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SummaryD-cyclins represent components of cell cycle machinery. To test the efficacy of targeting D-cyclins in cancer treatment, we engineered mouse strains that allow acute and global ablation of individual D-cyclins in a living animal. Ubiquitous shutdown of cyclin D1 or inhibition of cyclin D-associated kinase activity in mice bearing ErbB2-driven mammary carcinomas triggered tumor cell senescence, without compromising the animals’ health. Ablation of cyclin D3 in mice bearing Notch1-driven T cell acute lymphoblastic leukemias (T-ALL) triggered tumor cell apoptosis. Such selective killing of leukemic cells can also be achieved by inhibiting cyclin D associated kinase activity in mouse and human T-ALL models. Inhibition of cyclin D-kinase activity represents a highly-selective anticancer strategy that specifically targets cancer cells without significantly affecting normal tissues.

Fopx3(+) Treg safeguard against autoimmune diseases and immune pathology. The extrathymic conversion of naïve T cells into Foxp3(+) regulatory T cells can be achieved in vivo by the delivery of strong-agonist ligands under subimmunogenic conditions. Tolerogenic vaccination with strong-agonist mimetopes of self-antigen to promote self-antigen specific tolerance may represent the most specific and safest means of preventing autoimmunity. This review discusses the requirements for induction of dominant tolerance exerted by Foxp3(+) Tregs in autoimmunity with special emphasis on their impact to interfere with T1D. The future goals are the understanding of self-non-self discrimination at the cellular and molecular level, which should then enable investigators to develop clinical vaccination protocols that specifically interfere with unwanted immune responses.

Two major T lymphocyte lineages--alphabeta and gammadelta T cells--develop in the thymus from common precursors. Differentiation of both lineages requires signals coming from TCRs. Development of alphabeta T cells is driven at early stages by signaling from the pre-TCR, most likely in a ligand-independent fashion, and later--by signals delivered by alphabetaTCRs binding to their ligands--classical or non-classical MHC molecules. gammadelta lineage cells likewise require TCR signaling for their differentiation. Recent work from several groups suggests that TCR signaling not only ensures the developmental progression towards alphabeta and gammadelta lineages but that signal strength instructs lineage fate: weaker TCR signal results in alphabeta and stronger--in gammadelta lineage commitment. However, as most gammadeltaTCRs remain orphan receptors, it is still debated whether strong signals from gammadeltaTCRs in development are generated in a ligand-dependent manner (as in the case of alphabetaTCRs), ligand-independent manner (as for pre-TCR) or both. Here we summarize evidence supporting a possible role for ligands in gammadelta T cell lineage commitment and the generation of gammadelta sublineages.

αβ and γδ T cells develop in the thymus from a common precursor. Although lineages initially were defined by the type of TCR they express, it soon became clear that the TCR type per se does not play a deterministic role in the lineage decision, since in various transgenic and knockout models, as well as in a small fraction of cells in wt mice, the TCRγδ can drive the differentiation of αβ lineage cells and the TCRαβ can drive differentiation of γδ lineage cells. Thus until recently it was unclear what determines lineage choice and at which stage the two lineages diverge. Recent observations suggest that TCR signal strength determines lineage fate and that lineage choice is made at or shortly after the first TCR-controlled checkpoint. While it is clear that the decision between αβ and γδ lineages is made at the first TCR-controlled checkpoint and the αβ sublineages split off later, it is less clear whether γδ sublineages divert already at the first TCR-controlled checkpoint or later. Recent experiments support the former view.

