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In this issue of Immunity, Feyerabend et al. (2009) report that Delta-like 4, acting on Notch 1, prevents pro-T cells from differentiating into dendritic cells and B cells. In addition, in the absence of Notch 1, B cells in the thymus arose from a cell-extrinsic pathway.

DNA-labeling studies in alpha beta T cell receptor (TCR) transgenic mice show that the lifespan of immature CD4+8+ thymocytes is 3.5 days irrespective of whether they are selected for maturation or not. While nonselected cells die, the binding of the TCR to thymic major histocompatibility complex molecules rescues CD4+8+ cells from programmed cell death and induces first upregulation of the TCR level and then differentiation into CD4+8- or CD4-8+ cells in the absence of any cell division. When most CD4+8+ thymocytes express a selectable transgenic TCR the formation of mature cells with high TCR levels is 10-20 times as efficient as observed in normal mice, yet still only 20% of the CD4+8+ cells become mature. This is due to the limited availability of selecting 'niches': most CD4+8+ thymocytes with a selectable transgenic TCR will undergo maturation when they represent only 5% or less of all CD4+8+ cells.

ONE mechanism ensuring self tolerance of T cells is the clonal deletion of thymocytes bearing αβ T-cell receptors1–4. The stage of thymocyte development at which the interaction with antigen-presenting cells (APCs) leads to deletion, however, has not been determined directly. Indirect evidence suggests that intrathymic APCs induce deletion of CD4+8+ thymocytes3–6 (which die by apoptosis7) but deletion at less8 and more mature9 developmental stages has also been implied. It is also not clear if clonal elimination of thymocytes can be triggered by peripheral antigens carried on extrathymic APCs migrating through the thymus10. Here we show antigen-specific induction of apoptosis in CD4+8+ thymocytes cultured in suspension, by thymic as well as splenic APCs. Thus the recognition of antigen by CD4+8+ thymocytes may lead to deletion, suggesting that this is the central mechanism of tolerance induction, which is not limited by the antigen-presenting ability of the thymic stroma.

During their development, T cells are rescued from apoptotic cell death to follow distinct lineage fates. Recent data concerned with the role of the Notch transmembrane receptor in these events are interpreted to show that Notch promotes survival, but contrary to earlier reports has no function in lineage commitment.

Lymphocyte development has always attracted the interest of researchers looking at it either as an easily accessible model of lineage determination and cellular differentiation in higher organisms or as the basis for an understanding of the immune system as such. The present collection of reviews contains examples of both categories, without of course comprehensively covering this vast area of research.

Compelling evidence suggests that Foxp3-expressing CD25(+)CD4(+) regulatory T cells (Treg) are generated within the thymus as a separate lineage. However, Foxp3(+)CD4(+) Treg can also be generated de novo in a TGF-beta-dependent process from naive T cells by TCR triggering. Recently, we have shown that naturally occurring, but not in vitro TGF-beta-induced Foxp3(+) Treg display stable Foxp3 expression that was associated with selective demethylation of an evolutionarily conserved element within the Foxp3 locus named TSDR (Treg-specific demethylated region). Here, we report that inhibition of DNA methylation by azacytidine, even in absence of exogenous TGF-beta, not only promoted de novo induction of Foxp3 expression during priming, but also conferred stability of Foxp3 expression upon restimulation. Most notably, such stable Foxp3 expression was found only for cells displaying enhanced TSDR demethylation. In contrast, in vitro TSDR methylation diminished its transcriptional activity. Foxp3(+) Treg generated in vivo by DEC-205-mediated targeting of agonist ligands to dendritic cells showed long-term survival in the absence of the inducing antigen and exhibited efficient TSDR demethylation. Together, our data suggest that TSDR is an important methylation-sensitive element regulating Foxp3 expression and demonstrate that epigenetic imprinting in this region is critical for establishment of a stable Treg lineage.

Harald von Boehmer describes how he used mice transgenic for T cell receptor αβ to identify T cell receptor–dependent cellular selection and lineage commitment as mechanisms responsible for generating an effective and self-tolerant adaptive immune system.

alphabeta and gammadelta T cell lineages develop in the thymus from a common precursor. It is unclear at which stage of development commitment to these lineages takes place and in which way T cell receptor signaling contributes to the process. Recently, it was demonstrated that strong TCR signals favor gammadelta lineage development, whereas weaker TCR signals promote alphabeta lineage fate. Two models have been proposed to explain these results. The first model suggests that commitment occurs after TCR expression and TCR signaling directly instructs lymphocytes to adopt one or the other lineage fate. The second model suggests that commitment occurs before TCR expression and that TCR signaling merely confirms the lineage choice. By tracing the fate of single T cell precursors, this study shows that there is no commitment to either the alphabeta or gammadelta lineage before TCR expression and that modulation of TCR signaling in progeny of a single TCR-expressing cell changes lineage commitment.

