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Distinct T Cell Developmental Consequences in Humans and Mice Expressing Identical Mutations in the DLAARN Motif of ZAP-70

This information is current as Melissa E. Elder, Suzanne Skoda-Smith, Theresa A. of September 29, 2021. Kadlecek, Fengling Wang, Jun Wu and Arthur Weiss J Immunol 2001; 166:656-661; ; doi: 10.4049/jimmunol.166.1.656 http://www.jimmunol.org/content/166/1/656 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Distinct T Cell Developmental Consequences in Humans and Mice Expressing Identical Mutations in the DLAARN Motif of ZAP-701

Melissa E. Elder,2* Suzanne Skoda-Smith,¶ Theresa A. Kadlecek,§ Fengling Wang,* Jun Wu,† and Arthur Weiss‡§¶

The protein tyrosine kinase, ZAP-70, is pivotally involved in transduction of Ag-binding signals from the TCR required for T cell activation and development. Defects in ZAP-70 result in SCID in humans and mice. We describe an infant with SCID due to a novel ZAP-70 mutation, comparable with that which arose spontaneously in an inbred mouse colony. The patient inherited a homozygous missense mutation within the highly conserved DLAARN motif in the ZAP-70 kinase domain. Although the mutation

only modestly affected protein stability, catalytic function was absent. Despite identical changes in the amino acid sequence of Downloaded from ZAP-70, the peripheral T cell phenotypes of our patient and affected mice are distinct. ZAP-70 deficiency in this patient, as in other humans, is characterized by abundant nonfunctional CD4؉ T cells and absent CD8؉ T cells. In contrast, ZAP-70-deficient mice lack both major T cell subsets. Although levels of the ZAP-70-related protein tyrosine kinase, Syk, may be sufficiently increased in human thymocytes to rescue CD4 development, survival of ZAP-70-deficient T cells in the periphery does not appear to be dependent on persistent up-regulation of Syk expression. The Journal of Immunology, 2001, 166: 656–661. http://www.jimmunol.org/ inding of Ag by the TCR initiates intracellular signaling ylates SLP-76 and LAT, triggering distal pathways that bring pathways that lead to transcription of genes required for about T cell activation (12–15). Studies in humans and gene-tar- B T cell activation. Propagation of Ag-binding signals from geted mouse models have confirmed the importance of ZAP-70 to the TCR to the nucleus is dependent on activation of the nonre- TCR signaling (16–20). An autosomal recessive form of SCID in ceptor cytoplasmic protein tyrosine kinases (PTKs),3 Lck and humans has been described due to mutations within the kinase ZAP-70 (reviewed in Refs. 1–3). PTK activation is a rapid and domain of ZAP-70 that abolish protein expression (16–18). In hu- critical event that results in phosphorylation of numerous down- mans, ZAP-70 deficiency is characterized by absent CD8ϩ T cells stream molecules, including phospholipase C␥1 (PLC␥1), linker and normal numbers of nonfunctional CD4ϩ T cells in the periph- of activated T cells (LAT), Src homology 2 domain-containing eral blood. This unusual phenotype suggests that ZAP-70 is critical by guest on September 29, 2021 76-kDa leukocyte protein (SLP-76), and Vav (reviewed in Refs. for CD8ϩ T cell development but may be dispensable for selection 2–7). These tyrosine phosphorylation reactions are required for of CD4ϩ T lymphocytes in the thymus. The presence of peripheral 2ϩ ϩ mobilization of intracellular free calcium ([Ca ]i) and activation CD4 T cells in ZAP-70-deficient patients contrasts with their of the Ras/mitogen-activated protein kinase (MAPK) and phos- absence in ZAP-70 knockout mice, which are blocked at the phatidylinositol 3-kinase pathways, and culminate in T cell acti- CD4ϩCD8ϩ double-positive (DP) stage of thymocyte differentia- vation and initiation of T cell-specific responses (reviewed in Refs. tion (20). Recently, a point mutation within the highly conserved 1 and 3–6). DLAARN motif