hospital-acquired in a young patient with a suspected IEI, and despite the absence of clinical signs of severity (including an absence of both pneumonia and encephalitis), P1 was pre-emptively treated with intravenous acyclovir, leading to resolution of the varicella virus infection. At the age of 11 months, P1 had a lower respiratory tract infection caused by parainfluenza virus 3 (PIV3), documented by PCR. She also experienced another lower respiratory tract infection caused by *Chlamydia pneumoniae*, and various other respiratory tract infections presumed to be viral. She did not require intubation for any of these respiratory tract infections. At the age of three years, she presented simultaneous disseminated infections due to *M. avium* and *Histoplasma capsulatum*. These infections were treated with amikacin, rifampicin, isoniazid, ethambutol, levofloxacin, and liposomal amphotericin B. During the episode of BCG-osis, P1 also displayed leukopenia, anemia, and thrombocytopenia. At the age of four years she presented with vesicular lesions of the right upper limb. However, PCR tests for VZV and HSV were negative. At the age of five years, P1 presented pneumonia due to *M. avium intracellulare*. Clarithromycin was added to the antimycobacterial treatment regimen. At the age of six years the patient suffered rhinosinusitis leading to nasal obstruction. *Acinetobacter baumanii* and *M. intracellulare* subsp. *chimaera* were documented on culture of a sinus sample. During this episode, the patient displayed hepatosplenomegaly and pancytopenia associated, on bone marrow biopsy, with epithelioid granuloma giant multinucleated cells without hemophagocytic lymphohistiocytosis (HLH). The patient was treated with liposomal amphotericin, levofloxacin, clarithromycin, and ethambutol. She suffered failure to thrive, from infancy (–2 to –3 SD for weight and height). Between the ages of two and four years, P1 suffered recurrent abdominal pain, with an enlarged liver and abdominal distention. Digestive tract endoscopy results were normal. Abdominal and pelvic CT-scan results were normal, with the exception of organomegaly. P1 had ventricular septal defects and café au lait spots on the skin, from birth. In addition to BCG, she was vaccinated against diphtheria, tetanus, hepatitis A, hepatitis B, *Haemophilus influenzae* type b, seasonal flu, pneumococcus (Prevenar 13), poliomyelitis (Salk), and rotavirus (Rotarix), and had received one dose of measles-mumps-rubella vaccine. Serological tests for HIV were negative. Chromosome 22q11.2 deletion syndrome was ruled out by fluorescence *in situ* hybridization. Blood karyotype was normal. The primary fibroblasts of P1 displayed normal sensitivity to mitomycin C and gave normal results in FANCD2-monoubiquitination assays,¹⁷⁸,¹⁷⁹ ruling out Fanconi anemia. Plasma alpha-feto protein concentration was in the normal range. The oxidative burst in PNM cells (dihydrorhodamine test), and ADA and PNP activities in plasma were normal. A brain CT-scan at the age of four years was normal. P1 is currently being treated with subcutaneous recombinant IFN-γ (Imukin), polyvalent subcutaneous immunoglobulin, levofloxacin, ethambutol, clarithromycin, trimethoprim-sulfamethoxazole, iron, folic acid, vitamin D and calcium. She is well on this treatment. The mother of P1 had no history of unusually severe infectious diseases. She did not display adverse reaction to BCG vaccination.

The patient from kindred B (P2, II.1) is a girl born in 2015 and living in Turkey (Figure 1A). She was vaccinated with BCG at birth, and presented left axillary lymphadenitis (BCG-itis) without organomegaly at the age of five months. She was successfully treated for six months with isoniazid, rifampicin, and ethambutol. At the age of one year, she presented respiratory infection due to influenza B virus that did not require oxygen support. At the age of five years, P2 was referred to the hospital for intermittent fever and a rash of three months' duration. She was treated with oral antibiotics for upper respiratory tract infection, and with colchicine for suspected familial Mediterranean fever. She had been on intramuscular antibiotics for two weeks when she was diagnosed with pneumonia, without identification of the causal microbe. On admission, she had tachypnea, but was in good general condition with no fever. Lymphadenopathies were observed in both axillary regions on clinical examination. A chest CT-scan revealed multiple lymphadenopathies in both axillary regions, in the mediastinal and hilar regions, and a mass-like lesion in the left lower lobe of the lungs. Abdominal CT-scan was normal, with no organomegaly. Antibiotic treatment with ceftriaxone and clindamycin was continued for pneumonia. A peripheral smear and bone marrow aspiration were performed; the results excluded leukemia and HLH. A left shift and leukocytosis with neutrophilia were found. Cultures of blood and nasopharyngeal fluid were negative. Three fasting gastric fluid aspiration specimens were sent for staining for acid-fast bacilli (AFB) and mycobacterial culture, but the results were negative on all three occasions. A tuberculin skin test (TST) gave a 17 mm induration, and the Quantiferon test was negative. Due to a lack of improvement, antituberculosis treatment was initiated with moxifloxacin, rifampicin, isoniazid, pyrazinamide and ethambutol. Samples of the lung mass and mediastinal lymphadenopathies were sent for histological analysis and mycobacterial culture. Cultures revealed *M. avium intracellulare*. The histopathology report showed a few giant cells and spindle-shaped histiocytes. P2 was treated with azithromycin, ethambutol, and rifabutin. The patient had papular hyperemic rashes on her hands and feet during clinical follow-up and was referred to a dermatologist. The cutaneous biopsy results obtained from the rashes were consistent with acute generalized exanthematous pustulosis. At the age of five years, the patient was infected with SARS-CoV-2, causing mild cold-like symptoms with no need for hospitalization. This patient has never been vaccinated against COVID-19. P2 is currently doing well. The father, mother, and brother of P2 had no history of unusually severe infectious diseases, including adverse effects of live vaccines.

Informed consent was obtained in Argentina and Turkey, respectively, in accordance with local regulations and with institutional review board (IRB) approval. Experiments were conducted in Australia, Canada, France, Qatar, and the United States of America, in accordance with local regulations and with the approval of the IRB of the Rockefeller University and INSERM, for the United States of America and France, respectively. Healthy controls were recruited in Argentina, France, Turkey, and the United States of America.

**Both IRF1 variants are predicted to be deleterious**

IRF1 encodes interferon regulatory factor 1 protein (IRF1), a transcription factor induced by IFN-γ, the deficiency of which in mice underlies susceptibility to various pathogens, including mycobacteria<sup>43–57</sup> (Table S2). IRF1 is a 325-amino acid transcription factor composed of an amino-terminal DNA-binding domain (DBD) that can bind to DNA interferon-stimulated response elements (ISRE) or
positive regulatory domain I (PRDI) motifs,<sup>58</sup> an intermediate segment containing a putative nuclear localization sequence (NLS), and a C-terminal IRF association domain 2 (IAD2), which is crucial for transcriptional activity<sup>59-62</sup> (Figure 1G). Both the c.385C>T and c.103C>T variants are predicted to be loss-of-function (pLOF). Indeed, both result in the creation of a premature stop codon, p.R129* and p.Q35*, respectively (Figure 1G). The CADD score of the two variants is 38, well above the 99% MSC<sup>38,180</sup> of *IRF1* at 8.2 (Figure 1I). An analysis of public databases, including ExAC, gnomAD v2.1.1 or v3.1.1,<sup>181</sup> BRAVO/TOPmed freeze 8,<sup>182</sup> UK Bio-bank,<sup>183</sup> ATAV,<sup>184</sup> the Greater Middle East variome,<sup>185</sup> and the Turkish variome<sup>186</sup> containing WES or whole-genome sequencing (WGS) data from more than 250,000 individuals showed that the p.Q35* variant is private, whereas p.R129* is found in the heterozygous state in two individuals. All the non-synonymous coding or structural variants of *IRF1* listed in public WES and WGS databases are present in the heterozygous state, with the exception of one homozygous missense variant (c.199G>C, p.A67P, rs137993322) in the TOPmed<sup>182</sup> database with a gnomAD MAF of 1.2e-4 and a CADD score below that of p.R129* and p.Q35* (Figure 1I). We identified no other homozygous or compound heterozygous non-synonymous *IRF1* coding variants in our in-house database of 20,000 WES or WGS from patients with severe infectious diseases. The cumulative frequency of heterozygous pLOF variants in gnomAD is also low (4.3x10<sup>-5</sup>) (Figure 1I). Consistently, the consensus negative selection (CoNeS) score of *IRF1* is low, suggesting that this gene is under negative selection, consistent with either recessive or dominant inheritance<sup>187</sup> (Figure S1D). These findings further suggest that homozygosity for the pLOF private *IRF1* variants identified here was causal for MSMD in P1 and P2.

## Impaired development of lymphoid and myeloid cells in *Irfr1*<sup>-/-</sup> mice

IRF1-knockout mice (*Irfr1*<sup>-/-</sup>) were first described in 1993.<sup>114</sup> Consistent with the results obtained for IRF1-deficient patients, *Irfr1*<sup>-/-</sup> mice with various genetic backgrounds (C57BL/6 and 129/Sv) display impaired development of NK cells, and T<sub>H</sub>1 cells, together with a predominance of naive cells among CD8<sup>+</sup> T cells (Table S5).<sup>43,55,56,188,189</sup> However, other leukocyte subsets do not seem to have been analyzed in these mice. We studied the immunophenotypes of *Irfr1*<sup>-/-</sup> mice with a C57BL/6 background by isolating leukocytes from the bone marrow, spleen, and lung. We replicated the reported decrease in the absolute numbers of NK and CD8<sup>+</sup> T cells in C57BL/6 *Irfr1*<sup>-/-</sup> mice (Figures S3E and S3F). The development of myeloid DCs has been little studied, and with only rudimentary markers, in mice, but the available results suggest an impaired maturation of myeloid DCs,<sup>190</sup> whereas ILC2 maturation has not been studied. We found that cDC1 counts were low in the spleen and lungs of *Irfr1*<sup>-/-</sup> mice, whereas cDC2 counts in the lungs of *Irfr1*<sup>-/-</sup> mice were unaffected, and those in the spleen were only slightly lower than those of WT mice (Figures S3E and S3F). Spleen pDC counts were normal in *Irfr1*<sup>-/-</sup> mice (Figure S3F). The number of mature ILC2 in the lungs was low, whereas the number of immature ILC2 progenitors in the bone marrow was normal in *Irfr1*<sup>-/-</sup> mice (Figure S3G). Overall, IRF1 deficiency appears to impede the development of the same lymphoid and myeloid cell subsets in humans and mice, resulting in deficiencies of cDC1, ILC2, NK cells, and CD8<sup>+</sup> T cells.

