Supplemental Material
The supplemental material includes:
1) Supplemental Methods 2) Supplemental Table 1-5 3) Supplemental Figure 1-8
1 Methods
2 Study Design
3 We conducted a prospective, open-label study of B cells in SLE patients (referring to
4 the first cohort). All SLE patients were recruited from Renji Hospital and fulfilled the
5 1997 revised classification criteria of the American College of Rheumatology
6 (ACR),1 and 16 patients met the ACR criteria for lupus nephritis [LN].2 Clinical and
7 laboratory data were measured at baseline and at week 4, 12, 24. Clinical assessments
8 included changes in SELENA–SLEDAI score, resolution of organ involvement
9 presented at the time of enrollment, and laboratory variables including
10 immunoglobulin A/M/G, complement C3 and C4, anti-nuclear antibody spectrum,
11 anti-dsDNA antibodies, anti-nucleosome antibodies, and urinary protein excretion,
12 and changes in corticosteroid and immunosuppressant dose. Peripheral blood was also
13 collected at the same time. During the study, corticosteroid doses were tapered at the
14 discretion of the treating physicians, and immunosuppressants were also increased or
15 decreased based on the decision of physicians. Patients were classified into remission
16 group and flair/resistant group at week 24, as defined by SELENA–SLEDAI scores.
17 Patients in remission group had a SELENA–SLEDAI scores ≤ 4, without any clinical
18 manifestations after 24 weeks of treatment. Patients whose SELENA–SLEDAI scores
19 increased ≥ 3 (flair)3 or SELENA–SLEDAI scores ≥ 4, excluding serology alone, on
20 ≥ 2 consecutive visits (persistently active disease, PAD)4 were classified into
21 flair/resistant group. The study was commenced in January 2015, and concluded in
22 May 2017. 23 Participants
24 The study recruited 3 male and 51 female newly-onset SLE patients aged 18–65
25 years with moderate-to-severe disease activity, without any treatment background of
26 corticosteroids, antimalarials, or immunosuppressants before. Exclusion criteria
27 included a history of treatment with corticosteroids, antimalarials, or
28 immunosuppressants, active infection (including hepatitis B or C virus, Epstein-Barr
29 virus, human immunodeficiency virus or Mycobacterium tuberculosis infection), a
30 history of malignancy, combined with other autoimmune diseases, and pregnancy or
31 lactation in females. These newly-onset SLE patients were mainly used for phenotype,
32 B cell cytokine production and the follow-up studies. Another 50 active SLE patients
33 were recruited for functional and molecular studies. 33 rheumatoid arthritis patients,
34 who fulfilled the ARA 1987 reversed criteria for the classification of rheumatoid
35 arthritis [RA]5 without using corticosteroids, antimalarials and immunosuppressants
36 before were included as disease control; 30 healthy donors aged 18–65 years were
37 recruited as healthy controls.
38 A second cohort of 19 newly-onset SLE patients were recruited from February
39 2017 to January 2019 in Renji Hospital. Frozen peripheral blood mononuclear cells
40 (PBMCs) were taken from this cohort via Biobank with a purpose to validate the
41 relationship between the frequency of circulating AtM and disease activity.
42
43 Cell isolation 44 PBMCs were collected by Lymphoprep (Axis-Shield) density gradient centrifugation
45 of heparinized blood or buffy coat. Primary human B cells or CD4+ T were purified
46 from PBMCs by using a MiniMACS column and CD19 beads or CD4 beads
47 (Miltenyi Biotec) by positive magnetic selection. For fluorescence-activated cell
hi 48 sorting (FACS) sorting, CD24-/loCD20 atypical memory B cells (AtMs) and
+ + + 49 CD24 CD20 CD27 conventional memory B cells (CMs) were sorted on a sorter
50 (MoFlo Astrios, Beckman Coulter). The purity of sorted B cells or B cell subsets was
51 routinely more than 95%.
52
53 Cell culture
54 Complete medium consisted of RPMI-1640 containing L-alanyl-L-glutamine
55 dipeptide supplemented with 10% FBS, 5 x 10-5 M of 2-ME (Sigma), and antibiotics
56 (penicillin 100 U/ml, streptomycin 100 µg/ml, Gibco BRL). To study T-bet+ B cell
57 proliferation and differentiation in vitro, purified B cells from healthy donors were
58 labeled with Carboxyfluorescein succinimidyl ester (CFSE, Biolegend) and cultured
59 in complete medium and stimulated with 100 ng/mL human IFNγ (cat. 285-IF-100,
60 R&D Systems), 1 µg/mL R848 (cat. tlrl-r848 Invivogen) or in combination for 5
61 days. For certain experiments, 0.1nM, 1nM and 10 nM Rapamycin (cat. S1039,
62 Selleck) was added in the culture. The expressions of p-S6 and T-bet in B cells were
63 detected by flow cytometry. To measure the changes of TBX21 mRNA in stimulated
64 B cells, the cells were collected 6 hours after culture and TBX21 mRNA was
65 determined by qRT-PCR. 66
67 Plasma cell differentiation
68 Purified AtMs and CMs from active lupus patients were plated at 1~5 × 104 cells per
69 well in 96-well round-bottom plate and stimulated with 10 ng/mL human IL-2 (cat.
70 202-IL-010, R&D), human IL-10 10ng/mL (cat. 200-10, PeproTech) and 5 µg/mL
71 CpG2006 (cat. tlrl-2006, Invivogen), with or without 0.01nM and 0.1nM Rapamycin.
72 At day 7, cells were collected and the differentiation of plasma cells was determined
73 by flow cytometry. Cell-free supernatants were collected and stored at -80°C for total
74 IgA/IgM/IgG and auto-antibody detection.
75
76 B Cell apoptosis and proliferation assay
77 Purified AtMs and CMs from active lupus patients were sorted and plated at 1~5 ×
4 78 10 per well in complete medium for indicated time. For B cell proliferation analysis,
79 cells were sorted and labelled with CFSE in complete medium for 72h in the absence
80 or presence of conditions bellow: Goat F(ab’)2 anti-human Kappa -UNLB 10µg/mL
81 (cat. 2062-01, Southern Biotech) and Goat F(ab’)2 anti-human Lambda-UNLB
82 10µg/mL (cat. 2072-01, Southern Biotech), CD40L (human) (multimeric) (rec)
83 0.5µg/mL (cat. AG-40B-0010, AdipoGen), R848 1µg/mL, CpG2006 5µg/mL (cat.
