A
C
Contro
l RASLE
0
20
40
60
80
100
% C
D4+
T ce
lls /
PBM
C
Contro
l RASLE
0
20
40
60
80
100
%C
D8+
T ce
lls /
PBM
C
Contro
l RASLE
0
20
40
60
80
100
CD
45R
O+
/ CD
4+ T
cells
B
DN
A1
DNA2
CD
4
CD8
Bea
ds
Cisplatin
CD
14
CD3 CD
45R
O
CD45RA
DN
A1
DNA2
CD
4
CD8B
eads
Cisplatin
CD
45
CD3
CD
45R
O
CD4
Supplementary Figure 1: Identification of major T cell subsets in mass cytometry data.A) Example of biaxial gating for T cell populations in the AMP mass cytometry data. B) Example of biaxial gating for T cell populations in the BWH validation cohort mass cytometry data. C) Quantification of CD4+ T cells, CD8+ T cells, and memory CD4+ T cells in the AMP cohort. Error bars show median±interquartile range.
96.9
98.8 82.4
Memory CD4+ T cells 39.8
Memory CD4+ T Cells 59.9
CD4+ Cells64.6
CD8+ Cells25.759.8
99.8
97.1
CD4+ cells 65.9
B
HLA
-DR
ICOS
PD-1hi CXCR5- CD4+ T cellsD
12 1 13 11 8 2 14 7 10 15 5 6 9 3 4C
lust
er A
Clu
ster
B
CD57TbetGranzyme BHLA-DRCD64PU.1CCR2FoxP3HeliosCD25CD27PD-1KLRG1CD69GATA3TCRgdRORgTCCR7CD94AHRCD161CXCR3CD127CTLA4CD28ICOS
A
−2 0 2Row Z−Score
Metacluster 4
Metacluster
C p = 0.21 **
Contro
lSLE
0.00
0.01
0.02
0.03
0.04
Contro
lSLE
0.00
0.01
0.02
0.03
0.04
Met
aclu
ster
4 -
Clu
ster
B
Met
aclu
ster
4 -
Clu
ster
AMetacluster 4
HLA
-DR
exp
ress
ion
Cluster A
Cluster B
Supplementary Figure 2: Phenotypic features of cells in metacluster 4.A) Heatmap of marker expression in FlowSOM metaclusters as in Figure 1D including the 2 clusters (cluster A, cluster B) that comprise metacluster 4. B) FlowSOM map demonstrating level of expression of HLA-DR in individual clusters. C) Abundance of the clusters that comprise metacluster 4, cluster A and cluster B, in SLE patients and controls as in Figure 1C. Error bars show median±interquartile range. **p<0.01 by Mann-Whitney test. D) Example contour plot of expression of HLA-DR and ICOS on gated PD-1hi CXCR5- CD4+ T cells in mass cytometry data.
0 10 20 300
20
40
60
SLEDAI
0 10 20 300
5
10
15
SLEDAI
0 10 20 300
5
10
15
SLEDAI
0 10 20 300
1
2
3
SLEDAI
0 10 20 300
3
6
9
SLEDAI
0 10 20 300.0
0.5
1.0
1.5
SLEDAI
0 10 20 300
20
40
60
SLEDAI
0 10 20 300
5
10
15
SLEDAI
r=0.46p=0.029
% P
D-1
very
hig
h CXC
R5ne
g / m
CD
4%
PD
-1ve
ry h
igh C
XCR
5+ / m
CD
4
% P
D-1
hi C
XCR
5neg /
mC
D4
% P
D-1
hi C
XCR
5+ / m
CD
4
% P
D-1
inte
rmed
iate
CXC
R5ne
g / m
CD
4%
PD
-1in
term
edia
te C
XCR
5+ / m
CD
4
(Tph
cel
l gat
e)(T
fh c
ell g
ate)
% P
D-1
tota
l pos
itive
CXC
R5ne
g / m
CD
4%
PD
-1to
tal p
ositi
ve C
XCR
5+ / m
CD
4
r=0.49p=0.018
r=0.10p=0.66
r=0.41p=0.052
r=-0.10p=0.66
r=0.38p=0.094
r=-0.23p=0.28
r=0.12p=0.58
Supplementary Figure 3: Correlation of PD-1+ T cell populations with lupus disease activity.Correlation between lupus disease activity by SELENA-SLEDAI and the frequency of different CD4+ T cell populations defined based on expression of CXCR5 and level of expression of PD-1 (n=21 patients). Spearman correlation statistics shown.
