supplementary materials for · theresia e. b. stradal, nima rezaei, kaan boztug* *corresponding...

immunology.sciencemag.org/cgi/content/full/5/49/eabc3979/DC1

Supplementary Materials for

The cytoskeletal regulator HEM1 governs B cell

development and prevents autoimmunity

Elisabeth Salzer, Samaneh Zoghi, Máté G. Kiss, Frieda Kage, Christina Rashkova, Stephanie Stahnke, Matthias Haimel, René Platzer, Michael Caldera, Rico Chandra Ardy, Birgit Hoeger, Jana Block, David Medgyesi, Celine Sin, Sepideh Shahkarami, Renate Kain, Vahid Ziaee, Peter Hammerl, Christoph Bock, Jörg Menche, Loïc Dupré,

Johannes B. Huppa, Michael Sixt, Alexis Lomakin, Klemens Rottner, Christoph J. Binder, Theresia E. B. Stradal, Nima Rezaei, Kaan Boztug*

*Corresponding author. Email: [email protected]

Published 10 July 2020, Sci. Immunol. 5, eabc3979 (2020)

DOI: 10.1126/sciimmunol.abc3979

The PDF file includes:

Materials and Methods Fig. S1. Defective WRC complex assembly and stability. Fig. S2. T cell phenotype and function in human HEM1 deficiency. Fig. S3. Extended B cell immunophenotyping in human HEM1 deficiency. Fig. S4. Detailed characterization of Hem1–/– mice. Fig. S5. Single-cell RNA-seq analyses of splenic T and B cell subclusters. Fig. S6. Extended characterization of B cells and comparison with WASP-deficient B cells. Table S2. List of discriminating features for PCA of healthy donor and HEM1-deficient T cells upon fibronectin stimulation. Table S3. T cell immunophenotyping in HEM1-deficient individuals compared with age-matched reference values. Table S4. List of discriminating features for PCA of healthy donor and HEM1-deficient B cells upon IgM stimulation. Table S5. B cell immunophenotyping in HEM1-deficient individuals compared with age-matched reference values. Table S6. List of top differentially regulated genes in B cell subclusters as identified by 10× single-cell sequencing. Table S7. List of top differentially regulated genes in T cell subclusters as identified by 10× single-cell sequencing. Table S8. GO term enrichment of HEM1-deficient B cell functions derived from the top candidates from table S6. Table S9. GO term enrichment of HEM1-deficient T cell functions derived from the top candidates from table S7. Full Western blot scans for all figures.

References (76–80) Other Supplementary Material for this manuscript includes the following: (available at immunology.sciencemag.org/cgi/content/full/5/49/eabc3979/DC1)

Table S1 (Microsoft Excel format). Figure raw data.

Materials and Methods

Homozygosity mapping

Homozygous sections are mapped using H3M2 (Homozygosity Heterogeneous Hidden Markov

Model) software, allowing screening for variants segregating with the patient phenotype as

previously described (65).

Sanger sequencing

Sanger sequencing was used to validate the HEM1 variant NM_005337:c.385C>T, p.(Arg129Trp)

in exon 5 from WES in the affected patient and his family members. This was done by designing

specific primers for the variant found: Fw1: 5’- GGGGAAAGAAGGAGATCCAG -3’, Rv1: 5’-

GGCATCAATTTCCTTTGCAT -3’.

Sample preparation for 10X single-cell RNA-seq data

Homogenized and RBC depleted spleen cell lysates of two 5 weeks old (1 Hem1+/+ and 1 Hem1-

/-) and four 10 week (4 Hem1+/+ and 4 Hem1-/-) mice were adjusted to a concentration 1 million

per milliliter cells and the sample preparation was performed according to manufacturer’s

instructions using Chromium™ Single Cell 3' (for 5 week-old mice) or 5 (for 10 week-old mice)

Library & Gel Bead Kit v2, Cat. 120237, together with Chromium™ Single Cell A Chip Kit, Cat.

120236 and Chromium™ i7 Multiplex Kit, 96 rxns PN-120262. In brief, single-cell RNA-seq was

performed using the 10x Genomics Chromium Single Cell Controller with the Chromium Single

Cell 3' V2 Kit following the manufacturer’s instructions. After quality control, libraries were

sequenced on the Illumina HiSeq 4000 platform in 2x75bp paired-end mode. Raw sequencing data

were processed with Cell Ranger v1.3.0 (10x Genomics) for demultiplexing and alignment to the

GRCh38 human reference genome. The processed data were analyzed further using the R statistics

software and various Bioconductor packages. Specifically, we used Seurat v1.4.0.1461 to load pre-

processed results from Cell Ranger into R and to perform quality control (removing cells with less

than 1,000 genes or mitochondrial content greater than 90%).

Immunophenotyping on peripheral blood mononuclear cells (PBMCs)

Immunophenotyping was performed on an LSR-Fortessa (BD Biosciences). Antibodies were used

as follows: 2µl CD3-APCH7 (SK7), 3µl CD4-BV605 (RPA-T4), 3µl CD8-FITC (HIT8a), 3µl

'

3µl CD8-PECy7 (SFCI21Thy2D3), 2µl CD16-PECy7 (3G8), 0.7µl CD19-BV510 (SJ25C1), 3µl

CD20-PerCPCy5.5 (2H7), 3µl CD20-APC (2H7), 3µl CD21-PE (B-ly4), 2µl CD23-AF700 (M-

L233), 0.7µl CD24-BV786 (ML5) , 3µl CD25-PE (M-A251), 3µl CD27-PE-Cy7 (M-T271), 3µl

CD27-V450 (M-T271), 3µl CD27 BV605 (M-T271), 3µl CD27-PE (M-T271), 3µl CD38-PECy7

(HIT2), 1µl CD38-BV711 (HIT2), 3µl CD83-FITC (1G10), 3µl CD45RA-AF700 (HI100), 4µl

CD56-V450 (B159), 2µl CD95-PECy7 (DX2), 2µl CD197-PE-CF594 (150503), 3µl, CD278-

PerCPCy5.5 (DX29), 3µl CD279-PE (EH12.1), 2µl FCRL5 (CD307e)- BV605 (509F6), 20µl

TCRgd-PE (11F2), 2µl IgM-BV421 (G20-127), 3µl IgD-FITC (IA6-2), 3µl IgM-APC (G20-

127), 2µl IgM-V450 (G20-127), 5µl RoRgT-AF647 (Q21-559), all from BD Bioscience. 3µl

CD4-PerCPCy5.5 (RPA-T4), 3µl CD11a-FITC (HI111), 4µl CD11c-V450 (3.9), 2µl CD14-

PECy5 (61D3), 2µl CD19-PerCPCy5.5 (HIB19), 2µl CD28-APCo (CD28.2), 2µl CD31-APC

(WM59), 3µl CD45-PerCPCy5.5 (HI30), 3µl CD57-PE (TBO1), 2µl CD244-PE (eBioC1.7),

2.5µl FOXP3-FITC (PCH101), 2µl IL17A-V450 (eBio64DEC17) all from eBioscience. 3µl CD4

PE-Cy7 (SFCI12T4D11), 3µl CD4-PE (13B8.2), 3µl CD16-FITC (3G8), 2µl CD19-PECy7 (J3-

119), 3µl TCR v-alpha24-PECy7 (C15), 3µl TCR v-beta-11-FITC (C21) from Beckman Coulter.

2 µl CD8-V450 (RPA-T8) and 3µl CD43-BV605 (IG10) from BD Horizon and 20µl TCRab-

FITC (WT31) from Becton Dickinson and 3µl CD185-APC (51505) from R&D Systems. Tbet-

PerCP-Cy5.5 (4B10) was purchased from BioLegend and 5µl were used per reaction. Patient B

cells, gated on CD19+, were also stained with anti-BAFFR-FITC (#558081) and anti-CD274

APC (PD-L1, # 563741), Cholera toxin subunit B conjugate AF488 (#V-34403, invitrogen).

For pAKT and pS6 assay, MACS-sorted B cells of the patient were stimulated with CD40L (#

6420-CL-025/CF R&D) and IL4 (#200-04-5µG peprotech) for 30 minutes in 37 degrees and then

cells were fixed and permeabilized by standard BD phosphoflow protocol stained with with pS6

(Ser235-236) Alexa Fluor 647 (N7-548, #560435), pAKT (Ser473)-PE (M89-61, #560378).

Feeder cell-mediated T-cell expansion

Feeder T-cell expansion was done as previously described (64). Obtained expanded T-cells were

evaluated by flow cytometry for expression of CD4, CD8. Patient and healthy donor PBMCs

were isolated using Ficoll density centrifugation.

Freshly isolated PBMCs were incubated with EBV supernatant and on the following day 1 µg/ml

cyclosporin A was added to the cells. Cells were maintained in complete RPMI medium (Gibco)

with 10% FCS, 1% Hepes and 1% PenStrep (all from Invitrogen).

CRISPR-mediated gene deletion in Jurkat cell lines

gRNAs targeting HEM-1 were designed as described (75) and cloned into a lentiCRISPRv2

vector (addgene). Jurkat cells (ATCC) were cultivated according to the supplier’s instructions.

Lentivirus production and cell infection were performed according to standard procedures.

Single cell clones were generated by limiting dilution and evaluated for loss of HEM1. Editing at

genomic position was confirmed using capillary sequencing and subsequent upload into tide

(http://tide.nki.nl/, accessed November, 2017). Only clones with frameshift mutations in both

alleles of more than 90% were used for further experiments. Following gRNA targeting

sequences were used for cloning:

sgNCKAP1L_Ex5_f: TGTCACGGATTGAAGATCGG,

sgNCKAP1L_Ex5_r: CCGATCTTCAATCCGTGACA,

sgNCKAP1L_Ex5_f: TGACAGAAGTAAAATGACTG,

sgNCKAP1L_EX5_r: CAGTCATTTTACTTCTGTCA,

sgNCKAP1L_Ex9_f: CAAACTGCTAAACAGGTCCT,

sgNCKAP1L_Ex9_r: AGGACCTGTTTAGCAGTTTG,

sgNCKAP1L_Ex9.2_f: ACGGATAAGGGTGATGTAGA,

sgNCKAP1L_Ex9.2_r: TCTACATCACCCTTATCCGT,

sgNCKAP1L_Ex11_f: GTCAACGGCGGCAATTTCTG,

sgNCKAP1L_Ex11_r: CAGAAATTGCCGCCGTTGAC,

Sequencing Primers:

sgNCKAP1L_Ex5_f: CCCTGACTTTTGTTCCAATTTT,

sgNCKAP1L_Ex5_r: TAGTTCAAACCCGTGTTGTTTC,

sgNCKAP1L_Ex9.1_f: GGAGCGCTGGATTATCAGTAAG,

sgNCKAP1L_Ex9.1_r: AGAGTCTGTGCTCTTAACCTCCA,

sgNCKAP1L_Ex9.2_f: CTTCTCTTCTCACAGATGGCCT,

sgNCKAP1L_Ex9.2_r: ATTGTGTCCCTAAAACCCAGTG,

sgNCKAP1L_Ex11_f: TGGTCTTGAACTCCTGACCTC,

EBV-LCL generation

sgNCKAP1L_Ex11_r: GATACAAAAGGTTTTGGGATACTC

Generation of Hem1/Hem2 knock-out in B16-F1 cells by CRISPR/Cas9-mediated genome

editing

CRISPR/Cas9-mediated gene disruption of Hem2 was performed using the CRISPR guide

sequence 5´- GACGCCCCGGTCGTTGAGGA -3´. For description of Hem2 knock-out clone

generation and phenotypes (21). Subsequently, Hem1 was additionally disrupted using the

CRISPR guide sequence 5´-CTCACGATCCTGAATGACCG -3´. Hem1 gene removal was

performed in the background of Hem2 null clone #21 (ref (21)) and two clones selected for

further analyses (clone #7 and #8). CRISPR/Cas9-mediated genome editing was essentially

performed as described previously (21, 23).

Transfection of B16-F1 melanoma cells

B16-F1 mouse melanoma cells (ATCC CRL-6323) were grown in DMEM (4.5 g/l glucose;

Invitrogen, Germany) with 10% FCS (PAA Laboratories, Austria) and 2 mM L-glutamine

(Thermo Fisher Scientific) and incubated at 37°C in the presence of 7.5% CO2. Transfections

were carried out using JetPrime transfection reagent (Polyplus, VWR). Parental B16-F1 cells

were transfected using 0.5 µg DNA and 1 µl JetPrime (3 cm culture dishes), whereas in case of

knock-out cells equal transfection efficiency was achieved using 1 µg DNA and 2 µl JetPrime.

Plasmids and site-directed mutagenesis

pEGFP-vectors were purchased from Clontech Inc. (Mountain View, CA, USA). EGFP-Hem2 was

generated and kindly provided by Dr. Anika Steffen(21). Hem1 wildtype sequence was generated

using IMAGE Clone IRAK961M1081G2 (imaGenes, Germany), and subcloned into a pEGFP-C

vector backbone. Site-directed mutagenesis was used to introduce point mutations of interest into

both Hem1 and Hem2 using the respective EGFP-tagged plasmid as templates. Standard PCR

reactions were performed using Phusion high-fidelity DNA polymerase (New England Biolabs)

employing forward primers as follows: 5-CAACTTTGACTTCACTTGGAGCTACCTGGAC-3´

for EGFP-Hem1-R129W; 5´-CAACTTTGACTTCACTGCGAGCTACCTGGAC-3´ for EGFP-

Hem1-R129A; 5´-CAACTTTG ACTTCACTGAGAGCTACCTGGAC-3´ for EGFP-Hem1-

Western blotting

1-3 × 106 of patient or healthy donor EBV-immortalized B cells, feeder-expanded T cells,

PBMCs or Jurkat cells were lysed in 40-60 µl in RIPA buffer (50mM HEPES, 150mM NaCl,

10% glycerol, 1% Triton-X100, 2mM EDTA, 1% Sodium Deoxycholate, 50mM NaF, 10mM

NaO3V4), supplemented with PMSF (10658793, Fisher scientific) and complete™ Protease

Inhibitor Cocktail, EDTA-free (Roche, #11873580001). Lysates were loaded on 7.5-17,5%

acrylamide-bisacrylamide gels and blotted by wet transfer overnight at 120 mA at 4 °C or 1 hour

at 100V on PVDF membrane, except for BRK1, which was blotted on nitrocellulose membrane

(PVDF - #T830.1, Roth; Nitrocellulose – #10600018, GE Healthcare). Blocking was performed

with 5% BSA (8076.4, Roth) in TBS-Tween. Afterwards, membranes were incubated with

primary antibodies in Tris-buffered saline with 0.5% Tween and 5% BSA overnight at 4 °C and

after washing three times, membranes were incubated with respective secondary antibodies at

~21 °C for 1 h. Blots were developed using ECL (Thermo Scientific) and films (GE healthcare).

Western antibodies were as follows: Hem-1 (#HPA039490, Atlas antibodies), WAVE2 (#3659,

Cell Signaling) Hem-2 (#ab126061, Abcam), Abi-1 (#39444, Cell Signaling), Abi-2 (#ab108340,

Abcam), Phospho-p44/42 MAPK (Erk1/2) Thr202/Tyr204 (#4370 S, Cell Signaling

Technology), p44/42 MAPK (Erk1/2) 137F5 (#4695 S, Cell Signaling Technology), BRK1

(#SAB4301207, Sigma Aldrich), CYFIP1 (#ab108220, Abcam), HSP 90⍺/ß F-8 (sc-13119,

Santa Cruz Biotechnology), GAPDH (sc-32233, Santa Cruz Biotechnology). Secondary anti-

rabbit antibody was purchased from Cell Signaling (#7074S) and anti-mouse was purchased

from Becton Dickinson (#554002).

Immunoprecipitation experiments

GFP-Trap_A (ChromoTek) was used to immunoprecipitate EGFP-tagged proteins. Various

constructs of interest were ectopically expressed in B16-F1 cells. We used B16-F1 wildtype cells

here to explore specific Hem1 or Hem2 variants in regard to binding to other WAVE complex

subunits (such as R129W), because expression of these loss-of-function mutants was incapable

of restoring WAVE regulatory complex subunits expression in Hem1/Hem2 knock-outs.

R129E; and 5´-GAACTTTGATTTAACATGGAACT ACTTAGACTTG-3´ for EGFP-Hem2-

K131W. All constructs were sequence-verified.