Background & Aims: Disruption of the enteric glial cell (EGC) network is an early pathologic feature in Crohn's disease. To determine the contribution of antigen-specific CD8 and CD4 T cells to the breakdown of the EGC network, we studied specific autoimmune targeting of an ectopic antigen expressed by EGCs. Methods: Transgenic mice (GFAP-HA), which express the influenza hemagglutinin (HA) in EGCs, were either crossed with mice transgenic for a T-cell receptor (TCR) specific for a major histocompatibility complex (MHC) class I epitope of HA (CL4-TCR) or were adoptively transferred with conventional CL4 T cells. These were compared with GFAP-HA mice transferred with conventional T cells specific for an MHC class II epitope of HA (6.5). Results: Both CD8 and CD4 T-cell subtypes were activated in vivo in an antigen-specific manner; however, they differed substantially in their ability to expand in the mesenteric lymph nodes, trigger proinflammatory cytokines, and induce autoimmune damage in the intestine. Direct presentation of antigen, provided by lymph node stromal cells, caused the activation and deletion of CD8 T cells. This mechanism of T-cell tolerance did not affect CD4 T cells, which produced antigen-specific lethal autoimmunity. Conclusions: Our observations support a recently identified mechanism of peripheral T-cell tolerance that specifically protects against autoimmunity mediated by conventional CD8 T cells. Furthermore, we show that conventional CD4 T cells are not affected by this mechanism of tolerance, and their targeting of EGCs produces lethal intestinal autoimmunity.

T cells developing in the thymus are derived from hematopoietic stem cells (HSCs) in the bone marrow (BM). Understanding the developmental steps linking multipotent HSCs to intrathymic T lineage-committed progenitors is important for understanding cancer in T lineage cells, improving T cell reconstitution after BM transplantation, and designing gene-therapy approaches to treat defective T cell development or function. Such an understanding may also help ameliorate immunological defects in aging. This review covers the differentiation steps between HSCs and committed T cell progenitors within the thymus.

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 γδTCR or the αβTCR has a major but nonexclusive impact on whether cells will become CD4 − CD8 − γδ or CD4 + CD8 + αβ lineage cells. Pre-TCR signals synergize with moderate Notch signals to generate αβ lineage cells. Relatively strong signals by the γδTCR (or early expressed αβTCR) in the absence of Notch signaling are sufficient to yield γδ lineage cells. However, relatively weak signals of the latter two receptors combined with strong Notch signaling result in the formation of αβ lineage cells that generate a diverse αβTCR repertoire in pre-TCR-deficient mice. It remains to be determined whether TCR and/or Notch signals instruct or confirm predetermined lineage fate.

Summary Mechanisms of dominant tolerance have evolved within the mammalian immune system to prevent inappropriate immune responses. CD4 + CD25 + regulatory T (T reg ) cells have emerged as central constituents of this suppressive activity. By using multiphoton intravital microscopy in lymph nodes (LNs) of anesthetized mice, we have analyzed how cytotoxic T lymphocytes (CTLs) interact with antigen-presenting target cells in the presence or absence of activated T reg cells. Nonregulated CTLs killed their targets at a 6.6-fold faster rate than regulated CTLs. In spite of this compromised effector activity, regulated CTLs exhibited no defect in proliferation, induction of cytotoxic effector molecules and secretory granules, in situ motility, or ability to form antigen-dependent conjugates with target cells. Only granule exocytosis by CTLs was markedly impaired in the presence of T reg cells. This selective form of regulation did not require prolonged contact between CTLs and T reg cells but depended on CTL responsiveness to transforming growth factor-β. CTLs quickly regained full killing capacity in LNs upon selective removal of T reg cells. Thus, T reg cells reversibly suppress CTL-mediated immunity by allowing acquisition of full effector potential but withholding the license to kill.

Signals transduced by Notch receptors are indispensable for T cell specification and differentiation of alphabeta T lineage cells. However, the role of Notch signals during alphabeta versus gammadelta T lineage decision remains controversial. Here, we addressed this question by employing a clonal analysis of CD4(-)CD8(-) (DN) progenitor potential to position the divergence of alphabeta and gammadelta T cell lineages to the late DN2 to DN3 developmental stages. Accordingly, alphabeta and gammadelta precursor frequencies within these T cell progenitor subsets were determined, both in the presence and absence of Notch signaling through Delta-like 1. Notch signals were found to be critical for the DN to CD4(+)CD8(+) (DP) transition, irrespective of the identity (pTalphabeta or gammadelta) of the inducing T cell receptor complex, whereas gammadelta T cells developed from gammadeltaTCR-expressing T cell progenitors in the absence of further Notch ligand interaction. Collectively, our findings demonstrate a differential, stage-specific requirement for Notch receptor-ligand interactions in the differentiation of alphabeta and gammadelta T cells from T cell progenitors.