Antigen-specific transplantation tolerance in the absence of immunosuppressive drugs is a rarely achieved goal. Immune responses to Y chromosome-encoded transplantation antigens (HY) can have life-threatening consequences in the clinic. Here, we have adopted a procedure developed in T cell antigen receptor (TCR)-transgenic mice to convert naïve T cells into male-specific Foxp3 + regulatory T cells (Tregs) in WT female mice. For this purpose, female mice were infused by osmotic minipumps with a single class II MHC-presented HY peptide and Tregs visualized by tetramer staining. As a result, animals developed Treg-mediated long-term tolerance to all HY transplantation antigens, irrespective of whether they were recognized by CD4 or CD8 T cells, on skin or hematopoietic grafts from male donors.

Some regulatory T cells express the Foxp3 transcription factor and such Tregs have an essential function of preventing autoimmune disease in man and mouse. Foxp3 binds to Forkhead motifs of about 1100 genes and the strength of binding increases when Foxp3-expressing T cells are stimulated by PMA and ionomycin. In Foxp3-expressing T cell hybridomas, Foxp3 binding to DNA does not lead to the activation or suppression of genes which becomes only visible after T cell activation. These findings are in line with observations by others that Foxp3 exerts important functions through association with T cell receptor-dependent transcription factors in a DNA-binding complex.

In each organism, the immune system must acquire the ability to distinguish self from nonself. Experiments in T cell receptor transgenic mice indicate that this process involves the selection of lymphocytes in the thymus.

We examined the immunohistological aspects of the H-Y specific T cell receptor (TcR) alpha/beta transgene expression in the thymus of male and female transgenic (Tg) mice. Virtually all thymocytes expressed the beta transgene in both the male and female thymus. Expression of accessory molecules (co-receptors) in Tg mice deviated from control mice. In the male Tg thymus, CD8 expression was either low or absent on both cortical and medullary thymocytes. In contrast, in the thymus of female mice, CD8+ cells were found both in the cortex and in the medulla. The majority of medullary thymocytes was bright CD8+. This is in clear contrast to the CD8 distribution in control B6 mice, where only a few percent of medullary cells are CD8+. Similarly, the proportion of cells expressing CD4 antigens was reduced in the cortex and medulla of the thymus from male Tg mice, as compared to the thymus of female Tg mice and B6 control mice. Comparative analysis of the stromal cell types of the thymic microenvironments in the three groups of mice revealed that the cortical thymic microenvironment of male Tg mice differed, compared to that of female Tg mice. In particular, the deep cortex showed a closely packed meshwork of epithelial reticular cells. Moreover, H-2Db molecules (which are the restricting elements for the Tg TcR alpha/beta) were abnormally expressed in the thymic cortex of male mice. The cortical microenvironment in female mice, on the other hand, appeared normal. Together, the data indicate that TcR alpha/beta transgene expression in male mice leads to an aberrant co-receptor expression in both cortical and medullary lymphoid cells as well as an abnormal composition of the cortical microenvironment. Both phenomena may be the consequence of "negative selection" of developing H-Y-specific T cells, as it occurs only in the male Tg thymus. The absence of the H-Y antigen, but presence of the restricting element H-2Db in the thymic cortex of female mice, leads to accumulation of CD8+ in the medulla, a phenomenon interpreted as "positive selection".

The ontogeny of T cells in T-cell receptor (TCR) transgenic mice, which express a transgenicαβheterodimer, specific for the male (H-Y) antigen in association with H-2Db, was determined. The transgenicαchain was expressed on about 10% of the fetal thymocytes on day 14 of gestation. About 50% of day-15 fetal thymocytes expressed bothαandβtranschains and virtually all fetal thymocytes expressed the transgenicαβheterodimer by day 17. The early expression of the transgenic TCR on CD4-8-thymocytes prevented the development ofγδcells, and led to accelerated growth of thymocytes and an earlier expression of CD4 and CD8 molecules. Up to day 17, no significant differences in T-cell development could be detected between female and male thymuses. By day 18 of gestation, the male transgenic thymus contained more CD4-8-thymocytes than the female transgenic thymus. The preponderance of CD4-8-thymocytes in the male transgenic thymus increased until birth and was a consequence of the deletion of the CD4+8+thymocytes and their CD4-8+precursors. By the time of birth, the male transgenic thymus contained half the number of cells as the female transgenic thymus. The deletion of autospecific precursor cells in the male transgenic mouse began only at day 18 of gestation, despite the fact that the ligand could already be detected by day 16.The preferential accumulation of CD4-8+T cells, which expressed a high density of the transgenic TCR, occurred only after birth and was .obvious in 6-week-old female thymus. These data support the hypothesis that the positive selection of T cells expressing this transgenic heterodimer may involve two steps, i.e., the commitment of CD4+8+thymocytes to the CD4-8+lineage following the interaction of the transgenic TCR with restricting major histocompatibility molecules, followed by a slow conversion of CD4+8+thymocytes into CD4-8+T cells.In normal mice, the precursors of CD+4+8 and single positive thymocytes have the CD4-8-CD3-J11d+(or M1/69+) phenotype. Because of the early expression of the transgenicαβheterodimer, this population was not detected in adult transgenic mice. All CD4-8-M1/ 69+cells expressed the transgenic receptor associated with CD3 and could be readily grown in media containing T-cell lectins and interleukin 2.

