# FLT3L governs the development of partially overlapping hematopoietic lineages in humans and mice

Mana Momenilandi, Romain Lévy, Steicy Sobrino, Jingwei Li, Chantal Lagresle-Peyrou, Hossein Esmaeilzadeh, Antoine Fayand, Corentin Le Floc'h, Antoine Guérin, Erika Della Mina, et al.

<figure><img src="image_1.png" /></figure>

To cite this version:

Mana Momenilandi, Romain Lévy, Steicy Sobrino, Jingwei Li, Chantal Lagresle-Peyrou, et al. FLT3L governs the development of partially overlapping hematopoietic lineages in humans and mice. Cell, 2024, 187 (11), pp.2817-2837.e31. 10.1016/j.cell.2024.04.009. pasteur-04589107

HAL Id: pasteur-04589107

https://pasteur.hal.science/pasteur-04589107

Submitted on 27 May 2024

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Distributed under a Creative Commons Attribution 4.0 International License
# FLT3L governs the development of partially overlapping hematopoietic lineages in humans and mice

Mana Momenilandi, Romain Lévy, Steicy Sobrino, Jingwei Li, Chantal Lagresle-Peyrou, Hossein Esmaeilzadeh, Antoine Fayand, Corentin Le Floc'h, Antoine Guérin, Erika Della Mina, et al.

► To cite this version:

Mana Momenilandi, Romain Lévy, Steicy Sobrino, Jingwei Li, Chantal Lagresle-Peyrou, et al. FLT3L governs the development of partially overlapping hematopoietic lineages in humans and mice. Cell, 2024, 187 (11), pp.2817-2837.e31. 10.1016/j.cell.2024.04.009. pasteur-04589107

HAL Id: pasteur-04589107

https://pasteur.hal.science/pasteur-04589107v1

Submitted on 27 May 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Cell

# FLT3L governs the development of partially overlapping hematopoietic lineages in humans and mice

## Graphical abstract

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

## Authors

Mana Momenilandi, Romain Lévy, Steicy Sobrino, ..., Xavier Bossuyt, Jean-Laurent Casanova, Vivien Béziat

## Correspondence

vivien.beziat@inserm.fr

## In brief

Adult humans with inherited, complete FLT3L deficiency enable valuable insights into the role of the hematopoietic growth factor FLT3L in human hematopoiesis, revealing that FLT3L deficiencies impair monocyte, dendritic, and B cell development but not NK cell development. This study reveals key differences in the role of FLT3L in humans versus mice.

## Highlights

* Human FLT3L deficiency underlies bone-marrow failure and infectious diseases

* Human and mouse FLT3L deficiencies impair dendritic and B cell development

* FLT3L deficiency impairs monocyte development in humans but not mice

* FLT3L deficiency impairs NK cell development in mice but not in humans

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

# FLT3L governs the development of partially overlapping hematopoietic lineages in humans and mice

## Graphical abstract

<figure><img src="image_2.png" /></figure>

## Authors

Mana Momenilandi, Romain Lévy, Steicy Sobrino, ..., Xavier Bossuyt, Jean-Laurent Casanova, Vivien Béziat

## Correspondence

vivien.beziat@inserm.fr

## In brief

Adult humans with inherited, complete FLT3L deficiency enable valuable insights into the role of the hematopoietic growth factor FLT3L in human hematopoiesis, revealing that FLT3L deficiencies impair monocyte, dendritic, and B cell development but not NK cell development. This study reveals key differences in the role of FLT3L in humans versus mice.

## Highlights

* Human FLT3L deficiency underlies bone-marrow failure and infectious diseases

* Human and mouse FLT3L deficiencies impair dendritic and B cell development

* FLT3L deficiency impairs monocyte development in humans but not mice

* FLT3L deficiency impairs NK cell development in mice but not in humans

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

# FLT3L governs the development of partially overlapping hematopoietic lineages in humans and mice

Mana Momenilandi,<sup>1,2</sup> Romain Lévy,<sup>1,2,3,59</sup> Steicy Sobrino,<sup>4,5,59</sup> Jingwei Li,<sup>6,7,59</sup> Chantal Lagresle-Peyrou,<sup>2,8,59</sup>

Hossein Esmaeilzadeh,<sup>9,10,59</sup> Antoine Fayand,<sup>1,2,11,60</sup> Corentin Le Floc'h,<sup>1,2,60</sup> Antoine Guérin,<sup>12,13,60</sup>

Erika Della Mina,<sup>12,13,60</sup> Debra Shearer,<sup>6,7,60</sup> Ottavia M. Delmonte,<sup>14,60</sup> Ahmad Yatim,<sup>15,60</sup> Kevin Mulder,<sup>16,17,60</sup>

*(Author list continued on next page)*

<sup>1</sup>Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM UMR 1163, Necker Hospital for Sick Children, Paris, France

<sup>2</sup>Paris Cité University, Imagine Institute, Paris, France

<sup>3</sup>Pediatric Hematology-Immunology and Rheumatology Unit, Necker Hospital for Sick Children, AP-HP, Paris, France

<sup>4</sup>Laboratory of Chromatin and Gene Regulation During Development, Paris Cité University, UMR1163 INSERM, Imagine Institute, Paris, France

<sup>5</sup>Laboratory of Human Lymphohematopoiesis, INSERM, Imagine Institute, Paris, France

<sup>6</sup>Jake Gittlen Laboratories for Cancer Research, Pennsylvania State University College of Medicine, Hershey, PA, USA

<sup>7</sup>Department of Pathology and Laboratory Medicine, Pennsylvania State University College of Medicine, Hershey, PA, USA

<sup>8</sup>Biotherapy Clinical Investigation Center, Groupe Hospitalier Universitaire Ouest, AP-HP, INSERM, Paris, France

<sup>9</sup>Allergy Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

<sup>10</sup>Department of Allergy and Clinical Immunology, Namazi Hospital, Shiraz University of Medical Sciences, Shiraz, Iran

<sup>11</sup>Sorbonne University, AP-HP, Tenon Hospital, Department of Internal Medicine, Paris, France

<sup>12</sup>Garvan Institute of Medical Research, Darlinghurst, NSW, Australia

<sup>13</sup>St. Vincent's Clinical School, Faculty of Medicine, University of NSW, Sydney, NSW, Australia

<sup>14</sup>Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

<sup>15</sup>St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY, USA

<sup>16</sup>Gustave Roussy Cancer Campus, Villejuif, France

<sup>17</sup>Paris-Saclay University, Ile-de-France, France

<sup>18</sup>Dahdaleh Institute of Genomic Medicine, McGill University, Montreal, QC, Canada

<sup>19</sup>Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada

<sup>20</sup>Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey, PA, USA

<sup>21</sup>Department of Medical Biotechnology, School of Medicine, Zanjan University of Medical Sciences (ZUMS), Zanjan, Iran

<sup>22</sup>School of Mathematics, Institute for Research in Fundamental Sciences (IPM), Tehran, Iran

<sup>23</sup>Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden

*(Affiliations continued on next page)*

## SUMMARY

FMS-related tyrosine kinase 3 ligand (FLT3L), encoded by FLT3LG, is a hematopoietic factor essential for the development of natural killer (NK) cells, B cells, and dendritic cells (DCs) in mice. We describe three humans homozygous for a loss-of-function FLT3LG variant with a history of various recurrent infections, including severe cutaneous warts. The patients' bone marrow (BM) was hypoplastic, with low levels of hematopoietic progenitors, particularly myeloid and B cell precursors. Counts of B cells, monocytes, and DCs were low in the patients' blood, whereas the other blood subsets, including NK cells, were affected only moderately, if at all. The patients had normal counts of Langerhans cells (LCs) and dermal macrophages in the skin but lacked dermal DCs. Thus, FLT3L is required for B cell and DC development in mice and humans. However, unlike its murine counterpart, human FLT3L is required for the development of monocytes but not NK cells.

## INTRODUCTION

Hematopoiesis is the process of blood cell formation from self-renewing hematopoietic stem cells (HSCs).¹ HSC differentiation

is driven by molecular cues leading to a gradual commitment to specific lineages and subsets through phenotypically well-defined stages. Recent advances suggest that lineage commitment occurs along an increasingly stable unipotent trajectory,

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

# FLT3L governs the development of partially overlapping hematopoietic lineages in humans and mice

Mana Momenilandi,<sup>1,2</sup> Romain Lévy,<sup>1,2,3,59</sup> Steicy Sobrino,<sup>4,5,59</sup> Jingwei Li,<sup>6,7,59</sup> Chantal Lagresle-Peyrou,<sup>2,8,59</sup>

Hossein Esmaeilzadeh,<sup>9,10,59</sup> Antoine Fayand,<sup>1,2,11,60</sup> Corentin Le Floc'h,<sup>1,2,60</sup> Antoine Guérin,<sup>12,13,60</sup>

Erika Della Mina,<sup>12,13,60</sup> Debra Shearer,<sup>6,7,60</sup> Ottavia M. Delmonte,<sup>14,60</sup> Ahmad Yatim,<sup>15,60</sup> Kevin Mulder,<sup>16,17,60</sup>

*(Author list continued on next page)*

<sup>1</sup>Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM UMR 1163, Necker Hospital for Sick Children, Paris, France

<sup>2</sup>Paris Cité University, Imagine Institute, Paris, France

<sup>3</sup>Pediatric Hematology-Immunology and Rheumatology Unit, Necker Hospital for Sick Children, AP-HP, Paris, France

<sup>4</sup>Laboratory of Chromatin and Gene Regulation During Development, Paris Cité University, UMR1163 INSERM, Imagine Institute, Paris, France

<sup>5</sup>Laboratory of Human Lymphohematopoiesis, INSERM, Imagine Institute, Paris, France

<sup>6</sup>Jake Gittlen Laboratories for Cancer Research, Pennsylvania State University College of Medicine, Hershey, PA, USA

<sup>7</sup>Department of Pathology and Laboratory Medicine, Pennsylvania State University College of Medicine, Hershey, PA, USA

<sup>8</sup>Biotherapy Clinical Investigation Center, Groupe Hospitalier Universitaire Ouest, AP-HP, INSERM, Paris, France

<sup>9</sup>Allergy Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

<sup>10</sup>Department of Allergy and Clinical Immunology, Namazi Hospital, Shiraz University of Medical Sciences, Shiraz, Iran

<sup>11</sup>Sorbonne University, AP-HP, Tenon Hospital, Department of Internal Medicine, Paris, France

<sup>12</sup>Garvan Institute of Medical Research, Darlinghurst, NSW, Australia

<sup>13</sup>St. Vincent's Clinical School, Faculty of Medicine, University of NSW, Sydney, NSW, Australia

<sup>14</sup>Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

<sup>15</sup>St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY, USA

<sup>16</sup>Gustave Roussy Cancer Campus, Villejuif, France

<sup>17</sup>Paris-Saclay University, Ile-de-France, France

<sup>18</sup>Dahdaleh Institute of Genomic Medicine, McGill University, Montreal, QC, Canada

<sup>19</sup>Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada

<sup>20</sup>Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey, PA, USA

<sup>21</sup>Department of Medical Biotechnology, School of Medicine, Zanjan University of Medical Sciences (ZUMS), Zanjan, Iran

<sup>22</sup>School of Mathematics, Institute for Research in Fundamental Sciences (IPM), Tehran, Iran

<sup>23</sup>Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden

*(Affiliations continued on next page)*

## SUMMARY

FMS-related tyrosine kinase 3 ligand (FLT3L), encoded by FLT3LG, is a hematopoietic factor essential for the development of natural killer (NK) cells, B cells, and dendritic cells (DCs) in mice. We describe three humans homozygous for a loss-of-function FLT3LG variant with a history of various recurrent infections, including severe cutaneous warts. The patients' bone marrow (BM) was hypoplastic, with low levels of hematopoietic progenitors, particularly myeloid and B cell precursors. Counts of B cells, monocytes, and DCs were low in the patients' blood, whereas the other blood subsets, including NK cells, were affected only moderately, if at all. The patients had normal counts of Langerhans cells (LCs) and dermal macrophages in the skin but lacked dermal DCs. Thus, FLT3L is required for B cell and DC development in mice and humans. However, unlike its murine counterpart, human FLT3L is required for the development of monocytes but not NK cells.

## INTRODUCTION

Hematopoiesis is the process of blood cell formation from self-renewing hematopoietic stem cells (HSCs).¹ HSC differentiation

is driven by molecular cues leading to a gradual commitment to specific lineages and subsets through phenotypically well-defined stages. Recent advances suggest that lineage commitment occurs along an increasingly stable unipotent trajectory,

<figure><img src="image_4.png" /></figure>
Mathieu Mancini,¹⁸,¹⁹,⁶⁰ Darawan Rinchai,¹⁵,⁶⁰ Adeline Denis,⁵ Anna-Lena Neehus,¹,² Karla Balogh,⁶,⁷ Sarah Brendle,⁶,⁷,²⁰

Hassan Rokni-Zadeh,²¹ Majid Changi-Ashtiani,²² Yoann Seeleuthner,¹,² Caroline Deswarte,¹,² Boris Bessot,²,⁸

Cassandre Cremades,¹ Marie Materna,¹,² Axel Cederholm,²³ Masato Ogishi,¹⁵ Quentin Philippot,¹,² Omer Beganovic,<fnref n="24" />

Mania Ackermann,<fnref n="25" />,<fnref n="26" />,<fnref n="27" /> Margareta Wuyts,<fnref n="28" /> Taushif Khan,<fnref n="29" /> Sébastien Fouéré,<fnref n="30" /> Florian Herms,<fnref n="31" /> Johan Chanal,<fnref n="32" />

Boaz Palterer,¹⁴ Julie Bruneau,<fnref n="33" /> Thierry J. Molina,<fnref n="33" /> Stéphanie Leclerc-Mercier,<fnref n="33" /> Jean-Luc Prétet,<fnref n="34" /> Leila Youssefian,<fnref n="35" />

Hassan Vahidnezhad,<fnref n="36" />,<fnref n="37" />,<fnref n="38" /> Nima Parvaneh,<fnref n="39" /> Kristl G. Claeys,<fnref n="40" />,<fnref n="41" /> Rik Schrijvers,<fnref n="42" /> Marine Luka,<fnref n="43" /> Philippe Pérot,<fnref n="44" />

Jacques Fourgeaud,<fnref n="45" />,<fnref n="46" /> Céline Nourrisson,<fnref n="47" />,<fnref n="48" /> Philippe Poirier,<fnref n="47" />,<fnref n="48" /> Emmanuelle Jouanguy,¹,²,¹⁵

Stéphanie Boisson-Dupuis,¹,²,¹⁵ Jacinta Bustamante,¹,²,¹⁵,<fnref n="49" /> Luigi D. Notarangelo,¹⁴ Neil Christensen,⁶,⁷

Nils Landegren,²³,<fnref n="50" /> Laurent Abel,¹,²,¹⁵ Nico Marr,<fnref n="29" />,<fnref n="51" /> Emmanuelle Six,⁸ David Langlais,¹⁸,¹⁹,<fnref n="52" /> Tim Waterboer,<fnref n="53" />

(Author list continued on next page)

(Affiliations continued on next page)

with a limited amount of lateral transition during the successive progenitor steps.²,³ Inborn errors of human hematopoiesis can underlie a broad spectrum of non-exclusive phenotypes, ranging from various cell deficits, including anemia, thrombocytopenia, and leukopenia, to various hematopoietic malignancies, including myeloid and lymphoid leukemias. Mutations impairing the fitness of HSCs and early progenitors result in a broad impairment of hematopoiesis, as exemplified by reticular dysgenesis, which was first reported in 1959.⁴,⁵ Other inborn errors selectively block later stages of hematopoiesis, such as the

development of granulocytes (severe congenital neutropenia) and B cells (agammaglobulinemia), two conditions widely considered to be the first two inborn errors of immunity to be described in 1950⁶⁻⁸ and 1952, respectively.⁹,¹⁰

The hematopoietic growth factor Fms-like tyrosine kinase 3 ligand (FLT3L), encoded by FLT3LG, operates via its receptor FLT3.¹¹ Both molecules were identified in mice and humans in the early 1990s.¹²⁻¹⁷ FLT3 is expressed on the surface of early hematopoietic precursors. FLT3L is broadly expressed, with levels highest in stromal bone marrow (BM) cells and T

<footnote n="24">²⁴Laboratoire d'Onco-hématologie, Necker Hospital for Sick Children, AP-HP, Paris, France</footnote>

<footnote n="25">²⁵Hannover Medical School, Department of Pediatric Pulmonology, Allergology and Neonatology, Hannover, Germany</footnote>

<footnote n="26">²⁶Hannover Medical School, Cluster of Excellence RESIST (EXC 2155), Hannover, Germany</footnote>

<footnote n="27">²⁷Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), Hannover, Germany</footnote>

<footnote n="28">²⁸Department of Microbiology and Immunology, Clinical and Diagnostic Immunology, KU Leuven, Leuven, Belgium</footnote>

<footnote n="29">²⁹Research Branch, Sidra Medicine, Doha, Qatar</footnote>

<footnote n="30">³⁰Groupe Hospitalier Saint-Louis, Lariboisière, Fernand-Widal, CeGIDD, AP-HP, Paris, France</footnote>

<footnote n="31">³¹Dermatology Department, Paris-Cité University, INSERM 976, Saint Louis Hospital, Paris, France</footnote>

<footnote n="32">³²Dermatology Department, Cochin Hospital, INSERM U1016, AP-HP, Paris, France</footnote>

<footnote n="33">³³Department of Pathology, Necker Hospital for Sick Children, AP-HP, Paris-Cité University, Paris, France</footnote>

<footnote n="34">³⁴Papillomavirus National Reference Center, Besançon Hospital, Besançon, France</footnote>

<footnote n="35">³⁵Department of Pathology and Laboratory Medicine, UCLA Clinical Genomics Center, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA</footnote>

<footnote n="36">³⁶Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA</footnote>

<footnote n="37">³⁷Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA</footnote>

<footnote n="38">³⁸Center for Applied Genomics, Children's Hospital of Philadelphia, Philadelphia, PA, USA</footnote>

<footnote n="39">³⁹Department of Pediatrics, Children's Medical Center, Tehran University of Medical Sciences, Tehran, Iran</footnote>

<footnote n="40">⁴⁰Department of Neurology, University Hospitals Leuven, Leuven, Belgium</footnote>

<footnote n="41">⁴¹Laboratory for Muscle Diseases and Neuropathies, Department of Neurosciences, KU Leuven, and Leuven Brain Institute (LBI), Leuven, Belgium</footnote>

<footnote n="42">⁴²Department of Microbiology, Immunology and Transplantation, Allergy and Clinical Immunology Research Group, KU Leuven, Leuven, Belgium</footnote>

<footnote n="43">⁴³Labtech Single-Cell@Imagine, Imagine Institute, INSERM UMR 1163, 75015 Paris, France</footnote>

<footnote n="44">⁴⁴Pathogen Discovery Laboratory, Institut Pasteur, Paris Cité University, Paris, France</footnote>

<footnote n="45">⁴⁵Paris Cité University, URP 7328 FETUS, Paris, France</footnote>

<footnote n="46">⁴⁶Microbiology Department, AP-HP, Necker Hospital for Sick Children, Paris, France</footnote>

<footnote n="47">⁴⁷Clermont Auvergne University, INSERM U1071, M2iSH, USC INRAE 1382, CHU Clermont-Ferrand, 3IHP, Department of Parasitology-Mycology, Clermont-Ferrand, France</footnote>

<footnote n="48">⁴⁸National Reference Center for Cryptosporidiosis, Microsporidia and Other Digestive Protozoa, Clermont-Ferrand, France</footnote>

<footnote n="49">⁴⁹Study Center for Primary Immunodeficiencies, Necker Hospital for Sick Children, AP-HP, Paris, France</footnote>

