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,,¶
Departments of
*
Pediatrics,
Microbiology and Immunology, and
Medicine, and
Howard Hughes Medical Institute, University of California, San Francisco, CA 94143; and
¶ Department of Pediatrics, University of Florida, Gainesville, FL 32610
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 (PLC1), linker
of activated T cells (LAT), Src homology 2 domain-containing 76-kDa
leukocyte protein (SLP-76), and Vav (reviewed in Refs.
2, 3, 4, 5, 6, 7). These tyrosine phosphorylation reactions are
required for mobilization of intracellular free calcium
([Ca2+]i) and activation
of the Ras/mitogen-activated protein kinase (MAPK) and
phosphatidylinositol 3-kinase pathways, and culminate in T cell
activation and initiation of T cell-specific responses (reviewed in
Refs. 1 and 3, 4, 5, 6). One PTK critical for induction of Ag-specific T cell responses is ZAP-70. Recruitment of ZAP-70 to the TCR and its subsequent phosphorylation and activation, largely by Lck, is essential for all downstream signaling events (8, 9, 10, 11). Activated ZAP-70 phosphorylates SLP-76 and LAT, triggering distal pathways that bring about T cell activation (12, 13, 14, 15). Studies in humans and gene-targeted mouse models have confirmed the importance of ZAP-70 to TCR signaling (16, 17, 18, 19, 20). An autosomal recessive form of SCID in humans has been described due to mutations within the kinase domain of ZAP-70 that abolish protein expression (16, 17, 18). In humans, ZAP-70 deficiency is characterized by absent CD8+ T cells and normal numbers of nonfunctional CD4+ T cells in the peripheral blood. This unusual phenotype suggests that ZAP-70 is critical for CD8+ T cell development but may be dispensable for selection of CD4+ T lymphocytes in the thymus. The presence of peripheral CD4+ T cells in ZAP-70-deficient patients contrasts with their absence in ZAP-70 knockout mice, which are blocked at the CD4+CD8+ double-positive (DP) stage of thymocyte differentiation (20). Recently, a point mutation within the highly conserved DLAARN motif in the zap70 gene that results in the amino acid conversion R464C was described in inbred Strange (ST) mice (21). Although R464C affects ZAP-70 expression, its principal effect is to abrogate ZAP-70 catalytic activity (21). Like zap70 gene-targeted animals, the affected ST mice are devoid of mature T cells.
In this report, we describe an infant with SCID who is homozygous for the comparable DLAARN mutation (R465C) but who has normal numbers of circulating CD4+ T cells. The biochemical consequences of this ZAP-70 mutation on T cell activation are examined, and the role of Syk expression in survival of human ZAP-70-deficient T cells in the periphery is assessed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The patient was a 10-mo-old Caucasian male, the only child of second-degree cousins, who presented for immunologic evaluation after developing Pneumocystis carinii pneumonia at the age of 7 mo. Serum IgG was 63 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. In vitro lymphocyte-proliferative responses were absent to PHA, PWM, and Con A. At 1 year of age, the patient received a T cell-depleted bone marrow transplant from his mother. He later developed a non-EBV-associated large B cell lymphoma that responded to treatment with combination chemotherapy. Subsequently, the patient underwent successful retransplantation with mobilized peripheral blood stem cells from his father.
T cell culture and in vitro proliferative assays
Because the availability of the patient’s PBMC for study was limited, many experiments were performed with patient and normal T cell lines (TCL). For production of TCL, PBMC were grown for 2–4 wk in RPMI-C media (RPMI plus 20% human serum; NABI, Boca Raton, FL) with 0.5 ng/ml PMA (Sigma, St. Louis, MO), 1 µM ionomycin (Calbiochem, La Jolla, CA), and 20 IU/ml IL-2 (Boehringer Mannheim, Indianapolis, IN). Immunofluorescence analysis confirmed that the TCL were CD3+. For some experiments, CD8+ T cells were depleted from normal TCL using magnetic beads (Dynal Biotech, Lake Success, NY). In vitro lymphocyte proliferative assays were done as previously described (22).