We demonstrate that all-trans retinoic acid (RA) induces FoxP3+ adaptive T regulatory cells (A-Tregs) to acquire a gut-homing phenotype (α4β7+ CC chemokine receptor 9+) and the capacity to home to the lamina propria of the small intestine. Under conditions that favor the differentiation of A-Tregs (transforming growth factor–β1 and interleukin 2) in vitro, the inclusion of RA induces nearly all activated CD4+ T cells to express FoxP3 and greatly increases the accumulation of these cells. In the absence of RA, A-Treg differentiation is abruptly impaired by proficient antigen presenting cells or through direct co-stimulation. In the presence of RA, A-Treg generation occurs even in the presence of high levels of co-stimulation, with RA attenuating co-stimulation from interfering from FoxP3 induction. The recognition that RA induces gut imprinting, together with our finding that it enhances A-Treg conversion, differentiation, and expansion, indicates that RA production in vivo may drive both the imprinting and A-Treg development in the face of overt inflammation.

At two checkpoints, T cell development is controlled by T cell receptor (TCR) signaling, which determines survival and lineage commitment. At the first of these checkpoints, signaling by the pre-TCR, the gammadeltaTCR or the alphabetaTCR has a major but nonexclusive impact on whether cells will become CD4-CD8- gammadelta or CD4+CD8+ alphabeta lineage cells. Pre-TCR signals synergize with moderate Notch signals to generate alphabeta lineage cells. Relatively strong signals by the gammadeltaTCR (or early expressed alphabetaTCR) in the absence of Notch signaling are sufficient to yield gammadelta lineage cells. However, relatively weak signals of the latter two receptors combined with strong Notch signaling result in the formation of alphabeta lineage cells that generate a diverse alphabetaTCR repertoire in pre-TCR-deficient mice. It remains to be determined whether TCR and/or Notch signals instruct or confirm predetermined lineage fate.

There is increasing evidence that agonist ligand presentation either intrathymically or extrathymically plays a crucial if not essential role in the generation of regulatory T cells (Tregs). Thus, it is possible to induce Tregs of any desired specificity in vivo. The same goal can be achieved in vitro by expanding antigen-specific CD4+ T cells and retrovirally transducing them. In contrast, in vitro expansion of Tregs is limited to antigens that have resulted in Treg generation in vivo. Antigen-specific Tregs can be used in cellular therapy with the goal to prevent autoimmune disease or even to interfere with established autoimmunity. The latter requires that the Tregs can suppress effector cells that have already caused harm, which is possible because of the antigen-dependent homing properties of Tregs, i.e. these cells can accumulate in antigen-draining lymph nodes and exit into inflamed tissue. Generally, the in vivo interference is dependent on cytokines such as transforming growth factor-beta and interleukin-10 that were dispensable in in vivo analysis of immunosuppression. The precise mechanisms of suppression remain enigmatic, however, but may be further elucidated by the molecular analysis of suppressed versus non-suppressed T cells.

Thymic precursors expressing the pre-T cell receptor (TCR), the gammadeltaTCR, or the alphabetaTCR can all enter the CD4+ 8+ alphabeta lineage, albeit with different efficacy. Here it is shown that proliferation and differentiation of precursors with the different TCRs into alphabeta lineage cells require Notch signaling at the DN3 stage of thymic development. At the DN4 stage, Notch signaling still significantly contributes to the generation of alphabeta T cells. In particular, in alphabeta lineage commitment, the pre-TCR synergizes more efficiently with Notch signals than the other two TCRs, whereas gammadeltaTCR-expressing cells can survive and expand in the absence of Notch signals, even though Notch signaling enhances their proliferation. These observations suggest a new model of alphabeta versus gammadelta lineage choice in which lineage fate is determined by the extent of synergy between TCR and Notch signaling and in which the evolutionarily recent advent of the cell-autonomously signaling pre-TCR increased the efficacy of alphabeta T cell generation.

The chromosomal translocation t(7;9) in human T-cell acute lymphoblastic leukaemia (T-ALL) results in deregulated expression of a truncated, activated form of Notch 1 (TAN1) under the control of the T-cell receptor-beta (TCRB) locus. Although TAN1 efficiently induces T-ALL in mouse models, t(7;9) is present in less than 1% of human T-ALL cases. The recent discovery of novel activating mutations in NOTCH1 in more than 50% of human T-ALL samples has made it clear that Notch 1 is far more important in human T-ALL pathogenesis than previously suspected.

