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Haemopoiesis and early T-cell differentiation
trends reduced by PDT triggers membrane- nuclease actwatmn. Preliminary Jonathan D. Asbu,ell ts at the associated reactions that are early exl~enments show that polyamme Laboratory of lnlnlune Cell Bto,r~g3. synthesis may by invol, ed w,th the National Institutes of Hea:..,. signals of cell death by apoptosls. C. Carrera (San Diego) reported apoptotlc response of nondw,dmg ~3ethesda, MD 20892, USA; Nathaa on 2-chlorodeoxyadcnosine (2- lymphG :ytes to DNA damage. A. Begger ts at the Dwtsu.n of Hematology/Oncolog); Dept of CdA), a new class of antlmetabolite Me&cme and Btocbenustry, Cancer that i~ proving to be h:ghly effec- For the future? There is httle doubt that much Researct~ Center, Case Westera tive m the treatment of chronic lymphoid mahgnancles. The drug is progress has been made towards Reserve Umverslt3.; Cleveland, OH actwe against proliferating and understanding the process of PCD 44106, USA; John A. Ctdlowskt l~ at nonprohferatmg cells, and causes and, slgmficantly, towards the ap- the Dept of Physiology and DNA strand breaks, NAD con- phcation of this knowledge m chm- Btocbemtstr'y, Unn,erstty of North sumpnon and eventual interphase cally relevant situations. Reahmc Carohna School of Me&tree, Chapel death by apoptosis. Although the treatments for various tumnurs and Hdl, NC 275•6, USA; David P. Lane oligonucleosomal DNA fragmen- lymphoproliferative disorders may ts at the Umverslty of Dundee Cancer tation caused by 2-CdA can be pre- not be imminent, but they are now Researcb Campaign Laboratories, vented by treatment with phorboi no longer remote. The wealth of Dundee, UK DD1 4HN; Stanley ]. ester m wtro, the mmal DNA data presented at this meeting was Korsmeyer ts at the Hoz~ard Hugbes strand breaks are unaffected. This not intended to declare victory in Medical lnstmlte Research Labsuggests that activation of protein the war against cancer, but to indi- oratories, V,~a.shtngton Umverslty kinase C interferes with a DNA- cate that valuable weapons are School of [vle&cme, St Louis, MO 631 i0, USA. damage signal that precedes endo- being added to the armour,/.
Haemopoiesis and early T-cell differentiation Sophie Ezine and Rhodri Ceredig Lineage commitment ts a fundamental process that is initiated during the early stages of embryogenesis. Ultimately, this leads to the ge~,eration of the humoral and cellular arms of the immune system. In a recent workshop*, A fundamental quesuon in the : esearcbers from the diverse fields study of T-cell biology is the t~sue of embryology, haematology and of whether T cells are born or made. Before the complex forces of immunology gathered to address topic of early T-cell negative and positive selection can the differentiation. Imprint a pattern of self/noi~self discrimination on developing thymocytes, an apparently undifferentiated stem cell must commit itself to becoming an immature T cell, rather than a B cell or one og the other diverse components of the mature lymphoid system. The spectrum of work discussed at this meeting reflected the compartmentahzar~on of thi~ process, which begins in the haemopoietic tissue and fimshes m the thymus.
cells migrate to the fetal Iwer and colonize the bone marrow before birth. However, stem cells can also be found at other embryonic s~tes, such as the para-aortic splanchnopleura (PAS). Indeed, F. DleterlenLi&re (Nogent-sur-Marne) reported that PAS isolated from mouse emt-. . . . . . d.oy a of ge~rarlon fi.e. at the 20 somlte pmr stage) can give
Early haemopoiesis Totipotent haemopoletic stem cells are first detectable in the yolk sac. During development, these
*The Phihppe Laudat Conference on 'Haemopoiesis and early T-cell differentiation' was held at Le Bischenberg, Strasbourg, France, 5-9 September 1993.