## Lack of severe viral infections in IRF1-deficient patients

In addition to its known role in IFN-γ-related immunity, the antiviral role of IRF1 has also been extensively studied. The ablation<sup>70,76,88,89</sup> and overexpression<sup>90,91</sup> of the *IRF1* gene have been shown to be associated with susceptibility and resistance to viral infections, respectively, *in vivo* in mice<sup>50,51,92-94</sup> (Table S2) and *in vitro* in human cell lines.<sup>76,88,90</sup> Indeed, human IRF1 is induced by IFN-α/β,<sup>76,191</sup> but not by IFN-λ.<sup>76</sup> Inborn errors of IFN-α/β immunity underlie various severe viral diseases in humans.<sup>2,3,140-145,155,192,193</sup> However, neither P1 nor P2 presented life-threatening viral infections (see Case reports for P1 and P2). The patients had experienced benign upper respiratory infections caused by *Parainfluenza virus* 3 (P1), influenza virus (P2), and SARS-CoV-2 (P2). The last of these infections is of particular importance as even subtle defects of type IFN-α/β immunity underlie critical COVID-19 pneumonia.<sup>3,136-140</sup> One of the major mechanisms of SARS-CoV-2 virulence depends on the ability of this virus to induce only small amounts of IFN-α/β, rendering the amounts of this cytokine produced all the more important to the infected individual.<sup>3,194,195</sup> Phage immunoprecipitation-sequencing (PhIP-Seq) confirmed that both patients has been exposed to multiple DNA and RNA viruses (Figure 7A and Table S4). Thus, both these patients with IRF1 deficiency controlled many common, and even virulent viruses well.

## Genetics

P1 and P2 were genotyped from DNA extracted from whole blood, with the Genome-Wide Human SNP Array 6.0 and/or WES with SureSelect Human All Exon V6 from Agilent. *IRF1* exon 3 was amplified from genomic DNA with the following primers (5'-TGGTCTGTTTAAGCCAGCCTC-3' and 5'-CAGAAACACAAGTCTGCCACC-3'), and exon 5 with the following primers (5'-TTCCACCTCTCACCAAGAACC-3' and 5'-CAGAGAAGGTATCAGGGCTGG-3') both at a Tm of 60°C, with the GoTaq DNA Polymerase (#M3005, Promega). Amplicons were then sequenced by the Sanger sequencing method with Big Dye Terminator v3.1 (Thermo Fisher Scientific), and subjected to capillary electrophoresis (#A30469, Applied Biosystems 3500xL system, Thermo Fisher Scientific). The genotype of P1 was checked with DNA extracted from whole blood, granulocytes, EBV-B cells, HVS-T cells, SV40-fibroblasts, primary fibroblasts, and induced pluripotent stem cells (iPSCs). The genotype of her mother was checked with DNA extracted from whole blood and granulocytes. The genotype of P2 was checked with DNA extracted from whole blood, granulocytes, primary fibroblasts, SV40-fibroblasts and MDMs, and the genotypes of her relatives were checked with DNA extracted from whole blood.
## Cell culture and stimulation

HEK293T cells and SV40-fibroblasts were cultured in Dulbecco/Vogt modified Eagle's minimal essential medium (DMEM, #61965059, Gibco) supplemented with decomplemented 10% fetal bovine serum (FBS, #10270098, Gibco), and EBV-B cells were cultured in RPMI medium (RPMI 1640, #61870044, Gibco) supplemented with 10% decomplemented FBS. HVS-T cells were cultured with an equal mixture of RPMI and Panserin 401 (#P04-710401, Pan Biotech) supplemented with 10% decomplemented FBS, 1.2% Glutamax (#35050061, Gibco), gentamycin, and 100 IU/mL recombinant interleukin-2 (Aldesleukin, Novartis). T cell blasts were cultured in ImmunoCult-XF T cell Exp Medium (#10981, Stemcell) in the presence of IL-2 and primed every two weeks with ImmunoCult Human CD3/CD28/CD2 T cell Activator (#10970, Stemcell). All cells were grown at 37°C, under an atmosphere containing 5% CO₂. HEK293T cells were plated at a density of 600,000 cells per well, in six-well plates. For CD274 (PD-L1) induction, SV40-fibroblasts were plated at a density of 200,000 cells per well, in six-well plates, with 2 mL DMEM-10% FBS per well, and were left unstimulated or were stimulated the following day with 10², 10³, or 10⁴ IU/mL recombinant IFN-γ (IFN-γ, Imukin, Horizon Pharma). Forty-eight hours after stimulation, cells were harvested with trypsin, and stained as described below. For p-STAT1 induction, SV40-fibroblasts were starved overnight in DMEM-1% FBS medium, and were left unstimulated or were stimulated for 20 min with 10³ IU/mL recombinant IFN-γ (Imukin, Horizon Pharma), IFN-2αb (Introna, MSD), or 1 ng/mL IFN-β (Miltenyi Biotec). The same protocol was used for p-STAT1 induction in EBV-B cells, except that the cells were starved in RPMI-1% FBS medium and stimulated with 10⁵ IU/mL IFN-γ or IFN-α2b, or 10 ng/mL IFN-β (Miltenyi Biotec). The reaction was stopped by adding cold PBS, and the cells were harvested with trypsin and directly stained, as described below.

## Site-directed mutagenesis, transient and stable transfection

Empty vector (EV) and a plasmid containing the DDK-tagged *IRF1* cDNA were obtained from a commercial source (#RCPS100001 and #RC203500, respectively, Origene). Constructs carrying single-nucleotide mutant alleles were generated from this plasmid by mutagenesis with appropriate primers, with the Pfu Ultra II Fusion HS DNA (#600674, Agilent) polymerase, followed by digestion with *DpnI* (#R0176L, New England Biolab). For assessments of the re-initiation of translation, methionine codons were mutated to alanine codons (ATG>GCG). Plasmids were amplified in competent *E. coli* cells (#C3019H, New England Biolab) and purified with a maxiprep kit (#12663, Qiagen). HEK293T cells were transiently transfected with the various constructs at a concentration of 2.5 µg/mL, with the Lipofectamine LTX kit (#15338100, Thermo Fisher Scientific) in accordance with the manufacturer's instructions. Retroviral plasmids and vectors were prepared as previously described, with primers for site-directed mutagenesis or deletion, and were produced in Phoenix cells.¹⁹⁶

## Western blotting and EMSA

Total protein extracts were prepared by mixing cells with modified radioimmunoprecipitation assay buffer supplemented with protease inhibitors (EDTA-free Complete, Roche) and phosphatase inhibitor cocktail (PhosphoStop, Roche), 0.1 mM dithiothreitol (DTT; Life Technologies), 10⁻³ mM Na₃VO₄, and 1 mM PMSF, and incubating for 40 min on ice. The cytoplasmic and nuclear contents of the cells were separated with NE-PER nuclear and cytoplasmic extraction reagents (#78835, Thermo Fisher Scientific). Equal amounts of protein, according to a Bradford protein assay (#5000002, Biorad), were resolved by SDS-PAGE in a Criterion TGX 10% or 12% precast gel (Biorad) and the bands obtained were transferred to a nitrocellulose membrane (#1704159 and #1704157, Biorad). Membranes were probed with antibodies directed against IRF1 (unconjugated, clone D5E4, #8478, Cell Signaling; or unconjugated, polyclonal, #11335-1-AP, Proteintech), IRF8 (unconjugated, goat polyclonal, #sc-6058, Santa Cruz; or unconjugated, clone D20D8, #5628, Cell Signaling), IRF9 (unconjugated, rabbit polyclonal, #sc-496, Santa Cruz; or unconjugated, rabbit polyclonal, #14167-1-AP, ProteinTech), IRF3 (unconjugated, clone D9J5Q, #10949, Cell Signaling), STAT1 (unconjugated, clone 1, #610115, Beckton-Dickinson), pSTAT1 (unconjugated, clone 4a, #612232, Beckton-Dickinson), STAT2 (unconjugated, clone B-3, #sc-514193, Santa Cruz), MX1 (unconjugated, polyclonal, #13750-1-AP, ProteinTech), ISG15 (HRP-conjugated, clone F-9, sc-166755, Santa-Cruz), DDK-tag (HRP-conjugated, clone M2, #A8592, Sigma-Aldrich), GBP1 (unconjugated, clone 1B1, #sc-53857, Santa-Cruz, #A8592, Sigma-Aldrich), APOL3 (unconjugated, clone EPR8238(2), #ab154869, Abcam), RARRES3 (unconjugated, rabbit polyclonal, #12065-1-AP, ProteinTech), vinculin (unconjugated, clone EPR8185, #ab129002, Abcam; or HRP-conjugated, clone 7F9, #sc-73614-HRP, Santa Cruz), and lamin A/C (HRP-conjugated, clone E-1, #sc-376248-HRP, Santa Cruz). Unconjugated antibodies were detected by incubation with goat anti-mouse or rabbit IgG (H + L)-HRP-conjugated antibodies (#1706516 or #1706515, respectively, Biorad). Binding was detected by incubation with the Clarity Western ECL substrate (Biorad, #1705061) or Super-Signal West Femto (Thermo Fisher Scientific, #34096) with ChemiDoc MP (Biorad). The Spectra Multicolor Broad Range Protein Ladder (#26623, Thermo Fisher Scientific) or the Chameleon Duo Prestained Protein Ladder (#928-6000, Licor) was used to provide molecular weight markers. Membranes were stripped in Restore Western Blot Stripping Buffer (#21063, Thermo Fisher Scientific).

