Dysregulation of Epstein-Barr Virus Infection in Hypomorphic ZAP70 Mutation

Dysregulation of Epstein-Barr Virus Infection in Hypomorphic ZAP70 Mutation Abstract Background Some patients with genetic defects develop Epstein-Barr virus (EBV)-associated lymphoproliferative disorder (LPD)/lymphoma as the main feature. Hypomophic mutations can cause different clinical and laboratory manifestations from null mutations in the same genes. Methods We sought to describe the clinical and immunologic phenotype of a 21-month-old boy with EBV-associated LPD who was in good health until then. A genetic and immunologic analysis was performed. Results Whole-exome sequencing identified a novel compound heterozygous mutation of ZAP70 c.703-1G>A and c.1674G>A. A small amount of the normal transcript was observed. Unlike ZAP70 deficiency, which has been previously described as severe combined immunodeficiency with nonfunctional CD4+ T cells and absent CD8+ T cells, the patient had slightly low numbers of CD8+ T cells and a small amount of functional T cells. EBV-specific CD8+ T cells and invariant natural killer T (iNKT) cells were absent. The T-cell receptor repertoire, determined using next generation sequencing, was significantly restricted. Conclusions Our patient showed that a hypomorphic mutation of ZAP70 can lead to EBV-associated LPD and that EBV-specific CD8+ T cells and iNKT cells are critically involved in immune response against EBV infection. Epstein-Barr virus, hypomorphic mutation, lymphoproliferative disorder, T-cell receptor repertoire, whole-exome sequencing, ZAP70 Epstein-Barr virus (EBV) infects the majority of the population worldwide. Primary EBV infection is asymptomatic or occasionally causes infectious mononucleosis. Following primary infection, although EBV is not eliminated in memory B cells for the lifetime of the host, EBV is maintained latently and is controlled by the host immune response [1]. In this setting, EBV-specific T cells play important roles [1–3]. Indeed, in human immunodeficiency virus-infected or posttransplant patients, impaired T-cell response allows EBV-infected cells to become proliferating blasts, which can result in lymphoproliferative disease (LPD) or lymphoma [4, 5]. Although humanized mouse models have contributed to revealing the immune response against EBV infection [6], the details remain unclear. Some types of genetic defects are known and have been recently described for EBV-associated LPD/lymphoma as the main feature [7–11]. These disorders include signaling lymphocytic activation molecule-associated protein (SAP) deficiency [7, 8], interleukin-2 inducible tyrosine kinase (ITK) deficiency [9], CD27 deficiency [10], X-linked immunodeficiency with magnesium defect, EBV infection, and neoplasia (XMEN) disease [11]. These primary immunodeficiencies (PIDs) provided new insights into the roles of T-cell receptor (TCR) and associated costimulatory signals, and evidence for the critical involvement of invariant natural killer T (iNKT) cells in immune responses against EBV infection [2, 3]. Here we describe an EBV-LPD patient associated with a hypomorphic mutation of zeta-chain associated protein kinase, 70 kDa (ZAP70). This case demonstrated the pivotal roles of T-cell recognition and iNKT cells in the control of EBV. ZAP70 is a nonreceptor tyrosine kinase, which is a key component of the TCR signal transduction pathway [12, 13]. ZAP deficiency has been described as severe combined immunodeficiency (SCID) with nonfunctional CD4+ T cells and absent CD8+ T cells [14]. The patient was in good health until EBV infection with a few functional CD4+ and CD8+ T cells. However, dysregulation of EBV infection was revealed by cytomolecular analysis, including TCR repertoire analysis using next generation sequencing. MATREIALS AND METHODS Ethical Considerations Informed consent was obtained from the patient’s parents. The study was conducted in accordance with the Helsinki Declaration and was approved by the ethics board of the University of Toyama and Tokyo Medical and Dental University. Genetic Analysis Whole-exome sequencing was performed using genomic DNA from whole blood of the patient and his parents as described elsewhere [15]. In brief, exome capture was carried out using a SureSelect Human All Exon V5 kit (Agilent technologies, Santa Clara, CA), and massively parallel sequencing was performed using a HiSeq 2000 platform (Illumina, San Diego, CA) with 100-bp paired-end reads. The data were processed with an in-house constructed analysis pipeline, which conducted the alignment of the reads with Burrows-Wheeler aligner 0.5.8 [16], counting of variant allele numbers with Samtools [17], and annotation with ANNOVAR [18]. Identified variants were filtered using dbSNP131, an in-house SNP database, and the Human Genetic Variation Database (http://www.hgvd.genome.med.kyoto-u.ac.jp/). Predicted functional effects of variants were determined using SIFT [19], PhyloP [20], PolyPhen2 [21], and MutationTaster [22]. In order to validate the results, polymerase chain reaction (PCR) using primers listed in Supplementary Table 1 and Sanger sequencing were performed. RT-PCR and Detection of Splicing Product RNA was extracted from peripheral blood mononuclear cells (PBMCs) according to standard methods and cDNA was prepared using SuperScript VILO (Invitrogen, Carlsbad, CA). PCR was performed using cDNA. The PCR products were cloned using TOPO TA cloning kit (Life Technologies, Carlsbad, CA) and independent clones were sequenced. Primers are listed in Supplementary Table 1. Flow Cytometry PBMCs were stained with fluorochrome-conjugated antibodies. Stained cells were analyzed using BD LSRFortessa (BD Biosciences, San Jose, CA) and the data processed using FlowJo software (Tree Star, Ashland, OR). For lymphocyte phenotyping, monoclonal antibodies used to stain cell surface are listed in Supplementary Table 2. For EBV-specific CD8+ T cells, PBMCs were incubated with Clear Back (MBL, Nagoya, Japan) to block the Fc receptors, and stained with HLA-A*24:02 EBV mix tetramer-phycoerythrin (PE) (MBL), followed by PE-Vio770-conjugated anti-CD8 (clone BW135/80, Miltenyi Biotec, Bergisch Gladbach, Germany). The patient had HLA-A*24:02. For intracellular ZAP70 staining, PBMCs were labeled with VioGreen-conjugated anti-CD3 (clone BW264/56, Miltenyi Biotec), VioBlue-conjugated anti-CD4 (clone M-T466, Miltenyi Biotec), and PE-Vio770-conjugated anti-CD8. Then, cells were fixed and permeabilized with Fixation/Permeabilization kit (eBioscience, San Diego, CA), washed in permeabilization buffer (eBioscience), and stained with PE-conjugated anti-ZAP70 (clone 1E7.2, eBioscience). Functional Analysis For calcium flux analysis, PBMCs were loaded with 2 μM Fluo-4 AM (Life Technologies) for 45 minutes at 37°C and stained for CD4 and CD8. Cells were stimulated with mouse anti-human CD3 (1 μg/mL; clone UCHT1, BD Pharmingen, San Diego, CA) and goat anti-mouse antibodies (BD Pharmingen), or ionomycin (8 μg/mL; Life Technologies). The analysis was performed by flow cytometry and kinetic plots using FlowJo software. For T-cell proliferation analysis, PBMCs were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE 3 μM; eBioscience) for 5 minutes at room temperature and stimulated for 4 days with anti-CD3/CD28 activating Dynabeads (Life Technologies, Oslo, Norway), or phorbol myristate acetate (PMA, 10 ng/mL; Sigma-Aldrich) and ionomycin (0.25 μg/mL; Sigma-Aldrich). Then, cells were stained for CD4 and CD8, and analyzed by flow cytometry. Immunoblot and Immunoprecipitation Analysis The entire coding region of wild-type (WT) ZAP70 cDNA was subcloned in a pcDNA3 vector (Invitrogen, Waltham, MA). The mutant ZAP70 expression vectors were generated by site-directed mutagenesis. The plasmids containing WT or mutant ZAP70 genes were transfected into P116 cells using Lipofectamine LTX Reagent (Thermo Fisher Scientific, Waltham, MA), according to the manufacturer’s protocol. At 24 hours after transfection, the cells were subjected to analysis. For immunoblot analysis, cells were stimulated with mouse anti-human CD3 (1 μg/mL) and goat anti-mouse antibodies for 5 minutes. For immunoprecipitation analysis, Dynabeads M-280 sheep anti-rabbit IgG (Life Technologies, Carlsbad, CA) was used. Immunoblot and immunoprecipitation analysis was performed using the following antibodies: anti-ZAP70 (ab134509; Abcam, Cambridge, UK), anti-SLP76 (ab109254; Abcam), anti-pY113 SLP76 (ab75990; Abcam), and anti-CD3ζ (ab226475; Abcam). TCR Repertoire Analysis cDNA from PBMCs was amplified using the HTBI-M reagent system (iRepertoire, Huntsville, AL) according to the manufacturer’s protocol, which include nested primers targeting each of the V and J elements for the first round of PCR and communal primers for the second round of PCR. After gel purification, the resulting product were sequenced using a MiSeq platform (Illumina). The data were processed with a provided pipeline (iRepertoire). For each sequence, copy number, complementary determining region 3 (CDR3) length, V and J usage, N addition and V and J trimming were determined. Shannon entropy was calculated [23]. TRECs Analysis T-cell receptor excision circles (TRECs) quantification