NATURE IMMUNOLOGY VOLUME 6 NUMBER 7 JULY 2005 641

The cell surface receptor Notch is involved in lineage fate decisions in a variety of

tissues in various species1. Three papers in this issue of Nature Immunology deal with the effect of Notch signaling on the diffe-rentiation of lymphoid cells. Tan et al.2 and Sambandam et al.3 show that the presence of intrathymic early T lineage precursors (ETPs) but not the phenotypically similar pluripotent c-Kit+Sca-1+ precursors in bone marrow and blood essentially depend on active Notch. This finding raises new issues regarding the nature of thymic immigrants. Minter et al.4 report on a requirement for Notch-dependent transcrip-tion in the polarization of interferon-γ-pro-ducing T helper type 1 (TH1) effector cells. This work may afford new ways of interfering with disease caused by TH1 cells. Overall, these findings reinforce the idea that Notch exerts stringent control over lineage commitment at various levels of T cell maturation.

Notch receptors control differentiation and proliferation in response to ligands on neighboring cells1. In mammals, Notch recep-tor ligands are referred to as Delta-like 1–4 (DL1–DL4) as well as Jagged. Ligand binding triggers a series of proteolytic cleavages that release the intracellular portion of the Notch receptor (ICN) from the plasma membrane. This ICN then localizes to the nucleus, where it binds to the CSL transcription factor, which is also known as RBP-Jκ1, CBF-1, suppressor of hairless and Lag-1 in various species. In the absence of ICN, CSL represses transcription by interacting with various corepressors. When ICN binds to CSL, it recruits the coactivator mastermind-like 1 (MAML1), which binds to

ICN in the ICN-CSL-DNA complex, thereby converting the CSL complex into a tran-scriptional activator. The genes regulated by Notch-dependent transcription include Hes1 as well as Ptcra, which are important in T cell development.

Modulation of Notch signaling can be achieved by various approaches, of which three have been used in the three new reports2–4. Lunatic fringe (L-Fng), a glycosyltransferase, interferes with Notch receptor–ligand bind-ing. The γ-secretase inhibitors (GSIs) prevent proteolytic cleavage by the γ-secretase complex containing presenilin and nicastrin, which cleave membrane-bound Notch after ligand

binding. Finally, transgenic expression of a dominant negative MAML1 that can still bind to ICN but lacks the activation domain prevents Notch-dependent transcription5 (Fig. 1).

ETPs characterized by high expression of the c-Kit receptor and low expression of the interleukin 2α receptor chain (CD25) were considered to represent thymic immigrants because of their potential to generate T cells and B cells as well as some myeloid cells and because cells with a similar phenotype are present in blood6,7. ETPs have also been defined as cells lacking lineage markers (sur-face molecules present on more-differentiated lymphoid cells) and having high expression of

Notch in lymphopoiesis and T cell polarizationHarald von Boehmer

Notch is essential for T lineage cell differentiation. Three new papers look at when Notch signaling is required and how it affects different cell types.

Harald von Boehmer is at the Dana-Farber

Cancer Institute, Harvard Medical School, Boston,

Massachusetts 02115, USA.

e-mail: harald_von_boehmer@dfci.harvard.edu

Figure 1 ICN-dependent transcription and inhibition. Notch ligands (DSL) bind to extracellular Notch, resulting in cleavages of the Notch receptor that is critically dependent on the γ-secretase complex containing nicastrin and presenilin. ICN localizes to the nucleus, transforming the transcriptional repressor CSL into a transcriptional activator by recruiting MAML1 and possibly other coactivators. Studies have used L-Fng, which glycosylates the extracellular portion of Notch and thereby interferes with DSL binding2; GSIs, which prevent the proteolytic cleavage and therefore the generation of ICN4; and dominant negative MAML1 (dnMAML1), which interferes with activation of CSL3. MP, metalloprotease. Adapted from ref. 17. Reprinted from Curr. Opin. Chem. Biol., 6, Nam, Y., Aster, J.C. & Blacklow, S.C., Notch signaling as a therapeutic target, 501–509, 2002, with permission from Elsevier.

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642 VOLUME 6 NUMBER 7 JULY 2005 NATURE IMMUNOLOGY

A plague of autoantibodiesVigo Heissmeyer, K Mark Ansel & Anjana Rao

Hypermutation of antibody-producing B cells occurs in germinal centers, but how autoimmunity from the generation of potentially self-reactive antibodies is avoided has remained puzzling. Characterization of a new mouse mutant, sanroque, indicates previously unknown tolerance mechanisms act at this stage.

Vigo Heissmeyer, K. Mark Ansel and Anjana Rao are with the Department of Pathology, Harvard Medical School and the CBR Institute for Biomedical Research, Boston, Massachusetts 02115, USA. e-mail: arao@cbr.med.harvard.edu

