Insight into the basis of autonomousimmunoreceptor activationRichard Berry1, Zhenjun Chen2, James McCluskey2 and Jamie Rossjohn1

1 The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences,

Monash University, Clayton, Victoria 3800, Australia2 Department of Microbiology & Immunology, University of Melbourne, Parkville, Victoria 3010, Australia

Review

Expression of the pre-T cell receptor (pTCR) by immaturethymocytes is crucial for T cell development. The pTCRcomprises an invariant pre-Ta chain that pairs with anewly rearranged TCRb chain and CD3 signaling com-ponents. Despite its similarity to the mature abTCR,which binds to specific peptide-loaded major histocom-patibility molecules, the pTCR functions in a ligand-independent manner. Precisely how pTCR functionsautonomously has remained a source of intense debate.Recently, the structure of the extracellular domain ofthe pTCR has been determined, providing insight intothe mechanism of pTCR autonomous signaling. In thisreview, we reflect on the current understanding of pTCRfunction and draw comparisons to the mechanismsemployed by the mature abTCR and the related pre-Bcell receptor.

The pre-T cell receptor as a developmental checkpointMature ab T cell receptors (TCRs) are heterodimeric inte-gral membrane proteins present on the surface of T-lymphocytes. abTCRs can function in the recognition offoreign antigenic peptides that are presented on the sur-face of virally infected cells by class I major histocompati-bility (MHC-I) molecules. TCR engagement in secondarylymphoid tissue results in the initiation of intracellularsignaling cascades via the associated CD3 subunits andleads to activation and clonal expansion to produce a cohortof T cells with specificity towards the same antigen. Inorder to generate an enormous repertoire of receptorscapable of recognizing a myriad of structurally diverseantigens, TCR gene elements undergo somatic recombina-tion. In this process, the variable regions of these receptorgenes are generated from random combinations of anumber of possible variable (V), diverse (D) and joining(J) gene segments.

The generation of functional rearranged abTCR genesrelies on a complex developmental pathway. In this pro-cess, the gene encoding the TCRb chain is firstly rear-ranged during the double negative (DN) 3 stage [1]. At thistime, immature thymocytes lack expression of TCRa andinstead express an invariant pre-Ta chain that pairs withnascent TCRb chains to form the pre-T cell receptor(pTCR) (Figure 1a) [2]. Assembly of pTCR on the cellsurface results in signals from the associated CD3 subunits

Corresponding authors: McCluskey, J. (jamesm1@unimelb.edu.au); Rossjohn, J.(jamie.rossjohn@monash.edu).

1471-4906/$ – see front matter � 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.it.2011.01

that trigger the termination of pTa expression, cessation ofTCRb rearrangement (allelic exclusion) [3,4] and matura-tion to the DN IV stage [5]. The expression of pTCRappears to be a crucial step in T cell development becausepTa�/� mice have approximately 1–10% of the normalnumbers of thymocytes [3] and these commonly expressmore than one rearranged TCRb chain [4]. Following thematuration of DN cells that successfully generate TCRb

chains by in-frame rearrangements (a process termed b-selection), thymocytes undergo TCRa gene rearrangementand ultimately expand, proliferate and mature intoCD4+CD8+ double positive (DP) cells [6]. In this reviewwe highlight recent developments in our understandingof the mechanism responsible for pTCR signaling andpay particular attention to the recently determinedcrystal structure of the pTCR extracellular domain thathas provided a new insight into the concepts of pTCRoligomerization and autonomous signaling [7].

The relationship between oligomerization andautonomous signalingDespite its similarity to the abTCR, no bona fide pTCRligand has been identified. This, coupled with reportssuggesting class I and class II MHCs are dispensable forb-selection [8,9] has led to suggestions that the pTCRfunctions in a ligand-independent manner. This hypothe-sis is consistent with reports suggesting that the pTCR isconstitutively endocytosed [10] in a fashion similar to thatof stimulated abTCR [11].