rise both to IgM-secretlng plasma cells and to the Bla B-cell subset (IgMb-ghtB220dullCD5÷) after grafting into severe combined immunodeficiency (SCID) m,ce 1. PAS develop into B cells m vitro after ten days m the presence of lnterleukin 7 (IL-7), IL-3, stem-cell factor {SCF), 2-mercaptoethanol and the S17 stromal-cell line; and, in hmitmg dilution assay, PAS generate myelo~d cells in the presence of IL-3 and SCF (A. Cumano, Pans). E. Dzlerzak (London) addressed the ongm and ;requency of haemopoienc cells during embryomc development. Using the spleen colony-forming unit (CFU-S) assay, it was possible to detect day-8 CFU-b m tbe yolk sac and m embryonic bodies in embryos at the 26-27 somite pair stage 2. Furthermore, this activity was found to be
© 1994, Elsevier Sc,ence [ td 0167-569 ~/q4/$07 00
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trends restricted to the aorta-gemtalndge-mesonephrons (AGM) area and peaked at the 38-39 somite pa1~ stage. This population ~.; haemopoletac cells has a frequency of -'00 CFU-S 10-~ cells, which is ,,mdar to the adult bone marrow. However, the origin of these pluripotent AGM stem cells remains to be determined, as does the issue of whether or not they represent the equivalent of the mammalian fetal hver or the avian yolk sac. It remains to be answered whether there are many independent sites of stem-cell generation or whether various regions are seeded by cells from a unique source. Attempts to generate lymphoid cells m vitro from embryonic stem cells ~ESCs' have so far been unsuccessful. A malor problem m these studies has been the lack of definition of early markers that are specific for the T-cell lineage. However, S. Cande:as (Denver) reported that ESCs produce embryomc bodies alter growth for 14-21 days in methyl cellulose. In the presence of IL-7, Ik-2 and the $17 stromal-cell line, these embryonic bodies can gwe rise to lymphoidlike coionies (Thyl.2 b"ght FcRII/III÷ CD44 +) that can be grown in suspension in the presence of IL-2. Germline transcripnon of the gene for the V138.2 T-cell receptor (TCR) occu-s m these ESC-derwed embryonic bodies, suggesting that an early evc.nt m T-cell commitment is occurring. Transcripts are also detected in day 14 embryonic liver and thymus, and a specific V138 rearrangement is observed fi~r~ day 16 in the thymus, but not m the fetal liver. These germhne t~'anscripts have been detected in pre-Tcell lines (T3 and T7), but not in pre-B-cel! lines (38B9, 22D2). Lymphocyte development I. Wetsssman (Stanford) emphasized that stem cells m Thyl.l mice are sttll defined as Thyl.ll°LinSea-l* av.d that !ong-term reconsntution of all blood lineages in lethally irradiated mice can be achieved with a~ few a; 20 of these cells. Moreover, although these cells may be c-kit*, it appears that SCF does not mediate cell prohfe,~ ation in the bone marrow. In this respect, cychng cells are less efficient
at repopulat,on and are probabl) closer in the dlfferentmtton pathway to cells that wdl be committed to one lineage of dafferentianon.
by rearrangement at the TCR -/ locus and downregulanon of c-kit protem expression. Interestingly, thymlc development is arrested at the CD44-CD25 ÷ TN stage m Tbymtc d2fferentlatlon RAG-l-knockout mice (A. Zlotmk, lntrathymlc differentmtion events Palo Alto and S. Tonegawa, were defined according to cell- Cambridge, MA). Backcrosses of surface markers and the re- these mice w~th m~ce transgenic for arrangements of TCR genes that TCR 13 lead to the generation of eventually lead to the generanon CD4"CD8 ÷ thymocytes that are of a complete CD3/TCR a13 com- TCR 13*a- and express surface plex on mature ffymocytes. Early CD3e (S. Tonegawa, Cambridge, CD41°CD8-CD3 - thymocytes, as de- MA). A possible role for the incomfined by K. Shortman {Melbournel, plete CD3/TCR 13 complex may be are the precursors of a13 and ~/8 in the transductlon of signals T cells. These cells are more active revolved in the transition to the in repopulanng the thymus than CD4+CD8 ÷ double-positive (DP) are mple-negatwe (TN) CD3-CD4-- stage. Indeed, earher experiments CD8- cells. Furthermore, CD4 I° indicated that CD3~ is expressed cells can gwe rise to dendritic on the cell surface of double-negacells (DCs) expressing CD8a, CD4, twe (DN;CD44-CD25-CD41OCD8 l°) Thyl, CD2 and the a subumt of thymocytes at low concentrations, the IL-2 receptor {CD25), when even before expression of the TCR inlected &rectly into the thymus. 13chain. Thus, since each cohort of develTo test the role of signal transoping T cells ,.s accompanied by a duction during the TN stage, daycohort of DCs, it appears that 14 fetal thymlc organ cultures from the thymus can support DC de- RAG-l-knockout mice were culvelopment. The CD4 I° precursor tured in the presence of anti-CD3 cells can grow on cortical epithelial antibodies (C. Levelt, Freiburg). cell-hnes tn vitro and are a hetero- After six days of culture, matugeneous populatmn. Although ration to the DP stage was restored, these cells can generate B cells together with downregulatlon of upon intravenous transfer into CD25 and a fivefold increase m lethally irradiated mice, they have absolute cell number. These data lost the abdlty to generate CFU-S suggest that maturation to the DP {Ref. 3). In this context, it is of stage is induced by signal transducinterest to note that previous tion through an immature CD3 reports have also demonstrated complex, which appears to involve expression of the CD4 gene in the the TCR 13 chain associated with a fetal liver4. newly described chain cf 33 kDa The sequennai dcvelopme~: of (H. yon Boehmer, Basel). Thymothe CD4 I° population ~s character- cytes from TCR a-knockout mice ized first by the expression of are TCR 13- and CD3- at day 15, CD44 and/or CD25 antigens and, hut are TCR ~_'÷ ar.d CD3 + at In a second step, by the expression day 16 (Ref. 5). The gene encoding of CD4/CDB. However, m order to gp33 is upregulated at this stage follow these stages of development, and is