t-cell development and function – a downunder experience
TRANSCRIPT
hymic stromal cells, in par-ticular epithelial cells, are a fundamental component of the inductive thymic micro-
environment. Key questions relate to thebasis of thymic epithelial cell diversity – whatare the genes regulating their development,and is there a common progenitor cell for thedifferent epithelial subsets?
The thymic environment andstromal-cell developmentSeveral groups reported a variety of ap-proaches to identify the genetic basis of thy-mus development. Nick Trede (Boston, MA),using N-ethyl-N-nitrosourea (ENU) mutage-nized Zebrafish, identified five complemen-tation groups of mutants in a gynogeneticdiploid screen and has mapped four ofthem. Phenotypically, they all appear toblock T-cell differentiation at a very imma-ture stage (secondary to either missing T-cellgenes or defects in thymic differentiation),because none of them express Rag or Ikarosin the thymus.
Representational difference analysis(RDA) has been used very successfully todissect the genetics of thymic development.Comparing mutant mice with arrest-pointsat different stages of development of thethymic microenvironment G. Hollander(Basel) showed that thymic epithelial ex-pression of the autoimmune regulator gene(Aire) is dependent on signals derived fromT cells. In the embryo, this is primarily fromCD441/2CD251 pre-T cells and is coincidentwith the time when the thymocyte-mediatedinduction of the normal thymic microenvi-ronment occurs. Furthermore, in adults, Aireexpression at the cortico-medullary junctionand medulla is upregulated by thymocytesspecifically undergoing negative selection.Using a similar RT-PCR driven RDA approach, A. Chidgey (Melbourne) identifieda panel of genes differentially expressed
between cortical and medullary epitheliumin the adult. Hoxa3 and Pax1 have been previously shown to individually effectearly thymic organogenesis. Using doubleHoxa3–Pax1 compound mutants, N. Manley(Augusta, GA) demonstrated these tran-scription factors acted in concert, with pro-found defects in the epithelium [reducedproliferation and interleukin 7 (IL-7), stemcell factor (SCF) and transforming growthfactor 1 (TGF-1) mRNA] and, as a conse-quence, a specific block in thymocyte differ-entiation at the double negative (DN) todouble positive (DP) transition [after T-cellreceptor b (TCR b)-selection].
C. Blackburn and A. Bennett (Edinburgh)have identified, isolated and characterized apopulation of cells with characteristics ex-pected of progenitor cells for thymic epithe-lium. Identified by the monoclonal antibody(mAb) markers MTS20 and MTS24, appar-ent markers of more primordial epithelium(J. Gill and M. Malin, Melbourne), these cellsexpress Whn (winged helix nude), Pax1 andHoxA3. MTS20/241 cells purified fromE12.5 thymic primordia conferred thymus
function on nude mice, indicating that theyare functional progenitors for thymic epithe-lium. The concept of ‘precursor thymic ep-ithelial cells’ expressing both cortical andmedullary markers, analogous to CD41CD81
thymocytes proposed some time ago, wasquestioned by M. Malin. They were not present in normal early embryonic develop-ment and a variety of experimental models.In particular the c-kit x ILRgc2/2 mice inwhich virtually no T cells develop, show aclear MTS 20/241 epithelial network and regions exclusively expressing cortical ormedullary markers. In this regard, Hans-Reimer Rodewald (Ulm), using chimericmixtures, demonstrated monoclonal expan-sion of MHC II1 medullary foci (stem cells?)in the developing embryo to form a series of islets rather than an interconnected mesh-work. Whether such foci represent the entiremedulla, including MHC II1 epithelium, isnot known. Clearly, the origin of cortical andmedullary epithelial cells and the identity ofcommon precursor cells is an important unresolved issue, particularly given the fact that defective thymic epithelium is acommon feature of autoimmune prone mice.
Lymphoid progenitors and lineagecommitmentThe development of thymic progenitors con-tinues to be controversial. It is now clear thatprecursors at least partially committed to the T-cell lineage can arise pre-thymically,completely independent of TCR gene re-arrangement. Elegant single cell assays of H. Kawamoto and T. Ikawa (Kyoto) showedthat such thymic-committed progenitors arehighly enriched in the Lin2c-kit1IL-7R1
population of E12 fetal liver, and that theseand the earliest intrathymic CD32CD42CD82
(TN) pre-T cells retain potential for bothnatural killer (NK) and dendritic cell (DC)lineage. Using this assay, they furthershowed that the T-cell progenitors developed
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T-cell development and function – a downunderexperience
Richard Boyd and Ann Chidgey
It is nearly 40 years since Jacques
Miller revealed the importance of
the thymus as a major primary
lymphoid organ. However, despite a
vast number of subsequent
publications confirming this, and
defining many facets of
thymopoiesis, it is clear that several
key issues have remained
unresolved. In order to review the
enormous advances made in
understanding the life of a T cell
from precursor to pathology, the
third ‘ThymOz’ meeting has been
held in Australia*.
