www.sciencemag.org/cgi/content/full/science.aap9310/DC1

Supplementary Material for An autoimmune disease variant of IgG1 modulates B cell activation and

differentiation

Xiangjun Chen, Xiaolin Sun, Wei Yang, Bing Yang, Xiaozhen Zhao, Shuting Chen, Lili He, Hui Chen, Changmei Yang, Le Xiao, Zai Chang, Jianping Guo, Jing He,

Fuping Zhang, Fang Zheng, Zhibin Hu, Zhiyong Yang, Jizhong Lou, Wenjie Zheng, Hai Qi, Chenqi Xu, Hong Zhang, Hongying Shan, Xujie Zhou, Qingwen Wang, Yi Shi,

Luhua Lai, Zhanguo Li*, Wanli Liu*

*Corresponding author. Email: liulab@tsinghua.edu.cn (W.L.); li99@bjmu.edu.cn (Z.L.)

Published 4 October 2018 as Science First Release DOI: 10.1126/science.aap9310

This PDF file includes:

Materials and Methods Figs. S1 to S8 Tables S1 to S4 References

1

Materials and Methods

Reagents and plasmids

WT and G390R variant ITT motif peptides (VPEYKNMIGQ, VPEYKNMIRQ), ITAM motif

peptide (ENLYEGLNLD), WT and G390R variant phospho-mIgG1-tail peptides

(KVKWIFSSVVELKQTLVPEY(*p)KNMIGQAP,

KVKWIFSSVVELKQTLVPEY(*p)KNMIRQAP) were purchased from Bankpeptide (Hefei,

China). Peptides were determined to 98% pure by mass spectrometry.

Fluorochrome-conjugated anti-mouse B220 (RA3-6B2), CD3ε (145-2C11), CD19 (1D3),

GL-7 (GL-7), IgM (II/41), and CD95 (15A7) antibodies were purchased from eBioscience,

rat anti-mouse CD38 (NIMR-5), IgD (11-26) antibodies from Southern Biotech, and biotin

conjugated rat anti-mouse CD138 (281-2) antibody from BD bioscience. Anti-mouse Ig

antibodies that were unconjugated or conjugated with biotin, fluorophore or peroxidase were

purchased from Jackson ImmunoResearch Laboratory. Plasmids for the expression of

IgG-BCR with WT, G390R or Y/F, Y/F-G390R variant mIgG-tail in primary B cells were

inserted in pMSCV backbone with mCherry driven by a dual-promoter. Plasmids for the

expression of B1-8 IgG1 heavy chain with cytoplasmic tail fused with a FRET based

phospho-ITT reporter were added in pcDNA3.1 backbone. The plasmid for the expression of

mEos3.1-Grb2 was inserted in the pMSCV backbone. Plasmids for the expression of

full-length (aa 1-217) or the SH2 domain (aa 56-150) of human Grb2 were added in

pGEX-6P-1 with Glutathione S-transferases (GST) tag at the N-terminus.

Mice

C57BL/6 (JAX664), C57BL/6-CD45.1 (JAX2014), bm12 (JAX1162) and μMT (JAX2288)

mice were purchased from the Jackson laboratory. The IGHG1T/T mouse was generated by

CRISPR/Cas9-based gene manipulation in C57BL/6 background and was backcrossed with

WT C57BL/6 for at least two generations. Mice were maintained in specific-pathogen free

conditions. Mouse experiments were performed according to governmental and institutional

guidelines for animal welfare.

Cell culture and infection with retrovirus

J558L cells were gifts from Dr. S. K. Pierce (NIAID, NIH, USA). They were cultured in

complete RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum, 2 mM

L-Glutamine (Gibco), 100 U/mL penicillin (Gibco), and 100 µg/mL streptomycin (Gibco).

Phospho-ITT activation reporter was transiently expressed in J558L cells by electroporation.

Splenic B cells from WT or G390R variant mice were purified by negative selection with

auto-MACS (Miltenyi). To express IgG-BCRs with WT, G390R, Y/F or Y/F-G390R

mIgG-tail variant in primary B cells, negatively purified B cells were first cultured in

medium with 10 μg/ml lipopolysaccharide (LPS) and 50 μM beta-mercaptoethanol for 24 h.

The activated B cells were then spin-infected for 1.5 h with retrovirus particles that were

prepared from the culture supernatant of Plat-E cells upon the transfection of pMSCV

plasmids. For the exogenous expression of mEos3.1-Grb2 or mCherry-Grb2, negatively

purified B cells from either WT or G390R mice were cultured with 10 μg/ml LPS (Sigma)

and 20 ng/ml recombinant mouse IL-4 (Sino Biological) for 72 h, and were then infected with

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retrovirus containing these two cDNAs respectively. Class-switched IgG1+ primary B cells

with expressing either mEos3.1-Grb2 or mCherry-Grb2 were used for single-particle tracking

and Grb2 recruitment experiments, respectively.

TaqMan probe based genotyping of rs117518546

SLE patients and healthy controls in this report were Chinese Han population, who were

enrolled with informed consent in qualifying university hospitals in Northern (Beijing) and

Southern (Shenzhen) China. The SLE patients fulfilling the 1997 revised classification

criteria of the American College of Rheumatology were identified for inclusion in this study.

