ITAM Multiplicity and Thymocyte Selection
2000; Cell Press; Volume: 12; Issue: 6 Linguagem: Inglês
10.1016/s1074-7613(00)80210-1
ISSN1097-4180
AutoresPaul E. Love, Elizabeth W. Shores,
Tópico(s)CAR-T cell therapy research
ResumoAn intriguing feature of many receptors that participate in immune responses is the presence of multiple subunits and/or motifs that mediate signal transduction. One of the best studied of these signaling motifs is the ITAM (immune-receptor tyrosine-based activation motif) (50Reth M Antigen receptor tail clue.Nature. 1989; 338: 383-384Crossref PubMed Scopus (1135) Google Scholar). Virtually all receptors that utilize ITAMs for signal transduction contain multiple copies of these motifs. These include some Fc receptors (49Ravetch J.V Kinet J.-P Fc receptors.Annu. Rev. Immunol. 1991; 9: 457-492Crossref PubMed Scopus (1254) Google Scholar), activating NK receptors (30Lanier L.L Corlis B.C Wu J Leong C Phillips J.H Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells.Nature. 1998; 391: 703-707Crossref PubMed Scopus (705) Google Scholar), PIR-A (paired immunoglobulin-like receptor-A) (27Kubagawa H Chen C.-C Ho L.H Shimada T Gartland L Mashburn C Uehara T Ravetch J Cooper M.D Biochemical nature and cellular distribution of the paired immunoglobulin-like receptors, PIR-A and PIR-B.J. Exp. Med. 1999; 189: 309-317Crossref PubMed Scopus (119) Google Scholar), the B cell antigen receptor (BCR) (28Kurosaki T Genetic analysis of B cell antigen receptor signaling.Annu. Rev. Immunol. 1999; 17: 555-592Crossref PubMed Scopus (361) Google Scholar), and the T cell antigen receptor (TCR) (26Klausner R.D Lippincott-Schwartz J Bonifacino J.S The T cell antigen receptor insights into organelle biology.Annu. Rev. Cell Biol. 1990; 6: 403-431Crossref PubMed Scopus (295) Google Scholar). Depending on the particular cell type and state of maturation, signals mediated through ITAMs can regulate cell survival, cell death, development, or effector functions. ITAMs consist of semiconserved sequences of amino acids that contain two appropriately spaced tyrosines (YXXL/I X6–8 YXXL/I; where X denotes nonconserved residues) (50Reth M Antigen receptor tail clue.Nature. 1989; 338: 383-384Crossref PubMed Scopus (1135) Google Scholar). Following receptor engagement, phosphorylation of ITAM tyrosine residues by Src family kinases represents one of the earliest events in the signaling cascade (reviewed in 75Weiss A T cell antigen receptor signal transduction a tale of tails and cytoplasmic protein tyrosine kinases.Cell. 1993; 73: 209-212Abstract Full Text PDF PubMed Scopus (466) Google Scholar, 74Wange R.L Samelson L.E Complex complexes signaling at the TCR.Immunity. 1996; 5: 197-205Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 54Rudd C.E Adaptors and molecular scaffolds in immune cell signaling.Cell. 1999; 96: 5-8Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). In general, phosphorylation of both tyrosines within an ITAM (diphosphorylation) is thought to be essential for signaling, as this is required for efficient recruitment of the tandem SH2 domain containing protein tyrosine kinases Syk and ZAP-70 to the receptor complex. Activation of Syk and/or ZAP-70 results in the recruitment and phosphorylation of proteins that couple immune receptors to downstream signaling pathways. While the importance of ITAMs for signal transduction is clear, it remains uncertain why immune receptors contain multiple ITAMs. In this review we discuss three possible mechanisms by which ITAM multiplicity may regulate signal transduction by immune receptors and examine how these regulatory mechanisms relate to TCR signaling and thymocyte selection. Perhaps the most straightforward function for ITAM multiplicity may be to facilitate signal amplification by increasing the local concentration of effector molecules (Figure 1A). ITAM-mediated signal amplification could result from simple quantitative effects of multiple ITAMs or could involve more complex mechanisms. For example, cooperative interactions between