Immunofluorescence analyses
Cell surface expression of CD3, CD4, CD8, and CD69 was determined using a FACScalibur (Becton Dickinson, San Jose, CA). Leu-4 (anti-CD3)-FITC, Leu-4-PerCP, Leu-2a (anti-CD8)-FITC, Leu-3 (anti-CD4)-FITC, Leu-3-PE, and anti-CD69-PE were obtained from Becton Dickinson, and anti-CD3-tricolor (TC) and anti-CD8-TC from Caltag (Burlingame, CA). CD3/4/8 expression on lymph node cells was determined in the University of Florida Clinical Laboratory (Gainesville, FL). CD69 expression was analyzed on resting PBMC and after incubation with PHA (Sigma) for 4 h at 37°C. Intracellular Syk expression was determined as described (23). Briefly, 0.25–2 x 106 PBMC were stained with Leu-3-FITC, washed, and fixed in PBS plus 4% paraformaldehyde. After incubation with 2.4G2 (Fc-blocking mAb), PBMC were permeabilized in PBS plus 1% BSA and 0.1% saponin and stained with FITC-conjugated 4D10.1 (anti-Syk mAb).
Immunoblotting assays
For determination of tyrosine phosphoproteins, 2 x
106 TCL or CD8-depleted TCL were incubated for 20
min on ice with Leu-3-biotin plus Leu-4-biotin or PBS alone, washed,
and cross-linked with avidin for 4 min at 37°C. Alternatively, 2
x 106 TCL or CD8-depleted TCL were incubated for
4 min with 235 (anti-CD3 IgM mAb; a gift of S. M. Fu,
University of 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, 10 mM NaF, 1 mM PMSF, 1 µg/ml pepstatin A, 10
µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM
Na3VO4) for 30 min at
4°C. Whole cell lysates were resolved by SDS-PAGE, transferred to
Immobilon-P (Millipore, Bedford, MA), blocked, and then probed with
4G10 (anti-phosphotyrosine mAb; Upstate Biotechnology, Lake Placid,
NY). After incubation with HRP-conjugated goat anti-mouse mAb
(Southern Biotechnology Associates, Birmingham, AL), phosphoproteins
were assayed using an ECL detection system (Amersham Pharmacia Biotech,
Piscataway, NJ). For analysis 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,
Philadelphia, 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 x 107 TCL
were incubated with anti-PLC1 mAb (Upstate
Biotechnology)-conjugated protein G-Sepharose beads (Amersham Pharmacia
Biotech) for 1 h at 4°C. Immunoprecipitates were fractionated by
SDS-PAGE and incubated first with either 4G10 or anti-PLC1 mAb
and then HRP-conjugated goat anti-mouse mAb before ECL.
For in vitro kinase assays, precleared extracts from 2.5 x
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 mM
MnCl2), and incubated with 10 µCi
[-32P]ATP (3000 Ci/mmol; NEN Life Science
Products, Boston, MA) and 3.5 µg GST-band III for 10 min at 25°C.
Membranes were treated with 1 M KOH before autoradiography or
immunoblotting, as described (24).
[Ca2+]i mobilization
TCL at 2 x 107 cells/ml rested overnight in RPMI-C were loaded with 3 µM Indo-1 (Molecular Probes, Eugene, OR) for 20 min at 37°C, washed, and resuspended at 5 x 106 cells/ml in HBSS plus 1% BSA, 1 mM CaCl2, and 0.5 mM MgCl2, as described (17, 19). Cells were stimulated with 235 mAb or Leu-4-biotin plus avidin. Fluorescence measurements were performed using a Hitachi 4500 spectrophotometer (Hitachi, San Jose, CA) at an excitation wavelength of 355 nm and emission wavelengths of 400 and 500 nm. To ensure loading of Indo-1, 1 mM ionomycin was added to the cell suspensions.