Some patients also had clinical variables of interest available. The controls were validated

cohorts of healthy individuals not suffering from SLE or any other autoimmune disease. The

SLE patients and the healthy controls were age and sex matched. This study was approved by

the ethics committee of Peking University People’s Hospital. Genomic DNA was prepared

from peripheral blood samples of all volunteers. Genotyping of rs117518546 was performed

by using TaqMan probe based Real-Time PCR system. TaqMan probe C:

5′-VIC-CAGGAACATGATCGGACAG-NFQ-3′, TaqMan probe T:

5′-FAM-CAGGAACATGATCAGACAG-NFQ-3′ (Life technology). Relative quantification

of probes levels was calculated (7500 Sequence Detection System Software, ABI). Allele and

genotype frequencies were compared between cases and controls. Pearson’s Chi-square test

was applied to assess the allelic distribution between cases and controls. Binary logistic

regression analysis was applied to assess the genotypic distribution between cases and

controls or clinical manifestations within patient’s cohort. Odds Ratios (ORs) with 95%

confidence intervals (CIs) were calculated to estimate the relative risks.

IgG subclass distribution analysis

SLE patients were divided into three groups on the basis of different SNP genotypes: 396G/G

(WT), R/G (Hetero), and R/R (G396R). The amounts (OD450 value) of different IgG

subclass ANA antibodies were measured by Human Anti-ANA Screen ELISA Kit (Abcam)

with different human IgG-subclass specific antibodies (mouse anti-human IgG1 (HP6001),

IgG2 (31-7-4) or IgG3 (HP6050) antibodies, SouthernBiotech). IgG subclass distribution was

evaluated by comparing the ratio of IgG subclass (OD450 value) for ANA respectively:

IgG1/IgG2, IgG1/IgG3, and IgG2/IgG3.

Influenza vaccination in human volunteers

Healthy volunteers were recruited with informed consent. This study was approved by the

ethics committee for biomedical studies of Tsinghua University. Volunteers who had reported

fevers in the two weeks preceding the experiment or who had received influenza vaccination

in the previous winter season were excluded during recruitment. Volunteers were genotyped

with genomic DNA from buccal swabs, and those of either WT or homozygous SNP carriers

were selected for the influenza vaccination experiment. Subjects were vaccinated by

intramuscular injection of one-dose (0.5 ml) commercial trivalent inactivated influenza virus

vaccine (Sanofi Pasteur) composed of influenza strains from A/Michigan/45/2015 (H1N1)

pdm09-like virus, A/Hong Kong/4801/2014 (H3N2)-like virus, and B/Brisbane/60/2008-like

virus (WHO recommended composition of influenza virus vaccines for the 2017-2018

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influenza season). Sera were collected before (on day 0) and after (on day 20) influenza

vaccination to assess the production of influenza-specific antibody responses.

Influenza-specific IgG subclass antibody titers were detected with ELISA in a double-blinded

manner. Briefly, influenza vaccine contents (2 μg/ml) were coated on the ELISA plate at 4 ºC

overnight. The ELISA plates were blocked and then incubated with serum in the dilution of

1:100 (pre-immu) and 1:500 (post-immu) for IgG1, and 1:40 (pre-immu) and 1:100

(post-immu) for IgG2. Mouse anti-human IgG1 (HP6001) or IgG2 (31-7-4) (SouthernBiotech)

subclass-specific primary antibodies were used for subsequent OD measurements. Serum

samples from WT and G396R homozygous volunteers were detected in the same plate to

control plate-to-plate variation.

IgG1-G390R knock-in mice construction

IgG1-G390R knock-in mice were constructed with C57BL/6 background. sgRNA target

sequences were screened from mouse IGHG1 genome DNA sequence with online tool

(http://crispr.mit.edu/) and two sgRNA targeted sequences were selected

(5′-CATGATTGGGCAAGCACCCT-3′, 5′-GTGGGGTATAGGTCACCAC-3′). The

homology-directed repair (HDR) template was of mouse IGHG1 sequence, which had two

800-bp homology arms flanking the G390R mutation site and with mutated protospacer

adjacent motif (PAM) sites. Cas9 mRNA and sgRNA were in vitro-transcribed by using

mMESSAGE mMACHINE T7 Ultra kit, MEGAshortscript High Yield Transcription kit and

MEGAclear kit (Thermo Fisher). Cas9 mRNA (10 ng/μl), sgRNA1 (3 ng/μl), sgRNA2 (3

ng/μl) and HDR template (10 ng/μl) were mixed and delivered into zygotes by microinjection.

The generated mice harboring the IgG1-G390R variant (heterozygote) were verified by

sequencing the PCR products with murine genome DNA as the templates (Primers:

5′-CTATGCACTGCCCACATTGT-3′, 5′- GACACTGACATTGTGGGAACT-3′). The

acquired heterozygotes were backcrossed to C57BL/6 for at least two generations and then

the IGHG1T/T homozygote mice were obtained by heterozygote crossing.

Autoimmune mouse model

For the bm12 splenocyte-induced autoimmune model, splenocytes (107 per mouse) from age-

and gender- matched bm12 mice were adoptively transferred to 6-8-week-old WT or G390R

mice. For apoptotic thymocyte-induced autoimmune model (21), thymocytes were first

isolated from WT B6 mice, exposed to UV irradiation for 30 min and were then cultured for 3

h. Over 99% of thymocytes underwent apoptosis as detected by Annexin V staining. WT or

G390R variant mice were immunized with apoptotic thymocytes (107 per mouse) through

intravenous injection for four times on a weekly basis. An additional immunization was

performed with genomic DNA from apoptotic mouse lymphocytes (20 μg per mouse) and

purified GST-SmD (aa 83-119) protein (20 μg per mouse) through intraperitoneal injection at

week 5.

In vivo competitive assay

Splenic B cells from CD45.1 WT mice and CD45.2 WT or G390R mice were negatively

purified. The purity was over 95% as confirmed by flow cytometry with anti-mouse B220

staining. CD45.1+ WT B cells were mixed with CD45.2+ WT or G390R B cells at a ratio of

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1:2. They were then adoptively transferred to μMT recipient mice (1.2×107 total B cells per

mouse). Twenty-four hours after adoptive transfer, recipient mice were immunized with

apoptotic thymocytes (107 cells per mouse) by intravenous injection in a weekly basis. Five

days after the fourth injection, mice were sacrificed for B-cell-subset analysis by flow

cytometry.

