SYK expression endows human ZAP70-deficient CD8 T cells with residual TCR signaling

    SYK expression endows human ZAP70-deficient CD8 T cells with residual TCR signaling Fabian Hauck, Britta Blumenthal, Sebastian Fuchs, Christelle Lenoir, Emmanuel Martin, Carsten Speckmann, Thomas Vraetz, Wilma MannhardtLaakmann, Nathalie Lambert, Marine Gil, Stephan Borte, Marie Audrain, Klaus Schwarz, Annick Lim, Wolfgang W. Schamel, Alain Fischer, Stephan Ehl, Anne Rensing-Ehl, Capucine Picard, Sylvain Latour PII: DOI: Reference:

S1521-6616(15)30006-1 doi: 10.1016/j.clim.2015.07.002 YCLIM 7511

To appear in:

Clinical Immunology

Received date: Revised date: Accepted date:

21 May 2015 29 June 2015 1 July 2015

Please cite this article as: Fabian Hauck, Britta Blumenthal, Sebastian Fuchs, Christelle Lenoir, Emmanuel Martin, Carsten Speckmann, Thomas Vraetz, Wilma MannhardtLaakmann, Nathalie Lambert, Marine Gil, Stephan Borte, Marie Audrain, Klaus Schwarz, Annick Lim, Wolfgang W. Schamel, Alain Fischer, Stephan Ehl, Anne Rensing-Ehl, Capucine Picard, Sylvain Latour, SYK expression endows human ZAP70deficient CD8 T cells with residual TCR signaling, Clinical Immunology (2015), doi: 10.1016/j.clim.2015.07.002

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SYK expression endows human ZAP70-deficient CD8 T cells with residual TCR signaling

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Fabian Hauck, MD, PhD,a,b Britta Blumenthal, PhD,d,e Sebastian Fuchs, PhD,e,f Christelle Lenoir, MS,b

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Emmanuel Martin, PhD,b Carsten Speckmann, MD,e,g Thomas Vraetz, MD,g Wilma MannhardtLaakmann, MD,h Nathalie Lambert, MS,i Marine Gil, MS,i Stephan Borte, MD, PhD,j,k Marie Audrain, MD,l Klaus Schwarz, MD,m Annick Lim, MS,n Wolfgang W. Schamel, PhD,d,e Alain Fischer, MD,b,c,o,p

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Stephan Ehl, MD,e,g Anne Rensing-Ehl, MD,e Capucine Picard, MD, PhD, c,i, r,* Sylvain Latour, PhD,b,c,*

Dr. von Hauner Children’s Hospital, Ludwig-Maximilians-University, Munich, Germany;

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Laboratory of Lymphocyte Activation and Susceptibility to EBV Infection, INSERM UMR1163, Institut

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a

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IMAGINE, Paris, France; c

Paris Descartes University, Sorbonne Paris Cité, Imagine Institut, Paris, France;

Center for Chronic Immunodeficiency (CCI), University Medical Center, University of Freiburg,

Germany; f

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BIOSS Centre for Biological Signalling Studies, Faculty of Biology, University of Freiburg, Germany;

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d

Faculty of Biology, University of Freiburg, Freiburg, Germany

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Center for Pediatrics and Adolescent Medicine, University Medical Center, University of Freiburg,

Germany; h

Division of Pediatric Immunology and Rheumatology, Department of Pediatrics, University Hospital

Mainz, Germany; i

Study Center of Immunodeficiencies, Necker-Enfants Malades Hospital, Assistance Publique-Hôpitaux

de Paris (AP-HP), Paris, France;

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ImmunoDeficiencyCenter Leipzig (IDCL), St. Georg Hospital, Leipzig, Germany;

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Translational Centre for Regenerative Medicine (TRM), University Leipzig, Leipzig, Germany;

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Institute for Transfusion Medicine, University Hospital Ulm and Institute for Clinical Transfusion

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m

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Laboratoire d’Immunologie, CHU de Nantes, France;

Medicine and Immunogenetics Ulm, German Red Cross Blood Service, Baden-Württemberg-Hessen, Ulm, Germany;

Unité de Régulation Immunitaire et Vaccinologie, Institut Pasteur, Paris, France;

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Unité d’Immunologie et Hématologie Pédiatrique, Necker-Enfants Malades Hospital, AP-HP, Paris,

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n

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College de France, Paris , France

Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM UMR 1163, Institut

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r

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France;

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*

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IMAGINE, Necker Medical School, Paris, France.

These authors contributed equally.

Corresponding author: Sylvain Latour, PhD Laboratory of Lymphocyte Activation and Susceptibility to EBV Infection, Institut IMAGINE, 24 Boulevard du Montparnasse, Paris 75015 Cedex, France. Tel.: +33 1 42 75 43 03 Fax.: +33 1 42 75 42 21

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E-mail: [email protected].

