Search

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.

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

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.

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.

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.

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 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.

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.

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.

T lymphocytes respond to foreign antigens both by producing protein effector molecules known as lymphokines and by multiplying. Complete activation requires two signaling events, one through the antigen-specific receptor and one through the receptor for a costimulatory molecule. In the absence of the latter signal, the T cell makes only a partial response and, more importantly, enters an unresponsive state known as clonal anergy in which the T cell is incapable of producing its own growth hormone, interleukin-2, on restimulation. Our current understanding at the molecular level of this modulatory process and its relevance to T cell tolerance are reviewed.

Overlapping roles have been ascribed for T cell anergy, clonal deletion, and regulation in the maintenance of peripheral immunological tolerance. A measurement of the individual and additive impacts of each of these processes on systemic tolerance is often lacking. In this report we have used adoptive transfer strategies to tease out the unique contribution of T cell intrinsic receptor calibration (adaptation) in the maintenance of tolerance to a systemic self-antigen. Adoptively transferred naïve T cells stably calibrated their responsiveness to a persistent self-antigen in both lymphopenic and T cell–replete hosts. In the former, this state was not accompanied by deletion or suppression, allowing us to examine the unique contribution of adaptation to systemic tolerance. Surprisingly, adapting T cells could chronically help antigen-expressing B cells, leading to polyclonal hypergammaglobulinemia and pathology, in the form of mild arthritis. The helper activity mediated by CD40L and cytokines was evident even if the B cells were introduced after extended adaptation of the T cells. In contrast, in the T cell–replete host, neither arthritis nor autoantibodies were induced. The containment of systemic pathology required host T cell–mediated extrinsic regulatory mechanisms to synergize with the cell intrinsic adaptation process. These extrinsic mechanisms prevented the effector differentiation of the autoreactive T cells and reduced their precursor frequency, in vivo., Intrinsic adaptive mechanisms can keep T cells from responding to chronic antigenic stimulation (thus reducing the chances of autoimmunity), but cell-extrinsic mechanisms (such as clonal deletion) are needed to enforce true tolerance.

Sprouty (Spry) is known to be a negative feedback inhibitor of growth factor receptor signaling through inhibition of the Ras/MAPK pathway. Several groups, however, have reported a positive role for Spry involving sequestration of the inhibitory protein c-Cbl. Thus, Spry may have various functions in the regulation of receptor-mediated signaling depending on the context. In the immune system, the function of Spry is unknown. In this study, we investigated the role of Spry1 in T cell activation. Spry1, among the four mammalian homologs, was specifically induced by TCR signaling of CD4+ murine T cells. In fully differentiated Th1 clones, overexpressed Spry1 inhibited TCR signaling and decreased IL-2 production while reducing expression with specific siRNA transfection had the opposite effect, increasing IL-2 production. In contrast, in naive T cells, Spry1 overexpression enhanced TCR signaling, and increased proliferation and IL-2 production, while siRNA transfection again had the opposite effect, reducing IL-2 production following activation. The enhancing effect in naive cells was abrogated by preactivation of the T cells with Ag and APC, indicating that the history of exposure to Ag is correlated with a hierarchy of T cell responsiveness to Spry1. Furthermore, both the NF-AT and MAPK pathways were influenced by Spry1, implying a different molecular mechanism from that for growth factor receptor signaling. Thus, Spry1 uses a novel mechanism to bring about differential effects on TCR signaling through the same receptor, depending on the differentiation state of the T cell.

Background. The ability to achieve significant donor engraftment without fully myeloablative conditioning has revolutionized allogeneic stem cell transplantation. These nonmyeloablative approaches may allow extension of this potentially curative modality to an increasing number of patients including those with non-malignant diseases. Although a number of regimens have been explored, the optimal means of conditioning has not been determined. Methods. We previously demonstrated that rapamycin (RAPA) has the ability to promote T-cell tolerance even in the presence of costimulation. In the current study, we examine the ability of rapamycin or the calcineurin inhibitor cyclosporine A (CSA) to promote chimerism in a murine haploidentical bone marrow transplantation model. Mice were conditioned with 300 cGy and received either RAPA at 3 mg/kg/day IP, CSA at 20 mg/kg/day IP, or no immunosuppression starting on the day before the transplant and continued for 4 weeks. Results. There was no apparent toxicity, and animals maintained normal blood counts throughout. More importantly, long-term macrochimerism was observed only in the RAPA-treated group. Conclusions. These results establish a simple, nontoxic, irradiation-based regimen that facilitates engraftment without ablation. This strategy may prove useful in nonmalignant disorders such as hemoglobinopathies in which moderate levels of donor chimerism could prove curative.