Flow cytometry

Single-cell suspensions in MACS buffer (PBS, 1% FBS, 5 mM EDTA) were prepared before

staining. Primary cells from mouse spleen or bone marrow were first blocked with

anti-mouse CD16/CD32 (2.4G2, BD bioscience) antibody on ice for 20 min and then stained

with cell-marker specific antibodies on ice for 30 min. Cells were also stained with

propidium iodide (PI) to exclude the dead cells. Cells were washed and re-suspended to the

proper density with MACS buffer for LSRII analysis or AriaIII sorting. For IgG1+ plasma cell

staining, primary cells were first blocked and stained with fluorochrome-conjugated rat

anti-mouse B220 (RA3-6B2) and CD138 (281-2). After washing with MACS buffer once,

cells were fixed with 4% paraformaldehyde (PFA) for 20 min and then permeabilized with

PBS containing 0.1% Triton X-100 for 20 min. Subsequently, cells were stained with Alexa

Fluor 647-conjugated Fab fragment anti-mouse IgG1, Fc specific (Jackson ImmunoResearch)

on ice for 30 min.

Ig gene sequence analysis

Bone marrow plasma cells were sorted and pooled from bm12 splenocytes induced WT (n=3)

and G390R mice (n=3). Total RNA was isolated and cDNA was prepared. V region of Igγ1

was amplified by PCR with specific primers (22): MsVHE 5′-

GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG-3′; Cg inner Mix 5′-

GCTCAGGGAAATAGCCCTTGAC-3′. PCR products were cloned to pMD18-T vector and

single clones were sequenced. V region sequences were analyzed in ImMunoGeneTics tool

IMGT/V-QUEST (http://www.imgt.org/vquest/refseqh.html). The numbers of positively

charged amino acids in CDR3 were counted. The usages of VH and JH gene as well as the

length of CDR3 were subjected to statistical analysis by using two-way ANOVA.

NP immunization experiment

6-8-weeks-old age-matched WT and G390R mice were injected intraperitoneally with 5 μg

NP8-KLH and 20 μl Imject Alum (Thermo Fisher Scientific). Sera were collected on day 0

and weekly after immunization for the quantification of NP-specific antibody titers. Mice

were boosted on day 35 after the primary immunization with 5 μg of NP8-KLH. Different

NP-specific B-cell subsets were analyzed by flow cytometry at indicated time points.

Antibody detection in mouse serum

To detect the basal level of natural antibodies, 2 μg/ml goat anti-mouse kappa was coated on

the ELISA plate overnight at 4 ºC to capture antibodies in mouse sera. Different dilutions of

mouse sera from 7-week-old mice were added: IgM (1:8000), IgG1 (1:2000), IgG2b (1:4000),

IgG2c (1:500), IgG3 (1:3000), and IgE (1:500). ELISA plates were blocked with 0.3% gelatin

for 2 h in PBS and then incubated with serum for 1 h. Peroxidase-conjugated

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immunoglobulin subclass (IgM, IgG1, IgG2b, IgG2c, IgG3, IgE)-specific antibodies were

used for detection. For anti-dsDNA detection, deoxyribonucleic acid from calf thymus

(Sigma) was digested with S1 nuclease (Takara) and dialyzed in PBS (23). Calf thymus

dsDNA (3 μg/ml) was coated on ELISA plates with DNA-coating solution (Thermo Fisher)

overnight at 4 ºC. ELISA plates were coated with His-SmD (aa 83-119) proteins (2 μg/ml) for

the detection of SmD-specific autoantibodies. Autoantigen microarrays with mouse sera were

performed and analyzed in the Genomics and Microarray Core Facility, UT Southwestern

Medical Center. An autoantigen array super panel I containing 128 nuclear, cytoplasmic,

membrane and phospholipid autoantigens were used. Autoantigen-specific IgM, IgG1, and

IgG2b subclass antibodies were measured. Anti-nuclear antibodies (ANA) were quantified

through immunofluorescence with ANA detection slides coated with human epithelial larynx

carcinoma HEp-2 cells (Euroimmun) following the recommended protocols. For the

detection of NP-specific antibodies, ELISA plates were coated with 2 μg/ml NP8-BSA or

NP30-BSA.

Immunohistochemistry

To detect the deposition of immune complexes in glomeruli, kidneys were first harvested

from WT or G390R mice at week 6 after bm12 splenocyte induction, and fixed with 4%

paraformaldehyde. Samples were cryoprotected with 30% sucrose solution and then frozen in

O.C.T compound (Tissue-Tek) at −80 ºC. The kidneys were sliced using a Leica 3050

cryostat in 8-μm sections and attached to superfrost slides (Thermo Fisher). After rehydration,

sections were blocked with blocking buffer (100 mM Tris-HCl, pH 8.0, 0.3% Triton X-100, 2%

FBS, and 50 μg/ml goat non-specific IgG) for 30 minutes and then stained with Alexa Fluor

647-conjugated goat IgG anti-mouse IgG1, Fc specific (Jackson ImmunoResearch) in

staining buffer (100 mM Tris-HCl, pH 8.0, 0.3% Triton X-100) at 4 ºC for 12 hours. Slides

were washed and mounted with ProLong Gold Anti-fade Mountant containing 4′,

6-diamidino-2-phenylindole (DAPI) (Life Technologies). Sections were imaged with the

Olympus FLUOVIEW FV1000 confocal laser scanning microscope equipped with 4 lasers

(405 nm, 473 nm, 557 nm, and 635 nm) for fluorescence excitation, 2 photomultiplier tubes

(PMTs) for fluorescence detection, and a 10× objective lens. Images were processed and

analyzed using Image J (NIH, USA) and Image Pro Plus software (Media Cybernetics).

