Signal strength in thymic selection and lineage commitment

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Signal strength in thymic selection and lineage commitment Kristin A Hogquist During development, αβ T cells undergo positive or negative selection and CD4+/CD8+ lineage commitment — events that have a major impact on the functionality of the T cell repertoire. The precise mechanisms of these differentiative steps remain elusive. Research this year has focused on quantitative models of signaling. For positive selection, the timing and extent of ERK activation may be important. For lineage commitment, the extent of Lck recruitment and activation may be the decisive factor. Next, the search is on for the genes that commit the cell to the fate determined by these quantitative differences in signals. Addresses Center for Immunology, University of Minnesota, MMC 334, 420 Delaware Street SE, Minneapolis, MN 55455, USA; e-mail: [email protected] Current Opinion in Immunology 2001, 13:225–231 0952-7915/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations ITAM immune-receptor tyrosine-based activation motif MAPK mitogen-activated protein kinase NotchIC Notch-1 intracellular domain

binding kinetics (quantity) translates into a dramatic difference in cell fate (quality). Proximal events in TCR signaling

A key issue for thymic development is understanding which proximal steps occur during positive selection and how the accumulation of these signaling intermediates leads to a qualitative change in the signal perceived at the nuclear level. One of the most proximal events in TCR signal transduction is the phosphorylation of tyrosine residues in the immune-receptor tyrosine-based activation motifs (ITAMs). In mature T cells, high-affinity ligands stimulate the accumulation of the p23 phosphorylated form of the TCRζ chain [4,5]. A structural study of the TCRζ chain showed that this is due to the full phosphorylation of the tyrosines on all three ITAMs in that chain. [6••]. Lowaffinity ligands, on the other hand, resulted in accumulation of p21 [4,5], which was shown to be a form of the TCRζ chain with selective phosphorylation on only the second and third ITAMs. This led to the speculation that the duration of TCR ligation specifies which ITAMs are phosphorylated and, because p21 and p23 may recruit unique signaling effectors, this would result in positive or negative selection, respectively.

Introduction For many cell surface receptors, ligation initiates a cascade of biochemical events that alter the function or fate of the cell when the ligand is present above a particular threshold. For the TCR, however, things seem a little more complicated. TCR ligation appears to result in multiple cellular outcomes, depending on the extent of the ligation. Nowhere is this more relevant than in T cell development, where the outcomes are life or death. This review summarizes recent models for how the strength of a TCR signal impacts positive and negative selection, and lineage commitment.

Positive/negative selection: changing quantity to quality in TCR signaling The task of generating a T lymphocyte population that responds to foreign peptides presented by MHC, but not to self, is undertaken in the thymus. Positive selection is the result of TCR ligation of self-peptide−MHC complexes on epithelial cells of the cortex. Negative selection, which can also occur in the cortex, is likewise a result of TCR ligation of self-peptide−MHC. Although many models to account for this paradox have been considered, the affinity hypothesis has stood the test of time. Data for both MHC-class-Iand class-II-restricted receptors supported the notion that strong ligation of the TCR during development leads to negative selection whereas weak ligation leads to positive selection ([1,2]; reviewed in [3]). For the most part, positive-selection ligands have a faster dissociation rate from the TCR compared with negative-selection ligands, so naturally the field has focused on how this difference in

However, the experiments performed to date do not suggest that ITAMS selectively mediate positive or negative selection although they clearly amplify the TCR signal during thymic development [7–10]. In addition, ζ-chain phosphorylation in thymocytes is distinctly different from that in mature T cells. In thymocytes, low-affinity ligands resulted in the formation of both p23 and p21, albeit at lower levels compared with those induced by negativeselection ligands [11,12•]. This is consistent with experiments that showed that thymocytes can be more readily activated by low-affinity ligands than mature T cells [12•,13]. Although the molecular mechanisms responsible for this attenuation of the response to self as the thymocyte matures have yet to be defined, the results indicate that it may be misleading to study mature T cell signaling when the question relates to thymic selection. The above data suggest that, at very proximal points in the TCR signaling pathway, positive-selection signals are like negative-selection signals, only weaker. Consistent with this hypothesis is the fact that apparent ‘gradations’ in proximal TCR signaling can convert a negative-selection signal into a positive-selection one. Using D011 TCR transgenic mice expressing a TCRζ chain with no or only one ITAM, the weak negative selection observed in H-2b/d F1 mice could be converted to positive selection [9]. Furthermore, in HY TCR transgenic mice lacking the Tec family kinase genes, rlk and itk, CD8+ T cells were generated in the normally negative-selecting male environment

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Figure 1 (a) Threshold model

(b) Duration model

Signal intensity

Time Current Opinion in Immunology

Two models for differential signaling in response to low-affinity (solid) and high-affinity (dotted) interactions. (a) A simple threshold model postulates that weak interactions trigger a subset of the signals that strong interactions trigger. The different biological outcomes are due to different signaling pathways having lower or higher thresholds of initiation (indicated by the shading); for example, ERK activation for positive selection may have a lower threshold than p38 activation for negative selection. (b) A signal duration model predicts that highaffinity interactions trigger a strong but transient signal whereas lowaffinity ligands trigger a weak but sustained one. The different biological outcomes could potentially be due to different levels of nuclear accumulation of signaling molecules and activation of transcription factors.