different ITAMs could promote the phosphorylation of additional ITAMs by the recruitment of tyrosine kinases, as has been described for signaling through FcεRI and the TCR (33Lin S Cicala C Scharenberg A.M Kinet J.P The FcεRIβ subunit functions as an amplifier of Fc(epsilon)RIgamma-mediated cell activation signals.Cell. 1996; 85: 985-995Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 6Ashe J.M Wiest D.L Abe R Singer A ZAP-70 protein promotes tyrosine phosphorylation of T cell receptor signaling motifs (ITAMs) in immature CD4+8+ thymocytes with limiting p56lck.J. Exp. Med. 1999; 189: 1163-1168Crossref PubMed Scopus (26) Google Scholar). Second, ITAM multiplicity may promote signal discrimination. For example, specific ITAMs may preferentially couple immune receptors to distinct downstream signaling pathways (Figure 1B). In this regard it is important to appreciate that although ITAM sequences are conserved, they are not identical, and hence may be able to associate with distinct molecules or associate with the same effectors with different affinities. Third, multiple ITAMs could potentially negatively regulate signaling by immune receptors, depending on the extent or specific pattern of ITAM phosphorylation (Figure 1C). For example, monophosphorylated ITAMs could directly recruit inhibitory proteins, such as phosphatases, to the receptor complex. The tyrosine phosphatases SHP-1, SHP-2, and SHIP associate with a sequence (YxxL) that closely resembles a monophosphorylated ITAM (referred to as an immune-receptor tyrosine-based inhibitory motif [ITIM]) present in several inhibitory coreceptors, including FcγRIIB, CD22, and KIRs. The recruitment of tyrosine phosphatases to coreceptors that contain ITIMs has been shown to negatively regulate signaling through ITAM-containing activating receptors (72Vivier E Daeron M Immunoreceptor tyrosine-based inhibition motifs.Immunol. Today. 1997; 6: 286-291Abstract Full Text PDF Scopus (321) Google Scholar). Alternatively, ITAMs could act to sequester effector molecules in their inactive forms, making such molecules unavailable to other receptors, as has been suggested for FcεRI (68Torigoe C Inman J.K Metzger H An unusual mechanism for ligand antagonism.Science. 1998; 281: 568-572Crossref PubMed Scopus (132) Google Scholar). The mechanisms by which ITAM multiplicity may regulate immune function are not mutually exclusive, and each may be critical, depending on the cell type. The TCR contains several distinct signaling subunits with up to ten ITAMs distributed among these chains. Although the clonotypic (TCRα and TCRβ) chains are required for antigen recognition, they do not directly participate in signal transduction. Instead, TCR signaling is mediated by the invariant chains of the TCR:CD3-γ, -δ, -ε, and ζ chain. Each of the CD3 components contains a single ITAM within its cytoplasmic tail, and because there are two CD3ε chains in each TCR complex, the CD3 chains collectively contribute four ITAMs to the TCR. In contrast, ζ chain, which exists in the TCR as a disulfide-linked dimer, contains three tandem ITAMs; therefore, the ζζ homodimer contributes six ITAMs to the TCR complex. Phosphorylation of the TCR-ITAMs by Lck and Fyn and the subsequent recruitment and activation of ZAP-70 results in phosphorylation of effector molecules such as the adaptor proteins LAT and SLP-76, which link the TCR to PLC-γ1, Grb-2/Sos, and PI3K, resulting in the activation of the calcium and MAP kinase pathways (69van Leeuwen J.E Samelson L.E T cell antigen-receptor signal transduction.Curr. Opin. Immunol. 