nature immunology • volume 3 no 3 • march 2002 • http://immunol.nature.com

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The question of how an organism developshas preoccupied scientists for many centuries.As experimental tools have become moresophisticated, new approaches have been usedto address this question. Morphologicalchanges detected by the naked eye were sup-planted by investigations using microscopes.Cell movement, expansion and organizationinto structures within organs are still exam-ined today, but new experimental tools nowallow the study of molecular interactionsbetween the developing cell and its envi-ronment as well as the molecular changesthat occur within the cell. In this issue ofNature Immunology, Kaye and colleaguesuse the recently developed gene-chip tech-nology to identify a murine gene involvedin thymocyte differentiation1.

In most cases of development and differ-entiation, a precursor cell takes cues fromits surroundings to expand and, under cer-tain circumstances, migrate to the appropri-ate part of the body where it develops spe-cialized functions. During this process thecells acquire molecular sensors on theirsurface that can interact with other mole-cules present on neighboring cells, in theextracellular matrix or with soluble factorspresent in the microenvironment. Theseinteractions trigger a cascade of events inthe cytoplasm—which are collectivelyknown as signal transduction—and inducea variety of cytoplasmic and nuclear activ-ities. These include the release of granulecontent; increased or decreased ability totranslate mRNA and release of proteinsfrom their molecular traps (in the cyto-plasm); or activation, silencing or modula-tion of gene expression, fragmentation ofnuclear material and the entrance to or exitfrom cell cycle (in the nucleus).

Likewise, in the immune system someof the hematopoietic stem cells destined todevelop toward the T cell lineage migrate tothe thymus. Many years of extensive studyhave shown that these cells undergo dramat-ic changes in surface protein expression thatare associated with precise developmentalstages in the thymus. For example, changesin CD4 and CD8 expression define the earlydouble negative (DN, or CD4–CD8–), the

Thymocyte differentiation:it’s time to bend a littleDIMITRIS KIOUSSIS

The development of new experimental toolsis helping to unravel the molecular secrets ofcell differentiation.The identification, by genechip technology, of a HMG box proteincalled TOX has yielded valuable informationabout thymocyte differentiation.

intermediate double positive (DP, orCD4+CD8+) and the mature single positive(SP, or CD4+ or CD8+) stages. Changes inCD44 and CD25 expression that define theDN1, DN2, DN3 and DN4 developmentalstages within the DN population are alsouseful and informative. Numerous studies ofhow these phenotypic changes affect the

decisions taken by the developing thymocytehave indicated that the way the cells interprettheir environment is modified as differentia-tion proceeds, thus propelling the cell to anever more defined identity2,3.

When messages from the environmentreach the nucleus via signal transduction cas-cades, differential gene expression is induced.Obviously, differential gene activation deter-

mines what surface molecules are expressedin the next phase of differentiation, which, inturn, determines what new structures the cellcan recognize, where it will go and what itwill adhere to. Gene expression will alsodetermine what proteins are available for thesignaling machinery, thereby dictating thequality, duration and intensity of signals with-

in the cytoplasm. In this respect, it isbecoming more and more evident thatthese properties of signaling have pro-found effects on developmental decisions4.

Until now efforts have primarily focusedon how surface interactions between, forexample, TCR–major histocompatibilitycomplex (MHC), coreceptor-receptor andadhesion molecule–receptor, as well asproximal signaling, drive differentiation.Such approaches have enlivened the T celldevelopment field with raging debates overwhat is important during thymocyte differ-entiation and lineage commitment to γδand αβ TCR lineages or CD4 and CD8 lin-eages. But however important these studiesmay have been, they have presented thefield with ambiguous and sometimes con-tradictory results. Fewer attempts havebeen made to address events that occurwithin the nucleus.