was performed by real-time PCR using genomic DNA from whole blood of the patient as previously described [24]. Quantitation of DNA Copy Number In order to determine the ratio of X and Y chromosome, the copy numbers of IL2RG and SRY, which are on X and Y chromosome, respectively, were measured. Droplet digital PCR (ddPCR) was performed using QX200 Droplet Digital PCR System (Bio-Rad Laboratories, Hercules, CA). The primers and hydrolysis probes are listed in Supplementary Table 1. The IL2RG probes were labeled with HEX and SRY probe was labeled with FAM. Immunohistochemical Staining Pathological analysis was performed on cervical lymph node tissue. For immunohistochemistry, antibodies against cytoplasmic CD3, CD4, CD8, CD20 (Nichirei Biosciences, Tokyo, Japan), latent membrane protein 1 (LMP1, clone CS.1-4, DAKO, Tokyo, Japan), and Epstein-Barr virus nuclear 2 (EBNA2, clone PE2, DAKO) were used. EBV genome was detected by in situ hybridization using EBV-encoded RNA signals (EBERs; EBER PNA probe, DAKO). RESULTS Clinical Features A 21-month-old boy presented with fever, systemic lymphadenopathy, and facial paralysis. The patient was found to have enlarged spleen and EBV viremia (24000 copies/106 cells). He was born to nonconsanguineous Japanese parents and was in good health until he was admitted. He was immunized with live measles-rubella and BCG without adverse effect. His parents and 3 elder siblings were healthy except for the second brother. The brother died of acute encephalopathy at the age of 6 years; his CD8+ T-cell counts were within normal range. The patient was diagnosed with EBV-associated LPD, as described below. The patient temporarily responded to 2 mg/kg/day prednisolone; however, he developed mass lesions in his brain, liver, kidneys, and lungs at 24 months of age. They were refractory to multiagent chemotherapy, including anti-CD20 antibodies, cyclophosphamide, vincristine, pirarubicin, etoposide, high-dose methotrexate, high-dose cytarabine and intrathecal injection of methotrexate, cytarabine, and hydrocortisone. Subsequently, the mass lesion also appeared in his heart, which led to his death due to fetal atrioventricular block at 27 months of age. Pathological Findings Figure 1 shows histologic findings of the cervical lymph node before chemotherapy. Normal architecture of the lymph node was partially effaced and large and medium-sized lymphoid cell proliferation was observed. Large lymphoid cells were positive for CD20 and EBERs, while medium-sized lymphoid cells were positive for cytoplasmic CD3 and CD8 or CD4. Staining with EBNA2 and LMP1was positive, which indicated type III latency pattern. Figure 1. View largeDownload slide Pathological findings of the lymph node. A, Normal architecture of the cervical lymph node is partially effaced and large and medium-sized lymphoid cell proliferation is observed (H&E stain, ×100). Most of large lymphoid cells were positive for (B) EBV-encoded RNA (×400) and (C) Epstein-Barr virus nuclear 2 (×400). D, Few large lymphoid cells were positive for latent membrane protein 1 (×400). Figure 1. View largeDownload slide Pathological findings of the lymph node. A, Normal architecture of the cervical lymph node is partially effaced and large and medium-sized lymphoid cell proliferation is observed (H&E stain, ×100). Most of large lymphoid cells were positive for (B) EBV-encoded RNA (×400) and (C) Epstein-Barr virus nuclear 2 (×400). D, Few large lymphoid cells were positive for latent membrane protein 1 (×400). Immunodeficiency The immunological data are summarized in Table 1 [24,25]. Total lymphocyte counts were normal, but T-cell counts were low with decreased, but not absent, CD8+ T-cell counts (5%). Interestingly, CD8+ T-cell percentages were increased to 12% in total lymphocytes with a 1:2 CD4/CD8 ratio, when the patient was 24 months old. B- and NK-cell counts were almost within normal range. Before intravenous immunoglobulin, serum IgG, IgA, and IgM levels were elevated. He had protective titers of measles and rubella antibodies postvaccination; however, antibodies for EBV viral capsid antigen (VCA) IgG, VCA IgM, and EBNA antibodies were negative 3 weeks after onset of symptoms. Table 1. Immunophenotyping of the Patient Parameter, Units    Before Treatment  3 Months After Last Chemotherapy  Normal Valuesa  (21 Months)  (24 Months)  Lymphocytes, /μL    4480  640  3600–8900  T cells, % (/μL)  CD3+/Lym  34 (1520)  35 (220)  59–72 (2100–6400)  Helper T cells, % (/μL)  CD4+/Lym  27 (1210)  14 (88)  38–54 (1400–4800)  Naive CD4+ T cells, %  CD45RA+/CD3+CD4+  ND  4  77–89  Memory CD4+ T cells, %  CD45RO+/CD3+CD4+  ND  95  11–23  Recent thymic emigrants, %  CD31+/CD3+CD4+CD45RA+  ND  31  84–96  T follicular helper cells, %  CD45RO+CXCR5+/CD3+CD4+  ND  3  1–4  Regulatory T cells, %  CD25+CD127−/CD3+CD4+CCR4+  ND  13  15–31  Cytotoxic T cells, % (/μL)  CD8+/Lym  5 (220)  12 (74)  7–23 (390–2000)  Naive CD8+ T cells, %  CD45RA+/CD3+CD8+  ND  4  78–91  Memory CD8+ T cells, %  CD45RO+/CD3+CD8+  ND  96  9–22  Central memory T cells, %  CD62L+CCR7+/CD3+CD8+CD45RO+  ND  4  37–62  Effector memory T cells, %  CD62L−CCR7−/CD3+CD8+CD45RO+  ND  61  10–30  TCRδγ T cells, %  TCRαβ−TCRγδ+/CD3+  ND  20  1–13  Double negative T cells, %  CD4−CD8−/CD3+TCRαβ+  ND  21  1–2  Invariant NKT cells, %  TCR Vα24+TCR Vβ11+/CD3+  ND  0.00  0.01–0.12  B cells, % (/μL)  CD19+CD20+/Lym  36 (1610)  2 (14)b  8–22 (470–2000)  NK cells, % (/μL)  CD16+CD56+/Lym  27 (1210)  39 (250)  1–10 (100–1000)  IgG, g/L    16.01    5.53–9.71c  IgA, g/L    1.82    0.26–0.74c  IgM, g/L    2.82    0.35–0.81c  Measles IgG, IU/mL    2200 (protective)      Rubella IgG, IU/mL    93.0 (protective)      EBV VCA IgM    <10      EBV VCA IgG    <10      EBNA antibodies    <10      TREC, copies/μg DNA    Negative    3.5–8.1 × 103d  Parameter, Units    Before Treatment  3 Months After Last Chemotherapy  Normal Valuesa  (21 Months)  (24 Months)  Lymphocytes, /μL    4480  640  3600–8900  T cells, % (/μL)  CD3+/Lym  34 (1520)  35 (220)  59–72 (2100–6400)  Helper T cells, % (/μL)  CD4+/Lym  27 (1210)  14 (88)  38–54 (1400–4800)  Naive CD4+ T cells, %  CD45RA+/CD3+CD4+  ND  4  77–89  Memory CD4+ T cells, %  CD45RO+/CD3+CD4+  ND  95  11–23  Recent thymic emigrants, %  CD31+/CD3+CD4+CD45RA+  ND  31  84–96  T follicular helper cells, %  CD45RO+CXCR5+/CD3+CD4+  ND  3  1–4  Regulatory T cells, %  CD25+CD127−/CD3+CD4+CCR4+  ND  13  15–31  Cytotoxic T cells, % (/μL)  CD8+/Lym  5 (220)  12 (74)  7–23 (390–2000)  Naive CD8+ T cells, %  CD45RA+/CD3+CD8+  ND  4  78–91  Memory CD8+ T cells, %  CD45RO+/CD3+CD8+  ND  96  9–22  Central memory T cells, %  CD62L+CCR7+/CD3+CD8+CD45RO+  ND  4  37–62  Effector memory T cells, %  CD62L−CCR7−/CD3+CD8+CD45RO+  ND  61  10–30  TCRδγ T cells, %  TCRαβ−TCRγδ+/CD3+  ND  20  1–13  Double negative T cells, %  CD4−CD8−/CD3+TCRαβ+  ND  21  1–2  Invariant NKT cells, %  TCR Vα24+TCR Vβ11+/CD3+  ND  0.00  0.01–0.12  B cells, % (/μL)  CD19+CD20+/Lym  36 (1610)  2 (14)b  8–22 (470–2000)  NK cells, % (/μL)  CD16+CD56+/Lym  27 (1210)  39 (250)  1–10 (100–1000)  IgG, g/L    16.01    5.53–9.71c  IgA, g/L    1.82    0.26–0.74c  IgM, g/L    2.82    0.35–0.81c  Measles IgG, IU/mL    2200 (protective)      Rubella IgG, IU/mL    93.0 (protective)      EBV VCA IgM    <10      EBV VCA IgG    <10      EBNA antibodies    <10      TREC, copies/μg DNA    Negative    3.5–8.1 × 103d  Abbreviations: EBNA, Epstein-Barr nuclear antigen; EBV, Epstein-Barr virus; ND, not determined; NKT, natural killer T; TCR, T-cell receptor; TREC, T-cell receptor excision circle; VCA, viral capsid antigen. aAge-matched normal values in Japan as established by the laboratory performing the tests. b3 months after last anti-CD20 antibodies. cReference [25]. dReference [24]. View Large Table 1. Immunophenotyping of the Patient Parameter, Units    Before Treatment  3 Months After Last Chemotherapy  Normal Valuesa  (21 Months)  (24 Months)  Lymphocytes, /μL    4480  640  3600–8900  T cells, % (/μL)  CD3+/Lym  34 (1520)  35 (220)  59–72 (2100–6400)  Helper T cells, % (/μL)  CD4+/Lym  27 (1210)  14 (88)  38–54 (1400–4800)  Naive CD4+ T cells, %  CD45RA+/CD3+CD4+  ND  4  77–89  Memory CD4+ T cells, %  CD45RO+/CD3+CD4+  ND  95  11–23  Recent thymic emigrants, %  CD31+/CD3+CD4+CD45RA+  ND  31  84–96  T follicular helper cells, %  CD45RO+CXCR5+/CD3+CD4+  ND  3  1–4  Regulatory T cells, %  CD25+CD127−/CD3+CD4+CCR4+  ND  13  15–31  Cytotoxic T cells, % (/μL)  CD8+/Lym  5 (220)  12 (74)  7–23 (390–2000)  Naive CD8+ T cells, %  CD45RA+/CD3+CD8+  ND  4  78–91  Memory CD8+ T cells, %  CD45RO+/CD3+CD8+  ND  96  9–22  Central memory T cells, %  CD62L+CCR7+/CD3+CD8+CD45RO+  ND  4  37–62  Effector memory T cells, %  CD62L−CCR7−/CD3+CD8+CD45RO+  ND  61  10–30  TCRδγ T cells, %  TCRαβ−TCRγδ+/CD3+  ND  20  1–13  Double negative T cells, %  CD4−CD8−/CD3+TCRαβ+  ND  21  1–2  Invariant NKT cells, %  TCR Vα24+TCR Vβ11+/CD3+  ND  0.00  0.01–0.12  B cells, % (/μL)  CD19+CD20+/Lym  36 (1610)  2 (14)b  8–22 (470–2000)  NK cells, % (/μL)  CD16+CD56+/Lym  27 (1210)  39 (250)  1–10 (100–1000)  IgG, g/L    16.01    5.53–9.71c  IgA, g/L    1.82    0.26–0.74c  IgM, g/L    2.82    0.35–0.81c  Measles IgG, IU/mL    2200 (protective)      Rubella