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Human adaptive immunity rescues an inborn error of innate immunity

Laura Israel1,2, Ying Wang3, Katarzyna Bulek4, Erika Della Mina1,2, Zhao Zhang3,

Vincent Pedergnagna1,2 Maya Chrabieh1,2, Nicole A. Lemmens5,

Vanessa Sancho-Shimizu1,2,6, Marc Descatoire2,7, Théo Lasseau1,2, Elisabeth Israelsson8,

Lazaro Lorenzo1,2, Ling Yun1,2, Aziz Belkadi1,2, Andrew Moran9, Leonard E. Weisman10,

François Vandenesh11,12, Frederic Batteux13, Sandra Weller7, Michael Levin6,

Jethro Herberg6, Avinash Abhyankar14, Carolina Prando14, Yuval Itan14,

Willem van Wamel5, Capucine Picard1,2,15,16, Laurent Abel1,2,14, Damien Chaussabel8,

Xiaoxia Li4, Bruce Beutler3, Peter D. Arkwright9,

Jean-Laurent Casanova1,2,14,15,17,# and Anne Puel1,2,14,#

1 Laboratory of Human Genetics of Infectious Diseases, Necker Branch, UMR 1163, Necker Medical School, Imagine Institute, Paris, France, EU 2 Paris Descartes University, Paris, France, EU 3Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, Texas, USA 4 Department of Immunology, Cleveland Clinic, Cleveland, OH 44195, USA 5 Medical Microbiology and Infectious Diseases, CN Rotterdam, The Netherlands, EU 6 Paediatric Infectious Diseases Group, Division of Medicine, Imperial College London, London, UK 7Necker-Enfants Malades, INSERM U1151-CNRS UMR 8253, Sorbonne Paris Cité, Université Paris Descartes, Faculté de Médecine-Site Broussais, 75014 Paris, France, EU 8 Benaroya Research Institute at Virginia Mason, Seattle, Washington, USA 9 University of Manchester, Royal Manchester Children’s Hospital, Manchester M13 9WL, UK 10 Baylor College of Medicine, Texas Children's Hospital, Houston, TX, USA 11 UMR U1111 INSERM Université de Lyon, Lyon, France, EU 12 Hospices Civils de Lyon, Groupement Hospitalier Est, Lyon, France, EU 13 EA 1833, Faculté de Médecine, Université Paris Descartes, Sorbonne Paris-Cité, Service d'Immunologie Biologique, Hôpital Cochin, Paris, France, EU 14 St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, New York, USA 15 Pediatric Hematology-Immunology Unit, Assistance Publique Hôpitaux de Paris, Necker Hospital, Paris, France, EU 16 Study Center for Primary Immunodeficiencies, Assistance Publique Hôpitaux de Paris, Necker Hospital, Paris, France, EU 17 Howard Hughes Medical Institute, USA # Equal contributions Correspondence: JLC (jean-laurent.casanova@rockefeller.edu) or AP (anne.puel@inserm.fr)

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Summary

The molecular basis of incomplete penetrance of monogenic disorders is unclear. We

describe here eight related individuals with autosomal recessive TIRAP deficiency. The

proband had life-threatening staphylococcal disease in childhood, unlike the other seven

homozygotes. Responses to all TLR1/2, TLR2/6, and TLR4 agonists are impaired in the

fibroblasts and leukocytes of all TIRAP-deficient individuals. However, whole blood

response to TLR2/6 agonist staphylococcal lipoteichoic acid (LTA), is abolished only in the

index case, who is the only family member lacking LTA-specific Abs. This defective

response is reversed in the patient, but not in IRAK-4-deficient individuals, by the addition

of anti-LTA mAb. Moreover, anti-LTA mAb rescues the response of macrophages from

mice lacking TIRAP, but not TLR2 or MyD88. Acquired anti-LTA Abs therefore rescue

TLR2-dependent immunity to staphylococcal LTA in individuals with inherited TIRAP

deficiency, accounting for incomplete penetrance. Combined deficiency of TIRAP and anti-

LTA Abs underlies staphylococcal disease in the patient.

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Graphical abstract

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Introduction

Staphylococcus aureus is a Gram-positive bacterium that colonizes the skin and

nostrils of most healthy individuals (Li et al., 2015; Mulcahy and McLoughlin, 2016). It is

also a common cause of human diseases, ranging from minor cutaneous infections to

invasive hematogenous infections, as observed in children and young adults with

necrotizing staphylococcal pneumonitis (Gillet et al., 2002). Most severe staphylococcal

diseases remain unexplained. Various forms of acquired immunodeficiencies, including

infection with human immunodeficiency virus, hemodialysis, chemotherapy, intravenous

drug use, central catheters and vascular lines increase the likelihood of severe

staphylococcal disease (Lowy, 1998). The pathogenesis of invasive staphylococcal disease

is complex in such settings, often involving neutropenia, skin breaches, or both.

Alternatively, severe staphylococcal disease may result from various single-gene inborn

errors of immunity, including disorders of phagocytes in particular (Notarangelo, 2010;

Picard et al., 2015; Zhang et al., 2015).These disorders include disorders of the TLR and

IL-1R pathway, inborn errors of NF-κB, severe congenital neutropenia, chronic

granulomatous disease, leukocyte adhesion deficiency, and autosomal dominant hyper IgE

syndrome (Bousfiha et al., 2015; Minegishi et al., 2007; Picard et al., 2015). All these

defects affect phagocytes, albeit in different ways. The clinical features differ between

patients with staphylococcal diseases, reflecting the diversity of the underlying inborn

errors.

Autosomal recessive MyD88 and IRAK-4 deficiencies selectively impair signaling

via the TLR and IL-1R pathway (Picard et al., 2003; von Bernuth et al., 2008). More than

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80 patients worldwide have been diagnosed (reviewed in (Picard et al., 2010)). These

patients are susceptible to invasive bacterial infections, particularly those caused by

Streptococcus pneumoniae, but also those caused by S. aureus. They also develop

superficial staphylococcal infections (Picard et al., 2011). Leukocytes from patients with

deficiencies of IRAK-4 or MyD88 do not respond to activation via IL-1Rs or TLRs other

than TLR3, which signals via TRIF but not MyD88, and TLR4, which signals via TRIF or

MyD88 (Picard et al., 2003; Picard et al., 2010; von Bernuth et al., 2008). The molecular

and cellular basis of staphylococcal disease in these patients remains unknown. We

investigated this aspect, by testing the hypothesis that severe staphylococcal disease in

otherwise healthy children may be caused by mutations in other genes of the TLR and IL-

1R pathway (Casanova, 2015b)

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Results

Homozygous TIRAP R121W mutation in a child with severe staphylococcal

infection

We investigated a nine-year-old girl admitted to the intensive care unit with

pneumonia and sepsis caused by Panton Valentine Leukocidin (PVL)-producing S. aureus,

at the age of three months (P1, V.1, Figure 1A). She was born to consanguineous parents of

Pakistani descent living in the United Kingdom. The presumed complete genetic tree was

assessed by inferring relationships with King software based on genome-wide SNP

genotyping (Manichaikul et al., 2010) (Figure 1A and Supplementary Figure S1). Known

genetic etiologies of PIDs associated with life-threatening staphylococcal diseases were

excluded by whole-exome sequencing (WES) and Sanger sequencing. We searched for rare

variants (MAF<1%), and identified a homozygous substitution, c.361C>T, in exon 5 of

TIRAP, which encodes a TIR domain-containing adapter of TLR2 and TLR4 (Fitzgerald et

al., 2001; Horng et al., 2001; Yamamoto et al., 2002a). We confirmed the TIRAP mutation

by Sanger sequencing of the P1’s genomic DNA extracted from whole blood cells (WBC),

Epstein-Barr virus-immortalized B lymphocytes (EBV-B cells), and SV40 immortalized

fibroblasts (SV40 fibroblasts) (Figure 1B). This missense variant results in the replacement

of the arginine residue in position 121 of the TIR domain of TIRAP with a tryptophan

residue (R121W) (Figure 1C). The R121 residue of TIRAP has been strongly conserved

throughout evolution (Figure 1D). It is also conserved in 21 of the 26 human molecules

identified to date containing a TIR domain, other than TIRAP (e.g. R196 in MyD88,

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Supplementary Figure S2). In silico analysis with CADD and MSC predicted that the

R121W variation would be deleterious (Itan et al., 2016; Kircher et al., 2014). The TIRAP

gene is well conserved in the human population, with a neutrality index of 0.2346

(McDonald and Kreitman, 1991) and a GDI of 5.46 (Itan et al., 2015). No other rare or

common variants of TIRAP were found in P1. No rare bi-allelic mutations were found in

other genes of the TIR pathway, including individual TLRs and the other three TLR

adaptors (TRAM, SARM and TRIF). Moreover, no other candidate variant was identified in

the linkage regions defined by parametric multipoint linkage analysis (methods section) or

among the other 107 non-synonymous rare homozygous variants found in P1

(Supplementary Table 1). The patient was therefore homozygous for a potentially

deleterious rare allele of TIRAP.

Homozygosity for the very rare R121W variant in seven healthy relatives

This variant was not detected in 140 Pakistani individuals from the CEPH-HGD

panel (1,050 individuals from 52 ethnic groups). Eight heterozygous, but no homozygous

individuals were reported among 57,085 individuals from the Exome Aggregation

Consortium (ExAc) database (MAF=0.00007). This variant was detected, in the

heterozygous state, in three of 3,033 individuals (6,066 chromosomes) in our in-house

exome database. This patient was therefore homozygous for a very rare TIRAP missense

allele, with a minor allele frequency (MAF) of less than 1/10,000. Surprisingly however,

seven relatives of P1 were also found to be homozygous for the R121W TIRAP mutation,

including the proband’s father (aged 33 years), four of the father’s siblings (aged 20 to 28

years) and both his parents (aged 53 and 54 years) (Figure 1A). None of these individuals

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carried TIRAP polymorphisms. The proband’s mother and younger sister were

heterozygous for the R121W allele. None of these relatives, whether heterozygous or

homozygous for the TIRAP allele, had ever suffered from any severe infection, including

staphylococcal infections in particular. Thus, eight individuals from a consanguineous

Pakistani kindred, including a child with severe staphylococcal infection and seven

asymptomatic adults, were homozygous for a very rare and probably deleterious TIRAP

allele. These data suggested that P1 had autosomal recessive TIRAP deficiency, with

incomplete penetrance for severe staphylococcal disease. We therefore investigated

whether the eight individuals had TIRAP deficiency, and the mechanisms underlying

incomplete clinical penetrance.

Normal mRNA and protein expression of the R121W TIRAP allele

Human TIRAP encodes two isoforms (Figure 1C) generated by four different

transcripts. Two transcripts (NM_001039661.1 and NM_001318777) encode isoform A,

which contains 221 amino acids (aa), with a molecular weight (MW) of 23.8 kDa, whereas

the other two transcripts (NM_001318776 and NM_148910) encode isoform B, which

contains 235 aa and has a MW of 25.3 kDa. The R121W mutation is located in exon 5 of

reference transcript NM_001039661.1 (encoding a sequence present in all isoforms). We

assessed the impact of the mutation, by inserting the WT and R121W alleles into

pCDNA3.1-V5 vectors encoding a C-terminally tagged version of each of the isoforms of

TIRAP. Following the transient transfection of HEK 293T cells, the R121W allele yielded

levels of both isoforms (A and B) about 50% lower than those obtained with the WT allele

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(Figure 1E). This suggested that both R121W TIRAP isoforms were somewhat less stable

than the WT isoforms, at least when tagged molecules were overproduced in HEK 293T

cells. We then evaluated TIRAP mRNA levels for both isoforms by qPCR and RT-PCR in

SV40 fibroblasts from P1, her father (R121W/R121W), and her mother (R121W/WT), and

by qPCR in EBV-B cells from P1, in PBMCs from P1 and her aunt (R121W/R121W)

(Figure 1F and Supplementary Figure S3 and S4). Both P1, her father, mother, and her aunt

had mRNA levels within the normal range of the controls, with transcripts encoding

isoform A being mostly expressed in the three cell types tested, and in all individuals

tested. We were unable to detect the endogenous expression of either of the two TIRAP

isoforms by western blotting (WB) in SV40 fibroblasts from healthy controls, whereas

TIRAP protein levels in control EBV-B cells varied considerably between individuals

correlating with mRNA levels variability in controls (data not shown, Figure 1F). In

PBMCs from healthy controls only a 24 kDa protein, probably corresponding to isoform A,

was detected, consistent with qPCR results (Figure 1G). Homozygous family members had

normal levels of this isoform of TIRAP in PBMCs (Figure 1G). Overall, these data

suggested that the R121W mutation had no detectable impact on mRNA or protein levels in

circulating leukocytes.