[14•]. A ‘threshold model’ of thymic selection (Figure 1a) seems sufficient to explain these mutations: impaired TCR signal strength results in a shift from negative to positive selection and from positive selection to neglect.

The response of p38 and JNK to both ligands was unaffected. Therefore it appears that CD3δ may somehow regulate positive selection via a mechanism that is independent of its own cytoplasmic domain, possibly via controlling the ability of engaged receptors to associate with lipid rafts. Since negative selection was unaffected, this points to an important bifurcation at very proximal points in the TCR signal transduction pathway and is difficult to meld with a simple threshold model, where the most proximal TCR signals would differ only quantitatively. Another fascinating piece of the signaling puzzle that the paper by Werlen et al. [16••] provides is a comparison of the kinetics of ERK activation after engagement of positive- or negative-selection ligands. ERK activation has been identified as a potential point where the signals of positive and negative selection qualitatively diverge [19–21]. Werlen et al. [16••] reported that a high-affinity ligand stimulated a strong and transient activation of ERK. The low-affinity ligand stimulated a much weaker activation but this was sustained. A similar observation was made using the P14 TCR transgenic system (P Ohashi, personal communication). This would not have been predicted by a simple ‘threshold’ signaling model of thymic selection, where less-stable TCR−ligand interactions would result in a more transient biochemical signal (see Figure 1). That the lessstable TCR−ligand interactions led to a greater stable accumulation of signaling intermediates is the first real evidence of the quantity-to-quality conversion in thymocyte selection. This is especially exciting given the data from other biological systems, where transient and sustained ERK activation led to different cell fates [22,23].

The key role of ERK in thymic selection

On the other hand, two papers published this year [15••,16••] challenge a simple threshold model. Positive selection is profoundly deficient in CD3δ deficient mice [17]. Delgado et al. [15••] examined TCR signaling in such mice and found that ERK activation was severely impaired, but that activation of p38 and JNK was unaffected. In mice restored with a CD3δ molecule that lacked the cytoplasmic tail (and its ITAM motif), both positive selection and ERK activation were restored. This suggests that something about the extracellular and transmembrane regions of CD3δ are critical for TCR signaling in positive selection. Technically, this comparison was difficult because CD3δ deficient mice have very low levels of surface TCR. To control for this, the authors focused their analysis on TCRlo cells and this could be a concern about the work. However, Werlen et al. [16••] reported a similar result. Here, the effect of a mutation in the TCR α-chain connecting peptide (α-CPM) was studied. This mutation results in a selective abrogation of positive but not negative selection and, interestingly, prevents CD3δ from being recruited to the TCR−CD3 complex [18]. Werlen et al. [16••] showed that the α-CPM-mutant receptor responded normally to a negative-selection ligand but failed to induce ERK activation in response to a positive-selection ligand.

The importance of sustained TCR ligation in positive selection has been known for some time [24,25], so a sustained level of ERK activation makes sense. But how would lowaffinity ligands trigger sustained activation? Mariathasan and Ohashi have data to suggest that, because they do not trigger receptor internalization, low-affinity ligands can continue to trigger TCR signaling (S Mariathasan, P Ohashi, personal communication). Nevertheless, the ERK activation patterns are only correlative at this point. Whether ERK duration is the key to different outcomes remains to be determined experimentally. Finally, a recent report sheds light on the mechanism by which TCR signaling is linked to ERK activation in thymocytes [26••]. This study reports the targeted deletion of RasGRP, a novel guanine nucleotide exchange factor for Ras. Analysis of thymocytes from this mouse strain revealed a profound defect in positive selection, suggesting that Ras/MAPK (mitogen-activated protein kinase) activation is linked to TCR signaling via RasGRP. This molecule has a diacylglycerol (DAG)-binding domain and thus it is proposed that TCR engagement leads to the activation of phospholiase Cγ, resulting in accumulation of DAG in the plasma membrane and recruitment of RasGRP to activate Ras. It will be important to determine

Signal strength in thymic selection and lineage commitment Hogquist

if negative selection is also impaired in RasGRP deficient mice, since other data suggest that Ras/MAPK activation is not required for negative selection [19,20]. The importance of signaling context

In addition to TCR signaling, thymocytes integrate other signals from their microenvironment. The importance of this in thymic selection was illustrated by a recent study of thymic outcome when high- or low-affinity ligands for the OT-I TCR were expressed in thymic epithelial cells. Whereas the low-affinity ligand resulted in efficient positive selection, the high-affinity ligand did not result in efficient negative selection. Instead, TCR internalization and receptor editing were observed [27••]. The obligatory role that epithelial cells of the cortex play in positive selection has long been appreciated. That these cells did not promote negative selection may suggest that negative selection requires a type of co-stimulus that cortical epithelial cells do not provide and is consistent with the many experiments showing that negative selection requires a TCR co-stimulus (reviewed in [28]). Alternatively, epithelial cells may express a factor that actively prevents apoptosis, similar to that proposed to explain the response of receptor editing in B cells [29]. Regardless, it is clear that the thymocyte integrates signals from both the TCR and its environment during negative selection as well as positive selection. These environmental factors, although unidentified yet, play a critical role in the biological outcome.

