- Email: [email protected]
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immunity against the development of spontaneous mammary tumors in HER-2/neu transgenic mice. Gene Ther. 7, 703–706 Song, K. et al. (2000) Regulation of T-helper-1 versus T-helper-2 activity and enhancement of tumor immunity by combined DNA-based vaccination and nonviral cytokine gene transfer. Gene Ther. 7, 481–492 Song, K. et al. (2000) IL-12 plasmid-enhanced DNA vaccination against carcinoembryonic antigen (CEA) studied in immune-gene knockout mice. Gene Ther. 7, 1527–1535 Kim, J.J. et al. (1998) Modulation of amplitude and direction of in vivo immune responses by coadministration of cytokine gene expression cassettes with DNA immunogens. Eur. J. Immunol. 28, 1089–1103 Iwasaki, A. et al. (1997) The dominant role of bone marrow-derived cells in CTL induction following plasmid DNA immunization at different sites. J. Immunol. 159, 11–14 Torres, C.A. et al. (1997) Differential dependence on target site tissue for gene gun and intramuscular DNA immunization. J. Immunol. 158, 4529–4532 Casares, S. et al. (1997) Antigen presentation by dendritic cells after immunization with DNA encoding a MHC class II-restricted viral epitope. J. Exp. Med. 186, 1481–1486 Chattergoon, M.A. et al. (1998) Specific immune induction following DNA-based immunization
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Notch1 and T-cell development: insights from conditional knockout mice H. Robson MacDonald, Anne Wilson and Freddy Radtke Notch proteins influence cell-fate decisions in many developmental systems. Gain-of-function studies have suggested a crucial role for Notch1 signaling at several stages during lymphocyte development, including the B/T, αβ/γγ δ and CD4/CD8 lineage choices. Here, we critically re-evaluate these conclusions in the light of recent studies that describe inducible and tissue-specific targeting of the Notch1 gene.
H. Robson MacDonald* Anne Wilson Freddy Radtke The Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, 1066 Epalinges, Switzerland. *e-mail: hughrobson.macdonald@ isrec.unil.ch
Like other hematopoietic lineages, T cells are derived from a pluripotent hematopoietic stem cell (HSC) in the bone marrow (BM)1. During their development, T-cell precursors are confronted with at least three distinct cell-fate-specification events2–4. First, a common lymphoid precursor (CLP) must decide whether to adopt a T- or B-cell fate. Once the T-cell lineage is specified, a pro-T cell (or pre-T cell) in the thymus must then choose between the αβ versus γδ lineages. Finally, αβ-lineagecommitted precursor cells must differentiate along
either CD4 or CD8 lineages. These cell-fate choices are outlined in Fig. 1. In addition to the important role played by the various T-cell receptor (pre-TCR, αβTCR and γδTCR) and co-receptor molecules, other inductive signals are believed to be crucial in the lineage-commitment process. One molecule that has received a great deal of attention in this respect is Notch1. Indeed, numerous gain-of-function studies have implicated Notch1 in the T/B, αβ/γδ and CD4/CD8 lineage choices, as well as in survival and maturation of thymocytes5–10. Complementary loss-of-function approaches have not been possible until recently because conventional gene targeting of Notch1 results in an early embryonic lethal phenotype11,12. However, use of the Cre-loxP targeting strategy has allowed the development of conditional knockout mice in which Notch1 can be specifically inactivated
http://immunology.trends.com 1471-4906/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1471-4906(00)01828-7
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Fig. 1. Notch1 signaling during T-cell development. Hematopoietic stem cells (HSCs) give rise to a common lymphoid precursor (CLP; CD117+CD44+CD25−) that makes a first cell-fate decision giving rise to either pro-B cells (CD117+B220+CD19+CD43+) or pro-T cells (CD117+CD44+CD25+). The pro-T cell then matures further to a pre-T-cell stage (CD117loCD44−CD25+), where commitment to either αβ or γδ T-cell lineage occurs. Finally, αβ-lineage-committed thymocytes (CD4+CD8+) must choose between the mature CD4+ (CD4+CD8−) and CD8+ (CD4−CD8+) T-cell fates. Although gain-of-function studies suggest a decisive role for Notch1 in T/B-, αβ/γδ- and CD4/CD8-lineage decisions, more recent loss-of-function studies support a more restricted role for Notch1 in T-cell development (indicated in red).
either in BM precursors13 or in immature thymocytes14. In this review, we critically reevaluate the role of Notch1 in T-cell development and lineage commitment in the light of these recent genetargeting studies. Other aspects of Notch1 signaling in T-cell development have been reviewed elsewhere15–18. An introduction to Notch
Notch proteins are conserved transmembrane receptors containing epidermal growth factor (EGF)repeats in their ectodomain, which are implicated in ligand binding. The cytoplasmic domain harbors six ankyrin repeats and is involved in intracellular signaling. To date, four mammalian Notch homologs (Notch1–4), which interact with transmembranebound ligands such as Jagged1, Jagged2, Delta1, Delta-like3 and Delta-like4, have been identified19–21. The Notch receptor is synthesized as a precursor protein that is proteolytically processed during transport to the cell membrane22. The mature form of the Notch receptor is a heterodimer comprising an extracellular region containing the EGF repeats, and an intracellular subunit harboring a RAM23 domain together with ankyrin repeats and a PEST sequence. Notch family members have been shown to play crucial roles in binary cell-fate decisions mediated by lateral signaling, as well as in inductive cell-fate decisions in many developmental systems. In the lateral signaling model19, neighboring cells with an equipotent developmental capacity initially express both the Notch receptor and its ligand. Owing to an unknown mechanism, the concentrations of Notch receptors and ligands start to differ between neighboring cells. Small differences in receptor and/or ligand concentrations are amplified over time, http://immunology.trends.com
leading to cells that express the Notch receptor or its ligand exclusively. Notch-mediated signaling between such cells leads to the inhibition of one of two possible developmental fates in cells that receive the Notch signal. In the model of inductive cell-fate determination19, the Notch receptor and its ligand are expressed on two developmentally distinct cells. Notch signaling between these cells induces the cell receiving the Notch signal to differentiate into a particular cell lineage. For example, bipotential neural crest stem cells can be induced by Notch to adopt a glial (as opposed to neuronal) cell fate23. The relevance of inductive Notch signaling to T-cell development will be discussed in greater detail below. T/B-lineage commitment