Impaired subcellular localization of the TIRAP R121W isoforms

We assessed the subcellular distribution of the mutant TIRAP molecules.

Fluorescence microscopy of HEK 293 T cells transiently transfected with a pcDNA3.1

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vector encoding the WT form of TIRAP V5-tagged and labeled with Alexa Fluor 488-

conjugated anti-V5 antibodies showed the WT TIRAP to be present in precise small dots in

the cytoplasm, whereas transfection with the R121W TIRAP construct resulted in diffuse

cytoplasmic staining (Figure 2A). For the quantification of this subcellular localization

phenotype, we cotransfected cells with constructs encoding V5-tagged WT TIRAP and

either myc-tagged WT or R121W TIRAP, and then assessed the colocalization of the

encoded proteins by determining Pearson’s correlation coefficient (PCC) (Figure 2B).

TIRAP WT had a PCC of 0.85 for colocalization with itself, whereas its PCC for

colocalization with R121W was 0.25 (p < 0.0001). These results indicated that the R121W

mutation impaired the subcellular localization of TIRAP. We then investigated whether this

cellular localization defect had an impact on the colocalization of TIRAP with its partner,

MyD88. We obtained a PCC of 0.6 for the colocalization of TIRAP WT and MyD88,

whereas the PCC for the colocalization of TIRAP R121W and MyD88 was 0.2 (Figure 2C)

(p < 0.0001). These data suggested that the R121W mutation impaired TIRAP trafficking

within cells, thereby reducing the accessibility of this molecule to MyD88.

Impaired interaction of the TIRAP R121W isoforms with TLR2 and MyD88

Following stimulation, TLR2 or TLR4 recruits TIRAP, which recruits MyD88 to

the receptor complex through TIR domain interactions (Horng et al., 2002; Horng et al.,

2001; Yamamoto et al., 2002a; Yamamoto et al., 2002b). The TIR domain interactions

between MyD88 and TLR2 or TLR4 have been characterized in structural studies (Nada et

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al., 2012; Ohnishi et al., 2009). Four residues of MyD88 have been identified as essential

for this interaction, including R196, which is mutated (R196C) in three MyD88-deficient

patients (Picard et al., 2010; von Bernuth et al., 2008) The R196 residue of MyD88

corresponded to the R121 residue of TIRAP (Supplementary Figure S2) (Valkov et al.,

2011). Like the R196C mutation of MyD88, the R121W mutation of TIRAP seems to have

at most a modest impact on protein levels (Figure 1G). The R121W mutation may impair

the interaction of TIRAP with MyD88, thereby preventing downstream signaling and the

activation of target gene transcription. Moreover, the R121 residue is part of a long AB

loop that mediates direct binding to the TIR domain of TLR4 (Lin et al., 2012). The

R121W mutation may, therefore, also impair TIRAP interaction with TLR2 and TLR4. We

tested these hypotheses, by first performing in vitro co-immunoprecipitation assays in HEK

293T cells transfected with the WT and R121W TIRAP isoforms A and B, together with

TLR2 or MyD88. Both WT TIRAP isoforms interacted with TLR2, whereas this interaction

was strongly impaired when HEK 293T cells were transfected with the R121W TIRAP

isoforms (Figure 2D). Similarly, both WT TIRAP isoforms interacted with MyD88,

whereas this interaction was strongly impaired when HEK 293T cells were transfected with

either R121W TIRAP isoform (Figure 2E). We also tested TLR2/MyD88 co-

immunoprecipitation after PAM2CSK4 stimulation, in fibroblasts from healthy controls,

TIRAP heterozygous P1’s mother, and TIRAP homozygous P1 and her father (Figure 2F).

TLR2 and MyD88 co-precipitated upon TLR2 stimulation in both control and R121W

heterozygous fibroblasts, but not in R121W homozygous fibroblasts, whereas MyD88-

mutated fibroblasts (L93P/R196C) displayed an intermediate pattern. These data suggested

that the R121W mutation disrupted the interaction of both TIRAP isoforms with their

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TLR2 (and presumably TLR4) and MyD88 partners. These data, therefore, suggested that

individuals homozygous for R121W had a complete functional deficiency of TIRAP.

The R121W TIRAP allele is loss-of-function

The transient transfection of SV40 fibroblasts from P1 and her father with a

pcDNA3 vector encoding WT TIRAP isoform A or B induced IL-6 production to levels

similar to those in transfected control cells (Figure 3A). Given this high background, the

increase observed in response to TLR2 or TLR4 stimulation was only modest, in both cell

types (Supplementary Figures S5, S6, S7). In contrast, transfection of these SV40

fibroblasts with constructs encoding either of the R121W TIRAP isoforms had no effect

(Figure 3A). Overexpression of the WT MyD88 allele in SV40 fibroblasts from the control,

P1, her father or a MyD88-deficient patient led to the constitutive activation of IL-6

production, but the overproduction of WT TIRAP in MyD88-deficient fibroblasts did not

induce IL-6 production. Moreover, stable transfection of fibroblasts from P1 or her father

with lentiviral particles encoding WT TIRAP isoform A or B, or both A and B, rescued

constitutive IL-6 production, whereas no such rescue was observed after stable transfection

with the R121W TIRAP construct or the empty vector (data not shown). Finally, the

overproduction of each TIRAP WT isoform in HEK293T cells, as already shown for the

TIRAP isoform A, induced constitutive activation of the NF-κB pathway and IL-6

production (Figure 3B and (Horng et al., 2001)), whereas no such activation was observed

following the overproduction of the TIRAP R121W isoforms (Figure 3B). Collectively,

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these data showed that the R121W TIRAP allele is loss-of-function, at least in terms of

constitutive and MyD88-dependent IL-6 induction following overexpression in fibroblasts.

Impaired responses via TLR2 and TLR4 in fibroblasts homozygous for R121W

TLR2 heterodimerizes with TLR1 for the recognition of triacylated lipopeptides (Jin

et al., 2007), which are present in most Gram-positive bacteria, including S. aureus

(Kurokawa et al., 2009), and with TLR6 for the recognition of diacylated lipopeptides, also

present in S. aureus (Buwitt-Beckmann et al., 2006). It also polymerizes with TLR6 and

CD36 for the recognition of staphylococcal lipoteichoic acid (LTA) from most Gram-

positive bacteria, including S. aureus (Jimenez-Dalmaroni et al., 2009), as reviewed by

(Zahringer et al., 2008). TLR4 recognizes LPS from Gram-negative bacteria. Macrophages

and dendritic cells from TIRAP-deficient mice display abolished responses to the

stimulation of TLR1/2, TLR2/6, and TLR4, and splenocyte proliferation in response to LPS

is impaired (Horng et al., 2002; Yamamoto et al., 2002a). Human SV40 fibroblasts express

TLR2 at their surface (data not shown). We thus assessed their production of cytokines

upon stimulation with TLR2 and TLR4 agonists (PAM2CSK4 and FSL-1, two TLR2/6

synthetic agonists; purified LTA from S. aureus, another TLR2/6 agonist; PAM3CSK4, a

synthetic TLR1/2 agonist; LPS and synthetic lipid A, two TLR4 agonists). We detected

cytokine production upon stimulation by all these agonists. The response to PAM2CSK4,

was both TLR2- and TIRAP-dependent, as demonstrated by the inhibition of IL-6 induction

by siRNAs specific for TLR2 and TIRAP (Supplementary Figure S8), respectively, but not

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by scrambled siRNA. The responses to LPS and lipid A were also TLR4- and TIRAP-

dependent (data not shown). No or impaired IL-6 induction was observed in response to

PAM2CSK4, LTA, FSL-1, PAM3CSK4, LPS, lipid A, and heat-killed S. aureus (HKSA), in

SV40 fibroblasts from P1 and her father (both R121W/R121W), or in IRAK-4- and

NEMO-deficient cells (Figure 3C). However, unlike IRAK-4- and NEMO-deficient cells,

SV40 fibroblasts from P1 and her father responded normally to IL-1β. All SV40 fibroblasts

other than NEMO-deficient cells tested responded to TNF-α and poly(I:C) (a TLR3 agonist

in human fibroblasts) (Zhang et al., 2007). These data suggested that homozygosity for the

R121W allele abolished TLR1/2, TLR2/6 and TLR4 responses in human fibroblasts,

consistent with the findings reported for TIRAP-deficient mouse macrophages and

dendritic cells (Horng et al., 2002; Yamamoto et al., 2002a).

Impaired responses of phagocytes via TLR2 and TLR4 in TIRAP R121W

homozygotes

We investigated P1’s leukocyte responses to TLR2 and TLR4 agonists. CD62L (L-

selectin) is a transmembrane protein cleaved by the metalloproteinase TACE upon TLR

stimulation (Peschon et al., 1998). CD62L shedding in response to the stimulation of all

TLRs other than TLR3 is abolished in granulocytes from IRAK4- and MyD88-deficient

patients (von Bernuth et al., 2006). We investigated the impact of the R121W mutation on

CD62L shedding in six family members homozygous for the R121W mutation, after the

stimulation of granulocytes for 1hr with four TLR2 agonists (PAM2CSK4, FSL-1, LTA,

and PAM3CSK4) and one TLR4 agonist (LPS). The responses were compared with those of

granulocytes from two healthy controls and P1’s heterozygous mother. The six

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R121W/R121W individuals tested displayed impaired CD62L shedding in response to

stimulation with all TLR2 and TLR4 agonists tested, as demonstrated by comparison with

healthy controls, but their response to PMA was normal (Figure 3D). In addition, the

stimulation of PBMCs with various doses of PAM2CSK4, FSL-1, LTA, PAM3CSK4, and

LPS for 3hrs resulted in the abolition of intracellular labeling for IL-6 and TNF-α in CD14+

cells, particularly at low agonist concentrations (Figure 3E and Supplementary Figure S9).

Similarly, transcriptome analysis after the stimulation of whole blood for 2hrs suggested

that the induction of gene expression in response to PAM2CSK4, FSL-1, or LTA was

abolished and that the induction of gene expression in response to PAM3CSK4 or LPS was

impaired but not abolished (Supplementary Figure S10). Collectively, these data suggest

that granulocytes from TIRAP R121W homozygotes did not respond to the stimulation of

TLR1/2, TLR2/6, and TLR4, whereas their monocytes did not respond to TLR2/6 and

responded poorly to TLR1/2 and TLR4.

Residual responses to high concentrations of TLR agonists in TIRAP R121W

homozygous monocytes

After 3hrs of stimulation, the responses of monocytes from R121W homozygous

PBMCs to low doses (10 ng/ml) of PAM3CSK4 were abolished, but the levels of production

of both IL-6 and TNF-α in response to agonist stimulation at concentrations above 1 µg/ml

were similar to those of healthy controls, suggesting that there were TIRAP-independent

responses to high concentrations of PAM3CSK4 and, possibly, other TLR2 agonists, at least

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in some cell types (Figure 3E and Supplementary Figure S9). An effect of contaminants is

unlikely, as the agonist used was synthetic. Moreover, low levels of TNF-α induction were

observed in response to LPS stimulation in CD14+ cells from the R121W PBMCs tested.

