# Human IRF1 governs macrophagic IFN-γ immunity to mycobacteria

Jérémie Rosain, Anna-Lena Neehus, Jérémy Manry, Rui Yang, Jérémie Le Pen, Wassim Daher, Zhiyong Liu, Yi-Hao Chan, Natalia Tahuil, Özden Türel, et al.

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► To cite this version:

Jérémie Rosain, Anna-Lena Neehus, Jérémy Manry, Rui Yang, Jérémie Le Pen, et al. Human IRF1 governs macrophagic IFN-γ immunity to mycobacteria. Cell, 2023, 186 (3), pp.621 - 645.e33. 10.1016/j.cell.2022.12.038. pasteur-04130575

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# Human IRF1 governs macrophagic IFN-γ immunity to mycobacteria

## Graphical abstract

<figure><img src="image_3.png" /></figure>

## Authors

Jérémie Rosain, Anna-Lena Neehus,
Jérémy Manry, ...,
Jean-Laurent Casanova, Philippe Gros,
Jacinta Bustamante

## Correspondence

jeremie.rosain@institutimagine.org (J.R.),
casanova@rockefeller.edu (J.-L.C.),
jacinta.bustamante@inserm.fr (J.B.)

## In brief

Studies in humans with interferon regulatory factor 1 (IRF1) deficiency reveal differences in how type I and type II interferons protect humans against different types of pathogens.

## Highlights

* Inherited complete human IRF1 deficiency underlies severe mycobacterial disease

* Human IRF1 is essential for IFN-γ-dependent macrophagic immunity to mycobacteria

* Human IRF1 is essential for IFN-γ- and STAT1-dependent immunity to mycobacteria

* Human IRF1 is largely redundant for IFN-α/β-dependent antiviral intrinsic immunity

<figure><img src="image_4.png" /></figure>
Article

# Human IRF1 governs macrophagic IFN-γ immunity to mycobacteria

Jérémie Rosain,<fnref n="1" />,<fnref n="2" />,* Anna-Lena Neehus,<fnref n="1" />,<fnref n="2" />,<fnref n="3" />,⁵² Jérémy Manry,<fnref n="1" />,<fnref n="2" />,<fnref n="2" /> Rui Yang,<fnref n="4" />,⁵² Jérémie Le Pen,<fnref n="5" />,⁵² Wassim Daher,<fnref n="6" />,<fnref n="7" />,⁵²

Zhiyong Liu,<fnref n="4" />,⁵² Yi-Hao Chan,<fnref n="4" />,⁵² Natalia Tahuil,<fnref n="8" />,⁵² Özden Türel,<fnref n="9" />,⁵² Mathieu Bourgey,<fnref n="10" />,<fnref n="11" />,⁵² Masato Ogishi,<fnref n="4" />,⁵²

Jean-Marc Doisne,<fnref n="12" />,<fnref n="13" />,⁵² Helena M. Izquierdo,<fnref n="14" />,⁵² Takayoshi Shirasaki,<fnref n="15" />,⁵² Tom Le Voyer,<fnref n="1" />,<fnref n="2" />,⁵² Antoine Guérin,¹⁶,¹⁷,⁵²

Paul Bastard,<fnref n="1" />,<fnref n="2" />,<fnref n="4" />,¹⁸,⁵² Marcela Moncada-Vélez,<fnref n="4" />,⁵² Ji Eun Han,<fnref n="4" />,⁵³ Taushif Khan,¹⁹,⁵³ Franck Rapaport,<fnref n="4" />,⁵³

Seon-Hui Hong,<fnref n="5" /> Andrew Cheung,<fnref n="5" /> Kathrin Haake,<fnref n="3" /> Barbara C. Mindt,²⁰,²¹,²² Laura Pérez,²³ Quentin Philippot,<fnref n="1" />,<fnref n="2" />

Danyel Lee,<fnref n="1" />,<fnref n="2" />,<fnref n="4" /> Peng Zhang,<fnref n="4" /> Darawan Rinchai,<fnref n="4" /> Fatima Al Ali,¹⁹ Manar Mahmoud Ahmad Ata,¹⁹ Mahbuba Rahman,¹⁹

Jessica N. Peel,<fnref n="4" /> Søren Heissel,²⁴ Henrik Molina,²⁴ Yasemin Kendir-Demirkol,<fnref n="4" />,²⁵ Rasheed Bailey,<fnref n="4" /> Shuxiang Zhao,<fnref n="4" />

Jonathan Bohlen,<fnref n="1" />,<fnref n="2" /> Mathieu Mancini,<fnref n="10" />,²⁰,²¹ Yoann Seeleuthner,<fnref n="1" />,<fnref n="2" /> Marie Roelens,²⁶,²⁷ Lazaro Lorenzo,<fnref n="1" />,<fnref n="2" />

Camille Soudée,<fnref n="1" />,<fnref n="2" /> María Elvira Josefina Paz,²⁸ María Laura González,²⁹ Mohamed Jeljeli,³⁰ Jean Soulier,³¹,³²,³³

(Author list continued on next page)

(Affiliations continued on next page)

## SUMMARY

Inborn errors of human IFN-γ-dependent macrophagic immunity underlie mycobacterial diseases, whereas inborn errors of IFN-α/β-dependent intrinsic immunity underlie viral diseases. Both types of IFNs induce the transcription factor IRF1. We describe unrelated children with inherited complete IRF1 deficiency and early-onset, multiple, life-threatening diseases caused by weakly virulent mycobacteria and related intramacrophagic pathogens. These children have no history of severe viral disease, despite exposure to many viruses, including SARS-CoV-2, which is life-threatening in individuals with impaired IFN-α/β immunity. In leukocytes or fibroblasts stimulated *in vitro*, IRF1-dependent responses to IFN-γ are, both quantitatively and qualitatively, much stronger than those to IFN-α/β. Moreover, IRF1-deficient mononuclear phagocytes do not control mycobacteria and related pathogens normally when stimulated with IFN-γ. By contrast, IFN-α/β-dependent intrinsic immunity to nine viruses, including SARS-CoV-2, is almost normal in IRF1-deficient fibroblasts. Human IRF1 is essential for IFN-γ-dependent macrophagic immunity to mycobacteria, but largely redundant for IFN-α/β-dependent antiviral immunity.

## INTRODUCTION

The discovery of inborn errors of immunity (IEI) underlying severe infectious diseases delineates the essential vs. redundant func-

tions of the corresponding human genes in host defense *in natura*, while clarifying the pathogenesis of these infections. <fnref n="1" />⁻<fnref n="3" /> Mendelian susceptibility to mycobacterial disease (MSMD) is the most extensively studied monogenic susceptibility to a single type of

<footnote n="1">¹Laboratory of Human Genetics of Infectious Diseases, Inserm U1163, 75015 Paris, France</footnote>

<footnote n="2">²Paris Cité University, Imagine Institute, 75015 Paris, France</footnote>

<footnote n="3">³Institute of Experimental Hematology, REBIRTH Center for Regenerative and Translational Medicine, Hannover Medical School, 30625 Hannover, Germany</footnote>

<footnote n="4">⁴St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York, NY 10065, USA</footnote>

<footnote n="5">⁵Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA</footnote>

<footnote n="6">⁶Infectious Disease Research Institute of Montpellier (IRIM), Montpellier University, 34090 Montpellier, France</footnote>

<footnote n="7">⁷Inserm, IRIM, CNRS, UMR9004, 34090 Montpellier, France</footnote>

<footnote n="8">⁸Department of Immunology, Del Niño Jesus Hospital, San Miguel de Tucuman, T4000 Tucuman, Argentina</footnote>

<footnote n="9">⁹Department of Pediatric Infectious Disease, Bezmialem Vakif University Faculty of Medicine, 34093 İstanbul, Turkey</footnote>

<footnote n="10">¹⁰Dahdaleh Institute of Genomic Medicine, McGill University, Montreal, QC H3A 0G1, Canada</footnote>

<footnote n="11">¹¹Canadian Centre for Computation Genomics, Montreal, QC H3A 0G1, Canada</footnote>

<footnote n="12">¹²Innate Immunity Unit, Institut Pasteur, 75015 Paris, France</footnote>

<footnote n="13">¹³Inserm U1223, 75015 Paris, France</footnote>

<footnote n="14">¹⁴Institut Curie, PSL Research University, Inserm U932, 75005 Paris, France</footnote>

<footnote n="15">¹⁵Department of Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7292, USA</footnote>
Serge Romana,³⁴ Anne-Sophie L'Honneur,³⁵ Marie Materna,¹,² Rubén Martínez-Barricarte,³⁶,³⁷ Mathieu Pochon,¹,²

Carmen Oleaga-Quintas,¹,² Alexandre Michev,¹,² Mélanie Migaud,¹,² Romain Lévy,¹,²,¹⁸ Marie-Alexandra Alyanakian,³⁸

Flore Rozenberg,³⁵ Carys A. Croft,¹²,¹³,²⁷ Guillaume Vogt,³⁹,⁴⁰ Jean-François Emile,⁴¹ Laurent Kremer,⁶,⁷

Cindy S. Ma,¹⁶,¹⁷,⁵⁴ Jörg H. Fritz,²⁰,²¹,²²,⁴²,⁵⁴ Stanley M. Lemon,¹⁵,⁵⁴ András N. Spaan,⁴,⁴³,⁵⁴ Nicolas Manel,¹⁴,⁵⁴

Laurent Abel,¹,²,⁴,⁵⁴ Margaret R. MacDonald,⁵,⁵⁴ Stéphanie Boisson-Dupuis,¹,²,⁴,⁵⁵ Nico Marr,¹⁹,⁴⁴,⁵⁵

Stuart G. Tangye,¹⁶,¹⁷,⁵⁵ James P. Di Santo,¹²,¹³,⁵⁵ Qian Zhang,¹,²,⁴,⁵⁵ Shen-Ying Zhang,¹,²,⁴,⁵⁵ Charles M. Rice,⁵,⁵⁵

Vivien Béziat,¹,²,⁴,⁵⁵ Nico Lachmann,³,⁴⁵,⁴⁶,⁵⁵ David Langlais,¹⁰,²⁰,⁴⁷,⁵⁵ Jean-Laurent Casanova,¹,²,⁴,⁴⁸,⁴⁹,⁵¹,⁵⁶,*

Philippe Gros,¹⁰,⁵⁰,⁵⁶ and Jacinta Bustamante¹,²,⁴,²⁶,⁵⁶,*

¹⁶Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia

¹⁷St. Vincent's Clinical School, Faculty of Medicine, University of NSW, Sydney, NSW 2052, Australia

¹⁸Pediatric Hematology-Immunology and Rheumatology Unit, Necker Hospital for Sick Children, Assistance Publique Hôpitaux de Paris (AP-HP), 75015 Paris, France

¹⁹Department of Immunology, Sidra Medicine, Doha, Qatar

²⁰Department of Microbiology and Immunology, McGill University, Montreal, QC H3A 0G1, Canada

²¹McGill University Research Centre on Complex Traits, McGill University, Montreal, QC H3A 0G1, Canada

²²FOCiS Centre of Excellence in Translational Immunology, McGill University, Montreal, QC H3A 0G1, Canada

²³Department of Immunology and Rheumatology, "J. P. Garrahan" National Hospital of Pediatrics, C1245 CABA Buenos Aires, Argentina

²⁴Proteomics Resource Center, The Rockefeller University, New York, NY 10065, USA

²⁵Umraniye Education and Research Hospital, Department of Pediatric Genetics, 34764 İstanbul, Turkey

²⁶Study Center for Primary Immunodeficiencies, Necker Hospital for Sick Children, AP-HP, 75015 Paris, France

²⁷Paris Cité University, 75006 Paris, France

²⁸Department of Pediatric Pathology, Del Niño Jesus Hospital, San Miguel de Tucuman, T4000 Tucuman, Argentina

²⁹Central Laboratory, Del Niño Jesus Hospital, San Miguel de Tucuman, T4000 Tucuman, Argentina

³⁰Cochin University Hospital, Biological Immunology Unit, AP-HP, 75014 Paris, France

³¹Inserm/CNRS U944/7212, Paris Cité University, 75006 Paris, France

³²Hematology Laboratory, Saint-Louis Hospital, AP-HP, 75010 Paris, France

³³National Reference Center for Bone Marrow Failures, Saint-Louis and Robert Debré Hospitals, 75010 Paris, France

³⁴Rare Disease Genomic Medicine Department, Paris Cité University, Necker Hospital for Sick Children, 75015 Paris, France

³⁵Department of Virology, Paris Cité University, Cochin Hospital, 75014 Paris, France

³⁶Division of Genetic Medicine, Department of Medicine, Vanderbilt Genetics Institute, Vanderbilt University Medical Center, Nashville, TN 37232, USA

³⁷Department of Pathology, Microbiology, and Immunology, Vanderbilt Center for Immunobiology, Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN 37232, USA

³⁸Immunology Laboratory, Necker Hospital for Sick Children, AP-HP, 75015 Paris, France

³⁹Inserm UMR1283, CNRS UMR8199, European Genomic Institute for Diabetes, Lille University, Lille Pasteur Institute, Lille University Hospital, 59000 Lille, France

⁴⁰Neglected Human Genetics Laboratory, Paris Cité University, 75006 Paris, France

⁴¹Pathology Department, Ambroise-Paré Hospital, AP-HP, 92100 Boulogne-Billancourt, France

⁴²Department of Physiology, McGill University, Montreal, QC H3A 0G1, Canada

⁴³Department of Medical Microbiology, University Medical Center Utrecht, Utrecht University, 3584CX Utrecht, the Netherlands

⁴⁴College of Health and Life Sciences, Hamad Bin Khalifa University, Doha, Qatar

⁴⁵Department of Pediatric Pulmonology, Allergology and Neonatology and Biomedical Research in Endstage and Obstructive Lung Disease, German Center for Lung Research, Hannover Medical School, 30625 Hannover, Germany

⁴⁶Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, 30625 Hannover, Germany

⁴⁷Department of Human Genetics, McGill University, Montreal, QC H3A 0G1, Canada

⁴⁸Department of Pediatrics, Necker Hospital for Sick Children, AP-HP, 75015 Paris, France

⁴⁹Howard Hughes Medical Institute, New York, NY 10065, USA

⁵⁰Department of Biochemistry, McGill University, Montreal, QC H3A 0G1, Canada

⁵¹Lead contact

⁵²These authors contributed equally

⁵³These authors contributed equally

⁵⁴These authors contributed equally

⁵⁵These authors contributed equally

⁵⁶These authors contributed equally

*Correspondence: jeremie.rosain@institut reimagine.org (J.R.), casanova@rockefeller.edu (J.-L.C.), jacinta.bustamante@inserm.fr (J.B.)
https://doi.org/10.1016/j.cell.2022.12.038

infection in otherwise healthy individuals with apparently normal
resistance to most other infections. Patients with MSMD are
selectively vulnerable to weakly virulent mycobacteria—bacillus
Calmette-Guérin (BCG) vaccines and environmental mycobacte-
ria (EM)—and, in some cases, *Mycobacterium tuberculosis* and

other intramacrophagic microorganisms.⁴⁻⁹ MSMD is typically
"isolated", but can occasionally be "syndromic", if associated
with at least one other key infectious or non-infectious clinical
phenotype.⁴ Mutations of 19 different genes can account for
MSMD: *CYBB*, *IFNG*, *IFNGR1*, *IFNGR2*, *IL12B*, *IL12RB1*,
IL12RB2, IL23R, NEMO, SPPL2A, and TBX21 for isolated MSMD; ISG15, JAK1, RORC, TYK2, USP18, and ZNFX1 for syndromic MSMD; and IRF8, STAT1, and TYK2 for isolated or syndromic MSMD depending on the mutation.<sup>4,6-8,10</sup> Allelic forms at these 19 loci define 35 genetic etiologies of MSMD. Eighteen of the 19 known MSMD-causing genes encode products involved in the production of interferon-γ (IFN-γ) (IFNG, IL12B, IL12RB1, IL12RB2, IL23R, ISG15, RORC, TBX21, and TYK2), cellular responses to IFN-γ (CYBB, JAK1, IFNGR1, IFNGR2, STAT1, and USP18), or both (IRF8, NEMO, and SPPL2A) (Figure S1A). ZNFX1 is the only gene for which the mechanism of MSMD is not yet understood.<sup>6</sup> Thus, MSMD is typically caused by IEI of IFN-γ immunity. These conditions display a high level of genetic and allelic heterogeneity but striking physiological homogeneity.<sup>11</sup>

However, only three etiologies of isolated MSMD are truly Mendelian, i.e. with complete clinical penetrance<sup>1</sup>: autosomal recessive (AR) complete IFN-γ, IFN-γR1, and IFN-γR2 deficiencies (Figure S1B).<sup>4,5,12-15</sup> These three disorders abolish IFN-γ activity. The corresponding patients have early-onset, disseminated, recurrent, multiple, and life-threatening mycobacterial infections.<sup>4,5,12</sup> The other etiologies of isolated MSMD have both incomplete clinical penetrance and less severe clinical features, with a later onset, a narrower range of pathogens, lower rates of recurrence, and better outcomes. Overall, lower penetrance and lesser severity are associated with higher levels of residual IFN-γ activity (Figure S1B). For instance, AR partial IFN-γR1 or IFN-γR2 deficiencies, which impair but do not abolish cellular responses to IFN-γ, are less severe than the complete forms.<sup>12,16</sup> AR complete IL-12Rβ1 and IL-12p40 deficiencies are also milder conditions; they abolish cellular responses to IL-12 and IL-23, reducing IFN-γ production to 1%-10% of normal levels.<sup>17-19</sup> AR complete IL-12Rβ2 and IL-23R deficiencies are even milder and less penetrant, as there is a selective defect of the response to IL-12 or IL-23.<sup>20,21,22</sup> Despite being recessively inherited complete deficiencies, these four conditions are less penetrant and severe than recessive defects of IFN-γ or its receptor.<sup>12,19,20,23</sup> Finally, homozygosity for *TYK2* p.P1104A, which impairs but does not abolish IFN-γ induction in response to IL-23 only, is associated with the lowest known penetrance of MSMD (below 0.5%).<sup>24</sup> Thus, studies of MSMD have revealed that human antimycobacterial immunity is a genetically controlled quantitative trait: the lower the level of IFN-γ activity, the more severe the disease and the higher its penetrance.<sup>16</sup>

Less is known about the cellular basis of human immunity to mycobacteria.¹ Studies of peripheral leukocytes from MSMD patients have suggested that some IFN-γ-producing lymphocyte subsets, either alone or in combination, are essential for antimycobacterial immunity.<sup>8,20,25-27</sup> These essential subsets include the combination of natural killer (NK) cells, γδT cells, and type 1 and type 2 innate lymphoid cells (ILC1 and ILC2) impaired in IL-12Rβ2 deficiency<sup>20</sup>; the invariant NK (iNKT) and mucosal-associated invariant T (MAIT) cells impaired in IL-23R.<sup>20,21,22</sup> RORγ/RORγT,<sup>25</sup> IL-12Rβ1, and TYK2 deficiencies<sup>20,24</sup>; the T<sub>H1</sub>* cells impaired in RORγ/RORγT, IRF8 and SPPL2a deficiencies<sup>25,26</sup>; the γδT cells impaired in RORγ/RORγT<sup>25</sup>; the γδ2<sup>+</sup> T cells impaired in IL-12Rβ1, and IL-23R deficiencies<sup>20,22</sup>; and the combination of NK, iNKT, MAIT, and Vδ2<sup>+</sup> γδT cells impaired in T-bet deficiency.<sup>8</sup>

These studies also suggested that IL-12- and IL-23-producing type 2 dendritic cells (DCs), which are impaired in both AR SPPL2a<sup>26</sup> and autosomal dominant (AD) IRF8 deficiencies,<sup>26,28</sup> are essential for antimycobacterial immunity. Myeloid cells induce IFN-γ in lymphoid cells via at least ISG15,<sup>27</sup> IL-12,<sup>20,24</sup> and IL-23,<sup>20,24</sup> promoting the development of IFN-γ-producing cells, such as T<sub>H1</sub>* cells.<sup>26</sup> They are also activated by IFN-γ, which is commonly considered more of a macrophage-activating factor<sup>29</sup> than an antiviral IFN. IFN-γ controls mycobacterial growth within macrophages via JAK1, STAT1, and the NAPDH oxidase complex, as revealed by studies of MSMD-causing JAK1, STAT1, and CYBB mutations<sup>9,30-35</sup>. We investigated the cellular and molecular basis of human immunity to mycobacteria further, by studying two unrelated children with a severe and unexplained form of isolated MSMD, combining diseases due to both BCG and *Mycobacterium avium* in early childhood.