Differentiation of hematopoietic cells from multipotential progenitors is regulated by multiple growth factors and cytokines. A prominent feature of these soluble factors is promotion of cell survival, in part mediated by expression of either of the anti-apoptotic proteins, BCL-2 and BCL-XL. The complex expression pattern of these frequently redundant survival factors during hematopoiesis may indicate a role in lineage determination. To investigate the latter possibility, we analyzed factor-dependent cell-Patersen (FDCP)-Mix multipotent progenitor cells in which we stably expressed BCL-2 or BCL-XL. Each factor maintained complete survival of interleukin-3 (IL-3)-deprived FDCP-Mix cells but, unexpectedly, directed FDCP-Mix cells along restricted and divergent differentiation pathways. Thus, IL-3-deprived FDCP-Mix BCL-2 cells differentiated exclusively to granulocytes and monocytes/macrophages, whereas FDCP-Mix BCL-XL cells became erythroid. FDCP-Mix BCL-2 cells grown in IL-3 were distinguished from FDCP-Mix and FDCP-Mix BCL-XL cells by a striking reduction in cellular levels of Raf-1 protein. Replacement of the BCL-2 BH4 domain with the related BCL-XL BH4 sequence resulted in a switch of FDCP-Mix BCL-2 cells to erythroid fate accompanied by persistence of Raf-1 protein expression. Moreover, enforced expression of Raf-1 redirected FDCP-Mix BCL-2 cells to an erythroid fate, and prohibited generation of myeloid cells. These results identify novel roles for BCL-2 and BCL-XL in cell fate decisions beyond cell survival. These effects are associated with differential regulation of Raf-1 expression, perhaps involving the previously identified interaction between BCL-2-BH4 and the catalytic domain of Raf-1.

Peripheral T cell tolerance may result from activation-induced cell death [1], anergy [1], and/or immune response modulation by regulatory T cells [2] . In mice that express a transgenic receptor specific for peptide 111–119 of influenza hemagglutinin presented by E d class II MHC molecules as well as hemagglutinin under control of the immunoglobulin-κ promoter, we have found that anergic T cells [3] can also have immunoregulatory function and secrete IL-10 [4]. In order to obtain information on molecular mechanisms involved in anergy and immunoregulation, we have compared expression levels of 1176 genes in anergic, naive, and recently activated CD4 + T cells of the same specificity by gene array analysis. The results provide a plausible explanation for the anergic phenotype in terms of proliferation, provide new information on the surface phenotype of in vivo-generated anergic CD4 + T cells, and yield clues with regard to new candidate genes that may be responsible for the restricted cytokine production of in vivo-anergized CD4 + T cells. The molecular fingerprints of such T cells should enable the tracking of this small population in the normal organism and the study of their role in immunoregulation.

Summary The development and function of lymphocytes depend upon their precise migration in response to chemoattractant cytokines, or chemokines. Two recent reports suggest that, during thymic β-selection, the binding of the chemokine CXCL12 to the receptor CXCR4 on thymocytes provides not only directional but also developmental cues.

The construction of various gene-deficient mice has facilitated the understanding of the role of various receptors and signaling pathways that control the generation of alphabeta lineage cells. A predominant role is occupied by the pre-TCR, which not only generates large numbers of alphabeta lineage cells but also controls TCRbeta allelic exclusion as well as commitment to the gammadelta lineage versus the alphabeta lineage.

