. 2023 Feb 2;186(3):621-645.e33.

doi: 10.1016/j.cell.2022.12.038.

Human IRF1 governs macrophagic IFN-γ immunity to mycobacteria

Jérémie Rosain¹, Anna-Lena Neehus², Jérémy Manry³, Rui Yang⁴, Jérémie Le Pen⁵, Wassim Daher⁶, Zhiyong Liu⁴, Yi-Hao Chan⁴, Natalia Tahuil⁷, Özden Türel⁸, Mathieu Bourgey⁹, Masato Ogishi⁴, Jean-Marc Doisne¹⁰, Helena M Izquierdo¹¹, Takayoshi Shirasaki¹², Tom Le Voyer¹³, Antoine Guérin¹⁴, Paul Bastard¹⁵, Marcela Moncada-Vélez⁴, Ji Eun Han⁴, Taushif Khan¹⁶, Franck Rapaport⁴, Seon-Hui Hong⁵, Andrew Cheung⁵, Kathrin Haake¹⁷, Barbara C Mindt¹⁸, Laura Pérez¹⁹, Quentin Philippot¹³, Danyel Lee²⁰, Peng Zhang⁴, Darawan Rinchai⁴, Fatima Al Ali¹⁶, Manar Mahmoud Ahmad Ata¹⁶, Mahbuba Rahman¹⁶, Jessica N Peel⁴, Søren Heissel²¹, Henrik Molina²¹, Yasemin Kendir-Demirkol²², Rasheed Bailey⁴, Shuxiang Zhao⁴, Jonathan Bohlen¹³, Mathieu Mancini²³, Yoann Seeleuthner¹³, Marie Roelens²⁴, Lazaro Lorenzo¹³, Camille Soudée¹³, María Elvira Josefina Paz²⁵, María Laura González²⁶, Mohamed Jeljeli²⁷, Jean Soulier²⁸, Serge Romana²⁹, Anne-Sophie L'Honneur³⁰, Marie Materna¹³, Rubén Martínez-Barricarte³¹, Mathieu Pochon¹³, Carmen Oleaga-Quintas¹³, Alexandre Michev¹³, Mélanie Migaud¹³, Romain Lévy³², Marie-Alexandra Alyanakian³³, Flore Rozenberg³⁰, Carys A Croft³⁴, Guillaume Vogt³⁵, Jean-François Emile³⁶, Laurent Kremer⁶, Cindy S Ma¹⁴, Jörg H Fritz³⁷, Stanley M Lemon¹², András N Spaan³⁸, Nicolas Manel¹¹, Laurent Abel²⁰, Margaret R MacDonald⁵, Stéphanie Boisson-Dupuis²⁰, Nico Marr³⁹, Stuart G Tangye¹⁴, James P Di Santo¹⁰, Qian Zhang²⁰, Shen-Ying Zhang²⁰, Charles M Rice⁵, Vivien Béziat²⁰, Nico Lachmann⁴⁰, David Langlais⁴¹, Jean-Laurent Casanova⁴², Philippe Gros⁴³, Jacinta Bustamante⁴⁴

