Lethal Influenza in Two Related Adults with Inherited GATA2 Deficiency.

The pathogenesis of life-threatening influenza A virus (IAV) disease remains elusive, as infection is benign in most individuals. We studied two relatives who died from influenza. We Sanger sequenced GATA2 and evaluated the mutation by gene transfer, measured serum cytokine levels, and analyzed circulating T- and B-cells. Both patients (father and son, P1 and P2) died in 2011 of H1N1pdm IAV infection at the ages of 54 and 31 years, respectively. They had not suffered from severe or moderately severe infections in the last 17 (P1) and 15 years (P2). A daughter of P1 had died at 20 years from infectious complications. Low B-cell, NK- cell, and monocyte numbers and myelodysplastic syndrome led to sequence GATA2. Patients were heterozygous for a novel, hypomorphic, R396L mutation leading to haplo-insufficiency. B- and T-cell rearrangement in peripheral blood from P1 during the influenza episode showed expansion of one major clone. No T-cell receptor excision circles were detected in P1 and P3 since they were 35 and 18 years, respectively. Both patients presented an exuberant, interferon (IFN)-γ-mediated hypercytokinemia during H1N1pdm infection. No data about patients with viremia was available. Two previously reported adult GATA2-deficient patients died from severe H1N1 IAV infection; GATA2 deficiency may predispose to life-threatening influenza in adulthood. However, a role of other genetic variants involved in immune responses cannot be ruled out. Patients with GATA2 deficiency can reach young adulthood without severe infections, including influenza, despite long-lasting complete B-cell and natural killer (NK) cell deficiency, as well as profoundly diminished T-cell thymic output.

Introduction

Influenza A virus (IAV) infection typically causes a self-limiting disease of the upper respiratory tract [1, 2]. Primary viral pneumonia (PVP) is a rare complication of seasonal influenza [2]. Severe influenza is more common in the course of pandemic than seasonal influenza. Yet, most individuals infected with the 2009 pandemic H1N1 (H1N1pdm) IAV experienced an uncomplicated flu and up to 75% of infections were even estimated to be subclinical [3-5]. However, in a small subset of patients, H1N1pdm infection rapidly progressed to PVP with respiratory failure. A minority of patients were admitted to intensive care units (ICUs), due to acute respiratory distress syndrome (ARDS) in most cases [1, 3, 5]. The case-fatality rate for symptomatic H1N1pdm illness was estimated to be, depending on the age groups, between 0.002 and 0.308% in Western countries [3, 6].

Inherited and acquired variability in host immune responses may influence susceptibility and outcome of IAV infection [7-9]. However, the molecular nature of such human factors has remained largely elusive. It was recently shown that single-gene inborn errors of immunity may underlie severe influenza. Indeed, IRF7 deficiency was associated with severe influenza in one child, and two adult patients with GATA2 deficiency died from severe H1N1 IAV infection [10-12]. It is interesting and intriguing that severe B- and T-cell deficiencies do not underlie severe influenza [9].

GATA2 deficiency, due to germline heterozygous mutations in GATA2, was first reported in 2011 [13-16]. The initial presentation of autosomal dominant (AD) GATA2 deficiency usually occurs in the second decade, but ranges from early childhood to late adulthood. The disease manifestations are variable, and characterized by an increased risk of infectious complications, which usually remains low until the third or fourth decade. Patients are susceptible to severe viral infections, particularly by human papillomaviruses (HPVs) and herpesviruses, to nontuberculous mycobacteria, and to severe fungal infections [13-18]. Patients have an increased risk of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic myelomonocytic leukemia [13, 14, 16], chronic neutropenia [12], aplastic anemia [19], primary lymphedema, sensorineural deafness, or pulmonary alveolar proteinosis [13-17].

The laboratory findings include profound monocytopenia, dendritic cell (DC) deficiency, including in particular a deficiency of interferon (IFN)-producing plasmacytoid DCs (pDCs), and B-cell and natural killer (NK) cell lymphopenia [11–14, 17, 18]. Two previously reported adult GATA2-deficient patients died from severe H1N1 IAV infection, although after other severe manifestations [11, 12]. One patient had previously suffered from disseminated mycobacterial and HPV infections [11]. The second patient had developed AML at the age of 13 years, and, after intensive chemotherapy complicated by pulmonary aspergillosis, she suffered from numerous infections; she died at the age of 18 from an H1N1 influenza infection in complete remission [12]. The pathogenesis of influenza in these patients is supposed to involve the lack of pDCs, although a role of other cells, including nonhematopoietic cells, cannot be excluded.

