CD45-Csk Phosphatase-Kinase Titration Uncouples Basal and Inducible T Cell Receptor Signaling during Thymic Development

Immunity

Article CD45-Csk Phosphatase-Kinase Titration Uncouples Basal and Inducible T Cell Receptor Signaling during Thymic Development Julie Zikherman,1,2,3 Craig Jenne,4 Susan Watson,4 Kristin Doan,1,2,3,5 William Raschke,6,7 Christopher C. Goodnow,8 and Arthur Weiss1,2,3,5,* 1Division

of Rheumatology Russell Medical Research Center for Arthritis 3Department of Medicine 4Department of Microbiology & Immunology 5Howard Hughes Medical Institute University of California San Francisco, San Francisco, CA 94143, USA 6Sidney Kimmel Cancer Center, San Diego, CA 92121, USA 7Virogenics, Inc., San Diego, CA 92104, USA 8John Curtin School of Medical Research, Australian Phenomics Facility, The Australian National University, Canberra 2601, Australia *Correspondence: [email protected] DOI 10.1016/j.immuni.2010.03.006 2Rosalind

SUMMARY

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, whereas 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 titration regulates basal but not inducible signaling. High physiologic expression of CD45 is thus required for two reasons—to downmodulate inducible TCR signaling during positive selection and to counteract Csk during basal TCR signaling. INTRODUCTION 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 (pMHCs) triggers inducible TCR signaling by coligating the TCR ab chains and the CD4 or CD8 coreceptors (Veillette et al., 1989a; Veillette et al., 1989b). The coreceptor-associated Src-family kinase (SFK) Lck then phosphorylates the immunoreceptor tyrosineactivating motifs (ITAMs) of TCR-associated CD3 and z chains (Kane et al., 2000; Veillette et al., 2002), triggering further downstream signaling events. 342 Immunity 32, 342–354, March 26, 2010 ª2010 Elsevier Inc.

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 z chain occurs in double positive (DP; CD4+CD8+) thymocytes and is critical for active downregulation of surface TCR expression (Myers et al., 2005; Nakayama et al., 1989). Although Lck, TCRa, and MHCs are all required to mediate z phosphorylation, nonselecting MHC backgrounds are sufficient to do so (van Oers et al., 1994, 1996a; Becker et al., 2007; Sosinowski et al., 2001). Such nonselecting signals through the TCR are distinct from those that drive positive selection. We use the term ‘‘basal’’ here to refer to nonselecting 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, thereby 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

Immunity Roles of CD45 and Csk during Thymic Development

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 z 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 downregulate 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). Hyperresponsive 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 have described a coding point mutation in CD45, lightning, which was identified during a mutagenesis screen. The lightning mutation reduces 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. RESULTS A CD45 Allele, lightning, Produces Low Protein Expression but Normal Splicing Flow cytometric screening of peripheral blood from ENU (N-ethyl-N-nitrosourea)-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. (Figures 1A and 1B 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 thymocytes, 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 available online). The lightning phenotype cosegregated 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 the 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 (L/+ and L/L) thymocytes were sequenced. A nonsynonymous 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 and Figures S1B and S1C). To determine why the lightning polymorphism produced low CD45 expression, we treated L/L and wild-type thymocytes with cycloheximide to inhibit new protein synthesis and assessed total CD45 protein expression over time. The lightning allele conferred reduced CD45 protein half-life, presumably because of 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 (Figures 1A and 1B). In subsequent studies, it was determined that the phenotypes of L/+ and Ptprc+/ mice were virtually indistinguishable and data from these two genotypes were combined in several figures. CD45 Plays a Dual Positive and Negative Regulatory Role during Positive Selection 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 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 TCRb expression identifies 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 TCRbint expression identifies preselection DP thymocytes that have not received a strong selecting signal and experience predominantly basal or nonselecting TCR signaling (Figures S2A– S2C). Ptprc / thymocytes exhibited a specific and complete developmental block at this checkpoint and failed to upregulate these markers (Mee et al., 1999; Figure 2B and Figure S2B). Not only was this positive selection checkpoint rescued by low CD45 expression, but enhanced selection was also seen in L/ and L/L thymi (Figures 2B–2D). 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. Immunity 32, 342–354, March 26, 2010 ª2010 Elsevier Inc. 343