Transfections were performed as described above but amounts of DNA and JetPrime reagent

were up-scaled to 10 cm dishes. Three confluent 10 cm dishes per construct were lysed using

ice-cold IP-buffer (140 mM KCl, 50 mM Tris-HCl (pH 7.4), 50 mM NaF, 10 mM Na4P2O7, 2

mM MgCl2) supplemented with 1% Triton-X100 and a mini complete protease inhibitor pill

(Roche). Lysates were centrifuged at 20,000 x g for 15 min at 4°C. 10 µl of each cell lysate was

mixed with SDS sample buffer referred to as input. 30 µl bead slurry was washed 3x and spun

down (2,500 x g for 2 min at 4°C). Subsequently, cell lysates were added to the beads and

incubated under constant mixing for 1h at 4°C. After centrifugation, 10 µl of each sample was

incubated with SDS sample buffer and used as control of unbound, EGFP-tagged protein fraction

(supernatant). Beads were washed 3x with ice-cold IP-buffer prior to addition of 25 µl 4xSDS

sample buffer. All samples were boiled at 95°C for 5 min and loaded onto respective SDS gels.

After blotting proteins onto PVDF membranes (Immobilon) according to standard procedures,

respective antibodies (anti-GFP, 101G4B2, self-made; anti-Sra1, Steffen et al., 2004; anti-pan-

WAVE and anti-Abi1as previously described (24)) were used to confirm co-

immunoprecipitations of WAVE complex components with ectopically expressed EGFP-tagged

Hem1 or Hem2 variants.

Data processing and statistical analyses of B16-F1 cell images

Brightness and contrast levels were adjusted using MetaMorph software (Molecular Devices

Corp., Sunnyvale, CA, USA). Images were further processed for figure preparation using Adobe

Photoshop CS4. Data analyses were carried out using MetaMorph, Excel 2010 (Microsoft) and

Sigma plot 12.0 (Systat Software).

TCR and BCR signaling analysis

Jurkat cells as well as patient or healthy donor derived T-cell lines were starved for up to 4 h in

RPMI medium (Invitrogen) containing 1% FCS (Sigma-Aldrich), 0.2 mM L-glutamine (Sigma-

Aldrich), 10 µg/ml penicillin/streptomycin (Sigma-Aldrich and 20 mM HEPES (Gibco). Cells

were put on ice for at least 20 min and ice-cold stimulation mix were prepared containing mAb

to CD3 (clone OKT-3, purified, 2 µg/ml) and mAb against CD28 (clone Leu28, purified, 1

µg/ml, BD Bioscience). Cells were stimulated for indicated time periods, washed in ice-cold

PBS and lysed in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA (all from Roth) and

complete protease inhibitor tablets (Roche), 1 µM orthovanadate, 50 mM NaF, 1% Triton X-100

(Thermo Scientific). Upon addition of RIPA buffer, samples were incubated on ice for 5 min,

centrifuged at 4°C at maximum speed and the supernatant transferred to new tubes and boiled for

5 min. Lysates were loaded on 8 or 10% acrylamide gels. Gels were run at constant voltage and

blotted overnight at constant 120 mA at 4°C or for 1h at 100V using a mini-protean wet blotting

system (BioRad). Immunoblotting was performed according the procedure explained above.

Prior to stimulation, B cells were serum starved for 3–4 h and stimulated with anti-IgM 500

ng/ml (#2020-01) for the indicated time points. Subsequent handling was performed as described

for TCR signaling analyses: p44/42 MAPK (ERK1/2) (clone 137F5), Phospho-p44/42

(Thr202/Tyr204) MAPK (ERK1/2), (clone D13.14.4E; all from Cell Signaling), pAKT, AKT,

GAPDH (clone 86C5, Santa Cruz Biotechnology).

B-LCLs derived from the patients and control donors were starved for about 6 hours and

stimulated with either ICAM-1 (1µg/ml, R&D Systems), IgM (25 µg/ml, Southern Biotech) or

Fibronectin (2µg/ml, R&D Systems) for the following time periods: 0, 30 seconds, 5 minutes and

20 minutes. 2x106 cells were used per reaction. Prior to the ICAM-1 stimulation, the cells were

pre-treated with MgCl2 (5mM) + EGTA (1,5mM) for 10 minutes at 37°C. The reaction was

stopped by putting the samples on ice and adding cold PBS. We studied AKT phosphorylation

with the above-mentioned antibodies.

Immunofluorescence studies (IF) on patient T cells and B cells

To assess T-cell spreading on Fibronectin, expanded T cells were resuspended in RPMI 10%

FCS and transferred to Fibronectin (2ug/ml) coated slides. After 10 min of incubation at 37°C,

cells were fixed with 4% PFA at 37°C for 10 min, permeabilized with BD Cytofix and stained

with Phalloidin-AF488 (#A12379, ThermoScientific), α-tubulin Ab (ab18251, Abcam) and

DAPI (#63351, Roth). GAR-AF647 (A21244, ThermoScientific) was used to reveal the α-

tubulin staining. Slides were imaged with a Zeiss LSM700 confocal microscope and a 63x oil-

immersion objective. Unsupervised analysis of cell morphology parameters was performed with

the CellProfilerTM software followed by correlation of extracted features using principal

component analysis. For quantification of cell polarity, the cells were classified into three

categories, namely “polarized”, “intermediate” and “non polarized”. Based on manual selection

of approximately 10-15 cells from each category, all other cells were evaluated using a machine-

learning pipeline that uses the previously extracted 82 parameters to classify the cells into the

predefined categories. In a parallel approach we used the same machine learning pipeline to

classify the cells based on 5 preselected, biologically relevant parameters, namely max Feret

diameter, form factor, perimeter, cell area and protrusion length. Stainings including high-

affinity LFA1-A24 (#363402, Biolegend UK Ltd.), were performed as described previously (76).

The following secondary antibodies were used: anti-rabbit AF647 (#A-21244, Invitrogen), anti-

mouse IgG1 AF555 (#A21127, Invitrogen).

For IF staining of B cells, similar protocol to T cells has been performed, the slides were coated

with 25 µg anti-human IgM (# 2020-01 SouthernBiotech) and B cells were MACS sorted by B

Cell Isolation Kit II, human (#130-091-151 Miltenyi) and stimulated on the slides for 30 minutes

in 37 degrees. Staining included Phalloidin, Foxo1 (#2880T, Cell signaling), anti-Rabbit IgG

AF555 (# A32732 Invitrogen) and DAPI.

Video microscopy of patient and healthy control EBV lymphoblastic B-cell lines

Healthy donor and P2 EBV LCL cells were exposed to a 0-250 ng/ml CCL19 gradient and

imaged by time-lapse recording with IX83 inverted Olympus microscope in 30 sec frame

intervals. The morphology of all isolated cells was evaluated on every frame and assigned to 5

morphotypes dominated by typical protrusions (lamellipodia, pseudopodia, extended lamella,

blebs, spikes). The percentage of time during which the cells displayed the indicated

morphologies were evaluated and shown as mean ± SEM of 3 videos per cell line.

TCR mediated LFA1 activation

100,000 Jurkat cells were plated with anti-CD3 (OKT3) at the concentrations of 1, 5 and 10

µg/ml for 10 minutes. Following the stimulation, the cells were incubated with LFA1 (clone a24,

dilution of 1:450) for another 10 minutes at 37°C (76). Later the cells were fixed with 4%

paraformaldehyde for 15 minutes in 4°C. Finally, secondary AF488 (anti-mouse IgG1) was

added for 1 hour and MFI was assessed by flow cytometry.

Primary cell isolation and immunophenotyping in Hem1-/- mice

Peripheral blood was collected via the vena cava and red blood cells were lysed in red blood cell

lysis buffer (Morphisto). Spleens were crushed through a 100 μm cell strainer (BD Biosciences)

and red blood cells were lysed in red blood cell lysis buffer. Bone marrow cell suspensions were

isolated by flushing femurs and tibias through a 26-gage needle with 1% FCS in DPBS (Sigma)

and red blood cells were lysed as stated above. Total viable cells were counted manually using a

hemocytometer or by CASY cell counter & analyzer. For flow cytometric staining, 1x106 cells

were added in a 96-well V-bottom plate (Thermo Scientific) and incubated with 2.5 µg/ml of a

blocking anti-CD16/32 antibody (Clone 90, eBioscience) or anti-CD16/32 APC (Clone 90,

eBioscience) diluted in DPBS (Sigma) supplemented with 1% FCS for 20 min at 4°C. After two

washing steps, cells were stained with the following monoclonal antibodies: anti-CD45R (B220)

PerCP-Cy5.5 (clone RA3-6B2, eBioscience), anti-CD43 PE (clone S7, BD Biosciences), anti-

CD23 eFluor450 (clone B3B4, eBioscience), anti-CD21/35 BV605 (clone 7G6, BD

Biosciences), anti-IgM APC (clone II/41, eBioscience) anti-IgD PE-Cy7 (clone 11-26C,

eBioscience), anti-CD11b AlexaFluor700 (clone M1/70, eBioscience), anti-Ly6C BV605 (clone

HK1.4, BioLegend), anti-.Ly6G PE (clone 1A8, BioLegend), anti-F4/80 PerCP-Cy5.5 (clone

BM8, BioLegend), anti-CD11c APC-eFluor780 (clone N418, eBioscience), anti-CD19 APC

(clone eBio1D3, eBioscience), anti-Igκ FITC (clone 197.1, BD Biosciences), anti-CD93 FITC

(clone AA4.1, e Bioscience), anti-CD93 eFluor450 (clone AA4.1, e Bioscience) biotinylated

anti-Igλ (clone RML-42, BioLegend), biotinylated anti-CD23 (clone B3B4, BD Pharmingen™),

biotinylated anti-CD138 (clone 281-2, BioLegend), anti-GL7 eFluor450 (clone GL-7,

eBioscience), anti-CD3e PE (clone 145-2C11, eBioscience), anti-CD4 FITC (clone GK1.5,

eBioscience), anti-CD8a APC (clone 53-6.7; eBioscience), anti-CD185 (CXCR5) APC (clone

SPRCL5, BD Biosciences), anti-CD279 (PD-1) (clone J43, eBioscience), mouse hematopoietic

lineage antibody cocktail FITC (17A2, eBioscience), anti-CD117 (c-kit) APC-eFluor 780 (clone

2B8, eBioscience), anti-Sca1 (Ly6A/E) PE-Cy7 (clone D7, eBioscience), anti-CD34 eFluor450

(clone RAM34, eBioscience), anti-CD127 (IL7-R) PE ( clone A7R34, eBiodcience), anti-CD135

(Flt3) APC-eFluor 710 (clone A2F10, eBioscience), streptavidin APC-eFluor 780 (eBioscience),

Anti-CD73 (#127204, Biolegend), anti-CD80 (#553769, BD biosciences), anti Phospho-Syk

(Tyr348) APC, (17-9014-42e, Bioscience), 7-AAD BD (#559925, Pharmingen)

All stains were carried out in DPBS (Sigma) supplemented with 1% FCS for 30 min at 4°C,

followed by two washing steps. Finally, to identify dead cells staining with 7-AAD viability

solution (eBiosciences) was performed.

Data were acquired on a LSRII Fortessa (BD Biosciences, Billerica, MA, USA) and were

analyzed using FlowJo software 10 (Tree Star, Ashland, OR, USA).

Histology

Male and female mice 8-18 weeks of age were sacrificed and organs isolated, fixed in 4% PFA

in PBS and embedded in paraffin. Sections were cut at 5 μm thickness, dewaxed and stained

either with hematoxylin and eosin or with Heidenhains azan trichrome (H&E; Merck). Electron

microscopy and kidney histology was performed as previously described(63).

Anti-dsDNA antibody and cytokine measurements

ELISA used to determine anti-dsDNA, the plates were coated with 5 µg/ml double-stranded calf

thymus genomic DNA (Sigma-Aldrich) for 2 h at room temperature. For detection, 1 µg/ml goat

anti–mouse IgM-HRPO (SouthernBiotech) or goat anti–mouse IgG-HRPO (Thermo Fisher

Scientific) and as a substrate TMB (Enzo Life Sciences) were used.

Transwell migration assay

50,000 PBMCs of patients, parents and healthy donors were plated in 50 µl of serum free RPMI

(plus 0.5% BSA) in the upper chamber of the 96 transwell plates (Corning CLS3388).

Chemokine (CXCL12 and CCL19, Bio-Techne) in 150 µl of the mentioned medium was added

at the following concentrations to the lower chamber, 50, 100, 200 and 500 ng/ml. 50,000 cells

of each individual were added to the lower chamber as the control for calculating the percentages

later on. The assay was performed in duplicates. The incubation time for migration was 4 hours.

After the incubation time the transwell insert was removed and migrated cells to the lower

chamber were stained with anti-CD3-APCH7, anti-CD4-PECy7, anti-CD8-V450, anti-CD19-

PerCPCy5.5 antibodies and assessed by FACS Fortessa.

T cell subsets

T-cell cytokine production was analyzed in 0.5–1x106 total PBMCs that were stimulated for 5 h

with Phorbol 12-myristate 13-acetate (PMA, 0.2 mM) and Ionomycin (1 ng/mL) with the

addition of Brefeldin A during the final 2.5 h. To identify T-cell subpopulations, surface staining

of chemokine receptors was performed by incubating the cells with fluorochrome-conjugated

antibodies for 15 min in PBS supplemented with 2% human serum at 37°C. Subsequently, the

cells were fixed, permeabilized and stained for intracellular cytokines or transcription factors,

followed by flow cytometry analysis. The following antibodies were used in a total volume of

20µl: 5µl Tbet- PerCPCy5.5 (4B10) and 1.2µl CCR6-BV605 (G034E3) from Biolegend, 0.8µl

CCR7-PE-CF594 (150503), 0.5µl CD3-APC-H7 (SK7), 0.5µl CD45RA-AF700 (HI100), 3µl

RORgt-AF647 (Q21-559), 1µl CD8-V500 (RPA-T8), 0.8µl CD4-BV421 (RPA-T4), 1.2µl

XCXR3-BV711 (1C6), 1.2 µl CD194-PECy7 (1G1), 0.6µl IL4-APC (MP4-25D2) and 2µl Cd25-

PE (M-A251) all from BD Pharmingen, 5µl GATA3-AF488 (TWAJ), 0.3µl IFNy-FITC (4S.B3)

and 2µl IL17A-eFluor450 (eBio64DEC17) from eBiosciences and 1µl CD4-PECy7

(SFCI12T4D11) from Beckman Coulter.

Luminex assay

Assessment of serum cytokines was performed using The ProcartaPlexTM Multiplex

Immunoassay from Invitrogen according to the manufactures instructions using undiluted serum.

The following cytokines were assessed: BAFF, IL-2, IL-4, soluble L-Selectin, IFN gamma, TNF

alpha, soluble VCAM-1, soluble ICAM-1, SDF-1 alpha, IL-7, IL-1 beta, IL-6, IL-8, IL-10, IL-

18, IL-23, TGF beta, IL-17A, IL-22, IL-12p70, CXCL13 (BCL), CXCL-10 (IP10), LIGHT.

Probably due to low serum levels, BAFF, IL-4, IFN gamma, IL-6, IL-8, IL-23, TGF beta, IL-

17A, IL-22 and LIGHT were below the detection limit, as can happen according to the

manufactures technical support.

BAFF stimulation

PBMCs from both patients and two control donors were incubated with 50ng/ml recombinant

BAFF (R&D Systems) at 37°C on a shaker for the following time periods: 0, 5 minutes and 15

minutes. After that the cells were washed and stained for FACS analysis with 1µl BAFFR-BV60

(11C1, BD OptiBuild™), 1µl CD19-BV510, 3µl IgD-FITC, 2µl CD27-PE, 2µl CD3-APC (SK7,

eBioscience), 1µl CD4-BV421 (RPA-T4, BD Pharmingen) and 1µl CD8-PECy7. 400 000 cells

were used per reaction.

CTLA-4 cycling assay

To stain for CTLA-4 cycling through T-cell surfaces, Jurkat wildtype or sgRNA HEM1-

knockdown cells were incubated at 37°C in the presence of anti-CTLA-4 BV421 antibody

(Biolegend, BNI3), for the indicated time periods. Cells were immediately placed on ice, fixed

with IC fixation buffer (Invitrogen) and analyzed by flow cytometry. OKT3-stimulated or

unstimulated cells were compared.