T
* The ThymOz III Meeting was held on Heron Island, Queensland,Australia, on 11–16 April 2000.
before the B-cell progenitors in the fetalliver. This TCR-independent lineage com-mitment was also evident in the thymuswith gd T cells being preferentially derivedfrom IL-7Ra1 rather than IL7Ra2 pro-T cells,at the expense of ab T cells (D. Raulet,Berkeley, CA).
Understanding of the role of Notch incell-fate decisions during thymopoiesis continues to evolve. Although T-cell devel-opment has been previously shown to be se-verely impaired in Notch 1-deficient mice1,myeloid and lymphoid DCs are normal inboth number and phenotype (R. MacDonaldand A. Wilson, Lausanne). This obviouslyquestions the proposed origin of thymic DCsfrom a common T/DC precursor, or at leastthe requirement for Notch-1 is a clear pointof divergence between these lineages. Notchsignalling is also involved during laterphases of T-cell selection, but as a conse-quence of thymocyte/epithelial interactions(E. Jenkinson, Birmingham, UK). Its ligands(jagged1, jagged 2, and delta-like-1) were ex-pressed in thymic epithelial cells, but not inany thymocytes nor in DCs. Indeed, one rea-son why DCs might be more specialized fornegative selection is their lack of ability tostimulate cells, even for survival, throughNotch. Notch signalling was also shown tooccur during positive selection by expres-sion of deltex (a downstream molecule ofnotch signalling) in pre- and post-selectionthymocytes in reaggregate cultures. The fateof cells following positive selection was ad-dressed by G. Anderson (Birmingham, UK)who showed that the extensive wave of pro-liferation of newly developed SP thymocyteswas IL-7 but not TCR2MHC-dependent.
One of the most controversial sessions onthymic development focussed on the influ-ence of steroids, in particular corticosteroids.G. Wick (Innsbruck) extended earlier evi-dence for the enzyme intermediates in steroidbiosynthesis and also showed the presenceof glucocorticoid receptors (GRs) on all thy-mocyte subsets. This would be consistentwith a role for GC signalling in thymopoiesis,which has been proposed to antagonize TCRactivation in T-cell selection2. The obligatorynature of glucocorticoid (GC) signalling,particularly for positive selection, however,was challenged by J. Purton and T. Cole(Melbourne). In vivo and in vitro analysis of
T-cell development in their GR2/2 mice, in-cluding positive and negative selection, wasindistinguishable from wild-type controls.This is also in clear contradiction to earlierstudies based on anti-sense inhibition of GRexpression3. The resolution of this dilemmaof GC involvement in thymic developmentis clearly warranted.
Peripheral T cellsPeripheral T-cell homeostasis and the im-portance of receptor specificity has gener-ated considerable debate in the recent times.It has been previously shown that naive Tcells undergo a slow ‘homeostasis-drivenproliferation’ under conditions of lym-phopenia, such as irradiation or antibody-mediated T-cell depletion4,5. C. Suhr (La Jolla,CA) presented data showing that peripheralT-cell contact with self-peptide, which isprobably equivalent to that used initially toinduce positive selection, was important forthis peripheral expansion in lymphopenicanimals. It has been thought that only memory-type cells are expanded under theseconditions, whereas T cells with a naive phenotype must come from the thymus. M. Bevan (Seattle, WA) presented workshowing that the naive cells actually divideat a slow rate and take on the memory phe-notype (expressing high levels of CD44,CD122 and Ly6c). These cells also have thefunctional properties of memory cytotoxic T
lymphocytes (CTL), such as the ability to killwhen isolated directly ex vivo. However,such cells should be viewed as falling into a‘false memory’ category because they can re-vert back to a naive phenotype. Nonetheless,the old notion that only memory cells canexpand under conditions of lymphopenia isincorrect with naive T cells also capable ofregeneration.
The interplay between CD81 CTL andhelper T (Th) cells has come under increas-ing scrutiny in recent times. The notion thatTh cells simply provide IL-2 for CTL expan-sion is overly simplistic. B. Heath (Mel-bourne) used a transgenic mouse model toexamine the role of Th cells in autoimmu-nity. His laboratory had previously shownthat CD41 and CD81 T cells needed to rec-ognize antigen presented by a common anti-gen presenting cell (APC) for effective CTLpriming. Heath presented data suggestingthat this was not necessary beyond the prim-ing phase. Th cells were nevertheless re-quired for effector CTL function leading to autoimmune tissue destruction. B. Scott(Clayton) extended this theme using datafrom a tumor model in TCR transgenic mice.Her data suggested that one role for Th cellsduring the CTL effector phase was to trafficinto peripheral tissues and actually promoteCTL infiltration into these sites.