A strength-of-signal model for lineage commitment Models based on signal strength or duration have also been put forward to account for commitment of thymocytes to the helper or cytotoxic lineage (reviewed in [30]). In lineage commitment, however, the co-receptors seem to have a greater influence than the TCR. For the most part, modifying the MHC−peptide ligand for a given receptor does not alter its lineage choice whereas modification of the coreceptor often does. For example, transgenic expression of CD8 in the class-Irestricted F5 TCR transgenic mouse strain led thymocytes to adopt the ‘CD4 fate’ [31]. This result was initially thought to lend support to a stochastic/selective model of lineage commitment, by assuming the enforced expression of CD8 ‘rescued’ cells that had stochastically adopted the inappropriate lineage. This interpretation was likely to be too simple, since enforced expression of CD4 in class-IIrestricted TCR transgenics generally did not result in a CD8 fate. Additionally, it was recently shown that lineage commitment in TCR-transgenics could occur with efficiency approaching 100% [32•]. Such high efficiency is incompatible with the idea of a stochastic fate commitment that results in 50% of the cells going to waste. Instead it appears that, for class-II-restricted receptors, genetic alterations

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that impair co-receptor function result in the CD8 fate [33]. For class-I-restricted receptors, genetic alterations that enhance co-receptor function result in the CD4 fate [34]. This has led to the consideration of a ‘quantitative’ signaling model in lineage commitment. Because CD4 recruits more Lck to the TCR signaling complex than CD8 does [35], Lck became an obvious candidate for involvement in the quantitative signaling aspect of lineage commitment. CD4 binds Lck approximately 20-times more avidly than CD8 does [36]. Additionally, in thymocytes the Lck-recruiting abilities of CD4 and CD8 are even more disparate because a large fraction of the CD8 molecules expressed in thymocytes are alternatively spliced and lack the Lck-binding site [37]. Indeed, transgenic expression of a genomic CD8 construct that allows the expression of both splice variants in the thymus did not result in commitment of F5 T cells to the CD4 fate, like the cDNA-based construct described above (which expressed only Lck-binding CD8) did [38••]. This strongly favors a model in which Lck signal strength plays a key role in lineage commitment. More-direct evidence for this model came from the analysis of transgenic strains bearing mutated forms of Lck. A constitutively active form of Lck led a class-I-restricted receptor (OT-I) to adopt the CD4 fate [39••,40]. Alternatively, a catalytically inactive form of Lck forced a class-II-restricted receptor (AND) to adopt the CD8 fate [39••]. In another approach, Legname et al. [41•] used a tetracycline-responsive promoter driving Lck, in combination with a transgenic strain expressing the tetracyline-responsive transactivator [41•]. In Lck-deficient animals, tetracycline treatment resulted in the preferential restoration of CD4+ but not CD8+ T cells. Individual CD4+ T cells had a broad and high level of Lck whereas CD8+ T cells had only a low level of Lck. These data suggest that CD8+ T cells are selected only from precursors with a low level of Lck, consistent with the model stated above. Other data from an in vitro experimental system suggested that lineage commitment was determined by the duration of TCR signaling, more than the ‘strength’ of the signal [42•]. This group showed that when CD4+CD8+ thymocytes were incubated with dendritic cells presenting a high-affinity antigen for 1.5 hours during a primary culture, the thymocytes developed into CD8+CD4− cells in a secondary re-aggregate culture. Alternatively, those incubated with the same ligand for 14 hours in the first culture developed into CD8−CD4+ cells in the second [42•]. Although this result is intriguing, it will be important to test whether the co-receptor alters the duration of TCR signaling in vivo, where thymocytes interact with low-affinity self ligands on epithelial cells. Another in vitro experimental system also studied the effect of signal ‘duration’ on lineage commitment. In this work, thymocytes were given activating signals (anti-TCR antibodies or pharmacological activators) then re-cultured