DNA isolation, PCR amplification, and sequencing
Genomic DNA was isolated from PBMC using standard SDS/proteinase K digestion and phenol/chloroform extraction. Total RNA was isolated from PBMC using TRIzol reagent (Life Technologies, Grand Island, NY). cDNA was synthesized from total RNA using oligo(dT) or random primers. RT-PCR was used to amplify ZAP-70 cDNAs. PCR primers used to generate nearly full length cDNAs (bp 33–1996; stop codon at bp 2067) were 5'-GGAGCTCAGCAGACACCAG-3' (primer A) and 5'-GTTACTACAGCCTGGCCAGCAA-3' (primer B), respectively. Nucleotides 1554–2178 of ZAP-70 were amplified separately using primers C (5'-GGGATGAAGTACCTGGAGGAGAAG-3') and D (5'-GTTGTCTCCACACACAGCTG-3'). ZAP-70 sequence from nucleotides 1554–1877 was amplified from genomic DNA using primers C and E (5'-GCCTTCATCGAGCAGGGCAAG-3'). PCR-amplified uncloned cDNA and genomic DNA products or cDNA and genomic DNA clones isolated from independent RT-PCR were sequenced manually using either the T7 Sequenase version 2.0 PCR product or the T7 Sequenase version 2.0 DNA sequencing kits (Amersham Pharmacia Biotech), respectively.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Flow cytometric analysis of the patient’s PBMC demonstrated
decreased percentages (38–42%), but normal numbers (>3500
cells/µl), of circulating CD3+ T cells (data
not shown). The percentage of peripheral blood
CD4+ T lymphocytes was mildly decreased for age
(37–40%), but the absolute number (>3000 cells/µl) was normal. In
contrast, the patient had few CD8+ T cells in
either his blood (<170 cells/µl) or a femoral lymph node (Fig. 1
). The absence of
CD8+ T lymphocytes in the peripheral circulation
suggested an abnormality of T cell development and, specifically, in
thymic selection. CD8 deficiency in humans is commonly associated with
defective ZAP-70 expression (16, 17, 18, 19). A diagnosis of a T
cell signal transduction abnormality such as ZAP-70 deficiency was
suggested by findings that the patient’s T cells were refractory to
anti-CD3 stimulation in vitro (425 cpm (patient) vs 92,993 cpm
(control)) but responded normally to PMA plus ionomycin (38,713 cpm
(patient) vs 26,342 cpm (control)), agents that bypass proximal TCR
signaling events by mimicking activation of Ras and
[Ca2+]i flux.
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To confirm that the patient had a functional defect in coupling of
the TCR to distal signaling pathways, we analyzed whether his TCL was
capable of triggering cytoplasmic PTK reactions on TCR engagement.
Ab-mediated cross-linking of CD3 or both CD3 and CD4 resulted in a
markedly abnormal protein tyrosine phosphorylation pattern in
comparison with a normal TCL (Fig. 2A) or CD8-depleted TCL (data
not shown). Although phosphorylation of Lck was similar to that
observed in a normal TCL, phosphorylation of SLP-76, LAT, Erk1/2,
PLC1, and was significantly decreased in the patient’s T cells.
However, phosphorylation of a 70-kDa protein that appeared to be ZAP-70
was detected, albeit at a lower level than that in normal lymphocytes.
Immunoblot analysis confirmed the presence of ZAP-70 protein in the
patient’s TCL, at levels only modestly less than that in a normal TCL
(Fig. 2B). Similar results were obtained using PBMC or
CD8-depleted TCL (data not shown). Detection of considerable ZAP-70
protein in our patient’s T cells contrasts with previous published
findings in ZAP-70-deficient children, all of whom lacked ZAP-70
protein by immunoblot (16, 17, 18, 19). Expression of other
signaling molecules was unaffected.
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The patient’s lymphocyte phenotype, pattern of in
vitro proliferation, and phosphotyrosine status following TCR ligation
all suggested a defect in ZAP-70, even though considerable protein was
demonstrable in his T cells. To substantiate our hypothesis that the
patient had a variant of ZAP-70 deficiency, sequencing of the coding
portion of his zap70 gene was performed. PCR amplification
of ZAP-70 cDNA products was done, and 30 independent clones, as well as
uncloned cDNA, were analyzed. The patient was homozygous for a single
nucleotide change (C to T) at bp 1602, which results in the amino acid
conversion R465C (Fig. 3). The presence
of two mutant zap70 alleles was confirmed in the patient’s
genome by sequencing both cloned and uncloned chromosomal DNA products
from the patient. Characterization of the informative region of the
zap70 gene from both parents demonstrated that they were
heterozygous for the same mutation (data not shown). A minority of
cloned cDNA products from the patient and both of his parents also
contained an 18-bp in-frame deletion from nucleotides 1101–1116 that
encode a portion of interdomain B of the ZAP-70 protein. This form was
also found in one of the two original ZAP-70 cDNA clones
(25) and does not include residues critical for binding of
Src homology 2-containing proteins (7, 24). This likely
represents an alternatively spliced ZAP-70 product.