Affiliations

¹ Laboratory of Human Genetics of Infectious Diseases, Inserm U1163, 75015 Paris, France; Paris Cité University, Imagine Institute, 75015 Paris, France. Electronic address: jeremie.rosain@institutimagine.org.
² Laboratory of Human Genetics of Infectious Diseases, Inserm U1163, 75015 Paris, France; Paris Cité University, Imagine Institute, 75015 Paris, France; Institute of Experimental Hematology, REBIRTH Center for Regenerative and Translational Medicine, Hannover Medical School, 30625 Hannover, Germany.
³ Laboratory of Human Genetics of Infectious Diseases, Inserm U1163, 75015 Paris, France; Paris Cité University, Imagine Institute, 75015 Paris, France; Paris Cité University, Imagine Institute, 75015 Paris, France.
⁴ St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York, NY 10065, USA.
⁵ Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA.
⁶ Infectious Disease Research Institute of Montpellier (IRIM), Montpellier University, 34090 Montpellier, France; Inserm, IRIM, CNRS, UMR9004, 34090 Montpellier, France.
⁷ Department of Immunology, Del Niño Jesus Hospital, San Miguel de Tucuman, T4000 Tucuman, Argentina.
⁸ Department of Pediatric Infectious Disease, Bezmialem Vakif University Faculty of Medicine, 34093 İstanbul, Turkey.
⁹ Dahdaleh Institute of Genomic Medicine, McGill University, Montreal, QC H3A 0G1, Canada; Canadian Centre for Computation Genomics, Montreal, QC H3A 0G1, Canada.
¹⁰ Innate Immunity Unit, Institut Pasteur, 75015 Paris, France; Inserm U1223, 75015 Paris, France.
¹¹ Institut Curie, PSL Research University, Inserm U932, 75005 Paris, France.
¹² Department of Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7292, USA.
¹³ Laboratory of Human Genetics of Infectious Diseases, Inserm U1163, 75015 Paris, France; Paris Cité University, Imagine Institute, 75015 Paris, France.
¹⁴ Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia; St. Vincent's Clinical School, Faculty of Medicine, University of NSW, Sydney, NSW 2052, Australia.
¹⁵ Laboratory of Human Genetics of Infectious Diseases, Inserm U1163, 75015 Paris, France; Paris Cité University, Imagine Institute, 75015 Paris, France; St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York, NY 10065, USA; Pediatric Hematology-Immunology and Rheumatology Unit, Necker Hospital for Sick Children, Assistance Publique Hôpitaux de Paris (AP-HP), 75015 Paris, France.
¹⁶ Department of Immunology, Sidra Medicine, Doha, Qatar.
¹⁷ Institute of Experimental Hematology, REBIRTH Center for Regenerative and Translational Medicine, Hannover Medical School, 30625 Hannover, Germany.
¹⁸ Department of Microbiology and Immunology, McGill University, Montreal, QC H3A 0G1, Canada; McGill University Research Centre on Complex Traits, McGill University, Montreal, QC H3A 0G1, Canada; FOCiS Centre of Excellence in Translational Immunology, McGill University, Montreal, QC H3A 0G1, Canada.
¹⁹ Department of Immunology and Rheumatology, "J. P. Garrahan" National Hospital of Pediatrics, C1245 CABA Buenos Aires, Argentina.
²⁰ Laboratory of Human Genetics of Infectious Diseases, Inserm U1163, 75015 Paris, France; Paris Cité University, Imagine Institute, 75015 Paris, France; St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York, NY 10065, USA.
²¹ Proteomics Resource Center, The Rockefeller University, New York, NY 10065, USA.
²² St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York, NY 10065, USA; Umraniye Education and Research Hospital, Department of Pediatric Genetics, 34764 İstanbul, Turkey.
²³ Dahdaleh Institute of Genomic Medicine, McGill University, Montreal, QC H3A 0G1, Canada; Department of Microbiology and Immunology, McGill University, Montreal, QC H3A 0G1, Canada; McGill University Research Centre on Complex Traits, McGill University, Montreal, QC H3A 0G1, Canada.
²⁴ Study Center for Primary Immunodeficiencies, Necker Hospital for Sick Children, AP-HP, 75015 Paris, France; Paris Cité University, 75006 Paris, France.
²⁵ Department of Pediatric Pathology, Del Niño Jesus Hospital, San Miguel de Tucuman, T4000 Tucuman, Argentina.