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Abstract Autosomal recessive human ZAP70 deficiency is a rare cause of combined immunodeficiency

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(CID) characterized by defective CD4 T cells and profound CD8 T cell lymphopenia. Herein, we report two novel patients that extend the molecular genetics, the clinical and functional phenotypes

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associated with the ZAP70 deficiency. The patients presented as infant-onset CID with severe infections caused by varicella zoster virus and live vaccines. Retrospective TCR excision circle newborn screening was normal in both patients. One patient carried a novel non-sense mutation

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(p.A495fsX75) ; the other a previously described misense mutation (p.A507V). In contrast to CD4 T

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cells, the majority of the few CD8 T cells showed expression of the ZAP70-related tyrosine kinase SYK that correlated with residual TCR signaling including calcium flux and degranulation. Our findings

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highlight the differential requirements of ZAP70 and SYK during thymic development, peripheral

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homeostasis as well as effector functions of CD4 and CD8 T cells.

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ZAP70 deficiency can present with severe live vaccine adverse events

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ZAP70 deficiency is not detected by TREC-based newborn screening

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SYK partially compensates for human ZAP70 deficiency

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Polyclonal SYK- CD4 T cells have severely impaired TCR signaling

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Oligoclonal SYKinter CD8 T cells feature residual TCR signaling

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Key words:

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CID, ZAP70, SYK, TCR signaling, TREC newborn screening, live vaccine adverse event

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1. Introduction

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Human autosomal recessive ζ-chain associated protein tyrosine kinase of 70 kDa (ZAP70)

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deficiency is a rare cause of combined immunodeficiency (CID) (1). Few cases have been reported up to date (Suppl. Tab. 1, Suppl. Fig. 1). The predominant immunological features are dysfunctional CD4 T cells and profound CD8 T cell lymphopenia leading to CD4T+CD8TlowB+NK+ CID with or without

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immune dysregulation and lymphoma (2).

The non receptor protein tyrosine kinase ZAP70 is a member of the spleen tyrosine kinase

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(SYK) family and is mainly expressed in T cells (3). Upon antigen recognition, the T cell antigen receptor (TCR) triggers the activation of the lymphocyte specific protein tyrosine kinase (LCK) that in

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turn phosphorylates immunoreceptor tyrosine based activation motifs (ITAMs) of the TCRαβ:CD3:ζcomplex. ZAP70 then binds to the phosphorylated CD3 and ζ chains and subsequently is activated by

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LCK-mediated phosphorylation (3, 4). Activated ZAP70 next phosphorylates core adaptor proteins

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and acts as T-cell master kinase by amplifying and diversifying the initial TCR signal (5,6).

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Most of our knowledge arrises from the Zap70-/- murine model and only few detailed TCR signaling studies have been carried out in ZAP70-deficient human T cells (7). Importantly, the phenotype of the prototypic ZAP70-deficient mouse model differs from its human counterparts, as it displays an absolute intrathymic block at the double positive (DP) to single positive (SP) transition and consequently is resulting in combined CD4 and CD8 T cell lymphopenia (8). Herein, we report the clinical and genetic features of two novel ZAP70-deficient patients and we further study the TCR signaling cascade from their peripheral T cells. We demonstrate that oligoclonal ZAP70-deficient CD8 T cells express SYK and display residual TCR signaling such as activating protein phosphorylation, calcium flux and degranulation, in contrast to polyclonal ZAP70deficient CD4 T cells in which SYK is only express in small proportion of cells and TCR signaling is abrogated.

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2. Materials and methods

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2.1 Approval

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We obtained written informed consent for participation in the study from the parents of the patients. The study was approved by the institutional review boards at the Necker-Enfants Malades Hospital, Paris, France and at the Center for Pediatrics and Adolescent Medicine, University Medical

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Center, University of Freiburg, Germany.

2.2 Genetics

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Genomic DNA and RNA were isolated from peripheral blood mononuclear cells (PBMCs). ZAP70 was

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amplified from DNA and RNA and sequenced as previously described (10).

2.3 Flow cytometry and cell sorting

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Cells were labeled according to standard protocols with previously published antibodies, data was recorded on a FACSCanto II (BD Biosciences®) and analyzed with FlowJo software (TreeStar®) (11). CD3+CD4+, CD3+CD8+TCRVβ8- and CD3+CD8+TCRVβ8+ T cells were sorted on a MoFlo Astrios (Beckman Coulter®) following standard procedures. For phosphoflow, cells were stimulated with OKT3 (10 µg/ml) and anti-CD28 (10 µg/mL) or pervanadate (200 µM) for 5 min. Cells were fixed (BD Biosciences®, Cytofix), stained for surface antigens, permeabilized (BD Biosciences®, Perm buffer III) and stained with anti-SYK (Miltenyi Biotec) or -ZAP70 antibodies (eBiosciences) or –phosphoZAP70/Syk, -phospho-p38 or -phospho-AKT from BD Biosciences® (BD Phosflow).

2.4 Cell culture, stimulation, and immunoblotting

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T cell blasts were induced from PBMCs with phorbol 12-myristate 13-acetate (PMA) (10 ng/mL) and ionomycin (1 µM) for two days, and were cultured in RPMI 1640 with 10% human serum and IL-2

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(100 U/mL) for additional eight days. 5 x 106 cells were stimulated with soluble OKT3 (1 µg/mL) crosslinked with rabbit anti-mouse IgG (2 µg/mL) or PMA (10 ng/ml) and ionomycin (1 µM) for the

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indicated times. Cells were lysed as previously described and immunoblotting was performed following standard protocols. Antibodies used have been published elsewhere previously, excepted

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anti-SYK antibodies from Cell Signaling Technology® (11, 12).