Calcium mobilization

Intracellular Ca2+ mobilization in primary B cells was monitored with ratiometric indo-1

indicator by flow cytometry. Primary B cells were incubated in HBSS buffer (with 1.26 mM

Ca2+) with 1 μM indo-1 and 1% FBS at 30 ºC for 30 min, washed and then re-suspended in

HBSS. During flow cytometic detection, basal levels of calcium were recorded for 30 s and

then 8 μg/ml or 4 μg/ml F(ab)2 fragment anti-mouse IgG (surrogate antigen) (Jackson

ImmunoResearch) was added to induce calcium mobilization in cells. The indo-1 ratio of

violet (390/40bp) to blue (525/50bp) fluorescence intensity was calculated in FlowJo.

Phospho-flow analysis

Class-switched IgG1+ primary B cells were stimulated with 5 μg/ml F(ab)2 fragment goat

anti-mouse IgG, Fc specific (Jackson ImmunoResearch) at 37 °C for different periods of time,

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and then fixed with 4% PFA. To measure the phosphorylation of intracellular signaling

molecules, cells were permeabilized with 90% methanol, blocked with 100 μg/ml

non-specific goat IgG and 2% FBS, and then stained with rabbit antibodies specific for

pErk1/2 (Thr202/Tyr204) (Cell Signaling, #4377), pS6 (Ser235/236) (Cell Signaling, #2211),

and pNF-κB p65 (Ser536) (Cell Signaling, #3033). Alexa Fluor 488-conjugated F(ab)2

fragment goat anti-rabbit IgG (Invitrogen) was used as the secondary antibody.

RNA isolation and qPCR

Total RNA was isolated from sorted cells by TRIzol reagent (Life technology) according to

the standard protocols. cDNA was prepared by reverse transcription-PCR (RT-PCR) with

RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher) following manufacturer’s

protocols. SYBR® Premix Ex Taq II (Tli RNaseH Plus) (Takara) was used for quantitative

PCR (qPCR) and each group was detected in triplicate. Primers for qPCR:

Pax5(5′-AGTCACAGCATAGTGTCTACAG-3′; 5′-ATGGAGTATGAGGAGCCC-3′),

Irf4(5′-CTCATCACAGCTCATGTGG-3′; 5′-CCTCAGGAAATGTCCAGTG-3′),

Blimp-1(5′-GCAAAGAGGTTATTGGCGT-3′; 5′-TGTAGACTTCACCGATGAGG-3′),

c-myb(5′-GAAAGTGCCTCACCAGCAAGGT-3′; 5′-CGAGCTTTCATGGTTGCTGGAAG

-3′), Bcl-6(5′-CAGAGATGTGCCTCCATACTGC-3′; 5′-

CTCCTCAGAGAAACGGCAGTCA-3′), GAPDH(5′-TGTGTCCGTCGTGGATCTGA-3′;

5′- TTGCTGTTGAAGTCGCAGGAG-3′).

TIRFM imaging

Surrogate antigen presenting planar lipid bilayers (PLBs) were prepared as previously

reported (8). After class-switched primary B cells were stained with Alexa Fluor

647-conjugated Fab fragment anti-mouse IgG, Fc specific, they were loaded on the 20 nM

F(ab)2 anti-mouse IgG surrogate antigen-presenting PLBs for 10 min and then fixed with 4%

PFA for 20 min. For intracellular immunofluorescence staining of phospho-Syk,

phospho-PI3K, and Btk, fixed cells were permeabilized as described above, followed by goat

non-specific IgG blocking. Cells were incubated with primary antibodies anti-phospho-Syk

(pY525/526) Ab (Cell Signaling, #2711), anti-phospho-PI3K p85 (pY458)/p55 (pY199) Ab

(Cell Signaling, #4228), rabbit anti-Btk Ab (BD Pharmingen, #56365) for 1 h , washed with

PBS and then stained with secondary antibody Alexa Fluor 488-conjugated F(ab)2 fragment

goat anti- rabbit IgG (Invitrogen). Chambered coverglass slides (Thermo Fisher Scientific)

with prepared samples were mounted on an Olympus IX-81 microscope equipped with Andor

iXon+ DU-897D electron-multiplying CCD camera, Olympus 100×1.49 NA objective lens,

and a TIRF port. TIRFM images were captured with a 100 ms exposure time. TIRFM images

were processed and analyzed using Image J (NIH, USA) and Image Pro Plus software (Media

Cybernetics).

Single-particle tracking

Retrovirus-infected class-switched IgG1+ primary B cells were labeled with Alexa Fluor

647-conjugated Fab fragment anti-mouse IgG, Fc specific (Jackson ImmunoResearch) and

were then loaded to PLBs presenting surrogate antigen F(ab)2 fragment anti-mouse IgM +

IgG (Jackson ImmunoResearch) for 5 min. IgG1+ B cells expressing mEos3.1-Grb2 were

7

selected for imaging. The Grb2 molecules were first photobleached with a high power 561

nm laser to eliminate the background signal. TIRFM images were then captured in the

time-lapse acquisition mode with a 30 ms exposure time, at 200 ms intervals for a total of 150

frames simultaneously illuminated with low power (2%) 405 nm laser and high-power (50%)

561 nm laser. The images were acquired within 25min for each group once cells were loaded

onto the chambered coverglass slides (Thermo Fisher Scientific). Single-particle-tracking

analysis of Grb2 molecule was performed using Matlab (Mathworks) codes as previously

reported (8). The track length of each trajectory was acquired to calculate the dwell time.