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The R465C mutation eliminates an invariant residue within a highly
conserved motif (DLAARN) that is essential for PTK enzymatic function
(26). Although R465C is a novel mutation in human ZAP-70
deficiency, the comparable lesion (R464C) was described recently as a
de novo mutation in ST mice (21). Analysis of TCR
signaling in ST thymocytes demonstrated that R464C abrogated ZAP-70
kinase activity but did not eliminate protein expression or tyrosine
phosphorylation (21). To determine whether 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 TCL, ZAP-70
protein from the patient’s TCL could not phosphorylate GST-band III or
undergo autophosphorylation after TCR ligation (Fig. 4). This finding suggested that, like the
comparable R464C mutation in ST mice, the principal effect of the R465C
mutation in humans is to abolish ZAP-70 kinase function.
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1 and the MAPKs, Erk1/2, was
significantly decreased in the patient’s TCL (Fig. 5A). Consistent with a defect
in PLC1 phosphorylation, no increase in
[Ca2+]i was observed in
the patient’s TCL after incubation with two different CD3 mAbs (Fig. 5B). Moreover, the patient’s T cells did not inducibly
express the activation marker, CD69, after TCR stimulation by PHA (Fig. 5C). CD69 expression is dependent on activation of the
Ras/MAPK pathway. These results demonstrate that, despite its
expression in the patient’s T lymphocytes, the mutant ZAP-70 could not
phosphorylate downstream substrates required for activation of the
calcium and Ras/MAPK pathways.
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Findings in previous ZAP-70-deficient patients have led to the
suggestion that Syk may compensate for loss of ZAP-70 in the thymus,
thereby rescuing development of some CD4+ T cells
(23). To determine whether Syk plays a role in the
survival of ZAP-70-deficient T cells in the peripheral circulation, we
analyzed intracellular Syk expression in PBMC at the single-cell level.
Immunofluorescence analysis revealed that 
5% of the patient’s
peripheral CD4+ T cells had detectable Syk
expression, in comparison with 2% of normal
CD4+ T cells (Fig. 6). The level of Syk observed in this
CD4+ T cell subpopulation was approximately
one-tenth that demonstrated in human B cells. The percentage of
CD4+ Sykintermedate T cells
increased to 13% in the patient’s TCL but remained unchanged in the
normal TCL (data not shown). However, despite increased Syk expression,
the patient’s cultured CD4+ T cells were
unresponsive to TCR-mediated stimuli. Although not identical, similar
findings were reported recently in cultured T cells from two siblings
homozygous for the mutation A507V, which abolishes both ZAP-70
expression and function (27). Despite increased Syk
expression and rescue of SLP-76/LAT/PLC1 phosphorylation and
[Ca2+]i mobilization
after long term culture, induction of downstream signaling events,
including Ras/MAPK activation and CD3-mediated IL-2 production,
remained markedly defective in TCL from those patients
(27).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We describe an infant with SCID due to a novel mutation in the zap70 gene that abrogates enzymatic function without appreciably affecting either expression or phosphorylation of ZAP-70. With the exception of one child who lacked ZAP-70 mRNA by Northern blot in whom the underlying mutation is not known (19) and another child with temperature-sensitive P80Q and M572L mutations (28), ZAP-70-deficient patients have inherited mutations within a small region of the ZAP-70 kinase domain that significantly affect both protein stability and catalytic activity. These included a 13-bp deletion resulting in a frame-shift after residue 503 and premature termination at amino acid 538 (16), two amino acid conversions, S518R (17) and A507V (27), and a splicing error that inserts LEQ into the protein after residue 541 (17, 18).