Methods

Routine Immunologic Assays

Serum IgG, IgA, IgM, and IgG subclasses were measured by means of nephelometry (Siemens Nephelometer, Germany). Lymphocyte subpopulations were analyzed by using flow cytometry (BD Biosciences).

Sanger Sequencing of GATA2

Amplification and sequencing of GATA2 in genomic DNA from blood samples was performed as previously described [11].

Plasmids, Directed Mutagenesis, Western Blot, and Luciferase Reporter Assay

Western blots of GATA2 were performed on HEK293T cells transfected with vectors containing GATA2-WT isoform 1 (Origene RC208554) and GATA2-WT isoform 2 (Origene RC208514); mutated vectors containing the variants R396L, R396Q, and R398W were generated by site-directed mutagenesis (QuikChange II XL Site-Directed Mutagenesis Kit: Agilent 200521-5), or empty vectors (EV).

Luciferase reporter assays were carried out in HEK293T cells transfected with EV and with vectors containing the GATA2-WT isoform 1 or the R396L, R396Q or R398W variants. Normalization of transfection efficiency was made using Renilla luciferase gene expression modified (kindly provided by Zhibing Zhang and Jerome Strauss) [20]. The reading of the samples was performed using a luminometer (Victor X4 model 2030, PerkinElmer).

Production of Cytokines after Polyclonal Activation

Production of cytokines after stimulation of peripheral blood mononuclear cells (PBMCs) with 10 ng/ml of phorbol myristate acetate (PMA; Sigma Chemical Co.) plus 1 μg/ml of ionomycin (Sigma Chemical Co.) was performed as previously reported with minimal modifications [21].

Analysis of IL-17A- and IFN-γ-Producing T Cells

Blood diluted in culture medium (1:1) was stimulated with 10 ng/ml PMA and 1 μg/ml ionomycin in the presence of 1 μl of GolgiPlug (BD Biosciences) for 4 h. Then, cells were washed and stained with anti-human monoclonal antibodies CD3-FITC (BD Biosciences) and CD8-PerCP (BD Biosciences) for 15 min at room temperature. Next, cells were washed, fixed, and permeabilized following the manufacturer’s instructions (BD Cytofix/Cytoperm). Finally, cells were intracellularly stained with anti-human monoclonal antibodies IL-17A-PE (eBioscience), IFN-γ-PE (BD Biosciences), or isotype-matched negative controls (BD Biosciences), washed, and analyzed on a FACSCanto II flow cytometer (BD Biosciences).

Analysis of Cytokines in Serum and Culture Supernatants

The levels of cytokines and chemokines in serum and culture supernatants were measured using flow cytometry-based bead array systems (BD Biosciences).

Identification of Influenza A Virus (IAV) and Measurement of Neutralizing Antibodies Against Influenza A Viruses

Influenza A H1N1pdm virus was detected in nasopharyngeal swabs using real-time polymerase chain reaction (PCR) AH1N1 assay (F. Hoffmann-La Roche Ltd., Basel, Switzerland) [22]. Titers of neutralizing Ab against H1N1pdm (strain A/California/07/09) and a seasonal IAV strain (A/Brisbane/59/07) were measured in serum samples from P1 and P2 after H1N1pdm infection according to standard protocols [23].

PCR Analysis of the Rearranged T-Cell Receptor and Immunoglobulin Genes

PCR analysis of the immunoglobulin heavy chain (IGH) and T-cell receptor gamma (TCRG) gene rearrangement was performed with commercial kits (Master Diagnóstica SL, Granada, Spain).

Signal Joint T-Cell Receptor Excision Circle Quantification

Signal joint T-cell receptor excision circle (sjTREC) levels were measured in gDNA from peripheral blood using a quantitative real-time PCR adaptation of the original technique [24].

This research has been performed in accordance with the Declaration of Helsinki. The protocols were approved by Clinical Research Ethics Committees of hospitals involved. Written informed consent for the study was obtained from their legal representative. For a detailed description of materials and methods, see the Supplementary Material.