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2.5 Ca2+ and Mg2+ flux

PBMCs were loaded with Indo-1-AM (5 µM) or MAG-Indo-1-AM (5 µM), stained with CD4-APC and

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CD8-PE antibodies, stimulated with OKT3 (1 µg/mL) crosslinked with rabbit anti-mouse IgG (10 µg/ml) for eight minutes and thereafter with ionomycin (1 µM) followed by EDTA (10 mM) as

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previously described (11, 13). Data was recorded on a FACS ARIA II (BD Biosciences®) and Ca2+ and

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Mg2+ flux were analyzed with FlowJo software 7.2.5 (TreeStar®) (11, 13).

2.6 Degranulation and induced apoptosis Degranulation was performed with T cell blasts at day 9 and day 22 of culture as previously described with the exception that anti-CD107b antibody was used (12). Activation-induced cell death was triggered by stimulating T cell blasts at day 9 of culture with coated anti-CD3 (clone OKT3) for 24h as described (11, 12).

2.7 TCR repertoire Total PBMC RNA was extracted, reverse transcribed and real-time quantitative analysis of the T cell repertoire with the immunoscope technique was determined as previously described (11). TCRVβ

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usage was analysed for CD3+CD4+ and CD3+CD8+ T cells by flow cytometry with the IOTest® Beta Mark (Beckman Coulter) following the manufacturer’s instructions.

Results

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3.

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3.1 Clinical phenotype and genotype

Patient 1 (P1) (Tab. 1) was born in France to first-degree consanguineous Kurdish parents. At eleven days, he was hospitalized with severe respiratory syncytial virus (RSV) bronchiolitis. At five months,

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he developed profuse rotavirus (RV) gastroenteritis and severe VZV dermatitis superinfected with Staphylococcus aureus. Despite intensive anti-viral aciclovir treatment, the VZV infection spread

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systemically resulting in acute respiratory distress syndrome what required mechanical ventilation complicated by multiple pneumatothoraces. Additional opportunistic pulmonary infections with

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Pneumocystis jirovecii and Stenotrophomonas maltophilia and central line-associated Staphylococcus aureus sepsis occured during ventilation.

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Genetic analysis of the ZAP70 gene in P1 revealed an as yet undescribed homozygous

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amorphic splice site mutation (g.24189G>C (IVS11-1G>C) leading to a c.1483-1495del13 and p.A495fsX75 ZAP70) causing a complete loss of protein expression (Fig. 1A, Suppl. Fig. 1). At nine

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months, P1 underwent hematopoietic stem cell transplantation (HSCT) from a matched unrelated donor (MUD). At day 8 post HSCT, he developed a skin graft-versus-host disease grade III that was controlled with immunosuppressants. At 2 years post-HSCT, he developed at the time of immunological reconstitution a hydrocephalia with intracranial hypertension and lymphocytic pericarditis that were controlled with immunosuppressants (steroids and sirolimus). Currently, at 3 years post-HSCT, he is fine and well on low doses of steroids, sirolimus, acetazolamide, IVIgG and prophylactic PenV and presents a stable donor chimerism of 98%. Patient 2 (P2) (Tab. 1) was born in Germany to first-degree consanguineous Turkish parents. From the age of six months, he presented with recurrent bronchial and pulmonary infections. At twelve months, he received one 4-valent (measles, mumps, rubella, and VZV) attenuated live vaccine

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and developed transient exanthema at the injection site. At 14 months, he was hospitalized with failure to thrive, oral thrush, generalized papulopustular exanthema and Acinetobacter baumannii

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sepsis. At 24 months, he was rehospitalized with pneumonia and progressively deteriorated with vaccination strain VZV encephalitis.

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P2 was found to carry an already reported homozygous ZAP70 g.24227C>T (c.1520C>T) missense mutation (9) that codes for an amorphic p.A507V ZAP70 protein (Fig. 3A and Suppl. Fig.1). In CD4+, CD8+TCRVβ8-, and CD8+TCRVβ8+ T cells of P2 sorted by FACS only the homozygous ZAP70

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g.24227C>T mutation was detected, thus excluding mosaicism or somatic reversion in the oligoclonal

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CD8 T cell population (data not shown). At thirty months, P2 underwent HSCT from a matched unrelated donor after reduced intensity conditioning. However, cerebrospinal fluid was tested

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positive for vaccination strain rubella and measles viruses and magnetic resonance imaging showed encephalic lesions in brainstem and thalamus. He deceased 8 days later due to recurrent breathing

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arrests.

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In summary, both patients presented the clinical picture of infant-onset CID characterized by severe and life threatening (opportunistic) bacterial, viral, and fungal infections and life limiting

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vaccination strain infections, respectively.