After 48 hrs of stimulation, IL-6 production by the PBMCs of all homozygous individuals

tested in response to PAM3CSK4 (1 µg/ml) was normal, whereas it was profoundly

impaired but not abolished in response to LPS (1 ng/ml) and lipid A (1µg/ml); responses to

PAM2SCK4 (1 µg/ml), FSL-1 (100 ng/ml), and LTA (1 µg/ml) were abolished (Figure 4A

and Supplementary Figure S11). These results confirmed that the R121W allele has a

deleterious effect on TLR2 and TLR4 responses in leukocytes. However, the normal

induction of IL-6 production observed in R121W/R121W monocytes in response to

TLR1/2 stimulation with PAM3CSK4, at late time points and high concentrations of agonist

(and, to a lesser extent, in response to stimulation with LPS and lipid A) further suggests

that TIRAP may be redundant in such circumstances, as we ruled out the possibility of the

TIRAP mutant protein having residual activity. Alternatively, PAM3CSK4 and LPS/lipid A

may also be recognized independently of TLR2 and TLR4, respectively. In the absence of

TLR2- or TLR4-deficient subjects, given that the R121W TIRAP allele is loss-of-function,

we can conclude that these late responses to high concentrations of TLR agonists were

probably either TIRAP- or TLR-independent.

Impaired whole-blood response to LTA and lack of anti-LTA Abs in P1

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In all individuals homozygous for the TIRAP R121W mutation tested, IL-6

production by whole blood in response to 48 hrs of stimulation of TLR2/6 with PAM2CSK4

and FLS-1 was impaired, whereas normal levels of IL-6 were produced in response to

stimulation of TLR1/2 with PAM3CSK4 and TLR4 with LPS and lipid A (Figure 4B and

Supplementary Figure S11). However, the induction of IL-6 via TLR2/6 in response to

LTA was reproducibly abolished in the whole blood of P1, whereas a normal response was

observed for the other TIRAP R121W homozygotes tested (Figure 4B). We investigated

this striking difference in cellular phenotype, which we considered to be of potential

clinical relevance as only P1 suffered from staphylococcal disease. The leukocyte response

to LTA response is mediated principally by TLR2/6 heterodimers acting in concert with

CD36 (Jimenez-Dalmaroni et al., 2009), but anti-LTA Abs have been shown to enhance

this response through the recognition of their invariant IgG domain by CD32 (Bunk et al.,

2010). The response to LTA was impaired in PBMCs, granulocytes, and monocytes from

all R121W homozygotes (including P1), but normal in the whole blood of all these

individuals other than P1. We therefore used enzyme-linked immunosorbent assays

(ELISA) to test for the presence of circulating anti-LTA Abs in the plasma of individuals

from the kindred of P1. P1 was the only member of this kindred for whom anti-LTA IgG

Abs were reproducibly undetectable, in 10 plasma samples taken between the ages of four

months and nine years. No maternal anti-LTA antibodies were thus detected in the plasma

of P1 when the infectious episode occurred. By contrast, the six homozygous TIRAP

R121W relatives tested and P1’s mother (R121W/WT) had high titers of anti-LTA Abs

(Figure 4C). As a control, P1 had high titers of Abs directed against H. influenzae,

pneumococcal polysaccharides, tetanus, diphtheria toxins, and against 68 other

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staphylococcal antigens, such as PVL and hemolysin-α (Supplementary Figure S12 and

Supplementary Table 2). Overall, P1 was unique among the eight R121W homozygotes in

three ways: her whole blood did not respond to LTA, she had no LTA-specific Abs in her

plasma, and she suffered from invasive staphylococcal disease.

Rescue of the whole-blood LTA response in P1 with LTA-specific mAbs

We investigated whether the lack of anti-LTA IgG was responsible for the lack of

whole-blood responses to LTA, by testing 26 healthy adults, 22 healthy children, 246 sick

children (67 with invasive staphylococcal infections and 179 with other infections), seven

patients with MyD88 or IRAK-4 deficiency (all of whom presented staphylococcal

disease), and 10 patients with severe PVL+ S. aureus pneumonia for the presence of

specific anti-LTA Abs (Figure 4C). All healthy adults and IRAK-4- and MyD88-deficient

patients, most of the children (62%), whether healthy (96%), with S. aureus infections

(72%) or with other types of infections (54%), and up to 60% of the patients with severe

PVL+ S. aureus pneumonia, had high levels of serum anti-LTA Abs. Three of the latter

patients had a normal whole-blood response to LTA stimulation, one to eight months after

infection (data not shown). These results showed that the age at which serum anti-LTA IgG

antibodies appear varies in the population, even after staphylococcal disease, consistent

with previous reports (Wergeland et al., 1989) . They also showed that a lack of anti-LTA

IgG does not influence the whole-blood response to LTA in TIRAP-expressing individuals.

We then restored P1’s PBMCs IL-6 production in response to LTA stimulation by addition

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of LTA-responsive control plasma, suggesting that circulating anti-LTA Abs could rescue

TIRAP deficiency (Supplementary Figure S13). To test this hypothesis, we added a

chimeric mouse anti-LTA mAb to whole blood from healthy controls, P1, her aunt, and an

IRAK-4-deficient patient, or to PBMCs from healthy controls and P1 (Figure 4D and 4E

and Supplementary Figure S14). This restored P1’s whole blood and PBMC IL-6

production in response to LTA stimulation, whereas an isotypic control mAb (directed

against RSV) had no effect. Monocytes were the main cells producing IL-6 upon LTA

stimulation in the presence of anti-LTA mAb (Supplementary Figure S15) (Bunk et al.,

2010). Among the R121W homozygotes, the specific lack of anti-LTA Abs in the plasma

of P1 therefore accounts for the abolition of IL-6 induction in the whole blood of this

specific individual in response to LTA stimulation via TLR2/6. The only TIRAP

homozygote to display catastrophic staphylococcal disease (P1) mounted no antibody

response to LTA and her whole blood was unable to respond to LTA. The LTA-specific

mAb did not rescue the response of IRAK-4-deficient cells, suggesting that the response of

these cells is mediated by the canonical TLR2-MyD88 pathway. No TLR2-and MyD88-

deficient individuals were however available for study.

Anti-LTA mAb restores the LTA response in TIRAP-deficient mouse macrophages

We tested this model in mice. Like MyD88- and TLR2-deficient human

macrophages, TIRAP-deficient mouse macrophages do not respond to TLR2 stimulation

(Horng et al., 2001; Yamamoto et al., 2002a). We investigated whether the addition of

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exogenous anti-LTA antibodies restored the response to TLR stimulation. We first

determined whether such Abs were present in mouse sera. No anti-LTA Abs were detected

by ELISA in the 15 mice used for the study (data not shown). Mouse peritoneal

macrophages from WT B6, Tirap-/-, Myd88-/- and Tlr2-/- mice (n>3) were then treated with

LTA or low doses of PAM3CSK4 for 24 hrs, and IL-6 and TNFα production were assessed

in the presence or absence of exogenous anti-LTA or isotype control Abs. As observed in

cells from P1, anti-LTA Abs restored the production of IL-6 and TNFα in response to LTA,

but not PAM3CSK4, in TIRAP-deficient mouse macrophages (Figure 4F and

Supplementary Figure S16), whereas no such restoration was observed with the isotype

control mAb. MyD88- and TLR2-deficient macrophages did not respond in this way to the

addition of anti-LTA mAb. These findings unequivocally demonstrate that the TIRAP-

independent mechanism compensating for TIRAP deficiency in the presence of anti-LTA

Abs in mice, and, by inference, in humans, is dependent not only on IRAK4 but also on

both TLR2 and MyD88. Collectively, these data establish a plausible mechanism for the

occurrence of staphylococcal disease in P1 but not in any of the other seven TIRAP-

deficient individuals. The lack of LTA IgG does not compensate for TIRAP deficiency,

preventing leukocyte responses to staphylococcal LTA via TLR2/6, thereby accounting for

invasive staphylococcal disease.

Discussion

21

We describe here a large family with autosomal recessive TIRAP deficiency. Eight

family members are homozygous for the rare TIRAP R121W mutation affecting the TIR

domain. TIRAP has been shown to play a crucial role in the TLR2- and TLR4-mediated

and MyD88-dependent pathways in mice, as an adapter between these two TLRs and

MyD88. Our data show that the R121W TIRAP allele is loss-of-function, as opposed to

hypomorphic, because it cannot interact with TLRs upstream and MyD88 downstream.

TIRAP-deficient mouse macrophages display impaired activation of NF-κB in response to

the stimulation of TLR2 and TLR4 by MALP2, PGN (peptidoglycans) (TLR2/6), BLP

(bacterial lipopeptides) (TLR1/2) and LPS, respectively. LPS-induced splenocyte

proliferation and cytokine production are also impaired in TIRAP-deficient mice, which are

resistant to LPS-induced septic shock (Horng et al., 2002; Yamamoto et al., 2002a).

Consistently, fibroblasts, granulocytes, and monocytes from TIRAP-deficient humans do

not respond to TLR2/6 agonists PAM2SCK4, FSL-1, and LTA. However, residual activation

of the TLR2 pathway was recently reported in mouse TIRAP-deficient macrophages, in

response to high concentrations of PAM3CSK4 (TLR1/2) and MALP2 (TLR2/6) (Kenny et

al., 2009). Moreover, prolongation or enhancement of the interaction between mouse TLR2

and its agonists PAM2CSK4 (TLR2/6) and PAM3CSK4 (TLR1/2) can induce TIRAP-

independent responses, whereas MyD88 remains strictly required for all TLR2-dependent

responses (Cole et al., 2010). These findings suggest that the response of R121W/R121W

monocytes to high concentrations of or prolonged stimulation with PAM3CSK4 may be due

to TIRAP-independent responses mediated via TLR1/2 and MyD88. Likewise, residual

responses to LPS/lipid A in the patients may be TLR4-and MyD88-dependent. Our study

thus provides the first comprehensive description of inherited TIRAP deficiency in humans,

22

highlighting the essential role of this protein for TLR2/6 signaling. Our results also confirm

that TIRAP is less critical than MyD88 and IRAK-4 for TLR1/2 and TLR4 responses, as it

can be bypassed by increasing agonist concentration or the duration of stimulation.

The mouse TLR2 pathway plays an important role in host defense against S. aureus

(Takeuchi et al., 2000). Humans with inherited IRAK-4 and MyD88 deficiencies are also

prone to staphylococcal disease (reviewed in (Picard et al., 2010)), indicating that the

human TIR signaling pathway is essential for protective immunity to S. aureus. The

specific contribution of individual human TLRs and IL-1Rs has remained unclear

(Casanova et al., 2011). We show that homozygosity for the loss-of-function TIRAP

R121W mutant allele severely impairs, but does not abolish responses to TLR1/2 and TLR4

stimulation, whereas it abolishes responses to TLR2/6 stimulation. Impaired responses to

TLR2 are much more likely than impaired responses to TLR4 to have contributed to the

development of staphylococcal disease in P1, as S. aureus is a Gram-positive bacterium

that contains both TLR1/2 and TLR2/6 agonists, including LTA in particular, but does not

produce LPS (Lowy, 1998). However, the TIRAP mutation alone is not sufficient to

account for P1’s staphylococcal disease, as none of the other seven affected relatives had

had any serious staphylococcal infections, despite frequent exposure to these commensal

bacteria. The bacterial load in the initial inoculum, or the nature of the staphylococcus, may

influence the ability of TIRAP-deficient individuals to mount a protective immune

response. In that regard, it may be relevant that P1 was infected by a PVL-positive strain of

S. aureus; these relatively rare staphylococci are known to be more pathogenic in humans

(Gillet et al., 2002). The observed clinical differences may also reflect the young age at

23

which P1encountered this pathogenic strain of S. aureus. The proband has been doing well

for the last nine years, perhaps in part thanks to her robust Ab response to all

staphylococcal antigens tested other than LTA. She is however on antibiotic prophylaxis

(without IgG substitution, which would provide LTA-specific Abs, Supplementary Figure

S17), making it impossible to draw firm conclusions concerning the efficacy of her current

immune response to staphylococci.

