immunology an autoimmune disease variant of igg1 … · immunology an autoimmune disease variant of...
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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: [email protected] (W.L.); [email protected] (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
chan
ge (
log2
)
WT
G39
0R
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.
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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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