The recently determined crystal structure of the pTCRextracellular domain has provided a structural basis forthe ligand-independence of pTCR signaling [7]. In thecrystal structure and in solution, the pTCR was observedto form a head-to-tail ‘superdimer’ where two pTa-TCRb

heterodimers associate in an anti-parallel fashion(Figure 1b). In this arrangement, the pTa domain is sand-wiched between the constant b (Cb) domain of the samepTCR monomer and the variable b (Vb) domain of theinteracting partner [7]. A hydrophobic cluster that wouldotherwise be solvent-exposed (and hence unstable) in theVb domain mediates this arrangement. Interestingly, theresidues that are involved in pTCR superdimerization arehighly conserved across the Vb gene family and are essen-tial for pre-TCR function, as judged by intracellular reten-tion of pTCR evident by immunofluorescence microscopy.These findings provided an immediate explanation for howthe invariant pTa chain is able to pair only with correctly

.007 Trends in Immunology, April 2011, Vol. 32, No. 4 165

[()TD$FIG]

(a)

(b)(i) (ii)

pTα

pTα

TCRβTCRβ

αβTCR pre-TCR

Vα

Cα

Vβ

Cβ

ε δ ε δ

CD3εδ

ε γ

CD3εγ

TCRα TCRβ

CD3εγ

Vβ

Cβ

CD3ζζ CD3ζζ

TCRβ

Pre-Tα

pTα

ε γ

Immunoglobulindomain

Transmembrane(TM) domain

ITAM

Key:

Acidic TM residue

Basic TM residue

Electrostaticinteraction

TRENDS in Immunology

Figure 1. Structure and organization of the pTCR and abTCR. (a) Domain structure of abTCR and pTCR. Each receptor is shown in monomeric form with the associated CD3

subunits. The electrostatic interactions between charged transmembrane residues that drive assembly are depicted. (b) Arrangement of the pTCR head-to-tail superdimer

on the cell surface. It is currently unknown whether the pTCR acts exclusively in cis (I) or potentially in trans (II). Charged residues (yellow) previously implicated in pTCR

oligomerization are primarily found at surface exposed loops where they are available for interaction with CD3 (not shown).

Review Trends in Immunology April 2011, Vol. 32, No. 4

rearranged TCRb chains despite their highly diverse na-ture. In this way, the pTa chain appears to act as amolecular sensor, or chaperone, that signals autonomouslyto stimulate thymocyte development following successfulTCR b gene arrangement.

A role for oligomerization in pTCR function is not a newconcept. The extracellular domain of pTCR has beenreported to oligomerize spontaneously in the absence ofligand [12]. This oligomerization has been reported to occurin the absence of TCRb and was reported to be dependenton the presence of charged residues at positions 22, 24, 102and 117 in the pTa domain. Further support for this modelwas recently suggested by germ-line elimination ofthe charge of these residues, which results in impairedb-selection andTCR repertoire formation [13]. Intriguingly,in the structure of the pTCR superdimer, these chargedresiduesarenotpresentat thedimer interface. Instead, theyare located in solvent-exposed loops, one of which,by analogy to the ab TCR, has been implicated as aninteraction site for CD3 [14]. Thus, it is possible that thehigher surface expression and impaired b-selection of cellscontaining mutated pTCR lacking the charged residues is aconsequence of disruption of pTCR–CD3 interactions.How the pTa domain forms oligomers in the absence of aTCRb chain and the role of charged pTa residues in thisprocess remains unclear. Although the charged residuesthat were originally targeted were studied because theyare conserved in human and mouse pTa, a recent studythat describes the identification of sauropsidian pTa

highlighted the fact that these residues are replaced witha remarkable variety of amino acids of different chemicalproperties, including some that are hydrophobic [15].

166

These findings argue against an important role for thesecharged residues in pTCR dimerization. Interestingly, theTrp46 residue present at the superdimer interface of thepTCR and suggested to be important for dimerization isconserved in all species analyzed except for lizard. Thus itappears that the trigger for pTCR signaling (superdimer-ization) is mediated by residues conserved both within theTCRb gene family and during evolution. By utilizing such amechanism, the pTCR is able to sense successful TCRb generearrangements irrespective of their ultimate antigen spec-ificity.