<footnote n="50">⁵⁰Centre for Molecular Medicine, Department of Medicine, Karolinska Institutet, Stockholm, Sweden</footnote>

<footnote n="51">⁵¹College of Health and Life Sciences, Hamad Bin Khalifa University, Doha, Qatar</footnote>

<footnote n="52">⁵²Department of Human Genetics, McGill University, Montreal, QC, Canada</footnote>

<footnote n="53">⁵³Infections and Cancer Epidemiology, Infection, Inflammation and Cancer Program, German Cancer Research Center (DKFZ), Heidelberg, Germany</footnote>

<footnote n="54">⁵⁴Laboratory of Inborn Errors of Immunity, Department of Microbiology, Immunology and Transplantation, KU Leuven, Leuven, Belgium</footnote>

<footnote n="55">⁵⁵Department of Pediatrics, Leuven University Hospitals, Leuven, Belgium</footnote>

<footnote n="56">⁵⁶Specialized Immunology Laboratory of Dr. Shahrooei, Tehran, Iran</footnote>
Mathieu Mancini,¹⁸,¹⁹,⁶⁰ Darawan Rinchai,¹⁵,⁶⁰ Adeline Denis,⁵ Anna-Lena Neehus,¹,² Karla Balogh,⁶,⁷ Sarah Brendle,⁶,⁷,²⁰

Hassan Rokni-Zadeh,²¹ Majid Changi-Ashtiani,²² Yoann Seeleuthner,¹,² Caroline Deswarte,¹,² Boris Bessot,²,⁸

Cassandre Cremades,¹ Marie Materna,¹,² Axel Cederholm,²³ Masato Ogishi,¹⁵ Quentin Philippot,¹,² Omer Beganovic,²⁴

Mania Ackermann,²⁵,²⁶,²⁷ Margareta Wuyts,²⁸ Taushif Khan,²⁹ Sébastien Fouéré,³⁰ Florian Herms,³¹ Johan Chanal,³²

Boaz Palterer,¹⁴ Julie Bruneau,³³ Thierry J. Molina,³³ Stéphanie Leclerc-Mercier,³³ Jean-Luc Prétet,³⁴ Leila Youssefian,³⁵

Hassan Vahidnezhad,³⁶,³⁷,³⁸ Nima Parvaneh,³⁹ Kristl G. Claeys,⁴⁰,⁴¹ Rik Schrijvers,⁴² Marine Luka,⁴³ Philippe Pérot,⁴⁴

Jacques Fourgeaud,⁴⁵,⁴⁶ Céline Nourrisson,⁴⁷,⁴⁸ Philippe Poirier,⁴⁷,⁴⁸ Emmanuelle Jouanguy,¹,²,¹⁵

Stéphanie Boisson-Dupuis,¹,²,¹⁵ Jacinta Bustamante,¹,²,¹⁵,⁴⁹ Luigi D. Notarangelo,¹⁴ Neil Christensen,⁶,⁷

Nils Landegren,²³,⁵⁰ Laurent Abel,¹,²,¹⁵ Nico Marr,²⁹,⁵¹ Emmanuelle Six,⁸ David Langlais,¹⁸,¹⁹,⁵² Tim Waterboer,⁵³

(Author list continued on next page)

²⁴Laboratoire d'Onco-hématologie, Necker Hospital for Sick Children, AP-HP, Paris, France

²⁵Hannover Medical School, Department of Pediatric Pulmonology, Allergology and Neonatology, Hannover, Germany

²⁶Hannover Medical School, Cluster of Excellence RESIST (EXC 2155), Hannover, Germany

²⁷Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), Hannover, Germany

²⁸Department of Microbiology and Immunology, Clinical and Diagnostic Immunology, KU Leuven, Leuven, Belgium

²⁹Research Branch, Sidra Medicine, Doha, Qatar

³⁰Groupe Hospitalier Saint-Louis, Lariboisière, Fernand-Widal, CeGIDD, AP-HP, Paris, France

³¹Dermatology Department, Paris-Cité University, INSERM 976, Saint Louis Hospital, Paris, France

³²Dermatology Department, Cochin Hospital, INSERM U1016, AP-HP, Paris, France

³³Department of Pathology, Necker Hospital for Sick Children, AP-HP, Paris-Cité University, Paris, France

³⁴Papillomavirus National Reference Center, Besançon Hospital, Besançon, France

³⁵Department of Pathology and Laboratory Medicine, UCLA Clinical Genomics Center, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

³⁶Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

³⁷Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

³⁸Center for Applied Genomics, Children's Hospital of Philadelphia, Philadelphia, PA, USA

³⁹Department of Pediatrics, Children's Medical Center, Tehran University of Medical Sciences, Tehran, Iran

⁴⁰Department of Neurology, University Hospitals Leuven, Leuven, Belgium

⁴¹Laboratory for Muscle Diseases and Neuropathies, Department of Neurosciences, KU Leuven, and Leuven Brain Institute (LBI), Leuven, Belgium

⁴²Department of Microbiology, Immunology and Transplantation, Allergy and Clinical Immunology Research Group, KU Leuven, Leuven, Belgium

⁴³Labtech Single-Cell@Imagine, Imagine Institute, INSERM UMR 1163, 75015 Paris, France

⁴⁴Pathogen Discovery Laboratory, Institut Pasteur, Paris Cité University, Paris, France

⁴⁵Paris Cité University, URP 7328 FETUS, Paris, France

⁴⁶Microbiology Department, AP-HP, Necker Hospital for Sick Children, Paris, France

⁴⁷Clermont Auvergne University, INSERM U1071, M2iSH, USC INRAE 1382, CHU Clermont-Ferrand, 3IHP, Department of Parasitology-Mycology, Clermont-Ferrand, France

⁴⁸National Reference Center for Cryptosporidiosis, Microsporidia and Other Digestive Protozoa, Clermont-Ferrand, France

⁴⁹Study Center for Primary Immunodeficiencies, Necker Hospital for Sick Children, AP-HP, Paris, France

⁵⁰Centre for Molecular Medicine, Department of Medicine, Karolinska Institutet, Stockholm, Sweden

⁵¹College of Health and Life Sciences, Hamad Bin Khalifa University, Doha, Qatar

⁵²Department of Human Genetics, McGill University, Montreal, QC, Canada

⁵³Infections and Cancer Epidemiology, Infection, Inflammation and Cancer Program, German Cancer Research Center (DKFZ), Heidelberg, Germany

⁵⁴Laboratory of Inborn Errors of Immunity, Department of Microbiology, Immunology and Transplantation, KU Leuven, Leuven, Belgium

⁵⁵Department of Pediatrics, Leuven University Hospitals, Leuven, Belgium

⁵⁶Specialized Immunology Laboratory of Dr. Shahrooei, Tehran, Iran

(Affiliations continued on next page)

with a limited amount of lateral transition during the successive progenitor steps.²,³ Inborn errors of human hematopoiesis can underlie a broad spectrum of non-exclusive phenotypes, ranging from various cell deficits, including anemia, thrombocytopenia, and leukopenia, to various hematopoietic malignancies, including myeloid and lymphoid leukemias. Mutations impairing the fitness of HSCs and early progenitors result in a broad impairment of hematopoiesis, as exemplified by reticular dysgenesis, which was first reported in 1959.⁴,⁵ Other inborn errors selectively block later stages of hematopoiesis, such as the

development of granulocytes (severe congenital neutropenia) and B cells (agammaglobulinemia), two conditions widely considered to be the first two inborn errors of immunity to be described in 1950⁶⁻⁸ and 1952, respectively.⁹,¹⁰

The hematopoietic growth factor Fms-like tyrosine kinase 3 ligand (FLT3L), encoded by FLT3LG, operates via its receptor FLT3.¹¹ Both molecules were identified in mice and humans in the early 1990s.¹²⁻¹⁷ FLT3 is expressed on the surface of early hematopoietic precursors. FLT3L is broadly expressed, with levels highest in stromal bone marrow (BM) cells and T
Florent Ginhoux,<sup>16,17</sup> Cindy S. Ma,<sup>12,13</sup> Stuart G. Tangye,<sup>12,13</sup> Isabelle Meyts,<sup>54,55</sup> Nico Lachmann,<sup>25,26,27</sup> Jiafen Hu,<sup>6,7</sup> Mohammad Shahrooee,<sup>28,56,61</sup> Xavier Bossuyt,<sup>28,57,61</sup> Jean-Laurent Casanova,<sup>1,2,3,15,58,61</sup> and Vivien Béziat<sup>1,2,15,61,62,*</sup>

lymphocytes.<sup>17,18</sup> FLT3L is a transmembrane molecule that can act as a soluble cytokine after proteolytic release of its extracellular domain (EC).<sup>16,19,20</sup> Soluble FLT3L binds FLT3 to form signal-transducing homodimers, inducing the proliferation of early hematopoietic precursors.<sup>21</sup> In mice, *Flt3lg* knockout is associated with ~40% lower leukocyte counts in the blood and the spleen, ~80% lower splenic natural killer (NK) cell counts, ~70% lower splenic B cell counts, and an almost complete absence of conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs) in lymphoid and non-lymphoid tissues.<sup>22-25</sup> To date, the essential and redundant roles of human FLT3L remain unclear because humans with genetic defects resulting in a deficiency of the ligand or its receptor have never been described.

## RESULTS

### Three siblings with viral, bacterial, and fungal infections

We studied three siblings (P1, P2, and P3) born in 1989, 1991, and 1995, respectively, to consanguineous Iranian parents (Figure 1A; Table S1). From the age of 4 years, they suffered from recurrent episodes of viral, bacterial, and fungal infections. The bacterial infections included pneumonia, otitis media, pharyngitis, and cellulitis, leading to conductive hearing loss with confirmed bilateral otosclerosis in P2 and P3. The patients presented viral infections, including chronic oral herpes simplex virus (HSV) infection, high levels of asymptomatic Epstein-Barr virus (EBV) viremia (P2 and P3), high levels of human *Parechovirus* 1 (HPEV-1, *Parechovirus A*) replication in blood (P2) and stools (P2 and P3), and human papillomavirus (HPV)-driven skin lesions, including recalcitrant and disseminated HPV2<sup>+</sup> common warts (P1-P3), HPV5<sup>+</sup> epidermodysplasia verruciformis (P3), and HPV7<sup>+</sup> genital warts (P3) (Figures 1B and S1A). In 2022, P2 had mild COVID-19 after two injections of the Sinopharm whole inactivated vaccine. All patients had recurrent diarrhea from early infancy and failure to thrive. However, P2 and P3 excreted high loads of microsporidia (*Enterocytozoon bieneusi*) and displayed signs suggestive of chronic cholangitis on ultrasound (i.e., biliary tract dilation) at the ages of 27 and 23 years, respectively. P1 and P3 developed seronegative polyarthritis at the age of 10 years. None of the three patients had any allergies or cancers. P1 died following a car accident at the age of 29 years. P2 and P3 are alive and are currently 32 and 28 years old, respectively. None of these patients suffered an adverse reaction after vaccination with several live attenuated vaccines in accordance with the national immunization program in Iran (i.e., following vaccination with bacillus Calmette-Guérin [BCG],

oral polio, and measles, mumps, and rubella [MMR] vaccines). The three patients, thus, displayed broad susceptibility to infection, with HPV in particular.

### A homozygous frameshift variant of FLT3LG

We performed whole-exome sequencing (WES) for the entire family (the three patients, both parents, and two healthy siblings aged 38 and 35 years). The high percent homozygosity of P1 (5.5%), P2 (3.2%), and P3 (5.8%) on WES confirmed parental consanguinity. We performed a linkage analysis on WES data for the entire kindred, testing the hypothesis of an autosomal recessive (AR) trait with complete penetrance. Significant evidence of linkage was obtained for six regions (logarithm of the odds [LOD] score > 2.5) (Figure 1C). Within the linked regions, non-synonymous variants that were both biallelic and rare (minor allele frequency [MAF] < 0.01; Figures S1B and S1C) were detected for only seven protein-coding genes. Only one of these variants was predicted to be loss-of-function (pLOF), and this variant had never been reported in the homozygous state in any public database. This variant, confirmed by targeted Sanger sequencing (Figure S1D), was a single-nucleotide homozygous frameshift deletion leading to a premature stop codon (p.Ser118-Alafs*23) in the FLT3LG gene (c.343delC). *In silico* analysis predicted the variant to be damaging, with a combined annotation-dependent depletion (CADD) score of 34, well above the mutation significance cutoff (MSC) of 3.13 for this gene (Figure 1D).<sup>27,28</sup> The variant was reported in the heterozygous state, with an allele frequency of 0.00001987 in gnomAD V3.1.2<sup>29</sup> and 0.00015 in our in-house Iranian cohort of more than 6,000 individuals. Neither this nor any other pLOF variant was reported in the homozygous state in public databases.<sup>29-33</sup> There is only one missense FLT3LG variant present in the homozygous state in public databases (p.Ser22Pro), with an allele frequency of 0.00005920 in gnomAD V3.1.2 (Figure 1D). Accordingly, the CoNeS score of FLT3LG is consistent with a gene under strong negative selection (Figure 1E).<sup>26</sup> Moreover, we showed that *Flt3lg*<sup>-/-</sup> mice were vulnerable to the murine papillomavirus (MmuPV1) (Figure S2). These findings suggested that the homozygous c.343delC (p.Ser118Alafs*23) variant of FLT3LG might underlie the clinical phenotypes of these three patients.

### Human FLT3LG encodes at least seven transcripts

The FLT3LG mRNA is detectable in most tissues<sup>34-37</sup> but its levels are highest in T cells. Alternative splicing events are frequent for the FLT3LG mRNA, as attested by RNA sequencing (RNA-seq) data obtained with human naive CD8<sup>+</sup> T cells (Figure S3A), but the relative abundance and function of the

<footnote n="57"><sup>57</sup>Department of Laboratory Medicine, University Hospitals Leuven, Leuven, Belgium</footnote>

<footnote n="58"><sup>58</sup>Howard Hughes Medical Institute, New York, NY, USA</footnote>

<footnote n="59"><sup>59</sup>These authors contributed equally</footnote>

<footnote n="60"><sup>60</sup>These authors contributed equally</footnote>

<footnote n="61"><sup>61</sup>These authors contributed equally</footnote>

<footnote n="62"><sup>62</sup>Lead contact</footnote>

<footnote>*Correspondence: vivien.beziat@inserm.fr</footnote>

<footnote>https://doi.org/10.1016/j.cell.2024.04.009</footnote>
Florent Ginhoux,<sup>16,17</sup> Cindy S. Ma,<sup>12,13</sup> Stuart G. Tangye,<sup>12,13</sup> Isabelle Meyts,<sup>54,55</sup> Nico Lachmann,<sup>25,26,27</sup> Jiafen Hu,<sup>6,7</sup> Mohammad Shahrooee,<sup>28,56,61</sup> Xavier Bossuyt,<sup>28,57,61</sup> Jean-Laurent Casanova,<sup>1,2,3,15,58,61</sup> and Vivien Béziat<sup>1,2,15,61,62,*</sup>

lymphocytes.<sup>17,18</sup> FLT3L is a transmembrane molecule that can act as a soluble cytokine after proteolytic release of its extracellular domain (EC).<sup>16,19,20</sup> Soluble FLT3L binds FLT3 to form signal-transducing homodimers, inducing the proliferation of early hematopoietic precursors.<sup>21</sup> In mice, *Flt3lg* knockout is associated with ~40% lower leukocyte counts in the blood and the spleen, ~80% lower splenic natural killer (NK) cell counts, ~70% lower splenic B cell counts, and an almost complete absence of conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs) in lymphoid and non-lymphoid tissues.<sup>22-25</sup> To date, the essential and redundant roles of human FLT3L remain unclear because humans with genetic defects resulting in a deficiency of the ligand or its receptor have never been described.

## RESULTS

### Three siblings with viral, bacterial, and fungal infections

We studied three siblings (P1, P2, and P3) born in 1989, 1991, and 1995, respectively, to consanguineous Iranian parents (Figure 1A; Table S1). From the age of 4 years, they suffered from recurrent episodes of viral, bacterial, and fungal infections. The bacterial infections included pneumonia, otitis media, pharyngitis, and cellulitis, leading to conductive hearing loss with confirmed bilateral otosclerosis in P2 and P3. The patients presented viral infections, including chronic oral herpes simplex virus (HSV) infection, high levels of asymptomatic Epstein-Barr virus (EBV) viremia (P2 and P3), high levels of human *Parechovirus* 1 (HPEV-1, *Parechovirus A*) replication in blood (P2) and stools (P2 and P3), and human papillomavirus (HPV)-driven skin lesions, including recalcitrant and disseminated HPV2<sup>+</sup> common warts (P1-P3), HPV5<sup>+</sup> epidermodysplasia verruciformis (P3), and HPV7<sup>+</sup> genital warts (P3) (Figures 1B and S1A). In 2022, P2 had mild COVID-19 after two injections of the Sinopharm whole inactivated vaccine. All patients had recurrent diarrhea from early infancy and failure to thrive. However, P2 and P3 excreted high loads of microsporidia (*Enterocytozoon bieneusi*) and displayed signs suggestive of chronic cholangitis on ultrasound (i.e., biliary tract dilation) at the ages of 27 and 23 years, respectively. P1 and P3 developed seronegative polyarthritis at the age of 10 years. None of the three patients had any allergies or cancers. P1 died following a car accident at the age of 29 years. P2 and P3 are alive and are currently 32 and 28 years old, respectively. None of these patients suffered an adverse reaction after vaccination with several live attenuated vaccines in accordance with the national immunization program in Iran (i.e., following vaccination with bacillus Calmette-Guérin [BCG],

oral polio, and measles, mumps, and rubella [MMR] vaccines). The three patients, thus, displayed broad susceptibility to infection, with HPV in particular.

### A homozygous frameshift variant of FLT3LG

We performed whole-exome sequencing (WES) for the entire family (the three patients, both parents, and two healthy siblings aged 38 and 35 years). The high percent homozygosity of P1 (5.5%), P2 (3.2%), and P3 (5.8%) on WES confirmed parental consanguinity. We performed a linkage analysis on WES data for the entire kindred, testing the hypothesis of an autosomal recessive (AR) trait with complete penetrance. Significant evidence of linkage was obtained for six regions (logarithm of the odds [LOD] score > 2.5) (Figure 1C). Within the linked regions, non-synonymous variants that were both biallelic and rare (minor allele frequency [MAF] < 0.01; Figures S1B and S1C) were detected for only seven protein-coding genes. Only one of these variants was predicted to be loss-of-function (pLOF), and this variant had never been reported in the homozygous state in any public database. This variant, confirmed by targeted Sanger sequencing (Figure S1D), was a single-nucleotide homozygous frameshift deletion leading to a premature stop codon (p.Ser118-Alafs*23) in the FLT3LG gene (c.343delC). *In silico* analysis predicted the variant to be damaging, with a combined annotation-dependent depletion (CADD) score of 34, well above the mutation significance cutoff (MSC) of 3.13 for this gene (Figure 1D).<sup>27,28</sup> The variant was reported in the heterozygous state, with an allele frequency of 0.00001987 in gnomAD V3.1.2<sup>29</sup> and 0.00015 in our in-house Iranian cohort of more than 6,000 individuals. Neither this nor any other pLOF variant was reported in the homozygous state in public databases.<sup>29-33</sup> There is only one missense FLT3LG variant present in the homozygous state in public databases (p.Ser22Pro), with an allele frequency of 0.00005920 in gnomAD V3.1.2 (Figure 1D). Accordingly, the CoNeS score of FLT3LG is consistent with a gene under strong negative selection (Figure 1E).<sup>26</sup> Moreover, we showed that *Flt3lg*<sup>-/-</sup> mice were vulnerable to the murine papillomavirus (MmuPV1) (Figure S2). These findings suggested that the homozygous c.343delC (p.Ser118Alafs*23) variant of FLT3LG might underlie the clinical phenotypes of these three patients.