in the zap70 gene that results in the amino acid One PTK critical for induction of Ag-specific T cell responses is conversion R464C was described in inbred Strange (ST) mice (21). ZAP-70. Recruitment of ZAP-70 to the TCR and its subsequent Although R464C affects ZAP-70 expression, its principal effect is phosphorylation and activation, largely by Lck, is essential for all to abrogate ZAP-70 catalytic activity (21). Like zap70 gene-tar- downstream signaling events (8–11). Activated ZAP-70 phosphor- geted animals, the affected ST mice are devoid of mature T cells. In this report, we describe an infant with SCID who is homozy- gous for the comparable DLAARN mutation (R465C) but who has Departments of *Pediatrics, †Microbiology and Immunology, and ‡Medicine, and normal numbers of circulating CD4ϩ T cells. The biochemical §Howard Hughes Medical Institute, University of California, San Francisco, CA 94143; and ¶Department of Pediatrics, University of Florida, Gainesville, FL 32610 consequences of this ZAP-70 mutation on T cell activation are examined, and the role of Syk expression in survival of human Received for publication July 17, 2000. Accepted for publication September 27, 2000. ZAP-70-deficient T cells in the periphery is assessed. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Materials and Methods 1 This work was supported in part by National Institutes of Health Grant M01 Patient RR01271, UCSF Pediatric Clinical Research Center, and National Institutes of Health Grants 1 P01 AI45865-01 and 5 P60 AR20684. The patient was a 10-mo-old Caucasian male, the only child of second- 2 Address correspondence and reprint requests to Dr. Melissa E. Elder, Box 0105, degree cousins, who presented for immunologic evaluation after develop- University of California, San Francisco, CA 94143. E-mail address: elderm@peds. ing Pneumocystis carinii pneumonia at the age of 7 mo. Serum IgG was 63 ucsf.edu mg/dl (normal range, 250–690 mg/dl) with normal IgM and IgA for age. Initial blood count revealed 10,600 leukocytes/mm3 and 80% lymphocytes. 3 Abbreviations used in this paper: PTK, protein tyrosine kinase; PLC␥1, phospho- lipase C␥1; LAT, linker of activated T cells; SLP-76, Src homology 2 domain-con- In vitro lymphocyte-proliferative responses were absent to PHA, PWM, 2ϩ and Con A. At 1 year of age, the patient received a T cell-depleted bone taining 76-kDa leukocyte protein; [Ca ]i, intracellular free calcium; MAPK, mito- gen-activated protein kinase; DP, double-positive; ST mice, Strange mice; TCL , T marrow transplant from his mother. He later developed a non-EBV-asso- cell line. ciated large B cell lymphoma that responded to treatment with combination

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 657 chemotherapy. Subsequently, the patient underwent successful retransplan- an excitation wavelength of 355 nm and emission wavelengths of 400 and tation with mobilized peripheral blood stem cells from his father. 500 nm. To ensure loading of Indo-1, 1 mM ionomycin was added to the cell suspensions. T cell culture and in vitro proliferative assays DNA isolation, PCR amplification, and sequencing Because the availability of the patient’s PBMC for study was limited, many experiments were performed with patient and normal T cell lines (TCL). Genomic DNA was isolated from PBMC using standard SDS/proteinase K For production of TCL, PBMC were grown for 2–4 wk in RPMI-C media digestion and phenol/chloroform extraction. Total RNA was isolated from (RPMI plus 20% human serum; NABI, Boca Raton, FL) with 0.5 ng/ml PBMC using TRIzol reagent (Life Technologies, Grand Island, NY). PMA (Sigma, St. Louis, MO), 1 ␮M ionomycin (Calbiochem, La Jolla, cDNA was synthesized from total RNA using oligo(dT) or random prim- CA), and 20 IU/ml IL-2 (Boehringer Mannheim, Indianapolis, IN). Immu- ers. RT-PCR was used to amplify ZAP-70 cDNAs. PCR primers used to ϩ nofluorescence analysis confirmed that the TCL were CD3 . For some generate nearly full length cDNAs (bp 33–1996; stop codon at bp 2067) ϩ experiments, CD8 T cells were depleted from normal TCL using mag- were 5Ј-GGAGCTCAGCAGACACCAG-3Ј (primer A) and 5Ј-GTTACTA netic beads (Dynal Biotech, Lake Success, NY). In vitro lymphocyte pro- CAGCCTGGCCAGCAA-3Ј (primer B), respectively. Nucleotides 1554– liferative assays were done as previously described (22). 