84 tlrl-2006, Invivogen), or human IL-2 10ng/mL (cat. 202-IL-010, R&D), human IL-10
85 10ng/mL (cat. 200-10, PeproTech) combined with CpG2006 5µg/mL. Cells viability
86 and apoptosis were assessed with an apoptosis detection kit (containing Annexin
87 VI/Propidium Iodide), according to the manufacturer’s instructions (eBiosciences). 88
89 T cell proliferation assay
+ + 90 CD4 T cells were sorted from healthy donors and labelled with CFSE. Then CD4 T
4 91 cells were plated at 5 × 10 cells per well in 96-well round-bottom plate pre-coated
92 with anti human CD3 (clone OKT3, Biolegend), in the absence or presence of AtMs
93 and CMs sorted from lupus patients at a ratio of 1:1, with a final volume of 200µL
94 complete medium. On day 5, cells were first stained with Zombie YellowTM Dye
+ 95 (Biolegend) to eliminate dead cells and the proliferation of CD4 T cells was detected
96 by flow cytometry.
97
98 Flow cytometry and antibodies
99 PBMCs or enriched B cells were routinely stained with fluorochrome-labelled
100 antibodies in staining buffer (PBS with 5% FBS, 2mM EDTA and 0.09% NaN3). For
101 intracellular cytokine staining, cells were stimulated with 50 ng/ml phorbol
102 12-myristate 13-acetate (cat. P8139, PMA) (Sigma) plus 1 µg/mL Ionomycin (cat.
103 407952, Sigma) in the presence of 5 µg/mL Brefeldin A (Biolegend) or with
104 additional 5 µg/mL CpG2006 (cat. tlrl-2006, Invivogen) for 5 h. Then cells were
105 washed, and stained with Zombie YellowTM Dye (Biolegend) to eliminate dead cells.
106 After surface staining with antibodies against CD20, CD24 and CD27, then cells were
107 fixed, permeabilized and stained for the detection of intracellular cytokines IL-6 and
108 TNF-α according to the manufacturer’s instructions (BD Biosciences). For
109 intracellular staining of transcription factors, cells were fixed and permeabilized with 110 the Transcription Factor Buffer Set (cat. 00-5523-00, eBioscience). For BCR
111 crosslinking experiment, PBMCs were first stained for surface markers (CD20, CD24
112 and CD27). Cells were then incubated at 37 ̊C for 30 min in a volume of 200 µL
113 complete medium before adding Goat F(ab’)2 anti-human Kappa/Lambda at a final
114 concentration of 10 µg/mL and incubated at 37 ̊C for 5 min. After that, cells were
115 immediately fixed with the same volume of 4% Paraformaldehyde, and permeabilized
116 and intracellularly stained with a BD Cytofix/Cytoperm kit according to the
117 manufacturer’s instructions (BD Biosciences). Data were acquired using a LSR
118 Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree
119 Star).
120 Antibodies against the following molecules were obtained from BD Biosciences:
121 CD4 (clone OKT4), CD11a (clone G43-25B), CD19 (clone SJ25C1), CD38 (clone
122 HB7), CD69 (clone FN50), CD70 (clone KI-24), CD98 (clone UM7F8),
123 CD268/BAFFR (clone 11C1), CCR10 (clone 1B5), CXCR5 (clone RF8B2),
124 p-Syk(Y348) (clone Y348), p-Syk(Y352) (clone n3kobu5), p-PLCγ2(Y759) (clone
125 K86-689.37), PAX5 (clone 1H9), BCL6 (clone K112-91), BATF (clone S39-1060),
126 XBP-1s (clone Q3-695), Caspase-3 (clone C92-605),and Isotype antibodies (clone
127 MOPC-21). Antibodies against the following molecules were obtained from
128 Biolegend: CD3 (clone UCHT1), CD11c (clone Bu15), CD20 (clone 2H7), CD21
129 (clone Bu32), CD22 (clone HIB22), CD24 (clone ML5), CD25 (clone BC96), CD27
130 (clone O323), CD62L (clone DREG56), CD72 (clone 3F3), CD95/FAS (clone DX2),
131 CD267/TACI (clone 1A1), CD269/BCMA (clone 19F2), CD272/BTLA (clone 132 MIH26), CD360/IL-21R (clone 17A12), CCR5 (clone HEK/1/85a), CCR7 (clone
133 G043H7), CXCR3 (clone G025H7), CXCR4 (clone 12G5), FcRL3 (clone H5/FcRL3),
134 FcRL4 (clone 413D12), HLADR (clone L243), ICOSL (clone 2D3), NFATc1 (clone
135 7A6), PD1 (clone EH12.2H7), PDL1 (clone 29E.2A3), and Bcl-2 (clone 100).
136 Antibodies against the following molecules were obtained from eBioscience: CD32
137 (clone 6C4), CD38 (clone HIT2), CD44 (clone IM7), CD80 (clone 2D10.4), CD86
138 (clone IT2.2), CD217/IL-17RA (clone 424LTS), EOMES (clone WD1928), FcRL5
139 (clone 509F6), HELIOS (clone 22F6), LT2/LILRB1 (clone HP-F1), ILT4/LILRB2
140 (clone 42D1), p-S6(S235/236) (clone cupk43k), p-Akt(S473) (clone SDRNR),
141 p-mTOR (S2448) (clone MRRBY), p-Btk(Y551/511) (clone M4G3LN), T-bet (clone
142 eBio4B10), IRF4 (clone 3E4), and IL-6 (clone MQ2-13A5), and TNF-α (clone
143 MAb11). Antibodies against c-Myc (clone D84C12), FOXO1 (clone C29H4), and
144 NFATc2 (clone D4381) were obtained from Cell Signaling Technology. Antibodies
145 against HIF1α (clone 241812) and Siglec6 (clone 767329) were from R&D Systems.