A more likely hypothesis is based on the observation that P1 was the only TIRAP-

deficient individual in the kindred to display a complete lack of whole-blood TLR2/6-

dependent responses to staphylococcal LTA. This lack of response was causally related to

the specific absence of circulating Abs against LTA in P1, as the phenotype was not only

specific to this patient and absent from the other seven TIRAP-deficient relatives, but also

complemented by the addition of LTA-specific mAbs in P1. Monocytes are responsible for

the response of TIRAP-deficient whole blood and PBMC to LTA in the presence of LTA-

specific Abs. Moreover, TIRAP-deficient mouse macrophages responded to LTA via TLR2

in the presence of LTA-specific Abs. This Ab defect may, again, reflect the young age of

P1, as LTA Ab levels gradually appear and rise during childhood and teenage, with great

inter-individual variability (Dryla et al., 2005; Wergeland et al., 1989). If this is, indeed, the

case, then the lack of staphylococcal disease during childhood in the other R121W

homozygotes may have resulted from the staphylococci encountered being insufficiently

virulent to cause disease, an earlier Ab response to LTA (triggered by any Gram-positive

bacterium), or both. The normal levels of anti-LTA Abs in IRAK-4- and MyD88-deficient

children (and the six TIRAP-deficient individuals other than P1 tested) indicate that a

24

deficiency of TLR signaling pathways does not account for the lack of anti-LTA Ab

production in P1. Furthermore, IRAK-4-deficient patients display a lack of whole-blood IL-

6 production in response to LTA (Supplementary Figure S14), despite presenting high titers

of endogenous anti-LTA Abs, and even in the presence of exogenous anti-LTA mAb,

indicating that the response to LTA mediated by anti-LTA Ab is TIRAP-independent but

IRAK-4-dependent. Moreover, mouse macrophages lacking MyD88 or TLR2 are not

rescued by LTA-specific Abs, unlike TIRAP-deficient macrophages, for their responses to

LTA. The TIRAP-independent pathway rescued by LTA-specific Abs is therefore also

TLR2- and MyD88-dependent. This rescue is probably mediated via the Fc receptor CD32

(Bunk et al., 2010), although CD32-deficient humans and mice were not tested.

The continued lack of anti-LTA Abs in P1 is probably intrinsic and remains

unexplained. However, this is not a unique feature, as approximately half of children under

the age of 9 years and with no history of staphylococcal disease tested here had no

detectable anti-LTA Abs. We did not test P1’s serum before disease onset, but the observed

Ab defect is unlikely to be a consequence of staphylococcal disease, as infection should

have triggered the Ab response and other children with staphylococcal disease (even with

an identical clinical phenotype to this patient) produce such Abs. This defect is not the sole

cause of the staphylococcal disease observed in P1, as children with various forms of Ab

deficiency, including agammaglobulinemia, are not prone to such staphylococcal diseases

(Conley et al., 2009). The combined effects of a lack of anti-LTA Abs and the inherited

TIRAP deficiency, i.e. of an acquired adaptive deficiency and an inborn innate deficiency,

are therefore the mechanism underlying staphylococcal disease in this single patient

25

(Casanova et al., 2014). In the other individuals, IgG-dependent, adaptive immunity to LTA

operates as a somatic modifier, selectively rescuing the inborn error of TIRAP, which

disrupts TLR2-, TIRAP-, and MyD88-dependent innate immunity to LTA and

staphylococci. This specific observation does not explain why staphylococcal (and

pneumococcal) diseases become less frequent from adolescence onward in patients with

inherited MyD88 or IRAK-4 deficiency, whose cells did not respond to LTA in the

presence of LTA-specific Abs. However, it suggests that other mechanisms of acquired

immunity may be at work. In these patients, adaptive immunity is more likely to

compensate (via pathways other than IRAK-4-MyD88) than to rescue (via the IRAK-4-

MyD88 pathway itself) innate immunity. Regardless, we have shown that the clinical

penetrance of an inborn error of innate immunity may be determined by adaptive immunity.

This is consistent with human twin studies, which revealed a poor heritability of adaptive

immunity, when compared with innate immunity (Brodin et al., 2015; Casanova, 2015a).

The somatic diversity of adaptive immune responses can compensate or even rescue inborn

errors of innate immunity.

Author contributions

L.I., A.P. and J.L.C. designed the study and wrote the manuscript. L.I., Y.W., K.B.,

E.D.M., Z.Z., M.C., T.L., E.L., L.L., L.Y., A.M. and S.W. performed the experiments.

V.P., A.B., A.A., C.Pr. and Y.I conducted exome, linkage and statistical analysis. P.D.A.,

L.E.W., M.L., and J.H. provided patient’s material and reagents. V.S.S, M.D., F.V., C.Pi.,

L.A., D.C., X.L., and B.B. provided expertise and feedbacks. J.L.C., A.P. and L.A secured

26

funding. P.D.A conducted the medical follow up, provided expertise, feedbacks and critical

reading. All authors critically reviewed the manuscript.

Acknowledgments

We thank the patient and her family for participating in the study and all the

members of both branches of the laboratory for discussions. We thank Jacinta Bustamante

and Shen-Ying Zhang for providing the plasma samples, Sylvanie Fahy, Jérome Flatot,

Martine Courat, Lahouari Amar and Yelena Nemiroskaya for their assistance. We also

thank the members of the imaging platform, Raphaëlle Desvaux and Nicolas Goudin for

their help. We thank Biosynexus Incorporated, Gaithersburg, MD USA for providing us

with pagibaximab (an anti-LTA monoclonal Ab). The Laboratory of Human Genetics of

Infectious Diseases is supported by grants from the European Research Council (ERC-

2010-AdG-268777), Institut National de la Santé et de la Recherche Médicale, Université

Paris Descartes, the Imagine Institute, l’Agence Nationale de la Recherche (grant ANR-

2012-BSV3-0003-02), the St. Giles Foundation, the National Center for Research

Resources, and the National Center for Advancing Sciences (NCATS) grant number

8UL1TR000043 from the National Institutes of Health, and The Rockefeller University.

This work was partly supported by the Innovative Medicines Initiative Joint Undertaking

under grant agreement n◦[115523], COMBACTE, resources of which are composed of

financial contributions from the European Union's 7th Framework Program (FP7/2007-

2013) and EFPIA companies in kind contribution. Laura Israel was supported by Fondation

27

pour la Recherche Médicale. Carolina Prando was supported by the Thrasher Research

Fund and Yuval Itan was supported by the AXA Research Fund.

28

References

Bousfiha, A., Jeddane, L., Al-Herz, W., Ailal, F., Casanova, J.L., Chatila, T., Conley, M.E.,Cunningham-Rundles, C., Etzioni, A., Franco, J.L., et al. (2015). The 2015 IUIS PhenotypicClassificationforPrimaryImmunodeficiencies.JClinImmunol35,727-738.Brodin, P., Jojic, V., Gao, T., Bhattacharya, S., Angel, C.J., Furman,D., Shen-Orr, S., Dekker, C.L.,Swan,G.E.,Butte,A.J.,etal. (2015).Variation in thehuman immunesystem is largelydrivenbynon-heritableinfluences.Cell160,37-47.Bunk,S.,Sigel,S.,Metzdorf,D.,Sharif,O.,Triantafilou,K.,Triantafilou,M.,Hartung,T.,Knapp,S.,andvonAulock,S.(2010).InternalizationandcoreceptorexpressionarecriticalforTLR2-mediatedrecognitionoflipoteichoicacidinhumanperipheralblood.JImmunol185,3708-3717.Buwitt-Beckmann, U., Heine, H.,Wiesmuller, K.H., Jung, G., Brock, R., Akira, S., and Ulmer, A.J.(2006).TLR1-andTLR6-independentrecognitionofbacteriallipopeptides.JBiolChem281,9049-9057.Casanova,J.L.(2015a).Humangeneticbasisofinterindividualvariabilityinthecourseofinfection.ProcNatlAcadSciUSA112,E7118-7127.Casanova, J.L. (2015b). Severe infectious diseases of childhood as monogenic inborn errors ofimmunity.ProcNatlAcadSciUSA112,E7128-7137.Casanova, J.L., Abel, L., andQuintana-Murci, L. (2011). Human TLRs and IL-1Rs in host defense:natural insights fromevolutionary,epidemiological, andclinical genetics.AnnuRev Immunol 29,447-491.Casanova, J.L.,Conley,M.E.,Seligman,S.J.,Abel, L.,andNotarangelo, L.D. (2014).Guidelines forgeneticstudiesinsinglepatients:lessonsfromprimaryimmunodeficiencies.JExpMed211,2137-2149.Cole,L.E.,Laird,M.H.,Seekatz,A.,Santiago,A.,Jiang,Z.,Barry,E.,Shirey,K.A.,Fitzgerald,K.A.,andVogel, S.N. (2010). Phagosomal retention of Francisella tularensis results in TIRAP/Mal-independentTLR2signaling.JLeukocBiol87,275-281.Conley, M.E., Dobbs, A.K., Farmer, D.M., Kilic, S., Paris, K., Grigoriadou, S., Coustan-Smith, E.,Howard, V., and Campana, D. (2009). Primary B cell immunodeficiencies: comparisons andcontrasts.AnnuRevImmunol27,199-227.Dryla,A.,Prustomersky,S.,Gelbmann,D.,Hanner,M.,Bettinger,E.,Kocsis,B.,Kustos,T.,Henics,T.,Meinke, A., andNagy, E. (2005). Comparison of antibody repertoires against Staphylococcusaureusinhealthyindividualsandinacutelyinfectedpatients.ClinDiagnLabImmunol12,387-398.Fitzgerald, K.A., Palsson-McDermott, E.M., Bowie, A.G., Jefferies, C.A., Mansell, A.S., Brady, G.,Brint,E.,Dunne,A.,Gray,P.,Harte,M.T.,etal. (2001).Mal (MyD88-adapter-like) is required forToll-likereceptor-4signaltransduction.Nature413,78-83.Gillet, Y., Issartel, B., Vanhems, P., Fournet, J.C., Lina, G., Bes,M., Vandenesch, F., Piemont, Y.,Brousse,N.,Floret,D.,etal. (2002).AssociationbetweenStaphylococcusaureusstrainscarryinggene for Panton-Valentine leukocidin and highly lethal necrotising pneumonia in youngimmunocompetentpatients.Lancet359,753-759.Horng, T., Barton, G.M., Flavell, R.A., and Medzhitov, R. (2002). The adaptor molecule TIRAPprovidessignallingspecificityforToll-likereceptors.Nature420,329-333.Horng,T.,Barton,G.M.,andMedzhitov,R.(2001).TIRAP:anadaptermoleculeintheTollsignalingpathway.NatImmunol2,835-841.

