IMMUNOLOGY

An autoimmune disease variant ofIgG1 modulates B cell activationand differentiationXiangjun Chen1, Xiaolin Sun2*, Wei Yang3*, Bing Yang1*, Xiaozhen Zhao2,Shuting Chen1, Lili He1, Hui Chen4, Changmei Yang1, Le Xiao1, Zai Chang3,Jianping Guo2, Jing He2, Fuping Zhang5, Fang Zheng6, Zhibin Hu7, Zhiyong Yang8,Jizhong Lou4, Wenjie Zheng9, Hai Qi10, Chenqi Xu11, Hong Zhang12, Hongying Shan13,Xu-jie Zhou12, Qingwen Wang13, Yi Shi14,15, Luhua Lai16, Zhanguo Li2†, Wanli Liu1,17†

The maintenance of autoreactive B cells in a quiescent state is crucial for preventingautoimmunity. Here we identify a variant of human immunoglobulin G1 (IgG1) with a Gly396→Argsubstitution (hIgG1-G396R), which positively correlates with systemic lupus erythematosus.In induced lupus models, murine homolog Gly390→Arg (G390R) knockin mice generateexcessive numbers of plasma cells, leading to a burst of broad-spectrum autoantibodies.Thisenhanced production of antibodies is also observed in hapten-immunized G390Rmice, as wellas in influenza-vaccinated human G396R homozygous carriers.This variant potentiates thephosphorylation of the IgG1 immunoglobulin tail tyrosine (ITT) motif.This, in turn, alters theavailability of phospho-ITT to trigger longer adaptor protein Grb2 dwell times in immunologicalsynapses, leading to hyper–Grb2–Bruton’s tyrosine kinase (Btk) signaling upon antigenbinding.Thus, the hIgG1-G396R variant is important for both lupus pathogenesis and antibodyresponses after vaccination.

Autoimmune diseases such as systemic lupuserythematosus (SLE) are characterizedby the presence of large numbers of self-reactive antibodies that induce depositionof immune complexes (ICs), leading to in-

flammation and tissue damage (1). Autoreactivityis pervasive in the antibody repertoire of humanB cells across different developmental stages (2).It is especially enriched in the peripheral immuno-globulin G–positive (IgG+) memory B cell poolbut is efficiently diminished in the plasma cellcompartment in healthy individuals (2–4). How-ever, these checkpoints fail in patients withautoimmune diseases. It remains unclear howautoreactive IgG+ B cells are maintained in aquiescent state under physiological immunehomeostasis and how these checkpoints arebroken in pathological conditions. IgG–B cellreceptor (IgG-BCR) potently enhances memoryIgG antibody responses via the evolutionarilyconserved cytoplasmic tail of membrane-bound

IgG (mIgG-tail) (5–10). The mIgG-tail amplifiesBCR signaling via its phospho–immunoglobulintail tyrosine (ITT) motif, which recruits theadaptor protein Grb2 to enhance Ca2+ mobiliza-tion, synergistically with Bruton’s tyrosine kinase(Btk) and phospholipase C–g2 (PLC-g2) (5, 11).Here we identified a single-nucleotide poly-

morphism (SNP) rs117518546, which results ina glycine-to-arginine substitution at codon 396in human IgG1 (hIgG1-G396R) (fig. S1A and tableS1). This SNP was common in East Asian pop-ulations (fig. S1B) and was significantly corre-lated with susceptibility to SLE (tables S2 andS3). The G396R variant frequency was subs-tantially enriched in SLE patients comparedwith that of criteria-matched controls in threeindependent cohorts from multiclinical centersin China (1786 healthy controls versus 1838 SLEpatients in total, Pearson’s chi-square test andbinary logistic regression analysis, P = 6.0 × 10−5).Furthermore, the G396R variant was associated

with a more severe disease phenotype, includingearlier onset, multiple organ involvement, andhigher SLE disease activity; specifically aggra-vated autoantibody production; and inflammation(Fig. 1A and table S4). The variant drove an auto-antibody subclass profile shift toward IgG1-isotypepredominance in G396R patients (Fig. 1B). Thus,hIgG1-G396R is a risk locus for SLE.We generated knockin mice harboring the