FRET measurements

A phospho-ITT activation biosensor was constructed according to the following steps: the

Grb2 SH2 domain was fused with an N-terminal Cerulean fluorescent protein, and the

mIgG-tail ITT motif was fused with a C-terminal circularly permutated Venus (cpV)

construct. These fluorophores were separated by a flexible linker similar to the previously

reported Src biosensor (24). For photobleaching-based dequenching FRET experiments,

J558L cells expressing phospho-ITT activation biosensor with pre-stained BCRs were loaded

onto the chambers with 20 nM NP8-BSA antigens on the PLBs. Cells were allowed to react

for 5 min and then dequenching FRET images were captured by TIRFM as previously

reported (9). mCerulean was excited with a 405 nm laser and visualized using 483/32 nm

bandpass filter. cpVenus was excited with a 488 nm laser line and visualized with a 525/50

nm bandpass filter. FRET efficiencies were calculated as (DQ-Q)/DQ, DQ and Q represented

dequenched and quenched mCerulean fluorescence intensities, respectively. For dynamic

measurements of FRET change upon cell activation, multi-wavelength timelapse TIRFM

images were acquired once J558L cells with pre-stained BCRs were placed onto chambers.

Three channels were recorded: the donor channel, FRET channel (405 nm excitation and

525/50 nm emission), and BCR channel. Imaging consisted of a 100 ms exposure time for a

512×512 pixel image with a 5 s interval for time-lapse acquisition. The mCerulean-to-FRET

channel fluorescence intensity ratio was calculated and normalized to the value in the initial

frame. All images were analyzed with Image J (NIH, USA) and representative images were

processed with Image-Pro Plus (Media Cybernetics).

Grb2 recruitment in B cells from SLE patients

Human PBMCs from SLE patients with WT or G396R homozygous genotypes were isolated

on Ficoll-Hypaque and were then frozen in FBS containing 10% DMSO. PBMCs were

thawed and cultured in RPMI 1640 medium for 1 h prior to each experiment. PBMCs were

first incubated with human FcR-blocking reagent (Miltenyi) and were then labeled with FITC

anti-human CD20 antibody (2H7, BD bioscience), Alexa Fluor 568-conjugated Fab fragment

anti-human IgG1, Fc specific, and Alexa Fluor 405-conjugated Fab fragment anti-human IgM,

Fc specific. PBMCs were suspended in HBSS and were then loaded onto chambered

coverglass slides (Thermo Fisher Scientific) containing planar lipid bilayers with biotin

anti-human kappa and lambda light chain antibody (Invitrogen) (10 nM each) . PBMCs were

fixed with 4% paraformaldehyde after 4 min with surrogate antigens and were then

permeabilized with 0.2% Triton X-100 for 20 min and blocked with 100 μg/ml goat

non-specific IgG for 1 h. PBMCs were subsequently incubated with mouse anti-Grb2

8

antibody followed by the staining with Alexa Fluor 647-conjugated goat anti-mouse IgG1

antibody (Jackson ImmunoResearch). The formation of BCR microclusters and the

recruitment of Grb2 in the immunological synapses of IgM-B cells or IgG1-B cells (CD20+,

IgG1+) were imaged with TIRFM and were then analyzed with Image J, Image-Pro Plus, and

Matlab as previously reported (8).

Protein expression and purification

The full length or SH2 domain of mouse Grb2 (residues 60–152) in a pGEX-6P-1 vector

were expressed as a GST tagged fusion protein in E.coli Rosetta (DE3). Protein expression

was induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 20 ºC for 12 h.

For protein purification, the bacterial pellet was suspended in lysis buffer (50 mM Tris-HCl,

pH 8.0 and 200 mM NaCl), and was lysed by sonication. After centrifugation lysed cells at

10,000 xg for 30 min, the supernatant was incubated with ProteinIso GST Resin (Transgen) at

4 ºC for 3 h and was then eluted with elution buffer (50 mM Tris-HCl, pH 8.0, 200 mM NaCl,

and 20 mM reduced glutathione). GST protein was separated by proteolytic cleavage with

TEV protease overnight at room temperature. The protein was further purified by

anion-exchange chromatograpahy with a NaCl gradient (0–1.0 M NaCl in 50 mM Tris-HCl,

pH 8.0) and molecular-sieve chromatography with a Superdex 200 column at 4 ºC. The final

purity was validated by SDS-PAGE.

Isothermal titration calorimetry

To measure the binding affinities of WT and G390R variants of mIgG1-tail to Grb2, we

performed ITC measurements. Purified full-length Grb2 protein was first dialyzed in ITC

buffer (50 mM Tris-HCl, pH 8.0, and 200 mM NaCl). WT or G390R-variant

phospho-mIgG-tail peptides were dissolved in ITC buffer. The concentration of protein and

peptides were measured based on their respective UV 280 nm absorption. The

thermodynamic measurements of binding reaction were achieved with MicroCal ITC200

calorimeter at 25 ºC. The phospho-peptides (0.9 mM) were titrated into an experimental cell

containing Grb2 protein (30 µM). Each titration comprised 20 injections of 0.4 μl for the 1st

injection and 2 μl for the rest injections. Corresponding “peptide to buffer” controls were

detected for background correction. Raw data was processed and fitted with MicroCal Origin

7.0 software in a “One Set of Sites” model.