EMSA was performed by incubating 10 µg of nuclear protein lysate on ice for 30 min with an IRD700-conjugated ISRE probe (5'-GATCGGGAAAGGGAAACCGAAACTGAA-3') designed on the basis of the ISRE motif from the *ISG15* promoter. For supershift assays, nuclear protein lysates were incubated for 30 min on ice with 3 µg anti-DDK antibody (clone M2, #14793S, Cell Signaling) or the corresponding isotype (#2729S, Cell Signaling). Protein/oligonucleotide mixtures were then subjected to electrophoresis in 12.5% acrylamide/bis-acrylamide 37.5:1 gels in 0.5% TBE migration buffer for ~120 min at 200 mA and 4°C in the dark. Binding was detected with the Licor Odyssey CLx system (Li-Cor, Lincoln). Images were analyzed with Imagine Lab 6.0.1 build 34 (Bio-Rad Laboratories).
## Confocal microscopy

SV40-fibroblasts were plated on chambered coverslips (#80826, iBidi). The following day, they were left unstimulated or were stimulated for the indicated times with $10^3$ IU/mL IFN-γ (Imukin, Horizon Pharma). Cells were fixed by incubation for 15 min in 4% formaldehyde in PBS, pH 7.4 at 37°C. The cells were then incubated overnight at 4°C with primary antibody (unconjugated, clone D5E4, #8478, Cell Signaling). They were washed three times in PBS, stained by incubation with secondary antibodies for 1 h at room temperature (goat anti-rabbit IgG Alexa Fluor 555, #A21429), and left in Pro-Long Gold with DAPI (#P36931, Thermo Fisher Scientific). Cells were then visualized by confocal microscopy (×63 oil immersion lens, SP8 gSTED, Leica). Images were analyzed with Fiji software.

## Luciferase assay

We used two different ISRE-luciferase reporter assays: (i) an assay using the ISRE3 reporter plasmid (pGL4.10[luc2] backbone, Promega #E6651), which contains, as previously described,¹⁶⁴ three repeats of the ISRE sequence (5'-GGAAAGGGAAACC GAAACTGAA-3') separated by spacers designed on the basis of the ISRE motif from the *ISG15* promoter, (ii) an assay using the ISRE5 reporter plasmid, which contains, as previously described,¹⁴¹ five repeats of the ISRE sequence 5'-GGGAAAGTGAAACTA-3'. HEK293T cells were transiently transfected, in 96-well plates, with the (ISRE) reporter plasmid (100 ng/well and 100 µL DMEM-10% FBS medium), the pRL-SV40 vector (Promega, #E2231, 40 ng/well) and the *IRF1* WT or mutant p.CMV6 plasmid (100 ng/well), with the Lipofectamine LTX kit (Thermo Fisher Scientific, #15338-100), according to the manufacturer's instructions. Cells were used for the ISRE assay with the Dual-Luciferase system kit (Promega #E1980), according to the manufacturer's protocol, 24 h after transfection. Signal intensity was determined with a Victor X4 plate reader (Perkin Elmer). Experiments were performed in triplicate, and dual reporter activity is expressed as the fold-induction relative to cells transfected with the empty vector.

## RT-qPCR and cDNA

RNA was extracted with the RNeasy Plus Mini Kit (#74136, Qiagen) or Quick-RNA Micro-Prep Kit (#R1051, Zymo). Any remaining genomic DNA was removed by extraction on a column or by DNase digestion. RNA was reverse-transcribed with the SuperScript II Reverse Transcriptase (#18064014, Thermo Fisher Scientific) and oligo(dT)₁₂⁻₁₈ (#18418012, Thermo Fisher Scientific) or with the High-Capacity RNA-to-cDNA Kit (#4387406, Applied Biosystems), according to the manufacturer's protocol. qPCR was performed on cDNA with TaqMan Fast Universal PCR Master Mix (2X), no AmpErase UNG (#4352042, Thermo Fisher Scientific) on a 7500 Real-Time PCR System (Applied Biosystems) or Taqman VIIA7, with the following probes, all from Thermo Fisher Scientific: *IRF1* exons 3–4 (#Hs00971960_m1), *IRF1* exons 8–9 (#Hs00971965_m1), *GBP4* (#Hs00364728_m1), *APOL3* (#Hs00758274_m1), and *GUSB* (#1702016).

## Flow cytometry on cell lines

SV40-fibroblasts were incubated with the PE-Dazzle-594-PD-L1 (CD274) antibody (clone 29E.2A3, #329732, BioLegend), or the corresponding isotype (#400358, BioLegend). For pSTAT1 staining, the cells were starved overnight in DMEM-1% FBS. For intracellular staining, $10^6$ cells were washed with PBS-2% FBS- 2 mM EDTA buffer, fixed by incubation for 10 min at 37°C with Fix Buffer I (#557870, Beckton Dickinson) and permeabilized by incubation for 20 min at 4°C with Phosflow Perm Buffer III (#558050, Beckton Dickinson). Cells were then incubated with PE-coupled STAT1 (clone 1, #558537, Beckton Dickinson) or PE- or AF647-conjugated anti-pSTAT1 antibody (clone 4a, # 612564 or #612597, Beckton Dickinson), the corresponding isotype (#554680 and #565363, respectively, Beckton Dickinson), or unconjugated IRF1 (clone D5E4, #8478, Cell Signaling) or the corresponding isotype, for detection with PE-conjugated goat anti-rabbit antibody (#A10542, Thermo Fisher Scientific). All non-fibroblastic cells were also stained with the Aqua Dead Cell Stain kit (#L34957, Thermo Fisher Scientific). Cells were acquired on a Beckman Coulter Gallios flow cytometer and analyzed with FlowJo Software.

## Mass spectrometry on primary fibroblasts

Primary fibroblasts were plated at a density of 180,000 cells/mL in 12 mL DMEM supplemented with 10% FBS in T75 flasks. The following day, they were starved overnight and incubated for 24 h with or without $10^3$ IU/mL IFN-γ (Imukin, Horizon Pharma). The following day, cells were harvested with trypsin, washed once in PBS, and total protein was extracted by incubation for 40 min on ice (with vortexing for 15 s every 10 min) in the following SDS-free RIPA buffer: 150 mM NaCl, 1% NP40 (#85124, Thermo Fisher Scientific), 50 mM Tris, 1 mM EDTA, and EDTA-free protease inhibitor (#04693132001, Roche). A volume of 80–120 µL buffer was used, depending on the size of the pellet. The mixture was then centrifuged at 16,800 g for 10 min at 4°C and the protein-containing supernatant was collected. The concentration of protein in the supernatant was determined with the Pierce BCA Protein Assay Kit (#23225, Thermo Fisher Scientific) and the standard test tube protocol. Protein integrity was assessed with a colloidal blue staining kit (#LC6025, Thermo Fisher Scientific). Samples were then stored at –80°C before further processing.

The cysteine residues were then reduced and alkylated with DTT and IAA. Proteins were precipitated in ice-cold acetone and the resulting pellet was dissolved in 200 mM EPPs pH 8.5 supplemented with 1 µg sequencing grade trypsin. Samples were digested by incubation overnight at room temperature. An additional 1 µg of trypsin was then added and the temperature was increased to 50°C for 1 h. Peptides were labeled by incubation with TMTpro reagent for 1 h at room temperature. Stoichiometry and labeling efficiency were checked, hydroxylamine was added to quench the reaction and the peptides were pooled. TMTpro peptides were first
separated into two fractions by custom-made SCX column. Each of these two fractions was then further separated into eight frac-
tions on high-pH reversed-phase spin columns, and the eight fractions were then concatenated to obtain a total of 12 fractions. The
peptide fractions were separated by HPLC for 2 h with a linear gradient on a 250 mm\*75 µm EasySpray column connected to an Easy
nLC 1200 HPLC machine, with an orbitrap Fusion Lumos mass spectrometer operating in DDA MS2 positive mode used for analysis.
Spectra were queried against the human proteome (downloaded from http://uniprot.org/ on 2019/02/12) at an FDR of 1% with Se-
quest HT through Proteome Discoverer v. 2.5. A spectral purity of 75% was required to ensure adequate confidence in quantification.
Protein abundances were processed further in the Perseus statistical software environment. Abundances were log₂-transformed and
normalized against the median intensity for each sample. The mass spectrometry proteomics data have been deposited with the
ProteomeXchange Consortium via the PRIDE¹⁹⁷ partner repository with the dataset identifier PXD037759.