2178 of ZAP-70 were amplified separately using primers C (5Ј-GGGAT GAAGTACCTGGAGGAGAAG-3Ј)andD(5Ј-GTTGTCTCCACACA Immunofluorescence analyses CAGCTG-3Ј). ZAP-70 sequence from nucleotides 1554–1877 was Cell surface expression of CD3, CD4, CD8, and CD69 was determined amplified from genomic DNA using primers C and E (5Ј-GCCTTCATC using a FACScalibur (Becton Dickinson, San Jose, CA). Leu-4 (anti-CD3)- GAGCAGGGCAAG-3Ј). PCR-amplified uncloned cDNA and genomic FITC, Leu-4-PerCP, Leu-2a (anti-CD8)-FITC, Leu-3 (anti-CD4)-FITC, DNA products or cDNA and genomic DNA clones isolated from indepen- Leu-3-PE, and anti-CD69-PE were obtained from Becton Dickinson, and dent RT-PCR were sequenced manually using either the T7 Sequenase anti-CD3-tricolor (TC) and anti-CD8-TC from Caltag (Burlingame, CA). version 2.0 PCR product or the T7 Sequenase version 2.0 DNA sequencing kits (Amersham Pharmacia Biotech), respectively. CD3/4/8 expression on lymph node cells was determined in the University Downloaded from of Florida Clinical Laboratory (Gainesville, FL). CD69 expression was analyzed on resting PBMC and after incubation with PHA (Sigma) for 4 h Results at 37°C. Intracellular Syk expression was determined as described (23). Absent peripheral CD8ϩ T cells and defective CD4ϩ T cell Briefly, 0.25–2 ϫ 106 PBMC were stained with Leu-3-FITC, washed, and fixed in PBS plus 4% paraformaldehyde. After incubation with 2.4G2 (Fc- proliferation to TCR-mediated stimuli blocking mAb), PBMC were permeabilized in PBS plus 1% BSA and 0.1% Flow cytometric analysis of the patient’s PBMC demonstrated de- saponin and stained with FITC-conjugated 4D10.1 (anti-Syk mAb). Ͼ creased percentages (38–42%), but normal numbers ( 3500 cells/ http://www.jimmunol.org/ ϩ Immunoblotting assays ␮l), of circulating CD3 T cells (data not shown). The percentage of peripheral blood CD4ϩ T lymphocytes was mildly decreased for For determination of tyrosine phosphoproteins, 2 ϫ 106 TCL or CD8- age (37–40%), but the absolute number (Ͼ3000 cells/␮l) was nor- depleted TCL were incubated for 20 min on ice with Leu-3-biotin plus ϩ Leu-4-biotin or PBS alone, washed, and cross-linked with avidin for 4 min mal. In contrast, the patient had few CD8 T cells in either his at 37°C. Alternatively, 2 ϫ 106 TCL or CD8-depleted TCL were incubated blood (Ͻ170 cells/␮l) or a femoral lymph node (Fig. 1). The ab- for 4 min with 235 (anti-CD3 IgM mAb; a gift of S. M. Fu, University of sence of CD8ϩ T lymphocytes in the peripheral circulation sug- Virginia, Charlottesville, VA). Cells were lysed in Nonidet P-40 buffer (1% Nonidet P-40 plus 10 mM Tris (pH 7.6), 150 mM NaCl, 0.5 mM EDTA, gested an abnormality of T cell development and, specifically, in 10 mM NaF, 1 mM PMSF, 1 ␮g/ml pepstatin A, 10 ␮g/ml leupeptin, 1 thymic selection. CD8 deficiency in humans is commonly associ-

␮ by guest on September 29, 2021 g/ml aprotinin, and 1 mM Na3VO4) for 30 min at 4°C. Whole cell lysates ated with defective ZAP-70 expression (16–19). A diagnosis of a were resolved by SDS-PAGE, transferred to Immobilon-P (Millipore, Bed- T cell signal transduction abnormality such as ZAP-70 deficiency ford, MA), blocked, and then probed with 4G10 (anti-phosphotyrosine was suggested by findings that the patient’s T cells were refractory mAb; Upstate Biotechnology, Lake Placid, NY). After incubation with HRP-conjugated goat anti-mouse mAb (Southern Biotechnology Associ- to anti-CD3 stimulation in vitro (425 cpm (patient) vs 92,993 cpm ates, Birmingham, AL), phosphoproteins were assayed using an