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in the presence of IL-7, with or without further TCR activation [43•]. Continued TCR activation led to the generation of CD4 single-positive cells whereas the absence of TCR signaling led to CD8 single-positive cells. Several groups have shown that CD8 is initially downregulated more than CD4, generating a transient CD4+CD8lo population. The authors speculate that TCR signal duration is influenced by this decrease in CD8, thus only class-I-restricted receptors would experience a signal cessation and this forces a ‘reversion’ to the CD8+ lineage. Again, it will be important to confirm these results in vivo, where T cells are responding to low-affinity self-ligands. This paper also concluded that CD4+CD8lo intermediate population is the true target of lineage commitment. However, this is difficult to reconcile with the fact that this population is detected in some class-I-restricted TCR transgenics but not others and its presence seems to depend on the affinity of the TCR for self [44]. Several recent studies suggest that one of the downstream signaling pathways implicated in lineage commitment is the Ras/Raf/MAPK pathway. Some studies utilizing pharmacological inhibitors of the MAPK, ERK, showed an effect on CD4+ T cell development but not CD8+ cells [40,45,46]. These results formed the basis for a model of differential ERK activation being a deciding factor in lineage commitment. However, CD8+ cell differentiation was blocked quite effectively by ERK inhibitors in another study [47] and by transgenic expression of a dominant-negative form of MEK [48] or genetic deletion of ERK1 [49]. These discrepancies may have to do with the specificity or timing of the inhibition mechanism; we await more-precise experimental approaches to resolve them.

Notch activity in lineage commitment Like in the case of positive and negative selection, we are still left with the question of how the quantitative differences in Lck activity translate into qualitative differences in fate. Genetic approaches have suggested that Notch signaling may contribute to lineage commitment. Examination of mice expressing a constitutively active Notch-1 intracellular domain (NotchIC) led Robey et al. [50] to hypothesize that Notch-1 signaling promotes CD8+ cell commitment and disfavors CD4+ cell commitment. NotchIC also triggers bcl-2 expression and it was later suggested that the effect of Notch on lineage choice is an indirect effect of bcl-2 disregulation [51]. However, it seems that the effect of Notch on lineage commitment cannot be solely attributed to bcl-2, since NotchIC and bcl-2 have distinct and separable functions in mice expressing both as transgenes [52•]. Nonetheless, the effect of NotchIC on lineage commitment may not be simple to understand. Another NotchIC transgene was reported this year (similar but not identical to the one reported earlier) and it was shown to enhance both CD4+ and CD8+ lineage commitment [53•]. The disparate results from mice expressing similar transgenes point to

the drawbacks of using ‘dominant’ signaling mutants, where the biological effect may critically depend on the level of gene expression and may also initiate signals that are normally not activated. Indeed, mice with an induced deletion in the Notch-1 gene showed no effect on lineage commitment (HR MacDonald, F Radtke, personal communication). In this study, Cre/lox-mediated gene deletion of Notch-1 at the late double-negative (CD4−CD8−) stage was employed to circumvent the requirement for Notch-1 at the early double-negative stage. A thorough analysis of these mice revealed no requisite role for Notch-1 in lineage commitment or thymocyte survival. This study is difficult to reconcile with the reported effect of an anti-sense Notch-1 [42•] but does not necessarily contradict the studies of NotchIC mice. Both Notch-2 and -3 are also expressed in the thymus and may function in a redundant manner in the absence of Notch-1. In this regard it is interesting that a Notch-3 IC transgene did not alter lineage commitment [54]. Nevertheless, the products of lineage commitment (single-positive thymocytes) express Notch-regulated genes (such as deltex and Hes-1) whereas double-positive (CD4+CD8+) thymocytes do not, arguing that Notch signaling does occur during this stage of development [52•,53•]. Interestingly, both CD4+ and CD8+ single-positive cells express Notch-regulated genes, suggesting that a model where Notch-1 signaling exclusively favors the CD8+ cell fate may be too simplistic. A recent report describing a ‘helper deficient’ or ‘HD’ mouse strain provided very strong evidence that, eventually, quantitative signals translate into irreversible gene expression changes that determine lineage fate. This spontaneously arising mutation resulted in the lineage commitment of cells exclusively to the CD8+ lineage [55••]. Understanding the mutated target gene may well provide a point from which investigators can work ‘back’ and thereby understand the mechanism of lineage commitment. Diametric CD4 and CD8 gene expression has typically been used as the hallmark of lineage commitment because they are relatively straightforward to measure. Recall, however, that lineage commitment is much more than a co-receptor gene regulation issue. During lineage commitment, the thymocyte undergoes major genetic changes to allow different effector functions [56]. A better knowledge of this genetic program will empower the field with more stringent criteria for defining lineage commitment and may clarify some of the controversy and ambiguity in this field.

Conclusions In summary, two TCR-initiated biochemical events stand out as important in lineage decisions in the thymus: the Ras/MAPK pathway for positive and negative selection, and co-receptor-associated kinase activity for lineage commitment. We are likely to see ever-more elegant and clever approaches to manipulating these pathways applied to the

Signal strength in thymic selection and lineage commitment Hogquist

study of the thymus — particularly in terms of MAPK activation, where it appears that duration as well as signal strength may be important. Clearly, however, these TCRderived signals need to be understood in the context of the critical microenvironmental signals that stromal cells impart to developing thymocytes. This aspect of thymus biology is now in its infancy and hopefully further study will bring exciting developments in the years to come.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest •• of outstanding interest 1.