3.2 Immunological phenotype Immunological phenotypes of P1 and P2 are summarized in Tab. 1. TCR excision circle (TREC) and kappa-deleting recombination excision circle (KREC, only determined for P2) newborn screening retrospectively performed from neonatal Guthrie cards with cutoff-levels used in prospective newborn screening programs were normal. At the age of seven months and two years, respectively, both patients showed profound CD8 T cell lymphopenia while CD4 T cell, regulatory T cell (only determined for P1), NK, and B cell counts were normal. αβ and γδ T cell proportions were normal,

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but P1 lacked invariant natural killer T (iNKT) and mucosa associated invariant T (MAIT) cells (not determined for P2).

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Thymic output, as judged by recent thymic emigrants (RTEs) corresponding to CD4+CD45RA+CD31+ T cells (Tab. 1), was diminished and there was loss of naivety in both the CD4 and

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CD8 T cell compartments. TCR clonality analysis of total T cells for P1 by immunoscope technique showed an unbiased TCRαβ and TCRγδ repertoire with gaussian-like length distribution due to random P- and N-nucleotide additions with the exception of  T cells in which the proportion of cells

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with TCRVd2 and TCRVg9 are decreased (data not shown). Similar biased repertoire in  T cells was

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reported in a LCK-deficient patient (11). For P2, TCRVβ expression analysis by flow cytometry showed an unbiased repertoire for CD4 but a biased repertoire for CD8 T cells with TCRVβ1 and TCRVβ8

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predominating (data not shown). Proliferation capacity of CD4 and CD8 T cells of P1 and P2 was abrogated (Tab. 1 and data not shown). The serum of P2 was also tested (before BMT) for the

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presence of antoantibodies. 24 different specificities were tested (including ANA) and were found to

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3.3 TCR signaling

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be negative or below the reference values (data not shown).

To study the role of ZAP70 in TCR signaling, T cell blasts from an healthy control (Ctr.) and P1 were obtained by PMA and ionomycin stimulation to bypass the anticipated proximal ZAP70 signaling defect. The proportion of CD8+ T cells was very low before stimulation and only slightly increased after culture following stimulation with PMA/ionomycin (Table 1, Fig. 2A and C). First, the expression of different molecules involved in the TCR signaling, including the ZAP70 and SYK kinases, the SRC family kinases LCK and FYN, the TEC family kinase interleukin-2-inducible T cell kinase (ITK), the signaling molecule linker for activation of T cells (LAT), the phospholipase Cγ1 (PLCγ1), the protein kinase Cθ (PKCθ), and the extracellular signal-regulated kinase 1 and 2 (ERK1/2) was examined in the T cell blasts of P1. As expected, ZAP70 expression was not detectable. The majority

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of the other signaling molecules were normally expressed, with the exception of SYK which was overexpressed, as well as LAT and PKCθ but to a lesser extent (Fig. 1A). To screen for proximal TCR

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signaling, global tyrosine phosphorylation analysis by immunoblotting was performed in CD3stimulated T-cell blasts and found to be defective (Fig. 1B). Analysis of specific tyrosine

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phosphorylated substrates of the TCR signaling was further examined and showed as expected that phosphorylation of ZAP70 (pZAP70) was absent, hardly any phosphorylation of LAT (pLAT) was detectable, and phosphorylation of PLCγ1 and PKCθ (pPLCγ1 and pPKCθ) were weakened and

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delayed (Fig. 1C). More distal TCR signals were next evaluated. Firstly, activation of the nuclear factor of activated T cells c2 (NFATc2) and the ERK1/2 kinases pathway were analyzed and found to be

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defective as revealed by the absence of NFATc2 dephosphorylation and ERK1/2 phosphorylation (Fig. 1C). Secondly, TCR-induced second messengers were analyzed in CD4 and CD8 T cells from PBMCs of

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P1 (Fig. 2B). CD4 T cells showed no detectable Mg2+ and Ca2+ flux and had a lower Mg2+ baseline. Of note, CD8 T cells displayed an intermediate Ca2+ flux, while Mg2+ flux could not be analysed due to

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scarcity of the cell population. Consistent with the intermediate Ca2+ flux in CD8 T cells, TCR-induced

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degranulation of CD8 T cell blasts was not completely abrogated since a few CD8+CD107b+ were detectable corresponding to cells that have degranulated (Fig. 2C-E). However, activation induced-

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cell death of T cell blasts from P1 (which mostly contained CD4 T cells) was absent and not detected (Supplementary Fig. 2).

To corroborate these findings, ZAP70 and SYK expression were analyzed by flow cytometry in T cells of P2, no ZAP70 but residual SYK expression was detected (Fig 3A). When gating on CD4 and CD8 T cells, P2 showed a majority of CD8 T cells expressing SYK (87.8%) while a minor fraction of CD4 T cells (17.6 %) expressed high levels of SYK (SYKhigh) (14) (Fig 3B and C). To analyze TCR signaling by flow cytometry, T cells were stimulated with anti-CD3/CD28 or treated with the protein tyrosine phophatase inhibitor pervanadate. CD4 T cells did not display phosphorylated (pZAP70) after antiCD3/CD28 or pervanadate. However, after pervanadate, ZAP70-deficient CD8 T cells of P2 showed a slight signal increase after staining with the pZAP70 antibody that is known to crossreact with pSYK

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(pY352) (Fig. 3D). Further downstream signaling events were also analyzed in T cells of P2. Following anti-CD3/CD28 stimulation, CD4 T cells did not show phosphorylation of the mitogen activated