ECL de- (control)) but responded normally to PMA plus ionomycin (38,713 tection system (Amersham Pharmacia Biotech, Piscataway, NJ). For anal- ysis of other proteins, 4G10 blots were stripped and probed with the following Abs: 2F3.2 (anti-ZAP-70); 4D10.1, 1F6 (anti-Lck); anti-Fyn (a gift from A. Veilette, McGill University, Montreal, Canada); 6B10.2 (anti- ␨); anti-SLP-76 (a gift from G. Koretzky, University of Pennsylvania, Phil- adelphia, PA); anti-LAT (a gift from L. Samelson, National Institutes of Health, Bethesda, MD); and anti-phospho-MAPK (New England Biolabs, Beverly, MA). Protein blots of ZAP-70 were also done using PBMC extracts. Immunoprecipitation and in vitro kinase assays Precleared extracts of 2.5 ϫ 107 TCL were incubated with anti-PLC␥1 mAb (Upstate Biotechnology)-conjugated protein G-Sepharose beads (Amersham Pharmacia Biotech) for1hat4°C. Immunoprecipitates were fractionated by SDS-PAGE and incubated first with either 4G10 or anti- PLC␥1 mAb and then HRP-conjugated goat anti-mouse mAb before ECL. For in vitro kinase assays, precleared extracts from 2.5 ϫ 107 TCL were immunoprecipitated with rabbit anti-human ZAP-70-coated protein A-Sepharose beads, washed in kinase buffer (10 mM Tris (pH 7.4), with 10 ␮ ␥ 32 mM MnCl2), and incubated with 10 Ci [ - P]ATP (3000 Ci/mmol; NEN Life Science Products, Boston, MA) and 3.5 ␮g GST-band III for 10 min at 25°C. Membranes were treated with 1 M KOH before autoradiography or immunoblotting, as described (24).

2ϩ [Ca ]i mobilization 7 TCL at 2 ϫ 10 cells/ml rested overnight in RPMI-C were loaded with 3 ϩ ␮M Indo-1 (Molecular Probes, Eugene, OR) for 20 min at 37°C, washed, FIGURE 1. Lack of peripheral CD8 T cells in a patient with SCID. ϫ 6 PBMC and isolated lymph node cells obtained from the patient and normal and resuspended at 5 10 cells/ml in HBSS plus 1% BSA, 1 mM CaCl2, and 0.5 mM MgCl , as described (17, 19). Cells were stimulated with 235 individuals were stained with Leu-3 (CD4)-PE and Leu-2a (CD8)-FITC, 2 ϩ ϩ mAb or Leu-4-biotin plus avidin. Fluorescence measurements were per- and analyzed by flow cytometry. Percentages of CD4 and CD8 T cells formed using a Hitachi 4500 spectrophotometer (Hitachi, San Jose, CA) at are indicated in italics. 658 IDENTICAL MUTATIONS IN ZAP-70 IN HUMANS AND MICE WITH SCID cpm (patient) vs 26,342 cpm (control)), agents that bypass proxi- gested a defect in ZAP-70, even though considerable protein was mal TCR signaling events by mimicking activation of Ras and demonstrable in his T cells. To substantiate our hypothesis that the 2ϩ [Ca ]i flux. patient had a variant of ZAP-70 deficiency, sequencing of the cod- ing portion of his zap70 gene was performed. PCR amplification of Deficient tyrosine phosphorylation is not due to absent ZAP-70 ZAP-70 cDNA products was done, and 30 independent clones, as protein in the patient’s T cells well as uncloned cDNA, were analyzed. The patient was homozy- To confirm that the patient had a functional defect in coupling of gous for a single nucleotide change (C to T) at bp 1602, which the TCR to distal signaling pathways, we analyzed whether his results in the amino acid conversion R465C (Fig. 3). The presence TCL was capable of triggering cytoplasmic PTK reactions on TCR of two mutant zap70 alleles was confirmed in the patient’s genome engagement. Ab-mediated cross-linking of CD3 or both CD3 and by sequencing both cloned and uncloned chromosomal DNA prod- CD4 resulted in a markedly abnormal protein tyrosine phosphor- ucts from the patient. Characterization of the informative region of ylation pattern in comparison with a normal TCL (Fig. 2A)or the zap70 gene from both parents demonstrated that they were CD8-depleted TCL (data not shown). Although phosphorylation of heterozygous for the same mutation (data not shown). A minority Lck was similar to that observed in a normal TCL, phosphorylation of cloned cDNA products from the patient and both of his parents of