- Email: [email protected]
T cell receptor-mediated signs and signals governing T cell development
seminars in I M M U N OL OG Y, Vol 11, 1999: pp. 227]237 Article No. smim.1999.0179, available online at http:rrwww.idealibrary.com on
T cell receptor-mediated signs and signals governing T cell development Nicolai S. C. van Oers
gd . in a non-covalent association with a group of invariant proteins ŽCD3 g , d , « and TCR z .. These invariant subunits are responsible for efficient assembly and surface expression of the various TCR complexes Žreviewed in ref 1.. Additionally, the invariant chains contain a conserved signaling motif that functions to translate effective ligand binding into intracellular biochemical signals Žreviewed in ref 2.. This motif, termed the immune tyrosine based activation motif ŽITAM. wYxxLx Ž6-8.YxxLx, is present as a single copy in CD3 g , d , and « and as three copies in TCR z .3 The signals propagated through the ITAMs ultimately converge in the nucleus, resulting in the induction of various biological responses such as differentiation, proliferation, programmed cell death, cytokine release, andror cytolytic functions. A considerable wealth of knowledge now exists about the mechanisms of TCR-mediated signaling Žreviewed in ref 4.. The central feature of this pathway is the modulation of the tyrosine phosphorylation status of many effector molecules through the activation of several families of protein tyrosine kinases ŽPTKs. Žreviewed in ref 5.. The Src-family of PTKs are proposed to initiate TCR-induced signal transduction by phosphorylating pairs of tyrosine residues present in the ITAMs ŽFigure 2.. Once phosphorylated, the ITAMs form a consensus binding motif for the two Src-homology 2 ŽSH2. domains of the SykrZAP-70 family of PTKs. The recruitment of SykrZAP-70 results in the combined activation of both families of kinases, culminating in the phosphorylation of many additional effector molecules such as LAT, SLP-76, vav, and PLC-g 1.4 These changes lead to a well-established elevation in intracellular calcium and the activation of ras and its downstream MAPK cascades Žreviewed in refs 6 and 7.. The following review will examine the roles of three families of PTKs in TCR signal transduction, with particular emphasis on their functions during thymopoeisis. Several of the more recently identified regulatory molecules will also be reviewed, again with an em-
The developmental fate of T cells is largely controlled by the nature and success of signals mediated by the pre-T cell receptor (TCR) and TCR complexes. These intracellular signals are regulated by cascades of protein tyrosine phosphorylations initiated following ligand binding to the preTCR or TCR complexes. The phosphorylation cascades are primarily orchestrated by two distinct families of protein tyrosine kinases (PTKs), the Src- and the Syk r ZAP-70families. Germline gene targeting experiments, several human immunodeficiencies, and somatic cell mutants have all contributed to our understanding of how these families of kinases coordinate their actions to promote signaling. Upon activation, the PTKs transmit their signals to a number of newly described adaptor proteins including LAT, SLP-76, and vav, among others. The following review combines results derived from different experimental strategies to examine the contributions of the PTKs and the adaptor molecules to pre-TCR and TCR signaling processes. Key words: adaptor proteins r protein tyrosine kinases r signal transduction r T cell receptor Q1999 Academic Press
Introduction THE PRE-T CELL RECEPTOR Žpre-TCR. and T cell receptor ŽTCR. complexes are multi-subunit complexes that mediate the differentiation and expansion of both ab and gd T lineage cells during T cell development ŽFigure 1.. The complexes themselves comprise the ligand binding subunits Žpre-Tarb ; ab ;
From the Center for Immunology and the Department of Microbiology, UT Southwestern Medical Center, Room NA7.201, 6000 Harry Hines Blvd., Dallas, TX 75235-9093, USA Q1999 Academic Press 1044-5323r 99 r 040227q 11 $30.00r 0
227
N. S. C. van Oers
Figure 1. Pre-T Cell Receptor and T cell Receptor Complexes. The immune tyrosine-based activation motifs are represented as darkened rectangles. The pre-TCR complex appears to have a very weak association with the TCR z subunit.
to convert the CD25q CD44 lory cells into a CD25yCD44 lory subpopulation that begins expressing the CD4 and CD8 coreceptor molecules. The CD4yCD8y to CD4qCD8q transition coincides with the termination of pre-Ta expression, and initiation of rearrangements at the TCR a locus. A successful pairing and surface expression of the ab TCR complex allows the CD4qCD8q thymocytes to undergo the stringent processes of positive and negative selection.8 The consequences of these TCR-mediated selection events results in the establishment of a peripheral T cell repertoire that is restricted by selfMHC molecules.
phasis on TCR-mediated signal transduction processes necessary for T cell development.
T cell development The majority of ab T cells, a subset of gd T cells, and certain NK cell populations develop in the thymus. For ab T cells, this developmental process follows a highly ordered series of selection events that are phenotypically defined with the specific expression of certain cell surface molecules such as CD3, CD4, CD8, CD25, and CD44. A simplified overview of this process is illustrated in Figure 3. The entire process of selection will be covered in great detail in the many accompanying review articles in this volume. Following the arrival of precursor cells into the thymus, the TCR b locus undergoes a rearrangement process. A successfully rearranged TCR b gene product subsequently pairs with the pre-TCR a subunit, and this complex is expressed on the surface of cells that lack the CD4 and CD8 coreceptor molecules ŽFigure 3.. Such CD4y CD8y cells can be further subdivided into four distinct subpopulations on the basis of CD25 and CD44 expression. Signals generated through the pre-TCR complex are proposed
TCR r CD3 itams The generation of the peripheral T cell repertoire is contigent upon the nature and success of the TCR signaling pathways that are activated during thympoeisis. The main functions of the ITAMs of the TCR complex are to convey appropriate ligand binding events into intracellular signals. As previously descibed, the TCR complex comprises upwards of ten ITAMs, with six contributed by the TCR z subunit ŽFigure 1.. Given the presence of ten ITAMs, the 228
Signals governing T cell development
Figure 2. Model of Protein Tyrosine Kinase Activation by the T Cell Receptor ITAMs. Following TCR interactions with peptidermajor histocompatibility complexes, the Src-kinases are activated, resulting in the phosphorylation of the tyrosine residues in the ITAMs. This promotes the recruitment of the SykrZAP-70 family of kinases to the TCR complex, in interactions mediated by the two SH2 domains of the kinase with the doubly phosphorylated tyrosines in the ITAMs. The association of SykrZAP-70 with the phosphorylated ITAMs allows for their subsequent phosphorylation and activation. Cross-communication between the two families of kinases promotes the phosphorylation of multiple downstream effector molecules.
phosphorylation of increasing numbers of ITAMs may contribute quantitatively to the strength of signaling. The concept of quantitative signaling with increasing TCRrCD3 ITAMs was originally described for cell lines expressing chimeric molecules with the extracellular domain of CD8 and an intracellular region containing either one, two, or three TCR z ITAMs.9 However, many studies have also described qualitative differences between the various phosphorylated TCRrCD3 ITAMs and their abilities to interact with downstream effector molecules Žreviewed in ref 5.. The issues of qualitative vs quantitative signaling differences may have important ramifications for T cell selection processes in the thymus, and these have yet to be resolved. With regards to thymopoeisis, some apparent distinctions between the various invariant chains have
been described. For example, the targeted disruption of the CD3 « allele Žalso affects CD3 g and d transcription. results in a complete development arrest of both ab and gd T cells at the CD4yCD8y stage, similar to mice lacking the RAG genes.10,11 In contrast, the targeted disruption of the TCR z molecule results in a much less severe development block.12 ] 14 Thus, CD4qCD8q thymocytes lacking surface TCR expression are still apparent in these mice, although at numbers that vary from two- to 30-fold less than age matched, wild-type littermates. These results would imply that pre-TCR signaling is still functional in TCR z-null mice, consistent with the concept that the TCR, and perhaps the pre-TCR, contain two independent signal transduction modules wTCR z vs CD3 g , d , and « x.15 Alternatively, TCR z may not be required for the assembly of the pre-TCR complex. It 229
N. S. C. van Oers
Figure 3. Overview of T Cell Development. The development of ab T cells in the thymus follows a well-defined series of development changes phenotypically characterised by the expression of various cell surface markers, as indicated. The effects of the targeted disruptions of various effector molecules on this developmental pathway are high-lighted below the pathways. A solid bar indicates a complete developmental arrest, whereas a dashed bar implies an incomplete developmental block.
is interesting to note that normal T cell development can be restored in the TCR z knock-out mice by introducing TCR z transgenes comprising zero, one, or all three z ITAMs.16 These studies demonstrated that the TCR z ITAMs are not essential for thymopoeisis, but z is important for proper TCR assembly and surface expression. In fact, only in a well-defined ab trangenic TCR background ŽH-Y TCR transgenic mice. does it become obvious that the number of TCR z ITAMs can contribute in a quantitative capacity to positive and negative selection.17 A second distinction between the various invariant chains is the presence of a pool of constitutively tyrosine phosphorylated TCR z subunits in murine thymocytes and peripheral T cells.18,19 This constitutive phosphorylation, resulting in the formation of a 21-kDa phosphorylated form of TCR z , is unique to z as none of the other CD3 subunits are constitutively phosphorylated. The constitutive phosphorylation of TCR z is, in part, a consequence of ongoing TCR
interactions with peptiderMHC molecules expressed in the thymus.18,20 As discussed below, both the Lck and ZAP-70 protein tyrosine kinases ŽPTKs. are required for the maintenance of the constitutively phosphorylated TCR z subunits.21,22 In addition to the 21-kDa form of TCR z , a second phosphorylated form of TCR z Žapparent molecular mass of 23 kDa. can be induced following receptorrligand interactions.20,23 ] 25 Notably, a strong and provocative correlation has been established between the induction of particular phosphorylated forms of TCR z Ž21 vs 23 kDa. and the functional responsiveness of T cells Žreviewed in ref 23.. The presence or absence of these distinct phosphorylated forms of TCR z may also have relevance to the processes of positive and negative selection in the thymus.