A B
0 20 40 600
5
10
15
Age
% T
ph c
ells
/ m
emC
D4
Black/A
frican
America
nW
hite
Asian
Unkno
wn or n
ot rep
orted
0
5
10
15%
Tph
cel
ls /
mem
CD
4r=-0.32p=0.005
Supplementary Figure 4: Tph cell frequencies in SLE patients according to race, ethnicity, and age.A) Tph cell frequency in SLE patients grouped by self-reported race or ethnicity. Error bars show mean±SD. B) Correlation between Tph cell frequency and age in SLE patients. Spearman correlation statistics shown.
Hispan
ic
Not Hisp
anic
Unkno
wn or n
ot rep
orted
0
5
10
15
% T
ph c
ells
/ m
emC
D4
ATph cells
CXC
R3
mea
n ex
pres
sion
PD-1negative memCD4+ Tfh cells
CXC
R5
mea
n ex
pres
sion
B
CXC
R3
mea
n ex
pres
sion
CXC
R3
mea
n ex
pres
sion
CC
R2
mea
n ex
pres
sion
CC
R2
mea
n ex
pres
sion
D E
0
40
80
120
160
CC
R2
mea
n ex
pres
sion
0
20
40
60
80
100
% C
CR
2+
0
20
40
60
80
100
% C
XCR
5+
0
10
20
30
40
CXC
R5
mea
n ex
pres
sion
Con
trol
RA
SLE
Con
trol
RA
SLE
PD-1neg Tph
Con
trol
RA
SLE
Con
trol
RA
SLE
PD-1neg Tph
Con
trol
RA
SLE
Con
trol
RA
SLE
PD-1neg Tfh
Con
trol
RA
SLE
Con
trol
RA
SLE
PD-1neg Tfh
Cnsns
* ****nsns
ns **** nsns ********
0
20
40
60
0
20
40
60
80
0
20
40
60
80 ns**** * ns**** ns * ns*
0
20
40
60
0
1
2
3
4
5
Tph cells
Contro
l
New-on
set S
LE
Establi
shed
SLE
0
1
2
3
4
5
Contro
l
New-on
set S
LE
Establi
shed
SLE
Contro
l
New-on
set S
LE
Establi
shed
SLE
Contro
l
New-on
set S
LE
Establi
shed
SLE
Contro
l
New-on
set S
LE
Establi
shed
SLE
Contro
l
New-on
set S
LE
Establi
shed
SLE
ns ***** ns * ns ns **** **
Supplementary Figure 5: Chemokine receptor expression on Tph cells in SLE.A) Mean expression of CXCR3 on Tph cells, Tfh cells, and PD-1neg memory CD4+ T cells in mass cytometry analysis of samples from the BWH validation cohort. B) Mean expression of CCR2 and frequency of CCR2+ cells in PD-1neg memory CD4+ T cells and Tph cells from control (n=25), RA (n=25), and lupus nephritis (n=27) donors in the AMP cohort. C) Mean expression of CXCR5 and frequency of CXCR5+ cells in PD-1neg memory CD4+ T cells and Tfh cells as in (B). Tfh cells are CXCR5+ by definition. D) Mean expression of CCR2 on Tph cells and PD-1neg memory CD4+ T cells as in (A). E) Mean expression of CXCR5 on Tfh cells as in (A). Error bars show mean±SD. * p<0.05,** p<0.01, *** p<0.001, **** p<0.0001 by Kruskal–Wallis with Dunn’s multiple comparisons test (A-E) statistics shown.
PD-1negative memCD4+
B
-
Supplementary Figure 6: RNA-seq sorting and interferon scores in T cell populations.A) Example of gating for sorting T cell populations for RNA-seq. B) The interferon score was calculated for each T cell population from each donor using the RNA-seq transcriptomes. Plot shows the IFN score for controls (blue, n=4-5), RA patients (green, n=4-5), and SLE patients (red, n=6) for each T cell population. Error bars show mean±SD.
Naive cells Tregs Tfh cells Tph cells-1.0
-0.5
0.0
0.5
1.0
IFN
sco
re
Control
RA
SLE
A
Single cells
Live CD3+cells
Memory CD4+ Naive CD4+ Treg
Non Treg TfhTph
Lymphocytes
nsns
0.0
0.1
0.2
0.3
0.4
0.5
IL10
rela
tive
expr
essi
on
PD1 - - hi hi - + - + CXCR5
Naive c
ontro
l
Naive S
LE
Treg c
ontro
l
Treg S
LE
Tfh co
ntrol
Tfh SLE
Tph co
ntrol
Tph S
LE0
5
10
15
20IL10
FPK
M
Supplementary Figure 7: Expression of IL10 and IFNG in SLE Tph cells A) Expression of IL10 and IFNG in T cell subsets in RNA-seq data. B) IL10 expression by qPCR in PMA+ionomycin stimulated memory CD4+ T cell subsets from SLE patients (n=4). Error bars show mean±SD (G). Mann Whitney test comparing Tph cells or Tfh cells from controls andSLE patients (A), or Kruskal–Wallis with Dunn’s multiple comparisons test (B).