transported with the I~ chain it ~s necessary for CD4 I° cells to to the cell surface. A cell line dedownregulate the CD4 antigen and ri,ed from SCID mice, transfected become TN. Thus the following with the gene for the TCR 13 chain, tr2nsltions may be detected: expresses cell-surface 13 protein in c.klt+÷CD44+CD25- -, CD44+CD25+ assoclanon with CD3~ and signals thymic precursors (TCR 13 and in response to crosshnkmg with -¢ in germhne configuratlon) antI-TCR/CD3 antibodies. Immature CD44-CD25 + [60% of the cells T-cell lines have only the 13/33kDa express the recombinatmn-activathetero&mer, and signal transducmg gene 1 (RAG-i), as detected by tion may involve p56 I~k at this m sttu hybridization (H.R. stage. Thus, aithouDh DP thymoMacDonald, Lausanne), reflecting cytes are observed in RAG-1/pS6 t'~ the start of TCR 13 rearrangement]. double-knockout mice crossed These steps are also characterized to mice transgemc for TCR 13,
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trends the total yield of the thymus is (nly 10×106 cells. If the p56 kk protein is restored, then the thymus yield increases to 288 × 10 6, suggesting that p56 kk is essential for DP expansion rather than development. Commitment to the a[3 or ~8 T cell DN lineages probably occurs at the TN CD44-CD25 ÷ stage, since their immediate successors (CD44-CD25-CD41°CD81°) are the precursors of CD4÷CD8*TCR a~* DP thymocytes, which generate either CD4 or CD8 single posit,ve (SP) mature T cells6. Within the DP ~x[3÷ thymocytes, the second wave of RAG-1 expresston is detected in 80% of the cells and this corresponds re the time of transcrtption of the TCR ~ chain (H.R. MacDonald, Lausanne). Tbymlc selection
The generation of mature T cells from DP thymocytes proceeds through a stage of positive selecnon. J.J.T. Owen (Birmingham, UK) has used fetal thymlc organ cultures to study the minimal stromal-cell reqmrement for T-cell development7. Day-14 fetal thymocytes, selected on the basis of CD45 express~on, develop to the SP s¢age only if the full stroma is present and nor ff rhvmw emrhehal cell~ (TECs) or fetal mesenchyme or fibroblast (3T3) cells alone are present. Furthermore, there ~s a requirement for cortical epithelial cells expressing major histocompatlbihty complex (MHC) class II molecules in conjunctmn with either fetal mesenchyme or anotber source of fibroblasts. ,-.e comblnanon of TEC-denved IL-7 and SCF, along with fibroblast-denved IL-lc( and granulocyte-macrophage colony-snmulatmg factor (GMCSF), provide early signals for thymocyte mteracnons with TECs. Only acnvated DP cells will mature to SP cells in this system, and engagement of the TCR thereby provides a rescue signal from the alternanve fate of apoptosis. The thymocytc s~gnalling pathway and the TEC ligand have yet to be /efined. - An evaluation of murine thymoc~te development by C. Penit (Pans), using bromodeoxyuridine incorporation ,o trace a cohort of
developing thymocytes, demonstrated the sequential generation first of CD4 ÷ SP from TCR h' DP cells, followed by CD8 + SP cells (Ref. 8). The downregulanon of CD8 molecules (to leave CD~,÷ SP) occurs 72h before the downregulanon of heat-stable ant,gen (HSA). Similarly, the genetanon of CD8 ÷ cells follows the same sequence of events, but HSA ehmmanon begins 24h after CD4 downregulanon. The importance of HSA m thymopmesls was addressed by K. Humphries (Vancouver) using mice transgemc for HSA. Mice that overexpressed HSA had only one tenth of the total number of thymocytes compared with nontransgenic littermates. This was due to depletion of DP and SP, but not DN, thymocytes. Moreover, CD8 + T cells in the periphery were a]so reduced tenfold. Development of these defects appeared to coincide with the onset of negative sekcnon, suggesting that HSA-mediated s~gnals may have an effect on the efficiency of this process 9. Apoptosis Triggering of the TCR/CD3 complex m thymocytes and activated peripheral T cells may lead to induction of apoptosis, a form of nrnc, ramm~cl cell death IPCDI_ Another rathway for PCD-mductton in activated mature T cells involves antibody-mediated crosslinking of the 48kDa cell-surface melecule APO-1/Fas. In contrast to the TCR/CD3 complex, which can mediate prohferanon and call death, APO-1 has been shown to function primarily as a "death receptor'. K-M. Debatm (Heidelberg) reported on the APO-1 system in normal and mahgnant human T cells. In the thymus, APO-I is present on TECs and differentially expressed on thymocyte subpopulations. DP TCR- or TCR I° cells are also APO-I+; while SP TCR h' cells arc APO=I-. Thus, as the level of the TCR increases, cell-surface APO-1 expression is downregulate& By contrast, the opposite correlation is found for expression of the Bcl-2 protein, which is upregulated in TCRh' cells. Only a small percentage of APO-1 ÷ thymocytes are sensitive to apoptosls med,ated by anti-APO-1
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antibodies 1tl vitro. This suggests that there ~s a reqmrement for lnducnon of apopto~zs m th)mocytes, whereas pre-T-cetls spontaneously &e. Mature T cells have a low level of APO-I cxpressmn, which is strongly upregulated after actwanon. However, APO-I+ act,vated T cells are resistant to apoptosis ,nduced by ann-APO-1 m an early phase after activation. Apoptos~s-sensmve and -resistant phenotypes are also found m APO-I" malignant T cells. Thus, adult T-cell leukaemm (A'IL) cells, whmh have the same phenotype as acttvated mature T cells, are exqmsltely susceptible to apoptos~s medinted by ann-APO-1 annbo&es m. By contrast, APO-I" acute lymphoblasnc leukaemta T cells (T-ALL) show a variable ~ensmv W. In these cells, sensitivity or resistance to antt-APO-l-me&ated apoptosls is mdependent of phenotype, and of the level of APO-1 on the cell surface end Bcl-2 protein expression. Therefore, defining the suscepnbihty to APO-l-mediated apoptosis at the molecular level ,s a slgmficant goal m these lymphoprohferatwe &sorders. Gene regulation during