Protein tyrosine kinases Three families of protein tyrosine kinases ŽPTKs., the 230
Signals governing T cell development
Src-, Syk-, and Tec-families, have important and distinct functional contributions to T cell development. Lck and Fyn, two members of the Src-family of PTKs, are primarily restricted in expression to T cells.26 They have unique N-terminal sequences followed by a Src-homology 3 ŽSH3., a Src-homology 2 ŽSH2., and a kinase domain. Although these kinases are targeted to the plasma membrane through myristylation and palmitylation modifications at their NH 2-terminus, a proportion of Lck molecules are found associated with the CD4 and CD8 coreceptor molecules while Fyn can complex with the TCR, albeit at low stoichiometry.27 The important contributions of the CD4 and CD8 coreceptor molecules to T cell development are reviewed elsewhere in this issue. A combination of biochemical and genetic data has elegantly demonstrated a principle role for Lck in initiating TCR-signaling cascades by phosphorylating the ITAMs in the TCR complex 21,28 Žreviewed in ref 27.. This pathway was initially uncovered following the description of a Lck-deficient leukemic cell that was unable to initiate TCR-mediated signaling processes unless reconstituted with Lck.29 With regards to thymopoeisis, Lck is expressed in all thymocyte subsets, and numerous experiments have revealed a role for Lck in pre-TCR and TCR-signaling Žreviewed in refs 30 and 31.. In brief, mice rendered deficient in Lck have a 5]20 fold reduction in thymic cellularity and a severe paucity of both CD4qCD8q and mature single positive T cells.32 The mature T cells that can develop in these mice are unable to respond effectively to TCR stimulations.32 Such problems are primarily related to the fact that Lck is required for initiating TCR signal transduction by phosphorylating the ITAMs and for phosphorylating and activating the SykrZAP-70 family of PTKs following their high affinity binding to the phosphorylated ITAMs ŽFigure 2..33 ] 35 As a consequence, very little constitutive phosphorylation of the TCR z molecules can be detected in thymocytes from Lcknull mice.21 Moreover, no inducible phosphorylation of TCRrCD3 ITAMs or ZAP-70 is noted in these knock-out animals. Fyn is a second Src-family member that functions in the TCR signaling cascade, in part, by phosphorylating the TCR ITAMs. In contrast to Lck, Fyn is expressed at 10-fold higher levels in mature single positive thymocytes relative to CD4qCD8q cells.26 Interestingly, Fyn-deficient mice have normal numbers of thymocytes and peripheral T cells.36,37 The single positive thymocytes and mature T cells in Fyn-
null mice are hypo-responsive following TCR crosslinking, as evidenced by their poor proliferative responses and low mobilisation of intracellular calcium.36,37 Consistent with the knock-out animals, mice expressing a dominant negative Fyn transgene only have a defect in single positive TCR-mediated signal transduction.38 This defect is partly a consequence of reduced IL-2 production by the stimulated T cells, suggesting that Fyn couples to the IL-2 pathway.36,37 The inability of Lck to promote signaling in the absence of Fyn may relate to the unique substrate specificity of Fyn. Thus, Fyn can selectively phosphorylate both Pyk2, a member of the focal adhesion family of PTKs, and Fyb ŽSLAP130., a Fyn and SLP-76 associating signaling protein.39 ] 41 This selectivity suggests a bifurcation point in the functions of Fyn and Lck and may indicate a unique contribution for Fyn in IL-2 production in mature T cells through a pathway involving Fyb ŽSLAP130.. Mice rendered deficient in both Lck and Fyn have a complete ab T cell developmental arrest at the CD4yCD8y ŽCD25y CD44y . stage of thympoeisis.42,43 Taken together, the aforementioned data would suggest that Lck is the primary Src-family PTK responsible for T cell development although Fyn can partially compensate for Lck in pre-TCR and TCR signaling. The requirements for Fyn and Lck in gd and NK cell development are discussed in the accompanying review articles. Syk and ZAP-70 are members of a second family of PTKs that function as critical mediators of pre-TCR and TCR signaling, with ZAP-70 having a predominant role in mature T cells Žreviewed in ref 31.. Defined by the presence of two NH 2 terminal SH2 domains and a COOH-terminal kinase domain, ZAP70 is localised as a diffuse band under the plasma membrane.27,44 The two SH2 domains form a high affinity interaction with the doubly phosphorylated ITAMs. Upon TCR ligation, both kinases can associate with the tyrosine phosphorylated ITAMs of the pre-TCR and TCR complexes, and, in turn, are tyrosine phosphorylated.45 ] 47 The phosphorylations on ZAP-70 increase the intrinsic catalytic activity of the kinase and allow for the binding of additional signaling proteins Žvav, Lck., or subsequent attenuators of signaling Žc-cbl, SHP-1.. Both ZAP-70 and Syk are expressed in developing thymocytes, with ZAP-70 upregulated during the CD4qCD8q to the single positive transition, while Syk undergoes a concomitant down-modulation following pre-TCR signaling.46,48 The functional requirements for ZAP-70 in TCRmediating signaling processes were elucidated in sev231
N. S. C. van Oers
eral severe combined immunodeficiency ŽSCID. patients that harbored mutations in the ZAP-70 gene.49 ] 51 Phenotypically, immature CD4qCD8q thymocytes from such patients could only mature into CD4q T cells, and these cells were functionally incompetent to transduce TCR-mediated signals. Thus, TCR cross-linking resulted in a very poor induction of tyrosine phosphoproteins, demonstrating a central role for ZAP-70 in initiating TCR signal transduction cascades. Mice lacking ZAP-70 or mice harboring a catalytically inactive ZAP-70 molecule have normal numbers of CD4qCD8q thymocytes that are unable to undergo either positive or negative selection processes, such that no CD4q or CD8q cells are generated.22,52,53 The phenotypes described for the ZAP-70 deficiencies are largely explained by the requirements of ZAP-70 to phosphorylate several key signalingradaptor substrates including LAT, SLP-76, and vav.5,54 Moreover, the two SH2 domains of ZAP-70 are required for maintaining the constitutive and inducible phosphorylations of the TCR z chain.22 It is interesting to note that the results with the mice differ somewhat from that described for the human SCIDs, and this may relate to the differences in Syk expression and function, as described below. In contrast to ZAP-70, Syk-deficient mice have a completely normal pattern of ab T cell development and a selective impairment of certain gd lineage T cells.55,56 The targeted disruption of both ZAP-70 and Syk results in a complete attenuation of thymocyte development at the CD4yCD8y stage, with the majority of cells in these mice maintaining a CD25qCD44yrlo phenotype, analogous to the subsets in LckrFyn- and Rag-null mice.31 Such findings again demonstrate the existence of functional redundancy within a given family of PTKs. Thus, Syk can partially compensate for the absence of ZAP-70 by supporting pre-TCR signal transduction.31 The failure of Syk to promote differentiation of thymocytes to the single positive stage in mice may relate to the marked down-regulation of Syk expression that occurs in double positive thymocytes.46,48 This is consistent with the ability of Syk to support complete T cell development and TCR-mediated signaling when over-expressed as a transgene in ZAP-70-null mice.57 One important distinction between ZAP-70 and Syk is the observation that Syk has a much higher intrinsic enzymatic activity and much less dependence on Srcfamily kinases for its activation.58,59 Itk is a member of the TecrBtk family of PTKs that is also implicated in thymopoeisis. This family of kinases has several functional domains including a
NH 2 terminal plextrin homology ŽPH. domain, followed by a SH3, SH2, and a kinase domain. The PH domain is involved in localising these PTKs to the cellular membrane by interacting with the phospholipid PIP3 . Mice deficient in Itk have a small reduction in single positive thymocytes, an effect more obviously noted in a ab TCR transgenic background.60 Moreover, the mature T cells in these mice have reduced TCR-mediated proliferative responses largely as a consequence of reduced IL-2 production. It will be interesting to determine whether Itk connects to the FynrFyb pathway for IL-2 production. Mutations in the B cell family member, Btk, results in X-linked agammaglobulinemia in humans and X-linked immunodeficiency in mice.61 These extremely severe B cell phenotypes relative to the mild T cell defects in Itk-deficient mice may suggest the presence of additional Tec family members in T cells that can facilitate thymopoeisis. The exact role of Itk in TCRsignaling requires further investigation.
Signaling molecules The preliminary events of pre-TCR- or TCR-mediated PTK activation must be successfully relayed to appropriate downstream substrates. A theme common to many different signaling systems is the recruitment and activation of selected enzymes through the use of scaffold, anchoring, andror adaptor proteins.62 T cells are no exception, with an exciting number of newly identified and essential adaptor molecules having recently been identified Žreviewed in ref 4.. One key T cell adapter protein Žalso in NK cells, mast cells, and basophils. is termed LAT Žlinker for activation of T cells..54 LAT is a 36]38-kDa palmitylated transmembrane glycoprotein that becomes heavily tyrosine phosphorylated following TCR stimulation.54 At least 6 tyrosine residues are phosphorylated in LAT, most by ZAP-70, and these phosphorylated sequences form consensus binding motifs for the SH2 domains of phosphopholipase C gamma 1 ŽPLC-g 1., the Grb2 adapter protein, and Grap ŽGrb2-like. ŽFigure 4..54 Thus, LAT serves as an adaptor protein by connecting the PTKs to downstream substrates. The crucial role of LAT in T cell signaling was established with an analysis of a LAT-deficient Jurkat T cell line.63 Cross-linking the TCR on these cells revealed a normal ITAM and ZAP-70 phosphorylation without any subsequent mobilisation of intracellular calcium or ras activation.63 Additionally, overexpression of a mutant LAT bearing tyrosine to phenylalanine substi232
Signals governing T cell development
tutions reduces the magnitude of NF-AT driven transcriptional reporter constructs.54 Given the fact that the highest expression levels of LAT are in the thymus Žassessed by Northern blotting., LAT-deficient mice are likely to have a complete block in ab and gd T cell development. The lack of intracellular calcium mobilisation in the LAT-deficient cells is likely a consequence of the failure to recruit and phosphorylate PLC-g 1. Interestingly PLC-g 1, an enzyme essential for the activation of the phosphatidylinositol pathway, was one of the first well-char-
acterised phosphorylated substrates in T cells Žreviewed in ref 7.. Activated PLC-g 1 hydrolyses phosphatidylinositol 4,5 bisphosphate into the second messengers IP3 and diacylglycerol ŽDAG., which are, in turn, responsible for the well-characterised intracellular calcium increases and PKC activation, respectively.6 The calcium increase is required to activate the serinerthreonine phosphatase calcineurin, which, in turn, activates an important IL-2 transcription factor, nuclear factor of activated T cells ŽNFAT.. A second critical molecule recruited to phosphory-
Figure 4. T Cell Receptor Signaling Cascades. Following the initiation of TCR signaling, a number of downstream adaptor proteins are phosphorylated. One key adaptor protein phosphorylated by ZAP-70 is LAT. The phosphorlation sites on LAT serve as consensus binding motifs for the SH2 domains of PLC-g 1 and Grb2. The subsequent phosphorylation and activation of PLC-g 1 results in the release of intracellular calcium stores and the activation of PKC. Since Grb2 forms complexes with SLP-76 and SOS, the recruitment of Grb2 to LAT provides a mechanism for relocating SOS to the membrane, facilitating ras activation and the downstream MAPK pathway. The re-localisation andror phosphorylation of SLP-76 is also necessary for both ras activation and PLC-g 1 phosphorylation. This may be partly attributed to the association of SLP-76 with vav, a guanine nucleotide exchanger on the rhorracrCDC42 family of GTPases. The actions of vav may regulate actin polymerisation and cytoskeletal re-organisation. Vav can also form an independent complex with phosphorylated ZAP-70, and this may also contribute to vav activation. Finally, Fyn may couple to the IL-2 pathway through its association with a SLP-76 interacting protein, termed Fyb, or SLAP-130.
233
N. S. C. van Oers
pre-TCR a expression were apparent in the SLP-76 null animals, the absence of SLP-76 most probably impairs pre-TCR signaling. The ability of the dominant negative MAPKK to block thymocyte development is consistent with a requirement for SLP-76 to promote ras activation in early thymocyte development. In mature T cells, the mechanism by which SLP-76 couples to IL-2 production is not clear but certainly involves another TCR-induced phosphoprotein, the vav proto-oncogene Žreviewed ref 5.. Vav functions as a guanine nucleotide exchange factor ŽGEF. for the rhorracrCDC42 families of GTPases, and also contains a PH domain, and two SH3 domains at its COOH terminus interspersed by an SH2 domain. Following TCR ligation, vav becomes tyrosine phosphorylated and also forms two independent complexes with phosphorylated ZAP-70 or SLP-76 through its SH2 domain. Overexpression of wild-type vav can significantly increase both the basal and TCR-induced NFAT and IL-2-driven transcriptional activity.73 Notably, the combined overexpression of vav and SLP-76 can synergistically potentiate the aforementioned transcriptional activity. As described earlier, PLC-g 1 is not phosphorylated in SLP-76 deficient cells. This may be a consequence of the absence of vav association, since vav may be required to promote cytoskeletal reorganisation through its GEF functions. With regards to T cell development, vav-deficient mice have been generated by a RAG-complementation approach, and these mice exhibit a 10]20-fold reduction in all thymocyte subsets.74 ] 76 Moreover, these cells have severely reduced proliferative responses to TCR stimuli. It remains to be established why the phenotype of the SLP-76 deficient mice is much more severe than that of the vav-null mice, but may relate to the ability of SLP-76 to associate with several additional phosphorylated proteins.68 As mentioned earlier, SLP-76-deficient T cells cannot mobilise intracellular calcium. In contrast, most of the data with vav-deficient thymocytes indicates that a normal calcium flux can be elicited following TCR cross-linking. Taken together, these results would imply that SLP-76 may couple to multiple downstream effector molecules. Alternatively, a vav homologue may also be expressed in hematopoeitic cells and this may partially compensate for the absence of vav.