1999; 11: 242-248Crossref PubMed Scopus (213) Google Scholar). The potential for signal regulation by multiple ITAMs is perhaps most critical in situations in which fine-tuning of the signaling response is required. The complex process by which immature T cells undergo selection in the thymus clearly represents such a situation. The TCR repertoire expressed by mature T cells is largely devoid of autoreactivity and is restricted to recognizing foreign peptides in the context of self-MHC molecules. In contrast, the TCR repertoire of immature thymocytes is vast, largely due to V(D)J recombination. The process of removing overt self-reactivity while imparting self-MHC restriction to the mature TCR repertoire is thought to operate via selective processes regulated primarily by the affinity/avidity of the TCR for thymic ligands (self-MHC and self-peptide) and the ensuing TCR-mediated signals (reviewed in 21Jameson S.C Hogquist K.A Bevan M.J Positive selection of thymocytes.Annu. Rev. Immunol. 1995; 13: 93-126Crossref PubMed Scopus (531) Google Scholar, 56Sebzda E Mariathasan S Ohteki T Jones R Bachmann M.F Ohashi P Selection of the T cell repertoire.Annu. Rev. Immunol. 1999; 17: 829-874Crossref PubMed Scopus (409) Google Scholar). Because V(D)J recombination generates a random assortment of TCRs, the majority of immature CD4+CD8+ (DP) thymocytes will express TCRs lacking specificity for self-ligands (self-MHC + self-peptide) in the thymus, fail to receive signals required for survival, and die by a process termed "death by neglect." The remaining thymocytes undergo one of two antithetical fates, both of which paradoxically require TCR recognition of thymic ligands. Thymocytes expressing TCRs that have a relatively high avidity for thymic ligands receive signals that result in their deletion (physically or functionally deleted from the T cell repertoire). On the other hand, thymocytes expressing TCRs that have a lower avidity for thymic ligands receive quantitatively or qualitatively distinct signals that allow for their survival and eventual development to mature, functional, single-positive (CD4+CD8− or CD4−CD8+) T cells. The affinity of a TCR for its selecting ligand appears to be extremely low, and it is difficult to envision how such interactions might achieve an activation threshold sufficient to rescue them from death by neglect. An explanation that may reconcile this finding lies in the potential of multiple TCR ITAMs to amplify signals originating from low affinity/avidity interactions. One approach to assessing the role of ITAM multiplicity in thymocyte selection has involved the construction of mice selectively lacking individual TCR signal transducing chains or their ITAMs and examining the effect of these genetic alterations on thymocyte selection. In depth analysis of mice lacking endogenous ζ chain has pointed to an important role for ITAM multiplicity and signal amplification during thymocyte selection. In the absence of ζ chain, TCR surface expression is markedly reduced (35Liu C.-P Uda R She J Sancho J Wang B Weddell G Loring J Kurahara C Dudley E.C Hayday A Terhorst C Huang M Abnormal T cell development in CD3ζ−/− mutant mice and identification of a novel T cell population in the intestine.EMBO J. 1993; 12: 4863-4875PubMed Google Scholar, 36Love P.E Shores E.W Johnson M.D Tremblay M.E Lee J Grinberg A Huang S.P Singer A Westphal H T cell development in mice that lack the ζ chain of the T cell antigen receptor complex.Science. 1993; 261: 918-921Crossref PubMed Scopus (193) Google Scholar, 38Malissen M Gillet A Rocha B Trucy J Vivier E Boyer C Kontgen F Brun N Mazza G Spanopoulou E et al.T cell development in mice lacking the CD3-ε gene.EMBO J. 1993; 12: 4347-4355PubMed Google Scholar, 42Ohno H Aoe T Taki S Kitamura D Ishida Y Rajewsky K Saito T Development and functional impairment of T cells in mice lacking CD3ζ chains.EMBO J. 1993; 12: 4357-4366PubMed Google Scholar). In addition, ζ−/− mice contain very few mature thymocytes and peripheral T cells, suggesting an impairment in positive selection. When the specificity of the TCRs expressed by these mature T cells was examined, the cells were found to be autoreactive, suggesting that negative selection was also impaired (32Lin S.Y Ardouin L Gillet A Malissen M Malissen B The single positive T cells found in CD3.0−/− mice overtly react with self-major histocompatibility complex molecules upon restoration of normal surface density of T cell receptor-CD3 complex.J. Exp. Med. 1997; 185: 707-715Crossref PubMed Scopus (42) Google Scholar). Because surface TCR expression is extremely low in ζ chain null mice, it was not possible to directly evaluate the role of ζ chain ITAMs on thymocyte selection in these mice. Therefore, ζ−/− mice were reconstituted with transgenes encoding ζ chain variants containing 3, 2, 1, or 0 ITAMs (59Shores E.W Huang K Tran T Lee E Grinberg A Love P.E Role of TCR ζ chain in T cell development and selection.Science. 