Deep flow cytometry phenotyping and ex vivo naive CD4⁺ T cell polarization experiments

Cryopreserved PBMCs and their subpopulations were analyzed with a 28-color flow cytometry panel, as previously described.¹⁹⁸ PBMCs were collected from P1 at the age of five years and from P2 at the age of six years. Both patients were receiving broad-spectrum antimycobacterial drugs and P1 was also receiving recombinant IFN-γ. Cells were also labeled with anti-CD4 (APC-Cy7, RPA-T4, #557871, BD Pharmingen), anti-CD45RA (BV605, HI100, #562886, BD Horizon), and anti-CCR7 (AF700, 100,503, #561143, BD Pharmingen) antibodies, and naive (defined as CD45RA⁺CCR7⁺CD4⁺) T cells were isolated (>98% purity) with a FACS Aria cell sorter (BD Biosciences). Isolated cells were then cultured with T cell activation and expansion beads (TAE; anti-CD2/CD3/CD28; Miltenyi Biotec) + IL-2 (50 IU/mL, Millipore) to allow proliferation to occur, over a period of seven days. The cells were then subcultured with TAE beads alone (T<sub>H0</sub>) or under T<sub>H1</sub> (IL-12 [50 ng/mL; R&D Systems]), T<sub>H2</sub> (IL-4 [1 IU/mL; Thermo Fisher Scientific]), T<sub>H9</sub> ([100 IU/mL; Thermo Fisher Scientific], TGF-β [2.5 ng/mL; R&D Systems]), or T<sub>H17</sub> (TGF-β [2.5 ng/mL; R&D Systems], IL-1β [50 ng/mL; Peprotech], IL-6 [50 ng/mL; PeproTech], IL-21 [50 ng/mL; PeproTech], IL-23 [50 ng/mL; Thermo Fisher Scientific]) polarizing conditions. After five days of culture, the supernatant was used for assessments of the secretion of IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IFN-γ, and TNF with a cytometric bead array (BD Biosciences). Once the supernatant had been collected, the cells were stimulated with PMA (100 ng/mL)-ionomycin (750 ng/mL) for 6 h, with brefeldin A (10 µg/mL) added after the first 2 h of incubation. For the assessment of intracellular cytokine production, cells were stained for IFN-γ (BUV737, 4S.B3, #564620, BD Horizon), TNF (PerCP, Mab11, #502924, BioLegend), IL-9 (PE, MH9A3, #560807, BD Pharmingen), IL-13 (BV421, JES10-5A2, #563580, BD Horizon), IL-4 (AF488, 8D4-8, #500710, BioLegend), IL-17A (BV510, BL168, #512330, BioLegend), IL-17F (BV650, O33-782, #562264, BD Horizon), IL-2 (BV750, MQ1-17H12, #566361, BD Horizon), and IL-21 (eF660, eBio3A3-N2, #50-7219-42, Thermo Fisher Scientific), and analyzed by FACS (BD FACSymphony High-Speed Cell Analyzer A3). A previously described gating strategy was used.¹⁹⁸

Immunophenotyping of MAIT, iNKT, and γδ T cells

The immunophenotyping of MAIT, iNKT, and γδ T cells was performed as previously described⁸ on cryopreserved PBMCs from P1 prepared from a sample collected at the age of three years; and as previously described¹⁰ on cryopreserved PBMCs from P2 prepared from a sample collected at the age of six years. Both patients were receiving broad-spectrum antimycobacterial drugs. Briefly, staining was performed in the presence of Fcblock (Miltenyi Biotec), with Zombie-NIR live-dead exclusion dye (#423105, BioLegend), anti-CD3-Alexa532 (Clone UCHT1, # 58-0038-42, Thermo Fisher Scientific), anti-γδTCR-FITC (#11-9959-42, Thermo Fisher Scientific), anti-Vδ2-APC-Fire750 (#331420, BioLegend), anti-CD56-BV605 (clone 5.1H11, #362538, BioLegend), anti-CD4-BV750 (#5663656, BD Biosciences), anti-CD8a-BV510 (clone RPA-T8, #301047, BioLegend), anti-Vα7.2-BV711 (clone 3C10, #351731, BioLegend), anti-Vα24-Jα18-PE-Cy7 (clone 6B11, #342912, BioLegend), anti-Vδ1-Vioblue (#30-100-555, Miltenyi Biotec), anti-CD161-PE (clone HP-3G10, #339938, BioLegend) and anti-Vβ11-APC (Miltenyi Biotec) antibodies. Cells were analyzed with an Aurora cytometer (Cytek). The gating strategy for MAIT, iNKT, γδ1⁺, and γδ2⁺ T cells has been described elsewhere.⁵,⁷,⁹

ILC immunophenotyping

ILC, T, B, and NK cells were immunophenotyped on cryopreserved PBMCs from P1 and P2 prepared from samples collected at the ages of four and six years, respectively. Both patients were receiving broad-spectrum antimycobacterial drugs and P1 was also receiving recombinant IFN-γ. Briefly, biotinylated anti-human CD1a (biotin, HI149, #300112, BioLegend), CD14 (biotin, 61D3, #13-0149-82, Invitrogen), CD34 (biotin, 4H11, #316404, BioLegend), CD123 (biotin, 6H6, #306004, BioLegend), CD203c (biotin, FR3-16A11, #130-092-345, Miltenyi Biotec), CD303 (biotin, AC144, #130-090-691, Miltenyi Biotec), FcεRIα (biotin, AER-37(CRA-1), #334606, BioLegend), TCRαβ (biotin, IP26, #306704, BioLegend) and TCRγδ (biotin, B1, #555716, BD Biosciences) antibodies were used, in combination with streptavidin BUV661 (#565081, BD Biosciences), for lineage staining, along with anti-human CD4 FITC (OKT4, #317408, BioLegend), CD336 PerCP-eFluor710 (NKp44, 44.189, #46-3369-42), EOMES PE (WD1928, #12-4877-42, Invitrogen), CD8a PE-CF594 (RPA-T8, #562282, BD Biosciences), CD127 PE-Cy7 (eBioRDR5, #25-1278-42, Invitrogen), CD294 (CRTh2) AF647 (BM16, #558042, BD Biosciences), CD161 AF700 (HP-3G10, #339942, BD Biosciences), CD94 APC-Fire750 (DX22, #305518, BioLegend), CD335 (NKp46) BV421 (9E2/NKp46, #564065, BD Biosciences), CD45RA BV570 (HI100, #304132, BioLegend), CD117 BV605 (104D2, #313218, BioLegend), CD3 BV650 (UCHT1, #563852, BD Biosciences), CD7 BV711 (M-T701, #564018, BD Biosciences), T-bet BV786 (O4-46, #564141, BD Biosciences), CD19 BUV395 (SJ25C1, #563549, BD Biosciences), CD16 BUV496 (3G8, #564653, BD Biosciences), CD25 BUV563 (2A3, #565699, BD Biosciences), CD56 BUV737 (NCAM16.2, #564447, BD Biosciences) and CD45 BUV805 (HI30, #612891, BD Biosciences) antibodies. Human IgG from serum (Sigma-
<figure><img src="image_65.png" /></figure>

Aldrich) was used to block Fc receptors before staining. Extracellular staining was performed in Brilliant Stain Buffer (BD Biosciences). Dead cells were excluded with the fixable viability dye eFluor 506 (Invitrogen). Transcription factors were stained with the FoxP3 staining buffer set (Invitrogen), in accordance with the manufacturer's instructions. Samples were acquired on a Symphony A5 cytometer (BD Biosciences) with FACSDiva 8 software and were analyzed with FlowJo v.10 (BD Biosciences).

## Mass cytometry on fresh whole blood and cryopreserved PBMCs

Whole-blood mass cytometry was performed with two different panels, on two different blood samples from P2 collected two months apart, at the age of six years. P2 was receiving broad-spectrum antimycobacterial drugs. The first panel was that of the Maxpar Direct Immune Profiling Assay (#201325, Fluidigm), used according to the manufacturer's instructions. The second was a custom-produced panel, the content of which is detailed in STAR Methods. Marked cells were frozen at -80°C after overnight dead-cell staining, and acquisition was performed on a Helios machine (Fluidigm). All the samples were processed within 24 h of sampling. Data analysis was performed with OMIQ software. Mass cytometry on cryopreserved PBMCs was performed as previously described⁸ on two different samples collected at the ages of three and five years. The antibodies indicated in STAR Methods were used. The gating strategy was as previously described.⁸

## Whole-blood activation ELISA for cytokines and BCG-assay

Venous blood samples from healthy controls, P1 (aged 3 years), and P2 (aged 6 years) were collected in tubes containing heparin.¹⁹⁹,²⁰⁰ Both patients were receiving broad-spectrum antimycobacterial drugs. These samples were diluted 1:2 in RPMI 1640 (Gibco) supplemented with 100 IU/mL penicillin and 100 µg/mL streptomycin (Gibco). We then dispensed 1 mL of each diluted blood sample into each of five wells (1 mL/well) of a 48-well plate (Nunc). These samples were incubated for 48 h at 37°C, under an atmosphere containing 5% CO₂/95% air, and under various activation conditions: with medium alone, with live BCG (*M. bovis*-BCG, Pasteur substrain) at a MOI of 20 BCG cells/leukocyte, or with BCG plus recombinant (rh) IL-12 (20 ng/mL; R&D Systems), or BCG plus IFN-γ (Imukin, Horizon Pharma). The supernatants were then collected and subjected to ELISA. IFN-γ BCG release assay on cryopreserved PBMCs was performed as previously described⁸,¹⁰.

## ELISA

Supernatants from whole-blood stimulation experiments were used for determinations of IL-12p40 (#DP400, R&D Systems), IL-12p70 (#HS120, R&D Systems), and IFN-γ (#DIF50, R&D Systems), in accordance with the manufacturer's protocol.

## Phage immunoprecipitation-sequencing (PhIP-Seq)

A plasma sample was collected from P1 at the age of three years (before immunoglobulin administration) and from P2 at the age of six years. For antibody profiling by phage immunoprecipitation-sequencing (PhIP-Seq),²⁰¹ plasma samples from both patients and controls were assayed and data were analyzed as previously described,⁵,²⁰² but with the following modifications. We calculated species-specific significance cutoff values to estimate the minimum number of enriched, non-homologous peptides required to consider a sample seropositive (as previously described²⁰¹) with an in-house dataset and a generalized linear model. For each sample, we calculated virus-specific scores by dividing the counts of enriched, non-homologous peptides by the estimated cutoff score. These adjusted virus scores were used for the heatmap plot (Figure 7A). In addition to studying the patients reported here, we also calculated and plotted the mean antibody responses for a pediatric control cohort of lean individuals without infectious or immunological disease (n = 111; age range: 7 to 15 years; median age: 11.0 years) described in a previous study.²⁰³,²⁰⁴ Pooled human plasma for IVIg (Privigen CSL Behring AG) and human IgG-depleted serum (Molecular Innovations, Inc.) served as additional controls. All research on human subjects was performed after informed written consent had been obtained or after the samples had been rendered anonymous. The procedures were approved by the Institutional Research Ethics Boards of Sidra Medicine.

## Viral infection experiments

In viral infection experiments (as indicated), SV40-fibroblasts were subjected to pretreatment for 16 h with the indicated doses of IFN-α2b (Introna, MSD) or IFN-γ (Imukin, Horizon Pharma).

