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The kinase-phosphatase pair Csk and CD45 reciprocally regulate phosphorylation of the inhibitory tyrosine of the Src family kinases Lck and Fyn. T cell receptor (TCR) signaling and thymic development require CD45 expression but proceed constitutively in the absence of Csk. Here we show that relative titration of CD45 and Csk expression reveals distinct regulation of basal and inducible TCR signaling during thymic development. Low CD45 expression is sufficient to rescue inducible TCR signaling and positive selection, while high expression is required to reconstitute basal TCR signaling and beta selection. CD45 has a dual positive and negative regulatory role during inducible but not basal TCR signaling. By contrast, Csk plays a positive regulatory role during basal but not inducible signaling. High physiologic expression of CD45 is thus required for two reasons, to down-modulate inducible TCR signaling during positive selection, and to counteract Csk during basal TCR signaling.
The T cell receptor (TCR) and its signal transduction machinery are required during thymic selection in order to establish a functional and tolerant T cell repertoire (Starr et al., 2003). Antigen peptide bound to major histocompatibility molecules (pMHC) triggers inducible TCR signaling by coligating the TCR αβ chains and the CD4 or CD8 coreceptors (Veillette et al., 1989a; Veillette et al., 1989b). The co-receptor-associated Src-family kinase (SFK) Lck then phosphorylates the immunoreceptor tyrosine activating motifs (ITAMs) of TCR-associated CD3 and ζ chains (Kane et al., 2000; Veillette et al., 2002), triggering further downstream signaling events.
Although such inducible signal transduction has been extensively studied, ‘basal’ signals that occur during thymic development also depend upon TCR signaling machinery but are less well-defined. For example, constitutive phosphorylation of the TCR ζ chain occurs in double positive (CD4+CD8+; DP) thymocytes and is critical for active downregulation of surface TCR expression (Myers et al., 2005; Nakayama et al., 1989). Although Lck, TCRα, and MHC are all required to mediate ζ phosphorylation, non-selecting MHC backgrounds are sufficient to do so (Ito et al., 2002; van Oers et al., 1994, 1996a; Becker et al., 2007; Sosinowski et al., 2001). Such non-selecting signals through the TCR are distinct from those that drive positive selection. We use the term ‘basal’ here to refer to non-selecting TCR signals, and ‘inducible’ to refer to selecting TCR signals.
Lck activity is tightly regulated by its location as well as by changes in conformation and enzyme activity that are controlled by phosphorylation of its inhibitory and activation loop tyrosines, respectively (Palacios and Weiss, 2004). Reciprocal regulation of the inhibitory tyrosine of Lck is imposed by the kinase-phosphatase pair Csk and CD45, setting a threshold on TCR signals (Chow et al., 1993; Hermiston et al., 2009; Veillette et al., 2002).
Csk is a ubiquitously expressed kinase whose only substrate is the inhibitory tyrosine of the SFKs (Veillette et al., 2002). Conditional deletion of Csk in T cells produces thymocytes that autonomously progress through beta selection and positive selection checkpoints in the absence of TCR expression (Imamoto and Soriano, 1993; Nada et al., 1993; Schmedt et al., 1998; Schmedt and Tarakhovsky, 2001). This phenotype is entirely dependent upon Lck and Fyn, confirming that the function of Csk in T cells is to negatively regulate these SFKs (Schmedt and Tarakhovsky, 2001).
CD45 is a receptor-like tyrosine phosphatase abundantly expressed on all nucleated hematopoietic cells and is required for both TCR signaling and T cell development (Byth et al., 1996; Kishihara et al., 1993; Mee et al., 1999; Stone et al., 1997). CD45-deficient (Ptprc−/−) mice are characterized by a partial block at the preTCR beta-selection checkpoint and a near-complete block at positive selection. Lck inhibitory tyrosine 505 (Y505) is constitutively hyperphosphorylated in Ptprc−/− thymocytes, whereas an Lck Y505F transgene rescues both TCR signaling and T cell development in these animals, establishing this tyrosine as a physiologically relevant substrate and Lck as a critical mediator of CD45 function (Pingel et al., 1999; Seavitt et al., 1999; Stone et al., 1997). In addition, the loss of constitutive ζ phosphorylation in Ptprc−/− DP thymocytes and the impaired beta-selection phenotype of Ptprc−/− mice together suggest a requirement for CD45 in basal TCR signaling as well (Byth et al., 1996; Stone et al., 1997).
In addition to its positive regulatory role, CD45 has also been shown to down-regulate TCR signaling. Ptprc+/− animals in the context of a restricted TCR repertoire exhibit enhanced positive selection (Wallace et al., 1997), Several groups have generated a series of CD45 single isoform transgenic mice with subphysiologic CD45 expression (Kozieradzki et al., 1997; McNeill et al., 2007; Ogilvy et al., 2003). Hyper-responsive TCR signaling relative to wild-type was observed at intermediate CD45 expression (McNeill et al., 2007). It has been suggested that dephosphorylation of the activation loop tyrosine of the SFKs might mediate the negative regulatory role of CD45 (Ashwell and D'Oro, 1999; McNeill et al., 2007). However, it remains unclear whether these phenotypes relate purely to CD45 expression level or might be confounded by the influence of abnormal isoform expression (McNeill et al., 2007; Salmond et al., 2008).