Production of a monovalent streptavidin imaging platform equipped with a STAR635P-

conjugated IgM-reactive monoclonal antibody

Monovalent streptavidin (mSav) was prepared with some adaptions as described (77–79). The

pET21a(+) expression vectors encoding either for the “dead” (i.e. biotin non-binding) or the

“alive” (i.e. biotin binding) streptavidin subunit were a kind gift from Alice Ting (Stanford

University, USA). We substituted the hexa-histidine tag on the “alive” streptavidin subunit with

a hexa-glutamate tag to allow for purification via cation exchange chromatography. The

sequence of the “dead” streptavidin subunit was C-terminally extended with a hexa-histidine tag

for attachment to lipid bilayers containing 18:1 DGS-NTA(Ni) (79).

Both, dead and alive streptavidin subunits were expressed in Escherichia coli (BL-21) for 4 h at

37°C and refolded from protein inclusion bodies as described (77–79). After refolding in vitro

[described in (Howarth and Ting, 2008)], the streptavidin tetramer mixture (containing D4,

A1D3, A2D2, A3D1 and A4) was concentrated in a ultrafiltration cell (10 kDa cut off, Merck).

After buffer exchange to 10 mM Tris-HCl, pH 8.0 with Amicon Ultra-4 centrifugal filters (10

kDa cut off, Merck), the mixture of tetramers was purified by anion exchange chromatography

(MonoQ 5/50 GE Healthcare Life Sciences) using a column gradient from 0.1 to 0.4 M NaCl

over 90 ml. mSav was eluted with 0.22 M NaCl, concentrated (Amicon Ultra-4 centrifugal

filters, 10 kDa cut off) and further purified via gel filtration (Superdex 200 10/300 GE

Healthcare Life Sciences). The protein was again concentrated to ~1 mg ml-1 and stored in 1x

PBS supplemented with 50 % glycerol at -20 °C.

The IgM (BCR) reactive monoclonal antibody MHM-88-biotin (BioLegend) was conjugated

with Abberior STAR 635P NHS ester (Abberior) according to the manufacturer´s instructions.

The STAR635P-conjugated mAb MHM-88-biotin was purified from an excess of STAR635P

via gel filtration (Superdex-200 10/300; GE Healthcare Life Sciences), concentrated using

Amicon Ultra-4 centrifugal filters (10 kDa cut-off, Merck) and stored in 1x PBS supplemented

with 50 % glycerol at -20°C. Protein to dye ratio of the STAR635PP-conjugated MHM-88-biotin

mAb was 1.8 as determined by spectrophotometry at 280 nm and the corresponding absorption

maximum of the dye (638 nm).

To generate a 1:1 conjugate of mSav and STAR635P-MHM-88-biotin, we incubated both mSAv

with a 5-fold molar excess of STAR635P-MHM-88-biotin for 30 minutes at room temperature in

PBS. While the resulting mSav:STAR635P-MHM88-biotin complex can bind to DGS-NTA(Ni)

on SLBs, the excess STAR635P-MHM88-biotin cannot. Human AF488-conjugated ICAM-1-

12xHis was produced as described (80).

Preparation of functionalized glass-supported lipid bilayers (SLBs)

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-dioleoyl-sn-glycero-3-[N(5-

amino-1-carboxypentyl)iminodiacetic acid] succinyl (nickelsalt) [DGS-NTA(Ni)], and 1,2-

dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000]

(ammonium salt) (both from Avanti Polar Lipids), were pre-mixed in a 97.5:2:0.5 molar ratio in

chloroform (Merck), vacuum-dried overnight in a desiccator, re-suspended in 10 ml PBS and

sonicated under nitrogen at 120–170 W in a water bath sonicator (Q700, QSonica). Vesicles

were centrifuged for 4 h at 37,000 rpm (25°C) and for 8 hours at 43,000 rpm (4°C) with an

ultracentrifuge (Sorvall RC M150GX, Thermo Fisher Scientific) and a fixed angle rotor

(S150AT-0128, Thermo Fisher Scientific) to pellet non-unilamellar lipid vesicles. Glass slides

(24 x 50 mm or 22 x 64 mm, #1.5, VWR) were surface-treated with plasma (ZEPTO, electronic

diener) for 10 minutes and glued with picodent twinsil extrahart (Picodent) to the bottom of 8- or

16-well LabTek chambers (Nunc). Slides were exposed to a tenfold-diluted lipid vesicle

suspension (in PBS) for 15 minutes at room temperature and rinsed with 15 ml PBS (room

temperature). Subsequently, His-tagged proteins were incubated for 75 minutes at room

temperature and in the dark. Before imaging, functionalized SLBs were rinsed with 15 ml PBS to

remove unbound proteins. PBS was exchanged for imaging buffer [HBSS (Life technologies)

supplemented with 0.4 mg ml-1 ovalbumin (Merck) as well as 2 mM CaCl2 and 2 mM MgCl2

(Merck)] shortly before adding the cells to the imaging chamber.

Fig. S1. Defective WRC complex assembly and stability. (A) Image of skin lesions on the forehead of P1. (B)

Capillary sequencing confirmation of the missense mutation in NCKAP1L encoding HEM1 (c.C385T,

p.Arg129Trp). (C) Amino acid sequence conservation of HEM1/HEM2 among mice and humans. (D, E) Cropped

immunoblot analysis of HEM1, WAVE2, ABI1, GAPDH or HSP90 in feeder-expanded T cells and EBV-LCL

transformed B cells of healthy donor (HD), patients (P1, P2), their parents. Expression of WRC members in patient

and control cells. (F) Parental B16-F1 control cells were transfected with either EGFP alone or EGFP-tagged Hem1-

R129W or EGFP-Hem2-R131W, as indicated. Phalloidin staining on the left confirms that all cells harbor regular

lamellipodia presumably driven by endogenous WRC. Note that neither EGFP alone (negative control) nor Hem1-

R129W are capable of localizing to the tips of these lamellipodia formed, unlike Hem2-K131W, which targets to

these structures as expected for functional WRC (arrow in magnified inset on the right). These data suggest that

Hem1-R129W is incapable of incorporating into functional WRCs even in the presence of lamellipodia, whereas

Hem2-K131W does not appear to be functionally compromised. Scale bar is 10 µm in overview images and 3 µm in

insets (bottom, right). (G) Representative examples of Hem1/Hem2 knock-out clone #7 expressing various EGFP-

tagged constructs as indicated in the figure. Cells were counterstained for their actin cytoskeleton using phalloidin.

Scale bar is 20μm. Bar chart depicts the fraction of cells with or without lamellipodia or ruffles, respectively. N

gives the number of cells analyzed. Images on the bottom right show examples of phenotypes representing the

categorization used for quantification (right, top). Scale bar is 10μm. (H) Representative images (top) and

quantification (bottom) of expanded T cells spreading on fibronectin-coated surfaces stained with phalloidin and

DAPI and imaged with a Zeiss LSM700 confocal microscope and a 63x oil-immersion objective. Scale bar, 10 µm.

(I) F-actin intensities in T cells derived from Patients (P1, P2), parents and healthy donors (HD) following adherence

on fibronectin-coated slides.(J) Principal component analysis of cell morphology parameters extracted from

immunofluorescence images using CellProfilerTM. Patient (blue contour) and healthy donor (red contour) T cells can

be distinguished on the basis of these parameters (list of parameters in table S3).

CD3+CD4+CD4+ CMCD4+ EM

CD4+ Naive

CD4+ TEMRA

CD27+CD28+ CMEM1

EM2EM3

EM4

CD27+CD28+ Naive

CD4+ TEMRA E

CD4+ TEMRA pE1

CD4+ TEMRA pE20

50

100

CD

4+

CD8+CD8+ CMCD8+ EM

CD8+ Naive

CD8+ TEMRA

CD27+CD28+ CMEM1

EM2EM3

EM4

CD27+CD28+ NaiveCD8+ TEMRA E

CD8+ TEMRA pE1

CD8+ TEMRA pE20

50

100C

D8+

No chemokine

CXCL12 50ng/ml

CXCL12 100ng/ml

CXCL12 200ng/ml

CXCL12 500ng/ml0 50

100

TCD

4+ CXCL12

% control

No chemokine

CXCL12 50ng/ml

CXCL12 100ng/ml

CXCL12 200ng/ml

CXCL12 500ng/ml0 50

100

TCD

8+ CXCL12P

1

P2

Father

HD

1

AB

% control

0

5000

10000

15000

20000

wt

KO

2.9K

O2.15

KO

6.2K

O6.5

MFI LFA1

secondary alonecd3 (1)cd3 (5)

cd3 (10)unstim

ulated

C

0 50

100% Tcells

FAS

mediated apoptosis expanded T cells

D

ND_Ctrl

ND_FAS_low

ND_FAS_highP2_Ctrl

P2_FAS_low

P2_FAS_highP1_Ctrl

P1_FAS_low

P1_FAS_high

Father_Ctrl

Father_FAS_low

Father_FAS_high

Mother_Ctrl

Mother_FAS_low

Mother_FAS_high aliveearly apoptosis

late apoptosis

unstim

unstim10stim10

unstim30stim30

0 10 20 30 40

% cycling clells

wt

ko2-15

ko2-9

ko6-2

ko6-5

CTLA

-4 cycling sgHE

M1-Jurkat T cells

E

F

Nomalized to Mode

CD

95 PE-Cy7

0-10 3

10 310 4

10 5

gated on CD

3+TCR

αβ+CD

4-CD

8- Tcells

HD

1

HD

2

P1 P2

Shp_Ctrl

Ggated on C

D19+

0-10 3

10 310 4

10 5

Nomalized to Mode

10 110 2

10 310 4

10 5

gated on CD

3+CD

4+CX

CR

5+

Nomalized to Mode

HD

1

HD

2

P1

P2

Shp_C

trl

HD

1

HD

2

P1

P2

Shp_C

trl

PD

1-AP

CP

DL1-A

PC

H

I

TFH4.57

0-10 3

10 310 4

10 5

0

-10 3

10 3

10 4

HD

1

CXCR5-APC

CD

45RA

-AF700

TFH7.34

0-10 3

10 310 4

10 5

0

-10 3

10 3

10 4

HD

2

CD

45RA

-AF700

CXCR5-APC

TFH11.3

0-10 3

10 310 4

10 5

0

-10 3

10 3

10 4

HD

3CD

45RA

-AF700

CXCR5-APC

TFH3.32

0-10 3

10 310 4

10 5

0

-10 3

10 3

10 4

P1C

D45R

A-A

F700

CXCR5-APC

TFH1.96

0-10 3

10 310 4

10 5

0

-10 3

10 3

10 4

Father

CD

45RA

-AF700

CXCR5-APC

TFH7.83

0-10 3

10 310 4

10 5

0

-10 3

10 3

10 4

P2C

D45R

A-A

F700CXCR5-APC

Gated on C

D3+C

D4+:

Patients

Age-m

atched HD

Supplementary Figure 2


0-10 3

10 310 4

10 50

-10 310 3

10 410 5

0-10 3

10 310 4

10 50

-10 310 3

10 410 5

CD

4

P2age-matched

HD

CD

8C

D3+C

D28 stim

ulation

norm. to mode

Violet proliferation dye (VP

D450)

day 0day 4

HDsHem1HDsHem1HDsHem1

0 10 20 30

%positive


0 10 20 30


0 20 40 60


0 10 20 30

%positive

%positive

%positive

Th2-enrichedTh1-enriched

Th17-enrichedN

aive

Gata3

RO

Rγt

TbetG

ata3R

ORγt

TbetG

ata3R

ORγt

TbetG

ata3R

ORγt

Tbet

Percent

Percent

Fig. S2. T cell phenotype and function in human HEM1 deficiency. (A) Transwell migration of CD4+ and CD8+

T cells from P1, P2, father and a healthy control, 50 to 500 ng/ml of CXCL12 (as indicated on the graphs) were used

as chemoattractant. The percentage of migrating cells was calculated relative to a control (for further details see

methods section). (B) Active LFA1 intensity following stimulation with anti-CD3 (OKT3) with different

concentrations (indicated in brackets) in WT and HEM1-/- Jurkat cells, assessed by flow cytometry. (C)

Quantification plots of CD4+ and CD8+ T-cell subpopulations in P1, P2 and 4 different age-matched healthy

controls. Patients values are shown in black and age-matched healthy controls are shown in pink (left). (D) Flow

cytometry plots of T follicular helper cells (TfH, CD3+CD4+CXCR5+CD45RA-) for P1, P2, father and 3 healthy

donors. (E) Plots representing percentages of Th1, Th2 and Th17-enriched T-cell subsets by 3 major lineage specific

transcription factors (Tbet for Th1, Gata3 for Th2 and RORγt for Th17) in patients and two age-matched healthy

donors. (F) FAS mediated apoptosis in feeder-expanded T cells of P1, P2, healthy control and parents (left) and FAS

expression on the CD3+TCRαβ+, CD4- and CD8-double negative T cells in both patients, shipment control and 2

healthy donors (right). (G) Expression of PD-1 (left) on the surface of TfH cells and PDL1 (right) on the surface of

CD19+ cells of patients, shipment control and healthy controls. (H) CTLA4 cycling assay in CRISPR/Cas9 HEM1

knock-out Jurkat T cells. (I) patient-derived T cell proliferation at day 0 and 4 days after stimulation with CD3 and

CD28 stained with violetproliferation dye and gated for CD3+/CD4+ or CD3+CD8+ T cells.

Fig. S3. Extended B cell immunophenotyping in human HEM1 deficiency. (A) Gating strategy for B-cell

immunophenotyping. (B) Flow cytometry plots of B-cell subpopulations: naïve (IgD+CD27–), memory non-

switched (IgD+CD27+) and memory-switched (IgD–CD27+), (IgD–CD27–) double-negative B cells. Further,

transitional (CD19+CD24+CD38+) and innate-like (CD19+CD27–IgD+CD21lowCD38low) B cells are depicted for

P1, P2, age-matched and adult healthy controls according to the gating strategy mentioned in section A.

Fig. S4. Detailed characterization of Hem1–/–

mice. (A) Hem1-/- mouse generation by Cre/lox recombination

leading to deletion of exons four and five according to (25). (B) Expression of WAVE complex members in

hematopoietic cells from Hem1+/+ and Hem1-/- mouse. (C) Body weight plots of from Hem1+/+, Hem1+/- and Hem1-/-

mouse. (D) Neutrophil, monocyte, dendritic cell, macrophage counts in Hem1+/+, Hem1-/-, Hem1-/- spleen. (E)

Number of Ly6Chi and Ly6Clo expressing cells as well as neutrophils in peripheral blood. (F) Immunohistochemistry

staining of lung, liver and lymph node sections displaying granulomatous lesions. (G) LSK and progenitor counts in

Hem1+/+, Hem1-/-, Hem1-/- spleen. (H) T-cell counts of total, CD4+ and CD8+ T cells in the spleen. (I) Absolute

numbers of B220+ B cells and ratio of mature (CD21+CD23+) and immature (CD21low CD23-) B cells of wildtype

(Hem1+/+), heterozygous (Hem1+/-) or knockout (Hem1-/-) mice in bone marrow; representative FACS plots (left),

corresponding quantifications (right). (J) Flow cytometry plots of gated B220+ B cells (left), and frequency as well

as absolute numbers of B220+ B cells in the spleen of wildtype (Hem1+/+), heterozygous (Hem1+/-) or knockout

(Hem1-/-) mice (right). (K) Immature (CD19+IgD-CD93-), mature and transitional B cells (CD19+IgD+) assessed

using a previously described gating strategy40. One out of two representative experiments shown; every dot

corresponds to one mouse.

Fig. S5. Single-cell RNA-seq analyses of splenic T and B cell subclusters. Characterization of T- and B-cell sub-

clusters (A) Leiden clustering including sub-clustering of T and B-cells of pooled Hem1+/+ and Hem1-/- spleen cells

identifies further subsets. (B) Expression of genes identified through differential expression analysis for high density

T-cell sub-clusters are shown for Hem1+/+ and Hem1-/-. (C) Entropy distribution of T/B cells in 5-week vs. 10-week-

old mice depicting significant differences and directionality in Hem1-/- compared to Hem1+/+ mice.