## RESULTS

### Two unrelated children with severe mycobacterial diseases

We studied two unrelated children, P1 and P2. Both of them displayed severe forms of MSMD, with not only BCG disease, but also *M. avium* complex disease at a very young age, with four episodes of mycobacterial disease in P1, and two episodes in P2, before the age of six years in both cases (Figures 1A–1E). Phenotypes of this severity are seen almost exclusively in patients with recessive complete defects of IFN-γ, either of the receptor chains or STAT1. Both P1 and P2 had no history of unusually severe viral illness and the only other infection observed, histoplasmosis, is caused by an intramacrophagic fungus that has already been reported in other patients with MSMD<sup>4–6,8</sup> (see Case reports for P1 and P2). We performed whole-exome sequencing (WES) on the two patients. The ethnicity of the two patients was confirmed by principal component analysis (PCA) on the WES data<sup>36</sup> (Figure S1C). The homozygosity rates for P1 and P2 were 2.74% and 4%, respectively, suggesting that the parents were probably first- or second-degree cousins.<sup>37</sup> The parents being healthy, we tested the hypothesis of an AR disorder and considered homozygous variants on autosomes. The prevalence of MSMD is about 10<sup>-5</sup>. We therefore considered variants with a minor allele frequency (MAF) below 0.003 in gnomAD v2.1.1. We then selected non-synonymous and essential splice-sites variants predicted to have a combined annotation depletion-dependent (CADD) score above the 99% mutation significance cut-off (MSC),<sup>38</sup> for genes with a gene damage index (GDI) below the cutoff of 13.36 for inborn errors with AR inheritance<sup>39</sup> (Figure 1F). We also filtered out false-positive rare variants, which were absent or rare in public databases but had a frequency above 1% in our in-house cohort (the “blacklist”).<sup>40</sup> We found no homozygous candidate copy number variants (CNVs) in known MSMD genes.<sup>41</sup> We identified 26 rare homozygous single-nucleotide variants (SNVs) in 25 genes in P1, and 17 homozygous SNVs in 17 genes in P2 (Figure 1F and Table S1). In test of the hypothesis of genetic homogeneity, we found only one common gene: *IRF1*. Both patients displayed homozygous transitions predicted to be nonsense in the *IRF1* canonical transcript (NM_002198.2,<sup>42</sup> GRCh37):
A

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

B

<figure><img src="image_7.png" /></figure>

C

<figure><img src="image_8.png" /></figure>

D

<figure><img src="image_9.png" /></figure>

E

<figure><img src="image_10.png" /></figure>

F

<figure><img src="image_11.png" /></figure>

J

<figure><img src="image_12.png" /></figure>

G

<figure><img src="image_13.png" /></figure>

H

<figure><img src="image_14.png" /></figure>

I

<figure><img src="image_15.png" /></figure>

K

<figure><img src="image_16.png" /></figure>

L

<figure><img src="image_17.png" /></figure>

(legend on next page)
c.385C>T (p.R129*) in P1, and c.103C>T (p.Q35*) in P2 (Figure 1G). Sanger sequencing confirmed that P1 was homozygous for p.R129* and P2 was homozygous for p.Q35*, whereas asymptomatic relatives were heterozygous (Figures 1A and 1H). These findings suggest that MSMD in P1 and P2 resulted from homozygosity for these rare nonsense IRF1 variants.

## The overexpressed IRF1 p.Q35* and p.R129* mutants are loss-of-function

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 mycobacteria43–57 (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,⁵⁸ an intermediate segment containing a putative nuclear localization sequence (NLS), and a C-terminal IRF association domain 2 (IAD2), which is crucial for transcriptional activity59–62 (Figure 1G). Both the c.385C>T and c.103C>T variants are predicted to be loss-of-function (pLOF) and are rare or private in public databases (Figures 1E–1I and supplemental information). We studied the impact of the *IRF1* variants by transiently transfecting human embryonic kidney (HEK)293 T cells with plasmids encoding wild-type (WT) *IRF1* (NM_002198.2), mutants p.R129* and p.Q35*, or p.A67P cDNAs. We used two previously described LOF mutants isolated from human tumors as negative controls: (1) the missense mutant p.W11R⁶³ and (2) a frameshift mutant resulting in the deletion of exons 7 and 8⁶⁴ (hereafter referred to as Δ7-8). The proteins encoded by the constructs were left untagged or were tagged with DDK at the C terminus (Figure S1E). Immunoblotting of cell extracts showed that the WT-DDK, p.W11R-DDK, and p.A67P-DDK proteins were produced at a molecular weight (MW) slightly above 50 kDa, as expected, whereas the p.R129-DDK and the Δ7-8-DDK proteins had a lower MW (Figure 1J). These results indicate that the p.R129-DDK cDNA encodes a truncated protein. Both p.R129-DDK and Δ7-8-DDK gave bands of higher intensity than the WT protein, consistent

with an absence of the C-terminal degradation domain.⁶⁵ Immunoblotting with a monoclonal antibody (mAb) directed against the C terminus of IRF1 showed no re-initiation of translation with the p.R129* cDNA (Figure 1J). However, immunoblotting of the p.Q35* protein showed this protein to have a slightly lower MW than the WT protein when probed with either the anti-IRF1 or anti-DDK mAb, suggesting that a re-initiation of translation had occurred. Two ATG codons downstream of p.Q35 and upstream of p.R129 (p.M85 and p.M111) were predicted to be potential translation re-initiation sites in analyses in silico. We mutated the corresponding methionine (ATG) codon to an alanine (GCG) codon. With the WT-DDK cDNA template, the mutation of p.M85 (WT/M85A-DDK), p.M111 (WT/M111A-DDK), or both (WT/M85A/M111A-DDK) had no effect on the MW of the protein produced (Figure 1J). Conversely, with the p.Q35* cDNA template, the mutation of p.M85 (p.Q35*/M85A-DDK) abolished protein detection, whereas the mutation of p.M111 (p.Q35*/M111A-DDK) did not. The mutation of both methionine (p.Q35*/M85A/M111A-DDK) residues abolished protein production (Figure 1J). Consistently, the deletion of all amino acids upstream of M85A (p.M1_A84del) resulted in the production of a protein of the same MW as p.Q35* (Figure 1J). Overall, these results suggest that translation is re-initiated for the p.Q35* variant, resulting in the production of a protein lacking the first 84 amino-terminal amino acids of the DBD. Both the mutant proteins from the patients localized to the nucleus (Figure S1G). An electrophoretic mobility shift assay (EMSA) with an ISRE probe found that the p.Q35* protein, like the previously described p.W11R mutant protein,⁶³ did not bind DNA, whereas the p.R129-DDK mutant which retained the DBD was able to bind DNA, resulting in a band of higher mobility than was observed for the WT, consistent with the lower MW of the mutant protein (Figures 1K and S1H). We then assessed the transcriptional activity of IRF1 in a dual luciferase assay with two different plasmids containing two different ISRE repeats. The p.A67P-DDK cDNA induced luciferase to WT levels, whereas the p.R129* and p.Q35* mutants and two negative controls (p.W11R-DDK⁶³ and Δ7-8⁶⁴) did not (Figures 1L and S1I). Overall, these results suggest that the p.R129* (P1)

<caption>**Figure 1. Homozygous complete loss-of-function IRF1 variant in patients with severe MSMD**</caption>

(A) Pedigrees of the two consanguineous kindreds. M = mutated; WT = wild-type.

(B) Chest CT scan (P1) showing pulmonary infection.

(C) Hematoxylin and eosin staining of a lymph node (P1) during *M. avium* and *H. capsulatum* infections, showing a multinucleated giant cell.

(D) Chest X-ray for P2 showing pulmonary *M. avium* infection.

(E) Hematoxylin-eosin-saffron stain of a lung biopsy (P2) showing a giant cell engulfing another cell by phagocytosis (arrow).

(F) WES analysis of P1 and P2.

(G) IRF1 protein with the DNA-binding domain (DBD), nuclear localization sequence (NLS), and IRF-associated domain type 2 in blue (IAD2). Ho = homodimerization domain; He = heterodimerization domain.

(H) Electropherograms of representative IRF1 nucleotide sequences.

(I) CADD score vs. minor allele frequency (MAF) for the heterozygous state in gnomAD for variants *IRF1* (left), and (right) for a homozygous variant from BRAVO/TOPmed (p.A67P), and the variants present in patients. Missense variants are indicated by blue circles and predicted loss-of-function variants are indicated by red triangles.

(J) Western blot of total lysate from HEK293T cells with and without transfection with various C-DDK-tagged *IRF1* cDNAs or with empty vector (EV). NT = not transfected. Representative data from two independent experiments.

(K) EMSA on nuclear extract of HEK293T cells transfected with EV, WT, or mutant *IRF1* cDNAs, incubated with an ISRE probe. Representative data from two independent experiments.

(L) Dual luciferase ISRE3 reporter activity of HEK293T cells transfected with EV or mutant *IRF1* cDNAs. Data from 3 to 7 independent experiments performed in triplicate. Bars represent the mean and SD.

Statistical analysis by Student's t test. ns = not significant, p > 0.05; ****p < 0.0001.

See also Figure S1.
<figure><img src="image_18.png" /><figcaption>**Figure 2.** IRF1 mRNA and protein levels in cells from the two patients</figcaption></figure>

(A–J) qPCR for IRF1 normalized against GUSB and the mean value for controls (CTLs) for cDNA from (A) primary fibroblasts, (B) SV40-fibroblasts, (C) EBV-B cells, (D) iPSC-derived macrophages (iPSC-MΦ cells), (E) HSV-T cells, (F) monocytes-derived macrophages (MDMs), and (G) T cell blasts. Bars represent the mean and SD. Western blot for indicated protein in total lysate from (H) SV40-fibroblasts, (I) EBV-B cells, or (J) iPSC MΦ, with and without IFN-γ stimulation. Data from 2 to 3 independent experiments are shown.

(K) IRF1 staining and intracellular flow cytometry on SV40-fibroblasts with and without IFN-γ stimulation.

(L) Flow cytometry with intracellular IRF1 staining on SV40-fibroblasts retrotransduced with an empty vector (EV) or WT IRF1 cDNA. The data shown are representative of 2–3 independent experiments. Statistical analysis by Mann-Whitney tests. ns = not significant, *p* > 0.05; \*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.0001. See also Figure S2.

and p.Q35* (P2) variants impair IRF1 production and abolish its transcriptional activity.

## IRF1 protein expression is impaired in cells of the patients

IRF1 is produced ubiquitously in humans,⁶⁶ but its levels are highest in hematopoietic cells.⁶⁶,⁶⁷ We used non-hematopoietic (primary fibroblasts, and simian virus 40 [SV40]-immortalized fibroblasts) and hematopoietic (Epstein-Barr virus-immortalized B lymphocytes [EBV-B cells]), T cell blasts, *Herpesvirus saimiri*-transformed T (HVS-T) cells, monocyte-derived macrophages (MDMs), and tissue macrophages derived from an induced pluripotent stem cell (iPSC) line⁶⁸,⁶⁹ (iPSC-MΦ) to assess the impact of the p.R129* and p.Q35* IRF1 variants on endogenous IRF1 mRNA and protein levels. We performed quantitative RT-PCR (qRT-PCR) with two sets of probes and found much lower levels of IRF1 mRNA in the cells of P1 than in the corresponding cells

from healthy controls, primary fibroblasts (6-fold lower; Figure 2A), SV40-fibroblasts (10-fold lower; Figure 2B), EBV-B cells (3-fold lower; Figure 2C), iPSC-MΦ (>10-fold lower; Figure 2D), and HVS-T cells (4-fold lower; Figure 2E), suggesting that the IRF1 transcript underwent nonsense-mediated mRNA decay in the cells of P1. Conversely, IRF1 mRNA levels in the cells of P2 (primary fibroblasts, SV40-fibroblasts, EBV-B cells, MDMs, and T cell blasts) were only slightly lower than those in control cells (Figures 2A–2C, 2F, and 2G). We then assessed IRF1 protein levels by immunoblotting and flow cytometry, with a mAb specific for the C terminus of IRF1. Primary and SV40-fibroblasts, EBV-B cells, HVS-T cells, and iPSC-MΦ from P1 contained no detectable endogenous IRF1 protein (Figures 2H–2L and S2A–S2F). In addition, no truncated IRF1 protein was detected in SV40-fibroblasts from P1 after pretreatment with IFN-γ, even with a polyclonal antibody and prolonged exposure of the immunoblot (Figure S2F). The protein was barely detectable in primary and
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SV40-fibroblasts from P2 following pretreatment with IFN-γ (Figures 2H, 2K, and S2B), either by flow cytometry or after prolonged exposure of the immunoblot. The protein detected in the cells of P2 had a MW corresponding to that expected following a re-initiation of translation. Other transcription factors, such as IRF3, IRF8, IRF9, and STAT1, were produced by the cells of P1 and P2 (Figure 2H–2J and S2A–S2F). The impaired basal levels of IRF1 protein production in the SV40-fibroblasts from both patients were corrected by stable transduction with the WT IRF1 cDNA (Figure 2L). Together, these findings suggest that homozygosity for the IRF1 p.R129* variant results in nonsense-mediated decay of the IRF1 transcript and a lack of detectable protein, whereas homozygosity for the IRF1 p.Q35* variant leads to a partial escape of mRNA nonsense-mediated decay, due to a re-initiation of translation, with low levels of production of an N-terminally truncated protein. P1 and P2 thus display an absence of IRF1 protein (P1) or the production of an abnormal IRF1 protein (P2) in hematopoietic and non-hematopoietic cells. These results further suggest that both patients have AR complete IRF1 deficiency.

### IRF1 deficiency alters the development of some innate or innate-like leukocyte subsets

We investigated the role of human IRF1 in leukocyte development, by analyzing blood cells from P1 and P2. A complete blood count (CBC) showed that both P1 and P2 had counts of polymorphonuclear neutrophils (PMN), basophils (PMB), eosinophils (PME) and peripheral blood mononuclear cells (PBMCs) that were normal for age (Figures 3A and S3A). Conventional flow cytometry also showed normal counts of monocyte subsets in P2 (Figure 3B). Both patients had low percentages and counts of circulating conventional type 1 dendritic cells (cDC1) (>10-fold decrease) and cDC2 (~5-fold decrease), whereas the counts and percentages of plasmacytoid DCs (pDCs) were at the lower end of the normal range for both patients (Figures 3C, S3B, and S3C). NK cell counts in both patients were 80% lower than those in age-matched controls. The NK cytopenia was explained by a depletion of both CD56dim and NK CD56bright NK cells (Figures 3A, 3C, and S3B). The innate lymphoid cell compartment was also affected, with a decrease in the percentages of innate lymphoid cell precursors (ILCP) (~10-fold) and ILC2 (~5-fold) in both patients (Figures 3C and S3D). The frequencies and counts of iNKT cells were normal in both patients, whereas the percentage of MAIT cells was normal in P1 and slightly low in

P2 (Figures 3C and S3B). Counts of γδT cells were slightly lower in P2 than in age-matched controls (Figure 3C). The frequencies of Vδ1⁺ and Vδ2⁺ T cells as a proportion of total T cells were normal and low, respectively, in both patients (Figure S3B).

### IRF1 deficiency alters the development of certain adaptive leukocyte subsets

Both patients also had ~95% fewer naive CD8⁺ T cells than controls, and an excess of memory and T EMRA cells (Figure 3C and Table S3), consistent with the homeostatic expansion of CD8⁺ T cells following exposure to infectious agents. CD4⁺ T cell counts were normal in both patients, but the numbers of recent thymic emigrant CD4⁺ T cells were only a third those in controls (Figures 3A and 3C and Table S3). Counts and percentages of T_H1, T_H2, T_H17, T_H1*, and Treg cells were also normal in both patients, whereas TF_H cell counts were high in P2 (Figures 3C and S3B). B cell counts were in the normal range in both patients, with normal frequencies of transitional B cells, memory B cells, and plasmablast cells (Figures 3A, 3C, and S3B, and Table S3). The percentage of IgA⁺ memory B cells, was, however, higher than that in healthy controls, consistent with the abnormally high levels of plasma IgA detected in P1 (Figure S3B). However, both plasma IgA levels and IgA⁺ cell proportion were normal in P2. Both P1 and P2 had detectable antibodies against protein antigens and selected pneumococcal polysaccharide antigens in the serum, and normal serum levels of total IgG, and IgM (Table S4). Both patients displayed impaired development of myeloid dendritic cells (cDC1 and, to a much lesser extent, cDC2), innate lymphoid cells (ILC2 and ILCP), and IFN-γ-producing lymphoid cells (NK cells, naive CD8⁺ T cells and, to a much lesser extent, naive CD4⁺ T cells). Impaired development of cDC1, ILC2, NK, and CD8⁺ T cells were confirmed in IRF1-knockout (KO) mice (see supplemental information, Table S5, and Figures S3E and S3F).