ITT motif phosphorylation

For phospho-tyrosine immunoblot-based detection, purified WT, G390R-variant and

Y/F-variant GST-mIgG-tail proteins were used as the substrates. Protein substrates (0.75 μM),

active Lyn kinase (10 nM) (Sino Biological), and Adenosine 5’-triphosphate (ATP) disodium

solution (0.2 mM) (Sigma) were added into the 50 μl reaction buffer (25 mM HEPES, pH 7.4,

150 mM NaCl, 10 mM MgCl2, 10 mM MnCl2, 1.25 mM DTT, and 3 μM Na3VO4). The

reaction was performed at 30 ºC for 60 min. Anti-p-Tyr100 (Cell signaling, #9417) and

anti-GST (N100/13, Neuromab) antibody was used for the phospho-tyrosine and GST

immunoblot detection respectively. For the ITT motif phosphorylation by constitutively

active Syk (Syk-Y352D), 2×106 293T cells expressing constitutively active Syk or

BLNK-mCherry were lysed with 200 μl of lysis buffer (0.5% Triton X-100, 25 mM HEPES,

9

pH 7.4, 150 mM NaCl, 10m M MgCl2, 10 mM MnCl2, 1 mM PMSF, 2 mM Na3VO4, and 1%

protease inhibitor cocktail (Sigma)) and the supernatant was collected. Fifty microliters of

supernatant with active Syk was added to 50 μl of reaction buffer (0.4 mM ATP) with 1.5 μM

protein substrates or BLNK-mCherry, and allowed to react at 30 ºC for 60 min. Then 15 μl of

ProteinIso GST Resin (Transgen) was added to the mixture and incubated at 4°C for

additional 1 h. The Resin was subsequently washed and boiled in NuPAGE LDS sample

buffer (Invitrogen). Immunoblot detection of ITT motif phosphorylation was the same as Lyn

kinase assay. BLNK phosphorylation was detected with phospho-BLNK (Tyr96) antibody

(Cell signaling, #3601) and BLNK antibody (Cell signaling, #3602).

For ATP consumption based luminescence detection, WT, G390R-variant ITT motif peptides,

and ITAM-motif peptides were used as the substrates. Peptide substrates (50 μg/ml), active

Lyn kinase (15 nM) (Sino Biological), and ATP (0.5 μM) were added into a 50 μl of reaction

buffer. The reaction was performed at 30 ºC for 60 min. The remaining amount of ATP in the

reaction buffer was detected with an ATP quantification kit (beyotime).

To analyze the ITT motif tyrosine phosphorylation of endogenous IgG1, 3×107 Ramos cells

expressing WT or G390R IgG1-BCR were harvested and suspended in 600 μl of HBSS. Cells

were kept on ice (unstimulated) or pre-warmed at 37 ºC for 5 min, and were then stimulated

with 10 μg/ml goat anti-mouse IgG antibody for 5 min at 37 ºC. Subsequently, cells were

lysed for 20 min on ice with lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM

EDTA, 2 mM Na3VO4, 2% NP40, 1 mM PMSF, and 1% protease inhibitor cocktail (Sigma)).

Lysates were pelleted at 12,000 xg for 10 min at 4 ºC and the supernatants were collected for

immunoprecipitation. Twenty microliters of Protein A/G agarose beads (0.5 ml agarose/2.0

ml) (Santa Cruz) were then added. After an incubation of 8-12 h with mild agitation at 4 ºC,

Protein A/G agarose beads were washed with lysis buffer and were boiled in NuPAGE LDS

sample buffer to release the bound proteins. Immunoblots were performed and biotin-anti

pY100 antibody (Cell Signaling Technology) was then used to detect the ITT-tyrosine

phosphorylation.

MD simulations and MM/GBSA Calculations

To characterize the binding of ITT motif to the active Lyn kinase domain, we used the

structure of the active human Lyn kinase domain (PDB code: 3A4O) as the starting point. As

there are no reported structures of the peptide substrate-bound kinase domain of Lyn or other

Src family kinases, we referred to the complex structure of peptide substrate and ATP

analog-bound activated insulin receptor tyrosine kinase (PDB code: 1IR3) as a guide for

model building. ATP and Mg2+ ions were first docked to the active human Lyn kinase domain,

and then the activation loop was integrated according to the activation loop structure of

insulin receptor tyrosine kinase. The ITT motif peptide (TLVPEYKNMIGQAP, residues

380-393) was modeled into the active site using insulin receptor tyrosine kinase as the

template, with N- and C- termini of the substrate peptide in an extended conformation using

RosettaDesign (25).

All energy minimizations and molecular dynamics simulations were performed with

Amber14 using Amber ff99SB parameters and the TIP3P for water molecules. Parameters for

the phosphorylated Tyr397 were from the work of Homeyer et al (26). All the systems were

first minimized with positions of atoms in the proteins and ATP/Mg2+ restrained for 5000

10

steps and then without restraints for 2,000 steps. Then the whole system was heated from 100

K to 300 K and then maintained at 300 K using the weak-coupling algorithm at constant

volume. MD simulations were then performed at constant pressure with isotropic position

scaling for 100 ns, using time-steps of 2 fs. The van der Waals cutoff was set to 12 Å and

long-range electrostatic forces were calculated by the Particle Mesh Ewald (PME) method

(27).

The values of binding free energy of each substrate peptide to Lyn kinase domain were

calculated by MM/GBSA approach. Snapshots were taken from each 100-ns trajectory every

10 ps for MM/GBSA calculation. The van der Waals, electrostatics and internal energies of

peptides, kinase domain and complexes were calculated by sander module. The polar

solvation free energies were calculated by generalized Born (GB) approach with salt

concentration was set to 0.2 M. MD simulation experiments were performed in the High

Performance Computing Platform of the Center for Life Science, Beijing, China. MD

simulations data of G390R ITT motif:Lyn kinase complex structure are available in the

GitHub (https://github.com/proteincraft/Lyn_GR/).

11

Fig. S1. Human rs117518546 SNP leads to a G396R variant in the conserved

cytoplasmic tail of membrane bound IgG1 heavy chain

(A) Schematic of the human IgG1-BCR showing the ITT motif (green) and G396 residue (red)

at Y+5 in the mIgG-tail. (B) Minor allele frequency (MAF) of rs117518546 in different

populations according to the 1000 Genomes database. (C) Conserved sequence of the

mIgG-tail in different species and IgG subtypes.