Results

Case Reports

We studied three related Caucasian patients from a nonconsanguineous family from and living in Spain (Fig. 1a and Table S1). The proband (P1) was born in 1953. He had a history of chronic neutropenia. A diagnosis of refractory anemia secondary to MDS with dyserythropoiesis, dysgranulopoiesis, and dysmegakaryopoiesis with abundant macrophages and no reticulin fibrosis in bone marrow (BM) aspirates was made when he was 30 years old. Splenectomy was performed 1 year after. He suffered one episode of pneumonia at the age of 37 years, and mild recurrent upper and lower respiratory tract infections between ages 37 and 44. He was doing well without medication, when in January 2011, at the age of 54, he was hospitalized for PVP by H1N1pdm IAV. He was treated with oseltamivir, but the disease evolved in the next 3 days to ARDS. Viral pneumonia was complicated by secondary infection by Klebsiella pneumoniae and Acinetobacter baumannii, and he died 28 days after admission due to refractory septic shock. No necropsy or further BM analyses were performed.

Fig. 1

Novel missense mutation in GATA2. a Pedigree of the family of the GATA2-deficient patients. Patients (P1, P2, and P3) and their relatives are indicated by a black square or circle. GATA2 genotypes at residue 396 (R396L, mutants; wt, wild-type; E?, unknown) are indicated. The index patient is indicated by an arrow. b Electropherograms showing a heterozygous G>T substitution at nucleotide 16913 (exon 7) of GATA2 in P1. c Alignment of the portion of the human GATA2 molecule containing residue 396 and the corresponding regions in other species. Residue 396 is indicated in gray and by a thick arrow. Other residues in this region, found to be mutated (T354M, N371K, R396Q, R396W, R398W, and R398Q) in previously reported patients with autosomal dominant GATA2 deficiency are indicated by a thin arrow

One month earlier, the son of P1 (P2) had died due to severe H1N1pdm infection. P2 also had a history of MDS with dyserythropoiesis, dysgranulopoiesis, and dysmegakaryopoiesis, and a perianal abscess, diagnosed when he was 15 years old. At the age of 16 years, he was hospitalized for one episode of pneumonia. At the age of 31, he was admitted to the hospital due to flu-like symptoms lasting for 3 days. He presented with acute respiratory insufficiency. An X-ray showed an infiltrate in the left lower lobe. The patient was treated with levofloxacin and ceftriaxone. In a few hours, his condition evolved to severe respiratory failure with progressive pulmonary infiltrates. Empirical treatment with oseltamivir was started, and pharyngeal swabs were later positive for H1N1pdm IAV. Three days later, he was admitted to the ICU because of ARDS. Oseltamivir was withdrawn, and intravenous zanamivir was instituted. The infection led to death 3 days later due to refractory hypoxemia, despite the use of prone positioning ventilation and recruitment maneuvers. Unfortunately, extracorporeal membrane oxygenation was not available. No necropsy or further BM analyses were performed.

The daughter of P1 (P3) had developed flu-like symptoms with pulmonary interstitial infiltrates in October 2005, at the age of 17. Lung biopsy showed interstitial fibrosis and focal alveolar proteinosis with presence of abundant foamy macrophages. BM biopsy showed no abnormalities, except a high percentage of macrophages (84%). She died at the age of 20 from complications of SLE-like syndrome management (Table S1). No GATA2 deficiency-related diseases were observed in the other relatives.

GATA2 Deficiency in Three Patients

Blood samples from P1, obtained when he was 54 years, 6 days after hospital admission for H1N1 infection, were recruited to be included in a survey aimed to study the role of genetic variability in the severity of IAV [25, 26]. Routine immunological analysis showed neutropenia, monocytopenia, and a nearly complete absence of peripheral NK and CD20+ B-cells. No immunological analysis had been performed on P2 during the flu episode. Historical immunological analysis from P1, P2, and P3 at the ages of 43, 21, and 13 years, respectively, showed severely reduced numbers of B-cells and monocytes; P3 also had severely reduced numbers of NK cells (Table 1). On the basis of these data, familial GATA2 deficiency was suspected. By the Sanger method, we found a novel missense heterozygous R396L mutation in GATA2 in the three patients. The mutation was not observed in their healthy relatives (Fig. 1a, b). We did not find the R396L mutation in public database (dbSNP, 1000 genomes), in 55 healthy Caucasian individuals and in 1022 individuals from 52 ethnic groups from the HGDP-CEPH panel. Residue 396 is highly conserved across species (Fig. 1c). In silico analyses performed by means of PolyPhen-2 and PROVEAN/SIFT showed that the damaging effect of the R396L mutation is highly probable. Mutations in the zinc finger-2 domain, particularly R398W (one of the most frequent mutations causing GATA2 deficiency), R398Q, R396W, and R396Q have been reported in several independent studies [12, 13, 16, 17, 19], underscoring the key role of these residues on GATA2 function. The novel R396L mutation suggests that the residue R396 at GATA2 may be a mutational hotspot.