lated LAT is Grb2, an adapter protein that itself complexes to several other key regulatory molecules including sos-of-sevenless ŽSOS., SLP-76 ŽSH2 domain leukocyte protein of 76 kDa., and the c-cbl protooncogene ŽFigure 4..6,54,64 Grb2 contains one SH2 domain flanked on either side by a SH3 domain. The SH3 domains of Grb2 can bind to proline rich peptide sequences in both SOS and SLP-76. SOS is an important guanine nucleotide exchanger for the ras family of small GTPases, functioning to activate ras. Activated ras has a central role in IL-2 gene activation by regulating the MAPK ŽErk. pathway, and this pathway has been reviewed elsewhere.6 With regards to thymopoeisis, an activated form of ras, when expressed as a transgene in Rag-deficient embryonic stem cells, is capable of promoting the differentiation of ab T cells to the CD4qCD8q stage.65 Since ras connects to the MAPK pathway, these results are also consistent with the observations that a dominant negative MAPKK, when expressed in fetal thymic organ cultures, blocks the transition of thymocytes from double negative to double position cells.66 Using the Lck proximal promoter to express a dominant negative ras transgene in thymocytes, a specific block in positive but not negative selection was revealed.67 Grb2 also associates with SLP-76, a recently identified T and NK cell specific molecule that contains three tyrosine residues located near the NH 2terminus, a central proline rich stretch, and a COOH-terminal SH2 domain.64,68,69 Following TCR cross-linking, ZAP-70 phosphorylates SLP-76 on the three NH 2-terminal tyrosines, presumably following the association of SLP-76rGrb2 with phosphorylated LAT. In mature T cells, overexpression of SLP-76 potentiates TCR-mediated IL-2 gene induction while a mutant form lacking either the three NH 2-terminal tyrosines or a functional SH2 domain can attenuate IL-2 responses.69 Interestingly, T cell lines lacking SLP-76 have revealed a requirement for SLP-76 in PLC-g 1 phosphorylation and ras activation, but not in the TCR-induced phosphorylation of most other proteins.70 These results would suggest that the Grb2rSOS pathway is not sufficient to activate ras. The importance of SLP-76 in thymopoeisis was recently described with the generation of two independent SLP-76 deficient mice.71,72 The thymocyte subpopulations in these mice revealed a complete developmental arrest within the CD4yCD8y subset, at the transition point from CD44yCD25q to CD44yCD25y cells. This is the same developmental arrest point as the LckrFyn and ZAP-70rSyk double knock-out mice. As TCR b gene rearrangements and
Protein tyrosine phosphatases The pre-TCR and TCR-mediated signals are coordi234
Signals governing T cell development
Concluding remarks
nately regulated by the actions of PTKs and protein tyrosine phosphatases ŽPTPases.. Although many PTPases will likely be identified as key regulators of antigen receptor signal transduction, two well-characterised ones include CD45 and SHP-1. CD45 is a transmembrane PTPase that comprises two intracellular PTPase domains and is expressed on almost all hematopoeitic cells.77 Several isoforms have been identified on T cells as a consequence of regulated processes of alternative splicing. CD45 primarily functions as a positive regulator of antigen receptor signal transduction by activating the Src-kinases Lck and Fyn through the dephosphorylation of the negative regulatory tyrosine residue in these. The importance of CD45 in T cell development was evident with the 4]10-fold reduction in the numbers of CD4qCD8y and CD4yCD8q thymocytes and peripheral T cells in mice lacking either all CD45 isoforms or CD45-exon 6-null mice.78,79 The CD4qCD8q thymocytes from the CD45-null mice are refractory to anti-TCR mAb-induced negative selection and those mature T cells that do develop display reduced TCRmediated proliferative responses.78,79 This is largely explained by the fact that Fyn and Lck are hyperphosphorylated at their COOH-terminal negative regulatory tyrosine in CD45-null thymocytes. As a consequence, these kinases are relatively inactive resulting in hypo-phosphorylation of the TCR ITAMs and a relatively poor recruitment of ZAP-70 to the TCR complex.80. What remains unclear at present in how T cell development can proceed to the CD4qCD8q stage in the absence of CD45. SHP-1 is a hematopoeitic specific PTPase that appears to down-modulate antigen receptor signaling systems.77 Two naturally occurring SHP-1 mutations were identified in mice Žmotheaten mice. several years ago. Phenotypically, the thymocytes from these mice have a normal developmental profile. Functionally, both thymocytes and peripheral T cells from these SHP-1 mutant mice hyper-proliferate in response to anti-TCR stimulations.1,81 Additionally, the levels of the constitutively and inducibly phosphorylated proteins in these thymocytes are markedly elevated relative to normal mice.81 With regards to mechanism, the absence of SHP-1 results in the hyper-activation of the Src-family kinases, leading to increased basal and TCR-inducible phosphorylation levels of the ITAMs and ZAP-70. SHP-1 has also been reported to complex ZAP-70, and this may be an additional means of regulating antigen responsiveness.82
The processes of antigen receptor signal transduction are emerging as well-defined biochemical pathways involving a coordinated regulation of PTK and PTPase activities. A new set of exciting signalingradaptor proteins have recently been identified in these signaling cascades. Thus, a combination of cloning reports, gene knock-out results, and studies with somatic cell mutants have revealed essential roles for the LAT, SLP-76, and vav molecules. The exact functional mechanisms of these and other signaling molecules during pre-TCR and TCR-signaling will emerge rapidly. Over the next few years, novel activators and attenuators of antigen receptor signals will likely be identified, many of which will have significant functional contributions to ab , gd , and NK cell development.
Acknowledgements I would like to thank Dr T.C. Ayi, Mr. B. Tohlen, and Mrs. A. Feulner for their reading of this manuscript. This work was supported in part by a grant from the NIH ŽAI4295301A1. and Fikes support funding from UT Southwestern Medical Center.
References 1. Ashwell JD, Klausner RD Ž1990. Genetic and mutational analysis of the T-cell antigen receptor. Ann Rev Immunol 8:139]167 2. Weiss A Ž1993. T cell antigen receptor signal transduction: a tale of tails and cytoplasmic protein-tyrosine kinases. Cell 73:209]212 3. Cambier JC Ž1995. New nomenclature for the Reth motif Žor ARH1rTAMrARAMrYXXL.. Immunol Today 16:110 4. Rudd CE Ž1999. Adaptors and molecular scaffolds in immune cell signaling. Cell 96:5]8 5. Wange RL, Samelson LE Ž1996. Complex complexes: signaling at the TCR. Immunity 5:197]205 6. Cantrell D Ž1996. T cell antigen receptor signal transduction pathways. Ann Rev Immunol 14:259]274. 7. Weiss A, Littman DR Ž1994. Signal transduction by lymphocyte antigen receptors. Cell 76:263]274 8. Jameson SC, Bevan MJ Ž1998. T-cell selection. Curr Opin Immunol 10:214]219 9. Irving BA, Chan AC, Weiss A Ž1993. Functional characterization of a signal transducing motif present in the T cell receptor z chain. J Exp Med 177:1093]1103. 10. Malissen M, Gillet A, Ardoiun L, Bouvier G, Trucy J, Ferrier P, Viver E, Malissen B Ž1995. Altered T cell development in mice with a targeted mutation of the CD3-« gene. EMBO J 14:4641]4653 11. Tanaka Y, Ardoiun L, Gillet A, Lin S-Y, Magnan A, Malissen B, Malissen M Ž1995. Early T-cell development in CD3-deficient mice. Immunol Rev 148:171]199
235
N. S. C. van Oers
12. Love PE, Shores EW, Johnson MD, Tremblay ML, Lee EJ, Grinberg A, Huang SP, Singer A, Westphal H Ž1993. T cell development in mice that lack the z chain of the T cell antigen receptor compex. Science 261:918]921 13. Liu C-P, Ueda R, She J, Sancho J, Wang B, Weddell G, Loring J, Kurahara C, Dudley EC, Hayday A, Terhorst C, Huang M Ž1993. Abnormal T cell development in CD3-zyry mice and identification of a novel T cell population in the intestine. EMBO J 12:4863]4875 14. Malissen M, Gillet A, Rocha B, Trucy J, Brun N, Mazza G, Spanopoulou E, Guy-Grand D, Malissen B Ž1993. T cell development in mice lacking the CD3-zrh gene. EMBO J 12:4347]4355 15. Wegener A-MK, Letourneur F, Hoeveler A, Brocker T, Luton F, Malissen B Ž1992. The T cell receptorrCD3 complex is composed of at least two autonomous transduction modules. Cell 68:83]95 16. Shores EW, Huang K, Tran T, Lee E, Grinberg A, Love PE Ž1994. Role of TCR z chain in T cell development and selection. Science 266:1047]1050 17. Shores EW, Tran T, Grinberg A, Sommers CL, Shen H, Love PE Ž1997. Role of multiple TCR z chain signaling motifs in selection of the T cell repertoire. J Exp Med 185:893]900 18. Nakayama T, Singer A, Hsi ED, Samelson LE Ž1989. Intrathymic signalling in immature CD4qCD8q thymocytes results in tyrosine phosphorylation of the T-cell receptor zeta chain. Nature 341:651]654 19. van Oers NSC, Tao W, Watts JD, Johnson P, Aebersold R, Teh H-S Ž1993. Constitutive tyrosine phosphorylation of the T cell receptor ŽTCR. z subunit: Regulation of TCR-associated protein kinase activity by TCR z . Mol Cell Biol 13:5771]5780 20. van Oers NSC, Killeen N, Weiss A Ž1994. ZAP-70 is constitutively associated with tyrosine phosphorylated TCR z in murine thymocytes and lymph node T cells. Immunity 1:675]685 21. van Oers NSC, Killeen N, Weiss A Ž1996. Lck regulates the tyrosine phosphorylation of the TCR subunits and ZAP-70 in murine thymocytes. J Exp Med 183:1053]1062 22. Kadlecek TA, van Oers NSC, Lefrancois L, Olson S, Finlay D, Chu DH, Connolly K, Killeen N, Weiss A Ž1998. Differential requirements for ZAP-70 in TCR signaling and T cell development. J Immunol 161:4688]4694 23. Jameson SC, Bevan MJ Ž1995. T cell receptor antagonists and partial agonists. Immunity 2:1]11 24. Sloan-Lancaster J, Shaw AS, Rothbard JB, Allen PM Ž1994. Partial T cell signaling: Altered phospho-z and lack of ZAP-70 recruitment in APL-induced T cell anergy. Cell 79:913]922 25. Madrenas J, Wange RL, Wang JL, Isakov N, Samelson LE, Germain RN Ž1995. z phosphorylation without ZAP-70 activation induced by T cell receptor antagonists or partial agonists. Science 267:515]518 26. Olszowy MW, Leuchtmann PL, Veillette A, Shaw AS Ž1995. Comparison of p56 l ck and p59 f y n protein expression in thymocyte subsets, peripheral T cells, NK cells, and lymphoid cell lines. J Immunol 155:4236]4240 27. Chan AC, Desai D, Weiss A Ž1994. Role of protein tyrosine kinases and protein tyrosine phosphatases in T cell antigen receptor signaling. Ann Rev Immunol 12:555]592 28. Iwashima M, Irving BA, van Oers NSC, Chan AC, Weiss A Ž1994. Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science 263:1136]1139 29. Straus D, Weiss A Ž1992. Genetic evidence for the involvement of the Lck tyrosine kinase in signal transduction throught the T cell antigen receptor. Cell 70:585]593 30. Anderson SJ, Perlmutter RM Ž1995. A signaling pathway governing early thymocyte maturation. Immunol Today 16:99]105 31. Cheng AM, Cahn AC Ž1997. Protein tyrosine kinases in thymocyte development. Curr Opin Immunol 9:528]533