IgG, IU/mL    93.0 (protective)      EBV VCA IgM    <10      EBV VCA IgG    <10      EBNA antibodies    <10      TREC, copies/μg DNA    Negative    3.5–8.1 × 103d  Parameter, Units    Before Treatment  3 Months After Last Chemotherapy  Normal Valuesa  (21 Months)  (24 Months)  Lymphocytes, /μL    4480  640  3600–8900  T cells, % (/μL)  CD3+/Lym  34 (1520)  35 (220)  59–72 (2100–6400)  Helper T cells, % (/μL)  CD4+/Lym  27 (1210)  14 (88)  38–54 (1400–4800)  Naive CD4+ T cells, %  CD45RA+/CD3+CD4+  ND  4  77–89  Memory CD4+ T cells, %  CD45RO+/CD3+CD4+  ND  95  11–23  Recent thymic emigrants, %  CD31+/CD3+CD4+CD45RA+  ND  31  84–96  T follicular helper cells, %  CD45RO+CXCR5+/CD3+CD4+  ND  3  1–4  Regulatory T cells, %  CD25+CD127−/CD3+CD4+CCR4+  ND  13  15–31  Cytotoxic T cells, % (/μL)  CD8+/Lym  5 (220)  12 (74)  7–23 (390–2000)  Naive CD8+ T cells, %  CD45RA+/CD3+CD8+  ND  4  78–91  Memory CD8+ T cells, %  CD45RO+/CD3+CD8+  ND  96  9–22  Central memory T cells, %  CD62L+CCR7+/CD3+CD8+CD45RO+  ND  4  37–62  Effector memory T cells, %  CD62L−CCR7−/CD3+CD8+CD45RO+  ND  61  10–30  TCRδγ T cells, %  TCRαβ−TCRγδ+/CD3+  ND  20  1–13  Double negative T cells, %  CD4−CD8−/CD3+TCRαβ+  ND  21  1–2  Invariant NKT cells, %  TCR Vα24+TCR Vβ11+/CD3+  ND  0.00  0.01–0.12  B cells, % (/μL)  CD19+CD20+/Lym  36 (1610)  2 (14)b  8–22 (470–2000)  NK cells, % (/μL)  CD16+CD56+/Lym  27 (1210)  39 (250)  1–10 (100–1000)  IgG, g/L    16.01    5.53–9.71c  IgA, g/L    1.82    0.26–0.74c  IgM, g/L    2.82    0.35–0.81c  Measles IgG, IU/mL    2200 (protective)      Rubella IgG, IU/mL    93.0 (protective)      EBV VCA IgM    <10      EBV VCA IgG    <10      EBNA antibodies    <10      TREC, copies/μg DNA    Negative    3.5–8.1 × 103d  Abbreviations: EBNA, Epstein-Barr nuclear antigen; EBV, Epstein-Barr virus; ND, not determined; NKT, natural killer T; TCR, T-cell receptor; TREC, T-cell receptor excision circle; VCA, viral capsid antigen. aAge-matched normal values in Japan as established by the laboratory performing the tests. b3 months after last anti-CD20 antibodies. cReference [25]. dReference [24]. View Large The immunophenotypic analysis of T-cell subpopulations revealed markedly decreased proportions of both naive CD4+ and CD8+ T cells. We also found increased TCRγδ+ T-cell and TCRαβ+ double-negative T-cell counts, and decreased iNKT-cell counts, suggesting aberrant development of T cells. In particular, iNKT cells were nearly absent (Figure 2A). EBV-specific CD8+ T-cell counts were severely diminished (Figure 2B), consistent with the histologic findings of latency type III [26]. Because these data were obtained after chemotherapy, to distinguish whether the absence of iNKT cells was due to the chemotherapy or the underlying disease, we measured iNKT-cell counts in 2 groups of subjects: healthy controls and disease controls during or after chemotherapy and/or anti-CD20 antibodies administration. iNKT-cells counts were not different in those 2 groups, strongly suggesting that the patient had the underlying disease (Supplementary Figure 1). TREC levels were negative. Figure 2. View largeDownload slide Invariant natural killer T (iNKT) cells and Epstein-Barr virus (EBV)-specific CD8+ T cells. A, T-cell receptor (TCR) Vα24+ Vβ11+ iNKT cells gated on CD3+ T cells (left, healthy control subject; right, patient). B, HLA-A*24:02 EBV mix Tetramer+ EBV-specific cells gated on CD8+ T cells (left, healthy control subject; right, patient). Figure 2. View largeDownload slide Invariant natural killer T (iNKT) cells and Epstein-Barr virus (EBV)-specific CD8+ T cells. A, T-cell receptor (TCR) Vα24+ Vβ11+ iNKT cells gated on CD3+ T cells (left, healthy control subject; right, patient). B, HLA-A*24:02 EBV mix Tetramer+ EBV-specific cells gated on CD8+ T cells (left, healthy control subject; right, patient). Identification of ZAP70 Mutation The immunological data suggested that the patient had a specific susceptibility to EBV. We hypothesized that this susceptibility was caused by a single gene disorder, and therefore we performed whole-exome sequencing using DNA samples from the patient and his parents. As a result of filtering called variants, 5 candidate variants were identified, including 2 novel variants that are compound heterozygous ZAP70 mutation c.703-1G>A and c.1674G>A (p.Met558Ile) (Supplementary Table 3). The other 3 variants were not considered to be disease-causing genes because these were not related to immune system and the variants were not predicted as damaging using functional prediction algorithms. We confirmed the compound heterozygous ZAP70 mutations by Sanger sequencing (Figure 3A and 3B and Supplementary Figure 2A). The c.703-1G>A mutation was present in his father and the c.1674G>A mutation was present in his mother (Figure 3B). Sequencing of RT-PCR product from an RNA sample revealed a splice variant lacking exon 6 (Supplementary Figure 2B). Exon 6 includes the binding site of CD3 immunoreceptor tyrosine-based activation motifs (Supplementary Figure 2C), and Met558 is highly conserved (Supplementary Figure 2D). These findings suggest the compound heterozygous ZAP70 mutation c.703-1G>A and c.1674G>A is disease causing. Figure 3. View largeDownload slide Genetic analysis of compound heterozygous mutation of ZAP70. A, Schematic ZAP70 with N-SH2 domain, C-SH2 domain, and kinase domain. The position of exon 6 and M558 are shown in black. Arrows point to c.703-1G>A and c.1674G>A variants. B, Sanger sequencing of the family’s DNA. Wild-type c.703-1 or c.1674 positions are highlighted in blue. Heterozygous c.703-1G>A or c.1674G>A variants are highlighted in red. C, Flow cytometric analysis of ZAP70 expression in T cells, CD4+ T cells, and CD8+ T cells. Numbers in plots indicate the difference in mean fluorescence intensity. Figure 3. View largeDownload slide Genetic analysis of compound heterozygous mutation of ZAP70. A, Schematic ZAP70 with N-SH2 domain, C-SH2 domain, and kinase domain. The position of exon 6 and M558 are shown in black. Arrows point to c.703-1G>A and c.1674G>A variants. B, Sanger sequencing of the family’s DNA. Wild-type c.703-1 or c.1674 positions are highlighted in blue. Heterozygous c.703-1G>A or c.1674G>A variants are highlighted in red. C, Flow cytometric analysis of ZAP70 expression in T cells, CD4+ T cells, and CD8+ T cells. Numbers in plots indicate the difference in mean fluorescence intensity. Reduced Expression of ZAP70 Protein and Functional Defects in Mutant ZAP70 ZAP70 protein expression in T cells was analyzed using flow cytometry. ZAP70 expression was reduced in CD4+ and CD8+ T cells from the patient (Figure 3C). There was no difference in ZAP70 expression between CD4+ and CD8+ T cells in the patient. To determine the impact of ZAP70 mutants, ZAP70-deficient Jurkat P116 cells were transiently transduced with a vector that encoded WT ZAP70 (ZAP70WT), Met558Ile variant (ZAP70M558I), or exon 6-deleted ZAP70 (ZAP70Δexon 6). The expression levels of the 2 mutant ZAP70 proteins were reduced, suggesting degradation of the mutant protein (Figure 4A). We then analyzed TCR signaling downstream of ZAP70. Whereas CD3 cross-linking induced significant phosphorylation of SLP76 in ZAP70WT-transfected cells, this were significantly diminished in ZAP70M558I or ZAP70Δexon 6-transfected cells (Figure 4A). Furthermore, immunoprecipitation analysis showed that ZAP70Δexon 6 protein failed to bind CD3ζ chain (Figure 4B). These results indicate that the identified ZAP70 mutations are loss of function mutations due to their inability to transduce TCR signaling. Figure 4. View largeDownload slide Impaired functions of ZAP70 proteins in P116 cells transduced with ZAP70 mutants. A, Immunoblot analysis of P116 cells expressing ZAP70WT, ZAP70M558I, or ZAP70Δexon 6 stimulated with CD3 cross-linking. B, Immunoblot analysis of whole-cell lysates (WCL) and anti-CD3ζ chain immunoprecipitation. Figure 4. View largeDownload slide Impaired functions of ZAP70 proteins in P116 cells transduced with ZAP70 mutants. A, Immunoblot analysis of P116 cells expressing ZAP70WT, ZAP70M558I, or ZAP70Δexon 6 stimulated with CD3 cross-linking. B, Immunoblot analysis of whole-cell lysates (WCL) and anti-CD3ζ chain immunoprecipitation. Detection of Wild Type Allele Although the patient had compound heterozygous ZAP70 mutations and reduced expression of ZAP70, several of the findings were different from typical ZAP70 deficiency reported previously; that is, they did not meet the criteria of SCID (http://esid.org/Working-Parties/Registry/Diagnosis-criteria) or the milder CD8+ T-cell lymphopenia. Whole-exome sequencing revealed the ZAP70 mutations to be 50% of the allele frequency, and ddPCR revealed the X and Y chromosome ratio to be 1:1, which did not suggest reversion mosaicism or maternal T-cell engraftment. In order to explore the cause of the hypomorphic phenotype, we hypothesized that a small amount of normal splicing might occur despite the splice site mutation. We cloned PCR products from cDNA spanning exon 3 to 14 of ZAP70 and analyzed 45 independent clones by Sanger sequencing (Supplementary Figure 2E). Twenty clones were derived from aberrant splicing without exon 6, and 22 clones had missense mutation (c.1674G>A). The most remarkable result was that 3 clones were derived from normal splicing and did not have missense mutation (c.1674G>A). Assessment of Lymphocyte Function Signal transduction through the TCR/CD3 complex was examined in each CD4+ or CD8+ T-cell populations. First, TCR-mediated calcium mobilization was analyzed (Figure 5A and 5B). CD3 cross-linking induced a small and delayed free intracellular Ca2+ increase in the patient’s CD4+ and CD8+ T cells. In contrast, ionomycin, which is non-TCR-mediated stimulation, induced free intracellular Ca2+ increase to the same degree as in the control T cells. Second, T-cell proliferation after stimulation of the TCR was analyzed (Figure 5C and 5D). While PMA/ionomycin induced sufficient proliferation of patient T cells, anti-CD3/CD28 induced proliferation of a few but sufficient number of T cells, especially CD4+ T cells. These results demonstrate that most T cells were nonfunctional but a small proportion of T cells were normally functional, consistent with the detection of the wild-type allele. Figure 5. View largeDownload slide Signal transduction through the T-cell receptor (TCR)/CD3 complex in CD4+ or CD8+ T cells. A, Calcium mobilization induced by CD3 cross-linking (left) and ionomycin (right) in CD4+ T cells (gray, control; black, patient). B, Calcium mobilization in CD8+ T cells. C, Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled proliferation induced by anti-CD3/CD28 (left) and phorbol myristate acetate (PMA)/ionomycin (right) in CD4+ T cells. Numbers in plots indicate percent divided cells. D, CFSE-labeled proliferation in CD8+ T cells. Figure 5. View largeDownload slide Signal transduction through the T-cell receptor (TCR)/CD3 complex in CD4+ or CD8+ T cells. A, Calcium mobilization induced by CD3 cross-linking (left) and ionomycin (right) in CD4+ T cells (gray, control; black, patient). B, Calcium mobilization in CD8+ T cells. C, Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled proliferation induced by anti-CD3/CD28 (left) and phorbol myristate acetate (PMA)/ionomycin (right) in CD4+ T cells. Numbers in plots indicate percent divided cells. D, CFSE-labeled proliferation in CD8+ T cells. TCR Repertoire The existence of hypofunctional T cells led us to expect the slight skewing of TCR repertoire, as previously described in typical ZAP70 deficiency patients [14]. TCR CDR3 sequences were analyzed using next generation sequencing; this showed T-cell receptor β variable (TRBV) usage and CDR3 length were slightly skewed (Supplementary Figure 3A, 3B, and 3C). Unexpectedly, T cells from the patient had significantly skewed V-J combinations with expansion of TRBV6-5/TRBJ2-7, TRBV30/TRBJ1-2, TRBV18/TRBJ2-7, and TRBV6-6/TRBJ2-7 (Supplementary Figure 4A). Reduced diversity and uneven distribution were observed (Supplementary Figure 4B, and 4C). Junctional diversity was largely maintained (Supplementary Figure 3D). DISCUSSION We identified novel hypomorphic mutations of ZAP70 in a patient that did not manifest SCID but EBV-associated LPD, most likely following primary EBV infection. His laboratory findings indicated that impaired immunity against EBV might have been associated with the development of LPD. There are broadly similar laboratory findings between our patient and PID patients predisposed to EBV-associated LPD/lymphoma described previously. First, T cells, particularly naive T cells, were decreased but not absent, and TCR or associated costimulatory signals were impaired. Other T-cell immunodeficiencies present different clinical manifestations. PID patients without T cells most often contract other infectious diseases, which are life-threatening, before EBV infections [27]. Although SCID patients can develop EBV-associated LPD/lymphoma, it is rare as a main feature [28]. PID patients with impaired lymphocyte cytotoxicity, represented by familial hemophagocytic lymphohistiocytosis (HLH), develop fulminant infectious mononucleosis (FIM) or HLH due to an uncontrolled overwhelming hypercytokinemia produced by activated CD8+ T cells and NK cells [8]. SAP deficiency patients often develop FIM. The reason can be partially explained by the pathology of SAP deficiency, including reduced CD8+ T-cell and NK-cell cytotoxicity [8]. ZAP70 plays a pivotal role in TCR signal transduction, which is not directly related to lymphocyte cytotoxicity [12, 13]. In our patient, the development of EBV-associated LPD may have resulted from a limited resistance against the pathogen, including live vaccine strains and impaired recognition of EBV-infected cells. Lack of EBV-specific CD8+ T cells and negative titers of EBV antibodies support a globally impaired T-cell recognition of EBV antigen. Second, iNKT cell counts were remarkably decreased. A reduced number of iNKT cells has been reported in deficiencies of SAP [7, 8], ITK [9], CD27 [10], coronin-1A [29], and cytidine 5′ triphosphate synthase 1 (CTPS1) [30]. iNKT cells can directly and rapidly recognize EBV-infected cells through CD1d-mediated activation, and mediate direct cytotoxicity, which is especially critical during the earlier stage of EBV infection [31]. iNKT cells are indirectly responsible for controlling EBV infection through NK-cell, T-cell, and dendritic-cell activation by the production of interferon-γ and interleukin-2 [32]. ZAP70 is required for iNKT-cell development during positive selection, and iNKT cells are absent in Zap70 null mice [33]. Although the counts of iNKT cells in human ZAP70 deficiency is controversial [3, 34], these findings can help explain the remarkably decreased iNKT cells in the patient. The third similar manifestations between our patient and PID patients predisposed to EBV-associated LPD/lymphoma is that B-cell counts and development were normal or less impaired. The majority of EBV-infected cells are B cells, and the presence of B cells is important for EBV infection [1]. Dysgammaglobulinemia is often observed, but it is a result of impaired T-cell function or EBV infection by itself [7–9, 29, 30]. Hypomorphic ZAP70 mutation resulted in different clinical and laboratory manifestations from those of null mutation. Similar findings have been described in the CORON1A gene. While loss of function mutations of CORON1A are associated with T−B+NK+ SCID [35], hypomorphic mutations lead to PID predisposed to EBV-associated LPD/lymphoma [29]. Hypomorphic mutations due to normal splicing have been reported in some diseases [36, 37], including one ZAP70 patient [38]. This patient had had recurrent infections since infancy, and at the last follow-up, at 9 years of age, he was well without transplantation. Severe EBV infection was not observed; however, he developed severe varicella-zoster virus infection. Although the reason for the difference in susceptibility to EBV between our patient and the previously reported patient remains unclear, it may reflect clinical exposure or the impact of genetic defects, including iNKT-cell differentiation and TCR signal transduction. The TCR repertoire analysis in great detail using next generation sequencing showed significant restriction of the TCR repertoire, with reduced diversity and uneven distribution in the patient, indicating abnormal T-cell generation and the nonrandom usage of V, D, and J elements. These results confirm and extend previous findings of ZAP70 involvement at the immature single-positive thymocyte stage to the double- positive thymocyte stage [14]. Restriction of the TCR repertoire may contribute that EBV-specific CDR3 sequences could not be produced by chance, whereas other pathogen-specific CDR3 sequences could be produced, including measles and rubella. This hypothesis is supported by the observation that EBV-specific antibodies were absent whereas measles and rubella-specific antibodies were present in the patient. If many antigen-specific CDR3 sequences are known across a diverse HLA type, this analysis could be used to evaluate antigen-specific T cells [39–42]. Alterations in ZAP70 result in a wide spectrum of clinical features. While loss of function mutations of ZAP70 lead to SCID [12], hypomorphic mutations of ZAP70 seem to be associated with autoimmune disease [43, 44]. We identified novel hypomorphic mutations of ZAP70 and described a selective dysregulation of EBV infection. Our findings extend the spectrum of clinical features of ZAP70 mutations and indicate pivotal roles of T-cell recognition and iNKT cells in immune response against EBV. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Acknowledgments. We thank Dr Hideki Muramatsu for professional technical assistance. Financial support. This work was supported by the Research on Measures for Intractable Disease Project, and grants from Ministry of Education, Culture, Sports, Science and Technology of Japan (JSPS KAKENHI: grant number 26461570); and the Ministry of Health, Labour and Welfare of Japan (grant number H26-Nanchi-071). Potential conflicts of interest. All authors: No reported conflicts of interest. 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Akar HH, Patiroglu T, Akyildiz BNet al.   Silent brain infarcts in two patients with zeta chain-associated protein 70 kDa (ZAP70) deficiency. Clin Immunol  2015; 158: 88– 91. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Infectious Diseases Oxford University Press