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Figure 1. A Single Phe to Ser Substitution in an Extracellular Fibronectin Repeat of the CD45 lightning Allele Reduces Protein HalfLife (A and B) DP thymocytes from an allelic series of mice generated by crossing wild-type, lightning, and Ptprc null alleles were stained for surface CD45 and assessed by flow cytometry. Representative histograms as well as CD45 MFI (mean fluorescence intensity) are plotted (genotypes in all figures: / = Ptprc / ; WT = Ptprc+/+; L = Lightning allele). (C) DP thymocytes from allelic series were permeabilized, stained for CD45, and assessed by flow cytometry. Representative histograms are plotted. (D) Quantitative real-time PCR of Ptprc cDNA from two L/L and one wild-type thymi are normalized to GAPDH. Transcript amounts are expressed in relative units (wild-type CD45 = 1). Data are the average of three technical replicates. (E) Sequencing of PCR-amplified Ptprc cDNA fragments from L/L and L/+ thymi. Sequence flanking mutated residue F503 is shown. (F) Allelic series thymocytes incubated with CHX for varying time points. Flow cytometric assessment of permeabilized, stained cells for total CD45 protein content plotted with respect to baseline levels is shown. Results are representative of three independent experiments.

L/+ Wild-type : Lightning : Wild-type : Lightning :

Although low CD45 expression in L/L and L/ thymi promoted generation of postselection DP thymocytes, SP4 thymocyte number as well as thymic cellularity and total DP number was reduced (Figures 2F–2H; data not shown). Reduction in SP4 production was not due to redirection into the SP8 lineage (data not shown). Rather, the reduction in cell number between the DP postselection and the SP4 stages of thymic development suggests deletion due to enhanced negative selection in L/L and L/ thymi (Figures 2D and 2H). 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 thymocytes, 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 preselection DP survival also influence this phenotype. Low CD45 Expression Transforms Positive into Negative Selection In order to study the functional consequences of CD45 expression upon positive and negative selection directly, we assessed 344 Immunity 32, 342–354, March 26, 2010 ª2010 Elsevier Inc.

thymic development 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 exhibited severely impaired positive selection in the context of the OTII transgenic background with nearly absent SP4 T cell production (Figures S2E and S2F; Mee et al., 1999). Unexpectedly, the development of SP4 thymocytes was significantly reduced in OTII allelic series mice expressing low CD45 (Figures S2E and S2F). 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). These OTII L/L and L/ T cells displayed alternate Va and Vb 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, we assessed thymic cellularity of OTII allelic series thymi and compared it to that of Ptprc / OTII thymi (Figures S2I and S2J). 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

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Figure 2. Dual Positive and Negative Regulatory Roles for CD45 during Positive Selection (A) Representative plots of allelic series thymocytes costained for CD4 and CD8 for identification of DN, DP, and SP subsets. (B) Representative plots of allelic series DP thymocytes costained for TCRb and CD5. Gating to identify postselection thymocytes (TCRbhi CD5hi) is based upon the developmental block in Ptprc / thymocytes. (C and D) Quantification of relative and absolute number of TCRbhi CD5hi postselection DP thymocytes as gated in (B). Values are the mean of ten biological replicates ± SEM. (E) Competitive chimeras were generated with a 1:1 mix of L/L and WT donor bone marrow. Thymi were stained for pre- and postselection DP thymocytes with CD4, CD8, TCRb, and CD5, as well as 45.1 and 45.2 allotype markers. Bar graphs represent a ratio of postselection/preselection DP subsets. Data are normalized to a 1:1 input ratio at the preselection DP subset. Values in the graphs are the mean of five biological replicates ± SEM. (F) Quantification of SP4/DP ratios from allelic series thymi as stained and gated in (A). (G) Quantification of absolute DP cell counts from allelic series thymi as stained and gated in (A). (H) Quantification of absolute SP4 cell counts from allelic series thymi as stained and gated in (A). Values in (F)–(H) are the mean of ten biological replicates ± SEM. In this figure, ‘‘HET’’ refers to both L/+ and Ptprc+/ genotype. Statistical analysis with unpaired t tests was performed; *p < 0.05; **p < 0.005; ***p < 0.0005.