### Impairment of the lymphoid cell transcriptomes of IRF1-deficient patients

We analyzed the leukocyte development by performing single-cell RNA-seq and/or cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) on cryopreserved PBMCs. Integrated sample clustering of the various immune subsets identified 13 different major lymphoid subsets: mature (CD56dim) and immature (CD56bright) NK cells, naive CD4⁺ and CD8⁺ T cells,

<caption>**Figure 3.** Phenotyping of peripheral blood leukocytes and single-cell PBMCs from IRF1-deficient patients</caption>

(A) Monitoring of lymphoid cell numbers in the whole blood or cryopreserved PBMCs of patients.
(B) Counts for monocyte subsets by mass cytometry on fresh whole-blood cells. Bars represent the mean and SEM.
(C) Counts of myeloid and lymphoid subsets in fresh whole blood. Bars represent the mean and SEM.
(D) UMAP clustering for controls (CTLs) and samples from the IRF1-deficient patients (2x P1 and 1x P2) profiled by scRNA-seq or CITE-seq. General lineage populations are annotated.
(E) Subclustering of T and NK cells to define cell subtypes. Overlay of 10,000 PBMCs from CTLs (gray) and 10,000 PBMCs from an IRF1-deficient patient (red).
(F) As in (E), B cell subclustering and cell-type annotation, together with an overlay of the patients' and CTLs cells.
(G) Proportions of each cell type expressed as a percentage of total lymphocytes. P1.1 corresponds to the first scRNA-seq analysis for P1 and P1.2 corresponds to the CITE-seq performed later on. Bars represent the mean and SEM.
(H) Module score analysis comparing the expression of genes with IRF1-binding sites within 10 kb of their transcription start site (top panel), and genes differentially expressed between patients and controls and predicted to have ISRE motifs in their promoters (bottom panel). Cohen's *d* effect size estimates are shown for every significant variation of module expression. They were obtained by comparing the module score distribution in every cell subset in Wilcoxon signed-rank tests (*q* value ≤ 0.05).
See also Figure S3 and Table S6.
activated CD4⁺ T cells, central memory T cells, effector memory T cells, cytotoxic T cells, MAIT and NKT cells, Treg, and naive and memory B cells (Figures 3D–3F). Consistent with the results of mass and flow cytometry, quantification of these subsets revealed low percentages of CD56dim NK cells, naive CD4⁺ and CD8⁺ T cells, and an expansion of the population of effector memory T cells (Figures 3E–3G). P1 also had an abnormally large fraction of IgA⁺ B cells, this feature being most pronounced at the age of three years (Figure 3G and Table S6). We performed differential gene expression analysis for each lymphoid subset to detect changes in the transcriptional profiles of the cells of both patients. Interestingly, some genes were found to be differentially expressed in specific cells, but a sizable proportion of genes were differentially expressed in multiple subsets (Table S6). Gene ontology and pathway enrichment analysis indicated lower levels of expression for genes associated with terms such as leukocyte activation, defense response, cytokine signaling, cytotoxicity, and response to IFN in cells from the patients (Table S6). Moreover, an enrichment in genes with ISRE motifs in their promoters was observed for the genes underexpressed in patients, but not for those overexpressed in patients. We investigated the possible dysregulation of ISRE-containing gene expression by performing a module score analysis in which we compared the expression of sets of genes between control and patient cells and calculated effect size. We began the analysis by analyzing chromatin immunoprecipitation sequencing (ChIP-seq) on IFN-γ-stimulated mouse bone marrow-derived macrophages for the list of human orthologous genes binding IRF1. We found that IRF1-binding genes were significantly underexpressed in all subsets, this underexpression being strongest in memory and cytotoxic T cells and NK cells (Figure 3H). Underexpression was also more pronounced for differentially expressed genes with ISRE motifs in their promoters. However, IFNG expression was normal across lymphoid subsets (Table S6). Overall, these results suggest that IRF1 deficiency impairs the development of T and NK cells and the expression of target genes involved in immune activation.

## Mild impairment of IFN-γ production in IRF1-deficient lymphoid cells

Given the impaired development of IFN-γ-producing lymphocytes and antigen-presenting DCs, we hypothesized that impaired IFN-γ production underlies mycobacterial disease in both these patients. We first investigated the IFN-γ secretion pathway in cell lines derived from patient cells. ISG15, the secretion of which is essential for IFN-γ production by NK cells,²⁷ was induced to a similar extent in the SV40-fibroblasts of both patients and those of healthy controls (Figure S4A). The early response to IL-12—an inducer of IFN-γ production—which can be assessed by evaluating STAT4 phosphorylation, was also similar in the HVS-T cells of P1 and controls (Figure S4B). Moreover, IFN-γ production by HVS-T cells following stimulation with CD3/CD2/CD28 beads or PMA-ionomycin was similar for cells from P1 and controls (Figure S4C). We then studied the response to BCG of whole-blood samples from patients. IFN-γ was secreted by the peripheral leukocytes of both patients in response to BCG alone or BCG plus IL-12, and its levels in whole blood were similar to those of healthy local and travel controls

(Figure 4A). Consistent with the results for whole blood, the stimulation of PBMCs from both patients with BCG in the presence or absence of IL-12 or IL-23 resulted in normal total intracellular IFN-γ production (Figure 4B). The clustering of IFN-γ-producing cells indicated that most of these cells were CD4⁺ and CD8⁺ T cells. However, the Vδ2⁺ cell and NK cell subsets of innate or innate-like adaptive lymphoid cells also displayed low levels of IFN-γ production in both patients (Figures 4C and S4D) consistent with their reduced circulating number. We then analyzed the *in vitro* differentiation of naive CD4⁺ T cells from P1 after initial expansion with anti-CD2/CD3/CD28 mAb-coated beads and IL-2, followed by culture under T~H~0, T~H~1, T~H~2, T~H~9, or T~H~17 polarizing conditions. The production of IL-2 under T~H~0-polarizing conditions and the production of IFN-γ and TNF under T~H~1-polarizing conditions were strongly impaired in the IRF1-deficient naive CD4⁺ T cells of P1 (Figures 4D and S4E). As a control, the production of IL-5 and IL-13 by IRF1-deficient naive CD4⁺ T cells under T~H~2-polarizing conditions was normal (Figures 4D and S4E). Overall, IFN-γ production was impaired in NK and Vδ2⁺ cells, mostly due to their quantitative defects, and in T~H~1 cells upon non-mycobacterial stimulation, but the levels of production for this cytokine remained normal in CD4⁺ T cells, CD8⁺ T cells, and whole blood cells stimulated with BCG.

## Impaired response to IFN-γ of IRF1-deficient fibroblasts

IRF1 is a protein found predominantly in the nucleus in the resting state.⁷⁰,⁷¹ The levels of production of this protein regulate its activity.⁷²⁻⁷⁵ IRF1 can bind as a monomer, homodimer, or heterodimer to ISRE motifs in target genes, inducing their expression, as shown in human³¹,⁷²,⁷⁶,⁷⁷ and mouse cells.⁷³ IFN-γ is the strongest known stimulus increasing IRF1 protein levels, through the direct induction of IRF1 mRNA synthesis³¹,⁵⁹ and translation,⁷²⁻⁷⁵ in a STAT1-dependent manner, within 2 to 3 h.⁷⁶,⁷⁸ We confirmed these findings for mRNA induction, translation, and increases in gene expression following IFN-γ stimulation in SV40-fibroblasts (Figures S5A–S5C). We then investigated whether IRF1 deficiency impaired cellular responses to IFN-γ. As expected, the phosphorylation of STAT1 after 20 min of stimulation with IFN-γ, as determined by flow cytometry, was normal in EBV-B cells and SV40-fibroblasts from both patients (Figures S5D and S5E). We then comprehensively profiled the transcriptional response of the primary fibroblasts of patients after 30 min, 2 h, and 8 h of stimulation with IFN-γ, by RNA sequencing (RNA-seq). Primary fibroblasts from healthy controls displayed differential expression relative to non-stimulated conditions for 7 genes at 30 min, 343 genes at 2 h, and 1,484 genes at 8 h of stimulation, with *IRF1* among the differentially regulated genes at all these time points (Figures S5F and S5G, and Table S7). We found that 20% of these genes were expressed differently between patients and controls after 8 h. However, this percentage was significantly lower at 2 h (13%, p value 0.0012), and after 30 min of stimulation (0%). We also found that, relative to controls, the cells of both patients produced 30% and 40% less RNA for IFN-γ inducible genes at 2 h and 8 h, respectively (Figure S5H). Among genes differentially expressed in cells of both patients, an enrichment in the expression of genes with ISRE motifs in their proximal promoters was observed at both 2 and 8 h (Figure 5A), but it was stronger at the 8-h time point (p value 1e-42) than at 2 h (p value 1e-15) (Figure 5A). The impaired induction of
<figure><img src="image_28.png" /><figcaption>B Adult CTLs ▪ Age CTLs ▲ P1 ▼ P2</figcaption></figure>

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<figure><img src="image_44.png" /><figcaption>Figure 4. Production of IFN-γ by the lymphoid cells of IRF1-deficient patients</figcaption></figure>

(A) Induction of IFN-γ secretion in a whole-blood assay, for controls (CTLs), IL-12Rβ1-deficient patients, and patients. Bars represent the mean.

(B) Intracellular flow cytometry on IFN-γ⁺ PBMCs after stimulation with IL-12, IL-23, or BCG. Bars represent the mean. Technical duplicates of the same experiment are shown for P1 and P2.

(C) UMAP analysis of intracellular T-bet and IFN-γ expression by intracellular spectral flow cytometry across various PBMC subsets. Lymphoid subsets based on surface marker expression (upper panel), and their levels of IFN-γ and T-bet expression (lower panel).

(D) Cytokine levels in the supernatant of naive CD4⁺ T cells in polarizing conditions. Bars represent the median.
See also Figure S4.
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<figure><img src="image_46.png" /><figcaption>**Figure 5.** Response to IFN-γ of IRF1-deficient fibroblasts</figcaption></figure>

(A) HOMER de novo motif analysis of the genes differentially expressed in the primary fibroblasts after IFN-γ stimulation.

(B) qRT-PCR for *GBP4* (normalized against *GUSB*) in SV40-fibroblasts with or without retrotransduction with EV or WT *IRF1* cDNA and with or without stimulation IFN-γ. Data from 2 to 6 independent experiments are shown. Bars represent the mean and SD.

(C) Immunoblots in SV40-fibroblasts with and without stimulation with IFN-γ.

(D) Mass spectrometry on lysates of primary fibroblasts with and without IFN-γ stimulation. On the right, heatmaps for proteins (1) positively induced after stimulation with a log₂FC > 1 over the mean in the non-stimulated state for controls (2) and a log₂FC < 0.5 over the mean in the non-stimulated state for patients. On the left, 10th–90th percentiles for all proteins positively induced after stimulation with a log₂FC > 1 over the mean value in the non-stimulated state for controls. See also Figure S5 and Table S7.

GBP4 transcription in response to IFN-γ was confirmed in SV40-fibroblasts from both patients (Figure 5B) and was rescued by stable transduction with a retroviral vector overexpressing WT *IRF1* (Figure 5B). The amounts of protein generated for four IRF1 target genes—as demonstrated by RNA-seq (APOL3, GBP1, RARRES3, and CD274), immunoblotting or flow cytometry of SV40-fibroblasts or primary fibroblasts from patients—were also lower than those for control cells (Figures 5C and S5I–S5K). The impaired induction of CD274, the ligand of PD1,⁷⁹ was corrected in patients’ SV40-fibroblasts by transduction of WT *IRF1* cDNA (Figures S5J and S5K). We also confirmed the impaired induction of several proteins, including GBP1, GBP4, and APOL3, in the primary fibroblasts of both patients, after 24 h of stimulation with IFN-γ, as shown by mass spectrometry (Figure 5D). The mass of inducible protein was found to be 30% smaller in the patients’ cells (Figure 5D). These results suggest that IRF1 controls the enhancement of the second wave of response to IFN-γ, downstream of STAT1.

## Impaired response to IFN-γ in IRF1-deficient myeloid cells

We then studied cellular responses to IFN-γ in leukocytes from patients. IL-12p40 secretion into whole blood in response to BCG or BCG plus IFN-γ was in the control range for both patients

(Figure S6A). By contrast, IL-12p70 induction in response to IFN-γ was impaired in both patients (Figure S6B). Impaired IL-12p70 induction probably contributed to the disseminated histoplasmosis observed in P1, as such infections are frequently reported in patients with complete IL-12Rβ1 or IL-12p40 deficiencies.¹²,¹⁹,²³ Patients with such deficiencies are also prone to isolated episodes of mycobacterial disease, but they almost never present recurrent episodes, contrasting with observations for P1 and P2. Impaired IL-12p70 production is, therefore, unlikely to explain the recurrence of mycobacterial disease in these two patients. Mononuclear myeloid cells are the best studied and classically defined effectors of IFN-γ-induced immunity.²⁹ They are also the only cells in which mycobacteria can replicate.⁸⁰ We therefore comprehensively profiled the transcriptome of mononuclear myeloid cells from patients after IFN-γ stimulation, by RNA-seq on iPSC-MΦ (P1) and MDMs (P2). After 8 h of stimulation, 73 genes were found to display differential expression in the myeloid cells of both patients relative to controls, with all but one of these genes downregulated in the patients (Figures 6A, S6C, S6D, and Table S7). The expression of known MSMD genes was normal, except for *IL12RB1* and *ZNFX1*, which displayed mild downregulation (~4-fold). Conversely, the genes strongly expressed in controls but with
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much lower levels of expression (10- to 500-fold) in both patients included several genes known to be involved in cell-autonomous defense against intracellular pathogens, such as members of the GBP family<sup>81,82</sup> (chr1p22), members of the APOL family (chr22q12, including APOL3<sup>83</sup>), IDO1,<sup>84</sup> and RARRES3<sup>70,85</sup> (Figure 6A). The genes differentially expressed in the mononuclear myeloid cells of both patients were significantly enriched in (1) genes with an ISRE motif in their proximal promoter (Figure 6B) and (2) loci to which IRF1 is known to bind, as detected by ChIP-seq in resting<sup>86</sup> and IFN-γ-activated myeloid cells<sup>73</sup> (Figure S6E). We confirmed the impairment of GBP4 and APOL3 induction by IFN-γ, using qRT-PCR on iPSC-MΦ from P1 and MDMs from P2 (Figure S6F). The impairment of APOL3, GBP1, IDO1, and RARRES3 protein production in response to IFN-γ stimulation was confirmed by western blotting on iPSC-MΦ from P1 and MDMs from P2 (Figure 6C). ZNFX1 expression was normal (Figure 6C). The oxidative burst in response to stimulation with IFN-γ and/or PMA was reduced in MDMs from P2 (Figure 6D). We then analyzed IFN-γ-dependent immunity to intramacrophagic pathogens in IRF1-deficient myeloid cells. The pretreatment with IFN-γ of WT THP1 cells reduced the intracellular growth of Salmonella Typhimurium (Figures 6E, 6F, and S6G) and Mycobacterium abscessus relative to non-stimulated conditions (Figure 6G). By contrast, IFN-γ had no effect in THP1 IRF1-KO cells<sup>87</sup> or in THP1 IFN-γR1<sup>KO</sup> and STAT1<sup>KO</sup> cells (Figures 6F, 6G, and S6G). Similar findings were obtained for the MDMs of P2 for M. abscessus infection (Figures 6H and S6H). Together, these results suggest that human IRF1 governs IFN-γ-dependent macrophage activation and resistance to intracellular pathogens.

## Mildly impaired responses to IFN-α/β in IRF1-deficient cells

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> Neither P1 nor P2 presented life-threatening viral infections (Case reports for P1 and P2 and *supplemental information*). Phage immunoprecipitation-sequencing (PhIP-Seq) confirmed that both patients has been exposed to multiple DNA and RNA viruses (Figure 7A

and Table S4). Stimulation with IFN-α/β induces IRF1, albeit at a level lower than observed after stimulation with IFN-γ.<sup>59,74–76,78</sup> We confirmed these findings in human cells and also found, surprisingly, that IFN-α/β-dependent IRF1 induction was ISGF3-independent (i.e., STAT2 and IRF9-independent) but GAS-dependent (i.e., STAT1-dependent) (Figures S7A–S7C). We analyzed antiviral IFN-α/β immunity in the patients’ cells by flow cytometry, RNA-seq, and in viral growth assays. The early response to IFN-α/β in SV40-fibroblasts and EBV-B cells, assessed by evaluating STAT1 phosphorylation after 20 min of stimulation with IFN-α2b and IFN-β, was normal in both patients (Figure S7D). We then studied the transcriptomic response of primary fibroblasts stimulated with IFN-α2b for 30 min, 2 h, or 8 h. We compared these responses to those of the cells of patients with complete deficiencies of IRF9, STAT1, or IFNAR1. Primary fibroblasts from healthy controls displayed differential expression relative to unstimulated conditions for 110 genes, 667 genes, and 1,093 genes for these three time points, respectively, with IRF1 significantly upregulated at all these time points (Figures 7B and 7C, and Table S7). The impaired induction of various interferon-stimulated genes ([ISGs]; for 2%, 10%, and 8% of the genes induced in controls) was observed in the patients’ cells at these three time points. Quantitatively, 81% and 71% of the mRNA levels of IFN-α inducible genes in controls were correctly induced in both patients, at 2 and 8 h, respectively (Figure 7D). The corresponding percentages were 24% and 54% in IRF9-deficient cells, and 8% and 23% in STAT1-deficient cells, for the same time points. Most of the ISGs dysregulated in IRF1-deficient cells have an ISRE motif in their promoters (Figure 7E). A subset of these ISGs also displayed impaired induction in IRF1-deficient cells but not in IRF9-deficient cells, defining these genes as GAF-IRF1-dependent (Figure 7E and Table S7).

## Normal antiviral activity of IFN-α/β and IFN-γ in IRF1-deficient cells

We assessed the possible impact of these findings on the replication of several viruses in SV40-fibroblasts after pretreatment with IFN-α2b. In these conditions, the cells of the patients controlled the replication of encephalomyelitis virus (EMCV), influenza A virus (IAV), hepatitis A virus (HAV), herpes simplex virus type 1 (HSV-1), human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2), yellow fever live-attenuated viral vaccine (YF17D-venus), SARS-CoV-2, and vesicular stomatitis virus

### Figure 6. IFN-γ immunity in IRF1-deficient macrophages

(A) Heatmaps showing genes differentially expressed (|log₂(FC)| > 1 and adj. p value <0.05) in CTL cells after 8 h of stimulation with 10³ IU/mL IFN-γ, and differentially expressed in iPSC-MΦ (P1) and MDMs (P2) (|log₂(FC)| > 1 and adj. p value <0.05).