12

13

Fig. S2. The G390R knock-in mouse generated by CRISPR-Cas9 system

(A) Alignment of the IGHG1 DNA sequence from NCBI database with the sequencing data of the

G390R knock-in mouse. Annotated are the sgRNA target sequences and protospacer adjacent motif

(PAM). Mutated alleles are highlighted. G390R variant site (red box) and ITT motif (blue box) are

marked. (B) Alignment of sequencing data with sequence from NCBI database in predicted potential

off-target sites and other IgG subclasses (IgG2b, IgG2c, and IgG3). (C) Basal levels of natural IgM,

IgG1, IgG2b, IgG2c, IgG3, IgE antibodies in the serum of non-immunized 6-week old WT (n=3) and

G390R (n=3) mice. Bars indicate means. NS, not significant (unpaired two-tailed t-test). Data are

representative of two independent experiments.

14

15

Fig. S3. The G390R variant promotes autoantibody production in induced autoimmune model

mice and in aged mice

(A) Anti-dsDNA and anti-SmD antibody dynamics in WT (n=7) and G390R (n=5) mice after bm12

splenocyte induction. Two-way ANOVA was used for statistical analysis between WT and G390R in

terms of the dynamics of the autoantibody responses over time. (B) Autoantigen microarray analyses

of IgG subclass autoantibodies in serum from WT (n=5) and G390R (n=5) mice, respectively. (C)

Anti-SmD antibodies in week 5 serum of WT and G390R mice after weekly intravenous injection of

107 apoptotic thymocytes for four times. (D and E) Immunofluorescence of ANA with HEp-2

reactivity in week 5 serum (D) and deposited IgG ICs in glomeruli at week 6 (E) from apoptotic

thymocytes induced mice (WT, n=3 or 5; G390R, n=3 or 4,). (F) Anti-dsDNA and anti-SmD

antibody levels in 25-week old aged mice without autoimmune induction (WT, n=8 or 6; G390R,

n=8). At least three sections were analyzed for each sample in (E). Unpaired two-tailed t-test in (C)

to (F). Bars indicate means. NS, not significant. Data are representative of two independent

experiments.

16

17

Fig. S4. G390R mice generate more plasma cells in induced autoimmune models

(A) Flow cytometric analyses of GC, Mem B cells and plasma cells in bm12-induced mice (WT, n=6;

G390R, n=7) 6 weeks after induction. (B and C) Flow cytometric analyses of IgG1+ GC B cells,

Mem B cells, SP and BM plasma cells in apoptotic thymocytes immunized (6 weeks after

immunization) or 25-week aged WT (n=5 or 4) and G390R (n=4) mice without autoimmune

induction. (D) Schematic diagram of the in vivo competitive model. Purified 4×106 CD45.1+ WT B

cells and 8×106 CD45.2+ WT or G390R B cells were mixed and adoptively transferred to B cell

deficient (μMT) mice at day 0, followed by four weekly injections of 107 apoptotic thymocytes,

indicated by arrows. (E) The CD45.1 (WT) and CD45.2 (WT or G390R) B cell populations in spleen

naïve B (B220+), plasma cell (B220lo, CD138+) and IgG1+ plasma cells in the competitive model

(WT, n=6; G390R, n=7). Comparative analysis of competition index of WT and G396R groups in

splenic plasma cells and IgG1+ plasma cells. The formula for Competition index calculation is

indicated, which is calculated by dividing the plasma subset/naïve B cell ratio in the CD45.2

population by the same ratio in the CD45.1 population. (F) The length distribution of Igγ1 CDR3 in

the IgG1+ bone marrow plasma cells (WT, n=64; G390R, n=66) from bm12 splenocyte-induced WT

(n=3) and G390R mice (n=3). (G) IgH V gene and J gene usage in IgG1+ bone marrow plasma cells

as (F). (H) Statistical comparison of NP-specific IgG1 and IgG2c antibody affinity maturation in WT

(n=4 or 3) and G390R (n=4 or 3) mice after NP immunization. Affinity maturation is defined as the

ratio of anti-NP8/anti-NP30 antibody titers. Unpaired two-tailed t-test, (A) to (C), (E), and (H). Bars

indicate means ± SEM. Two-way ANOVA, (F) and (G). Data are representative of two independent

experiments.

18

19

Fig. S5. The IgG1-G390R variant elevates IgG-BCR signaling

(A) Flow cytometry shows the efficiency (means ± SEM) of class-switched IgG1+ WT and G390R

primary B cells on day 4 after 10 μg/ml LPS and 25 ng/ml IL-4 stimulation. (B) Cell surface

expression levels of IgG1-BCR in class-switched WT and G390R primary B cells. (C) The size of

contact area with surrogate antigen presenting PLBs was compared in IgG1+ class-switched WT and

G390R primary B cells with different concentrations of antigen stimulation. (D and E) Recruitment

and phosphorylation of the IgG-BCR downstream signaling molecule Syk (D) and PI3K (p85) (E) in

B cell immunological synapses (IS). Representative total internal reflection fluorescence microscopy

(TIRFM) images are shown and total immunofluorescence intensity (FI) was quantified. (F) Calcium

mobilization in primary B cells expressing either WT or G390R IgG1-BCR after surrogate antigen

stimulation (arrow). (G) Synaptic recruitment of Btk in IgG1+ class-switched WT and G390R

primary B cells after membrane bound antigen stimulation. (H) Phosphorylation kinetics of Erk, S6

and NF-κB (p65) in IgG1+ primary cells after BCR crosslinking. (I) IgM-BCRs accumulation and

Grb2 recruitment in IS of IgM+ primary B cells from WT or G396R homozygous SLE patients after

antigen stimulation. (J) Representative TIRFM images of IgG1-BCR microclusters from the

activated human IgG1+ primary B cells. FI and microcluster size were quantified. Dots indicate

individual measurements (n>850). Bars denote means. Unpaired two-tailed t-test, (C) to (E), (G), (I),

and (J). Two-way ANOVA, (F) and (H). NS, not significant. Data are representative of at least two

independent experiments.