murine homolog mIgG1-G390R (denoted asIghg1T/T or G390R mice) (figs. S1C and S2, Aand B). Under normal conditions, there wereno significant differences in terms of naturalantibodies or other evident phenotypes betweenwild-type (WT) and G390Rmice (fig. S2C). In thebm12 splenocyte–inducible lupus model (12),the levels of IgG1 subclass anti–double-strandedDNA (dsDNA), anti–Smith D (SmD), and anti-nuclear antibodies (ANA)were notably increasedin G390R mice (Fig. 1, C and D, and fig. S3A).Autoantigen microarrays further confirmed aclear increase in IgG1 but not IgM and IgG2bautoantibody production in G390R mice (Fig.1E and fig. S3B). Moreover, G390R mice showedenlarged glomeruli and substantial IgG1 depositscontaining ICs along with moderate IgG2b dep-osition (Fig. 1F). Similar results were also ob-served in a second lupusmodel by using apoptoticthymocytes (fig. S3, C to E). In aged mice, theG390R variant facilitated autonomous IgG1 auto-antibody production (fig. S3F). Thus, the G390Rvariant promotes autoantibody production dur-ing autoimmune disease progression.Six weeks after the induction of autoimmunity

with bm12 splenocytes, there was a fourfold in-crease in the number of IgG1+ germinal center(GC) B cells in G390R mice compared with thatof WTmice (Fig. 2A and fig. S4A). IgG1+ memoryB cells only mildly increased in G390Rmice (Fig.2B). Notably, there was an almost sixfold increasein IgG1+ plasma cells in both the spleens and bonemarrow of G390R mice (Fig. 2, C to E, and fig.S4A). This phenomenon was also seen in modelsof apoptotic thymocyte-induced lupus and aging(fig. S4, B andC). In a competitivemodel, inwhichWT or G390R B cells were adoptively transferredinto B cell–deficient hosts (fig. S4D), more IgG1+

plasma cells were differentiated in the G390Rgroup than in the WT group relative to the in-ternal control. The competition index for theG390R group was 50% higher than that for the

RESEARCH

Chen et al., Science 362, 700–705 (2018) 9 November 2018 1 of 6

1Ministry of Education Key Laboratory of Protein Sciences, Center for Life Sciences, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Institute forImmunology, School of Life Sciences, Tsinghua University, Beijing 100084, China. 2Department of Rheumatology and Immunology, Peking University People's Hospital, Beijing Key Laboratoryfor Rheumatism and Immune Diagnosis (BZ0135), Peking-Tsinghua Center for Life Sciences, State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100044, China.3School of Life Sciences, Tsinghua University, Beijing 100084, China. 4Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. 5KeyLaboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. 6Department of Immunology, School of BasicMedicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. 7Department of Epidemiology, Center for Global Health, School of Public Health,Nanjing Medical University, Nanjing 211166, China. 8Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143, USA. 9Department of Rheumatologyand Clinical Immunology, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100730, China. 10Tsinghua-Peking Centerfor Life Sciences, Laboratory of Dynamic Immunobiology, School of Medicine, Tsinghua University, Beijing 100084, China. 11State Key Laboratory of Molecular Biology, Shanghai ScienceResearch Center, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China. 12RenalDivision, Peking University First Hospital, Peking University Institute of Nephrology, Key Laboratory of Renal Disease, Ministry of Health of China, Beijing 100034, China. 13Department ofRheumatism and Immunology, Peking University Shenzhen Hospital, Shenzhen 518036, China. 14CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology,University of Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100101, China. 15Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science,Chinese Academy of Sciences, Beijing 100101, China. 16BNLMS, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, and Peking-Tsinghua Center for Life Sciencesat College of Chemistry and Molecular Engineering, Center for Quantitative Biology, Peking University, Beijing 100871, China. 17Beijing Key Lab for Immunological Research on ChronicDiseases, Beijing 100084, China.*These authors contributed equally to this work.†Corresponding author. Email: liulab@tsinghua.edu.cn (W.L.); li99@bjmu.edu.cn (Z.L.)