(B) HOMER de novo motif analysis of the genes differentially expressed in the iPSC-MΦ (P1), and in MDMs (P2); after IFN-γ stimulation.

(C) Western blot of protein extracts in iPSC-MΦ and MDMs, with and without stimulation with IFN-γ.

(D) Extracellular H₂O₂ release for MDMs from CTLs and P2 (technical duplicates mean ± SD).

(E) HLA-DR expression by flow cytometry, using THP1-Φ with and without IFN-γ stimulation.

(F) Gentamicin protection assay performed on PMA-differentiated THP1-Φ with and without IFN-γ pretreatment and Salmonella Typhimurium-GFP (Stm-GFP) infection. Results are expressed as the proportion of Salmonella Typhimurium<sup>high</sup> on Salmonella Typhi<sup>+</sup>. Representative results from 2 to 3 independent experiments.

(G) CFU assays on PMA-differenciated THP1-Φ with and without IFN-γ pretreatment, following infection by M. abscessus. All replicates from n = 3 independent experiments are displayed. Bars represent the mean and SD.

(H) Flow cytometry analyses on MDMs with and without IFN-γ pretreatment, following infection for 24 h with M. abscessus-tdTomato. Bars represent the mean and SD.

See also Figure S6 and Table S7.
<figure><img src="image_55.png" /></figure>

A

<figure><img src="image_56.png" /></figure>

B

Primary fibroblasts
IFN-α2b 2 hours

<figure><img src="image_57.png" /></figure>

C

Primary fibroblasts
IFN-α2b 8 hours

<figure><img src="image_58.png" /></figure>

E

de novo motif
IFN-α2b 2h
IFN-α2b 2h

<figure><img src="image_59.png" /></figure>

<figure><img src="image_60.png" /></figure>

<figure><img src="image_61.png" /></figure>

<figure><img src="image_62.png" /></figure>

<figure><img src="image_63.png" /></figure>

J

%SARS-CoV-2+ cells
 norm. to untreated

<figure><img src="image_64.png" /></figure>

(legend on next page)
Indiana (VSV) as effectively as control cells, whereas viral replication continued unabated in IFN-αR1<sup>-/-</sup>, IRF9<sup>-/-</sup>, STAT2<sup>-/-</sup> or STAT1<sup>-/-</sup> cells (Figures 7F-7K and S7F-S7I), except for EMCV and VSV, for which greater susceptibility was observed with a low dose of IFN-α (Figures 7G and 7H). We conducted similar experiments with IFN-γ, which was initially described as an antiviral molecule<sup>95-98</sup> before being identified as the macrophage-activating factor.<sup>29</sup> We found that IRF1-deficient cells had a lower susceptibility to infection following IFN-γ pretreatment, like control cells, for all viruses other than IAV, EMCV, and VSV, for which resistance was weaker than in control cells (Figures 7F-7K and S7F-S7I). Collectively, these results suggest that the antiviral response of IRF1-deficient cells to IFN-α and IFN-γ is only mildly impaired and sufficient to protect IRF1-deficient fibroblasts against viral infection, consistent with the absence of severe viral illnesses in these two patients.

DISCUSSION

We report here that AR complete IRF1 deficiency is a genetic etiology of isolated and severe MSMD. The two unrelated IRF1-deficient patients of Latin American and Turkish ancestries described here experienced recurrent early-onset life-threatening mycobacterial diseases due to multiple mycobacteria (BCG, *M. avium*) despite treatment with multiple antimycobacterial drugs, and even treatment with recombinant IFN-γ in the case of P1. Remarkably, IRF1 deficiency was found to be as clinically severe as AR complete deficiencies of IFN-γ, IFN-γR1, or IFN-γR2.<sup>4,5,12</sup> Patients with these deficiencies display adverse reactions to BCG when vaccinated at birth, followed by relapses of BCG disease and/or disseminated disease caused by EM.<sup>4,5,12</sup> Disseminated infection with *M. avium* before six years of age is a hallmark of a lack of IFN-γ immunity due to inherited defects of the IFN-γ response pathway,<sup>4,5,12,35</sup> whereas *M. avium* disease at a later age may be due to profound deficiencies as a result of the production of anti-IFN-γ auto-antibodies,<sup>99,100</sup> or a severe progressive quantitative defect of IFN-γ myeloid target cells caused by GATA2 deficiency.<sup>101,102</sup> Given the severity of the clinical phenotype in these two unrelated patients, the clinical penetrance of inherited IRF1 deficiency for MSMD is probably complete. Consistently, *Irf1*<sup>-/-</sup> mice are susceptible to both BCG<sup>52</sup> and *M. tuberculosis*,<sup>53,54</sup> and other intramacrophagic pathogens.<sup>103-107</sup> Myeloid cells

are classically considered to be the effector cells for IFN-γ,²⁹
and this cytokine is also their most potent activator. ¹⁰⁸ Our find-
ings indicate that IRF1 governs the response to IFN-γ, down-
stream of STAT1, in mononuclear myeloid cells. IRF1 controls
the potent induction of several genes encoding intracellular com-
ponents known to be effectors of resistance to intracellular path-
ogens, such as a protein from the GBP family, ⁸¹⁻⁸³,¹⁰⁴,¹⁰⁹,¹¹⁰
IDO1,⁸⁴ RARRES3/PLAAT4,⁸⁵ and APOL3.⁸³,¹¹¹ Our work ex-
tends the list of known IFN-γ-inducible IRF1-dependent genes
in mice<sup>73</sup> to human genes with no known ortholog in mice,<sup>112</sup
such as RARRES3<sup>85</sup> and APOL3.⁸³,¹¹¹ Inherited deficiencies of
some of these IFN-γ- and IRF1-dependent effector genes may
underlie MSMD or tuberculosis. IRF1 deficiency underlies severe
and isolated MSMD, as in patients with AR IFN-γ, IFN-γR1, or
IFN-γ2 deficiencies<sup>4,5,12</sup> whose mononuclear myeloid cells
cannot respond correctly to IFN-γ.

We also found additional quantitative and qualitative deficiencies of IFN-γ-producing lymphocytes in the two IRF1-deficient patients, consistent with reported findings for *Irf1*<sup>-/-</sup> mice.<sup>50,55,56,105,113-115</sup> The observed deficiencies included an impairment of the development of NK cells and naive αβ CD8<sup>+</sup> T cells, and impaired IFN-γ production by residual NK cells and γδT<sup>+</sup> T cells. However, the leukocytes of both patients produced normal total amounts of IFN-γ upon exposure to mycobacteria *in vitro*, suggesting that the abnormal counts or function of IFN-γ-producing lymphoid cell subsets in peripheral blood made only a marginal contribution to mycobacterial disease. IRF1 deficiency impairs the development of myeloid DCs, predominantly that of cDC1, the counts of which were low in the blood of both IRF1-deficient patients and in the tissues of *Irf1*<sup>-/-</sup> mice. Our results suggest that IRF1 is essential for myeloid cell maturation, through an as yet unknown mechanism, possibly involving interaction with IRF8.<sup>26,28,116,117</sup> Indeed, human IRF8 governs DC development in a gene dosage-dependent manner, as illustrated by AD and AR IRF8 deficiencies.<sup>26,28,116-118</sup> We also documented low levels of IL-12p70 induction in both patients, which is more likely to result from direct binding to the *IL12A* locus, as previously shown in murine myeloid cells,<sup>119,120</sup> rather than a decrease in myeloid cell number. Consistently, patients with AD PU.1 deficiency, who have low circulating myeloid cell counts, have a normal capacity to produce IL-12.<sup>121</sup> Overall, in addition to their profoundly impaired cellular responses to IFN-γ, the patients with IRF1

Figure 7. IFN-α and IFN-γ-driven antiviral immunity in the cells of IRF1-deficient patients

(A) Antiviral antibody responses to species for which at least one sample tested seropositive by PhIP-Seq. "IVIG" correspond to the mean response for samples from pooled patients on IVIGs and "pediatric CTLs" to pediatric controls. A hierarchical clustering of samples based on antiviral antibody levels is shown at the top.

(B and C) Heatmap showing genes differentially expressed (|log2(FC)|> 1 and adj. p value <0.05) in CTL cells after 2 h (B) or 8 h (C) of IFN-α2b stimulation, and differentially expressed in primary fibroblasts from P1 (|log2(FC)|> 1 and adj. p value <0.05) relative to CTLs. Genes differentially expressed in P1 and P2 relative to the control group at 2 and 8 h from among those 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 after Benjamini-Hochberg correction in controls, and with a |log2(FC)|> 1 and adj. p value <0.05 after correction in patients relative to controls.

(D) RNA-sequencing of IFN-α2b-inducible genes (logFC>2 in controls) in primary fibroblasts with and without IFN-α2b stimulation.

(E) HOMER *de novo* motif analysis of genes differentially expressed in primary fibroblasts with IFN-α2b stimulation.

(F–K) Influenza A virus (IAV), (G) encephalomyocarditis virus (EMCV), (H) vesicular stomatitis Indiana virus (VSV), (I) HSV-1, (J) SARS-CoV-2, and (K) HIV-2 infection of SV40-fibroblasts after pretreatment with IFN-α2b or IFN-γ. All viral infections were performed in 2–3 independent experiments. Bars represent the mean and SD.

See also Figure S7 and Table S7.
deficiency have a mild impairment of IFN-γ production, which
may have contributed to their MSMD.

By contrast, neither patient presented any severe viral diseases,
despite low levels of ILCP, NK cells, and naive CD8⁺ T cells. Pa-
tients with severe combined immunodeficiency (SCID) caused
by LOF mutations of *IL2RG* or *JAK3* who undergo hematopoietic
stem cell transplantation do not reconstitute a normal pool of pe-
ripheral ILC and NK cells.<sup>122</sup> They are not prone to viral infections
other than human papillomavirus-driven common and flat warts,
perhaps due to the persistence of deficiencies of keratinocytes
or antigen-presenting cells in the skin.<sup>123</sup> Isolated deficiencies of
ILCs and NK cells in patients with IEIs, such as GINS1, MCM4,
or MCM10 deficiency,<sup>122,124-126</sup> can lead to various degrees of
susceptibility to viral diseases, mostly caused by cytomegalovirus
(CMV). These IEIs are caused by genes with expression profiles
not restricted to NK cells or ILCs, and the defect may be
broader.<sup>122,124-126</sup> Patients with inherited CD8, TAP1, TAP2,
TAPASIN, or β2-microglobulin deficiencies, all of whom have
low levels of HLA-I in all cell types tested and low counts of blood
CD8⁺ T cells, are not prone to viral infections either.<sup>127-133</sup> IEIs im-
pairing CD8⁺ T cell effector or expansion functions can underlie
susceptibility to EBV.<sup>134</sup> P1 had not encountered CMV or EBV,
but P2 had been exposed to CMV and had controlled its replica-
tion and tested positive for EBV by PCR. Overall, the low but non-
zero counts of circulating antiviral lymphoid cell subsets in the two
patients with IRF1 deficiency have so far proved sufficient to
ensure immunity against the many viruses encountered in child-
hood. Innate and adaptive leukocytic immunity to viruses is
evidently affected by complete IRF1 deficiency, albeit with no
apparent clinical consequences, implying that other antiviral
mechanisms can compensate for this relatively broad lymphoid
deficiency.

More surprisingly, cell-intrinsic, IFN-α/β immunity is IRF1-in-
dependent for the nine viruses we have tested *in vitro*. The
contrast between AR STAT1 and IRF1 deficiencies is striking in
this respect, as the two patients with IRF1 deficiency did not suf-
fer from the life-threatening viral diseases seen in patients with
AR complete STAT1 deficiency.<sup>34,35</sup> Despite the impaired induc-
tion of a subset of target genes in fibroblasts stimulated with IFN-
α/β, the patients displayed no severe viral diseases, even upon
infection with SARS-CoV-2, probably the most potent known
sensor of IFN-α/β deficiency.<sup>135-140</sup> Consistently, the cells of
the patients controlled the replication of the nine RNA and
DNA viruses tested *in vitro*, unlike cells from patients with AR
STAT1, STAT2, IRF9, or IFN-αR1 deficiency. All IEIs affecting
the response to IFN-α/β underlying susceptibility to acute viral
diseases (i.e., AR IFN-αR1, IFN-αR2, STAT1, STAT2, or IRF9 de-
ficiencies) are caused by deficiencies leading to a complete
abolition of ISGF3 function in response to stimulation with
IFN-α/β.<sup>140-147</sup> These observations suggest that functional
ISGF3 (composed of STAT1, STAT2, and IRF9) is essential for
IFN-α/β intrinsic immunity, whereas IRF1 (formerly known as
ISGF2<sup>59,148</sup>) is largely redundant for such immunity *in vivo*, as
demonstrated by viral replication levels. We found that human
IRF1 was driven by GAF but not ISGF3 complexes in response
to IFN-α/β, but the GAF/STAT1-dependent induction of IRF1,
and of subsequent target ISGs such as GBP, RARRES3, and
APOL3, was not required for immunity to many viruses. By

contrast, *Irf1*-deficient mice have been reported to be suscepti-
ble to a number of viral infections.<sup>50,51,57,93,94</sup> The lack of IRF1 in
mice may, nevertheless, be partly compensated by other IRFs
for antiviral immunity.<sup>149</sup> IRF1 is not induced after stimulation
with IFN-λ.<sup>76</sup> In addition, humans with a complete deficiency of
IL-10Rβ display a complete lack of response to IFN-λ and are
apparently not susceptible to infectious diseases,<sup>150,151</sup> except
fulminant viral hepatitis A.<sup>152</sup> Overall, the natural course of infec-
tious diseases in the known patients with inherited deficiencies
of IRF1 (this study), IRF3,<sup>153,154</sup> IRF7,<sup>140,142</sup> or IRF9<sup>141,155</sup> sug-
gests that IRF1 is essential for IFN-γ-dependent myeloid antimy-
cobacterial immunity, whereas IRF3, IRF7, and IRF9 are essen-
tial for IFN-α/β-dependent antiviral immunity.

## Limitations of the study

Our molecular, cellular, immunological, and clinical studies of two unrelated patients with inherited IRF1 deficiency do not exclude the possibility of predisposition to unusual and/or severe clinical diseases caused by viruses that have not been tested *in vitro*, or that have not been encountered by the patients *in vivo*, or both, or even such diseases caused by seemingly benign viruses in different infection conditions (e.g., high levels of inoculum, atypical route of infection, infection before vaccination), in these or other IRF1-deficient patients. Any susceptibility to viruses in these patients might be due to impaired IRF1-dependent IFN-α/β immunity, or due to the deficit of cytotoxic NK and CD8+ T cells, or both. In particular, the long-term outcome of infection with viruses capable of latency, such as those of the Herpesviridae, is unknown.

## STAR★METHODS

Detailed methods are provided in the online version of this paper
and include the following:

* KEY RESOURCES TABLE

* RESOURCE AVAILABILITY

* Lead contact

* Materials Availability

* Data and code availability

* EXPERIMENT MODEL AND SUBJECT DETAILS

* METHOD DETAILS

* Case reports for P1 and P2

* Both *IRF1* variants are predicted to be deleterious

* Impaired development of lymphoid and myeloid cells in *Irf1*⁻/⁻ mice

* Lack of severe viral infections in IRF1-deficient patients

* Genetics

* Cell culture and stimulation

* Site-directed mutagenesis, transient and stable transfection

* Western blotting and EMSA

* Confocal microscopy

* Luciferase assay

* RT-qPCR and cDNA

* Flow cytometry on cell lines

* Mass spectrometry on primary fibroblasts
* Deep flow cytometry phenotyping and *ex vivo* naive CD4⁺ T cell polarization experiments

* Immunophenotyping of MAIT, iNKT, and γδ T cells

* ILC immunophenotyping

* Mass cytometry on fresh whole blood and cryopreserved PBMCs

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

* ELISA

* Phage immunoprecipitation-sequencing (PhIP-Seq)

* Viral infection experiments

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

* RNA-seq

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

* Infection of macrophages with bacteria and mycobacteria

* CITE-seq

* Generation of single-cell suspensions from murine blood and tissues for flow cytometry

* Flow cytometry on mouse cells

## QUANTIFICATION AND STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.cell.2022.12.038.

## ACKNOWLEDGMENTS

We thank the patients and their families, all members of the laboratory, Stephen Elledge (for the PhIP-Seq phage display viral library), and Jose Mauricio Ayala, Véronique Cholette, and Rebecca Bellworthy. The Laboratory of Human Genetics of Infectious Diseases is supported in part by grants from Inserm, Paris Cité University, the St. Giles Foundation, The Rockefeller University, the Center for Clinical and Translational Science (UL1TR001866), the National Center for Research Resources and the National Center for Advancing Sciences, the National Institutes of Health (NIH), (R01AI095983, R01AI088364, R01AI163029, and U19AI162568), the National Institute of Allergy and Infectious Diseases, the French National Research Agency (ANR) under the "Investments for the future" program (ANR-10-IAHU-01), the Integrative Biology of Emerging Infectious Diseases Laboratory of Excellence (ANR-10-LABX-62-IBEID), the French Foundation for Medical Research (FRM) (EQU201903007798), the ANRS Nord-Sud (ANRS-COV05), ANRS (ECTZ170784-ANRS0073), GENVIR (ANR-20-CE93-003), GENMSMD (ANR-16-CE17-0005-01), AABIFNCOV (ANR-20-CO11-0001), GenMIS-C (ANR-21-COVR-0039), SUNLIVE (ANR-19-CE15-0012-01), MAFMACRO (ANR-22-CE92-0008) grants, Ecos-NORD (ECOS N°C19S01), the Fisher Center for Alzheimer's Research Foundation, the Meyer Foundation, the JPB Foundation, the European Union's Horizon 2020 research and innovation program (824110, EASI-Genomics), the Square Foundation, Grandir-Fonds de solidarité pour l'Enfance, the Fondation du Souffle, the SCOR Corporate Foundation for Science, the French Ministry of Higher Education, Research, and Innovation (MESRI-COVID-19), and REACTing-INSERM. The Laboratory of Virology and Infectious Disease was supported in part by the NIH (R01AI091707-10 to C.M.R.). J.L.P. was supported by the Francois Wal-lace Monahan Postdoctoral Fellowship at The Rockefeller University and the European Molecular Biology Organization Long-Term Fellowship (ALTF 380-2018). N. Marr was supported by Sidra Medicine and the Qatar National Research Fund (NPRP9-251-3-045). The Yale Center for Mendelian Genomics (UM1HG006504) was funded by the National Human Genome Research Institute, the Yale GSP Coordinating Center (U24 HG008956), and the Yale High-Performance Computing Center (S10OD018521). This research was partly supported by Calcul Québec, Compute Canada Canadian Institutes of Health