A0.000989.5e−05 1.5e−060.72

1.6

2.0

2.4

5w 10wKO T Cells

Entro

py Cluster01

C

5w 10wWT T Cells

5w 10w 5w 10wKO B Cells WT B Cells

Top genes Cluster 0a

Top genes Cluster 0b

Top genes Cluster 0c

Top genes Cluster 0d

cell_sub_ontolog _class

UMAP1

UM

AP2

100%80%60%40%20%

80%60%40%20%

100%80%60%40%20%

100%80%60%40%20%

40

20

0.5

1.0

1.5

10

20

15

10

5

stat

us KOWT

stat

us KOWT

stat

us KOWT

stat

us KOWT

B


0:00 0:30 1:00 1:30 2:00 2:30 min

HD

P2

CC

L19

CC

L19

Lamellipodia Extended lamella Blebs Spikes

0

20

40

60

0

40

60

% T

ime

% T

ime

HD1 HD2 P1 P2

HD1 HD2 P1 P2

20

% T

ime

HD1 HD2 P1 P2

Extended lamella

0

20

40

60Blebs

0

20

40

60Spikes

0

20

40

60

% T

ime

% T

ime

HD1 HD2 P1 P2HD1 HD2 P1 P2

A

D

TL IRMmSav:biotin-MHM88-abSTAR635P

HD10 min

400 25000grey value

P210 min

0 5 10 15 20 25 30 350

20

40

60HD2

P2

C

0 10 20 300

2000

4000

6000

0 10 20 300

2000

4000

6000

HD3

0 5 10 15 20 25 30 350

20

40

60

HD3

WASP WASP

HD2

P2

IKZF4

IKZF1 PAX5

IRF8

CD79A

−0.2

−0.1

0.0

0.1

0.2

0.3

CCNT1

PPP1R1AGSS

ZNF385AIKZF4

OR6C75

ACSL4IKZF1

SPI1

CELA1

KRT86

SDR9C7AVIL

ALAD

KIF5A

PRPF40B

LACRT

HOXC10

PDE1B

C1QL4NAB2

PAX5

ARHGAP45NPFF

VDR

MBNL1

TMCC3

METTL7B

ASIC1DCD

GTSF1

ALAS1IR

F8GCLC

MMP19CBFB

AQP6

OR6C3

ESYT1

CD79A

TARBP2

TNS2TBK1

SLC39

A5

CALCOCO1

KRT83DHH

SHMT2

GRASP

ANKRD33

PRICKLE

1

ORMDL2OS9

LMBR1L

ITGA7IL2

2

SLC4A

8EBF1

POU6F1

FCHO1BIN

2

R3HDM2

BAZ2A

ZNF641

PRKAG1

INHBE

CD63ARF3

NEMP1

DNAJC22RDH5

NR4A1

NXPH4

CCND3

OR10AD1

OR10P1CSAD

KRT77RAC2

H2AC6AQP5

SMARCC2

CCDC65AACS

ADIPOR2

RUFY3

TUBA1A

NDUFA4L

2

KCNH3

CXCL2TBCK

GLI1

RDH16

ADCY6ITSN2

FASN

PIK3CA

ZNF814

DESI2

ACTL7A

Gene

Cor

rela

tion

Top 100 correlates per dataset for NCKAP1L in CRISPR DepMap 19Q4 B

Area

syn

apse

STA

R63

5 (µ

m2 )

Inte

grat

ed d

ensi

ty S

TAR

635P

(AU

)

Area

syn

apse

STA

R63

5 (µ

m2 )

Inte

grat

ed d

ensi

ty S

TAR

635P

(AU

)

Time (minutes) Time (minutes)

Time (minutes) Time (minutes)

All B cell clusters

1a clusters

1b clusters

stat

us KOWT

stat

us KOWT

stat

us KOWT

0.2

0.2

0.1

0.1

0.1

0.0

20%

15%

10%

5%

20%

15%

10%

5%

20%25%30%

15%10%5%

Pseudopodia

Pseudopodia

Lamellipodia

E

D F


Fig. S6. Extended characterization of B cells and comparison with WASP-deficient B cells. (A) Depmap

analysis of the gene expression profiles of Hem1-/- cell lines. (B) Healthy donor (top) and patient (bottom) B cells

following IgM stimulation using a planar-supported lipid bilayer functionalized with a monovalent streptavidin

platform presenting biotinylated and abSTAR635P-conjugated MHM-88 mAbs, white light (left), IRM (middle) and

IgM (right). Scale bar: 5µm (C) EBV-LCL cells were exposed to a 0-250 ng/ml CCL19 gradient and imaged by

time-lapse recording. Representative frames of one video out of 3 performed for each of the 2 healthy donors and

the 2 PID patients. Positions of individual cells are marked by color dots on 30 sec frame intervals. The morphology

of all isolated cells was evaluated on every frame and assigned to 5 morphotypes dominated by typical protrusions

(lamellipodia, pseudopodia, extended lamella, blebs, spikes). The graphs represent the percentage of time during

which the cells displayed the indicated morphologies. The values represent mean ± SEM of 3 videos per cell line. 46

healthy donor cells and 47 HEM1-mutant patient cells were analyzed over 144 frames with a time interval of 5 sec,

and respective quantification. (D) Area of IgM at the synapse (Area STAR635P)/µm2 over time in P2 and HD2

sorted B cells on planar-supported lipid bilayer, illustrates accumulation of IgM at the immune synapse (left),

Integrated density of IgM (AU) over time illustrates intensity of IgM at the immune synapse (right) (HD2-dark blue,

patient 2-red). (E) As in D for HD3 (green) and WASP-deficient patient. (F) Representative graphs for Foxo1 target

genes regulating cell cycle progression (Rbl2, Cdk1, Cdkn1b, Myc), DNA damage (Ddb1) and apoptosis (Bcl6,

Bcl2l11, Batf) in all B-cell subclusters (top), subcluster 1a (middle) and subcluster 1b (bottom).

Table S2. List of discriminating features for PCA of healthy donor and HEM1-deficient T cells

upon fibronectin stimulation. Featurename CohensD

AreaShape_MeanRadius 0.440732

AreaShape_MinorAxisLength 0.713254

AreaShape_MaxFeretDiameter 0.580766

Granularity_11_Tubulin 0.236738


Granularity_1_Phalloidin 0.248792

Granularity_2_Phalloidin 0.092951




Intensity_IntegratedIntensityEdge_Tubulin 0.539787

Intensity_LowerQuartileIntensity_Tubulin 0.322067

Intensity_MassDisplacement_DAPI 0.226098

Intensity_MaxIntensity_DAPI 0.267065

Intensity_MaxIntensity_Phalloidin 0.420668

Intensity_MaxIntensity_Tubulin 0.646242

Intensity_MeanIntensityEdge_Phalloidin 0.218751

Intensity_MinIntensityEdge_Phalloidin 0.018598

Intensity_StdIntensity_DAPI 0.133218

Neighbors_SecondClosestDistance_Adjacent 0.074912

Neighbors_SecondClosestObjectNumber_Adjacent 0.144991

RadialDistribution_MeanFrac_Phalloidin_1of8 0.000698



RadialDistribution_RadialCV_Phalloidin_1of8 0.413741





RadialDistribution_RadialCV_Tubulin_3of8 0.03634





RadialDistribution_ZernikeMagnitude_Phalloidin_1_1 0.431538


















RadialDistribution_ZernikeMagnitude_Tubulin_3_1 0.206701











Texture_Correlation_Phalloidin_3_01 0.099257

Texture_Correlation_Phalloidin_3_03 0.090446

Texture_Correlation_Tubulin_3_01 0.321256

Texture_Correlation_Tubulin_3_03 0.270227

Texture_Entropy_Phalloidin_3_01 0.616071

Texture_InfoMeas1_Phalloidin_3_01 0.183153

Texture_InfoMeas1_Tubulin_3_03 0.104116

Table S2. (Continued) FillopodiaLength 0.397042

FillopodiaNumber 0.000305

FillopodiaBranches 0.087134

FillopodiaLength_HighRes 0.351666

FillopodiaNumber_HighRes 0.442775

FillopodiaBranches_HighRes 0.319007

AreaShape_MaxFeretDiameter 0.417268

Granularity_2_DAPI 0.239459




Intensity_LowerQuartileIntensity_DAPI 0.001289

Intensity_LowerQuartileIntensity_Tubulin 0.10074

Intensity_MassDisplacement_Phalloidin 0.08559

Intensity_MaxIntensityEdge_Phalloidin 0.490208

Intensity_MaxIntensity_DAPI 0.267463

Intensity_MeanIntensity_Phalloidin 0.348926

Intensity_MinIntensityEdge_Phalloidin 0.282791

Intensity_StdIntensityEdge_DAPI 0.129291

Intensity_StdIntensity_DAPI 0.255537

Intensity_StdIntensity_Tubulin 0.486011

Number_Object_Number 0.122991

ObjectSkeleton_NumberNonTrunkBranches_Skeletonized_Cells 0.460189

ObjectSkeleton_NumberTrunks_RemoveHoles 0.439762

ObjectSkeleton_NumberTrunks_Skeletonized_Cells 0.415254

ObjectSkeleton_TotalObjectSkeletonLength_RemoveHoles 0.35692

Texture_Correlation_DAPI_3_00 0.245044



Texture_DifferenceVariance_DAPI_3_03 0.310876

Texture_InfoMeas1_DAPI_3_03 0.318958

Texture_InfoMeas2_DAPI_3_01 0.125286

Texture_InverseDifferenceMoment_DAPI_3_02 0.204222

AreaShape_MeanRadius 0.307049

Intensity_IntegratedIntensityEdge_Phalloidin 0.505984

Intensity_IntegratedIntensityEdge_Tubulin 0.657101

Intensity_LowerQuartileIntensity_Phalloidin 0.423541

Intensity_MADIntensity_Phalloidin 0.467002

Intensity_MedianIntensity_Tubulin 0.600504

Number_Object_Number 0.181822

Table S3. T cell immunophenotyping in HEM1-deficient individuals compared with age-matched

reference values.

POPULATION PATIENT 1

(5Y)

PATIENT 2

(7Y)

FATHER

(40Y)

2-5 Y*

REFERENC

E

5-10 Y*

>16 Y*

LYMPHOCYTES (ALC) 3.17 ↑5.6 3.19 (1.4-5.5) (1.2-4.7) (1.2-4.1)

T LYMPHOCYTES CD3+ 2.00661 3.6288 2.07669 (0.85-4.3) (0.77-4.0) (0.78-3.0)

HELPER T CELLS CD3+CD4+ 0.85273 1.092 1.50249 (0.5-2.7) (0.4-2.5) (0.5-2.0)

NAÏVE CD3+CD4+CD45RA+CD27+

↓0.1125603 ↓0.13322 0.28246812 (0.3-2.3) (0.2-2.5) (0.1-2.3)

EFFECTOR MEMORY CD3+CD4+CD45RA-CD27-

↑0.1176767 0.140868 0.21335358 (0.0035-0.089)

(0.003-0.17) (0.013-0.22)

CENTRAL MEMORY CD3+CD4+CD45RA-CD27+

0.61908198 ↑0.81354 1.00366332 (0.16-0.66) (0.0037-0.51) (0.18-1.1)

TEMRA CD3+CD4+CD45RA+CD27-

↑0.17819838

↑0.7752864 0.020726068

(0.000061-0.016)

(0.000025-0.025)

(0.000098-0.068)

CYTOTOXIC T CELLS CD3+CD8+

0.51354 1.3944 0.140041 (0.2-1.8) (0.2-1.7) (0.2-1.2)

NAÏVE CD3+CD8+CD45RA+CD27+

0.16535988 ↓0.3248952 0.062878409

(0.053-1.1) (0.042-1.3) (0.016-1.0)

EFFECTOR MEMORY CD3+CD8+CD45RA-CD27-

0.06881436 0.12145224 0.010208989

(0.024-0.59) (0.045-0.41) (0.04-0.64)

CENTRAL MEMORY CD3+CD8+CD45RA-CD27+

↑0.10116738

↑0.1715112 0.046073489

(0.0043-0.064)

(0.0061-0.043)

(0.0047-0.12)

TEMRA CD3+CD8+CD45RA+CD27-

↓0.00184874

↓0.0055776 0.000266078

(0.025-0.53) (0.057-0.34) (0.025-0.28)

CD3+TCRAB+ 1.63119324 3.213168 1.90271378 (0.6-4.3) (0.6-3-7) (0.7-2.8)

CD3+TCRAB+CD4+CD8- 1.016233389

1.417007088

1.729566826

(0.4-2.8) (0.36-2.8) (0.3-2.4)

CD3+TCRAB+CD4-CD8+ 0.487726779

↑1.62264984

0.139088377

(0.17-1.7) (0.21-1.5) (0.12-1.7)

CD3+TCRAB+CD4+CD8+ (DPT)

0.02887212 0.082257101

0.007040041

(0.0033-0.043)

(0.0036-0.018)

(0.0023-0.06)

CD3+TCRAB+CD4-CD8- (DNT) 0.098524072

0.091896605

0.02758935 (0.016-0.14) (0.01-0.1) (0.0069-0.074)

CD3+TCRGD+ 0.20241084 0.143052 0.067788138

(0.027–0.96) (0.027–0.96) (0.025–0.2)

CD3+TCRGD+CD4+CD8- 0.007408237

0.001244552

0.002040423

(0.001–0.021) (0.00051–0.015)

(0.00012–0.0076)

CD3+TCRGD+CD4-CD8+ 0.095537916

0.065374764

0.016675882

(0.0019–0.2) (0.0051–0.069)

(0.0018–0.094)

CD3+TCRGD+CD4+CD8+ 0.000145736

↓0 3.32162E-05

(0.000038–0.0045)

(0.000034–0.0011)

(0.000035–0.0011)

CD3+TCRGD+CD4-CD8- 0.099383722

0.076389768

0.049010824

(0.021–0.85) (0.033–0.54) (0.019–0.17)

RECENT THYMIC EMIGRANTS ↓0.07069131

↓0.0721812 0.16377141 (0.19-2.6) (0.2-1.7) (0.05–2.4)

INK-T (CD3+CD16+/-CD56+) 0.174033 0.31696 0.042108 (0.015-0.25) (0.012-0.34) (0.023–0.41)

REGULATORY T CELLS CD3+ CD4+CD25++CD127

↓0.01230262

0.022878553

n.d. (0.039–0.15) (0.02–0.27) (0.025–0.18)

B LYMPHOCYTES (CD19+)

↑0.3487 ↑0.9688 0.33814 (0.18-1.3) (0.10-0.80) (0.064-0.82)

NK CELLS (CD3-CD16+/-CD56+) 0.234897 0.11872 0.110055 (0.061-0.51) (0.070-0.59) (0.10-1.2)

Table legend: T lymphocytes (CD3+). B lymphocytes (CD19+). natural killer (NK) cells (CD3)CD16+ and ⁄ or CD56+). helper T lymphocytes (CD3+CD4+). cytotoxic T lymphocytes (CD3+CD8+). NK-T cells (CD3+ CD16+and ⁄ orCD56+). naive helper T lymphocytes (CD3+CD4+CD45RA+CD27+). terminally differentiated helper T lymphocytes (CD3+CD4+CD45 RA+CD27-). central memory helper T lymphocytes (CD3+CD4+CD45RA-CD27+). effector memory helper T lymphocytes (CD3+CD4+CD45RA-CD27-). naive cytotoxic T lymphocytes (CD8+CD45RA+CD197+CD27+). terminally differentiated cytotoxic T lymphocytes (CD8+CD45RA+CD197-CD27-). central memory cytotoxic T lymphocytes (CD8+CD45RA+CD197+CD27+). effector memory cytotoxic T lymphocytes (CD8+CD45 RA+CD197-CD27-). recent thymic emigrants (CD3+ CD4+CD45RA+CD31+) and regulatory T cells (CD3+ CD4+CD25++CD127-). We also analysed the expression of T cell receptor (TCR)-ab and TCR-cd on the CD4+ and on the CD8+ T lymphocytes. as well as the expression of CD185 (=CXCR5) on CD3+CD4+CD45RO+ T lymphocytes. * reference values taken from Schartoje et al. 2012 (29). red above age-matched reference. blue below age-matched reference.

Table S4. List of discriminating features for PCA of healthy donor and HEM1-deficient B cells

upon IgM stimulation.

Feature

AreaShape_Area

AreaShape_Compactness

AreaShape_Eccentricity

AreaShape_Extent

AreaShape_FormFactor

AreaShape_MajorAxisLength

AreaShape_MaxFeretDiameter

AreaShape_MaximumRadius

AreaShape_MeanRadius

AreaShape_MedianRadius

AreaShape_MinFeretDiameter

AreaShape_MinorAxisLength

AreaShape_Orientation

AreaShape_Perimeter

AreaShape_Solidity

Intensity_IntegratedIntensityEdge_Phaloidin

Intensity_IntegratedIntensity_Phaloidin

Intensity_LowerQuartileIntensity_Phaloidin

Intensity_MADIntensity_Phaloidin

Intensity_MassDisplacement_Phaloidin

Intensity_MaxIntensityEdge_Phaloidin

Intensity_MaxIntensity_Phaloidin

Intensity_MeanIntensityEdge_Phaloidin

Intensity_MeanIntensity_Phaloidin

Intensity_MedianIntensity_Phaloidin

Intensity_MinIntensityEdge_Phaloidin

Intensity_MinIntensity_Phaloidin

Intensity_StdIntensityEdge_Phaloidin

Intensity_StdIntensity_Phaloidin

Intensity_UpperQuartileIntensity_Phaloidin

Table S5. B cell immunophenotyping in HEM1-deficient individuals compared with age-matched

reference values.