Table 1

Leukocyte count and lymphocyte subpopulations

	P154 years	P143 years	P221 years	P313 years	S-H1N1pdm(N = 6)	HC(N = 29)
Leukocyte count (cells/μl)
Lymphocytes	900	4730	1530	2060	1355 (955–1880)a	2666 (1640–3410)a
Monocytes	290	0-183	10-70	0–100	645 (371–1042)a	601 (445–777)a
Neutrophils	420	870	660	4660	5208 (3070–7610)a	4531 (2790–6810)a
Lymphocyte subpopulations (%)b
T-cells
CD3+	95	92	84	96	66 (46–77)	72 (65–81)
CD3+CD4+	49	42	41	29	46 (30–60)	45 (35–56)
CD45RA− (CD3+CD4+)c	34		73	39	49 (34–62)	48 (30–76)
CD3+CD8+	64	63	45	68	19 (11–29)	25 (18–32)
CD4+CD8+ (CD3)	20.3				1.40 (0.4–2.2)	2.0 (0.7–2.4)
TCR γδ	7	10	6	19	2 (1–8)	4 (1–9)
Tregd	1.4				6.4 (4.7–8.7)	6.5 (4.9–8.2)
CD3+CD56+	62		33	24	4 (2–11)	6 (2–11)
CD3+CD57+	66	50	29	19	7 (2–17)	12 (4–26)
B-cells
CD19+	5.4	0	0,8	1,7	23 (10–48)	12 (8.0–18)
CD20+	0.2	0	0,7	1,6	21 (8–46)	12 (8–17)
CD19+CD27+	5.4				8.3 (1.7–23)	3.2 (1.6–4.7)
NK cells
CD3−CD16+	0.3	0.6			11 (8–15)	14 (8–22)
CD3−CD56+	0		12	0.8	9 (6–13)	13 (7–19)
CD3−CD57+	0.2		11	0.6	4.2 (1.5–7.5)	7 (2–13)

S-H1N1pdm patients with primary viral pneumonia and severe acute respiratory failure due to H1N1pdm infection, HC adult healthy controls, Treg regulatory T-cells

aValues are mean (percentiles 10–90)

bIn the S-H1N1pdm group, values are mean (range); in the HC group, values are mean (percentiles 10–90)

cCD45RA− (CD3+CD4+) cells were CD45R0+

dTreg were estimated as the percentage of CD4+ T-cells expressing CD25highCD127−/low (see also Fig. S2). P1 had 6.2 Treg cell/μl, a 92% reduction compared with the median values observed in healthy controls (78 Treg/μl; percentile 10–90 and 38–136 Treg/μl)

Expression and Function of R396L GATA2 Allele in HEK293T Cells

The protein expression of isoforms 1 and 2 of GATA2 in HEK293T-transfected cells was shown to be slightly reduced in the mutant GATA2 R396L, R396Q, and R398W alleles compared to the WT (Fig. 2a and Fig. S1A-B).

Fig. 2

Expression and transcriptional activity of the R396L allele in overexpression system. a Expression of GATA2 protein. HEK293T cells were transfected with an empty vector (EV), a construct encoding a GATA2 WT (wild type) DDK-tagged or mutated GATA2 variants (R396L, R396Q, R398W) for 48 h. Protein expression of GATA2 isoforms 1 (480 amino acids (aa)) and 2 (466 aa) was evaluated by western blotting with an anti-GATA2 antibody and an anti-DDK tag. GAPDH was included for normalization. NT corresponds to no transfected cells. The results shown are representative of three independent assays. b Functional characterization of the R396L allele. GATA2-mediated luciferase activity was measured in HEK293T cells. The GATA2-dependent transactivation potential was evaluated with GATA2 luciferase reporter vector. Cells were transiently transfected with (+) or without (−) promyelocytic leukemia protein (PML), empty vector (EV), and different GATA2-vectors (WT, R396L, R396Q, or R398W). The results shown are representative of three independent assays, each performed in triplicate

Only GATA2 isoform 1 has transcriptional activity. The promyelocytic leukemia protein (PML) gene is a member of the tripartite motif (TRIM) family and potentiates transactivation activity of GATA2. HEK293T cells transfected with GATA2-WT showed a functional activity that was enhanced when the cells were co-transfected with PML. However, the functions of GATA2-R396L, GATA2-R396Q, and GATA2-R398W mutants were severely impaired and did not increase with co-expression of PML (Fig. 2b), suggesting that R396L is dysfunctional and hypomorphic.