Research (CIHR) Project Grant to D. Langlais. (#168959) and a CIHR Foundation Grant (to P.G.). D. Langlais was also supported by an FRQS Chercheur-Boursier Junior 1 Award and the Calgary Foundation for Innovation John R. Evans Leaders Fund. P.G. is supported by a Distinguished James McGill Professorship award from McGill University. S.M.L is supported by the NIH: R01-AI103083 and R01-AI150095. J.R. was supported by *poste d'accueil Inserm*". J.R., P.B., and T.L.V were supported by the MD-PhD program of the Imagine Institute by the Bettencourt Schueller Foundation. N.L received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (852178 grant), German Research Foundation, (DFG) under Germany's Excellence Strategy—EXC 2155—project number 390874280 and REBIRTH "Förderung aus Mitteln des Niedersächsischen Vorab". A.-L.N. was supported by the international PhD program of the Imagine Institute, by the Bettencourt Schueller Foundation and the *fin de thèse* FRM program (FDT202204015102). R.Y. was supported by the Sackler Center for Biomedicine and Nutrition, the Shapiro-Silverberg Fund for the Advancement of Translational Research at the Center for Clinical and Translational Science of the Rockefeller University, and the Research Grant Program from the Immune Deficiency Foundation. D. Lee. was supported by FRMfellowship (FDM202006011282). C.S.M was supported by an Early-Mid Career Research Fellowship from the Department of Health of the New South Wales Government of Australia. S.G.T was supported by an NHMRC Leadership 3 Investigator Grant (1176665) and NHMRC grant (1113904). M.O. was supported by the David Rockefeller Graduate Program, the Funai Foundation for Information Technology, the Honjo International Scholarship Foundation, and the New York Hideo Noguchi Memorial Society. This work was supported by grants from ANRS (ECTZ118797), Sidaction (20-2-AEQ-12822-2), and FRM (EQU202103012774) to N. Manel, and H.I. was supported by fellowships from *Institut Curie*, Seneca Foundation (20941/PD/18), and ANRS (ECTZ171453). A.N.S. was supported in part by the European Union's Horizon 2020 research and innovation program (789645 Marie Sklodowska-Curie grant). Y.-H.C. is supported by an A'STAR International Fellowship. J. Bohlen is an EMBO postdoctoral fellow. We thank the NIH Tetramer Core Facility (NTCF) for providing the 5-OP-RU-loaded MR1 tetramer, which was developed jointly with Dr. James McCluskey, Dr. Jamie Rossjohn, and Dr. David Fairlie.

## AUTHORS CONTRIBUTIONS

J.R., J. Bustamante, and J.-L.C. conceived the study, designed the experiments, interpreted the data, and drafted the manuscript. J.R., J. Bustamante., Y.S., and L.A. analyzed WES. J.R., A.-L.N., R.Y., J.L.P., M.B., W.D., M. Mancini., D. Lee D. Langlais, J.-M.D, A.G., M.O., P.B., M.M.-V., J.E.H., L.L., H.M.I., Z.L., Y.-H.C., A.C., T.L.V., T.S., Y.K.-D., R.B., J. Bohlen, Q.P., J.N.P, S.H., H.M., S.Z., C.S., M. Materna., M.P., M. Migaud., A.M., C.-O.Q, V.B., S.B-D., J.-F.E., and C.A.C, L.P., J.S., M. Roelens, and M.-A.A performed experiments on cells, analyzed them, and generated figures. N.T., O.T., M.B., L.P., M.E.J.P., M.L.G., and J. Bustamante recruited patients. J.M., F. Rapaport, D.R., and P.Z. analyzed RNA-seq experiments. A-S.L. and F. Rozenberg performed serological tests for virology. T.K., F.A.A., M.M.A.A, M. Rahman, and N. Marr performed or analyzed PhIP-Seq. K.H. and N.L. provided iPSC cells. D. Langlais, B.C.M., and J.H.F. performed experiments in mice. R.M.B., M.O., and R.L. analyzed data. M.Migaud, and S.R. provided resources. A.N.S., S.R., J.H.F., L.A., S.B.-D., N. Marr, N. Mancini, M.R.M., N.L., V.B., C.S.M, S.G.T., J.P.D.S., A.N.S., Q.Z., S.Y.Z., J.-F.E., L.K., S.M.L., G.V., C.M.R., J.H.F., D. Langlais, J.-L.C., P.G., and J. Bustamante supervised experiments or analyses. All the authors discussed, revised, and approved the manuscript.

## DECLARATION OF INTERESTS

J.-L.C. serves on the scientific advisory boards of ADMA Biologics Inc., Kymera Therapeutics, and Elixiron Immunotherapeutics.

Received: April 1, 2022

Revised: November 22, 2022

Accepted: December 19, 2022

Published: February 2, 2023
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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Antibodies</td><td></td><td></td></tr><tr><td>Human IRF1 (clone D5E4)</td><td>Cell Signaling</td><td>Cat# 8478, RRID:AB_10949108</td></tr><tr><td>Human IRF1 (rabbit polyclonal)</td><td>Proteintech</td><td>Cat# 11335-1-AP, RRID:AB_2877759</td></tr><tr><td>Human IRF8 (goat polyclonal)</td><td>Santa Cruz</td><td>Cat# sc-6058, RRID:AB_649510</td></tr><tr><td>Human IRF8 (clone D20D8)</td><td>Cell signaling</td><td>Cat# 5628, RRID:AB_10828231</td></tr><tr><td>Human IRF9 (rabbit polyclonal)</td><td>Santa Cruz</td><td>Cat# sc-496, RRID:AB_2127709</td></tr><tr><td>Human IRF9 (rabbit polyclonal)</td><td>Proteintech</td><td>Cat# 14167-1-AP, RRID:AB_2296227</td></tr><tr><td>Human IRF3 (clone D9J5Q)</td><td>Cell Signaling</td><td>Cat# 10949, RRID:AB_2797733</td></tr><tr><td>Human STAT1 (clone 1)</td><td>Beckton-Dickinson</td><td>Cat# 610115, RRID:AB_397521</td></tr><tr><td>Human pSTAT1 (clone 4a)</td><td>Beckton-Dickinson</td><td>Cat# 612232, RRID:AB_399555</td></tr><tr><td>Human STAT2 (clone B-3)</td><td>Santa Cruz</td><td>Cat# sc-514193, RRID:AB_2810271</td></tr><tr><td>Human MX1 (polyclonal)</td><td>ProteinTech</td><td>Cat# 13750-1-AP, RRID:AB_2266768</td></tr><tr><td>Human ISG15 (clone F-9)</td><td>Santa-Cruz</td><td>Cat# sc-166755, RRID:AB_2126308</td></tr><tr><td>Human vinculin (clone EPR8185)</td><td>Abcam</td><td>Cat# ab129002, RRID:AB_11144129</td></tr><tr><td>Human vinculin (clone 7F9)</td><td>Santa Cruz</td><td>Cat# sc-376248-HPR, RRID:AB_10991536</td></tr><tr><td>Anti-mouse IgG (H + L)-HRP-conjugated</td><td>Bio-Rad</td><td>Cat# 170-6516, RRID:AB_11125547</td></tr><tr><td>Anti-rabbit IgG (H + L)-HRP-conjugated</td><td>Bio-Rad</td><td>Cat# 170-6515, RRID:AB_11125142</td></tr><tr><td>Human DDK-tag (clone M2)</td><td>Sigma-Aldrich</td><td>Cat# A8592, RRID:AB_439702</td></tr><tr><td>Human DDK (clone M2)</td><td>Cell Signaling</td><td>Cat# 14793, RRID:AB_2572291</td></tr><tr><td>Human Isotype rabbit</td><td>Cell Signaling</td><td>Cat# 2729, RRID:AB_1031062)</td></tr><tr><td>Human GBP1 (clone 1B1)</td><td>Santa-Cruz</td><td>Cat# sc-53857, RRID:AB_2109333</td></tr><tr><td>Human APOL3 (clone EPR8238)</td><td>Abcam</td><td>Cat# ab154869</td></tr><tr><td>Human RARRES3 (rabbit polyclonal)</td><td>ProteinTech</td><td>Cat# 12065-1-AP, RRID:AB_2175704</td></tr><tr><td>anti-rabbit IgG Alexa Fluor 555</td><td>Thermo Fisher Scientific</td><td>Cat# A-21429, RRID:AB_2535850</td></tr><tr><td>PE-Dazzle-594- Human PD-L1 (CD274) antibody (clone 29E.2A3)</td><td>BioLegend</td><td>Cat# 329732, RRID:AB_2616889</td></tr><tr><td>PE-Dazzle-594-Mouse IgG2b, κ Isotype Ctrl Antibody</td><td>BioLegend</td><td>Cat# 400358</td></tr><tr><td>PE-Human STAT1 (clone 1)</td><td>Beckton-Dickinson</td><td>Cat# 558537, RRID:AB_647231</td></tr><tr><td>PE-Human pSTAT1 (clone 4a)</td><td>Beckton-Dickinson</td><td>Cat# 612564, RRID:AB_399855</td></tr><tr><td>AF647-Human pSTAT1 (clone 4a)</td><td>Beckton-Dickinson</td><td>Cat# 612597, RRID:AB_399880)</td></tr><tr><td>PE Mouse IgG1, κ Isotype Control</td><td>Beckton-Dickinson</td><td>Cat# 554680, RRID:AB_395506</td></tr><tr><td>PE Mouse IgG2a, κ Isotype Control</td><td>Beckton-Dickinson</td><td>Cat# 565363, RRID:AB_2869665</td></tr><tr><td>PE-conjugated goat anti-rabbit</td><td>Thermo Fisher Scientific</td><td>Cat# A10542, RRID:AB_2534042</td></tr><tr><td>Human IFN-γ-BUV737 clone 4S.B3</td><td>BD Horizon</td><td>Cat# 564620, RRID:AB_2869591</td></tr><tr><td>Human TNF-PerCP clone Mab11</td><td>BioLegend</td><td>Cat# 502924, RRID:AB_2561288</td></tr><tr><td>Human IL-9-PE clone MH9A3</td><td>BD Pharmingen</td><td>Cat# 560807, RRID:AB_2033985</td></tr><tr><td>Human IL-13-BV421 clone JES10-5A2</td><td>BD Horizon</td><td>Cat# 563580, RRID:AB_2738290</td></tr><tr><td>Human IL-4-AF488 clone 8D4-8</td><td>BioLegend</td><td>Cat# 500710, RRID:AB_1877131</td></tr><tr><td>Human IL-17A-BV510 clone BL168</td><td>BioLegend</td><td>Cat# 512330, RRID:AB_2562745</td></tr><tr><td>Human IL-17F-BV650 clone O33-782</td><td>BD Horizon</td><td>Cat# 564264, RRID:AB_2869555</td></tr><tr><td>Human IL-2-BV750 clone MQ1-17H12</td><td>BD Horizon</td><td>Cat# 566361, RRID:AB_2739710</td></tr><tr><td>Human IL-21-eF660 clone eBio3A3-N2</td><td>Thermo Fisher Scientific</td><td>Cat# 50-7219-42, RRID:AB_10598202</td></tr><tr><td>Human aCD3-Alexa532 (Clone UCHT1)</td><td>Thermo Fisher Scientific</td><td>Cat# 58-0038-42, RRID:AB_11218675</td></tr><tr><td>Human γδTCR-FITC (clone)</td><td>Thermo Fisher Scientific</td><td>Cat# 11-9959-42, RRID:AB_10669049</td></tr><tr><td>Human Vδ2-APC-Fire750)</td><td>BioLegend</td><td>Cat# 331420, RRID:AB_2687326</td></tr></tbody></table>

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<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>Human CD56-BV605 (clone 5.1H11)</td><td>BioLegend</td><td>Cat# 362538, RRID:AB_2565856</td></tr><tr><td>Human CD4-BV750</td><td>BD Biosciences</td><td>Cat# 566356, RRID:AB_2744426</td></tr><tr><td>Human CD8a-BV510 (clone RPA-T8)</td><td>BioLegend</td><td>Cat# 301047, RRID:AB_2561378</td></tr><tr><td>Human Vz7.2-BV711 (clone 3C10)</td><td>BioLegend</td><td>Cat# 351731, RRID:AB_2629679</td></tr><tr><td>Human Vz24-Jz18-PE-Cy7 (clone 6B11)</td><td>BioLegend</td><td>Cat# 342912, RRID:AB_2562230</td></tr><tr><td>Human Vδ1-Vioblue</td><td>Miltenyi Biotec</td><td>Cat# 30-100-555</td></tr><tr><td>Human CD161-PE (clone HP-3G10)</td><td>BioLegend</td><td>Cat# 339938, RRID:AB_2564141</td></tr><tr><td>Human Vβ11-APC (Miltenyi Biotec)</td><td>Miltenyi Biotec</td><td>Cat# 130-125-508</td></tr><tr><td>Human CD1a-biotin (clone HI149)</td><td>BioLegend</td><td>Cat# 300112, RRID:AB_389344</td></tr><tr><td>Human CD14-biotin (clone 61D3)</td><td>Invitrogen</td><td>Cat# 13-0149-82, RRID:AB_466373</td></tr><tr><td>Human CD34-biotin (clone 4H11)</td><td>BioLegend</td><td>Cat# 316404</td></tr><tr><td>Human CD123-biotin (clone 6H6)</td><td>BioLegend</td><td>Cat# 306004, RRID:AB_314578</td></tr><tr><td>Human CD203c-biotin (clone FR3-16A11)</td><td>Miltenyi Biotec</td><td>Cat# 130-092-345, RRID:AB_615067</td></tr><tr><td>Human CD303-biotin (clone AC144)</td><td>Miltenyi Biotec</td><td>Cat# 130-090-691, RRID:AB_244166</td></tr><tr><td>Human FcεRIα-biotin (clone AER-37 CRA-1)</td><td>BioLegend</td><td>Cat# 334606, RRID:AB_2571885</td></tr><tr><td>Human TCRαβ-biotin (clone IP26)</td><td>BioLegend</td><td>Cat# 306704, RRID:AB_314632</td></tr><tr><td>Human TCRγδ-biotin (clone B1)</td><td>BD Biosciences</td><td>Cat# 555716, RRID:AB_396060</td></tr><tr><td>Human CD4 FITC (clone OKT4)</td><td>BioLegend</td><td>Cat# 317408, RRID:AB_571951</td></tr><tr><td>Human CD336 PerCP-eFluor710 (clone 44.189)</td><td>Thermo Fisher Scientific</td><td>Cat# 46-3369-42, RRID:AB_2573749</td></tr><tr><td>Human EOMES PE (clone WD1928)</td><td>Thermo Fisher Scientific</td><td>Cat# 12-4877-42, RRID:AB_2572615</td></tr><tr><td>Human CD8a PE-CF594 (clone RPA-T8)</td><td>BD Biosciences</td><td>Cat# 562282, RRID:AB_11154052</td></tr><tr><td>Human CD127 PE-Cy7 (clone eBioRDR5)</td><td>Thermo Fisher Scientific</td><td>Cat# 25-1278-42, RRID:AB_1659672</td></tr><tr><td>Human CD294 AF647 (clone BM16)</td><td>BD Biosciences</td><td>Cat# 558042, RRID:AB_2112699</td></tr><tr><td>Human CD161 AF700 (clone HP-3G10)</td><td>BioLegend</td><td>Cat# 339942, RRID:AB_2565870</td></tr><tr><td>Human CD94 APC-Fire750 (clone DX22)</td><td>BioLegend</td><td>Cat# A305-518A, RRID:AB_2773751</td></tr><tr><td>Human CD335 BV421 (clone 9E2/NKp46)</td><td>BD Biosciences</td><td>Cat# 564065, RRID:AB_2738572</td></tr><tr><td>Human CD45RA BV570 (clone HI100)</td><td>BioLegend</td><td>Cat# 304132, RRID:AB_2563813</td></tr><tr><td>Human CD117 BV605 (clone 104D2)</td><td>BioLegend</td><td>Cat# 313218, RRID:AB_2562025</td></tr><tr><td>Human CD3 BV650 (clone UCHT1)</td><td>BD Biosciences</td><td>Cat# 563852</td></tr><tr><td>Human CD7 BV711 (clone M-T701)</td><td>BD Biosciences</td><td>Cat# 564018, RRID:AB_2738544</td></tr><tr><td>Human T-bet BV786 (clone O4-46)</td><td>BD Biosciences</td><td>Cat# 564141, RRID:AB_2738615</td></tr><tr><td>Human CD19 BUV395 (clone SJ25C1)</td><td>BD Biosciences</td><td>Cat# 563549, RRID:AB_2738272</td></tr><tr><td>Human CD16 BUV496 (clone 3G8)</td><td>BD Biosciences</td><td>Cat# 564653, RRID:AB_2744294</td></tr><tr><td>Human CD25 BUV563 (clone 2A3)</td><td>BD Biosciences</td><td>Cat# 565699, RRID:AB_2744341</td></tr><tr><td>Human CD56 BUV737 (clone NCAM16.2)</td><td>BD Biosciences</td><td>Cat# 564447, RRID:AB_2744432</td></tr><tr><td>Human CD45 BUV805 (clone HI30)</td><td>BD Biosciences</td><td>Cat# 612891, RRID:AB_2870179</td></tr><tr><td>Human CD11c (S-HCL-3)</td><td>BioLegend</td><td>Cat# 371523, RRID:AB_2814332</td></tr><tr><td>Human CD141 (M80)</td><td>BioLegend</td><td>Cat# 344127, RRID:AB_2832671</td></tr><tr><td>Human CD161 (HP-3G10)</td><td>BioLegend</td><td>Cat# 339949, RRID:AB_2832665</td></tr><tr><td>Human CD14 (Me5E2)</td><td>BioLegend</td><td>Cat# 301857, RRID:AB_2800735</td></tr><tr><td>Human CD16 (3G8)</td><td>BioLegend</td><td>Cat# 302063, RRID:AB_2800737</td></tr><tr><td>Human CD19 (HIB19)</td><td>BioLegend</td><td>Cat# 302263, RRID:AB_2800740</td></tr><tr><td>Human CD1c (L161)</td><td>BioLegend</td><td>Cat# 331549, RRID:AB_2832653</td></tr><tr><td>Human CD28 (CD28.2)</td><td>BioLegend</td><td>Cat# 302961, RRID:AB_2800750</td></tr><tr><td>Human CD370 (CLEC9A) (8F9)</td><td>BioLegend</td><td>Cat# 353811, RRID:AB_2876671</td></tr><tr><td>Human CD38 (HB-7)</td><td>BioLegend</td><td>Cat# 356639, RRID:AB_2814303</td></tr><tr><td>Human CD3e (UCHT1)</td><td>BioLegend</td><td>Cat# 300477, RRID:AB_2800722</td></tr><tr><td>Human CD4 (RPA-T4)</td><td>BioLegend</td><td>Cat# 300565, RRID:AB_2800724</td></tr></tbody></table>