Overexpression of intracellular Notch plays an important role in the generation of human acute lymphoblastic T cell leukemia (T-ALL). In mouse models, it was shown that Notch-dependent T-ALL required pre-TCR signaling. Here we show that pre-TCR signaling is required to condition mice for Notch-dependent transformation but that it is not required to sustain malignant growth of T-ALL. In contrast to previous studies, we found that disease development does not require pre-TCR but that it can be accelerated in Rag2-/- mice by transient mimicking of pre-TCR signals. (Blood. 2006;108:305-310)

In order to exploit regulatory T cells in a clinical setting it is desirable to be able to generate such cells by a variety of antigens that elicit unwanted immune responses. This goal has been achieved by targeting antigen to dendritic cells under subimmunogenic conditions which results in the conversion of naive Foxp3 negative T cells into Foxp3-expressing regulatory T cells that are indistinguishable from what has been referred to as natural Treg. Such cells have the ability to interfere with immunity at early as well as late stages of the immune response during which effector cells have already been formed. This suggests that Treg cannot only be exploited to prevent immune responses but also to interfere with already established immunity.

There is sufficient evidence to suggest that tumor growth elicits specific immune responses, including CD8(+) and CD4(+) T cell responses that may delay tumor growth and could potentially be harnessed to eradicate cancer. Nevertheless the frequent outcome of cancer is lethality associated with uncontrolled growth and dissemination of tumor cells. The failure of the immune response may be naturally programmed and related to a specific subpopulation of CD4(+)CD25(+) regulatory T cells, whose function is to protect us against autoimmunity. Recent investigations have shed light on the in vivo behavior and functions of these cells. It is becoming evident that a major impact of these cells is on the cytolytic action of specific CD8(+) T cells that target the tumor. Inhibition of cytotoxicity is dependent on TGF-beta signaling by the effector cells. Thus, targeting immune regulation may provide a promising approach to the immune therapy of cancer. This approach however could also have unexpected deleterious consequences, as surprising new observations indicate that regulatory T cells can also delay tumor growth by independent mechanisms that relate to their cross talk with the innate immune response to cancer.

Publisher Summary This chapter focuses on the generation of CD25+ suppressor cells by exogenous antigens in the fully mature immune system, as well as with their in vivo stability and function. Evidence suggests that CD25+ suppressor cells represent a suitable tool to induce antigen-specific immunologic tolerance in the fully mature immune system in the absence of general immune suppression, which often has undesired side effects ranging from increased risk of infection to development of life-threatening lymphoma. The fact that CD4+25+ FoxP3-expressing suppressor cells have an essential role in preventing the early onset of autoimmune disease in mammals, and the fact that these cells can be artificially induced through subimmunogenic presentation of TCR agonist ligands, opens new possibilities to exploit these cells to induce specific tolerance in the fully mature immune system. This may become a powerful tool in the prevention of allergies and transplant rejection. Inducing such cells by organ-specific antigens, may become an effective means to prevent autoimmunity in patients. However, such attempts have had success in animal models of disease but not in clinical trials.

At two consecutive ‘checkpoints' in their development, T cells have to be rescued from programmed cell death and to choose between distinct lineage fates; recent results show that the Notch transmembrane receptor can significantly influence T-cell development at both of these points.

Today immunologists are well aware of positive and negative selection of developing T cells. This was very different almost three decades ago when MHC-restricted Ag recognition by T cells represented an enormous puzzle in the absence of data on the molecular nature of Ag recognition by T cells. It

Targeting of antigens to immature dendritic cells has been shown to result in antigen-specific T-cell tolerance in vivo. In the INS-HA/TCR-HA transgenic mouse model for type 1 diabetes, we tested the potential of the dendritic cell-specific monoclonal antibody DEC-205 conjugated to the hemagglutinin (HA) antigen (DEC-HA) to prevent disease onset. Whereas untreated INS-HA/TCR-HA mice all develop insulitis, and approximately 40% of these mice become diabetic, repeated injection of newborn mice with DEC-HA protected almost all mice from disease development. Histological examination of the pancreata revealed significant reduction of peri-islet infiltrations in DEC-HA-treated mice, and the islet structure remained intact. Moreover, HA-specific CD4+ T-cells from anti-DEC-HA-treated INS-HA/TCR-HA mice exhibited increased expression of Foxp3, cytotoxic T-lymphocyte-associated antigen-4, and the immunosuppressive cytokines interleukin-10 and transforming growth factor-beta. The findings indicate that targeting of the HA antigen to immature dendritic cells in vivo leads to a relative increase of antigen-specific Foxp3+ regulatory T-cells that suppress the development of type 1 diabetes. Our results provide a basis for the development of novel strategies focusing on prevention rather than treatment of autoimmune diseases.