### Human FLT3LG encodes at least seven transcripts

The FLT3LG mRNA is detectable in most tissues<sup>34-37</sup> but its levels are highest in T cells. Alternative splicing events are frequent for the FLT3LG mRNA, as attested by RNA sequencing (RNA-seq) data obtained with human naive CD8<sup>+</sup> T cells (Figure S3A), but the relative abundance and function of the

<footnote n="57"><sup>57</sup>Department of Laboratory Medicine, University Hospitals Leuven, Leuven, Belgium</footnote>

<footnote n="58"><sup>58</sup>Howard Hughes Medical Institute, New York, NY, USA</footnote>

<footnote n="59"><sup>59</sup>These authors contributed equally</footnote>

<footnote n="60"><sup>60</sup>These authors contributed equally</footnote>

<footnote n="61"><sup>61</sup>These authors contributed equally</footnote>

<footnote n="62"><sup>62</sup>Lead contact</footnote>

<footnote>*Correspondence: vivien.beziat@inserm.fr</footnote>

<footnote>https://doi.org/10.1016/j.cell.2024.04.009</footnote>
<figure><img src="image_6.png" /><figcaption>**Figure 1.** Three patients homozygous for a frameshift FLT3LG variant</figcaption></figure>

(A) Pedigree showing the familial segregation of the c.343delC (p. Ser118Alafs*23) FLT3LG allele. Solid black symbols indicate patients with FLT3L deficiency. Symbols linked with a double line indicate consanguinity. M, mutant; WT, wild type.

(B) Cutaneous warts on the hands of P1, P2, and P3.

(C) Genome-wide linkage analysis on gDNA from 7 family members, assuming AR inheritance with complete penetrance. All 6 linked regions with a high LOD score are shown in blue and positioned on chromosomes 7, 19, and 20; one of the regions on chromosome 19 included FLT3LG (red arrow).

(D) Allele frequency and CADD score for the only FLT3LG variant reported in the homozygous state in public databases (showed in blue). The c.343delC variant is indicated in red. The dotted line corresponds to the mutation significance cutoff (MSC) for FLT3LG.

(E) CoNeS score of FLT3LG is consistent with an AR trait.²⁶

See also Figures S1 and S2.

alternative full-length mRNA transcripts have been studied in less detail in human cells than in mice.³⁵,³⁸,³⁹ The patients' variant is present in only some of these alternative splicing variants. We therefore studied the expression of FLT3LG transcripts in T cell blasts from healthy donors (HDs) (Figure S3B). We found that there were at least seven alternative transcripts (T1–T7), with frequencies between 4% and 34% of total FLT3LG mRNA. Together, the canonical T1/T2 transcripts accounted for only ≈34% of the total FLT3LG transcripts in control T cell blasts (Figures S3B and S3C). The first four coding exons (exons 2 to 5), which encode the first 114 amino acids of the canonical functional protein encoded by T1/T2, are conserved in all seven transcripts. The biological activity of FLT3L is conferred by the EC,¹⁷ so some non-canonical transcripts (T3–T7) may be expressed and functional.

**Two soluble isoforms of FLT3L are released by cleavage or secretion**

We investigated whether the seven FLT3LG transcripts were expressed in a HEK293T overexpression system. RT-qPCR revealed that all seven transcripts were expressed at similar levels (Figure S3D). By flow cytometry, we observed strong cell-surface expression of the canonical protein encoded by T1/T2, weak cell-surface expression of the proteins encoded by T3, T4, and T5, and no detectable cell-surface expression of the proteins encoded by T6 and T7 (Figure S3E). Western blotting (WB) on total cell extracts detected high levels of a protein with a mo-

lecular weight (MW) higher than predicted from the amino-acid sequences of T1/T2 and T7, probably due to posttranslational modifications (Figure S3F). T3, T4, and T5 protein levels were lower than those for T1/T2 and T7 (Figure S3F). No protein was detected for T6 (Figure S3F). Furthermore, immunoblotting with an N-terminal antibody or enzyme-linked immunosorbent assay (ELISA) revealed that only T1/T2 and T7 gave rise to detectable amounts of protein in the supernatant of HEK293T cells 48 h post transfection (Figures S3F and S3G). Release of the EC by enzymatic cleavage¹⁹ resulted in the truncated canonical protein encoded by T1/T2 having a lower MW in the supernatant than in the whole-cell extract (Figure S3F). This cleaved protein could not be detected with the anti-V5 antibody (against a C-terminal tag), whereas the T7 protein, which was secreted due to the lack of the cleavage site and the transmembrane domain (TM) (skipping of exon 7), was detectable (Figure S3F). Thus, these data show that human FLT3LG encodes two main isoforms. The first (the canonical protein encoded by T1 or T2) is expressed at the cell membrane and can be released into the supernatant by enzymatic cleavage. The second (encoded by T7) is secreted due to the absence of a TM.

**Only one transmembrane FLT3L and the secreted FLT3L are functional**

As a protein was detected for five isoforms (encoded by T1/T2, T3, T4, T5, and T7), we investigated the functional activity of these proteins *in vitro*. K562 cells stably transduced with FLT3,
<figure><img src="image_5.png" /><figcaption>**Figure 1.** Three patients homozygous for a frameshift FLT3LG variant</figcaption></figure>

(A) Pedigree showing the familial segregation of the c.343delC (p. Ser118Alafs*23) FLT3LG allele. Solid black symbols indicate patients with FLT3L deficiency. Symbols linked with a double line indicate consanguinity. M, mutant; WT, wild type.

(B) Cutaneous warts on the hands of P1, P2, and P3.

(C) Genome-wide linkage analysis on gDNA from 7 family members, assuming AR inheritance with complete penetrance. All 6 linked regions with a high LOD score are shown in blue and positioned on chromosomes 7, 19, and 20; one of the regions on chromosome 19 included FLT3LG (red arrow).

(D) Allele frequency and CADD score for the only FLT3LG variant reported in the homozygous state in public databases (showed in blue). The c.343delC variant is indicated in red. The dotted line corresponds to the mutation significance cutoff (MSC) for FLT3LG.

(E) CoNeS score of FLT3LG is consistent with an AR trait.²⁶

See also Figures S1 and S2.

alternative full-length mRNA transcripts have been studied in less detail in human cells than in mice.³⁵,³⁸,³⁹ The patients' variant is present in only some of these alternative splicing variants. We therefore studied the expression of FLT3LG transcripts in T cell blasts from healthy donors (HDs) (Figure S3B). We found that there were at least seven alternative transcripts (T1–T7), with frequencies between 4% and 34% of total FLT3LG mRNA. Together, the canonical T1/T2 transcripts accounted for only ≈34% of the total FLT3LG transcripts in control T cell blasts (Figures S3B and S3C). The first four coding exons (exons 2 to 5), which encode the first 114 amino acids of the canonical functional protein encoded by T1/T2, are conserved in all seven transcripts. The biological activity of FLT3L is conferred by the EC,¹⁷ so some non-canonical transcripts (T3–T7) may be expressed and functional.

**Two soluble isoforms of FLT3L are released by cleavage or secretion**

We investigated whether the seven FLT3LG transcripts were expressed in a HEK293T overexpression system. RT-qPCR revealed that all seven transcripts were expressed at similar levels (Figure S3D). By flow cytometry, we observed strong cell-surface expression of the canonical protein encoded by T1/T2, weak cell-surface expression of the proteins encoded by T3, T4, and T5, and no detectable cell-surface expression of the proteins encoded by T6 and T7 (Figure S3E). Western blotting (WB) on total cell extracts detected high levels of a protein with a mo-

lecular weight (MW) higher than predicted from the amino-acid sequences of T1/T2 and T7, probably due to posttranslational modifications (Figure S3F). T3, T4, and T5 protein levels were lower than those for T1/T2 and T7 (Figure S3F). No protein was detected for T6 (Figure S3F). Furthermore, immunoblotting with an N-terminal antibody or enzyme-linked immunosorbent assay (ELISA) revealed that only T1/T2 and T7 gave rise to detectable amounts of protein in the supernatant of HEK293T cells 48 h post transfection (Figures S3F and S3G). Release of the EC by enzymatic cleavage¹⁹ resulted in the truncated canonical protein encoded by T1/T2 having a lower MW in the supernatant than in the whole-cell extract (Figure S3F). This cleaved protein could not be detected with the anti-V5 antibody (against a C-terminal tag), whereas the T7 protein, which was secreted due to the lack of the cleavage site and the transmembrane domain (TM) (skipping of exon 7), was detectable (Figure S3F). Thus, these data show that human FLT3LG encodes two main isoforms. The first (the canonical protein encoded by T1 or T2) is expressed at the cell membrane and can be released into the supernatant by enzymatic cleavage. The second (encoded by T7) is secreted due to the absence of a TM.

**Only one transmembrane FLT3L and the secreted FLT3L are functional**

As a protein was detected for five isoforms (encoded by T1/T2, T3, T4, T5, and T7), we investigated the functional activity of these proteins *in vitro*. K562 cells stably transduced with FLT3,
<figure><img src="image_7.png" /><figcaption>Figure 2. The patients' FLT3LG mutation is loss-of-expression and loss-of-function in vitro</figcaption></figure>

(A) Schematic representation of the two functional FLT3L isoforms. The different domains are indicated. The position of the p.Ser118Alafs*23 mutation is indicated in red, and that of the p.Ser22Pro mutation is indicated in black. mFLT3L (mFL), membrane-bound FLT3L; sFLT3L (sFL), secreted FLT3L; SP, signal peptide; EC, extracellular domain; TM, transmembrane domain; IC, intracellular domain.

(B) Surface FLT3L expression in an overexpression system, as assessed by FACS on HEK293T cells 48 h after transfection with an empty vector (EV), or a vector encoding the WT sFL or mFL isoform, or the indicated FLT3LG variants.

(C) WB analysis of cell-culture supernatants or cell lysates obtained from HEK293T cells, as in (B).

(D) Soluble FLT3L protein determination by ELISA on the cell-culture supernatant of HEK293T cells, as in (B) and (C). Bars indicate the mean and SD.

(E) Phospho-FLT3 (pFLT3) levels in K562 cells transduced with FLT3, as assessed by WB, after 5 min stimulation with the supernatant collected from HEK293T cells 48 h after transfection. Recombinant human FLT3L (rhFLT3L) was used as a positive control. All the data shown are representative of three independent experiments.

See also Figures S3A–S3J.

the only known receptor of FLT3L, were stimulated with the sup-
ernatant of HEK293T cells transfected with an empty vector
(EV) or a construct containing one of the six coding sequences
(Figure S3H). With this system, consistent with the findings for
the supernatant of transfected HEK293T cells, the supernatants
of T1/T2 and T7 induced FLT3 phosphorylation to levels similar
to those achieved with a commercial human recombinant
FLT3L used as a positive control. As expected, no FLT3 phos-
phorylation was detected after the stimulation of K562 cells
with the supernatant of HEK293T cells transfected with T3, T4,
T5, or T6 (Figure S3H). In addition, despite weak expression at
the cell surface (Figure S3E), cells transfected with T3, T4, or
T5 were unable to induce FLT3 phosphorylation in K562 cells
when the two cell lines were incubated together to allow contact
between cells (Figure S3I). Thus, only the isoforms encoded by
T1/T2 and T7 were functional, both of which contain amino-
acid residues that are affected in the patients. We named the ca-
nonical surface-expressed FLT3L encoded by T1 or T2 mem-
brane-bound FLT3L (mFLT3L) (mFL) and the secreted FLT3L
growth factor encoded by T7 secreted FLT3L (sFLT3L) (sFL)
(Figure 2A).

The p.Ser118Alafs*23 variant is LOF in vitro

We assessed the impact of the p.Ser118Alafs*23 variant found in
the patients on FLT3L function *in vitro*. We transfected HEK293T
cells with an EV or a vector encoding mFL or sFL, with the wild-
type (WT), p.Ser118Alafs*23 (S118Afs), or p.Ser22Pro (S22P)
variant. In this system, RT-qPCR revealed that mRNA levels
were similar for all constructs (Figure S3J). However, unlike the
WT mFL or sFL, the p.Ser118Alafs*23 variant resulted in an
absence of both protein isoforms in flow cytometry experiments
and WBs on total cell extracts or cell-culture supernatants
(Figures 2B and 2C). A re-initiation of translation downstream
of the premature stop codons was excluded by immunoblotting
total cell extracts or cell-culture supernatant with a C-terminal
antibody against the V5 tag (Figure 2C). The absence of
p.Ser118Alafs*23 protein shedding or secretion into the super-
natant was confirmed by ELISA (Figure 2D). Furthermore, the su-
pernatant of HEK293T cells transfected with the mutant mFL or
sFL was unable to induce FLT3 phosphorylation in the K562 re-
porter assay (Figure 2E). As expected, both the membrane and
secreted isoforms carrying the p.Ser22Pro variant (the only cod-
ing variant found in the homozygous state in public databases)
<figure><img src="image_6.png" /><figcaption>Figure 2. The patients' FLT3LG mutation is loss-of-expression and loss-of-function in vitro</figcaption></figure>

(A) Schematic representation of the two functional FLT3L isoforms. The different domains are indicated. The position of the p.Ser118Alafs*23 mutation is indicated in red, and that of the p.Ser22Pro mutation is indicated in black. mFLT3L (mFL), membrane-bound FLT3L; sFLT3L (sFL), secreted FLT3L; SP, signal peptide; EC, extracellular domain; TM, transmembrane domain; IC, intracellular domain.

(B) Surface FLT3L expression in an overexpression system, as assessed by FACS on HEK293T cells 48 h after transfection with an empty vector (EV), or a vector encoding the WT sFL or mFL isoform, or the indicated FLT3LG variants.

(C) WB analysis of cell-culture supernatants or cell lysates obtained from HEK293T cells, as in (B).

(D) Soluble FLT3L protein determination by ELISA on the cell-culture supernatant of HEK293T cells, as in (B) and (C). Bars indicate the mean and SD.

(E) Phospho-FLT3 (pFLT3) levels in K562 cells transduced with FLT3, as assessed by WB, after 5 min stimulation with the supernatant collected from HEK293T cells 48 h after transfection. Recombinant human FLT3L (rhFLT3L) was used as a positive control. All the data shown are representative of three independent experiments.

See also Figures S3A–S3J.

the only known receptor of FLT3L, were stimulated with the sup-
ernatant of HEK293T cells transfected with an empty vector
(EV) or a construct containing one of the six coding sequences
(Figure S3H). With this system, consistent with the findings for
the supernatant of transfected HEK293T cells, the supernatants
of T1/T2 and T7 induced FLT3 phosphorylation to levels similar
to those achieved with a commercial human recombinant
FLT3L used as a positive control. As expected, no FLT3 phos-
phorylation was detected after the stimulation of K562 cells
with the supernatant of HEK293T cells transfected with T3, T4,
T5, or T6 (Figure S3H). In addition, despite weak expression at
the cell surface (Figure S3E), cells transfected with T3, T4, or
T5 were unable to induce FLT3 phosphorylation in K562 cells
when the two cell lines were incubated together to allow contact
between cells (Figure S3I). Thus, only the isoforms encoded by
T1/T2 and T7 were functional, both of which contain amino-
acid residues that are affected in the patients. We named the ca-
nonical surface-expressed FLT3L encoded by T1 or T2 mem-
brane-bound FLT3L (mFLT3L) (mFL) and the secreted FLT3L
growth factor encoded by T7 secreted FLT3L (sFLT3L) (sFL)
(Figure 2A).

The p.Ser118Alafs*23 variant is LOF in vitro

We assessed the impact of the p.Ser118Alafs*23 variant found in
the patients on FLT3L function *in vitro*. We transfected HEK293T
cells with an EV or a vector encoding mFL or sFL, with the wild-
type (WT), p.Ser118Alafs*23 (S118Afs), or p.Ser22Pro (S22P)
variant. In this system, RT-qPCR revealed that mRNA levels
were similar for all constructs (Figure S3J). However, unlike the
WT mFL or sFL, the p.Ser118Alafs*23 variant resulted in an
absence of both protein isoforms in flow cytometry experiments
and WBs on total cell extracts or cell-culture supernatants
(Figures 2B and 2C). A re-initiation of translation downstream
of the premature stop codons was excluded by immunoblotting
total cell extracts or cell-culture supernatant with a C-terminal
antibody against the V5 tag (Figure 2C). The absence of
p.Ser118Alafs*23 protein shedding or secretion into the super-
natant was confirmed by ELISA (Figure 2D). Furthermore, the su-
pernatant of HEK293T cells transfected with the mutant mFL or
sFL was unable to induce FLT3 phosphorylation in the K562 re-
porter assay (Figure 2E). As expected, both the membrane and
secreted isoforms carrying the p.Ser22Pro variant (the only cod-
ing variant found in the homozygous state in public databases)
<figure><img src="image_8.png" /><figcaption>Figure 3. Biological significance of the FLT3LG allele and rescue of the phenotype</figcaption></figure>

(A) Total mRNA was extracted from the T cell blasts of three HDs, a heterozygous control (the father), P2, and P3, and EBV-B cells from three HDs, both healthy siblings, P2, and P3. Total mRNA was subjected to RT-qPCR for the assessment of FLT3LG expression with two different probes. Data are displayed as 2⁻Δct values normalized against the expression of endogenous GUS. Bars represent the mean values and SD.

(B) Soluble FLT3L determination by ELISA in plasma samples from 11 HDs (all HDs were adults, male or female, and from multiple ethnic groups), the three heterozygous family members, P2, and P3. Bars indicate the mean and SD.

(C) Soluble FLT3L determination by ELISA on supernatant samples collected from T cell blasts from three adult HDs, the heterozygous father, P2, and P3. The medium (Med) from the T cell culture was used as a negative control. Bars represent the mean and SD.

(D) WB analysis of cell lysates obtained from T cell blasts from three unrelated HDs, P2, and P3. Vinculin was used as a loading control.

(E–G) T cell blasts from patients and controls were either left non-transduced (NT) or were transduced with lentiviruses generated with an empty vector (EV) or with vectors containing the WT sFL or mFL cDNA.

(E) Cell-surface FLT3L expression assessed by flow cytometry with a monoclonal Ab (EP1140Y) shown for P2 and one HD.

(F) WB analysis of FLT3L on T cell blasts from P2, P3, and two HDs. Vinculin was used as a loading control. The red arrow shows the band corresponding to the endogenous FLT3L.

(G) Soluble FLT3L protein determination by ELISA on the cell-culture supernatant of T cell blasts from P2, P3, and two HDs. Bars represent mean with SD. See also Figure S3K.

were produced in normal amounts and were functional (Figures 2B–2E), suggesting that AR FLT3L deficiency is exceedingly rare globally. Together, these data confirm that the p.Ser118Alafs*23 variant is LOF *in vitro* due to an impairment of the expression of both mFL and sFL.