Role of Syk in CD4+ thymocyte selection in human ZAP-70 deficiency
The development of peripheral CD4+ T cells
in ZAP-70-deficient patients, which is not seen in mice deficient in
ZAP-70, has been attributed in part to the nature of the underlying
mutations that conceivably may allow for some residual ZAP-70 activity
in vivo. However, our patient provides the first description of
identical mutations in zap70 having disparate effects on T
cell development in humans vs mice. To explain these findings, we
suggest instead that the ability of Syk to contribute to pre-TCR and
TCR signaling is substantially different in humans as compared with
mice. Our conclusion is supported by recent observations that Syk is
expressed at highest levels in human and murine thymocytes during
stages of development in which pre-TCR signaling is required
(23). In contrast to mice, which down-regulate Syk
expression to peripheral T cell levels at the
CD44-CD25+ checkpoint,
human DP thymocytes express significant levels of Syk and do not
completely 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 thymocytes, but not peripheral T cells, from a
ZAP-70-deficient patient were capable of mobilizing
[Ca2+]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 characterized, 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 downstream substrates,
including PLC1, 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 transducing signals from both
the pre-TCR and TCR. However, under normal circumstances, Syk appears
to play a role for the most part in early 
T cell
differentiation, whereas ZAP-70 function is critical for positive and
negative selection in the thymus and T cell activation in the
periphery. Our results differ from a recent observation that a subset
of cultured primary T cells from SCID patients with a ZAP-70 null
mutation are capable of proximal TCR signaling as a result of markedly
increased Syk levels (27). We suggest that, despite
increased Syk expression in a subset of our patient’s TCL, the
catalytically inactive ZAP-70 mutant is able to compete favorably for
immunoreceptor tyrosine-based activation 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 immunoreceptor
tyrosine-based activation motif phosphorylation of TCR subunits in DP
cells limited in their expression of Lck (29). Although
not equivalent cell types, reduced phosphorylation was also
observed in our patient’s TCL, despite the presence of considerable
Lck and mutant ZAP-70 protein.
In summary, we suggest that human and murine DP thymocytes have different dependencies on ZAP-70 function during development, based on the T cell phenotypes of comparable ZAP-70 mutations. Despite similar effects on TCR signaling, a patient with a catalytically inactive ZAP-70 protein is able to produce large numbers of nonfunctional peripheral CD4+ T cells. The elevated levels of Syk in human DP thymocytes, in contrast to those in mice, may explain the differential consequences of identical ZAP-70 mutations.
Syk expression in ZAP-70-deficient peripheral T cells
Recent studies in mice demonstrate that survival of circulating T cells is diminished in the absence of TCR ligands (30, 31, 32), suggesting that TCR-mediated signals are required for maintenance of the peripheral T cell pool. However, survival of CD4+ T cells in the blood of ZAP-70-deficient patients would not appear to depend on ZAP-70-dependent TCR signaling or to up-regulation of Syk expression. It is doubtful that the presence of a Sykintermediate subset can account entirely for survival of the patient’s considerable number of circulating CD4+ T cells. Alternatively, the level of Syk activity required for peripheral T cell survival may be less than that required for propagation of TCR signals. Further investigation is required to determine whether viability of ZAP-70-deficient T cells is dependent on residual Syk function and whether these CD4+ T cells have a normal life span in the peripheral circulation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Melissa E. Elder, Box 0105, University of California, San Francisco, CA 94143.
3 Abbreviations used in this paper: PTK, protein tyrosine kinase; PLC1, phospholipase C1; LAT, linker of activated T cells; SLP-76, Src homology 2 domain-containing 76-kDa leukocyte protein; [Ca2+]i, intracellular free calcium; MAPK, mitogen-activated protein kinase; DP, double-positive; ST mice, Strange mice; TCL , T cell line.
Received for publication July 17, 2000. Accepted for publication September 27, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 in an SLP-76-deficient T cell. Science 281:413.1 and the Ras pathway. Immunity 9:617.[Medline]
chain. Cell 71:649.[Medline]
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