Evidence suggests that regulatory T cells expressing the transcription factor Foxp3 develop extrathymically and intrathymically. Mechanisms of extrathymic induction require further scrutiny, especially as proliferation and/or phenotypic changes of preexisting suppressor cells must be distinguished from true de novo generation. Here we report the conversion of truly naive CD4(+) T cells into suppressor cells expressing Foxp3 by targeting of peptide-agonist ligands to dendritic cells and by analysis of Foxp3 expression at the level of single cells. We show that conversion was achieved by minute antigen doses with suboptimal dendritic cell activation. The addition of transforming growth factor-beta or the absence of interleukin 2 production, which reduces proliferation, enhanced the conversion rate. In addition, regulatory T cell populations induced in subimmunogenic conditions could subsequently be expanded by delivery of antigen in immunogenic conditions. The extrathymic generation and proliferation of regulatory T cells may contribute to self-tolerance as well as the poor immunogenicity of tumors and may be exploited clinically to prevent or reverse unwanted immunity.

CD4+CD25+ T-cells can be used to interfere with spontaneous autoimmune diseases such as type 1 diabetes. However, their low frequency and often unknown specificity represent major obstacles to their therapeutic use. Here we have explored the fact that ectopic expression of the transcription factor Foxp3 can confer a suppressor phenotype to naïve CD4+ T-cells. We found that retroviral transduction of polyclonal CD4 T-cells with FoxP3 was not effective in interfering with established type 1 diabetes. Thus, more subtle and more organ-specific regulation might be required to prevent type 1 diabetes, as well as to avoid systemic immunosuppression. However, a single injection of 105 FoxP3-transduced T-cells with specificity for islet antigen stabilized and reversed disease in mice with recent-onset diabetes. By comparing FoxP3-transduced T-cells with various antigen specificities, it became clear that the in vivo effect correlated with specific homing to and activation in pancreatic lymph nodes and not with in vitro suppressor activity or cytokine production. Our results complement recent results on in vitro–amplified antigen-specific T-cells in ameliorating type 1 diabetes and suggest that FoxP3 transduction of expanded T-cells might achieve the same goal.

Although autoimmune diseases can be initiated by immunization with a single antigen, it is not clear whether a single self antigen is essential for the initiation and, perhaps, the perpetuation of spontaneous autoimmunity. Some studies have suggested that insulin may represent an essential autoantigen in type 1 diabetes. Here we show that unlike tolerance to glutamic acid decarboxylase, tolerance to transgenically overexpressed preproinsulin 2 substantially reduced the onset and severity of type 1 diabetes in nonobese diabetic mice. However, some mice still developed type 1 diabetes, suggesting that insulin is a key, but not absolutely essential, autoantigen. The results are consistent with the idea that the human IDDM2 locus controls susceptibility to type 1 diabetes by regulating intrathymic preproinsulin expression.

The induction of antigen-specific tolerance in the mature immune system of the intact organism has met with limited success. Therefore, nonspecific immunosuppression has been the treatment of choice to prevent unwanted immunity. Here, it is shown that prolonged subcutaneous infusion of low doses of peptide by means of osmotic pumps transforms mature T cells into CD4+25+ suppressor cells that can persist for long periods of time in the absence of antigen and confer specific immunologic tolerance upon challenge with antigen. The described procedure resembles approaches of tolerance induction used decades ago, induces tolerance in the absence of immunity, and holds the promise to become an effective means of inducing antigen-specific tolerance prospectively, whereas its power to suppress already ongoing immune responses remains to be determined.

The 89th Boehringer Ingelheim Fonds International Titisee Conference—‘From Allergy to Cancer: New Perspectives for Therapeutic Vaccination’—took place between 17 and 21 March 2004 and was organized by R. Valenta and T. Brocker. The meeting brought together a diverse spectrum of scientists concerned with understanding the function of individual immune system components and exploiting this basic knowledge to manipulate the immune response. ![][1] The immune system has been evolutionarily selected such that it can execute wanted immune reactions and prevent unwanted immunity. There has been steady progress in understanding the essential role of the various mechanisms that result in immunity—or the lack thereof—and this knowledge is beginning to bear fruit as shown by the successful manipulation of immunity when natural processes fail. One key component of the immune system are the lymphocytes, each of which is equipped with a unique antigen‐binding receptor that is carefully selected in order to match the needs of the individual organism. Developing T lymphocytes are screened for the specificity of their randomly generated receptors through processes termed positive and negative selection in the thymus (hence the name T lymphocytes; von Boehmer et al , 2003). This not only eliminates (by inducing apoptotic cell death) cells with receptors that are specific for self‐antigens, but also positively selects (by avoiding premature death) cells that are able to detect pieces of foreign proteins when presented by the individual's own major histocompatibility (MHC)‐encoded molecules. Class II MHC proteins present peptides from proteins that are endocytosed (such as bacterial proteins), whereas class I MHC molecules present peptides from proteins that are made inside the cell (such as viral proteins). There is also a process named cross‐presentation through which proteins from a variety of different cells can be taken up and presented by class II or class I MHC molecules of antigen presenting cells … [1]: /embed/graphic-1.gif