positive selection. We conclude that reducing CD45 expression can transform positive into negative selection in the context of a restricted TCR repertoire. High CD45 Expression Is Required to Rescue Basal TCR Signaling 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 and Figure S2A). Identity of the cells in the putative DN4 gate was confirmed with intracellular staining for TCRb (Figure S3). Ptprc / thymi, as previously reported, exhibited a partial block at this critical transition (Figures 3A and 3B). In contrast to the positive selection checkpoint, high expression of CD45 was required to fully correct this partial block in beta selection (Figures 3A and 3B). 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 liganddependent TCR signals are wired differently. This in turn Immunity 32, 342–354, March 26, 2010 ª2010 Elsevier Inc. 345

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Figure 3. High CD45 Expression Is Required to Rescue Beta Selection and Basal Signaling during Thymic Development (A) Representative plots of allelic series DN thymocytes (dump staining for CD4, CD8, CD3, CD19, gd, NK1.1, pNK, CD11b, and CD11c; Gr1 was performed to remove contaminating cell lineages from the gate) stained with CD44 and CD25 to identify subsets DN 1-4 (clockwise from top left). DN3 gate is CD44 CD25+ and DN4 gate is CD44 CD25 . (B) Quantification of DN4/DN3 ratios from allelic series thymi as stained and gated in (A). Values are the mean of six biological replicates ± SEM, normalized to wild-type. (C) Competitive chimeras were generated with a 1:1 mix of L/L and WT donor bone marrow. Thymi were stained for DN3 and DN4 subsets with the method outlined in Figure 3A, as well as with 45.1 and 45.2 allotype markers. Bar graphs represent ratio of DN4/DN3 subsets. Data are normalized to a 1:1 input ratio at the DN3 subset. Values in the graphs are the mean of three biological replicates ± SEM. (D) Representative histograms of surface marker expression on unstimulated DP thymocytes from the allelic series. Experimental samples from the allelic series (black histogram) are each overlayed upon wild-type (gray shaded histogram) for comparison. (E and F) Quantification of MFI of CD5 and TCRb surface levels respectively from pre-selection DP thymocytes as gated in Figure S2. Values are the mean of six biological replicates ± SEM, normalized to wild-type. (G) TCR-associated z chain was immunoprecipitated from whole-cell lysates of resting allelic series thymi. Immunoprecipitates (IPs) were subsequently blotted for total z and total phosphotyrosine levels. p21 z is depicted. Blots are representative of three independent experiments. In this figure, ‘‘HET’’ refers to both L/+ and Ptprc+/ genotype. Statistical analysis with unpaired t tests was performed; *p < 0.05; **p < 0.005; ***p < 0.0005.

suggests that nonselecting (‘‘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 nega346 Immunity 32, 342–354, March 26, 2010 ª2010 Elsevier Inc.

tively regulates TCR signaling and serves as a well-defined marker of TCR signal intensity in thymocytes (Azzam et al., 1998). CD5 expression on preselection DP thymocytes depends upon Lck, z chain, and nonselecting MHC expression (Azzam et al., 1998; Dutz et al., 1995). Preselection DP thymocytes

Immunity Roles of CD45 and Csk during Thymic Development

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Figure 4. Differential Influence of CD45 Expression on Inducible TCR Signaling in DP and SP Thymocytes

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from Ptprc / mice exhibited low CD5 expression, whereas high CD45 expression was required to reconstitute normal CD5 expression (Figures 3D and 3E). Whereas positive selection is enhanced by low expression of CD45, basal signaling is impaired. TCR surface expression is actively downregulated on wildtype DP thymocytes. This is due in part to a posttranslational mechanism that requires a basal TCR signal to produce constitutive phosphorylation of TCR-associated z-chain (Myers et al., 2005; Nakayama et al., 1989). This signal has been shown to depend upon nonselecting 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 (Figures 3D and 3F). Consistent with this observation, basal z 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 z, downregulate the TCR, and upregulate CD5 constitutively upon preselection 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. Distinct Effects of CD45 upon Inducible TCR Signaling in DP and SP Thymocytes In order to directly determine the effect of CD45 expression upon TCR signal strength in the thymus, we assayed biochemical responses to TCR stimulation such as Erk phosphorylation