146 Antibodies against IFNAR (clone REA124) and BLIMP1 (clone 3h2E8) were from
147 Miltenyi Biotec and Novus Biologicals, respectively.
148
149 ELISA and Luminex assays
150 Human IgM/IgA/IgG were measured by ELISA with appropriate antibody pairs (IgM:
151 Coating antibody, Goat anti-human IgM, cat. 2020-01; Detecting antibody, Mouse
152 Anti-human IgM-HRP, cat. 9020-05. IgA: Coating antibody, Goat anti-human H+L,
153 cat. 2010-01; Detecting antibody: Goat anti-human IgA-HRP, cat. 2050-05. IgG: 154 Coating antibody, Goat anti-human H+L, cat. 2010-01; Detecting antibody, Goat
155 anti-human IgG-HRP, cat. 2040-05. All antibodies were from Southern Biotech).
156 Titers of anti-nucleosome antibody and anti-dsDNA were measured by ANUA and
157 anti-dsDNA kits (EUROIMMUN). Concentrations of cytokines in the plasma were
158 detected by the multiplexed Luminex xMAP assay according to the manufacturer’s
159 instructions (eBioscience).
160
161 Quantitative real-time PCR
162 Total RNA was extracted from B cells by 1,000 µL TRIzol (Invitrogen). RNA
163 extraction was performed according to instructions provided by manufacturer. RNA
164 was then reverse transcribed (Takara) and analyzed by qPCR. qPCR was performed
165 on DNA using the following oligonucleotide primers: TBX21 (forward,
166 5′-TTGAGGTGAACGACGGAGAG-3′; reverse,
167 5′-CCAAGGAATTGACAGTTGGGT-3′), GAPDH (forward,
168 5′-CCCATCACCATCTTCCAGGA-3′; reverse,
169 5′-TTGTCATACCAGGAAATGAGC-3′). Samples were normalized to GAPDH and
170 represented as fold change using the ΔΔCT method.
171
172 RNA sequencing and data processing
173 AtMs and CMs were sorted from 6 active SLE patients without using any prednisone,
174 hydroxychloroquine or immunosuppressants before. CMs from 5 healthy donors were
4 175 also sorted. 2~10 × 10 cells per sample were used for RNA-seq. RNA was isolated 176 by TRIzol (Invitrogen). The RNA quality and integrity were analyzed by Qubit 2.0
177 (Life Technologies, USA) and Bioanalyzer 2100 (Agilent, Germany). For library
178 preparation, 1 µg total RNA were captured by NEBNext Oligo d(T)25 beads (NEB,
179 USA), sheared to fragments of ~250bp, and reverse transcribed by NEBNext RNA
180 first and Strand Synthesis Module second (NEB, USA). The products were
181 end-repaired, A-tailed and ligated to Illumina sequencing adapters and amplified by
182 PCR. The sequencing library was qualified by Qubit 2.0 (Life technologies, USA)
183 and Bioanalyzer 2100 (Agilent, Germany), then sequenced on Illumina Hiseq X-Ten
184 with 2×150 bp paired-end sequencing, which was controlled by Hiseq Control
185 Software (HCS).
186
187 Differential analysis
188 Limma+voom were used for differential analysis between cell groups.6 Unpaired and
189 paired sample test were used for comparisons of CMs-HD versus CMs-SLE, and
190 CMs-SLE versus AtMs-SLE, respectively. P value was adjusted by the
191 Benjamini-Hochberg method and P<0.05 was considered to be statistically
192 significant.
193
194 Principal component analysis (PCA)
195 Kennel PCA was used to perform multivariate statistic analysis on the mRNA profile
196 datasets (log2rpkm). Filtering was first done to keep the mRNA data of the top genes
197 (might be several thousand) in terms of the variation across samples (or dissimilarity 198 across groups, where dissimilarity is defined as sum of the between-group variations
199 divided by sum of the within-group variations). This filtered data was then used as
200 input for kennel PCA, where loadings (correlations between samples and components)
201 and scores (contributions of the genes to components) of the components were
202 produced. We chose the components which could properly partition our samples
203 (using loadings of the components) and identified the key genes for the selected
204 components (using the scores of the components). 3D PCA show was implemented
205 through an R function scatter3d from package car.
206
207 Heatmap
208 For heatmap drawing, log2 scale of the RPKM values (fragments per kilobase of
209 transcript per million mapped reads) of the significant genes were used as input for
210 heatmap generation. The genes were visualized using hierarchically clustered heat
211 map, which was drawn by pheatmap in the R package software. P value was adjusted
212 by Benjamini-Hochberg (BH) (P-adj). Significant genes with P-adj <0.05 were
213 selected to show the variations across groups. Z-scores from RNA-seq data were used
214 to draw the heatmaps.
215
216 Gene Set Enrichment Analysis (GSEA)
217 GSEA v3.0 (Broad Institute, PreRanked mode) was used for enrichment analysis. To
218 be consistent with the way for identifying significant genes, we used the t-statistic
219 output from limma as the metrics for ranking. 1,000 gene set permutations was set as 220 default, and gene sets were obtained through collecting pathways from GO,
221 REACTOME, KEGG and REACTOME. A gene set with a FDR q value <0.05 would
222 be considered as significantly enriched.
223
224 Multiplexed immunohistochemistry
225 Formalin-fixed paraffin embedded blocks of kidney biopsies from LN patients and
226 peritumors were collected from department of renal pathology, Renji Hospital. Slides
227 taken from paraffin blocks prepared with kidney biopsies were fixed in 4%
228 paraformaldehyde less than 12 hours. All slides were reviewed histologically by
229 hematoxylin and eosin (HE) staining. For TSA staining, tissue sections were firstly
230 deparaffinized, rehydrated and then treated with microwave antigen retrieval,
231 followed by incubating of these slides in 3% H2O2 to block endogenous peroxidase
232 activity. Nonspecific binding sites were blocked with normal goat serum for 15min
233 after that. And then tissue sections were incubated with primary antibodies overnight
234 at 4°C and HRP-conjugated secondary antibodies for 20min at room temperature.
235 Finally, the slides were visualized using the OPAL dye. The following
236 antibodies/fluorescent dyes were repeated by the same procedure in order, namely:
237 anti-CD20 (clone L26, eBioscience)/Opal-570, anti-T-bet (clone poly RB, Santa Cruz
238 Biotechnology)/Opal-690, anti-p-S6 (clone D57.2.2E, Cell Signaling)/Opal-540,
239 DAPI (SIGMA). Slides were captured by using the PerkinElmer Vectra platform. And
240 cells were phenotyped into different categories under the markers of interest: B cells 241 (CD20+), AtMs (CD20+T-bet+), CD20+T-bet- B cells, other immune cells
242 (CD20-DAPI+).
