BTK drives neutrophil activation for sterilizing antifungal immunity

We describe a previously unappreciated role for Bruton’s tyrosine kinase (BTK) in fungal immune surveillance against aspergillosis, an unforeseen complication of BTK inhibitors (BTKi) used for treating B cell lymphoid malignancies. We studied BTK-dependent fungal responses in neutrophils from diverse populations, including healthy donors, patients who were treated with BTKi, and X-linked agammaglobulinemia patients. Upon fungal exposure, BTK was activated in human neutrophils in a TLR2-, Dectin-1-, and FcγR-dependent manner, triggering the oxidative burst. BTK inhibition selectively impeded neutrophil-mediated damage to Aspergillus hyphae, primary granule release, and the fungus-induced oxidative burst by abrogating NADPH oxidase subunit p40phox and GTPase RAC2 activation. Moreover, neutrophil-specific Btk deletion in mice enhanced aspergillosis susceptibility by impairing neutrophil function, not recruitment or lifespan. Conversely, GM-CSF partially mitigated these deficits by enhancing p47phox activation. Our findings underline the crucial role of BTK signaling in neutrophils for antifungal immunity and provide a rationale for GM-CSF use to offset these deficits in patients who are susceptible.

intravascular and extravasated pulmonary neutrophils of Aspergillus-infected mice at the indicated times post-infection (n = 3-4).(D) Numbers of live myeloid phagocyte subsets in the lung of Aspergillus-infected mice at the indicated days post-infection (n = [3][4][5][6].(E) Percentage of dead cells for the indicated myeloid phagocyte subsets in the lung of Aspergillus-infected mice at day 2 post-infection (n = 5-6).(F) Concentrations of the indicated cytokines and chemokines in the mouse lung by Luminex-based assay at steady state and day 1 post-infection (n = 6-9).Lin: lineage (CD3/CD19/NK1.1).Quantitative data are means ± SEM. *P<0.05,**P<0.01,determined using two-sided unpaired t test (E) or 2-way ANOVA with Šidák's multiple comparisons test (F).Supplemental Figure 3. Ibrutinib inhibits Aspergillus hyphal damage, but not conidial uptake or intracellular killing, in human neutrophils.(A-B) Healthy donor neutrophils were treated with vehicle or ibrutinib and analyzed for their ability to associate with or kill A. fumigatus FLARE conidia (n = 4).(A) Percentage of neutrophils associated with total (live + dead) A. fumigatus conidia, thus quantifying "% association".(B) Percentage of healthy donor neutrophils associated with dead conidia, thus quantifying "% killing".(C) Vehicle-or ibrutinib-treated healthy donor neutrophils were co-incubated with A. fumigatus conidia at the indicated effector-target ratios as indicated and the neutrophils' ability to inhibit conidial germination was quantified relative to the wells not containing neutrophils (n = 4).(D) A. fumigatus hyphal damage by vehicle-or ibrutinib-treated healthy donor neutrophils at the indicated effector-target ratios (n = 6).(E) Neutrophil extracellular traps are dispensable for healthy donor neutrophil-induced A. fumigatus hyphal damage.Neutrophils were exposed to vehicle or DNase (100U/ml) at the indicated effector-target ratios (n = 6).Quantitative data are mean ± SEM (C, D, E) or depicted as box and whisker plots with values ranging from minimum to maximum (A, B).Ibrutinib concentration, 250 nM.Af: Aspergillus fumigatus.*P<0.05,**P<0.01,determined using two-sided paired t test (D).Supplemental Figure 4. BTK plays an essential role in Aspergillus hyphal damage but is dispensable for conidial uptake or intracellular killing by neutrophils.(A) Percentage of neutrophils isolated from lymphoma patients, before or at day 3 after treatment initiation with ibrutinib or acalabrutinib, which are associated with A. fumigatus FLARE reporter conidia (n = 3).(B) Aspergillus hyphal damage induced by neutrophils isolated from lymphoma patients, before or at day 3 after treatment initiation with ibrutinib (n = 8) or acalabrutinib (n = 5), at the indicated effector-target ratios.(C) Percentage of healthy donor or XLA neutrophils that contain (live + dead) A. fumigatus conidia, thus quantifying "% association" (left panel).Percentage of healthy donor or XLA neutrophils that contain dead conidia, thus quantifying "% killing" (right panel).(n = 3-9) (D) A. fumigatus hyphal damage induced by neutrophils isolated from healthy donors or from XLA patients (n = 9), at the indicated effector-target ratios.(E) WT and Btk -/-mice were infected with A. fumigatus (Af) FLARE reporter conidia and at day 2 post-infection, neutrophils were examined for their ability to associate with or kill conidia in the lung (n = 9-10).(F) Percentage of bone marrow neutrophils from the indicated strains that contain (live + dead) A. fumigatus conidia, thus quantifying "% association" (left panel).Percentage of bone marrow neutrophils from the indicated strains that contain dead conidia, thus quantifying "% killing" (right panel) (n = 3-4).Quantitative data are mean ± SEM.Box and whisker plots depict values ranging from minimum to maximum (C).BTKi: BTK inhibitor; XLA: X-linked agammaglobulinemia; Af: Aspergillus fumigatus.*P<0.05,**P<0.01,***P<0.001,determined using two-sided paired t test (B), or two-sided unpaired t test (D).Supplemental Figure 5: Acalabrutinib inhibits p40 phox phosphorylation in human neutrophils.Immunoblot analysis of p40 phox phosphorylation (at T154) in human neutrophils upon stimulation with serum-opsonized heat-killed Aspergillus conidia at the indicated time points.Representative immunoblot images (top panels) and quantified pixel density values (lower panels) are shown.The ratio of pixel density for phosphorylated p40 phox vs. total p40 phox (bottom left) and fold-change of the ratios (i.e., phosphorylated p40 phox to total p40 phox ) relative to unstimulated neutrophils (bottom right) are shown.Each dot depicts an individual healthy donor (n = 8).Acalabrutinib concentration, 2.5 µM.*P<0.05,**P<0.01,****P<0.0001,determined using two-sided paired t test, or two-sided Wilcoxon test (20 min, bottom right panel).