29

Itan, Y., Shang, L., Boisson, B., Ciancanelli, M.J., Markle, J.G., Martinez-Barricarte, R., Scott, E.,Shah, I., Stenson, P.D., Gleeson, J., et al. (2016). The mutation significance cutoff: gene-levelthresholdsforvariantpredictions.NatMethods13,109-110.Itan,Y.,Shang,L.,Boisson,B.,Patin,E.,Bolze,A.,Moncada-Velez,M.,Scott,E.,Ciancanelli,M.J.,Lafaille,F.G.,Markle,J.G.,etal.(2015).Thehumangenedamageindexasagene-levelapproachtoprioritizingexomevariants.ProcNatlAcadSciUSA112,13615-13620.Jimenez-Dalmaroni,M.J.,Xiao,N.,Corper,A.L.,Verdino,P.,Ainge,G.D.,Larsen,D.S.,Painter,G.F.,Rudd, P.M., Dwek, R.A., Hoebe, K., et al. (2009). Soluble CD36 ectodomain binds negativelychargeddiacylglycerolligandsandactsasaco-receptorforTLR2.PLoSOne4,e7411.Jin,M.S.,Kim,S.E.,Heo,J.Y.,Lee,M.E.,Kim,H.M.,Paik,S.G.,Lee,H.,andLee,J.O.(2007).CrystalstructureoftheTLR1-TLR2heterodimerinducedbybindingofatri-acylatedlipopeptide.Cell130,1071-1082.Kenny, E.F., Talbot, S., Gong,M., Golenbock, D.T., Bryant, C.E., andO'Neill, L.A. (2009).MyD88adaptor-likeisnotessentialforTLR2signalingandinhibitssignalingbyTLR3.JImmunol183,3642-3651.Kircher,M.,Witten,D.M.,Jain,P.,O'Roak,B.J.,Cooper,G.M.,andShendure,J. (2014).Ageneralframeworkforestimatingtherelativepathogenicityofhumangeneticvariants.NatGenet46,310-315.Kurokawa,K., Lee,H.,Roh,K.B.,Asanuma,M.,Kim,Y.S.,Nakayama,H., Shiratsuchi,A.,Choi, Y.,Takeuchi,O.,Kang,H.J.,etal. (2009).TheTriacylatedATPBindingClusterTransporterSubstrate-bindingLipoproteinofStaphylococcusaureusFunctionsasaNativeLigandforToll-likeReceptor2.JBiolChem284,8406-8411.Li, Z., Peres, A.G., Damian, A.C., and Madrenas, J. (2015). Immunomodulation and DiseaseTolerancetoStaphylococcusaureus.Pathogens4,793-815.Lin,Z.,Lu, J.,Zhou,W.,andShen,Y. (2012).Structural insights intoTIRdomainspecificityofthebridgingadaptorMalinTLR4signaling.PLoSOne7,e34202.Lowy,F.D.(1998).Staphylococcusaureusinfections.NEnglJMed339,520-532.Manichaikul, A.,Mychaleckyj, J.C., Rich, S.S., Daly, K., Sale,M., and Chen,W.M. (2010). Robustrelationshipinferenceingenome-wideassociationstudies.Bioinformatics26,2867-2873.McDonald, J.H., and Kreitman, M. (1991). Adaptive protein evolution at the Adh locus inDrosophila.Nature351,652-654.Minegishi,Y.,Saito,M.,Tsuchiya,S.,Tsuge,I.,Takada,H.,Hara,T.,Kawamura,N.,Ariga,T.,Pasic,S.,Stojkovic,O.,etal.(2007).Dominant-negativemutationsintheDNA-bindingdomainofSTAT3causehyper-IgEsyndrome.Nature448,1058-1062.Mulcahy,M.E.,andMcLoughlin,R.M.(2016).Host-BacterialCrosstalkDeterminesStaphylococcusaureusNasalColonization.TrendsMicrobiol24,872-886.Nada,M.,Ohnishi,H., Tochio,H.,Kato,Z.,Kimura,T.,Kubota,K., Yamamoto,T.,Kamatari, Y.O.,Tsutsumi, N., Shirakawa, M., et al. (2012). Molecular analysis of the binding mode ofToll/interleukin-1receptor(TIR)domainproteinsduringTLR2signaling.MolImmunol52,108-116.Notarangelo,L.D.(2010).Primaryimmunodeficiencies.JAllergyClinImmunol125,S182-194.Ohnishi,H.,Tochio,H.,Kato,Z.,Orii,K.E.,Li,A.,Kimura,T.,Hiroaki,H.,Kondo,N.,andShirakawa,M.(2009).StructuralbasisforthemultipleinteractionsoftheMyD88TIRdomaininTLR4signaling.ProcNatlAcadSciUSA106,10260-10265.Peschon, J.J., Slack, J.L., Reddy, P., Stocking, K.L., Sunnarborg, S.W., Lee, D.C., Russell, W.E.,Castner,B.J.,Johnson,R.S.,Fitzner,J.N.,etal.(1998).Anessentialroleforectodomainsheddinginmammaliandevelopment.Science282,1281-1284.

30

Picard,C.,Al-Herz,W.,Bousfiha,A.,Casanova,J.L.,Chatila,T.,Conley,M.E.,Cunningham-Rundles,C.,Etzioni,A.,Holland,S.M.,Klein,C.,etal.(2015).PrimaryImmunodeficiencyDiseases:anUpdateon theClassification from the InternationalUnionof Immunological Societies ExpertCommitteeforPrimaryImmunodeficiency2015.JClinImmunol35,696-726.Picard,C.,Casanova,J.L.,andPuel,A.(2011).InfectiousdiseasesinpatientswithIRAK-4,MyD88,NEMO,orIkappaBalphadeficiency.ClinMicrobiolRev24,490-497.Picard,C.,Puel,A.,Bonnet,M.,Ku,C.L.,Bustamante,J.,Yang,K.,Soudais,C.,Dupuis,S.,Feinberg,J.,Fieschi,C.,etal.(2003).PyogenicbacterialinfectionsinhumanswithIRAK-4deficiency.Science299,2076-2079.Picard, C., von Bernuth, H., Ghandil, P., Chrabieh, M., Levy, O., Arkwright, P.D., McDonald, D.,Geha,R.S., Takada,H., Krause, J.C., etal. (2010).Clinical featuresandoutcomeofpatientswithIRAK-4andMyD88deficiency.Medicine(Baltimore)89,403-425.Takeuchi,O.,Hoshino,K.,andAkira,S. (2000).Cuttingedge:TLR2-deficientandMyD88-deficientmicearehighlysusceptibletoStaphylococcusaureusinfection.JImmunol165,5392-5396.Valkov,E.,Stamp,A.,Dimaio,F.,Baker,D.,Verstak,B.,Roversi,P.,Kellie,S.,Sweet,M.J.,Mansell,A.,Gay,N.J., et al. (2011).Crystal structureof Toll-like receptor adaptorMAL/TIRAP reveals themolecular basis for signal transduction and disease protection. Proc Natl Acad Sci U S A 108,14879-14884.von Bernuth, H., Ku, C.L., Rodriguez-Gallego, C., Zhang, S., Garty, B.Z., Marodi, L., Chapel, H.,Chrabieh,M.,Miller,R.L.,Picard,C.,etal.(2006).AfastprocedureforthedetectionofdefectsinToll-likereceptorsignaling.Pediatrics118,2498-2503.von Bernuth, H., Picard, C., Jin, Z., Pankla, R., Xiao, H., Ku, C.L., Chrabieh, M., Mustapha, I.B.,Ghandil, P., Camcioglu, Y., et al. (2008). Pyogenic bacterial infections in humans with MyD88deficiency.Science321,691-696.Wergeland,H.I.,Haaheim,L.R.,Natas,O.B.,Wesenberg,F.,andOeding,P. (1989).Antibodies tostaphylococcalpeptidoglycananditspeptideepitopes,teichoicacid,andlipoteichoicacidinserafromblooddonorsandpatientswithstaphylococcalinfections.JClinMicrobiol27,1286-1291.Yamamoto,M.,Sato,S.,Hemmi,H.,Sanjo,H.,Uematsu,S.,Kaisho,T.,Hoshino,K.,Takeuchi,O.,Kobayashi, M., Fujita, T., et al. (2002a). Essential role for TIRAP in activation of the signallingcascadesharedbyTLR2andTLR4.Nature420,324-329.Yamamoto, M., Sato, S., Mori, K., Hoshino, K., Takeuchi, O., Takeda, K., and Akira, S. (2002b).Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that preferentially activatestheIFN-betapromoterintheToll-likereceptorsignaling.JImmunol169,6668-6672.Zahringer,U.,Lindner,B.,Inamura,S.,Heine,H.,andAlexander,C.(2008).TLR2-promiscuousorspecific? A critical re-evaluation of a receptor expressing apparent broad specificity.Immunobiology213,205-224.Zhang,S.Y.,Jouanguy,E.,Ugolini,S.,Smahi,A.,Elain,G.,Romero,P.,Segal,D.,Sancho-Shimizu,V.,Lorenzo, L., Puel, A., et al. (2007). TLR3 deficiency in patientswith herpes simplex encephalitis.Science317,1522-1527.Zhang,Y.,Su,H.C.,andLenardo,M.J.(2015).Genomicsisrapidlyadvancingprecisionmedicineforimmunologicaldisorders.NatImmunol16,1001-1004.

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Figure legends

Figure 1: TIRAP R121W is a rare mutation located in the TIR domain of TIRAP with a

modest impact at the mRNA and protein level

A. Pedigree of the kindred, with allele segregation. Genotypes are reported on the

family tree: the patient (P1), her father, four aunts and uncles, and the two paternal

grandparents were sequenced and carry the homozygous R121W mutation of TIRAP.

Each generation is identified with a roman numeral (from I to V), and each

individual with an Arabic numeral (from left to right). The patient (P1) with clinical

infectious disease is shown in black and indicated with an arrow. The relationships

between individuals in gray were extrapolated according to the estimated kinship

coefficients (supplementary figure S1)

B. Electrophoregram of TIRAP genomic DNA sequences from a healthy control and

the patient (III.1). The R121W mutation replaces a cytosine with a thymine residue

in exon 5 (c.C797T).

C. Schematic representation of TIRAP, showing the PIP2 domain, the PEST domain,

the TIR domain and the coding exons. The R121W mutation in the TIR domain is

indicated in red.

D. Conservation of the R121 amino acid in the TIR domain of TIRAP in 16 other

species.

E. Expression of the WT and mutant TIRAP genes in transfected cell lines. HEK293T

cells were transfected with both TIRAP isoforms (A and B), as the WT and with the

R121W mutation. Whole-cell extracts were obtained 48 hrs after transfection and

32

evaluated by western blotting with a mouse monoclonal anti-TIRAP antibody and an

antibody raised against the V5 tag. (n=3)

F. Relative quantitative PCR performed to measure the levels of the transcripts

encoding TIRAP isoforms A and B in SV40 fibroblasts (five controls, P1’s, P1

father’s and mother’s cells), PBMCs (9 controls, P1, and P1 aunt’s cells) and EBV-B

cells (5 controls and P1). SV40 fibroblasts stably transfected with the cDNA

encoding either isoform A or B were used as controls to test probe specificity.

TIRAP mRNA levels encoding isoform A or B were expressed relative to GAPDH

mRNA levels using the 2ΔΔC(t) method. For each probe, normalization was made to

one of the controls which had been set to 1. Error bars represent SD (n=2).

G. TIRAP protein levels in PBMCs from two controls, P1’s father and P1, as

determined by western blotting using an anti-TIRAP mouse mAb. Anti-GAPDH Ab

was used as a protein load control. Quantification was performed with the Image J

software (n=1).

Figure 2: The R121W mutation impairs TIRAP cellular localization and interaction with its

partners MyD88 and TLR2

A. HEK293T cells were transfected with a construct encoding a V5-tagged WT or

mutant isoform A of TIRAP for 36 hrs and then labeled with an Alexa Fluor 488-

conjugated anti-V5 antibody. Analyses were carried out by ApoTome fluorescence

microscopy. The images shown are representative of three independent experiments

and correspond to >95% of all the images obtained (30 images acquired pre

experiment)

33

B. Cells were cotransfected with constructs encoding TIRAP WT-V5 and either myc-

tagged WT TIRAP or myc-tagged R121W TIRAP. V5-tagged proteins were labeled

with Alexa Fluor 488-conjugated anti-V5 antibodies and myc-tagged proteins were

labeled with Alexa Fluor 555-conjugated anti-myc antibodies. Colocalization

analysis was performed with Image J software, by calculating Pearson’s correlation

coefficient (PCC). Three independent experiments were performed and 30 cells

were analyzed in each experiment. Error bars represent SD.

C. Cells were cotransfected with constructs encoding myc-tagged MyD88 and either

V5-tagged WT TIRAP or V5-tagged R121W TIRAP. V5-tagged proteins were

labeled with Alexa Fluor 488-conjugated anti-V5 antibodies and myc-tagged

proteins were labeled with Alexa Fluor 555-conjugated anti-myc antibodies.

Colocalization analysis was carried out with Image J software, by calculating

Pearson’s correlation coefficient (PCC). Three independent experiments were

performed and 30 cells were analyzed in each experiment. Error bars represent SD.

D. HEK293T cells were cotransfected with constructs encoding both the TIRAP A and

B isoforms with a V5 tag, WT or mutated, and with a construct encoding HA-

tagged TLR2. HA immunoprecipitation and V5 immunoblotting were then

performed on total cell extracts (n=2).

E. Cells were cotransfected with constructs encoding both TIRAP isoforms (A and B

isoforms), with a V5 tag, WT or mutated, and with a construct encoding M2-tagged

MyD88. M2 immunoprecipitation and V5 immunoblotting were then performed on

total cell extracts (n=2).

34

F. Co-immunoprecipitation between TLR2 and MyD88 without or after stimulation

with PAM2CSK4 (10µg/ml) for 10 and 30min, in SV-40 fibroblasts from a control

(TIRAP WT/WT), the heterozygous P1’s mother (TIRAP R121W/WT), a MyD88-

mutated patient (MyD88 L93P/R196C), and TIRAP homozygous P1 and her father

(TIRAP R121W/R121W) (n=1)

Figure 3: TIRAP R121W allele is loss of function

A. IL-6 induction in SV40 fibroblasts from a healthy control, P1, her father, and

MyD88-deficient cells, upon transfection with WT or mutant TIRAP isoforms. IL-6

production was measured by ELISA in cells transfected with an empty vector, WT

or mutant isoforms A plus B, or MyD88-deficient cells transfected with WT

MyD88 (n=3). Error bars represent SEM.