The earliest stage of lymphoid development where Notch1 has been reported to play a role is the T/B-lineage decision. It is widely accepted that BM-derived CLPs give rise to either B or T cells (in addition to natural killer cells and possibly lymphoid dendritic cells, which will not be discussed further here). In adult mice, B-cell development normally proceeds in the BM, whereas T-cell development is largely restricted to the thymus and (to a lesser extent) intestine. Whether commitment of CLPs to the T-cell lineage occurs in the BM or only after migration to the thymus or intestine is currently unknown. Two independent lines of evidence implicate Notch1 in the binary lineage decision of a CLP to develop into a B cell or a T cell. First, retrovirally transduced BM cells that overexpress the constitutively active Notch1 intracellular domain (Notch IC) do not develop into B cells in the BM of lethally irradiated recipients9. Instead, immature T cells (mainly of the CD4+CD8+ phenotype) accumulate in the BM of these chimeric mice. In reciprocal experiments13, conditional inactivation of a loxP-flanked Notch1 gene in BM precursors [using a Cre recombinase transgene driven by an interferon α (IFN-α)-responsive Mx promoter] results in an early block in T-cell development (at or before the pro-T-cell stage) in the thymus of lethally irradiated wild-type (wt) recipients. In this situation, instead of T cells, immature B cells of Notch1−/− origin (with a phenotype indistinguishable from those normally found in BM) accumulate in the irradiated wt thymus. Taken together, these complementary gain-offunction and loss-of-function studies clearly indicate that varying levels of Notch1 expression not only control the development of T and B cells, but also promote ectopic development of either lineage at the expense of the other. By analogy with the role of Notch genes in invertebrate systems20, the simplest interpretation of these data would be that Notch1 provides a critical signal that determines the outcome of a binary (T or B) cell-fate decision by a
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Fig. 2. Distinct developmental fates of Notch1−/− common lymphoid precursors (CLPs) in the thymus and intestine. According to this model, CLPs migrate from the bone marrow to either the thymus or intestine, where they encounter epithelial cells that express a ligand for the Notch1 receptor, such as Jagged1 and/or Jagged2. In the wild-type (wt) situation, the Notch1–Jagged interaction instructs CLPs towards the T-cell lineage in both the thymus and the intestine. In the thymus, CLPs deficient for the Notch1 receptor cannot be instructed to adopt a T-cell fate and therefore adopt a B-cell fate by default. In the intestine, Notch1−/− CLPs cannot be instructed to become T cells; however, in contrast to the thymus, they cannot adopt a default B-cell fate (owing to the intestinal microenvironment that is not permissive for B-cell development) and therefore persist as developmentally arrested cells.
CLP. Because the absence of Notch1 blocks T-cell development and promotes ectopic B-cell development, whereas Notch1 overexpression promotes ectopic T-cell development at the expense of B cells, B-cell development from the CLP might represent the default pathway that occurs in the absence of Notch1 signaling. According to this scenario, inductive signaling via Notch1 would be necessary for CLPs to adopt a T-cell fate. Interestingly, a low level of steady state B-cell lymphopoiesis occurs in the normal thymus24, consistent with the possibility that an instructive mechanism of T-cell-fate specification from bipotential precursors is inherently slightly leaky. In addition to the thymus, the epithelium of the intestinal mucosa is a site of T-cell development. Recent evidence indicates that extrathymic T-cell development in the gut takes place in specialized structures known as cryptopatches, located in the lamina propria25. Interestingly, conditional inactivation of Notch1 in BM precursors (using the http://immunology.trends.com
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inducible Mx–Cre transgene described above) results in failure to reconstitute the extrathymic T-cell compartment of the intestinal epithelium in irradiated mice. This indicates that Notch1 is essential for T-cell-fate specification in both the gut and the thymus26. In contrast to the thymus, however, no ectopic development of Notch1−/− immature B cells can be demonstrated in the gut, consistent with earlier studies that suggest that the gut microenvironment is not permissive for B-cell development25. Analysis of Notch1−/− putative CLPs (defined by expression of CD117) in the intestinal epithelium and lamina propria led to the surprising conclusion that Notch1 deficiency has almost no effect on CLP numbers in the gut26. By contrast, CLPs are drastically reduced (although still detectable) in the thymi of mice reconstituted with Notch1−/− BM precursors. Taken together, the contrasting effects of Notch1 deficiency in the thymus and the gut can be integrated into a unified model for the role of Notch1 in T/B lineage commitment. As illustrated in Fig. 2, bipotential CLPs normally migrate from the BM to the thymus or intestine, where they receive a critical Notch1 signal that instructs them to adopt a T-cell fate. In the absence of Notch1 signaling, thymic CLPs cannot develop as T cells but instead adopt a Bcell fate by default. By contrast, because microenvironmental cues supporting ectopic B-cell development are absent in the gut, Notch1−/− CLPs cannot adopt a T- nor a B-cell fate. This scenario is consistent with the presence of immature Notch1−/− B cells in the thymus but not the gut. Moreover, it explains the increased numbers of residual Notch1−/− CLPs in the gut compared with the thymus, because CLPs unable to adopt either a B- or T-cell fate would presumably persist as developmentally arrested cells. Molecular control of T/B-cell fate determination by Notch1 signaling
The mechanism by which Notch1 controls T/B-cellfate specification in a CLP remains to be elucidated. However, based on the known molecular targets of Notch signaling, a plausible model can be formulated. As illustrated schematically in Fig. 3, engagement of Jagged (a ligand for Notch1 expressed by thymic epithelial cells) leads to proteolytic cleavage of Notch IC at (or close to) the transmembrane domain by a γ-secretase22. Upon translocation to the nucleus, Notch IC heterodimerizes with the helix–loop–helix (HLH) transcription factor recombination signal binding protein Jκ (RBP-Jκ), thereby converting it from a repressor to an activator. It is possible that the Notch IC–RBP-Jκ complex acts as a transcriptional activator of T-cell lineage-specific genes and thereby provides an instructional signal that directs CLPs towards a T-cell fate. Potential downstream target genes of Notch IC–RBP-Jκ signaling include a family of basic HLH (bHLH) transcription factors known as
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Fig. 3. Molecular control of T-cell-fate specification by Notch1 signaling. In this hypothetical model, the interaction of ligands such as Jagged1 and/or Jagged2 expressed on thymic epithelial cells (TECs) with the Notch1 receptor expressed on common lymphoid precursors (CLPs) leads to activation and cleavage of the Notch1 receptor at (or close to) its transmembrane domain. Two signaling pathways are simultaneously activated. Translocation of the intracellular subunit of the Notch1 receptor (Notch IC) into the nucleus and its heterodimerization with the helix–loop–helix (HLH) transcription factor recombination signal binding protein Jκ (RBP-Jκ) converts RBP-Jκ from a transcriptional repressor into an activator, thereby leading to transcription of T-lineage-specific target genes (still to be identified) and instruction of CLPs towards the T-cell lineage. Simultaneously, a second pathway is activated through Deltex leading to inhibition of E2A-encoded transcription factors (such as E47 and/or E12), which are essential for B-cell development. This pathway involves the inhibition of Rasactivated Jun N-terminal kinase (JNK).