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Abstract

Abstract Background Some patients with genetic defects develop Epstein-Barr virus (EBV)-associated lymphoproliferative disorder (LPD)/lymphoma as the main feature. Hypomophic mutations can cause different clinical and laboratory manifestations from null mutations in the same genes. Methods We sought to describe the clinical and immunologic phenotype of a 21-month-old boy with EBV-associated LPD who was in good health until then. A genetic and immunologic analysis was performed. Results Whole-exome sequencing identified a novel compound heterozygous mutation of ZAP70 c.703-1G>A and c.1674G>A. A small amount of the normal transcript was observed. Unlike ZAP70 deficiency, which has been previously described as severe combined immunodeficiency with nonfunctional CD4+ T cells and absent CD8+ T cells, the patient had slightly low numbers of CD8+ T cells and a small amount of functional T cells. EBV-specific CD8+ T cells and invariant natural killer T (iNKT) cells were absent. The T-cell receptor repertoire, determined using next generation sequencing, was significantly restricted. Conclusions Our patient showed that a hypomorphic mutation of ZAP70 can lead to EBV-associated LPD and that EBV-specific CD8+ T cells and iNKT cells are critically involved in immune response against EBV infection. Epstein-Barr virus, hypomorphic mutation, lymphoproliferative disorder, T-cell receptor repertoire, whole-exome sequencing, ZAP70 Epstein-Barr virus (EBV) infects the majority of the population worldwide. Primary EBV infection is asymptomatic or occasionally causes infectious mononucleosis. Following primary infection, although EBV is not eliminated in memory B cells for the lifetime of the host, EBV is maintained latently and is controlled by the host immune response [1]. In this setting, EBV-specific T cells play important roles [1–3]. Indeed, in human immunodeficiency virus-infected or posttransplant patients, impaired T-cell response allows EBV-infected cells to become proliferating blasts, which can result in lymphoproliferative disease (LPD) or lymphoma [4, 5]. Although humanized mouse models have contributed to revealing the immune response against EBV infection [6], the details remain unclear. Some types of genetic defects are known and have been recently described for EBV-associated LPD/lymphoma as the main feature [7–11]. These disorders include signaling lymphocytic activation molecule-associated protein (SAP) deficiency [7, 8], interleukin-2 inducible tyrosine kinase (ITK) deficiency [9], CD27 deficiency [10], X-linked immunodeficiency with magnesium defect, EBV infection, and neoplasia (XMEN) disease [11]. These primary immunodeficiencies (PIDs) provided new insights into the roles of T-cell receptor (TCR) and associated costimulatory signals, and evidence for the critical involvement of invariant natural killer T (iNKT) cells in immune responses against EBV infection [2, 3]. Here we describe an EBV-LPD patient associated with a hypomorphic mutation of zeta-chain associated protein kinase, 70 kDa (ZAP70). This case demonstrated the pivotal roles of T-cell recognition and iNKT cells in the control of EBV. ZAP70 is a nonreceptor tyrosine kinase, which is a key component of the TCR signal transduction pathway [12, 13]. ZAP deficiency has been described as severe combined immunodeficiency (SCID) with nonfunctional CD4+ T cells and absent CD8+ T cells [14]. The patient was in good health until EBV infection with a few functional CD4+ and CD8+ T cells. However, dysregulation of EBV infection was revealed by cytomolecular analysis, including TCR repertoire analysis using next generation sequencing. MATREIALS AND METHODS Ethical Considerations Informed consent was obtained from the patient’s parents. The study was conducted in accordance with the Helsinki Declaration and was approved by the ethics board of the University of Toyama and Tokyo Medical and Dental University. Genetic Analysis Whole-exome sequencing was performed using genomic DNA from whole blood of the patient and his parents as described elsewhere [15]. In brief, exome capture was carried out using a SureSelect Human All Exon V5 kit (Agilent technologies, Santa Clara, CA), and massively parallel sequencing was performed using a HiSeq 2000 platform (Illumina, San Diego, CA) with 100-bp paired-end reads. The data were processed with an in-house constructed analysis pipeline, which conducted the alignment of the reads with Burrows-Wheeler aligner 0.5.8 [16], counting of variant allele numbers with Samtools [17], and annotation with ANNOVAR [18]. Identified variants were filtered using dbSNP131, an in-house SNP database, and the Human Genetic Variation Database (http://www.hgvd.genome.med.kyoto-u.ac.jp/). Predicted functional effects of variants were determined using SIFT [19], PhyloP [20], PolyPhen2 [21], and MutationTaster [22]. In order to validate the results, polymerase chain reaction (PCR) using primers listed in Supplementary Table 1 and Sanger sequencing were performed. RT-PCR and Detection of Splicing Product RNA was extracted from peripheral blood mononuclear cells (PBMCs) according to standard methods and cDNA was prepared using SuperScript VILO (Invitrogen, Carlsbad, CA). PCR was performed using cDNA. The PCR products were cloned using TOPO TA cloning kit (Life Technologies, Carlsbad, CA) and independent clones were sequenced. Primers are listed in Supplementary Table 1. Flow Cytometry PBMCs were stained with fluorochrome-conjugated antibodies. Stained cells were analyzed using BD LSRFortessa (BD Biosciences, San Jose, CA) and the data processed using FlowJo software (Tree Star, Ashland, OR). For lymphocyte phenotyping, monoclonal antibodies used to stain cell surface are listed in Supplementary Table 2. For EBV-specific CD8+ T cells, PBMCs were incubated with Clear Back (MBL, Nagoya, Japan) to block the Fc receptors, and stained with HLA-A*24:02 EBV mix tetramer-phycoerythrin (PE) (MBL), followed by PE-Vio770-conjugated anti-CD8 (clone BW135/80, Miltenyi Biotec, Bergisch Gladbach, Germany). The patient had HLA-A*24:02. For intracellular ZAP70 staining, PBMCs were labeled with VioGreen-conjugated anti-CD3 (clone BW264/56, Miltenyi Biotec), VioBlue-conjugated anti-CD4 (clone M-T466, Miltenyi Biotec), and PE-Vio770-conjugated anti-CD8. Then, cells were fixed and permeabilized with Fixation/Permeabilization kit (eBioscience, San Diego, CA), washed in permeabilization buffer (eBioscience), and stained with PE-conjugated anti-ZAP70 (clone 1E7.2, eBioscience). Functional Analysis For calcium flux analysis, PBMCs were loaded with 2 μM Fluo-4 AM (Life Technologies) for 45 minutes at 37°C and stained for CD4 and CD8. Cells were stimulated with mouse anti-human CD3 (1 μg/mL; clone UCHT1, BD Pharmingen, San Diego, CA) and goat anti-mouse antibodies (BD Pharmingen), or ionomycin (8 μg/mL; Life Technologies). The analysis was performed by flow cytometry and kinetic plots using FlowJo software. For T-cell proliferation analysis, PBMCs were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE 3 μM; eBioscience) for 5 minutes at room temperature and stimulated for 4 days with anti-CD3/CD28 activating Dynabeads (Life Technologies, Oslo, Norway), or phorbol myristate acetate (PMA, 10 ng/mL; Sigma-Aldrich) and ionomycin (0.25 μg/mL; Sigma-Aldrich). Then, cells were stained for CD4 and CD8, and analyzed by flow cytometry. Immunoblot and Immunoprecipitation Analysis The entire coding region of wild-type (WT) ZAP70 cDNA was subcloned in a pcDNA3 vector (Invitrogen, Waltham, MA). The mutant ZAP70 expression vectors were generated by site-directed mutagenesis. The plasmids containing WT or mutant ZAP70 genes were transfected into P116 cells using Lipofectamine LTX Reagent (Thermo Fisher Scientific, Waltham, MA), according to the manufacturer’s protocol. At 24 hours after transfection, the cells were subjected to analysis. For immunoblot analysis, cells were stimulated with mouse anti-human CD3 (1 μg/mL) and goat anti-mouse antibodies for 5 minutes. For immunoprecipitation analysis, Dynabeads M-280 sheep anti-rabbit IgG (Life Technologies, Carlsbad, CA) was used. Immunoblot and immunoprecipitation analysis was performed using the following antibodies: anti-ZAP70 (ab134509; Abcam, Cambridge, UK), anti-SLP76 (ab109254; Abcam), anti-pY113 SLP76 (ab75990; Abcam), and anti-CD3ζ (ab226475; Abcam). TCR Repertoire Analysis cDNA from PBMCs was amplified using the HTBI-M reagent system (iRepertoire, Huntsville, AL) according to the manufacturer’s protocol, which include nested primers targeting each of the V and J elements for the first round of PCR and communal primers for the second round of PCR. After gel purification, the resulting product were sequenced using a MiSeq platform (Illumina). The data were processed with a provided pipeline (iRepertoire). For each sequence, copy number, complementary determining region 3 (CDR3) length, V and J usage, N addition and V and J trimming were determined. Shannon entropy was calculated [23]. TRECs Analysis T-cell receptor excision