HD1 HD2 P1 P2 Age-matched HD 2-5Y 5-10Y >16Y

IgD+CD27- Naïve B 37.6 41.8 63.6 66.5 75.1 75.3 (54.0–88.4) 65.7 (47.3–77.0) 65.5 (48.4-79.7)

IgD+CD27+ Non-switched Memory B 4.78 1.65 5.16 4.43 2.54 10.0 (2.7–19.8) 11.0 (5.2–20.4) 11.1 (7.0–23.8)

IgD-CD27+ Switched Memory 42 37.4 11.4 13.8 5.21 21.5 (7.8-37.1) 27.1 (18.6-46.7) 28.3 (17.5-46.5)

CD21loCD38lo innate-like 8.18 15.4 23.6 21 8.95 2.7 (1.7-5.4) 4.6 (2.3-10.0) 4.4 (2.2 -10.0)

CD21+CD38hi Transitional Warnatz 4.04 4.7 2.73 3.47 7.18

IgD+IgM+ 38.3 39 66.3 67.5 72.1

IgD-IgM- 26.6 29.6 16.1 10.8 6.86

CD27-IgM+ B cells 27.8 30.2 57 51.9 55

CD27-IgM+CD24+CD38- Memory B 8.6 4.84 3.73 3.29 1.68

CD27-IgM+ CD24-CD38- Mature B 67.1 73 78.6 68.5 75.8

Mature B - CD24-CD38-CD21-IgM+ 5.23 5.8 27.4 27.1 9.26

Mature B - FM 87.7 86.5 50.2 49.8 81

Mature B - MZ 0 0 0 0 0.02 0

Mature B - T2 0 0 0 0 0

Mature B - T1 0 0 0 0 0

CD27-IgM+CD24+CD38+ Transitional B 7.02 3.43 2.45 4.03 8.28

Transitional B - FM 0 0 0 0 0

Transitional B -MZ 0 0 0 0 0

Transitional B - T2 81 58.8 36.2 24.2 32.7

Transitional B - T1 17.2 41.2 40.6 65.2 53.4

Taken from "B Cell Subsets in Healthy Children:

Reference Values for Evaluation of B Cell

Maturation Process in Peripheral Blood" Piatosa

et al Cytometry Part B (Clinical Cytometry)

78B:372–381 (2010) (35)

Table S6. List of top differentially regulated genes in B cell subclusters as identified by 10× single-

cell sequencing.

names scores logfoldchanges pvals pvals_adj rank FALSE TRUE exp_diff

B2m 34.15181 14.11283 6.1E-190 8.5E-186 97 4.163026 13.92961 9.766588

H2-Ab1 22.64221 12.32605 8.2E-101 5.66E-97 96 10.29833 18.84206 8.54373

Tmsb4x 20.14099 34.72802 3.09E-80 1.43E-76 97 20.13953 44.21116 24.07163

Ly6a 19.99108 4.891176 5.19E-80 1.8E-76 92 1.580791 4.749356 3.168565

H2-Q7 19.88554 2.949646 1.69E-78 4.68E-75 80 0.518549 1.832618 1.314069

Wdr89 19.25346 2.845833 1.38E-74 3.19E-71 77 0.201154 0.956223 0.755069

Cd52 18.49977 11.84196 1.44E-69 2.84E-66 93 8.888912 17.097 8.208084

Serpina3g 18.34494 3.695384 2.52E-67 4.35E-64 88 0.129534 1.026609 0.897075

Eef1g 17.8255 3.951239 1.33E-65 2.05E-62 89 2.076876 4.690987 2.614112

Gm11808 17.5437 2.408428 3.83E-64 5.3E-61 66 0.42467 1.337339 0.912669

Malat1 17.40641 38.20204 7.07E-64 8.9E-61 89 29.28174 55.76137 26.47963

Blvrb 17.33208 2.623529 2.37E-61 2.74E-58 71 0.437758 1.478112 1.040354

Uqcrh 16.72455 2.434325 8.04E-59 8.56E-56 68 1.856554 3.407725 1.551171

Cd72 16.649 2.740821 2.5E-57 2.31E-54 73 0.649629 1.962232 1.312603

Cox7a2l 16.37908 2.128168 1.66E-56 1.43E-53 60 1.149938 2.345064 1.195126

Cox4i1 15.92157 2.635848 1.55E-53 1.26E-50 72 2.022053 3.73133 1.709278

Use1 15.83514 1.994903 5.23E-53 4.02E-50 54 0.393446 1.072103 0.678657

Sec61g 15.71395 1.987765 2.79E-52 2.03E-49 52 0.918384 1.941631 1.023247

Sh3bgrl3 15.65959 4.187589 7.09E-52 4.67E-49 81 4.337284 7.227468 2.890184

Atp5l 15.5748 2.019539 1.54E-51 9.71E-49 57 1.253504 2.411159 1.157655

Psmb9 15.5001 1.993486 7.17E-51 4.32E-48 53 0.5338 1.337339 0.803539

Il9r 15.37805 3.226145 1.79E-49 1.03E-46 78 0.065128 0.488412 0.423284

AW112010 15.25744 2.858289 1.15E-48 6.11E-46 76 0.231245 1.060086 0.828841

Pfdn5 15.17293 2.048791 1.88E-49 1.04E-46 58 2.217951 3.551931 1.33398

H2-D1 14.97801 4.252138 6.02E-48 2.98E-45 75 5.276381 8.218884 2.942503

Eif3e 14.84978 1.805217 2.17E-47 1.03E-44 44 0.946826 1.87382 0.926994

Gm2000 14.73168 2.171283 1.5E-46 6.72E-44 62 0.224856 0.758798 0.533943

Psmb1 14.73052 1.807487 1.5E-46 6.72E-44 45 0.839035 1.722747 0.883711

H2-K1 14.25131 3.545734 4.99E-44 2.16E-41 71 4.393137 6.839485 2.446348

Zeb2 14.22683 2.974309 5.49E-43 2.23E-40 69 0.115829 0.675536 0.559708

Cfp 14.21612 3.490643 7.37E-43 2.92E-40 68 0.068013 0.582833 0.514819

Gimap4 14.21233 1.897093 1.48E-43 6.22E-41 49 0.68446 1.539056 0.854596

Psmb8 14.0436 2.063764 1.55E-42 5.94E-40 59 1.509068 2.755365 1.246296

Nme1 14.01224 2.364694 4.25E-42 1.59E-39 64 0.260305 0.928755 0.66845

Cox6c 13.95437 1.930879 4.49E-42 1.64E-39 50 1.026175 2.057511 1.031336

H2-Eb1 13.85902 6.09518 8.48E-42 3.01E-39 64 11.48846 15.7133 4.224846

Psenen 13.77051 1.520629 2.49E-41 8.61E-39 20 0.395507 0.872103 0.476596

Hmgn3 13.73165 3.143406 2.27E-40 7.5E-38 61 0.049155 0.36824 0.319085

Cox6b1 13.65675 1.728649 1.75E-40 5.9E-38 39 1.000618 1.902146 0.901528

Rac2 13.5577 2.814216 4.32E-40 1.39E-37 60 3.13211 5.044635 1.912525

Sys1 13.39201 1.754466 3.82E-39 1.17E-36 41 0.235779 0.640343 0.404564

Serf2 13.26091 2.425059 1.98E-38 5.95E-36 58 2.821105 4.452361 1.631256

Tma7 13.23722 1.633338 2.68E-38 7.9E-36 33 0.984646 1.824893 0.840247

Txn1 13.13239 2.406811 2.75E-37 7.76E-35 55 0.294106 1.034335 0.740229

Eif3k 13.10718 1.591804 8.98E-38 2.59E-35 27 1.277102 2.174249 0.897147

Snrnp25 12.99251 1.998286 6.51E-37 1.73E-34 52 0.202906 0.641202 0.438296

Nedd8 12.98198 1.406858 4.62E-37 1.28E-34 12 0.636645 1.212017 0.575372

Myl6 12.96959 2.16931 9.87E-37 2.58E-34 51 1.567704 2.894421 1.326717

H2-Oa 12.94598 1.476601 6.12E-37 1.66E-34 17 1.119332 1.908155 0.788822

Grb2 12.9108 1.511941 1.2E-36 3.07E-34 19 0.463417 0.986266 0.522849

Ctss 12.80992 1.561806 3.56E-36 8.97E-34 24 1.473207 2.391416 0.918209

H2-DMa 12.79413 1.550395 3.83E-36 9.48E-34 23 1.606142 2.539056 0.932914

Minos1 12.72555 1.622794 1.36E-35 3.25E-33 31 0.377061 0.879828 0.502767

Mrpl52 12.71515 1.510082 9.5E-36 2.31E-33 18 0.621393 1.239485 0.618092

Tonsl 12.68556 1.955561 2.01E-35 4.71E-33 45 0.151381 0.490987 0.339606

Btf3 12.60242 2.21656 4.32E-35 9.97E-33 44 2.417663 3.881545 1.463882

Ubb 12.52945 2.751292 8.12E-35 1.81E-32 43 4.083368 5.975966 1.892598

Nap1l1 12.44813 1.808804 4.5E-34 9.73E-32 41 0.705791 1.524464 0.818672

Capg 12.43444 1.724749 3.59E-34 7.88E-32 38 0.963005 1.848927 0.885922

Table S6. (Continued)

Slc25a4 12.38478 1.820542 7.71E-34 1.62E-31 39 0.299464 0.803433 0.503969

Naca 12.36275 2.767898 6.41E-34 1.37E-31 39 3.475577 5.367382 1.891805

Rbm3 12.28279 1.896182 2E-33 4.12E-31 38 1.384893 2.496996 1.112103

Sub1 12.25099 3.042099 7.47E-33 1.52E-30 37 1.39458 3.257511 1.862931

Cox7c 12.16432 1.585649 7.91E-33 1.59E-30 26 1.163644 2.029185 0.865541

Gm9493 12.15356 1.797363 9.69E-33 1.92E-30 35 0.159728 0.471245 0.311517

Gngt2 12.09862 1.677512 2.05E-32 4E-30 34 0.318322 0.787983 0.46966

Atp5h 12.07816 1.542971 2.23E-32 4.28E-30 22 1.049361 1.857511 0.80815

Oaz1 12.06675 2.451035 2.76E-32 5.24E-30 32 3.154575 4.818026 1.66345

Tmem243 11.90447 1.397089 1.29E-31 2.41E-29 10 0.497424 0.992275 0.494851

Irf8 11.77144 1.767951 7.64E-31 1.41E-28 30 1.060284 2.00515 0.944866

Ffar2 11.76355 3.651984 1.72E-30 3.09E-28 28 0.023702 0.263519 0.239818

Pold4 11.67754 1.60431 1.52E-30 2.77E-28 28 1.156121 2.030901 0.87478

Uqcrq 11.60933 1.425882 3.68E-30 6.45E-28 15 0.44899 0.925322 0.476332

Cyba 11.5795 2.540618 6.82E-30 1.15E-27 24 2.487943 4.177682 1.689739

Ndufa2 11.57466 1.298091 4.97E-30 8.6E-28 4 0.75134 1.322747 0.571407

H2-T22 11.56804 1.392101 5.4E-30 9.24E-28 9 0.424979 0.871245 0.446265

Pld4 11.55545 2.000729 1.11E-29 1.85E-27 23 0.347486 0.979399 0.631914

Eif3m 11.48863 1.28106 1.18E-29 1.94E-27 2 0.575124 1.060944 0.485821

Psme2 11.43103 1.531076 2.88E-29 4.64E-27 21 0.560594 1.154506 0.593913

Calr 11.34449 1.458517 6.29E-29 9.9E-27 16 0.610367 1.197425 0.587058

Chmp4b 11.30871 1.64483 1.09E-28 1.7E-26 19 0.198887 0.523605 0.324718

Erp44 11.26186 1.613834 1.87E-28 2.88E-26 18 0.205894 0.530472 0.324578

Ccnd2 11.25155 2.856625 3.35E-28 4.93E-26 14 0.129843 0.695279 0.565436

Hint1 11.25042 1.576055 2.06E-28 3.13E-26 16 0.960016 1.758798 0.798782

Tomm20 11.23705 1.375697 2.08E-28 3.13E-26 8 0.486088 0.964807 0.478719

Slc25a5 11.22102 1.714582 2.63E-28 3.92E-26 14 1.018343 1.917597 0.899254

Pomp 11.08021 1.403828 1.14E-27 1.64E-25 11 0.464448 0.941631 0.477183

Tmem256 11.06441 1.752368 1.45E-27 2.07E-25 12 0.181575 0.513305 0.33173

Stat1 11.03757 1.633136 1.75E-27 2.47E-25 11 0.386336 0.901288 0.514952

Ccdc12 10.97055 1.281108 3.16E-27 4.42E-25 3 0.446929 0.862661 0.415732

2010107E04Rik 10.95613 1.270359 3.54E-27 4.91E-25 1 0.706616 1.246352 0.539736

Snrpf 10.89376 1.416615 7.78E-27 1.06E-24 8 0.662923 1.255794 0.592871

Arpc2 10.84056 1.620526 1.4E-26 1.89E-24 7 1.709913 2.703004 0.993091

Cox8a 10.81287 1.706971 1.75E-26 2.34E-24 6 1.87366 2.944206 1.070546

Eif3h 10.74577 1.422622 3.07E-26 4.05E-24 5 1.382523 2.197425 0.814902

Ctsh 10.6987 1.313595 5.46E-26 7.13E-24 4 1.06317 1.742489 0.679319

Ndufb5 10.66185 1.228734 7.52E-26 9.64E-24 0 0.481657 0.896137 0.41448

Atp5k 10.65526 1.358484 8.42E-26 1.07E-23 1 0.31183 0.661803 0.349972

H2-Aa 10.65481 5.604219 7.1E-26 9.18E-24 1 16.88541 20.76996 3.884549

Lat2 10.6236 1.298304 1.14E-25 1.43E-23 0 0.381698 0.762232 0.380533

Table S7. List of top differentially regulated genes in T cell subclusters as identified by 10× single-

cell sequencing.