VSV (Indiana strain) replication experiments were performed as previously described.¹⁴⁵ Briefly, 1.25 × 10⁵ SV40-fibroblasts per well were plated in 24-well plates, in DMEM supplemented with 10% FBS. Cells were infected with VSV at a MOI of 0.1, in DMEM supplemented with 2% FBS. Cells and supernatants were obtained at various time points (1, 8, 24 and 48 h) and frozen. VSV titers were determined by calculating the 50% endpoint (TCID₅₀), as described by Reed and Muench,²⁰⁵ following the inoculation of Vero cell cultures in 96-well plates.

For HIV infections, the HIV-1 reporter virus HIV-GFP env-nef- was NL4-3 ΔenvΔnef encoding GFP in nef¹⁵⁷ and the HIV-2 reporter virus was ROD9 ΔenvΔnef encoding GFP in nef.¹⁵⁸ Viral particles were produced by transfecting 293FT cells in six-well plates with 3 µg DNA and 8 µL TransIT-293 (Mirus Bio) per well: for VSV-G-pseudotyped HIV-1, 0.2 µg CMV-VSVG, 0.2 µg HXB2²⁰⁶ and 2.6 µg HIV-GFP env-nef-; for VSV-G-pseudotyped HIV-2, 0.4 µg CMV-VSVG and 2.6 µg HIV-2ROD9ΔenvΔnef GFP. One day after transfection, the medium was removed, and fresh medium was added. Viral supernatants were harvested the following day, passed through a filter with 0.45 µm pores, and frozen at -80°C. SV40-fibroblasts were plated in a 96-well plate at a density of 2.5 × 10⁴/mL in 200 µL of DMEM supplemented with 10% FCS per well. The following day, the cells were left unstimulated or were
stimulated by overnight incubation with 10³ IU/mL IFN-α2a (#11343506, Immunotools), or 10 or 10³ IU/mL of IFN-γ (Imukin, Horizon Pharma). Cells were washed and then incubated with infected viral supernatants in the presence of 1 µg/mL protamine. Two days later, the GFP expression of living cells was assessed on a BD FACSVers flow cytometer.

The SARS-CoV-2 NYC isolate (GenBank OM345241) was obtained from a de-identified patient in July 2020. The virus isolate was amplified through six-to seven-day passages in Caco-2 cells at 37°C. After each passage, virus-containing supernatant was harvested, clarified by centrifugation (3,000 x g for 10 min), and filtered through a 0.22 µm-mesh disposable vacuum filter system. The passage 3 stock, used in this study, had a titer of 3.4 x 10⁶ PFU/mL, as determined on Vero E6 cells with a 1% methylcellulose overlay, as previously described.²⁰⁷ SV40-fibroblasts stably transduced with ACE2 were used to seed 96-well plates at a density of 7,000 cells per well in the presence or absence of the indicated doses of IFN-α2b (Introna; MSD) or IFN-γ. The cells were infected with SARS-CoV-2 16 h later, by adding the 0.1 µL of viral inoculum to the media (final volume 110 µL) and centrifuging the cells for 5 min at 500 x g at room temperature. The infections were conducted in four replicates (separate wells). The cells were fixed, 24 h post-infection, with neutral buffered formalin at a final concentration of 10%, stained for SARS-CoV-2 with an anti-N antibody at a dilution of 1:3000 (#GTX135357; GeneTex), then with an Alexa Fluor 647-conjugated secondary antibody (#A-21245; Invitrogen) and 1 µg/mL Hoechst 33,342 (#H3570; Invitrogen). Plates were imaged with ImageXpress micro XL and analyzed with MetaXpress (Molecular Devices).

The YF17D-venus reporter virus expressing the Venus fluorescent protein was generated as described by Yi et al.¹⁵⁹; the titer of this virus was 1.8 x 10⁶ TCID₅₀/mL on Huh-7.5 cells. SV40-fibroblasts were used to seed 96-well plates at a density of 8,000 cells/well in the presence or absence of 10 or 10³ IU/mL IFN-α2b (Introna; MSD), or 10³ IU/mL of IFN-γ (Imukin, Horizon Pharma). After incubation for 16 h, the medium was removed and the cells were inoculated with 50 µL YF17D-venus diluted in Opti-MEM (#51985-091; Gibco). The dilution used was determined empirically (1/3 2) so as to obtain >90% YF17D-venus positive IFNAR1-deficient cells at 3 dpi (MOI ≈ 0.3; titer determined on Huh-7.5 cells). After inoculation for 1 h, the inoculum was removed and 100 µL phenol-free medium (FluoroBrite DMEM; #A1896701; Gibco) supplemented with 10% FBS was added. Infections were performed with six replicates (separate wells). Three hours post-infection, the nuclei of the cells were stained by incubation with 1 µg/mL Hoechst 33,342 (#H3570; Invitrogen). Live imaging and quantification were performed with a BioTek Cytation 7 microscope.

For hepatitis A virus assays, SV40-fibroblasts were used to seed 12-well plates. They were incubated at 37°C for 24 h, and were then treated with IFN-α2b (10³ IU/mL) or IFN-γ (10³ IU/mL), or left untreated for 16 h before infection with the HM175/18f-NLuc reporter virus (6 x10³ genome equivalents/well).⁷⁰ The cells were incubated for another 72 h, then harvested and assayed for nanoluciferase (NLuc) activity, as previously described.⁷⁰ The replication of influenza A virus (IAV) was assessed as previously described at a MOI of 10, with the A/California/4/2009 strain.²⁰⁸ The HSV-1 replication experiment was performed as previously described¹⁴¹,¹⁴⁵,¹⁶⁵ with a MOI of 0.001.

The EMCV replication experiment was performed on SV-40 fibroblasts plated at a density of 5x10⁴ cells per well in 48-well plates. The cells were subjected to pretreated with the indicated concentrations of IFN-α2b or IFN-γ, or were left untreated, for 16 h before infection. Cells were then incubated with EMCV at a MOI of 0.01 for 1 h, washed twice in PBS, and then transferred to fresh complete DMEM only, or complete DMEM supplemented with the indicated concentrations of IFN-α2b or IFN-γ. Virus samples were collected 1 h post-infection (hpi), 12 hpi, 24 hpi, and 48 hpi. Total RNA was extracted from the mixture of cells and supernatant with the Quick-RNA Micro-Prep Kit (#R1051, Zymo Research), according to the manufacturer's protocol. Reverse transcriptase-PCR was performed with random hexamers (#18080-051, Invitrogen). The viral titer was determined with SYBR Green qPCR methods (#4385612, Applied Biosystems) for EMCV 3D, using the previously described primers.¹⁶¹,²⁰⁹ with β-glucuronidase (GUSB) as the housekeeping gene for normalization. The results are expressed according to the ΔΔCt method, as recommended by the manufacturer.

## iPSC-derived macrophages (iPSC-MΦ) and MDMs

The iPSC clones C16 and C11 derived from a healthy control, and the iPSC clones derived from a patient with complete STAT1 deficiency have been described elsewhere.⁶⁹,¹⁶²,¹⁶³ We obtained iPSCs from P1 by reprogramming primary fibroblasts with a “4-in-1” third generation SIN lentiviral vector containing the original Yamanaka factors, as previously described.²¹⁰ Cells were maintained on irradiated CF1 mouse embryonic fibroblasts (#A34180 or #A34181) cultured in KnockOut DMEM (#10829018) supplemented with 20% KnockOut Serum Replacement medium (#10828028), GlutaMAX Supplement (#35050061), penicillin-streptomycin (#15140122), MEM non-essential amino acids solution (#11140050), and 2-mercaptoethanol (#31350010), all from Thermo Fisher Scientific. Cultures were split weekly with collagenase IV (#17104-019, Gibco). Pluripotency was confirmed by an embryonic stem cell-like morphology, and the expression of SSEA-A, TRA1-60 and alkaline phosphatase. The karyotype was shown to be normal by R-banding and SNP array techniques. We then obtained macrophages from the iPSCs (iPSC-MΦ) as follows: the medium was depleted of bFGF for seven days, and embryonic bodies were allowed to form by incubation on an orbital shaker in the presence of ROCK inhibitor (#1254, Biotechnne).¹⁶²,¹⁶³ After five days, EBs were collected and transferred to differentiation medium composed of STEMdiff APEL2 Medium (#5275, Stem Cell Technologies) supplemented with 50 ng/L recombinant human M-CSF and 25 ng/mL IL-3 (Peprotech).¹⁶²,¹⁶³ The medium was replaced weekly, and it was possible to harvest monocytic cells from the supernatant after 21 days, for terminal differentiation in RPMI supplemented with 10% FBS, 1% PS and 50 ng/mL M-CSF for 10 days.¹⁶²,¹⁶³

MDMs were prepared as previously described.²¹¹ Briefly CD14⁺ cells were isolated from PBMCs by positive selection with anti-CD14 Micro-Beads (Miltenyi Biotec). Cells were cultured in 12-well plates, in RPMI 1640 containing 10% FBS and M-CSF (50 ng/mL,
R&D Systems). On day 7, IL-4 (50 ng/mL, R&D Systems) was added, and the cells were incubated for a further seven days for the completion of MDM differentiation. Primary fibroblasts, iPSC-MΦ, or MDMs were left unstimulated or were stimulated with 10³ IU/mL recombinant IFN-γ (Imukin, Horizon Pharma).

