouse T helper cell clone (TH1) with concanavalin A in the absence of antigen presenting cells (APC) in vitro results in the induction of a hyporesponsive state known as T cell clonal anergy. This state is characterized by a decrease in proliferation following stimulation with antigen and APC resulting from a decrease in the production of IL-2. Production of the lymphokines IL-3/granulocyte macrophage colony stimulating factor and IFN-gamma is also reduced, although to a lesser degree. Stimulation of such anergic cells with IL-2 results in proliferation and a complete reversal of the state. We demonstrate that this reversal is not due to the outgrowth of a subpopulation of cells that had escaped anergy induction, but rather occurs in all the cells. Anergy also dissipated spontaneously, although much more slowly, in the absence of T cell antigen receptor occupancy. Finally, we show that a similar state can be produced by normal activation with antigen and APC if IL-2 and other factors are removed at 16-20 h. These results indicate that the anergic state is not a permanent change in the TH1 cell. Anergy induction appears to be a consequence of the inability of the cell to divide extensively following stimulation through the antigen-specific receptor. We propose a model to explain these results in terms of a relatively stable negative regulatory factor.

Amino acid residues that are critical in maintaining the framework structure of immunoglobulin heavy- and light-chain variable (V) regions are strongly conserved in the V alpha and V beta proteins of the alpha beta T-cell antigen receptor (TCR alpha beta). Consequently, it has been proposed that TCR alpha beta has a conformation similar to that of an immunoglobulin Fab fragment and that the regions of the TCR homologous to the three immunoglobulin complementarity-determining regions (CDRs 1, 2, and 3) bind to the peptide antigen-major histocompatibility complex (MHC) molecule ligand. A single amino acid substitution in the predicted CDR1 of the V beta 3 protein of certain mouse strains dramatically altered TCR alpha beta usage in an antigen-specific MHC-restricted immune response but did not abrogate V beta 3 specificity for the superantigens minor lymphocyte stimulatory locus (Mls)c and staphylococcal enterotoxin A (SEA). The results confirm the importance of the V beta CDR1 in antigen-MHC molecule recognition, supporting the Fab-like structural model of TCR alpha beta, and provide further evidence that conventional antigen-MHC recognition and superantigen recognition are mediated by distinct regions of the TCR beta chain. They also suggest that allelic polymorphism may be a significant source of diversity in the TCR repertoire.

Although peripheral naive cells only secrete IL-2 upon primary stimulation, their presumptive immediate precursors, HSAlow CD4+8- thymocytes, can produce a large amount of the set of lymphokines usually associated with preactivated or memory CD4+ lymphocytes: IL-4, IL-5, IL-10 and gamma-IFN. This phenotype can be attributed to true virgin thymocytes and not only to recirculating lymphocytes, because it is found in newborn thymuses and in fetal thymic organ culture. This mature stage of CD4+8- thymocytes is itself preceded by an immature stage (HSAhigh) where only IL-2 and small amounts of gamma-IFN can be elicited by the combination of calcium ionophore and phorbol ester, but not by TCR cross-linking. CD8+4- thymocytes pass through a similar immature HSAhigh stage, where their pattern of lymphokine secretion is not yet differentiated from that of CD4+8- HSA high thymocytes. The subsets acquire their specific profiles at the HSAlow stage. We propose that recent thymic CD4+8- emigrant cells include a significant proportion of Th0 type cells, and that their role is critical to prime the immune system for IL-4 production, as well as to explain the longstanding observations of synergy between helper cell subpopulations in the periphery.

Peripheral CD4+ and CD8+ T lymphocytes carry out different functions during immune reactions, partly as a result of the distinct patterns of lymphokines that they secrete upon stimulation. Using thymic cells from adult and newborn mice as well as from fetal organ cultures, we show here that this functional differentiation occurs inside the thymus and is completed during the single positive stage by the time the T-cell receptor becomes fully coupled to the intracellular activation pathways leading to lymphokine secretion. Surprisingly, CD4+8- thymocytes differ from their immediate progeny, naive peripheral CD4+ cells, in that they secrete a broader range of lymphokines, including interleukins 4, 5 and 10 and gamma-interferon, and more closely resemble immunologically experienced (activated or memory) CD4+ lymphocytes.

We describe a novel biphasic regulation of Il2 transcription in naive CD4+ T cells. Few (∼5%) CD4+ T cells transcribe Il2 within 6 h of anti-TCR-β plus anti-CD28 stimulation (early phase). Most naive CD4+ T cells do not initiate Il2 transcription until after an additional ∼12 h of T cell stimulation (late phase). In comparison, essentially all previously activated (Pre-Ac) CD4+ T cells that transcribe Il2 do so with an early-phase response. Late-phase Il2 expression mostly requires c-Rel, CD28, and TNFR signaling. In contrast, early-phase transcription is only partly c-Rel and CD28 dependent and TNFR independent. There was also increased stable DNA accessibility at the Il2 locus and elevated c-Rel expression in resting Pre-Ac CD4+ cells. Upon T cell activation, a faster and greater increase in DNA accessibility as well as c-Rel nuclear expression were observed in Pre-Ac CD4+ cells relative to naive CD4+ T cells. In addition, both acetylated histone H3 and total H3 decreased at the Il2 locus upon rechallenge of Pre-Ac CD4+ T cells, whereas increased acetylated histone H3 with no change in total H3 was observed following activation of naive CD4+ T cells. We propose a model in which nucleosome disassembly facilitates rapid initiation of Il2 transcription in CD4+ T cells, and suggest that a threshold level of c-Rel must be reached for Il2 promoter activity in both naive and Pre-Ac CD4+ T cells. This is provided, at least partially, by TNFR signaling during priming, but not during recall.