Supplemental Figure 6. Engagement of pattern recognition receptors activates BTK signaling and promotes anti-Aspergillus functions of healthy donor neutrophils. (A)
Zymosan stimulates BTK phosphorylation in neutrophils during whole-blood stimulation.Representative FACS histogram depicts BTK phosphorylation on live neutrophils in whole human blood (top panel).Quantitative summary of phosphorylated BTK mean fluorescence intensity (MFI) (bottom panel).(B) TLR2, FcgR or Dectin-1 inhibition leads to abrogated BTK phosphorylation in healthy human neutrophils upon stimulation by serumopsonized heat-killed Aspergillus conidia.Representative FACS histograms (upper panels) and mean fluorescence intensity (MFI) summary data (lower panels) for phosphorylated BTK (at Y223) in healthy donor neutrophils at baseline and at the indicated timepoints after stimulation with serum-opsonized heat-killed Aspergillus conidia, in the presence of vehicle or inhibitors, in the case of TLR2 (left panels), Dectin-1 (right panels), or isotype and blocking antibodies, in the case of FcgR (middle panels) (n = 5).(C) TLR2 ligation enhances damage of serum-opsonized A. fumigatus hyphae by healthy human neutrophils.Neutrophils were added at an effector:target ratio of 8:1 (n = 5-7).Data depict fold change in hyphal killing relative to the "medium alone" neutrophil controls, for each of the indicated agonists.Each dot represents an individual healthy donor.*P<0.05,**P<0.01determined using two-sided paired t test (A, B) or two-sided Welch's t test (C).