Here we described a coding point mutation in CD45, lightning, which was identified during a mutagenesis screen. The lightning mutation reduced surface expression of CD45 without perturbing alternative splicing, thereby uncoupling expression and isoform effects. We identified differential requirements for CD45 during basal and inducible TCR signaling and demonstrated distinct regulation of these processes by Csk. We conclude that high physiologic expression of CD45 in the thymus serves two independent functions. First, high expression of CD45 is required to counteract phosphorylation of the inhibitory tyrosine of Lck by Csk in order to promote basal signaling. Second, although low expression of CD45 is sufficient to rescue inducible TCR signaling in DP thymocytes, high expression of CD45 dampens inducible TCR signaling in DP thymocytes by dephosphorylating the activation loop tyrosine of Lck, thereby expanding the dynamic range of TCR-ligand interactions at a critical tolerance checkpoint.
Flow cytometric screening of peripheral blood from ENU-mutagenized C57BL/6 mice (Nelms and Goodnow, 2001) identified a mutant strain, lightning, characterized by low surface expression (15% wild-type) of CD45 on all nucleated hematopoietic lineages. (Figure 1A, B and data not shown). Analysis of CD45 expression in lethally irradiated hosts reconstituted with lightning bone marrow identified this trait as hematopoietic and cell intrinsic (data not shown). Intracellular staining of permeabilized lightning thymocytes revealed that the total cellular pool of CD45 was also substantially reduced relative to wild-type, suggesting that the expression phenotype is not attributable to a defect in protein transport (Figure 1C). Importantly, tightly regulated isoform splicing of CD45, which occurs in a cell-type and developmental stage-specific program (McNeill et al., 2004), was unperturbed in lightning mice (Figure S1A).
The lightning phenotype cosegregates with markers flanking the Ptprc gene encoding CD45 on chromosome 1 (data not shown). In contrast to CD45 protein expression, Ptprc transcript expression in lightning thymocytes revealed no reduction relative to wild-type (Figure 1D), arguing that the lightning mutation must lie within the Ptprc transcript rather than in the regulatory region of the Ptprc gene. Ptprc cDNAs from heterozygous and homozygous lightning thymocytes were sequenced. A non-synonymous T-C point mutation in the coding region was identified. This mutation results in a Phe-Ser substitution in an evolutionarily conserved packing residue F503 (CQFYV) of the most membrane-proximal extracellular fibronectin type-III repeat of CD45 (Figure 1E, S1B, C).
To determine why the lightning polymorphism produced low CD45 expression, lightning homozygous (L/L) and wild-type thymocytes were treated with cycloheximide to inhibit new protein synthesis, and total CD45 protein expression was assessed over time in. The lightning allele conferred reduced CD45 protein half-life, presumably due to impaired protein stability and increased turnover (Figure 1F).
We took advantage of the lightning phenotype to generate an allelic series of mice with varied CD45 expression by breeding the lightning strain to null and wild-type Ptprc lines (Figure 1A, B). In subsequent studies, it was determined that the phenotypes of lightning/+ (L/+) and Ptprc+/− mice were virtually indistinguishable and data from these two genotypes were combined in several figures.
We studied the effect of varied expression of CD45 upon positive selection. Consistent with prior reports, Ptprc−/− thymocytes exhibited a severe block in the transition from double positive (DP) to single positive (SP) thymic stages, corresponding to impaired positive selection. Very low expression of CD45 (L/− = 7% of wild-type) was sufficient to rescue positive selection and reconstitute SP thymic subsets in the allelic series (Figure 2A).
Upregulation of surface CD69, CD5, and TCRβ expression identify DP thymocytes that have received an inducible or positive selection signal through the TCR (Swat et al., 1993; Azzam et al., 1998; Punt et al., 1996). Conversely, CD5lo CD69lo TCRβint expression identifies pre-selection DP thymocytes that have not received a strong selecting signal and experience predominantly basal or non-selecting TCR signaling (Figure S2A, B, C). Ptprc−/− thymocytes exhibited a specific and complete developmental block at this checkpoint and failed to upregulate these markers (Mee et al., 1999; Figure 2B, S2B). Not only is this positive selection checkpoint rescued by low CD45 expression, but enhanced selection was seen in L/− and L/L thymi (Figure 2B, C, D). Competitive repopulation experiments confirmed that this reflects a cell intrinsic advantage (Figure 2E). Higher levels of CD45 resulted in fewer positively selected thymocytes. These results suggest that in addition to its obligate positive regulatory role at this checkpoint, high CD45 expression inhibits positive selection, unmasking a negative regulatory role.