Organ-specific autoimmune disease can be caused by αβ T cells that have escaped self-tolerance induction. Here we show that one of the causes of escape from self-tolerance is the coexpression of two different T cell receptors by the same cell, which can occur in up to 30% of all T cells in normal mice and can lead to low-level surface expression of an autospecific TCR. We found that double receptor–expressing T cells can escape tolerance even to ubiquitously expressed antigens but can nevertheless induce autoimmune diabetes when the relevant protein is expressed in pancreatic tissue. Such diabetogenic T cells are absent, however, among T cells expressing the autospecific TCR as the sole receptor.

The development of pre-T-cells with productive T-cell receptor beta (TCR beta) rearrangements can be furthered by each of the pre-T-cell receptors (pre-TCR), the alpha beta TCR as well as the gamma delta TCR, albeit by distinct mechanisms. While the gamma delta TCR affects CD4-8- precursor cells irrespective of their TCR beta rearrangement status both the pre-TCR and the alpha beta TCR select only cells with productive TCR beta genes for expansion and maturation. The alpha beta TCR is much less effective than the pre-TCR because of the paucity of TCR alpha proteins in TCR beta positive precursors.

The vast majority of T lymphocytes, with the exception of gut-associated, intraepithelial lymphocytes, differentiate and mature inside the thymus. Early T cell development is characterized by expansion and differentiation of thymocytes which do not yet express mature TCRs on their cell surface. Important events in early thymocyte development are controlled by a pre-TCR complex consisting of a conventional TCR beta chain and a novel transmembrane protein termed pre-TCR alpha (p T alpha chain) which are noncovalently associated with components of CD3. Recent studies of pre-TCR function have led to a better understanding of the molecular events in early thymocyte development.

In peripheral lymphoid tissues of TCR transgenic mice that express the nominal antigen (HY peptide plus H-2Db MHC) recognized by the transgenic TCR, there exist unusual CD4-CD8- and CD4-CD8low cells bearing the transgenic TCR. Here we show that, unlike TCR alpha beta T cells that are generated in the absence of nominal antigen, these unusual cells do not express endogenous TCR alpha genes, have maintained the TCR delta locus on both chromosomes, and can coexpress TCR alpha beta and TCR gamma delta chains on the cell surface. The latter is also true for CD4-CD8-, HSA+ TCR alpha beta + thymocytes in male and female TCR transgenic mice. The number of TCR alpha beta and TCR gamma delta coexpressing cells is increased in pre-TCR-deficient mice. The data indicate that the TCR alpha beta can replace the TCR gamma delta in the development of gamma delta lineage cells and that the pre-TCR interferes with the generation of gamma delta-expressing cells.

We have studied memory in T cell receptor (TCR) transgenic mice expressing a Db-restricted TCR specific for the male peptide (H-Y). CD8+ T cells from female TCR transgenic C57BL/6 (B6) mice were activated by transferring them Into X-irradiated male (B6 x bm12)F1 hybrid recipients. Subsequently, they were highly purified by cell sorting and transferred for various lengths of time into female B6 nu/nu recipient mice. Other nu/nu recipient mice received highly purified naive T cells expressing the transgenic TCR. The functional potential of naive and “memory” T cells was analyzed by stimulation with male cells In vivo. The results show that memory cells can be derived from activated T cells and persist in the absence of antigen for at least 13 weeks. Naive and memory T cells differ in that memory T cells give a more vigorous and sustained response than naive T cells.

Expression of a productive T-cell receptor (TCR) beta gene has profound consequences for T-cell development, preceding an increase in thymocyte number, appearance of CD4 and CD8 coreceptors and suppression of further TCR beta-gene rearrangement. Here Marcus Groettrup and Harald von Boehmer discuss the data obtained in various experimental models and describe a novel signal transducing receptor complex on pre-T cells, which may regulate the TCR beta-induced developmental changes.