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<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>Human CD45RA (HI100)</td><td>BioLegend</td><td>Cat# 304161, RRID:AB_2800763</td></tr><tr><td>Human CD45RO (UCHL1)</td><td>BioLegend</td><td>Cat# 304259, RRID:AB_2800766</td></tr><tr><td>Human CD56 (NCAM) (5.1H11)</td><td>BioLegend</td><td>Cat# 362561, RRID:AB_2814309</td></tr><tr><td>Human CD66b (6/40c)</td><td>BioLegend</td><td>Cat# 392913, RRID:AB_2832738</td></tr><tr><td>Human CD69 (FN50)</td><td>BioLegend</td><td>Cat# 310949, RRID:AB_2800809</td></tr><tr><td>Human CD8a (RPA-T8)</td><td>BioLegend</td><td>Cat# 301069, RRID:AB_2800729</td></tr><tr><td>Human TCR gd (B1)</td><td>BioLegend</td><td>Cat# 331233, RRID:AB_2814200</td></tr><tr><td>Human TCR Va7.2 (3C10)</td><td>BioLegend</td><td>Cat# 351737, RRID:AB_2819993</td></tr><tr><td>IgG1 k Isotype Ctl (MOPC-21).</td><td>BioLegend</td><td>Cat# 400185</td></tr><tr><td>89 Y Human CD45 (clone HI30) PBMC panel 1</td><td>Fluidigm</td><td>Cat# 3089003B, RRID:AB_2661851</td></tr><tr><td>113 In Human CD57 (clone HCD57) PBMC panel 1</td><td>BioLegend</td><td>Cat# 322302, RRID:AB_535988</td></tr><tr><td>115 In Human CD11c (clone Bu15) PBMC panel 1</td><td>BioLegend</td><td>Cat# 337202, RRID:AB_1236381</td></tr><tr><td>141 Pr Human CD33 (clone WM53) PBMC panel 1</td><td>BioLegend</td><td>Cat# 303410, RRID:AB_2074243</td></tr><tr><td>142 Nd Human CD19 (clone HIB19) PBMC panel 1</td><td>BioLegend</td><td>Cat# 302202, RRID:AB_314232</td></tr><tr><td>143 Nd Human CD45RA (clone HI100) PBMC panel 1</td><td>BioLegend</td><td>Cat# 304102, RRID:AB_314406</td></tr><tr><td>144 Nd Human CD141 (clone M80) PBMC panel 1</td><td>BioLegend</td><td>Cat# 344102, RRID:AB_2201808</td></tr><tr><td>145 Nd Human CD4 (clone RPA-T4) PBMC panel 1</td><td>BioLegend</td><td>Cat# 300502, RRID:AB_314070</td></tr><tr><td>146 Nd Human CD8 (clone RPA-T8) PBMC panel 1</td><td>BioLegend</td><td>Cat# 301002, RRID:AB_314120</td></tr><tr><td>147 Sm Human CD20 (clone 2H7) PBMC panel 1</td><td>BioLegend</td><td>Cat# 302302, RRID:AB_314250</td></tr><tr><td>148 Nd Human CD16 (clone 3G8) PBMC panel 1</td><td>BioLegend</td><td>Cat# 302014, RRID:AB_314214</td></tr><tr><td>149 Sm Human CD127 (clone A019D5) PBMC panel 1</td><td>Fluidigm</td><td>Cat# 3149011B, RRID:AB_2661792</td></tr><tr><td>150 Nd Human CD1c (clone L161) PBMC panel 1</td><td>BioLegend</td><td>Cat# 331502, RRID:AB_1088995</td></tr><tr><td>151 Eu Human CD123 (clone 6H6) PBMC panel 1</td><td>BioLegend</td><td>Cat# 306002, RRID:AB_314576</td></tr><tr><td>152 Sm Human CD66b (clone G10F5) PBMC panel 1</td><td>BioLegend</td><td>Cat# 305102, RRID:AB_314494</td></tr><tr><td>153 Eu Human PD-1 (clone EH12.2H7) PBMC panel 1</td><td>BioLegend</td><td>Cat# 329926, RRID:AB_11147365</td></tr><tr><td>154 Sm Human CD86 (clone IT2.2) PBMC panel 1</td><td>BioLegend</td><td>Cat# 305410, RRID:AB_314530</td></tr><tr><td>155 Gd Human CD27 (clone O323) PBMC panel 1</td><td>BioLegend</td><td>Cat# 302802, RRID:AB_314294</td></tr><tr><td>156 Gd Human CCR5 (clone J418F1) PBMC panel 1</td><td>BioLegend</td><td>Cat# 359102, RRID:AB_2562457</td></tr><tr><td>158 Gd Human CD117 (clone 104D2) PBMC panel 1</td><td>BioLegend</td><td>Cat# 313202, RRID:AB_314981</td></tr><tr><td>159 Tb Human CD24 (clone ML5) PBMC panel 1</td><td>BioLegend</td><td>Cat# 311102, RRID:AB_314851</td></tr><tr><td>160 Gd Human CD14 (clone M5E2) PBMC panel 1</td><td>BioLegend</td><td>Cat# 301810, RRID:AB_314192</td></tr><tr><td>161 Dy Human CD56 (clone B159) PBMC panel 1</td><td>BD Biosciences</td><td>Cat# 555513, RRID:AB_395903</td></tr><tr><td>162 Dy Human gdTCR (clone REA591) PBMC panel 1</td><td>Miltenyi</td><td>Cat# 130-122-291; RRID: AB_2801872</td></tr><tr><td>163 Dy Human CRTh2 (clone REA598) PBMC panel 1</td><td>Miltenyi</td><td>Cat# 130-122-305, RRID:AB_2801886</td></tr><tr><td>164 Dy Human CLEC12A (clone 50C1) PBMC panel 1</td><td>BioLegend</td><td>Cat# 353602, RRID:AB_10962440</td></tr><tr><td>165 Ho Human CCR6 (clone G034E3) PBMC panel 1</td><td>BioLegend</td><td>Cat# 353402, RRID:AB_10918625</td></tr><tr><td>166 Er Human CD25 (clone M-A251) PBMC panel 1</td><td>BioLegend</td><td>Cat# 356102, RRID:AB_2561752</td></tr><tr><td>167 Er Human CCR7 (clone G043H7) PBMC panel 1</td><td>BioLegend</td><td>Cat# 353256, RRID:AB_2814291</td></tr><tr><td>168 Er Human CD3 (clone UCHT1) PBMC panel 1</td><td>BioLegend</td><td>Cat# 300402, RRID:AB_314056</td></tr><tr><td>169 Tm Human CX3CR1 (clone 2A9-1) PBMC panel 1</td><td>BioLegend</td><td>Cat# 341602, RRID:AB_1595422</td></tr><tr><td>170 Er Human CD38 (clone HB-7) PBMC panel 1</td><td>BioLegend</td><td>Cat# 356602, RRID:AB_2561794</td></tr><tr><td>171 Yb Human CD161 (clone HP-3G10) PBMC panel 1</td><td>BioLegend</td><td>Cat# 339902, RRID:AB_1501090</td></tr><tr><td>172 Yb Human CD209 (clone 9E9A8) PBMC panel 1</td><td>BioLegend</td><td>Cat# 330102, RRID:AB_1134253</td></tr><tr><td>173 Yb Human CXCR3 (clone REA232) PBMC panel 1</td><td>Miltenyi</td><td>Cat# 130-108-022, RRID:AB_2655743</td></tr><tr><td>174 Yb Human HLADR (clone L243) PBMC panel 1</td><td>BioLegend</td><td>Cat# 307602, RRID:AB_314680</td></tr><tr><td>176 Yb Human CCR4 (clone 205,410) PBMC panel 1</td><td>R&DSystems</td><td>Cat# MAB1567, RRID:AB_2074395</td></tr><tr><td>209 BiHuman CD11b (clone ICRF44) PBMC panel 1</td><td>Fluidigm</td><td>Cat# 3209003B, RRID:AB_2687654</td></tr><tr><td>89 Y Human CD45 (clone HI30) PBMC panel 2</td><td>Fluidigm</td><td>Cat# 3089003B, RRID:AB_2661851</td></tr></tbody></table>

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<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>113 In Human HLA-ABC (clone W6/32) PBMC panel 2</td><td>BioLegend</td><td>Cat# 311402, RRID:AB_314871</td></tr><tr><td>115 In Human CD11c (clone Bu15) PBMC panel 2</td><td>BioLegend</td><td>Cat# 337202, RRID:AB_1236381</td></tr><tr><td>141 Pr Human CD33 (clone WM53) PBMC panel 2</td><td>BioLegend</td><td>Cat# 303410, RRID:AB_2074243</td></tr><tr><td>142 Nd Human CD19 (clone HIB19) PBMC panel 2</td><td>BioLegend</td><td>Cat# 302202, RRID:AB_314232</td></tr><tr><td>143 Nd Human CD45RA (clone HI100) PBMC panel 2</td><td>BioLegend</td><td>Cat# 304102, RRID:AB_314406</td></tr><tr><td>144 Nd Human CD141 (clone M80) PBMC panel 2</td><td>BioLegend</td><td>Cat# 344102, RRID:AB_2201808</td></tr><tr><td>145 Nd Human CD4 (clone RPA-T4) PBMC panel 2</td><td>BioLegend</td><td>Cat# 300502, RRID:AB_314070</td></tr><tr><td>146 Nd Human CD8 (clone RPA-T8) PBMC panel 2</td><td>BioLegend</td><td>Cat# 301002, RRID:AB_314120</td></tr><tr><td>147 Sm Human CLEC9A (clone 8F9) PBMC panel 2</td><td>BioLegend</td><td>Cat# 353802, RRID:AB_10983070</td></tr><tr><td>148 Nd Human CD16 (clone 3G8) PBMC panel 2</td><td>BioLegend</td><td>Cat# 302014, RRID:AB_314214</td></tr><tr><td>149 Sm Human FceR1a (clone AER-37) PBMC panel 2</td><td>BioLegend</td><td>Cat# 334602, RRID:AB_1227649</td></tr><tr><td>150 Nd Human CD1c (clone L161) PBMC panel 2</td><td>BioLegend</td><td>Cat# 331502, RRID:AB_1088995</td></tr><tr><td>151 Eu Human CD123 (clone 6H6) PBMC panel 2</td><td>BioLegend</td><td>Cat# 306002, RRID:AB_314576</td></tr><tr><td>152 Sm Human CD66b (clone G10F5) PBMC panel 2</td><td>BioLegend</td><td>Cat# 305102, RRID:AB_314494</td></tr><tr><td>153 Eu Human CD83 (clone HB15e) PBMC panel 2</td><td>BioLegend</td><td>Cat# 305302, RRID:AB_314510</td></tr><tr><td>154 Sm Human CD86 (clone IT2.2) PBMC panel 2</td><td>BioLegend</td><td>Cat# 305410, RRID:AB_314530</td></tr><tr><td>155 Gd Human CD27 (clone O323) PBMC panel 2</td><td>BioLegend</td><td>Cat# 302802, RRID:AB_314294</td></tr><tr><td>156 Gd Human PD-L1 (clone 29E.2A3) PBMC panel 2</td><td>BioLegend</td><td>Cat# 329711, RRID:AB_2228868</td></tr><tr><td>158 Gd Human CD163 (clone REA812) PBMC panel 2</td><td>Miltenyi</td><td>Cat# 130-122-293, RRID:AB_2801874</td></tr><tr><td>159 Tb Human CD103 (clone Ber-Act8) PBMC panel 2</td><td>BioLegend</td><td>Cat# 350202, RRID:AB_10639864</td></tr><tr><td>160 Gd Human CD14 (clone M5E2) PBMC panel 2</td><td>BioLegend</td><td>Cat# 301810, RRID:AB_314192</td></tr><tr><td>161 Dy Human CD56 (clone B159) PBMC panel 2</td><td>BD Biosciences</td><td>Cat# 555513, RRID:AB_395903</td></tr><tr><td>162 Dy Human CD64 (clone 10,1) PBMC panel 2</td><td>BioLegend</td><td>Cat# 305016, RRID:AB_2103461</td></tr><tr><td>163 Dy Human CD172a/b (clone SE5A5) PBMC panel 2</td><td>Fluidigm</td><td>Cat# 3163017B, RRID:AB_2864730</td></tr><tr><td>164 Dy Human CD40 (clone HB14) PBMC panel 2</td><td>BioLegend</td><td>Cat# 334302, RRID:AB_1236384</td></tr><tr><td>166 Er Human CD169 (clone 7-239) PBMC panel 2</td><td>BioLegend</td><td>Cat# 346002, RRID:AB_2189031</td></tr><tr><td>167 Er Human CD117 (clone 104D2) PBMC panel 2</td><td>BioLegend</td><td>Cat# 313202, RRID:AB_314981</td></tr><tr><td>168 Er Human CD3 (clone UCHT1) PBMC panel 2</td><td>BioLegend</td><td>Cat# 300402, RRID:AB_314056</td></tr><tr><td>169 Tm Human CX3CR1 (clone 2A9-1) PBMC panel 2</td><td>BioLegend</td><td>Cat# 341602, RRID:AB_1595422</td></tr><tr><td>170 Er Human CD38 (clone HB-7) PBMC panel 2</td><td>BioLegend</td><td>Cat# 356602, RRID:AB_2561794</td></tr><tr><td>171 Yb Human CD207 (clone 1000) PBMC panel 2</td><td>BioLegend</td><td>Cat# 352202, RRID:AB_10898115</td></tr><tr><td>172 Yb Human CD206 (clone 44,607) PBMC panel 2</td><td>BioLegend</td><td>Cat# 321112, RRID:AB_571921</td></tr><tr><td>174 Yb Human HLADR (clone L243) PBMC panel 2</td><td>BioLegend</td><td>Cat# 307602, RRID:AB_314680</td></tr><tr><td>175 Lu Human Axl (clone 108,724) PBMC panel 2</td><td>R&DSystems</td><td>Cat# MAB154, RRID:AB_2062558</td></tr><tr><td>176 Yb Human CD209 (clone 9E9A8) PBMC panel 2</td><td>BioLegend</td><td>Cat# 330102, RRID:AB_1134253</td></tr><tr><td>209 BiHuman CD11b (clone ICRF44) PBMC panel 2</td><td>Fluidigm</td><td>Cat# 3209003B, RRID:AB_2687654</td></tr><tr><td>163Dy Human CXCR3 (clone G025H7) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3163004B, RRID:AB_2810969</td></tr><tr><td>152Sm Human TCRgd (clone 11F2) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3152008B, RRID:AB_2687643</td></tr><tr><td>142ND Human CD19 (clone HIB19) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3142001B, RRID:AB_2651155</td></tr><tr><td>144ND Human CD38 (clone HIT2) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3144014B, RRID:AB_2687640</td></tr><tr><td>151Eu Human CD123 (clone 6H6) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3151001B, RRID:AB_2661794</td></tr><tr><td>153Eu Human Va7.2 (clone 3C10) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3153024B, RRID:AB_2891190</td></tr><tr><td>154Sm Human CD3 (clone UCHT1) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3154003B, RRID:AB_2811086</td></tr></tbody></table>

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<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>155Gd Human CD45RA (clone HI100) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3155011B, RRID:AB_2810246</td></tr><tr><td>158Gd Human CD27 (clone L128) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3158010B, RRID:AB_2858231</td></tr><tr><td>159Tb Human CD1c (clone L161) Whole blood custom panel</td><td>Biolegend</td><td>Cat# 331502, RRID:AB_1088995</td></tr><tr><td>161Dy Human CLEC9A (clone 8F9) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3161018B, RRID:AB_2810252</td></tr><tr><td>164Dy Human CD161 (clone HP-3G10) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3164009B, RRID:AB_2687651</td></tr><tr><td>168Er Human CD8 (clone SK1) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3168002B, RRID:AB_2892771</td></tr><tr><td>170Er Human iNKT (clone 6B11) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3170015B</td></tr><tr><td>175Lu Human CCR4 (clone L291H4) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3175035A, RRID:AB_2921320</td></tr><tr><td>174Yb Human CD4 (clone RPA-T4) Whole blood custom panel</td><td>Biolegend</td><td>Cat# 300502, RRID:AB_314070</td></tr><tr><td>162Dy Human CD21 (clone REA940) Whole blood custom panel</td><td>Miltenyi Biotec Inc.</td><td>Cat# 130-124-315, RRID:AB_2811646</td></tr><tr><td>165Ho Human NKG2C (clone REA205) Whole blood custom panel</td><td>Miltenyi Biotec Inc.</td><td>Cat# 130-122-278, RRID:AB_2801859</td></tr><tr><td>148ND Human CD20 (clone 2H7) Whole blood custom panel</td><td>Biolegend</td><td>Cat# 302302, RRID:AB_314250</td></tr><tr><td>173Yb Human HLA-DR (clone L243) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3173005B, RRID:AB_2810248</td></tr><tr><td>156Gd Human CCR10 (clone REA326) Whole blood custom panel</td><td>Miltenyi Biotec Inc.</td><td>Cat# 130-122-317, RRID:AB_2801898</td></tr><tr><td>089Y Human CD45 (clone HI30) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3089003B, RRID:AB_2661851</td></tr><tr><td>116Cd Human CD66b (clone QA17A51) Whole blood custom panel</td><td>Biolegend</td><td>Cat# 396902, RRID:AB_2814367</td></tr><tr><td>141Pr Human CCR6 (clone G034E3) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3141003A, RRID:AB_2687639</td></tr><tr><td>143ND Human CD127 (clone A019D5) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3143012B, RRID:AB_2810240</td></tr><tr><td>147Sm Human CD11c (clone Bu15) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3147008B, RRID:AB_2687850</td></tr><tr><td>149Sm Human CD25 (clone 2A3) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3149010B, RRID:AB_2756416</td></tr><tr><td>150ND Human NKVFS1 (clone NKVFS1) Whole blood custom panel</td><td>Bio-Rad</td><td>Cat# MCA2243GA, RRID:AB_323743</td></tr><tr><td>167Er Human CCR7 (clone G043H7) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3167009A, RRID:AB_2858236</td></tr><tr><td>169Tm Human NKG2A (clone Z199) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3169013B, RRID:AB_2756426</td></tr><tr><td>171Yb Human CXCR5 (clone RF8B2) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3171014B, RRID:AB_2858239</td></tr><tr><td>166Er Human CD24 (clone ML5) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3166007B, RRID:AB_2661803</td></tr><tr><td>145ND Human CD31 (clone WM59) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3145004B, RRID:AB_2737262</td></tr><tr><td>160Gd Human CD14 (clone M5E2) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3160001B, RRID:AB_2687634</td></tr></tbody></table>