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Chen et al., Science 362, 700–705 (2018) 9 November 2018 2 of 6

NS

NSNS

A

C

B

D

F

E8.2 10.8 11.5 23.1 20.722.2 14.7 11.4 18.8

Contro

ls

Arthrit

is

Seros

itis

Rayna

ud's

Prote

inuria

Anti-d

sDNA

Anti-S

SA

Anti-S

SB

Anti-r

-RNP

0

20

40

60

80

100

120WTHeteroG396R

Per

cent

age

(%)

P<0.0001

P=0.0444

WT

Heter

o

G396R

0.0

0.5

1.0

1.5

Rat

io

P=0.0048

P=0.0033

WT

Heter

o

G396R

0.0

0.5

1.0

1.5

2.0

2.5

3.0

P=0.0911

P=0.0322

WT

Heter

o

G396R

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

NS

IgG1/IgG2 IgG1/IgG3 IgG2/IgG3

IgG1

IgG2b

0

0.5

1.0

1.5

2.0

2.5

3.0

AN

A M

FI

(x 1

03 ) P<0.0001

P=0.0364

WTG390R

IgG1

IgG2b

0

1.0

2.0

3.0

4.0

IgG

dep

ositi

on M

FI (

x 10

3 )

P<0.0001P=0.0183

WTG390R

10

IgM IgG1

WT G390R1 2 3 4 5 1 2 3 4 5

WT G390R1 2 3 4 5 1 2 3 4 5

-2

Log2

(sig

nal i

nten

sity

)

IgM

IgG1

IgG2b

IgG2c

IgG3

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

Ant

i-ds

DN

A (

OD

490) WT

G390R

DAPI ANA Merge

WT

G39

0R

WT G390R

Fig. 1. An SLE-correlated SNP variant increases autoantibody produc-tion. (A) Genotypic correlation analysis of the human IgG1-G396R variant withclinical manifestations in SLE patients (279 controls and 251 SLE patients,as detailed in table S4).The percentages indicate the portion of G396Rhomozygotes. Hetero, heterozygous; SSA, Sjögren’s syndrome–relatedantigenA;SSB,Sjögren’s syndrome–relatedantigenB; r-RNP, ribonucleoproteins.(B) Ratio of IgG subclass for ANA in WT (n = 22), Hetero (n = 19), and G396R(n = 13) SLE patients. One-way analysis of variance (ANOVA), IgG1/IgG2(P = 0.0017), IgG1/IgG3 (P = 0.0552), and IgG2/IgG3 (P = 0.9268).(C) Anti-dsDNA antibodies detected in serum from bm12-induced WT (n = 7)and G390R (n = 5) mice 3 weeks after induction. OD490, optical density at a

wavelength of 490 nm. (D) Immunofluorescence of IgG1+ ANA with HEp-2reactivity in week 3 serum from bm12-induced mice (WT, n = 6; G390R, n = 6).Mean fluorescence intensity (MFI) of IgG1 and IgG2b subclass ANA wasquantified. DAPI,4′,6-diamidino-2-phenylindole. (E) Autoantigenmicroarraywasused to detect autoantibodies in serum as in (C). (F) Immunofluorescenceof deposited IgG1 ICs in mice glomeruli at week 6 after induction. Statisticalanalysis of MFI of IgG1 and IgG2b ICs in glomeruli.The scale bar represents20 mm in (D) and (F). Unpaired two-tailed Student’s t tests, (B) to (D) and (F).NS, not significant. Red bars indicate means. At least three sections wereanalyzed for each sample in (F). Data are representative of at least twoindependent experiments in (B) to (D) and (F).

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WT group (fig. S4E). Thus, increased B cell dif-ferentiation upon antigen stimulation, espe-cially plasma cell generation, may be responsiblefor excessive autoantibody production inG390R mice.Positively charged amino acids in IgH

complementarity-determining region 3 (CDR3)are associated with antibody autoreactivity (13),and indeed, these types of amino acids wereenriched in IgG1+ plasma cells from G390R micecompared with those from WT mice (Fig. 2F).Moreover, the length of the Igg1 CDR3, whichpotentially predicts autoreactivity enrichments(2), was prominently increased in the G390Rpopulation (fig. S4F). We also observed reducedIg variable (V) gene VH1 and joining (J) gene JH1usage, but increased VH5, VH14, and JH4 usagein G390R IgG1+ plasma cells (fig. S4G).The enhanced antibody production by the

hIgG1-G396R variant may also have effects onphysiological humoral responses to non-self anti-gens. Thus, we conducted influenza vaccinationexperiments in healthy humanWT controls andG396R homozygous carriers (Fig. 3A). The G396Rvariant significantly potentiated the generationof influenza virus–specific IgG1 (Fig. 3B) andonly showed a relative minor effect on IgG2production. We also confirmed these effects in4-hydroxy-3-nitrophenylacetyl (NP) eight-keyholelimpet hemocyanin (KLH)–immunized mice (Fig.3C). NP-specific IgG1 antibody responses were