The importance of pTCR extracellular domainsAlthough the pTCR superdimerization model provides anelegant explanation for how successful TCR b gene ar-rangement is monitored, it is at odds with reports thattransgenicmice expressing amutant pTa chain lacking theextracellular domain can proceed past the b-selectioncheckpoint [16,17]. Indeed, these reports, together withthe lack of sequence conservation in the cytoplasmicdomains of the pre-Ta, suggest that only the transmem-brane domain of the pre-Ta might be important for func-tion. How then can these findings be reconciled? Oneexplanation stems from the finding that at the DN 3 stage,thymocytes have an extremely low signaling threshold foractivation [18]. It is most likely, therefore, that transgenicexpression of elevated levels of normally non-functionalpTCR chains, including variants lacking the Vb domain[19], provide enough signal to bypass b-selection. Thisinterpretation is consistent with findings that expressionof TCRa at the pTCR checkpoint in the absence of endoge-nous pTa resulted in a marked deficiency but not total

Review Trends in Immunology April 2011, Vol. 32, No. 4

ablation of proliferation, survival and differentiation ofab T lymphocyte precursors [20]. Furthermore, undercompetitive conditions, a hybrid molecule consisting ofthe extracellular domain of pTa and the intracellularand cytoplasmic domains of TCRa was considerably moreefficient at mediating the DN 3 to DN 4 transition, sup-porting the suggestion of an important role for the pTa

extracellular domain in T-lymphocyte development [20].

Extracellular pTCR organizationOne interestingquestion raisedby the structureof thepTCRsuperdimer is whether the molecule can act in cis and intrans. Although there is little evidence with regard to thisissue, a cis arrangement appears to be the most likelybecause the pTCR is internalized and localized constitutive-ly in lysozymes in the absence of cell–cell interactions[10,12]. However, this does not preclude the possibility ofthe pTCR acting in trans in some instances. Intriguingly, acis arrangement would constrain the pTCR to lie side-on tothe membrane. This is in stark contrast to the conventionalassumption that the abTCR extends away from the mem-brane; one report, however, suggests that the latter orien-tation is incorrect [21] and the length of the stalks linkingthe immunoglobulin domains to themembrane are relative-ly long and likely to be flexible enough to allow a side-onarrangement of the abTCR.A flattened view of the pre- andmature TCR on the membrane is consistent with sugges-tions that the CD3ed and CD3eg subunits are positioned onthe same, rather than opposite, sides of the TCR [22].

pTCR stoichiometry and assembly with CD3A major driving force for assembly between abTCR andCD3 is via polar interactions between specific chargedresidues within the transmembrane domains [23]. Eachof the three basic TCR transmembrane residues interactswith a pair of acidic residues present in the three CD3signaling dimers (Figure 1a). Furthermore, assembly takesplace in a defined order, with CD3ed first recruited to theTCRa, followed by CD3eg interacting with TCRb andfinally the association of CD3zz with TCRa [23]. Both ofthe positively charged arginine and lysine residues presentin the TCRa transmembrane domain are absolutely con-served in the pTa chain, suggesting pTCR-CD3 assemblymimics that of abTCR-CD3 assembly [24]. However, un-like abTCR, the absence of CD3d does not affect pTCRassembly, surface expression or function [25].

Thus, we suggest that the stoichiometry of the function-al pTCR is most likely to consist of two copies each of pTa,TCRb, CD3ed, CD3eg and CD3zz. This would result in theclose association of 20 ITAM motifs, in contrast to only 10ITAMs in the abTCR–CD3 complex. This arrangementmight aid facilitation of the ligand-independent autono-mous signals generated by the pTCR via the concentrationof signaling motifs and associated kinases. Such a mecha-nism has been widely reported to play a role in abTCRsignaling [26,27].

The pTCR cytoplasmic tails and intracellular signalingThe mature TCRa and TCRb chains have unusually shortcytoplasmic regions of only three amino acids that containno identifiable signaling motif. Instead, signal transduction

is initiated exclusively by the ITAMs of the associated CD3subunits. However, the cytoplasmic region in murine andhuman pTa is considerably larger (30 and 114 amino acids,respectively). The biological significance of the pTa cyto-plasmic region remains controversial with some reportssuggesting it is dispensable for T cell development [16,28]whereas others find it to be crucial [29]. However, elevatedexpression levels of mutated pTa transgenes might explainthis discrepancy in a manner similar to that describedabove. The size of the pTa cytoplasmic tail is considerablydiminished in sauropsids and some mammals [15]. Thus, itis possible that this region has recently evolved new signal-ing functions in order to finely tune or provide greatercontrol over signaling outcomes. Indeed, a poly(proline-ar-ginine)-rich sequence present in the cytoplasmic portion ofhuman pTa has been reported to bind to CIN85 and CMSadaptor proteins [30] that regulate cytoskeletal rearrange-ments and T cell polarization [31,32]. Deletion of this bind-ing motif has a negative impact on pTCR-mediated calciummobilization and NFAT transcriptional activity [30].