Alam SM, Travers PJ, Wung JL, Nasholds W, Redpath S, Jameson SC, Gascoigne NRJ: T-cell receptor affinity and thymocyte positive selection. Nature 1996, 381:616-620.

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Madrenas J, Wange RL, Wang JL, Isakov N, Samelson LE, Germain RN: Zeta phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonists. Science 1995, 267:515-518.

Update Correia-Neves et al. [57••] recently reported the clever design of a mouse strain with limited TCR diversity. They created this strain by crossing a TCRβ transgenic strain to a TCRα minilocus transgenic strain, which can only rearrange a single Vα to one of two Jα gene segments. This created a mouse strain whose limited TCR gene diversity is broad but manageable, in terms of study. They used single-cell PCR and high-throughput sequencing to catalog the specificity of the T cell repertoire in these mice. By comparing the sequences expressed in thymocytes immediately before and after thymic selection, they showed the severity with which selection constricts the repertoire. Their analysis also revealed a surprising influence of CDR3 and peptide on lineage commitment, with single residue changes causing a switch from class I to class II restriction. The recently reported analysis of mice that have decreased levels of the adaptor protein Grb2 lends further support to the model that positive and negative selection invoke activation of different MAP kinase pathways. Gong et al. [58••] report that mice heterozygous for Grb2 have impaired activation of the JNK and p38 MAP kinases but normal activation of ERK. Interestingly, these mice showed an impaired response in several models of negative selection whereas positive selection was normal. A careful quantitative analysis of MAP kinases showed a distinctly lower threshold for activation of ERK compared with JNK and p38. This prompted the authors to support a simple threshold model (see Figure 1) where weak signals trigger ERK activation and positive selection (light gray area in Figure 1) but only strong signals trigger JNK and p38 activation and negative selection (dark gray area in Figure 1). This model may still be too simplistic, however, since its unclear why weaker signals (like positive selection) would not be more readily impaired by partial signal inhibition (such as the decreased Grb2 levels provide) and why low-affinity ligands seem capable of triggering efficient JNK and p38 activation [16••]. An interesting possibility raised by this work is that positive selection signaling proceeds in a Grb2-independent manner, which is consistent with the new finding that ras activation and positive selection in thymocytes use a RasGRP-dependent pathway [26••].

Acknowledgements I thank Maureen McGargill and Steve Jameson for thoughtful discussion and for reading the manuscript. This work was supported by grants from the National Institutes of Health.

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6. ••

van Oers NS, Tohlen B, Malissen B, Moomaw CR, Afendis S, Slaughter C: The 21 and 23-kD forms of TCR zeta are generated by specific ITAM phosphorylation. Nat Immunol 2000, 1:322-328. This paper defined the molecular basis of the p21 and p23 phosphorylated forms of the TCR ζ chain, using site-directed mutagenesis and mass spectrometry. The p21 form is generated by phosphorylation of four tyrosines located in the second and third immune-receptor tyrosine-based activation motifs (ITAMs). The p23 form is fully phosphorylated at all three ITAMs. Both forms were shown to be dependent upon the binding of ZAP-70. 7.

Shores EW, Tran T, Grinberg A, Sommers CL, Shen H, Love PE: Role of the multiple T cell receptor (TCR)-zeta chain signaling motifs in selection of the T cell repertoire. J Exp Med 1997, 185:893-900.

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10. van Oers NS, Love PE, Shores EW, Weiss A: Regulation of TCR signal transduction in murine thymocytes by multiple TCR zetachain signaling motifs. J Immunol 1998, 160:163-170. 11. Smyth LA, Williams O, Huby RD, Norton T, Acuto O, Ley SC, Kioussis D: Altered peptide ligands induce quantitatively but not qualitatively different intracellular signals in primary thymocytes. Proc Natl Acad Sci USA 1998, 95:8193-8198. 12. Lucas B, Stefanova I, Yasutomo K, Dautigny N, Germain RN: • Divergent changes in the sensitivity of maturing T cells to structurally related ligands underlies formation of a useful T cell repertoire. Immunity 1999, 10:367-376. This paper, along with [13], showed that thymocytes are uniquely capable of being activated by low-affinity ligands. As the cells matured, they maintained an equal sensitivity to high-affinity ligands but lost the ability to respond to low-affinity ligands. This was surprising, given the low surface TCR level on thymocytes, but was consistent with the biological function, in that thymocytes must respond to self during positive selection but mature T cells must not respond to self. 13. Davey GM, Schober SL, Endrizzi BT, Dutcher AK, Jameson SC, Hogquist KA: Pre-selection thymocytes are more sensitive to TCR stimulation than mature T cells. J Exp Med 1998, 188:1867-1874. 14. Schaeffer EM, Broussard C, Debnath J, Anderson S, McVicar DW, • Schwartzberg PL: Tec family kinases modulate thresholds for thymocyte development and selection. J Exp Med 2000, 192:987-1000. This paper analyzed mice deficient for the kinase genes, rlk and itk. Deletion of these kinases resulted in decreased calcium mobilization and ERK activation, and had graded effects on positive and negative selection. Importantly, in double-deficient mice, positive selection of CD8+ T cells was observed in HY male mice. This conversion from negative to positive selection by TCR signal diminution supports a threshold model of thymic selection. 15. Delgado P, Fernandez E, Dave V, Kappes D, Alarcon B: CD3 delta •• couples T-cell receptor signalling to ERK activation and thymocyte positive selection. Nature 2000, 406:426-430. CD3δ deficient mice, which have a severe defect in positive selection, were found to lack the ability to activate ERK whereas JNK or p38 activation was