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protein kinase p38 and the serine-threonine kinase AKT, while they responded to pervanadate. In contrast, CD8 T cells showed residual phosphorylation of p38 and AKT after anti-CD3/CD28 and full

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phosphorylation after pervanadate (Fig. 3D). In isolated P2 T cells, stimulation with crosslinked antiCD3 antibody did not induce a global Ca2+ flux (Supplementary Fig. 3). However, a minority of cells responded, but as no CD4 and CD8 staining was included, differential analysis of CD4 versus CD8 T

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cells was not possible. Because P1 CD8 T cells displayed an intermediate Ca2+ flux, it can be

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speculated that the small cell population responding in P2 are also CD8 T cells. Taken together, these results indicate that TCR-induced signaling in ZAP70-deficient CD4 T

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cells is almost completely abrogated, while some proximal and distal signals like Ca2+ flux, p38 and AKT activation and degranulation are partially preserved in ZAP70-deficient, SYK-expressing CD8 T

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cells.

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4. Discussion

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The majority of reported ZAP70-deficient patients presented with infant CID, but immune dysregulation, malignancy, and late-onset CID have been described as well (2, 10, 15). We report two

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novel infant-onset CID presentations with severe VZV and lethal live vaccine infections, respectively, thus expanding the clinical spectrum of ZAP70 deficiency. Thirteen human ZAP70 mutations including one hypomorphic mutation and one case of unknown genomic cause with undetectable mRNA have

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been described (Suppl. Tab. 1). We now add on a novel autosomal recessive splice site mutation (p.A495fsX75) that abrogates ZAP70 expression. We find normal retrospective TREC and KREC copy

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numbers in the original newborn screening Guthrie cards associated with polyclonal CD4, but oligoclonal CD8 T cells. Human ZAP70 deficiency does not interfere with V(D)J-recombination and

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positive selection of CD4 SP T cells, but more selectively disturbes CD8 SP T cell lineage development, and therefore can not be detected by TREC-based newborn screening as already reported (16). At

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the time of diagnosis however, both patients presented as CD4T+CD8TlowB+NK+ CID phenotype with

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reduced thymic output and loss of T cell naivety. Like in other TCR signaling disorders such as LCK and ITK deficiency (F. Hauck and S. Latour, unpublished data) thymic output is suboptimal and seems to

Tcells (11).

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decline during infancy, and in the setting of recurrent infections is associated with a decline of naïve

The prototypic ZAP70-deficient mouse model differs from its human counterparts as it displays an absolute intrathymic block at the DP to SP transition and consequently has combined CD4 and CD8 lymphopenia (8). Murine studies have shown that SYK is involved in the early stages of T-cell development in the thymus and SYK expression is efficiently downregulated by pre-TCR signaling (17). This is different in humans since SYK is expressed until the CD4 SP stage as well as in ZAP70deficient HTLV-1-transformed thymocytes, indicating that SYK can mediate TCR signaling and CD4 SP T cell development (18). However, what drives the residual CD8 T cell development and how

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peripheral ZAP70-deficient CD4 T cells receive homeostatic TCR signaling remains controversial (9, 19, 20).

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We observed that the majority of CD8 peripheral ZAP70-deficient T cells expressed intermediate amounts of SYK while a minor proportion of CD4 T cells expressed high amounts of SYK

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(SYKhigh). Culture-expanded ZAP70-deficient SYKhigh CD4 T cells with alternative TCR signaling have been described before (9) (Suppl. Tab. 1). Interestingly, human effector CD4 T cells can express a SYK:FcRγ chain complex that replaces the ZAP70:ζ-chain complex for TCR signaling (14). Thus, the

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minor fraction of ZAP70-deficient SYKhigh CD4 T cells might be effector T cells that have re-expressed

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SYK in the secondary lymphoid organs. The major fraction of polyclonal CD4 T cells, which were negative for SYK (described above) exhibited abortive and weak TCR signaling, that might however,

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be sufficient for peripheral homeostatic proliferation. By contrast and for the first time, we showed that the majority of peripheral and oligoclonal ZAP70-deficient CD8 T cells expressed SYK and

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generated rapid and intermediate-strength TCR signaling allowing residual TCR-mediated effector

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functions of CD8 T cells as shown their ability to degranulate. Saini and colleagues showed that in ZAP70-deficient mice, harboring an inducible Zap70

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transgene, CD4 SP lineage choice timely preceded the one of CD8 SP and was induced by priming TCR signals that were dependent on LCK and standard amounts of additional signaling components. CD8 SP lineage choice took place thereafter and depended on prolonged and increasingly strong TCR signals that resulted from developmentally controlled downregulation of the inhibitory receptor CD5 and upregulation of the TCR and ZAP70 (21). Our data and previous studies of ZAP70-deficient patients pinpoint to a similar sequential CD4 and CD8 SP lineage choice in human T cell development that is governed by increasing TCR signaling strength (10, 18). In ZAP70-deficient human HTLV-1 transformed thymocytes, CD4 SP T cell development was less dependent on ZAP70 than that of CD8 SP T cells, as the absence of ZAP70 could be compensated by SYK, for TCR signaling and sufficient for the development of polyclonal CD4 T cells only (18). However as SYK was downregulated in peripheral CD4 T cells, the SYK-mediated TCR signaling capacity was lost thereafter (17, 18). In

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contrast, SYK was not able to fully restore the development of SP CD8 T cells, only enabling the development of a few peripheral CD8 T cells. Importantly, we observed that these peripheral ZAP70-

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deficient CD8 T cells expressed SYK and exhibited rapid and intermediate-strength TCR signal most likely provided by SYK. This further argues for differential requirements of ZAP70 for TCR signalling

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during thymic development and peripheral homeostasis as well as effector functions of CD4 and CD8

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T cells.