almost twofold higher in G390Rmice and three-fold higher during antigen recall responses (Fig.3D). Affinity maturation did not appear to besignificantly affected (fig. S4H). The increase inantibody production was in line with the en-hanced numbers of NP-specific IgG1+ GC B andplasma cells in G390R mice (Fig. 3, E and F).Thus, this variant enhances antibody productionin both autoimmune disorders and in physiolog-ical humoral responses upon vaccination.Because IgG1+ light-zone GC B cells in bm12-

induced G390R mice up-regulated plasma cellfate favoring transcription profiles (Fig. 4A), wethen investigated the membrane-proximal signal-ing upon B cell activation (8, 14). BCR micro-cluster formation, the synaptic recruitment ofGrb2 and Btk, calcium mobilization, and thephosphorylation of downstream signaling mol-ecules [Erk, S6, and nuclear factor–kB (NF-kB)]were significantly enhanced, indicating heightenedsignaling mediated by the variant (Fig. 4, B and C,and fig. S5, A to H). This effect was also observedin IgG1+ primary B cells from the peripheral bloodof G396R homozygotes compared with thosefromWTSLEpatients (Fig. 4Dand fig. S5, I and J).In a fluorescence resonance energy transfer

(FRET)–based phospho-ITT activation reporter(fig. S6, A to C), G390R variants showed sig-nificantly higher FRET ratio changes comparedwith WT upon antigen stimulation (Fig. 4E).Thus, the G390R variant promotes the activation

of the phospho-ITT–Grb2 signaling module.Additional single-molecule imaging experimentsrevealed a more confined Grb2 motion in theimmunological synapse of the G390R variantcompared with that of WT primary B cells (Fig.4F). Thus, two potential models were proposedto explain the excessive activation of phospho-ITT–Grb2 signaling module: Increased Grb2binding to the phospho-ITT motif and/or in-creased ITT-motif phosphorylation. Isothermaltitration calorimetry assays demonstrated sim-ilar binding between the Grb2 protein and eitherthe WT or G390R phospho-ITT motif peptide(fig. S7A). In the secondmodel, both Syk and Lyntyrosine kinases are involved in the phosphoryl-ation of the ITT motif (11). G390R ITT phospho-rylation by Syk was only mildly higher (fig. S7, Band C). Substantially enhanced phosphorylationof the G390R ITT motif by Lyn was observedin vitro (Fig. 4G and fig. S7D) and in vivo (fig. S7E).Finally, to identify the structural basis for

excessive G390R ITT motif phosphorylation byLyn, we carried out molecular dynamics simu-lations of the Lyn kinase domain bound to eitherthe WT or the G390R ITT motif. We constructedcomplex models of substrate peptide and kinasedomain based on the structure of the activehuman Lyn kinase domain (Protein Data Bank3A4O) (15). The antiparallel b sheet formed bythe substrate peptide and the activation loop ofkinase domain persisted for the length of each

Chen et al., Science 362, 700–705 (2018) 9 November 2018 3 of 6

WT

G39

0R

GL7 IgG1

Fas

12.6 ± 1.31.0 ± 0.2

21.7 ± 1.42.0 ± 0.2

SP GC B IgG1+ SP GC BA

C

4.2 ± 0.5

11.0 ± 0.8

0.29 ± 0.04

0.71 ± 0.05

CD

138

B220 IgG1

SP PC IgG1+ SP PC

B

0

1

2

3

4

5

6

7

IgG

1+ G

C B

cel

ls (

X10

5 ) P<0.0001

WT G390R

0.0

0.5

1.0

1.5

2.0

2.5

SP

IgG

1+ P

C (

X10

5 ) P<0.0001

WT G390R

WT

G39

0R

D

0.16 ± 0.02

0.36 ± 0.04

8.6 ± 2.9

20.0 ± 3.2

CD

138

B220 IgG1

BM PC IgG1+ BM PC

0

2

4

6

8

10

12

14

IgG

1+ P

C (

/104

BM

cel

ls)

P=0.0005

WT G390R

WT

G39

0R

0

3

6

9

12

15

IgG

1+ M

em B

cel

ls (

X10

5 ) P=0.0309

WT G390R

2.6 ± 0.5

7.7 ± 1.0

IgD IgG1

CD

38

IgG1+ SP Mem B

WT

G39

0R

E

F

WT0

20

40

60

80

100

120Mem BPlasma

Rel

ativ

e P

erce

ntag

e (%

)