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unaffected. Ca2+ mobilization, phospholipase-Cγ and LAT phosphorylation, and the level of phospho-TCRζ associated with lipid rafts were all affected. Expression of a tail-less CD3δ restored both ERK activation and positive selection, suggesting that CD3δ controls downstream signaling by coupling the engaged TCR complex to lipid rafts, in a manner independent of its own cytoplasmic domain. 16. Werlen G, Hausmann B, Palmer E: A motif in the αβ T cell receptor •• controls positive selection by modulating the ERK pathway. Nature 2000, 406:422-426. A mutation in the TCR α-chain connecting peptide region profoundly affects positive but not negative selection. Here it was shown that this mutant, which fails to associate with CD3δ, failed to activate ERK whereas p38 or JNK activation was unaffected. The recruitment of LAT, Lck, ZAP-70 and phospho-TCRζ to lipid rafts was also shown to be impaired. These data are consistent with those in reference [15••] and suggest that CD3δ is involved in generating an intact ‘signalosome’ required for positive selection. This paper also showed a sustained ERK activation generated by TCR engagement of low-affinity ligands and a transient ERK activation generated by highaffinity ligands. The connecting-peptide mutation completely abrogated the sustained ERK activation but had no effect on the transient activation. 17.

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20. Sugawara T, Moriguchi T, Nishida E, Takahama Y: Differential roles of ERK and p38 MAP kinase pathways in positive and negative selection of T lymphocytes. Immunity 1998, 9:565-574. 21. Sharp LL, Schwarz DA, Bott CM, Marshall CJ, Hedrick SM: The influence of the MAPK pathway on T cell lineage commitment. Immunity 1997, 7:609-618. 22. Marshall CJ: Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 1995, 80:179-185. 23. York RD, Yao H, Dillon T, Ellig CL, Eckert SP, McCleskey EW, Stork PJ: Rap1 mediates sustained MAP kinase activation induced by nerve growth factor. Nature 1998, 392:622-626. 24. Wilkinson RW, Anderson G, Owen JJ, Jenkinson EJ: Positive selection of thymocytes involves sustained interactions with the thymic microenvironment. J Immunol 1995, 155:5234-5240. 25. Kisielow P, Miazek A: Positive selection of T cells: rescue from programmed cell death and differentiation require continual engagement of the T cell receptor. J Exp Med 1995, 181:1975-1984. 26. Dower NA, Stang Sl, Bottorff DA, Ebinu JO, Dickie P, Ostergaard HL, •• Stone JC: RasGRP is essential for mouse thymocyte differentiation and TCR signaling. Nat Immunol 2000, 1:317-321. The phenotype of mice lacking the Ras activator, RasGRP, was shown in this paper. A profound defect in positive selection was reported, with little or no defect in the generation of double-positive precursors. ERK activation induced by phorbol myristate acetate or anti-CD3 was completely abrogated in such mice, suggesting that the TCR is linked to ERK activation via a nonredundant diacylglycerol/RasGRP mechanism. 27. McGargill MA, Derbinski JM, Hogquist KA: Receptor editing in •• developing T cells. Nat Immunol 2000, 1:336-341. This paper reports mice expressing both a class-I-restricted TCR and its target antigen. Typically this scenario results in clonal deletion of developing thymocytes. Here, however, it was demonstrated that thymocytes underwent TCR internalization and receptor editing. Thus it appears that, like immature B cells, immature T cells can undergo receptor editing or clonal deletion. The authors speculated that receptor editing occurred instead of clonal deletion in this situation because the antigen was expressed by cortical epithelial cells. 28. Amsen D, Kruisbeek AM: Thymocyte selection: not by TCR alone. Immunol Rev 1998, 165:209-229. 29. Sandel PC, Monroe JG: Negative selection of immature B cells by receptor editing or deletion is determined by site of antigen encounter. Immunity 1999, 10:289-299. 30. Singer A, Bosselut R, Bhandoola A: Signals involved in CD4/CD8 lineage commitment: current concepts and potential mechanisms. Semin Immunol 1999, 11:273-281.