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5. Conclusion

ZAP70 deficiency is not detected by TREC-based newborn screening and can present as infant-onset

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CID with severe systemic VZV or live vaccine complications. SYK partially compensates for human ZAP70 deficiency. Most of polyclonal peripheral CD4 T cells did not express SYK and displayed

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severely impaired TCR signaling. However, oligoclonal peripheral CD8 T cells expressed SYK and showed residual TCR signaling, that might have overcome the signaling threshold necessary for their

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development and might endow them with partial effector functions.

Conflict of interest and financial disclosure There were no conflicts of interest to report in this study.

Acknowledgements We thank the patients and their famillies for participating in this study. We acknowledge the contribution of all collaborators involved in this trans-center study. This work was supported by grants from INSERM, ANR (ANR-08-MIEN-012-01, ANR-2010-MIDI-005-02 and ANR-10-IAHU-01),

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Fondation ARC (France), the European Research Council (ERC-2009-AdG_20090506 n°FP7-249816), the German Research Council/DFG (HA5967/1-1 and EXC294), the German Federal Ministry of

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Education and Research (Bundesministerium fuer Bildung und Forschung 1 EO 0803). F.H. was was fellowship recipient of the IMAGINE Foundation (MD/PhD-Programme) and the German Federal

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Ministry of Education and Research (BMBF 01EO1303 and BMBF 1315883). S. L. is a senior scientist

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of CNRS (France). E. M. was supported by the ANR (France) and the Ligue contre le cancer (France).

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Picard C, Dogniaux S, Chemin K, Maciorowski Z, Lim A, Mazerolles F, et al. Hypomorphic mutation of ZAP70 in human results in a late onset immunodeficiency and no autoimmunity. Eur J Immunol 2009; 39:1966-76.

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Hauck F, Randriamampita C, Martin E, Gerart S, Lambert N, Lim A, et al. Primary T-cell immunodeficiency with immunodysregulation caused by autosomal recessive LCK deficiency. J Allergy Clin Immunol 2012; 130:1144-52 e11.

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Martin E, Palmic N, Sanquer S, Lenoir C, Hauck F, Mongellaz C et al. CTP synthase 1 deficiency in humans reveals its central role in lymphocyte proliferation. Nature. 2014; 510:288-92

13.

Li F, Chaigne-Delande B, Kanellopoulou C, Davis JC, Matthews HF, Douek DC et al.

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Second messenger role for Mg2+ revealed by human T-cell immunodeficiency. Nature. 2011; 475:471-6. Krishnan S, Warke VG, Nambiar MP, Tsokos GC, Farber DL. The FcR gamma subunit and Syk kinase replace the CD3 zeta-chain and ZAP-70 kinase in the TCR signaling complex of human effector CD4 T cells. J Immunol 2003; 170:4189-95.

15.

Newell A, Dadi H, Goldberg R, Ngan BY, Grunebaum E, Roifman CM. Diffuse large B-cell lymphoma as presenting feature of Zap-70 deficiency. J Allergy Clin Immunol 2011; 127:51720.

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Grazioli S, Bennett M, Hildebrand KJ, Vallance H, Turvey SE, Junker AK. Limitation of TRECbased newborn screening for ZAP70 Severe Combined Immunodeficiency. Clin Immunol 2014; 153:209-10.

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Chu DH, van Oers NS, Malissen M, Harris J, Elder M, Weiss A. Pre-T cell receptor signals are responsible for the down-regulation of Syk protein tyrosine kinase expression. J Immunol 1999; 163:2610-20.

18.

Gelfand EW, Weinberg K, Mazer BD, Kadlecek TA, Weiss A. Absence of ZAP-70 prevents signaling through the antigen receptor on peripheral blood T cells but not on thymocytes. J Exp Med 1995; 182:1057-65.

19.

Elder ME, Skoda-Smith S, Kadlecek TA, Wang F, Wu J, Weiss A. Distinct T cell developmental consequences in humans and mice expressing identical mutations in the DLAARN motif of ZAP-70. J Immunol 2001; 166:656-61.

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Toyabe S, Watanabe A, Harada W, Karasawa T, Uchiyama M. Specific immunoglobulin E responses in ZAP-70-deficient patients are mediated by Syk-dependent T-cell receptor signalling. Immunology 2001; 103:164-71.

21.

Saini M, Sinclair C, Marshall D, Tolaini M, Sakaguchi S, Seddon B. Regulation of Zap70 expression during thymocyte development enables temporal separation of CD4 and CD8 repertoire selection at different signaling thresholds. Sci Signal 2010; 3:ra23.