²⁶ Central Laboratory, Del Niño Jesus Hospital, San Miguel de Tucuman, T4000 Tucuman, Argentina.
²⁷ Cochin University Hospital, Biological Immunology Unit, AP-HP, 75014 Paris, France.
²⁸ Inserm/CNRS U944/7212, Paris Cité University, 75006 Paris, France; Hematology Laboratory, Saint-Louis Hospital, AP-HP, 75010 Paris, France; National Reference Center for Bone Marrow Failures, Saint-Louis and Robert Debré Hospitals, 75010 Paris, France.
²⁹ Rare Disease Genomic Medicine Department, Paris Cité University, Necker Hospital for Sick Children, 75015 Paris, France.
³⁰ Department of Virology, Paris Cité University, Cochin Hospital, 75014 Paris, France.
³¹ Division of Genetic Medicine, Department of Medicine, Vanderbilt Genetics Institute, Vanderbilt University Medical Center, Nashville, TN 37232, USA; Department of Pathology, Microbiology, and Immunology, Vanderbilt Center for Immunobiology, Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
³² Laboratory of Human Genetics of Infectious Diseases, Inserm U1163, 75015 Paris, France; Paris Cité University, Imagine Institute, 75015 Paris, France; Pediatric Hematology-Immunology and Rheumatology Unit, Necker Hospital for Sick Children, Assistance Publique Hôpitaux de Paris (AP-HP), 75015 Paris, France.
³³ Immunology Laboratory, Necker Hospital for Sick Children, AP-HP, 75015 Paris, France.
³⁴ Innate Immunity Unit, Institut Pasteur, 75015 Paris, France; Inserm U1223, 75015 Paris, France; Paris Cité University, 75006 Paris, France.
³⁵ Inserm UMR1283, CNRS UMR8199, European Genomic Institute for Diabetes, Lille University, Lille Pasteur Institute, Lille University Hospital, 59000 Lille, France; Neglected Human Genetics Laboratory, Paris Cité University, 75006 Paris, France.
³⁶ Pathology Department, Ambroise-Paré Hospital, AP-HP, 92100 Boulogne-Billancourt, France.
³⁷ Department of Microbiology and Immunology, McGill University, Montreal, QC H3A 0G1, Canada; McGill University Research Centre on Complex Traits, McGill University, Montreal, QC H3A 0G1, Canada; FOCiS Centre of Excellence in Translational Immunology, McGill University, Montreal, QC H3A 0G1, Canada; Department of Physiology, McGill University, Montreal, QC H3A 0G1, Canada.
³⁸ St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York, NY 10065, USA; Department of Medical Microbiology, University Medical Center Utrecht, Utrecht University, 3584CX Utrecht, the Netherlands.
³⁹ Department of Immunology, Sidra Medicine, Doha, Qatar; College of Health and Life Sciences, Hamad Bin Khalifa University, Doha, Qatar.
⁴⁰ Institute of Experimental Hematology, REBIRTH Center for Regenerative and Translational Medicine, Hannover Medical School, 30625 Hannover, Germany; Department 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; Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, 30625 Hannover, Germany.
⁴¹ Dahdaleh Institute of Genomic Medicine, McGill University, Montreal, QC H3A 0G1, Canada; Department of Microbiology and Immunology, McGill University, Montreal, QC H3A 0G1, Canada; Department of Human Genetics, McGill University, Montreal, QC H3A 0G1, Canada.
⁴² Laboratory of Human Genetics of Infectious Diseases, Inserm U1163, 75015 Paris, France; Paris Cité University, Imagine Institute, 75015 Paris, France; St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York, NY 10065, USA; Department of Pediatrics, Necker Hospital for Sick Children, AP-HP, 75015 Paris, France; Howard Hughes Medical Institute, New York, NY 10065, USA. Electronic address: casanova@rockefeller.edu.
⁴³ Dahdaleh Institute of Genomic Medicine, McGill University, Montreal, QC H3A 0G1, Canada; Department of Biochemistry, McGill University, Montreal, QC H3A 0G1, Canada.
⁴⁴ Laboratory of Human Genetics of Infectious Diseases, Inserm U1163, 75015 Paris, France; Paris Cité University, Imagine Institute, 75015 Paris, France; St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York, NY 10065, USA; Study Center for Primary Immunodeficiencies, Necker Hospital for Sick Children, AP-HP, 75015 Paris, France. Electronic address: jacinta.bustamante@inserm.fr.