An analogous mechanism in pre-BCR?B cell receptors (BCRs) also function in the recognition ofantigens but differ from TCRs in that their targets areunprocessed proteins that do not require presentation byMHC molecules. Following antigen engagement, BCRsstimulate B cell proliferation and differentiation to gener-ate a population of antibody-secreting plasma B cells andmemory B cells. From a structural perspective, BCRs areessentially membrane-bound antibodies and are composedof two identical heavy chains (HC) and light chains (LC)linked by disulfide bonds. Like their thymic counterparts,BCRs undergo somatic recombination and, consequently,B-lymphocytes express a surrogate chain that acts toensure fidelity of the HC gene rearrangement [33–38].In the pre-BCR (pBCR), the light chain is replaced by l5and VpreB chains, which occupy positions normally asso-ciated with the CL and VL chains, respectively (Figure 2a).Unlike the light chain domains, the N-terminus of l5 andthe C-terminus of VpreB, which together make up thesurrogate light chain (SLC), contain unique regions thatare crucial for pBCR function [39–41].

At first sight, the pBCR appears to utilize signalingmechanisms similar to those described for the pTCR; theyboth oligomerize and this oligomerization is mediated bythe surrogate chains and leads to ligand-independent sig-naling [42]. However, upon closer inspection the mechan-isms by which this occurs are quite distinct. In the pTCR,the surrogate chain makes contacts with conserved resi-dues within the Vb and is thereby able to bind to anycorrectly folded TCRb chain. In the pBCR, the uniqueregions of l5 and VpreB extend over the top of the focalpoint for sequence diversity, the HC complementaritydetermining region (CDR)3 [42]. By interacting with themost variable region, the SLC is able to influence the VH

repertoire present in mature B-lymphocytes. These find-ings explain why not all of the in-frame rearranged HCs inmice are capable of pairing with the SLC [43].

In addition to their role in sensing HC rearrangements,the l5 and VpreB unique regions mediate pBCR oligomeri-zation [42]. The pBCR is considered to formextended chains

167

[()TD$FIG]

CD79

VL1VL1

CL1CL1

VH1VH1

CH2 CH2

CH3 CH3

CH4 CH4

CH1CH1

VH1VH1

CH1CH1

A B

CD79

CH2 CH2

CH3 CH3

CH4 CH4 A B

λ5 λ5

Heavychain

VpreB

Vpre

BHeavychain

Surrogatelight chain

BCR pre-BCR(a)

(b)

Lightchain

(c)

Signal ++ ++ ++

+ ++

Inactiveoligomer

Inactivemonomer

Active/primedmonomer

Active oligomer

Signal - -

Multi-valentantigen

TRENDS in Immunology

Figure 2. Structure, organization and signaling of the BCR and pBCR. (a) Domain structure of BCR and pBCR. Each receptor is shown in monomeric form with the associated

CD79 subunits. (b) Model of pBCR on the cell surface. pBCR oligomerizes via the VpreB and l5 unique regions (red and orange respectively) leading to autonomous

signaling. (c) Model of the dissociation-activation model of BCR signaling. In resting B cells the majority of BCRs form inactive oligomers with a small percentage being

present as active monomers in order to maintain a low tonic survival signal. In the presence of multi-valent ligands, BCRs are tethered in an active oligomeric assembly that

prevents the reformation of inactive BCR oligomers. The multi-valent antigen is depicted as a yellow triangle.

Review Trends in Immunology April 2011, Vol. 32, No. 4

on the cell surface via such cis interactions (Figure 2b). Thisoligomerization is not required to place the SLC in closeproximity to the newly rearranged VH, as is the case forpTCR. Instead, such an arrangement could activate intra-cellular signaling cascades via the concentration of CD79ITAM motifs and associated signaling molecules.

