Peprotech], IL-1β [50 ng/mL, #200-01B-10, Peprotech], IL-6 [50 ng/mL, #200-06-20, PeproTech], IL-21 [50 ng/mL, #200-21, Peprotech], IL-23 [50 ng/mL, #200-23-10, PeproTech]) polarizing conditions. After five days of polarization, the cells were stimulated with PMA (100 ng/mL, #P8139-1MG, Sigma Aldrich)-ionomycin (750 ng/mL, #I0634-1MG, Sigma Aldrich) for 6 h, with brefeldin A (10 mg/mL, #B7651-5MG, Sigma Aldrich) added after the first 2 h of incubation. For the assessment of intracellular cytokine production, cells were incubated with conjugated monoclonal antibodies directed against IFN-γ (BUV737, clone 4S.B3, #564620, BD Horizon), TNF (PerCP, clone Mab11, #502924, BioLegend), IL-9 (PE, clone MH9A3, #560807, BD Pharmingen), IL-13 (BV421, clone JES10-5A2, #563580, BD Horizon), IL-4 (AF488, clone 8D4-8, #500710, BioLegend), IL-17A (BV510, clone BL168, #512330, BioLegend), IL-17F (BV650, clone O33-782, #564264, BD Horizon), IL-2 (BV750, clone MQ1-17H12, #566361, BD Horizon), IL-21 (eF660, clone eBio3A3-N2, #50-7219-42, Thermo Fisher Scientific), and IL-22 (PerCP e710, clone 22URTI, #46-7229-42, eBioscience). All cells were also stained with the Zombie UV fixable viability dye kit (#423107, BioLegend). Cells and beads were acquired on a BD FACSymphony A5 Cell Analyzer (BD bioscience) and analyzed with FlowJo Software. A previously described gating strategy was used (109).

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

We used 3 µm-thick sections of formalin-fixed paraffin-embedded blocks of tissue from cutaneous biopsies performed on patients (warts and normal skin), healthy relatives (normal skin) and healthy donors (normal skin). Slides were first baked for 2 h in a slide oven until all the visible wax had disappeared. They were then dewaxed in a meta-xylene bath for 20 min and rehydrated in a series of descending concentrations of ethanol (100%, 70% and 50%; 10 min each). Slides were washed for five minutes in water and then incubated in antigen retrieval solution for 30 min at 96°C followed by 10 min at room temperature. They were then washed for 10 min in pure water and 10 min in PBS. A PAP pen was used to encircle the samples on each slide, and the slides were then blocked by incubation with 100 µL superblock solution per sample for 45 min in a hydration chamber. Staining was achieved in two steps: a mixture of antibodies diluted in PBS-BSA 0.5% was first applied to the slides, which were then incubated overnight at 4°C in a hydration chamber. The slides were then washed with PBS for 10 minutes and incubated with a second mixture at room temperature for 1.5 h. Our 29-metal antibody IMC panel was designed to evaluate the immune infiltrate present in healthy skin and at the site of infection. It made it possible to study several cell subsets simultaneously, including APCs (DCs, macrophages, monocytes, Langerhans cells), lymphocytes (CD4, CD8, Trm, γδ, Treg, and B cells), neutrophils and keratinocytes (Abs summarized in STAR Methods). Each antibody used in our mixture was previously validated on tonsils, by IMC for conjugated antibodies purchased directly from Fluidigm and by both immunofluorescence and IMC for in-house conjugated antibodies. All conjugations were performed according to the Fluidigm protocol. The stained slides were washed for 16 min in 0.2% Triton-X100 and 16 min in PBS. They were then stained with Intercalator-Ir in Maxpar PBS 1:400 for 30 min at room temperature in a hydration chamber, washed in pure water for 4 min and air-dried for at least 20 min. Finally, IMC data corresponding to pre-established regions of interest (ROIs) were acquired as raw .mcd files with the Hyperion imaging system (Fluidigm).

## IMC data analysis

IMC data preprocessing and segmentations were performed with the Steinbock toolkit.¹²⁵ The preprocessing steps led to the generation of a single .tif file per ROI. Segmentation masks were generated with the deep learning model Deepcell using the iridium channel as the nuclear signal and vimentin, pancytokeratin, CD45, E-cadherin and HLA-DR as cytoplasmic signals. Signal spillover was subsequently compensated as previously described.¹²⁶ Segmented and compensated data were then analyzed with Qupath software.¹²⁷ In Qupath, cells with an area of 15 µm² were filtered out from downstream analysis. In each remaining cell, various signal data were calculated for each channel (mean, median, minimum and maximum, giving the range, and standard deviation). Based on the vimentin, cytokeratin, CD45, CD4, E-cadherin, CD68, CD8a, CD3 and Langerin channels for an image of healthy skin from a healthy donor, we manually annotated about 10 to 15 cells of each of the following cell subsets: CD4⁺ T cells, CD8⁺ T cells, macrophages, Langerhans cells, other CD45⁺ cells, keratinocytes and other CD45⁻ cells. These annotations were then used to train a random forest cell classifier directly in Qupath. This classifier was then applied to other images with occasional adjustments based on new annotations. In parallel, for quantification, we defined two ROIs in each image: the epidermis, identified with the pancytokeratin and E-cadherin channels, and the 200 µm of subepidermal dermis. This approach enabled us to analyze the composition of the epidermis and the subepidermal dermis accurately for each sample.

## Mouse papillomavirus (MmuPV1) infection

All mouse work was approved by the Institutional Animal Care and Use Committee of Pennsylvania State University's College of Medicine (COM), and all methods were performed in accordance with guidelines and regulations. The mice were housed (2–5 mice/cage) in sterile cages within sterile filter hoods in the COM BL2 animal core facility. Infectious mouse papillomavirus (MmuPV1) was isolated from lesions on the tails of mice from our previous studies.¹²⁸,¹²⁹ In brief, papillomas scraped from the tails of the mice were homogenized in phosphate-buffered saline (1×PBS) with a Polytron homogenizer (Brinkman PT10-35) at maximum speed for three minutes, with chilling in an ice bath. The homogenate was spun in a bench centrifuge at 10,000 rpm, the supernatant was decanted into Eppendorf tubes, and the resulting mouse papillomavirus suspension was stored in glycerol at -80°C (1:1,V/V). C57BL/6, Rag1⁻/⁻, and Flt3lg⁻/⁻ (4–6 weeks old) mice were obtained from Jackson Laboratories and reared in the animal core facility of PSUCOM. Three female Flt3lg-heterozygous mice in the B6 background were purchased from the Mutant Mouse Resource & Research Centers
(MMRRC). Heterozygous female and male mice (from F1 breeding) were crossed to acquire homozygous *Flt3lg*⁻/⁻ mice, and this knockout was confirmed with the recommended genotyping method. In brief, three mixed primers were used to distinguish between the mutant and wild type: a mutant reverse primer 5' ATT TGT CAC GTC CTG CAC GAC G 3'; a common primer 5' TGG CAG CTG AAG TGA CTG AC 3'; and a wild-type reverse primer 5' AAG CCA AAG CTG GAT GAC AG 3'. We used six female and five male *Flt3lg*⁻/⁻ homozygotes in this study. Mice (4–8 weeks old) were sedated i.p. with 0.1 ml/10 g body weight ketamine/xylazine mixture (100 mg/10 mg in 10 ml double-distilled H₂O) and their skin was wounded at two sites (tail and muzzle), as previously described.¹³⁰ Twenty-four hours after wounding, the mice were again anesthetized and challenged with infectious virus (suspensions of 1 × 10⁹ viral DNA genomes/ infected site) at the wound sites. Infection was monitored twice weekly at the muzzle and tail wound sites and progress was documented photographically for each animal.¹²⁸⁻¹³⁰ Two, three, two and four *Flt3lg*⁻/⁻ mice of both sexes were killed three, four, six and eight weeks, respectively, after viral infection. *Rag1*⁻/⁻ and C57BL/6 mice were also killed at weeks three, four, six, and eight weeks post infection for comparisons at particular time points. The infected tissues were harvested for additional *in situ* analyses.