Zamoyska R, Derham P, Gorman SD, von Hoegen P, Bolen JB, Veillette A, Parnes JR: Inability of CD8 alpha′′ polypeptides to associate with p56lck correlates with impaired function in vitro and lack of expression in vivo. Nature 1989, 342:278-281.

38. Salmon P, Mong M, Kang X-J, Cado D, Robey E: The role of CD8 •• alpha′′ in the CD4 versus CD8 lineage choice. J Immunol 2000, 163:5312-5318. Normal thymocytes express a mixture of alternatively spliced forms of CD8: one that can bind Lck, the other that cannot. CD8 transgenes that were previously shown to permit the development of ‘mismatched’ CD4+ T cells were generated using a cDNA construct that could express only CD8 with the Lck-binding site (see [31]). In this paper, the authors used a genomic construct that allowed both the Lck-binding and -nonbinding forms to be synthesized. Such a construct did not permit the development of CD4+ T cells and strongly suggests that a reduced Lck recruitment is important in CD8+ lineage commitment. 39. Hernandez-Hoyos G, Sohn SJ, Rothenberg EV, Alberola-Ila J: Lck •• activity controls CD4/CD8 T cell lineage commitment. Immunity 2000, 12:313-322. In this paper, Lck activity in thymocytes was reduced or enhanced by the transgenic expression of active or inactive forms of Lck. The active form forced class-I-restricted thymocytes to adopt the CD4+ lineage whereas the inactive form forced class-II-restricted thymocytes to adopt the CD8+ lineage. The reciprocal crosses did not alter lineage commitment. These data suggest that the dose of Lck alters the TCR signal in a manner that dictates lineage. Since CD4 and CD8 recruit high and low levels of Lck, respectively, these data would suggest an instructive role for the co-receptors in lineage commitment. 40. Sharp LL, Hedrick SM: Commitment to the CD4 lineage mediated by extracellular signal-related kinase mitogen-activated protein kinase and Lck signaling. J Immunol 1999, 163:6598-6605. 41. Legname G, Seddon B, Lovatt M, Tomlinson P, Sarner N, Tolaini M, • Williams K, Norton T, Kioussis D, Zamoyska R: Inducible expression of a p56Lck transgene reveals a central role for Lck in the differentiation of CD4 SP thymocytes. Immunity 2000, 12:537-546. This paper reports a tetracycline-based system for the inducible expression of Lck. The induced restoration of Lck in Lck-deficient mice had a significant effect on CD4+ lineage commitment but less effect on the CD8+ lineage. By intracellular staining, the authors showed that CD8+ cells were selected from precursors expressing a uniformly low level of Lck, compared with CD4+ cells. These data are consistent with a model where the level of Lck recruited to the signaling complex dictates cell lineage. 42. Yasutomo K, Doyle C, Miele L, Germain RN: The duration of antigen • receptor signalling determines CD4+ versus CD8+ T-cell lineage fate. Nature 2000, 404:506-510. A two-step experimental system, consisting of a co-culture of thymocytes with dendritic cells followed by a re-aggregate of the remaining thymocytes with stromal cells, was employed to dissect issues of timing in lineage commitment. The length of time that thymocytes were exposed to antigen in the first culture led to different outcomes in the second culture, suggesting a role for TCR signal duration in lineage commitment.

Signal strength in thymic selection and lineage commitment Hogquist

43. Brugnera E, Bhandoola A, Cibotti R, Yu Q, Guinter TI, Yamashita Y, • Sharrow SO, Singer A: Coreceptor reversal in the thymus: signaled CD4+CD8+ thymocytes initially terminate CD8 transcription even when differentiating into CD8+ T cells. Immunity 2000, 13:59-71. Another two-step in vitro system was reported here, in which thymocytes were cultured initially with cross-linking antibodies or drugs, then cultured with or without IL-7 in a second step. The authors propose a model that places importance on the signal cessation of class-I-restricted thymocytes as they initially downregulate CD8, causing them to re-express it at a high level (co-receptor reversal). Class-II-restricted cells would not experience a signal cessation when CD8 is downregulated and the model proposes that persistent TCR signaling leads to CD4+ cell differentiation. 44. Ohashi PS, Zinkernagel RM, Leuscher I, Hengartner H, Pircher H: Enhanced positive selection of a transgenic TCR by a restriction element that does not permit negative selection. Int Immunol 1993, 5:131-138. 45. Shao H, Wilkinson B, Lee B, Han PC, Kaye J: Slow accumulation of active mitogen-activated protein kinase during thymocyte differentiation regulates the temporal pattern of transcription factor gene expression. J Immunol 1999, 163:603-610. 46. Bommhardt U, Basson MA, Krummrei U, Zamoyska R: Activation of the extracellular signal-related kinase/mitogen-activated protein kinase pathway discriminates CD4 versus CD8 lineage commitment in the thymus. J Immunol 1999, 163:715-722. 47.