Why does the pBCR employ a different mechanism?The question remains of why have the pTCR andpBCR evolved different mechanisms to perform the taskof monitoring successful gene rearrangement? One expla-nation could lie in subtle differences in the functionalrequirements, e.g. to bias or not to bias the resultingrepertoire. Alternatively, the differences that are apparentin these pre-immunoreceptors might reflect the alternateapproaches employed by their mature counterparts. TheabTCRmost likely exists on the cell surface as amixture of

168

monomers, dimers and higher order multimers [44,45]. Itcan be activated via interaction with only a single pMHCligand [46] and, indeed, a conformational change that couldpotentially transmit this signal from the TCR to CD3 ecto-domains has been reported [14]. However, signaling isconsiderably enhanced by multivalent ligands [26] andis induced by antibodies that crosslink CD3 [47], suggest-ing oligomerization does play a key role in at least enhanc-ing the TCR signal. Further, TCRs are highly clusteredin the immunological synapse in response to antigen-presenting cell stimulation [48]. In contrast, signaling bythe mature BCR and, in particular, the role of oligomeri-zation is quite different (Figure 2c). For example, the BCRcan be activated only by multivalent and not monovalentligands [49]. The BCR has a propensity to form oligomerson the cell surface, but these appear to represent an auto-inhibited form of the receptor in which the ITAM domains

Review Trends in Immunology April 2011, Vol. 32, No. 4

are inaccessible [50]. Instead, antigen ligation results in ashift towards the more active monomer but only lowvalency ligands prevent the reformation of closed oligo-mers [50]. This mechanism does not require the precisespacing or positioning of antigenic epitopes and, thus, isideally suited to the BCR, which can be activated by aplethora of structurally distinct soluble and membrane-embedded ligands [51,52].

Summary and future perspectivesThe past few years have seen considerable advances in ourunderstanding of themechanism responsible for pTCR andpBCR function. It is now clear that pTCR does not require asoluble ligand for activation. Instead, pTCR autonomoussignaling is intrinsically linked to the formation of a ‘super-dimer’ on the cell surface. This arrangement allows theinvariant pTa chain to act as a molecular sensor to probethe success of TCRb gene rearrangement in a way thatdoes not bias the mature repertoire. Further studies arerequired to understand the sub-cellular localization of thepTCR superdimer and how this self-association leads tothe activation of the associated CD3 subunits. Given thatderegulation of pTCR function can lead to lymphomagen-esis and that, notably, some human T cell acute leukemiclymphomas express the pTCR [53], the basic understand-ing of pTCR function will provide the basis for the design ofpTCR-targeted therapeutics.

AcknowledgmentsWe thank past and present members of the team who contributed to ourunderstanding of pre-TCR assembly. This work was supported by grantsfrom the National Health and Medical Research Council of Australia andthe Australian Research Council (ARC). J.R. is supported by an ARCFederation Fellowship.

References1 Godfrey, D.I. et al. (1993) A developmental pathway involving four

phenotypically and functionally distinct subsets of CD3-CD4-CD8-triple-negative adult mouse thymocytes defined by CD44 and CD25expression. J. Immunol. 150, 4244–4252

2 Groettrup, M. et al. (1993) A novel disulfide-linked heterodimer on pre-T cells consists of the T cell receptor beta chain and a 33 kdglycoprotein. Cell 75, 283–294

3 Xu, Y. et al. (1996) Function of the pre-T-cell receptor alpha chain inT-cell development and allelic exclusion at the T-cell receptor betalocus. Proc. Natl. Acad. Sci. U.S.A. 93, 2169–2173

4 Aifantis, I. et al. (1997) Essential role of the pre-T cell receptor in allelicexclusion of the T cell receptor beta locus. Immunity 7, 601–607

5 Fehling, H.J. et al. (1995) Crucial role of the pre-T-cell receptor alphagene in development of alpha beta but not gamma delta T cells.Nature375, 795–798

6 Hoffman, E.S. et al. (1996) Productive T-cell receptor beta-chain generearrangement: coincident regulation of cell cycle and clonality duringdevelopment in vivo. Genes Dev. 10, 948–962

7 Pang, S.S. et al. (2010) The structural basis for autonomousdimerization of the pre-T-cell antigen receptor. Nature 467, 844–848

8 Koller, B.H. et al. (1990) Normal development of mice deficient in beta2 M, MHC class I proteins, and CD8+ T cells. Science 248, 1227–1230