(A) TCR-stimulated, fixed, and permeabilized thymocytes were stained for phospho-Erk and costained for CD4 and CD8 so that DP and SP subsets could be identified. Data were collected by flow cytometry. Histograms depict intracellular phospho-Erk in DP and SP subsets from wild-type (gray shaded histogram) and L/L (black line) thymi. Data are representative of at least eight independent experiments. (B) Intracellular calcium was assessed by flow cytometry over time in Fluo-4-loaded thymocytes after TCR or ionomycin stimulation. DP thymocytes were gated by size. (C) Allelic series thymocytes were stimulated and stained for intracellular phospho-Erk, CD4, and CD8 as described in (A). Quantification of cells which fall into the positive half of a bimodal phospho-Erk distribution (as depicted in [A]). PMA stimulation produced identical upregulation of Erk phosphorylation in all genotypes (data not shown). Values are the mean of three independent experiments ± SEM.

and calcium flux. L/L DP thymocytes displayed enhanced Erk phosphorylation relative to the 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 hyperresponsive calcium flux to TCR stimulation as well (Figure 4B). Importantly, PMAinduced Erk-phosphorylation and ionomycin-induced calcium flux were comparable across genotypes (Figures 4A and 4B). To determine whether altered TCR surface expression on L/L DP thymocytes accounted for our results, we reassessed Erkphosphorylation in the context of staining for surface TCRb expression. By gating upon matched TCR surface expression in WT and L/L thymocytes, we demonstrated that TCR hyperresponsiveness in L/L DP thymocytes was independent of TCR surface expression (Figures S4B and S4C). 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 was 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 wildtype levels. TCR signaling in both subsets was 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, we performed extensive dose titrations 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 Immunity 32, 342–354, March 26, 2010 ª2010 Elsevier Inc. 347

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Figure 5. CD45 Differentially Controls Lck and Fyn Regulatory Tyrosine Phosphorylation (A) Whole-cell lysates of resting allelic series thymocytes were blotted with Ab to the inhibitory and activating tyrosines of Lck (Lck505/Src416). Src416 Ab binds activating tyrosines of all SFKs; the lower band represents p56 Lck and the upper band represents p59 Fyn. Total Lck is stained as a loading control. (B and C) Whole-cell lysates of purified DP (C) or SP4 (D) thymocytes from L/L and WT mice were blotted with Ab as described in (A). (D) Fyn IPs from whole-cell lysates of resting allelic series thymocytes were blotted with Ab to the inhibitory and activating tyrosines of Fyn (Src 527/Src 416). Total Fyn was stained as a loading control. Blots in this figure are representative of at least three independent experiments.

response in DP thymocytes during the critical positive selection checkpoint. This difference is likely to have important consequences for thymic repertoire selection. Such marked decreased responsiveness in the dose response studies for higher levels of CD45 was not seen in the more mature SP4 population. CD45 Differentially Affects Lck and Fyn Regulatory Tyrosine Phosphorylation To determine the biochemical mechanism by which CD45 dose perturbs basal and inducible signaling, we assayed site-specific phosphorylation of the SFKs in thymocytes. Unstimulated whole-cell lysates were probed for phosphorylation at the activating (Y394) and inhibitory (Y505) tyrosines of Lck, the T cellspecific SFK. With reduced CD45 expression, progressive hyperphosphorylation at both sites was seen (Figure 5A). Dual hyperphosphorylation of both tyrosines was observed in purified L/L DP and SP thymic subsets (Figures 5B and 5C). 348 Immunity 32, 342–354, March 26, 2010 ª2010 Elsevier Inc.

The relative sensitivity of these two phosphorylation sites to CD45 dose was different. Much higher CD45 expression was required to dephosphorylate the activating tyrosine of Lck, Y394, suggesting it is a poorer affinity substrate. Nevertheless, we noted that even inhibitory Y505 required high CD45 expression for maximal dephosphorylation (Figure 5A). To assess whether Fyn and Lck phosphorylation are differentially regulated, we immunoprecipitated Fyn from thymic lysates of the allelic series and probed it for phosphorylation at the activating and inhibitory tyrosines. High CD45 expression promotes dephosphorylation of the activation loop tyrosine of Lck, but has the opposite effect on the Fyn activation loop tyrosine (Figures 5A and 5D). Supraphysiologic CD45 Enhances Basal and Inhibits Inducible TCR Signaling ‘‘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 produced a mouse (‘‘HE’’) in which thymocytes express 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 postselection DP subsets, respectively (Figures S5A and S5B). Supraphysiologic ‘‘HE’’ CD45-expressing mice fall on an extended 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 demonstrated enhanced beta selection, with impaired DP selection. Progressive dephosphorylation of both activating and inhibitory tyrosines of Lck was detected in the presence of supraphysiologic CD45 expression, supporting both tyrosines as CD45 substrates (Figures S5C–S5E). Consistent with positive selection, inducible TCR signaling was inhibited in ‘‘HE’’ thymocytes in contrast to L/L thymocytes (Figures S5F and S5G). 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 wildtype CD45 expression exists along a continuum of biochemical, signaling, and developmental phenotypes, rather than at a peak or trough. The Negative Regulatory Role of CD45 during Thymic Development Is Independent of Fyn 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,