G390R

P=0.0018

0 1 20

20

40

60

80WTG390R

Per

cent

age

(%)

P<0.0001

Positively charged residues

Fig. 2. The IgG1-G390R variant promotes plasma cell accumulationin induced autoimmune models. (A to D) Flow cytometric analysesof IgG1+ GC B cells, memory B (Mem B) cells, spleen (SP), and bonemarrow (BM) plasma cells (PC) in WT (n = 6) and G390R (n = 7) mice atweek 6 after bm12 splenocyte induction. GC B cells (GL7+, Fas+) werepregated on B220+ cells. Percentage (means ± SEM) of B cell subsetsand corresponding comparison of cell numbers (right) are indicated.(E) The relative percentage (means ± SEM) of IgG1+ plasma cells and

memory B cells in fate-selected cells of WT in comparison to those ofG390R mice. (F) Frequency (means ± SEM) of Igg1 CDR3 with differentnumbers of positively charged amino acids in IgG1+ bone marrowplasma cells (WT CDR3, n = 64; G390R CDR3, n = 66) from bm12-inducedWT (n = 3) and G390R mice (n = 3). Unpaired two-tailed Student’st tests, (A) to (E). Two-way ANOVA, (F). Bars indicate means.Representative data are from at least two independent experimentsin (A) to (F).

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trajectory. Additionally, the Asp-Phe-Gly (DFG)motif and aC helix of the Lyn kinase domainwere maintained in an active conformation (Fig.4H and fig. S7, F and G). Notably, the Arg390

residue in the G390R ITT motif formed an ad-ditional hydrogen bond with the backbone car-bonyl of Asn290 in Lyn kinase domain, whichwas not available for the Gly390 residue in theWT ITT motif (Fig. 4H). Furthermore, the dis-tance between the hydroxyl group of catalyticresidue Tyr385 and g-phosphate group of adeno-sine triphosphate (ATP) was significantly shorterfor the G390R variant than for WT (Fig. 4I andfig. S7H), suggesting more stable contacts andpotentially more effective phosphotransfer for

the G390R variant. Indeed, the binding freeenergy of the G390R variant was consistentlylower than that of the WT ITT motif (Fig. 4H).Together, these computational results suggestthe higher binding ability of the G390R variantto active Lyn kinase.The G390R variant potentiates ITT-motif

phosphorylation and alters the availability of thephospho-ITT motif, thereby triggering a signifi-cantly longer dwell time of Grb2 in the immuno-logical synapse. This, in turn, changes the synapticGrb2 recruitment from a “recruit-and-escape”model to a “recruit-and-confine”model, in whichthe initially recruited but subsequently escapedGrb2 can be recaptured by the proximal phospho-

ITT motifs within the synapse (fig. S8). Thisresults in excessive G390R variant IgG1+ B cellactivation and differentiation to plasma cellsproducing increased IgG1 antibodies in the con-text of both autoimmune disorders and phys-iological humoral responses upon infection orvaccination. This enhancement of G390R variantIgG1+ B cells may have some bona fide effects onthe production of other IgG subclass antibodiesby potential mechanisms: (i) Enhanced IgG1+

B cell activation may promote the function ofT follicular helper cells (16) to potentiate theactivation and differentiation of other IgGsubclass B cells; (ii) increased IgG1+ antibodiesmay drive the activation and proliferation of

Chen et al., Science 362, 700–705 (2018) 9 November 2018 4 of 6

0

2

4

6

8

10

12

Fol

d ch

ange

Inf

luen

za

spec

ific

IgG

1 A

b tit

er

WT G396R

P=0.0094

IgG1

P=0.3957

WT G396R

Fol

d ch

ange

Inf

luen

za

spec

ific

IgG

2 A

b tit

er

0

2

4

6

8

10

12

14

16

IgG2B D

0.0

0.2

0.4

0.6

0.8

1.0

NP

+ Ig

G1+

GC

B c

ell

num

ber

(×10

5 )

P=0.0011

WT G390R

0

10

20

30

40

NP

+Ig

G1+

PC

/ 10

6 B

M c

ells

P=0.0009

WT G390R0

0.5

1.0

1.5

2.0

NP

+Ig

G1+

Mem

B c

ell

num

ber

(×10

4 )

P=0.3182

WT G390R

E

0

1.0

2.0

3.0

4.0

5.0

SP

NP

+Ig

G1+

PC

nu

mbe

r (×

103 )

