analysis (PCA) of expression features followed
by K-means clustering to identify two clusters
that broadly separated donors by case-control
status (Fig. 4E), severity of SLEDAI score (Fig.
4F), and along principal component 1 (PC1).
Cases in the High cluster had significantly
higher inferred activation of monocytes rela-
tive to cases in the Low cluster (PWilcoxon<6.20×
10 –^9 ;fig.S5C).PC1correlatedmostwithgenes
in the Panup, Myeup, and Bupmodules, includ-
ing the myeloid-specific expression ofIFITM3,
a gene previously described to stratify pediatric
SLE cases ( 3 ) (Fig. 4E). To assess the correspon-
dence between molecular clusters and clinicalfeatures, we projected 94 held-out cases each
to a molecular cluster on the basis of expres-
sion features (Fig. 4G). Cases assigned to the
High cluster were enriched for disease flare
(15/19 flare cases, fig. S5D) and portended a
factor of 5 increase in the odds of having anti-
Smith antibodies (Padjusted:Fisher< 0.05; Fig. 4H).Perezet al.,Science 376 , eabf1970 (2022) 8 April 2022 6 of 13
Fig. 3. Type 1 interferonÐ
stimulated gene expression
of myeloid cells in SLE.
(A) Heatmap of pseudobulk gene
expression profiles of 302 differ-
entially expressed genes detected
in at least one of 11 cell types.
For each gene, colored row bars
indicate cell types in which it
was differentially expressed.
Colored columns indicate cell
type, case-control status, and
ancestry. Labeled modules were
identified using hierarchical
clustering. (B) Top GSEA
(gene set enrichment analysis)
pathway enrichment results for
each module. Each dot color
represents the–log(q) value;
dot size represents the number of
genes overlapping with the gene
ontology. (C) Identification of
six myeloid cell types including
classical, nonclassical, and
complement-expressing non-
classical monocytes (cM, ncM,
ncMcomp), conventional
type 1, conventional type 2,
and plasmacytoid dendritic cells
(cDC1, cDC2, pDC). (D) Marker
genes used for annotating
each cell type. (E) Percentages of
myeloid cells (yaxis) versus
case-control status and ancestry
(xaxis) for each myeloid sub-
population. Myeloid subpopula-
tions with significant percentage
changes between cases and
controls are highlighted. *P<
0.01, ***P< 0.0001 (WLS); blue
bar indicates significant meta-
analysis by FisherÕs method.
(FandG) RNA velocity stream
plots for cM (right UMAP)
and ncM and ncMcomp(left
UMAP) subpopulations colored
by the average expression of
Myeupgenes enriched for type 1
ISGs (F) and the relative
density of cells from SLE cases
versus healthy controls (G).
(H) Distribution of the degree of
inferred activation for individuals
across disease activities (HC, healthy controls; inactive, SLEDAI between 0 and 4; active, SLEDAI of 5 or more). (I) Average inferred activation across cells per sample (yaxis)
versus disease activity (xaxis) for Asian (left) and European (right) samples separately. ECTL, European control; ESLE, European case; ACTL, Asian control; ASLE, Asian case.