243
244 Statistical analysis
245 Statistical analyses were performed using GraphPad Prism 6.0. Statistical tests
246 included paired t test, Pearson’s correlation test and ordinary one-way ANOVA with
247 Holm-Sidak’s multiple comparison test when values were normally distributed, and
248 Spearman’s rank correlation, paired nonparametric test (Wilcoxon matched-pairs
249 signed rank test and Friedman test with Dunn’s multiple comparisons test) and
250 unpaired nonparametric test (Mann-Whitney test and Kruskal-Wallis test with Dunn’s
251 multiple comparisons test) when the values were not normally distributed. All tests
252 were carried out as 2-tailed tests. P<0.05 was considered to be statistically significant.
253 Data availability
254 The NCBI accession number for the RNA-seq experiments reported in this
255 manuscript is BioProject PRJNA505362.
256
257 Study approval
258 This study was approved by Shanghai Renji Hospital Ethics Committee (No.
259 2013-126) and all participants provided written informed consent. The study was
260 conducted in accordance with the principles expressed in the Declaration of Helsinki.
261 262 Patient and Public Involvement statement
263 At what stage in the research process were patients/the public first involved in the research
264 and how?
265 A: Lupus patients were involved in this research when this study started from January 2015.
266 After signing written consent, peripheral blood was collected and clinical data were recorded.
267 33 RA patients and 30 healthy donors were also recruited and their peripheral blood was
268 collected during this period.
269 How were the research question(s) and outcome measures developed and informed by their
270 priorities, experience, and preferences?
271 A: Patients were involved in the original research and actively contributed to identifying the
272 issue of inconsistent reporting, the need for guidance, and the research question.
273 How were patients/the public involved in the design of this study?
274 A: Patients/the public were not involved in the design of this study.
275 How were they involved in the recruitment to and conduct of the study?
276 A: Patients were involved in the conduct of the study by actively providing clinical
277 manifestations, treatment adjustment and self-feelings during the study.
278 Were they asked to assess the burden of the intervention and time required to participate in
279 the research? 280 A: Patients were not asked to assess the burden of the intervention and time required to
281 participate in the research.
282
283 Supplementary references
284 1 Hochberg MC. Updating the American College of Rheumatology revised 285 criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 286 1997;40:1725. 287 2 Dooley MA, Aranow C, Ginzler EM. Review of ACR renal criteria in 288 systemic lupus erythematosus. Lupus 2004;13:857-60. 289 3 Buyon JP, Petri MA, Kim MY, et al. The effect of combined estrogen and 290 progesterone hormone replacement therapy on disease activity in systemic 291 lupus erythematosus: a randomized trial. Ann Intern Med 2005;142:953-62. 292 4 Nikpour M, Urowitz MB, Ibanez D, et al. Frequency and determinants of flare 293 and persistently active disease in systemic lupus erythematosus. Arthritis 294 Rheum 2009;61:1152-8. 295 5 Arnett FC, Edworthy SM, Bloch DA, et al. The American Rheumatism 296 Association 1987 revised criteria for the classification of rheumatoid arthritis. 297 Arthritis Rheum 1988;31:315-24. 298 6 Law CW, Chen Y, Shi W, et al. voom: Precision weights unlock linear model 299 analysis tools for RNA-seq read counts. Genome Biol 2014;15:R29.
300 Supplemental Table 1. Basic clinical characteristics of SLE patients, RA patients and healthy donors.
A-SLE B-RA C-HD P value
(n=54) (n=33) (n=30) P value A VS B A VS C B VS C
Age 32.3±1.62 (18~63) 50.2±1.84 (20~71) 34.1±1.53 (21~54) <0.0001 <0.0001 ns <0.0001
Sex (% female) 51 (94.4%) 29 (87.9%) 28 (93.3%) 0.524
WBC (×109/L) 3.51±0.27 (1.46~10.7) 5.94±0.28 (3.1~9.64) 6.17±0.28 (3.5~10.6) <0.0001 <0.0001 <0.0001 ns
RBC (×1012/L) 3.81±0.12 (0.71~5.9) 4.26±0.07 (3.17~5.05) 4.48±0.06 (3.85~5.11) <0.0001 0.012 <0.0001 ns
Hb (g/L) 104.6±3.1 (26~113) 120.0±2.8 (89~146) 134.4±2.1 (105~155) <0.0001 0.009 <0.0001 0.005
PLT (×109/L) 164.0±10.4 (34~450) 272.3±17.0 (173~663) 239.1±8.2 (169~341) <0.0001 <0.0001 <0.0001 ns
IgA (g/L) 2.69±0.15 (0.85~6.75) 2.32±0.20 (0.27~5.24) 1.77±0.17 (0.04~3.51) <0.001 ns 0.002 ns
IgM (g/L) 1.30±0.09 (0.27~3.12) 1.31±0.12 (0.05~2.96) 1.24±0.17 (0.01~3.20) 0.734
IgG (g/L) 24.69±1.22 (4.20~40.40) 15.71±1.12 (4.90~33.40) 12.62±0.88 (0.50~18.9) <0.0001 <0.0001 <0.0001 ns
C3 (g/L) 0.56±0.31 (0.10~1.54)
C4 (g/L) 0.088±0.07 (0.018~0.33)
Anti-dsDNA (log value) 1.99±0.89 (0.57~4.10)
SLEDAI 14.28±1.05 (2~33) ANA+ (%) 54 (100%)
Anti-SSA+ (%) 33 (61.1%)
Anti-SSB+ (%) 12 (22.2%)
Anti-U1RNP+ (%) 30 (55.6%)
Anti-Sm+ (%) 12 (22.2%)
Anti-rRNP+ (%) 8 (14.8%)
Anti-ANUA 3.10±2.30 (0.00~8.77)
ACL+ (%) 4 (7.40%)
Fever (%) 15 (27.8%)
Rash (%) 37 (68.5%)
Alpecia (%) 13 (24.1%)
Arthritis (%) 25 (46.3%)
Oral ulcers (%) 8 (14.8%)
Cutaneous vasculitis (%) 12 (22.2%)
Raynaud’s (%) 2 (3.7%)
Leukopenia and/or Thrombocytopenia (%) 45 (83.3%) Anemia (%) 30 (55.6%)
Serositis (%) 19 (35.2%)
NPSLE (%) 4 (7.4%)
LN (%) 16 (29.6%)
Urine protein of LN patients (mg/24h) 2731.7±757.8 (500.0~10231.8)
Supplemental Table 2. Basic clinical characteristics of the second SLE cohort.