PMID: 36736301
PMCID: PMC9907019
DOI: 10.1016/j.cell.2022.12.038

Human IRF1 governs macrophagic IFN-γ immunity to mycobacteria

Jérémie Rosain et al. Cell. 2023.

. 2023 Feb 2;186(3):621-645.e33.

doi: 10.1016/j.cell.2022.12.038.

Authors

Affiliations

¹ Laboratory of Human Genetics of Infectious Diseases, Inserm U1163, 75015 Paris, France; Paris Cité University, Imagine Institute, 75015 Paris, France. Electronic address: jeremie.rosain@institutimagine.org.
² Laboratory of Human Genetics of Infectious Diseases, Inserm U1163, 75015 Paris, France; Paris Cité University, Imagine Institute, 75015 Paris, France; Institute of Experimental Hematology, REBIRTH Center for Regenerative and Translational Medicine, Hannover Medical School, 30625 Hannover, Germany.
³ Laboratory of Human Genetics of Infectious Diseases, Inserm U1163, 75015 Paris, France; Paris Cité University, Imagine Institute, 75015 Paris, France; Paris Cité University, Imagine Institute, 75015 Paris, France.
⁴ St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York, NY 10065, USA.
⁵ Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA.
⁶ Infectious Disease Research Institute of Montpellier (IRIM), Montpellier University, 34090 Montpellier, France; Inserm, IRIM, CNRS, UMR9004, 34090 Montpellier, France.
⁷ Department of Immunology, Del Niño Jesus Hospital, San Miguel de Tucuman, T4000 Tucuman, Argentina.
⁸ Department of Pediatric Infectious Disease, Bezmialem Vakif University Faculty of Medicine, 34093 İstanbul, Turkey.
⁹ Dahdaleh Institute of Genomic Medicine, McGill University, Montreal, QC H3A 0G1, Canada; Canadian Centre for Computation Genomics, Montreal, QC H3A 0G1, Canada.
¹⁰ Innate Immunity Unit, Institut Pasteur, 75015 Paris, France; Inserm U1223, 75015 Paris, France.
¹¹ Institut Curie, PSL Research University, Inserm U932, 75005 Paris, France.
¹² Department of Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7292, USA.
¹³ Laboratory of Human Genetics of Infectious Diseases, Inserm U1163, 75015 Paris, France; Paris Cité University, Imagine Institute, 75015 Paris, France.
¹⁴ Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia; St. Vincent's Clinical School, Faculty of Medicine, University of NSW, Sydney, NSW 2052, Australia.
¹⁵ Laboratory of Human Genetics of Infectious Diseases, Inserm U1163, 75015 Paris, France; Paris Cité University, Imagine Institute, 75015 Paris, France; St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York, NY 10065, USA; Pediatric Hematology-Immunology and Rheumatology Unit, Necker Hospital for Sick Children, Assistance Publique Hôpitaux de Paris (AP-HP), 75015 Paris, France.
¹⁶ Department of Immunology, Sidra Medicine, Doha, Qatar.
¹⁷ Institute of Experimental Hematology, REBIRTH Center for Regenerative and Translational Medicine, Hannover Medical School, 30625 Hannover, Germany.
¹⁸ Department of Microbiology and Immunology, McGill University, Montreal, QC H3A 0G1, Canada; McGill University Research Centre on Complex Traits, McGill University, Montreal, QC H3A 0G1, Canada; FOCiS Centre of Excellence in Translational Immunology, McGill University, Montreal, QC H3A 0G1, Canada.
¹⁹ Department of Immunology and Rheumatology, "J. P. Garrahan" National Hospital of Pediatrics, C1245 CABA Buenos Aires, Argentina.
²⁰ Laboratory of Human Genetics of Infectious Diseases, Inserm U1163, 75015 Paris, France; Paris Cité University, Imagine Institute, 75015 Paris, France; St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York, NY 10065, USA.
²¹ Proteomics Resource Center, The Rockefeller University, New York, NY 10065, USA.
²² St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York, NY 10065, USA; Umraniye Education and Research Hospital, Department of Pediatric Genetics, 34764 İstanbul, Turkey.
²³ Dahdaleh Institute of Genomic Medicine, McGill University, Montreal, QC H3A 0G1, Canada; Department of Microbiology and Immunology, McGill University, Montreal, QC H3A 0G1, Canada; McGill University Research Centre on Complex Traits, McGill University, Montreal, QC H3A 0G1, Canada.
²⁴ Study Center for Primary Immunodeficiencies, Necker Hospital for Sick Children, AP-HP, 75015 Paris, France; Paris Cité University, 75006 Paris, France.