SLP-76, LAT, Erk1/2, PLC␥1, and ␨ was significantly de- also contained an 18-bp in-frame deletion from nucleotides 1101– creased in the patient’s T cells. However, phosphorylation of a 1116 that encode a portion of interdomain B of the ZAP-70 pro- 70-kDa protein that appeared to be ZAP-70 was detected, albeit at tein. This form was also found in one of the two original ZAP-70 a lower level than that in normal lymphocytes. Immunoblot anal- cDNA clones (25) and does not include residues critical for bind- ysis confirmed the presence of ZAP-70 protein in the patient’s ing of Src homology 2-containing proteins (7, 24). This likely Downloaded from TCL, at levels only modestly less than that in a normal TCL (Fig. represents an alternatively spliced ZAP-70 product. 2B). Similar results were obtained using PBMC or CD8-depleted TCL (data not shown). Detection of considerable ZAP-70 protein R465C renders ZAP-70 catalytically inactive in our patient’s T cells contrasts with previous published findings The R465C mutation eliminates an invariant residue within a in ZAP-70-deficient children, all of whom lacked ZAP-70 protein highly conserved motif (DLAARN) that is essential for PTK en- by immunoblot (16–19). Expression of other signaling molecules zymatic function (26). Although R465C is a novel mutation in http://www.jimmunol.org/ was unaffected. human ZAP-70 deficiency, the comparable lesion (R464C) was described recently as a de novo mutation in ST mice (21). Analysis A novel DLAARN motif mutation is identified in the patient’s of TCR signaling in ST thymocytes demonstrated that R464C ab- zap70 gene rogated ZAP-70 kinase activity but did not eliminate protein ex- The patient’s lymphocyte phenotype, pattern of in vitro prolifera- pression or tyrosine phosphorylation (21). To determine whether tion, and phosphotyrosine status following TCR ligation all sug- the patient’s homozygous R465C mutation had similar effects on ZAP-70 function, we analyzed the enzymatic activity of his mutant ZAP-70 protein in an in vitro kinase assay using the exogenous substrate, GST-band III (24). In contrast to findings in a normal by guest on September 29, 2021

FIGURE 2. Defective induction of PTK-mediated events in the pa- FIGURE 3. Defective TCR signaling in the patient’s cells is due to a tient’s T cells. A, Tyrosine phosphoprotein cascade. Patient (Pt) and normal homozygous missense mutation in ZAP-70 mRNA. Sequence of uncloned (Nl) TCL were incubated with 235 mAb (␣CD3) or Leu-4-biotin plus ZAP-70 cDNA from the patient (right) compared with normal ZAP-70 Leu-3-biotin (␣CD3/␣CD4) and avidin. Unstimulated (Ϫ) and stimulated cDNA (left).ACtoTchange at bp 1602 results in the amino acid (ϩ) lysates were analyzed by 4G10 (anti-phosphotyrosine mAb) immuno- conversion R465C within the DLAARN motif of the ZAP-70 kinase do- blot. Ordinate, Positions of mol wt (MW) standards in kilodaltons. B, Ex- main. The mutation was demonstrable in independent cDNA clones and pression of T cell-signaling molecules. 4G10 blots were stripped and re- was confirmed to be homozygous in the patient’s uncloned and cloned probed with Abs specific to ZAP-70, SLP-76, Syk, Fyn, Lck, LAT, or ␨. genomic DNA. The Journal of Immunology 659

TCL, ZAP-70 protein from the patient’s TCL could not phosphor- (27). Despite increased Syk expression and rescue of SLP-76/ ␥ 2ϩ ylate GST-band III or undergo autophosphorylation after TCR li- LAT/PLC 1 phosphorylation and [Ca ]i mobilization after long gation (Fig. 4). This finding suggested that, like the comparable term culture, induction of downstream signaling events, including R464C mutation in ST mice, the principal effect of the R465C Ras/MAPK activation and CD3-mediated IL-2 production, re- mutation in humans is to abolish ZAP-70 kinase function. mained markedly defective in TCL from those patients (27). We subsequently confirmed that the mutant ZAP-70 was cata- lytically inactive by evaluating