Supplemental Figure 7. GM-CSF boosts human neutrophil ROS production upon
Aspergillus stimulation, while IFN-g and G-CSF do not.(A) Summary data of AUC of luminol-amplified chemiluminescence RLU (expressed as AUC) in neutrophils isolated from ibrutinib-or acalabrutinib-treated lymphoma patients that were stimulated with serum-opsonized live or heat-killed (HK) A. fumigatus (Af) conidia, in the presence of increasing concentrations of IFN-g (n = 3).(B) Summary data of AUC of luminol-amplified chemiluminescence RLU (expressed as AUC) in vehicle-or ibrutinib-treated healthy donor neutrophils that were stimulated with serum-opsonized live or HK Af conidia, in the presence of increasing G-CSF concentrations (n = 3).(C) Summary data of AUC of luminol-amplified chemiluminescence RLU (expressed as AUC) in healthy donor neutrophils that were stimulated with serum-opsonized live or HK Af conidia, in the presence of increasing GM-CSF concentrations (n = 5).The baseline GM-CSF-unexposed AUC data are also depicted for the baseline "-IBR" data in Figure 7B, as the experiments with/without ibrutinib and GM-CSF were conducted at the same time.(D) Summary RLU AUC data for vehicle-or ibrutinib-treated healthy donor neutrophils (left; n = 5) or in neutrophils isolated from ibrutinib-or acalabrutinib-treated lymphoma patients (right; n = 3) that were stimulated with PMA in the presence of the indicated increasing GM-CSF concentrations.PMA, phorbol-12-myristate-13-acetate; IFN-g: interferon g; G-CSF: granulocyte colonystimulating factor.Ibrutinib concentration, 250 nM.Each dot represents an individual healthy donor or patient.BTKi: BTK inhibitor.**P<0.01,***P<0.001determined using repeated measures one-way ANOVA with Dunnett's multiple comparisons test (B; C, live Af) or Friedman's test with Dunn's multiple comparison test (C, HK Af).Supplemental Figure 8. Model of BTK-dependent promotes neutrophil activation in response to Aspergillus fumigatus.Recognition of Aspergillus fumigatus PAMPs by the fungal PRRs TLR2, Dectin-1 and/or FcγR leads to phosphorylation of BTK.Once activated, BTK mediates ROS production through phosphorylation of the p40 phox subunit of the NADPH oxidase complex, triggering translocation of the cytosolic components (p40 phox , p47 phox, and p67 phox ) to complex with gp91 phox , p22 phox and RAC2 at the phagosomal membrane.The activated NADPH oxidase complex catalyzes the oxidation of NADPH, leading to ROS generation.BTK also promotes the activation of RAC2 to drive the release of primary granules harboring anti-microbial molecules such as myeloperoxidase.Both neutrophil effector functions, i.e.ROS production and primary granule release are suppressed by the BTK inhibitors ibrutinib and acalabrutinib.The suppressed ROS production can be rescued with the FDA-approved cytokine granulocyte macrophagecolony stimulating factor (GM-CSF), which acts through phosphorylation of p47 phox to bypass the BTK inhibition-induced neutrophil deficit in ROS production.PAMPs: pathogenassociated molecular patterns; BTK: Bruton's tyrosine kinase; PRRs: pattern recognition receptors; TLR2: toll-like receptor 2; FcγR: Fc receptors for IgG; ROS: reactive oxygen species; NADPH: nicotinamide adenine dinucleotide phosphate oxidase; RAC2: Rac Family Small GTPase 2. The illustration was created with BioRender.com.

Generation of the A. fumigatus B-5233:mRFP strain
To generate the A. fumigatus B-5233:mRFP strain, protoplasts were generated using Trichoderma harzianum lysing enzyme (MilliporeSigma).A construct consisting of the gpdA promotor from Aspergillus nidulans, mRFP, and the ptrA resistance marker (3) was inserted into the genome ectopically via polyethylene glycol mediated transformation (4,5).Protoplasts were selected for on media containing 4 µg/ml pyrithiamine hydrobromide (MilliporeSigma).Pyrithiamine resistant conidia were screened by flow cytometry, comparing red fluorescence in the ECD-A channel to that of B-5233 wildtype, and the W72310:mRFP control strain (6).Growth of the A. fumigatus B-5233:mRFP strain was comparable to the wild-type A. fumigatus B-5233 strain by quantifying their radial growth on gut microbiota medium (GMM) agar.
Percentage germination inhibition of conidia in wells containing neutrophils was enumerated relative to the wells containing Aspergillus without neutrophils.