We then performed luciferase assays in transient co-transfections with PML and combined vectors of GATA2-WT and GATA2-R396L, as well as vectors of GATA2-R398W and GATA2-R396Q as negative controls, in different amounts mimicking heterozygosity (Fig. 3a). We observed a nonsignificant decrease in GATA2-mediated relative luciferase activity with increasing levels of the GATA2-R396L, GATA2-R398W, and GATA2-R396Q mutants. Similar results were obtained with constant amounts of the GATA2-WT vector and different amounts of GATA2 R396L, R396Q, and R398W mutants (Fig. 3b). Interestingly, GATA2-mediated relative luciferase activity was observed to decrease at higher concentrations of the R396L vector, which could suggest promoter competition. However, when statistical analysis was performed, the difference of GATA2-mediated relative luciferase activity between this point and the activity of the WT alone was not found to be significant, indicating that the observed decrease in relative luciferase activity does not fall into the loss of function category, which could be expected in a dominant-negative model. These results suggest that haplo-insufficiency could be the mechanism of GATA2 deficiency caused by the R396L, R398W, and R396Q mutations.

Fig. 3

Analysis of the impact of heterozygosity for the R396L allele in overexpression system. GATA2-mediated luciferase activity was measured in HEK293T cells. a Transient co-transfection of cells with promyelocytic leukemia protein (PML) (+) and different amounts of vectors (empty vector (EV), wild type (WT), R396L, EV/WT, EV/R396L, WT/R396L, EV/R396Q, WT/R396Q, EV/R398W, or WT/R398W). b Transient co-transfection of cells with PML and constant amount of the GATA2-WT vector with decreasing amounts of GATA2 mutant (R396L, R396Q, or R398W) vectors were used in the experiment. The results shown are representative of three independent assays, each performed in triplicate. Statistical analysis was performed using GraphPad Prism version 5.03 by means of an unpaired t test (Wilcoxon matched-pairs signed-rank test). Data were considered significant when p < 0.01

Analysis of B and T Lymphocytes

A nearly normal proportion (5.4% of lymphocytes), although at reduced absolute numbers, of peripheral CD19+ B-cells (49 CD19+ B-cells/μl; P10–P90 normal values in our healthy adult controls are 135–500 CD19+ B-cells/μl) was observed in P1 during the H1N1pdm infection (Table 1, at age 54 years). However, all B-cells were found to be CD19lowCD20−CD27+++CD24−CD38+++IgD−IgM− plasmacytoid B-cells (Fig. 4a). CD4 T-cell lymphopenia with an inverted CD4/CD8 ratio and a severe deficiency of regulatory T-cells (Treg) were also observed (Table 1 and Fig. S2). The ratio of naïve to memory (CD45RA+/CD45RA−) CD4 T-cells was found to be within normal values. However, very high numbers of CD3+CD56+ and CD3+CD57+ T-cells were found, probably by accumulation of terminal effector CD8+ T-cells. Historical analysis from P2 and P3 showed severely reduced numbers of peripheral B-cells, a normal ratio of CD45RA+/CD45RA− CD4+ T-cells, and high numbers of CD3+CD56+ cells. Unfortunately, no biological material was available to analyze peripheral DCs.

Fig. 4

Peripheral B lymphocytes and production of antibody against influenza A virus in patient 1 during H1N1pdm infection. a B-cell phenotype from P1, from one patient with severe acute respiratory failure due to H1N1pdm infection (S-H1N1) and from one healthy control (HC). Plasmacytoid B-cells are indicated by rectangles. Acquisition was stopped when 20,000 lymphocytes were gated based on CD45 expression and side scattering. b Measurement of neutralizing antibodies against H1N1pdm (thin line) and a seasonal IAV strain (dashed line) in P1. Influenza human antibody standard 09/194 to A/California/07/09-like viruses with a neutralization titer of 1/516 (The National Institute for Biological Standards and Control, UK) was used for titer normalization. Seroconversion is considered to occur when the neutralization titer increase is higher than fourfold. Microneutralization titers at day 4 were lower than 1/8 (the limit of detection of the analysis). c Spectratyping analysis of the B-cell receptor heavy chain (IGH) repertoire in P1 and in an adult healthy control (HC)