Published online 29 May 2005: doi:10.1038/ni1214

c-Kit and medium to high expression of CD24 (ref. 8). However, unlike extrathymic c-Kithi cells, ETPs have rather limited B cell potential despite the fact that the ETP subset includes a fraction of cells with the Flt-3 receptor3 that in the bone marrow is present on immature lymphocyte precursors9. Analyses of the Notch dependence of ETPs and extrathymic lymphoid precursors in bone marrow and blood of transgenic mice expressing domi-nant negative MAML1 and L-Fng produced the somewhat unexpected finding that ETPs were mostly absent from the thymus2,3, whereas extrathymic precursors of lympho-cytes were not affected3. Moreover, thymi from the transgenic mice contained plenty of B cells that were derived from Notch-inhibited precursors, as reported earlier for Notch-defi-cient mice10. So, as for thymic immigrants, it is back to square one, especially because most of the c-Kitlo cells in a normal thymus express lineage markers3. There are several possible solutions to this dilemma. First, the thymus is colonized by a small number of c-Kithi immigrants that immediately undergo Notch signaling after entering the thymus. Second, the thymus is colonized by Notch signal-ing–dependent precursors such as ETPs and distinct precursors for B cells that can still be inhibited by Notch signaling and are not contained in the ETP subset. ETPs committed to the T lineage, however, have so far escaped detection outside the thymus in adult mice. Moreover, dominant negative MAML1 has no effect on extrathymic lymphocyte precur-sors3. Finally, playing ‘devil’s advocate’, one could argue that thymic immigrants repre-sent lineage marker–positive lymphocyte

precursors with T cell and B cell potential11 that under the influence of Notch signaling inefficiently dedifferentiate into ETPs, as has been described for B220+CD19– precursors from Pax5-deficient mice, cultured on DL1 Notch ligand– expressing stromal cells12,13. Such cells downregulate B220, upregulate c-Kit and eventually lose Flt-3 expression12. These proposals seem sufficiently provocative to stimulate further search for the elusive thy-mic immigrant.

The manuscript by Minter et al.4 follows up on observations concerning Notch signal-ing in the polarization of T cells into TH1 or TH2 effector cells14. Here, loss of Notch func-tion was achieved by the use of GSIs in vivo or in vitro before exposure of naive T cells to TH1- or TH2-polarizing conditions in cul-ture. Whereas TH1 polarization was inhibited by GSI, TH2 polarization was not affected. Notably, the pretreatment did not reduce cell proliferation during the first 96 hours in which interferon-γ secretion was already abolished. Thus, cytokine production rather than pro-liferation was affected by the reduction in ICN. Because the transcription factor T-bet is indispensable for TH1 commitment15, the researchers investigated whether ICN directly upregulates expression of the gene encoding T-bet (Tbx21), which was found to be absent from cells pretreated with GSI. Indeed, chro-matin precipitation using antibodies to Notch1 demonstrated a Notch-CSL complex in ICN-transduced T cells that was bound to a putative consensus CSL binding site in the Tbx21 pro-moter. The apparent requirement for Notch signaling for TH1 polarization suggested that in vivo GSI treatment may ameliorate

experimental autoimmune encephalitis in a mouse model of multiple sclerosis. Indeed, pretreatment with GSI delayed the onset of disease and continued treatment suppressed the severity of disease. Although these studies seem convincing regarding an essential func-tion for ICN in TH1 polarization, there are caveats because of discrepant data obtained using disparate ICN constructs and animal models, as discussed4. Although these apparent conflicts and possible adaptive mechanisms that compensate for Notch deficiency need to be resolved, the introduction of Notch into the field of T cell differentiation16 has provided healthy controversy and excellent research tools for delineating lineage fate from early precursors of T cells to T effector cells.

1. Artavanis-Tsakonas, S., Rand, M.D. & Lake, R.J. Science 284, 770–776 (1999).

2. Tan, J.B., Visan, I., Yuan, J.S. & Guidos, C.J. Nat. Immunol. 6, 671–679 (2005).

3. Sambandam, A. et al. Nat. Immunol. 6, 663–670 (2005).

4. Minter, L.M. et al. Nat. Immunol. 6, 680–688 (2005).

5. Nam, Y., Weng, A.P., Aster, J.C. & Blacklow, S.C. J. Biol. Chem. 278, 21232–21239 (2003).

6. Allman, D. et al. Nat. Immunol. 4, 168–174 (2003).7. Schwarz, B.A. & Bhandoola, A. Nat. Immunol. 5, 953–

960 (2004).8. Porritt, H.E. et al. Immunity 20, 735–745 (2004).9. Sitnicka, E. et al. J. Exp. Med. 198, 1495–1506

(2003).10. Radtke, F. et al. Immunity 10, 547–558 (1999).11. Martin, C.H. et al. Nat. Immunol. 4, 866–873

(2003).12. Hoflinger, S. et al. J. Immunol. 173, 3935–3944

(2004).13. Schmitt, T.M. & Zuniga-Pflucker, J.C. Immunity 17,

749–756 (2002).14. Amsen, D. et al. Cell 117, 515–526 (2004).15. Szabo, S.J. et al. Cell 100, 655–669 (2000).16. Robey, E. et al. Cell 87, 483–492 (1996).17. Nam, Y., Aster, J.C. & Blacklow, S.C. Curr. Opin. Chem.

Biol. 6, 501–509 (2002).

The immune system spends an enor-mous portion of its ‘budget’ on security

measures to control its own aggressiveness.

Almost as critical as repelling invaders isprotecting healthy tissues from wanton attacks by activated lymphocytes. An impressive num-ber of regulatory mechanisms, collectively called ‘tolerance’, have evolved to prevent autoimmunity1. Some of these mechanisms involve deletion of self-reactive cells, whereas others impose compliance on potentially ‘thuggish’ factions of effector cells that are marshaled for immune defense. In a recent

issue of Nature2, Vinuesa et al. describe a previously unknown tolerance mechanism that, by restricting T cell help, specifically prevents autoantibody production by self-reactive B cells.

The authors derived a mouse strain with a salient autoimmune phenotype by backcrossing founder mice made mutant by ethylnitrosourea and screening the homozygous offspring for high titers of

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