Aspergillus fumigatus hyphal damage by neutrophils
The killing efficacy of neutrophils against hyphae was assessed by the XTT-based colorimetric assay.The day before the experiment, Aspergillus conidia were harvested, counted, and adjusted to concentration of 2x10 6 /ml in RPMI-1640 (Corning, Cat# 10-041-CV) & 1% penicillin-streptomycin (Corning, Cat# 30-002-CI) with 20% fetal bovine serum (R&D Systems, Cat# S11150).Conidial suspensions were added at 2x10 5 , 1x10 5 , 0.5x10 5 or 0.25x10 5 conidia per well of a flat-bottom 96-well plate and placed in a 37°C incubator with 5% CO2 for 12 hours to allow for hyphal formation.After the 12-hour incubation, human neutrophils from healthy volunteers or patients were isolated from whole blood, counted and adjusted to 8x10 6 cells/ml in RPMI-1640 + 1% P/S.The hyphae were opsonized with 20% human serum from healthy donors for 30 minutes and co-incubated with 4x10 5 neutrophils per well (to create effector to target ratios of 2:1, 4:1, 8:1, and 16:1) for 2 hours.6 technical replicates were used for each target ratio.Non-neutrophil-containing hyphal wells in triplicate, were used as reference controls.After spinning the plates at 3200g for 10 minutes, the supernatants were stored for granule analysis by ELISA, and the remaining neutrophils were hypo-tonically lysed with deionized water.The remaining hyphae were washed with deionized water and incubated with 0.5 mg/ml XTT (Fischer Scientific, Cat# J61726) and 40 µg/ml 2,3-dimethoxy-5-methyl-1,4-benzoquinone coenzyme Q0 (Alfa Aesar, Cat# B24777) for 1 hour at 37°C.Absorbance was read at 450 nm wavelength with 660 nm wavelength correction using a microplate reader (Agilent, BioTek Synergy H1), and percentage hyphal damage was calculated relative to non-neutrophilcontaining wells.
To assess the role of neutrophil extracellular traps in A. fumigatus hyphal damage, opsonized hyphae were co-incubated with vehicle or 200 U/ml DNase (Roche, Cat# 4716728001), and 4x10 5 healthy donor neutrophils per well were added.Hyphal damage was assessed as described above.
To assess the role of TLR2 or Dectin-1 agonists in boosting A. fumigatus hyphal damage by neutrophils, the neutrophils were resuspended at 8x10 6 cells/ml in RPMI-1640 + 1% P/S, and were treated with 10 µg/mL Pam3CSK4 (TLR2 agonist; Invivogen, Cat# tlrl-pms) or 0.2 mg/mL b-glucan particles (Dectin-1 agonist; (gift from Dr. Yamanaka at Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan) for 30 minutes at 37°C.For FcgR stimulation, 4x10 5 neutrophils were added per well to wells containing immobilized immune complex for 30 minutes at 37°C.To immobilize the immune complexes, wells of flat-bottomed 96-well were coated with 20 µg/ml human serum albumin (Milli-poreSigma, Cat# A1653) in 50 mM carbonate buffer, pH 9.4 (MilliporeSigma, Cat# S7795) overnight at 4°C, washed with PBS + 0.05% Tween-20, blocked with 10% BSA (Fisher Scientific, Cat# BP9705-100) in PBS for 1 hour at room temperature and coated with 10 µg/ml anti-HSA antibody (Invitrogen, Cat# MA5-29022) for one additional hour.Subsequent to the agonist treatment, the primed neutrophils were added atop opsonized hyphae at 4x10 5 neutrophils per well, and the hyphal damage was assessed as described above.