Scientists studying T cell developmentare beginning to appreciate the importanceof the nuclear proteins that control chro-matin structure and, by extension, regulatedifferential gene expression. Some studieshave tried to identify molecular messengerswith nuclear targets and determine whatgenes they target (outside-in)5,6. Otherattempts have concentrated on genes that aredifferentially expressed in an attempt to con-nect the molecular mechanisms that underlietheir unique expression profile to signalingand surface interactions (inside-out)7,8. Kaye

and colleagues use such an approach to dissectT cell lineage commitment1.

Kaye and colleagues describe the isolationof a gene that encodes a protein called TOX(for thymus HMG box)1. This protein carries adomain that has attracted a lot of attentionfrom the field of chromatin structure and generegulation. This domain binds DNA (in asequence- or structure-specific manner) and

Figure 1. Possible disruption of nucleosomes. (a) Theinitial interactions of HMG-containing proteins (H) withDNA may disrupt the structure of nucleosomes due to theirability to introduce a bend in the backbone of the nucleicacid. (b) As the DNA unravels new protein-binding sites areexposed on the DNA and the recruitment of additionalnuclear factors (X and Y) starts. (c) These factors can inter-act, but they may be too far apart from each other. HMG boxprotein (H) exerts its bending activity and forces the two fac-tors (X and Y) closer together.Additional domains present inthese HMG proteins can have activating functions or canrecruit other proteins. (d) The final result is that a newremodeled region of the chromatin is formed where previ-ously only nucleosomes existed.

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http://immunol.nature.com • march 2002 • volume 3 no 3 • nature immunology 215

causes a bend in the nucleic acid backbone(Fig. 1). Proteins containing HMG boxes thatregulate genes specific to T cells have beendescribed before and include T cell factor(TCF), lymphoid enhancer factor (Lef) andHBP1 (HMG box-containing protein 1)9–11. Asthe special architectural arrangement of DNAand associated proteins is thought to be impor-tant in the regulation of gene expression, aprotein that can impose structural changes,such as bending, would be a major player inthis process (Fig. 1). This property and thepattern of TOX expression (which is highest inthe thymus) prompted the authors to examineits role in thymocyte differentiation1.

Apart from the strangely high amounts ofTOX expressed at the very early stages of thy-mocyte development (DN1 and DN2) itsexpression pattern suggested an involvementin the DP to SP transition phase1. From hereon its role becomes less clear, as mature naïvecells responding to antigen no longer modu-late its expression1.

More striking, however, are the results fromtransgenic mice that express TOX. Althoughsuch mice do not show overt changes in theiroverall thymus cellularity, they show anincrease in the production of CD8+ SP thymo-cytes at the expense of SP CD4+ T cell numbers.These CD8+ SP T cells also appear to expressTCRs that, under normal circumstances, arepreferentially expressed on CD4+ SP T cells.What is even more intriguing is the fact that theappearance of these cells is unaffected by theabsence of MHC class I. One is tempted toargue that TOX over-expression has removedthe need for CD4 coreceptor signaling, so thateven those cells that would normally be lostbecause of CD4 down-regulation are now res-

cued. Nevertheless, in the absence of evidencethat such cells may have been selected on MHCclass II it is too early to draw firm conclusions.

Kaye and colleagues propose that TOXaffects lineage commitment and, when over-expressed, leads to the CD8 pathway of dif-ferentiation, regardless of TCR specificity1.There is no question that, in this case, genet-ic manipulation has caused a distortion in lin-eage commitment1; similar perturbations inlineage commitment have been reported inother cases, such as with CSK (COOH-termi-nal Src kinase)–deficient mice12. In the articleby Kaye and colleagues1, genetic manipula-tion disrupted TOX expression both in quan-titative terms (over 20 times more protein wasfound in the thymus of TOX-transgenic com-pared to wild-type mice) and in qualitativeterms (the fine developmental regulation ofTOX expression during the DN1→DN4 andDN4→DP→SP transitions was lost) so thatuniformly high expression occurred through-out development. In addition, because highTOX expression was maintained continuous-ly, it is likely that downstream targets werealso activated or silenced, which induced thephenotypic repercussions observed1.