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<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>176Yb Human CD56 (clone NCAM16.2) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3176008B, RRID:AB_2661813</td></tr><tr><td>172Yb Human CD57 (clone HNK-1) Whole blood custom panel</td><td>Biolegend</td><td>Cat# 359602, RRID:AB_2562403</td></tr><tr><td>150ND Human KIR3DL1L2 (clone REA970) Whole blood custom panel</td><td>Miltenyi Biotec Inc.</td><td>Cat# 130-126-489, RRID:AB_2889458</td></tr><tr><td>146ND Human IgD (clone IA6-2) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3146005B, RRID:AB_2811082</td></tr><tr><td>209BiHuman CD16 (clone 3G8) Whole blood custom panel</td><td>Fluidigm</td><td>Cat# 3209002B, RRID:AB_2756431</td></tr><tr><td>eFluor 450 Murine CD3ε (clone 145-2C11)</td><td>Thermo Fisher Scientific</td><td>Cat# 48-0031-82, RRID:A14714</td></tr><tr><td>BUV395 Murine CD4 (clone GK1.5)</td><td>BD Bioscience</td><td>Cat# 563790, RRID:AB_2738426</td></tr><tr><td>PE Murine CD5 (clone 53-7.3)</td><td>Thermo Fisher Scientific</td><td>Cat# 12-0051-82, RRID:AB_465523</td></tr><tr><td>eFluor 615 Murine CD8α (clone 53-6.7)</td><td>Thermo Fisher Scientific</td><td>Cat# 47-0081-82, RRID:AB_1272185</td></tr><tr><td>Brilliant Violet 650 Murine CD11b (clone M1/70)</td><td>Thermo Fisher Scientific</td><td>Cat# 416-0112-82</td></tr><tr><td>PE Murine CD11b (clone M1/70)</td><td>Thermo Fisher Scientific</td><td>Cat# 12-0112-82, RRID:AB_2734869</td></tr><tr><td>eFluor 450 Murine CD11b (clone M1/70)</td><td>Thermo Fisher Scientific</td><td>Cat# 48-0112-82, RRID:AB_1582236</td></tr><tr><td>FITC Murine CD11c (clone N418)</td><td>Thermo Fisher Scientific</td><td>Cat# 11-0114-82, RRID:AB_464940</td></tr><tr><td>PE Murine CD11c (clone N418)</td><td>Thermo Fisher Scientific</td><td>Cat# 12-0114-82, RRID:AB_465552</td></tr><tr><td>eFluor 450 Murine CD11c (clone N418)</td><td>Thermo Fisher Scientific</td><td>Cat# 48-0114-82, RRID:AB_1548654</td></tr><tr><td>PE Murine CD19 (clone eBio1D3)</td><td>Thermo Fisher Scientific</td><td>Cat# 12-0193-82, RRID:AB_657659</td></tr><tr><td>Alexa Fluor 660 Murine CD19 (clone eBio1D3)</td><td>Thermo Fisher Scientific</td><td>Cat# 606-0193-82, RRID:AB_2896251</td></tr><tr><td>eFluor 450 Murine CD19 (clone eBio1D3)</td><td>Thermo Fisher Scientific</td><td>Cat# 48-0193-82, RRID:AB_2734905</td></tr><tr><td>Alexa Fluor 488 Murine CD25 (clone PC61.5)</td><td>Thermo Fisher Scientific</td><td>Cat# 53-0251-82, RRID:AB_763472</td></tr><tr><td>Brilliant Violet 711 Murine CD25 (clone PC61.5)</td><td>Thermo Fisher Scientific</td><td>Cat# 407-0251-82</td></tr><tr><td>PerCP-Cyanine5.5 Murine CD26 (clone H194-112)</td><td>Thermo Fisher Scientific</td><td>Cat# 45-0261-82, RRID:AB_1548738</td></tr><tr><td>Brilliant Violet 785 Murine CD44 (clone IM7)</td><td>BioLegend</td><td>Cat# 103059, RRID:AB_2571953</td></tr><tr><td>Brilliant Violet 785 Murine CD45 (clone 30-F11)</td><td>BioLegend</td><td>Cat# 103149, RRID:AB_2564590</td></tr><tr><td>PE/Cyanine7 Murine CD45 (clone 30-F11)</td><td>BioLegend</td><td>Cat# 103114, RRID:AB_312979</td></tr><tr><td>Brilliant Violet 711 Murine CD45 (clone 30-F11)</td><td>BioLegend</td><td>Cat# 103147, RRID:AB_2564383</td></tr><tr><td>Brilliant Violet 650 Murine CD45 (clone 30-F11)</td><td>BioLegend</td><td>Cat# 103151, RRID:AB_2565884</td></tr><tr><td>PE Murine CD45R (B220) (clone RA3-6B2)</td><td>Thermo Fisher Scientific</td><td>Cat# 12-0452-82, RRID:AB_465671</td></tr><tr><td>Brilliant UV 395 Murine CD45R (B220) (clone RA3-6B2)</td><td>Thermo Fisher Scientific</td><td>Cat# 363-0452-82</td></tr><tr><td>eFluor 450 Murine CD45R (B220) (clone RA3-6B2)</td><td>Thermo Fisher Scientific</td><td>Cat# 48-0452-82, RRID:AB_1548761</td></tr><tr><td>eFluor 450 Murine CD49b (clone DX5)</td><td>Thermo Fisher Scientific</td><td>Cat# 48-5971-82, RRID:AB_10671541</td></tr><tr><td>PE/Dazzle 594 Murine CD62L (clone MEL-14)</td><td>BioLegend</td><td>Cat# 104448, RRID:AB_2566163</td></tr><tr><td>BV786 Murine CD64 (clone X54-5/7.1)</td><td>BD Biosciences</td><td>Cat# 741024; RRID:AB_2740644</td></tr><tr><td>APC Murine CD86 (clone GL-1)</td><td>BioLegend</td><td>Cat# 105012, RRID:AB_493342</td></tr><tr><td>Brilliant Violet 510 Murine CD90.2 (Thy-1.2) (clone 53-2.1)</td><td>BioLegend</td><td>Cat# 140319, RRID:AB_2561395</td></tr><tr><td>APC Murine CD117 (c-Kit) (clone 2B8)</td><td>Thermo Fisher Scientific</td><td>Cat# 17-1171-82, RRID:AB_469430</td></tr><tr><td>Brilliant Violet 711 Murine CD127 (clone A7R34)</td><td>BioLegend</td><td>Cat# 135035, RRID:AB_2564577</td></tr><tr><td>Brilliant Violet 650 Murine CD127 (clone A7R34)</td><td>BioLegend</td><td>Cat# 135043, RRID:AB_2629681</td></tr><tr><td>PE Murine CD135 (Flt3) (clone A2F10)</td><td>BioLegend</td><td>Cat# 135306, RRID:AB_1877217</td></tr><tr><td>APC Murine CD172a (SIRPa) (clone P84)</td><td>Thermo Fisher Scientific</td><td>Cat# 17-1721-82, RRID:AB_10733158</td></tr><tr><td>PE/Cyanine7 Murine CD317 (BST2, PDCA-1) (clone 927)</td><td>BioLegend</td><td>Cat# 348416, RRID:AB_2716221</td></tr><tr><td>PE/Dazzle 594 Murine F4/80 (clone BM8)</td><td>BioLegend</td><td>Cat# 123146, RRID:AB_2564133</td></tr><tr><td>PE Murine FcεR1α (clone 36,951)</td><td>Thermo Fisher Scientific</td><td>Cat# 12-5898-82, RRID:AB_466028</td></tr><tr><td>eFluor 450 Murine FcεR1α (clone 36,951)</td><td>Thermo Fisher Scientific</td><td>Cat# 48-5898-82, RRID:AB_2574086</td></tr><tr><td>FITC Murine FOXP3 (clone FJK-16s)</td><td>Thermo Fisher Scientific</td><td>Cat# 11-5773-82, RRID:AB_465243</td></tr></tbody></table>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>eFluor 660 Murine GATA3 (clone TWAJ)</td><td>Thermo Fisher Scientific</td><td>Cat# 50-9966-42, RRID:AB_10596663</td></tr><tr><td>PE-Cyanine7 Murine KLRG1 (clone 2F1)</td><td>Thermo Fisher Scientific</td><td>Cat# 25-5893-82, RRID:AB_1518768</td></tr><tr><td>Biotin Murine Integrin α4β7 (clone DATK32)</td><td>BioLegend</td><td>Cat# 120612, RRID:AB_11203892</td></tr><tr><td>Alexa Fluor 488 Murine Ly-6A/E (Sca-1) (clone E13-161.7)</td><td>BioLegend</td><td>Cat# 122516, RRID:AB_756201</td></tr><tr><td>Murine Ly6C (clone AL-21)</td><td>BD Biosciences</td><td>Cat# 553104, RRID:AB_394628</td></tr><tr><td>PE Murine Ly6G/Ly6C (Gr-1) (clone RB6-8C5)</td><td>Thermo Fisher Scientific</td><td>Cat# 12-5931-82, RRID:AB_466045</td></tr><tr><td>eFluor 450 Murine Ly6G/Ly6C (Gr-1) (clone RB6-8C5)</td><td>Thermo Fisher Scientific</td><td>Cat# 48-5931-82, RRID:AB_1548788</td></tr><tr><td>PerCP-Cy5.5 Murine Ly6G (clone 1A8)</td><td>BD Biosciences</td><td>Cat# 560602; RRID:AB_1727563</td></tr><tr><td>BUV805 Murine MHC Class II (I-A/I-E) (clone M5/114)</td><td>BD Biosciences</td><td>Cat# 748844; RRID:AB_2873247</td></tr><tr><td>PE-Cyanine7 Murine NK1.1 (clone PK136)</td><td>Thermo Fisher Scientific</td><td>Cat# 25-5941-82, RRID:AB_469665</td></tr><tr><td>eFluor 450 Murine NK1.1 (clone PK136)</td><td>Thermo Fisher Scientific</td><td>Cat# 48-5941-82, RRID:AB_2043877</td></tr><tr><td>PerCP-Cyanine5.5 Murine NK1.1 (clone PK136)</td><td>Thermo Fisher Scientific</td><td>Cat# 45-5941-82, RRID:AB_914361</td></tr><tr><td>PE Murine Siglec-F (clone E50-2440)</td><td>BD Biosciences</td><td>Cat# 562068; RRID:AB_394341</td></tr><tr><td>PerCP-eFluor 710 Murine IL-33R (ST2) (clone RMST2-2)</td><td>Thermo Fisher Scientific</td><td>Cat# 46-9335-82, RRID:AB_2573883</td></tr><tr><td>eFluor 450 Murine TCRβ (clone H57-597)</td><td>Thermo Fisher Scientific</td><td>Cat# 48-5961-82, RRID:AB_11039532</td></tr><tr><td>PE Murine TCRβ (clone H57-597)</td><td>Thermo Fisher Scientific</td><td>Cat# 12-5961-82, RRID:AB_466066</td></tr><tr><td>eFluor 450 Murine TCR γ/δ (clone eBioGL3)</td><td>Thermo Fisher Scientific</td><td>Cat# 48-5711-82, RRID:AB_2574071</td></tr><tr><td>PE Murine TCR γ/δ (clone eBioGL3)</td><td>Thermo Fisher Scientific</td><td>Cat# 12-5711-82, RRID:AB_465934</td></tr><tr><td>eFluor 450 Murine TER-119 (clone TER-119)</td><td>Thermo Fisher Scientific</td><td>Cat# 48-5921-82, RRID:AB_1518808</td></tr><tr><td>PE-Cyanine7 Murine TER-119 (clone TER-119)</td><td>Thermo Fisher Scientific</td><td>Cat# 25-5921-82, RRID:AB_469661</td></tr><tr><td>Murine XCR-1 (clone ZET)</td><td>BioLegend</td><td>Cat# 148204, RRID:AB_2563843</td></tr></tbody></table>

Bacterial and virus strains

<table><tr><td>Salmonella enterica subsp. enterica serovar Typhimurium GFP</td><td>ATCC</td><td>14028GFP</td></tr><tr><td>Mycobacterium abscessus sensu stricto, strain CIP104536T, smooth with pTEC27</td><td>Bernut et al.<sup>156</sup></td><td>N/A</td></tr><tr><td>expanded T7 Virscan phage library</td><td>S. Elledge (Brigham and Women's Hospital and Harvard University Medical School, Boston, MA, USA)</td><td>VirScan Phage Library, version 3</td></tr><tr><td>Vesicular stomatitis virus Indiana (VSV)</td><td>Bastard et al.<sup>145</sup></td><td>N/A</td></tr><tr><td>Human immunodeficiency viruses-1 (HIV-1) reporter virus (NL4-3 ΔenvΔnef encoding GFP in nef)</td><td>Bhargava et al.<sup>157</sup></td><td>N/A</td></tr><tr><td>Human immunodeficiency viruses-2 (HIV-2) reporter virus (ROD9 ΔenvΔnef encoding GFP in nef)</td><td>Manel et al.<sup>158</sup></td><td>N/A</td></tr><tr><td>Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) NYC isolate</td><td>Zhang et al.<sup>140</sup></td><td>GenBank OM345241</td></tr><tr><td>Yellow-fever vaccine virus-venus (YF17D-venus) reporter</td><td>Yi et al.<sup>159</sup></td><td>N/A</td></tr><tr><td>Influenza A virus A/California/4/2009 (IAV)</td><td>Manicassamy et al.<sup>160</sup></td><td>N/A</td></tr><tr><td>Herpes simplex virus 1 (HSV-1)</td><td>ATCC</td><td>VR-1493</td></tr><tr><td>Hepatitis A virus (HAV) reporter virus (HM175/18f-NLuc)</td><td>Yamane et al.<sup>70</sup></td><td>N/A</td></tr><tr><td>Encephalomyocarditis virus (EMCV)</td><td>Gao et al.<sup>161</sup></td><td>N/A</td></tr></table>