## MmuPV1 DNA copy number analyses

Linearized MmuPV1 genomic DNA was used for standard curve determination with a probe qPCR system. The primer pairs (5' GGTTGCGTCGGAGAACATATAA 3' and 5' CTAAAGCTAACCTGCCACATATC 3') for E2 amplification were used together with the probe (5' 6-FAM-TGCCCTTCA/ZEN/GTGGGTTGAGGACAG 3'-IBFQ). Each reaction contained 9 µL ultrapure water, 5 pmol of each primer, 9 µL Brilliant III qPCR Master Mix (Agilent) and 2 µL DNA template. The PCR conditions were: initial denaturation at 95°C for 3 min, then 40 cycles at 95°C for 5 seconds and 60°C for 10 seconds on an Agilent AriaMx. Viral copy numbers were converted into an equivalent DNA load as follows: 1 ng viral DNA = 1.2 × 10⁸ copies (http://cels.uri.edu/gsc/cndna.html). For the tissue samples, 40 ng total DNA was used for qPCR. All samples were tested at least in duplicate. Viral DNA titers were calculated from a standard curve.

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

Serum was collected from mice at the end of the experiment. ELISA assay was performed for antibodies against MmuPV1 L1. 0.5 µg MmuPV1 L1 VLPs in 50 µL 1X PBS/well was coated in the wells of Nunc® MaxiSorpTM 96-well microtiter plates (Thermo Fisher Scientific, Waltham MA), by incubation at room temperature for 30 minutes. For antibody against MmuPV1 E4 protein, 0.5 µg KLH-conjugated MmuPV1 E4 peptide (PKTTPPRRELFPPTPLTQPP, synthesized by China Peptide)/well in 50 µL bicarbonate (pH 9.6) buffer was incubated overnight with the plates at 37°C. Coated plates were blocked by incubation with PBS/5% dry milk powder for 1 hour at room temperature. ELISA was then performed as previously described.⁹⁸ Each of the serum samples was diluted 1:100 or more in PBS/5% dry milk and the dilutions were added to the 96-well plates, which were then incubated for 1 hour at room temperature. Negative controls were either sera collected from non-infected mice or minus primary antibodies. The plates were washed five times with washing buffer (0.05% (v/v) Tween 20 in 1×PBS), and we then added goat anti-mouse IgG (H+L)-alkaline phosphatase (AP) (Southern Biotech) secondary antibody diluted 1:2000 in 5% dry milk powder and incubated the plates for one hour at room temperature. The plates were washed five times with washing buffer, and 100 µL of 1 mg/mL pNPP (para-nitrophenyl phosphate), a substrate for alkaline phosphatase (AP), was added to each well for color development, and absorbance at 405 nm/450 nm was measured with a Fisher Microplate Reader. A mouse monoclonal antibody against MmuPV1L1 (MPV.A4) that neutralizes MmuPV1 infection *in vitro* and *in vivo*⁹⁷ was used as the positive control for L1 detection.

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

Infected tissue biopsy specimens were fixed in 10% neutral buffered formalin and embedded in paraffin. Adjacent sequential sections were cut for hematoxylin and eosin (H&E) staining, *in situ* hybridization (ISH), RNA *in situ* hybridization (RNA-ISH), and immunohistochemistry (IHC), as described in previous studies.⁹⁴,¹²⁹,¹³¹ For ISH, a biotin-labeled 3913 bp EcoRV/ BamH1 subgenomic fragment from MmuPV1 was used as the probe for the detection of MmuPV1 DNA in tissues.¹²⁸ Access to target DNA was obtained by incubation with 0.2 mg/mL pepsin in 0.1 N HCl at 37°C for 8 min. The sections were washed thoroughly. The biotinylated probe was then applied and the sections were heated at 95°C for 5 min to dissociate the target and probe DNA. Re-annealing was allowed to occur for 2 hours at 37°C. Target-bound biotin was detected with a streptavidin AP conjugate and a colorimetric reaction in BCIP/ NBT. For ISH, the nuclei were counterstained with Nuclear Fast Red (Vector Laboratories). Viral RNA was detected in formalin-fixed, paraffin-embedded (FFPE) sections with RNAscope technology (Advanced Cell Diagnostics, Inc.), with custom probes binding to the second exon of the E1E4 ORF (nt 3139-3419), RNAscope® 2.5 VS Probe- V-MusPV-E4, according to the manufacturer's protocol. The bound probes were detected by colorimetric staining in the RNAscope 2.5 HD Assay – BROWN, with hematoxylin counterstaining. For IHC, an in-house rabbit anti-MmuPV1 E4 polyclonal antibody (generously provided by Dr. John Doorbar's laboratory) or an anti-MmuPV1 L1 antibody (an in-house mouse monoclonal antibody, MPV.B9) was used on FFPE sections. The bound antibody was detected with the ImmPRESS anti-rabbit IgG polymer system (Vector # MP-7801) or the M.O.M.® (Mouse on Mouse) ImmPRESS® HRP (peroxidase) Polymer Kit (Vector # MP-2400) with the ImmPACT® NovaRED® Substrate (SK-4805). Before mounting, the slides were counterstained with 50% hematoxylin Gill's No. 1 solution (Sigma-Aldrich) and 0.02% ammonium hydroxide solution (Sigma-Aldrich).¹³¹
(MMRRC). Heterozygous female and male mice (from F1 breeding) were crossed to acquire homozygous *Flt3lg*⁻/⁻ mice, and this knockout was confirmed with the recommended genotyping method. In brief, three mixed primers were used to distinguish between the mutant and wild type: a mutant reverse primer 5' ATT TGT CAC GTC CTG CAC GAC G 3'; a common primer 5' TGG CAG CTG AAG TGA CTG AC 3'; and a wild-type reverse primer 5' AAG CCA AAG CTG GAT GAC AG 3'. We used six female and five male *Flt3lg*⁻/⁻ homozygotes in this study. Mice (4–8 weeks old) were sedated i.p. with 0.1 ml/10 g body weight ketamine/xylazine mixture (100 mg/10 mg in 10 ml double-distilled H₂O) and their skin was wounded at two sites (tail and muzzle), as previously described.¹³⁰ Twenty-four hours after wounding, the mice were again anesthetized and challenged with infectious virus (suspensions of 1 × 10⁹ viral DNA genomes/ infected site) at the wound sites. Infection was monitored twice weekly at the muzzle and tail wound sites and progress was documented photographically for each animal.¹²⁸⁻¹³⁰ Two, three, two and four *Flt3lg*⁻/⁻ mice of both sexes were killed three, four, six and eight weeks, respectively, after viral infection. *Rag1*⁻/⁻ and C57BL/6 mice were also killed at weeks three, four, six, and eight weeks post infection for comparisons at particular time points. The infected tissues were harvested for additional in situ analyses.