32. Molina TJ, Kishihara K, Siderovski DP, van Ewijk W, Narendran A, Timms E, Wakeham A, Paige CJ, Hartmann K-U, Veillette A, Davidson D, Mak TW Ž1992. Profound block in thymocyte development in mice lacking p56lck. Nature 357:161]164 33. Chan AC, Dalton M, Johnson R, Kong G-H, Wang T, Thoma R, Kurosaki T Ž1995. Activation of ZAP-70 kinase activity by phosphorylation of tyrosine 493 is required for lymphocyte antigen receptor function. EMBO J 14:2499]2508 34. Watts JD, Affolter M, Krebs DL, Wange RL, Samelson LE, Aebersold R Ž1994. Identification by electrospray ionization mass spectromoetry of the sites of tyrosine phosphorylation induced in activated Jurkat T cells on the protein tyrosine kinase ZAP-70. J Biol Chem 269:29520]29529 35. Wange RL, Guitian R, Isakov N, Watts JD, Aebersold R, Samelson LE Ž1995. Activating and inhibitory mutations in adjacent tyrosines in the kinase domain of ZAP-70. J Biol Chem 270:18730]18733 36. Stein PL, Lee H-M, Rich S, Soriano P Ž1992. pp59 fyn mutant mice display differential signaling in thymocytes and peripheral T cells. Cell 70:741]750 37. Appleby MW, Gross JA, Cooke MP, Levin SD, Qian X, Perlmutter RM Ž1992. Defective T cell receptor signaling in mice lacking the thymic isoform of p59 f y n . Cell 70:751]763 38. Cooke MP, Abraham KM, Forbush KA, Perlmutter RM Ž1991. Regulation of T cell receptor signaling by a src family protein-tyrosine kinase Žp59 fyn .. Cell 65:281]292 39. Qian D, Lev S, van Oers NSC, Schlessinger J, Weiss A Ž1997. Tyrosine phosphorylation of Pyk2 is selectively regulated by Fyn during TCR signaling. J Exp Med 185:1253]1259 40. Musci MA, Hendricks-Taylor LR, Motto DG, Paskind M, Kamens J, Turck CW, Koretzky GA Ž1997. Molecular cloning of SLAP-130, an SLP-76-associated substrate of the T cell antigen rceptor stimulated protein tyrosine kinases. J Biol Chem 272:11674]11677 41. daSilva AJ, Li Z, de Vera C, Canto E, Findell P, Rudd CE Ž1997. Cloning of a novel T-cell protein FYB that binds FYN and SH2-domain-containing leukocyte protein 76 and modulate interleukin 2 production. Proc Natl Acad Sci USA 94:7493]7498 42. Groves T, Smiley P, Cooke MP, Forbush K, Perlmutter RM, Guidos CJ Ž1996. Fyn can partially substitute for Lck in T lymphocyte development. Immunity 5:417]428 43. van Oers NSC, Lowin-Kropf B, Finlay D, Connolly K, Weiss A Ž1996. ab T cell development is abolished in mice lacking both Lck and Fyn protein tyrosine kinases. Immunity 5:429]436 44. Huby RDJ, Iwashima M, Weiss A, Ley SC Ž1997. ZAP-70 protein tyrosine kinase is constitutively targeted to the T cell cortex independently of its SH2 domains. J Cell Biol 137:1639]1649 45. Chan AC, Iwashima M, Turck CW, Weiss A Ž1992. ZAP-70: A 70kD protein tyrosine kinase that associates with the TCR z chain. Cell 71:649]662 46. Chan AC, van Oers NSC, Tran A, Turka L, Law C-L, Ryan JC, Clark EA, Weiss A Ž1994. Differential expression of ZAP-70 and Syk protein tyrosine kinases, and the role of this family of protein tyrosine kinases in T cell antigen receptor signalling. J Immunol 152:4758]4766 47. van Oers NSC, von Boehmer H, Weiss A Ž1995. The pre-TCR complex is functionally coupled to the TCR z subunit. J Exp. Med 182:1585]1590 48. Chu DH, van Oers NSC, Malissen M, Harris J, Elder M, Weiss A Ž1999. Pre-T cell receptor signals are responsible for the downregulation of Syk protein tyrosine kinase expression. Submitted 49. Arpaia E, Shahar M, Dadi H, Cohen A, Roifman CM Ž1994. Defective T cell receptor signaling and CD8q thymic selection in humans lacking ZAP-70 kinase. Cell 76:947]958
236
Signals governing T cell development
50. Chan AC, Kadlecek TA, Elder ME, Filipovich AH, Kuo W-L, Iwashima M, Parslow TG, Weiss A Ž1994. ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency. Science 264:1599]1601 51. Elder ME, Lin D, Clever J, Chan AC, Hope TJ, Weiss A, Parslow T Ž1994. Human severe combined immunodeficiency due to a defect in ZAP-70, a T cell tyrosine kinase. Science 264:1596]1599 52. Negishi I, Motoyama N, Nakayama K-I, Nakayama K, Senju S, Hatakeyama S, Zhang Q, Chan AC, Loh DY Ž1995. Essential role for ZAP-70 in both positive and negative selection of thymocytes. Nature 376:435]438 53. Wiest DL, Ashe JM, Howcroft TK, Lee H-M, Kemper DM, Negishi I, Singer DS, Singer A, Abe R Ž1997. A spontaneously arising mutation in the DLAARN motif of murine ZAP-70 abrogates kinase activity and arrests thymocyte development. Immunity 6:663]671 54. Zhang W, Sloan-Lancaster J, Kitchen J, Trible RP, Samelson LE Ž1998. LAT: The ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation. Cell 92:83]92 55. Turner M, Mee PJ, Costello PS, Williams O, Price AA, Duddy LP, Furlong MT, Geahlen RL, Tybulewicz VLJ Ž1995. Perinatal lethality and blocked B cell development in mice lacking the tyrosine kinase Syk. Nature 378:298]302 56. Cheng AM, Rowley B, Pao W, Hayday A, Bolen JB, Pawson T Ž1995. Syk tyrosine kinase required for mouse viability and B cell development. Nature 378:303]306 57. Gong Q, White L, Johnson R, White M, Negishi I, Thomas M, Chan AC Ž1997. Restoration of thymocyte development and function in zap-70yry mice by the Syk protein tyrosine kinase. Immunity 7:369]377 58. Latour S, Chow LML, Veillette A Ž1996. Differential intrinsic enzymatic activity of Syk and ZAP-70 protein tyrosine kinases. J Biol Chem 271:22782]22790 59. Chu D, Spits H, Peyron J-F, Rowley RB, Bolen JB, Weiss A Ž1996. The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling. EMBO J 15:6251]6261 60. Liao XC, Littman DR Ž1995. Altered T cell development in mice lacking Itk. Immunity 3:757]769 61. Rawlings DJ, Witte ON Ž1994. Bruton’s tyrosine kinase is a key regulator in B cell development. Immunol Rev 138:105]119 62. Pawson T, Scott JD Ž1997. Signaling though scaffold, anchoring, and adaptor proteins. Science 278:2075]2080 63. Finco TS, Kadlecek T, Zhang W, Samelson LE, Weiss A Ž1998. LAT is required for TCR-edmiated activation of PLC-g1 and the ras pathway. Immunity 9:617]626 64. Jackman JK, Motto DG, Sun Q, Tanemoto M, Turck CW, Peltz GA, Koretzky GA, Findell PR Ž1995. Molecular cloning of SLP-76, a 76 kDa tyrosine phosphoprotein associated with Grb2 in T cells. J Biol Chem 269:7029]7032 65. Swat W, Shinkai Y, Cheng H-L, Davidson L, Alt FW Ž1996. Activated ras signals differentiation and expansion of CD4 q CD8 q thymocytes. Proc Natl Acad Sci USA 93:4683]4687 66. Crompton T, Gilmour KC, Owen MJ Ž1996. The MAP kinase pathway controls differentiaton from double-negative to double positive thymocyte. Cell 86:243]251 67. Swan KA, Alberola-IIa J, Gross JA, Appleby MW, Forbush KA, Thomas JF, Perlmutter RM Ž1995. Involvement of p21ras distinguishes positive and negative selection in thymocytes. EMBO J 14:276]285
68. Motto DG, Ross SE, Wu J, Hendricks-Taylor LR, Koretzky GA Ž1996. Implication of the GRB2-associated phosphoprotein SLP-76 in T cell receptor interleukin 2 production. J Exp Med 183:1937]1943 69. Wardenburg JB, Fu C, Jackman JK, Flotow H, Wilkinson SE, Williams DH, Johnson R, Kong G, Chan AC, Findell P Ž1996. Phosphorylation of SLP76 by the ZAP-70 protein tyrosine kinase is required for T cell receptor function. J Biol Chem 271:19641]19644 70. Yablonski D, Kuhne MR, Kadlecek T, Weiss A Ž1998. Uncoupling of nonreceptor tyrosine kinases from PLC-g1 in an SLP-76-deficient T cell. Science 281:413]416 71. Clements JL, Yang B, Ross-Barta SE, Eliason SL, Hrstka RF, Williamson RA, Koretzky GA Ž1998. Requirment for the leukocyte-specific adapter protein SLP-76 for normal T cell development. Science 281:416]419 72. Pivniouk V, Tsitsikov E, Swinton P, Rathbun G, Alt FW, Geha RS Ž1998. Impaired viability and profound block in thymocyte development in mice lacking the adaptor protein SLP-76. Cell 94:229]238 73. Wu J, Katzav S, Weiss A Ž1995. A functional T-cell receptor signaling pathway is required for p95 vav activity. Mol Cell Biol 15:4337]4346 74. Tarakhovsky A, Turner M, Schaal S, Mee PJ, Duddy LP, Rajewsky K, Tybulewicz VLJ Ž1995. Defective antigen receptor-mediated proliferation of B and T cells in the absence of vav. Nature 374:467]470 75. Zhang R, Alt FW, Davidson L, Orkin SH, Swat W Ž1995. Defective signaling through the B and T cell antigen receptors in lymphoid cells lacking the vav proto-oncogene. Nature 374:470]473 76. Fischer KD, Zmuldzinas A, Gardner S, Barbacid M, Bernstein A, Guidos C Ž1995. Defective T cell receptor signaling and positive selection of Vav-deficient CD4qCD8q thymocytes. Nature 374:474]477 77. Neel BG Ž1997. Role of phosphatases in lymphocyte activation. Curr Opin Immunol 9:405]420 78. Blyth KF, Conroy LA, Howlett S, Smith AJH, May J, Alexander DR, Holmes N Ž1996. CD45-null transgenic mice reveal a positive regulatory role for CD45 in early thymocyte development, in the selection of CD4qCD8q thymocytes, and in B cell maturation. J Exp Med 183:1707]1718 79. Kishihara K, Penninger J, Wallace VA, Kundig TM, Kawai K, Wakeham A, Timms E, Pfeffer K, Ohashi PS, Thomas ML, Furlonger C, Paige CJ, Mak TW Ž1993. Normal B cell development but impaired T cell maturation in CD45-exon 6 protein tyrosine phosphatase deficient mice. Cell 74:143]156 80. Stone JD, Conroy LA, Byth KF, Hederer RA, Howlett S, Takemoto Y, Holmes N, Alexander DR Ž1997. Aberrant TCR-mediated signaling in CD45-null thymocytes involves dysfunctional regulation of Lck, Fyn, TCR-zeta, and ZAP-70. J Immunol 158:5773]82 81. Pani G, Fischer K-D, Mlinaric-Rascan I, Siminovitch KA Ž1996. Signaling capacity of the T cell antigen receptor is negatively regulated by the PTP1C tyrosine phosphatase. J Exp Med 184:839]852 82. Plas DR, Johnson R, Pingel JT, Matthews RJ, Dalton M, Roy G, Chan AC, Thomas M Ž1996. Direct regulation of ZAP-70 by SHP-1 in T cell antigen receptor signaling. Science 272:1173]1176
237
T cell receptor-mediated signs and signals governing T cell development Nicolai S. C. van Oers