²⁵ Department of Pediatric Pathology, Del Niño Jesus Hospital, San Miguel de Tucuman, T4000 Tucuman, Argentina.
²⁶ Central Laboratory, Del Niño Jesus Hospital, San Miguel de Tucuman, T4000 Tucuman, Argentina.
²⁷ Cochin University Hospital, Biological Immunology Unit, AP-HP, 75014 Paris, France.
²⁸ Inserm/CNRS U944/7212, Paris Cité University, 75006 Paris, France; Hematology Laboratory, Saint-Louis Hospital, AP-HP, 75010 Paris, France; National Reference Center for Bone Marrow Failures, Saint-Louis and Robert Debré Hospitals, 75010 Paris, France.
²⁹ Rare Disease Genomic Medicine Department, Paris Cité University, Necker Hospital for Sick Children, 75015 Paris, France.
³⁰ Department of Virology, Paris Cité University, Cochin Hospital, 75014 Paris, France.
³¹ Division of Genetic Medicine, Department of Medicine, Vanderbilt Genetics Institute, Vanderbilt University Medical Center, Nashville, TN 37232, USA; Department of Pathology, Microbiology, and Immunology, Vanderbilt Center for Immunobiology, Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
³² Laboratory of Human Genetics of Infectious Diseases, Inserm U1163, 75015 Paris, France; Paris Cité University, Imagine Institute, 75015 Paris, France; Pediatric Hematology-Immunology and Rheumatology Unit, Necker Hospital for Sick Children, Assistance Publique Hôpitaux de Paris (AP-HP), 75015 Paris, France.
³³ Immunology Laboratory, Necker Hospital for Sick Children, AP-HP, 75015 Paris, France.
³⁴ Innate Immunity Unit, Institut Pasteur, 75015 Paris, France; Inserm U1223, 75015 Paris, France; Paris Cité University, 75006 Paris, France.
³⁵ Inserm UMR1283, CNRS UMR8199, European Genomic Institute for Diabetes, Lille University, Lille Pasteur Institute, Lille University Hospital, 59000 Lille, France; Neglected Human Genetics Laboratory, Paris Cité University, 75006 Paris, France.
³⁶ Pathology Department, Ambroise-Paré Hospital, AP-HP, 92100 Boulogne-Billancourt, France.
³⁷ Department of Microbiology and Immunology, McGill University, Montreal, QC H3A 0G1, Canada; McGill University Research Centre on Complex Traits, McGill University, Montreal, QC H3A 0G1, Canada; FOCiS Centre of Excellence in Translational Immunology, McGill University, Montreal, QC H3A 0G1, Canada; Department of Physiology, McGill University, Montreal, QC H3A 0G1, Canada.
³⁸ St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York, NY 10065, USA; Department of Medical Microbiology, University Medical Center Utrecht, Utrecht University, 3584CX Utrecht, the Netherlands.
³⁹ Department of Immunology, Sidra Medicine, Doha, Qatar; College of Health and Life Sciences, Hamad Bin Khalifa University, Doha, Qatar.
⁴⁰ Institute of Experimental Hematology, REBIRTH Center for Regenerative and Translational Medicine, Hannover Medical School, 30625 Hannover, Germany; Department 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; Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, 30625 Hannover, Germany.
⁴¹ Dahdaleh Institute of Genomic Medicine, McGill University, Montreal, QC H3A 0G1, Canada; Department of Microbiology and Immunology, McGill University, Montreal, QC H3A 0G1, Canada; Department of Human Genetics, McGill University, Montreal, QC H3A 0G1, Canada.
⁴² Laboratory of Human Genetics of Infectious Diseases, Inserm U1163, 75015 Paris, France; Paris Cité University, Imagine Institute, 75015 Paris, France; St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York, NY 10065, USA; Department of Pediatrics, Necker Hospital for Sick Children, AP-HP, 75015 Paris, France; Howard Hughes Medical Institute, New York, NY 10065, USA. Electronic address: casanova@rockefeller.edu.
⁴³ Dahdaleh Institute of Genomic Medicine, McGill University, Montreal, QC H3A 0G1, Canada; Department of Biochemistry, McGill University, Montreal, QC H3A 0G1, Canada.
⁴⁴ Laboratory of Human Genetics of Infectious Diseases, Inserm U1163, 75015 Paris, France; Paris Cité University, Imagine Institute, 75015 Paris, France; St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York, NY 10065, USA; Study Center for Primary Immunodeficiencies, Necker Hospital for Sick Children, AP-HP, 75015 Paris, France. Electronic address: jacinta.bustamante@inserm.fr.