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Figure 6. Fyn Accounts for Differential Regulation of Inducible TCR Signaling by CD45 in DP and SP4 Thymic Subsets (A and B) Histograms depict intracellular phosphoErk in TCR-stimulated DP and SP subsets from L/L (black line) or Ptprc+/+ (gray shaded histogram) mice on either a Fyn+/+ or Fyn / genetic background. (C–F) Graphs represent quantification of data from (A and B) along with the addition of L/+ Fyn+/+ and L/+ Fyn / mice. Data are representative of two independent experiments.

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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 did not influence basal Lck phosphorylation in L/L or L/+ thymi (Figure S6A). We assessed progression from DP to SP4 subset and between preselection CD5loTCRbint and postselection CD5hiTCRbhi DP subsets in Fyn-deficient L/L animals to evaluate positive selection (Figures S6B and S6C). We found that Fyn deficiency had 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 are not mediated by Fyn. We also assessed basal signaling in Fyn-deficient L/L thymocytes by evaluating CD5 and TCRb expression on the surface of preselection DP thymocytes. As described earlier, these

markers were down and upregulated 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 z-phosphorylation, previously demonstrated to depend upon Lck, was normal in Fyn-deficient thymi (data not shown). This is consistent with a CD45dependent, 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.

Differential Dependence upon Fyn and Lck in TCR Signaling in DP and SP Subsets 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 nonredundant 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 (Figures 6A, 6C, and 6E), loss of Fyn expression had a profound effect upon TCR signaling in SP4 thymocytes from allelic series mice (Figures 6B, 6D, and 6F). 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 (Figures 4C and 6). Thus, signaling through Fyn Immunity 32, 342–354, March 26, 2010 ª2010 Elsevier Inc. 349

Immunity Roles of CD45 and Csk during Thymic Development

A

Ptprc-/-

L/-

C L/L

Csk+/+ L/+

Csk+/L/L

Ptprc+/+

L/+

L/L 0μg αCD3ε Csk+/+ 1μg αCD3ε

5μg αCD3ε

% max

Csk+/PMA TCRβ

B

Phospho-Erk

D p=0.20

***

***

p=0.27

p=0.35

** p=0.88

accounts for the TCR signaling differences in allelic series DP and SP4 thymic subsets. Importantly, L/L Fyn / DP and SP4 thymocytes were each hyperresponsive relative to Fyn / thymocytes, suggesting that Lck rather than Fyn mediates the negative regulatory role of CD45 during inducible TCR signaling (Figures 4 and 6). This is consistent with a unique role for CD45 in dephosphorylating the activation loop tyrosine of Lck but not Fyn (Figure 5). Csk Uncouples CD45 Effect on Basal and Inducible Signaling during Development 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 relocalized upon TCR stimulation, we reasoned that reducing Csk dosage (Csk+/ ) in allelic series mice ought to affect basal but not inducible signaling. Furthermore, we examined 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 TCRb and CD5 in preselection DP thymocytes. We observed rescue of these basal signaling phenotypes in allelic series 350 Immunity 32, 342–354, March 26, 2010 ª2010 Elsevier Inc.

Figure 7. Csk Dose Reduction Uncouples Basal and Inducible Signaling during Thymic Development (A) Representative histograms of TCRb expression on preselection DP thymocytes from L/ and L/L mice on either a Csk+/+ or Csk+/ genetic background (black line). Ptprc / Csk+/+ sample (gray shaded histogram) is plotted for reference. (B) Quantification of data presented in (A) along with the addition of L/+ Csk+/+ and L/+ Csk+/ mice. Values are the mean of at least three independent experiments ± SEM, normalized to the wild-type. (C) Histograms depict intracellular phospho-Erk in TCR-stimulated DP subsets from L/L or L/+ (black line) or Ptprc+/+ (gray shaded histogram) mice on either a Csk+/+ or Csk+/ genetic background. (D) Quantification of data presented in (A) along with the addition of Ptprc / , Csk+/ , L/ Csk+/+, and L/ Csk+/ mice. Values are the mean of at least three independent experiments ± SEM. p values listed on the graph are nonsignificant and intended to demonstrate absence of rescue by Csk dose reduction. Statistical analysis with unpaired t tests was performed; *p < 0.05; **p < 0.005; ***p < 0.0005.