gd . in a non-covalent association with a group of invariant proteins ŽCD3 g , d , « and TCR z .. These invariant subunits are responsible for efficient assembly and surface expression of the various TCR complexes Žreviewed in ref 1.. Additionally, the invariant chains contain a conserved signaling motif that functions to translate effective ligand binding into intracellular biochemical signals Žreviewed in ref 2.. This motif, termed the immune tyrosine based activation motif ŽITAM. wYxxLx Ž6-8.YxxLx, is present as a single copy in CD3 g , d , and « and as three copies in TCR z .3 The signals propagated through the ITAMs ultimately converge in the nucleus, resulting in the induction of various biological responses such as differentiation, proliferation, programmed cell death, cytokine release, andror cytolytic functions. A considerable wealth of knowledge now exists about the mechanisms of TCR-mediated signaling Žreviewed in ref 4.. The central feature of this pathway is the modulation of the tyrosine phosphorylation status of many effector molecules through the activation of several families of protein tyrosine kinases ŽPTKs. Žreviewed in ref 5.. The Src-family of PTKs are proposed to initiate TCR-induced signal transduction by phosphorylating pairs of tyrosine residues present in the ITAMs ŽFigure 2.. Once phosphorylated, the ITAMs form a consensus binding motif for the two Src-homology 2 ŽSH2. domains of the SykrZAP-70 family of PTKs. The recruitment of SykrZAP-70 results in the combined activation of both families of kinases, culminating in the phosphorylation of many additional effector molecules such as LAT, SLP-76, vav, and PLC-g 1.4 These changes lead to a well-established elevation in intracellular calcium and the activation of ras and its downstream MAPK cascades Žreviewed in refs 6 and 7.. The following review will examine the roles of three families of PTKs in TCR signal transduction, with particular emphasis on their functions during thymopoeisis. Several of the more recently identified regulatory molecules will also be reviewed, again with an em-
The developmental fate of T cells is largely controlled by the nature and success of signals mediated by the pre-T cell receptor (TCR) and TCR complexes. These intracellular signals are regulated by cascades of protein tyrosine phosphorylations initiated following ligand binding to the preTCR or TCR complexes. The phosphorylation cascades are primarily orchestrated by two distinct families of protein tyrosine kinases (PTKs), the Src- and the Syk r ZAP-70families. Germline gene targeting experiments, several human immunodeficiencies, and somatic cell mutants have all contributed to our understanding of how these families of kinases coordinate their actions to promote signaling. Upon activation, the PTKs transmit their signals to a number of newly described adaptor proteins including LAT, SLP-76, and vav, among others. The following review combines results derived from different experimental strategies to examine the contributions of the PTKs and the adaptor molecules to pre-TCR and TCR signaling processes. Key words: adaptor proteins r protein tyrosine kinases r signal transduction r T cell receptor Q1999 Academic Press
Introduction THE PRE-T CELL RECEPTOR Žpre-TCR. and T cell receptor ŽTCR. complexes are multi-subunit complexes that mediate the differentiation and expansion of both ab and gd T lineage cells during T cell development ŽFigure 1.. The complexes themselves comprise the ligand binding subunits Žpre-Tarb ; ab ;
From the Center for Immunology and the Department of Microbiology, UT Southwestern Medical Center, Room NA7.201, 6000 Harry Hines Blvd., Dallas, TX 75235-9093, USA Q1999 Academic Press 1044-5323r 99 r 040227q 11 $30.00r 0
227
N. S. C. van Oers
Figure 1. Pre-T Cell Receptor and T cell Receptor Complexes. The immune tyrosine-based activation motifs are represented as darkened rectangles. The pre-TCR complex appears to have a very weak association with the TCR z subunit.
to convert the CD25q CD44 lory cells into a CD25yCD44 lory subpopulation that begins expressing the CD4 and CD8 coreceptor molecules. The CD4yCD8y to CD4qCD8q transition coincides with the termination of pre-Ta expression, and initiation of rearrangements at the TCR a locus. A successful pairing and surface expression of the ab TCR complex allows the CD4qCD8q thymocytes to undergo the stringent processes of positive and negative selection.8 The consequences of these TCR-mediated selection events results in the establishment of a peripheral T cell repertoire that is restricted by selfMHC molecules.
phasis on TCR-mediated signal transduction processes necessary for T cell development.
T cell development The majority of ab T cells, a subset of gd T cells, and certain NK cell populations develop in the thymus. For ab T cells, this developmental process follows a highly ordered series of selection events that are phenotypically defined with the specific expression of certain cell surface molecules such as CD3, CD4, CD8, CD25, and CD44. A simplified overview of this process is illustrated in Figure 3. The entire process of selection will be covered in great detail in the many accompanying review articles in this volume. Following the arrival of precursor cells into the thymus, the TCR b locus undergoes a rearrangement process. A successfully rearranged TCR b gene product subsequently pairs with the pre-TCR a subunit, and this complex is expressed on the surface of cells that lack the CD4 and CD8 coreceptor molecules ŽFigure 3.. Such CD4y CD8y cells can be further subdivided into four distinct subpopulations on the basis of CD25 and CD44 expression. Signals generated through the pre-TCR complex are proposed
TCR r CD3 itams The generation of the peripheral T cell repertoire is contigent upon the nature and success of the TCR signaling pathways that are activated during thympoeisis. The main functions of the ITAMs of the TCR complex are to convey appropriate ligand binding events into intracellular signals. As previously descibed, the TCR complex comprises upwards of ten ITAMs, with six contributed by the TCR z subunit ŽFigure 1.. Given the presence of ten ITAMs, the 228
Signals governing T cell development
Figure 2. Model of Protein Tyrosine Kinase Activation by the T Cell Receptor ITAMs. Following TCR interactions with peptidermajor histocompatibility complexes, the Src-kinases are activated, resulting in the phosphorylation of the tyrosine residues in the ITAMs. This promotes the recruitment of the SykrZAP-70 family of kinases to the TCR complex, in interactions mediated by the two SH2 domains of the kinase with the doubly phosphorylated tyrosines in the ITAMs. The association of SykrZAP-70 with the phosphorylated ITAMs allows for their subsequent phosphorylation and activation. Cross-communication between the two families of kinases promotes the phosphorylation of multiple downstream effector molecules.
phosphorylation of increasing numbers of ITAMs may contribute quantitatively to the strength of signaling. The concept of quantitative signaling with increasing TCRrCD3 ITAMs was originally described for cell lines expressing chimeric molecules with the extracellular domain of CD8 and an intracellular region containing either one, two, or three TCR z ITAMs.9 However, many studies have also described qualitative differences between the various phosphorylated TCRrCD3 ITAMs and their abilities to interact with downstream effector molecules Žreviewed in ref 5.. The issues of qualitative vs quantitative signaling differences may have important ramifications for T cell selection processes in the thymus, and these have yet to be resolved. With regards to thymopoeisis, some apparent distinctions between the various invariant chains have
been described. For example, the targeted disruption of the CD3 « allele Žalso affects CD3 g and d transcription. results in a complete development arrest of both ab and gd T cells at the CD4yCD8y stage, similar to mice lacking the RAG genes.10,11 In contrast, the targeted disruption of the TCR z molecule results in a much less severe development block.12 ] 14 Thus, CD4qCD8q thymocytes lacking surface TCR expression are still apparent in these mice, although at numbers that vary from two- to 30-fold less than age matched, wild-type littermates. These results would imply that pre-TCR signaling is still functional in TCR z-null mice, consistent with the concept that the TCR, and perhaps the pre-TCR, contain two independent signal transduction modules wTCR z vs CD3 g , d , and « x.15 Alternatively, TCR z may not be required for the assembly of the pre-TCR complex. It 229
N. S. C. van Oers
Figure 3. Overview of T Cell Development. The development of ab T cells in the thymus follows a well-defined series of development changes phenotypically characterised by the expression of various cell surface markers, as indicated. The effects of the targeted disruptions of various effector molecules on this developmental pathway are high-lighted below the pathways. A solid bar indicates a complete developmental arrest, whereas a dashed bar implies an incomplete developmental block.
is interesting to note that normal T cell development can be restored in the TCR z knock-out mice by introducing TCR z transgenes comprising zero, one, or all three z ITAMs.16 These studies demonstrated that the TCR z ITAMs are not essential for thymopoeisis, but z is important for proper TCR assembly and surface expression. In fact, only in a well-defined ab trangenic TCR background ŽH-Y TCR transgenic mice. does it become obvious that the number of TCR z ITAMs can contribute in a quantitative capacity to positive and negative selection.17 A second distinction between the various invariant chains is the presence of a pool of constitutively tyrosine phosphorylated TCR z subunits in murine thymocytes and peripheral T cells.18,19 This constitutive phosphorylation, resulting in the formation of a 21-kDa phosphorylated form of TCR z , is unique to z as none of the other CD3 subunits are constitutively phosphorylated. The constitutive phosphorylation of TCR z is, in part, a consequence of ongoing TCR
interactions with peptiderMHC molecules expressed in the thymus.18,20 As discussed below, both the Lck and ZAP-70 protein tyrosine kinases ŽPTKs. are required for the maintenance of the constitutively phosphorylated TCR z subunits.21,22 In addition to the 21-kDa form of TCR z , a second phosphorylated form of TCR z Žapparent molecular mass of 23 kDa. can be induced following receptorrligand interactions.20,23 ] 25 Notably, a strong and provocative correlation has been established between the induction of particular phosphorylated forms of TCR z Ž21 vs 23 kDa. and the functional responsiveness of T cells Žreviewed in ref 23.. The presence or absence of these distinct phosphorylated forms of TCR z may also have relevance to the processes of positive and negative selection in the thymus.