9 Grusby, M.J. and Glimcher, L.H. (1995) Immune responses in MHCclass II-deficient mice. Annu. Rev. Immunol. 13, 417–435

10 Panigada, M. et al. (2002) Constitutive endocytosis and degradation ofthe pre-T cell receptor. J. Exp. Med. 195, 1585–1597

11 Liu, H. et al. (2000) On the dynamics of TCR:CD3 complex cell surfaceexpression and downmodulation. Immunity 13, 665–675

12 Yamasaki, S. et al. (2006) Mechanistic basis of pre-T cell receptor-mediated autonomous signaling critical for thymocyte development.Nat. Immunol. 7, 67–75

13 Ishikawa, E. et al. (2010) Germ-line elimination of electric charge onpre-T-cell receptor (TCR) impairs autonomous signaling for b-selectionand TCR repertoire formation. Proc. Natl. Acad. Sci. U.S.A. 107,19979–19984

14 Beddoe, T. et al. (2009) Antigen ligation triggers a conformationalchange within the constant domain of the alphabeta T cell receptor.Immunity 30, 777–788

15 Smelty, P. et al. (2010) Identification of the pre-T-cell receptor a chainin nonmammalian vertebrates challenges the structure-function of themolecule. Proc. Natl. Acad. Sci. U.S.A. 107, 19991–19996

16 Gibbons, D. et al. (2001) The biological activity of natural and mutantpTalpha alleles. J. Exp. Med. 194, 695–703

17 Irving, B.A. et al. (1998) Thymocyte development in the absence of pre-T cell receptor extracellular immunoglobulin domains. Science 280,905–908

18 Haks, M.C. et al. (2003) Low activation threshold as a mechanismfor ligand-independent signaling in pre-T cells. J. Immunol. 170,2853–2861

19 Krimpenfort, P. et al. (1989) T cell depletion in transgenic micecarrying a mutant gene for TCR-beta. Nature 341, 742–746

20 Borowski, C. et al. (2004) Pre-TCRalpha and TCRalpha are notinterchangeable partners of TCRbeta during T lymphocytedevelopment. J. Exp. Med. 199, 607–615

21 Mitra, A.K. et al. (2004) Supine orientation of a murine MHC class Imolecule on the membrane bilayer. Curr. Biol. 14, 718–724

22 Kuhns, M.S. et al. (2010) Evidence for a functional sidedness to thealphabetaTCR. Proc. Natl. Acad. Sci. U.S.A. 107, 5094–5099

23 Call, M.E. et al. (2002) The organizing principle in the formation of theT cell receptor-CD3 complex. Cell 111, 967–979

24 Berger, M.A. et al. (1997) Subunit composition of pre-T cell receptorcomplexes expressed by primary thymocytes: CD3 delta is physicallyassociated but not functionally required. J. Exp. Med. 186, 1461–

146725 Kosugi, A. et al. (1997) Subunit composition of the pre-T-cell receptor

complex analysed by monoclonal antibody against the pre-T-cellreceptor alpha chain. Immunology 91, 618–622

26 Abastado, J.P. et al. (1995) Dimerization of soluble majorhistocompatibility complex-peptide complexes is sufficient foractivation of T cell hybridoma and induction of unresponsiveness.J. Exp. Med. 182, 439–447

27 Minguet, S. et al. (2007) Full activation of the T cell receptor requiresboth clustering and conformational changes atCD3. Immunity 26, 43–54

28 Fehling, H.J. et al. (1997) Restoration of thymopoiesis in pT alpha-/-mice by anti-CD3epsilon antibody treatment or with transgenesencoding activated Lck or tailless pT alpha. Immunity 6, 703–714

29 Aifantis, I. et al. (2002) A critical role for the cytoplasmic tail of pTalphain T lymphocyte development. Nat. Immunol. 3, 483–488

30 Navarro, M.N. et al. (2007) Identification of CMS as a cytosolic adaptorof the human pTalpha chain involved in pre-TCR function. Blood 110,4331–4340

31 Tibaldi, E.V. and Reinherz, E.L. (2003) CD2BP3, CIN85 and thestructurally related adaptor protein CMS bind to the same CD2cytoplasmic segment, but elicit divergent functional activities. Int.Immunol. 15, 313–329