CD4+CD25+ regulatory T cells (Treg cells) control immune responsiveness to a large variety of antigens. The isolation and therapeutic manipulation of Treg cells requires the use of reliable surface receptors that are selectively up-regulated in Treg cells. On the basis of global gene expression studies, we identified neuropilin-1 (Nrp1) as a specific surface marker for CD4+CD25+ Treg cells. Nrp1, a receptor involved in axon guidance, angiogenesis, and the activation of T cells, is constitutively expressed on the surface of CD4+CD25+ Treg cells independently of their activation status. In contrast, Nrp1 expression is down-regulated in naive CD4+CD25- T cells after TCR stimulation. Furthermore, CD4+Nrp1(high) T cells express high levels of Foxp3 and suppress CD4+CD25- T cells. Thus, Nrp1 constitutes a useful surface marker to distinguish Treg cells from both naive and recently activated CD4+CD25+ non-regulatory T cells.

Publisher Summary This chapter focuses on T-cell development with special emphasis on antigen receptor-controlled differentiation programs. Development of T cells is tightly associated with the adaptation of adaptive immune system to self major histocompatibility complex (MHC)-peptide complexes. Shortly, before T-cell development could be approached by molecular techniques, hypotheses on the control of development correctly predicted positive selection that results in the alignment of functional commitment and receptor specificity of mature T cells and negative selection as essential component of self–nonself discrimination. Immunologists who have a greater interest in immune function and self–nonself discrimination by the immune system find themselves confronted with exciting questions concerning the regulation of ectopic antigen expression and its impact on negative selection, as well as new aspects of immunoregulation carried out by another lineage of CD4 + /CD25 + T cells. The interplay of the innate and adaptive immune system has come much more into focus with the recognition of lineages of T cells that are selected by nonclassical MHC molecules.

Experiments in nonobese diabetic (NOD) mice that lacked expression of glutamic acid decarboxylase (GAD) in β cells have suggested that GAD represents an autoantigen essential for initiating and maintaining the diabetogenic immune response. Several attempts of inducing GAD-specific recessive tolerance to support this hypothesis have failed. Here we report on successful tolerance induction by expressing a modified form of GAD under control of the invariant chain promoter resulting in efficient epitope display. In spite of specific tolerance insulitis and diabetes occurred with normal kinetics indicating that GAD is not an essential autoantigen in the pathogenesis of diabetes.

Spontaneous tumors frequently express antigens that can be recognized by the immune system but nevertheless manage to evade immune surveillance. To better understand the mechanism of evasion, we followed CD8 and CD4 T cells reacting against a subcutaneously growing tumor, modified to express influenza hemagglutinin (HA) as surrogate tumor antigen. Adoptive transfer of 8,000 antigen-specific CD8 T cells was sufficient to protect against challenge with 1x10(6) tumor cells, while larger numbers of T cells rejected established tumors. HA-specific CD4 T cells could not reject tumors on their own but helped rejection by CD8 T cells. Rejection of the tumor coincided with prolonged survival of expanded antigen-specific CD8 and CD4 T cells, while a failing anti-tumor response was accompanied by transient expansion followed by rapid elimination of antigen-specific T cells. Thus, a highly immunogenic tumor can evade immune surveillance because of an insufficient number of tumor-specific T cells and antigen overload, resulting in exhaustion of the immune response. In this scenario, adoptive immunotherapy rather than vaccination promises successful treatment.

Humans expressing a defective form of the transcription factor AIRE (autoimmune regulator) develop multiorgan autoimmune disease. We usedaire- deficient mice to test the hypothesis that this transcription factor regulates autoimmunity by promoting the ectopic expression of peripheral tissue– restricted antigens in medullary epithelial cells of the thymus. This hypothesis proved correct. The mutant animals exhibited a defined profile of autoimmune diseases that depended on the absence of aire in stromal cells of the thymus. Aire-deficient thymic medullary epithelial cells showed a specific reduction in ectopic transcription of genes encoding peripheral antigens. These findings highlight the importance of thymically imposed “central” tolerance in controlling autoimmunity.

T cell receptor agonists can induce the differentiation of regulatory T (T(R)) cells. We report here that the immunoglobulin kappa-controlled expression of an agonist in different cell types correlated with the phenotype of the generated T(R) cells. We found that aberrant expression on thymic stroma yielded predominantly CD4(+)CD25(+) T(R) cells, which--under physiological conditions--may be induced by ectopically expressed organ-specific antigens and thus prevent organ-specific autoimmunity. Expression of the agonist antigen by nonactivated hematopoietic cells produced mostly CD4(+)CD25(-) T(R) cells. This subset can be derived from mature monospecific T cells without "tutoring" by other T cells and can be generated in the absence of a functioning thymus. Suppression of CD4(+) T cell proliferative responses by both CD25(+) and CD25(-) subsets was interleukin 10 (IL-10) independent and was overcome by IL-2. These data suggest that distinct pathways can be exploited to interfere with unwanted immune responses.