Hes genes27. One member of this family (Hes1) might be of particular interest in T-cell-fate specification because thymic development of Hes1−/− precursor cells is arrested before the pro-T-cell stage28, a phenotype reminiscent of Notch1−/− precursors13. A second independent consequence of Notch1 signaling involves activation of the cytoplasmic protein Deltex (Fig. 3). Interestingly, Deltex has been reported to repress the transcriptional activity of the E2A gene products E12 and E47 (Ref. 29), which are essential for B-cell development30. Hence, in addition to an instructive role in promoting T-cell-lineage commitment via RBP-Jκ, Notch1 might simultaneously repress the expression of B-lineagespecific genes via Deltex.
αβ T cells. It is still controversial whether γδ TCR and pre-TCR signaling alone are sufficient to direct pro-T cells towards the γδ or αβ lineages, respectively31–33. Nonetheless, there is evidence that other inductive factors (including Notch1) might be involved in cell-fate specification at this particular developmental stage. Two types of experiments have led to the suggestion that Notch1 influences the αβ/γδ-lineage decision6. First, BM precursor cells hemizygous for Notch1 (Notch1+/−) give rise preferentially to γδ cells when they develop in the thymus of mixed chimeras in the presence of wt precursors (Notch1+/+). Second, overexpression of Notch IC under the control of the proximal lck promoter in transgenic mice overcomes the block in αβ T-cell development imposed by the absence of TCRβ (and hence a pre-TCR). Collectively, these data have been interpreted to mean that Notch1 signaling favors the development of αβ lineage cells at the expense of the γδ lineage6. Although suggestive, neither of these experimental approaches is definitive, and complementary loss-offunction studies will ultimately be required to clarify the role of Notch1 in αβ/γδ-lineage commitment. In this regard, the absolute requirement for Notch1 in T-cell-fate specification of CLPs (see above) complicates the situation and dictates that tissuespecific inactivation of Notch1 at a subsequent stage of thymic development will be necessary to address this issue directly. This problem is complicated further by the fact that there is no consensus as to the developmental stage where αβ- and γδ-T-cell lineages diverge. Nevertheless, a study in which Notch1 was inactivated at the late pre-T (CD25+CD44−) stage of development (using a Cre recombinase transgene driven by a CD4 mini-gene promoter–enhancer–silencer cassette) did not reveal any detectable effect on absolute numbers of αβ or γδ cells in the thymus14. Thus, a conservative conclusion would be that any essential role for Notch1 in αβ/γδlineage commitment must occur before the late pre-T-cell stage of development. Tissue-specific inactivation of Notch1 at earlier developmental stages in the thymus (using Cre recombinase transgenes driven by lck or CD2 promoters) should provide a definitive answer to the question of whether Notch1 signaling controls αβ/γδ-lineage commitment.
αβ/γγδ -lineage commitment
A second stage of T-cell development that implicates Notch1 signaling is the αβ/γδ-lineage decision. Following commitment to the T-cell lineage, immature pro-T cells begin to rearrange and express their TCRγ, -δ and -β genes. Pro-T cells that successfully rearrange TCRγ and TCRδ will express a γδ TCR and be eligible to develop further as γδ T cells. By contrast, productive TCRβ gene rearrangement will lead to expression of a pre-TCR (consisting of a TCRβ chain associated with an invariant pTα chain), which is compatible with further development as http://immunology.trends.com
CD4/CD8-lineage commitment
A final stage of T-cell development that has been linked to Notch1 signaling is the CD4/CD8-lineage decision. After successfully re-arranging TCRβ and expressing a pre-TCR, αβ-lineage-committed pre-T cells expand and progress to the CD4+CD8+ double-positive (DP) stage of development. At this point, successful TCRα re-arrangement leads to expression of an αβ TCR that can be positively or negatively selected as DP thymocytes progress to the mature CD4+CD8− single-positive (CD4+ SP) and
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Table 1. Evidence that Notch1 influences CD4/CD8-lineage commitment, maturation and survivala Experimental system
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Selective increase in CD8+ SP thymocytes
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Increased resistance to glucocorticoid-induced apoptosis
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Increased resistance to activation-induced apoptosis
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Decreased maturation of CD8+ SP thymocytes
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Transgenic mice overexpressing Notch IC
Increased maturation of CD4+ SP and CD8+ SP thymocytes
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aAbbreviations: FTOC, fetal thymus organ culture; Notch IC, Notch1 intracellular domain; SP, single positive.