A B
Naive c
ontro
l
Naive S
LE
Treg c
ontro
l
Treg S
LE
Tfh co
ntrol
Tfh SLE
Tph co
ntrol
Tph S
LE
0
50
100
150
IFNG
FPK
M
nsns
A
DN
A1
DNA2
CD
45
CD19
Bea
ds
Cisplatin
CD
14
CD3
CD
27
CD38
CD
20
CD38
DN
A1
DNA2
CD
3
CD19
Bea
ds
Cisplatin
CD
45
CD19
CD
11c
CD19
CD
16
CD14
CD
21
CXCR5
B
C
0 20 40 60 800
5
10
15
% CD21- CXCR5- / B cells0 20 40 60 80
0
5
10
15
% CD21- CXCR5- / B cells
% T
ph c
ells
/ m
emC
D4+ T
cel
ls
% T
fh c
ells
/ m
emC
D+ T
cel
ls
r = 0.60p = 0.0009
r = 0.67p < 0.0001r = 0.60p = 0.0016
r = 0.15p = 0.46
r = 0.32p = 0.0047r = 0.05p = 0.81
E
0 10 20 30 400
20
40
60
80
CD11c+ / B cells
CD
21- C
XCR
5- / B
cells
Dr = 0.78p < 0.0001
0
10
20
30
% C
D11
c+ C
D21
- / B c
ells
G
Plasmablasts
B Cells
B Cells
F
CD
21
CD11c
CD11c+ CD21-
Supplementary Figure 8: Tph cells correlate with CD11c+ B cells.A) Gating strategy for B cells and plasmablasts in the AMP mass cytometry data using the B cell panel. B) Gating strategy for CD11c+ B cells in the AMP mass cytometry data using the myeloid panel. C) Example visualization of CD21 and CXCR5 on B cells gated as in (A) in the AMP mass cytometry data. D) Correlation between frequency of CD11c+ B cells and CD21- CXCR5- B cells in all patients in the AMP cohort. E) Correla-tion between the frequency of Tph cells or Tfh cells and CD21- CXCR5- B cells in the AMP cohort (black = controls, green = RA patients, red = SLE patients). Spearman correlation statistics show (D,E). F) Example of detection of CD11c+ CD21- B cells in co-cultures of memory B cells from controls co-cultured with indicated CD4+ T cell subsets from SLE patients G) Quantification of CD11c+ B cells among B cells (excluding plasmab-lasts) in co-cultures as in (F). Pooled data from 6 donors in 6 different experiments. Error bars show mean±SD.
Tph cells Tfh cells
0
50
100
150
PRDM1
Naive c
ontro
l
Naive R
A
Naive S
LE
Treg c
ontro
l
Treg R
A
Treg S
LE
Tfh co
ntrol
Tfh RA
Tfh SLE
Tph co
ntrol
Tph R
A
Tph S
LE0
5
10
SOX4
Naive c
ontro
l
Naive R
A
Naive S
LE
Treg c
ontro
l
Treg R
A
Treg S
LE
Tfh co
ntrol
Tfh RA
Tfh SLE
Tph co
ntrol
Tph R
A
Tph S
LE
Naive c
ontro
l
Naive R
A
Naive S
LE
Treg c
ontro
l
Treg R
A
Treg S
LE
Tfh co
ntrol
Tfh RA
Tfh SLE
Tph co
ntrol
Tph R
A
Tph S
LE0
10
20
30
40
50BCL6
ns ns ns ** ns ns
Supplementary Figure 9: Expression of BCL6, PRDM1, and SOX4 in circulating Tph and Tfh cells. Expression of BCL6, PRDM1, and SOX4 in T cell subsets in RNA-seq data. Error bars show mean±SD. * p<0.05,** p<0.01, *** p<0.001, **** p<0.0001 by Mann-Whitney test comparing Tph cells or Tfh cells from controls and SLE patients.