32 Dustin, M.L. et al. (1998) A novel adaptor protein orchestrates receptorpatterning and cytoskeletal polarity in T-cell contacts.Cell 94, 667–677

33 Kudo, A. andMelchers, F. (1987) A second gene, VpreB in the lambda 5locus of the mouse, which appears to be selectively expressed in pre-Blymphocytes. EMBO J. 6, 2267–2272

34 Lanig, H. et al. (2004) Three-dimensional modeling of a pre-B-cellreceptor. Mol. Immunol. 40, 1263–1272

35 Minegishi, Y. et al. (1999) Novel mechanisms control the folding andassembly of lambda5/14.1 and VpreB to produce an intact surrogatelight chain. Proc. Natl. Acad. Sci. U.S.A. 96, 3041–3046

36 Pillai, S. and Baltimore, D. (1987) Formation of disulphide-linkedmu 2omega 2 tetramers in pre-B cells by the 18K omega-immunoglobulinlight chain. Nature 329, 172–174

37 Sakaguchi, N. and Melchers, F. (1986) Lambda 5, a new light-chain-related locus selectively expressed in pre-B lymphocytes. Nature 324,579–582

38 Tsubata, T. and Reth,M. (1990) The products of pre-B cell-specific genes(lambda5 andVpreB) and the immunoglobulinmuchain forma complexthat is transported onto the cell surface. J. Exp. Med. 172, 973–976

169

Review Trends in Immunology April 2011, Vol. 32, No. 4

39 Bradl, H. et al. (2003) Interaction of murine precursor B cell receptorwith stroma cells is controlled by the unique tail of lambda 5 andstroma cell-associated heparan sulfate. J. Immunol. 171, 2338–2348

40 Gauthier, L. et al. (2002) Galectin-1 is a stromal cell ligand of the pre-Bcell receptor (BCR) implicated in synapse formation between pre-B andstromal cells and in pre-BCR triggering. Proc. Natl. Acad. Sci. U.S.A.99, 13014–13019

41 Ohnishi, K. andMelchers, F. (2003) The nonimmunoglobulin portion oflambda5 mediates cell-autonomous pre-B cell receptor signaling. Nat.Immunol. 4, 849–856

42 Bankovich, A.J. et al. (2007) Structural insight into pre-B cell receptorfunction. Science 316, 291–294

43 ten Boekel, E. et al. (1997) Changes in the V(H) gene repertoire ofdeveloping precursor B lymphocytes in mouse bone marrow mediatedby the pre-B cell receptor. Immunity 7, 357–368

44 Lillemeier, B.F. et al. (2010) TCR and Lat are expressed on separateprotein islands on T cell membranes and concatenate duringactivation. Nat. Immunol. 11, 90–96

45 Schamel,W.W. et al. (2005) Coexistence ofmultivalent andmonovalentTCRs explains high sensitivity and wide range of response. J. Exp.Med. 202, 493–503

170

46 Irvine, D.J. et al. (2002) Direct observation of ligand recognition byT cells. Nature 419, 845–849

47 Van Wauwe, J.P. et al. (1980) OKT3: a monoclonal anti-humanT lymphocyte antibody with potent mitogenic properties.J. Immunol. 124, 2708–2713

48 Grakoui, A. et al. (1999) The immunological synapse: a molecularmachine controlling T cell activation. Science 285, 221–227

49 Minguet, S. et al. (2010) Low-valency, but not monovalent,antigens trigger the B-cell antigen receptor (BCR). Int. Immunol.22, 205–212

50 Yang, J. and Reth, M. (2010) Oligomeric organization of the B-cellantigen receptor on resting cells. Nature 467, 465–469

51 Harwood, N.E. and Batista, F.D. (2008) New insights into theearly molecular events underlying B cell activation. Immunity 28,609–619

52 Kim, Y.M. et al. (2006) Monovalent ligation of the B cell receptorinduces receptor activation but fails to promote antigenpresentation. Proc. Natl. Acad. Sci. U.S.A. 103, 3327–3332

53 Bellavia, D. et al. (2002) Combined expression of pTalpha and Notch3in T cell leukemia identifies the requirement of preTCR forleukemogenesis. Proc. Natl. Acad. Sci. U.S.A. 99, 3788–3793