Protein tyrosine kinases Three families of protein tyrosine kinases ŽPTKs., the 230
Signals governing T cell development
Src-, Syk-, and Tec-families, have important and distinct functional contributions to T cell development. Lck and Fyn, two members of the Src-family of PTKs, are primarily restricted in expression to T cells.26 They have unique N-terminal sequences followed by a Src-homology 3 ŽSH3., a Src-homology 2 ŽSH2., and a kinase domain. Although these kinases are targeted to the plasma membrane through myristylation and palmitylation modifications at their NH 2-terminus, a proportion of Lck molecules are found associated with the CD4 and CD8 coreceptor molecules while Fyn can complex with the TCR, albeit at low stoichiometry.27 The important contributions of the CD4 and CD8 coreceptor molecules to T cell development are reviewed elsewhere in this issue. A combination of biochemical and genetic data has elegantly demonstrated a principle role for Lck in initiating TCR-signaling cascades by phosphorylating the ITAMs in the TCR complex 21,28 Žreviewed in ref 27.. This pathway was initially uncovered following the description of a Lck-deficient leukemic cell that was unable to initiate TCR-mediated signaling processes unless reconstituted with Lck.29 With regards to thymopoeisis, Lck is expressed in all thymocyte subsets, and numerous experiments have revealed a role for Lck in pre-TCR and TCR-signaling Žreviewed in refs 30 and 31.. In brief, mice rendered deficient in Lck have a 5]20 fold reduction in thymic cellularity and a severe paucity of both CD4qCD8q and mature single positive T cells.32 The mature T cells that can develop in these mice are unable to respond effectively to TCR stimulations.32 Such problems are primarily related to the fact that Lck is required for initiating TCR signal transduction by phosphorylating the ITAMs and for phosphorylating and activating the SykrZAP-70 family of PTKs following their high affinity binding to the phosphorylated ITAMs ŽFigure 2..33 ] 35 As a consequence, very little constitutive phosphorylation of the TCR z molecules can be detected in thymocytes from Lcknull mice.21 Moreover, no inducible phosphorylation of TCRrCD3 ITAMs or ZAP-70 is noted in these knock-out animals. Fyn is a second Src-family member that functions in the TCR signaling cascade, in part, by phosphorylating the TCR ITAMs. In contrast to Lck, Fyn is expressed at 10-fold higher levels in mature single positive thymocytes relative to CD4qCD8q cells.26 Interestingly, Fyn-deficient mice have normal numbers of thymocytes and peripheral T cells.36,37 The single positive thymocytes and mature T cells in Fyn-
null mice are hypo-responsive following TCR crosslinking, as evidenced by their poor proliferative responses and low mobilisation of intracellular calcium.36,37 Consistent with the knock-out animals, mice expressing a dominant negative Fyn transgene only have a defect in single positive TCR-mediated signal transduction.38 This defect is partly a consequence of reduced IL-2 production by the stimulated T cells, suggesting that Fyn couples to the IL-2 pathway.36,37 The inability of Lck to promote signaling in the absence of Fyn may relate to the unique substrate specificity of Fyn. Thus, Fyn can selectively phosphorylate both Pyk2, a member of the focal adhesion family of PTKs, and Fyb ŽSLAP130., a Fyn and SLP-76 associating signaling protein.39 ] 41 This selectivity suggests a bifurcation point in the functions of Fyn and Lck and may indicate a unique contribution for Fyn in IL-2 production in mature T cells through a pathway involving Fyb ŽSLAP130.. Mice rendered deficient in both Lck and Fyn have a complete ab T cell developmental arrest at the CD4yCD8y ŽCD25y CD44y . stage of thympoeisis.42,43 Taken together, the aforementioned data would suggest that Lck is the primary Src-family PTK responsible for T cell development although Fyn can partially compensate for Lck in pre-TCR and TCR signaling. The requirements for Fyn and Lck in gd and NK cell development are discussed in the accompanying review articles. Syk and ZAP-70 are members of a second family of PTKs that function as critical mediators of pre-TCR and TCR signaling, with ZAP-70 having a predominant role in mature T cells Žreviewed in ref 31.. Defined by the presence of two NH 2 terminal SH2 domains and a COOH-terminal kinase domain, ZAP70 is localised as a diffuse band under the plasma membrane.27,44 The two SH2 domains form a high affinity interaction with the doubly phosphorylated ITAMs. Upon TCR ligation, both kinases can associate with the tyrosine phosphorylated ITAMs of the pre-TCR and TCR complexes, and, in turn, are tyrosine phosphorylated.45 ] 47 The phosphorylations on ZAP-70 increase the intrinsic catalytic activity of the kinase and allow for the binding of additional signaling proteins Žvav, Lck., or subsequent attenuators of signaling Žc-cbl, SHP-1.. Both ZAP-70 and Syk are expressed in developing thymocytes, with ZAP-70 upregulated during the CD4qCD8q to the single positive transition, while Syk undergoes a concomitant down-modulation following pre-TCR signaling.46,48 The functional requirements for ZAP-70 in TCRmediating signaling processes were elucidated in sev231
N. S. C. van Oers
eral severe combined immunodeficiency ŽSCID. patients that harbored mutations in the ZAP-70 gene.49 ] 51 Phenotypically, immature CD4qCD8q thymocytes from such patients could only mature into CD4q T cells, and these cells were functionally incompetent to transduce TCR-mediated signals. Thus, TCR cross-linking resulted in a very poor induction of tyrosine phosphoproteins, demonstrating a central role for ZAP-70 in initiating TCR signal transduction cascades. Mice lacking ZAP-70 or mice harboring a catalytically inactive ZAP-70 molecule have normal numbers of CD4qCD8q thymocytes that are unable to undergo either positive or negative selection processes, such that no CD4q or CD8q cells are generated.22,52,53 The phenotypes described for the ZAP-70 deficiencies are largely explained by the requirements of ZAP-70 to phosphorylate several key signalingradaptor substrates including LAT, SLP-76, and vav.5,54 Moreover, the two SH2 domains of ZAP-70 are required for maintaining the constitutive and inducible phosphorylations of the TCR z chain.22 It is interesting to note that the results with the mice differ somewhat from that described for the human SCIDs, and this may relate to the differences in Syk expression and function, as described below. In contrast to ZAP-70, Syk-deficient mice have a completely normal pattern of ab T cell development and a selective impairment of certain gd lineage T cells.55,56 The targeted disruption of both ZAP-70 and Syk results in a complete attenuation of thymocyte development at the CD4yCD8y stage, with the majority of cells in these mice maintaining a CD25qCD44yrlo phenotype, analogous to the subsets in LckrFyn- and Rag-null mice.31 Such findings again demonstrate the existence of functional redundancy within a given family of PTKs. Thus, Syk can partially compensate for the absence of ZAP-70 by supporting pre-TCR signal transduction.31 The failure of Syk to promote differentiation of thymocytes to the single positive stage in mice may relate to the marked down-regulation of Syk expression that occurs in double positive thymocytes.46,48 This is consistent with the ability of Syk to support complete T cell development and TCR-mediated signaling when over-expressed as a transgene in ZAP-70-null mice.57 One important distinction between ZAP-70 and Syk is the observation that Syk has a much higher intrinsic enzymatic activity and much less dependence on Srcfamily kinases for its activation.58,59 Itk is a member of the TecrBtk family of PTKs that is also implicated in thymopoeisis. This family of kinases has several functional domains including a
NH 2 terminal plextrin homology ŽPH. domain, followed by a SH3, SH2, and a kinase domain. The PH domain is involved in localising these PTKs to the cellular membrane by interacting with the phospholipid PIP3 . Mice deficient in Itk have a small reduction in single positive thymocytes, an effect more obviously noted in a ab TCR transgenic background.60 Moreover, the mature T cells in these mice have reduced TCR-mediated proliferative responses largely as a consequence of reduced IL-2 production. It will be interesting to determine whether Itk connects to the FynrFyb pathway for IL-2 production. Mutations in the B cell family member, Btk, results in X-linked agammaglobulinemia in humans and X-linked immunodeficiency in mice.61 These extremely severe B cell phenotypes relative to the mild T cell defects in Itk-deficient mice may suggest the presence of additional Tec family members in T cells that can facilitate thymopoeisis. The exact role of Itk in TCRsignaling requires further investigation.
Signaling molecules The preliminary events of pre-TCR- or TCR-mediated PTK activation must be successfully relayed to appropriate downstream substrates. A theme common to many different signaling systems is the recruitment and activation of selected enzymes through the use of scaffold, anchoring, andror adaptor proteins.62 T cells are no exception, with an exciting number of newly identified and essential adaptor molecules having recently been identified Žreviewed in ref 4.. One key T cell adapter protein Žalso in NK cells, mast cells, and basophils. is termed LAT Žlinker for activation of T cells..54 LAT is a 36]38-kDa palmitylated transmembrane glycoprotein that becomes heavily tyrosine phosphorylated following TCR stimulation.54 At least 6 tyrosine residues are phosphorylated in LAT, most by ZAP-70, and these phosphorylated sequences form consensus binding motifs for the SH2 domains of phosphopholipase C gamma 1 ŽPLC-g 1., the Grb2 adapter protein, and Grap ŽGrb2-like. ŽFigure 4..54 Thus, LAT serves as an adaptor protein by connecting the PTKs to downstream substrates. The crucial role of LAT in T cell signaling was established with an analysis of a LAT-deficient Jurkat T cell line.63 Cross-linking the TCR on these cells revealed a normal ITAM and ZAP-70 phosphorylation without any subsequent mobilisation of intracellular calcium or ras activation.63 Additionally, overexpression of a mutant LAT bearing tyrosine to phenylalanine substi232
Signals governing T cell development
tutions reduces the magnitude of NF-AT driven transcriptional reporter constructs.54 Given the fact that the highest expression levels of LAT are in the thymus Žassessed by Northern blotting., LAT-deficient mice are likely to have a complete block in ab and gd T cell development. The lack of intracellular calcium mobilisation in the LAT-deficient cells is likely a consequence of the failure to recruit and phosphorylate PLC-g 1. Interestingly PLC-g 1, an enzyme essential for the activation of the phosphatidylinositol pathway, was one of the first well-char-
acterised phosphorylated substrates in T cells Žreviewed in ref 7.. Activated PLC-g 1 hydrolyses phosphatidylinositol 4,5 bisphosphate into the second messengers IP3 and diacylglycerol ŽDAG., which are, in turn, responsible for the well-characterised intracellular calcium increases and PKC activation, respectively.6 The calcium increase is required to activate the serinerthreonine phosphatase calcineurin, which, in turn, activates an important IL-2 transcription factor, nuclear factor of activated T cells ŽNFAT.. A second critical molecule recruited to phosphory-
Figure 4. T Cell Receptor Signaling Cascades. Following the initiation of TCR signaling, a number of downstream adaptor proteins are phosphorylated. One key adaptor protein phosphorylated by ZAP-70 is LAT. The phosphorlation sites on LAT serve as consensus binding motifs for the SH2 domains of PLC-g 1 and Grb2. The subsequent phosphorylation and activation of PLC-g 1 results in the release of intracellular calcium stores and the activation of PKC. Since Grb2 forms complexes with SLP-76 and SOS, the recruitment of Grb2 to LAT provides a mechanism for relocating SOS to the membrane, facilitating ras activation and the downstream MAPK pathway. The re-localisation andror phosphorylation of SLP-76 is also necessary for both ras activation and PLC-g 1 phosphorylation. This may be partly attributed to the association of SLP-76 with vav, a guanine nucleotide exchanger on the rhorracrCDC42 family of GTPases. The actions of vav may regulate actin polymerisation and cytoskeletal re-organisation. Vav can also form an independent complex with phosphorylated ZAP-70, and this may also contribute to vav activation. Finally, Fyn may couple to the IL-2 pathway through its association with a SLP-76 interacting protein, termed Fyb, or SLAP-130.
233
N. S. C. van Oers
pre-TCR a expression were apparent in the SLP-76 null animals, the absence of SLP-76 most probably impairs pre-TCR signaling. The ability of the dominant negative MAPKK to block thymocyte development is consistent with a requirement for SLP-76 to promote ras activation in early thymocyte development. In mature T cells, the mechanism by which SLP-76 couples to IL-2 production is not clear but certainly involves another TCR-induced phosphoprotein, the vav proto-oncogene Žreviewed ref 5.. Vav functions as a guanine nucleotide exchange factor ŽGEF. for the rhorracrCDC42 families of GTPases, and also contains a PH domain, and two SH3 domains at its COOH terminus interspersed by an SH2 domain. Following TCR ligation, vav becomes tyrosine phosphorylated and also forms two independent complexes with phosphorylated ZAP-70 or SLP-76 through its SH2 domain. Overexpression of wild-type vav can significantly increase both the basal and TCR-induced NFAT and IL-2-driven transcriptional activity.73 Notably, the combined overexpression of vav and SLP-76 can synergistically potentiate the aforementioned transcriptional activity. As described earlier, PLC-g 1 is not phosphorylated in SLP-76 deficient cells. This may be a consequence of the absence of vav association, since vav may be required to promote cytoskeletal reorganisation through its GEF functions. With regards to T cell development, vav-deficient mice have been generated by a RAG-complementation approach, and these mice exhibit a 10]20-fold reduction in all thymocyte subsets.74 ] 76 Moreover, these cells have severely reduced proliferative responses to TCR stimuli. It remains to be established why the phenotype of the SLP-76 deficient mice is much more severe than that of the vav-null mice, but may relate to the ability of SLP-76 to associate with several additional phosphorylated proteins.68 As mentioned earlier, SLP-76-deficient T cells cannot mobilise intracellular calcium. In contrast, most of the data with vav-deficient thymocytes indicates that a normal calcium flux can be elicited following TCR cross-linking. Taken together, these results would imply that SLP-76 may couple to multiple downstream effector molecules. Alternatively, a vav homologue may also be expressed in hematopoeitic cells and this may partially compensate for the absence of vav.