ISLEDAIAverage
Pseudoactivation
CTL Inactive
(<=4)Active
(>=5)CTL
SLEDAIInactive
(<=4)Active
(>=5)ABPBMC cM ncM cDC pDC T4 T8 NK Prolif B ProgCTL SLE Asian EuropeanPBDonorsAPOBEC3G
DDX*58,60,60L, EPSTI1GBP*1,3-5,
HLA-*C,DQB1
IFI*16,27,35,44,44L,6,H1IFIT*1,3,M2, IRF*7,9
ISG*15,20, JAK2
LY6E, MT2AMX*1,2, OAS*1-3,L
PARP*9,12,14, PLAC8
PSM*B9,E2, RNF213SAMD9,9L, STAT1,2
TAP1, VRK2APOBEC3AC1QB, CD300E
CX3CR1, CXCL10FCGR*1A,1B,3A
GIMAP*2,4,7
IFIT*2,M1,M3IFNGR1, LAIR1
LGALS*1,2,3BP
RNASE2, TMEM123FCRL5, BTG2GZF1, IER5L
IL1B, ILF3-AS1CCL*3,4
GABARAPL1
TCL1AVPREB3
SNORD3B-1Example GenesMDS2, CD1CGNLY, ITGAE
DNAJB1
KLR*G1,B1,C1AK5, PASK
LYPD2IGJ, VMO1RNU12Cell TypePan
upPan
downMye
upMyedownupTupBMin Max
Normalized Expression* * * * *-log q# ofgenes8(^1624)
32
10
(^2030)
(^4050)
Pan
up
Mye
up
Mye
down
upB Pan
down
upT
defense res. to virusInterferon Signaling
res. to external biotic stim.response to virus
defense res. to other org.res. to other organism
innate immune responseres. to biotic stimulus
type I IFN signaling pathwaycellular res.e to type I IFN
response to type I interferonIFN alpha/beta signaling
response to cytokinedefense response
Cytokine Sig. in Immune sys.immune effector process
cellular res. to cytokine stim.res. to interferon-gamma
cytokine-mediated sig. pathwaycellular res. to IFN-gamma
reg. of immune system processregulation of immune response
pos. reg. of imm. sys. processresponse to interferon-beta
neg. reg. of transcription by RNA pol IIcellular res. to external stim.
response to mechanical stimulusresponse to abiotic stimulus
AP-1 transcription factor networkpositive regulation of cell death
cis-reg. seq.-specific DNA bindingregulation of cell death
RNA pol II cis-reg. seq.-specific DNA bindingregulation of apoptotic process
regulation of programmed cell deathneg. reg. of biosynthetic process
negative regulation of viral transcriptioncell activation
pos. regulation of programmed cell deathToll-like receptor signaling pathway
ATF-2 transcription factor networkresponse to lipid
response to organic cyclic compoundcellular defense response
regulation of phosphorylationleukocyte activation
Imm. reg. int. btw Lymph. & non-Lymph. cellOsteoclast differentiation
response to oxygen-containing compoundmembrane fusion
regulation of phosphorus metabolic processimmune system development
regulation of phosphate metabolic processhemopoiesis
hematopoietic or lymph. organ developmentnegative regulation of apoptotic process
neg. regulation of programmed cell deathneg. reg. of apoptotic signaling pathway
regulation of apoptotic signaling pathwaynegative regulation of cell death
phosphatidylinosital 3-kinase reg. activitycellular response to fluid shear stress
hedgehog receptor activitysmoothened binding
hedgehog family protein bindingpre-mRNA branch point binding
Hedgehog signaling pathwayBasal cell carcinoma
Sig. events mediated by the Hedgehog fam.Kinesins
UMAP1
UMAP2
CD
0.5 1.0
CD14
FCGR3A
C1QA
CLEC9A
CLEC10A
LILRA4
cMncM
ncM
comp
cDC1cDC2pDC
E cM ncM ncMcomp cDC1 cDC2 pDC
Myeloid Percentage
(^10040)
30
20
10
0
90
80
70
60
0
1
2
3
4
5
6
50
ECTLESLEACTLASLEECTLESLEACTLASLEECTLESLEACTLASLEECTLESLEACTLASLEECTLESLEACTLASLEECTLESLEACTLASLE
15.0 2.0
6
4
2
0
12.5
10.0
7.5
5.0
2.5
1.5
1.0
0.5
0.0
Cell Type
DE
F
Myeloid express moduleio
n
5 10 15 20
Myeloid m expressionodule
5 10 15 20
UMAP1
UMAP2
Difference in density
SLE - CTL
G
-0.0. 000
025
Difference in density
SLE - CTL
0.1 0.0-0.1
Normalized Expression
UMAP1
UMAP2
ASN EUR
Inferred Activation
H
012 012
0.0
0.2
0.4
0.6
0.8 0.5
0.0
-0.5
0.5
0.0
-0.5
HC
Inactive
(SLEDAI<=4)
Active
(SLEDAI>=5)
Density
ASN EUR
RESEARCH | RESEARCH ARTICLE