Mariathasan S, Ho SS, Zakarian A, Ohashi PS: Degree of ERK activation influences both positive and negative thymocyte selection. Eur J Immunol 2000, 30:1060-1068.

48. Alberola-Ile J, Forbush KA, Seger R, Krebs EG, Perlmutter RM: Selective requirement for MAP kinase activity in thymocyte differentiation. Nature 1995, 373:620-623. 49. Pages G, Guerin S, Grall D, Bonino F, Smith A, Anjuere F, Auberger P, Pouyssegur J: Defective thymocyte maturation in p44 MAP kinase (ERK 1) knockout mice. Science 1999, 286:1374-1377. 50. Robey E, Chang D, Itano A, Cado D, Alexander H, Lans D, Weinmaster G, Salmon P: An activated form of Notch influences the choice between CD4 and CD8 T cell lineages. Cell 1996, 87:483-492. 51. Deftos ML, He YW, Ojala EW, Bevan MJ: Correlating Notch signaling with thymocyte maturation. Immunity 1998, 9:777-786. 52. Chang D, Valdez P, Ho T, Robey E: MHC recognition in thymic • development: distinct, parallel pathways for survival and lineage commitment. J Immunol 2000, 165:6710-6715. To determine whether bcl-2 upregulation accounts for the effect of Notch-1 signaling in lineage commitment, the effect of expressing both bcl-2 and NotchIC (Notch-1 intracellular domain) as transgenes was studied. The combination of Notch activity and bcl-2 expression was sufficient to allow the appearance of CD8+ lineage cells in TCRα-deficient double transgenics, but not in TCRα-deficient single transgenics. This suggests that survival and lineage commitment represent distinct, parallel pathways of positive selection.

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53. Deftos ML, Huang E, Ojala EW, Forbush KA, Bevan MJ: Notch1 • signaling promotes the maturation of CD4 and CD8 SP thymocytes. Immunity 2000, 13:73-84. This paper describes a constitutively active Notch-1 transgene and show that it promotes development of both CD4+ and CD8+ T cells. Several genes that are transcriptionally regulated by Notch-1 activity in T cells were also identified and shown to be expressed in both CD4+ and CD8+ lineage thymocytes. These results challenge a simple model whereby Notch-1 signaling leads exclusively to the CD8+ fate. 54. Bellavia D, Campese AF, Alesse E, Vacca A, Felli MP, Balestri A, Stoppacciaro A, Tiveron C, Tatangelo L, Giovarelli M et al.: Constitutive activation of NF-kappa B and T-cell leukemia/lymphoma in Notch3 transgenic mice. EMBO J 2000, 19:3337-3348. 55. Keefe R, Dave V, Allman D, Wiest D, Kappes DJ: Regulation of •• lineage commitment distinct from positive selection. Science 1999, 286:1149-1153. The recently derived ‘helper deficient’ or ‘HD’ mouse strain is studied in this paper. By crossing to various TCR-transgenic and MHC-deficient mouse strains, the authors show that the naturally occurring HD mouse strain has an intrinsic defect in thymic CD4+ lineage commitment. This defect is independent of positive selection, class-II-mediated antigen-presentation or CD4+ cell function. 56. Corbella P, Spanopoulou M, Mamalaki C, Tolaini M, Itano A, Lans D, Baltimore D, Robey E, Kioussis D: Functional commitment to helper T cell lineage precedes positive selection and is independent of T cell receptor MHC specificity. Immunity 1994, 1:269-276. 57. Correia-Neves M, Waltzinger C, Benoist C, D Mathis: The shaping of •• the T cell repertoire. Immunity 2001, 14:21-32. This paper reports a mouse strain that was engineered to have a limited T cell repertoire, with diversity only in the TCR α chain CDR3. By sequencing hundreds of TCR α chain genes from sorted thymocytes and lymph node populations, the authors were able to document the overall restrictions and patterns of the pre- and post-selection repertoires and compare CD4+ and CD8+ T cell populations. 58. Gong Q, Cheng AM, Akk AM, Alberola-Ila J, Gong G, Pawson T, •• Chan AC: Disruption of T cell signalling networks and development by Grb2 haploid insufficiency. Nat Immunol 2001, 2:29-35. This paper reports the analysis of Grb2+/– mice, which express decreased levels of this important adaptor protein. Thymocytes from these mice display reduced JNK and p38 activation in response to anti-CD3 but display normal ERK activation. Positive selection was normal in heterozygous mice but negative selection in response to antigen, superantigen or anti-CD3 was impaired, suggesting that negative selection requires JNK and p38 MAP kinases but positive selection does not. The authors also show that ERK is more efficiently activated by phorbol esters than JNK and p38 are. This fact is consistent with a model where weak TCR signals are able to trigger ERK activation and positive selection but where stronger TCR signals are required for JNK and p38 activation and negative selection.