While low CD45 expression in L/L and L/− thymi promoted generation of post-selection DP thymocytes, SP4 thymocyte number as well as thymic cellularity and total DP number was reduced (Figure 2F, G, H; data not shown). Reduction in SP4 production was not due to redirection into the SP8 lineage as the SP4/SP8 ratio was increased (data not shown). Rather, the reduction in cell number between the DP post-selection and the SP4 stages of thymic development suggests deletion due to enhanced negative selection in L/L and L/− thymi (Figure 2D, H). Indeed, reduced DP cell number is a hallmark of enhanced negative selection in mouse models characterized by exaggerated TCR signaling (Gomez et al., 2001; Thien et al., 2005). To assess this possibility, we stimulated L/L and wild-type DP thymocytes ex vivo and evaluated upregulation of the negative selection marker Nur77 (Calnan et al., 1995). Nur77 upregulation was enhanced in L/L DP thymocytes relative to wild-type, suggesting that, similar to positive selection, negative selection is enhanced by low CD45 expression (Figure S2D). Reduced DP cell number with low CD45 expression may therefore be attributed to both enhanced positive and negative selection. It remains possible that impaired beta selection or pre-selection DP survival also influence this phenotype.
In order to study the functional consequences of CD45 expression upon positive and negative selection directly, thymic development was assessed in the context of a fixed TCR repertoire by breeding the allelic series onto the class II-restricted OTII TCR transgenic line. Consistent with prior reports, Ptprc−/− mice exhibit severely impaired positive selection in the context of the OTII transgenic background with nearly absent SP4 T cell production (Figure S2E, F; Mee et al., 1999). Unexpectedly, the development of SP4 thymocytes was significantly reduced in OTII allelic series mice expressing low CD45 (Figure S2E, F). Production of mature peripheral CD4+ lymph node T cells was also impaired in OTII mice expressing low CD45, although less so than in the thymus (Figure S2G). However, these OTII L/L and L/− T cells displayed alternate Vα and Vβ usage, suggesting strong selection pressure in the thymus (Figure S2H).
Loss of OTII+ SP4 thymocytes in L/L and L/− mice may reflect either failed positive or enhanced negative selection. To distinguish between these possibilities, thymic cellularity of OTII allelic series thymi was assessed and compared to Ptprc−/− OTII thymi (Figure S2I, J). If reduced SP4 output from L/− and L/L thymi were due to impaired positive selection, thymic cell number ought to be high as in Ptprc−/− OTII mice. However, thymic cellularity in L/− and L/L OTII thymi was significantly reduced by more than 50% relative to Ptprc−/− thymi. This was true of littermate controls and irrespective of age (range 4–9 weeks, Figure S2K). These data suggest that in contrast to Ptprc−/− OTII mice, impaired SP4 output in L/− and L/L OT2 thymi reflects increased negative selection rather than failed positive selection. We conclude that reducing CD45 expression can transform positive into negative selection in the context of a restricted TCR repertoire.
Beta-selection, in contrast to positive selection, is a ligand-independent or basal signaling event. This checkpoint requires pre-TCR signaling and is completely dependent upon Lck and Fyn (Irving et al., 1998; van Oers et al., 1996b).
Progression from DN3 (double negative) to DN4 stages at the beta-selection checkpoint was assessed along the allelic series (Figure 3A, Figure S2A). Identity of the cells in the putative DN4 gate was confirmed with intracellular staining for TCRβ (Figure S3). Ptprc−/− thymi, as previously reported, exhibit a partial block at this critical transition (Figure 3A, B). In contrast to the positive selection checkpoint, high expression of CD45 is required to fully correct this partial block in beta-selection (Figure 3A, B). Competitive repopulation experiments unmasked a partial block in beta-selection in L/L thymocytes and demonstrate that this phenotype is cell intrinsic (Figure 3C). Distinct regulation of beta selection and positive selection checkpoints by CD45 suggests that basal and ligand-dependent TCR signals are differentially wired. This in turn suggests that non-selecting (‘basal’) signals during DP thymocyte development may also be distinctly regulated by CD45.
To clarify the role of CD45 during such basal signaling, surface markers on preselection DP thymocytes within the allelic series were characterized. CD5 is an inhibitory coreceptor that negatively regulates TCR signaling and serves as a well-defined marker of TCR signal intensity in thymocytes (Azzam et al., 1998). CD5 expression on pre-selection DP thymocytes depends upon Lck, ζ chain, and non-selecting MHC expression (Azzam et al., 1998; Dutz et al., 1995). Pre-selection DP thymocytes from Ptprc−/− mice exhibited low CD5 expression, whereas high CD45 expression was required to reconstitute normal CD5 expression (Figure 3D, E). Whereas positive selection is enhanced by low expression of CD45, basal signaling is impaired.
TCR surface expression is actively downregulated on wild-type DP thymocytes. This is due in part to a post-translational mechanism that requires a basal TCR signal to produce constitutive phosphorylation of TCR-associated ζ-chain (Myers et al., 2005; Nakayama et al., 1989). This signal has been shown to depend upon non-selecting MHC and Lck expression (Becker et al., 2007; van Oers et al., 1996a). In the absence of these factors, TCR expression is constitutively high in DP thymocytes. We observed that the TCR was not effectively downregulated upon thymocytes with low CD45 expression (Figure 3D, F). Consistent with this observation, basal ζ phosphorylation was very low in L/− and L/L animals and inversely correlated with surface TCR expression (Figure 3G and data not shown).