circles (TRECs) quantification was performed by real-time PCR using genomic DNA from whole blood of the patient as previously described [24]. Quantitation of DNA Copy Number In order to determine the ratio of X and Y chromosome, the copy numbers of IL2RG and SRY, which are on X and Y chromosome, respectively, were measured. Droplet digital PCR (ddPCR) was performed using QX200 Droplet Digital PCR System (Bio-Rad Laboratories, Hercules, CA). The primers and hydrolysis probes are listed in Supplementary Table 1. The IL2RG probes were labeled with HEX and SRY probe was labeled with FAM. Immunohistochemical Staining Pathological analysis was performed on cervical lymph node tissue. For immunohistochemistry, antibodies against cytoplasmic CD3, CD4, CD8, CD20 (Nichirei Biosciences, Tokyo, Japan), latent membrane protein 1 (LMP1, clone CS.1-4, DAKO, Tokyo, Japan), and Epstein-Barr virus nuclear 2 (EBNA2, clone PE2, DAKO) were used. EBV genome was detected by in situ hybridization using EBV-encoded RNA signals (EBERs; EBER PNA probe, DAKO). RESULTS Clinical Features A 21-month-old boy presented with fever, systemic lymphadenopathy, and facial paralysis. The patient was found to have enlarged spleen and EBV viremia (24000 copies/106 cells). He was born to nonconsanguineous Japanese parents and was in good health until he was admitted. He was immunized with live measles-rubella and BCG without adverse effect. His parents and 3 elder siblings were healthy except for the second brother. The brother died of acute encephalopathy at the age of 6 years; his CD8+ T-cell counts were within normal range. The patient was diagnosed with EBV-associated LPD, as described below. The patient temporarily responded to 2 mg/kg/day prednisolone; however, he developed mass lesions in his brain, liver, kidneys, and lungs at 24 months of age. They were refractory to multiagent chemotherapy, including anti-CD20 antibodies, cyclophosphamide, vincristine, pirarubicin, etoposide, high-dose methotrexate, high-dose cytarabine and intrathecal injection of methotrexate, cytarabine, and hydrocortisone. Subsequently, the mass lesion also appeared in his heart, which led to his death due to fetal atrioventricular block at 27 months of age. Pathological Findings Figure 1 shows histologic findings of the cervical lymph node before chemotherapy. Normal architecture of the lymph node was partially effaced and large and medium-sized lymphoid cell proliferation was observed. Large lymphoid cells were positive for CD20 and EBERs, while medium-sized lymphoid cells were positive for cytoplasmic CD3 and CD8 or CD4. Staining with EBNA2 and LMP1was positive, which indicated type III latency pattern. Figure 1. View largeDownload slide Pathological findings of the lymph node. A, Normal architecture of the cervical lymph node is partially effaced and large and medium-sized lymphoid cell proliferation is observed (H&E stain, ×100). Most of large lymphoid cells were positive for (B) EBV-encoded RNA (×400) and (C) Epstein-Barr virus nuclear 2 (×400). D, Few large lymphoid cells were positive for latent membrane protein 1 (×400). Figure 1. View largeDownload slide Pathological findings of the lymph node. A, Normal architecture of the cervical lymph node is partially effaced and large and medium-sized lymphoid cell proliferation is observed (H&E stain, ×100). Most of large lymphoid cells were positive for (B) EBV-encoded RNA (×400) and (C) Epstein-Barr virus nuclear 2 (×400). D, Few large lymphoid cells were positive for latent membrane protein 1 (×400). Immunodeficiency The immunological data are summarized in Table 1 [24,25]. Total lymphocyte counts were normal, but T-cell counts were low with decreased, but not absent, CD8+ T-cell counts (5%). Interestingly, CD8+ T-cell percentages were increased to 12% in total lymphocytes with a 1:2 CD4/CD8 ratio, when the patient was 24 months old. B- and NK-cell counts were almost within normal range. Before intravenous immunoglobulin, serum IgG, IgA, and IgM levels were elevated. He had protective titers of measles and rubella antibodies postvaccination; however, antibodies for EBV viral capsid antigen (VCA) IgG, VCA IgM, and EBNA antibodies were negative 3 weeks after onset of symptoms. Table 1. Immunophenotyping of the Patient Parameter, Units    Before Treatment  3 Months After Last Chemotherapy  Normal Valuesa  (21 Months)  (24 Months)  Lymphocytes, /μL    4480  640  3600–8900  T cells, % (/μL)  CD3+/Lym  34 (1520)  35 (220)  59–72 (2100–6400)  Helper T cells, % (/μL)  CD4+/Lym  27 (1210)  14 (88)  38–54 (1400–4800)  Naive CD4+ T cells, %  CD45RA+/CD3+CD4+  ND  4  77–89  Memory CD4+ T cells, %  CD45RO+/CD3+CD4+  ND  95  11–23  Recent thymic emigrants, %  CD31+/CD3+CD4+CD45RA+  ND  31  84–96  T follicular helper cells, %  CD45RO+CXCR5+/CD3+CD4+  ND  3  1–4  Regulatory T cells, %  CD25+CD127−/CD3+CD4+CCR4+  ND  13  15–31  Cytotoxic T cells, % (/μL)  CD8+/Lym  5 (220)  12 (74)  7–23 (390–2000)  Naive CD8+ T cells, %  CD45RA+/CD3+CD8+  ND  4  78–91  Memory CD8+ T cells, %  CD45RO+/CD3+CD8+  ND  96  9–22  Central memory T cells, %  CD62L+CCR7+/CD3+CD8+CD45RO+  ND  4  37–62  Effector memory T cells, %  CD62L−CCR7−/CD3+CD8+CD45RO+  ND  61  10–30  TCRδγ T cells, %  TCRαβ−TCRγδ+/CD3+  ND  20  1–13  Double negative T cells, %  CD4−CD8−/CD3+TCRαβ+  ND  21  1–2  Invariant NKT cells, %  TCR Vα24+TCR Vβ11+/CD3+  ND  0.00  0.01–0.12  B cells, % (/μL)  CD19+CD20+/Lym  36 (1610)  2 (14)b  8–22 (470–2000)  NK cells, % (/μL)  CD16+CD56+/Lym  27 (1210)  39 (250)  1–10 (100–1000)  IgG, g/L    16.01    5.53–9.71c  IgA, g/L    1.82    0.26–0.74c  IgM, g/L    2.82    0.35–0.81c  Measles IgG, IU/mL    2200 (protective)      Rubella IgG, IU/mL    93.0 (protective)      EBV VCA IgM    <10      EBV VCA IgG    <10      EBNA antibodies    <10      TREC, copies/μg DNA    Negative    3.5–8.1 × 103d  Parameter, Units    Before Treatment  3 Months After Last Chemotherapy  Normal Valuesa  (21 Months)  (24 Months)  Lymphocytes, /μL    4480  640  3600–8900  T cells, % (/μL)  CD3+/Lym  34 (1520)  35 (220)  59–72 (2100–6400)  Helper T cells, % (/μL)  CD4+/Lym  27 (1210)  14 (88)  38–54 (1400–4800)  Naive CD4+ T cells, %  CD45RA+/CD3+CD4+  ND  4  77–89  Memory CD4+ T cells, %  CD45RO+/CD3+CD4+  ND  95  11–23  Recent thymic emigrants, %  CD31+/CD3+CD4+CD45RA+  ND  31  84–96  T follicular helper cells, %  CD45RO+CXCR5+/CD3+CD4+  ND  3  1–4  Regulatory T cells, %  CD25+CD127−/CD3+CD4+CCR4+  ND  13  15–31  Cytotoxic T cells, % (/μL)  CD8+/Lym  5 (220)  12 (74)  7–23 (390–2000)  Naive CD8+ T cells, %  CD45RA+/CD3+CD8+  ND  4  78–91  Memory CD8+ T cells, %  CD45RO+/CD3+CD8+  ND  96  9–22  Central memory T cells, %  CD62L+CCR7+/CD3+CD8+CD45RO+  ND  4  37–62  Effector memory T cells, %  CD62L−CCR7−/CD3+CD8+CD45RO+  ND  61  10–30  TCRδγ T cells, %  TCRαβ−TCRγδ+/CD3+  ND  20  1–13  Double negative T cells, %  CD4−CD8−/CD3+TCRαβ+  ND  21  1–2  Invariant NKT cells, %  TCR Vα24+TCR Vβ11+/CD3+  ND  0.00  0.01–0.12  B cells, % (/μL)  CD19+CD20+/Lym  36 (1610)  2 (14)b  8–22 (470–2000)  NK cells, % (/μL)  CD16+CD56+/Lym  27 (1210)  39 (250)  1–10 (100–1000)  IgG, g/L    16.01    5.53–9.71c  IgA, g/L    1.82    0.26–0.74c  IgM, g/L    2.82    0.35–0.81c  Measles IgG, IU/mL    2200 (protective)      Rubella IgG, IU/mL    93.0 (protective)      EBV VCA IgM    <10      EBV VCA IgG    <10      EBNA antibodies    <10      TREC, copies/μg DNA    Negative    3.5–8.1 × 103d  Abbreviations: EBNA, Epstein-Barr nuclear antigen; EBV, Epstein-Barr virus; ND, not determined; NKT, natural killer T; TCR, T-cell receptor; TREC, T-cell receptor excision circle; VCA, viral capsid antigen. aAge-matched normal values in Japan as established by the laboratory performing the tests. b3 months after last anti-CD20 antibodies. cReference [25]. dReference [24]. View Large Table 1. Immunophenotyping of the Patient Parameter, Units    Before Treatment  3 Months After Last Chemotherapy  Normal Valuesa  (21 Months)  (24 Months)  Lymphocytes, /μL    4480  640  3600–8900  T cells, % (/μL)  CD3+/Lym  34 (1520)  35 (220)  59–72 (2100–6400)  Helper T cells, % (/μL)  CD4+/Lym  27 (1210)  14 (88)  38–54 (1400–4800)  Naive CD4+ T cells, %  CD45RA+/CD3+CD4+  ND  4  77–89  Memory CD4+ T cells, %  CD45RO+/CD3+CD4+  ND  95  11–23  Recent thymic emigrants, %  CD31+/CD3+CD4+CD45RA+  ND  31  84–96  T follicular helper cells, %  CD45RO+CXCR5+/CD3+CD4+  ND  3  1–4  Regulatory T cells, %  CD25+CD127−/CD3+CD4+CCR4+  ND  13  15–31  Cytotoxic T cells, % (/μL)  CD8+/Lym  5 (220)  12 (74)  7–23 (390–2000)  Naive CD8+ T cells, %  CD45RA+/CD3+CD8+  ND  4  78–91  Memory CD8+ T cells, %  CD45RO+/CD3+CD8+  ND  96  9–22  Central memory T cells, %  CD62L+CCR7+/CD3+CD8+CD45RO+  ND  4  37–62  Effector memory T cells, %  CD62L−CCR7−/CD3+CD8+CD45RO+  ND  61  10–30  TCRδγ T cells, %  TCRαβ−TCRγδ+/CD3+  ND  20  1–13  Double negative T cells, %  CD4−CD8−/CD3+TCRαβ+  ND  21  1–2  Invariant NKT cells, %  TCR Vα24+TCR Vβ11+/CD3+  ND  0.00  0.01–0.12  B cells, % (/μL)  CD19+CD20+/Lym  36 (1610)  2 (14)b  8–22 (470–2000)  NK cells, % (/μL)  CD16+CD56+/Lym  27 (1210)  39 (250)  1–10 (100–1000)  IgG, g/L    16.01    5.53–9.71c  IgA, g/L    1.82    0.26–0.74c  IgM, g/L    2.82    0.35–0.81c  Measles IgG, IU/mL    2200 (protective)      Rubella IgG, IU/mL    93.0 (protective)      EBV VCA IgM    <10      EBV VCA IgG    <10      EBNA antibodies    <10      TREC, copies/μg DNA    Negative    3.5–8.1 × 103d  Parameter, Units    Before Treatment  3 Months After Last Chemotherapy  Normal Valuesa  (21 Months)  (24 Months)  Lymphocytes, /μL    4480  640  3600–8900  T cells, % (/μL)  CD3+/Lym  34 (1520)  35 (220)  59–72 (2100–6400)  Helper T cells, % (/μL)  CD4+/Lym  27 (1210)  14 (88)  38–54 (1400–4800)  Naive CD4+ T cells, %  CD45RA+/CD3+CD4+  ND  4  77–89  Memory CD4+ T cells, %  CD45RO+/CD3+CD4+  ND  95  11–23  Recent thymic emigrants, %  CD31+/CD3+CD4+CD45RA+  ND  31  84–96  T follicular helper cells, %  CD45RO+CXCR5+/CD3+CD4+  ND  3  1–4  Regulatory T cells, %  CD25+CD127−/CD3+CD4+CCR4+  ND  13  15–31  Cytotoxic T cells, % (/μL)  CD8+/Lym  5 (220)  12 (74)  7–23 (390–2000)  Naive CD8+ T cells, %  CD45RA+/CD3+CD8+  ND  4  78–91  Memory CD8+ T cells, %  CD45RO+/CD3+CD8+  ND  96  9–22  Central memory T cells, %  CD62L+CCR7+/CD3+CD8+CD45RO+  ND  4  37–62  Effector memory T cells, %  CD62L−CCR7−/CD3+CD8+CD45RO+  ND  61  10–30  TCRδγ T cells, %  TCRαβ−TCRγδ+/CD3+  ND  20  1–13  Double negative T cells, %  CD4−CD8−/CD3+TCRαβ+  ND  21  1–2  Invariant NKT cells, %  TCR Vα24+TCR Vβ11+/CD3+  ND  0.00  0.01–0.12  B cells, % (/μL)  CD19+CD20+/Lym  36 (1610)  2 (14)b  8–22 (470–2000)  NK cells, % (/μL)  CD16+CD56+/Lym  27 (1210)  39 (250)  1–10 (100–1000)  IgG, g/L    16.01    5.53–9.71c  IgA, g/L    1.82    0.26–0.74c  IgM, g/L    2.82    0.35–0.81c  Measles IgG, IU/mL    2200 (protective)      Rubella IgG, IU/mL    93.0 (protective)      EBV VCA IgM    <10      EBV VCA IgG    <10      EBNA antibodies    <10      TREC, copies/μg DNA    Negative    3.5–8.1 × 103d  Abbreviations: EBNA, Epstein-Barr nuclear antigen; EBV, Epstein-Barr virus; ND, not determined; NKT, natural killer T; TCR, T-cell receptor; TREC, T-cell receptor excision circle; VCA, viral capsid antigen. aAge-matched normal values in Japan as established by the laboratory performing the tests. b3 months after last anti-CD20 antibodies. cReference [25]. dReference [24]. View Large The immunophenotypic analysis of T-cell subpopulations revealed markedly decreased proportions of both naive CD4+ and CD8+ T cells. We also found increased TCRγδ+ T-cell and TCRαβ+ double-negative T-cell counts, and decreased iNKT-cell counts, suggesting aberrant development of T cells. In particular, iNKT cells were nearly absent (Figure 2A). EBV-specific CD8+ T-cell counts were severely diminished (Figure 2B), consistent with the histologic findings of latency type III [26]. Because these data were obtained after chemotherapy, to distinguish whether the absence of iNKT cells was due to the chemotherapy or the underlying disease, we measured iNKT-cell counts in 2 groups of subjects: healthy controls and disease controls during or after chemotherapy and/or anti-CD20 antibodies administration. iNKT-cells counts were not different in those 2 groups, strongly suggesting that the patient had the underlying disease (Supplementary Figure 1). TREC levels were negative. Figure 2. View largeDownload slide Invariant natural killer T (iNKT) cells and Epstein-Barr virus (EBV)-specific CD8+ T cells. A, T-cell receptor (TCR) Vα24+ Vβ11+ iNKT cells gated on CD3+ T cells (left, healthy control subject; right, patient). B, HLA-A*24:02 EBV mix Tetramer+ EBV-specific cells gated on CD8+ T cells (left, healthy control subject; right, patient). Figure 2. View largeDownload slide Invariant natural killer T (iNKT) cells and Epstein-Barr virus (EBV)-specific CD8+ T cells. A, T-cell receptor (TCR) Vα24+ Vβ11+ iNKT cells gated on CD3+ T cells (left, healthy control subject; right, patient). B, HLA-A*24:02 EBV mix Tetramer+ EBV-specific cells gated on CD8+ T cells (left, healthy control subject; right, patient). Identification of ZAP70 Mutation The immunological data suggested that the patient had a specific susceptibility to EBV. We hypothesized that this susceptibility was caused by a single gene disorder, and therefore we performed whole-exome sequencing using DNA samples from the patient and his parents. As a result of filtering called variants, 5 candidate variants were identified, including 2 novel variants that are compound heterozygous ZAP70 mutation c.703-1G>A and c.1674G>A (p.Met558Ile) (Supplementary Table 3). The other 3 variants were not considered to be disease-causing genes because these were not related to immune system and the variants were not predicted as damaging using functional prediction algorithms. We confirmed the compound heterozygous ZAP70 mutations by Sanger sequencing (Figure 3A and 3B and Supplementary Figure 2A). The c.703-1G>A mutation was present in his father and the c.1674G>A mutation was present in his mother (Figure 3B). Sequencing of RT-PCR product from an RNA sample revealed a splice variant lacking exon 6 (Supplementary Figure 2B). Exon 6 includes the binding site of CD3 immunoreceptor tyrosine-based activation motifs (Supplementary Figure 2C), and Met558 is highly conserved (Supplementary Figure 2D). These findings suggest the compound heterozygous ZAP70 mutation c.703-1G>A and c.1674G>A is disease causing. Figure 3. View largeDownload slide Genetic analysis of compound heterozygous mutation of ZAP70. A, Schematic ZAP70 with N-SH2 domain, C-SH2 domain, and kinase domain. The position of exon 6 and M558 are shown in black. Arrows point to c.703-1G>A and c.1674G>A variants. B, Sanger sequencing of the family’s DNA. Wild-type c.703-1 or c.1674 positions are highlighted in blue. Heterozygous c.703-1G>A or c.1674G>A variants are highlighted in red. C, Flow cytometric analysis of ZAP70 expression in T cells, CD4+ T cells, and CD8+ T cells. Numbers in plots indicate the difference in mean fluorescence intensity. Figure 3. View largeDownload slide Genetic analysis of compound heterozygous mutation of ZAP70. A, Schematic ZAP70 with N-SH2 domain, C-SH2 domain, and kinase domain. The position of exon 6 and M558 are shown in black. Arrows point to c.703-1G>A and c.1674G>A variants. B, Sanger sequencing of the family’s DNA. Wild-type c.703-1 or c.1674 positions are highlighted in blue. Heterozygous c.703-1G>A or c.1674G>A variants are highlighted in red. C, Flow cytometric analysis of ZAP70 expression in T cells, CD4+ T cells, and CD8+ T cells. Numbers in plots indicate the difference in mean fluorescence intensity. Reduced Expression of ZAP70 Protein and Functional Defects in Mutant ZAP70 ZAP70 protein expression in T cells was analyzed using flow cytometry. ZAP70 expression was reduced in CD4+ and CD8+ T cells from the patient (Figure 3C). There was no difference in ZAP70 expression between CD4+ and CD8+ T cells in the patient. To determine the impact of ZAP70 mutants, ZAP70-deficient Jurkat P116 cells were transiently transduced with a vector that encoded WT ZAP70 (ZAP70WT), Met558Ile variant (ZAP70M558I), or exon 6-deleted ZAP70 (ZAP70Δexon 6). The expression levels of the 2 mutant ZAP70 proteins were reduced, suggesting degradation of the mutant protein (Figure 4A). We then analyzed TCR signaling downstream of ZAP70. Whereas CD3 cross-linking induced significant phosphorylation of SLP76 in ZAP70WT-transfected cells, this were significantly diminished in ZAP70M558I or ZAP70Δexon 6-transfected cells (Figure 4A). Furthermore, immunoprecipitation analysis showed that ZAP70Δexon 6 protein failed to bind CD3ζ chain (Figure 4B). These results indicate that the identified ZAP70 mutations are loss of function mutations due to their inability to transduce TCR signaling. Figure 4. View largeDownload slide Impaired functions of ZAP70 proteins in P116 cells transduced with ZAP70 mutants. A, Immunoblot analysis of P116 cells expressing ZAP70WT, ZAP70M558I, or ZAP70Δexon 6 stimulated with CD3 cross-linking. B, Immunoblot analysis of whole-cell lysates (WCL) and anti-CD3ζ chain immunoprecipitation. Figure 4. View largeDownload slide Impaired functions of ZAP70 proteins in P116 cells transduced with ZAP70 mutants. A, Immunoblot analysis of P116 cells expressing ZAP70WT, ZAP70M558I, or ZAP70Δexon 6 stimulated with CD3 cross-linking. B, Immunoblot analysis of whole-cell lysates (WCL) and anti-CD3ζ chain immunoprecipitation. Detection of Wild Type Allele Although the patient had compound heterozygous ZAP70 mutations and reduced expression of ZAP70, several of the findings were different from typical ZAP70 deficiency reported previously; that is, they did not meet the criteria of SCID (http://esid.org/Working-Parties/Registry/Diagnosis-criteria) or the milder CD8+ T-cell lymphopenia. Whole-exome sequencing revealed the ZAP70 mutations to be 50% of the allele frequency, and ddPCR revealed the X and Y chromosome ratio to be 1:1, which did not suggest reversion mosaicism or maternal T-cell engraftment. In order to explore the cause of the hypomorphic phenotype, we hypothesized that a small amount of normal splicing might occur despite the splice site mutation. We cloned PCR products from cDNA spanning exon 3 to 14 of ZAP70 and analyzed 45 independent clones by Sanger sequencing (Supplementary Figure 2E). Twenty clones were derived from aberrant splicing without exon 6, and 22 clones had