animals with Csk dose reduction (Figures 7A and 7B and Figures S7A and 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 (Figures S7C and S7D). We next assessed inducible TCR signaling in allelic series DP thymocytes and observed no effect or rescue with reduced Csk dosage (Figures 7C and 7D). Interestingly, we also observed that reduced thymic cellularity in L/L and L/ mice was not rescued by Csk dose reduction (Figure S7E). This implies that reduced cellularity is not due to impaired beta selection given that 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). DISCUSSION We have described a CD45 allele, lightning, characterized by a mutation in the protein’s extracellular domain. CD45 protein expression was reduced because of impaired protein stability

Immunity Roles of CD45 and Csk during Thymic Development

and increased turnover, but importantly, regulated isoform splicing was 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 coreceptor (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 (McNeill et al., 2007; Salmond et al., 2008), 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 identified were not due to an idiosyncratic effect of the lightning mutation. We have 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 has uncoupled 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 z-chain phosphorylation, and TCR surface expression) were 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 nonselecting MHC signals effectively recruit or require coreceptor. Indeed, although constitutive z-chain phosphorylation in DP thymocytes has been demonstrated to depend upon nonselecting 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. Indeed, consistent with this model we have demonstrated that reduced Csk dosage rescued 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 mM (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 has unmasked 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 have identified dual hyperphosphorylation of Lck at tyrosines 394 and 505 with decreasing doses of CD45. Importantly, we observed 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 rescued 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 did not affect inducible signaling in DP thymocytes, implying that phosphorylation of Lck Y505 does not mediate inducible signaling phenotypes of allelic series mice. Given that 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 hyperphosphorylation 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 because 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 nonredundant 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 Immunity 32, 342–354, March 26, 2010 ª2010 Elsevier Inc. 351

Immunity Roles of CD45 and Csk during Thymic Development

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 coreceptor-associated Lck. This would further enforce MHCdependence during thymic positive selection (Van Laethem et al., 2007). Also, such coreceptor 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 and, 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.

Cy5.5, PE-Cy5.5, PE-Cy7, Pacific Blue, APC, or Alexa 647 (eBiosciences or BD Biosciences) were used for FACS staining. Nur77 Ab (BD) and phosphoErk (197G2) (Cell Signaling) Ab were used for intracellular staining. Lck 505 (BD), Scr 416, Src 527, and Fyn Ab (Cell Signaling) were used for westerns as were phospho-tyrosine (4G10), Lck (1F6), and z (6B10) Ab prepared in our laboratory. Fyn Ab (Santa Cruz) was used for IP. Unconjugated CD33 (2C11) Ab (Harlan) and goat anti-armenian hamster IgG(H+L) Ab were used for TCR stimulation. Goat anti-rabbit /IgG-APC (Jackson Immunoresearch) and anti-Donkey Alexa 488 (Molecular Probes) Ab were used for secondary intracellular staining. Real-Time PCR and Sequencing cDNA was prepared with Trizol reagent protocol and random hexamer priming with SuperscriptIII kit (Invitrogen). Real-time PCR was performed with CD45 and GAPDH Taqman primers (ABI). PCR products were amplified from cDNA with 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). Flow Cytometry and Data Analysis 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 with FlowJo (v8.8.4) software (Treestar Incorporated). Statistical analysis with unpaired t tests was performed with Prism v4c (GraphPad Software). Structural modeling was performed with PyMOL version 1.1beta2 software (DeLano Scientific). Intracellular Staining for CD45 or TCRb Single-cell suspensions were surface-stained, fixed (BD cytofix), stained for intracellular CD45 or TCRb with fluorophore-conjugated Ab in saponin-based medium B (Caltag), and washed with Perm/Wash (BD Biosciences). CD45 Turnover Studies Single-cell suspensions of thymocytes were incubated at 37 C in complete media with 100 mg/ml cycloheximide (CHX; Sigma). At indicated time points, cells were stained for intracellular CD45 as described above. Intracellular Staining for Nur77 Thymocytes were stimulated with plate-bound CD33 with or without CD28 mAbs for 2 hr 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.