The article by Kaye and colleagues1 opensan exciting new avenue within the field ofthymocyte differentiation and future workshould clarify the precise points at whichTOX regulates thymocyte development. Myprediction is that, in most cases, no one sin-gle gene will determine the lineage commit-ment of a cell. It is unlikely that, during evo-lution, nature would have put all its eggs inone basket. Witness the redundancy identifiedin gene knock-out studies or the presence ofmultiple members of a gene family that can

“stand-in” when a relative is absent. A moreplausible scenario is the careful orchestrationof the expression of molecules that turn thecell in one direction or another. All thesemolecules probably contribute, to a certainextent, to lineage commitment, and changesin expression, amounts or timing are likely toensure that the precise events take place at thecorrect time. Undoubtedly, disrupting theexpression of any one of these moleculeswith the use of transgenes or knock-out dele-tions is bound to yield important and valuableinformation, such as that reported in the arti-cle by Kaye and colleagues1. We shouldalways, however, remain open to potentiallydifferent interpretations. Only the gradualaccumulation of information, the correlationbetween the different findings and construc-tive discussion of new data will lead to futurebreakthroughs in the understanding of howthymocyte development and lineage commit-ment works.

1. Wilkinson, B. et al. Nature Immunol. 3, 272–280 (2002).2. Robey, E. & Fowlkes, B. J. Annu. Rev. Immunol. 12, 675–705

(1994).3. Godfrey, D. I., Kennedy, J., Suda,T. & Zlotnik,A. J. Immunol. 150,

4244–4252 (1993).4. Basson, M.A. & Zamoyska, R. Immunol.Today 21, 509–514

(2000).5. Lee, G. R., Fields, P. E. & Flavell, R.A. Immunity 14, 447–459

(2001).6. Lopez-Rodriguez, C. et al. Immunity 15, 47–58 (2001).7. Hostert,A. et al. Immunity. 9, 497–508 (1998).8. Ellmeier,W., Sawada, S. & Littman, D. Annu. Rev. Immunol. 17,

523–554 (1999).9. Reya,T., Okamura, R. & Grosschedl, R. Cold Spring Harb. Symp.

Quant. Biol. 64, 133–140 (1999).10. Staal, F. J et al. Stem Cells 19, 165–179 (2001).11. Zhuma,T. et al. EMBO J. 18, 6396–6406 (1999).12. Schmedt, C. & Tarakhovsky,A. J. Exp. Med. 193, 815–826 (2001).

Division of Molecular Immunology, National Institutefor Medical Research,The Ridgeway London NW71AA, UK. (dkioussis@nimr.mrc.ac.uk)

Within the complex biology of immune sys-tem regulation, mechanisms that down-modu-late host immune responses are likely to beequally important as those that activate them.Proper regulation of immune responsesrequires the effective and timely action ofmechanisms that shut-off host immune

TS cells and immunetolerance induction: aregulatory renaissance?MARK B. FEINBERG AND GUIDO SILVESTRI

CD8+ TS cells induce antigen-specifictolerance.The TS cells may do this byincreasing the expression of inhibitoryreceptor ILT3 and ILT4 on DCs, renderingthese cells toleregenic to CD4 cells.

responses. In this way, uncontrolled lympho-cyte proliferation upon exposure to antigenicstimuli can be avoided and the generation ofaberrant immune responses that target hostantigens and lead to the development ofautoimmune diseases can be prevented. Theconcept of suppressor T (TS) cells was first

developed in the 1970s; it was envisioned thatthis subset of lymphocytes was responsible forthe active control, and ultimately the termina-tion, of immune responses1. TS cells becamedisfavored in the 1980s and early 1990s, main-ly because of difficulties in identifying a dis-tinct phenotype for antigen-specific TS cells.

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