The pre-T cell receptor (TCR) signals constitutively in the absence of putative ligands on thymic stroma and signal transduction correlates with translocation of the pre-TCR into glycolipid-enriched microdomains (rafts) in the plasma membrane. Here, we show that the pre-TCR is constitutively routed to lysosomes after reaching the cell surface. The cell-autonomous down-regulation of the pre-TCR requires activation of the src-like kinase p56(lck), actin polymerization, and dynamin. Constitutive signaling and degradation represents a feature of the pre-TCR because the gammadeltaTCR expressed in the same cell line does not exhibit these features. This is also evident by the observation that the protein adaptor/ubiquitin ligase c-Cbl is phosphorylated and selectively translocated into rafts in pre-TCR- but not gammadeltaTCR-expressing cells. A role of c-Cbl-mediated ubiquitination in pre-TCR degradation is supported by the reduction of degradation through pharmacological inhibition of the proteasome and through a dominant-negative c-Cbl ubiquitin ligase as well as by increased pre-TCR surface expression on immature thymocytes in c-Cbl-deficient mice. The pre-TCR internalization contributes significantly to the low surface level of the receptor on developing T cells, and may in fact be a requirement for optimal pre-TCR function.

Signals that emanate from the pre-T cell receptor (pre-TCR) regulate multiple processes required for the development of the alphabeta T cell lineage. In contrast to the gammadelta TCR, the pre-TCR localizes cell-autonomously to membrane rafts, where it appears to signal in a constitutive and ligand-independent manner. We addressed here the role played by structural features specific to the cytoplasmic domain of the pre-TCRalpha chain (pTalpha). More specifically, we examined a COOH-terminal proline-rich sequence that might play a role in signal transduction and a juxtamembrane cysteine residue that could be a target for palmitoylation, thus allowing spontaneous raft localization. Expression of pTalpha mutants in transgenic mice, retrovirally transduced T cell precursors and cell lines showed that the pTalpha cytoplasmic tail, in particular the proline-rich domain, plays a crucial role in pre-TCR signaling and T cell development. In contrast, the pTalpha juxtamembrane cysteine appeared to be dispensable for pre-TCR function.

Recent studies provide fresh insight into the mechanisms by which precursor cells are committed to and develop within the T-lymphocyte lineage. Precursor/product studies have identified developmental stages between that of the pluripotent hematopoietic stem cell and thymocytes committed to the T lineage. Specific ligands and signaling pathways interacting with the Notch-1 receptor and its ability to influence commitment within the lymphoid lineage have been described. Although the structural features or putative ligands endowing the pre-TCR with constitutive signaling capacity remain elusive, numerous distal mediators of pre-TCR signaling have been identified. It remains for the future to determine what roles they may have in survival, proliferation, lineage commitment and allelic exclusion of TCR genes. Receptor editing and lineage commitment of αβ T cells still represent controversial topics that need further study.

Type 1 diabetes is an organ-specific autoimmune disease that is mediated by autoreactive T cells. We show here that administration of a soluble dimeric peptide-major histocompatibility complex (pMHC) class II chimera (DEF) to prediabetic double-transgenic mice prevents the onset of disease or, in animals that are already diabetic, restores normoglycemia. The antidiabetogenic effects of DEF rely on the induction of anergy in splenic autoreactive CD4+ T cells via alteration of early T cell receptor signaling and stimulation of interleukin 10-secreting T regulatory type 1 cells in the pancreas. Soluble dimeric pMHC class II may be useful in the development of immunospecific therapies for type 1 diabetes.

Gut intraepithelial CD8 T lymphocytes (T-IEL) are distinct from thymus-derived cells and are thought to derive locally from cryptopatch (CP) precursors. The intermediate stages of differentiation between CP and mature T-IEL were not identified, and the local differentiation process was not characterized. We identified and characterized six phenotypically distinct lineage-negative populations in the CP and the gut epithelium: (a) we determined the kinetics of their generation from bone marrow precursors; (b) we quantified CD3-epsilon, recombination activating gene (Rag)-1, and pre-Talpha mRNAs expression at single cell level; (c) we characterized TCR-beta, -gamma, and -alpha locus rearrangements; and (d) we studied the impact of different mutations on the local differentiation. These data allowed us to establish a sequence of T cell precursor differentiation in the gut. We also observed that the gut differentiation varied from that of the thymus by a very low frequency of pre-Talpha chain mRNA expression, a different kinetics of Rag-1 mRNA expression, and a much higher impact of CD3 epsilon/delta and pre-Talpha deficiencies. Finally, only 3% of CP cells were clearly involved in T cell differentiation, suggesting that these structures may have additional physiological roles in the gut.