Biological samples

<table><tr><td>Peripheral blood mononuclear cells from indicated individuals</td><td>This manuscript</td><td>N/A</td></tr><tr><td>Plasma from indicated individuals</td><td>This manuscript</td><td>N/A</td></tr><tr><td>Biopsies from indicated individuals</td><td>This manuscript</td><td>N/A</td></tr></table>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3">Chemicals, peptides, and recombinant proteins</td></tr><tr><td>Protamine sulfate</td><td>Merck</td><td>Cat# P3369-10G</td></tr><tr><td>Recombinant interferon gamma-1b (Imukin)</td><td>Clinigen Healthcare France</td><td>Cat# 3400955776789</td></tr><tr><td>Recombinant interferon alpha-2b (Introna)</td><td>MSD France</td><td>Cat# 3400934956287</td></tr><tr><td>Aldesleukin (Proleukin)</td><td>Novartis</td><td>Cat# 3400956215867</td></tr><tr><td>Recombinant Human IFN-beta 1a (Mammalian) Protein</td><td>Bio-Techne</td><td>Cat# 11410-2</td></tr><tr><td>Collagenase IV</td><td>Gibco</td><td>Cat# 17104-019</td></tr><tr><td>DpnI</td><td>New England Biolab</td><td>Cat# R0176L</td></tr><tr><td>M-CSF</td><td>Peprotech</td><td>Cat# 300-25</td></tr><tr><td>IL-3</td><td>Peprotech</td><td>Cat# 200-03</td></tr><tr><td>M-CSF</td><td># 204-IL-010</td><td>Cat# 216-MC-010</td></tr><tr><td>IL-4</td><td>R&amp;D Systems</td><td>Cat# 204-IL-010</td></tr><tr><td>Pro-Long Gold with DAPI</td><td>Thermo Fisher Scientific</td><td>Cat #P36931</td></tr><tr><td>Aqua Dead Cell Stain kit</td><td>Thermo Fisher Scientific</td><td>Cat# L34957</td></tr><tr><td colspan="3">Critical commercial assays</td></tr><tr><td>SureSelect Human All Exon V6</td><td>Agilent</td><td>Cat# 5190-8864</td></tr><tr><td>Human SNP Array 6.0</td><td>Agilent</td><td>Cat# 901153</td></tr><tr><td>GoTaq DNA Polymerase</td><td>Promega</td><td>Cat# M3005</td></tr><tr><td>RNeasy Plus Mini Kit</td><td>Qiagen</td><td>Cat# 74136</td></tr><tr><td>Quick-RNA Micro-Prep Kit</td><td>Zymo</td><td>Cat# R1051</td></tr><tr><td>Universal PCR Master Mix (2X), no AmpErase UNG</td><td>Thermo Fisher Scientific</td><td>Cat# 4352042</td></tr><tr><td>RiboZero TruSeq Stranded Total RNA Library Prep Kit</td><td>Illumina</td><td>Cat# 20020596</td></tr><tr><td>ELISA IL-12p40</td><td>R&amp;D Systems</td><td>Cat# DP400</td></tr><tr><td>ELISA IL-12p70 HS</td><td>R&amp;D Systems</td><td>Cat# HS120</td></tr><tr><td>NE-PER nuclear and cytoplasmic extraction reagents</td><td>Thermo Fisher Scientific</td><td>Cat #78835</td></tr><tr><td>RNeasy Plus Mini Kit</td><td>Qiagen</td><td>Cat# 74136</td></tr><tr><td>Quick-RNA Micro-Prep Kit</td><td>Zymo</td><td>Cat# R1051</td></tr><tr><td>Super-Script II Reverse Transcriptase</td><td>Thermo Fisher Scientific</td><td>Cat# 18064014</td></tr><tr><td>High-Capacity RNA-to-cDNA Kit</td><td>Applied Biosystems</td><td>Cat# 4387406</td></tr><tr><td>TaqMan Fast Universal PCR Master Mix (2X), no AmpErase UNG</td><td>Thermo Fisher Scientific</td><td>Cat# 4352042,</td></tr><tr><td colspan="3">Deposited data</td></tr><tr><td>RNA sequencing on primary fibroblasts</td><td>This manuscript</td><td>gene expression omnibus (GEO: GSE218033)</td></tr><tr><td>RNA sequencing on iPSC-derived macrophages</td><td>This manuscript</td><td>gene expression omnibus (GEO: GSE218033)</td></tr><tr><td>RNA sequencing on monocyte-derived macrophages</td><td>This manuscript</td><td>gene expression omnibus (GEO: GSE218033)</td></tr><tr><td>Mass spectrometry on primary fibroblasts</td><td>This manuscript</td><td>ProteomeXchange (PXD037759)</td></tr><tr><td>scRNAseq on cryopreserved PBMCs</td><td>This manuscript</td><td>gene expression omnibus (GEO: GSE216489)</td></tr><tr><td colspan="3">Experimental models: Cell lines</td></tr><tr><td>HEK293T cells</td><td>ATCC</td><td>Cat# CRL-11268, RRID:CVCL_1926</td></tr><tr><td>THP1 WT</td><td>Song et al.<sup>87</sup></td><td>ATCC Cat# TIB-202, RRID:CVCL_0006</td></tr><tr><td>THP1 IRF1<sup>KO</sup></td><td>Song et al.<sup>87</sup></td><td>N/A</td></tr><tr><td>THP1 IFN-γR1<sup>KO</sup> clone 30</td><td>This manuscript</td><td>N/A</td></tr><tr><td>THP1 STAT1<sup>KO</sup> clone 13</td><td>This manuscript</td><td>N/A</td></tr><tr><td>iPSC healthy control clone 11</td><td>Ackermann et al.<sup>162</sup></td><td>hCD34-iPSC11</td></tr><tr><td>iPSC healthy control clone 16</td><td>Lachmann et al.<sup>163</sup></td><td>hCD34-iPSC16</td></tr><tr><td>iPSC P1 clone 10</td><td>This manuscript</td><td>N/A</td></tr><tr><td>iPSC P2 clone 3</td><td>This manuscript</td><td>N/A</td></tr><tr><td>iPSC STAT1</td><td>Haake et al.<sup>69</sup></td><td>iSTAT1_compl</td></tr></tbody></table>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3">Experimental models: Organisms/strains</td></tr><tr><td>C57BL/6 (B6) WT</td><td>This manuscript</td><td>In-house colony established from Jax # 000664</td></tr><tr><td>C57BL/6 (B6) IRF1<sup>-/-</sup></td><td>This manuscript</td><td>Jax # 002762 backcrossed to a B6 background</td></tr><tr><td colspan="3">Oligonucleotides</td></tr><tr><td>IRF1 genomic exon 3F</td><td>Thermo Fisher Scientific</td><td>TGGTCTGTTTAAGCCAGCCTC</td></tr><tr><td>IRF1 genomic exon 3R</td><td>Thermo Fisher Scientific</td><td>CAGAAACACAAGTCTGCCACC</td></tr><tr><td>IRF1 genomic exon 5F</td><td>Thermo Fisher Scientific</td><td>TTCCACCTCTCACCAAGAACC</td></tr><tr><td>IRF1 genomic exon 5R</td><td>Thermo Fisher Scientific</td><td>CAGAGAAGGTATCAGGGCTGG</td></tr><tr><td>IRD700-conjugated ISRE probe-F</td><td>Metabion</td><td>GATCGGGAAAGGGAAACCGAAACTGAA</td></tr><tr><td>IRD700-conjugated ISRE probe-R</td><td>Metabion</td><td>TCAGTTTCGGTTTCCCTTTCCCGATC</td></tr><tr><td>oligo(dT)<sub>12-18</sub></td><td>Thermo Fisher Scientific</td><td>Cat# 18418012</td></tr><tr><td>IRF1 exons 3-4 qPCR probe</td><td>Thermo Fisher Scientific</td><td>Cat# Hs00971960_m1</td></tr><tr><td>IRF1 exons 8-9</td><td>Thermo Fisher Scientific</td><td>Cat# Hs00971965_m1</td></tr><tr><td>GBP4</td><td>Thermo Fisher Scientific</td><td>Cat# Hs00364728_m1</td></tr><tr><td>APOL3</td><td>Thermo Fisher Scientific</td><td>Cat# Hs00758274_m1</td></tr><tr><td>GUSB</td><td>Thermo Fisher Scientific</td><td>Cat# 1702016</td></tr><tr><td>IRF1-p.M85A-DDK-F</td><td>Eurofins</td><td>CAACTTTCGCTGTGCCGCGAACTCCCTG CCAGAT</td></tr><tr><td>IRF1-p.M85A-DDK-R</td><td>Eurofins</td><td>ATCTGGCAGGGAGTTCGCGGCACAGCG AAAGTTG</td></tr><tr><td>IRF1-p.M111A-DDK-F</td><td>Eurofins</td><td>GCGAGTGTACCGGGCGCTTCCACCTCTC</td></tr><tr><td>IRF1-p.M111A-DDK-R</td><td>Eurofins</td><td>GAGAGGTGGAAGCGCCCGGTACACTCGC</td></tr><tr><td>IRF1-p.Q35*-F</td><td>Eurofins</td><td>GAGGAGATGATCTTCTAGATCCCATGGAAGC</td></tr><tr><td>IRF1-p.Q35*-R</td><td>Eurofins</td><td>GCTTCCATGGGATCTAGAAGATCATCTCCTC</td></tr><tr><td>IRF1-p.R129*-F</td><td>Thermo Fisher Scientific</td><td>GTCGAAGTCCAGCTGAGATGCTAAG</td></tr><tr><td>IRF1-p.R129*-R</td><td>Thermo Fisher Scientific</td><td>CTTAGCATCTCAGCTGGACTTCGAC</td></tr><tr><td>IRF1-p.R129-DDK-F</td><td>Thermo Fisher Scientific</td><td>GCTGGACTTCGACTTTCTTTCTTTCTCTG</td></tr><tr><td>IRF1-p.R129-DDK-R</td><td>Thermo Fisher Scientific</td><td>ACGCGTACGCGGCCGCTCGA</td></tr><tr><td>IRF1-p.W11R-DDK-F</td><td>Thermo Fisher Scientific</td><td>GCATGAGACCCCGGCTAGAGATG</td></tr><tr><td>IRF1-p.W11R-DDK-R</td><td>Thermo Fisher Scientific</td><td>CATCTCTAGCCGGGGTCTCATGC</td></tr><tr><td>IRF1-Δ7-8-F</td><td>Thermo Fisher Scientific</td><td>CTGGAGTCAGGGCCTGCTCC</td></tr><tr><td>IRF1-Δ7-8-R</td><td>Thermo Fisher Scientific</td><td>CTCTTGGAGCAGTCGGAGTGGC</td></tr><tr><td>IRF1-Δ7-8-DDK-F</td><td>Thermo Fisher Scientific</td><td>TTGAGTAGGTACCCCTTCCCATCCACGTTTG</td></tr><tr><td>IRF1-Δ7-8-DDK-R</td><td>Thermo Fisher Scientific</td><td>ACGCGTACGCGGCCGCTCGA</td></tr><tr><td>IRF1-p.A67P-DDK-F</td><td>Thermo Fisher Scientific</td><td>GCCGATACAAACCAGGGGAAAAG</td></tr><tr><td>IRF1-p.A67P-DDK-R</td><td>Thermo Fisher Scientific</td><td>CTTTTCCCCTGGTTTGTATCGGC</td></tr><tr><td>IRF1-p.M1_A84del-F</td><td>Eurofins</td><td>ATGAACTCCCTGCCAGATAT</td></tr><tr><td>IRF1-p.M1_A84del-R</td><td>Eurofins</td><td>GGCGATCGCGGCGGCAGATC</td></tr><tr><td>IRF1 retrovirus F</td><td>Thermo Fisher Scientific</td><td>GATCCATTTAAATTCGAATTCATGCCCCATC ACTCGGATGCGC</td></tr><tr><td>IRF1 retrovirus R</td><td>Thermo Fisher Scientific</td><td>ATCGATACCGTCGACCTCGAGTTAAACCT TATCGTCGTCATC</td></tr><tr><td>sgRNA IFNGR1 exon 3F</td><td>Eurofins</td><td>CACCGACACATTCTACTCACCATCT</td></tr><tr><td>sgRNA IFNGR1 exon 3R</td><td>Eurofins</td><td>AAACAGATGGTGAGTAGAATGTGTA</td></tr><tr><td>sgRNA STAT1 exon 3F</td><td>Eurofins</td><td>CACCGTATTTGCAGCTCGTTTGTGG</td></tr><tr><td>sgRNA STAT1 exon 3R</td><td>Eurofins</td><td>AAACCCACAAACGAGCTGCAAATAA</td></tr></tbody></table>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Recombinant DNA</strong></td></tr><tr><td>pCMV6-EV</td><td>Origene</td><td>Cat# RCPS100001</td></tr><tr><td>pCMV6-IRF1-WT-DDK</td><td>Origene</td><td>Cat# RC203500</td></tr><tr><td>pCMV6-IRF1-p.M85A-DDK</td><td>This manuscript</td><td>N/A</td></tr><tr><td>pCMV6-IRF1-p.M111A-DDK</td><td>This manuscript</td><td>N/A</td></tr><tr><td>pCMV6-IRF1-M85A/M111A-DDK</td><td>This manuscript</td><td>N/A</td></tr><tr><td>pCMV6-IRF1-p.Q35*</td><td>This manuscript</td><td>N/A</td></tr><tr><td>pCMV6-IRF1-p.Q35*/M85A-DDK</td><td>This manuscript</td><td>N/A</td></tr><tr><td>pCMV6-IRF1-p.Q35*/M111A-DDK</td><td>This manuscript</td><td>N/A</td></tr><tr><td>pCMV6-IRF1-p.Q35*/M85A/M111A-DDK</td><td>This manuscript</td><td>N/A</td></tr><tr><td>pCMV6-IRF1-p.R129*</td><td>This manuscript</td><td>N/A</td></tr><tr><td>pCMV6-IRF1-p.R129-DDK</td><td>This manuscript</td><td>N/A</td></tr><tr><td>pCMV6-IRF1-p.W11R-DDK</td><td>This manuscript</td><td>N/A</td></tr><tr><td>pCMV6-IRF1-Δ7-8</td><td>This manuscript</td><td>N/A</td></tr><tr><td>pCMV6-IRF1-Δ7-8-DDK</td><td>This manuscript</td><td>N/A</td></tr><tr><td>pCMV6-IRF1-p.A67P-DDK</td><td>This manuscript</td><td>N/A</td></tr><tr><td>pCMV6-IRF1-p.M1_A84del</td><td>This manuscript</td><td>N/A</td></tr><tr><td>pGL4.10[luc2] backbone with three GGAA AGGGAAACCGAAACTGAA repeats</td><td>Guerin et al.<sup>164</sup></td><td>Cat# E6651</td></tr><tr><td>pGL4.10[luc2] backbone with five GGGAAAGTGAAACTA repeats</td><td>Hernandez et al.<sup>165</sup></td><td>N/A</td></tr><tr><td>pRL-SV40</td><td>Promega</td><td>Cat# E2231</td></tr><tr><td>lentiCRISPR v2</td><td>Addgene</td><td>Cat# 52961</td></tr><tr><td>psPAX2</td><td>Addgene</td><td>Cat# 12260</td></tr><tr><td>pCMV-VSV-G</td><td>Addgene</td><td>Cat# 8454</td></tr><tr><td>pHXB2-Env</td><td>NIH-AIDS Reagent Program</td><td>Cat# 1069</td></tr></tbody></table>

Software and algorithms

<table><tr><td>Philip-stat</td><td>Larman et al.<sup>166</sup></td><td>https://github.com/lasersonlab/Philip-stat</td></tr><tr><td>Bowtie for alignment of PhIP-Seq raw reads</td><td>Larman et al.<sup>166</sup></td><td>http://bowtie-bio.sourceforge.net/index.shtml</td></tr><tr><td>R</td><td>The R Project for Statistical Computing</td><td>https://www.r-project.org</td></tr><tr><td>DESeq2</td><td>Love et al.<sup>167</sup></td><td>https://bioconductor.org/packages/release/bioc/html/DESeq2.html</td></tr><tr><td>Uni-form Mani-fold Approximation and Projection (UMAP)</td><td>Becht et al.<sup>168</sup></td><td>v.0.3.5</td></tr><tr><td>BVAtools</td><td>https://bitbucket.org/muggic/bvatools</td><td>https://bitbucket.org/muggic/bvatools</td></tr><tr><td>Cell Ranger</td><td>10X Genomics</td><td>v3.0.1 for scRNA and v6.0.1 for CITE-seq</td></tr><tr><td>DoubletFinder package</td><td>McGinnis et al.<sup>169</sup></td><td>v2.0.3</td></tr><tr><td>Seurat R package</td><td>Stuart et al.<sup>170</sup></td><td>v4.0.2</td></tr><tr><td>MAST</td><td>Finak et al.<sup>171</sup></td><td>https://github.com/RGLab/MAST</td></tr><tr><td>gProfiler R package</td><td>Raudvere et al.<sup>172</sup></td><td>https://biit.cs.ut.ee/gprofiler/gost</td></tr><tr><td>FastQC</td><td>https://www.bioinformatics.babraham.ac.uk/projects/fastqc/</td><td>https://www.bioinformatics.babraham.ac.uk/projects/fastqc/</td></tr><tr><td>STAR</td><td>Dobin et al.<sup>173</sup></td><td>v2.7.3a</td></tr><tr><td>RSeQC</td><td>Wang et al.<sup>174</sup></td><td>v0.11.2</td></tr><tr><td>HTSeq-count</td><td>Anders et al.<sup>175</sup></td><td>v3.26.8</td></tr><tr><td>edgeR package</td><td>McCarthy et al.<sup>176</sup></td><td>N/A</td></tr><tr><td>HOMER</td><td>Heinz et al.<sup>177</sup></td><td>v4.11</td></tr></table>
RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jean-Laurent Casanova (casanova@mail.rockefeller.edu).

Materials Availability

All raw and processed data and biological materials, including immortalized cell lines from patients, are available upon request from the lead contact under a Material/Data Transfer Agreement with Inserm or the Rockefeller University.

Data and code availability

* RNA-seq, single-cell RNA-seq, and CITE-seq data have been deposited at GEO and are publicly available as of the date of publication. Accession numbers are listed in the **key resources table**. The mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium via the PRIDE partner repository and datasets identifier is listed in the **key resources table**. Original western blot images, flow cytometry data, mass spectrometry data, and microscopy data reported in this paper will be shared by the **lead contact** upon request.

* This paper does not report original code.

* Any additional information required to reanalyze the data reported in this paper is available from the [lead contact](#) upon request.

EXPERIMENT MODEL AND SUBJECT DETAILS

Patient 1 (P1, kindred A) is an eight-year-old girl born to consanguineous parents originating from and living in Argentina (Figure 1A). She was vaccinated with BCG at birth. At the age of five months, she was hospitalized for lymphadenitis and hepatosplenomegaly. BCG was detected in a lymph node biopsy and bone marrow aspirate by culture, attesting to disseminated BCG disease (BCG-osis). P1 was treated with a combination of amikacin, rifampicin, isoniazid, ethambutol, and levofloxacin for two years. At the age of three years, she had fever and abdominal pain. Computed tomography (CT) revealed hepatomegaly, and mediastinal and abdominal lymphadenopathies (Figure 1B). Mediastinal lymph node biopsy revealed granulomatous lymphadenitis (Figure 1C), and culture yielded *M. avium*. During this episode, PCR also detected *Histoplasma* sp. in the blood. P1 was treated with rifampicin, isoniazid, ethambutol, levofloxacin for two years for mycobacterial disease, and with amphotericin B followed by voriconazole for prophylaxis. At the age of four years, IFN-γ treatment was initiated following a suspected reactivation of mycobacterial disease. At the age of five years, P1 was hospitalized for pneumonia. *M. avium* was again identified in a sputum sample. Clarithromycin was added to the patient's IFN-γ and antimycobacterial treatment. At the age of seven years, P1 had rhinitis causing upper respiratory obstruction. *Mycobacterium intracellulare* subsp. *chimaera* was documented on a nasal swab. During this episode, P1 also had hepatosplenomegaly and pancytopenia. Bone marrow biopsy showed epithelioid granulomas with giant multinucleated cells. P1 was treated with lipo-somal amphotericin, levofloxacin, clarithromycin, and ethambutol. She is now eight years old and clinically well on immunoglobulins, recombinant IFN-γ, levofloxacin, clarithromycin, cotrimoxazole, and itraconazole.

Patient 2 (P2, kindred B) is a seven-year-old girl born to consanguineous parents originating from and living in Turkey (Figure 1A). She was vaccinated with BCG at two months of age. She presented with left axillary lymphadenitis at five months old. BCG was grown from the abscess, attesting to local-regional BCG-infection (BCG-itis). P2 was successfully treated with isoniazid, rifampicin, and ethambutol for six months. At the age of five years, she presented fever, digital clubbing, and rash. A chest computed tomography (CT)-scan revealed multiple mediastinal and hilar lymphadenopathies, and a lung mass (Figure 1D). Biopsy specimens from the lymphadenopathies and the lung mass contained multinucleated giant cells and spindle-shaped histiocytes (Figure 1E). *M. avium* was cultured from these lesions. P2 has since been treated with azithromycin, ethambutol, and rifabutin.

Mice used in this study (C57BL/6 (B6) mice and *Irf1*<sup>-/-</sup> mice were all male.

METHOD DETAILS

Case reports for P1 and P2

The patient from kindred A (P1, II.1) is a girl born in 2014 and living in Tucuman, in the North East of Argentina (Figure 1A). She was vaccinated with BCG at birth. At the age of one month, she presented impetigo and a local abscess at the site of vaccination with BCG. At the age of five months, she was hospitalized with fever, hepatosplenomegaly, and intra-abdominal and mediastinal adenomegaly. *Mycobacterium bovis*-BCG was cultured from lymph node and bone marrow aspiration fluids. P1 also had digestive symptoms, with diarrhea and intestinal subocclusion, and a cutaneous eruption. BCG-osis was successfully treated with a combination of amikacin, rifampicin, isoniazid, ethambutol, and levofloxacin. At the age of eight months, during hospitalization for intestinal malrotation surgery, P1 was found to have vesicular lesions on the scalp, trunk, perianal region, vulva, and lower limbs, but no signs of meningoencephalitis or pneumonia. The Tzanck test was positive, suggesting infection with varicella-zoster virus (VZV). Infection with this virus was confirmed by positive PhIP-Seq (Figure 7A) and serological testing for VZV (Table S4). Given that VZV was