## MmuPV1 DNA copy number analyses

Linearized MmuPV1 genomic DNA was used for standard curve determination with a probe qPCR system. The primer pairs (5' GGTTGCGTCGGAGAACATATAA 3' and 5' CTAAAGCTAACCTGCCACATATC 3') for E2 amplification were used together with the probe (5' 6-FAM-TGCCCTTCA/ZEN/GTGGGTTGAGGACAG 3'-IBFQ). Each reaction contained 9 µL ultrapure water, 5 pmol of each primer, 9 µL Brilliant III qPCR Master Mix (Agilent) and 2 µL DNA template. The PCR conditions were: initial denaturation at 95°C for 3 min, then 40 cycles at 95°C for 5 seconds and 60°C for 10 seconds on an Agilent AriaMx. Viral copy numbers were converted into an equivalent DNA load as follows: 1 ng viral DNA = 1.2 × 10⁸ copies (http://cels.uri.edu/gsc/cndna.html). For the tissue samples, 40 ng total DNA was used for qPCR. All samples were tested at least in duplicate. Viral DNA titers were calculated from a standard curve.

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

Serum was collected from mice at the end of the experiment. ELISA assay was performed for antibodies against MmuPV1 L1. 0.5 µg MmuPV1 L1 VLPs in 50 µL 1X PBS/well was coated in the wells of Nunc® MaxiSorpTM 96-well microtiter plates (Thermo Fisher Scientific, Waltham MA), by incubation at room temperature for 30 minutes. For antibody against MmuPV1 E4 protein, 0.5 µg KLH-conjugated MmuPV1 E4 peptide (PKTTPPRRELFPPTPLTQPP, synthesized by China Peptide)/well in 50 µL bicarbonate (pH 9.6) buffer was incubated overnight with the plates at 37°C. Coated plates were blocked by incubation with PBS/5% dry milk powder for 1 hour at room temperature. ELISA was then performed as previously described.⁹⁸ Each of the serum samples was diluted 1:100 or more in PBS/5% dry milk and the dilutions were added to the 96-well plates, which were then incubated for 1 hour at room temperature. Negative controls were either sera collected from non-infected mice or minus primary antibodies. The plates were washed five times with washing buffer (0.05% (v/v) Tween 20 in 1×PBS), and we then added goat anti-mouse IgG (H+L)-alkaline phosphatase (AP) (Southern Biotech) secondary antibody diluted 1:2000 in 5% dry milk powder and incubated the plates for one hour at room temperature. The plates were washed five times with washing buffer, and 100 µL of 1 mg/mL pNPP (para-nitrophenyl phosphate), a substrate for alkaline phosphatase (AP), was added to each well for color development, and absorbance at 405 nm/450 nm was measured with a Fisher Microplate Reader. A mouse monoclonal antibody against MmuPV1L1 (MPV.A4) that neutralizes MmuPV1 infection *in vitro* and *in vivo*⁹⁷ was used as the positive control for L1 detection.

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

Infected tissue biopsy specimens were fixed in 10% neutral buffered formalin and embedded in paraffin. Adjacent sequential sections were cut for hematoxylin and eosin (H&E) staining, *in situ* hybridization (ISH), RNA *in situ* hybridization (RNA-ISH), and immunohistochemistry (IHC), as described in previous studies.⁹⁴,¹²⁹,¹³¹ For ISH, a biotin-labeled 3913 bp EcoRV/ BamH1 subgenomic fragment from MmuPV1 was used as the probe for the detection of MmuPV1 DNA in tissues.¹²⁸ Access to target DNA was obtained by incubation with 0.2 mg/mL pepsin in 0.1 N HCl at 37°C for 8 min. The sections were washed thoroughly. The biotinylated probe was then applied and the sections were heated at 95°C for 5 min to dissociate the target and probe DNA. Re-annealing was allowed to occur for 2 hours at 37°C. Target-bound biotin was detected with a streptavidin AP conjugate and a colorimetric reaction in BCIP/ NBT. For ISH, the nuclei were counterstained with Nuclear Fast Red (Vector Laboratories). Viral RNA was detected in formalin-fixed, paraffin-embedded (FFPE) sections with RNAscope technology (Advanced Cell Diagnostics, Inc.), with custom probes binding to the second exon of the E1E4 ORF (nt 3139-3419), RNAscope® 2.5 VS Probe- V-MusPV-E4, according to the manufacturer's protocol. The bound probes were detected by colorimetric staining in the RNAscope 2.5 HD Assay – BROWN, with hematoxylin counterstaining. For IHC, an in-house rabbit anti-MmuPV1 E4 polyclonal antibody (generously provided by Dr. John Doorbar's laboratory) or an anti-MmuPV1 L1 antibody (an in-house mouse monoclonal antibody, MPV.B9) was used on FFPE sections. The bound antibody was detected with the ImmPRESS anti-rabbit IgG polymer system (Vector # MP-7801) or the M.O.M.® (Mouse on Mouse) ImmPRESS® HRP (peroxidase) Polymer Kit (Vector # MP-2400) with the ImmPACT® NovaRED® Substrate (SK-4805). Before mounting, the slides were counterstained with 50% hematoxylin Gill's No. 1 solution (Sigma-Aldrich) and 0.02% ammonium hydroxide solution (Sigma-Aldrich).¹³¹
## Immunofluorescent staining of *Enterocytozoon bieneusi*

Indirect immunofluorescence staining was performed on stool smears positive for *Enterocytozoon bieneusi*. Between steps, slides were washed three times in PBS - 0.05 % Tween® 20 (PBS-T). Smears were blocked by overnight incubation at room temperature with 5% (m/v) powdered milk in PBS-T. Serum samples diluted 1/10,000 in 1% powdered milk, 1 % Tween® 20 in PBS were then added and the smears were incubated for 1 h. The smears were then incubated with FITC-conjugated anti-human IgA (Inova Diagnostics, 504045) for 30 minutes. The mounting solution was then added and the slides were examined at x630 magnification under a Leitz fluorescence microscope.