EXPERIMENTAL PROCEDURES Mice 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 six generation. Mutagenesis and mapping were performed as previously described (Nelms and Goodnow, 2001). HE, Ptprc / , Csk / , Fyn / mice, 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 a 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. Bone Marrow Chimeras 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 postirradiation. Further experiments were performed as described below. Antibodies and Reagents Murine CD3, CD4, CD5, CD8, CD25, CD44, panCD45, CD45RA, RB, RC, B220, CD45.1, CD45.2, CD69, CD19, gd, NK1.1, pNK, CD11b, CD11c, Gr, TCRb, Va2, and Vb5 Ab conjugated to FITC, PE-Texas Red, PE, PerCP-

352 Immunity 32, 342–354, March 26, 2010 ª2010 Elsevier Inc.

Thymocyte Stimulation, Calcium Flux, and Intracellular Phospho-Erk Staining These assays were performed as previously described (Zikherman et al., 2009). Surface staining of fixed, stimulated thymocytes with TCRb-FITC antibody prior to permeabilization allowed simultaneous assessment of phosphoErk and TCR expression. Immunoprecipitation and Immunoblotting Immunoprecipitation and immunoblotting were performed as previously described (Zhu et al., 2008). Purified cell populations of SP4 and DP thymocytes were obtained by sorting. SUPPLEMENTAL INFORMATION Supplemental Information includes seven figures and can be found with this article online at doi:10.1016/j.immuni.2010.03.006. ACKNOWLEDGMENTS We would like to thank A. Roque for assistance with animal husbandry, N. Killeen and M. Hermiston for critical reading of the manuscript, and members of the Weiss lab for helpful discussions. We would like to thank P. Soriano and T. Mak for providing valuable mice used in these studies. We are grateful to V. Vedantham for help with real-time PCR and to T. Freedman for modeling

Immunity Roles of CD45 and Csk during Thymic Development

of the lightning polymorphism. This work was supported by an Arthritis Foundation Postdoctoral Grant to J.Z.; an ACR Rheumatology Investigator Award to J.Z.; National Institutes of Health (NIH) RO1 AI74847 held by J. Cyster to C.J.; NIH RO1 AI52127 to C.G.; and NIH PO1 AI35297 to A.W. Received: April 13, 2009 Revised: November 23, 2009 Accepted: December 29, 2009 Published online: March 25, 2010 REFERENCES Appleby, M.W., Gross, J.A., Cooke, M.P., Levin, S.D., Qian, X., and Perlmutter, R.M. (1992). Defective T cell receptor signaling in mice lacking the thymic isoform of p59fyn. Cell 70, 751–763. Ashwell, J.D., and D’Oro, U. (1999). CD45 and Src-family kinases: And now for something completely different. Immunol. Today 20, 412–416. Azzam, H.S., Grinberg, A., Lui, K., Shen, H., Shores, E.W., and Love, P.E. (1998). CD5 expression is developmentally regulated by T cell receptor (TCR) signals and TCR avidity. J. Exp. Med. 188, 2301–2311. Barnden, M.J., Allison, J., Heath, W.R., and Carbone, F.R. (1998). Defective TCR expression in transgenic mice constructed using cDNA-based alphaand beta-chain genes under the control of heterologous regulatory elements. Immunol. Cell Biol. 76, 34–40. Becker, A.M., DeFord-Watts, L.M., Wuelfing, C., and van Oers, N.S. (2007). The constitutive tyrosine phosphorylation of CD3zeta results from TCR-MHC interactions that are independent of thymic selection. J. Immunol. 178, 4120–4128. Brdicka, T., Pavlistova´, D., Leo, A., Bruyns, E., Korı´nek, V., Angelisova´, P., Scherer, J., Shevchenko, A., Hilgert, I., Cerny´, J., et al. (2000). Phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), a novel ubiquitously expressed transmembrane adaptor protein, binds the protein tyrosine kinase csk and is involved in regulation of T cell activation. J. Exp. Med. 191, 1591–1604. Byth, K.F., Conroy, L.A., Howlett, S., Smith, A.J., May, J., Alexander, D.R., and Holmes, N. (1996). CD45-null transgenic mice reveal a positive regulatory role for CD45 in early thymocyte development, in the selection of CD4+CD8+ thymocytes, and B cell maturation. J. Exp. Med. 183, 1707–1718.

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