High expression of CD45 is required to phosphorylate ζ, downregulate the TCR, and upregulate CD5 constitutively upon pre-selection DP thymocytes. CD45 does not appear to have a substantial negative regulatory role during basal signaling. In contrast, low CD45 is sufficient not only to rescue but also to enhance inducible TCR signaling during positive selection. Hence, a negative role for CD45 is evident in inducible but not basal signaling in DP thymocytes.
In order to directly determine the effect of CD45 expression upon TCR signal strength in the thymus, biochemical responses to TCR stimulation such as Erk phosphorylation and calcium flux were assayed. L/L DP thymocytes displayed enhanced Erk phosphorylation relative to wild-type, whereas SP4 subsets exhibited relatively impaired signaling (Figure 4A). This result demonstrates opposing effects of CD45 in distinct thymic subsets and was evident in mixed bone marrow chimeras, confirming that this signaling phenotype is cell intrinsic (Figure S4A). L/L DP thymocytes displayed hyper-responsive calcium flux to TCR stimulation as well (Figure 4B). Importantly, PMA-induced Erk-phosphorylation and ionomycin-induced calcium flux were comparable across genotypes (Figure 4A, B).
To determine whether altered TCR surface expression on L/L DP thymocytes accounted for our results, Erk-phosphorylation was re-assessed in the context of staining for surface TCRβ expression. By gating upon matched TCR surface expression in WT and L/L thymocytes, we demonstrated that TCR hyper-responsiveness in L/L DP thymocytes was independent of TCR surface expression (Figure S4B, C).
TCR-induced Erk phosphorylation was assayed across a range of CD45 expression in DP and SP4 thymocytes (Figure 4C). In both thymic subsets, CD45 expression is absolutely required for inducible TCR signaling (Stone et al., 1997). Low expression of CD45 was sufficient to reconstitute signaling in DP thymocytes, whereas higher expression of CD45 was required to fully rescue signaling in SP4 thymocytes to wild type levels. TCR signaling in both subsets is dampened by high CD45 expression, suggesting that CD45 plays a negative regulatory role during inducible signaling.
To determine the qualitative effect of CD45 expression on the dose response curve, extensive dose titrations were performed across the allelic series (Figure S4D). By “flattening” (or reducing) the slope of the dose response curve (Figure S4D), high CD45 expression might expand the ‘dynamic range’ of antigen response in DP thymocytes during the critical positive selection checkpoint. This difference is likely to have important consequences for thymic repertoire. Such decreased responsiveness in the dose response studies for higher levels of CD45 was not seen in the more mature SP4 population.
To determine the biochemical mechanism by which CD45 dose perturbs basal and inducible signaling, site-specific phosphorylation of the SFKs in thymocytes was assayed. Unstimulated whole cell lysates were probed for phosphorylation at the activating (Y394) and inhibitory (Y505) tyrosines of the T cell-specific SFK, Lck. With reduced CD45 expression, progressive hyperphosphorylation at both sites is seen (Figure 5A). Dual hyper-phosphorylation of both tyrosines was observed in purified L/L DP and SP thymic subsets (Figure 5B, C).
The relative sensitivity of these two sites to CD45 dose is different. Much higher CD45 expression was required to dephosphorylate the activating tyrosine of Lck, Y394, suggesting it is a poorer affinity substrate. Nevertheless, even inhibitory Y505 requires high CD45 expression for maximal dephosphorylation.
To assess whether Fyn and Lck phosphorylation are differentially regulated, Fyn was immunoprecipitated from thymic lysates of the allelic series and probed for phosphorylation at the activating and inhibitory tyrosines. High CD45 expression dephosphorylates the activation loop tyrosine of Lck, but has the opposite effect on the Fyn activation loop tyrosine (Figure 5A, D).
‘HE’ mice express supraphysiologic amounts of CD45 on the surface of hematopoietic cells and were generated by expressing a properly splicing CD45 ‘H’ transgene under the control of the LFA-1 promoter in addition to endogenous ‘E’ CD45 alleles (Virts et al., 2003). Two copies of the H transgene locus superimposed on both copies of the wild-type CD45 alleles produce a mouse in which thymocytes express approximately 125% of normal CD45 on their surface.
We probed endogenous beta-selection and positive selection in HE mice by assessing the transition between DN3 and DN4 thymic subsets, and between pre- and post-selection DP subsets respectively (Figure 5SA, B). Supraphysiologic ‘HE’ CD45-expressing mice fall on a continuum with the allelic series and appear to have a dichotomous effect on these processes relative to low CD45-expressing mice. Whereas L/L and L/− thymi exhibit impaired beta-selection, but enhanced DP selection, ‘HE’ thymi demonstrate enhanced beta-selection, with impaired DP selection.