P=0.0151

G390RWT

0

20

40

60

80

100 P=0.0005

0

0.2

0.4

0.6

0.8

1.0 P=0.0160

G390RWT

SP

NP

+Ig

G1+

PC

nu

mbe

r (×

104 )

NP

+Ig

G1+

PC

/ 10

6 B

M c

ells

G390RWT

F

WTHomozygous

G396RHomozygous

Genotyping

Pre-immuSera

Post-immuSera

Influenza vaccination

Day 20Day 0

Day 1

A C NP8-KLHi.p. immunization

(Day 0)

9.0 ± 0.34.3 ± 0.2

NP

IgG1

47.2 ± 1.646.1 ± 3.5

GL-7

CD

38

32.0 ± 6.3 57.1 ± 2.7

IgG1

NP

27.3 ± 5.115.0 ± 4.0

IgG1

NP

WT G390R WT G390R

WT G390R WT G390R

NP+ IgG1+ GC B

NP+ IgG1+ Mem B

NP+ IgG1+ SP PC

NP+ IgG1+ BM PC

14Day 0 7

GC B Mem BPlasma

35 42

NP8-KLHi.p.

NP8-KLHRecallSera

Sera

IgM

IgG1

IgG2b

IgG2c

IgG3

89

101112131415161718

NP

-spe

cific

ant

ibod

y tit

er (

log2

)

P=0.0158 WTG390R

NSNS

NS

NS

IgM

IgG1

IgG2b

IgG2c

IgG3

89

101112131415161718

P=0.0097

NS

NS

NSNS

WTG390R

Fig. 3. The IgG1-G390R variant facilitates IgG1 antibody production inphysiological humoral responses. (A) Schematic diagram of the humaninfluenza vaccination study.WTand G396R homozygous healthy volunteerswere influenza vaccinated on day 1. Pre- and postimmune sera were collectedon day 0 and day 20. (B) Influenza-specific IgG1 and IgG2 antibodyresponses in vaccinatedWT (n = 6) andG396R (n = 3) healthy volunteers. Foldchange of antibody titers on day 20 to day 0was quantified. (C) Diagrammaticrepresentation of the NP8-KLH immunization in mice. GC B, memory B,and plasma cells were analyzed on days 7 (GC B) and 14 (memory B andplasma cells). (D) NP-specific antibody titers in WT (n = 7 or 3) and G390R

(n = 5) mice on days 14 (left) and 42 (right) after NP8-KLH immunization.(E) Flow cytometric analyses of NP-specific IgG1+ GC (pregated on B220+,GL7+, CD38−) on day 7, memory B cells (pregated on B220+, IgG1+, NP+),spleen and bone marrow plasma cells (pregated on B220lo, CD138+) onday 14 in NP8-KLH immunized WT (n = 4 or 5) and G390R (n = 4) mice.Percentage (means ± SEM) and B cell–subset numbers were compared.(F) Spleen and bonemarrow plasma cell numbers in NP recall responses (WT,n = 3; G390R, n = 3). Unpaired two-tailed Student’s t tests, (C) to (F). Redbars indicatemeans. NS, not significant. Data in (C) to (F) are representative ofat least two independent experiments.

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Chen et al., Science 362, 700–705 (2018) 9 November 2018 5 of 6

A C

B

E

F

-6

-4

-2

0

2

4

6F

old

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log2

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WT

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Bcl

6

Irf4

Blim

p1

c-m

yb

Pax

5

IgG1+ LZ GC B

0 5 10 150

0.5

1.0

1.5

2.0

2.5

3.0

Time (min)

mC

herr

y-G

rb2

Tot

al F

I (×

107 )

WTG390R

P<0.0001P=0.0002

P=0.0164

NS

Ctrl WT

G390R

0

1

2

3

4

5

6

Dw

ell t

ime

(log

2, s

)

P<0.0001H

I

25kD

35kD

Y/F WT G390R

p-ITT

Lyn - +- + - +35kD

25kD

anti-p-Tyr100

GST-mIgG-tail

anti-GST

GST

p-ITT/GSTRatio0.04 0.22 0.08 1 0.11 3.17

G

ITT motif ITT motif peptidepeptide

Lyn kinase domain

ATP

MgMg2+2+

Tyr 385

Arg 390

Asn 290Asn 290

Arg 390Arg 390

H bondH bond

Asn 290

ΔGbind (WT)=

ΔGbind (G390R)=

-39.8 ± 8.3 kcal mol-1

-47.0 ± 6.8 kcal mol-1

pTyr 397

Activation loop

Lys 139

Glu 164αC helix

DFGmotif

0 2 4 6 8 10 12 14 16 18 200.00

0.05

0.10

0.15

0.20

WTG390R

Distance (Å)

