NATURE IMMUNOLOGY VOLUME 8 NUMBER 2 FEBRUARY 2007 119

Shaping a helper T cell identityHarinder Singh

Cell fate ‘decisions’ involving multipotential progenitors seem to be ‘dictated’ by threshold activities of lineage-determining transcription factors. Such ‘decisions’ are now shown to be reinforced by the induction of secondary transcription factors that act in concert with the primary regulators.

Naive CD4+ helper T cells differentiate into T helper type 1 (TH1) or TH2 cells that

in turn regulate immune responses to a vari-ety of pathogens based on the expression of genes encoding specific cytokines1. TH1 cells characteristically express interferon-γ (IFN-γ), whereas TH2 cells produce interleukin 4 (IL-4) and IL-5. From a developmental standpoint, that differentiation system enables the analysis of cell fate ‘choice’ in the context of an immune response. Stimulation of naive T cells through their antigen receptors along with cytokine or other extracellular signals directs their differ-entiation along the TH1 or TH2 pathway. The genes encoding IFN-γ (Ifng) and IL-4 (Il4) have been subjected to extensive molecular analy-sis. Those experiments have resulted in the characterization of key transcription factors, cis-acting regulatory sequences and epigenetic modifications that regulate Ifng or Il4 expres-sion during the course of TH1 or TH2 cell dif-ferentiation2. A key finding is that both genes are maintained in a poised or primed state in naive T cells. Therefore, cellular differentiation involves the concerted activation and repres-sion of alternative lineage-specific cytokine genes. In this issue of Nature Immunology, Djuretic et al. explore the molecular mecha-nisms by which Ifng is optimally activated and Il4 is simultaneously repressed after naive T cells are induced to differentiate into TH1 effector cells3. Their principal finding is that the transcription factor T-bet, the chief TH1 cell fate determinant, induces the expression of a secondary transcription factor, Runx3. The

two regulatory proteins bind to cis elements in the Ifng and Il4 loci in differentiating TH1 cells, activating the former gene while repress-ing the latter. This study nicely illustrates a general developmental principle that has been encountered in the context of other cell fate ‘choices’ in the hematopoietic system (Fig. 1).

The authors use both gain-of function and loss-of-function approaches to establish their main conclusions3. In so doing, they identify previously unknown regulatory functions for Runx3, which has been shown before to be

pivotal in the generation of CD8+ T cells by repressing Cd4 (ref. 4). Runx3 is shown here to be induced specifically in TH1-polarizing conditions and its expression is dependent on T-bet. Runx3-deficient TH1 cells are found to be impaired in their expression of IFN-γ. Conversely, ectopic expression of Runx3 promotes IFN-γ expression in nonpolar-izing conditions. However, Runx3, unlike T-bet, does not induce IFN-γ expression when ectopically expressed in differentiating TH2 cells. Collectively, these results establish that

Harinder Singh is in the Department of Molecular

Genetics and Cell Biology, Howard Hughes Medical

Institute, The University of Chicago, Chicago, Illinois

60637, USA.

e-mail: hsingh@uchicago.edu

T-bet

PU.1

EBF

Runx3

Egr-1, Egr-2

Pax5

Ifng (TH1)

Il4 (TH2)

Csf1r (macrophage)

Il1r1 (neutrophil)

Cd79a (B cell)

Csf1r (macrophage)

?

Primary regulator

Secondary regulator

Lineage-specifictarget genes

Nab2

Figure 1 Gene-regulatory networks orchestrating three distinct cell fate ‘choices’ in the immune system. Each network is composed of a primary and a secondary regulator. The expression and function of the latter is contingent on the former. Both regulators function in a concerted way to activate lineage appropriate genes. One or both simultaneously repress alternative lineage genes. Green lines, gene activation inputs; red lines, gene repression inputs.

N E W S A N D V I E W S©

2007

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

eim

mun

olog

y

120 VOLUME 8 NUMBER 2 FEBRUARY 2007 NATURE IMMUNOLOGY

T-bet is a primary regulator of Ifng expression and Runx3 cooperates with T-bet to promote the activity of the TH1 lineage–specific gene. Thus, Runx3 can be viewed as a component of a positive-feedback circuit that reinforces TH1 cellular identity induced by T-bet.

Runx3-deficient TH1 cultures also manifest an increased frequency of cells expressing both IFN-γ and IL-4, indicating a failure to prop-erly silence the alternative lineage cytokine gene. Notably, when expressed along with T-bet in developing T cells, Runx3 promotes repression of Il4. Finally, gene repression is partially dependent on a silencer element in Il4, which contains binding sites for T-bet and Runx3 (discussed below). These experiments provide compelling evidence that T-bet and Runx3 repress the alternative lineage Il4 dur-ing TH1 differentiation. Thus, TH1 identity is ‘collaboratively’ shaped by T-bet and Runx3 by accentuation of one set of molecular features and elimination of an alternative set.

How do T-bet and Runx3 cooperate to activate Ifng while repressing Il4? The authors provide key molecular clues, but the mechanistic details remain to be eluci-dated. T-bet and Runx3 bind the promoter of Ifng, and although crosslinking of T-bet in vivo is modulated by Runx3, it is not strictly dependent on the latter. Their results lead the authors to suggest that T-bet can initi-ate alterations in the chromatin structure of the Ifng promoter that then enable Runx3, after its induction, to bind the promoter and augment gene activation. Thus, T-bet may function as a ‘pioneer transcription factor’

and Runx3 action in TH1 cells is contingent on T-bet.