Analysis of Antibodies

Immunoglobulin levels (IgG, IgA, IgM, and IgG subclasses) were normal in the three patients. IgG antibodies (Ab) against Epstein-Barr virus, cytomegalovirus, herpes simplex virus, varicella-zoster virus, and rubella virus were detected in serum obtained during the influenza episodes from P1 (aged 54 years) and P2 (31 years), although these viral infections had been unremarkable or silent. Surprisingly, neutralizing Ab titers against H1N1pdm increased from < 1:8 to 1:37 and 1:55 in serum samples from P1 taken at days 4, 12, and 19, respectively, after admission. Interestingly, similar titers of neutralizing antibodies against the previous annual H1N1 strain were also detected (Fig. 4b). Neutralizing Ab against H1N1pdm were not detected in an acute serum sample from P2 taken 3 days after hospital admission, although he had serum antibodies against a seasonal strain. No later samples from P2 were available.

Analysis of B-Cell Clonality

High numbers of peripheral plasmacytoid B-cells were observed in P1 in spite that he had no peripheral B-cells for at least 11 years. However, a high number of plasma cells were observed in BM from patient P1 and from previously reported patients, which would account for maintenance of antibody production [19, 27]. Spectratyping analysis of IGH repertoire in DNA from peripheral blood from P1 obtained during the flu episode showed a restricted, pauciclonal pattern with the presence of one unusually abundant fragment length. These results suggest that the observed peripheral, plasmacytoid, B-cells result from the expansion of one major clone (Fig. 4c). Unfortunately, no later samples were available for characterizing the antibody produced by this major clone.

Functional Analysis of T Lymphocytes

Roughly half of GATA2-deficient patients show CD4+ T-cell lymphopenia, and accumulation of CD56+ terminal effector CD8+ T-cells, usually with an effector memory RA (EMRA) phenotype [18, 27, 28]. Only a few studies have analyzed T-cell function in GATA2-deficient patients [11, 27, 29]. T-cell proliferative responses in PBMC from P3 were severely impaired, but they normalized upon addition of a co-stimulus, suggesting that the defect was due to the absence of peripheral monocytes/macrophages (Fig. S3) [27]. Analysis of IFN-γ- and IL-17A-producing T-cells in response to polyclonal activation, performed during the flu episode, showed that P1 had a strongly increased expansion of IFN-γ-producing CD4+ and CD8+ T-cells (Fig. 5a). PBMC from P1 also showed a strong IFN-γ production after polyclonal activation (Fig. 5b). These results contrast with those from Bigley et al. [11], who reported low IFN-γ production by GATA2-deficient cells in response to LPS.

Fig. 5

Analysis of peripheral T lymphocytes. a Analysis of IFN-γ- and IL-17A-producing T-cells in response to polyclonal activation in P1, in patients suffering from severe acute respiratory failure due to H1N1pdm infection (S-H1N1pdm; N = 5) and in healthy controls (HC, N = 5). b Production of IFN-γ, IL-2, IL-4, and IL-17A by PBMC after polyclonal activation. The experiments were performed when the patient was attended at the intensive care unit during the severe H1N1pdm infection. c Spectratyping analysis of the T-cell receptor gamma gene (TCRG) repertoire. Analysis performed in DNA from whole blood from P1 and P3 at different ages and from one representative healthy control (HC)

Analysis of T-Cell Clonality

Less than half of GATA2-deficient patients show abnormal T-cell receptor rearrangement patterns [19]. Spectratyping analysis of TCRG gene rearrangement in DNA obtained from whole blood from P1 during the flu episode showed a pauciclonal pattern with expansion of a major clone. A severely reduced TCRG pattern was already detected in P1 at the ages of 35 and 44 years (Fig. 5c). The same analysis in DNA from P3 at the ages of 13 and 18 years showed a slightly reduced pattern of rearrangement (Fig. 5c).

sjTREC Quantification

TRECs were only previously analyzed in eight pediatric GATA2-deficient patients under 18 years of age, showing normal values in most patients [30]. No TRECs were showed in P1 at the age of 35, 44, and 54 years (Table 2). A low number of TRECs were observed in blood samples from P3 at the age of 13 years, whereas no TRECs were observed in samples obtained when he was 18 years old (Table 2). Overall, our data would suggest that T-cell thymic output is more impaired in GATA2-deficient patients than previously thought. Memory T-cells against conserved epitopes of IAV, as those previously described [31, 32], could have expanded in patient P1. Unfortunately, no samples to test IAV-specific responses were available.