22.

Fuchs S, Rensing-Ehl A, Speckmann C, Bengsch B, Schmitt-Graeff A, Bondzio I, et al. Antiviral and regulatory T cell immunity in a patient with stromal interaction molecule 1 deficiency. J Immunol 2012; 188:1523-33.

23.

Bryceson YT, Pende D, Maul-Pavicic A, Gilmour KC, Ufheil H, Vraetz T, et al. A prospective evaluation of degranulation assays in the rapid diagnosis of familial hemophagocytic syndromes. Blood 2012; 119:2754-63.

24.

Rigaud S, Fondaneche MC, Lambert N, Pasquier B, Mateo V, Soulas P, et al. XIAP deficiency in humans causes an X-linked lymphoproliferative syndrome. Nature 2006; 444:110-4.

25.

Lim A, Baron V, Ferradini L, Bonneville M, Kourilsky P, Pannetier C. Combination of MHCpeptide multimer-based T cell sorting with the Immunoscope permits sensitive ex vivo

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quantitation and follow-up of human CD8+ T cell immune responses. J Immunol Methods 2002; 261:177-94.

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Figure 1. Defective TCR signaling in T cell blasts of patient P1.

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Figure Legends

A, Expression of SYK family kinases ZAP70 and SYK, SRC family kinases LCK and FYN, TEC family kinase ITK, and signaling molecules LAT, PLCG1 (PLC1), PKCtheta (PKCθ)and ERK1/2 in T cells of P1 and a

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control healthy donor (Ctr.) by immunoblotting. B, Kinetics of anti-CD3 and PMA plus ionomycin (P+I)

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stimulations of T cells of P1 and Ctr. Cell lysates were analyzed for their content in tyrosine phosphorylated proteins by immunoblotting with anti-phosphotyrosine antibody (P-Tyr) with ACTIN as loading control . C, same as in B with the exception that cell lysates were analyzed for

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phosphorylated forms (p-) of ZAP70, LAT, PLCG1, PKCtheta, NFATC2, and ERK1/2 with ACTIN as

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loading control.

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Figure 2. Residual TCR signaling in CD8+ T cells of patient P1. A, Dot plots of control (red and blue) and P1 (green and orange) primary CD4 and CD8 T cells from PBMCs of patient P1 and a control healthy donor (Ctr.). B, the corresponding curves of Mg2+ and Ca2+ flux kinetics after stimulation with anti-CD3 (1), cross-linker (2), and ionomycin (3) or quenching with EDTA (4). C, Dot plots of CD4 and CD8 T cell blasts at day 22 of culture. D, Representative dot blots of CD8 and CD107b, a marker of degranulation T cell blasts at day 22 of culture after stimulation with 3 µg/ml of anti-CD3 antibodies. E, CD3-induced degranulation from T cell blasts at day 22 of culture. The proportion (%) of CD8+CD107b+ cells were calculated from flow-cytometry dot plots as shown in C. Numbers in dot plots correspond to percentages of cells in gates.

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Figure 3. Expression of SYK and residual TCR signaling in primary CD8+ T cells of patient P2. A. Expression of ZAP70 and SYK in primary CD3+ T cells from PBMCs of patient P2 and a control

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healthy donor (Ctr.) shown by flow cytometry histograms. B, Dot plots of CD4 and CD8 T cells among the CD3+ T cells of P1 and Ctr. Percentages of cells in the gates are indicated. C, Expression of ZAP70

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and SYK in primary CD4+ and CD8+ T cells, same as in A. Percentages of cells expressing SYK are indicated. D, Analysis of phosphorylation of ZAP/SYK (pZAP70/SYK), AKT (pAKT) and p38 MAP (pp38) kinases in CD4 and CD8 T cells in response to anti-CD3 plus anti-CD28 (aCD3/CD28) stimulation by

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flow cytometry. Histograms corresponding to unstimulated (No stim.) are in red, to aCD3/CD28 in

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green, and the non-specific tyrosine activator pervanadate in blue.

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ACCEPTED MANUSCRIPT Table 1. Genotype, phenotype and clinical course of ZAP70-deficient patients. P1 (7 months)

P2 (2 years)

gDNA

g.24189G>C (IVS11-1G>C)

g.24227C>T

cDNA

c.1483-1495del13

c.1520C>T

Protein

p.A495fsX75

p.A507V

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Newborn screening (copies/µl)2 TREC

150 (>100)

KREC

n.d.

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+

3

3,600 (2,300-5,400)

4,464 (1,900-5,900)

1,757 (1,400-3,700)

4,248 (1,400-4,300)

1,658 (700-2,200)

72 (500-1,700)

72 (490-1,300)

3 (0-10)

0.5 (0-10)

96 (90-100)

98 (90-100)

0 (n.d.)

n.d.

0 (n.d.)

n.d.

6

12 (58.8-83.0)

19.9 (65.0-79.5)

7

5.4 (3.6-9.0)

n.d.