lymphopoiesis During the early ~ra~e~ nf T-cell dlfferennatlon, funcnonal TCR variable regions are assembled somatically from germline gene segments. Transcription of TCR genes depends on lymphoid-specific regulatory elements, including the recently identified enhancers of TCR [3 and TCR (x transcrlpnon. These enhancer elements are composed of domains that bind both to T-cell specific and to ubiqmtous nuclear proteins. Yet, while enhancers are revolved in the expression of TCR genes, there is no direct evidence for thmr role m V-D-J recombmatmn. To address the issue of gene regulauon, M. Capone (Marseille)II analysed transgemc mice carrying germhne V-gene constructs linked either to the TCR [3 or TCR cl gene enhancers (EIB or Ea). In I" cells of independently derived mouse lines. the transgemc constructs underwent high rates of site-specific rearrangements. Furthermore, the rearrangements of El3-contammg
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tTends transgenes compared with Eta-contaming transgenes began at different stages of T-cell differentiation m embryomc and adult thymuses. In E/8-transgeni: lines, DI3-J/8 rearrangements were detected from day 14, whde high levels of rearrangement in Et,-transgenic hnes were not detected until day 17. These results imply that El3 and Ea confer distinct timing on the rearrangement events. Furthermore, m one EI3-transgemc line (EI~2), the levels of DI~-J[3 rearrangements were high :n early D~,r thymocytes and ~+ thymocytes. By contrast, m Ea3 and Ea6 mkce, although high levels of DI~-JI3 rear~angemea.; were detected in immature SP cells, the levels of rearrangement m "¢~" thymocyres were low. Stmdar analyses on VI3gone rearrangements led to the demonstration that sequences within El3 and Ea confer tissueand stage-specificitT to the V-D-J recombination events affecting adjacent gone segments. Germline transcription of the J[3 segments within the transgenes occurred before substantial D-J rearrangements could be detected during Tcell differentiation. These results concur w~th the idea that transcription through a germline segment is a prerequisite for recombination. interestingi); germiine JI3 transcripts could also be detected m B cells and T8 T cells m the absence of V-D-J recombmanon, suggesnng that simple transcription of a gone segment is, by itself, not sufficient to mduce recomhmanon. Signal trdnsductton Usir,g a polymerase cham fetenon (PCR) based on degenerate oligol:ucleotlde sequences for conserved reg',ons of all tyrosine kinases, L. Berg (Cambridge, MA) has cloned novel tyrosme kmases from neonatal mouse thymocytes. The gene for one of these, tsk (T cell-specific k.nase), is expressed by thymocytes as early as fetal day 14 and con6nues to be expressed through T-ce~' ontogeny. This gene is also expressed at 10-20-fold higher le*els m adult thymocvtes than m resting peripheral T cells. Tsk is a member of a new famdy of tyrosine kmases that includes the B-cell klnase, Btk, which is
defecnvc in X-linked agammaglobuhnaemia (XLA) in humans and xid in mice. In patients w~th XLA, B-cell development is arrested at the pre-B stage in the bone marrog. Interestingly, Tsk and Btk are approximately 60% identical m amino acid sequence and, in particular, share strong homology in the N-terminal 'unique' region known to be the site of the xld mutation, Together with the expression data, the homology to Btk strongly suggests a role for Tsk m T-cell development. The tsk gone maps to chromosome 11 in the mouse, and is closely hnked to a cluster of lymphokine genes (GM-CSF, IL-3, iL-13), whereas the human gone maps to chromosome 5q. The sequence of tsk and an analysis of tsk expression were published last year L'. We were unable to discuss all of the snmulanng presentanons given al this workshop due to space hmitanons Th~s meeting was sponsored by INSERM, with generous donation, from AMGEN a~d Systemlx. Sophie Ezme ts at U.345 INSERM, hlstttut Necker, 156 rue de Vaugtrard, 75730 Parts Cddex 15,
Immunology
France; Rbodr~ Ceredtg is at U.184 INSERM, CNRS LGME, Instztut de Cbzrnie Biologzque, 11 rue Humann, 67085 Strasbourg Cddex, France. References 1 Godin, I.E., Garcsa-Porrero,J.A., Coutmho, A. etal. (I 993) Nature 364, 67-70 2 Medvmsky,A.L. Samoyhna,N.L., Muller, A.M. and Dzierzak, E.A. (1993) Nature 364, 64--67 3 Ardavm, C., Wu, L., Ll, C.L. and Shortman, K. (1993) Nature 362, 761-763 4 Haars, R., Conradt, P., Mdmer, 1. and Wagner, H. (1991) Stand. J. Immunol. 34, 253-258 5 Groettrup, M. and yon Boehmer, H. (1993) Immunol. Today 14, 610--614 6 Godffrey,D.I. and Zlotnlk, A. (1993) Immunol. Today 14, 547-553 7 Jenkmson, E.J., Anderson, G. and Owen, ].].T. (1992) J. Exp. Med. 176, 845-853 8 Lucas, B., Vasseur, E and Pemt, C. (1993) J. lmmunol. 151, 4574--4582 9 Hough, M.R., Takeb E, Humphries, R.K. and Kay, R. (19941J. Exp. Med. 179, 177-184 10 Deb~tin, K-M., Goldman, C.K., Waldmann, T.A. and Krammer, P.H. (1993) Blood 81, 2972-2977 11 Capone, M., Watrin, F., Fernex, C. etal. (1993) EMBO J. 12, 4335-4346 12 Heyeck, S.D. and Berg, L.J. {1993) Proc. Natl Acad. So. USA 90, 669-673
T o d a y is t h e forum for discussion Viewpoint
The immunological community thrives on the constant generation of new hypotheses, reformed speculation and debate. T h r o u g h the viewpoint section, I m m u n o l o g y Today is committed to the presentation of novel and challenging concepts across the whole spectrum of immunological research. Naturally, the majority of viewpoint articles are volunteered to tht Fditor by the authors themselves and we wish to e m o u r a g e this practice. Spontaneously submitted manus~,Hpts a-e not always considered for pubhcation. Therefor'e, we encourage you to contact the Editor in the first instance with a brief s u m m a r y of the focus and content of any proposed article. Once commissioned, all viewpoint manuscripts undergo peer review: inevitably some will still be rejected, however the impact of those that are accepted is enhanced significantly.