We describe a novel signal-transducing protein complex, which consists of the T cell receptor (TCR) beta chain that is disulfide linked to a 33 kd glycoprotein and noncovalently associated with proteins of the CD3 complex on the surface of the pre-T cell line SCB.29. This 33 kd glycoprotein, provisionally designated gp33, represents neither of the known TCR chains and has escaped previous detection because it labels poorly by surface iodination. This glycoprotein is absent from the surface of mature T cell lines. A TCR beta complex with identical molecular masses before and after reduction can be immunoprecipitated from surface-iodinated large thymocytes of TCR alpha-deficient mice. The novel gp33-TCR beta complex may be entirely or partly responsible for control of early T cell development exerted by the TCR beta protein.

Exclusion and inclusion of T cell receptor (TCR) genes were analyzed in alpha beta TCR transgenic mice. Both transgenes are expressed unusually early on the surface of CD4-8-, HSA+, IL-2R- thymocytes. These progenitor cells give rise to progeny, which at the single-cell level contains endogenous alpha but not beta TCR-RNA as well as protein, in addition to products encoded by the transgenes. Thus, the surface expression of an alpha beta TCR does not prevent further alpha TCR rearrangement in immature thymocytes that still transcribe RAG-1 and RAG-2 genes. Reduced levels of RAG-1 and RAG-2 RNA are detectable only in CD4+8+ TCR high cells, which result from positive selection in the thymus. The results suggest that a developing T cell may try different alpha beta TCRs for binding to thymic MHC ligands, and that recombination at the alpha locus ceases only after positive selection.

Mice transgenic for the T-cell receptor (TCR) were instrumental to the understanding that developing alpha beta T cells undergo programmed cell death unless the TCR beta gene properly rearranges and produces receptors of appropriate specificity. The delineation of developmental pathways for 'conventional' alpha beta T cells has resulted in the recognition of a different lineage of alpha beta T cells that develop in the thymus, the function of which remains to be elucidated.

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.

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.

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.

In mammals, lymphocytes develop from hematopoietic stem cells throughout life. The exact branchpoint of irreversible commitment to the lymphocyte lineage is not known, and developmental stages committed to the T and B lymphocyte lineages are being defined. The T cell lineage branches further into cells with ~8 or a8 T cell receptors (TCRs) for antigen. The a8 TCR lineage is subdivided by surface markers and functional potential into CD4+8helper and CD4-8+ killer lineages, which are discussed here. CD4+ He/per and CDl Connolly et al., 1988): cross-linking of either a CD4 or CD8 coreceptor with the a8 TCR by the same MHC molecule is required for efficient activation of T cells (Emmrich et al., 1986; Gabert et al., 1987). For some time, it has not been clear whether CD4+ and CD8’ T cells can each express both class I and class II MHC-specific TCRs. If they can, those cells with a “mismatched” combination of coreceptor and a6 TCR (which would bind to different MHC molecules) would be wasted because these cells could not be efficiently activated. The alternative possibility is that the formation of the CD4+ helper and CD8+ killer lineages is crucially dependent on the specificity of the a8 TCR for class II and class I MHC molecules, respectively. To probe the latter idea, the contribution of a particular receptor (of defined antigen specificity) to the formation of CD4 and CD8 lineages needed to be examined. Because of the great TCR diversity, this could not be done in normal mice but required TCR transgenic mice. The results of the transgenic experiments showed, first, that binding of an up TCR to the polymorphic part of thymic MHC molecules in the absence of the specific peptide antigen is essential for the generation of functional CD4+ helper and CD8+ killer lineage cells from nonfunctional CD4+8+ intrathymic precursors. Second, a CISSS l-specific TCR exclusively supports the development of mature CD8+, while a class II-specific TCR exclusively supports the development of mature CD4+ lineage Cells (von Boehmer, 1990). These differentiation Steps are called positive selection. Minireview

T cells express α,β heterodimeric receptor proteins which are involved in the MHC-restricted binding of antigen (1,2). The nucleotide sequences of T-cell receptor genes(3-6) have not provided an obvious explanation for the 'MHC-ness' of T-cell specificity or the different specificity of T-cell subsets. The binding of α,β heterodimeric T-cell receptors has not yet been studied in solution so it is possible that T cells express additional molecules that are involved in antigen binding. Recent experimental data(7), reviewed in our last issue (Immunol. Today 1986, 7, 308-311) indicate that the α,β heterodimer alone is responsible for the MHC-restricted specificity of T cells. Here, Harald von Boehmer discusses the implications of these data and presents a model for the selection of α,β heterodimeric T-cell receptors during T-cell differentiation in the thymus.