T cell receptor engagement in the absence of proper accessory signals leads to T cell anergy. E3 ligases are involved in maintaining the anergic state. However, the specific molecules responsible for the induction of anergy have yet to be elucidated. Using microarray analysis we have identified here early growth response gene 2 (Egr-2) and Egr-3 as key negative regulators of T cell activation. Overexpression of Egr2 and Egr3 was associated with an increase in the E3 ubiquitin ligase Cbl-b and inhibition of T cell activation. Conversely, T cells from Egr3(-/-) mice had lower expression of Cbl-b and were resistant to in vivo peptide-induced tolerance. These data support the idea that Egr-2 and Egr-3 are involved in promoting a T cell receptor-induced negative regulatory genetic program.

Reports on the status of DNA methylation of the IFN-γ gene during T cell development in human and mouse have presented somewhat contradictory results. In this study we demonstrate in the mouse that methylation of the IFN-γ promoter inhibits its transcriptional activity, and define a small hypomethylated region in T cells that correlates with transcription. The IFN-γ promoter was also hypomethylated in NK cells, but not in B cells or nonhemopoietic tissues. Surprisingly, unlike the promoters of the IL-2 and IL-4 genes, the IFN-γ promoter was hypomethylated in naive CD4+ and CD8+ T cells, and in this form from very early in T cell development. A population of non-B, non-T, non-NK cells containing the hypomethylated promoter was also found in the bone marrow. The hypomethylated state appears stable until peripheral CD4+ T cells differentiate in response to Ag and APC. After T cell stimulation in vitro under Th2 conditions, but far less so under Th1 conditions, CD4+ cells display a more methylated IFN-γ promoter, which may contribute to the lack of expression of IFN-γ in these preactivated cells. Our experiments support a new model of IFN-γ chromatin structural changes in murine T cell development that differs from what has been previously published for human T cells.

The in vivo T cell response to persistent Ag contains a hyporesponsive phase following an initial expansion and subsequent partial deletion of the responding cells. The mechanism(s) responsible for this tolerance process is poorly understood. In this study, we describe a new paired transgenic model (TCR and Ag), which within 7–14 days produces 20–40 million hyporesponsive T cells. This state is characterized by an 85–95% reduction in all cytokine production, an impairment of re-expression of CD25 and CD69, and a desensitization of the proliferative response to Ag. TCR levels were normal, and in vivo mixing experiments showed no evidence for active suppression. The hyporesponsiveness partially dissipated without proliferation when the cells were transferred into a non-Ag-bearing host. If the second host expressed Ag, the T cells initially regained responsiveness, but then slowly entered an even deeper state of tolerance characterized by an additional 7- to 10-fold lowering of cytokine production and a greater desensitization of proliferation. Surprisingly, this readaptation took place with the same level of Ag presentation, suggesting that other parameters can influence the tolerance threshold. Both the readjustment in sensitivity and the reversal without Ag convincingly demonstrate for the first time a truly adaptive tolerance process in CD4+ T cells in vivo.

We have isolated a full-length cDNA clone (thymic stromal origin (TSO)-1C12) from a SCID thymus library using a probe from a PCR-based subtractive library enriched for sequences from fetal thymic stromal cells. TSO-1C12 mRNA is expressed mainly in the thymic cortex and is highly enriched in SCID thymus. Expression per cell is highest during fetal thymus development and decreases after day 16. Antipeptide Abs immunoprecipitated a hydrophobic, plasma membrane glycoprotein (thymic stromal cotransporter, TSCOT) whose translated sequence has weak homology to bacterial antiporters and mammalian cation cotransporters with 12 transmembrane domains. TSCOT represents a new member of this superfamily that is highly expressed in thymic cortical epithelial cells.