CD4−CD8+ SP (CD8+ SP) stages34. The αβ TCR on mature CD8+ SP and CD4+ SP cells recognizes peptides presented by MHC class I or class II, respectively. Therefore, it is possible that specific TCRαβ signaling (in conjunction with related signaling through lck-associated CD4 or CD8 coreceptors) is sufficient in itself to direct DP cells to the CD4 or CD8 lineage. Alternatively, it is possible that CD4/CD8-lineage determination in DP cells occurs independently of TCR–MHC interactions, and that the latter are required only to ensure the survival of cells with an appropriate match of coreceptor and TCR specificity. In agreement with the latter hypothesis, several reports have implicated Notch1 signaling either in the CD4/CD8-lineage commitment event or in the subsequent maturation and survival of CD4+ SP and/or CD8+ SP cells. These studies (summarized in Table 1) are largely based on gain-of-function approaches in which Notch IC is overexpressed either in DP thymoma cells or in transgenic mice. The conclusions of these studies have been seriously challenged by a report14 where the Notch1 gene has been inactivated in a tissue-specific fashion in CD25+CD44− late pre-T cells (using a CD4 minigene-driven Cre-recombinase transgene). Surprisingly, inactivation of Notch1 signaling at this relatively early stage has no effect on the subsequent steady-state development of DP or mature CD4+ SP and CD8+ SP thymocytes. Moreover, bromodeoxyuridine (BrdU) turnover studies and competitive mixed BM chimeras provide no evidence that Notch1 influences the rate of maturation or survival of CD4+ SP or CD8+ SP cells. Finally, in contrast to conclusions reached using DP thymoma cell lines, Notch1 deficiency in DP thymocytes does not affect their sensitivity to spontaneous or glucocorticoid-induced apoptosis. The conclusions reached on the basis of tissuespecific Notch1 inactivation in immature thymocytes14 thus conflict sharply with earlier reports suggesting that Notch1 is important for CD4/CD8-lineage commitment, maturation and http://immunology.trends.com
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survival. In attempting to reconcile these discrepancies, it should be noted that overexpression of Notch IC in transgenic mice or thymoma cell lines (Table 1) might activate signaling pathways that are not normally controlled by Notch1 under physiological conditions. For example, Notch IC might activate signaling that is normally mediated via Notch2 or Notch3 (both of which are expressed in the thymus)35. In this context, overexpression of the cytoplasmic domain of Notch3 in immature thymocytes of transgenic mice (under the control of the proximal lck promoter) does not specifically affect CD4/CD8-lineage commitment, although it does result in an increase in absolute numbers of thymocytes36. Nevertheless, it remains a formal possibility that Notch signaling is (at least partially) redundant for CD4/CD8lineage commitment, such that a loss-of-function mutation of Notch1 alone would have no apparent phenotype. Whatever the explanation, it is clear that a unique role for Notch1 in CD4/CD8-lineage commitment, maturation or survival can be excluded on the basis of the conditional genetargeting data. Concluding remarks
Notch1 has been implicated in three distinct (and sequential) lineage-specification events during T-cell development. In particular, signaling via Notch1 has been postulated to: (1) direct the differentiation of a bipotential CLP towards a T- (as opposed to a B-) cell fate; (2) favor the differentiation of pro-T (or pre-T) cells towards the αβ (as opposed to γδ) lineage; (3) favor the differentiation, maturation or survival of DP thymocytes towards the CD8+ SP and/or CD4+ SP lineages. All of these putative fate-determining properties of Notch1 are supported by gain-of-function studies that involve overexpression of Notch IC in BM precursors, transgenic mice or thymoma cell lines. However, as summarized in Fig. 1, recent loss-of-function studies in inducible and tissue-specific Notch1 knockout mice only confirm a critical role for Notch1 signaling in T/B-cell-fate specification. By contrast, these studies exclude an essential role for Notch1 in CD4/CD8lineage commitment and restrict its putative role (if any) in αβ/γδ-lineage commitment to an early developmental stage. Taken together with other studies37 indicating that inducible inactivation of Notch1 in BM precursors does not affect the development of myeloid, erythroid or other lymphoid (natural killer or thymic dendritic cell) lineages, it is tempting to speculate that the only non-redundant function of Notch1 in the hematopoietic system is to direct bipotential CLPs to the T-cell lineage. Future derivation (and intercrossing) of mutant mouse strains deficient in genes encoding other members of the Notch family and their respective ligands should help to clarify the general role of Notch signaling in hematopoiesis.
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the vertebrate nervous system. Curr. Opin. Genet. Dev. 7, 659–665 Tomita, K. et al. (1999) The bHLH gene Hes1 is essential for expansion of early T cell precursors. Genes Dev. 13, 1203–1210 Ordentlich, P. et al. (1998) Notch inhibition of E47 supports the existence of a novel signaling pathway. Mol. Cell. Biol. 18, 2230–2239 Bain, G. et al. (1997) Both E12 and E47 allow commitment to the B cell lineage. Immunity 6, 145–154 Kang, J. and Raulet, D.H. (1997) Events that regulate differentiation of αβ TCR+ and γδ TCR+ T cells from a common precursor. Semin. Immunol. 9, 171–179 MacDonald, H.R. and Wilson, A. (1998) The role of the T-cell receptor (TCR) in αβ/γδ lineage commitment: clues from intracellular TCR staining. Immunol. Rev. 165, 87–94 Robey, E. and Fowlkes, B.J. (1998) The αβ versus γδ T-cell lineage choice. Curr. Opin. Immunol. 10, 181–187 Kisielow, P. and von Boehmer, H. (1995) Development and selection of T cells: facts and puzzles. Adv. Immunol. 58, 87–209 Felli, M.P. et al. (1999) Expression pattern of Notch1, 2 and 3 and Jagged1 and 2 in lymphoid and stromal thymus components: distinct ligandreceptor interactions in intrathymic T cell development. Int. Immunol. 11, 1017–1025 Bellavia, D. et al. (2000) Constitutive activation of NF-κB and T-cell leukemia/lymphoma in Notch3 transgenic mice. EMBO J. 19, 3337–3348 Radtke, F. et al. (2000) Notch1 deficiency dissociates the intrathymic development of dendritic cells and T cells. J. Exp. Med. 191, 1085–1094 Yasutomo, K. et al. (2000) The duration of antigen receptor signalling determines CD4+ versus CD8+ T cell lineage fate. Nature 404, 506–510
TB vaccines: progress and problems Peter Andersen Tuberculosis (TB) is the biggest killer worldwide of any infectious disease, a situation worsened by the advent of the HIV epidemic and the emergence of multi-drug resistant strains of Mycobacterium tuberculosis. The existing vaccine, Mycobacterium bovis bacille Calmette–Guérin (BCG), has proven inefficient in several recent field trials. There is currently intense research using cutting-edge vaccine technology to combat this ancient disease. However, it is necessary to understand why BCG has failed before we can rationally develop the next generation of vaccines. Several hypotheses that might explain the failure of BCG and the strategies designed to address these shortcomings are discussed.