PMID: 36736301
PMCID: PMC9907019
DOI: 10.1016/j.cell.2022.12.038

Abstract

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.

Keywords: IRF1; Mycobacterium; inborn errors of immunity; interferon-stimulated gene; interferon-γ; macrophages; viruses.

PubMed Disclaimer

Conflict of interest statement

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

Figures

**Figure 1 –. Homozygous complete loss-of-function *IRF1* variant in patients with severe MSMD.**
**(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 blotof 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 3–7 independent experiments performed in triplicate. Bars represent the mean and standard deviation (SD). Statistical analysis by Student’s t-test. ns = not significant, p > 0.05; ****p < 0.0001.

**Figure 2 –. IRF1 mRNA and protein levels in cells from the two patients.**
Quantitative PCR 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-derived MΦ, with and without IFN-γ stimulation. Data from 2–3 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.

**Figure 3 –. Phenotyping of peripheral blood leukocytes and single-cell PBMCs from IRF1-deficient patients.**
**(A)** Monitoring of lymphoid cell numbers in the whole blood or PBMCs of patients. **(B)** Counts for monocyte subsets by mass cytometry on fresh whole-blood cells. **(C)** Counts of myeloid and lymphoid subsets in fresh whole blood. **(D)** UMAP clustering for 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. **(H)** Module score analysis comparing the expression of genes with IRF1-binding sites within 10 kb of their TSS (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).

**Figure 4 –. Production of IFN-γ by the lymphoid cells of IRF1-deficient patients.**
**(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 naïve CD4⁺ T cells in polarizing conditions.

**Figure 5 –. Response to IFN-γ of IRF1-deficient fibroblasts.**
**(A)** HOMER *de novo* motif analysis of the genes differentially expressed in the primary fibroblasts after IFN-γ stimulation. **(B)** RT-qPCR 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 2–6 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 (i) positively induced after stimulation with a log₂FC>1 over the mean in the non-stimulated state for controls (ii) and a log₂FC<0.5 over the mean in the non-stimulated state for patients. On the left, 10^th-90^th percentiles for all proteins positively induced after stimulation with a log₂FC>1 over the mean value in the non-stimulated state for controls.

**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 hours 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 ± SD). **(E)** HLA-DR expression by flow cytometry, using THP1-Ф with and without IFN-γ stimulation. **(F)** Gentamicin protection assay performed on PMA-differentiated THP1-Ф with and without IFN-γ pretreatment and *Salmonella* Typhimurium-GFP (Stm-GFP) infection. Results are expressed as the proportion of *Salmonella* Typhimurium^high on Salmonella Typhi⁺. Representative results from 2–3 independent experiments. **(G)** CFU assays on PMA-differenciated THP1-Ф with and without IFN-γ pretreatment, following infection by *Mycobacterium abscessus*. All replicates from n=3 independent experiments are displayed. **(H)** Flow cytometry analyses on MDMs with and without IFN-γ pretreatment, following infection for 24 hours with *M. abscessus*-tdTomato.

**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. Heatmap showing genes differentially expressed (|log₂(FC)| > 1 and adj. p-value < 0.05) in CTL cells after 2 hours **(B)** or 8 hours **(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 hours from among those differentially expressed at both timepoints 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)** 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.

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Human IRF1 governs macrophagic IFN-γ immunity to mycobacteria

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Human IRF1 governs macrophagic IFN-γ immunity to mycobacteria

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