ZAP-70-dependent phosphoryla- Discussion tion events in the patient’s T lymphocytes. In contrast to a TCR- Novel human ZAP-70 mutation stimulated normal TCL, tyrosine phosphorylation of PLC␥1 and the MAPKs, Erk1/2, was significantly decreased in the patient’s We describe an infant with SCID due to a novel mutation in the TCL (Fig. 5A). Consistent with a defect in PLC␥1 phosphoryla- zap70 gene that abrogates enzymatic function without appreciably 2ϩ affecting either expression or phosphorylation of ZAP-70. With the tion, no increase in [Ca ]i was observed in the patient’s TCL after incubation with two different CD3 mAbs (Fig. 5B). Moreover, the exception of one child who lacked ZAP-70 mRNA by Northern patient’s T cells did not inducibly express the activation marker, blot in whom the underlying mutation is not known (19) and an- CD69, after TCR stimulation by PHA (Fig. 5C). CD69 expression other child with temperature-sensitive P80Q and M572L mutations is dependent on activation of the Ras/MAPK pathway. These re- (28), ZAP-70-deficient patients have inherited mutations within a sults demonstrate that, despite its expression in the patient’s T small region of the ZAP-70 kinase domain that significantly affect lymphocytes, the mutant ZAP-70 could not phosphorylate down- both protein stability and catalytic activity. These included a 13-bp stream substrates required for activation of the calcium and Ras/ deletion resulting in a frame-shift after residue 503 and premature Downloaded from MAPK pathways. termination at amino acid 538 (16), two amino acid conversions, S518R (17) and A507V (27), and a splicing error that inserts LEQ Survival of the patient’s T cells in the circulation is not due to into the protein after residue 541 (17, 18). up-regulation of Syk expression Role of Syk in CD4ϩ thymocyte selection in human ZAP-70 Findings in previous ZAP-70-deficient patients have led to the sug- deficiency gestion that Syk may compensate for loss of ZAP-70 in the thy- http://www.jimmunol.org/ ϩ mus, thereby rescuing development of some CD4ϩ T cells (23). To The development of peripheral CD4 T cells in ZAP-70-deficient determine whether Syk plays a role in the survival of ZAP-70- patients, which is not seen in mice deficient in ZAP-70, has been deficient T cells in the peripheral circulation, we analyzed intra- attributed in part to the nature of the underlying mutations that cellular Syk expression in PBMC at the single-cell level. Immu- conceivably may allow for some residual ZAP-70 activity in vivo. nofluorescence analysis revealed that ϳ5% of the patient’s However, our patient provides the first description of identical mu- peripheral CD4ϩ T cells had detectable Syk expression, in com- tations in zap70 having disparate effects on T cell development in parison with ϳ2% of normal CD4ϩ T cells (Fig. 6). The level of humans vs mice. To explain these findings, we suggest instead that Syk observed in this CD4ϩ T cell subpopulation was approxi- the ability of Syk to contribute to pre-TCR and TCR signaling is mately one-tenth that demonstrated in human B cells. The percent- substantially different in humans as compared with mice. Our con- by guest on September 29, 2021 age of CD4ϩ Sykintermedate T cells increased to 13% in the pa- clusion is supported by recent observations that Syk is expressed at tient’s TCL but remained unchanged in the normal TCL (data not highest levels in human and murine thymocytes during stages of shown). However, despite increased Syk expression, the patient’s development in which pre-TCR signaling is required (23). In con- cultured CD4ϩ T cells were unresponsive to TCR-mediated stim- trast to mice, which down-regulate Syk expression to peripheral T Ϫ ϩ uli. Although not identical, similar findings were reported recently cell levels at the CD44 CD25 checkpoint, human DP thymo- in cultured T cells from two siblings homozygous for the mutation cytes express significant levels