Peter Andersen Dept of TB Immunology, Statens Seruminstitut, 5 Artillerivej, DK-2300 Copenhagen S, Denmark. e-mail: [email protected]
Tuberculosis (TB) is a major global health problem that claims more than two million lives each year1. In developing countries, the incidence of TB has always been high and in industrialized countries the disease has re-emerged as a public health problem2. This development has been accelerated by the advent of the
HIV epidemic and the increasing incidence of multi-drug-resistant strains of Mycobacterium tuberculosis. The current TB vaccine, Mycobacterium bovis bacille Calmette–Guérin (BCG), was developed almost a century ago and, although this vaccine has proved inefficient, in several field trials, it is still in widespread use3. In 1993, TB was declared a global health emergency by the World Health Organization (Press Release: WHO attacks global neglect of tuberculosis crisis. Millions dying from ‘low priority’ disease). This statement came after more than two decades of international neglect of this important disease. Since then, funding for TB research has increased steadily and, today, TB immunology and vaccinology are two of the most rapidly evolving fields in medical research. Furthermore, TB research has benefited in recent
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immunity against the development of spontaneous mammary tumors in HER-2/neu transgenic mice. Gene Ther. 7, 703–706 Song, K. et al. (2000) Regulation of T-helper-1 versus T-helper-2 activity and enhancement of tumor immunity by combined DNA-based vaccination and nonviral cytokine gene transfer. Gene Ther. 7, 481–492 Song, K. et al. (2000) IL-12 plasmid-enhanced DNA vaccination against carcinoembryonic antigen (CEA) studied in immune-gene knockout mice. Gene Ther. 7, 1527–1535 Kim, J.J. et al. (1998) Modulation of amplitude and direction of in vivo immune responses by coadministration of cytokine gene expression cassettes with DNA immunogens. Eur. J. Immunol. 28, 1089–1103 Iwasaki, A. et al. (1997) The dominant role of bone marrow-derived cells in CTL induction following plasmid DNA immunization at different sites. J. Immunol. 159, 11–14 Torres, C.A. et al. (1997) Differential dependence on target site tissue for gene gun and intramuscular DNA immunization. J. Immunol. 158, 4529–4532 Casares, S. et al. (1997) Antigen presentation by dendritic cells after immunization with DNA encoding a MHC class II-restricted viral epitope. J. Exp. Med. 186, 1481–1486 Chattergoon, M.A. et al. (1998) Specific immune induction following DNA-based immunization
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through in vivo transfection and activation of macrophages/antigen-presenting cells. J. Immunol. 160, 5707–5718 Ulmer, J.B. et al. (1996) Generation of MHC class Irestricted cytotoxic T lymphocytes by expression of a viral protein in muscle cells: antigen presentation by non-muscle cells. Immunology 89, 59–67 Fu, T.M. et al. (1997) Priming cytotoxic T lymphocytes by DNA vaccines: requirement for professional antigen presenting cells and evidence for antigen transfer from myocytes. Mol. Med. 3, 362–371 Corr, M. et al. (1999) In vivo priming by DNA injection occurs predominantly by antigen transfer. J. Immunol. 163, 4721–4727 Kwissa, M. et al. (2000) Efficient vaccination by intradermal or intramuscular inoculation of plasmid DNA expressing hepatitis B surface antigen under desmin promoter/enhancer control. Vaccine 18, 2337–2344 Widera, G. et al. (2000) Increased DNA vaccine delivery and immunogenicity by electroporation in vivo. J. Immunol. 164, 4635–4640 Haddad, D. et al. (2000) Plasmid vaccine expressing granulocyte–macrophage colonystimulating factor attracts infiltrates including immature dendritic cells into injected muscle. J. Immunol. 165, 3772–3781 Pizetsky, D.S. (2000) The antigenic properties of bacterial DNA in normal and aberrant immunity. Springer Semin. Immunopathol. 22, 153–166
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51 Gilkeson, G.S. et al. (1996) Modulation of renal disease in autoimmune NZB/NZW mice by immunization with bacterial DNA. J. Exp. Med. 183, 1389–1397 52 Coon, B. et al. (1999) DNA immunization to prevent autoimmune diabetes. J. Clin. Invest. 104, 189–194 53 Garren, H. and Steinman, L. (2000) DNA vaccination in the treatment of autoimmune diseases. Curr. Dir. Autoimmun. 2, 203–216 54 Calarota, S.A. et al. (1999) Immune responses in asymptomatic HIV-1-infected patients after HIVDNA immunization followed by highly active antiretroviral treatment. J. Immunol. 163, 2330–2338 55 Le, T.P. et al. (2000) Safety, tolerability and humoral immune responses after intramuscular administration of a malaria DNA vaccine to healthy adult volunteers. Vaccine 18, 1893–1901 56 Kalka, C. et al. (2000) Vascular endothelial growth factor (165) gene transfer augments circulating endothelial progenitor cells in human subjects. Circ. Res. 86, 1198–1202 57 Baumgartner, I. et al. (1998) Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia. Circulation 97, 1114–1123 58 Kim, J.J. et al. (2000) Chemokine gene adjuvants can modulate immune responses induced by DNA vaccines. J. Interferon Cytokine Res. 20, 487–498
Notch1 and T-cell development: insights from conditional knockout mice H. Robson MacDonald, Anne Wilson and Freddy Radtke Notch proteins influence cell-fate decisions in many developmental systems. Gain-of-function studies have suggested a crucial role for Notch1 signaling at several stages during lymphocyte development, including the B/T, αβ/γγ δ and CD4/CD8 lineage choices. Here, we critically re-evaluate these conclusions in the light of recent studies that describe inducible and tissue-specific targeting of the Notch1 gene.
H. Robson MacDonald* Anne Wilson Freddy Radtke The Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, 1066 Epalinges, Switzerland. *e-mail: hughrobson.macdonald@ isrec.unil.ch
Like other hematopoietic lineages, T cells are derived from a pluripotent hematopoietic stem cell (HSC) in the bone marrow (BM)1. During their development, T-cell precursors are confronted with at least three distinct cell-fate-specification events2–4. First, a common lymphoid precursor (CLP) must decide whether to adopt a T- or B-cell fate. Once the T-cell lineage is specified, a pro-T cell (or pre-T cell) in the thymus must then choose between the αβ versus γδ lineages. Finally, αβ-lineagecommitted precursor cells must differentiate along
either CD4 or CD8 lineages. These cell-fate choices are outlined in Fig. 1. In addition to the important role played by the various T-cell receptor (pre-TCR, αβTCR and γδTCR) and co-receptor molecules, other inductive signals are believed to be crucial in the lineage-commitment process. One molecule that has received a great deal of attention in this respect is Notch1. Indeed, numerous gain-of-function studies have implicated Notch1 in the T/B, αβ/γδ and CD4/CD8 lineage choices, as well as in survival and maturation of thymocytes5–10. Complementary loss-of-function approaches have not been possible until recently because conventional gene targeting of Notch1 results in an early embryonic lethal phenotype11,12. However, use of the Cre-loxP targeting strategy has allowed the development of conditional knockout mice in which Notch1 can be specifically inactivated
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Fig. 1. Notch1 signaling during T-cell development. Hematopoietic stem cells (HSCs) give rise to a common lymphoid precursor (CLP; CD117+CD44+CD25−) that makes a first cell-fate decision giving rise to either pro-B cells (CD117+B220+CD19+CD43+) or pro-T cells (CD117+CD44+CD25+). The pro-T cell then matures further to a pre-T-cell stage (CD117loCD44−CD25+), where commitment to either αβ or γδ T-cell lineage occurs. Finally, αβ-lineage-committed thymocytes (CD4+CD8+) must choose between the mature CD4+ (CD4+CD8−) and CD8+ (CD4−CD8+) T-cell fates. Although gain-of-function studies suggest a decisive role for Notch1 in T/B-, αβ/γδ- and CD4/CD8-lineage decisions, more recent loss-of-function studies support a more restricted role for Notch1 in T-cell development (indicated in red).