Zusammenfassung 1. 1. Humanserum verlangsamt die elektrophoretische Beweglichkeit von einer Reihe von Saugetierzellen, die durch einen schnellen Teilungsrhythmus gekennzeichnet sind (z. B. Knochenmark, Milz), nicht aber von Zellen, die nicht mehr teilungsfahig (periphere Blutzellen) sind. 2. 2. Die verlangsamende Wirkung zeigen ca. 95 Prozent der bisher untersuchten Humanseren und 3 von 6 ausgetesteten Kaninchenseren. Neugeborenenserum sowie die Seren von Kalb, Meerschweinchen, Ratte und Maus zeigen keine starkere Membranwirkung. Bei den Spendern von nicht aktiven Seren besteht keine Abhangigkeit von den Blutobergruppen, vom Rhesusfaktor oder dem Gesundheitszustand. 3. 3. Unsere Versuche sprechen dafur, daβ die elektrophoretische Beweglichkeit von L-Zellen auf Grund einer Adsorption von 2 Faktoren aus dem frischen Humanserum an die Zellmembran verlangsamt wird. Der eine (A1F1) entspricht immunelektrophoretisch einemy γG-Globulin. Der 2. Faktor (A2F1) ergibt immunelektrophoretisch ebenfalls eine γG-Bande. Beide Faktoren sind nicht dialysabel und werden durch 10 Min. langes Erhitzen auf 55 ° zerstort. 4. 4. Die Membranwirkung des Humanserums tritt erst im nachuterinen Leben voll auf. Die Kombination von A1F1 mit Nabelschnurserum zeigt aber, daβ der in A2F1 befindliche 2. Faktor bereits im Nabelschnurserum vorhanden sein durfte. 5. 5. Die Wirkung der beiden Faktoren auf die Zellmembran erinnert an das Antikorper-Komplement-System. Es konnte jedoch gezeigt werden, daβ zumindest kein vollstandiges Komplementsystem an der Reaktion beteiligt ist. 6. 6. Ein Zusammenhang zwischen dem in A2F1 erhaltenen Faktor und der von Muller-Eberhard entdeckten C′1 q-Komponente des Komplements wird diskutiert. 7. 7. Es besteht kein direkter Zusammenhang zwischen der die elektrophoretische Beweglichkeit verlangsamenden Wirkung und dem zytotoxischen Effekt des Humanserums auf Gewebekulturen und andere Saugetierzellsysteme.

A sensitive culture system for measuring lymphocyte transformation under physiological conditions by thymidine incorporation into DNA has been developed to study mouse and chick cell responses to mitogens. Both phytohemagglutinin (PHA) and pokeweed mitogen (PWM) stimulated thymus and spleen lymphocytes. Reduced but definite responses were obtained with lymph nodes, but negligible response with bone marrow cells. Thymocytes of newborn mice did not respond to PHA, but responded well to PWM. PHA responsiveness of thymocytes increased with aging until 12 weeks of postnatal life and then decreased in older animals. The level of background thymidine incorporation increased with advancing age. Spleen cells of 2-week-old mice were transformed by PHA and PWM, but in contrast to mouse thymus there was no decrease in older animals. Neonatal thymectomy of mice reduced the response of spleen cells to both PHA and PWM, especially in younger animals. The reduction was almost complete in the case of the PHA response, but only partial with the PWM response. Spleen cells from bursectomised chickens, checked for absence of B cell function, still responded well to both PWM and PHA. The results suggest PHA is a marker for T-lymphocytes in a certain “mature” stage of differentiation. PWM appears to stimulate a wider spectrum of cells.