## QUANTIFICATION AND STATISTICAL ANALYSIS

Descriptive statistics were used for analysis (e.g., median, mean, SD, and minimum and maximum values on plots, as described in the figure legends). Data were analyzed using GraphPad Prism software v.10.1.0 (GraphPad Software). The statistical significance of quantitative differences between groups was assessed in two-tailed unpaired Mann-Whitney *U*-tests, Anova with Bonferroni adjustment, or Wilcoxon rank sum test, as indicated. P values are indicated only for statistically significant comparisons. All data are expressed as the mean ± s.d. calculated from at least three independent experiments unless otherwise stated. For the scRNA-Seq data, pseudobulk principal component analysis and gene-set enrichment analysis were performed as described in the methods.
<figure><img src="image_47.png" /></figure>

## Immunofluorescent staining of *Enterocytozoon bieneusi*

Indirect immunofluorescence staining was performed on stool smears positive for *Enterocytozoon bieneusi*. Between steps, slides were washed three times in PBS - 0.05 % Tween® 20 (PBS-T). Smears were blocked by overnight incubation at room temperature with 5% (m/v) powdered milk in PBS-T. Serum samples diluted 1/10,000 in 1% powdered milk, 1 % Tween® 20 in PBS were then added and the smears were incubated for 1 h. The smears were then incubated with FITC-conjugated anti-human IgA (Inova Diagnostics, 504045) for 30 minutes. The mounting solution was then added and the slides were examined at x630 magnification under a Leitz fluorescence microscope.

## QUANTIFICATION AND STATISTICAL ANALYSIS

Descriptive statistics were used for analysis (e.g., median, mean, SD, and minimum and maximum values on plots, as described in the figure legends). Data were analyzed using GraphPad Prism software v.10.1.0 (GraphPad Software). The statistical significance of quantitative differences between groups was assessed in two-tailed unpaired Mann-Whitney *U*-tests, Anova with Bonferroni adjustment, or Wilcoxon rank sum test, as indicated. P values are indicated only for statistically significant comparisons. All data are expressed as the mean ± s.d. calculated from at least three independent experiments unless otherwise stated. For the scRNA-Seq data, pseudobulk principal component analysis and gene-set enrichment analysis were performed as described in the methods.
Supplemental figures

<figure><img src="image_47.png" /></figure>

<table><thead><tr><th>Chr</th><th>Gene</th><th>Position</th><th>Variant</th><th>Consequence</th><th>Global frequency</th><th>Homozygotes reported in databases</th><th>CADD</th><th>MSC</th></tr></thead><tbody><tr><td>19</td><td>PLK5</td><td>1529418</td><td>C/T</td><td>Missense</td><td>0.003129</td><td>4</td><td>7.6</td><td>3.313</td></tr><tr><td>19</td><td>YJU2</td><td>4254465</td><td>G/C</td><td>Missense</td><td>0.0007810</td><td>0</td><td>16.9</td><td></td></tr><tr><td>19</td><td>SHD</td><td>4283068</td><td>G/A</td><td>Missense</td><td>0.0005313</td><td>0</td><td>14.5</td><td>7.476</td></tr><tr><td>19</td><td>CCDC61</td><td>46519975</td><td>G/T</td><td>Missense</td><td>0</td><td>0</td><td>28</td><td>8.756</td></tr><tr><td>19</td><td>HRC</td><td>49657711</td><td>CAT/-</td><td>Inframe</td><td>0.005227</td><td>27</td><td>14</td><td>3.313</td></tr><tr><td>19</td><td>FLT3LG</td><td>49982166</td><td>C/-</td><td>Frameshift</td><td>0.00001187</td><td>0</td><td>34</td><td>3.313</td></tr><tr><td>20</td><td>NFATC2</td><td>50048790</td><td>G/A</td><td>Missense</td><td>0.001245</td><td>4</td><td>11.2</td><td>3.313</td></tr></tbody></table>

<figure><img src="image_48.png" /><figcaption>Figure S1. Three patients with FLT3L deficiency, related to Figure 1</figcaption></figure>

(A) Images of the HPV infection phenotype in P2 and P3, showing severe cutaneous warts in two different anatomical areas of the two patients. The common warts on the hands and feet of P2 and P3 were HPV2+. Note the numerous HPV5+ flat warts on the abdomen of P3, suggesting atypical epidermodysplasia verruciformis (lower right picture).

(B) An analysis of whole-exome sequencing (WES) data identified only seven genes carrying a biallelic rare coding variant within the linked region, assuming recessive inheritance with complete penetrance.

(C) Rare biallelic variants within the linked region. FLT3LG was the only one of the seven genes related to the immune system and never reported in the homozygous state in a database of sequences obtained from healthy donors (gnomAD v2.1.1).

(D) The chromatogram shows the Sanger sequencing results for all the patients and for healthy family members, including heterozygous carriers and two homozygous WT individuals for FLT3LG. The site of the mutation is indicated by a dashed red rectangle.
Supplemental figures

<figure><img src="image_48.png" /></figure>

<table><thead><tr><th>Chr</th><th>Gene</th><th>Position</th><th>Variant</th><th>Consequence</th><th>Global frequency</th><th>Homozygotes reported in databases</th><th>CADD</th><th>MSC</th></tr></thead><tbody><tr><td>19</td><td>PLK5</td><td>1529418</td><td>C/T</td><td>Missense</td><td>0.003129</td><td>4</td><td>7.6</td><td>3.313</td></tr><tr><td>19</td><td>YJU2</td><td>4254465</td><td>G/C</td><td>Missense</td><td>0.0007810</td><td>0</td><td>16.9</td><td></td></tr><tr><td>19</td><td>SHD</td><td>4283068</td><td>G/A</td><td>Missense</td><td>0.0005313</td><td>0</td><td>14.5</td><td>7.476</td></tr><tr><td>19</td><td>CCDC61</td><td>46519975</td><td>G/T</td><td>Missense</td><td>0</td><td>0</td><td>28</td><td>8.756</td></tr><tr><td>19</td><td>HRC</td><td>49657711</td><td>CAT/-</td><td>Inframe</td><td>0.005227</td><td>27</td><td>14</td><td>3.313</td></tr><tr><td>19</td><td>FLT3LG</td><td>49982166</td><td>C/-</td><td>Frameshift</td><td>0.00001187</td><td>0</td><td>34</td><td>3.313</td></tr><tr><td>20</td><td>NFATC2</td><td>50048790</td><td>G/A</td><td>Missense</td><td>0.001245</td><td>4</td><td>11.2</td><td>3.313</td></tr></tbody></table>

<figure><img src="image_49.png" /><figcaption>Figure S1. Three patients with FLT3L deficiency, related to Figure 1</figcaption></figure>

(A) Images of the HPV infection phenotype in P2 and P3, showing severe cutaneous warts in two different anatomical areas of the two patients. The common warts on the hands and feet of P2 and P3 were HPV2+. Note the numerous HPV5+ flat warts on the abdomen of P3, suggesting atypical epidermodysplasia verruciformis (lower right picture).

(B) An analysis of whole-exome sequencing (WES) data identified only seven genes carrying a biallelic rare coding variant within the linked region, assuming recessive inheritance with complete penetrance.

(C) Rare biallelic variants within the linked region. FLT3LG was the only one of the seven genes related to the immune system and never reported in the homozygous state in a database of sequences obtained from healthy donors (gnomAD v2.1.1).