We cloned and sequenced a mouse gene encoding a new type of membrane bound serine protease (epithin) containing a multidomain structure. The initial cDNA clone was found previously in a polymerase chain reaction (PCR)-based subtractive library generated from fetal thymic stromal cells, and the message was shown to be highly expressed in a thymic epithelial nurse cell line. A clone isolated from a severe combined immunodeficiency (SCID) thymus library and extended to its full length at the 5' end with the RACE technique contains an open reading frame of 902 amino acids. Based on the sequence of this clone, the predicted protein structure is a type II membrane protein with a C-terminal serine protease domain linked to the membrane by four low density lipoprotein receptor modules and two CUB domains. High message expression by northern blotting was detected in intestine, kidney, lung, SCID, and Rag-2(-/-) thymus, and 2-deoxyguanosine-treated fetal thymic rudiment, but not in skeletal muscle, liver, heart, testis, and brain. Sorted MHC class II+ and II- fetal thymic stromal cells were positive for expression by reverse transcriptase-PCR, whereas CD45(+) thymocytes were not. The gene was found in chicken and multiple mammalian species under low stringency Southern hybridization conditions. Under high stringency conditions, only a single gene per haploid genome was identified in the mouse. This gene, Prss14 (protease, serine, 14), was mapped to mouse chromosome 9 and is closely linked to the Fli1 (Friend leukemia integration 1) gene.

Full activation of T cells requires signaling through the T-cell antigen receptor (TCR) and additional surface molecules interacting with ligands on the antigen-presenting cell. TCR recognition of agonist ligands in the absence of accessory signals frequently results in the induction of a state of unresponsiveness termed anergy. However, even in the presence of costimulation, anergy can be induced by TCR partial agonists. The unique pattern of early receptor-induced tyrosine phosphorylation events induced by partial agonists has led to the hypothesis that altered TCR signaling is directly responsible for the development of anergy. Here we show that anergy induction is neither correlated with nor irreversibly determined by the pattern of early TCR-induced phosphorylation. Rather, it appears to result from the absence of downstream events related to interleukin 2 receptor occupancy and/or cell division. This implies that the anergic state can be manipulated independently of the precise pattern of early biochemical changes following TCR occupancy, a finding with implications for understanding the induction of self-tolerance and the use of partial agonist ligands in the treatment of autoimmune diseases.

Publisher Summary This chapter reviews the studies of two-signal models of lymphocyte activation. The two-signal model for B cell activation was first proposed by Bretscher and Cohn in 1968. There were two possible mechanisms by which the unresponsive state was induced. One was a consequence of the way in which the T cell antigen receptor was cross-linked: planar membrane presentation and chemical fixation of the proteins on the antigen presenting cell surface could both be hindering the mobility of the major histocompatibility complex molecules and thus preventing the formation of a proper array of receptor aggregates for signal transduction. The second possibility was that something was missing. Second idea fitted nicely with the two-signal model for B cell activation. Fehon in 1949 published his ideas on high dose paralysis where he concluded that too much antigen turns off the antibody-secreting cell. This idea was pursued by Dresser, who in a classic paper in 1962 concluded that it was not necessarily the dose of antigen that was critical, but its form of presentation to the immune system.

The adaptive immune system is a vertebrate anticipatory organ—a quality it shares with the nervous system. It is designed to learn to recognize molecular structures on pathogens quickly and to remember them for most of the life of the individual organism. It achieves this goal by continually generating a broad array of recognition receptors through an elaborate randomized gene rearrangement process. As a consequence of this, the system inevitably generates many receptors that recognize self-components. To deal with this problem, the immune system has evolved mechanisms to prevent it from making self-destructive responses. These mechanisms, referred to as the “process of tolerance induction,” are the topic of this collection. There are several categories of adjustments that the immune system makes in order to achieve a state of tolerance (Schwartz 2008). The first takes place during development, when newly generated T cells and B cells test their receptors for recognition of antigens in their immediate environment. Reactive cells are censored by a deletional, a receptor editing, or a tuning mechanism. These processes are referred to as “negative selection” or “central tolerance.” It takes place in the bone marrow for B cells and the thymus for T cells. After the lymphocytes mature and move into the circulation, they can encounter new self-antigens in secondary lymphoid organs such as the spleen and lymph nodes. At this point, the context in which the antigen is presented becomes important for determining the outcome of the response. The lymphocyte requires secondary signals (costimulation or help) in addition to occupancy of its antigen-specific receptor in order to make a positive response. In the absence of these signals, the lymphocytes become hyporesponsive (anergic) or die. In addition, there exists a lineage of dominant CD4+ regulatory cells called “natural T regulatory cells” (nTregs) that have been selected for recognition of self-antigens in the thymus and that can dampen down early immune responses if they include such ligands. Finally, even if the lymphocyte does make a positive response against an inappropriate antigen, it can often correct this mistake by inactivating further responses through negative feedback. This can result in the induction of unresponsiveness or alteration of the nature of the effector class of the response to prevent tissue destruction. Such immunoregulation can be mediated by new cells (such as induced Tregs) that dampen down immune responses with bystander suppression, or through cytokine release that selectively inhibits the generation of effector cells of a particular type such as interleukin (IL)–4 preventing the differentiation of T cells of the Th1 or Th17 phenotype. As long as the elicited immune response does not cause damage to the tissues, the organism is said to be tolerant. In this broad definition of tolerance, the equilibrium is viewed as a physiological state in which an intact immune system fails to react destructively against the individual that harbors it.