31.3 (83.5-94.7)

42.6 (71.5-84.2)

37.9 (4.8-13.3)

49.8 (14.9-25.5)

26.2 (0.2-2.7)

6.7 (0.5-2.2)

4.6 (0.1-1.5)

0.9 (0.0-0.9)

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7,200 (3,400-9,000)

T cells (/µl) +

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CD3 T cells +

CD4 T cells +

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CD8 T cells

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T cells (%) +

TCR γδ T cells +

+

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TCR αβ T cells +

+

Vα24 Vβ11 CD161 iNKT cells +

+

Vα7.2 CD161 MAIT cells +

+

+

CD4 CD45RA CD31 RTE +

58 (>8) 57 (>6)

Lymphocytes (/µl) CD45 lymphocytes

+

low

CD4 CD25 CD127

TReg

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ZAP70 genotype1

4

5

+

+

+

+

+

-

+

+

9

+

-

-

-

10

+

+

-

-

+

+

+

+

4.1 (68.7-96.6)

1.1 (57.1-91.4)

+

-

+

+

1.4 (3.1-11.6)

2.1 (5.9-22.3)

CD4 CD45RA CCR7 /CD27 TN

8

CD4 CD45RA CCR7 /CD27 TCM CD4 CD45RA CCR7 /CD27 TEM

CD4 CD45RA CCR7 /CD27 TEMRA CD8 CD45RA CCR7 /CD27 TN CD8 CD45RA CCR7 /CD27 TCM

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-

-

-

49.7 (0.1-7.1)

22,7 (0.3-6.8)

+

+

-

-

44.8 (0.2-12.7)

50,4 (0.6-16.4)

PHA (6.25 µg/ml)

0.85 (>50)

defective

OKT3 (50 ng/ml)

0.25 (>30)

defective

13

0.25 (>10)

CD8 CD45RA CCR7 /CD27 TEM CD8 CD45RA CCR7 /CD27 TEMRA

T cell proliferation (cpm x 103)

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-7

-5

+

792 (160-950)

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CD3 CD16 CD56 NK cells

n.d.

39 (>80)

NK cells (/µl) +

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0.25 (>10)

PMA (10 M) + ionomycin (10 M)

-

n.d.

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VZV antigen (n.d.)

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Tetanus toxoid (300 ng/ml)

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B cells (/µl) +

1,872 (610-2,600)

Serum immunoglobulins (g/l) IgM

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Viral infections

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IgA

1,134 (390-1,400)

0.87 (0.36-1.04)

0.56 (0.52-2.20)

0.61 (3.5-11.8)

3.99 (5.4-13.4)

0.1 (0.36-1.65)

1.84 (0.30-1.88)

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IgG

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CD19 B cells

634 (130-720)

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RSV bronchiolitis 18

recurrent UAI

17

19

RV gastroenteritis

recurrent LAI

VZV dermatitis,

aMMRV

pneumonitis, sepsis

post-vaccinal rash

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aMMRV reactivation dermatitis aM pneumonitis aMRV encephalitis

Bacterial infections S. aureus dermatitis

recurrent UAI

S. aureus sepsis

recurrent LAI

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ACCEPTED MANUSCRIPT S. maltophilia pneumonia

A. baumanii sepsis

P. jirovecii penumonitis

C. albicans mucositis

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Fungal infections

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n.d.: not determined, gDNA, cDNA and protein reference sequences NG_007727.1, NM_001079.3 and 2 3 NP_001070.2 are used, the original newborn Guthrie cards were retrieved, age-matched best-fitting normal 1, 2 4 values are indicated in parentheses and are derived from references , iNKT: invariant natural killer T cells, 5 6 7 8 MAIT: mucosa-associated invariant T cells, RTE: recent thymic emigrants, TReg: regulatory T cells, TN: naive T 9 10 11 cells, TCM: central memory T cells, TEM: effector memory T cells, TEMRA: exhausted memory-like CD45RA 12 13 expressing T cells, T cell proliferation was analysed by CSFE-dilution, P1 had received two 5-valent dead and 14 15 one 7-valent conjugate vaccine at the ages of two and four months, VZV: varicella zoster virus, P1 had 16 17 experience VZV wildtype infection over two months, RSV: respiratory syncytial virus, UAI: upper airway 18 19 20 infection, RV: rotavirus, LAI: lower airway infection, aMMRV: attenuated measles/mumps/rubella/varicella vaccination strains.

Shearer WT, Rosenblatt HM, Gelman RS, Oyomopito R, Plaeger S, Stiehm ER, et al. Lymphocyte subsets in healthy children from birth through 18 years of age: the Pediatric AIDS Clinical Trials Group P1009 study. J Allergy Clin Immunol 2003; 112:973-80.

2.

van Gent R, van Tilburg CM, Nibbelke EE, Otto SA, Gaiser JF, Janssens-Korpela PL, et al. Refined characterization and reference values of the pediatric T- and B-cell compartments. Clin Immunol 2009; 133:95-107.

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Highlights:  ZAP70 deficiency can present with severe live vaccine adverse events  ZAP70 deficiency is not detected by TREC-based newborn screening  SYK partially compensates for human ZAP70 deficiency  Polyclonal SYK- CD4 T cells have severely impaired TCR signaling  Oligoclonal SYKinter CD8 T cells feature residual TCR signaling

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