Progressive dephosphorylation of both activating and inhibitory tyrosines of Lck is detected in the presence of high CD45 expression, supporting both tyrosines as CD45 substrates (Figure S5C, D, E).
Consistent with positive selection, inducible TCR signaling is inhibited in ‘HE’ thymocytes in contrast to L/L thymocytes (Figure S5F, G). This demonstrates the negative regulatory role of CD45 during inducible TCR signaling.
The addition of the high-expressing ‘HE’ CD45 line to the allelic series serves to establish that the biochemical, signaling and developmental consequences of perturbing CD45 dose are independent of the lightning mutation and relate instead purely to protein expression. Furthermore, physiologic wild-type CD45 expression exists along a continuum of biochemical, signaling, and developmental phenotypes, rather than at a peak or trough.
The two SFKs expressed in T cells, Lck and Fyn, are partially redundant but also have distinct physiological roles (Appleby et al., 1992; Stein et al., 1992; Zamoyska et al., 2003). In order to understand which of these two CD45 substrates mediate the basal and inducible phenotypes of allelic series thymocytes, we bred the lightning allele into the Fyn-deficient genetic background.
We analyzed thymic development, signaling and SFK phosphorylation in Fyn-deficient allelic series thymi. Fyn deficiency does not influence basal Lck phosphorylation in L/L or L/+ thymi (Figure S6A). We assessed progression from DP to SP4 subset and between pre-selection CD5loTCRβint and post-selection CD5hiTCRβhi DP subsets in Fyn-deficient L/L animals to evaluate positive selection (Figure S6B, C). We found that Fyn deficiency has no effect upon these phenotypes in allelic series mice. Therefore, both the effect of CD45 upon Lck phosphorylation and the negative regulatory role of CD45 in inducible TCR signaling is not mediated by Fyn.
We also assessed basal signaling in Fyn-deficient L/L thymocytes by evaluating CD5 and TCRβ expression on the surface of pre-selection DP thymocytes. As described earlier, these markers are down and up-regulated respectively on L/L thymocytes as a result of impaired basal signaling intensity. We found that these phenotypes were also completely unaffected by Fyn deficiency (Figure S6C and data not shown). Moreover, basal ζ-phosphorylation, previously demonstrated to depend upon Lck, is normal in Fyn-deficient thymi (data not shown). This is consistent with a CD45-dependent, Fyn-independent basal signaling pathway in DP thymocytes and identifies a critical distinction between the physiological roles of partially redundant SFKs Fyn and Lck. Taken together, these data suggest that Lck rather than Fyn mediates the effect of CD45 expression upon these phenotypes. However, this does not rule out a redundant role for Fyn which is masked by physiologic Lck expression.
The expression patterns of Fyn and Lck differ among T cell subsets. Specifically, it has been reported that Fyn is significantly upregulated after the DP stage of thymic development (Olszowy et al., 1995). Although Fyn lacks a non-redundant role in DP thymocytes during basal or inducible TCR signaling (Figure S6; Mamchak et al., 2008; Zamoyska et al., 2003), we hypothesized that Fyn might contribute to signaling differences between DP and SP4 thymocytes in allelic series mice (Figure 4C).
To test this hypothesis, we assayed TCR signal transduction in allelic series thymocytes deficient for Fyn. Although Fyn deficiency minimally influenced signaling in DP thymocytes (Figure 6A, C, E), loss of Fyn expression had a profound effect upon TCR signaling in SP4 thymocytes from allelic series mice (Figure 6B, D, F). In the absence of Fyn, SP4 signaling was significantly dampened in all allelic series mice, and differential regulation of DP and SP4 signaling by CD45 was no longer evident (Figure 4C, ,6).6). Thus, signaling through Fyn accounts for the TCR signaling differences in allelic series DP and SP4 thymic subsets. Importantly, L/L Fyn−/− DP and SP4 thymocytes were each hyper-responsive relative to Fyn−/− thymocytes, suggesting that Lck rather than Fyn mediates the negative regulatory role of CD45 during inducible TCR signaling (Figure 4, ,6).6). This is consistent with a unique role for CD45 in dephosphorylating the activation loop tyrosine of Lck but not Fyn.
Csk and CD45 reciprocally regulate the inhibitory tyrosines of the SFKs and are uniquely specialized to target this substrate. Csk activity is, in turn, regulated by its dynamic localization. Csk is constitutively recruited to lipid rafts, in part, by the adaptor PAG. Upon receptor stimulation PAG is rapidly dephosphorylated and Csk is released to the cytoplasm (Brdicka et al., 2000; Kawabuchi et al., 2000). Because Csk is dynamically re-localized upon TCR stimulation, we reasoned that reducing Csk dosage (Csk+/−) in allelic series mice ought to affect basal but not inducible signaling. Furthermore, we asked whether the negative regulatory role of CD45 was an indirect result of dephosphorylating the inhibitory tyrosine of Lck, or rather an independent and direct effect mediated by dephosphorylation of the activation loop tyrosine of Lck. We hypothesized that if the latter were true, reducing Csk dosage and thus phosphorylation of the inhibitory tyrosine of Lck, ought not to affect inducible signaling by allelic series DP thymocytes.