1,,~,,notogrroaar 154
Vol.is no 41994
Haemopoiesis and early T-cell differentiation Sophie Ezine and Rhodri Ceredig Lineage commitment ts a fundamental process that is initiated during the early stages of embryogenesis. Ultimately, this leads to the ge~,eration of the humoral and cellular arms of the immune system. In a recent workshop*, A fundamental quesuon in the : esearcbers from the diverse fields study of T-cell biology is the t~sue of embryology, haematology and of whether T cells are born or made. Before the complex forces of immunology gathered to address topic of early T-cell negative and positive selection can the differentiation. Imprint a pattern of self/noi~self discrimination on developing thymocytes, an apparently undifferentiated stem cell must commit itself to becoming an immature T cell, rather than a B cell or one og the other diverse components of the mature lymphoid system. The spectrum of work discussed at this meeting reflected the compartmentahzar~on of thi~ process, which begins in the haemopoietic tissue and fimshes m the thymus.
cells migrate to the fetal Iwer and colonize the bone marrow before birth. However, stem cells can also be found at other embryonic s~tes, such as the para-aortic splanchnopleura (PAS). Indeed, F. DleterlenLi&re (Nogent-sur-Marne) reported that PAS isolated from mouse emt-. . . . . . d.oy a of ge~rarlon fi.e. at the 20 somlte pmr stage) can give
Early haemopoiesis Totipotent haemopoletic stem cells are first detectable in the yolk sac. During development, these
*The Phihppe Laudat Conference on 'Haemopoiesis and early T-cell differentiation' was held at Le Bischenberg, Strasbourg, France, 5-9 September 1993.
rise both to IgM-secretlng plasma cells and to the Bla B-cell subset (IgMb-ghtB220dullCD5÷) after grafting into severe combined immunodeficiency (SCID) m,ce 1. PAS develop into B cells m vitro after ten days m the presence of lnterleukin 7 (IL-7), IL-3, stem-cell factor {SCF), 2-mercaptoethanol and the S17 stromal-cell line; and, in hmitmg dilution assay, PAS generate myelo~d cells in the presence of IL-3 and SCF (A. Cumano, Pans). E. Dzlerzak (London) addressed the ongm and ;requency of haemopoienc cells during embryomc development. Using the spleen colony-forming unit (CFU-S) assay, it was possible to detect day-8 CFU-b m tbe yolk sac and m embryonic bodies in embryos at the 26-27 somite pair stage 2. Furthermore, this activity was found to be
© 1994, Elsevier Sc,ence [ td 0167-569 ~/q4/$07 00
1,,,n,,otogy Today
15 1
rot. Is No. 4 1994
trends restricted to the aorta-gemtalndge-mesonephrons (AGM) area and peaked at the 38-39 somite pa1~ stage. This population ~.; haemopoletac cells has a frequency of -'00 CFU-S 10-~ cells, which is ,,mdar to the adult bone marrow. However, the origin of these pluripotent AGM stem cells remains to be determined, as does the issue of whether or not they represent the equivalent of the mammalian fetal hver or the avian yolk sac. It remains to be answered whether there are many independent sites of stem-cell generation or whether various regions are seeded by cells from a unique source. Attempts to generate lymphoid cells m vitro from embryonic stem cells ~ESCs' have so far been unsuccessful. A malor problem m these studies has been the lack of definition of early markers that are specific for the T-cell lineage. However, S. Cande:as (Denver) reported that ESCs produce embryomc bodies alter growth for 14-21 days in methyl cellulose. In the presence of IL-7, Ik-2 and the $17 stromal-cell line, these embryonic bodies can gwe rise to lymphoidlike coionies (Thyl.2 b"ght FcRII/III÷ CD44 +) that can be grown in suspension in the presence of IL-2. Germline transcripnon of the gene for the V138.2 T-cell receptor (TCR) occu-s m these ESC-derwed embryonic bodies, suggesting that an early evc.nt m T-cell commitment is occurring. Transcripts are also detected in day 14 embryonic liver and thymus, and a specific V138 rearrangement is observed fi~r~ day 16 in the thymus, but not m the fetal liver. These germhne t~'anscripts have been detected in pre-Tcell lines (T3 and T7), but not in pre-B-cel! lines (38B9, 22D2). Lymphocyte development I. Wetsssman (Stanford) emphasized that stem cells m Thyl.l mice are sttll defined as Thyl.ll°LinSea-l* av.d that !ong-term reconsntution of all blood lineages in lethally irradiated mice can be achieved with a~ few a; 20 of these cells. Moreover, although these cells may be c-kit*, it appears that SCF does not mediate cell prohfe,~ ation in the bone marrow. In this respect, cychng cells are less efficient
at repopulat,on and are probabl) closer in the dlfferentmtton pathway to cells that wdl be committed to one lineage of dafferentianon.