either in BM precursors13 or in immature thymocytes14. In this review, we critically reevaluate the role of Notch1 in T-cell development and lineage commitment in the light of these recent genetargeting studies. Other aspects of Notch1 signaling in T-cell development have been reviewed elsewhere15–18. An introduction to Notch
Notch proteins are conserved transmembrane receptors containing epidermal growth factor (EGF)repeats in their ectodomain, which are implicated in ligand binding. The cytoplasmic domain harbors six ankyrin repeats and is involved in intracellular signaling. To date, four mammalian Notch homologs (Notch1–4), which interact with transmembranebound ligands such as Jagged1, Jagged2, Delta1, Delta-like3 and Delta-like4, have been identified19–21. The Notch receptor is synthesized as a precursor protein that is proteolytically processed during transport to the cell membrane22. The mature form of the Notch receptor is a heterodimer comprising an extracellular region containing the EGF repeats, and an intracellular subunit harboring a RAM23 domain together with ankyrin repeats and a PEST sequence. Notch family members have been shown to play crucial roles in binary cell-fate decisions mediated by lateral signaling, as well as in inductive cell-fate decisions in many developmental systems. In the lateral signaling model19, neighboring cells with an equipotent developmental capacity initially express both the Notch receptor and its ligand. Owing to an unknown mechanism, the concentrations of Notch receptors and ligands start to differ between neighboring cells. Small differences in receptor and/or ligand concentrations are amplified over time, http://immunology.trends.com
leading to cells that express the Notch receptor or its ligand exclusively. Notch-mediated signaling between such cells leads to the inhibition of one of two possible developmental fates in cells that receive the Notch signal. In the model of inductive cell-fate determination19, the Notch receptor and its ligand are expressed on two developmentally distinct cells. Notch signaling between these cells induces the cell receiving the Notch signal to differentiate into a particular cell lineage. For example, bipotential neural crest stem cells can be induced by Notch to adopt a glial (as opposed to neuronal) cell fate23. The relevance of inductive Notch signaling to T-cell development will be discussed in greater detail below. T/B-lineage commitment
The earliest stage of lymphoid development where Notch1 has been reported to play a role is the T/B-lineage decision. It is widely accepted that BM-derived CLPs give rise to either B or T cells (in addition to natural killer cells and possibly lymphoid dendritic cells, which will not be discussed further here). In adult mice, B-cell development normally proceeds in the BM, whereas T-cell development is largely restricted to the thymus and (to a lesser extent) intestine. Whether commitment of CLPs to the T-cell lineage occurs in the BM or only after migration to the thymus or intestine is currently unknown. Two independent lines of evidence implicate Notch1 in the binary lineage decision of a CLP to develop into a B cell or a T cell. First, retrovirally transduced BM cells that overexpress the constitutively active Notch1 intracellular domain (Notch IC) do not develop into B cells in the BM of lethally irradiated recipients9. Instead, immature T cells (mainly of the CD4+CD8+ phenotype) accumulate in the BM of these chimeric mice. In reciprocal experiments13, conditional inactivation of a loxP-flanked Notch1 gene in BM precursors [using a Cre recombinase transgene driven by an interferon α (IFN-α)-responsive Mx promoter] results in an early block in T-cell development (at or before the pro-T-cell stage) in the thymus of lethally irradiated wild-type (wt) recipients. In this situation, instead of T cells, immature B cells of Notch1−/− origin (with a phenotype indistinguishable from those normally found in BM) accumulate in the irradiated wt thymus. Taken together, these complementary gain-offunction and loss-of-function studies clearly indicate that varying levels of Notch1 expression not only control the development of T and B cells, but also promote ectopic development of either lineage at the expense of the other. By analogy with the role of Notch genes in invertebrate systems20, the simplest interpretation of these data would be that Notch1 provides a critical signal that determines the outcome of a binary (T or B) cell-fate decision by a
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Fig. 2. Distinct developmental fates of Notch1−/− common lymphoid precursors (CLPs) in the thymus and intestine. According to this model, CLPs migrate from the bone marrow to either the thymus or intestine, where they encounter epithelial cells that express a ligand for the Notch1 receptor, such as Jagged1 and/or Jagged2. In the wild-type (wt) situation, the Notch1–Jagged interaction instructs CLPs towards the T-cell lineage in both the thymus and the intestine. In the thymus, CLPs deficient for the Notch1 receptor cannot be instructed to adopt a T-cell fate and therefore adopt a B-cell fate by default. In the intestine, Notch1−/− CLPs cannot be instructed to become T cells; however, in contrast to the thymus, they cannot adopt a default B-cell fate (owing to the intestinal microenvironment that is not permissive for B-cell development) and therefore persist as developmentally arrested cells.
CLP. Because the absence of Notch1 blocks T-cell development and promotes ectopic B-cell development, whereas Notch1 overexpression promotes ectopic T-cell development at the expense of B cells, B-cell development from the CLP might represent the default pathway that occurs in the absence of Notch1 signaling. According to this scenario, inductive signaling via Notch1 would be necessary for CLPs to adopt a T-cell fate. Interestingly, a low level of steady state B-cell lymphopoiesis occurs in the normal thymus24, consistent with the possibility that an instructive mechanism of T-cell-fate specification from bipotential precursors is inherently slightly leaky. In addition to the thymus, the epithelium of the intestinal mucosa is a site of T-cell development. Recent evidence indicates that extrathymic T-cell development in the gut takes place in specialized structures known as cryptopatches, located in the lamina propria25. Interestingly, conditional inactivation of Notch1 in BM precursors (using the http://immunology.trends.com
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inducible Mx–Cre transgene described above) results in failure to reconstitute the extrathymic T-cell compartment of the intestinal epithelium in irradiated mice. This indicates that Notch1 is essential for T-cell-fate specification in both the gut and the thymus26. In contrast to the thymus, however, no ectopic development of Notch1−/− immature B cells can be demonstrated in the gut, consistent with earlier studies that suggest that the gut microenvironment is not permissive for B-cell development25. Analysis of Notch1−/− putative CLPs (defined by expression of CD117) in the intestinal epithelium and lamina propria led to the surprising conclusion that Notch1 deficiency has almost no effect on CLP numbers in the gut26. By contrast, CLPs are drastically reduced (although still detectable) in the thymi of mice reconstituted with Notch1−/− BM precursors. Taken together, the contrasting effects of Notch1 deficiency in the thymus and the gut can be integrated into a unified model for the role of Notch1 in T/B lineage commitment. As illustrated in Fig. 2, bipotential CLPs normally migrate from the BM to the thymus or intestine, where they receive a critical Notch1 signal that instructs them to adopt a T-cell fate. In the absence of Notch1 signaling, thymic CLPs cannot develop as T cells but instead adopt a Bcell fate by default. By contrast, because microenvironmental cues supporting ectopic B-cell development are absent in the gut, Notch1−/− CLPs cannot adopt a T- nor a B-cell fate. This scenario is consistent with the presence of immature Notch1−/− B cells in the thymus but not the gut. Moreover, it explains the increased numbers of residual Notch1−/− CLPs in the gut compared with the thymus, because CLPs unable to adopt either a B- or T-cell fate would presumably persist as developmentally arrested cells. Molecular control of T/B-cell fate determination by Notch1 signaling