Pro

babi

lity

0 2 4 6 8 10 12

1.1

1.2

1.3

1.4 WTG390R

Time (min)

Nor

mal

ized

Rat

io(m

Cer

ulea

n/F

RE

T)

P<0.0001

WT

G39

0R

0 min 2 min 4 min 8 min 12 min

1.0

1.2

1.4

Ratio

Normalized mCerulean/FRET channel Ratio

0

1.0

2.0

3.0

4.0

5.0

6.0

7.0 P=0.0048

WT G396R0

1.0

2.0

3.0

4.0

5.0

6.0

Rec

ruite

d G

rb2

Tot

al F

I (×

106 )

P=0.0372

WT G396R

WT

G39

6R

CD20 IgG1 Grb2 Merge

NS

0.0

2.0

4.0

6.0

8.0

BC

R T

otal

FI (

×10

7 )

0 5 10 20Antigen (nM)

WT

G390R

P<0.0001P=0.0072P<0.0001

D

BCR

WT G390R

mCherry-Grb2

WT

G39

0R

0 min 5 min 10 min 15 min

Fig. 4. The IgG1-G390R variant induces excessive IgG-BCR signaling bypotentiating phospho-ITT–Grb2 signaling module. (A) Transcriptionprofile of Bcl6, Irf4, Blimp1, c-myb, and Pax5 in IgG1+ light-zone GC B cells(B220+, GL-7+, Fas+, CD86hi, CXCR4lo) from WT (n = 3) and G390R (n = 3)mice upon immunization. (B) Total fluorescence intensity (FI) of synapticaccumulated BCRs were compared in IgG1+ class-switched WTand G390Rprimary B cells with different concentrations of antigen stimulation. (C)Dynamics of Grb2 recruitment to the immunological synapse in WTandG390R class-switched IgG1+ primary B cells (n > 13). (D) Synaptic IgG1-BCRaccumulation and Grb2 recruitment in IgG1+ B cells from the peripheral bloodof WT (n = 7) and G396R homozygous (n = 5) SLE patients after surrogateantigen stimulation. (E) Heat map indicates ratio dynamics of mCerulean tocpV (FRETchannel) fluorescence intensity in cells expressing FRET-basedphospho-ITTactivation biosensor with WTor G390R ITTmotifs after antigenstimulation. Ratio dynamics were quantified, and ratios were normalized to theinitial frame upon activation. (F) Trajectories of mEos3.1-Grb2 (colored) in

immunological synapse (gray). Dwell time of mEos3.1-Grb2 in activated IgG1+

class-switched WTor G390R primary B cells (n > 10) are calculated andcompared, with resting WTcells as control. (G) In vitro phosphorylation assaywith purified glutathione S-transferase (GST)–fused mIgG-tail protein tocompare the phosphorylation of WTor G390R ITTmotif by active Lyn kinase.p, phosphorylated. (H) Predicted ITTmotif peptide binding modes with theactive Lyn kinase domain. A snapshot of G390R ITTmotif:Lyn kinase complexstructure from molecular dynamics simulations and molecular mechanics–generalized Born surface area (MM-GBSA) binding free energy (DGbind)are shown. (I) Probability distributions of the distance between the hydroxylgroup of catalytic residue Tyr385 and the g-phosphate group of ATP in 100-nssimulations with WTor G390R ITTmotif peptide docking to the active Lynkinase domain in 50 to 100 ns of the simulations.The scale bars represent1.6 mm in (C) to (F). Unpaired two-tailed Student’s t tests in (B) to (D) and (F).Two-way ANOVA in (E). Red bars indicate means. NS, not significant. Dataare representative of three independent experiments in (A) to (G).

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dendritic cells, which then enhance general B cellactivation and differentiation (17); (iii) IgG1+

B cells may undergo class switching again toother IgG subclasses (18); (iv) IgG1+ B cellsmay produce inflammatory cytokines such asinterleukin-12 (IL-12) and tumor necrosis factor–a(TNF-a) to regulate the differentiation of otherIgG-subclass plasma cells (19); and (v) excessiveIgG1 antibodies may promote antigen presenta-tion to activate B cells producing other IgG-subclass antibodies (20). Thus, we contend thatIgG-BCR not only promotes enhanced memoryresponses but also leads to the developmentand relapse of autoimmune diseases whendysregulated.