by rearrangement at the TCR -/ locus and downregulanon of c-kit protem expression. Interestingly, thymlc development is arrested at the CD44-CD25 ÷ TN stage m Tbymtc d2fferentlatlon RAG-l-knockout mice (A. Zlotmk, lntrathymlc differentmtion events Palo Alto and S. Tonegawa, were defined according to cell- Cambridge, MA). Backcrosses of surface markers and the re- these mice w~th m~ce transgenic for arrangements of TCR genes that TCR 13 lead to the generation of eventually lead to the generanon CD4"CD8 ÷ thymocytes that are of a complete CD3/TCR a13 com- TCR 13*a- and express surface plex on mature ffymocytes. Early CD3e (S. Tonegawa, Cambridge, CD41°CD8-CD3 - thymocytes, as de- MA). A possible role for the incomfined by K. Shortman {Melbournel, plete CD3/TCR 13 complex may be are the precursors of a13 and ~/8 in the transductlon of signals T cells. These cells are more active revolved in the transition to the in repopulanng the thymus than CD4+CD8 ÷ double-positive (DP) are mple-negatwe (TN) CD3-CD4-- stage. Indeed, earher experiments CD8- cells. Furthermore, CD4 I° indicated that CD3~ is expressed cells can gwe rise to dendritic on the cell surface of double-negacells (DCs) expressing CD8a, CD4, twe (DN;CD44-CD25-CD41OCD8 l°) Thyl, CD2 and the a subumt of thymocytes at low concentrations, the IL-2 receptor {CD25), when even before expression of the TCR inlected &rectly into the thymus. 13chain. Thus, since each cohort of develTo test the role of signal transoping T cells ,.s accompanied by a duction during the TN stage, daycohort of DCs, it appears that 14 fetal thymlc organ cultures from the thymus can support DC de- RAG-l-knockout mice were culvelopment. The CD4 I° precursor tured in the presence of anti-CD3 cells can grow on cortical epithelial antibodies (C. Levelt, Freiburg). cell-hnes tn vitro and are a hetero- After six days of culture, matugeneous populatmn. Although ration to the DP stage was restored, these cells can generate B cells together with downregulatlon of upon intravenous transfer into CD25 and a fivefold increase m lethally irradiated mice, they have absolute cell number. These data lost the abdlty to generate CFU-S suggest that maturation to the DP {Ref. 3). In this context, it is of stage is induced by signal transducinterest to note that previous tion through an immature CD3 reports have also demonstrated complex, which appears to involve expression of the CD4 gene in the the TCR 13 chain associated with a fetal liver4. newly described chain cf 33 kDa The sequennai dcvelopme~: of (H. yon Boehmer, Basel). Thymothe CD4 I° population ~s character- cytes from TCR a-knockout mice ized first by the expression of are TCR 13- and CD3- at day 15, CD44 and/or CD25 antigens and, hut are TCR ~_'÷ ar.d CD3 + at In a second step, by the expression day 16 (Ref. 5). The gene encoding of CD4/CDB. However, m order to gp33 is upregulated at this stage follow these stages of development, and is transported with the I~ chain it ~s necessary for CD4 I° cells to to the cell surface. A cell line dedownregulate the CD4 antigen and ri,ed from SCID mice, transfected become TN. Thus the following with the gene for the TCR 13 chain, tr2nsltions may be detected: expresses cell-surface 13 protein in c.klt+÷CD44+CD25- -, CD44+CD25+ assoclanon with CD3~ and signals thymic precursors (TCR 13 and in response to crosshnkmg with -¢ in germhne configuratlon) antI-TCR/CD3 antibodies. Immature CD44-CD25 + [60% of the cells T-cell lines have only the 13/33kDa express the recombinatmn-activathetero&mer, and signal transducmg gene 1 (RAG-i), as detected by tion may involve p56 I~k at this m sttu hybridization (H.R. stage. Thus, aithouDh DP thymoMacDonald, Lausanne), reflecting cytes are observed in RAG-1/pS6 t'~ the start of TCR 13 rearrangement]. double-knockout mice crossed These steps are also characterized to mice transgemc for TCR 13,
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trends the total yield of the thymus is (nly 10×106 cells. If the p56 kk protein is restored, then the thymus yield increases to 288 × 10 6, suggesting that p56 kk is essential for DP expansion rather than development. Commitment to the a[3 or ~8 T cell DN lineages probably occurs at the TN CD44-CD25 ÷ stage, since their immediate successors (CD44-CD25-CD41°CD81°) are the precursors of CD4÷CD8*TCR a~* DP thymocytes, which generate either CD4 or CD8 single posit,ve (SP) mature T cells6. Within the DP ~x[3÷ thymocytes, the second wave of RAG-1 expresston is detected in 80% of the cells and this corresponds re the time of transcrtption of the TCR ~ chain (H.R. MacDonald, Lausanne). Tbymlc selection