(D) The chromatogram shows the Sanger sequencing results for all the patients and for healthy family members, including heterozygous carriers and two homozygous WT individuals for FLT3LG. The site of the mutation is indicated by a dashed red rectangle.
<figure><img src="image_49.png" /><figcaption>Figure S2. FLT3L-deficient mice are susceptible to cutaneous MmuPV1 infections, related to Figure 1</figcaption></figure>

We used the mouse papillomavirus (MmuPV1) infection model in congenic wild type (WT) and *flt3lg*<sup>-/-</sup> on C57BL/6 background. The MmuPV1 virus causes skin wart lesions in athymic nude or *Rag1*<sup>-/-</sup> C57BL/6 mice but not in C57BL/6 WT mice.

(A) Representative images of tail and muzzle lesions (red circles) from RAG1-deficient (middle) and *flt3lg*<sup>-/-</sup> mice (bottom) 3 weeks post infection with MmuPV1. Images of a wild-type B6 mouse (top) are also shown for comparison. We infected the muzzle and tail of 11 (5 males and 6 females) *flt3lg*<sup>-/-</sup> mice, 6 (3 males and 3 females) *Rag1*<sup>-/-</sup> mice, and 10 (5 males and 5 females) WT B6 mice with MmuPV1. *Rag1*<sup>-/-</sup> mice have no T or B cells. We monitored skin lesions between 2 and 8 weeks before the animals were killed. None of the WT C57BL/6 mice developed visible skin lesions, consistent with the findings of previous studies. All three *Rag1*<sup>-/-</sup> mice developed skin warts 3 weeks post infection. These lesions persisted until the end of monitoring. *Flt3lg*<sup>-/-</sup> mice were tested for infection and killed at various time points (weeks 3, 4, and 6) after infection. All *Flt3lg*<sup>-/-</sup> mice began to develop visible lesions 3 weeks post infection. The lesions began to regress around week 5 and disappeared during week 6.

(B) Infected tissues from *Flt3lg*<sup>-/-</sup> mice were harvested at different time points for additional tests, including histology (H&E staining), immunohistochemistry (IHC), and *in situ* hybridization (ISH). Tail tissues from *Flt3lg*<sup>-/-</sup> mice displayed epidermal hyperplasia, with vacuolated cells and parakeratosis. Strong MmuPV1 DNA and RNA (E1E4) signals, equivalent to those observed in infected *Rag1*<sup>-/-</sup> mice, were detected in infected *Flt3lg*<sup>-/-</sup> mice by *in situ* hybridization 3 weeks after infection. Using antibodies against E4 and L1, we detected strong viral protein signals in the lesions. Viral signals (DNA/RNA/protein) were absent from the tail tissues of *flt3lg*<sup>-/-</sup> and WT C57BL/6 mice displaying regression 6 weeks after viral infection (data not shown). Representative images of a wart from a RAG1-deficient animal (bottom) and a wart (middle) and healthy skin (top) from the same FLT3L-deficient animal are shown. The data shown are representative of all the mice tested.

(C) PCR measuring MmuPV1 viral load in the skin lesions of *Flt3lg*<sup>-/-</sup> and *Rag1*<sup>-/-</sup> mice 3-6 weeks post infection. Intralesional viral load was determined by qPCR for MmuPV1 E2. In *Flt3lg*<sup>-/-</sup> mice, the viral DNA load was high in week 3 post infection, began to decrease in week 4, and was undetectable in week 6. In *Rag1*<sup>-/-</sup> mice, the viral DNA load was high at all tested time points.

(D) ELISA determining the levels of anti-MmuPV1 E4 and L1 antibodies in WT or *Flt3lg*<sup>-/-</sup> mouse plasma sampled 3-8 weeks post infection. Normal anti-MmuPV1 L1 and E4 Ab levels were detected in *Flt3lg*<sup>-/-</sup>. Thus, *Flt3lg*<sup>-/-</sup> mice can mount an anti-MmuPV1 T cell and B cell response, but their control and clearance of infection are impaired. WT, wild type; Neg, negative control. 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 differences between the negative control, WT, and *Flt3lg*<sup>-/-</sup> mice. *p* ≤ 0.05, **p** < 0.001, ***p** < 0.0001; ns, not significant.
<figure><img src="image_50.png" /><figcaption>Figure S2. FLT3L-deficient mice are susceptible to cutaneous MmuPV1 infections, related to Figure 1</figcaption></figure>

We used the mouse papillomavirus (MmuPV1) infection model in congenic wild type (WT) and *flt3lg*<sup>-/-</sup> on C57BL/6 background. The MmuPV1 virus causes skin wart lesions in athymic nude or *Rag1*<sup>-/-</sup> C57BL/6 mice but not in C57BL/6 WT mice.

(A) Representative images of tail and muzzle lesions (red circles) from RAG1-deficient (middle) and *flt3lg*<sup>-/-</sup> mice (bottom) 3 weeks post infection with MmuPV1. Images of a wild-type B6 mouse (top) are also shown for comparison. We infected the muzzle and tail of 11 (5 males and 6 females) *flt3lg*<sup>-/-</sup> mice, 6 (3 males and 3 females) *Rag1*<sup>-/-</sup> mice, and 10 (5 males and 5 females) WT B6 mice with MmuPV1. *Rag1*<sup>-/-</sup> mice have no T or B cells. We monitored skin lesions between 2 and 8 weeks before the animals were killed. None of the WT C57BL/6 mice developed visible skin lesions, consistent with the findings of previous studies. All three *Rag1*<sup>-/-</sup> mice developed skin warts 3 weeks post infection. These lesions persisted until the end of monitoring. *Flt3lg*<sup>-/-</sup> mice were tested for infection and killed at various time points (weeks 3, 4, and 6) after infection. All *Flt3lg*<sup>-/-</sup> mice began to develop visible lesions 3 weeks post infection. The lesions began to regress around week 5 and disappeared during week 6.

(B) Infected tissues from *Flt3lg*<sup>-/-</sup> mice were harvested at different time points for additional tests, including histology (H&E staining), immunohistochemistry (IHC), and *in situ* hybridization (ISH). Tail tissues from *Flt3lg*<sup>-/-</sup> mice displayed epidermal hyperplasia, with vacuolated cells and parakeratosis. Strong MmuPV1 DNA and RNA (E1E4) signals, equivalent to those observed in infected *Rag1*<sup>-/-</sup> mice, were detected in infected *Flt3lg*<sup>-/-</sup> mice by *in situ* hybridization 3 weeks after infection. Using antibodies against E4 and L1, we detected strong viral protein signals in the lesions. Viral signals (DNA/RNA/protein) were absent from the tail tissues of *flt3lg*<sup>-/-</sup> and WT C57BL/6 mice displaying regression 6 weeks after viral infection (data not shown). Representative images of a wart from a RAG1-deficient animal (bottom) and a wart (middle) and healthy skin (top) from the same FLT3L-deficient animal are shown. The data shown are representative of all the mice tested.