Measurement of human neutrophil degranulation in response to Aspergillus fumigatus hyphae
The supernatants of human neutrophils co-incubated with preformed hyphae, were harvested and used for downstream enzyme-linked immunosorbent-assay (ELISA) experiments to measure neutrophil degranulation response against Aspergillus hyphae.We used the human myeloperoxidase DuoSet ELISA kit (R&D Systems, Cat# DY3174), human lactoferrin ELISA kit (Abcam, Cat# ab108882) and human MMP-9 DuoSet ELISA kit (R&D Systems, Cat# DY911) according to the manufacturer's instructions to measure myeloperoxidase (primary granules), lactoferrin (secondary granules) and MMP-9 (tertiary granules) respectively.Absorbance of the ELISA plates was read at 450 nm wavelength with 570 nm wavelength correction using a microplate reader (Agilent, BioTek Synergy H1).

Analysis of BTK expression in human neutrophils
To assess BTK transcript levels in human neutrophils, previously published data were sourced from the Gene Expression Omnibus (GEO) database (accession number GSE145033) (7); these data were acquired using RNA from healthy human neutrophils isolated from 10 male and 10 female donors (7).The raw read counts were downloaded from the GEO database and relevant attributes, including "Gene Symbol" and "Transcript Length" were retrieved using biomaRt (8,9).Subsequently, expression was determined using edgeR (10).
For zymosan stimulation in human whole blood, 1 mg/ml zymosan was directly added to heparinized whole blood, along with LIVE/DEAD fixable blue stain (Thermo Fisher Scientific, Cat# L34962) and a mouse antibody against human CD15 (clone HI98, BioLegend, Cat# 301908) which were added, mixed, and incubated at 37°C for 20 minutes.Subsequently, the cells in whole blood were fixed by adding 2 ml of prewarmed Phosflow Lyse/Fix buffer (BD Biosciences, Cat# 558049) for 10 minutes in a water bath at 37°C with intermittent mixing.After centrifugation at 350g for 6 minutes, Lyse/Fix buffer was removed, and the cells were washed with ice-cold phosphate-buffered saline (PBS).Cells were then permeabilized, stained, and analyzed as above.
Quantification was obtained by densitometry image analysis using FIJI (13).