Table 2

Quantification of sjTRECs.

Patient	Age (years)	sjTRECsa
P1	35	0
	44	0
	54	0
P3	13	844
	18	0
Healthy donors (N=15)	10-15	>1500
Healthy donors (N=30)	18-65	> 100

aThe results are expressed as sjTRECs per 100ng of gDNA from whole blood.

Serum Cytokine and Chemokine Levels during H1N1pdm Infection

Hypercytokinemia has been reported to play a direct role in the development of severe ARDS secondary to IAV infection [1, 8]. Serum levels of interferon gamma-induced protein 10 (IP-10, CXCL10) and monokine induced by gamma interferon (MIG, CXCL9) as well as IFN-γ, monocyte chemotactic protein-1 (MCP-1, CCL2), or IL-8 (CXCL8) were strongly elevated in P1 and P2 in the course of H1N1pdm infection. This increase was particularly evident when cytokine/chemokine levels were compared with those observed in other patients with severe H1N1pdm infection from our (Fig. 6) or other previously reported studies (Table S2). The pattern of serum cytokines/chemokines was also similar to that observed in patients with severe H5N1 or H7N9 IAV infection (Table S2). High levels of IL-6 and IL-10 were also observed in P1 and P2.

Fig. 6

Serum cytokine and chemokine levels. Sera from P1 (filled square) and P2 (filled triangle) were obtained 6 and 4 days after admission to the intensive care unit, respectively. Samples from patients with severe acute respiratory failure due to H1N1pdm infection (S-H1N1pdm; N = 6) obtained 5.4 days (range 3–7) after hospital admission and seven healthy controls (HC) are also included. No differences were observed when serum levels of IL-1-β, IL-2, IL-4, IL-12p70, IL-17A, TNF-α, and CCL5 (RANTES) were compared

Discussion

We report here three relatives with AD GATA2 deficiency, two of whom, with no recent history of severe infections, died of influenza during the 2009 H1N1pdm at 1-month intervals. Patients carried a novel, R396L, germline heterozygous mutation of the GATA2 gene. This mutant protein was expressed normally yet was functionally hypomorphic, underlying haplo-insufficiency in heterozygotes. The severity of the deficiency is illustrated by the death of the three patients from infectious complications, including two deaths from influenza.

P1 and P2 presented an exuberant IFN-γ-mediated hypercytokinemia during H1N1pdm infection. Accumulating evidence suggests that hypercytokinemia, particularly of IFN-γ, IL-6, IL-8, CCL2, CXCL9, and/or CXCL10 (an IFN-γ-mediated signature), plays a direct role in ARDS development and in IAV-mediated lung pathology and mortality, and that this effect is independent of viral titers [1, 33–35]. No data about viremia or inflammatory response in the other GATA2-deficient patients with fatal influenza were reported [11, 12]. Plasma levels of Th1 cytokines were also found to be high in GATA2-deficient patients and severe Epstein-Barr virus (EBV) disease with a marked EBV viremia [36]. Hemophagocytic lymphohistiocytosis (HLH) secondary to an EBV-positive lymphoma [36] severe herpes simplex virus infection [37] and disseminated Mycobacterium kansasii infection (Rodríguez-Gallego et al. unpublished results) developed in GATA2-deficient patients. HLH is a deadly systemic hyperinflammatory condition described as a cytokine storm, particularly of Th1 cytokines [38]. Taken together, our and previous data suggest that, in spite of the profound defects in mononuclear cells, hyperinflammatory responses (particularly Th1-mediated) may develop in GATA2-deficient patients. Abundant tissue-resident macrophages in lung, BM, and healthy skin can be observed in GATA2-deficient patients lacking circulating monocytes [11, 17, 39]. In fact, abundant macrophages had been previously observed in BM biopsies (P1 and P3) and lung biopsies (P3) from our patients. Although alveolar macrophages were found to be, at least partially, dysfunctional, these cells might offer an alternative route of antigen presentation in the absence of DC [17, 39]. Treg deficiency could underlie the exuberant IFN-γ-mediated inflammatory response and immunopathology in these patients [40, 41]. Alternatively, a role of genetic variants involved in pathogen sensing and/or inflammatory responses to infection cannot be ruled out [42]. However, in the absence of data about the patient’s viremia, we cannot exclude that overwhelming IAV infection is due to the observed deficiencies in mononuclear cells and underlies such an inflammatory response and ARDS in our patients.