(C) PCR measuring MmuPV1 viral load in the skin lesions of *Flt3lg*<sup>-/-</sup> and *Rag1*<sup>-/-</sup> mice 3-6 weeks post infection. Intralesional viral load was determined by qPCR for MmuPV1 E2. In *Flt3lg*<sup>-/-</sup> mice, the viral DNA load was high in week 3 post infection, began to decrease in week 4, and was undetectable in week 6. In *Rag1*<sup>-/-</sup> mice, the viral DNA load was high at all tested time points.

(D) ELISA determining the levels of anti-MmuPV1 E4 and L1 antibodies in WT or *Flt3lg*<sup>-/-</sup> mouse plasma sampled 3-8 weeks post infection. Normal anti-MmuPV1 L1 and E4 Ab levels were detected in *Flt3lg*<sup>-/-</sup>. Thus, *Flt3lg*<sup>-/-</sup> mice can mount an anti-MmuPV1 T cell and B cell response, but their control and clearance of infection are impaired. WT, wild type; Neg, negative control. 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 differences between the negative control, WT, and *Flt3lg*<sup>-/-</sup> mice. *p* ≤ 0.05, **p** < 0.001, ***p** < 0.0001; ns, not significant.
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HEK293T cells

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Cell-cell contact experiment

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Figure S3. *FLT3LG* encodes two functional isoforms, related to Figures 2 and 3

(A) *FLT3LG* mRNA splicing events detected by RNA-seq in human CD8⁺ T cells. Mapping of the coverage of raw sequence reads onto the *FLT3LG* gene is shown, together with the number of reads spliced between exons; only splicing events with more than 2% of the maximum coverage in each cell type are shown. The major exons detected in RNA-seq are depicted, and the structures of transcripts T1–T7 are shown; blue rectangles represent exons and connecting lines indicate introns.

(B) The proportion of the indicated *FLT3LG* transcripts obtained by topo cloning on cDNA generated from expanded T cell blasts from healthy donors (HDs) (n = 2), P3, and the heterozygous mother (M, mother).

(C) Schematic representation of the 7 principal *FLT3LG* transcripts (>4% of total transcripts) detected by topo cloning on cDNA from an HD. The position of the c.343delC mutation is indicated by a red arrow. The various exons are represented by boxes. The different colors correspond to the different domains of the FLT3L protein. UTR, untranslated region; SP, signal peptide; EC, extracellular domain; TM, transmembrane domain; IC, intracellular domain. Alternative coding sequences (alt. coding seq.) due to frameshifts are defined relative to the canonical isoform (T1/T2).

(D) Proportions of total mRNA corresponding to the *FLT3LG* transcript variants. HEK293T cells were transfected with an empty plasmid or a plasmid containing a sequence corresponding to the indicated *FLT3LG* transcripts. After 24 h, *FLT3LG* mRNA levels were assessed by RT-qPCR. Data are presented as 2⁻⁰⁻⁺ values normalized against endogenous GUS control gene expression. The data shown are representative of three independent experiments. NT, not transfected. Bars indicate mean values and the SD.

(E) Cell-surface FLT3L expression as assessed by FACS on HEK293T cells, as in (D).

(F) Total FLT3L protein levels on western blots (WBs) of protein extracts or culture supernatant from HEK293T cells, as in (D) and (E).

(G) Soluble FLT3L protein determination by ELISA on the supernatant of HEK293T cells, as in (D)–(F). Bars indicate the mean and SD.

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

(I) HEK293T cells were transfected with an empty plasmid or a plasmid containing a sequence encoding the indicated FLT3L transcript. Phospho-FLT3 (pFLT3) levels in K562 cells transduced with FLT3, as assessed by western blotting, after stimulation for 5 min in contact with the HEK293T cells 48 h after transfection. A recombinant human FLT3L (rhFLT3L) was used as a positive control.

(J) Proportions of total mRNA corresponding to the *FLT3LG* transcript variants, as assessed by RT-qPCR on mRNA extracted from HEK293T cells 48 h after transfection with an empty vector (EV), or a vector encoding the WT soluble or membrane-bound FLT3L isoform, or the indicated *FLT3LG* variant. Data are presented as 2⁻⁰⁻⁺ values normalized against endogenous *GUS* control gene expression. Bars indicate the mean and SD.

(K) 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. Total mRNA was extracted, and total *FLT3LG* mRNA levels were determined by RT-qPCR. Data are displayed as 2⁻⁰⁻⁺ values normalized against endogenous *GUS* control gene expression. Bars indicate the mean and SD. HD, healthy donor. The data shown are representative of three independent experiments performed.
Figure S3. *FLT3LG* encodes two functional isoforms, related to Figures 2 and 3

(A) *FLT3LG* mRNA splicing events detected by RNA-seq in human CD8⁺ T cells. Mapping of the coverage of raw sequence reads onto the *FLT3LG* gene is shown, together with the number of reads spliced between exons; only splicing events with more than 2% of the maximum coverage in each cell type are shown. The major exons detected in RNA-seq are depicted, and the structures of transcripts T1–T7 are shown; blue rectangles represent exons and connecting lines indicate introns.

(B) The proportion of the indicated *FLT3LG* transcripts obtained by topo cloning on cDNA generated from expanded T cell blasts from healthy donors (HDs) (n = 2), P3, and the heterozygous mother (M, mother).

(C) Schematic representation of the 7 principal *FLT3LG* transcripts (>4% of total transcripts) detected by topo cloning on cDNA from an HD. The position of the c.343delC mutation is indicated by a red arrow. The various exons are represented by boxes. The different colors correspond to the different domains of the FLT3L protein. UTR, untranslated region; SP, signal peptide; EC, extracellular domain; TM, transmembrane domain; IC, intracellular domain. Alternative coding sequences (alt. coding seq.) due to frameshifts are defined relative to the canonical isoform (T1/T2).

(D) Proportions of total mRNA corresponding to the *FLT3LG* transcript variants. HEK293T cells were transfected with an empty plasmid or a plasmid containing a sequence corresponding to the indicated *FLT3LG* transcripts. After 24 h, *FLT3LG* mRNA levels were assessed by RT-qPCR. Data are presented as 2⁻⁰⁻⁺ values normalized against endogenous GUS control gene expression. The data shown are representative of three independent experiments. NT, not transfected. Bars indicate mean values and the SD.

(E) Cell-surface FLT3L expression as assessed by FACS on HEK293T cells, as in (D).

(F) Total FLT3L protein levels on western blots (WBs) of protein extracts or culture supernatant from HEK293T cells, as in (D) and (E).

(G) Soluble FLT3L protein determination by ELISA on the supernatant of HEK293T cells, as in (D)–(F). Bars indicate the mean and SD.

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

(I) HEK293T cells were transfected with an empty plasmid or a plasmid containing a sequence encoding the indicated FLT3L transcript. Phospho-FLT3 (pFLT3) levels in K562 cells transduced with FLT3, as assessed by western blotting, after stimulation for 5 min in contact with the HEK293T cells 48 h after transfection. A recombinant human FLT3L (rhFLT3L) was used as a positive control.