## Abolition of endogenous FLT3L production

We tested the impact of the p.Ser118Alafs*23 genotype on the expression of endogenous FLT3LG in the patients’ plasma and cells. FLT3L expression in leukocytes is strongest in T lymphocytes.³⁹ We analyzed FLT3L expression in T cell blasts and EBV-transformed B cells. RT-qPCR showed that the p.Ser118Alafs*23 mutation did not affect endogenous FLT3LG mRNA levels (Figure 3A). Accordingly, in our cDNA cloning experiment with T cell blasts (Figure S3B), the distribution of transcripts (T1–T7) was similar in the patients and controls. However, FLT3L was undetectable by ELISA in the patients’ T cell blast culture supernatants and plasma, whereas it was detected in the controls and the heterozygous family members, in whom FLT3L levels were similar (Figures 3B and 3C). WBs of whole-cell lysate and flow cytometry for cell-surface expression detected low levels of FLT3L in control T cell blasts but not in T cell blasts from the patients (Figures 3D and 3E). We then stably transduced T cell blasts from P2 and P3 with a lentiviral vector encoding the WT mFL and sFL (Figures 3E–3G and S3K). As expected, only the WT mFL construct restored FLT3L expression at the cell surface of the patients’ T cell blasts, as demonstrated by flow cytometry (Figure 3E). WBs and ELISA showed that the WT mFL isoform restored FLT3L expression in cell lysates and cell-culture supernatants (Figures 3F and 3G). The WT sFL isoform restored protein detection by ELISA in the supernatant but not by WB on total cell lysates (Figures 3F and 3G), possibly due to immediate secretion of the protein. Endogenous FLT3L in plasma or the supernatant of T cell blasts from controls was unable to induce detectable FLT3
<figure><img src="image_7.png" /><figcaption>Figure 3. Biological significance of the FLT3LG allele and rescue of the phenotype</figcaption></figure>

(A) Total mRNA was extracted from the T cell blasts of three HDs, a heterozygous control (the father), P2, and P3, and EBV-B cells from three HDs, both healthy siblings, P2, and P3. Total mRNA was subjected to RT-qPCR for the assessment of FLT3LG expression with two different probes. Data are displayed as 2⁻Δct values normalized against the expression of endogenous GUS. Bars represent the mean values and SD.

(B) Soluble FLT3L determination by ELISA in plasma samples from 11 HDs (all HDs were adults, male or female, and from multiple ethnic groups), the three heterozygous family members, P2, and P3. Bars indicate the mean and SD.

(C) Soluble FLT3L determination by ELISA on supernatant samples collected from T cell blasts from three adult HDs, the heterozygous father, P2, and P3. The medium (Med) from the T cell culture was used as a negative control. Bars represent the mean and SD.

(D) WB analysis of cell lysates obtained from T cell blasts from three unrelated HDs, P2, and P3. Vinculin was used as a loading control.

(E–G) T cell blasts from patients and controls were either left non-transduced (NT) or were transduced with lentiviruses generated with an empty vector (EV) or with vectors containing the WT sFL or mFL cDNA.

(E) Cell-surface FLT3L expression assessed by flow cytometry with a monoclonal Ab (EP1140Y) shown for P2 and one HD.

(F) WB analysis of FLT3L on T cell blasts from P2, P3, and two HDs. Vinculin was used as a loading control. The red arrow shows the band corresponding to the endogenous FLT3L.

(G) Soluble FLT3L protein determination by ELISA on the cell-culture supernatant of T cell blasts from P2, P3, and two HDs. Bars represent mean with SD. See also Figure S3K.

were produced in normal amounts and were functional (Figures 2B–2E), suggesting that AR FLT3L deficiency is exceedingly rare globally. Together, these data confirm that the p.Ser118Alafs*23 variant is LOF in vitro due to an impairment of the expression of both mFL and sFL.

## Abolition of endogenous FLT3L production

We tested the impact of the p.Ser118Alafs*23 genotype on the expression of endogenous FLT3LG in the patients’ plasma and cells. FLT3L expression in leukocytes is strongest in T lymphocytes.³⁹ We analyzed FLT3L expression in T cell blasts and EBV-transformed B cells. RT-qPCR showed that the p.Ser118Alafs*23 mutation did not affect endogenous FLT3LG mRNA levels (Figure 3A). Accordingly, in our cDNA cloning experiment with T cell blasts (Figure S3B), the distribution of transcripts (T1–T7) was similar in the patients and controls. However, FLT3L was undetectable by ELISA in the patients’ T cell blast culture supernatants and plasma, whereas it was detected in the controls and the heterozygous family members, in whom FLT3L levels were similar (Figures 3B and 3C). WBs of whole-cell lysate and flow cytometry for cell-surface expression detected low levels of FLT3L in control T cell blasts but not in T cell blasts from the patients (Figures 3D and 3E). We then stably transduced T cell blasts from P2 and P3 with a lentiviral vector encoding the WT mFL and sFL (Figures 3E–3G and S3K). As expected, only the WT mFL construct restored FLT3L expression at the cell surface of the patients’ T cell blasts, as demonstrated by flow cytometry (Figure 3E). WBs and ELISA showed that the WT mFL isoform restored FLT3L expression in cell lysates and cell-culture supernatants (Figures 3F and 3G). The WT sFL isoform restored protein detection by ELISA in the supernatant but not by WB on total cell lysates (Figures 3F and 3G), possibly due to immediate secretion of the protein. Endogenous FLT3L in plasma or the supernatant of T cell blasts from controls was unable to induce detectable FLT3
<figure><img src="image_9.png" /><figcaption>C</figcaption></figure>

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

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

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

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

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

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

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

(legend on next page)
<figure><img src="image_8.png" /><figcaption>B</figcaption></figure>

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

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

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

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

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

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

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

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

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

(legend on next page)
phosphorylation in our K562 reporter assay (not shown) and could not be used to assess FLT3L activity at more physiological levels. Thus, homozygosity for the p.Ser118Alafs*23 variant underlies complete FLT3L deficiency in these patients. FLT3L expression can be restored by transducing the patients' cells with the WT mFL or sFL cDNA.

## FLT3L deficiency is associated with hypoplastic BM

Flt3lg-knockout mice have low levels of hematopoietic progenitors, especially DC progenitors (DCPs) and B cell precursors (BcellPs), in their BM.<sup>24,40</sup> We first studied BM smears from P2 and P3 to characterize the impact of FLT3L deficiency on cellularity and progenitor populations. BM cellularity was low in both patients, with lipid structures observed in the smears (Figure 4A). A myelogram revealed general medullary hypoplasia, of the myeloid lineage in particular, resulting in an increase in the proportions of lymphocytes and late erythroblasts (acidophilic erythroblasts) (Table S1). The proportion of cells of the granulocyte lineage among the nucleated cells detected in the BM myelogram was about 40% lower, in both patients, than the mean value for nine unrelated aged-matched HDs, whereas the proportion of lymphocytes was more than 50% higher (Figure 4B). A biopsy from P2 showed that his BM was hypocellular (ratio hematopoietic cells/adipocytes, [10%–20%] versus 60% for an age-matched control individual). It was also characterized by a granulocytic lineage that was highly hypoplastic relative to the erythroblastic or megakaryocytic lineages (Figures 4C and S4A). A diagnosis of BM failure was therefore established. In addition, the BM mononuclear cells (BMNCs) of the patients had a low capacity for generating myeloid and erythroid progenitors (EryPs), as shown by an ~8-fold decrease in the number of colony-forming units-granulocyte macrophage (CFU-GM) and burst-forming units-erythoid (BFU-E) in standard differentiation

assays (Figure 4D). Together, these data show that the BM of the FLT3L-deficient patients was severely hypoplastic, probably due to an impairment of hematopoietic stem and progenitor cell (HSPC) differentiation or maintenance.

## FLT3L deficiency results in a major decrease in HSPCs

Consistent with the findings reported above, standard flow cytometry showed that the percentage of CD34<sup>+</sup>Lin<sup>-</sup> HSPCs among BMNCs was much lower in P2 than in controls (~9-fold lower than the mean value for HDs) (Figure 4E, left). We then obtained an enriched population in residual CD34<sup>+</sup> HSPCs from P2's BM, on which we performed extensive phenotyping to characterize its CD34<sup>+</sup> HSC and progenitor content (Figures 4E, S4B, and S4C). We found that the proportions of multipotent progenitors (MPPs), granulocyte-monocyte progenitors (GMPs), and DCPs were slightly lower than those in HDs, whereas those of megakaryocyte-erythroid progenitors (MEPs) were higher in P2 than in controls (Figures 4E and S4C). The frequencies of common myeloid progenitors (CMPs), multipotent lymphoid progenitors (MLPs), and B/NK cell progenitors (BNKPs) were within the normal range (Figures 4E and S4C). We then used a strategy described elsewhere<sup>41</sup> to perform a complementary analysis of the frequencies of circulating hematopoietic precursors in P2 and P3, based on an analysis of the CD34<sup>+</sup>Lin<sup>-</sup> population in peripheral blood mononuclear cells (PBMCs) (Figures 4F and S4D). As in the BM, the frequency of CD34<sup>+</sup> HSPCs among PBMCs was lower in the patients than in 19 unrelated controls (~9-fold lower) (Figure 4F, left). The frequencies of HSC/MPP and MLPs among the rare HSPCs were in the normal range (P2) or were lower (P3) than those in controls. The frequencies of the CMP/MEP subsets were high in both patients. GMP levels were low, and DCPs were absent in both patients. BNKPs were undetectable in both patients (Figures 4F and S4D).

## Figure 4. Bone-marrow and hematopoietic progenitors in FLT3L-deficient patients

(A) Light microscopy images of bone marrow smears, for P2, P3, and one age-matched adult HD, after May-Grumwald-Giemsa (MGG) staining. The nucleated cells appear violet. The round, unstained areas correspond to adipocytes, dissolved by MGG staining.

(B) Scatter dot plots visualizing the proportions of the indicated hematopoietic lineages among total nucleated cells from P2, P3, and HDs. Bars represent the mean and SD. Two-tailed Mann-Whitney U tests were performed to assess the difference between the patients and nine HDs (4 males and 5 females, aged 23–43, all from European ancestries). *p ≤ 0.05; ns, not significant.

(C) Bone biopsy for P2 and an age-matched control, showing hypocellularity in P2. At higher magnification of P2's slide, we observed mostly mature erythroblasts and megakaryocytes, whereas the granulocytic lineage appeared largely hypoplastic.

(D) Counts of hematopoietic progenitors (CFU and BFU) in a colony-forming assay performed 13 days after culture with cells from P2, P3, and an adult HD. The pie charts display the proportions of the total colonies corresponding to CFU-GM and BFU-E in indicated individuals.

(E) Left: frequency of CD34<sup>+</sup> hematopoietic stem and progenitor cells (HSPCs) from the BM samples of P2 and 5 adult HDs (all males, aged 24–43). Right: frequency of hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), multipotent lymphoid progenitors (MLPs), common myeloid progenitors (CMPs), megakaryocyte-erythroid progenitors (MEPs), granulocyte-monocyte progenitors (GMPs), dendritic cell progenitors (DCPs), and B/NK cell progenitors (BNKPs) in CD34<sup>+</sup>Lin<sup>-</sup> cells from P2 and 5 HDs. Data are presented as individual values (min to max). Boxplots show the median and interquartile range (IQR).

(F) Left: frequency of CD34<sup>+</sup> cells from the PBMCs of P2, P3, and 19 HDs. Right: frequency of HSCs/MPPs, MLPs, CMPs/MEPs, GMPs/DCPs, DCPs, and BNKPs in P2, P3, and 13 HDs (all HDs were adults, male or female, and from multiple ethnic groups). Data are presented as individual values (min to max). Boxplots show the median and IQR. Two-tailed Mann-Whitney U tests were performed to assess the difference between the patients and the controls. *p ≤ 0.05, **p ≤ 0.01; ns, not significant. The value for a single time point was used for each individual in all statistical analyses; however, the graphs for the patients display different values measured at different time points.

(G and H) Single-cell RNA sequencing on CD34<sup>-</sup>-enriched HSPCs from the bone marrow of P2 and two HDs (one male, one female, aged 44 and 53, respectively).

(G) Unsupervised analysis of CD34<sup>+</sup> HSPCs from HDs (HD1: 7,604 cells and HD2: 7,298 cells) and P2 (262 cells), represented as two-dimensional uniform manifold approximation and projection (UMAP) plots. All HSCs, HSC and HSC-enriched; MPP; MLP; ImP1 and ImP2, immature myeloid progenitors; NeutroP, neutrophil progenitor; MonoDCP, monocyte and dendritic cell progenitor; BcellIP, B cell progenitor; Cycling_BcellIP, cycling B cell progenitors; MEP, megakaryocyte and erythrocyte progenitor; EryP, erythroid progenitor; MkP, megakaryocyte-committed progenitor; EoBasMastP, eosinophil, basophil, and mast cell progenitors; NA, not annotated.

(H) Proportion of each HSPC subpopulation among the CD34<sup>+</sup> HSPC shown in (G). Bars represent the mean and SD. See also Figure S4.
phosphorylation in our K562 reporter assay (not shown) and could not be used to assess FLT3L activity at more physiological levels. Thus, homozygosity for the p.Ser118Alafs*23 variant underlies complete FLT3L deficiency in these patients. FLT3L expression can be restored by transducing the patients' cells with the WT mFL or sFL cDNA.

## FLT3L deficiency is associated with hypoplastic BM

Flt3lg-knockout mice have low levels of hematopoietic progenitors, especially DC progenitors (DCPs) and B cell precursors (BcellPs), in their BM.<sup>24,40</sup> We first studied BM smears from P2 and P3 to characterize the impact of FLT3L deficiency on cellularity and progenitor populations. BM cellularity was low in both patients, with lipid structures observed in the smears (Figure 4A). A myelogram revealed general medullary hypoplasia, of the myeloid lineage in particular, resulting in an increase in the proportions of lymphocytes and late erythroblasts (acidophilic erythroblasts) (Table S1). The proportion of cells of the granulocyte lineage among the nucleated cells detected in the BM myelogram was about 40% lower, in both patients, than the mean value for nine unrelated aged-matched HDs, whereas the proportion of lymphocytes was more than 50% higher (Figure 4B). A biopsy from P2 showed that his BM was hypocellular (ratio hematopoietic cells/adipocytes, [10%–20%] versus 60% for an age-matched control individual). It was also characterized by a granulocytic lineage that was highly hypoplastic relative to the erythroblastic or megakaryocytic lineages (Figures 4C and S4A). A diagnosis of BM failure was therefore established. In addition, the BM mononuclear cells (BMNCs) of the patients had a low capacity for generating myeloid and erythroid progenitors (EryPs), as shown by an ~8-fold decrease in the number of colony-forming units-granulocyte macrophage (CFU-GM) and burst-forming units-erythoid (BFU-E) in standard differentiation

assays (Figure 4D). Together, these data show that the BM of the FLT3L-deficient patients was severely hypoplastic, probably due to an impairment of hematopoietic stem and progenitor cell (HSPC) differentiation or maintenance.

## FLT3L deficiency results in a major decrease in HSPCs

Consistent with the findings reported above, standard flow cytometry showed that the percentage of CD34<sup>+</sup>Lin<sup>-</sup> HSPCs among BMNCs was much lower in P2 than in controls (~9-fold lower than the mean value for HDs) (Figure 4E, left). We then obtained an enriched population in residual CD34<sup>+</sup> HSPCs from P2's BM, on which we performed extensive phenotyping to characterize its CD34<sup>+</sup> HSC and progenitor content (Figures 4E, S4B, and S4C). We found that the proportions of multipotent progenitors (MPPs), granulocyte-monocyte progenitors (GMPs), and DCPs were slightly lower than those in HDs, whereas those of megakaryocyte-erythroid progenitors (MEPs) were higher in P2 than in controls (Figures 4E and S4C). The frequencies of common myeloid progenitors (CMPs), multipotent lymphoid progenitors (MLPs), and B/NK cell progenitors (BNKPs) were within the normal range (Figures 4E and S4C). We then used a strategy described elsewhere<sup>41</sup> to perform a complementary analysis of the frequencies of circulating hematopoietic precursors in P2 and P3, based on an analysis of the CD34<sup>+</sup>Lin<sup>-</sup> population in peripheral blood mononuclear cells (PBMCs) (Figures 4F and S4D). As in the BM, the frequency of CD34<sup>+</sup> HSPCs among PBMCs was lower in the patients than in 19 unrelated controls (~9-fold lower) (Figure 4F, left). The frequencies of HSC/MPP and MLPs among the rare HSPCs were in the normal range (P2) or were lower (P3) than those in controls. The frequencies of the CMP/MEP subsets were high in both patients. GMP levels were low, and DCPs were absent in both patients. BNKPs were undetectable in both patients (Figures 4F and S4D).

## Figure 4. Bone-marrow and hematopoietic progenitors in FLT3L-deficient patients

(A) Light microscopy images of bone marrow smears, for P2, P3, and one age-matched adult HD, after May-Grumwald-Giemsa (MGG) staining. The nucleated cells appear violet. The round, unstained areas correspond to adipocytes, dissolved by MGG staining.

(B) Scatter dot plots visualizing the proportions of the indicated hematopoietic lineages among total nucleated cells from P2, P3, and HDs. Bars represent the mean and SD. Two-tailed Mann-Whitney U tests were performed to assess the difference between the patients and nine HDs (4 males and 5 females, aged 23–43, all from European ancestries). *p ≤ 0.05; ns, not significant.

(C) Bone biopsy for P2 and an age-matched control, showing hypocellularity in P2. At higher magnification of P2's slide, we observed mostly mature erythroblasts and megakaryocytes, whereas the granulocytic lineage appeared largely hypoplastic.

(D) Counts of hematopoietic progenitors (CFU and BFU) in a colony-forming assay performed 13 days after culture with cells from P2, P3, and an adult HD. The pie charts display the proportions of the total colonies corresponding to CFU-GM and BFU-E in indicated individuals.

(E) Left: frequency of CD34<sup>+</sup> hematopoietic stem and progenitor cells (HSPCs) from the BM samples of P2 and 5 adult HDs (all males, aged 24–43). Right: frequency of hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), multipotent lymphoid progenitors (MLPs), common myeloid progenitors (CMPs), megakaryocyte-erythroid progenitors (MEPs), granulocyte-monocyte progenitors (GMPs), dendritic cell progenitors (DCPs), and B/NK cell progenitors (BNKPs) in CD34<sup>+</sup>Lin<sup>-</sup> cells from P2 and 5 HDs. Data are presented as individual values (min to max). Boxplots show the median and interquartile range (IQR).

(F) Left: frequency of CD34<sup>+</sup> cells from the PBMCs of P2, P3, and 19 HDs. Right: frequency of HSCs/MPPs, MLPs, CMPs/MEPs, GMPs/DCPs, DCPs, and BNKPs in P2, P3, and 13 HDs (all HDs were adults, male or female, and from multiple ethnic groups). Data are presented as individual values (min to max). Boxplots show the median and IQR. Two-tailed Mann-Whitney U tests were performed to assess the difference between the patients and the controls. *p ≤ 0.05, **p ≤ 0.01; ns, not significant. The value for a single time point was used for each individual in all statistical analyses; however, the graphs for the patients display different values measured at different time points.

(G and H) Single-cell RNA sequencing on CD34<sup>+</sup>-enriched HSPCs from the bone marrow of P2 and two HDs (one male, one female, aged 44 and 53, respectively).

(G) Unsupervised analysis of CD34<sup>+</sup> HSPCs from HDs (HD1: 7,604 cells and HD2: 7,298 cells) and P2 (262 cells), represented as two-dimensional uniform manifold approximation and projection (UMAP) plots. All HSCs, HSC and HSC-enriched; MPP; MLP; ImP1 and ImP2, immature myeloid progenitors; NeutroP, neutrophil progenitor; MonoDCP, monocyte and dendritic cell progenitor; BcellIP, B cell progenitor; Cycling_BcellIP, cycling B cell progenitors; MEP, megakaryocyte and erythrocyte progenitor; EryP, erythroid progenitor; MkP, megakaryocyte-committed progenitor; EoBasMastP, eosinophil, basophil, and mast cell progenitors; NA, not annotated.