Anergy is a mechanism of T lymphocyte tolerance induced by antigen receptor stimulation in the absence of co-stimulation. Anergic T cells were shown to have a defect in antigen-induced transcription of the interleukin-2 gene. Analysis of the promoter indicated that the transcription factor AP-1 and its corresponding cis element were specifically down-regulated. Exposure of anergic T cells to interleukin-2 restored both antigen responsiveness and activity of the AP-1 element.

Three models have been proposed to explain the inability of T lymphocytes to respond to self-antigens (reviewed in 1): (a) self-reactive T cells are present but are prevented from functioning by suppressor T cells (suppression); (b) self-reactive T cells are present but have been functionally inactivated following interaction with host antigens (clonal anergy); and (c) self-reactive T cells are physically deleted (clonal deletion). A growing body of conclusive evidence indicates that clonal deletion is a major mechanism of tolerance induction for those antigens expressed in the thymus, the site of T cell development.2–5 It is difficult, however, to understand how this mechanism could account for tolerance to tissue-specific antigens, expressed in low amounts outside of the thymus. A potential resolution to this paradox may be found in recent studies that provide in vivo evidence for a nondeletional mechanism of clonal anergy6–8 that may operate outside of the thymus. The characteristics of the anergy observed in vivo are strikingly similar to those described for the induction of unresponsiveness in vitro for type 1 CD4+ T cell clones. Here we review our results on the induction of anergy in T cell clones and present new data on the mechanism by which it is maintained.

Nature Immunology 6, 472–480 (2005). On page 476, the top panel of Figure 5c was incorrect. The correct figure is provided here.

The thymic preference for CD4+ T cells over CD8+ T cells is often attributed to a default pathway favouring CD4+ T cells [1] or to homeostatic mechanisms [2,3]. It is also clear, however, that T-cell receptor (TCR) preferences for major histocompatibility complex (MHC) class I versus class II binding will strongly influence an individual clone's skewing to the CD4 or CD8 subset [4]. The variable region of each TCRα chain (Vα) studied to date is found to be overrepresented in either CD4+[5–7] or CD8+ cells [7–9], suggesting that each Vα element can interact more favourably with either MHC class I or class II molecules [10]. Indeed, TCRs appear to have an intrinsic ability to interact with MHC molecules [11,12], and single amino acid residues present in germline-encoded complementarity determining region 1 (CDR1) and CDR2 of the Vα element can be responsible for determining MHC specificity [13]. Interestingly, the degree of CD4/CD8 skewing is variable among different mouse strains [14,15] and in human populations [16]. Here, we have shown that polymorphism in CD4/CD8 skewing between B6 and BALB/c mice is determined by the stem cell genotype and not by environmental effects, and that it maps in or near the TCR α-chain complex, Tcra. This was confirmed by comparing Tcrab with Tcraa or Tcrac haplotypes in congenic mice. We propose that the array of Vα genes in various Tcra haplotypes exerts influence over the proportion of CD4 and CD8 subsets generated and may account in part for the observed thymic skewing. Thus, while it has been suggested that the TCR genes have been selected by evolution for MHC binding, our results further indicate selection for class II MHC preference.

In situ hybridization was used to investigate the expression of T cell receptor (TCR) alpha, beta, and gamma mRNAs in developing fetal and adult precursor thymocytes. gamma transcription was observed at the earliest time tested (day 12), followed by beta 12 h later, and TCR alpha on day 16. The early beta transcripts appeared to be from unrearranged or incompletely rearranged (D-J-C) beta loci. V beta region transcription was first detectable on day 14 and transcription of different V beta genes was induced at different times. These results delineate a schedule sequence of TCR gene activation, which begins within 1 d after entry of stem cells into the fetal thymus.