lated LAT is Grb2, an adapter protein that itself complexes to several other key regulatory molecules including sos-of-sevenless ŽSOS., SLP-76 ŽSH2 domain leukocyte protein of 76 kDa., and the c-cbl protooncogene ŽFigure 4..6,54,64 Grb2 contains one SH2 domain flanked on either side by a SH3 domain. The SH3 domains of Grb2 can bind to proline rich peptide sequences in both SOS and SLP-76. SOS is an important guanine nucleotide exchanger for the ras family of small GTPases, functioning to activate ras. Activated ras has a central role in IL-2 gene activation by regulating the MAPK ŽErk. pathway, and this pathway has been reviewed elsewhere.6 With regards to thymopoeisis, an activated form of ras, when expressed as a transgene in Rag-deficient embryonic stem cells, is capable of promoting the differentiation of ab T cells to the CD4qCD8q stage.65 Since ras connects to the MAPK pathway, these results are also consistent with the observations that a dominant negative MAPKK, when expressed in fetal thymic organ cultures, blocks the transition of thymocytes from double negative to double position cells.66 Using the Lck proximal promoter to express a dominant negative ras transgene in thymocytes, a specific block in positive but not negative selection was revealed.67 Grb2 also associates with SLP-76, a recently identified T and NK cell specific molecule that contains three tyrosine residues located near the NH 2terminus, a central proline rich stretch, and a COOH-terminal SH2 domain.64,68,69 Following TCR cross-linking, ZAP-70 phosphorylates SLP-76 on the three NH 2-terminal tyrosines, presumably following the association of SLP-76rGrb2 with phosphorylated LAT. In mature T cells, overexpression of SLP-76 potentiates TCR-mediated IL-2 gene induction while a mutant form lacking either the three NH 2-terminal tyrosines or a functional SH2 domain can attenuate IL-2 responses.69 Interestingly, T cell lines lacking SLP-76 have revealed a requirement for SLP-76 in PLC-g 1 phosphorylation and ras activation, but not in the TCR-induced phosphorylation of most other proteins.70 These results would suggest that the Grb2rSOS pathway is not sufficient to activate ras. The importance of SLP-76 in thymopoeisis was recently described with the generation of two independent SLP-76 deficient mice.71,72 The thymocyte subpopulations in these mice revealed a complete developmental arrest within the CD4yCD8y subset, at the transition point from CD44yCD25q to CD44yCD25y cells. This is the same developmental arrest point as the LckrFyn and ZAP-70rSyk double knock-out mice. As TCR b gene rearrangements and
Protein tyrosine phosphatases The pre-TCR and TCR-mediated signals are coordi234
Signals governing T cell development
Concluding remarks
nately regulated by the actions of PTKs and protein tyrosine phosphatases ŽPTPases.. Although many PTPases will likely be identified as key regulators of antigen receptor signal transduction, two well-characterised ones include CD45 and SHP-1. CD45 is a transmembrane PTPase that comprises two intracellular PTPase domains and is expressed on almost all hematopoeitic cells.77 Several isoforms have been identified on T cells as a consequence of regulated processes of alternative splicing. CD45 primarily functions as a positive regulator of antigen receptor signal transduction by activating the Src-kinases Lck and Fyn through the dephosphorylation of the negative regulatory tyrosine residue in these. The importance of CD45 in T cell development was evident with the 4]10-fold reduction in the numbers of CD4qCD8y and CD4yCD8q thymocytes and peripheral T cells in mice lacking either all CD45 isoforms or CD45-exon 6-null mice.78,79 The CD4qCD8q thymocytes from the CD45-null mice are refractory to anti-TCR mAb-induced negative selection and those mature T cells that do develop display reduced TCRmediated proliferative responses.78,79 This is largely explained by the fact that Fyn and Lck are hyperphosphorylated at their COOH-terminal negative regulatory tyrosine in CD45-null thymocytes. As a consequence, these kinases are relatively inactive resulting in hypo-phosphorylation of the TCR ITAMs and a relatively poor recruitment of ZAP-70 to the TCR complex.80. What remains unclear at present in how T cell development can proceed to the CD4qCD8q stage in the absence of CD45. SHP-1 is a hematopoeitic specific PTPase that appears to down-modulate antigen receptor signaling systems.77 Two naturally occurring SHP-1 mutations were identified in mice Žmotheaten mice. several years ago. Phenotypically, the thymocytes from these mice have a normal developmental profile. Functionally, both thymocytes and peripheral T cells from these SHP-1 mutant mice hyper-proliferate in response to anti-TCR stimulations.1,81 Additionally, the levels of the constitutively and inducibly phosphorylated proteins in these thymocytes are markedly elevated relative to normal mice.81 With regards to mechanism, the absence of SHP-1 results in the hyper-activation of the Src-family kinases, leading to increased basal and TCR-inducible phosphorylation levels of the ITAMs and ZAP-70. SHP-1 has also been reported to complex ZAP-70, and this may be an additional means of regulating antigen responsiveness.82
The processes of antigen receptor signal transduction are emerging as well-defined biochemical pathways involving a coordinated regulation of PTK and PTPase activities. A new set of exciting signalingradaptor proteins have recently been identified in these signaling cascades. Thus, a combination of cloning reports, gene knock-out results, and studies with somatic cell mutants have revealed essential roles for the LAT, SLP-76, and vav molecules. The exact functional mechanisms of these and other signaling molecules during pre-TCR and TCR-signaling will emerge rapidly. Over the next few years, novel activators and attenuators of antigen receptor signals will likely be identified, many of which will have significant functional contributions to ab , gd , and NK cell development.
Acknowledgements I would like to thank Dr T.C. Ayi, Mr. B. Tohlen, and Mrs. A. Feulner for their reading of this manuscript. This work was supported in part by a grant from the NIH ŽAI4295301A1. and Fikes support funding from UT Southwestern Medical Center.
References 1. Ashwell JD, Klausner RD Ž1990. Genetic and mutational analysis of the T-cell antigen receptor. Ann Rev Immunol 8:139]167 2. Weiss A Ž1993. T cell antigen receptor signal transduction: a tale of tails and cytoplasmic protein-tyrosine kinases. Cell 73:209]212 3. Cambier JC Ž1995. New nomenclature for the Reth motif Žor ARH1rTAMrARAMrYXXL.. Immunol Today 16:110 4. Rudd CE Ž1999. Adaptors and molecular scaffolds in immune cell signaling. Cell 96:5]8 5. Wange RL, Samelson LE Ž1996. Complex complexes: signaling at the TCR. Immunity 5:197]205 6. Cantrell D Ž1996. T cell antigen receptor signal transduction pathways. Ann Rev Immunol 14:259]274. 7. Weiss A, Littman DR Ž1994. Signal transduction by lymphocyte antigen receptors. Cell 76:263]274 8. Jameson SC, Bevan MJ Ž1998. T-cell selection. Curr Opin Immunol 10:214]219 9. Irving BA, Chan AC, Weiss A Ž1993. Functional characterization of a signal transducing motif present in the T cell receptor z chain. J Exp Med 177:1093]1103. 10. Malissen M, Gillet A, Ardoiun L, Bouvier G, Trucy J, Ferrier P, Viver E, Malissen B Ž1995. Altered T cell development in mice with a targeted mutation of the CD3-« gene. EMBO J 14:4641]4653 11. Tanaka Y, Ardoiun L, Gillet A, Lin S-Y, Magnan A, Malissen B, Malissen M Ž1995. Early T-cell development in CD3-deficient mice. Immunol Rev 148:171]199
235
N. S. C. van Oers
12. Love PE, Shores EW, Johnson MD, Tremblay ML, Lee EJ, Grinberg A, Huang SP, Singer A, Westphal H Ž1993. T cell development in mice that lack the z chain of the T cell antigen receptor compex. Science 261:918]921 13. Liu C-P, Ueda R, She J, Sancho J, Wang B, Weddell G, Loring J, Kurahara C, Dudley EC, Hayday A, Terhorst C, Huang M Ž1993. Abnormal T cell development in CD3-zyry mice and identification of a novel T cell population in the intestine. EMBO J 12:4863]4875 14. Malissen M, Gillet A, Rocha B, Trucy J, Brun N, Mazza G, Spanopoulou E, Guy-Grand D, Malissen B Ž1993. T cell development in mice lacking the CD3-zrh gene. EMBO J 12:4347]4355 15. Wegener A-MK, Letourneur F, Hoeveler A, Brocker T, Luton F, Malissen B Ž1992. The T cell receptorrCD3 complex is composed of at least two autonomous transduction modules. Cell 68:83]95 16. Shores EW, Huang K, Tran T, Lee E, Grinberg A, Love PE Ž1994. Role of TCR z chain in T cell development and selection. Science 266:1047]1050 17. Shores EW, Tran T, Grinberg A, Sommers CL, Shen H, Love PE Ž1997. Role of multiple TCR z chain signaling motifs in selection of the T cell repertoire. J Exp Med 185:893]900 18. Nakayama T, Singer A, Hsi ED, Samelson LE Ž1989. Intrathymic signalling in immature CD4qCD8q thymocytes results in tyrosine phosphorylation of the T-cell receptor zeta chain. Nature 341:651]654 19. van Oers NSC, Tao W, Watts JD, Johnson P, Aebersold R, Teh H-S Ž1993. Constitutive tyrosine phosphorylation of the T cell receptor ŽTCR. z subunit: Regulation of TCR-associated protein kinase activity by TCR z . Mol Cell Biol 13:5771]5780 20. van Oers NSC, Killeen N, Weiss A Ž1994. ZAP-70 is constitutively associated with tyrosine phosphorylated TCR z in murine thymocytes and lymph node T cells. Immunity 1:675]685 21. van Oers NSC, Killeen N, Weiss A Ž1996. Lck regulates the tyrosine phosphorylation of the TCR subunits and ZAP-70 in murine thymocytes. J Exp Med 183:1053]1062 22. Kadlecek TA, van Oers NSC, Lefrancois L, Olson S, Finlay D, Chu DH, Connolly K, Killeen N, Weiss A Ž1998. Differential requirements for ZAP-70 in TCR signaling and T cell development. J Immunol 161:4688]4694 23. Jameson SC, Bevan MJ Ž1995. T cell receptor antagonists and partial agonists. Immunity 2:1]11 24. Sloan-Lancaster J, Shaw AS, Rothbard JB, Allen PM Ž1994. Partial T cell signaling: Altered phospho-z and lack of ZAP-70 recruitment in APL-induced T cell anergy. Cell 79:913]922 25. Madrenas J, Wange RL, Wang JL, Isakov N, Samelson LE, Germain RN Ž1995. z phosphorylation without ZAP-70 activation induced by T cell receptor antagonists or partial agonists. Science 267:515]518 26. Olszowy MW, Leuchtmann PL, Veillette A, Shaw AS Ž1995. Comparison of p56 l ck and p59 f y n protein expression in thymocyte subsets, peripheral T cells, NK cells, and lymphoid cell lines. J Immunol 155:4236]4240 27. Chan AC, Desai D, Weiss A Ž1994. Role of protein tyrosine kinases and protein tyrosine phosphatases in T cell antigen receptor signaling. Ann Rev Immunol 12:555]592 28. Iwashima M, Irving BA, van Oers NSC, Chan AC, Weiss A Ž1994. Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science 263:1136]1139 29. Straus D, Weiss A Ž1992. Genetic evidence for the involvement of the Lck tyrosine kinase in signal transduction throught the T cell antigen receptor. Cell 70:585]593 30. Anderson SJ, Perlmutter RM Ž1995. A signaling pathway governing early thymocyte maturation. Immunol Today 16:99]105 31. Cheng AM, Cahn AC Ž1997. Protein tyrosine kinases in thymocyte development. Curr Opin Immunol 9:528]533