To test these hypotheses, we generated Csk+/− allelic series animals. We assessed basal signaling by assaying expression of TCRβ and CD5 in preselection DP thymocytes. We observed rescue of these basal signaling phenotypes in allelic series animals with Csk dose reduction (Figure 7A, 7B, S7A, S7B). Interestingly, the rescue produced by halving Csk dose precisely corresponded to a doubling of CD45 expression such that, for instance, Csk+/− L/− thymocytes resembled L/L thymocytes. We assessed beta-selection in these mice and saw similar rescue of this ligand-independent signaling checkpoint (Figure S7C, D).
We next assessed inducible TCR signaling in allelic series DP thymocytes and observed no effect or rescue with reduced Csk dosage (Figure 7C, D). Interestingly, we also observed that reduced thymic cellularity in L/L and L/− mice is not rescued by Csk dose reduction (Figure S7E). This implies that reduced cellularity is not due to impaired beta-selection since the latter process is rescued by Csk haploinsufficiency.
Taken together these data suggest that Csk regulates basal but not inducible signaling in DP thymocytes. This in turn implies that basal signaling is regulated by CD45 exclusively through its effects on the inhibitory tyrosine of the SFKs. In contrast, the negative regulatory role of CD45 during inducible TCR signaling in DP thymocytes is independent of the inhibitory tyrosine, suggesting that the direct dephosphorylation of the activation loop tyrosine of Lck by CD45 mediates this effect. Simple haploinsufficiency of Csk in the context of the allelic series uncouples the contribution of inhibitory and activation loop tyrosines of Lck to basal and inducible signaling respectively (Figure S7F).
We describe a CD45 allele, lightning, characterized by a mutation in the protein’s extracellular domain. Expression was reduced due to impaired protein stability and increased turnover, but importantly, regulated isoform splicing is unperturbed and the polymorphism is structurally insulated from the cytoplasmic phosphatase domain.
CD45 isoform expression is tightly regulated and may affect protein function through differential dimerization or differential association with the CD4 co-receptor (Dornan et al., 2002; Xu and Weiss, 2002). In contrast to previously published series of CD45 transgenic lines in which single isoforms of CD45 are expressed at a range of doses, the lightning allelic series titrates protein expression while preserving isoform splicing. By including both Ptprc+/− animals and the high-expressing CD45 ‘HE’ line in our studies, we conclude that the phenotypes we identify were not due to an idiosyncratic effect of the lightning mutation.
We demonstrated distinct requirements for CD45 in basal and inducible TCR signaling during thymic development that are obscured in the context of complete CD45 deficiency. Whereas Ptprc−/− mice have previously revealed an absolute requirement for CD45 in positive selection, and a partial requirement during beta-selection (Byth et al., 1996; Kishihara et al., 1993; Mee et al., 1999), the lightning allelic series uncouples these processes, revealing that low CD45 doses are sufficient to rescue the former, but high doses are required to correct the latter. Additional indicators of impaired basal signaling in Ptprc−/− thymocytes (CD5 expression, constitutive ζ-chain phosphorylation, and TCR surface expression) are rescued only with high CD45 expression. In contrast, biochemical indicators of inducible TCR signal intensity in DP thymocytes showed rescue of CD45-dependent defects even at very low CD45 expression.
Inducible signaling via MHC presentation of antigen recruits coreceptor-associated Lck to the TCR. In contrast, neither beta-selection, nor basal or weak non-selecting MHC signals effectively recruit or require coreceptor. Indeed, while constitutive ζ-chain phosphorylation in DP thymocytes has been demonstrated to depend upon non-selecting MHC and Lck, it is coreceptor independent (Becker et al., 2007; van Oers et al., 1996a; van Oers et al., 1993). We propose therefore that independent pools of CD4-associated and ‘free’ Lck may be differentially regulated by CD45 and independently mediate basal and inducible signaling (Falahati and Leitenberg, 2007; Leitenberg et al., 1996).
The distinct requirements for CD45 expression during basal and inducible signaling might also relate to the dynamic localization of Csk (Brdicka et al., 2000; Kawabuchi et al., 2000). High amounts of Csk bound to PAG and localized to Lck in lipid rafts in the basal state might require high expression of CD45 to counteract Lck Y505 phosphorylation, whereas loss of Csk from lipid rafts upon TCR stimulation might explain why even low doses of CD45 are sufficient to mediate inducible TCR signaling (Figure S7F). Indeed, consistent with this model we have demonstrated that reduced Csk dosage rescues basal but not inducible signaling phenotypes in L/− and L/L DP thymocytes.
CD45 is expressed at extremely high amounts on the surface of hematopoietic cells, by some estimates up to 25 µM (data not shown). Yet, the reason for this apparent overabundance remains uncertain, especially when no definitive physiologically relevant ligand has been identified in 20 years of searching. We propose that one reason why so much CD45 is required on the surface of cells may be to counteract membrane-associated Csk in the basal state.