of Syk and do not completely A507V, which abolishes both ZAP-70 expression and function down-regulate this PTK to levels seen in peripheral T lymphocytes until after positive selection has commenced (23). Consistent with these observations, TCR-stimulated HTLV-1-transformed DP thy- mocytes, but not peripheral T cells, from a ZAP-70-deficient pa- 2ϩ tient were capable of mobilizing [Ca ]i and phosphorylating Syk (19). In contrast, DP thymocytes from ZAP-70-deficient mice are unable to initiate TCR signaling events (20, 21). As a consequence of its relative abundance in human vs murine DP thymocytes, Syk likely is able to partially compensate for loss of ZAP-70 during positive selection only in ZAP-70-deficient patients. Although ZAP-70-deficient T cells are polyclonal by Southern analysis of TCR-␤ usage (16), their TCR repertoires have not been character- ized, including any bias toward auto- vs allo-reactivity. Findings in our patient support the notion that the fundamental role of ZAP-70 in TCR signaling is phosphorylation of down- stream substrates, including PLC␥1, SLP-76, and LAT. Although ZAP-70 is most important for initiation of downstream signaling events in peripheral T cells, findings in ZAP-70-deficient patients suggest that Syk, at levels detectable in the thymus, is capable of FIGURE 4. R465C abrogates ZAP-70 catalytic activity. Whole cell ly- sates of patient (Pt) and normal (Nl) TCL stimulated with 235 (anti-CD3 transducing signals from both the pre-TCR and TCR. However, IgM mAb) were immunoprecipitated with rabbit anti-human ZAP-70- under normal circumstances, Syk appears to play a role for the coated beads and incubated with GST-band III and [␥-32P]ATP before most part in early ␣␤ T cell differentiation, whereas ZAP-70 func- autoradiography in an in vitro kinase assay. Blots were stripped and re- tion is critical for positive and negative selection in the thymus and probed with 2F3.2 (anti-ZAP-70 mAb) or anti-GST Ab. T cell activation in the periphery. Our results differ from a recent 660 IDENTICAL MUTATIONS IN ZAP-70 IN HUMANS AND MICE WITH SCID

motif binding, thus inhibiting Syk phosphorylation of downstream effectors critical to activation of the calcium and Ras/MAPK pathways. Findings in ST and ZAP-70-knockout thymocytes suggest that another important function of ZAP-70 is to promote immunore- ceptor tyrosine-based activation motif phosphorylation of TCR subunits in DP cells limited in their expression of Lck (29). Al- though not equivalent cell types, reduced ␨ phosphorylation was also observed in our patient’s TCL, despite the presence of con- siderable Lck and mutant ZAP-70 protein. In summary, we suggest that human and murine DP thymocytes have different dependencies on ZAP-70 function during develop- FIGURE 6. Immunofluorescence analysis of intracellular Syk expres- ment, based on the T cell phenotypes of comparable ZAP-70 mu- sion. Patient and normal PBMC were stained with either anti-CD19 (B cell) tations. Despite similar effects on TCR signaling, a patient with mAb or Leu-3 (anti-CD4)-PE plus anti-CD3-tricolor, permeabilized, and a catalytically inactive ZAP-70 protein is able to produce large stained with 4D10.1-FITC (anti-Syk); 4D10.2-FITC plus competitor pep- numbers of nonfunctional peripheral CD4ϩ T cells. The elevated tide (anti-Syk ϩ pep); or an isotype-matched staining control (control Ig), before analysis by flow cytometry. Percentages of gated CD3ϩCD4ϩSykϩ levels of Syk in human DP thymocytes, in contrast to those in cells are indicated in italics. mice, may explain the differential consequences of identical

ZAP-70 mutations. Downloaded from observation that a subset of cultured primary T cells from SCID patients with a ZAP-70 null mutation are capable of proximal TCR Syk expression in ZAP-70-deficient peripheral T cells signaling as a result of markedly increased Syk levels (27). We Recent studies in mice demonstrate that survival of circulating T suggest that, despite increased Syk expression in a subset of our cells is diminished in the absence of TCR ligands (30–32), sug-