B. NF-κB luciferase assay in HEK293T cells expressing stable overexpression of NF-

κB driven luciferase reporter plasmid upon transfection with WT TIRAP isoform A

or B, or mutant TIRAP isoforms A or B. (n=3). Error bars represent SEM

C. SV40 transformed fibroblasts from healthy controls, P1, her father, an IRAK-4-

deficient patient and NEMO-deficient fibroblasts were stimulated with TLR1/2

(PAM3CSK4), TLR2/6 (PAM2CSK4, purified LTA-SA, FSL-1), TLR3 (poly(I:C))

and TLR4 (LPS and lipid A) ligands, in addition to heat killed S. aureus (HKSA)

IL-1β (IL-1R) and TNF-α (TNF-αR). Supernatants were collected 24 hrs after

stimulation and IL-6 production was assessed by ELISA. (n=3). Error bars represent

SEM.

35

D. Granulocyte L-selectin shedding assessed in several family members (III.1, IV.1-5

and V.1) after one hour of stimulation with PAM2CSK4, PAM3CSK4, FSL-1,

purified LTA, LPS and PMA as an activation control. Mean fluorescence intensity

was determined by flow cytometry after the extracellular staining of CD62L. * p

values < 0.05 (unpaired t-test). (n=1)

E. PBMCs from several family members (III.1, IV.1 to 5 and V.1) were stimulated

with various doses of PAM2CSK4, FSL-1, LTA, PAM3CSK4 and LPS for 3 hrs,

subjected to extracellular staining with anti-CD3, anti-CD19 and anti-CD14

antibodies and intracellular staining for IL-6. The proportion of CD14+ cells

positive for IL-6 is shown. Acquisition was performed on 10,000 CD14+ cells. Each

data point represent the mean of all individuals +/- SEM

Figure 4: The lack of anti-LTA Abs in P1’s plasma accounts for defective LTA whole

blood response

A. PBMCs from controls, P1, her father (II.1) and aunt (II.2) were stimulated with

PAM3CSK4 (1 µg/ml), PAM2CSK4 (1 µg/ml), FSL-1 (100 ng/ml), LTA (1 µg/ml),

and LPS (10 ng/ml) for 48 hrs. The supernatant were tested, by ELISA, for IL-6

production. Representative results, from one of two experiments carried out, are

shown.

B. IL-6 production by whole blood was assessed by ELISA in I.1, I.2, II.2, II.3, II.4,

II.5, II.6 and III.1 after 48 hrs of stimulation with PAM2CSK4, FSL-1, LTA,

PAM3CSK4 and LPS. The assay was performed several times and each point

represents the mean for one individual. Whole blood from P1 was tested five times

for FSL-1 stimulation, six for LTA, seven for PAM3CSK4 and LTA and eight times

36

for PAM2CSK4. Whole blood from other individuals was tested once (I.1), twice

(I.2, II.2, II.3, II.4) or three times (II.1) for each stimulus.

C. Tests for the presence of specific anti-LTA antibodies in plasma detected by ELISA,

dilution 1/100 is shown but tests were performed at 1/20, 1/100, 1/500 plasma

dilution.

D. Complementation of the defect of IL-6 production in response to LTA stimulation in

whole blood from the patient, by the addition of monoclonal anti-LTA antibodies

(pagibaximab) (0,5 or 1 mg/ml). Comparison with the isotypic control (0,5 or 1

mg/ml), (palivizumab, anti-RSV). Results from one representative experiment of the

two carried out are shown. (p=0.001, unpaired t-test). Error bars represent SEM.

E. Complementation of the impaired IL-6 production in response to LTA stimulation in

PBMCs from P1 by the addition of anti-LTA mAbs. IL-6 production by controls’ or

P1’s PBMCs after 48hrs of stimulation with 1µg/ml of PAM3CSK4 or 1 µg/ml of

LTA in the presence of 10% of FCS, 1mg/ml of exogenous isotype control mAbs or

1mg/ml of anti-LTA mAbs. Error bars represent SEM (n=4).

F. Peritoneal mouse macrophages from WT B6, Myd88-/-, Tiraptor/tor and TLR2lan/lan

(n>3) were stimulated with PAM3CKS4 (40ng/ml) or LTA (1ug/ml) or left

unstimulated and complemented with 0,5mg/ml of either anti-LTA Abs

(Pagibaximab) or isotype control (anti RSV, Synagis). IL-6 production was assessed

by ELISA. Error bars represent SEM

37

Supplementary legends : Figure S1: Familial relationship. A. Within-family relationships estimated with KING. X-

axis represents the expected kinship coefficient for a non-consanguineous family and y-axis

represents the estimated kinship coefficient in this family. Each dot represents the kinship

coefficient between two members of the family. Black dots represent an estimated kinship

coefficient concordant with the expected kinship coefficient for this kind of relationship.

Red dots represent an estimated kinship coefficient discordant with the expected kinship

coefficient for this kind of relationship. Pedigree constructed according to the estimated

kinship coefficients.

Figure S2: Alignment of the human TIRAP protein with other human TIR domain-

containing molecules

Figure S3: TIRAP RT-PCR ranging from 27 to 33 cycles, on RNA extracted from SV40-

fibroblasts from P1 and her father, compared to healthy control. Primer specific for

transcripts encoding isoform A or B were used.

Figure S4: Quantitative PCR performed to measure the levels of the transcripts encoding

TIRAP isoforms A and B in SV40 fibroblasts (five controls, P1’s, P1 mother’s and father’s

cells), EBV-B cells (5 controls and P1) and PBMCs (9 controls, P1, and P1 aunt’s cells).

SV40 fibroblasts stably transfected with either the cDNA encoding isoform A or B were

used as controls to test probe specificity. The percentages of the transcripts encoding

isoform A or B were normalized to the total amount of all transcripts (encoding for both

isoforms A and B, set at 100%.) Error bars represent SD.

Figures S5-S7: Complementation study in SV40 fibroblasts from P1 (S5), her father (S6)

and a control (S7), after stable transfection with lentiviral particles encoding the WT or

38

mutant A or B isoform of TIRAP, or both. Particles containing luciferase instead of TIRAP

were used for mock transfection. IL-6 production was assessed by ELISA.

Figure S8: IL-6 mRNA levels assessed by RT-qPCR and expressed relative to GUS mRNA

levels by the 2ΔΔC(t) method, in SV40 fibroblasts from a healthy control. Cells were

stimulated for 4 hrs with PAM2SCK4 or TNF-α, 24 hrs after transfection with siRNAs

against TLR2 and TIRAP.

Figure S9: PBMCs from several family members (III.1, IV.1-5 and V.1) were stimulated

with various doses of PAM2CSK4, PAM3CSK4, LTA, FSL-1, and LPS for 3 hrs, subjected

to extracellular staining with anti-CD3, anti-CD19 and anti-CD14 antibodies and

intracellular staining for TNF-α. The proportion of CD14+ cells positive for TNF-α is

shown. Acquisition was performed on 10,000 CD14+ cells. Each data point represents the

mean of all individuals +/- SEM

Figure S10: Transcriptome analysis performed on whole blood after 3 hrs of stimulation

with PAM2CSK4, FSL-1, LTA, PAM3CSK4, LPS or PMA ionomycin. The results for P1

and her mother (R121W/WT) are compared with those for one healthy control and one

IRAK-4-deficient patient.

Figure S11: PBMCs and whole blood from healthy controls, P1 and her father were

stimulated with lipid A (1µg/ml), LPS (10ng/ml) and PMA/ionomycin for 48 hrs. IL-6

production was assessed by ELISA. Error bars represent SD.

Figure S12: Assessment by luminex of antibodies against 68 staphyloccocal antigens, (6

only are shown in the figure, all results are summarized on supplementary table 2), in 6

different plasma from P1 (between 1 to 8 years old), 5 homozygous relatives, 6 IRAK-4 or

MyD88 deficient patients and 11 sex and aged matched controls. Bonferroni’s multiple

39

comparison testing indicated that for none of the antigen tested, the patient showed

significantly lower level of antibodies than controls.

Figure S13: PBMCs from P1 and two controls were left non-stimulated or stimulated by

PAM3CKS4 or LTA in the presence of fetal calf serum (FCS) or plasma from each

individual. IL-6 production was assessed by ELISA. Error bars represent SD.

Figure S14: IL-6 production by whole blood was assessed in a healthy control and an

IRAK4-deficient patient upon 48 hrs of stimulation with PAM3CSK4 or LTA supplemented

with 0.5mg/ml of either a control isotype antibody (anti-RSV, Synagis) or with anti-LTA

(Pagibaximab) monoclonal antibodies.

Figure S15: IL-6 production by controls’ PBMCs, monocytes, and monocyte-depleted

PBMCs after 24 hrs of stimulation with 1µg/ml of PAM3CSK4 or 1µg/ml of LTA in the

presence of medium alone, or medium supplemented with 1 mg/ml of either anti-LTA mAb

or with a isotype control mAb (anti-RSV).

Figure S16: Peritoneal mouse macrophages from WT B6, Myd88-/-, Tiraptor/tor and

TLR2lan/lan (n>3) were stimulated with PAM3CKS4 (40ng/ml) or LTA (1ug/ml) or left

unstimulated and complemented with 0,5mg/ml of either anti-LTA Abs (Pagibaximab) or

isotype control (anti RSV, Synagis). TNF-α production was assessed by ELISA. Error bars

represent SEM.

Figure S17: Assessment by ELISA of levels of anti-LTA antibodies in 16 different batches

of commercially available IVIG compared to healthy controls (n=10) and P1. Error bars

represent SEM.

1

Supplementary tables legends:

Supplementary table 1: List of genes presenting a non-synonymous rare homozygous

variation in P1 exome data.

Supplementary table 2 : IgG levels against 68 recombinant S. aureus proteins were

measured in serum samples using a bead-based flow cytometry technique in 6 different

plasma from the patient (between 1 to 8 years old), 5 homozygous relatives, 6 IRAK-4

or MyD88 deficient patients and 11 aged matched controls. The table summarize mean

and SD for each antigen in each plasma category. Bonferroni’s multiple comparison

testing was performed and p value for significant differences compared to controls are

indicated.

Supplementary table 3: Metrics and coverage data for the three exomes sequenced

2

Supplementary tables:

Supplementary table 1 :

nomenclature chr allele gene AA

protein

pos

1 c.542G>A 1 G/A ENSG00000132681 (ATP1A4) R/Q 181

2 c.1150A>G 1 T/C ENSG00000137992 (DBT) S/G 384

3 c.125G>A 1 G/A ENSG00000213088 (DARC) G/D 42

4 c.1693T>C 1 A/G ENSG00000134242 (PTPN22) W/R 565

5 c.2009G>A 1 G/A ENSG00000169174 (PCSK9) G/E 670

6 c.344G>A 1 C/T ENSG00000143278 (F13B) R/H 115

7 c.1601A>G 1 T/C ENSG00000198734 (F5) Q/R 534

8 c.7480T>C 1 A/G ENSG00000066279 (ASPM) Y/H 2494

9 c.49A>G 2 A/G ENSG00000163599 (CTLA4) T/A 17

10 c.380T>C 2 A/G ENSG00000135899 (SP110) L/S 127

11 c.733G>A 2 C/T ENSG00000170820 (FSHR) A/T 245

12 c.1853G>A 2 C/T ENSG00000170820 (FSHR) S/N 618

13 c.3448T>C 2 A/G ENSG00000169432 (SCN9A) W/R 1150

14 c.41C>G 3 C/G ENSG00000173402 (DAG1) S/W 14

15 c.743T>C 3 T/C ENSG00000145192 (AHSG) M/T 248

16 c.767G>C 3 G/C ENSG00000145192 (AHSG) S/T 256

17 c.163A>G 4 T/C ENSG00000145384 (FABP2) T/A 55

18 c.53C>A 4 C/A ENSG00000154277 (UCHL1) S/Y 18

19 c.1805G>T 4 C/A ENSG00000174125 (TLR1) S/I 602

20 c.3568G>A 5 G/A ENSG00000095015 (MAP3K1) A/T 1190

21 c.46G>A 5 G/A ENSG00000169252 (ADRB2) G/R 16

22 c.79G>C 5 G/C ENSG00000169252 (ADRB2) E/Q 27

23 c.412G>A 5 G/A ENSG00000168685 (IL7R) V/I 138

24 c.197T>C 5 T/C ENSG00000168685 (IL7R) I/T 66

3

25 c.2893G>A 5 C/T ENSG00000138829 (FBN2) V/I 965

26 c.10411G>A 5 G/A ENSG00000164199 (GPR98) E/K 3471

27 c.122C>A 5 G/A ENSG00000258864 (CTC-554D6.1) A/E 41

28 c.5465T>A 5 T/A ENSG00000134982 (APC) V/D 1822

29 c.1630A>C 5 A/C ENSG00000112964 (GHR) I/L 544

30 c.989C>T 6 G/A ENSG00000204525 (HLA-C) A/V 330

31 c.982G>A 6 C/T ENSG00000204525 (HLA-C) V/I 328

32 c.925A>G 6 T/C ENSG00000204525 (HLA-C) M/V 309

33 c.302G>A 6 C/T ENSG00000234745 (HLA-B) S/N 101

34 c.47G>C 6 C/G ENSG00000204525 (HLA-C) G/A 16

35 c.11T>C 6 A/G ENSG00000234745 (HLA-B) M/T 4

36 c.1136T>C 6 A/G ENSG00000146070 (PLA2G7) V/A 379

37 c.1105A>G 6 T/C ENSG00000204525 (HLA-C) T/A 369

38 c.991A>G 6 T/C ENSG00000204525 (HLA-C) M/V 331

39 c.872A>C 6 T/G ENSG00000204525 (HLA-C) Q/P 291

40 c.41C>G 6 G/C ENSG00000234745 (HLA-B) S/W 14

41 c.44C>G 6 G/C ENSG00000234745 (HLA-B) A/G 15

42 c.49C>G 6 G/C ENSG00000234745 (HLA-B) L/V 17

43 c.292G>T 6 C/A ENSG00000234745 (HLA-B) D/Y 98

44 c.610G>C 6 C/G ENSG00000234745 (HLA-B) E/Q 204

45 c.363C>G 6 G/C ENSG00000234745 (HLA-B) S/R 121

46 c.355C>T 6 G/A ENSG00000234745 (HLA-B) L/F 119

47 c.5T>G 6 A/C ENSG00000234745 (HLA-B) L/R 2

48 c.560A>T 6 T/A ENSG00000234745 (HLA-B) E/V 187

49 c.22G>A 6 C/T ENSG00000204525 (HLA-C) A/T 8

50 c.559G>C 6 C/G ENSG00000234745 (HLA-B) E/Q 187

51 c.910C>G 6 G/C ENSG00000112619 (PRPH2) Q/E 304

52 c.206A>C 6 T/G ENSG00000234745 (HLA-B) E/A 69

53 c.929G>A 6 C/T ENSG00000112619 (PRPH2) R/K 310

4

54 c.594G>T 6 G/T ENSG00000078401 (EDN1) K/N 198

55 c.829C>G 6 G/C ENSG00000204525 (HLA-C) Q/E 277

56 c.142T>G 6 A/C ENSG00000234745 (HLA-B) S/A 48

57 c.205G>A 6 C/T ENSG00000234745 (HLA-B) E/K 69

58 c.71C>T 7 C/T ENSG00000184408 (KCND2) S/L 24

59 c.553A>C 7 T/G ENSG00000188050 (RNF133) I/L 185

60 c.1642G>A 7 G/A ENSG00000257743 (RP11-1220K2.2) E/K 548

61 c.236T>C 7 T/C ENSG00000211746 (TRBV19) I/T 79

62 c.785C>T 7 G/A ENSG00000257138 (TAS2R38) A/V 262

63 c.894T>G 7 T/G ENSG00000164867 (NOS3) D/E 298

64 c.1388T>C 7 A/G ENSG00000004948 (CALCR) L/P 463

65 c.1408C>T 7 G/A ENSG00000122512 (PMS2) P/S 470

66 c.1739T>C 7 A/G ENSG00000105929 (ATP6V0A4) M/T 580

67 c.2200T>G 8 T/G ENSG00000042832 (TG) S/A 734

68 c.5995C>T 8 C/T ENSG00000042832 (TG) R/W 1999

69 c.2564C>G 9 G/C ENSG00000165271 (NOL6) A/G 855

70 c.494G>T 9 G/T ENSG00000137124 (ALDH1B1) G/V 165

71 c.1091G>A 9 C/T ENSG00000095397 (DFNB31) R/H 364

72 c.6011C>G 10 C/G ENSG00000107736 (CDH23) T/S 2004

73 c.244G>C 10 G/C ENSG00000107736 (CDH23) G/R 82

74 c.457T>C 10 T/C ENSG00000165730 (STOX1) Y/H 153

75 c.145A>G 10 A/G ENSG00000043591 (ADRB1) S/G 49

76 c.1165G>C 10 G/C ENSG00000043591 (ADRB1) G/R 389

77 c.361C>T 11 C/T ENSG00000150455 (TIRAP) R/W 121

78 c.328A>C 11 A/G ENSG00000109861 (CTSC) I/L 110

79 c.775A>G 11 T/C ENSG00000172638 (EFEMP2) I/V 259

80 c.388C>T 12 G/A ENSG00000121318 (TAS2R10) P/S 130

81 c.242C>G 12 G/C ENSG00000197870 (PRB3) P/R 81

82 c.1751C>T 12 C/T ENSG00000182326 (C1S) A/V 584

5

83 c.603G>T 13 G/T ENSG00000123171 (CCDC70) E/D 201

84 c.7397T>C 13 T/C ENSG00000139618 (BRCA2) V/A 2466

85 c.1120T>G 13 A/C ENSG00000123191 (ATP7B) S/A 374

86 c.1270G>C 13 C/G ENSG00000123191 (ATP7B) V/L 424

87 c.2129T>C 13 A/G ENSG00000123191 (ATP7B) V/A 710

88 c.1565G>A 13 C/T ENSG00000123191 (ATP7B) R/K 522

89 c.2126G>A 14 G/A ENSG00000100714 (MTHFD1) R/Q 709

90 c.259G>A 15 C/T ENSG00000159337 (PLA2G4D) V/I 87

91 c.1061T>C 15 T/C ENSG00000140463 (BBS4) I/T 354

92 c.1331A>C 16 A/C ENSG00000179588 (ZFPM1) E/A 444

93 c.538G>A 16 C/T ENSG00000121270 (ABCC11) G/R 180

94 c.1066A>G 17 T/C ENSG00000108578 (BLMH) I/V 356

95 c.154G>T 17 G/T ENSG00000132518 (GUCY2D) A/S 52

96 c.188A>G 19 T/C ENSG00000131398 (KCNC3) D/G 63

97 c.7211G>C 20 C/G ENSG00000130702 (LAMA5) R/P 2404

98 c.385A>G 20 A/G ENSG00000171867 (PRNP) M/V 129

99 c.2339A>C 20 T/G ENSG00000075043 (KCNQ2) N/T 780

100 c.112A>G 21 T/C ENSG00000180509 (KCNE1) S/G 38

101 c.185A>G 22 A/G ENSG00000211647 (IGLV5-48) E/G 62

102 c.226A>G 22 A/G ENSG00000211647 (IGLV5-48) S/G 76

103 c.227G>A 22 G/A ENSG00000211647 (IGLV5-48) S/N 76

104 c.1049A>G 22 T/C ENSG00000100299 (ARSA) N/S 350

105 c.1172C>G 22 G/C ENSG00000100299 (ARSA) T/S 391

106 c.466A>G X A/G ENSG00000101981 (F9) T/A 156

107 c.97T>C X T/C ENSG00000147100 (SLC16A2) S/P 33

6

Supplementary table 2 :

Controls (n=11) IRAK4-/- or MyD88-/- (n=6) Patient Tirap-/- Tirap -/- healthy relatives (n=6)

ANTIGEN Mean SD Mean SD diff from controls Mean SD diff from

controls Mean SD diff from controls

lukS 7858 4788 10201 4213 ns 16101 1685 ** 15158 4463 * fnbpB5 1603 2194 643 387 ns 2586 1952 ns 1002 1339 ns CHIPS 13332 7139 18752 2287 ns 15310 3108 ns 17858 2357 ns eapH2 8513 6242 17449 4792 * 15821 5272 ns 12783 6814 ns clfBN2,3 7252 4927 7166 4749 ns 3198 1055 ns 7484 3602 ns fnbpA1 3719 4580 2577 2803 ns 259 219 ns 5206 3337 ns flip-R 6752 7105 7395 2962 ns 30.8 41.3 ns 4300 2863 ns SEM 979 1617 1827 1274 ns 128 132 ns 2606 2558 ns ETA 928 1560 3251 4680 ns 589 919 ns 6189 6222 ns SEH 1666 2642 4729 6702 ns 120 88.3 ns 1604 2893 ns sdrD 993 1515 2348 2668 ns 829 270 ns 1183 623 ns fndpA2 625 673 2850 3697 ns 2468 576 ns 4533 2618 ** SCIN 6051 5072 9487 6094 ns 14700 2490 ** 10059 4400 ns TSST-1 5464 7542 11846 9159 ns 857 1426 ns 12967 3838 ns fnbpA3 1771 2428 3706 3870 ns 850 438 ns 4711 3087 ns hlgB 9996 4257 12800 2716 ns 11915 2507 ns 13878 2583 ns Nuc 4187 4733 12337 6345 ns 14860 3065 * 10325 8195 ns fnbpA5 1095 1540 3063 3626 ns 2579 2280 ns 2592 2670 ns SEB 2315 3328 3566 3790 ns 11940 9488 * 11248 8091 ns SEA 2333 2946 7571 6999 ns 6581 3120 ns 12030 4061 *** lukE 11775 6025 15911 4041 ns 12915 2182 ns 17219 3311 ns lukD 10921 5598 11314 3694 ns 9710 1359 ns 14191 3657 ns eap 6709 3166 14967 2424 *** 13178 2035 *** 13154 1843 *** clfAN2,3 2260 2512 3886 5438 ns 8120 4415 * 5536 2257 ns A-tox 8050 5053 13513 5196 ns 13210 2040 ns 13791 4941 ns ssl-1 5833 5231 11074 8742 ns 1980 1616 ns 9521 5242 ns sasH 406 516 415 584 ns 213 138 ns 269 338 ns ssl-3 9652 6770 12954 3787 ns 6048 4741 ns 14085 3514 ns ssl-5 3274 2055 4918 2768 ns 2344 1132 ns 4184 1800 ns ssl-9 10101 5194 15332 2994 ns 9048 4923 ns 14444 5669 ns fnbpA6 561 432 1458 2632 ns 134 47.8 ns 1024 629 ns ssl-10 6309 4233 8170 3254 ns 2594 1361 ns 7962 1364 ns ssl-11 1589 1999 1792 2021 ns 85.7 79.7 ns 1125 1401 ns sec 7475 8773 14402 6989 ns 9714 7676 ns 15402 4819 ns etb 182 246 2672 5129 ns 79 60.7 ns 454 745 ns seo 608 720 2156 1622 * 338 266 ns 1246 536 ns fnbpB 562 847 285 255 ns 2300 1506 ** 946 948 ns fnbpB1 699 1123 472 531 ns 158 85.2 ns 372 310 ns isdA 14551 4645 17305 2466 ns 18029 1641 ns 13815 8321 ns