The mechanism by which Notch1 controls T/B-cellfate specification in a CLP remains to be elucidated. However, based on the known molecular targets of Notch signaling, a plausible model can be formulated. As illustrated schematically in Fig. 3, engagement of Jagged (a ligand for Notch1 expressed by thymic epithelial cells) leads to proteolytic cleavage of Notch IC at (or close to) the transmembrane domain by a γ-secretase22. Upon translocation to the nucleus, Notch IC heterodimerizes with the helix–loop–helix (HLH) transcription factor recombination signal binding protein Jκ (RBP-Jκ), thereby converting it from a repressor to an activator. It is possible that the Notch IC–RBP-Jκ complex acts as a transcriptional activator of T-cell lineage-specific genes and thereby provides an instructional signal that directs CLPs towards a T-cell fate. Potential downstream target genes of Notch IC–RBP-Jκ signaling include a family of basic HLH (bHLH) transcription factors known as
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Fig. 3. Molecular control of T-cell-fate specification by Notch1 signaling. In this hypothetical model, the interaction of ligands such as Jagged1 and/or Jagged2 expressed on thymic epithelial cells (TECs) with the Notch1 receptor expressed on common lymphoid precursors (CLPs) leads to activation and cleavage of the Notch1 receptor at (or close to) its transmembrane domain. Two signaling pathways are simultaneously activated. Translocation of the intracellular subunit of the Notch1 receptor (Notch IC) into the nucleus and its heterodimerization with the helix–loop–helix (HLH) transcription factor recombination signal binding protein Jκ (RBP-Jκ) converts RBP-Jκ from a transcriptional repressor into an activator, thereby leading to transcription of T-lineage-specific target genes (still to be identified) and instruction of CLPs towards the T-cell lineage. Simultaneously, a second pathway is activated through Deltex leading to inhibition of E2A-encoded transcription factors (such as E47 and/or E12), which are essential for B-cell development. This pathway involves the inhibition of Rasactivated Jun N-terminal kinase (JNK).
Hes genes27. One member of this family (Hes1) might be of particular interest in T-cell-fate specification because thymic development of Hes1−/− precursor cells is arrested before the pro-T-cell stage28, a phenotype reminiscent of Notch1−/− precursors13. A second independent consequence of Notch1 signaling involves activation of the cytoplasmic protein Deltex (Fig. 3). Interestingly, Deltex has been reported to repress the transcriptional activity of the E2A gene products E12 and E47 (Ref. 29), which are essential for B-cell development30. Hence, in addition to an instructive role in promoting T-cell-lineage commitment via RBP-Jκ, Notch1 might simultaneously repress the expression of B-lineagespecific genes via Deltex.
αβ T cells. It is still controversial whether γδ TCR and pre-TCR signaling alone are sufficient to direct pro-T cells towards the γδ or αβ lineages, respectively31–33. Nonetheless, there is evidence that other inductive factors (including Notch1) might be involved in cell-fate specification at this particular developmental stage. Two types of experiments have led to the suggestion that Notch1 influences the αβ/γδ-lineage decision6. First, BM precursor cells hemizygous for Notch1 (Notch1+/−) give rise preferentially to γδ cells when they develop in the thymus of mixed chimeras in the presence of wt precursors (Notch1+/+). Second, overexpression of Notch IC under the control of the proximal lck promoter in transgenic mice overcomes the block in αβ T-cell development imposed by the absence of TCRβ (and hence a pre-TCR). Collectively, these data have been interpreted to mean that Notch1 signaling favors the development of αβ lineage cells at the expense of the γδ lineage6. Although suggestive, neither of these experimental approaches is definitive, and complementary loss-offunction studies will ultimately be required to clarify the role of Notch1 in αβ/γδ-lineage commitment. In this regard, the absolute requirement for Notch1 in T-cell-fate specification of CLPs (see above) complicates the situation and dictates that tissuespecific inactivation of Notch1 at a subsequent stage of thymic development will be necessary to address this issue directly. This problem is complicated further by the fact that there is no consensus as to the developmental stage where αβ- and γδ-T-cell lineages diverge. Nevertheless, a study in which Notch1 was inactivated at the late pre-T (CD25+CD44−) stage of development (using a Cre recombinase transgene driven by a CD4 mini-gene promoter–enhancer–silencer cassette) did not reveal any detectable effect on absolute numbers of αβ or γδ cells in the thymus14. Thus, a conservative conclusion would be that any essential role for Notch1 in αβ/γδlineage commitment must occur before the late pre-T-cell stage of development. Tissue-specific inactivation of Notch1 at earlier developmental stages in the thymus (using Cre recombinase transgenes driven by lck or CD2 promoters) should provide a definitive answer to the question of whether Notch1 signaling controls αβ/γδ-lineage commitment.
αβ/γγδ -lineage commitment
A second stage of T-cell development that implicates Notch1 signaling is the αβ/γδ-lineage decision. Following commitment to the T-cell lineage, immature pro-T cells begin to rearrange and express their TCRγ, -δ and -β genes. Pro-T cells that successfully rearrange TCRγ and TCRδ will express a γδ TCR and be eligible to develop further as γδ T cells. By contrast, productive TCRβ gene rearrangement will lead to expression of a pre-TCR (consisting of a TCRβ chain associated with an invariant pTα chain), which is compatible with further development as http://immunology.trends.com
CD4/CD8-lineage commitment
A final stage of T-cell development that has been linked to Notch1 signaling is the CD4/CD8-lineage decision. After successfully re-arranging TCRβ and expressing a pre-TCR, αβ-lineage-committed pre-T cells expand and progress to the CD4+CD8+ double-positive (DP) stage of development. At this point, successful TCRα re-arrangement leads to expression of an αβ TCR that can be positively or negatively selected as DP thymocytes progress to the mature CD4+CD8− single-positive (CD4+ SP) and
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Table 1. Evidence that Notch1 influences CD4/CD8-lineage commitment, maturation and survivala Experimental system
Result
Ref.