(H) Proportion of each HSPC subpopulation among the CD34<sup>+</sup> HSPC shown in (G). Bars represent the mean and SD. See also Figure S4.
<figure><img src="image_17.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_29.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>F</figcaption></figure>

scRNA-seq on total PBMC

<figure><img src="image_31.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_32.png" /><figcaption>H</figcaption></figure>

scRNA-seq on HLA-DR⁺
sorted cells

<figure><img src="image_33.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_34.png" /></figure>

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<figure><img src="image_18.png" /><figcaption>A</figcaption></figure>

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<figure><img src="image_24.png" /></figure>

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<figure><img src="image_26.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_29.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_32.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_33.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

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(legend on next page)
## The residual HSPCs of FLT3L-deficient individuals are biased toward the megakaryocytic and erythrocytic lineages

We performed single-cell RNA-seq (scRNA-seq) on CD34⁺ HSPCs isolated from the BMNCs of P2 and two healthy controls matched for age and sex. The results were analyzed with a previously described clustering approach (Figures 4G and 4H).⁴² Due to the limited sample size, and the rarity of CD34⁺ cells in FLT3L-deficient patients, only a small number of HSPCs could be analyzed for P2. Nevertheless, this analysis confirmed the normal frequencies of HSCs and MPPs among P2's HSPCs and revealed a frequency bias toward the megakaryocytic-erythroid pathway, with a large proportion of immediate progenies of MEP cells, including megakaryocyte-committed progenitors (MkPs) and primitive erythroid progenitors (EryPs). Consistent with our flow cytometry analysis, the frequencies of myeloid progenitors (neutrophil progenitors [NeutroPs] and monocyte and DCPs [MonoDCPs], together corresponding to GMPs) were slightly lower in the patient than in the two controls. For lymphoid progenitors, MLP levels were slightly lower, and B cell progenitors (BcellPs and CyclingBcellPs) levels were considerably lower in the patient than in the two controls (Figures 4G and 4H). In a pseudobulk analysis, FLT3 mRNA levels were normal in P2's HSCs (the most immature cells), whereas they were low in bulk HSPC analysis, suggesting that the development or survival of FLT3⁺ progenitors may be poor in FLT3L-deficient individuals (Figure S4E). Overall, our data show that the rare BM and circulating HSPCs of the patients are enriched in MkPs and EryPs and that the frequencies of B cells, monocytes, granulocytes, and DC progenitors are low.

## Monocytopenia in FLT3L-deficient patients

We then investigated the impact of complete FLT3L deficiency on complete blood counts in all three patients over a period of 5 (P1), 9 (P2), and 19 (P3) years of follow-up. Hemoglobin (HB)

levels and red blood cell (RBC) counts were slightly low in all three patients and were considered to correspond to moderate normocytic anemia with normal reticulocyte counts (Figures 5A and S5A; Table S1). This moderate anemia was accompanied by low ferritin levels in P3, with a systematically high erythrocyte sedimentation rate (ESR), and hypergammaglobulinemia in all three patients (Figure S5A; Table S1). The observed anemia was therefore probably multifactorial, with iron deficiency in P3 and hemodilution due to hypergammaglobulinemia in all three patients. Total platelet counts and platelet morphology were normal but had decreased at last follow-up for P2 and P3 (Figure 5A; Table S1). Total white blood cell (WBC) counts oscillated between low and normal values (Figure 5A; Table S1). All the patients consistently had profound monocytopenia, except for P3, who presented normal values on rare occasions (Figure 5A; Table S1). The levels of eosinophils and basophils were in the normal range (Table S1). Neutrophil counts were in the lower part of the control range or, occasionally, lower (Figure 5A; Table S1). Total lymphocyte counts were normal (Figure 5A; Table S1). Thus, patients had normal platelet and lymphocyte counts, near-normal neutrophil and RBC counts, and monocytopenia.

## FLT3L deficiency impairs the differentiation of monocytes and DCs

We performed a deep immunophenotyping by conventional cytometry on fresh PBMCs and mass cytometry (cytometry by time of flight [CyTOF]) on fresh whole blood and BM (Figures 5B-5D and S5B-S5E; Table S1). Comparisons with healthy controls confirmed that the patients had profound monocytopenia in both the blood and BM, affecting all monocyte subsets, including classical (CD14⁺CD16⁻), intermediate (CD14⁺CD16⁺), and non-classical (CD14dimCD16⁺) monocytes (Figures 5B, S5B, and S5E). Both patients tested (P2 and P3) had almost no CD123⁺ pDCs or cDC2 in the blood and BM, and cDC1s were

<caption>**Figure 5.** Hematological profile, including the myeloid- and lymphoid-related immune compartments in the peripheral blood of FLT3L-deficient patients</caption>

(A) Absolute numbers for peripheral blood cell subsets in P1, P2, and P3 over time. The reference values for each parameter are indicated by shaded areas. WBC, white blood cell; RBC, red blood cell.

(B) Absolute counts for peripheral blood monocyte subsets assessed by CyTOF in P2, P3, and 11 HDs (all HDs were adults, male or female, and from multiple ethnic groups). Data are presented as individual values (min to max). Boxplots show the median and IQR. Two-tailed Mann-Whitney U tests were performed to assess the difference between patients and controls. *p* ≤ 0.05; ns, not significant. The value for a single time point for each individual was used for all statistical analyses. However, the graphs for the patients display different values measured at different time points.

(C) Absolute counts of peripheral blood dendritic cells assessed by CyTOF in P2, P3, and 12 HDs (all HDs were adults, male or female, and from multiple ethnic groups). Data presentation and statistics are as in (B).

(D) Flow-cytometry analysis of three subsets of circulating DCs assessed in P2, P3, and one HD after the acquisition of >10⁷ events, with more than 5 million live cells/sample. The first gating shows the HLA-DR⁺Lin⁻ (CD3, CD19, CD20, CD14, CD16, and CD56) population gated on live CD45⁺ cells after the removal of doublets.

(E) UMAP clustering for 5' scRNA-seq performed on total PBMCs from P2, P3, and six HDs (all HDs were adults, male or female, and from multiple ethnic groups). The 22 clusters were identified on the basis of cell marker expression analysis.

(F) Frequencies of the clusters shown in (E). Data are presented as individual values (min to max).

(G) UMAP clustering for 5' scRNA-seq performed on the flow cytometry-sorted HLA-DR⁺Lin⁻ (CD3, CD19, CD20, and CD56) populations from P2, P3, and two adult HDs (one male and one female, from the same ethnic group as the patients). The seven clusters targeted are identified on the basis of cell marker expression analysis.⁴³

(H) Frequencies of the clusters identified in (G). Data are presented as individual values (min to max).

(I) Volcano plot showing the results of a differential gene expression (DGE) analysis of the experiment in (G) and (H), comparing normalized gene expression levels between patients and controls. Genes with a log₂ fold change (log₂FC) in expression >1 or <-1, and a FDR < 0.0005 were considered significant. A color code indicates the level of significance.

See also Figure S5.
## The residual HSPCs of FLT3L-deficient individuals are biased toward the megakaryocytic and erythrocytic lineages

We performed single-cell RNA-seq (scRNA-seq) on CD34⁺ HSPCs isolated from the BMNCs of P2 and two healthy controls matched for age and sex. The results were analyzed with a previously described clustering approach (Figures 4G and 4H).⁴² Due to the limited sample size, and the rarity of CD34⁺ cells in FLT3L-deficient patients, only a small number of HSPCs could be analyzed for P2. Nevertheless, this analysis confirmed the normal frequencies of HSCs and MPPs among P2's HSPCs and revealed a frequency bias toward the megakaryocytic-erythroid pathway, with a large proportion of immediate progenies of MEP cells, including megakaryocyte-committed progenitors (MkPs) and primitive erythroid progenitors (EryPs). Consistent with our flow cytometry analysis, the frequencies of myeloid progenitors (neutrophil progenitors [NeutroPs] and monocyte and DCPs [MonoDCPs], together corresponding to GMPs) were slightly lower in the patient than in the two controls. For lymphoid progenitors, MLP levels were slightly lower, and B cell progenitors (BcellPs and CyclingBcellPs) levels were considerably lower in the patient than in the two controls (Figures 4G and 4H). In a pseudobulk analysis, FLT3 mRNA levels were normal in P2's HSCs (the most immature cells), whereas they were low in bulk HSPC analysis, suggesting that the development or survival of FLT3⁺ progenitors may be poor in FLT3L-deficient individuals (Figure S4E). Overall, our data show that the rare BM and circulating HSPCs of the patients are enriched in MkPs and EryPs and that the frequencies of B cells, monocytes, granulocytes, and DC progenitors are low.

## Monocytopenia in FLT3L-deficient patients

We then investigated the impact of complete FLT3L deficiency on complete blood counts in all three patients over a period of 5 (P1), 9 (P2), and 19 (P3) years of follow-up. Hemoglobin (HB)

levels and red blood cell (RBC) counts were slightly low in all three patients and were considered to correspond to moderate normocytic anemia with normal reticulocyte counts (Figures 5A and S5A; Table S1). This moderate anemia was accompanied by low ferritin levels in P3, with a systematically high erythrocyte sedimentation rate (ESR), and hypergammaglobulinemia in all three patients (Figure S5A; Table S1). The observed anemia was therefore probably multifactorial, with iron deficiency in P3 and hemodilution due to hypergammaglobulinemia in all three patients. Total platelet counts and platelet morphology were normal but had decreased at last follow-up for P2 and P3 (Figure 5A; Table S1). Total white blood cell (WBC) counts oscillated between low and normal values (Figure 5A; Table S1). All the patients consistently had profound monocytopenia, except for P3, who presented normal values on rare occasions (Figure 5A; Table S1). The levels of eosinophils and basophils were in the normal range (Table S1). Neutrophil counts were in the lower part of the control range or, occasionally, lower (Figure 5A; Table S1). Total lymphocyte counts were normal (Figure 5A; Table S1). Thus, patients had normal platelet and lymphocyte counts, near-normal neutrophil and RBC counts, and monocytopenia.

## FLT3L deficiency impairs the differentiation of monocytes and DCs

We performed a deep immunophenotyping by conventional cytometry on fresh PBMCs and mass cytometry (cytometry by time of flight [CyTOF]) on fresh whole blood and BM (Figures 5B-5D and S5B-S5E; Table S1). Comparisons with healthy controls confirmed that the patients had profound monocytopenia in both the blood and BM, affecting all monocyte subsets, including classical (CD14⁺CD16⁻), intermediate (CD14⁺CD16⁺), and non-classical (CD14dimCD16⁺) monocytes (Figures 5B, S5B, and S5E). Both patients tested (P2 and P3) had almost no CD123⁺ pDCs or cDC2 in the blood and BM, and cDC1s were

<caption>**Figure 5.** Hematological profile, including the myeloid- and lymphoid-related immune compartments in the peripheral blood of FLT3L-deficient patients</caption>

(A) Absolute numbers for peripheral blood cell subsets in P1, P2, and P3 over time. The reference values for each parameter are indicated by shaded areas. WBC, white blood cell; RBC, red blood cell.

(B) Absolute counts for peripheral blood monocyte subsets assessed by CyTOF in P2, P3, and 11 HDs (all HDs were adults, male or female, and from multiple ethnic groups). Data are presented as individual values (min to max). Boxplots show the median and IQR. Two-tailed Mann-Whitney U tests were performed to assess the difference between patients and controls. *p* ≤ 0.05; ns, not significant. The value for a single time point for each individual was used for all statistical analyses. However, the graphs for the patients display different values measured at different time points.

(C) Absolute counts of peripheral blood dendritic cells assessed by CyTOF in P2, P3, and 12 HDs (all HDs were adults, male or female, and from multiple ethnic groups). Data presentation and statistics are as in (B).

(D) Flow-cytometry analysis of three subsets of circulating DCs assessed in P2, P3, and one HD after the acquisition of >10⁷ events, with more than 5 million live cells/sample. The first gating shows the HLA-DR⁺Lin⁻ (CD3, CD19, CD20, CD14, CD16, and CD56) population gated on live CD45⁺ cells after the removal of doublets.

(E) UMAP clustering for 5' scRNA-seq performed on total PBMCs from P2, P3, and six HDs (all HDs were adults, male or female, and from multiple ethnic groups). The 22 clusters were identified on the basis of cell marker expression analysis.

(F) Frequencies of the clusters shown in (E). Data are presented as individual values (min to max).

(G) UMAP clustering for 5' scRNA-seq performed on the flow cytometry-sorted HLA-DR⁺Lin⁻ (CD3, CD19, CD20, and CD56) populations from P2, P3, and two adult HDs (one male and one female, from the same ethnic group as the patients). The seven clusters targeted are identified on the basis of cell marker expression analysis.⁴³

(H) Frequencies of the clusters identified in (G). Data are presented as individual values (min to max).

(I) Volcano plot showing the results of a differential gene expression (DGE) analysis of the experiment in (G) and (H), comparing normalized gene expression levels between patients and controls. Genes with a log₂ fold change (log₂FC) in expression >1 or <-1, and a FDR < 0.0005 were considered significant. A color code indicates the level of significance.

See also Figure S5.
<figure><img src="image_37.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_38.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_39.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_40.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_41.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_42.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_43.png" /><figcaption>**Figure 6. B cell and NK cell features in FLT3L-deficient patients**</figcaption></figure>

(A) Absolute blood counts of peripheral B cell subsets, as defined by CyTOF, for P2, P3, and 20 HDs (all HDs were adults, male or female, and from multiple ethnic groups). Data are presented as individual values (min to max). Boxplots show the median and IQR. Two-tailed Mann-Whitney U tests were performed to assess the difference between the patients and the controls. \*p ≤ 0.05, \*\*p < 0.01; ns, not significant. The value for a single time point for each individual was used for all statistical analyses. However, the graphs for the patients display different values measured at different time points.

(B) Absolute blood counts and proportions among B cells of age-associated B cells (ABCs), as defined by CyTOF, for P2, P3, and 20 HDs (as in A). Data presentation and statistics are as in (A).

(C) Proportions of plasmablasts among the CD19⁺ B cells of BMNCs, as defined by CyTOF, for the BM samples of patients and four adult HDs. Data presentation and statistics are as in (A).

(D) Heatmap showing the relative values for antibody reactivity against 38 different HPV types measured in a Luminex-based serological analysis performed on plasma samples from P2, P3, and 20 adult controls (male or female, multiple ethnic groups; each row represents a single serum sample). MFI values above 200 were considered positive.

*(legend continued on next page)*
<figure><img src="image_37.png" /></figure>

A

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

B

<figure><img src="image_40.png" /></figure>

C

<figure><img src="image_41.png" /></figure>

D

<figure><img src="image_42.png" /></figure>

E

<figure><img src="image_43.png" /></figure>

F

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /><figcaption>Figure 6. B cell and NK cell features in FLT3L-deficient patients</figcaption></figure>

(A) Absolute blood counts of peripheral B cell subsets, as defined by CyTOF, for P2, P3, and 20 HDs (all HDs were adults, male or female, and from multiple ethnic groups). Data are presented as individual values (min to max). Boxplots show the median and IQR. Two-tailed Mann-Whitney U tests were performed to assess the difference between the patients and the controls. \*p ≤ 0.05, \*\*p < 0.01; ns, not significant. The value for a single time point for each individual was used for all statistical analyses. However, the graphs for the patients display different values measured at different time points.

(B) Absolute blood counts and proportions among B cells of age-associated B cells (ABCs), as defined by CyTOF, for P2, P3, and 20 HDs (as in A). Data presentation and statistics are as in (A).

(C) Proportions of plasmablasts among the CD19⁺ B cells of BMNCs, as defined by CyTOF, for the BM samples of patients and four adult HDs. Data presentation and statistics are as in (A).

(D) Heatmap showing the relative values for antibody reactivity against 38 different HPV types measured in a Luminex-based serological analysis performed on plasma samples from P2, P3, and 20 adult controls (male or female, multiple ethnic groups; each row represents a single serum sample). MFI values above 200 were considered positive.

(legend continued on next page)
undetectable by CyTOF and flow cytometry on multiple occasions (Figures 5C, 5D, and S5E; Table S1). Monocytopenia and the almost total absence of DCs were confirmed in a scRNA-seq experiment performed on total PBMCs (Figures 5E and 5F). We increased the resolution for myeloid antigen-presenting cells (APCs) by using fluorescence-activated cell sorting (FACS) to sort HLA-DR⁺Lin⁻ cells from the PBMCs of P2, P3, and two controls to achieve an enrichment in monocytes and DCs before scRNA-seq. Myeloid cell subsets were analyzed as previously described.43-45 The frequencies of non-classical monocytes and of all DCs among sorted APCs were low, suggesting that, despite their very low counts, classical monocytes nevertheless constituted the major myeloid source of APCs in the patient. This approach did not detect DC precursors (preDCs) in either of the patients tested (Figures 5G and 5H). We detected rare pDCs and cDC2s in both patients and cDC1s and cDC3s in P2 only, suggesting that the differentiation of these cells was severely impaired but not completely abolished (Figures 5G and 5H). A comparison of the differential gene expression (DGE) profiles of patients and healthy controls revealed alterations in the monocytes and cDC2s of the patients (Figure 5I). IRF1, STAT1, GBP5, and GBP2, which encode proteins involved in interferon (IFN) signaling pathways, were upregulated in the patients' classical monocytes (Figure 5I). IRF1 and STAT1 were also upregulated in the patients' cDC2 subset. In addition, genes involved in inflammatory responses, such as S100A8 and S100A9, were upregulated in the monocytes of the patients (Figure 5I). No significant alterations were observed in the patients' pDCs. Together, these data demonstrate a near-total absence of DCs in the blood of the patients. Transcriptomic analysis of the residual monocytes and the rare cDC2s found in patients suggested an inflammatory state, possibly reflecting persistent infections.

## FLT3L deficiency has no effect on T cell differentiation and function but impairs B cell differentiation

Despite a profound deficiency of multiple myeloid APCs, the patients' T cells developed and differentiated in vivo and responded to APC-independent stimuli in vitro and ex vivo (Figures S5C, S5D, and S6A-S6F; Table S1). By contrast, blood total B cell counts were very low in both patients (P2 and P3), with three subsets affected (transitional, naive, and memory B cells), whereas plasmablast counts were within the range for controls (Figure 6A). The frequency of transitional B cells among the residual B cells was very low, suggesting a central differentiation defect, whereas that of memory B cells was similar to that in controls. In addition, both patients had an abnormally high proportion of age-associated B cells (ABCs), accounting for >30% of naive B cells (Figures 6B and S7A).46,47 These findings were confirmed in our scRNA-seq analysis of PBMCs, and no

major differences in gene expression were observed between the B cells of controls and patients (Figure 5F). The B cell phenotypes were also confirmed in the BM (Figure S5E), except that the proportion of plasmablasts among the patients' BMNCs was low (Figure 6C). Consistent with a central production defect, all B cell maturation steps were severely impaired in the BM of the two patients (Figures 4G, 4H, and S7B). However, the IGH repertoire of sorted blood CD19⁺ B cells from the patients was diverse and without clonal expansion. Furthermore, V and J gene usage within unique CDR3 sequences was similar to that in the controls (Figures S7C and S7D), suggesting that the impaired B cell differentiation was not due to defects in the generation of productive IGH rearrangements. Surprisingly, the plasma concentrations of Ig classes and IgG subclasses were all normal, whereas IgA levels were high (Table S1). The patients were seropositive for many pathogens on conventional and VirScan serological testing (Figure S7E; Table S1). Their plasma IgA strongly stained E. bieneusi spores in a fluorescence microscopy experiment (Figure S7F). A multiplex serological analysis revealed that P2 and P3 were seropositive for 33 and 29 HPV types, respectively, whereas a group of 20 adult controls displayed seropositivity for a median of six HPV types (range: 1-21) (Figure 6D).48 The presence of anti-HPV IgG antibodies, which are T cell-dependent, suggests that anti-HPV immunity is impaired but not abolished in the patients, consistent with the MmuPV1 infection model (Figure S2). Interestingly, allo-hemagglutinin levels against group A were very low in the patients (tiers of 1/4 and 1/8 for P2 and P3, respectively), suggesting weak T cell-independent responses (Table S1). Similarly, serological tests for SARS-CoV-2 antibodies remained negative despite vaccination (Sinopharm) in both patients and documented COVID-19 in P2 (Table S1). Together, these data demonstrate that human FLT3L plays a crucial role in B cell differentiation and Ab responses, consistent with the recurrent bacterial infections of the lungs observed in patients.