(J) Proportions of total mRNA corresponding to the *FLT3LG* transcript variants, as assessed by RT-qPCR on mRNA extracted from HEK293T cells 48 h after transfection with an empty vector (EV), or a vector encoding the WT soluble or membrane-bound FLT3L isoform, or the indicated *FLT3LG* variant. Data are presented as 2⁻⁰⁻⁺ values normalized against endogenous *GUS* control gene expression. Bars indicate the mean and SD.

(K) 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. Total mRNA was extracted, and total *FLT3LG* mRNA levels were determined by RT-qPCR. Data are displayed as 2⁻⁰⁻⁺ values normalized against endogenous *GUS* control gene expression. Bars indicate the mean and SD. HD, healthy donor. The data shown are representative of three independent experiments performed.
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Bone biopsy immunostaining

Erythroblastic lin. (GPC)

Megakaryocytic lin. (FVIII)

Granulocytic lin. (MPO)

P2

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A

Bone biopsy immunostaining

Erythroblastic lin. (GPC)

Megakaryocytic lin. (FVIII)

Granulocytic lin. (MPO)

P2

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Figure S4. Bone phenotype and bone-marrow HSPC immunophenotyping, related to Figure 4

(A) Immunostaining on a bone biopsy specimen from P2 showing a predominance of the erythroblastic (glycophorin C [GPC]-positive) and megakaryocytic (factor VIII [FVIII]-positive) lineages, whereas the granulocytic lineage was hypoplastic (myeloperoxidase [MPO]-positive).

(B) Schematic representation of hematopoietic differentiation indicating the progenitor populations: hematopoietic stem cell (HSC), multipotent progenitor (MPP), multipotent lymphoid progenitor (MLP), common myeloid progenitor (CMP), megakaryocyte-erythroid progenitor (MEP), granulocyte-monocyte progenitor (GMP), dendritic cell progenitor (DCP), and B cell-NK progenitor (BNKP).

(C) Gating strategy for the analysis of HSPCs (CD34⁺ cell-enriched) from P2 and one HD.

(D) Gating strategy for the HSPC analysis on the blood of P2, P3, and one HD.

(E) Boxplot representing FL73 mRNA levels in the HSPCs and the All-HSC population for each HD (n = 2) and P2. Wilcoxon rank sum test with continuity correction was used for statistical comparison between the patient and the controls; **p < 0.001, *p < 0.01; ns, not significant.
Figure S4. Bone phenotype and bone-marrow HSPC immunophenotyping, related to Figure 4

(A) Immunostaining on a bone biopsy specimen from P2 showing a predominance of the erythroblastic (glycophorin C [GPC]-positive) and megakaryocytic (factor VIII [FVIII]-positive) lineages, whereas the granulocytic lineage was hypoplastic (myeloperoxidase [MPO]-positive).

(B) Schematic representation of hematopoietic differentiation indicating the progenitor populations: hematopoietic stem cell (HSC), multipotent progenitor (MPP), multipotent lymphoid progenitor (MLP), common myeloid progenitor (CMP), megakaryocyte-erythroid progenitor (MEP), granulocyte-monocyte progenitor (GMP), dendritic cell progenitor (DCP), and B cell-NK progenitor (BNKP).

(C) Gating strategy for the analysis of HSPCs (CD34⁺ cell-enriched) from P2 and one HD.

(D) Gating strategy for the HSPC analysis on the blood of P2, P3, and one HD.

(E) Boxplot representing FL73 mRNA levels in the HSPCs and the All-HSC population for each HD (n = 2) and P2. Wilcoxon rank sum test with continuity correction was used for statistical comparison between the patient and the controls; **p < 0.001, *p < 0.01; ns, not significant.
<figure><img src="image_69.png" /><figcaption>B</figcaption></figure>

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

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

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

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

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

<figure><img src="image_73.png" /><figcaption>E</figcaption></figure>

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**Figure S5. Blood and bone-marrow immunophenotyping, related to Figures 5 and 6**

(A) Frequency of lymphocytes and neutrophils among blood leukocytes, hemoglobin (HB) levels, and erythrocyte sedimentation rate (ESR) for P1, P2, and P3 at different ages. The reference values for each population are indicated by a gray area.

(B–D) CyTOF performed on whole blood from 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 controls. 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) Frequency of monocyte subpopulations.

(C) Absolute counts for total T cells and the two main subsets detected by CyTOF.

(D) Absolute counts for all the T cell subsets detected by CyTOF. CM, central memory; EM, effector memory; TEMRA, terminally differentiated effector memory T cell; RTE, recent thymic emigrant; Th, T helper cell; Tfh, T follicular helper; Treg, regulatory T cell; iNKT, invariant natural killer T cell; MAIT, mucosal-associated invariant T cell.

(E) CyTOF performed on total bone-marrow (BM) samples from P2, P3, and four healthy donors (HDs). The data show the proportions of the indicated BM leukocyte subsets within the indicated subsets. Data are presented as individual values (min to max). Boxplots indicate the median and IQR.
Figure S5. Blood and bone-marrow immunophenotyping, related to Figures 5 and 6

(A) Frequency of lymphocytes and neutrophils among blood leukocytes, hemoglobin (HB) levels, and erythrocyte sedimentation rate (ESR) for P1, P2, and P3 at different ages. The reference values for each population are indicated by a gray area.

(B–D) CyTOF performed on whole blood from 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 controls. 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) Frequency of monocyte subpopulations.

(C) Absolute counts for total T cells and the two main subsets detected by CyTOF.

(D) Absolute counts for all the T cell subsets detected by CyTOF. CM, central memory; EM, effector memory; TEMRA, terminally differentiated effector memory T cell; RTE, recent thymic emigrant; Th, T helper cell; Tfh, T follicular helper; Treg, regulatory T cell; iNKT, invariant natural killer T cell; MAIT, mucosal-associated invariant T cell.

(E) CyTOF performed on total bone-marrow (BM) samples from P2, P3, and four healthy donors (HDs). The data show the proportions of the indicated BM leukocyte subsets within the indicated subsets. Data are presented as individual values (min to max). Boxplots indicate the median and IQR.
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TCR-β clonality

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**Figure S6. T cell function and TCR sequencing, related to Figure 6**

(A) CDR3 amino-acid sequence length distributions for the T cell receptor (TCR)-β locus, based on the sequences of total gDNA extracted from total PBMCs from P2 and P3 in red, and four HDs in gray. No differences were found between patients and controls.

(B) TCR-β clonality and related expansion status. Each colored circle represents a unique TCR sequence, and the area of the circle represents the abundance of that TCR sequence. Black areas (small black circles) represent a TCR repertoire without clonal expansion. No differences were found between patients and controls.

(C) Carboxyfluorescein succinimidyl ester (CFSE) proliferation assay. CD3+ T cell proliferation in response to stimulation with mitogens and antigens. Red and black indicate the values for patients and healthy donors, respectively. The patients' cells responded weakly to PHA (phytohaemagglutinin) and had an impaired response to OKT3 and antigenic stimuli (tuberculin, tetanus toxin), probably reflecting the extremely low levels of antigen-presenting cells (APCs). NS, un-stimulated; Stim, stimulated.