SLE(n=19)
Age 34.6±15.3 (18~69)
Sex (% female) 18 (94.7%)
WBC (×109/L) 4.69±3.95 (1.71~18.09)
RBC (×1012/L) 3.69±0.55 (2.16~4.46)
Hb (g/L) 102.2±14.1 (65~122)
PLT (×109/L) 170.4±104.1 (61~441)
IgA (g/L) 3.57±1.76 (1.46~9.29)
IgM (g/L) 1.17±0.53 (0.47~2.32)
IgG (g/L) 20.18±5.79 (9.77~31.2)
C3 (g/L) 0.69±0.38 (0.20~1.71)
C4 (g/L) 0.115±0.118 (0.02~0.48)
Anti-dsDNA (log value) 2.01±0.60 (0.73~2.93)
SLEDAI 9.53±5.70 (2~23)
ANA+ (%) 19 (100%)
Anti-SSA+ (%) 11 (57.9%)
Anti-SSB+ (%) 0 (0.0%)
Anti-U1RNP+ (%) 8 (42.1%)
Anti-Sm+ (%) 6 (31.6%)
Anti-rRNP+ (%) 7 (36.8%)
Anti-ANUA 3.13±2.14 (0.07~8.53)
ACL+ (%) 1 (5.26%)
Fever (%) 5 (26.3%)
Rash (%) 11 (57.9%)
Alpecia (%) 4 (21.1%)
Arthritis (%) 3 (15.8%) Oral ulcers (%) 1 (5.26%)
Cutaneous vasculitis (%) 1 (5.26%)
Raynaud’s (%) 0 (0.0%)
Leukopenia and/or Thrombocytopenia (%) 13 (68.4%)
Anemia (%) 12 (63.2%)
Serositis (%) 7 (36.8%)
NPSLE (%) 0 (0.0%)
LN (%) 6 (31.6%)
Urine protein of LN patients (mg/24h) 3076.0±2650.4 (519.0~7764.3)
Supplemental Table 3. Clinical characteristics of SLE patients during 24 weeks of drug intervention (n=23). 0W 24W P value
WBC (×10^9/L) 3.44±0.42 (1.84~10.7) 5.47±0.44 (2.59~11.52) 0.012
RBC (×10^12/L) 3.91±0.12 (2.47~5.04) 4.40±0.08 (3.92~5.16) <0.001
Hb (g/L) 106.00±3.76 (70.0~135.0) 125.83±3.05 (112~158) 0.0001
PLT (×10^9/L) 172.9±19.2 (34~450) 230.2±14.2 (136~384) <0.001
IgA (g/L) 2.88±0.27 (0.85~6.75) 2.51±0.17 (1.28~4.91) 0.010
IgM (g/L) 1.46±0.16 (0.46~3.12) 1.26±0.12 (0.34~3.04) 0.344
IgG (g/L) 24.65±1.78 (4.2~40.2) 16.27±0.92 (9.4~24.1) <0.001
C3 (g/L) 0.54±0.07 (0.1~1.54) 0.81±0.04 (0.31~1.24) <0.0001
C4 (g/L) 0.08±0.02 (0.02~0.33) 0.14±0.02 (0.06~0.27) 0.031
Anti-dsDNA (log value) 2.14±0.19 (0.90~4.10) 1.54±0.16 (0.58~3.41) <0.0001
SLEDAI 13.78±1.60 (5~32) 4.65±0.68 (0~16) <0.0001
Fever (n) 7 2
Rash (n) 11 0
Alopecia (n) 7 0
Arthritis (n) 12 1
Oral ulcer (n) 1 0
Cutaneous vasculitis (n) 4 0
Raynaud’s (n) 1 0
Leukopenia/Thrombocytopenia (n) 20 7
Anemia (n) 12 1
Serositis (n) 7 1
NPSLE (n) 2 0
Urine protein of LN patients (mg/24h) 3285.6±1014.1 (500.0~8640.9) 1419.4±690.5 (115.4~5535.8)
Supplemental Table 4. Clinical responses to drug intervention in SLE patients after 24 weeks (n=23). Patient Age Sex Manifestations before treatment Manifestations at week 24 Use of drugs SLEDAI scores SLEDAI Treatment number before scores at result
treatment week 24
1# 23 F Aleukocytosis / Fever / Arthritis / Cutaneous Vasculitis / Serositis / 26 7 flare Aleukocytosis / Retinal vasculitis / Serositis / Prednisone/HCQ/MMF Proteinuria / High titers of dsDNA / Low complement High titers of dsDNA / Low complement
2# 45 F Aleukocytosis / Fever / Arthritis / NPSLE / High titers of dsDNA Fever / High titers of dsDNA / Low Prednisone/HCQ/ CTX 17 5 flare
complement
3# 30 M Aleukocytosis / Fever / Proteinuria / Hematuria / Pyuria / High titers of High titers of dsDNA Prednisone/HCQ/ CTX 18 2 remission
dsDNA / Low complement
4# 27 F Aleukocytosis / Rash / Arthritis / Serositis / High titers of dsDNA / Low High titers of dsDNA / Low complement Prednisone/HCQ/ MTX 12 4 remission
complement
5# 27 F Aleukocytosis / Thrombocytopenia / Rash /Arthritis / Serositis / Proteinuria / High titers of dsDNA Prednisone/HCQ/ AZA 25 6 resistance
Proteinuria / Hematuria / Pyuria / High titers of dsDNA / Low
complement
6# 27 F Aleukocytosis/ Fever / Arthritis / High titers of dsDNA / Low High titers of dsDNA / Low complement Prednisone/HCQ/ MTX 10 4 remission
complement
7# 38 F Aleukocytosis / Thrombocytopenia / Rash / Arthritis / Cutaneous High titers of dsDNA / Low complement Prednisone/HCQ/ CTX 24 4 remission
vasculitis / Proteinuria / High titers of dsDNA / Low complement
8# 18 F Rash / Alopecia / Oral ulcer / High titer of dsDNA / Low complement High titers of dsDNA / Low complement Prednisone/HCQ/ MTX 10 4 remission
9# 28 M Thrombocytopenia / Fever / Rash / Serositis / NPSLE / High titers of High titers of dsDNA / Low complement Prednisone/HCQ/ MTX 16 4 remission
dsDNA / Low complement
10# 34 F Aleukocytosis / Proteinuria / Pyuria / High titers of dsDNA Proteinuria/ Hematuria / High titers of dsDNA Prednisone/HCQ/ CTX 13 10 resistance 11# 28 F Aleukocytosis / Rash / Alopecia / Arthritis / High titers of dsDNA / Low No manifestation Prednisone/HCQ/ MMF 11 0 remission
complement