7

fnbpB2 2021 1901 2132 1722 ns 3835 1315 ns 4857 2757 ns fnbpB4 1504 2328 1809 2445 ns 543 239 ns 1235 1139 ns fnbpB6 438 596 704 477 ns 167 54.4 ns 1089 1081 ns fnbpB7 1448 2504 1716 2539 ns 440 177 ns 1194 1273 ns fnbpA7 741 832 1542 2410 ns 483 721 ns 1367 1279 ns sasC 154 124 329 416 ns 140 74.7 ns 310 181 ns flipR-L 5760 5261 9998 4771 ns 97 72.4 ns 4849 3099 ns esxB 259 299 247 225 ns 148 191 ns 159 147 ns prsA 621 908 2336 2516 ns 2727 3676 ns 572 308 ns atl2 7643 4486 14766 3511 * 17075 4651 ** 14688 3973 * lipase 11684 7289 17179 3296 ns 4671 3633 ns 10058 6773 ns sed 1528 2142 1757 684 ns 703 276 ns 516 308 ns fnbpA 2609 3450 3057 4941 ns 849 863 ns 2685 1722 ns sdrE 1541 1876 2450 2785 ns 1008 507 ns 2158 1399 ns SEE 253 620 3777 4716 ns 2646 1587 ns 5403 2913 ** SEJ 2491 3455 5448 1919 ns 1825 868 ns 2573 1698 ns isdH 695 920 796 623 ns 911 349 ns 740 728 ns sasG 850 1456 777 1042 ns 177 159 ns 1608 1932 ns sdrC 1478 1642 3970 3871 ns 897 835 ns 4118 3403 ns SEI 511 669 1499 1007 ns 298 145 ns 3369 3106 ** SEQ 745 1709 1484 1646 ns 74.7 41.6 ns 2098 1742 ns isaA 7943 5917 17129 6758 * 2210 1136 ns 6631 4743 ns lyt M 1678 2595 8325 6795 ** 900 631 ns 2280 1613 ns SER 312 573 725 832 ns 132 136 ns 1274 1705 ns Aly 1391 1447 3361 1938 ns 3427 1824 ns 2198 1016 ns pro Atl 1361 2225 4501 6461 ns 6402 3651 ns 3597 1229 ns SEG 493 596 1733 1107 ns 303 322 ns 3606 2476 *** eapH1 827 1128 2255 2254 ns 1471 1009 ns 1204 1083 ns SA2097 4.02 6.71 15.6 14.9 ns 25.6 18.8 * 8.42 8.23 ns

8

Supplementary table 3 :

P1 (III.1) II.5 I.1

Single/Paired-end Paired-end Paired-end Paired-end

Bait-set 50Mb 50Mb 71Mb

Total reads 164,759,220 184,566,394 132,090,076

Total reads aligned in the targeted regions 22,485,016 26,512,246 75,999,552

Mean coverage 44.97 53.02 107.04

Target bases covered by > 2X 99.4 99.6 99.7

Target bases covered by > 10X 92.3 94.6 98.5

Target bases covered by > 20X 75.8 81.5 95.2

Target bases covered by > 30X 58.4 66.1 90

A. B. C.

D.

Fig. 1

F.

C1 C2 Father P1

TIRAP

PBMCs

GAPDH

TIR

AP

norm

. to

GA

PD

H

G.

TIRAP

V5

GAPDH

HEK 293T cells E.

Ct r

+ A

Ct r

+ B

Ct r

+ A

&B

Ct r

P1

Fa t h

e r

Mo t h

e rC

t rP

1A

u n tC

t rP

1

0 . 1

1

1 0

1 0 0

1 0 0 0

1 0 0 0 0

TIR

AP

is

ofo

rms

A o

r B

mR

NA

le

ve

l

no

rma

liz

ed

to

GA

PD

H

I s o A

S V 4 0

f i b r o b l a s t s

P B M C s

I s o B

B E B V

A.

B.

IB: HA

IB: V5

IP: HA IB: V5

TLR2-HA + + + + +

TIRAP A WT-V5 + - - - -

TIRAP A mut-V5 - + - - -

TIRAP B WT-V5 - - + - -

TIRAP Bmut-V5 - - - + -

MyD88-M2 + + + + - - + -

TIRAP A WT-V5 + - - - + - - -

TIRAP A mut-V5 - + - - - + - -

TIRAP B WT-v5 - - + - - - - -

TIRAP B mut-v5 - - - + - - - -

IB: V5

IB: M2

IP: M2 IB: M2

IP: M2 IB: V5

TIRAPWT

TIRAPmut

C.

D. E.

TIRAPWT TIRAPWT Merge

TIRAPWT TIRAPmut Merge

TIRAPmut MyD88 Merge

TIRAPWT MyD88 Merge

Fig. 2

F.

A.

B.

Fig. 3

C.

MFI

NS PAM2 LTA FSL-1 LPS PMA

Granulocytes, 1h

102

103

PAM3

Controls Mother P1 R121W/R121W healthy

E.

D.

A W T B W T A R 1 2 1 W B R 1 2 1 W M o c k

0

2 1́ 0 0 4

4 1́ 0 0 4

6 1́ 0 0 4

8 1́ 0 0 4

NF

-kB

lu

cif

era

se

(u

nit

s)

2 9 3 T c e l l s

0 0 , 0 1 0 , 1 1

0

1 0

2 0

3 0

P A M 2 C S K 4 ( u g / m l )

% o

f IL

-6+

ce

lls

0 0 , 0 1 0 , 1 1 1 0

0

2 0

4 0

6 0

P A M 3 C S K 4 ( u g / m l )

C t r

R 1 2 1 W / R 1 2 1 W

0 0 , 1 1

0

2 0

4 0

6 0

8 0

L T A ( u g / m l )

0 0 , 0 1 0 , 1

0

1 0

2 0

3 0

4 0

5 0

F S L - 1 ( u g / m l )

% o

f IL

-6+

ce

lls

0 0 , 1 1

0

2 0

4 0

6 0

L P S ( n g / m l )

CD14+ cells, 3h

C o n t r o ls P 1 F a t h e r M y D 8 8 - / -

0

2 1́ 0 0 3

4 1́ 0 0 3

6 1́ 0 0 3

IL-6

(p

g/m

l)

E m p t y v e c t o r

T I R A P A + B W T

T I R A P A + B R 1 2 1 W

M y D 8 8

S V - 4 0 f i b r o b l a s t s

N S P A M 2

T L R 2

L T A

T L R 2

F S L 1

T L R 2

P A M 3

T L R 2

P o ly ( I: C )

T L R 3

L P S

T L R 4

L ip id A

T L R 4

H K S A IL - 1 b

IL 1 - R

T N F a

T N F R

1 0 0

1 0 1

1 0 2

1 0 3

1 0 4

1 0 5

1 0 6

IL-6

(p

g/m

l)

C T R

I R A K 4 - / -

N E M O - / -

P 1 ( R 1 2 1 W / R 1 2 1 W )

F a t h e r ( R 1 2 1 W / R 1 2 1 W )

S V 4 0 f i b r o b l a s t s

Fig. 4

B.

PBMCs, 48h A. D.

C.

Whole blood, 48h

N S P A M 3 P A M 2 F S L - 1 L T A L P S

1 0 0

1 0 1

1 0 2

1 0 3

1 0 4

1 0 5

IL-6

pg

/ml

C t r

R 1 2 1 W / R 1 2 1 W h e a l t h y

P 1

E.

N S P A M 3 P A M 2 F S L - 1 L T A L P S

1 0 0

1 0 1

1 0 2

1 0 3

1 0 4

1 0 5

1 0 6

IL-6

(p

g/m

l)

C t r

R 1 2 1 W / R 1 2 1 W h e a l t h y

P 1

****

ns

C +

( a d u l t s )

C +

( c h i ld r e n )

C h i ld r e n

w i t h o t h e r

in f e c t io n

C h i ld r e n

w i t h S t a p h

in f e c t io n s

IR A K 4 - / -

M y D 8 8 - / -

P 1 R 1 2 1 W / R 1 2 1 W

h e a l t h y

0 . 0

0 . 5

1 . 0

1 . 5

2 . 0

OD

a n t i - L T A i n p l a s m a

N S P A M 3 L T A N S P A M 3 L T A N S P A M 3 L T A

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

4 0 0 08 0 0 0

W T

M y D 8 8 - / -

T L R 2 - / -

T I R A P - / -

m e d i a + a n t i - L T A A b + C o n t r o l I s o t y p e A b

p = 0 . 0 1 4

n sIL

-6 (

pg

/ml)

F.

C t r A u n t P 1 C t r A u n t P 1 C t r A u n t P 1

1 0 1

1 0 2

1 0 3

1 0 4

1 0 5

IL-6

(p

g/m

l)

N S

P A M 3 C S K 4

L T A

W h o l e b l o o d + a n t i - L T A A b s + i s o t y p e

* *

C t r P 1 C t r P 1 C t r P 1

1 0 0

1 0 1

1 0 2

1 0 3

1 0 4

1 0 5

IL-6

(p

g/m

l)

F C S + a n t i - L T A A b s + i s o t y p e

N S

P A M 3 C S K 4

L T A

Supp Fig.

S1.

S2.

S3.

Ct r

+ A

Ct r

+ B

Ct r

+ A

&B

Ct r

P1

Fa t h

e r

Mo t h

e rC

t rP

1A

u n tC

t rP

1

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

% m

RN

A o

f e

ac

h p

rob

e

no

rma

liz

e t

o G

AP

DH

I s o A

I s o B

S V - 4 0

f i b r o b l a s t s

P B M C s B E B V

C+

P1

Father

27 30 33 cycles 27 30 33

Isoform A Isoform B

SV-40 fibroblasts

S4.

S5.

Non transfected Mock Iso A Iso B Iso A+B Iso A mut Iso B mut Iso A+B mut

IL-6

(pg/

ml)

Patient's SV-40 fibroblasts NS PAM2 LPS TNFα

102

103

104

105

101

100

S7.

Non transfected

Mock Iso A Iso B Iso A+B Iso A mut Iso B mut Iso A+B mut

IL-6

(pg/

ml)

Father's SV-40 fibroblasts

102

103

104

105

101

100

Non transfected Mock Iso A Iso B Iso A+B Iso A mut Iso B mut Iso A+B mut

IL-6

(pg/

ml)

Control’s SV-40 fibroblasts

102

103

104

101

100

S6.

Supp Fig.

Supp Fig.

S10.

PAM2

C+

IRA

K4-

/- P1

Mot

her

FSL-1

C+

IRA

K4-

/- P1

Mot

her

LTA

C+

IRA

K4-

/- P1

Mot

her

PAM3

C+

IRA

K4-

/- P1

Mot

her

LPS

C+

IRA

K4-

/- P1

Mot

her

C+

IRA

K4-

/- P1

Mot

her

PMA

S9. S8.

0

5

10

15

20

Scrambled siTIRAP siTLR2

IL-6

rela

ted

to G

US

SV-40 fibroblasts NS PAM2 TNFα

Supp Fig.

S11.

S12.

C t r P 1 F a t h e r C t r P 1 F a t h e r

1 0 0

1 0 1

1 0 2

1 0 3

1 0 4

1 0 5

N S

l i p i d A

L P S

P M A i o n o

IL-6

(p

g/m

l)

P B M C s W h o l e b l o o d

Supp Fig.

S13.

S14.

1 0 0

1 0 1

1 0 2

1 0 3

1 0 4

1 0 5

N SP A M 3 C S K 4

L T A

plasma FCS P1 Ctr1 Ctr2 FCS P1 Ctr1 Ctr2PBMCs Ctr P1

IL-6

(p

g/m

l)

Whole blood +Anti-LTA Abs +isotype Whole blood +Anti-LTA Abs +isotype

C+ IRAK4-/-

IL-6

(pg

/ml)

WB, 48H NS PAM3 LTA

102

100

101

103

104

105

Supp Fig.

S15.

m e d ia a n t i - L T A is o m e d ia a n t i - L T A is o m e d ia a n t i - L T A is o

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

1 0 0 0 0

1 5 0 0 0

IL-6

(p

g/m

l)

N S

P A M 3

L T A

P B M C s M o n o c y t e s M o n o - d e p l e t e d P B M C s

S16.

N S P A M 3 C S K 4 L T A N S P A M 3 C S K 4 L T A N S P A M 3 C S K 4 L T A

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 0

4 0 0 0

8 0 0 0W T

M y D 8 8 - / -

T L R 2 - / -

T I R A P - / -

m e d i a + a n t i - L T A A b + C o n t r o l I s o t y p e A b

p = 0 . 0 0 4

n s

TN

Fa

(p

g/m

l)

S17.

C o n t r o l s

( n = 1 0 )

P a t i e n t

( R 1 2 1 W / R 1 2 1 W )

IV Ig s

( n = 1 6 )

0

1

2

3

4

OD