Active RAC2 pull-down assay
Human neutrophils from healthy volunteers were isolated by negative immunomagnetic separation using the EasySep™ Direct Human Neutrophil Isolation Kit (STEMCELL Technologies, Cat# 19666) per kit instructions.Cells were resuspended at 44x10 6 /ml in 1x HBSS and either 10 million cells aliquoted with HBSS media control, vehicle (DMSO) or 2.5 µM ibrutinib to pretreat the cells for 30 minutes at 37°C.Unstimulated cells were then lysed on ice using 2x assay/lysis buffer kit component (Abcam, Cat# 21162) with added protease inhibitor (MilliporeSIGMA, Cat# P8340) and phosphatase inhibitors (Milli-poreSIGMA, Cat# P2850 & Cat# P0044).For cells designated to undergo stimulation, opsonized zymosan at 1 mg/ml (MilliporeSigma, Cat# Z4250) was added and stimulated for 30 minutes at 37°C with shaking at 120 rpm.After 30 minutes the cells were lysed on ice using the same 2x assay/lysis buffer kit component containing protease and phosphatase inhibitors.All cell lysates were then spun at 9300g at 4°C for 10 minutes after which supernatant was transferred to new tubes.50 µl of cleared neutrophil lysates were saved with 10 ml of 6x SDS sample buffer from each sample for total RAC2 detection.Positive and negative control lysates were supplemented with 10 mM EDTA, then 10 ml of 10 mM GTPgS or 10 ml of 100 mM GDP and incubated for 8 minutes at 30°C.Nucleotide with a gene number between 100-2500 and a mitochondrial gene proportion < 0.1 were selected for downstream analysis.The matrices were then normalized by the LogNormalize method.The FindVariableFeatures() function was used to select the top 2,000 variable genes, with the vst selection method.Scaling was performed by the function ScaleData() regressing out the mitochondrial gene content.Principal component analysis (PCA) and clustering were then performed on the scaled data.UMAP (version 0.2.7.0) was utilized for visualization.After cell types were identified using marker genes in the dataset corresponding to each sample, they were integrated.Genes that were shared among all datasets were identified for downstream integration.Anchors were identified with the FindIntegrationAnchors() function, and these anchors were used to integrate the cells together with the function IntegrateData().To study neutrophil cell-state trajectories, we used the analysis toolkit Monocle3, which is implemented as an R package (version 0.2.3.0).A principal graph was learned on the UMAP projection of the cells with the learn_graph() function.To generate a pseudotime axis, the cells were then ordered with the order_cells() function.Gene scores are using the AddModuleScore() function.
For isolation of neutrophils from lungs, whole lungs were removed and transferred to a petri dish with 0.5 ml of 3 mg/ml collagenase type IV (Worthington-Biochem, Cat# LS004189) containing 40 U/ml DNase I recombinant (Roche Diagnostics, Cat# 4716728001) in PBS with 5% fetal bovine serum (R&D Systems, Cat# S11150).Lungs were then minced using a razor blade for approximately 5 minutes per lung to achieve a slurry of <0.5 mm pieces, transferred to a 50 ml conical tube containing 7 ml collagenase solution and incubated at 37°C, in a shaking water bath, for 1 hour at 180 rpm.Following this step, the slurry was passed through an 18G needle and then filtered through a 100 µm cell strainer.After washing, cells were subjected to RBC lysis with ACK lysis buffer (Quality Biological, Cat# 118-156-721) and two further wash and filter steps were performed.For neutrophil enrichment the cell suspension was subjected to positive immunomagnetic selection using anti-Ly6G microbeads (Miltenyi Biotec, Cat# 130-120-337).For isolation of neutrophils from bronchoalveolar lavage, lungs and trachea were surgically exposed and a small incision made in the trachea to allow insertion of a catheter (BD Biosciences, Cat# 381444).700 µl of PBS was passed into the lungs and collected; this step was repeated a further three times to flush cells from the lower respiratory tract.

Histology
For hematoxylin and eosin (H&E) and Grocott's methenamine silver (GMS) staining, mouse lungs were harvested and placed in 10% formalin for 24 to 48 hours.Formalin was then replaced with 70% ethanol to store until paraffin embedding.Sections of the lungs were prepared and stained with H&E and/or GMS (Histoserv Inc., Germantown, MD).For histological analysis of ibrutinib-treated mice, the animals were infected with 3 x 10 7 conidia, and the lungs were harvested at day 4 post-infection.To analyze germinating conidia, multiple regions of interest per mouse were randomly chosen and germination was enumerated.Germination frequency was reported as the fraction of conidia which exhibit germination.
Measurement of cytokines, chemokines, and b-D-glucan in mouse lung homogenates Mouse lungs were harvested from naïve mice or at day 2 post-pulmonary Aspergillus infection and homogenized in PBS containing 0.5% Tween 20 and protease inhibitor cocktail (Roche, Cat# COEDTAF-RO), using an Omni-Tip TM homogenizer (Omni International, Cat# TH115-PCR).Homogenates were clarified by centrifugation and subsequent passage through a 0.22 µm filter.The cytokines and chemokines were measured using a multiplexed bead-based assay system (Luminex Corporation, Austin, TX) as described previously (16), while (1,3)-beta-D-glucan was measured using a GLUCATELL kit (Associates of Cape Cod Inc., Cat# GT-003) according to kit instructions for the end-point assay.Briefly, for Luminex analysis, individual Luminex bead sets were coupled to the analyte-specific capture antibodies according to the manufacturer's protocols, and biotinylated polyclonal antibodies were used at twice the recommended concentrations for a classical ELISA.Subsequently, in a 50 µl volume, the assay was run with 1,200 beads per set of the examined analytes, and the plates were read where >50 beads per bead The reporter FLARE Af293-dsRed conidia were generated as above, described under the "Generation of fluorescent Aspergillus reporter (FLARE) conidia" section.Bone marrow neutrophils were isolated as described under "Isolation of neutrophils from mouse bone marrow, lungs and bronchoalveolar lavage" (15).Neutrophils were co-incubated with the opsonized FLARE conidia for 3 hours at a 1:1 multiplicity of infection (MOI).Subsequently, the neutrophils were stained using a human anti-Ly6G antibody (clone 1A8, Bi-oLegend, Cat# 127628) and analyzed by flow cytometry to define Ly6G + neutrophils that are associated with conidia (dsRed/mRFP -AF633 + or dsRed/mRFP + AF633 + ), neutrophils that contain live (dsRed + AF633 + or mRFP + AF633 + ) or killed conidia (dsRed -AF633 + or mRFP -AF633 + ), and bystander neutrophils that have not engaged with conidia (dsRed - or mRFP -and AF633 -).Flow cytometry was performed using a 5-laser BD LSR Fortessa II (BD Biosciences, Franklin Lakes, NJ).Data were collected using FACS Diva and analyzed using FlowJo (BD Biosciences, Franklin Lakes, NJ).