## FLT3L deficiency has a minor impact on NK cell development

We then investigated the impact of FLT3L deficiency on NK cells, a subset severely affected by such deficiency in the mouse model.24,25 Total blood NK cell counts were normal for P3 but slightly low in P2 (Figure 6E). The frequencies of patients' CD56bright NK cells and the distribution of CD56dim NK cell differentiation subsets, based on killer-cell immunoglobulin-like receptor (KIR) and NKG2A expression, were normal (Figures 6F and 6G).49,50 In P3, 30% the CD56dim NK cells were NKG2C⁺CD57⁺, suggesting that patients with FLT3L deficiency can develop adaptive NK cells (Figure 6G). We detected no NKG2C expression in P2 (Figure 6G), who was homozygous for a

<footnote>(E) Absolute counts of total NK cells and CD56bright NK cells, and the proportion of CD56bright cells among NK cells from P2, P3, and 16 HDs (all HDs were adults, male or female, and from multiple ethnic groups). Data presentation and statistics are as in (A).</footnote>

<footnote>(F) Percentages of CD56dim NK cell differentiation subsets, as defined on the basis of pan-KIR and NKG2A expression detected by flow cytometry,⁴⁹ for P2, P3, and 16 HDs (as in E). Data presentation and statistics are as in (A).</footnote>

<footnote>(G) Flow-cytometry analysis showing that the CD56dim NK cell subsets of the two patients include adaptive NK cells. This subset can be identified as NKG2C⁺/−CD161⁻CD57⁺ cells. Two HDs were included as examples. HD1 has no adaptive NK cells. HD2 has a large subset of adaptive NK cells. Note that P2 is homozygous for a frequent deletion in *KLRC2*, which encodes NKG2C.⁵⁰
See also Figures S5E, S6, and S7.</footnote>
undetectable by CyTOF and flow cytometry on multiple occasions (Figures 5C, 5D, and S5E; Table S1). Monocytopenia and the almost total absence of DCs were confirmed in a scRNA-seq experiment performed on total PBMCs (Figures 5E and 5F). We increased the resolution for myeloid antigen-presenting cells (APCs) by using fluorescence-activated cell sorting (FACS) to sort HLA-DR⁺Lin⁻ cells from the PBMCs of P2, P3, and two controls to achieve an enrichment in monocytes and DCs before scRNA-seq. Myeloid cell subsets were analyzed as previously described.<sup>43-45</sup> The frequencies of non-classical monocytes and of all DCs among sorted APCs were low, suggesting that, despite their very low counts, classical monocytes nevertheless constituted the major myeloid source of APCs in the patient. This approach did not detect DC precursors (preDCs) in either of the patients tested (Figures 5G and 5H). We detected rare pDCs and cDC2s in both patients and cDC1s and cDC3s in P2 only, suggesting that the differentiation of these cells was severely impaired but not completely abolished (Figures 5G and 5H). A comparison of the differential gene expression (DGE) profiles of patients and healthy controls revealed alterations in the monocytes and cDC2s of the patients (Figure 5I). IRF1, STAT1, GBP5, and GBP2, which encode proteins involved in interferon (IFN) signaling pathways, were upregulated in the patients' classical monocytes (Figure 5I). IRF1 and STAT1 were also upregulated in the patients' cDC2 subset. In addition, genes involved in inflammatory responses, such as S100A8 and S100A9, were upregulated in the monocytes of the patients (Figure 5I). No significant alterations were observed in the patients' pDCs. Together, these data demonstrate a near-total absence of DCs in the blood of the patients. Transcriptomic analysis of the residual monocytes and the rare cDC2s found in patients suggested an inflammatory state, possibly reflecting persistent infections.

## FLT3L deficiency has no effect on T cell differentiation and function but impairs B cell differentiation

Despite a profound deficiency of multiple myeloid APCs, the patients' T cells developed and differentiated in vivo and responded to APC-independent stimuli in vitro and ex vivo (Figures S5C, S5D, and S6A-S6F; Table S1). By contrast, blood total B cell counts were very low in both patients (P2 and P3), with three subsets affected (transitional, naive, and memory B cells), whereas plasmablast counts were within the range for controls (Figure 6A). The frequency of transitional B cells among the residual B cells was very low, suggesting a central differentiation defect, whereas that of memory B cells was similar to that in controls. In addition, both patients had an abnormally high proportion of age-associated B cells (ABCs), accounting for >30% of naive B cells (Figures 6B and S7A).<sup>46,47</sup> These findings were confirmed in our scRNA-seq analysis of PBMCs, and no

major differences in gene expression were observed between the B cells of controls and patients (Figure 5F). The B cell phenotypes were also confirmed in the BM (Figure S5E), except that the proportion of plasmablasts among the patients' BMNCs was low (Figure 6C). Consistent with a central production defect, all B cell maturation steps were severely impaired in the BM of the two patients (Figures 4G, 4H, and S7B). However, the IGH repertoire of sorted blood CD19⁺ B cells from the patients was diverse and without clonal expansion. Furthermore, V and J gene usage within unique CDR3 sequences was similar to that in the controls (Figures S7C and S7D), suggesting that the impaired B cell differentiation was not due to defects in the generation of productive IGH rearrangements. Surprisingly, the plasma concentrations of Ig classes and IgG subclasses were all normal, whereas IgA levels were high (Table S1). The patients were seropositive for many pathogens on conventional and VirScan serological testing (Figure S7E; Table S1). Their plasma IgA strongly stained *E. bieneusi* spores in a fluorescence microscopy experiment (Figure S7F). A multiplex serological analysis revealed that P2 and P3 were seropositive for 33 and 29 HPV types, respectively, whereas a group of 20 adult controls displayed seropositivity for a median of six HPV types (range: 1-21) (Figure 6D).<sup>48</sup> The presence of anti-HPV IgG antibodies, which are T cell-dependent, suggests that anti-HPV immunity is impaired but not abolished in the patients, consistent with the MmuPV1 infection model (Figure S2). Interestingly, allo-hemagglutinin levels against group A were very low in the patients (tiers of 1/4 and 1/8 for P2 and P3, respectively), suggesting weak T cell-independent responses (Table S1). Similarly, serological tests for SARS-CoV-2 antibodies remained negative despite vaccination (Sinopharm) in both patients and documented COVID-19 in P2 (Table S1). Together, these data demonstrate that human FLT3L plays a crucial role in B cell differentiation and Ab responses, consistent with the recurrent bacterial infections of the lungs observed in patients.

## FLT3L deficiency has a minor impact on NK cell development

We then investigated the impact of FLT3L deficiency on NK cells, a subset severely affected by such deficiency in the mouse model.<sup>24,25</sup> Total blood NK cell counts were normal for P3 but slightly low in P2 (Figure 6E). The frequencies of patients' CD56<sup>bright</sup> NK cells and the distribution of CD56<sup>dim</sup> NK cell differentiation subsets, based on killer-cell immunoglobulin-like receptor (KIR) and NKG2A expression, were normal (Figures 6F and 6G).<sup>49,50</sup> In P3, 30% the CD56<sup>dim</sup> NK cells were NKG2C<sup>+</sup> CD57<sup>+</sup>, suggesting that patients with FLT3L deficiency can develop adaptive NK cells (Figure 6G). We detected no NKG2C expression in P2 (Figure 6G), who was homozygous for a

<footnote>(E) Absolute counts of total NK cells and CD56<sup>bright</sup> NK cells, and the proportion of CD56<sup>bright</sup> cells among NK cells from P2, P3, and 16 HDs (all HDs were adults, male or female, and from multiple ethnic groups). Data presentation and statistics are as in (A).</footnote>

<footnote>(F) Percentages of CD56<sup>dim</sup> NK cell differentiation subsets, as defined on the basis of pan-KIR and NKG2A expression detected by flow cytometry,<sup>49</sup> for P2, P3, and 16 HDs (as in E). Data presentation and statistics are as in (A).</footnote>

<footnote>(G) Flow-cytometry analysis showing that the CD56<sup>dim</sup> NK cell subsets of the two patients include adaptive NK cells. This subset can be identified as NKG2C<sup>+</sup>-CD161<sup>-</sup>CD57<sup>+</sup> cells. Two HDs were included as examples. HD1 has no adaptive NK cells. HD2 has a large subset of adaptive NK cells. Note that P2 is homozygous for a frequent deletion in *KLRC2*, which encodes NKG2C.<sup>50</sup></footnote>

<footnote>See also Figures S5E, S6, and S7.</footnote>
<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /><figcaption>**Figure 7.** Skin phenotype of FLT3L-deficient patients</figcaption></figure>

(A) Imaging mass cytometry (IMC) on paraffin-embedded skin biopsy specimens from HDs or healthy skin (HS) and a wart from P2. In the upper panel, the epidermis and dermis were defined based on cytokeratin and E-cadherin expression. The images in the middle panel show the staining for CD3, CD8, CD68, and

(legend continued on next page)
<figure><img src="image_46.png" /><figcaption>**Figure 7.** Skin phenotype of FLT3L-deficient patients</figcaption></figure>

(A) Imaging mass cytometry (IMC) on paraffin-embedded skin biopsy specimens from HDs or healthy skin (HS) and a wart from P2. In the upper panel, the epidermis and dermis were defined based on cytokeratin and E-cadherin expression. The images in the middle panel show the staining for CD3, CD8, CD68, and

(legend continued on next page)
frequent deletion in the associated gene.⁵¹ However, 40% of P2's CD56dim NK cells displayed strong CD57 expression and a downregulation of CD161 (Figure 6G), suggesting an adaptive NK cell response to human cytomegalovirus (HCMV), despite NKG2C gene deletion.⁵⁰,⁵² Both patients were seropositive for HCMV, but no viral replication was detected by PCR (Table S1). Normal NK cell differentiation was confirmed in our scRNA-seq experiment and by CyTOF immunophenotyping of the patients' BM samples (Figures 5E, 5F, and S5E). Together, these data show that human FLT3L deficiency does not impair NK cell differentiation, allowing the normal development of adaptive NK cell responses.

## Normal LC and dermal macrophage levels but a low frequency of dermal DCs

Finally, the broad susceptibility of the patients to HPVs prompted us to assess the patients' cutaneous immunity in greater detail. We used imaging mass cytometry (IMC) and conventional immunohistochemistry (IHC) to study healthy skin (HS) and wart samples from the patients (P2 and P3). We found that the patients' epidermis contained normal numbers of Langerhans cells (LCs) (Figures 7A and 7B), suggesting that the differentiation of these cells was not impaired in the absence of the hematopoietic growth factor FLT3L, consistent with their earlier, yolk-sac-derived embryonic origin in mice.⁴⁰,⁵³ LCs were also detected in the epidermis of warts from P2 and P3 (Figures 7A and 7B). CD4⁺ and CD8⁺ T cells and macrophages were present in normal numbers in healthy dermis from the patients (Figures 7A and 7B) and were detected below the infected epidermis in the lesions. We increased the cellular resolution by also performing scRNA-seq on both healthy and infected skin from the patients, after HLA-I⁺CD45⁺ leukocyte enrichment by FACS. With the exception of cDCs, the levels of which were extremely low in the patients, all the leukocyte subsets detected, including LCs and macrophages, were present at frequencies similar to those in controls (Figures 7C and 7D). These data suggest that the skin HPV phenotype is not driven by a quantitative decrease in dermal macrophages or epidermal LCs, instead probably resulting from a loss of dermal DCs.

## DISCUSSION

We describe human FLT3L deficiency, a new inborn error of immunity (IEI) underlying a broad infectious phenotype in three affected siblings. The FLT3LG gene was identified in mice and humans in 1993¹⁵ and 1994,¹⁷ respectively. In FLT3L-deficient mice, the cellularity of the BM is slightly lower than normal.²⁴,²⁵,⁴⁰ By contrast, FLT3L-deficient humans have a profoundly hypo-

plastic BM with an overall decrease in CD34⁺ HSPC counts. The frequencies of megakaryocyte-erythrocyte progenitors (MEPs) among the residual HSPCs of patients are abnormally high, as in the mouse model.⁵⁴ The frequency of myeloid progenitors (GMPs) was low in the patients but unaffected in *Flt3lg⁻/⁻* mice.⁵⁴ Both humans and mice with FLT3L deficiency display a depletion of DCPs.⁴⁰ In humans, the proportion of early lymphoid progenitors (MLPs and BNKPs) in the BM of the FLT3L-deficient patients remained within or near the control range, whereas the proportions of B cell progenitors were low. In *Flt3lg⁻/⁻* mice, the proportion of common lymphoid progenitors (CLPs) and B cell precursors (B-CFUs) was remarkably low in the BM.²⁴,⁴⁰,⁵⁴⁻⁵⁶ Thus, consistent with the model proposed by Tsapogas et al. based on mouse data,¹¹ the high proportion of MEPs among FLT3L-deficient HSPCs suggests that the early progenitors, which were not sufficiently stimulated by FLT3L signals, preferentially differentiate along megakaryocyte/erythrocyte pathways. However, this bias allows significant levels of differentiation into most mature hematopoietic subsets, enabling the patients to survive into adulthood, at least with the additional support of modern medicine. The same patients would probably have died at an early age before the advent of vaccination and anti-infectious medicine.

The differences between FLT3L-deficient mice and humans are more apparent in mature hematopoietic cells. Platelet counts were normal in the blood of the FLT3L-deficient patients, but these patients presented slight, probably multifactorial, anemia not observed in the mouse model.²⁴ As in mice,²⁴ blood leukocyte cellularity was only moderately low, and neutrophil and total lymphocyte counts were normal or near normal. Conversely, by contrast to the mouse model,²⁴ three patients displayed severe blood monocytopenia at most of the time points tested. As in mice,²²,²³,⁴⁰,⁵⁷ the differentiation of all types of DCs was severely affected in the patients but not completely abolished, with cDCs and pDCs barely detectable in the blood, BM, and skin. As in mice,²⁴,²⁵ T cell counts, including those for αβ and γδ T cells, were normal or near normal. Accordingly, all *in vitro* APC-independent assays showed T cell function to be normal. The patients had very few mature circulating B cells (~90% fewer than the controls), and this deficit appeared to be more profound than that in mice (~70%).²⁴,²⁵ The residual B cells included very few transitional B cells, and there was an accumulation of ABCs suggestive of an exhaustion of B cell production.⁵⁸ However, like *Flt3lg⁻/⁻* mice, the patients presented normal immunoglobulin production and even the production of excessive amounts of IgA (hyper-IgA), despite the low proportion of plasmablasts observed in the BM. Finally, by contrast to Flt3l-deficient mice,²⁴ the patients had normal or near-normal NK cell counts

langerin. Finally, the images in the lower panel show Langerhans cells (LCs), macrophages (Mac), CD4, and CD8 T cells after segmentation and identification by a machine-learning approach.

(B) Quantification of the main immune subsets in the epidermis (LCs) and dermis (Mac, CD4, and CD8 T cells) after identification by a machine-learning approach applied to biopsy specimens from six different HDs (adults, male or female and from multiple ethnic groups), with comparison to HS and warts from two patients. Data are presented as individual values (min to max). Boxplots show the median with IQR. No statistical analysis was performed here.

(C) The frequencies of each immune subset after 5' scRNA-seq on skin biopsy specimens of HS from three HDs (adults, male or female, and from multiple ethnic groups), P2 and P3, and a wart from P2. All the principal clusters targeted are identified on the basis of cell marker expression analysis. Data presentation are as in (B).

(D) UMAP clustering for the immune subsets shown in (C).
frequent deletion in the associated gene.⁵¹ However, 40% of P2's CD56dim NK cells displayed strong CD57 expression and a downregulation of CD161 (Figure 6G), suggesting an adaptive NK cell response to human cytomegalovirus (HCMV), despite NKG2C gene deletion.⁵⁰,⁵² Both patients were seropositive for HCMV, but no viral replication was detected by PCR (Table S1). Normal NK cell differentiation was confirmed in our scRNA-seq experiment and by CyTOF immunophenotyping of the patients' BM samples (Figures 5E, 5F, and S5E). Together, these data show that human FLT3L deficiency does not impair NK cell differentiation, allowing the normal development of adaptive NK cell responses.

## Normal LC and dermal macrophage levels but a low frequency of dermal DCs

Finally, the broad susceptibility of the patients to HPVs prompted us to assess the patients' cutaneous immunity in greater detail. We used imaging mass cytometry (IMC) and conventional immunohistochemistry (IHC) to study healthy skin (HS) and wart samples from the patients (P2 and P3). We found that the patients' epidermis contained normal numbers of Langerhans cells (LCs) (Figures 7A and 7B), suggesting that the differentiation of these cells was not impaired in the absence of the hematopoietic growth factor FLT3L, consistent with their earlier, yolk-sac-derived embryonic origin in mice.⁴⁰,⁵³ LCs were also detected in the epidermis of warts from P2 and P3 (Figures 7A and 7B). CD4⁺ and CD8⁺ T cells and macrophages were present in normal numbers in healthy dermis from the patients (Figures 7A and 7B) and were detected below the infected epidermis in the lesions. We increased the cellular resolution by also performing scRNA-seq on both healthy and infected skin from the patients, after HLA-I⁺CD45⁺ leukocyte enrichment by FACS. With the exception of cDCs, the levels of which were extremely low in the patients, all the leukocyte subsets detected, including LCs and macrophages, were present at frequencies similar to those in controls (Figures 7C and 7D). These data suggest that the skin HPV phenotype is not driven by a quantitative decrease in dermal macrophages or epidermal LCs, instead probably resulting from a loss of dermal DCs.

## DISCUSSION

We describe human FLT3L deficiency, a new inborn error of immunity (IEI) underlying a broad infectious phenotype in three affected siblings. The FLT3LG gene was identified in mice and humans in 1993¹⁵ and 1994,¹⁷ respectively. In FLT3L-deficient mice, the cellularity of the BM is slightly lower than normal.²⁴,²⁵,⁴⁰ By contrast, FLT3L-deficient humans have a profoundly hypo-

plastic BM with an overall decrease in CD34⁺ HSPC counts. The frequencies of megakaryocyte-erythrocyte progenitors (MEPs) among the residual HSPCs of patients are abnormally high, as in the mouse model.⁵⁴ The frequency of myeloid progenitors (GMPs) was low in the patients but unaffected in *Flt3lg⁻/⁻* mice.⁵⁴ Both humans and mice with FLT3L deficiency display a depletion of DCPs.⁴⁰ In humans, the proportion of early lymphoid progenitors (MLPs and BNKPs) in the BM of the FLT3L-deficient patients remained within or near the control range, whereas the proportions of B cell progenitors were low. In *Flt3lg⁻/⁻* mice, the proportion of common lymphoid progenitors (CLPs) and B cell precursors (B-CFUs) was remarkably low in the BM.²⁴,⁴⁰,⁵⁴⁻⁵⁶ Thus, consistent with the model proposed by Tsapogas et al. based on mouse data,¹¹ the high proportion of MEPs among FLT3L-deficient HSPCs suggests that the early progenitors, which were not sufficiently stimulated by FLT3L signals, preferentially differentiate along megakaryocyte/erythrocyte pathways. However, this bias allows significant levels of differentiation into most mature hematopoietic subsets, enabling the patients to survive into adulthood, at least with the additional support of modern medicine. The same patients would probably have died at an early age before the advent of vaccination and anti-infectious medicine.