12# 26 F Aleukocytosis / Fever / Rash / Alopecia / Cutaneous Proteinuria / Hematuria / Pyuria / High titers Prednisone/HCQ/ CTX 32 16 flare
Vasculitis / Serositis / Proteinuria / Hematuria / Pyuria / High titers of of dsDNA / Low complement
dsDNA / Low complement
13# 47 F Aleukocytosis / Rash / Alopecia / Arthritis / High titers of dsDNA / Low High titers of dsDNA / Low complement Prednisone/HCQ/ MTX 12 4 remission
complement
14# 28 F Aleukocytosis / Rash / High titers of dsDNA / Low complement High titers of dsDNA / Low complement Prednisone/HCQ/ CsA 8 4 remission
15# 28 F Thrombocytopenia / Alopecia / High titers of dsDNA High titers of dsDNA 5 2 remission Prednisone/HCQ/ CsA
16# 37 F Cutaneous vasculitis / Arthritis / Serositis / Proteinuria / High titers of High titers of dsDNA Prednisone/HCQ/ 22 2 remission
dsDNA / Low complement KF506
17# 18 F Rash / High titers of dsDNA / Low complement High titers of dsDNA / Low complement Prednisone/HCQ 6 4 remission
18# 40 F Aleukocytosis / Arthritis / High titer of dsDNA High titers of dsDNA Prednisone/HCQ/ MTX 6 2 remission
19# 35 F Aleukocytosis / High titers of dsDNA / Low complement Aleukocytosis / High titers of dsDNA / Low Prednisone/HCQ/ CsA 5 5 flare
complement
20# 28 F Aleukocytosis / Arthritis / High titers of dsDNA / Low complement Arthritis / High titers of dsDNA / Low Prednisone/HCQ/ 8 8 flare
complement FK506
21# 30 F Aleukocytosis / Arthritis / Serositis / Proteinuria / High titers of dsDNA Low complement Prednisone/HCQ/ 16 2 remission
/ Low complement FK506
22# 28 F Aleukocytosis / Fever / Rash / Alopecia / High titers of dsDNA / Low Fever / High titers of dsDNA / Low Prednisone/HCQ/ MTX 10 5 flare
complement complement
23# 43 F Aleukocytosis /Alopecia / High titers of dsDNA / Low complement High titers of dsDNA / Low complement Prednisone/HCQ 5 4 remission HCQ: Hydroxychloroquine MMF: Mycophenolate mofetil CsA: Cyclosporine MTX: Methotrexate CTX: Cyclophosphamide AZA: Azathioprine FK506: Tacrolimus Supplemental Table 5. Clinical characteristics of lupus nephritis patients (n=27).
LN (n=27) Sex (F,%) 27 (100%) Age (y) 35.1±1.7 (24~59)
Ⅲ (%) 3 (11%) Ⅳ (%) 7 (26%) Ⅴ (%) 4 (15%)
Ⅲ+Ⅴ (%) 7 (26%) Ⅳ+Ⅴ (%) 6 (22%) Urine protein (mg/24h) 2649.4±371.0 (100.0~7023.2) Activity Index 4.7±0.5 (1~11) Chronic Index 4.4±0.4 (2~8) Scr (μmol/L) 64.92±4.37 (37.0~125.0) BUN (mmol/L) 7.12±0.55 (1.76~14.5) UA (μmol/L) 341.74±20.13 (186.0~578.0) ALB (g/L) 31.19±1.04 (20.1~41.5) Anti-dsDNA (IU/mL) 63.95±7.23 (3.02~100) C3 (g/L) 0.53±0.04 (0.148~1.11) C4 (g/L) 0.08±0.01 (0.015~0.25) IgA (g/L) 2.91±0.27 (1.27~6.85) IgM (g/L) 0.91±0.10 (0.17~2.0) IgG (g/L) 13.39±1.30 (5.8~36.4) ANUA (IU/mL) 2.56±0.35 (0.066~6.63) SLEDAI 11.5±1.1 (4~28) Renal SLEDAI 6.2±0.9 (0~16)
A B
C 150 r=0.435 400 r=0.357 D P=0.001 P=0.009 300 100 r=0.995 200 25 P<0.0001 50
IL-6 (pg/mL) IL-6 20 IL-18 (pg/mL) IL-18 100 AtMs%B hi 15 0 0 0 10 20 30 0 10 20 30 10
CD20 AtMs %B AtMs %B - 5 0 250 r=0.599 250 r=0.497 CD24 0 5 10 15 20 25 P<0.0001 P<0.001 + - hi 200 200 CD11c CD24 CD20 AtMs%B 150 150 (pg/mL) (pg/mL)
100 γ 100 IFN- α IFN- 50 50 0 0 0 10 20 30 0 10 20 30 AtMs %B AtMs %B
r=0.584 r=-0.468 F r=0.607 r=-0.465 2.0 25 2.0 E 25 P=0.009 P=0.043 P=0.006 P=0.045
20 1.5 20 1.5 15 15 1.0 1.0 C3(g/L) 10 C3(g/L) 10 SLEDAI SLEDAI 0.5 0.5 5 5 0 0 0 0 0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25 - hi + - hi + - hi CD24-CD20hi AtMs%B CD24 CD20 AtMs%B CD11c CD24 CD20 AtMs%B CD11c CD24 CD20 AtMs%B
Supplementary Figure 1. Correlations between circulating AtMs%B and clinical parameters in newly-onset SLE patients. (A) Correlations between circulating AtMs%B and concentrations of serum complement 3 (C3) and C4 (n=54). (B) Correlations between circulating AtMs%B and blood hemoglobin concentration (n=54). (C) Correlations between circulating AtMs%B and plasma concentrations of pro-inflammatory cytokines IL-6, IL-18, IFN-α and IFN-γ (n=52). (D) Correlation between circulating CD24-CD20hi AtMs%B and CD11c+CD24-CD20hi AtMs%B from the second newly-onset SLE patient cohort (n=19). (E-F) Correlations between circulating CD24-CD20hi AtMs%B (E) or CD11c+CD24-CD20hi AtMs%B (F) and SLEDAI scores and serum C3 concentrations in the second newly-onset SLE patient cohort (n=19). P values were determined by Spearman’s rank correlation (A-F). A AtMs-SLE vs CMs-SLE B AtMs-SLE vs CMs-SLE Endocytosis Upregulated genes RNA and protein synthesis Downregulated genes