The mechanisms by which T-bet and Runx3 repress Il4 seem to be different. In this case, the authors suggest that T-bet and Runx3 coopera-tively bind to a composite DNA element in the Il4 silencer. Gel-shift assays and coimmunopre-cipitation analysis are consistent with that pos-sibility. However, why in this gene context the binding of T-bet and Runx3 together results in repression remains to be explored. It has been suggested before that Runx3 functions as a repressor as well as an activator in the context of the CD4-versus-CD8 T cell fate ‘choice’5. The dual regulatory capabilities of Runx3 are resur-rected during TH1 versus TH2 differentiation.

These results nicely fit with an emerging general model for cell fate determination in the hematopoietic system6. Hematopoietic multi-potential progenitors have low mixed-lineage gene expression patterns7, a phenomenon called ‘transcriptional priming’. Consequently, cell fate specification depends on the induc-tion as well as repression of subsets of alter-native lineage-specific genes. In the context of macrophage differentiation, the primary transcription factor PU.1 induces the expres-sion of a secondary transcription factor, Egr-2, and the two function in concert to positively regulate a large set of macrophage genes, including Csf1r, which encodes the receptor c-Fms6. Egr-2 and the related family member Egr-1 interact with the corepressor Nab2 to repress neutrophil genes (Fig. 1). Notably, in the context of early B cell differentiation, the primary cell fate determinant EBF induces the

expression of a secondary determinant, Pax5 (ref. 8). The function of Pax5 in activating B lineage genes is contingent on EBF. For Cd79a (also called mb-1), EBF seems to function as a ‘pioneer transcription factor’ and participates in alterations of chromatin structure and DNA demethylation at the Cd79a promoter9. Those epigenetic modifications enable Pax5 to bind to the Cd79a promoter and stimulate high tran-scription of Cd79a. Pax5 also represses myeloid lineage genes, including Csf1r10. It remains to be determined if EBF functions along with Pax5 to repress alternative lineage myeloid genes during B cell differentiation. In conclusion, although mechanistic analyses will probably demonstrate molecular diversity, it seems that the fundamental architecture of gene regula-tory networks that are used to orchestrate dis-tinct lineage-specific gene expression patterns in the immune system is conserved. Such an architecture may also be used in a wide array of developmental contexts.

COMPETING INTERESTS STATEMENTThe author declares that he has no competing financial interests.

1. Szabo, S.J., Sullivan, B.M., Peng, S.L. & Glimcher, L.H. Annu. Rev. Immunol. 21, 713–758 (2003).

2. Murphy, K.M. & Reiner, S.L. Nat. Rev. Immunol. 2, 933–944 (2002).

3. Djuretic et al. Nat. Immunol. 8, 145–153 (2007). 4. Taniuchi, I. et al. Cell 111, 621–633 (2002).5. Yarmus, M. et al. Proc. Natl. Acad. Sci. USA 103,

7384–7389 (2006).6. Laslo, P. et al. Cell 126, 755–766 (2006).7. Hu, M. et al. Genes Dev. 11, 774–785 (1997).8. Medina, K.L. et al. Dev. Cell 7, 607–617 (2004).9. Maier, H. et al. Nat. Immunol. 5, 1069–1077 (2004).10. Mikkola, I., Heavey, B., Horcher, M. & Busslinger, M.

Science 297, 110–113 (2002).

More promiscuity resulting in more toleranceDietmar Zehn & Michael J Bevan

Tolerance to self is engendered by multiple mechanisms. Lymph node stromal cells are now found to contribute to self-tolerance by their endogenous expression of peripheral tissue antigens.

The evolution of an adaptive immune system, which is based on the clonal expression of

somatically generated receptors to recognize foreign antigens, necessitated the coevolution of ways to remove cells with harmful reactivity to self antigens. Purging the repertoire of auto-

reactive T lymphocytes is especially important for self-tolerance and may occur both during their differentiation in the thymus (referred to as ‘central tolerance’)1 and after they leave the thymus to circulate throughout the body (‘peripheral tolerance’)2 (Fig. 1). Central tol-erance easily deals with overt reactivity to ubiquitous self antigens, as antigen encounter prevents immature thymocytes from complet-ing their differentiation. Inducing tolerance to peripheral tissue–restricted antigens (PTAs) poses a greater problem, however. As shown by Lee and colleagues in this issue of Nature

Immunology3, the complexity and intriguing nature of the means of enforcing self-tolerance is still emerging.

It was a revelation that the ‘ectopic’ thymic expression of many peripheral genes (or trans-genes driven by promoters with tissue-restricted activity) was not ‘background noise’ but was a perfectly evolved way of inducing central tol-erance to PTAs or developmentally regulated self antigens4. In the thymus, the medullary epithelial cells ‘promiscuously’ express a broad range of genes otherwise unique to organs such as the eye, pancreas and many others. As a

Dietmar Zehn and Michael J. Bevan are in the

Department of Immunology, Howard Hughes

Medical Center, University of Washington, Seattle,

Washington 98195, USA.

e-mail: mbevan@u.washington.edu

NEWS AND V IEWS©

2007

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

eim

mun

olog

y