The differences between FLT3L-deficient mice and humans are more apparent in mature hematopoietic cells. Platelet counts were normal in the blood of the FLT3L-deficient patients, but these patients presented slight, probably multifactorial, anemia not observed in the mouse model.²⁴ As in mice,²⁴ blood leukocyte cellularity was only moderately low, and neutrophil and total lymphocyte counts were normal or near normal. Conversely, by contrast to the mouse model,²⁴ three patients displayed severe blood monocytopenia at most of the time points tested. As in mice,²²,²³,⁴⁰,⁵⁷ the differentiation of all types of DCs was severely affected in the patients but not completely abolished, with cDCs and pDCs barely detectable in the blood, BM, and skin. As in mice,²⁴,²⁵ T cell counts, including those for αβ and γδ T cells, were normal or near normal. Accordingly, all *in vitro* APC-independent assays showed T cell function to be normal. The patients had very few mature circulating B cells (~90% fewer than the controls), and this deficit appeared to be more profound than that in mice (~70%).²⁴,²⁵ The residual B cells included very few transitional B cells, and there was an accumulation of ABCs suggestive of an exhaustion of B cell production.⁵⁸ However, like *Flt3lg⁻/⁻* mice, the patients presented normal immunoglobulin production and even the production of excessive amounts of IgA (hyper-IgA), despite the low proportion of plasmablasts observed in the BM. Finally, by contrast to *Flt3l*-deficient mice,²⁴ the patients had normal or near-normal NK cell counts

langerin. Finally, the images in the lower panel show Langerhans cells (LCs), macrophages (Mac), CD4, and CD8 T cells after segmentation and identification by a machine-learning approach.

(B) Quantification of the main immune subsets in the epidermis (LCs) and dermis (Mac, CD4, and CD8 T cells) after identification by a machine-learning approach applied to biopsy specimens from six different HDs (adults, male or female and from multiple ethnic groups), with comparison to HS and warts from two patients. Data are presented as individual values (min to max). Boxplots show the median with IQR. No statistical analysis was performed here.

(C) The frequencies of each immune subset after 5' scRNA-seq on skin biopsy specimens of HS from three HDs (adults, male or female, and from multiple ethnic groups), P2 and P3, and a wart from P2. All the principal clusters targeted are identified on the basis of cell marker expression analysis. Data presentation are as in (B).

(D) UMAP clustering for the immune subsets shown in (C).
and differentiation and displayed normal adaptive NK cell re-
sponses to HCMV. Thus, human FLT3L deficiency leads to
normal or near-normal platelet, RBC, neutrophil, and lymphocyte
counts, with normal T and near-normal NK cell differentiation,
but with a major deficit of B cells and monocytes and an almost
total absence of DCs of all types. The major differences between
the patients and the mouse model included profound monocyto-
penia and near-normal NK cell levels in the patients.

A striking phenotype of the patients was their severe and
broad HPV infections. Only a few IEIs underlie skin HPV infec-
tions with broad HPV susceptibility and high penetrance. These
IEIs include GATA2, CXCR4, and DOCK8 deficiencies, all of
which affect the counts or function of T cells and APCs.⁵⁹,⁶⁰ By
contrast, FLT3L deficiency does not impair T cell number or
function but leads to much lower counts of APCs, including
circulating monocytes and B cells. The immunophenotyping of
skin cells demonstrated the presence of very small numbers of
DCs in the patients' skin but near-normal and normal counts
of macrophages and LCs, respectively. These observations sug-
gest that the patients' susceptibility to cutaneous viruses is
driven principally by an absence of dermal DCs. The absence
of reported HPV susceptibility in patients with complete IRF8
deficiency, who lack both monocytes and DCs, is probably
due to the death of affected individuals in the absence of
HSC transplantation in early childhood, before exposure to
HPV.⁶¹⁻⁶³ The central role of DCs in immunity to HPV does not
exclude a role for LCs or macrophages. Indeed, consistent
with the delayed, but not abolished, control of MmuPV1 infection
in *Flt3lg*⁻¹ mice, the presence of antibodies against a wide
range of HPV types in patients (e.g., HPV1, HPV4, and HPV6)
in the absence of identified related symptoms suggests that
the patients develop a T cell-dependent response and can con-
trol most HPV infections. However, the characterization of pa-
tients with FLT3L deficiency strongly suggests a key non-redund-
dant role for dermal DCs in the optimal control of HPV infections.

The three patients also developed recurrent bacterial infec-
tions of the lungs. This phenotype is typical of B cell defects
and consistent with the very low B cell counts in the patients.
Defective B cell responses, despite normal or high Ig levels in
the serum, are also observed in other IEIs, such as AD STAT3,
AD or AR IL6ST, and AR ZNF341 deficiencies.⁶⁴⁻⁶⁸ FLT3L-defi-
cient patients also display intestinal colonization with *Micro-
sporidium Enterocytozoon bienusi*, a fungal pathogen generally
detected in the context of acquired immunodeficiency syndrome
(AIDS).⁶⁹,⁷⁰ As in FLT3L deficiency, patients with IEIs affecting
DC-CD4 crosstalk (HLA-II, CD40, and CD40L deficiencies)
have been reported to develop opportunistic fungal infections
due, in particular, to *Microsporidium* and *Cryptosporidium*.
This condition can underlie chronic cholangitis and may neces-
sitate liver transplantation in addition to BM transplantation.⁷¹⁻⁷⁶
*Microsporidium* colonization of the gut can, therefore, account
for the chronic cholangitis in the patients, which, together with
*Parechovirus* infection, can account for their chronic diar-
rhea.⁷⁷⁻⁸⁰ It is more difficult to explain the polyarthritis reported
in P1 and P3. It is tempting to speculate that the defective
myeloid differentiation in the patients disrupts the homeostasis
of osteoclasts, a cell type responsible for bone resorption that
originates from a differentiation pathway common to macro-

phages.⁸¹⁻⁸³ The identification and description of more FLT3L-
deficient patients and further studies of these patients are now
required to clarify the precise mechanism underlying this
phenotype.

The patients have a broad infectious phenotype, but the lack
of susceptibility to certain types of pathogens is surprising.
*Flt3lg*⁻¹ mice presented no unusual susceptibility to natural in-
fections in standard pathogen-free conditions.²⁴ However,
these mice were highly susceptible to *Toxoplasma gondii* and
HSV-1.⁸¹,⁸⁴ In addition, the *Flt3*-deficient *warmflash* mouse
strain is susceptible to murine cytomegalovirus (MCMV).⁸²
Despite their seropositivity for HSV-1 and HCMV, the two sur-
viving patients did not develop clinical disease. The FLT3L-
deficient patients did not develop mycobacterial infections,
despite vaccination with BCG and their clear impairment of
interleukin (IL)-12 and IFN-γ production *in vitro* in response to
BCG stimulation due to the almost total absence of APCs. Sus-
ceptibility to mycobacterial infection is a common feature of
known DC and monocyte deficiencies.⁴¹,⁶¹⁻⁶³,⁸³,⁸⁵⁻⁸⁹ Similarly,
given the rarity of pDCs in the patients, it is surprising that they
display no particular susceptibility to respiratory viruses, such
as the influenza virus or SARS-CoV-2, which were both docu-
mented in P2. Such susceptibility is a common feature of pa-
tients with IRF7 and TLR7 deficiencies, both of which impair
IFN-α production by pDCs.⁹⁰⁻⁹² The residual monocytes,
together with the rare residual DCs, were probably sufficient
to protect the FLT3L-deficient patients against these patho-
gens. The discovery of new patients will undoubtedly clarify
the spectrum of infectious susceptibility in FLT3L-deficient pa-
tients. Overall, our findings show that FLT3L plays a key role in
human hematopoiesis, with marked differences between
FLT3L-deficient patients and *flt3lg*⁻¹ mice and a surprising
viability into adulthood, given the profound multiple cytopenia,
including an almost total absence of DCs.

Limitations of the study

One limitation of our study is that we studied only three relatives
from one Iranian kindred. The discovery of other patients from
other kindreds and ancestries may reveal additional hematolog-
ical, infectious, or other phenotypes. Another limitation is that we
did not study our patients from infancy. The BM phenotype may
have been present at birth, may be a consequence of acceler-
ated senescence, or both.

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

● EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

● METHOD DETAILS

○ Human patients

○ Case reports

○ Whole-exome sequencing (WES) and linkage analysis (LA)

○ Sanger sequencing
and differentiation and displayed normal adaptive NK cell re-
sponses to HCMV. Thus, human FLT3L deficiency leads to
normal or near-normal platelet, RBC, neutrophil, and lymphocyte
counts, with normal T and near-normal NK cell differentiation,
but with a major deficit of B cells and monocytes and an almost
total absence of DCs of all types. The major differences between
the patients and the mouse model included profound monocyto-
penia and near-normal NK cell levels in the patients.

A striking phenotype of the patients was their severe and
broad HPV infections. Only a few IEIs underlie skin HPV infec-
tions with broad HPV susceptibility and high penetrance. These
IEIs include GATA2, CXCR4, and DOCK8 deficiencies, all of
which affect the counts or function of T cells and APCs.⁵⁹,⁶⁰ By
contrast, FLT3L deficiency does not impair T cell number or
function but leads to much lower counts of APCs, including
circulating monocytes and B cells. The immunophenotyping of
skin cells demonstrated the presence of very small numbers of
DCs in the patients' skin but near-normal and normal counts
of macrophages and LCs, respectively. These observations sug-
gest that the patients' susceptibility to cutaneous viruses is
driven principally by an absence of dermal DCs. The absence
of reported HPV susceptibility in patients with complete IRF8
deficiency, who lack both monocytes and DCs, is probably
due to the death of affected individuals in the absence of
HSC transplantation in early childhood, before exposure to
HPV.⁶¹⁻⁶³ The central role of DCs in immunity to HPV does not
exclude a role for LCs or macrophages. Indeed, consistent
with the delayed, but not abolished, control of MmuPV1 infection
in *Flt3lg*⁻¹ mice, the presence of antibodies against a wide
range of HPV types in patients (e.g., HPV1, HPV4, and HPV6)
in the absence of identified related symptoms suggests that
the patients develop a T cell-dependent response and can con-
trol most HPV infections. However, the characterization of pa-
tients with FLT3L deficiency strongly suggests a key non-reduc-
dant role for dermal DCs in the optimal control of HPV infections.

The three patients also developed recurrent bacterial infec-
tions of the lungs. This phenotype is typical of B cell defects
and consistent with the very low B cell counts in the patients.
Defective B cell responses, despite normal or high Ig levels in
the serum, are also observed in other IEIs, such as AD STAT3,
AD or AR IL6ST, and AR ZNF341 deficiencies.⁶⁴⁻⁶⁸ FLT3L-defi-
cient patients also display intestinal colonization with *Micro-
sporidium Enterocytozoon bienusi*, a fungal pathogen generally
detected in the context of acquired immunodeficiency syndrome
(AIDS).⁶⁹,⁷⁰ As in FLT3L deficiency, patients with IEIs affecting
DC-CD4 crosstalk (HLA-II, CD40, and CD40L deficiencies)
have been reported to develop opportunistic fungal infections
due, in particular, to *Microsporidium* and *Cryptosporidium*.
This condition can underlie chronic cholangitis and may neces-
sitate liver transplantation in addition to BM transplantation.⁷¹⁻⁷⁶
*Microsporidium* colonization of the gut can, therefore, account
for the chronic cholangitis in the patients, which, together with
*Parechovirus* infection, can account for their chronic diar-
rhea.⁷⁷⁻⁸⁰ It is more difficult to explain the polyarthritis reported
in P1 and P3. It is tempting to speculate that the defective
myeloid differentiation in the patients disrupts the homeostasis
of osteoclasts, a cell type responsible for bone resorption that
originates from a differentiation pathway common to macro-

phages.⁸¹⁻⁸³ The identification and description of more FLT3L-
deficient patients and further studies of these patients are now
required to clarify the precise mechanism underlying this
phenotype.

The patients have a broad infectious phenotype, but the lack
of susceptibility to certain types of pathogens is surprising.
*Flt3lg*⁻¹ mice presented no unusual susceptibility to natural in-
fections in standard pathogen-free conditions.²⁴ However,
these mice were highly susceptible to *Toxoplasma gondii* and
HSV-1.⁸¹,⁸⁴ In addition, the *Flt3*-deficient *warmflash* mouse
strain is susceptible to murine cytomegalovirus (MCMV).⁸²
Despite their seropositivity for HSV-1 and HCMV, the two sur-
viving patients did not develop clinical disease. The FLT3L-
deficient patients did not develop mycobacterial infections,
despite vaccination with BCG and their clear impairment of
interleukin (IL)-12 and IFN-γ production *in vitro* in response to
BCG stimulation due to the almost total absence of APCs. Sus-
ceptibility to mycobacterial infection is a common feature of
known DC and monocyte deficiencies.⁴¹,⁶¹⁻⁶³,⁸³,⁸⁵⁻⁸⁹ Similarly,
given the rarity of pDCs in the patients, it is surprising that they
display no particular susceptibility to respiratory viruses, such
as the influenza virus or SARS-CoV-2, which were both docu-
mented in P2. Such susceptibility is a common feature of pa-
tients with IRF7 and TLR7 deficiencies, both of which impair
IFN-α production by pDCs.⁹⁰⁻⁹² The residual monocytes,
together with the rare residual DCs, were probably sufficient
to protect the FLT3L-deficient patients against these patho-
gens. The discovery of new patients will undoubtedly clarify
the spectrum of infectious susceptibility in FLT3L-deficient pa-
tients. Overall, our findings show that FLT3L plays a key role in
human hematopoiesis, with marked differences between
FLT3L-deficient patients and *flt3lg*⁻¹ mice and a surprising
viability into adulthood, given the profound multiple cytopenia,
including an almost total absence of DCs.

Limitations of the study

One limitation of our study is that we studied only three relatives from one Iranian kindred. The discovery of other patients from other kindreds and ancestries may reveal additional hematological, infectious, or other phenotypes. Another limitation is that we did not study our patients from infancy. The BM phenotype may have been present at birth, may be a consequence of accelerated senescence, or both.

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

● EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

● METHOD DETAILS

○ Human patients

○ Case reports

○ Whole-exome sequencing (WES) and linkage analysis (LA)

○ Sanger sequencing
* Metagenomic next-generation sequencing (mNGS)

* Cell culture and isolation

* RNA-seq analyses

* mRNA extraction, cDNA synthesis, and assessment of gene expression by RT-PCR/RT-qPCR

* Characterization of FLT3LG mRNA splice variants by Topo cloning

* Cloning and mutagenesis

* Transient transfection

* Cell lysates and analysis of the FLT3L protein by western blotting

* Lentivirus transduction

* Soluble FLT3L determination

* Analysis of FLT3L expression by flow cytometry

* Deep immunophenotyping of whole blood

* Bone marrow experiments

* Bone biopsy evaluation

* VirScan-phage immunoprecipitation-sequencing (PhIP-Seq)

* Luminex anti-HPV serological tests

* High-throughput sequencing (HTS) of the human TCR repertoire

* High-throughput sequencing (HTS) of the human BCR repertoire

* Skin digestion and skin cell sorting

* Single-cell RNA-sequencing

* Single-cell RNA-sequencing analysis

* Whole-blood activation assay

* BCG stimulation assay

* T-cell proliferation assay

* *In-vitro* and *ex-vivo* CD3⁺TCRαβ⁺ CD4⁺ T-cell polarization experiment

* Skin tissue processing and imaging mass cytometry (IMC) data acquisition

* IMC data analysis

* Mouse papillomavirus (MmuPV1) infection

* MmuPV1 DNA copy number analyses

* Anti-MmuPV1 antibody detection in enzyme-linked immunosorbent assays (ELISA)

* *In situ* hybridization (ISH), RNA *in situ* hybridization (RNA-ISH), and immunohistochemistry (IHC) for MmuPV1 detection

* Immunofluorescent staining of *Enterocytozoon bieneusi*

## QUANTIFICATION AND STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.cell.2024.04.009.

## ACKNOWLEDGMENTS

We thank patients and family, the members of the HGID laboratory, and Jérôme Mégret (sorting facility of Necker Hospital). We thank Patrick Verschueren, Lucas Van Aelst, Tom Hillary, Vibeke Vergote, and Martin Hiele (University Hospitals Leuven) for clinical investigation of the patients; Jerome Tamburini, Rudy Birsen, and Clément Larrue for donating the K562 cell lines; Stephen Elledge for providing the VirScan phage library; and the National Facility for Autoimmunity and Serology Profiling at SciLifeLab for instrument support. This study was supported by the St. Giles Foundation, Rockefeller University, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris Cité University, National Center for Research Resources, National Institutes of Health (NIH) (R01AI143810), National Center for Advancing Sciences of the National Institutes of Health (UL1TR001866), French National Research Agency (ANR) (ANR-10-IAHU-01, ANR-21-CE15-0034, and ANR-10-LABX-62-IBEID), ITMO Cancer of Aviesan and INCa within the framework of the 2021–2030 Cancer Control Strategy, C1 grant from KU Leuven (C16/18/007), French Society of Dermatology (SFD), the Schlumberger Foundation for teaching and research (FSER) (FSER202302017062), Square Foundation, Stavros Niarchos Foundation (SNF) as part of its grant to the SNF Institute for Global Infectious Disease Research at The Rockefeller University, William E. Ford (General Atlantic’s chairman and chief executive officer), Gabriel Caillaux (General Atlantic’s co-

president, managing director, and head of business in EMEA), General Atlantic Foundation, ANRS—Maladies infectieuses emergentes (M. Momenilandi, ECTZ173053), American Association of Immunologists (A.G.), National Health and Medical Research Council of Australia (S.G.T. and C.S.M.), French Foundation for Medical Research (A.-L.N., FDT202204015102), Bettencourt-Scheller Foundation (A.-L.N.), Ecole de l'INSERM Liliane Bettencourt (Q.P.), David Rockefeller Graduate Program (M.O.), New York Hideyo Noguchi Memorial Society (M.O.), Funai Foundation for Information Technology (M.O.), Honjo International Scholarship Foundation (M.O.), New York Hideyo Noguchi Memorial Society (M.O.), Swedish Research Council and the Göran Gustafsson Foundation (N. Landegren), National Institute of Dental and Craniofacial Research program (J.H., R21DE028650), National Cancer Institute (J.H., R21CA271069 and R21 CA274265-01A1), National Institute of Allergy and Infectious Diseases (J.H. and N.C., R21AI121822), Penn State Cancer Institute Program Project Development Award Sponsored by the Highmark Community Health Reinvestment Fund and the Jake Gittlen Memorial Golf Tournament (J.H. and N.C.), FWO Vlaanderen (I.M., senior clinical researcher at FWO Vlaanderen, FWO G0B5120N; R.S., FWO 1805523N), European Research Council (I.M., no. 948959), and CSL Behring (I.M.). L.D.N. is supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, NIH, Bethesda (USA). Open access funding was provided by Rockefeller University.

## AUTHOR CONTRIBUTIONS

V.B., J.-L.C., X.B., M.S., and M. Momenilandi designed the research project; experimental work, including study design, resource provision, implementation of the study and data analysis were performed by M. Momenilandi, S.S., J.L., C.L.-P., A.F., C.L.F., A.G., E.D.M., D.S., A.Y., A.D., M. Mancini, A.-L.N., K.B., S.B., C.D., B.B., C.C., M. Materna, A.C., M.O., Q.P., O.B., M.A., M.W., T.K., S.B.-D., J. Bustamante, M.L., Philippe Pérot, J.F., C.N., Philippe Poirier, N.C., N. Landegren, N.M., E.S., T.W., F.G., C.S.M., S.G.T., N. Lachmann, and J.H.; bioinformatic and computational analyses were performed by M. Momenilandi, A.F., D.R., C.L.F., O.M.D., M.O., K.M., H.R.-Z., M.C.-A, Y.S., B.P., and D.L.; clinical studies, patient care, biological samples (including samples from the patients and HDs), and diagnostic laboratory tests were handled by R.L., H.E., S.F., F.H., J.C., J. Bruneau, T.J.M., S.L.-M., J.-L.P., N.P., K.G.C., R.S., I.M., and M.S.; L.Y., H.V., E.J., L.D.N., and L.A. provided knowledge, proficiency, and feedback; M. Momenilandi and V.B. drafted the manuscript; and the manuscript was revised by all authors.