NES NES
C
CMs-Baseline CMs-BCR crosslinking AtMs-Baseline #Cells AtMs-BCR crosslinking
ISO p-Syk(Y348) p-Syk(Y352) p-BTK(Y551/511) p-PLCγ2(Y759)
Supplementary Figure 2. Molecular feature of lupus AtMs. (A-B) GSEA identified that the pathways related in endocytosis were highly enriched (A) while pathways related in RNA and protein synthesis were down-regulated (B) in AtMs-SLE compared with CMs-SLE based on gene expression data from RNA-seq. NES: normalized enrichment score. (C) Flow cytometry analysis of the differentially expressed markers of BCR signalling between AtMs and CMs from the same SLE patients at baseline or stimulated by BCR crosslinking for 5 minutes. One representative experiment out of three was shown. PI3K/Akt molecules PIK3R3 AKT1 AKT2 P=0.004 P=0.011 P=3.63-e06 6 200 1500 150 4 1000 100 CPM 2 500 50 0 0 0 CMs AtMs CMs AtMs CMs AtMs Negative regulators of PI3K/Akt pathway PTEN PIK3IP1 CD300A P=0.053 P=6.39-e04 P=8.16-e04 250 400 25 200 300 20 150 15
CPM 200 100 10 50 100 5 0 0 0 CMs AtMs CMs AtMs CMs AtMs
Supplementary Figure 3. Expression of PI3K/Akt pathway-related genes in lupus AtMs and CMs. RNA-seq data were used to compare the expression profiles of PI3K/Akt pathway-related genes between AtMs and CMs from active SLE patients (n=6). CPM: counts per million. Limma+voom (paired samples test) was used for differential analysis and P values were adjusted by the Benjamini-Hochberg multiple test. mean ±SEM.Pvaluesweredeterminedbyone-way ANOVA withHolm-Sidak’s multiplecomparisonstest(B,pairedt(H). D,F)and expression inliveandnon-proliferating Bcellsintheabsenceorpresenceofindicated concentrationsof Rapamycin (Rapa)for5days(n=6).(G-H) Representativeflowcytometry plots(G)andaccumulateddata(H)toshowp-S6 T-bet cells (F) accumulated data(B)toshowp-S6 Supplementary flow cytometryplots(C,E)andaccumulateddataofthefrequencies ofZombie Yellow G
CFSE in healthydonorBcellsstimulated withthecombinationofIFNγandR848inabsenceorpresence 0.1nMand1 CFSE E C A
7.39
5.62 29.2 19.8 #Cells Zombie Yellow Figure 4.mTORC1 signalingwasimplicatedinthedifferentiation of AtMs. (A-B)Flowcytometryanalysis(A)and IFNγ+R848 26.1 p-S6 T-bet IFNγ+R848 p-S6 CFSE 49.0 38.2 24.5 26.8 CFSE 43.3 21.7 IFNγ+R848+Rapa 0.1nM + cellsamongCD11c 22.0 49.1 18.9 60.0 CD11c CD11c CD11c Isotype
IFNγ+R848+Rapa 0.1nM IFNγ+R848 IFNγ+R848+Rapa 1nM IFNγ+R848+Rapa 0.1nM 1.89 + - - T-bet T-bet T-bet 0.80 28.0 1.37 19.7 + - + (p-S6 (p-S6 (p-S6 - T-bet IFNγ+R848+Rapa 1nM + 57.1 + 19.0 15.9 65.0 + : 27.9%) 81.0 12.4 : 97.3%) : 98.0%) - B
cells, CD11c
IFNγ+R848+Rapa 1nM 1.49 1.01 22.8 F B 1.46 17.6
+ 100 - Proliferating cell % p-S6 % T-bet 20 40 60 80 20 40 60 0 45.9 48.6 52.6 46.0 85.7 7.97 + B - P=0.003 P=0.002 Isotype
- cells andCD11c livecells(D) IFNγ+R848 Rapa 0.1 ISO P=0.003 P=0.002 ISO D 1.47 0.27 5.47 0.36 P=0.960 P=0.047
Live cells % 100 20 40 60 80 0
Rapa 1( H and thefrequencyofliveproliferating - + + + P=0.002 100 T-bet T-bet %B p-S6 %B 20 40 60 20 40 60 80 0 0 nM) Rapamycin. Errorbarsindicated IFNγ+R848 Rapa 0.1 P=0.002 Non-proliferating Bcells + B P=0.010
cells. (C-F)Representative - -
P<0.0001 Rapa 1( IFNγ+R848 IFNγ+R848 P=0.006 Rapa 0.1 Rapa 0.1 nM) (nM) (nM)
A C
CMs-HD CMs-SLE AtMs-SLE CMs-HD CMs-SLE AtMs-SLE 2 3 PDCD1 1 2 BATF FCRL3 1 FOXO1 0 FCRL4 0 IKZF2 -1 FCRL5 -1 NFATC2 ILIRB1 Z-Score ILIRB2 Z-Score MAF SIGLEC6 NFIL3 BTLA NR4A3 CD22 TBX21 CD72 ZEB2
Dysfunctional receptors CD200
AtM High CREM
CD200R1 ATF3 ITGAX ZBTB32 CD19 MS4A1 JAZF1 CD32 LITAF HOPX
CD86 Dysfunction-related TFs IL21R MYBL1 CR2 TOX CD24 MAML2
CD27 ZEB1
CD40 ZBTB20 CD44 Development related CD70 TCF7 AtM Low CXCR5 TAF1A CCR7 TXNIP
Dysfunctional receptors
CMs B AtMs #Cells
ISO PD1 FcRL3 FcRL5 SIGLEC6 ILT2/LILRB1 Dysfunctional receptors #Cells
ILT4/LILRB2 BTLA(CD272) PDL1 CD22 CD72 FcRL4 Dysfunction-related TFs #Cells
NFATc2 BATF FOXO1 HELIOS HIF1α c-Myc
Supplementary Figure 5. AtMs displayed a dysfunctional phenotype. (A) Heatmap to show the differential gene expression profiles of dysfunctional receptor and B cell development-related molecules between lupus AtMs and lupus and healthy CMs. (B) Flow cytometry analysis to show differentially expressed dysfunctional receptors and dysfunction-related transcription factors between AtMs and CMs from the same SLE patient. One representative plot out of three to five was shown. (C) Heatmap to show the differential gene expression profiles of dysfunction-related transcription factors between lupus AtMs and lupus and healthy CMs. Developmental receptors