### RNA-seq

Primary fibroblasts were plated at a density of 700,000 cells per well in six-well plates, in 2 mL DMEM supplemented with 10% FBS. They were starved overnight in DMEM with 0.3% BSA (#A1595, Sigma-Aldrich). Terminally differentiated iPSC-MΦ were positively selected with anti-CD14 Micro-Beads (Miltenyi Biotec) to ensure that a pure population was obtained and >50,000 cells per well were plated in 12-well plates, in RPMI supplemented with 10% FBS and 100 ng/mL M-CSF. Primary fibroblasts, iPSC-MΦ, and MDMs were left unstimulated or were stimulated for 2 or 8 h with 10³ IU/mL recombinant IFN-γ (Imukin, Horizon Pharma). Primary fibroblasts were left unstimulated or were stimulated with 10³ IU/mL recombinant IFN-α2b (Introna, MSD). Total RNA was extracted from cells with the Quick-RNA Micro-Prep kit (#R1051, Zymo Research), and treated with DNAse (Zymo) to remove residual genomic DNA. rRNA and mitochondrial RNA (mRNA) were depleted. RNA-seq libraries were prepared with the Illumina RiboZero TruSeq Stranded Total RNA Library Prep Kit (Illumina) and sequenced on a NovaSeq machine in the 100- or 150-nt, paired-end configuration. The RNA-seq FASTQ files were first inspected with FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) to ensure that the raw data were of high quality. For each subject, the two FASTQ files generated were then mapped onto the human reference genome (Ensembl GRCh37 release 75) with STAR v.2.7.3a, in the two-pass mode.<sup>173</sup> The mapping quality of each BAM file was then evaluated with RSeQC.<sup>174</sup> Reads were quantified to generate gene-level feature counts from the read mapping, with HTSeq-count v.0.11.2.<sup>175</sup> We normalized the datasets with the functions DGEList and calcNormFactors from the edgeR version 3.26.8 package<sup>176</sup> implemented in R v.3.6.3. We retained only genes with at least 1 count-per-million (CPM) in at least two samples. We considered a gene to be differentially expressed between two sets of conditions if the log₂-fold-change between the two sets of conditions was greater than 1 (absolute value) and the adjusted p value was below 0.05, according to the calculations made with the DE-Seq function of the DESeq2 package version 1.24.0<sup>167</sup> implemented in R. Differential gene expression data were plotted on heatmaps with heat-map.2 implemented in R. We used the findMotifs.pl script from HOMER v4.11<sup>177</sup> with the human v.6.3 database and the parameter -len 12.

### Generation of THP1 cells with knockouts of IFN-γR1 or STAT1

THP1<sup>KO</sup> cells were prepared as previously described.<sup>8</sup> Briefly, the plasmid was digested with BsmBI. Annealed forward and reverse sgRNA for the target sequence (IFNGR1 exon 3 or STAT1 exon 3) was inserted into the pLentiCRISPRv2 plasmid digested with BsmBI. The resulting plasmid was used to transduce HEK293T cells in the presence of pCMV-VSV-G (#8454, Addgene), pHXB2 (#1069, NIH-AIDS Reagent Program), psPAX2 (#12260, Addgene), and pLentiCRISPRv2 (#52961, Addgene) with inserted sgRNA. After 24 h, the supernatant of HEK293T cells was filtered through a membrane with 45 µm pores, protamine was added to a final concentration of 8 µg/mL, and 100 µL of the viral supernatant was added to 200,000 THP1 WT cells in a 96-well round-bottomed plate. Spinoculation was performed for 2 h at 1200 x g. Two days later, selection on 5 ng/mL puromycin was initiated for five days. The cells were then cultured under single-clone conditions. After expansion IFN-γR1<sup>KO</sup> or STAT1<sup>KO</sup> clones completely deficient for the molecules concerned were identified on the basis of their (i) on complete lack of expression of IFN-γR1 or STAT1, respectively, on flow cytometry; (ii) complete lack of induction of HLA-DR after 24 h of stimulation with 10<sup>4</sup> IU/mL IFN-γ (Imukin, Horizon Pharma).

### Infection of macrophages with bacteria and mycobacteria

THP1 WT cells, the previously described IRF1<sup>KO</sup> cells,<sup>87</sup> or custom-generated IFN-γR1<sup>KO</sup> or STAT-1<sup>KO</sup> cells were cultured in FBS-supplemented RPMI. They differentiated into adherent macrophages with 48 h of PMA treatment (20 ng/mL, #P8139-1MG, Sigma Aldrich). The cells were then left unstimulated or were stimulated for 24 h with IFN-γ at a concentration of 10<sup>3</sup> IU/mL (Imukin, Horizon Pharma). Cells were then infected as previously described<sup>83</sup> with *Salmonella enterica* subsp. *enterica* serovar Typhimurium GFP (14028GFP, ATCC), in a gentamicin protection assay. Briefly, *Salmonella Typhimurium*-GFP was cultured overnight in LB broth supplemented with 100 µg/mL ampicillin. The overnight culture was then diluted 1:33 in the same medium and cultured for another 3 h until it reached exponential growth phase. The bacteria were then washed once with 1X PBS and used to infect cells at a MOI of 5–10. After 45 min, the medium was changed and the cells were incubated with gentamicin (#15750060, Thermo Fisher Scientific) at a concentration of 100 µg/mL for 1 h. The medium was then removed; the cells were gently washed three times with 1X PBS and incubated with RPMI supplemented with 10% FBS and 20 µg/mL gentamicin with or without 10<sup>3</sup> IU/mL IFN-γ for the indicated infection time. The cells were then treated with trypsin, and fixed by incubation with 4% paraformaldehyde (#30525-89-4, Santa Cruz) for 20 min at room temperature. Cells were acquired on a Beckman Coulter Gallios flow cytometer and analyzed with FlowJo Software. The infection of MDMs (differentiation with M-CSF and IL-4 (#216-MC-010 and #204-IL-010, respectively, R&D Systems)) with *M. abscessus*-tdTomato<sup>212</sup> and intracellular CFU determination were performed as previously described.<sup>213</sup> Infected MDMs were acquired on a Beckton Dickinson Fortessa flow cytometer and analyzed with FlowJo Software.

### CITE-seq

Single-cell RNA-seq profiling was performed on a first PBMC sample from P1 (P1.1 sampled at the age of three years), and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) was performed on PBMCs obtained from P1 (P1.2, sample
obtained at the age of five years), P2 (sample obtained at the age of six years), one pediatric control and two other healthy controls. Both patients were receiving broad-spectrum antimycobacterial drugs. P1 was also receiving recombinant IFN-γ at the age of five years but not at the age of three years. The frozen PBMCs were quickly thawed at 37°C and gently resuspended by serial additions of DMEM +10% heat-inactivated FBS, to obtain a final volume of 14 mL. The cell suspension was centrifuged at 300 x g for 5 min, and the cells were washed twice with 5 mL DMEM +10% HI-FBS each to remove cell debris. Cells were counted and viability was assessed with the LIVE/DEAD Viability kit (Thermo Fisher Scientific), according to the manufacturer's guidelines. Residual red blood cells were removed with 25 µL of the antibody cocktail and beads from the StemCell EasySep PBMC isolation kit. One million cells were resuspended in 1 mL Biolegend Cell Staining Buffer and passed through a 40 µm-mesh Flowmi cell strainer (Sigma) to remove aggregates. Cells were then centrifuged at 400 x g for 5 min and resuspended in 45 µL Biolegend Cell Staining Buffer supplemented with 5 µL Human TruStain FcX Fc block (Biolegend). Cells were then incubated for 30 min at 4°C with a cocktail of DNA-barcoded TotalSeq-B antibodies (Biolegend): CD11c (S-HCL-3), CD141 (M80), CD161 (HP-3G10), CD14 (Me5E2), CD16 (3G8), CD19 (HIB19), CD1c (L161), CD28 (CD28.2), CD370 (CLEC9A) (8F9), CD38 (HB-7), CD3e (UCHT1), CD4 (RPA-T4), CD45RA (HI100), CD45RO (UCHL1), CD56 (NCAM) (5.1H11), CD66b (6/40c), CD69 (FN50), CD8a (RPA-T8), TCR gd (B1), TCR Va7.2 (3C10), IgG1 k Isotype control (MOPC-21). Cells were washed twice in cell staining buffer, once in PBS +0.04% BSA, and resuspended at a concentration of 1000 cells/µL in PBS +0.04% BSA. The samples were loaded onto a 10X Genomics Chromium G chip and reverse transcription and library preparation were performed with the Chromium Single-Cell 3' Reagent Kits (v2 for P1.1, and v3.1 for the others), in accordance with the manufacturer's instructions. The quality of the cDNA and feature barcode library was assessed with a TapeStation (Agilent), before sequencing on the S4 flow cells of an Illumina NovaSeq 6000 sequencer.

Two additional control PBMC scRNA-seq datasets were obtained from another study that we performed.²¹⁴ For the analysis, we included scRNA-seq for P1.1 and 2 controls, and CITE-seq for 3 controls and the P1.2 and P2 samples. The analysis was performed as previously described.²¹⁴ Briefly, sequence read quality was assessed with BVATools (https://bitbucket.org/mugqic/bvatools). Cell Ranger (v3.0.1 for scRNA and v6.0.1 for CITE-seq) was used to map reads to the hg38 human reference genome assembly, to perform filtering, and to count barcodes and unique molecular indices (UMIs). Genes that were not expressed in any of the datasets were discarded. Cells with >20% mitochondrial genes were excluded. We filtered out low-quality cells and doublets, by excluding cells falling outside the [-1SD; +2.5SD] interval from the UMI and gene count distributions. We identified and removed any remaining doublets by manually removing cells that co-expressed markers for different cell types (CD14, CD79A, TRBC1, HBA2 and LILRA4). We then used DoubletFinder package v2.0.3 to identify and filter out any remaining cell doublets.¹⁶⁹ Once dead cells and doublets had been removed, the patient and control samples were analyzed together with the Seurat v4.0.2 R package¹⁷⁰ and cell clustering was performed by the Uni-form Mani-fold Approximation and Projection dimension reduction method¹⁶⁸ applied to the most variable genes, but excluding mitochondrial and ribosomal protein genes, together with sex-related genes as both patients were female. Based on the clustering pattern obtained, we performed subclustering for the large immune cell lineages separately (myeloid/ pDC, T/NK, and B cells) to identify any remaining doublets. Small residual cell clusters expressing multiple cell type-specific markers were, therefore, excluded. A final UMAP¹⁶⁸ clustering identified 14 different cells, including 11 T cells and 2 B cells; myeloid cells were excluded from further analyses because of the small numbers of these cells in the patient samples, probably due to the poor survival of these cells during sample freezing and shipping. The protein markers identified with CITE-seq were used to assign a cell-type identity to each cluster, in conjunction with the MAST approach¹⁷¹ to identify the marker genes for each cluster. Differential gene expression analysis was performed with MAST for each lymphoid cell cluster, by comparing the RNA levels in P1.2 and P2 with those in five healthy controls. Adjusted p values were estimated with the Benjamini-Hochberg (BH) procedure. Every significant differentially expressed gene (absolute FC ≥ 1.5 and adj-p value ≤ 0.001) was classified as down- or upregulated, extracted and used for downstream analysis. All the differential expression results are provided in Table S6. Enrichment analyses for GO biological processes, KEGG and Reactome pathways and Transfac motifs were performed separately on up- and downregulated differentially expressed genes and for each cell subpopulation, with the gProfiler R package and the g:SCS threshold method¹⁷²; all the enrichment results are provided in Table S6.