In spite that P1 lacked peripheral B-cells for at least 11 years, he was able to produce neutralizing antibodies against the novel H1N1pdm IAV strain and a previous annual H1N1 strain. Broadly neutralizing antibodies directed against conserved regions of the hemagglutinin stalk and receptor-binding domain were recently described [43-45]. Since this patient had not been vaccinated against H1N1pdm, our results suggest that one or a few clones of long-living memory B-cells against a previous IAV expanded in patient P1 and that these cells produced antibodies able to cross-react and to neutralize H1N1pdm. Specific Abs are recognized as the main factor to prevent IAV infection, whereas cell-mediated immunity is thought to be crucial for the control of an established infection [1, 31, 32, 46]. This is the rationale for influenza vaccination. However, patients lacking T- and/or B-cells are not prone to severe influenza, although vaccination is considered not to be effective [9]. In any event, Abs able to neutralize H1N1pdm were unable to fight the ongoing infection in P1. In contrast to B-cells, NK cells, DCs, and monocytes, T-cells are thought to be relatively well-preserved in patients with GATA2 deficiency [27, 30, 39]. Our results suggest that T-cells are more affected in GATA2-deficient patients than previously suspected and, like in other leukocyte subsets, the effect of GATA2 deficiency on T-cells is progressive. P1 did not suffer from severe viral, mycobacterial, or fungal infections despite complete B-cell deficiency for at least 11 years and the absence of thymic output of T-cells for at least 19 years. Overall, our data emphasize that GATA2-deficient patients may live for a long time off one’s immunological memories. Our data underscores the role that long-living memory T- and B-cells can play in the resistance to infection in adulthood, particularly in patients with late-onset primary or acquired immunodeficiencies.

Several primary immunodeficiency disorders (PIDs) predisposing to viral infections were previously reported [47-49]. However, severe PID affecting T- and B-cells, such as severe combined immunodeficiency or agammaglobulinemia, do not predispose to severe influenza [9]. This contrasts with parainfluenza and other viruses, which often kill children with inborn errors of T-cells [9]. No predisposition to severe influenza was reported in patients with either isolated NK cell deficiencies or DC deficiency [9, 47–50]. PIDs affecting IFN type I and II have not been reported to have severe influenza [9]. To our best knowledge, IRF7 deficiency and GATA2 deficiency are the only described single-gene inborn errors of immunity associated with lethal IAV infection so far. The only known patient with complete IRF7 deficiency developed life-threatening influenza at the age of 2.5 years [10]. Her leukocytes, including pDCs, as well as fibroblasts and iPSC-derived pulmonary epithelial cells, produced diminished amounts of type I IFNs. The child is now 7 years old and well with annual vaccination against influenza as her sole prophylaxis, suggesting that IRF7 deficiency might not impair immunity against secondary IAV infection. IRF7 is ubiquitously expressed, and so susceptibility to severe influenza in IRF7 deficiency cannot be ascribed only to a hematopoietic impairment. Patients with AD GATA2 deficiency have a large clinical phenotype including viral infectious disease. Due to the profound impairment in so many leukocyte subpopulations, including loss or diminished pDCs and NK, it is difficult to propose the mechanism underlying susceptibility to severe influenza in GATA2-deficient patients. Besides its role in hematopoiesis, GATA2 is expressed in endothelium and lymphatic valves, and it is also involved in adipogenesis [17, 28, 39]. However, the clinical infectious phenotype of GATA2 deficiency is reversed by allogenic hematopoietic stem cell transplantation [39], suggesting that hematopoietic anomalies are by themselves responsible for susceptibility to severe infection. Undoubtedly, the lack of pDCs and NK cells may contribute. However, unlike the IRF7-deficient patient, who survived life-threatening influenza in childhood, our and the two previously reported patients with GATA2 deficiency who died by IAV infection, with no hematologic malignancy at the moment of the IAV infection, died in adulthood [10-12]. Such deficiencies should be considered in selected patients with life-threatening flu, even in the absence of any personal or familial history. It is not known why some GATA2-deficient patients present with specific complications, particularly severe viral infections, while others (even family members with the same mutation) do not [17, 39]. A role of other genetic variants involved in innate immune responses cannot be ruled out. Our description of two adult relatives with AD GATA2 deficiency who died of flu, with no recent history of other severe infections, suggests that lethal flu may result from and actually reveal single-gene inborn errors of immunity, at least in some patients [51].

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