We recognize participants in Phase 1 of the Accelerating Medicines Partnership RA/SLE
Network, which includes: Jennifer H. Anolik1, William Apruzzese2, Arnon Arazi3, Ami Ben
Artzi4, John P. Atkinson5, Joan M. Bathon6, Celine C. Berthier7, David L. Boyle8, Michael
B. Brenner1, S. Louis Bridges9, Jill P. Buyon10, Vivian P. Bykerk11, John A. Carrino11,
Arnold Ceponis8, Adam Chicoine2, Robert Clancy10, Sean Connery12, Andrew C.
Cordle13, Melissa Cunningham14, Maria Dall’Era15, Robert B. Darnell16, Anne Davidson17,
Evan Der18, Betty Diamond17, Laura T. Donlin11, Andrea Fava19, Andrew Filer20, Gary S.
Firestein8, Chamith Y. Fonseka2,3, Lindsy Forbess4, Richard A. Furie21, Anqi Gao2,
Daniel Goldman19, Susan M. Goodman11, Ellen M. Gravallese2, Peter K. Gregersen17,
Rohit Gupta22, Joel M. Guthridge23, Nir Hacohen3, David A. Hildeman24, Michael
Holers25, Paul J. Hoover2, Diane Horowitz21, Raymond Hsu15, Laura B. Hughes9, Mariko
L. Ishimori4, Lionel B. Ivashkiv11, Judith A. James23, A. Helena Jonsson2, Ruba Kado7,
Ken C. Kalunian8, Diane L. Kamen14, Joshua Keegan2, Stephen Kelly26, Gregory Keras2,
Mattias Kretzler7, James A. Lederer2, Peter Lowry25, Holden Maecker22, Arthur M.
Mandelin27, Elena M. Massarotti2, Eric L. Matteson28, William J. McCune7, Mandy J.
McGeachy13, Maureen McMahon29, Fumitaka Mizoguchi2, Larry W. Moreland13, Oganna
Nwawka11, Dana E. Orange11, Meyeon Park15, Deborah Parks5, Fernanda Payan-
Schober12, William F. Pendergraft30, Harris R. Perlman27, Alessandra B. Pernis11,
Michelle Petri19, Mina Pichavant22, Costantino Pitzalis31, Chaim Putterman18, Deepak A.
Rao2, Soumya Raychaudhuri2,3, Christopher Ritchlin1, William H. Robinson22, Karen
Saloman-Escoto32, Daimon Simmons2, Kamil Slowikowski2,3, Hermant Suryawanshi33,
Darren A. Tabechian1, Ralf G. Thiele1, Thomas Tuschl33, Paul J. Utz22, Dia
Waguespack34, Gerald Watts2, Kevin Wei2, Michael Weisman4, Aaron Wise13, E. Steve
Woodle35, David Wofsy15, Fan Zhang2,3.
1University of Rochester Medical Center, Rochester, New York. 2Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts. 3Broad Institute of Massachusetts Institute and Technology and Harvard University, Cambridge, Massachusetts. 4Cedars-Sinai Medical Center, Los Angeles, California. 5Washington University School of Medicine, Saint Louis, Missouri. 6Columbia University, New York, New York. 7University of Michigan, Ann Arbor, Michigan. 8University of California, San Diego, La Jolla, California. 9University of Alabama at Birmingham, Birmingham, Alabama. 10New York University School of Medicine, New York, New York. 11Hospital for Special Surgery, New York, New York. 12Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, Texas.
13University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania. 14Medical University of South Carolina, Charleston, South Carolina.
15University of California San Francisco, San Francisco, California.
16New York Genome Center and the Rockefeller University, New York, New York. 17The Feinstein Institute for Medical Research, Northwell Health, Manhasset, New York.
18Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, New York.
19Johns Hopkins University, Baltimore, Maryland.
20NIHR Birmingham Biomedical Research Centre, University Hospitals Birmingham NHS Foundation Trust and University of Birmingham, Birmingham, UK
21Northwell Health, Great Neck, New York.
22Stanford University School of Medicine, Stanford, California.
23Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA. 24University of Cincinnati and Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio.
25University of Colorado School of Medicine, Aurora, Colorado.
26Barts Health NHS Trust, London, UK
27Northwestern University Feinberg School of Medicine, Chicago, Illinois.
28Mayo Clinic College of Medicine, Rochester, Minnesota.
29University of California Los Angeles, Los Angeles, California.
30The Integrative Medical Clinic of North Carolina, PLLC, Chapel Hill, North Carolina.
31William Harvey Research Institute, Queen Mary University of London, London, UK
32University of Massachusetts Medical School, Worcester, Massachusetts.
33Rockefeller University, New York, New York.
34University of Texas, Houston, Texas.
35University of Cincinnati College of Medicine, Cincinnati, Ohio.