(D) Production of interferon-gamma (IFN-γ) and tumor necrosis factor (TNF) after whole-blood activation with BCG, BCG + IL-12, or BCG + IL-23, as determined by ELISA. The patients' cells had an impaired response to BCG, probably reflecting the extremely low levels of APCs. NS, unstimulated; BCG, Bacille Calmette-Guerin; IL, interleukin; PMA; Phorbol 12-myristate 13-acetate.

(E) IFN-γ production after PBMC stimulation with various stimuli, as assessed by flow cytometry. Ctl, controls; P, patients.

(F) Sorted memory (left) or naive (right) CD4+ T cells from P2, P3, and healthy donors were either maintained in TH0 conditions (T cell activation and expansion [TAE] beads) or polarized under TH1 (TAE beads + IL-12) or TH17 (TAE beads + IL-1/IL-6/IL-21/IL-23/transforming growth factor β [TGF-β]) conditions. After 5 days of culture, the frequencies of cells positive for the cytokines indicated were assessed by intracellular staining and flow cytometry. Under all polarizing conditions tested, we found no significant difference between the patients' cells and healthy control cells.
**Figure S6. T cell function and TCR sequencing, related to Figure 6**

(A) CDR3 amino-acid sequence length distributions for the T cell receptor (TCR)-β locus, based on the sequences of total gDNA extracted from total PBMCs from P2 and P3 in red, and four HDs in gray. No differences were found between patients and controls.

(B) TCR-β clonality and related expansion status. Each colored circle represents a unique TCR sequence, and the area of the circle represents the abundance of that TCR sequence. Black areas (small black circles) represent a TCR repertoire without clonal expansion. No differences were found between patients and controls.

(C) Carboxyfluorescein succinimidyl ester (CFSE) proliferation assay. CD3+ T cell proliferation in response to stimulation with mitogens and antigens. Red and black indicate the values for patients and healthy donors, respectively. The patients' cells responded weakly to PHA (phytohaemagglutinin) and had an impaired response to OKT3 and antigenic stimuli (tuberculin, tetanus toxin), probably reflecting the extremely low levels of antigen-presenting cells (APCs). NS, un-stimulated; Stim, stimulated.

(D) Production of interferon-gamma (IFN-γ) and tumor necrosis factor (TNF) after whole-blood activation with BCG, BCG + IL-12, or BCG + IL-23, as determined by ELISA. The patients' cells had an impaired response to BCG, probably reflecting the extremely low levels of APCs. NS, unstimulated; BCG, Bacille Calmette-Guerin; IL, interleukin; PMA; Phorbol 12-myristate 13-acetate.

(E) IFN-γ production after PBMC stimulation with various stimuli, as assessed by flow cytometry. Ctl, controls; P, patients.

(F) Sorted memory (left) or naive (right) CD4+ T cells from P2, P3, and healthy donors were either maintained in TH0 conditions (T cell activation and expansion [TAE] beads) or polarized under TH1 (TAE beads + IL-12) or TH17 (TAE beads + IL-1/IL-6/IL-21/IL-23/transforming growth factor β [TGF-β]) conditions. After 5 days of culture, the frequencies of cells positive for the cytokines indicated were assessed by intracellular staining and flow cytometry. Under all polarizing conditions tested, we found no significant difference between the patients' cells and healthy control cells.
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B

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C

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A

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B

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C

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D

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E

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F

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Figure S7. B cell phenotype and VirScan assay, related to Figure 6

(A) CyTOF gating strategy for age-associated B cell (ABC) subsets in P2, P3, and HDs.

(B) Analysis of B cell precursors among total bone-marrow mononuclear (BMNCs) cells from P2, P3, and one age-matched HD.

(C) Heatmap illustrating the comparison of V and J gene usage frequencies in IGH productive rearrangements in P2, P3, and four healthy adult controls. The different colors (black to yellow) indicate the different frequencies of gene segment usage from the least (black) to the most frequently used (yellow). ANOVA with Bonferroni adjustment was used for statistical comparison; no significant differences were detected.

(D) Tree maps representing the diversity and clonality of the productive IGH repertoire from P2, P3, and two healthy adult controls. Each dot represents a single CDR3, and the size of the dot represents the relative frequency of that CDR3 in the entire population.

(E) VirScan profiles of the patients, showing the adjusted virus scores for the indicated samples from patients and controls, mock IP samples, IgG-depleted serum, and intravenous immunoglobulin (IVIg). The heatmap shows adjusted virus score values for each sample as a color gradient from blue (antibodies detected but below our significance cutoff values), through purple, to red (for adjusted virus score values above our significance cutoff values). The bar plot (bottom) illustrates the size of the Ab repertoire for a given sample, indicating the precise number of different species displaying peptide enrichment (dark blue) and the number of different species for which the adjusted virus score values exceeded the cutoff values for significance (light blue).

(F) *Enterocytozoon bieneusi* immunofluorescence staining performed on a positive fecal sample with 10,000-fold dilutions of serum from a negative control (not infected), a positive control (infected), P2, and P3. A FITC-coupled anti-IgA antibody was used as secondary antibody. The positive control was slightly positive at this dilution, whereas a clear delineated staining of the microsporidial wall was observed with serum from the patients.
Figure S7. B cell phenotype and VirScan assay, related to Figure 6

(A) CyTOF gating strategy for age-associated B cell (ABC) subsets in P2, P3, and HDs.

(B) Analysis of B cell precursors among total bone-marrow mononuclear (BMNCs) cells from P2, P3, and one age-matched HD.

(C) Heatmap illustrating the comparison of V and J gene usage frequencies in IGH productive rearrangements in P2, P3, and four healthy adult controls. The different colors (black to yellow) indicate the different frequencies of gene segment usage from the least (black) to the most frequently used (yellow). ANOVA with Bonferroni adjustment was used for statistical comparison; no significant differences were detected.

(D) Tree maps representing the diversity and clonality of the productive IGH repertoire from P2, P3, and two healthy adult controls. Each dot represents a single CDR3, and the size of the dot represents the relative frequency of that CDR3 in the entire population.

(E) VirScan profiles of the patients, showing the adjusted virus scores for the indicated samples from patients and controls, mock IP samples, IgG-depleted serum, and intravenous immunoglobulin (IVIg). The heatmap shows adjusted virus score values for each sample as a color gradient from blue (antibodies detected but below our significance cutoff values), through purple, to red (for adjusted virus score values above our significance cutoff values). The bar plot (bottom) illustrates the size of the Ab repertoire for a given sample, indicating the precise number of different species displaying peptide enrichment (dark blue) and the number of different species for which the adjusted virus score values exceeded the cutoff values for significance (light blue).

(F) *Enterocytozoon bieneusi* immunofluorescence staining performed on a positive fecal sample with 10,000-fold dilutions of serum from a negative control (not infected), a positive control (infected), P2, and P3. A FITC-coupled anti-IgA antibody was used as secondary antibody. The positive control was slightly positive at this dilution, whereas a clear delineated staining of the microsporidial wall was observed with serum from the patients.