20

Fig. S6. FRET based biosensor for the examination of phospho-ITT motif activation

(A) Schematic diagram showing the composition of the phospho-ITT motif activation reporter.

mCerulean and cpVenus are the FRET donor and receptor respectively. Reporter is fused to the C

terminus of the IgG transmembrane domain (TM) through flexible linker 1. Linker sequences are

listed. Biosensors with WT, G390R, and Y/F-ITT motif were constructed. mCerulean and cpVenus

yield high FRET in the quiescent state because of the close proximity between the N- and C-terminus

of the Grb2 SH2 domain, while phosphorylation of the ITT motif induces SH2 binding and in turn

increases the distance between mCerulean and cpVenus, thus decreasing the FRET. (B) FRET change

after NP8-BSA antigen stimulation in J558L cells expressing B1-8 IgG-BCRs fused with WT

phospho-ITT motif activation reporter. Representative TIRFM images and heat maps show the

fluorescence intensity change of mCerulean before and after photobleaching (BP and AP). FRET

efficiencies were calculated and compared. (C) FRET efficiencies of the reporter with the Y/F-ITT

motif were detected and calculated as in (B). Bars indicate means. NS, not significant (unpaired

two-tailed t-test). Data are representative of three independent experiments.

21

22

Fig. S7. G390R variant confers higher phosphorylation of ITT motif by Lyn kinase

(A) Isothermal titration calorimetry (ITC) of Grb2 binding to the WT or G390R phospho-mIgG-tail

peptide. Raw isothermal plot of binding (upper panel), integrated binding isotherm and fitted curve

(lower panel) using one-site binding model are shown. (B and C) ITT motif phosphorylation by a

constitutively active Syk (Syk*) in 293T cells. The phosphorylation of BLNK was provided as the

positive control for the kinase activity of the constitutively active Syk in (B). (D) Luciferin

luminescence indicates ATP consumption in an in vitro Lyn kinase assay with immunoreceptor

tyrosine-based activation motif (ITAM) peptide provided as a positive control on the left panel. WT

or G390R ITT motif peptides were provided as the substrates on the right panel. Bars denote means.

Unpaired two-tailed t-tests. (E) Comparison of ITT motif phosphorylation in Ramos B cells

expressing WT or G390R IgG1-BCRs after surrogate antigen stimulation. (F) The root mean square

deviation (RMSD) profile presented as a function of simulation time indicates the stability of

simulation systems. (G) The dynamic of distance between Lys 139 and Glu 164 during the

processing of the molecular dynamic simulation, which supports the stabilization of active kinase

domain conformation in the simulation. (H) The dynamic of distance between the hydroxyl group of

catalytic residue Tyr 385 and γ-phosphate group of ATP in 100-ns simulations with WT or G390R

ITT motif peptide docking to the active Lyn kinase domain. Data are representative of at least two

independent experiments in (A) to (E).

23

24

Fig. S8. The distinct recruitment models of Grb2 adaptor protein in the immunological

synapses of WT and G390R variant

Upon antigen engagement, ITT motif tyrosine in the cytoplasmic tail of membrane-bound IgG1 will

first be phosphorylated by active Lyn kinase in the B cell immunological synapse. The phospho-ITT

motifs then recruit and bind Grb2 in immunological synapse. Stably confined adaptor protein Grb2

serves as the platform for signalosome assembly with Btk and PLCγ2. Recruited Grb2 can also

escape or dissociate from phospho-ITT motifs after the initial binding, leading to non-effective

downstream signalosome assembly. This is described as the “recruit and escape” model of Grb2

recruitment in immunological synapse (A). The G390R variant potentiates ITT motif tyrosine

phosphorylation by active Lyn kinase, promoting the availability (or density) of phospho-ITT motifs

within the B cell immunological synapse. Thus, the dissociated Grb2 proteins can be rebound by the

proximal or nearby phospho-ITT motifs and are thus confined within the B cell immunological

synapse at high efficiency. This is described as the “recruit and confine” model of Grb2 recruitment

in immunological synapse correspondingly (B). The “recruit and escape” model is likely more

frequent in WT IgG1+ B cells (A). In contrast, the “recruit and confine” model is likely the dominant

paradigm in G390R IgG1+ B cells (B).

25

Table. S1. SNP locus rs117518546 in human IgG1

Table. S2. Demographic characteristics of SLE cohort and controls

Cohort I (People’s hospital in Beijing) and II (First hospital in Beijing) represent northern Chinese

population, and cohort III (Shenzhen Hospital in Shenzhen) represents southern Chinese population.

In cohort II, high numbers of male patients were enrolled into the studies to exclude the potential

effects of gender to the correlations between SLE and SNP rs117518546.

26

27

Table. S3. SNP allele frequency and genotype distribution analyses in controls

and SLE patients

OR: odds ratio. CI: confidence interval. Pearson’s Chi-square test and binary logistic

regression analysis were applied to assess the allelic and genotypic distribution

between cases and controls, respectively.

28

Table. S4. Correlation analysis of G396R variant and clinical manifestations in SLE

patients

Binary logistic regression analysis of the risk of human rs117518546 SNP for clinical

manifestations in SLE patients. OR: odds ratio. CI: confidence interval. SLEDAI: Systemic

Lupus Erythematosus Disease Activity Index. Binary logistic regression analysis to calculate

statistical significance.

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