The generation of mature T cells from DP thymocytes proceeds through a stage of positive selecnon. J.J.T. Owen (Birmingham, UK) has used fetal thymlc organ cultures to study the minimal stromal-cell reqmrement for T-cell development7. Day-14 fetal thymocytes, selected on the basis of CD45 express~on, develop to the SP s¢age only if the full stroma is present and nor ff rhvmw emrhehal cell~ (TECs) or fetal mesenchyme or fibroblast (3T3) cells alone are present. Furthermore, there ~s a requirement for cortical epithelial cells expressing major histocompatlbihty complex (MHC) class II molecules in conjunctmn with either fetal mesenchyme or anotber source of fibroblasts. ,-.e comblnanon of TEC-denved IL-7 and SCF, along with fibroblast-denved IL-lc( and granulocyte-macrophage colony-snmulatmg factor (GMCSF), provide early signals for thymocyte mteracnons with TECs. Only acnvated DP cells will mature to SP cells in this system, and engagement of the TCR thereby provides a rescue signal from the alternanve fate of apoptosis. The thymocytc s~gnalling pathway and the TEC ligand have yet to be /efined. - An evaluation of murine thymoc~te development by C. Penit (Pans), using bromodeoxyuridine incorporation ,o trace a cohort of
developing thymocytes, demonstrated the sequential generation first of CD4 ÷ SP from TCR h' DP cells, followed by CD8 + SP cells (Ref. 8). The downregulanon of CD8 molecules (to leave CD~,÷ SP) occurs 72h before the downregulanon of heat-stable ant,gen (HSA). Similarly, the genetanon of CD8 ÷ cells follows the same sequence of events, but HSA ehmmanon begins 24h after CD4 downregulanon. The importance of HSA m thymopmesls was addressed by K. Humphries (Vancouver) using mice transgemc for HSA. Mice that overexpressed HSA had only one tenth of the total number of thymocytes compared with nontransgenic littermates. This was due to depletion of DP and SP, but not DN, thymocytes. Moreover, CD8 + T cells in the periphery were a]so reduced tenfold. Development of these defects appeared to coincide with the onset of negative sekcnon, suggesting that HSA-mediated s~gnals may have an effect on the efficiency of this process 9. Apoptosis Triggering of the TCR/CD3 complex m thymocytes and activated peripheral T cells may lead to induction of apoptosis, a form of nrnc, ramm~cl cell death IPCDI_ Another rathway for PCD-mductton in activated mature T cells involves antibody-mediated crosslinking of the 48kDa cell-surface melecule APO-1/Fas. In contrast to the TCR/CD3 complex, which can mediate prohferanon and call death, APO-1 has been shown to function primarily as a "death receptor'. K-M. Debatm (Heidelberg) reported on the APO-1 system in normal and mahgnant human T cells. In the thymus, APO-I is present on TECs and differentially expressed on thymocyte subpopulations. DP TCR- or TCR I° cells are also APO-I+; while SP TCR h' cells arc APO=I-. Thus, as the level of the TCR increases, cell-surface APO-1 expression is downregulate& By contrast, the opposite correlation is found for expression of the Bcl-2 protein, which is upregulated in TCRh' cells. Only a small percentage of APO-1 ÷ thymocytes are sensitive to apoptosls med,ated by anti-APO-1
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antibodies 1tl vitro. This suggests that there ~s a reqmrement for lnducnon of apopto~zs m th)mocytes, whereas pre-T-cetls spontaneously &e. Mature T cells have a low level of APO-I cxpressmn, which is strongly upregulated after actwanon. However, APO-I+ act,vated T cells are resistant to apoptosis ,nduced by ann-APO-1 m an early phase after activation. Apoptos~s-sensmve and -resistant phenotypes are also found m APO-I" malignant T cells. Thus, adult T-cell leukaemm (A'IL) cells, whmh have the same phenotype as acttvated mature T cells, are exqmsltely susceptible to apoptos~s medinted by ann-APO-1 annbo&es m. By contrast, APO-I" acute lymphoblasnc leukaemta T cells (T-ALL) show a variable ~ensmv W. In these cells, sensitivity or resistance to antt-APO-l-me&ated apoptosls is mdependent of phenotype, and of the level of APO-1 on the cell surface end Bcl-2 protein expression. Therefore, defining the suscepnbihty to APO-l-mediated apoptosis at the molecular level ,s a slgmficant goal m these lymphoprohferatwe &sorders. Gene regulation during lymphopoiesis During the early ~ra~e~ nf T-cell dlfferennatlon, funcnonal TCR variable regions are assembled somatically from germline gene segments. Transcription of TCR genes depends on lymphoid-specific regulatory elements, including the recently identified enhancers of TCR [3 and TCR (x transcrlpnon. These enhancer elements are composed of domains that bind both to T-cell specific and to ubiqmtous nuclear proteins. Yet, while enhancers are revolved in the expression of TCR genes, there is no direct evidence for thmr role m V-D-J recombmatmn. To address the issue of gene regulauon, M. Capone (Marseille)II analysed transgemc mice carrying germhne V-gene constructs linked either to the TCR [3 or TCR cl gene enhancers (EIB or Ea). In I" cells of independently derived mouse lines. the transgemc constructs underwent high rates of site-specific rearrangements. Furthermore, the rearrangements of El3-contammg