We evaluated the effects on IRF1-binding genes in the cells of the patients by estimating module scores with Seurat's AddModuleScore function, using the default settings. Module score was determined for each cell by calculating the mean transcriptional activity of the genes from the module and then subtracting a mean expression value for a set of control genes. The list of IRF1-binding genes was obtained as follows: IRF1 binding sites were identified by ChIP-seq in IFN-γ-treated (3 h) mouse bone marrow-derived macrophages obtained in a previous study,⁷³ we then retain genes with IRF1 sites within 10 kb on either side of the transcription start site and performed a conversion to human orthologs. Control genes were randomly sampled from bins defined based on the observed level of expression for the genes in the module. The significance of differences between expression in the patients and control module expression was then assessed by comparing the corresponding module score distributions for each cell subset in a Wilcoxon signed rank-test approach (q value ≤ 0.05). We obtained a sample size-free evaluation of the difference in expression by calculating Cohen's SD effect size for every significant variant.

The scRNA-seq and CITE-seq datasets have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE216489.
## Generation of single-cell suspensions from murine blood and tissues for flow cytometry

All mice were kept in specific pathogen-free conditions and handled according to the guidelines and regulations of the Canadian Council on Animal Care. Experimental protocols were approved by the McGill University Institutional Animal Care Committee (protocol number 2018-8014). Groups of male C57BL/6 (B6) and *Irf1*<sup>-/-</sup> mice (originally from The Jackson Laboratory, reared in-house) were killed, and their blood was collected by cardiac puncture and transferred to heparin-containing tubes (Sarstedt). Blood was diluted 1:5 in PBS, centrifuged in a benchtop centrifuge and the red blood cells in the pellet were lysed with Red Blood Cell Lysing Buffer Hybri-Max (Sigma-Aldrich) according to the manufacturer's instructions. Cells were washed twice with FACS buffer (PBS supplemented with 2% heat-inactivated FBS) and resuspended in FACS buffer. Mouse lungs were processed as previously described<sup>215</sup> but with minor modifications. Briefly, lungs were perfused with 5 mL ice-cold PBS, isolated and finely minced. Lung pieces were digested by incubation in 2.5 mL lung digestion buffer (RPMI-1640 supplemented with 5% FBS, 0.2 mg/mL Liberase [Roche] and 0.1 mg/mL DNase I [Roche]) for 50 min at 37°C. Digests were homogenized by passage through a syringe (18 G needle) and a cell strainer with 70 µM pores. Cells were washed with PBS and red blood cells were lysed with Red Blood Cell Lysing Buffer Hybri-Max (Sigma-Aldrich). Cells were washed twice and resuspended in FACS buffer. For the generation of single-cell splenocyte suspensions, spleens were isolated, gently homogenized between the frosted ends of glass slides and digested by incubation in 3 mL spleen digestion buffer (HBSS supplemented with 10 mM HEPES, 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 1 mg/mL collagenase D [Roche] and 0.2 mg/mL DNase I [Roche]) for 30 min at 37°C. Cell suspensions were further homogenized by pipetting and incubated for an additional 15 min at 37°C. EDTA was added to a final concentration of 1 mM and the suspensions were incubated for another 10 min at room temperature. Splenocytes were collected by centrifugation. The pellet was washed with PBS and red blood cells were lysed with Red Blood Cell Lysing Buffer Hybri-Max (Sigma-Aldrich). Cells were washed twice and resuspended in FACS buffer. Bone marrow was isolated from femurs and tibiae and red blood cells were lysed with Red Blood Cell Lysing Buffer Hybri-Max (Sigma-Aldrich). Cells were washed twice and resuspended in FACS buffer.

## Flow cytometry on mouse cells

Pelleted cells were resuspended in 2.4G2 hybridoma supernatant diluted in FACS buffer and the resulting suspensions were incubated for 15 min on ice to block Fcγ receptors. Surface markers were then stained by incubation with the correspondingfluorochrome-labeled antibody dilutions in FACS buffer for 30 min on ice. Dead cells were excluded by staining with Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific) according to the manufacturer's instructions. Intracellular staining was performed with the FoxP3/Transcription factor staining kit (Thermo Fisher Scientific), in accordance with the manufacturer's protocol. Stained cell suspensions were acquired on a BD LSRFortessa Cell Analyzer (BD Biosciences) and data were analyzed with FlowJo v.10 software (BD Biosciences). All the antibodies used for murine flow cytometry analyses are listed in STAR Methods.

## QUANTIFICATION AND STATISTICAL ANALYSIS

Unless otherwise indicated in the figure legends, the statistics provided correspond to independent experiments. Mann-Whitney or Student's t tests were performed. In the relevant figures, n.s. indicates not significant, *p* > 0.05; **p** < 0.0001; ***p** < 0.001; **p** < 0.01; and *p* < 0.05. Analyses were performed with GraphPad software.
Supplemental figures

<figure><img src="image_66.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_67.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_68.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_69.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_70.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_71.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_72.png" /><figcaption>G</figcaption></figure>

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Figure S1. Identification of homozygous complete loss-of-function *IRF1* variants in patients with severe MSMD, related to Figure 1

(A) Cells involved in the production of and response to IFN-γ. Proteins for which a mutation of the corresponding gene has been recognized to cause isolated MSMD are depicted in blue, those responsible for syndromic MSMD are shown in red, and those that can cause either isolated MSMD or syndromic MSMD are depicted with crossed lines.

(B) Relative clinical severity of isolated MSMD forms vs. relative residual IFN-γ activity.

(C) Principal component analysis of WES data from the two patients and our in-house WES database.

(D) Consensus negative selection (CoNeS) of *IRF1*.

(E) Diagram of the various *IRF1* cDNA constructs used for transient overexpression experiments.

(F) RT-qPCR on HEK293T cells without (NT) and with transfection with empty vector (EV) or various *IRF1* cDNAs. The bars represent the mean. Data from two to four independent experiments are shown. Bars represent the mean.

(G) Western blot for DDK, IRF1, lamin A/C, and vinculin on cytoplasmic (C) and nuclear (N) extracts from HEK293T cells not transfected (NT) or transfected with EV or various IRF1 cDNAs. Data representative of three independent experiments are shown.

(H) EMSA and supershift of the nuclear extract of HEK293T cells transfected with EV or various IRF1 cDNAs with DDK tagging of the C terminus of the protein, incubated with a fluorescent ISRE probe. Representative data from two independent experiments are shown.

(I) Dual luciferase ISRE5 reporter activity of HEK293T cells transfected with EV or various IRF1 cDNAs. Bars represent the mean and SD. Data from three to six independent experiments are shown. Statistical analysis was performed with Student's t test. ns = not significant, p > 0.05; ****p < 0.0001.
<figure><img src="image_75.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_76.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_77.png" /><figcaption>D</figcaption></figure>

SV40-fibroblasts

<figure><img src="image_78.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_79.png" /><figcaption>E</figcaption></figure>

EBV-B cells

<figure><img src="image_80.png" /><figcaption>**Figure S2.** IRF1 protein levels in cells from IRF1-deficient patients, related to Figure 2</figcaption></figure>

(A) Western blot for IRF1, IRF3, IRF9, STAT1, and vinculin on total lysate from HVS-T cells from three healthy controls (CTLs), P1, and a patient with complete IRF9 deficiency. Data from a single experiment are shown.

(B) IRF1 expression in primary fibroblasts from three controls (CTLs), P1, P2, and a patient with complete IFN-γR1 deficiency, with and without 16 h of stimulation with 10³ IU/mL IFN-γ.

(C-E) Intracellular IRF1 (upper panel) and STAT1 (lower panel) levels, assessed by flow cytometry on (C) HVS-T cells (from a CTL and P1), (D) SV40-fibroblasts (from a CTL, P1, P2, and from a patient with complete STAT1 deficiency), or (E) EBV-B cells (from a CTL, P1, P2, and from a patient with complete STAT1 deficiency).

(F) IRF1 and STAT1 expression in SV40-fibroblasts from three CTLs, P1, and a patient with complete IFN-γR1 deficiency, with and without 16 h of stimulation with 10³ IU/mL IFN-γ, or in HEK293T cells transfected with p.R129-DDK IRF1 cDNA. Data representative of three independent experiments are shown.
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Figure S3. Leukocyte development in IRF1-deficient patients and in IRF1-deficient mice, related to Figure 3

(A) Monitoring of counts of leukocytes, monocytes and lymphocytes, polymorphonuclear neutrophils (PMN), polymorphonuclear eosinophils (PME), and polymorphonuclear basophils (PMB) in the blood of the patients, in red.

(B) Mass or flow cytometry analysis of cryopreserved PBMC subsets from CTLs, P1, and P2. Bars represent the mean and SEM.

(C) Flow cytometry analysis of dendritic cell (DC) subsets in fresh PBMCs from a CTL, P1, and P2.

(D) Flow cytometry immunophenotyping of live CD45⁺ lymphocytes from PBMCs, for ILCP (CD117⁺CRTh2⁺) and ILC2 (CRTh2⁺), in CTLs, P1, and P2.