Although very low doses of CD45 are sufficient to rescue inducible TCR signaling and positive selection, high amounts of CD45 dampen these processes. The allelic series unmasks a negative regulatory role for CD45 that is obscured in Ptprc−/− animals by its positive regulatory role.
CD45 has been postulated to negatively regulate TCR signaling by dephosphorylating the activating Lck tyrosine 394 (McNeill et al., 2007). Dual hyperphosphorylation of Lck at both inhibitory and activating tyrosines has been reported in many CD45 deficient cell lines and mice (Ashwell and D'Oro, 1999). Here we also identify dual hyperphosphorylation of Lck at tyrosines 394 and 505 with decreasing doses of CD45. Importantly, we observe progressive dephosphorylation at these sites with supraphysiologic expression of CD45, suggesting that any dimerization that occurs at high surface density is insufficient to completely inhibit phosphatase activity. Maximal Y394 phosphorylation at low CD45 expression levels correlates with maximal inducible TCR signaling in DP thymocytes. We suggest that in the presence of even low amounts of CD45, Y394 rather than Y505 phosphorylation “dominantly” controls the sensitivity of inducible TCR signaling.
Reducing Csk dosage rescues basal signaling in allelic series thymocytes and implies that CD45 is required to counteract Csk phosphorylation of Lck Y505 in the basal state. Lck Y505 phosphorylation dominantly controls Lck activity under these conditions. By contrast, reduced Csk dosage does not affect inducible signaling in DP thymocytes, implying that phosphorylation of Lck Y505 does not mediate inducible signaling phenotypes of allelic series mice. Since high CD45 expression dampens inducible TCR signaling, we conclude that CD45 negatively regulates Lck activity through an alternate substrate, most likely the activation loop tyrosine Y394. Our data support a model whereby Lck Y394 is directly dephosphorylated by CD45, independently of Csk and its unique substrate Lck Y505.
Although dual activating and inhibitory tyrosine phosphorylation of Lck has been observed in CD45 deficient T cells and T cell lines, other CD45-deficient hematopoietic cells, including B cells and macrophages, do not exhibit hyper-phosphorylation of SFK activation loop tyrosines (Zhu et al., 2008). This is more consistent with the regulation of Fyn activating tyrosine phosphorylation in our allelic series thymocytes, and suggests that Lck regulation by CD45 is unique among the SFKs. This may be the result of differential localization of the SFKs since Lck is uniquely associated with CD4 coreceptor and, consequently, with CD45 (Veillette et al., 1988). The activating tyrosine of Lck may only function as an important CD45 substrate at high CD45 doses and in close proximity such that Lck is uniquely regulated by CD45 with respect to other SFKs.
We have identified differential regulation of inducible signaling in DP and SP subsets by CD45 and have demonstrated that Fyn rather than Lck accounts for these differences. Importantly, Fyn does not play a non-redundant role in either basal signaling or negative regulation of inducible signaling by CD45. Unpublished data from our lab suggests that the CD45 requirement for signaling by peripheral T cells is similar to that in SP4 rather than DP thymocytes, implying that DP thymocytes are unique among T cells. Indeed, prior studies had suggested that very low levels of CD45 expression were sufficient to reconstitute thymic development, while much higher levels were required to rescue TCR signaling in peripheral T cells (Kozieradzki et al., 1997; McNeill et al., 2007; Ogilvy et al., 2003). We propose that this discrepancy is actually due to the distinct CD45 requirements of DP thymocytes and mature T cells which are in turn the result of differential roles of Fyn and Lck in these subsets.
This differential cell-type specific TCR ‘wiring’ has interesting implications for DP thymocyte signaling. Perhaps low Fyn expression forces DP thymocytes to be more dependent upon co-receptor-associated Lck. This would further enforce MHC-dependence during thymic positive selection (Van Laethem et al., 2007). As well, such co-receptor associated Lck is itself more tightly regulated by CD45 than “free” Lck (Falahati and Leitenberg, 2007). By dialing down activating Y394 phosphorylation of Lck at physiologic doses of CD45, the dynamic range of antigen recognition by DP thymocytes is enhanced at a crucial developmental checkpoint.
The lightning allelic series demonstrates that hypomorphic alleles of known genes can unmask previously obscured functions. Indeed, the physiologic relevance of CD45 dose is highlighted by recent studies demonstrating that mice expressing intermediate CD45 are resistant to fatal Ebola and Anthrax infections to which both wild type and CD45−/− mice succumb (Panchal et al., 2009a; Panchal et al., 2009b).
We conclude by suggesting that high physiologic expression of CD45 on the surface of T cells serves two functions: First, to counteract Csk activity during basal signaling. Second, to negatively regulate TCR signaling specifically in DP thymocytes, thereby expanding the dynamic range of ligand-receptor interactions at a critical developmental checkpoint. This provides a unique solution to the central problem of the adaptive immune system: how, in the face of a universe of unknown antigens, to generate working receptors that respond sensitively, but not too sensitively.