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tTends transgenes compared with Eta-contaming transgenes began at different stages of T-cell differentiation m embryomc and adult thymuses. In E/8-transgeni: lines, DI3-J/8 rearrangements were detected from day 14, whde high levels of rearrangement in Et,-transgenic hnes were not detected until day 17. These results imply that El3 and Ea confer distinct timing on the rearrangement events. Furthermore, m one EI3-transgemc line (EI~2), the levels of DI~-J[3 rearrangements were high :n early D~,r thymocytes and ~+ thymocytes. By contrast, m Ea3 and Ea6 mkce, although high levels of DI~-JI3 rear~angemea.; were detected in immature SP cells, the levels of rearrangement m "¢~" thymocyres were low. Stmdar analyses on VI3gone rearrangements led to the demonstration that sequences within El3 and Ea confer tissueand stage-specificitT to the V-D-J recombination events affecting adjacent gone segments. Germline transcription of the J[3 segments within the transgenes occurred before substantial D-J rearrangements could be detected during Tcell differentiation. These results concur w~th the idea that transcription through a germline segment is a prerequisite for recombination. interestingi); germiine JI3 transcripts could also be detected m B cells and T8 T cells m the absence of V-D-J recombmanon, suggesnng that simple transcription of a gone segment is, by itself, not sufficient to mduce recomhmanon. Signal trdnsductton Usir,g a polymerase cham fetenon (PCR) based on degenerate oligol:ucleotlde sequences for conserved reg',ons of all tyrosine kinases, L. Berg (Cambridge, MA) has cloned novel tyrosme kmases from neonatal mouse thymocytes. The gene for one of these, tsk (T cell-specific k.nase), is expressed by thymocytes as early as fetal day 14 and con6nues to be expressed through T-ce~' ontogeny. This gene is also expressed at 10-20-fold higher le*els m adult thymocvtes than m resting peripheral T cells. Tsk is a member of a new famdy of tyrosine kmases that includes the B-cell klnase, Btk, which is
defecnvc in X-linked agammaglobuhnaemia (XLA) in humans and xid in mice. In patients w~th XLA, B-cell development is arrested at the pre-B stage in the bone marrog. Interestingly, Tsk and Btk are approximately 60% identical m amino acid sequence and, in particular, share strong homology in the N-terminal 'unique' region known to be the site of the xld mutation, Together with the expression data, the homology to Btk strongly suggests a role for Tsk m T-cell development. The tsk gone maps to chromosome 11 in the mouse, and is closely hnked to a cluster of lymphokine genes (GM-CSF, IL-3, iL-13), whereas the human gone maps to chromosome 5q. The sequence of tsk and an analysis of tsk expression were published last year L'. We were unable to discuss all of the snmulanng presentanons given al this workshop due to space hmitanons Th~s meeting was sponsored by INSERM, with generous donation, from AMGEN a~d Systemlx. Sophie Ezme ts at U.345 INSERM, hlstttut Necker, 156 rue de Vaugtrard, 75730 Parts Cddex 15,
Immunology
France; Rbodr~ Ceredtg is at U.184 INSERM, CNRS LGME, Instztut de Cbzrnie Biologzque, 11 rue Humann, 67085 Strasbourg Cddex, France. References 1 Godin, I.E., Garcsa-Porrero,J.A., Coutmho, A. etal. (I 993) Nature 364, 67-70 2 Medvmsky,A.L. Samoyhna,N.L., Muller, A.M. and Dzierzak, E.A. (1993) Nature 364, 64--67 3 Ardavm, C., Wu, L., Ll, C.L. and Shortman, K. (1993) Nature 362, 761-763 4 Haars, R., Conradt, P., Mdmer, 1. and Wagner, H. (1991) Stand. J. Immunol. 34, 253-258 5 Groettrup, M. and yon Boehmer, H. (1993) Immunol. Today 14, 610--614 6 Godffrey,D.I. and Zlotnlk, A. (1993) Immunol. Today 14, 547-553 7 Jenkmson, E.J., Anderson, G. and Owen, ].].T. (1992) J. Exp. Med. 176, 845-853 8 Lucas, B., Vasseur, E and Pemt, C. (1993) J. lmmunol. 151, 4574--4582 9 Hough, M.R., Takeb E, Humphries, R.K. and Kay, R. (19941J. Exp. Med. 179, 177-184 10 Deb~tin, K-M., Goldman, C.K., Waldmann, T.A. and Krammer, P.H. (1993) Blood 81, 2972-2977 11 Capone, M., Watrin, F., Fernex, C. etal. (1993) EMBO J. 12, 4335-4346 12 Heyeck, S.D. and Berg, L.J. {1993) Proc. Natl Acad. So. USA 90, 669-673
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Vol.is no 41994