Two major findings on the involvementof regulatory T cells in autoimmunity were presented. A. Baxter (Sydney) and
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Box 1. The outstanding questions
• What is the functional hierarchy of the multiple progenitors, in terms of their propen-sity for T and non-T lineage cells, and what are the mechanisms that selectively allowtheir entry into the thymus?
• What is the cellular and molecular basis of the development of functional hetero-geneity in the thymic epithelial microenvironment – is there a common epithelialprecursor cell, and how do developing T cells regulate this?
• What are the thymus-specific signals for T-cell receptor (TCR) gene rearrangements,positive selection and capacity for emigration?
• What is the molecular basis to the profoundly different outcomes of engagement ofthe TCR with major histocompatibility (MHC)–peptide ligands at different stages ofT-cell maturation, and what role do glucocorticoids play?
• What determines the intrathymic development and lineage branching of NkT andCD25+ regulatory T cells, given that they are absent in the neonatal period? What isthe extent of their clinical importance?
• What is the cellular and molecular basis of the development and maintenance of theperipheral T-cell pool and the regulation of Th:Tc:DC interactions?
D. Godfrey’s group (Melbourne) reported onthe importance of the NK T cell defect innon-obese diabetic (NOD) mice, which underlies the susceptibility of these mice to disease. To study these highly potent im-munoregulatory cells more precisely, theyhave produced a congenic line of NOD micethat express the NK- and NKT-associated allelic marker NK1.1. A comparative studyof these mice to B6 and NK1.1 congenicBALB/c confirmed the parallel defects inboth cell types – in terms of their production,export and function in the NOD strain (K. Hammond, Melbourne; A. Baxter, Sydney). Reconstituting the NOD mice withrelatively low numbers of highly enrichedNK1.1+ T cells prevented the onset of dis-ease. S. Sakaguchi (Kyoto) showed thatCTLA-4 is not only a molecule transducingan attenuating signal to activated T cells, butalso a unique costimulatory molecule for activating CD251CD41 regulatory T cells to exert suppressive activity. CD251CD41
naturally anergic and suppressive T cellsconstitutively express CTLA-4, but in vivoblockade of this molecule elicits autoimmunedisease (for example, autoimmune gastritisin BALB/c mice) and in vitro blockade
abrogates CD251CD41 T-cell-mediated suppression. This finding on a novel role of CTLA-4 might explain why CTLA-4blockade is so effective in evoking tumor immunity (and exacerbating autoimmunity).
Concluding remarksThe current balance between continuallyevolving elegant in vitro and in vivo experi-mental systems in combination with sophis-ticated genetic analyses has continued toprovide the important impetus for furtherunderstanding T-cell development and thethymic microenvironment. Several major issues remain unresolved (Box. 1); definingthese processes at the molecular level, andthe transition of these findings to the clinicalsetting remain major challenges, and willprovide the focus of ThymOz IV, 2002.
We are greatly indebted to the many researchers
who have agreed to release their unpublished
findings, and apologize to those whose equally
interesting findings could not be included in
this brief summary of selected topics. We also
thank the personnel on Heron Island for creating
a stimulating environment for such an interactive
conference.
Richard Boyd and Ann Chidgey are at the Dept of Pathology and Immunology, MonashMedical School, Commercial Rd., Prahran, Victoria,Australia. (For further information on this andthe upcoming ThymOz IV meeting, please con-tact RB or AC at [email protected])
References1 Radtke, F. et al. (1999) Deficient T cell
fate-specification in mice with an inducedinactivation of Notch 1. Immunity 10, 547–558
2 Vacchio, M.S. et al. (1999) Thymus-derivedglucocorticoids set the thresholds for selectionby inhibiting TCR-mediated thymocyteactivation. J. Immunol. 1636, 1327–1333
3 Ashwell, J.D. et al. (2000) Glucocorticoids inT cell development and function. Annu. Rev.Immunol. 18, 309–345
4 Ernst, B. et al. (1999) The peptide ligandsmediating positive selection in the thymuscontrol T cell survival and homeostaticproliferation in the periphery. Immunity 11,173–181
5 Goldrath, A.W. and Bevan, M.J. (1999) Low-affinity ligands for the TCR driveproliferation of mature CD81 T cells inlymphopenic hosts. Immunity 11, 183–190
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