(E) Conventional flow cytometry counts of cDC1, cDC2, NK cells, ILC2, CD4⁺ T cells, Treg and CD8⁺ T cells, in the lungs of WT mice (n = 5), and IRF1-deficient mice (n = 5). Bars represent the mean and SD. \*p < 0.05; \*\*p < 0.001.

(F) Conventional flow cytometry counts of Ly6C⁺ monocytes, cDC1, cDC2, pDCs, NK cells, CD4⁺ T cells, Treg and CD8⁺ T cells in the spleen of WT mice (n = 5), and IRF1-deficient mice (n = 5). Bars represent the mean and SD. \*p < 0.05; \*\*p < 0.001.

(G) Conventional flow cytometry counts of CLP, CHILP, and ILC2P in the bone marrow of WT mice (n = 5), and IRF1-deficient mice (n = 5). Unpaired two-tailed t tests were used for the statistical analysis. Bars represent the mean and SD.
<figure><img src="image_88.png" /><figcaption>**Figure S4.** IFN-γ production pathway in P1 and P2, related to Figure 4</figcaption></figure>

(A) Western blot analysis of ISG15 and MX1 induction in the SV40-fibroblasts of three CTLs, P1, P2, one patient with complete ISG15-deficiency, and one patient with complete IFN-αR1 deficiency.

(legend continued on next page)
(B) pSTAT4 levels in HVS-T cells from two controls (CTLs), P1, and a patient with complete IL-12Rβ1 deficiency without stimulation (gray), and after 15 min of stimulation with 50 ng/mL IL-12 (solid black line), or 10⁵ IU/mL IFN-α2b (dotted black line). Data representative of two independent experiments are shown.

(C) Production of IFN-γ by the HVS-T cells of controls (CTLs), P1, and a patient with complete IFN-γ deficiency with and without 2 h of stimulation with brefeldin, anti-CD3/CD2/CD28 mAb-coated beads, or with PMA-ionomycin. Bars represent the mean and SD. Data from one experiment are shown.

(D) Analysis of IFN-γ⁺ cells on intracellular flow cytometry for various PBMC subsets, following 48 h of stimulation with IL-12, IL-23, or BCG. Bars represent the mean and SEM. Technical duplicates of the same experiment are shown for P1 and P2.

(E) Intracellular flow cytometry analysis of IFN-γ, TNF, IL-9, IL-13, IL-4, IL-17A, IL-17F, IL-2, and IL-21 levels in naive CD4⁺ T cells from P1 and controls after expansion for 5 days with anti-CD2/CD3/CD28 mAb-coated beads and IL-2, followed by culture for 7 days in polarizing conditions, and stimulation with PMA-ionomycin in the presence of brefeldin for 6 h.
<figure><img src="image_89.png" /></figure>

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### F Primary fibroblasts

IFN-γ 2 hours

<figure><img src="image_98.png" /></figure>

### G Primary fibroblasts

IFN-γ 8 hours

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**Figure S5. Response to IFN-γ in non-hematopoietic cells from P1 and P2, related to Figure 5**

(A and B) IRF1 RT-qPCR (A) and western blot for IRF1, pSTAT1, STAT1 and vinculin (B) on SV40-fibroblasts with and without stimulation with 10³ IU/mL IFN-γ. Data from one experiment are shown.

(C) Confocal microscopy of IRF1 in SV40-fibroblasts of a CTL, P1, and a STAT1-deficient patient stimulated for the indicated times.

(D and E) Phosphorylation of STAT1, analyzed by staining and flow cytometry after 20 min of incubation with or without 10⁵ IU/mL IFN-γ, in EBV-B cells (D), or with or without 10³ IU/mL IFN-γ in SV40-fibroblasts (E) from controls (CTLs), the patients (P1 and P2), or patients with complete IFN-γR1 or STAT1 deficiency.

(F and G) Data representative of two independent experiments are shown. Heatmaps of genes differentially expressed (log₂FC) in primary fibroblasts from controls or patients, after (F) 2 or (G) 8 h of stimulation with 10³ IU/mL IFN-γ. We show the genes differentially expressed in patients relative to the control group at both time points (2 or 8 h), for the genes differentially expressed at both time points relative to non-stimulated fibroblasts in the control group, i.e., with a |log₂(FC)| > 1 and adj. p value <0.05 in controls, and with a |log₂(FC)| > 1 and adj. p value <0.05 in patients relative to controls at the corresponding time point. We used the IFN-γR1⁻/⁻ patient as a negative control.

(H) Amount of mRNA, assessed by RNA sequencing for IFN-α2b-inducible genes (logFC>2 in controls) in primary fibroblasts from the indicated individuals with and without 30 min, 2 h, and 8 h of stimulation with IFN-γ.

(I and J) Flow cytometry analysis of CD274 (PD-L1) expression with and without stimulation for 48 h with 10², 10³, or 10⁴ IU/mL IFN-γ in SV40-fibroblasts with and without stable retrotransduction with EV or WT IRF1 cDNA. Bars represent the mean and SD.

(K) Flow cytometry analysis of the CD274 expression of primary fibroblasts with and without 48 h of stimulation with 10² or 10³ IU/mL IFN-γ.
<figure><img src="image_100.png" /></figure>
**Figure S6. Response to IFN-γ in myeloid cells from P1 and P2, related to Figure 6**

(A and B) Induction of IL-12p40 (A) and IL-12p70 (B) secretion in whole-blood assays for local and travel controls (CTLs), an IL-12p40-deficient patient, P1, and P2, following stimulation with live BCG alone or in combination with IFN-γ (5,000 IU/mL). Bars represent the mean.

(C and D) Heatmaps of genes differentially expressed (log<sub>2</sub>(FC)) in iPSC-derived macrophages (iPSC-MΦ), and monocytes-derived macrophages (MDMs) from controls (CTL) or patients, after (C) 2 or (D) 8 h of stimulation with 10<sup>3</sup> IU/mL IFN-γ. We show the genes differentially expressed in patients relative to the control group at both time points (2 or 8 h), for genes differentially expressed at both time points relative to non-stimulated cells in the control group, i.e., with a |log<sub>2</sub>(FC)| > 1 and adj. p value <0.05 after Benjamini-Hochberg correction in controls, and with a |log<sub>2</sub>(FC)| > 1 and adj. p value <0.05 after correction in patients relative to controls at the corresponding time point.

(E) Fold-enrichment in dysregulated genes, as determined by RNA-seq, for the myeloid cells of both patients relative to IRF1 ChIP-seq results for resting human monocytes<sup>36</sup> and IFN-γ-activated murine bone marrow-derived macrophages.<sup>73</sup>

(F) RT-qPCR for *APOL3* and *GBP4* in iPSC-MΦ or MDMs with and without stimulation for 2 and 8 h with 10<sup>3</sup> IU/mL IFN-γ. The bars represent the mean.

(G) Examples of flow cytometry dot plots obtained for gentamicin protection assay performed on PMA-differentiated WT or IRF1<sup>KO</sup>, IFN-γR1<sup>KO</sup>, or STAT1<sup>KO</sup> THP1-derived macrophages (THP1-Φ) with and without 24 h of pretreatment with 10<sup>3</sup> IU/mL IFN-γ and infection with *Salmonella Typhimurium*-GFP (Stm-GFP) at a MOI of 6 for 8 h. The percent *Salmonella Typhimurium*<sup>+</sup> cells is displayed in black and the percent *Salmonella Typhimurium*<sup>high</sup> cells is displayed in red.

(H) Examples of flow cytometry dot plots obtained for Figure 6H.
A

<figure><img src="image_101.png" /></figure>

B IFN-α2b

<figure><img src="image_102.png" /></figure>

C

<figure><img src="image_103.png" /></figure>

D

<figure><img src="image_104.png" /></figure>

E

<figure><img src="image_105.png" /></figure>

F

<figure><img src="image_106.png" /></figure>

G

<figure><img src="image_107.png" /></figure>

H

<figure><img src="image_108.png" /><figcaption>**Figure S7.** Antiviral IFN-α/β and IFN-γ cell-intrinsic immunity of IRF1-deficient fibroblasts, related to Figure 7</figcaption></figure>

(A) RT-qPCR for *IRF1* and *MX1* in SV40-fibroblasts from the indicated controls or patients after 2 h of stimulation with 10³ IU/mL IFN-α2a or IFN-γ.

(B) *IRF1* levels determined by RNA sequencing, in primary fibroblasts stimulated for 30 min, 2 h, or 8 h with IFN-α2b or IFN-γ. Results are normalized against non-stimulated conditions.

(C) Western blot of IRF1, MX1, STAT1, STAT2, IRF9, and vinculin in SV40-fibroblasts from controls (CTLs), the patients (P1, P2), or patients with complete STAT1, STAT2, IRF9, IFN-αR1, or IFN-γR1 deficiencies stimulated for 4 h with IFN-γ or IFN-α2β.

(D) Phosphorylation of STAT1, as assessed by staining and flow cytometry, after 20 min of incubation with or without 10³ IU/mL IFN-α2b or 10 ng/mL IFN-β, in SV40-fibroblasts or EBV-B cells from two controls (CTLs), the patients (P1, P2), and patients with complete IFN-αR1 or STAT1 deficiency. Data representative of two to three experiments are shown.

(E) Venn diagram of genes positively induced in primary fibroblasts of CTLs (log2FC > 1) but less strongly induced (log2FC < -1) in IRF1-, STAT1-, or IRF9-deficient primary fibroblasts than in controls after 2 or 8 h of stimulation with IFN-α2b.

(F-H) Hepatitis A virus (HAV), (G) human immunodeficiency virus-1 (HIV-1), and (H) yellow fever vaccine virus-venus (YF17D-venus) replication in SV40-fibroblasts for the indicated controls or patients (P1 and P2), after pretreatment for 16 h with the indicated doses of IFN-α2b or IFN-γ. All viral infections were performed in two or three independent experiments.