names scores logfoldchanges pvals pvals_adj rank FALSE TRUE exp_diff

Ly6c2 33.42394 10.93219 2E-201 2.7E-197 96 1.245312 8.483668 7.238357

Ctsw 21.81617 2.386814 1.82E-97 1.26E-93 72 0.55747 1.589824 1.032354

Eef1g 21.78611 4.672163 2.73E-96 1.26E-92 91 2.452131 5.604271 3.15214

Klrd1 20.99863 2.712085 1.5E-90 5.18E-87 78 0.39668 1.432789 1.036109

Plac8 19.71289 4.502293 2.93E-79 8.11E-76 90 0.433378 2.587312 2.153933

Ms4a4c 19.59711 2.835699 7.27E-79 1.68E-75 80 0.180888 0.88191 0.701021

Wdr89 19.35934 2.862626 4.33E-77 8.57E-74 81 0.249888 1.119975 0.870087

Il2rb 18.82173 2.706898 9.85E-75 1.71E-71 77 0.750471 2.116206 1.365735

Ctla2a 18.81201 3.271044 6.06E-74 9.32E-71 82 0.272409 1.39196 1.119551

Gm11808 18.57191 2.633646 4.94E-72 6.21E-69 76 0.399551 1.398241 0.99869

Bcl2 18.33723 2.251271 5.37E-71 5.72E-68 69 0.975774 2.182789 1.207015

Epsti1 18.30539 2.213102 4.95E-71 5.71E-68 67 1.013369 2.212312 1.198942

Gzmm 17.849 6.013988 2.66E-65 2.63E-62 87 0.041005 1.309673 1.268668

Samd3 17.61364 3.429948 1.24E-64 1.14E-61 85 0.034904 0.324121 0.289217

Dnajc15 16.58976 1.833762 2.76E-59 2.25E-56 59 0.965994 1.917085 0.951092

Hcst 16.49436 2.046138 1.18E-58 9.06E-56 62 2.168596 3.496231 1.327635

Arl4c 16.06904 1.63452 1.13E-55 8.23E-53 50 0.816689 1.593593 0.776904

Gm2000 16.06147 2.327264 3.9E-55 2.7E-52 70 0.191297 0.721734 0.530437

Sell 15.89171 1.593914 1.04E-54 6.89E-52 47 0.953791 1.760678 0.806887

Gramd3 15.20066 1.590154 3.41E-50 2.14E-47 46 0.326873 0.771985 0.445112

Npm1 15.12223 3.7891 6.11E-50 3.68E-47 79 4.083445 6.694095 2.61065

1700025G04Rik 15.04549 2.433575 8.08E-49 4.66E-46 73 0.066667 0.316583 0.249916

B2m 14.626 6.607387 6.46E-47 3.58E-44 77 9.349215 13.92902 4.579805

Nme1 14.59045 2.202938 2.2E-46 1.17E-43 66 0.468551 1.322236 0.853685

Cox7a2l 14.56078 1.733019 2.39E-46 1.22E-43 55 1.85886 2.944724 1.085863

Ifitm10 14.45901 2.190761 1.65E-45 8.14E-43 65 0.100314 0.393216 0.292902

Fcgrt 14.31486 3.312247 3.18E-44 1.47E-41 72 0.035891 0.309673 0.273783

Gm9493 14.25437 1.797724 2.64E-44 1.26E-41 58 0.212203 0.599874 0.387672

Naca 14.04148 3.390531 1.72E-43 7.7E-41 71 5.849708 8.197236 2.347528

Klk8 14.02331 1.498428 3.79E-43 1.64E-40 43 0.600538 1.201633 0.601095

Use1 13.57541 1.430645 1.17E-40 4.77E-38 38 0.422432 0.882538 0.460106

Tonsl 13.54897 1.901775 1.77E-40 6.99E-38 60 0.177568 0.545854 0.368286

Txk 12.99684 1.265832 1.41E-37 5.28E-35 28 0.424406 0.820352 0.395946

Eif3k 12.90955 1.488899 3.69E-37 1.34E-34 42 1.866218 2.793342 0.927124

Bola2 12.76462 1.323845 2.29E-36 8.14E-34 33 0.712607 1.281407 0.5688

Sidt1 12.7414 1.487475 3.57E-36 1.23E-33 41 0.267205 0.619975 0.35277

Ostf1 12.61954 1.202039 1.19E-35 4E-33 23 0.92454 1.503769 0.579229

Eif3f 12.5257 2.014021 3.69E-35 1.22E-32 61 3.767878 5.146357 1.378479

Rras2 12.47497 1.534201 8.93E-35 2.88E-32 44 0.174518 0.439698 0.265181

Uqcrh 12.44001 1.713719 1.05E-34 3.3E-32 54 3.035801 4.189698 1.153898

Slc25a4 12.339 1.359061 3.55E-34 1.09E-31 35 0.559175 1.072236 0.513062

Btf3 12.29937 2.232093 5.38E-34 1.62E-31 58 3.673396 5.200377 1.526981

Nop10 12.16057 1.265015 2.93E-33 8.44E-31 27 1.347959 2.060302 0.712343

Tm6sf1 12.12978 1.689839 5.02E-33 1.42E-30 52 0.129206 0.36809 0.238885

Eif3e 12.1008 1.291906 6.22E-33 1.69E-30 31 1.499327 2.253141 0.753814

Ptpn18 12.09502 1.69493 6.08E-33 1.68E-30 53 3.258502 4.406407 1.147905

Malat1 12.08887 17.39454 6.82E-33 1.81E-30 53 19.98636 32.04334 12.05698

Serf2 12.03145 2.166346 1.23E-32 3.21E-30 52 4.314132 5.805276 1.491144

Tomm20 11.87513 1.255185 7.74E-32 1.95E-29 25 1.031135 1.66897 0.637835

Mrpl23 11.83688 1.126944 1.25E-31 3.09E-29 12 0.590938 1.015075 0.424138

Fau 11.62761 8.904607 1.25E-30 3.03E-28 49 27.03131 33.20352 6.172203

St3gal6 11.62094 1.629823 1.71E-30 3.95E-28 46 0.114132 0.317839 0.203707

Psmb1 11.61629 1.259921 1.47E-30 3.51E-28 26 1.207447 1.889447 0.682

Cd8b1 11.59978 2.383952 1.68E-30 3.95E-28 46 2.477524 4.059673 1.58215

2410015M20Rik 11.5933 1.109946 2.02E-30 4.59E-28 10 0.741409 1.215452 0.474044

Rnf138 11.56595 1.19766 2.79E-30 6.22E-28 21 0.309197 0.604899 0.295703

Zfp296 11.51063 3.535808 1.1E-29 2.33E-27 41 0.012023 0.131281 0.119258

Hspe1 11.49131 1.627561 5.79E-30 1.27E-27 42 1.683266 2.677136 0.99387

Atp5l 11.4367 1.449502 1.04E-29 2.25E-27 39 2.030058 2.947864 0.917806


Rnaset2b 11.4301 1.339738 1.39E-29 2.91E-27 34 0.260565 0.561558 0.300993

Nkg7 11.40084 4.062746 1.91E-29 3.95E-27 39 3.870794 6.667085 2.796291

Tmsb4x 11.34284 21.24658 2.97E-29 6.06E-27 38 40.8 55.52701 14.72701

Klra7 11.26101 6.48867 2E-28 3.96E-26 36 0.00332 0.261307 0.257987

Eif3m 11.26083 1.063232 7.52E-29 1.51E-26 7 0.904083 1.403894 0.499812

Rnaset2a 11.15374 1.280002 2.72E-28 5.3E-26 30 0.279946 0.579146 0.2992

Ms4a4b 11.11818 2.118446 3.36E-28 6.47E-26 34 3.742844 5.192839 1.449995

Gm10073 11.10464 1.164844 4.5E-28 8.54E-26 16 0.336025 0.639447 0.303422

Fcer1g 11.09501 3.69182 8.74E-28 1.57E-25 31 0.050516 0.512563 0.462047

G0s2 11.07709 2.44872 8.67E-28 1.57E-25 31 0.037147 0.187814 0.150667

Cxcr3 11.00609 1.453641 1.15E-27 2.03E-25 30 0.220547 0.516332 0.295784

Selplg 11.00164 1.1999 1.19E-27 2.09E-25 22 1.857873 2.597362 0.739488

Park7 10.84949 1.116768 6.06E-27 1.01E-24 11 1.02952 1.589824 0.560304

H2afj 10.70287 1.104351 2.77E-26 4.57E-24 9 1.44253 2.072864 0.630334

Cd3d 10.66115 1.749764 4.32E-26 7.03E-24 26 3.872858 5.07098 1.198122

Ifngr1 10.61926 1.167192 6.55E-26 1.05E-23 17 0.648452 1.11495 0.466498

Eif3h 10.61243 1.313016 7.09E-26 1.13E-23 24 2.445222 3.302136 0.856914

Ssr4 10.57957 1.040931 1.02E-25 1.6E-23 5 0.815882 1.279523 0.463641

Eef1d 10.54692 1.138146 1.39E-25 2.16E-23 13 1.92472 2.630653 0.705934

Eomes 10.51957 1.791811 1.98E-25 3.01E-23 21 0.116734 0.356784 0.24005

Pfdn5 10.47105 1.22838 3.04E-25 4.58E-23 20 2.686496 3.498116 0.811619

Slc25a5 10.46702 1.560634 3.15E-25 4.69E-23 19 1.497263 2.41897 0.921707

Cox6c 10.43197 1.375535 4.48E-25 6.59E-23 18 1.889637 2.745603 0.855966

Gm6133 10.31804 2.552712 2.17E-24 3.06E-22 16 0.023867 0.132538 0.10867

Hspa8 10.28795 2.7904 1.93E-24 2.78E-22 16 5.534051 7.464824 1.930773

Sec61g 10.2634 1.162762 2.46E-24 3.44E-22 15 1.353881 2.005653 0.651773

Eif3i 10.25167 1.031754 2.8E-24 3.88E-22 4 1.071422 1.594221 0.522799

Il10rb 10.19468 1.174074 5.28E-24 7.17E-22 13 0.192822 0.39196 0.199138

Zyx 10.15268 1.170614 7.51E-24 1.01E-21 12 0.572364 1.007538 0.435173

Cox4i1 10.08958 1.684586 1.39E-23 1.85E-21 11 3.316196 4.458543 1.142347

Pim2 10.00102 1.361377 3.73E-23 4.89E-21 10 0.220637 0.491206 0.270569

2010107E04Rik 9.988852 1.072568 3.75E-23 4.89E-21 8 1.295379 1.883794 0.588415

Ccl5 9.903649 22.49802 9.81E-23 1.26E-20 7 16.43006 32.0245 15.59444

Snrpf 9.898991 1.188747 8.96E-23 1.16E-20 7 1.331449 1.994975 0.663526

Npm3 9.883368 1.042617 1.06E-22 1.35E-20 6 0.722925 1.158291 0.435366

Vasp 9.855266 0.941681 1.38E-22 1.73E-20 0 0.857335 1.282663 0.425328

Fbl 9.780163 0.976884 2.9E-22 3.59E-20 2 0.549035 0.89196 0.342924

Sys1 9.772749 1.162707 3.28E-22 4.01E-20 3 0.221175 0.440955 0.219779

Atp5h 9.741666 1.272472 4.07E-22 4.94E-20 2 2.308659 3.130653 0.821995

Sp110 9.693188 0.976295 6.79E-22 8.17E-20 1 0.435891 0.729899 0.294009

Taf10 9.642398 1.028356 1.06E-21 1.27E-19 0 0.441364 0.75691 0.315546

Table S8. GO term enrichment of HEM1-deficient B cell functions derived from the top candidates

from table S6.

#term ID term descriptionobserved

gene count

background

gene count

false

discovery

rate

matching proteins in your network (labels)

GO:0019882antigen processing and

presentation14 63 1.29e-11

B2m,Calr,Ctss,H2-Aa,H2-Ab1,H2-D1,H2-DMa,H2-Eb1,H2-K1,H2-

Q7,March1,Psmb8,Psmb9,Psme2

GO:0002376 immune system process 49 1703 1.47e-10

B2m,Calr,Capg,Ccr7,Cd55,Cfp,Clec2i,Cr2,Ctsh,Ctss,Cxcr5,Cyba,

Ddit4,Ffar2,Foxp1,Grb2,H2-Aa,H2-Ab1,H2-D1,H2-DMa,H2-

Eb1,H2-K1,H2-

Q7,Ikzf3,Il16,Irf8,Kmt2e,Lat2,Malt1,March1,Myh9,Nckap1l,Pld4

,Psmb8,Psmb9,Psme2,Rabgap1l,S1pr1,Serpina3g,Siglecg,Stat1,

Tec,Tgfbr2,Tmod3,Tnfrsf13c,Trim35,Wasf2,Ywhaz,Zfp36l2


presentation of peptide antigen11 34 1.47e-10

B2m,Calr,Ctss,H2-Aa,H2-Ab1,H2-D1,H2-DMa,H2-Eb1,H2-K1,H2-

Q7,March1

GO:0002682regulation of immune system

process36 1165 3.97e-08

B2m,Calr,Ccr7,Cd37,Cd55,Cfp,Clec2i,Cr2,Ctsh,Cyba,Dtx1,Fcer2

a,Ffar2,Foxp1,H2-Aa,H2-Ab1,H2-D1,H2-DMa,H2-

K1,Ikzf3,Inpp4b,Lat2,Malt1,Nckap1l,Ndfip1,Nme1,Nme2,Rac2,

Stat1,Tec,Tespa1,Tgfbr2,Tnfrsf13c,Tsc22d3,Zfp36,Zfp36l2

GO:0006955 immune response 31 914 9.49e-08

B2m,Capg,Ccr7,Cd55,Cfp,Cr2,Ctsh,Ctss,Cxcr5,Cyba,Ffar2,Foxp1

,Grb2,H2-Aa,H2-Ab1,H2-D1,H2-DMa,H2-Eb1,H2-K1,H2-

Q7,Irf8,Lat2,Malt1,March1,Serpina3g,Siglecg,Stat1,Tec,Tnfrsf1

3c,Trim35,Ywhaz

GO:1903706 regulation of hemopoiesis 19 377 9.28e-07

B2m,Ccr7,Clec2i,Dtx1,Foxp1,H2-Aa,H2-

DMa,Ikzf3,Inpp4b,Malt1,Nckap1l,Ndfip1,Nme1,Nme2,Stat1,Te

spa1,Tgfbr2,Zfp36,Zfp36l2

GO:0002684positive regulation of immune

system process26 771 2.70e-06

B2m,Calr,Ccr7,Cd37,Cd55,Cfp,Clec2i,Cr2,Cyba,Fcer2a,Ffar2,Fox

p1,H2-Aa,H2-Ab1,H2-D1,H2-DMa,H2-

K1,Lat2,Malt1,Nckap1l,Rac2,Stat1,Tec,Tespa1,Tgfbr2,Tnfrsf13c

GO:1902105regulation of leukocyte

differentiation16 284 3.20e-06

Ccr7,Clec2i,Dtx1,Foxp1,H2-Aa,H2-

DMa,Ikzf3,Inpp4b,Malt1,Nckap1l,Ndfip1,Nme1,Nme2,Tespa1,

Tgfbr2,Zfp36l2

GO:0019884

antigen processing and

presentation of exogenous

antigen

7 26 3.93e-06 B2m,H2-Aa,H2-Ab1,H2-DMa,H2-Eb1,H2-K1,Psme2

GO:0050776 regulation of immune response 23 635 4.34e-06

B2m,Ccr7,Cd37,Cd55,Cfp,Clec2i,Cr2,Ctsh,Cyba,Fcer2a,Ffar2,H2-

Ab1,H2-D1,H2-DMa,H2-

K1,Lat2,Malt1,Nckap1l,Ndfip1,Rac2,Tec,Tespa1,Tnfrsf13c


response19 438 5.17e-06

B2m,Ccr7,Cd55,Cfp,Clec2i,Cr2,Cyba,Fcer2a,Ffar2,H2-Ab1,H2-

D1,H2-DMa,H2-K1,Lat2,Malt1,Nckap1l,Tec,Tespa1,Tnfrsf13c

GO:0002478



peptide antigen

6 20 1.99e-05 B2m,H2-Aa,H2-Ab1,H2-DMa,H2-Eb1,H2-K1

GO:0002822

regulation of adaptive immune

response based on somatic

recombination of immune

receptors built from

immunoglobulin superfamily

domains

11 146 3.00e-05B2m,Ccr7,Cd55,Fcer2a,H2-Ab1,H2-D1,H2-DMa,H2-

K1,Malt1,Ndfip1,Tnfrsf13c

GO:0002495


presentation of peptide antigen

via MHC class II

5 14 9.99e-05 H2-Aa,H2-Ab1,H2-DMa,H2-Eb1,March1

GO:0002824

positive regulation of adaptive

immune response based on

somatic recombination of

immune receptors built from

immunoglobulin superfamily

domains

9 102 9.99e-05 B2m,Ccr7,Fcer2a,H2-Ab1,H2-D1,H2-DMa,H2-K1,Malt1,Tnfrsf13c

GO:0045619regulation of lymphocyte

differentiation11 171 9.99e-05

Ccr7,Dtx1,H2-Aa,H2-

DMa,Ikzf3,Malt1,Nckap1l,Ndfip1,Tespa1,Tgfbr2,Zfp36l2

GO:0032101regulation of response to

external stimulus21 681 0.00013

Acp5,Adrb2,Calr,Ccr7,Cd37,Cd55,Ctss,Cyba,Ffar2,Foxp1,Gstp1,

Il16,Nckap1l,Ndfip1,Rac2,S1pr1,Siglecg,Stat1,Tec,Tgfbr2,Zfp36

GO:0048518positive regulation of biological

process77 5340 0.00014

Adrb2,Arpc2,B2m,Calr,Cbx7,Ccnd2,Ccr7,Cd37,Cd55,Cfp,Chmp4

b,Clec2i,Cr2,Ctsh,Ctss,Cxcr5,Cyba,Ddit4,Eif3e,Ell3,Fcer2a,Ffar2,

Foxn3,Foxp1,Grb2,Gstp1,H2-Aa,H2-Ab1,H2-D1,H2-DMa,H2-

K1,Hint1,Hmga1,Hmgn3,Ikzf3,Il16,Il9r,Irf8,Itsn2,Kmt2e,Lat2,Ma

cf1,Malt1,Myh9,Naca,Nckap1l,Ndfip1,Nme1,Nme2,Oaz1,Pold4

,Psme2,Ptges3,Rac2,Rapgef4,Rbm3,S1pr1,Slc25a4,Slc25a5,Stat

1,Sub1,Tec,Tespa1,Tgfbr2,Tmod3,Tmsb4x,Tnfrsf13c,Tonsl,Trim

35,Txn1,Txnip,Ubb,Ube2v1,Wasf2,Zeb2,Zfp36,Zfp36l2

GO:0048583regulation of response to

stimulus58 3552 0.00014

Acp5,Adrb2,Arhgef1,B2m,Calr,Ccr7,Cd37,Cd55,Cfp,Clec2i,Cr2,C

tsh,Ctss,Cyba,Ddit4,Dtx1,Ell3,Fcer2a,Ffar2,Foxp1,Grb2,Gstp1,H

2-Ab1,H2-D1,H2-DMa,H2-

K1,Hint1,Hmga1,Hmgn3,Il16,Inpp4b,Itsn2,Klhl24,Lat2,Ly6e,Mac

f1,Malt1,Nckap1l,Ndfip1,Pfdn5,Pik3ip1,Rac2,S1pr1,Sesn1,Sigle

cg,Slc25a4,Slc25a5,Stat1,Tec,Tespa1,Tgfbr2,Tnfrsf13c,Txn1,Ub

b,Ube2v1,Wdr96,Zeb2,Zfp36


GO:0050865 regulation of cell activation 18 520 0.00015

Adrb2,Ccr7,Cd37,Clec2i,Dtx1,H2-Aa,H2-Ab1,H2-

DMa,Ikzf3,Malt1,Nckap1l,Ndfip1,Rac2,Tec,Tespa1,Tgfbr2,Tnfrs

f13c,Zfp36l2

GO:0022900 electron transport chain 9 120 0.00024Cox4i1,Cox6c,Cox7a2l,Cox7c,Cox8a,Cyba,Sh3bgrl3,Uqcrh,Uqcr