REFERENCES AND NOTES

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ACKNOWLEDGMENTS

We thank S. K. Pierce, J. Lukszo, and K. Rajewsky for providingexperimental materials. We thank Q. Z. Li and I. Raman inMicroarray Core Facility, University of Texas Southwestern MedicalCenter, for support with the autoantigen microarray. We thankX. Wang, J. Wu, and Z. Wang for discussions. We thank P. Tolar,C. Wu, and D. Long for critical reading of this manuscript. Funding:This work is supported by funds from the National Natural Science

Foundation of China (8182500030, 81730043, and 81621002 toW.L. and 31530020 to Z.L.), Ministry of Science and Technology ofChina (2014CB542500-03 to W.L. and 2014CB541901 to J.G.), BeijingSci-Tech Program (Z171100000417007 to Z.L.), Beijing Nova Program(Z171100001117025 to X.S.), and Sanming Project of Medicine inShenzhen (SZSM201612009 to Q.W.). Author contributions:Conceptualization, X.C. and W.L.; funding acquisition, W.L., Z.L.,X.S., J.G., and Q.W.; investigation, X.C., B.Y., L.H., S.C., L.X.,C.Y., W.Y., X.Zha., J.G., J.H., X.Zho., H.Z., Q.W., and H.S.; methodology,X.C., X.S., and W.Y.; resources, W.Z., Y.S., Z.C., Z.S., Z.H., F.Zha.,F.Zhe., H.C., and J.L.; supervision, W.L., Z.L., and L.L.; writing–originaldraft, X.C. and W.Y.; and writing–review and editing, W.L., Z.L.,X.C., B.Y., X.S., L.L., Z.Y., H.Q., and C.X. All authors contributedto revising the manuscript. Competing interests: The authorsdeclare no financial or commercial conflicts of interest. Data andmaterials availability: All data described in this paper are presenteither in the main text or in the supplementary materials.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/362/6415/700/suppl/DC1Materials and MethodsFigs. S1 to S8Tables S1 to S4References (21–27)

11 September 2017; resubmitted 10 April 2018Accepted 19 September 2018Published online 4 October 201810.1126/science.aap9310

Chen et al., Science 362, 700–705 (2018) 9 November 2018 6 of 6

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An autoimmune disease variant of IgG1 modulates B cell activation and differentiation

Hai Qi, Chenqi Xu, Hong Zhang, Hongying Shan, Xu-jie Zhou, Qingwen Wang, Yi Shi, Luhua Lai, Zhanguo Li and Wanli LiuXiao, Zai Chang, Jianping Guo, Jing He, Fuping Zhang, Fang Zheng, Zhibin Hu, Zhiyong Yang, Jizhong Lou, Wenjie Zheng, Xiangjun Chen, Xiaolin Sun, Wei Yang, Bing Yang, Xiaozhen Zhao, Shuting Chen, Lili He, Hui Chen, Changmei Yang, Le

originally published online October 4, 2018DOI: 10.1126/science.aap9310 (6415), 700-705.362Science 

, this issue p. 700ScienceBruton's tyrosine kinase signaling after antigen binding.−Grb2−immunological synapses and hyper

enhanced IgG1 immunoglobulin tail tyrosine motif phosphorylation, triggering longer adaptor protein Grb2 dwell times in this SNP, as well as knockin mice, showed enhanced plasma cell accumulation and antibody production. This SNP

(hIgG1-G396R). This SNP was enriched in SLE patients and associated with increased disease severity. Humans with report the presence of a common IgG1 single-nucleotide polymorphism (SNP) in East Asian populations et al.Chen

positive autoreactive B cells in check are of intense interest.−the checkpoints that normally keep immunoglobulin G (IgG) titers of self-reactive antibodies. These result in immune complexes, inflammation, and tissue pathology. Consequently,

One common feature of autoimmune diseases like systemic lupus erythematosus (SLE) is the presence of highAn IgG1 SNP enhances autoimmunity

ARTICLE TOOLS http://science.sciencemag.org/content/362/6415/700

MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2018/10/03/science.aap9310.DC1

CONTENTRELATED http://stm.sciencemag.org/content/scitransmed/10/434/eaan2306.full

REFERENCES

http://science.sciencemag.org/content/362/6415/700#BIBLThis article cites 27 articles, 7 of which you can access for free

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