1994; 266: 1047-1050Crossref PubMed Scopus (116) Google Scholar, 60Shores E.W Flamand V Tran T Grinberg A Kinet J.P Love P.E FcεR1γ can support T cell development and function in mice lacking endogenous TCR ζ chain.J. Immunol. 1997; 159 (a): 222-230PubMed Google Scholar, 61Shores E.W Tran T Grinberg A Sommers C.L Shen H Love P.E Role of the multiple TCRζ signaling motifs in selection of the T cell repertoire.J. Exp. Med. 1997; 185 (b): 893-900Crossref PubMed Scopus (103) Google Scholar). These experiments demonstrated that the efficiency of both positive and negative selection correlated directly with the number of ζ chain ITAMs present in the TCR complex. These studies are also consistent with the idea that multiple ζ chain ITAMs act in a quantitative manner to regulate TCR signaling during thymocyte selection. Mice selectively lacking the CD3 components of the TCR (CD3-γ, -δ, -ε) have also been generated (39Malissen M Gillet A Ardouin L Bouvier G Trucy J Ferrier P Vivier E Malissen B Altered T cell development in mice with a targeted mutation of the CD3-ε gene.EMBO J. 1995; 14: 4641-4653PubMed Google Scholar, 11Dave V.P Cao Z Browne C Alarcon B Fernandez-Miguel G Lafaille J de la Hera A Tonegawa S Kappes D.J CD3δ deficiency arrests development of the αβ but not the γδ T cell lineage.EMBO J. 1997; 16: 1360-1370Crossref PubMed Scopus (156) Google Scholar, 12DeJarnette J Sommers C.L Huang K Woodside K.J Emmons R Katz K Shores E.W Love P.E Specific requirement for CD3, in T cell development.Proc. Natl. Acad. Sci. 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Indeed, in vitro studies indicate that individual TCR ITAMs may preferentially bind to different molecules or the same molecules with distinct affinities (19Isakov N Wange R.L Burgess W.H Watts J.D Aebersold R Samelson L.E ZAP-70 binding specificity to T cell receptor tyrosine-based activation motifs the tandem SH2 domains of ZAP-70 bind distinct tyrosine-based activation motifs with varying affinity.J. Exp. Med. 1995; 181: 375-380Crossref PubMed Scopus (161) Google Scholar, 44Osman N Lucas S.C Turner H Cantrell D A comparison of the interaction of Shc and the tyrosine kinase ZAP-70 with the T cell antigen receptor ζ chain tyrosine-based activation motif.J. Biol. Chem. 1995; 270: 13981-13986Crossref PubMed Scopus (75) Google Scholar, 45Osman N Turner H Lucas S Reif K Cantrell D.A The protein interactions of the immunoglobulin receptor family tyrosine-based activation motifs present in the T cell receptor ζ subunits and the CD3 γδε chains.Eur. J. 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Indeed, stimulation of thymocytes from mice that express ζ chains lacking all ITAMs were found to contain increased levels of phosphorylated CD3 chains compared with those expressing a wild-type (three ITAM) ζ chain. These data indicate that the CD3 ITAMs may partially compensate for the loss of ζ chain ITAMs and that ζ chain ITAMs probably do not play a specific role in thymocyte selection (70van Oers N Love P.E Shores E.W Weiss A Regulation of TCR signal transduction in thymocytes by multiple TCR-ζ chain signaling motifs.J. Immunol. 