Lightning mice were generated directly on the C57BL/6 genetic background during N-ethyl-N-nitrosourea mutagenesis screen conducted at Australian National University and backcrossed at least 6 generation. Mutagenesis and mapping as previously described (Nelms and Goodnow, 2001). HE mice, Ptprc−/−, Csk−/−, Fyn−/−, and OTII TCR transgenic mice were previously described (Barnden et al., 1998; Imamoto and Soriano, 1993; Kishihara et al., 1993; Stein et al., 1992; Virts et al., 2003). All mutant strains were fully backcrossed to C57BL/6 genetic background. Mice were used at 5–9 weeks of age for all experiments. All mice were housed in a specific pathogen free facility at UCSF according to the University Animal Care Committee and National Institutes of Health (NIH) guidelines.
Host mice (CD45.1 or CD45.1/CD45.2 heterozygotes) were lethally irradiated, reconstituted with lightning and/or wild-type bone marrow (CD45.1 or CD45.2), and analyzed at 6–10 weeks post-irradiation. Further experiments were performed as described below.
Murine CD3, CD4, CD5, CD8, CD25, CD44, panCD45, CD45RA, RB, RC, B220, CD45.1, CD45.2, CD69, CD19, γδ, NK1.1, pNK, CD11b, CD11c, Gr, TCRβ, Vα2, and Vβ5 Ab conjugated to FITC, PE-Texas Red, PE, PerCP-Cy5.5, PE-Cy5.5, PE-Cy7, Pacific Blue, APC, or Alexa 647 (eBiosciences or BD Biosciences); Nur77 Ab (BD); phospho-Erk (197G2), Scr 416, Src 527, and Fyn Ab (Cell Signaling); Fyn Ab for IP (Santa Cruz); Phospho-tyrosine (4G10), Lck (1F6), and ζ (6B10) Ab prepared in our laboratory; unconjugated CD3ε (2C11) Ab (Harlan); Lck505 and biotinylated CD3ε (2C11) Ab (BD Biosciences); biotinylated CD4 (GK5.1) Ab (UCSF Hybridoma Core); Goat anti-armenian hamster IgG(H+L) and goat anti-rabbit IgG Ab conjugated to either PE or APC (Jackson Immunoresearch); Anti-Donkey Alexa 488 (Molecular Probes).
cDNA was prepared using Trizol reagent protocol and random hexamer priming with SuperscriptIII kit (Invitrogen). Real time PCR was performed using CD45 and GAPDH Taqman primers (ABI). PCR products were amplified from cDNA using a series of nested primers (Operon) traversing the CD45 transcript and then sequenced (ELIM). Lightning mutation numbering (F503) is derived from the convention that the juxtamembrane wedge glutamate is E613 in mouse (Majeti et al., 2000).
Cells were stained with indicated Ab and analyzed on FACSCalibur (Becton Dickson) or CyAN ADP (DAKO) as previously described (Hermiston et al., 2005). Data analysis was performed using FlowJo (v8.8.4) software (Treestar Incorporated, Ashland, OR). Statistical analysis with unpaired t-tests was performed using Prism v4c (GraphPad Software, Inc). Structural modeling was performed using PyMOL version 1.1beta2 software (DeLano Scientific, Palo Alto, CA).
Single cell suspensions were surface stained, fixed (BD cytofix), stained for intracellular CD45 or TCRβ with fluorophore-conjugated Ab in saponin-based medium B (Caltag), and then washed with Perm/Wash (BD Biosciences).
Single cell suspensions of thymocytes were incubated at 37°C in complete media with 100 µg/ml cycloheximide (CHX; Sigma). At indicated time points, cells were stained for intracellular CD45 as described above.
Thymocytes were stimulated with plate-bound CD3ε with or without CD28 mAbs for 2 hours in 10% RPMI at 37°C. Cells were then fixed, permeabilized with MeOH, washed and stained first with Nur77 Ab, and then with secondary anti-Donkey Alexa 488 Ab along with staining for surface markers.
These assays were performed as previously described (Zikherman et al., 2009). Surface staining of fixed, stimulated thymocytes with TCRβ-FITC antibody prior to permeabilization allowed simultaneous assessment of phospho-Erk and TCR expression.
Performed as previously described (Zhu et al., 2008). Purified cell populations of SP4 and DP thymocytes were obtained by sorting.
We would like to thank Al Roque for assistance with animal husbandry, Nigel Killeen and Michelle Hermiston for critical reading of the manuscript, and members of the Weiss lab for helpful discussions. We would like to thank Phillipe Soriano and Tak Mak for providing valuable mice used in these studies. We are grateful to Vasanth Vedantham for help with real-time PCR and to Tanya Freedman for modeling of the lightning polymorphism.
Grant support : Arthritis Foundation Postdoctoral Grant to JZ; ACR Rheumatology Investigator Award to JZ; CJ was supported by NIH RO1 AI74847 held by Jason Cyster; NIH RO1 AI52127 to CG; NIH PO1 AI35297 to AW.
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