32. Molina TJ, Kishihara K, Siderovski DP, van Ewijk W, Narendran A, Timms E, Wakeham A, Paige CJ, Hartmann K-U, Veillette A, Davidson D, Mak TW Ž1992. Profound block in thymocyte development in mice lacking p56lck. Nature 357:161]164 33. Chan AC, Dalton M, Johnson R, Kong G-H, Wang T, Thoma R, Kurosaki T Ž1995. Activation of ZAP-70 kinase activity by phosphorylation of tyrosine 493 is required for lymphocyte antigen receptor function. EMBO J 14:2499]2508 34. Watts JD, Affolter M, Krebs DL, Wange RL, Samelson LE, Aebersold R Ž1994. Identification by electrospray ionization mass spectromoetry of the sites of tyrosine phosphorylation induced in activated Jurkat T cells on the protein tyrosine kinase ZAP-70. J Biol Chem 269:29520]29529 35. Wange RL, Guitian R, Isakov N, Watts JD, Aebersold R, Samelson LE Ž1995. Activating and inhibitory mutations in adjacent tyrosines in the kinase domain of ZAP-70. J Biol Chem 270:18730]18733 36. Stein PL, Lee H-M, Rich S, Soriano P Ž1992. pp59 fyn mutant mice display differential signaling in thymocytes and peripheral T cells. Cell 70:741]750 37. Appleby MW, Gross JA, Cooke MP, Levin SD, Qian X, Perlmutter RM Ž1992. Defective T cell receptor signaling in mice lacking the thymic isoform of p59 f y n . Cell 70:751]763 38. Cooke MP, Abraham KM, Forbush KA, Perlmutter RM Ž1991. Regulation of T cell receptor signaling by a src family protein-tyrosine kinase Žp59 fyn .. Cell 65:281]292 39. Qian D, Lev S, van Oers NSC, Schlessinger J, Weiss A Ž1997. Tyrosine phosphorylation of Pyk2 is selectively regulated by Fyn during TCR signaling. J Exp Med 185:1253]1259 40. Musci MA, Hendricks-Taylor LR, Motto DG, Paskind M, Kamens J, Turck CW, Koretzky GA Ž1997. Molecular cloning of SLAP-130, an SLP-76-associated substrate of the T cell antigen rceptor stimulated protein tyrosine kinases. J Biol Chem 272:11674]11677 41. daSilva AJ, Li Z, de Vera C, Canto E, Findell P, Rudd CE Ž1997. Cloning of a novel T-cell protein FYB that binds FYN and SH2-domain-containing leukocyte protein 76 and modulate interleukin 2 production. Proc Natl Acad Sci USA 94:7493]7498 42. Groves T, Smiley P, Cooke MP, Forbush K, Perlmutter RM, Guidos CJ Ž1996. Fyn can partially substitute for Lck in T lymphocyte development. Immunity 5:417]428 43. van Oers NSC, Lowin-Kropf B, Finlay D, Connolly K, Weiss A Ž1996. ab T cell development is abolished in mice lacking both Lck and Fyn protein tyrosine kinases. Immunity 5:429]436 44. Huby RDJ, Iwashima M, Weiss A, Ley SC Ž1997. ZAP-70 protein tyrosine kinase is constitutively targeted to the T cell cortex independently of its SH2 domains. J Cell Biol 137:1639]1649 45. Chan AC, Iwashima M, Turck CW, Weiss A Ž1992. ZAP-70: A 70kD protein tyrosine kinase that associates with the TCR z chain. Cell 71:649]662 46. Chan AC, van Oers NSC, Tran A, Turka L, Law C-L, Ryan JC, Clark EA, Weiss A Ž1994. Differential expression of ZAP-70 and Syk protein tyrosine kinases, and the role of this family of protein tyrosine kinases in T cell antigen receptor signalling. J Immunol 152:4758]4766 47. van Oers NSC, von Boehmer H, Weiss A Ž1995. The pre-TCR complex is functionally coupled to the TCR z subunit. J Exp. Med 182:1585]1590 48. Chu DH, van Oers NSC, Malissen M, Harris J, Elder M, Weiss A Ž1999. Pre-T cell receptor signals are responsible for the downregulation of Syk protein tyrosine kinase expression. Submitted 49. Arpaia E, Shahar M, Dadi H, Cohen A, Roifman CM Ž1994. Defective T cell receptor signaling and CD8q thymic selection in humans lacking ZAP-70 kinase. Cell 76:947]958
236
Signals governing T cell development
50. Chan AC, Kadlecek TA, Elder ME, Filipovich AH, Kuo W-L, Iwashima M, Parslow TG, Weiss A Ž1994. ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency. Science 264:1599]1601 51. Elder ME, Lin D, Clever J, Chan AC, Hope TJ, Weiss A, Parslow T Ž1994. Human severe combined immunodeficiency due to a defect in ZAP-70, a T cell tyrosine kinase. Science 264:1596]1599 52. Negishi I, Motoyama N, Nakayama K-I, Nakayama K, Senju S, Hatakeyama S, Zhang Q, Chan AC, Loh DY Ž1995. Essential role for ZAP-70 in both positive and negative selection of thymocytes. Nature 376:435]438 53. Wiest DL, Ashe JM, Howcroft TK, Lee H-M, Kemper DM, Negishi I, Singer DS, Singer A, Abe R Ž1997. A spontaneously arising mutation in the DLAARN motif of murine ZAP-70 abrogates kinase activity and arrests thymocyte development. Immunity 6:663]671 54. Zhang W, Sloan-Lancaster J, Kitchen J, Trible RP, Samelson LE Ž1998. LAT: The ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation. Cell 92:83]92 55. Turner M, Mee PJ, Costello PS, Williams O, Price AA, Duddy LP, Furlong MT, Geahlen RL, Tybulewicz VLJ Ž1995. Perinatal lethality and blocked B cell development in mice lacking the tyrosine kinase Syk. Nature 378:298]302 56. Cheng AM, Rowley B, Pao W, Hayday A, Bolen JB, Pawson T Ž1995. Syk tyrosine kinase required for mouse viability and B cell development. Nature 378:303]306 57. Gong Q, White L, Johnson R, White M, Negishi I, Thomas M, Chan AC Ž1997. Restoration of thymocyte development and function in zap-70yry mice by the Syk protein tyrosine kinase. Immunity 7:369]377 58. Latour S, Chow LML, Veillette A Ž1996. Differential intrinsic enzymatic activity of Syk and ZAP-70 protein tyrosine kinases. J Biol Chem 271:22782]22790 59. Chu D, Spits H, Peyron J-F, Rowley RB, Bolen JB, Weiss A Ž1996. The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling. EMBO J 15:6251]6261 60. Liao XC, Littman DR Ž1995. Altered T cell development in mice lacking Itk. Immunity 3:757]769 61. Rawlings DJ, Witte ON Ž1994. Bruton’s tyrosine kinase is a key regulator in B cell development. Immunol Rev 138:105]119 62. Pawson T, Scott JD Ž1997. Signaling though scaffold, anchoring, and adaptor proteins. Science 278:2075]2080 63. Finco TS, Kadlecek T, Zhang W, Samelson LE, Weiss A Ž1998. LAT is required for TCR-edmiated activation of PLC-g1 and the ras pathway. Immunity 9:617]626 64. Jackman JK, Motto DG, Sun Q, Tanemoto M, Turck CW, Peltz GA, Koretzky GA, Findell PR Ž1995. Molecular cloning of SLP-76, a 76 kDa tyrosine phosphoprotein associated with Grb2 in T cells. J Biol Chem 269:7029]7032 65. Swat W, Shinkai Y, Cheng H-L, Davidson L, Alt FW Ž1996. Activated ras signals differentiation and expansion of CD4 q CD8 q thymocytes. Proc Natl Acad Sci USA 93:4683]4687 66. Crompton T, Gilmour KC, Owen MJ Ž1996. The MAP kinase pathway controls differentiaton from double-negative to double positive thymocyte. Cell 86:243]251 67. Swan KA, Alberola-IIa J, Gross JA, Appleby MW, Forbush KA, Thomas JF, Perlmutter RM Ž1995. Involvement of p21ras distinguishes positive and negative selection in thymocytes. EMBO J 14:276]285
68. Motto DG, Ross SE, Wu J, Hendricks-Taylor LR, Koretzky GA Ž1996. Implication of the GRB2-associated phosphoprotein SLP-76 in T cell receptor interleukin 2 production. J Exp Med 183:1937]1943 69. Wardenburg JB, Fu C, Jackman JK, Flotow H, Wilkinson SE, Williams DH, Johnson R, Kong G, Chan AC, Findell P Ž1996. Phosphorylation of SLP76 by the ZAP-70 protein tyrosine kinase is required for T cell receptor function. J Biol Chem 271:19641]19644 70. Yablonski D, Kuhne MR, Kadlecek T, Weiss A Ž1998. Uncoupling of nonreceptor tyrosine kinases from PLC-g1 in an SLP-76-deficient T cell. Science 281:413]416 71. Clements JL, Yang B, Ross-Barta SE, Eliason SL, Hrstka RF, Williamson RA, Koretzky GA Ž1998. Requirment for the leukocyte-specific adapter protein SLP-76 for normal T cell development. Science 281:416]419 72. Pivniouk V, Tsitsikov E, Swinton P, Rathbun G, Alt FW, Geha RS Ž1998. Impaired viability and profound block in thymocyte development in mice lacking the adaptor protein SLP-76. Cell 94:229]238 73. Wu J, Katzav S, Weiss A Ž1995. A functional T-cell receptor signaling pathway is required for p95 vav activity. Mol Cell Biol 15:4337]4346 74. Tarakhovsky A, Turner M, Schaal S, Mee PJ, Duddy LP, Rajewsky K, Tybulewicz VLJ Ž1995. Defective antigen receptor-mediated proliferation of B and T cells in the absence of vav. Nature 374:467]470 75. Zhang R, Alt FW, Davidson L, Orkin SH, Swat W Ž1995. Defective signaling through the B and T cell antigen receptors in lymphoid cells lacking the vav proto-oncogene. Nature 374:470]473 76. Fischer KD, Zmuldzinas A, Gardner S, Barbacid M, Bernstein A, Guidos C Ž1995. Defective T cell receptor signaling and positive selection of Vav-deficient CD4qCD8q thymocytes. Nature 374:474]477 77. Neel BG Ž1997. Role of phosphatases in lymphocyte activation. Curr Opin Immunol 9:405]420 78. Blyth KF, Conroy LA, Howlett S, Smith AJH, May J, Alexander DR, Holmes N Ž1996. CD45-null transgenic mice reveal a positive regulatory role for CD45 in early thymocyte development, in the selection of CD4qCD8q thymocytes, and in B cell maturation. J Exp Med 183:1707]1718 79. Kishihara K, Penninger J, Wallace VA, Kundig TM, Kawai K, Wakeham A, Timms E, Pfeffer K, Ohashi PS, Thomas ML, Furlonger C, Paige CJ, Mak TW Ž1993. Normal B cell development but impaired T cell maturation in CD45-exon 6 protein tyrosine phosphatase deficient mice. Cell 74:143]156 80. Stone JD, Conroy LA, Byth KF, Hederer RA, Howlett S, Takemoto Y, Holmes N, Alexander DR Ž1997. Aberrant TCR-mediated signaling in CD45-null thymocytes involves dysfunctional regulation of Lck, Fyn, TCR-zeta, and ZAP-70. J Immunol 158:5773]82 81. Pani G, Fischer K-D, Mlinaric-Rascan I, Siminovitch KA Ž1996. Signaling capacity of the T cell antigen receptor is negatively regulated by the PTP1C tyrosine phosphatase. J Exp Med 184:839]852 82. Plas DR, Johnson R, Pingel JT, Matthews RJ, Dalton M, Roy G, Chan AC, Thomas M Ž1996. Direct regulation of ZAP-70 by SHP-1 in T cell antigen receptor signaling. Science 272:1173]1176
237