patient’s TCL, the catalytically inactive ZAP-70 mutant is able to gesting that TCR-mediated signals are required for maintenance of http://www.jimmunol.org/ compete favorably for immunoreceptor tyrosine-based activation the peripheral T cell pool. However, survival of CD4ϩ T cells in

FIGURE 5. Distal signaling path- by guest on September 29, 2021 ways are poorly activated in the pa- tient’s T cells. A, Phosphorylation of ZAP-70 substrates. Patient (Pt) and normal (Nl) whole cell lysates were immunoprecipitated (IP) with anti- PLC␥1 mAb-coated beads. Immuno- blots were probed with 4G10 (anti- phosphotyrosine) or anti-PLC␥1 mAb. Stripped whole cell lysate 4G10 blots were reprobed with anti- phospho-MAPK mAb to detect phos- pho-Erk1/2. B, Mobilization of 2ϩ [Ca ]i after TCR stimulation. Indo- 1-loaded TCL were stimulated with 235mAb(anti-CD3IgM)orLeu4(anti- CD3)-biotin plus avidin (Av). Iono- mycin (Iono) was added to ensure that the cells were loaded properly 2ϩ with Indo-1. Changes in [Ca ]i in nanomols over time (s) were deter- mined by fluorometry. C, CD69 ex- pression by immunofluorescence. PBMC were stained with anti- CD69-PE and Leu-4-PerCP before and after incubation with PHA. Per- centages of CD3ϩCD69ϩ cells are indicated in italics. The Journal of Immunology 661 the blood of ZAP-70-deficient patients would not appear to depend 15. Finco, T. S., T. Kadlecek, W. Zhang, L. E. Samelson, and A. Weiss. 1998. LAT ␥ on ZAP-70-dependent TCR signaling or to up-regulation of Syk is required for TCR-mediated activation of PLC 1 and the Ras pathway. Immu- nity 9:617. intermediate expression. It is doubtful that the presence of a Syk sub- 16. Elder, M. E., D. Lin, J. Clever, A. C. Chan, T. J. Hope, A. Weiss, and T. Parslow. set can account entirely for survival of the patient’s considerable 1994. Human severe combined immunodeficiency due to a defect in ZAP-70, a number of circulating CD4ϩ T cells. Alternatively, the level of Syk T cell tyrosine kinase. Science 264:1596. 17. Chan, A. C., T. A. Kadlecek, M. E. Elder, A. H. Filipovich, W.-L. Kuo, activity required for peripheral T cell survival may be less than that M. Iwashima, T. G. Parslow, and A. Weiss. 1994. ZAP-70 deficiency in an required for propagation of TCR signals. Further investigation is autosomal recessive form of severe combined immunodeficiency. Science 264: required to determine whether viability of ZAP-70-deficient T 1599. 18. Arpaia, E., M. Shahar, H. Dadi, A. Cohen, and C. M. Roifman. 1994. Defective cells is dependent on residual Syk function and whether these ϩ ϩ T cell receptor signaling and CD8 thymocyte selection in humans lacking CD4 T cells have a normal life span in the peripheral circulation. ZAP-70 kinase. Cell 76:947. 19. Gelfand, E. W., K. Weinberg, B. D. Mazer, T. A. Kadlecek, and A. Weiss. 1995. Absence of ZAP-70 prevents signaling through the antigen receptor on peripheral Acknowledgments blood T cells but not on thymocytes. J. Exp. Med. 182:1057. We thank Carmen White and Aniceto Gonzaga for technical assistance. 20. Negishi, I., N. Motoyama, K-I. Nakayama, K. Nakayama, S. Senju, S. Hatakeyama, Q. Zhang, A. C. Chan, and D. Y. Loh. 1995. Essential role for References ZAP-70 in both positive and negative selection of thymocytes. Nature 376:435. 1. Weiss, A., and D. R. Littman. 1994. Signal transduction by lymphocyte antigen 21. Wiest, D. L., J. M. Ashe, T. K. Howcroft, H-M. Lee, D. M. Kemper, I. Negishi, receptors. Cell 76:263. D. S. Singer, A. Singer, and R. Abe. 1997. A spontaneously arising mutation in 2. Wange, R. L., and L. E. Samelson. 1996. Complex complexes: signaling at the the DLAARN motif of murine ZAP-70 abrogates kinase activity and arrests TCR. Immunity 5:197. thymocyte development. Immunity 6:663.

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