The present study documents that stabilization of beta-catenin is sufficient to induce lesions reminiscent of prostate intraepithelial neoplasia (PIN). Such lesions were present in all compound mutant mice and all prostate acini expressing stabilized beta-catenin. High grade PIN-like lesions resembling early human prostate cancer were detected as early as 10 weeks of age. Surprisingly, stabilization of beta-catenin in other secretory epithelia including salivary, preputial, harderian, and mammary glands induced extensive squamous metaplasia and keratinization associated with terminal differentiation of the target cells, but failed to cause neoplastic transformation. Epidermal hyperplasia, hair follicle cysts, and odontomas were also observed. The prostatic lesions exhibited upregulation of c-myc, increased rate of cellular proliferation, loss of the Na-K-Cl co-transporter NKCC1, and expression of androgen receptor. Basal cell markers such as p63 and keratin 5 were not expressed by the masses of PIN-like lesions, but were present in small foci of proliferating beta-catenin expressing basal cells. Our observations indicate that beta-catenin stabilization is a crucial event for the initiation of PIN-like lesions, but induces squamous metaplasia rather than tumorigenesis in secretory epithelia other than the prostate.

Mammary anlagen are formed in the embryo as a derivative of the epidermis, a process that is controlled by Lef-1 and therefore possibly by β-catenin. To investigate the role of β-catenin signaling in mammary alveolar epithelium, we have stabilized endogenous β-catenin in differentiating alveolar epithelium through the deletion of exon 3 (amino acids 5–80) of the β-catenin gene. This task was accomplished in mice carrying a floxed β-catenin gene and a Cre transgene under control of the mammary-specific whey acidic protein (WAP) gene promoter or the mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Stabilized β-catenin was obtained during the first pregnancy, and its presence resulted in the dedifferentiation of alveolar epithelium followed by a transdifferentiation into epidermal and pilar structures. Extensive squamous metaplasia, but no adenocarcinomas, developed upon β-catenin activation during pregnancy and persisted throughout involution. These data demonstrate that the activation of β-catenin signaling induces a program that results in loss of mammary epithelial cell differentiation and induction of epidermal structures.

It has been suggested that anergic T cells may not be only inert cells but may rather play an active role, for example by regulating immune responses. We have previously reported the existence of “anergic” IL-10-producing CD4+T cells generatedin vivoby continuous antigenic stimulation. Using a gene transfer system where the antigen recognized by such T cells is expressed in skeletal muscle by two different DNA viral vectors, we show that these cells not only remain tolerant toward their cognate antigen but also can suppress the immune response of naïve T cells against the immunogenic adenoviral proteins. Furthermore, they can completely inhibit tissue destruction that takes place as a result of an immune response. The system presented here is unique in that the T cells have been anergizedin vivo, their antigen specificity and functional status are known, and the amount, form, and timing of antigen expression can be manipulated. This model will therefore permit us to carefully dissect the mechanisms by which these anergic T cells regulate the priming and/or effector function of naïve T cells.

Pre-T cell antigen receptor (pre-TCR) signaling plays a crucial role in the development of immature T cells. Although certain aspects of proximal pre-TCR signaling have been studied, the intermediate signal transducers and the distal transcription modulators have been poorly characterized. We report here a correlation between pre-TCR signaling and a biphasic rise in the cytosolic Ca2+ concentration. In addition, we show that constitutive pre-TCR signaling is associated with an increased rate of Ca2+ influx through store-operated plasma membrane Ca2+ channels. We show also that the biphasic nature of the observed pre-TCR-induced rise in cytosolic Ca2+ differentially modulates the activities of the transcription factors NF-kappaB and NFAT in developing T cells.

Concepts of specific immunotolerance were first introduced after the clonal selection theory gained acceptance. In early days Burnet and Lederberg formulated the simple concept that there was a fundamental difference between immature and mature, antigen-receptor bearing lymphocytes such that binding of antigen by the former would result in cell death, while binding to the latter would result in gain of effector function. At that time it was not known that immunocytes developed in distinct anatomical sites and thus would perhaps not encounter all self antigens as if they were circulating in the organism like mature lymphocytes. Even then this concept would fail to deal with proteins expressed only in adulthood. Nevertheless, this was the first theory of recessive tolerance that gained experimental support and by now is an established fact. A variant of that theme is the recently discovered receptor-deletion whereby immature B but not T cells change their receptors when confronted with antigen, perhaps because the antigen keeps the rearrangement process ongoing whereby already productive genes are deleted and new ones are being generated. Thus we have clonal or cellular deletion as well as receptor editing of immature lymphocytes as possible mechanism that affect immature lymphocytes whereby unresponsiveness is achieved without prior activation to effector function i.e. cytokine secretion, cytolytic activity or antibody production.