missense mutation (c.1674G>A). The most remarkable result was that 3 clones were derived from normal splicing and did not have missense mutation (c.1674G>A). Assessment of Lymphocyte Function Signal transduction through the TCR/CD3 complex was examined in each CD4+ or CD8+ T-cell populations. First, TCR-mediated calcium mobilization was analyzed (Figure 5A and 5B). CD3 cross-linking induced a small and delayed free intracellular Ca2+ increase in the patient’s CD4+ and CD8+ T cells. In contrast, ionomycin, which is non-TCR-mediated stimulation, induced free intracellular Ca2+ increase to the same degree as in the control T cells. Second, T-cell proliferation after stimulation of the TCR was analyzed (Figure 5C and 5D). While PMA/ionomycin induced sufficient proliferation of patient T cells, anti-CD3/CD28 induced proliferation of a few but sufficient number of T cells, especially CD4+ T cells. These results demonstrate that most T cells were nonfunctional but a small proportion of T cells were normally functional, consistent with the detection of the wild-type allele. Figure 5. View largeDownload slide Signal transduction through the T-cell receptor (TCR)/CD3 complex in CD4+ or CD8+ T cells. A, Calcium mobilization induced by CD3 cross-linking (left) and ionomycin (right) in CD4+ T cells (gray, control; black, patient). B, Calcium mobilization in CD8+ T cells. C, Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled proliferation induced by anti-CD3/CD28 (left) and phorbol myristate acetate (PMA)/ionomycin (right) in CD4+ T cells. Numbers in plots indicate percent divided cells. D, CFSE-labeled proliferation in CD8+ T cells. Figure 5. View largeDownload slide Signal transduction through the T-cell receptor (TCR)/CD3 complex in CD4+ or CD8+ T cells. A, Calcium mobilization induced by CD3 cross-linking (left) and ionomycin (right) in CD4+ T cells (gray, control; black, patient). B, Calcium mobilization in CD8+ T cells. C, Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled proliferation induced by anti-CD3/CD28 (left) and phorbol myristate acetate (PMA)/ionomycin (right) in CD4+ T cells. Numbers in plots indicate percent divided cells. D, CFSE-labeled proliferation in CD8+ T cells. TCR Repertoire The existence of hypofunctional T cells led us to expect the slight skewing of TCR repertoire, as previously described in typical ZAP70 deficiency patients [14]. TCR CDR3 sequences were analyzed using next generation sequencing; this showed T-cell receptor β variable (TRBV) usage and CDR3 length were slightly skewed (Supplementary Figure 3A, 3B, and 3C). Unexpectedly, T cells from the patient had significantly skewed V-J combinations with expansion of TRBV6-5/TRBJ2-7, TRBV30/TRBJ1-2, TRBV18/TRBJ2-7, and TRBV6-6/TRBJ2-7 (Supplementary Figure 4A). Reduced diversity and uneven distribution were observed (Supplementary Figure 4B, and 4C). Junctional diversity was largely maintained (Supplementary Figure 3D). DISCUSSION We identified novel hypomorphic mutations of ZAP70 in a patient that did not manifest SCID but EBV-associated LPD, most likely following primary EBV infection. His laboratory findings indicated that impaired immunity against EBV might have been associated with the development of LPD. There are broadly similar laboratory findings between our patient and PID patients predisposed to EBV-associated LPD/lymphoma described previously. First, T cells, particularly naive T cells, were decreased but not absent, and TCR or associated costimulatory signals were impaired. Other T-cell immunodeficiencies present different clinical manifestations. PID patients without T cells most often contract other infectious diseases, which are life-threatening, before EBV infections [27]. Although SCID patients can develop EBV-associated LPD/lymphoma, it is rare as a main feature [28]. PID patients with impaired lymphocyte cytotoxicity, represented by familial hemophagocytic lymphohistiocytosis (HLH), develop fulminant infectious mononucleosis (FIM) or HLH due to an uncontrolled overwhelming hypercytokinemia produced by activated CD8+ T cells and NK cells [8]. SAP deficiency patients often develop FIM. The reason can be partially explained by the pathology of SAP deficiency, including reduced CD8+ T-cell and NK-cell cytotoxicity [8]. ZAP70 plays a pivotal role in TCR signal transduction, which is not directly related to lymphocyte cytotoxicity [12, 13]. In our patient, the development of EBV-associated LPD may have resulted from a limited resistance against the pathogen, including live vaccine strains and impaired recognition of EBV-infected cells. Lack of EBV-specific CD8+ T cells and negative titers of EBV antibodies support a globally impaired T-cell recognition of EBV antigen. Second, iNKT cell counts were remarkably decreased. A reduced number of iNKT cells has been reported in deficiencies of SAP [7, 8], ITK [9], CD27 [10], coronin-1A [29], and cytidine 5′ triphosphate synthase 1 (CTPS1) [30]. iNKT cells can directly and rapidly recognize EBV-infected cells through CD1d-mediated activation, and mediate direct cytotoxicity, which is especially critical during the earlier stage of EBV infection [31]. iNKT cells are indirectly responsible for controlling EBV infection through NK-cell, T-cell, and dendritic-cell activation by the production of interferon-γ and interleukin-2 [32]. ZAP70 is required for iNKT-cell development during positive selection, and iNKT cells are absent in Zap70 null mice [33]. Although the counts of iNKT cells in human ZAP70 deficiency is controversial [3, 34], these findings can help explain the remarkably decreased iNKT cells in the patient. The third similar manifestations between our patient and PID patients predisposed to EBV-associated LPD/lymphoma is that B-cell counts and development were normal or less impaired. The majority of EBV-infected cells are B cells, and the presence of B cells is important for EBV infection [1]. Dysgammaglobulinemia is often observed, but it is a result of impaired T-cell function or EBV infection by itself [7–9, 29, 30]. Hypomorphic ZAP70 mutation resulted in different clinical and laboratory manifestations from those of null mutation. Similar findings have been described in the CORON1A gene. While loss of function mutations of CORON1A are associated with T−B+NK+ SCID [35], hypomorphic mutations lead to PID predisposed to EBV-associated LPD/lymphoma [29]. Hypomorphic mutations due to normal splicing have been reported in some diseases [36, 37], including one ZAP70 patient [38]. This patient had had recurrent infections since infancy, and at the last follow-up, at 9 years of age, he was well without transplantation. Severe EBV infection was not observed; however, he developed severe varicella-zoster virus infection. Although the reason for the difference in susceptibility to EBV between our patient and the previously reported patient remains unclear, it may reflect clinical exposure or the impact of genetic defects, including iNKT-cell differentiation and TCR signal transduction. The TCR repertoire analysis in great detail using next generation sequencing showed significant restriction of the TCR repertoire, with reduced diversity and uneven distribution in the patient, indicating abnormal T-cell generation and the nonrandom usage of V, D, and J elements. These results confirm and extend previous findings of ZAP70 involvement at the immature single-positive thymocyte stage to the double- positive thymocyte stage [14]. Restriction of the TCR repertoire may contribute that EBV-specific CDR3 sequences could not be produced by chance, whereas other pathogen-specific CDR3 sequences could be produced, including measles and rubella. This hypothesis is supported by the observation that EBV-specific antibodies were absent whereas measles and rubella-specific antibodies were present in the patient. If many antigen-specific CDR3 sequences are known across a diverse HLA type, this analysis could be used to evaluate antigen-specific T cells [39–42]. Alterations in ZAP70 result in a wide spectrum of clinical features. While loss of function mutations of ZAP70 lead to SCID [12], hypomorphic mutations of ZAP70 seem to be associated with autoimmune disease [43, 44]. We identified novel hypomorphic mutations of ZAP70 and described a selective dysregulation of EBV infection. Our findings extend the spectrum of clinical features of ZAP70 mutations and indicate pivotal roles of T-cell recognition and iNKT cells in immune response against EBV. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Acknowledgments. We thank Dr Hideki Muramatsu for professional technical assistance. Financial support. This work was supported by the Research on Measures for Intractable Disease Project, and grants from Ministry of Education, Culture, Sports, Science and Technology of Japan (JSPS KAKENHI: grant number 26461570); and the Ministry of Health, Labour and Welfare of Japan (grant number H26-Nanchi-071). Potential conflicts of interest. All authors: No reported conflicts of interest. 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The Journal of Infectious DiseasesOxford University Press

Published: Apr 19, 2018

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