CMs AtMs #Cells
ISO CD11a CD11c CD19 CD20 CD21 Developmental receptors #Cells
CD27 CD32 CD38 CD62L CD95 CD98 Activation/co-stimulatory receptors #Cells
CD40 CD44 CD69 CD70 CD86 HLADR Cytokine receptors #Cells
TACI(CD267) BAFFR(CD268) BCMA(CD269) IL-21R(CD360) IFNAR CD25(IL-2RA)
Chemokine receptors #Cells
CXCR3 CXCR4 CXCR5 CCR5 CCR7 CCR10
Supplementary Figure 6. Phenotypic characterization of AtMs by Flow cytometry. Flow cytometry analysis showed differentially expressed developmental receptors, activation and co-stimulatory receptors, cytokine receptors and chemokine receptors in AtMs compared to CMs from active SLE patients. For each marker, one representative plot out of three to five was shown. A Anti-apoptotic B CMs BCL2 PIM1
P=6.51-e06 P=0.011 AtMs 500 250 400 200 300 150 #Cells
CPM 200 100 100 50 0 0 PIM2 PIM3 ISO BCL2 P=2.03-e05 P=1.81-e05 800 1500 400 P=0.001 CMs AtMs 600 1000 300
CPM 400 500 200 200
MFI of BCL2 0 0 100 CMs AtMs CMs AtMs 0 CMs AtMs
C Pro-inflammatory Anti-inflammatory IL6 TNF IL10 TGFB1 P=2.85-e07 P=4.51-e04 P= 5.66-e04 P=1.73-e04 60 80 12.5 900
50 70 800 60 10.0 40 700 50 7.5 30 40 600 CPM 20 30 5.0 500
20 2.5 10 10 400 0 0 0 300 CMs AtMs CMs AtMs CMs AtMs CMs AtMs
D Medium PIB PIB+CpG Medium PIB PIB+CpG
CMs-SLE 0.99 22.15 50.66 1.49 22.95 83.26
AtMs-SLE 1.63 6.80 27.43 0.73 4.04 44.58 SSC
IL-6 TNF-α
Supplementary Figure 7. AtMs exhibited increased apoptosis potential and impaired ability to produce pro-inflammatory cytokines. (A) RNA-seq data were used to compare the expressions of apoptosis related genes between AtMs-SLE and CMs-SLE from active SLE patients (n=6). (B) Upper: representative flow cytometry histograms to show the different expression of BCL2 between lupus AtMs and CMs; Lower: comparison of the accumulated data of MFI of BCL2 expression between lupus AtMs and CMs (n=6). (C) RNA- seq data were used to compare the altered gene expression profiles of pro-inflammatory and anti-inflammatory factors between AtMs and CMs from active SLE patients (n=6). (D) Representative flow cytometry plots showed the capacity of AtMs and CMs from active SLE patients to produce IL-6 and TNF-α (n=20) . PBMCs were stimulated with PIB or PIB plus CpG for 5 hours and the cytokines were detected. PIB: PMA+Ionomycin+Brefeldin A. For A and C, Limma+voom (paired samples test) was used for differential analysis and P values were adjusted by the Benjamini-Hochberg multiple test. For B, P value was determined by paired t test. A B
CD20 T-bet 10 P=0.036 8 6 4 %nucleated Cells
+ 2 0
CD20 Peritumor LN
DAPI CD20 T-bet DAPI Lupus nephritis
200 P=0.075 2 150 B/mm + 100
CD20 50
0 Peritumor LN C 30 r=0.433 2.5 r=0.397 1.5 r=-0.394 P=0.024 2.0 P=0.040 P=0.042 20 1.5 1.0 1.0 C3 (g/L) SLEDAI 10 0.5 0.5 0 0.0 0.0 0 50 100 150 (log value) Anti-dsDNA 0 50 100 150 0 50 100 150 T-bet+ AtMs /mm2 T-bet+ AtMs /mm2 T-bet+ AtMs /mm2
D 150 50 r=-0.404 8000 r=0.454 r=0.603 P<0.001 40 P=0.037 6000 P=0.017 100 30 4000 20 50 ALB (g/L)
2 Scr (µmol/L) 10 000
0 0 0 0 50 100 150 Urine protein (mg/24h) 0 50 100 150 0 50 100 150 T-bet+ AtMs /mm2 T-bet+ AtMs /mm2 T-bet+ AtMs /mm2
20 r=0.489 800 r=0.525 P=0.010 P=0.005 15 600 10 400
(µmol/L) UA 2
BUN (mmol/L) 5 00 0 0 0 50 100 150 0 50 100 150 T-bet+ AtMs /mm2 T-bet+ AtMs /mm2
Supplementary Figure 8. CD20+T-bet+ AtMs infiltrated in the renal tissues of lupus nephritis patients. (A) Immunofluorescence staining of CD20 (red), T-bet (green) and DAPI (blue) in renal tissues of lupus nephritis patients. (B) Comparison of frequency of total B cells in nucleated cells and the density of B cells in renal tissues of LN (n=27) and peritumor (n=10). (C) Correlations between density of T-bet+ AtMs in renal tissues and SLEDAI scores, serum anti-dsDNA titers and complement 3 concentrations in LN patients (n=27). (D) Correlations between density of T-bet+ AtMs and the levels of serum albumin, urine proteins and renal function indicators including concentrations of serum creatinine, urea nitrogen and uric acid in LN patients (n=27). Error bars indicated mean ± SEM. P values were determined by Mann-Whitney test (B) and Spearman’s rank correlation (C and D).