1998; 160: 163-170PubMed Google Scholar). Moreover, the efficiency of thymocyte selection in H-Y TCR transgenic mice appeared similar in ζ chain variant mice regardless of whether their ζ chain contained only the membrane proximal or membrane distal ζ chain ITAM (61Shores E.W Tran T Grinberg A Sommers C.L Shen H Love P.E Role of the multiple TCRζ signaling motifs in selection of the T cell repertoire.J. Exp. Med. 1997; 185 (b): 893-900Crossref PubMed Scopus (103) Google Scholar). While these studies performed to date do not support a role for distinct ITAMs in selectively mediating signaling pathways required for positive versus negative selection, further experiments are needed to dissect the contribution of each TCR ITAM to selection. On the other hand, it may not be necessary to invoke qualitative differences in the signaling responses regulating positive and negative selection if quantitative differences in molecules such as transcription factors can control thymocyte fate. For example, low levels of specific transcription factors may be sufficient to activate genes controlling positive selection, whereas higher quantities of the same factors may be required to activate genes promoting negative selection. In this respect, the potential of multiple ITAMs to amplify small initial differences in signal intensity could be critical, as the difference in the affinity of a TCR for its positively and negatively selecting ligand can be relatively small. The multiple ITAM configuration of the TCR may also provide a means by which the TCR can transmit negative signals, thereby influencing the outcome of the selection process. Indeed, several recent experiments suggest that partially phosphorylated ITAMs may play a role in regulating signaling by the TCR. This idea initially arose from the discovery that stimulation of the TCR with "altered ligands" induces only partial phosphorylation of ζ chain (p21) and the recruitment of nonphosphorylated (inactive) ZAP-70. In contrast, stimulation of the TCR with agonist ligands results in more extensive phosphorylation of ζ chain (p23), which leads to the recruitment and subsequent activation of ZAP-70 (63Sloan-Lancaster J Shaw A.S Rothbard J.B Allen P.M Partial T cell signaling altered phospho-ζ, and lack of Zap70 recruitment in APL-induced T cell anergy.Cell. 1994; 79: 913-922Abstract Full Text PDF PubMed Scopus (575) Google Scholar, 37Madrenas J Wange R.L Isakov N Samelson L.E Germain R.N ζ-phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonists.Science. 1995; 267: 515-518Crossref PubMed Scopus (493) Google Scholar). Studies in which thymocytes were stimulated with positively and negatively selecting ligands revealed that stimulation with either ligand resulted in the formation p23 phospho-ζ and phosphorylated ZAP-70. However, the levels of both p23 phospho-ζ and ZAP-70 were higher when negatively selecting ligand was used to stimulate cells (64Smyth L.A Williams O Huby R.D Norton T Acuto O Ley S.C Kioussis D Altered peptide ligands induce quantitatively but not qualitatively different intracellular signals in primary thymocytes.Proc. Natl. Acad. Sci. USA. 1998; 95: 8193-8198Crossref PubMed Scopus (57) Google Scholar). These data are consistent with the idea that quantitative differences in ζ chain phosphorylation may regulate the outcome of selection. However, to date studies have only been performed on bulk populations of thymocytes. Thus, it is possible that qualitative differences in ζ chain phosphorylation patterns may promote positive versus negative selection, but such a conclusion will require examination at the single cell level. It has also been proposed that differential ζ chain phosphorylation may serve to negatively regulate TCR signaling. In this regard, experiments performed with transfected cell lines indicate that p21-ζ contains monophosphorylated ITAMs, whereas p23-ζ contains one or more diphosphorylated ITAMs (24Kersh E.N Shaw A.S Allen P.M Fidelity of T cell activation through multistep T cell receptor zeta phosphorylation.Science. 1998; 281: 572-575Crossref PubMed Scopus (293) Google Scholar). The implications of these data are intriguing, as recent studies have suggested a distinct role for monophosphorylated ζ chain ITAMs in initiating inhibitory signals (25Kersh E.N Kersh G.J Allen P.M Partially phosphorylated T cell receptor zeta molecules can inhibit T cell activation.J. Exp. Med. 1999; 190: 1627-1636Crossref PubMe
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