q

GO:0002474


presentation of peptide antigen

via MHC class I

5 20 0.00026 B2m,Calr,H2-D1,H2-K1,H2-Q7

GO:0009987 cellular process 140 12459 0.00026

Acap1,Acp5,Adrb2,Aff4,Arhgef1,Arpc2,Atp5h,Atp5k,B2m,B3gn

t2,B3gnt8,Blvrb,Brwd1,Btf3,Calr,Capg,Cbx7,Ccnd2,Ccr7,Cd37,C

hmp4b,Clec2i,Cox4i1,Cox6c,Cox7a2l,Cox7c,Cox8a,Cr2,Ctsh,Cts

s,Cxcr5,Cyba,Ddit4,Dtx1,Eef1g,Eif3e,Eif3h,Eif3k,Eif3m,Ell3,Erp4

4,Ffar2,Foxn3,Foxp1,Ggps1,Gm2000,Gngt2,Grb2,Grcc10,Gstp1,

H2-Ab1,H2-D1,H2-DMa,H2-K1,H2-

Q7,Herc4,Hint1,Hmga1,Hmgn3,Hvcn1,Ikzf3,Il16,Il9r,Inpp4b,Irf8,

Itsn2,Kcnq5,Klhl24,Kmt2e,Lat2,Ly6e,Macf1,Malt1,March1,Mrpl

52,Myh9,Myl6,Naca,Nap1l1,Nckap1l,Ndfip1,Ndufa2,Ndufb5,N

edd8,Neurl3,Nme1,Nme2,Oaz1,Pfdn5,Pgap1,Pld4,Pold4,Pomp,

Psenen,Psmb1,Psmb8,Psmb9,Ptges3,Rabgap1l,Rac2,Rapgef4,R

bm3,Rcsd1,Rnf167,S1pr1,Serpina3g,Sesn1,Sh3bgrl3,Siglecg,Skp

1a,Slc25a4,Slc25a5,Snrnp25,Snrpf,Snx30,St3gal1,Stat1,Sub1,Te

c,Tespa1,Tgfbr2,Tmod3,Tmsb4x,Tomm20,Tonsl,Trim35,Tubb4

b,Txn1,Txnip,Uba52,Ubb,Ube2v1,Uqcrh,Uqcrq,Use1,Wasf2,Yw

haz,Zeb2,Zfp36,Zfp36l2

GO:0050863 regulation of T cell activation 13 287 0.00026Ccr7,Clec2i,Dtx1,H2-Aa,H2-Ab1,H2-

DMa,Malt1,Nckap1l,Ndfip1,Rac2,Tespa1,Tgfbr2,Tnfrsf13c


activation15 396 0.00034

Ccr7,Clec2i,Dtx1,H2-Aa,H2-Ab1,H2-

DMa,Ikzf3,Malt1,Nckap1l,Ndfip1,Rac2,Tespa1,Tgfbr2,Tnfrsf13c

,Zfp36l2

GO:0030155 regulation of cell adhesion 19 624 0.00036

Adrb2,Arpc2,Calr,Ccr7,Cytip,Dtx1,Gstp1,H2-Aa,H2-Ab1,H2-

DMa,Macf1,Malt1,Nckap1l,Ndfip1,Rac2,S1pr1,Tespa1,Tgfbr2,T

nfrsf13c

GO:0050789 regulation of biological process 115 9594 0.00042 Acap1,Acp5,Adrb2,Aff4,Arhgef1,Arpc2,B2m,Brwd1,Btf3,Calr,Capg,Cbx7,Ccnd2,Ccr7,Cd37,Cd55,Cfp,Chmp4b,Clec2i,Cox7a2l,Cr2,Ctsh,Ctss,Cxcr5,Cxxc5,Cyba,Cytip,Ddit4,Dtx1,Eif3e,Eif3k,Ell3,Erp44,Fcer2a,Ffar2,Foxn3,Foxp1,Gngt2,Grb2,Grcc10,Gstp1,H2-Aa,H2-Ab1,H2-D1,H2-DMa,H2-K1,Hint1,Hmga1,Hmgn3,Hvcn1,Ikzf3,Il16,Il9r,Inpp4b,Irf8,Itsn2,Kcnq5,Klhl24,Kmt2e,Lat2,Ly6e,Macf1,Malt1,Myh9,Myl12b,Naca,Nckap1l,Ndfip1,Nedd8,Nme1,Nme2,Oaz1,Pfdn5,Pik3ip1,Pold4,Psenen,Psmb8,Psmb9,Psme2,Ptges3,Rab4b,Rabgap1l,Rac2,Rapgef4,Rbm3,Rnf167,S1pr1,Serpina3g,Sesn1,Sh3bgrl3,Siglecg,Slc25a4,Slc25a5,Stat1,Sub1,Tec,Tespa1,Tgfbr2,Tmod3,Tmsb4x,Tnfrsf13c,Tonsl,Trim35,Tsc22d3,Txn1,Txnip,Ubb,Ube2v1,Use1,Wasf2,Wdr96,Ywhaz,Zeb2,Zfp36,Zfp36l2

GO:1903037regulation of leukocyte cell-cell

adhesion12 264 0.00054 Ccr7,Dtx1,Gstp1,H2-Aa,H2-Ab1,H2-DMa,Malt1,Nckap1l,Ndfip1,Tespa1,Tgfbr2,Tnfrsf13c


activation16 479 0.00065 Ccr7,Cd37,Clec2i,Dtx1,H2-Aa,H2-Ab1,H2-DMa,Ikzf3,Malt1,Nckap1l,Ndfip1,Rac2,Tespa1,Tgfbr2,Tnfrsf13c,Zfp36l2

GO:0048534hematopoietic or lymphoid

organ development19 659 0.00065 B2m,Ccr7,Cr2,Cxcr5,Foxp1,H2-Ab1,H2-DMa,Irf8,Kmt2e,Malt1,Myh9,Nckap1l,Pld4,Rabgap1l,Siglecg,Tgfbr2,Tmod3,Wasf2,Zfp36l2


proliferation11 224 0.00065 Ccr7,Clec2i,Gstp1,H2-Aa,H2-Ab1,Ikzf3,Nckap1l,Ndfip1,Rac2,Tgfbr2,Tnfrsf13c

GO:0030097 hemopoiesis 18 600 0.00066 B2m,Ccr7,Cr2,Foxp1,H2-Ab1,H2-DMa,Irf8,Kmt2e,Malt1,Myh9,Nckap1l,Pld4,Rabgap1l,Siglecg,Tgfbr2,Tmod3,Wasf2,Zfp36l2

GO:0019886



peptide antigen via MHC class II

4 12 0.00078 H2-Aa,H2-Ab1,H2-DMa,H2-Eb1

GO:0042127regulation of cell population

proliferation32 1594 0.00082 Adrb2,Calr,Ccnd2,Ccr7,Cd37,Clec2i,Ctsh,Cyba,Ell3,Foxp1,Grb2,Gstp1,H2-Aa,H2-Ab1,Hmga1,Ikzf3,Naca,Nckap1l,Ndfip1,Nme1,Nme2,Pold4,Rac2,S1pr1,Slc25a5,Stat1,Tec,Tgfbr2,Tnfrsf13c,Trim35,Txnip,Zfp36

GO:1902600proton transmembrane

transport8 113 0.00086 Atp5h,Atp5k,Cox4i1,Cox6c,Cox7a2l,Cox7c,Cox8a,Hvcn1

GO:0002697regulation of immune effector

process13 335 0.00089 B2m,Ccr7,Cd37,Cd55,Fcer2a,Ffar2,Foxp1,H2-D1,H2-K1,Malt1,Ndfip1,Rac2,Stat1

GO:0048584positive regulation of response

to stimulus36 1922 0.00089 Adrb2,B2m,Calr,Ccr7,Cd55,Cfp,Clec2i,Cr2,Ctsh,Ctss,Cyba,Fcer2a,Ffar2,Foxp1,Grb2,H2-Ab1,H2-D1,H2-DMa,H2-K1,Hint1,Hmga1,Il16,Lat2,Macf1,Malt1,Nckap1l,Ndfip1,Rac2,S1pr1,Tec,Tespa1,Tgfbr2,Tnfrsf13c,Ubb,Ube2v1,Zeb2

GO:0050896 response to stimulus 86 6616 0.00091 Acap1,Acp5,Adrb2,Arhgef1,Arpc2,B2m,B3gnt2,Calr,Capg,Cbx7,Ccnd2,Ccr7,Cd37,Cd55,Cfp,Clec2i,Cox4i1,Cr2,Ctsh,Ctss,Cxcr5,Cyba,Ddit4,Dtx1,Erp44,Ffar2,Foxn3,Foxp1,Gngt2,Grb2,Gstp1,H2-Aa,H2-Ab1,H2-D1,H2-DMa,H2-Eb1,H2-K1,H2-Q7,Hint1,Hmga1,Hvcn1,Il16,Il9r,Irf8,Kmt2e,Lat2,Ly6a,Ly6e,Macf1,Malt1,March1,Naca,Nckap1l,Nedd8,Nme1,Nme2,Pold4,Psenen,Psmb9,Ptges3,Rac2,Rapgef4,Rbm3,Rcsd1,S1pr1,Serpina3g,Sesn1,Siglecg,Slc25a4,Slc25a5,Stat1,Sub1,Tec,Tgfbr2,Tnfrsf13c,Tomm20,Tonsl,Trim35,Tsc22d3,Txn1,Txnip,Ube2v1,Wasf2,Ywhaz,Zfp36,Zfp36l2


effector process10 196 0.00093 B2m,Ccr7,Cd37,Fcer2a,Ffar2,Foxp1,H2-D1,H2-K1,Malt1,Rac2

Table S9. GO term enrichment of HEM1-deficient T cell functions derived from the top candidates

from table S7.

#term ID term descriptionobserved

gene count

background

gene count

false

discovery

rate

matching proteins in your network (labels)

GO:0002376 immune system process 45 1703 1.91e-07

Actn1,B2m,Ccl5,Ccr7,Ccr9,Cd3d,Cd3g,Crip1,Cxcr3,Cyba,Ddit

4,Evl,Foxp1,Fyn,Gpr183,H2-D1,H2-K1,H2-Q6,H2-

Q7,Id2,Il2rb,Il7r,Itgal,Itgb2,Lef1,Lgals1,Myo1f,Nckap1l,Pag1,

Pecam1,Pglyrp1,Psmb10,Psmb8,Psmb9,Psme1,Psme2,Rgcc,S

1pr1,Satb1,Sell,Serpina3g,Tcf7,Tgfbr3,Zfp36l1,Zfp36l2

GO:0022900 electron transport chain 13 120 3.19e-07Cox4i1,Cox5a,Cox5b,Cox6c,Cox7a2,Cox7b,Cox7c,Cox8a,Cyb

a,Ndufv3,Sh3bgrl3,Uqcrh,Uqcrq

GO:0001775 cell activation 24 552 3.92e-07

B2m,Ccl5,Ccr7,Ccr9,Cd3d,Evl,Foxp1,Fyn,Gpr183,Id2,Il2rb,Il7

r,Itgal,Itgb2,Lef1,Lgals1,Myo1f,Psmb10,Rgcc,Satb1,Tcf7,Tspa

n32,Zfp36l1,Zfp36l2

GO:0002682regulation of immune system

process35 1165 3.92e-07

B2m,Ccl5,Ccr7,Ctla2a,Cxcr3,Cyba,Dtx1,Foxp1,Fyn,Gpr183,H

2-D1,H2-

K1,Hcst,Id2,Il6st,Il7r,Itgal,Itgb2,Lef1,Lgals1,Myo1f,Nckap1l,P

ag1,Pecam1,Peli1,Pglyrp1,Rac2,Rgcc,Sell,Tespa1,Tsc22d3,Ts

pan32,Zfp36,Zfp36l1,Zfp36l2

GO:0042110 T cell activation 16 244 1.01e-06B2m,Ccr7,Ccr9,Cd3d,Foxp1,Fyn,Gpr183,Il7r,Itgal,Itgb2,Lef1,

Psmb10,Satb1,Tcf7,Zfp36l1,Zfp36l2

GO:0045321 leukocyte activation 21 464 1.34e-06

B2m,Ccl5,Ccr7,Ccr9,Cd3d,Foxp1,Fyn,Gpr183,Id2,Il2rb,Il7r,Itg

al,Itgb2,Lef1,Lgals1,Myo1f,Psmb10,Satb1,Tcf7,Zfp36l1,Zfp36

l2

GO:0046649 lymphocyte activation 19 378 1.36e-06B2m,Ccr7,Ccr9,Cd3d,Foxp1,Fyn,Gpr183,Id2,Il2rb,Il7r,Itgal,It

gb2,Lef1,Lgals1,Psmb10,Satb1,Tcf7,Zfp36l1,Zfp36l2

GO:0046034 ATP metabolic process 13 162 2.37e-06Atp1b1,Atp5e,Atp5h,Atp5j,Atp5j2,Cox4i1,Cox5a,Cox5b,Cox

7c,Cox8a,Ndufv3,Uqcrh,Uqcrq


presentation9 63 5.06e-06

B2m,H2-D1,H2-K1,H2-Q6,H2-

Q7,Psmb8,Psmb9,Psme1,Psme2

GO:1902600 proton transmembrane transport 11 113 5.06e-06Atp5e,Atp5h,Atp5j,Cox4i1,Cox5a,Cox5b,Cox6c,Cox7a2,Cox

7b,Cox7c,Cox8a

GO:0002694 regulation of leukocyte activation 20 479 6.51e-06

Ccl5,Ccr7,Ctla2a,Dtx1,Gpr183,Id2,Il6st,Il7r,Itgal,Itgb2,Lgals1,

Nckap1l,Pag1,Peli1,Pglyrp1,Rac2,Tespa1,Tspan32,Zfp36l1,Zf

p36l2

GO:0030217 T cell differentiation 12 154 7.83e-06B2m,Ccr7,Ccr9,Cd3d,Foxp1,Gpr183,Il7r,Lef1,Satb1,Tcf7,Zfp3

6l1,Zfp36l2

GO:0051249 regulation of lymphocyte activation 18 396 7.86e-06Ccl5,Ccr7,Ctla2a,Dtx1,Gpr183,Id2,Il6st,Il7r,Itgal,Lgals1,Ncka

p1l,Pag1,Peli1,Pglyrp1,Rac2,Tespa1,Zfp36l1,Zfp36l2


system process25 771 9.10e-06

B2m,Ccl5,Ccr7,Cyba,Foxp1,Fyn,Gpr183,H2-D1,H2-

K1,Id2,Il6st,Il7r,Itgal,Itgb2,Lef1,Lgals1,Nckap1l,Pecam1,Peli1

,Pglyrp1,Rac2,Rgcc,Sell,Tespa1,Zfp36l1

GO:0009167purine ribonucleoside

monophosphate metabolic process13 196 9.10e-06

Atp1b1,Atp5e,Atp5h,Atp5j,Atp5j2,Cox4i1,Cox5a,Cox5b,Cox

7c,Cox8a,Ndufv3,Uqcrh,Uqcrq

GO:0042775mitochondrial ATP synthesis

coupled electron transport8 51 9.10e-06 Cox4i1,Cox5a,Cox5b,Cox7c,Cox8a,Ndufv3,Uqcrh,Uqcrq