Transgenic mice overexpressing Notch IC
Selective increase in CD8+ SP thymocytes
5
Thymoma transfected with Notch IC
Increased resistance to glucocorticoid-induced apoptosis
7
Thymoma transfected with Notch IC
Increased resistance to activation-induced apoptosis
8
Antisense Notch1 in re-aggregate FTOC
Decreased maturation of CD8+ SP thymocytes
38
Transgenic mice overexpressing Notch IC
Increased maturation of CD4+ SP and CD8+ SP thymocytes
10
aAbbreviations: FTOC, fetal thymus organ culture; Notch IC, Notch1 intracellular domain; SP, single positive.
CD4−CD8+ SP (CD8+ SP) stages34. The αβ TCR on mature CD8+ SP and CD4+ SP cells recognizes peptides presented by MHC class I or class II, respectively. Therefore, it is possible that specific TCRαβ signaling (in conjunction with related signaling through lck-associated CD4 or CD8 coreceptors) is sufficient in itself to direct DP cells to the CD4 or CD8 lineage. Alternatively, it is possible that CD4/CD8-lineage determination in DP cells occurs independently of TCR–MHC interactions, and that the latter are required only to ensure the survival of cells with an appropriate match of coreceptor and TCR specificity. In agreement with the latter hypothesis, several reports have implicated Notch1 signaling either in the CD4/CD8-lineage commitment event or in the subsequent maturation and survival of CD4+ SP and/or CD8+ SP cells. These studies (summarized in Table 1) are largely based on gain-of-function approaches in which Notch IC is overexpressed either in DP thymoma cells or in transgenic mice. The conclusions of these studies have been seriously challenged by a report14 where the Notch1 gene has been inactivated in a tissue-specific fashion in CD25+CD44− late pre-T cells (using a CD4 minigene-driven Cre-recombinase transgene). Surprisingly, inactivation of Notch1 signaling at this relatively early stage has no effect on the subsequent steady-state development of DP or mature CD4+ SP and CD8+ SP thymocytes. Moreover, bromodeoxyuridine (BrdU) turnover studies and competitive mixed BM chimeras provide no evidence that Notch1 influences the rate of maturation or survival of CD4+ SP or CD8+ SP cells. Finally, in contrast to conclusions reached using DP thymoma cell lines, Notch1 deficiency in DP thymocytes does not affect their sensitivity to spontaneous or glucocorticoid-induced apoptosis. The conclusions reached on the basis of tissuespecific Notch1 inactivation in immature thymocytes14 thus conflict sharply with earlier reports suggesting that Notch1 is important for CD4/CD8-lineage commitment, maturation and http://immunology.trends.com
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survival. In attempting to reconcile these discrepancies, it should be noted that overexpression of Notch IC in transgenic mice or thymoma cell lines (Table 1) might activate signaling pathways that are not normally controlled by Notch1 under physiological conditions. For example, Notch IC might activate signaling that is normally mediated via Notch2 or Notch3 (both of which are expressed in the thymus)35. In this context, overexpression of the cytoplasmic domain of Notch3 in immature thymocytes of transgenic mice (under the control of the proximal lck promoter) does not specifically affect CD4/CD8-lineage commitment, although it does result in an increase in absolute numbers of thymocytes36. Nevertheless, it remains a formal possibility that Notch signaling is (at least partially) redundant for CD4/CD8lineage commitment, such that a loss-of-function mutation of Notch1 alone would have no apparent phenotype. Whatever the explanation, it is clear that a unique role for Notch1 in CD4/CD8-lineage commitment, maturation or survival can be excluded on the basis of the conditional genetargeting data. Concluding remarks
Notch1 has been implicated in three distinct (and sequential) lineage-specification events during T-cell development. In particular, signaling via Notch1 has been postulated to: (1) direct the differentiation of a bipotential CLP towards a T- (as opposed to a B-) cell fate; (2) favor the differentiation of pro-T (or pre-T) cells towards the αβ (as opposed to γδ) lineage; (3) favor the differentiation, maturation or survival of DP thymocytes towards the CD8+ SP and/or CD4+ SP lineages. All of these putative fate-determining properties of Notch1 are supported by gain-of-function studies that involve overexpression of Notch IC in BM precursors, transgenic mice or thymoma cell lines. However, as summarized in Fig. 1, recent loss-of-function studies in inducible and tissue-specific Notch1 knockout mice only confirm a critical role for Notch1 signaling in T/B-cell-fate specification. By contrast, these studies exclude an essential role for Notch1 in CD4/CD8lineage commitment and restrict its putative role (if any) in αβ/γδ-lineage commitment to an early developmental stage. Taken together with other studies37 indicating that inducible inactivation of Notch1 in BM precursors does not affect the development of myeloid, erythroid or other lymphoid (natural killer or thymic dendritic cell) lineages, it is tempting to speculate that the only non-redundant function of Notch1 in the hematopoietic system is to direct bipotential CLPs to the T-cell lineage. Future derivation (and intercrossing) of mutant mouse strains deficient in genes encoding other members of the Notch family and their respective ligands should help to clarify the general role of Notch signaling in hematopoiesis.
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TB vaccines: progress and problems Peter Andersen Tuberculosis (TB) is the biggest killer worldwide of any infectious disease, a situation worsened by the advent of the HIV epidemic and the emergence of multi-drug resistant strains of Mycobacterium tuberculosis. The existing vaccine, Mycobacterium bovis bacille Calmette–Guérin (BCG), has proven inefficient in several recent field trials. There is currently intense research using cutting-edge vaccine technology to combat this ancient disease. However, it is necessary to understand why BCG has failed before we can rationally develop the next generation of vaccines. Several hypotheses that might explain the failure of BCG and the strategies designed to address these shortcomings are discussed.
Peter Andersen Dept of TB Immunology, Statens Seruminstitut, 5 Artillerivej, DK-2300 Copenhagen S, Denmark. e-mail: [email protected]
Tuberculosis (TB) is a major global health problem that claims more than two million lives each year1. In developing countries, the incidence of TB has always been high and in industrialized countries the disease has re-emerged as a public health problem2. This development has been accelerated by the advent of the
HIV epidemic and the increasing incidence of multi-drug-resistant strains of Mycobacterium tuberculosis. The current TB vaccine, Mycobacterium bovis bacille Calmette–Guérin (BCG), was developed almost a century ago and, although this vaccine has proved inefficient, in several field trials, it is still in widespread use3. In 1993, TB was declared a global health emergency by the World Health Organization (Press Release: WHO attacks global neglect of tuberculosis crisis. Millions dying from ‘low priority’ disease). This statement came after more than two decades of international neglect of this important disease. Since then, funding for TB research has increased steadily and, today, TB immunology and vaccinology are two of the most rapidly evolving fields in medical research. Furthermore, TB research has benefited in recent
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