## DECLARATION OF INTERESTS

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

Received: July 26, 2023

Revised: March 4, 2024

Accepted: April 10, 2024

Published: May 2, 2024

## REFERENCES

1. Orkin, S.H., and Zon, L.I. (2008). Hematopoiesis: an evolving paradigm for stem cell biology. *Cell* 132, 631–644. https://doi.org/10.1016/j.cell.2008.01.025.

2. Bao, E.L., Cheng, A.N., and Sankaran, V.G. (2019). The genetics of human hematopoiesis and its disruption in disease. *EMBO Mol. Med.* 11, e10316. https://doi.org/10.15252/emmm.201910316.

3. Ceredig, R., Rolink, A.G., and Brown, G. (2009). Models of haematopoiesis: seeing the wood for the trees. *Nat. Rev. Immunol.* 9, 293–300. https://doi.org/10.1038/nri2525.

4. Lagresle-Peyrou, C., Six, E.M., Picard, C., Rieux-Laucat, F., Michel, V., Ditadi, A., Demerens-de Chappedelaine, C.D., Morillon, E., Valensi, F., Simon-Stoos, K.L., et al. (2009). Human adenylate kinase 2 deficiency
* Metagenomic next-generation sequencing (mNGS)

* Cell culture and isolation

* RNA-seq analyses

* mRNA extraction, cDNA synthesis, and assessment of gene expression by RT-PCR/RT-qPCR

* Characterization of FLT3LG mRNA splice variants by Topo cloning

* Cloning and mutagenesis

* Transient transfection

* Cell lysates and analysis of the FLT3L protein by western blotting

* Lentivirus transduction

* Soluble FLT3L determination

* Analysis of FLT3L expression by flow cytometry

* Deep immunophenotyping of whole blood

* Bone marrow experiments

* Bone biopsy evaluation

* VirScan-phage immunoprecipitation-sequencing (PhIP-Seq)

* Luminex anti-HPV serological tests

* High-throughput sequencing (HTS) of the human TCR repertoire

* High-throughput sequencing (HTS) of the human BCR repertoire

* Skin digestion and skin cell sorting

* Single-cell RNA-sequencing

* Single-cell RNA-sequencing analysis

* Whole-blood activation assay

* BCG stimulation assay

* T-cell proliferation assay

* *In-vitro* and *ex-vivo* CD3⁺TCRαβ⁺ CD4⁺ T-cell polarization experiment

* Skin tissue processing and imaging mass cytometry (IMC) data acquisition

* IMC data analysis

* Mouse papillomavirus (MmuPV1) infection

* MmuPV1 DNA copy number analyses

* Anti-MmuPV1 antibody detection in enzyme-linked immunosorbent assays (ELISA)

* *In situ* hybridization (ISH), RNA *in situ* hybridization (RNA-ISH), and immunohistochemistry (IHC) for MmuPV1 detection

* Immunofluorescent staining of *Enterocytozoon bieneusi*

## QUANTIFICATION AND STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.cell.2024.04.009.

## ACKNOWLEDGMENTS

We thank patients and family, the members of the HGID laboratory, and Jérôme Mégret (sorting facility of Necker Hospital). We thank Patrick Verschueren, Lucas Van Aelst, Tom Hillary, Vibeke Vergote, and Martin Hiele (University Hospitals Leuven) for clinical investigation of the patients; Jerome Tamburini, Rudy Birsen, and Clément Larrue for donating the K562 cell lines; Stephen Elledge for providing the VirScan phage library; and the National Facility for Autoimmunity and Serology Profiling at SciLifeLab for instrument support. This study was supported by the St. Giles Foundation, Rockefeller University, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris Cité University, National Center for Research Resources, National Institutes of Health (NIH) (R01AI143810), National Center for Advancing Sciences of the National Institutes of Health (UL1TR001866), French National Research Agency (ANR) (ANR-10-IAHU-01, ANR-21-CE15-0034, and ANR-10-LABX-62-IBEID), ITMO Cancer of Aviesan and INCa within the framework of the 2021–2030 Cancer Control Strategy, C1 grant from KU Leuven (C16/18/007), French Society of Dermatology (SFD), the Schlumberger Foundation for teaching and research (FSER) (FSER202302017062), Square Foundation, Stavros Niarchos Foundation (SNF) as part of its grant to the SNF Institute for Global Infectious Disease Research at The Rockefeller University, William E. Ford (General Atlantic’s chairman and chief executive officer), Gabriel Caillaux (General Atlantic’s co-

president, managing director, and head of business in EMEA), General Atlantic Foundation, ANRS—Maladies infectieuses emergentes (M. Momenilandi, ECTZ173053), American Association of Immunologists (A.G.), National Health and Medical Research Council of Australia (S.G.T. and C.S.M.), French Foundation for Medical Research (A.-L.N., FDT202204015102), Bettencourt-Scheller Foundation (A.-L.N.), Ecole de l'INSERM Liliane Bettencourt (Q.P.), David Rockefeller Graduate Program (M.O.), New York Hideyo Noguchi Memorial Society (M.O.), Funai Foundation for Information Technology (M.O.), Honjo International Scholarship Foundation (M.O.), New York Hideyo Noguchi Memorial Society (M.O.), Swedish Research Council and the Göran Gustafsson Foundation (N. Landegren), National Institute of Dental and Craniofacial Research program (J.H., R21DE028650), National Cancer Institute (J.H., R21CA271069 and R21 CA274265-01A1), National Institute of Allergy and Infectious Diseases (J.H. and N.C., R21AI121822), Penn State Cancer Institute Program Project Development Award Sponsored by the Highmark Community Health Reinvestment Fund and the Jake Gittlen Memorial Golf Tournament (J.H. and N.C.), FWO Vlaanderen (I.M., senior clinical researcher at FWO Vlaanderen, FWO G0B5120N; R.S., FWO 1805523N), European Research Council (I.M., no. 948959), and CSL Behring (I.M.). L.D.N. is supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, NIH, Bethesda (USA). Open access funding was provided by Rockefeller University.

## AUTHOR CONTRIBUTIONS

V.B., J.-L.C., X.B., M.S., and M. Momenilandi designed the research project; experimental work, including study design, resource provision, implementation of the study and data analysis were performed by M. Momenilandi, S.S., J.L., C.L.-P., A.F., C.L.F., A.G., E.D.M., D.S., A.Y., A.D., M. Mancini, A.-L.N., K.B., S.B., C.D., B.B., C.C., M. Materna, A.C., M.O., Q.P., O.B., M.A., M.W., T.K., S.B.-D., J. Bustamante, M.L., Philippe Pérot, J.F., C.N., Philippe Poirier, N.C., N. Landegren, N.M., E.S., T.W., F.G., C.S.M., S.G.T., N. Lachmann, and J.H.; bioinformatic and computational analyses were performed by M. Momenilandi, A.F., D.R., C.L.F., O.M.D., M.O., K.M., H.R.-Z., M.C.-A, Y.S., B.P., and D.L.; clinical studies, patient care, biological samples (including samples from the patients and HDs), and diagnostic laboratory tests were handled by R.L., H.E., S.F., F.H., J.C., J. Bruneau, T.J.M., S.L.-M., J.-L.P., N.P., K.G.C., R.S., I.M., and M.S.; L.Y., H.V., E.J., L.D.N., and L.A. provided knowledge, proficiency, and feedback; M. Momenilandi and V.B. drafted the manuscript; and the manuscript was revised by all authors.

## DECLARATION OF INTERESTS

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

Received: July 26, 2023

Revised: March 4, 2024

Accepted: April 10, 2024

Published: May 2, 2024

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2. Bao, E.L., Cheng, A.N., and Sankaran, V.G. (2019). The genetics of human hematopoiesis and its disruption in disease. *EMBO Mol. Med.* 11, e10316. https://doi.org/10.15252/emmm.201910316.

3. Ceredig, R., Rolink, A.G., and Brown, G. (2009). Models of haematopoiesis: seeing the wood for the trees. *Nat. Rev. Immunol.* 9, 293–300. https://doi.org/10.1038/nri2525.

4. Lagresle-Peyrou, C., Six, E.M., Picard, C., Rieux-Laucat, F., Michel, V., Ditadi, A., Demerens-de Chappedelaine, C.D., Morillon, E., Valensi, F., Simon-Stoos, K.L., et al. (2009). Human adenylate kinase 2 deficiency
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6. Kostmann, R. (1950). Hereditär reticulos-en ny systemsjukdom. Sven. Läkartideningen 47, 2861–2868.

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8. Kostman, R. (1975). Infantile genetic agranulocytosis. A review with presentation of ten new cases. Acta Paediatr. Scand. 64, 362–368. https://doi.org/10.1111/j.1651-2227.1975.tb03847.x.

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12. Matthews, W., Jordan, C.T., Wiegand, G.W., Pardoll, D., and Lemischka, I.R. (1991). A receptor tyrosine kinase specific to hematopoietic stem and progenitor cell-enriched populations. Cell 65, 1143–1152. https://doi.org/10.1016/0092-8674(91)90010-V.

13. Rosnet, O., Marchetto, S., deLapeyriere, O., and Birnbaum, D. (1991). Murine FLT3, a gene encoding a novel tyrosine kinase receptor of the PDGFR/CSF1R family. Oncogene 6, 1641–1650.

14. Rosnet, O., Mattei, M.-G., Marchetto, S., and Birnbaum, D. (1991). Isolation and chromosomal localization of a novel FMS-like tyrosine kinase gene. Genomics 9, 380–385. https://doi.org/10.1016/0888-7543(91)90270-0.

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19. Fujita, K., Chakarov, S., Kobayashi, T., Sakamoto, K., Voisin, B., Duan, K., Nakagawa, T., Horiuchi, K., Amagai, M., Ginhoux, F., et al. (2019). Cell-autonomous FLT3L shedding via ADAM10 mediates conventional dendritic cell development in mouse spleen. Proc. Natl. Acad. Sci. USA 116, 14714–14723. https://doi.org/10.1073/pnas.1818907116.

20. Horiuchi, K., Morioka, H., Takaishi, H., Akiyama, H., Blobel, C.P., and Toyama, Y. (2009). Ectodomain shedding of FLT3 ligand is mediated by TNF-α converting enzyme. J. Immunol. 182, 7408–7414. https://doi.org/10.4049/jimmunol.0801931.

21. Toffalini, F., and Demoulin, J.-B. (2010). New insights into the mechanisms of hematopoietic cell transformation by activated receptor tyrosine kinases. Blood 116, 2429–2437. https://doi.org/10.1182/blood-2010-04-279752.

22. Durai, V., Bagadia, P., Briseño, C.G., Theisen, D.J., Iwata, A., Davidson, J.T., Gargaro, M., Fremont, D.H., Murphy, T.L., and Murphy, K.M. (2018). Altered compensatory cytokine signaling underlies the discrepancy between FLT3-/- and FLT3I-/- mice. J. Exp. Med. 215, 1417–1435. https://doi.org/10.1084/jem.20171784.

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24. McKenna, H.J., Stocking, K.L., Miller, R.E., Brasel, K., De Smedt, T., Maraskovsky, E., Maliszewski, C.R., Lynch, D.H., Smith, J., Pulendran, B., et al. (2000). Mice lacking flt3 ligand have deficient hematopoiesis affecting hematopoietic progenitor cells, dendritic cells, and natural killer cells. Blood 95, 3489–3497. https://doi.org/10.1182/blood.V95.11.3489.

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5. De Vaal, O.M., and Seynhaeve, V. (1959). Reticular dysgesenia. Lancet 2, 1123-1125. https://doi.org/10.1016/S0140-6736(59)90105-9.

6. Kostmann, R. (1950). Hereditär reticulos-en ny systemsjukdom. Sven. Läkartideningen 47, 2861-2868.

7. Kostmann, R. (1956). Infantile genetic agranulocytosis; agranulocytosis infantilis hereditaria. Acta Paediatr. Suppl. (Upps) 45, 1-78.

8. Kostman, R. (1975). Infantile genetic agranulocytosis. A review with presentation of ten new cases. Acta Paediatr. Scand. 64, 362-368. https://doi.org/10.1111/j.1651-2227.1975.tb03847.x.

9. Bruton, O.C. (1952). Agammaglobulinemia. Pediatrics 9, 722-728. https://doi.org/10.1542/peds.9.6.722.

10. Casanova, J.-L., and Hammarström, L. (2023). Foreword to the English translation of Kostmann's memoirs. J. Clin. Immunol. 43, 671-674. https://doi.org/10.1007/s10875-023-01446-2.

11. Tsapogas, P., Mooney, C.J., Brown, G., and Rolink, A. (2017). The cytokine FLT3-ligand in normal and malignant hematopoiesis. Int. J. Mol. Sci. 18, 1115. https://doi.org/10.3390/ijms18061115.

12. Matthews, W., Jordan, C.T., Wiegand, G.W., Pardoll, D., and Lemischka, I.R. (1991). A receptor tyrosine kinase specific to hematopoietic stem and progenitor cell-enriched populations. Cell 65, 1143-1152. https://doi.org/10.1016/0092-8674(91)90010-V.

13. Rosnet, O., Marchetto, S., deLapeyriere, O., and Birnbaum, D. (1991). Murine FLT3, a gene encoding a novel tyrosine kinase receptor of the PDGFR/CSF1R family. Oncogene 6, 1641-1650.

14. Rosnet, O., Mattei, M.-G., Marchetto, S., and Birnbaum, D. (1991). Isolation and chromosomal localization of a novel FMS-like tyrosine kinase gene. Genomics 9, 380-385. https://doi.org/10.1016/0888-7543(91)90270-0.

15. Lyman, S.D., James, L., Vanden Bos, T.V., de Vries, P., Brasel, K., Glianiak, B., Hollingsworth, L.T., Picha, K.S., McKenna, H.J., and Splett, R.R. (1993). Molecular cloning of a ligand for the flt3flk-2 tyrosine kinase receptor: A proliferative factor for primitive hematopoietic cells. Cell 75, 1157-1167. https://doi.org/10.1016/0092-8674(93)90325-K.

16. Lyman, S.D., James, L., Escobar, S., Downey, H., de Vries, P., Brasel, K., Stocking, K., Beckmann, M.P., Copeland, N.G., and Cleveland, L.S. (1995). Identification of soluble and membrane-bound isoforms of the murine flt3 ligand generated by alternative splicing of mRNAs. Oncogene 10, 149-157.

17. Lyman, S.D., James, L., Johnson, L., Brasel, K., de Vries, P., Escobar, S.S., Downey, H., Splett, R.R., Beckmann, M.P., and McKenna, H.J. (1994). Cloning of the human homologue of the murine flt3 ligand: a growth factor for early hematopoietic progenitor cells. Blood 83, 2795-2801. https://doi.org/10.1182/blood.V83.10.2795.2795.

18. Chklovskaia, E., Nissen, C., Landmann, L., Rahner, C., Pfister, O., and Wodnar-Filipowicz, A. (2001). Cell-surface trafficking and release of flt3 ligand from T lymphocytes is induced by common cytokine receptor β-chain signaling and inhibited by cyclosporin A. Blood 97, 1027-1034. https://doi.org/10.1182/blood.V97.4.1027.

19. Fujita, K., Chakarov, S., Kobayashi, T., Sakamoto, K., Voisin, B., Duan, K., Nakagawa, T., Horiuchi, K., Amagai, M., Ginhoux, F., et al. (2019). Cell-autonomous FLT3L shedding via ADAM10 mediates conventional dendritic cell development in mouse spleen. Proc. Natl. Acad. Sci. USA 116, 14714-14723. https://doi.org/10.1073/pnas.1818907116.

20. Horiuchi, K., Morioka, H., Takaishi, H., Akiyama, H., Blobel, C.P., and Toyama, Y. (2009). Ectodomain shedding of FLT3 ligand is mediated by TNF-α converting enzyme. J. Immunol. 182, 7408-7414. https://doi.org/10.4049/jimmunol.0801931.

21. Toffalini, F., and Demoulin, J.-B. (2010). New insights into the mechanisms of hematopoietic cell transformation by activated receptor tyrosine kinases. Blood 116, 2429-2437. https://doi.org/10.1182/blood-2010-04-279752.

22. Durai, V., Bagadia, P., Briseño, C.G., Theisen, D.J., Iwata, A., Davidson, J.T., Gargaro, M., Fremont, D.H., Murphy, T.L., and Murphy, K.M. (2018). Altered compensatory cytokine signaling underlies the discrepancy between FLT3-/- and FLT3I-/- mice. J. Exp. Med. 215, 1417-1435. https://doi.org/10.1084/jem.20171784.

23. Ginhoux, F., Liu, K., Helft, J., Bogunovic, M., Greter, M., Hashimoto, D., Price, J., Yin, N., Bromberg, J., Lira, S.A., et al. (2009). The origin and development of nonlymphoid tissue CD103+ DCs. J. Exp. Med. 206, 3115-3130. https://doi.org/10.1084/jem.20091756.

24. McKenna, H.J., Stocking, K.L., Miller, R.E., Brasel, K., De Smedt, T., Maraskovsky, E., Maliszewski, C.R., Lynch, D.H., Smith, J., Pulendran, B., et al. (2000). Mice lacking flt3 ligand have deficient hematopoiesis affecting hematopoietic progenitor cells, dendritic cells, and natural killer cells. Blood 95, 3489-3497. https://doi.org/10.1182/blood.V95.11.3489.

25. Gwin, K.A., Shapiro, M.B., Dolence, J.J., Huang, Z.L., and Medina, K.L. (2013). Hoxa9 and FLT3 signaling synergistically regulate an early checkpoint in lymphopoiesis. J. Immunol. 191, 745-754. https://doi.org/10.4049/jimmunol.1203294.

26. Rapaport, F., Boisson, B., Gregor, A., Béziat, V., Boisson-Dupuis, S., Bustamante, J., Jouanguy, E., Puel, A., Rosain, J., Zhang, Q., et al. (2021). Negative selection on human genes underlying inborn errors depends on disease outcome and both the mode and mechanism of inheritance. Proc. Natl. Acad. Sci. USA 118, e2001248118. https://doi.org/10.1073/pnas.2001248118.

27. Kircher, M., Witten, D.M., Jain, P., O'Roak, B.J., Cooper, G.M., and Shendure, J. (2014). A general framework for estimating the relative pathogenicity of human genetic variants. Nat. Genet. 46, 310-315. https://doi.org/10.1038/ng.2892.

28. Itan, Y., Shang, L., Boisson, B., Ciancanelli, M.J., Markle, J.G., Martinez-Barricarte, R., Scott, E., Shah, I., Stenson, P.D., Gleeson, J., et al. (2016). The mutation significance cutoff: gene-level thresholds for variant predictions. Nat. Methods 13, 109-110. https://doi.org/10.1038/nmeth.3739.

29. Chen, S., Francioli, L.C., Goodrich, J.K., Collins, R.L., Kanai, M., Wang, Q., Alföldi, J., Watts, N.A., Vittal, C., Gauthier, L.D., et al. (2022). A Genome-Wide Mutational Constraint Map Quantified from Variation in 76,156 Human Genomes. Preprint at bioRxiv. https://doi.org/10.1101/2022.03.20.485034.

30. Taliun, D., Harris, D.N., Kessler, M.D., Carlson, J., Szpiech, Z.A., Torres, R., Taliun, S.A.G., Corvelo, A., Gogarten, S.M., Kang, H.M., et al. (2021). Sequencing of 53,831 diverse genomes from the NHlib TOPMed Program. Nature 590, 290-299. https://doi.org/10.1038/s41586-021-03205-y.

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