GO:0030098 lymphocyte differentiation 14 243 1.20e-05B2m,Ccr7,Ccr9,Cd3d,Foxp1,Gpr183,Id2,Il7r,Lef1,Lgals1,Satb

1,Tcf7,Zfp36l1,Zfp36l2

GO:0048523negative regulation of cellular

process69 4336 5.24e-05

Actn1,Arhgef1,B2m,Bach2,Bhlhe40,Ccl5,Ccr7,Crlf3,Cst7,Ctla

2a,Cxcr3,Ddit4,Dtx1,Evl,Foxn3,Foxo1,Foxp1,Fyn,Gmfg,H2-

D1,H2-

K1,Id2,Ift80,Il2rb,Il6st,Il7r,Itm2b,Klf3,Lef1,Lgals1,Mtss1,Myo

1f,Nckap1l,Ndufa13,Pag1,Pecam1,Peli1,Pfdn5,Pglyrp1,Pik3i

p1,Pla2g16,Plac8,Rab3ip,Rapgef4,Rbm38,Rgcc,Rgs10,Rnf167

,S1pr1,Satb1,Serpina3g,Sesn1,Sh3bp5,Srgn,St6gal1,Stk38,Tb

xa2r,Tcf7,Tgfbr3,Trib2,Tsc22d3,Tspan32,Txn1,Txnip,Usp28,

Zbtb20,Zfp36,Zfp36l1,Zfp36l2

GO:0006123mitochondrial electron transport,

cytochrome c to oxygen5 15 7.83e-05 Cox4i1,Cox5a,Cox5b,Cox7c,Cox8a


GO:0006091generation of precursor

metabolites and energy14 295 7.85e-05

Cox4i1,Cox5a,Cox5b,Cox6c,Cox7a2,Cox7b,Cox7c,Cox8a,Cyba,Il6st,Ndufv3,

Sh3bgrl3,Uqcrh,Uqcrq

GO:0048519negative regulation of biological

process73 4771 8.41e-05

Actn1,Arhgef1,B2m,Bach2,Bhlhe40,Ccl5,Ccr7,Crlf3,Cst7,Ctla2a,Cxcr3,Cyba,

Ddit4,Dtx1,Evl,Foxn3,Foxo1,Foxp1,Fyn,Gmfg,H2-D1,H2-

K1,Id2,Ift80,Il2rb,Il6ra,Il6st,Il7r,Itm2b,Klf3,Lef1,Lgals1,Mtss1,Myo1f,Nckap1

l,Ndufa13,Numb,Oaz1,Pag1,Pecam1,Peli1,Pfdn5,Pglyrp1,Pik3ip1,Pla2g16,Pl

ac8,Rab3ip,Rapgef4,Rbm38,Rgcc,Rgs10,Rnf167,S1pr1,Satb1,Serpina3g,Ses

n1,Sh3bp5,Srgn,St6gal1,Stk38,Tbxa2r,Tcf7,Tgfbr3,Trib2,Tsc22d3,Tspan32,T

xn1,Txnip,Usp28,Zbtb20,Zfp36,Zfp36l1,Zfp36l2

GO:0050789 regulation of biological process 120 9594 9.90e-05

Actn1,Add3,Arhgap15,Arhgef1,Atp1b1,Auts2,B2m,Bach2,Bhlhe40,Bmyc,Bt

f3,Ccl5,Ccr7,Ccr9,Cd3d,Cd3g,Chchd2,Cox17,Crip1,Crlf3,Cst7,Ctla2a,Cxcr3,C

yba,Cytip,Ddit4,Dgka,Dtx1,Edf1,Evl,Foxn3,Foxo1,Foxp1,Fyn,Gmfg,Gng2,Gp

r183,H2-D1,H2-

K1,Hcst,Id2,Ift80,Igfbp4,Il2rb,Il6ra,Il6st,Il7r,Itgal,Itgb2,Itm2b,Klf3,Lef1,Lgals

1,Lsp1,Mtss1,Myo1f,Nckap1l,Ndufa13,Nsg2,Numb,Oaz1,Pag1,Pdk1,Pecam

1,Peli1,Pfdn5,Pglyrp1,Pik3ip1,Pla2g16,Plac8,Prr13,Psmb8,Psmb9,Psme1,Ps

me2,Rab3ip,Rac2,Ramp1,Rapgef4,Rbm38,Rgcc,Rgs10,Rnf167,S100a10,S100

a11,S100a13,S100a4,S1pr1,Satb1,Scml4,Sell,Serpina3g,Sesn1,Sh3bgrl3,Sh3

bp5,Srgn,Ssbp2,Ssh2,St6gal1,St8sia1,Stk38,Sub1,Sun2,Tbxa2r,Tcf7,Tespa1,

Tgfbr3,Tmsb4x,Tomm7,Trib2,Tsc22d3,Tspan32,Ttc28,Txn1,Txnip,Usp28,Zb

tb20,Zfp36,Zfp36l1,Zfp36l2

GO:0050794 regulation of cellular process 115 9045 9.90e-05

Actn1,Arhgap15,Arhgef1,Atp1b1,Auts2,B2m,Bach2,Bhlhe40,Bmyc,Btf3,Ccl

5,Ccr7,Ccr9,Cd3d,Cd3g,Chchd2,Cox17,Crip1,Crlf3,Cst7,Ctla2a,Cxcr3,Cyba,D

dit4,Dgka,Dtx1,Edf1,Evl,Foxn3,Foxo1,Foxp1,Fyn,Gmfg,Gng2,Gpr183,H2-

D1,H2-


1,Lsp1,Mtss1,Myo1f,Nckap1l,Ndufa13,Nsg2,Numb,Pag1,Pdk1,Pecam1,Peli1

,Pfdn5,Pglyrp1,Pik3ip1,Pla2g16,Plac8,Prr13,Psme1,Psme2,Rab3ip,Rac2,Ra

mp1,Rapgef4,Rbm38,Rgcc,Rgs10,Rnf167,S100a10,S100a11,S100a13,S100a4

,S1pr1,Satb1,Scml4,Sell,Serpina3g,Sesn1,Sh3bgrl3,Sh3bp5,Srgn,Ssbp2,Ssh2

,St6gal1,St8sia1,Stk38,Sub1,Sun2,Tbxa2r,Tcf7,Tespa1,Tgfbr3,Tmsb4x,Tom

m7,Trib2,Tsc22d3,Tspan32,Ttc28,Txn1,Txnip,Usp28,Zbtb20,Zfp36,Zfp36l1,

Zfp36l2

GO:0009987 cellular process 144 12459 0.00017

Actn1,Als2cl,Arhgap15,Arhgef1,Atp1b1,Atp5e,Atp5h,Atp5j,Atp5j2,Auts2,B

2m,Bach2,Bhlhe40,Btf3,Ccl5,Ccr7,Ccr9,Cd3d,Cd3g,Chchd2,Cnn3,Cox17,Cox

4i1,Cox5a,Cox5b,Cox6c,Cox7a2,Cox7b,Cox7c,Cox8a,Crip1,Crlf3,Ctsd,Cxcr3,

Cyba,Ddit4,Dgka,Dtx1,Edf1,Ephx1,Evl,Foxn3,Foxo1,Foxp1,Fyn,Galnt6,Ggt5,

Gmfg,Gng2,Gpr183,H2-D1,H2-K1,H2-

Q7,Hcst,Id2,Ift80,Il2rb,Il6ra,Il6st,Il7r,Itgal,Itgb2,Itm2b,Klf3,Krtcap2,Lef1,Lgal

s1,Lsp1,Mrpl33,Mrpl52,Mtss1,Myl6,Myo1f,Nckap1l,Ndufa1,Ndufa13,Nduf

b7,Ndufv3,Nsg2,Numb,Oaz1,Pag1,Pdk1,Pecam1,Peli1,Pfdn5,Pglyrp1,Pla2g1

6,Plac8,Pomp,Prr13,Psmb1,Psmb10,Psmb8,Psmb9,Ptpn18,Rab3ip,Rac2,Ra

mp1,Rapgef4,Rbm38,Rgcc,Rgs10,Rnf167,S100a10,S100a13,S1pr1,Satb1,Sc

ml4,Sec61b,Sell,Serpina3g,Sesn1,Sh3bgrl3,Sh3bp5,Slc17a9,Srgn,Ssbp2,Ssh

2,St6gal1,St8sia1,St8sia6,Stk38,Sub1,Sun2,Tbxa2r,Tcf7,Tespa1,Tgfbr3,Thad

a,Tmsb4x,Tomm7,Tspan32,Ttc28,Txn1,Txnip,Ubl5,Uqcrh,Uqcrq,Usp28,Zbt

b20,Zfp36,Zfp36l1,Zfp36l2

GO:0002474


presentation of peptide antigen via

MHC class I

5 20 0.00020 B2m,H2-D1,H2-K1,H2-Q6,H2-Q7

GO:0065007 biological regulation 124 10168 0.00020

Actn1,Add3,Als2cl,Arhgap15,Arhgef1,Atp1b1,Auts2,B2m,Bach2,Bhlhe40,B

myc,Btf3,Ccl5,Ccr7,Ccr9,Cd3d,Cd3g,Chchd2,Cnn3,Cox17,Crip1,Crlf3,Cst7,Ct

la2a,Cxcr3,Cyba,Cytip,Ddit4,Dgka,Dtx1,Edf1,Evl,Foxn3,Foxo1,Foxp1,Fyn,G

mfg,Gng2,Gpr183,H2-D1,H2-


1,Lsp1,Mtss1,Myo1f,Nckap1l,Ndufa13,Nsg2,Numb,Oaz1,Pag1,Pdk1,Pecam

1,Peli1,Pfdn5,Pglyrp1,Pik3ip1,Pla2g16,Plac8,Prr13,Psmb8,Psmb9,Psme1,Ps

me2,Rab3ip,Rac2,Ramp1,Rapgef4,Rbm38,Rgcc,Rgs10,Rnf167,S100a10,S100

a11,S100a13,S100a4,S1pr1,Satb1,Scml4,Sec61b,Sell,Serpina3g,Sesn1,Sh3b

grl3,Sh3bp5,Srgn,Ssbp2,Ssh2,St6gal1,St8sia1,Stk38,Sub1,Sun2,Tbxa2r,Tcf7,

Tespa1,Tgfbr3,Thada,Tmsb4x,Tomm7,Trib2,Tsc22d3,Tspan32,Ttc28,Txn1,T

xnip,Usp28,Zbtb20,Zfp36,Zfp36l1,Zfp36l2

GO:1903706 regulation of hemopoiesis 15 377 0.00020B2m,Ccl5,Ccr7,Ctla2a,Dtx1,Foxp1,Id2,Il7r,Lef1,Nckap1l,Pglyrp1,Tespa1,Zfp

36,Zfp36l1,Zfp36l2

GO:0042127regulation of cell population

proliferation34 1594 0.00021

Ccl5,Ccr7,Cox17,Cxcr3,Cyba,Foxo1,Foxp1,Fyn,Gpr183,Id2,Ift80,Il6ra,Il6st,Il

7r,Itgal,Lef1,Mtss1,Nckap1l,Peli1,Plac8,Rac2,Rbm38,Rgcc,S100a11,S100a13

,S1pr1,St6gal1,St8sia1,Tcf7,Tgfbr3,Tspan32,Txnip,Zfp36,Zfp36l1

GO:0050798 activated T cell proliferation 4 8 0.00021 Fyn,Itgal,Itgb2,Satb1


differentiation13 284 0.00021

Ccl5,Ccr7,Ctla2a,Dtx1,Foxp1,Id2,Il7r,Lef1,Nckap1l,Pglyrp1,Tespa1,Zfp36l1,Z

fp36l2

GO:0050863 regulation of T cell activation 13 287 0.00022 Ccl5,Ccr7,Ctla2a,Dtx1,Il6st,Il7r,Itgal,Lgals1,Nckap1l,Pag1,Peli1,Rac2,Tespa1

GO:0006955 immune response 24 914 0.00024B2m,Ccl5,Ccr7,Ccr9,Cd3d,Cd3g,Crip1,Cyba,Evl,Foxp1,Fyn,Gpr183,H2-D1,H2-

K1,H2-Q7,Itgal,Lef1,Lgals1,Myo1f,Pag1,Pglyrp1,Rgcc,Serpina3g,Tgfbr3

GO:0009605 response to external stimulus 36 1794 0.00033

Auts2,B2m,Bhlhe40,Ccl5,Ccr7,Ccr9,Cox4i1,Cxcr3,Cyba,Ddit4,Evl,Foxo1,Fox

p1,Fyn,Gpr183,H2-

K1,Id2,Itgb2,Lef1,Lsp1,Ly6a,Myo1f,Nckap1l,Peli1,Pglyrp1,Plac8,Psmb9,Rac2

,S1pr1,Satb1,Sell,Sesn1,Tbxa2r,Tspan32,Txnip,Zfp36

GO:0030155 regulation of cell adhesion 19 624 0.00033Ccl5,Ccr7,Cytip,Dtx1,Il6st,Il7r,Itgal,Lef1,Lgals1,Myo1f,Nckap1l,Pag1,Peli1,R

ac2,Rgcc,S100a10,S1pr1,St6gal1,Tespa1

GO:0030334 regulation of cell migration 22 805 0.00033Ccl5,Ccr7,Cxcr3,Evl,Foxp1,Gpr183,Il6st,Lef1,Myo1f,Nckap1l,Numb,Pecam1,

Rac2,Rapgef4,Rgcc,S1pr1,Sell,Ssh2,Sun2,Tbxa2r,Tgfbr3,Tmsb4x


differentiation10 171 0.00034 Ccr7,Ctla2a,Dtx1,Id2,Il7r,Nckap1l,Pglyrp1,Tespa1,Zfp36l1,Zfp36l2

GO:1904894positive regulation of receptor

signaling pathway via STAT7 72 0.00041 Ccl5,Crlf3,Fyn,Il6ra,Il6st,Il7r,Pecam1

GO:0007159 leukocyte cell-cell adhesion 6 48 0.00050 Ccl5,Itgal,Itgb2,Pecam1,Rac2,Sell

GO:0040012 regulation of locomotion 23 924 0.00075Ccl5,Ccr7,Cxcr3,Evl,Foxp1,Gpr183,Il6st,Lef1,Myo1f,Nckap1l,Numb,Pecam1,

Rac2,Rapgef4,Rgcc,S1pr1,Sell,Ssh2,St6gal1,Sun2,Tbxa2r,Tgfbr3,Tmsb4x

Full Western blot scans for all figures.

Figure 1C:

HEM1 in PBMCs:

WAVE2 in PBMCs:

ABI1 in PBMCs:

HSP90 in PBMCs:

HEM1 (upper box)/Abi1 (lower box) in Jurkat cells:

WAVE2 (upper scan)/GAPDH (lower scan) in Jurkat cells:

Figure 1D:

Figure 2D:

p-ERK in Jurkat cells:

Total ERK in Jurkat cells:

HSP90 in Jurkat cells:

Figure 7D

IgM stimulation: p-AKT in B-LCLs

IgM stimulation: Total AKT in B-LCLs

Fibronectin stimulation: p-AKT in B-LCLs

Fibronectin stimulation: Total AKT in B-LCLs

ICAM1 stimulation: p-AKT in B-LCLs

ICAM1 stimulation: Total AKT in B-LCLs

Figure S1D

HEM1 in expanded T cells:

WAVE2 in expanded T cells:

ABI1 in expanded T cells:

GAPDH in expanded T cells:

HEM1 in B-LCLs :

WAVE2 (upper box) and ABI1 (lower box) in B-LCLs:

GAPDH in B-LCLs:

Figure S1E

CYFIP1 in B-LCLs:

HSP90 in B-LCLs:

CYFIP1 (right) and Hsp90 (left) in PBMCs:

BRK1 in expanded T (upper box) cells and B-LCLs (lower box):

GAPDH in expanded T cells (upper box) and B-LCLs (lower box):

Figure S4B:

supplementary materials for · theresia e. b. stradal, nima rezaei, kaan boztug* *corresponding...

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