Aspergillus fumigatus hyphal damage by mouse neutrophils
Mouse neutrophils were used in the hyphal killing assay as specified above, under the "Aspergillus fumigatus hyphal damage by neutrophils" section.After hypotonic lysis of neutrophils, hyphae were washed with deionized water and incubated with 1X alamar-Blue TM (Invitrogen, Cat# DAL 1100) in 37°C incubator with 5% CO2 for 18 hours.alamar-Blue TM fluorescence was recorded using a microplate reader (Agilent, BioTek Synergy H1) and percentage hyphal damage was calculated relative to non-neutrophil-containing wells.

Measurement of mouse neutrophil degranulation in response to Aspergillus fumigatus
The supernatants of mouse bone-marrow neutrophils co-incubated with preformed hyphae, were harvested and used for downstream enzyme-linked immunosorbent-assay (ELISA) experiments to measure neutrophil degranulation response against Aspergillus hyphae.We used the mouse myeloperoxidase DuoSet ELISA kit (R&D Systems, Cat# DY3667) according to kit instructions to measure myeloperoxidase (MPO) from the supernatants taken after mouse neutrophil-Aspergillus hyphae co-culture.Absorbance of

For
single cell sequencing, sodium heparin or EDTA treated blood collected from three lymphoma patients, at one day prior and three days post-acalabrutinib treatment, was utilized.White blood cells were isolated with the Erythroclear Red Blood Cell Depletion Reagent Kit (STEMCELL Technologies, Cat# 01738) and neutrophils using EasySep TM Direct Human Neutrophil Isolation Kit (STEMCELL Technologies, Cat# 19666), according to manufacturer's protocol.Cells were washed with PBS with 0.02% bovine serum albumin (BSA), and utilized for single-cell RNA-seq.To obtain single-cell gel beads-in-emulsion (GEMs), we resuspended cells at a concentration of 1000 cells/µl and loading the mix on a Chromium Comptroller Instrument (10x Genomics).Single-cell cDNAs and libraries were prepared with a Chromium Single Cell 3′ Library & Gel Bead Kit v3.1 (10x Genomics, Cat# 1000121).Quality and quantity of the cDNAs were assessed on a 4200 TapeStation (Agilent Technologies) with High Sensitivity D5000 DNA Screen Tape (Agilent, Cat# 5067-5592).Libraries were diluted to the same molarity and pooled for sequencing on a NovaSeq6000 (Illumina) sequencer.Processing and analysis of single-cell RNA-seq dataIllumina run folders were demultiplexed and converted to FASTQ format with Cell Ranger mkfastq version 4.0.0 and Illumina bcl2fastq version 2.20.Reads were further counted and analyzed with Cell Ranger count version 4.0.0 and the refdata-gex-GRCh38-2020-A reference, to generate raw and filtered matrix files.Matrix files were imported into the R package Seurat version 4.0.1 for downstream processing.From the raw matrices, cells