www.sciencetranslationalmedicine.org/cgi/content/full/6/230/230ra46/DC1
Supplementary Materials for
Cell Distance Mapping Identifies Functional T Follicular Helper Cells in Inflamed Human Renal Tissue
Vladimir M. Liarski, Natalya Kaverina, Anthony Chang, Daniel Brandt, Denisse Yanez, Lauren Talasnik, Gianluca Carlesso, Ronald Herbst, Tammy O. Utset, Christine Labno,
Yahui Peng, Yulei Jiang, Maryellen L. Giger, Marcus R. Clark*
*Corresponding author. E-mail: [email protected]
Published 2 April 2014, Sci. Transl. Med. 6, 230ra46 (2014) DOI: 10.1126/scitranslmed.3008146
The PDF file includes:
Fig. S1. TFH ICOS+ cells in human LuN are limited to the renal tubulointerstitium. Fig. S2. Preliminary development and validation of automated CDM method. Fig. S3. Relationship between cell density and observed TFH–B cell conjugate rates. Fig. S4. Immunofluorescence analysis of MR biopsies reveals different patterns of TFH–B cell distribution. Fig. S5. Comparison of CDM results to a control random distribution model. Fig. S6. TFH ICOS+ cells in human LuN produce IL-21 but not IL-17. Fig. S7. Original antibody staining used for three-dimensional surface creation of TFH–B cell cognate pairs in LuN. Table S1. LuN patient cohort clinical and histological characteristics. Table S2. Digital image database used for CDM analysis. Table S3. Direct statistical comparison of CDM distributions between clinical biopsy groups. Table S4. Curve fit analysis for assessing kinetics of CDM distributions between cases.
Supplementary Materials:
Figure S1 – T FH ICOS+ cells in human LuN are limited to the renal tubulointerstitium.
Single-color immunohistochemistry staining for ICOS of human renal biopsy. Right
panel denotes magnification of boxed area in left panel. *Denotes glomeruli. Arrows
denote location of ICOS+ cells. Scale bars: 100 µm.
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Figure S2 – Preliminary development and validation of automated CDM method.
(a) Top row – Input images representing unaltered raw images obtained from multicolor
confocal immunofluorescent microscopy staining of human tonsil germinal center (GC)
for CD20, DAPI, ICOS, and CD4. Middle row – Interim stain signatures obtained after
applying manual algorithm to generate binary masks for each stain. Bottom row – Visual
comparison of results between the manual and automated algorithms performed on the
same digital high-power field (dHPF). Red – automated result; green – manual result;
yellow - areas of overlap. Scale bar: 20 m. (b) Quantitative comparison of manual
(open circle) and automated (closed circle) algorithms for distance mapping of human
tonsil tissue shown in (a). Fraction (%) of TFH cells (CD4+ICOS+ in left panel [n=2 dHPF
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from 2 biopsies] or CD4+PD-1+ [n=5 dHPFs from 2 biopsies] in right panel) at indicated
distance from nearest CD20+ B cell. Error bars denote SEM.
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Figure S3 – Relationship between cell density and o bserved T FH–B cell conjugate rates.
Effect of density segregation based on mean numbers of ICOS+ TFH cells per digital
high-power field (dHPF) for human lupus nephritis (a), and mixed cellular renal allograft
rejection cases (b). Density correction was based on mean cell number values across
dHPFs for each stain combination based on individual disease process. Frequency of
TFH to B cell with each distance relationship is illustrated. Each point represents the
mean absolute percentage of total TFH cells at a given minimum distance cutoff in a
distribution segregated by mean dHPF density as indicated. Error bars denote SEM.
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Figure S4 – Immunofluorescence analysis of MR biops ies reveals different patterns of T FH–B cell distribution.
Confocal multichannel immunofluorescent staining of mixed cellular renal allograft
rejection (MR) showing global distributions of TFH and B cells. The sample is stained for
CD4, ICOS, CD20, and DAPI with composite multiplexed images shown as indicated.
Scale bars: 20 µm.
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Figure S5 – Comparison of CDM results to a control random distribution model.
Comparison of CDM results to a control random distribution model. Comparison of
cumulative ICOS+ (a) or PD-1+ (b) TFH:B cell conjugate rates at specified distance
cutoffs against a theoretical random heteroconjugate distribution. The observed B cell
positions from analyzed human lupus nephritis cases were used as a reference point for
which a completely random TFH distribution was generated by computer algorithm for
each ROI, holding the number of respective cell nuclei constant. P-values, reflecting a
null hypothesis of the distributions being equal, were obtained for each digital high-
power field (dHPF) and were aggregated on a per-biopsy basis. Each point represents
the weighted average p-value based on the above for a single clinical biopsy. Horizontal
bars represent the mean p-value across each set of biopsies for a given distance cutoff
with error bars denoting SD. The y-axis scale is logarithmic. Dashed lines represent
logarithmic data points for a reference p-value of 0.05 and 0.005 reflecting appropriate
Bonferroni corrections.
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Figure S6 – TFH ICOS+ cells in human LuN produce IL-21 but not IL-17.
Confocal multichannel immunofluorescent staining of human tonsil germinal center (GC)
and lupus nephritis (LuN) cases (n=2 biopsies) showing CD3+ICOS+ TFH cells that are
also positive for IL-21 (a). Similar staining performed for IL-17 and CD3 of control
Crohn’s disease, tonsil, and LuN samples (n=5 biopsies) (b) showing lack of TH17-
lineage cells in LuN. Scale bars: 20 µm.
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Figure S7 – Original antibody staining used for thr ee-dimensional surface creation of T FH–B cell cognate pairs in LuN.
(a,b) Three-dimensional surface reconstruction obtained from single sections of human
lupus nephritis. DAPI surfaces, created with the default settings in Imaris, are illustrated
along with unaltered original stains for (a) CD3 (red), LFA1 (green), CD20 (magenta),
and MHC class II (blue) and (b) CD3 (red), LFA1 (green), ICAM1 (magenta), and MHC
class II (blue). Scale in µm indicated for each image.
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Supplementary tables:
Table S1 – LuN patient cohort clinical and histolog ic characteristics. ICOS+ ICOS-
Clinical Characteristics
Age, mean±SD years 31.3±14.4 32.3±12.5
Sex
Female 15 (78.9) 20 (87)
Male 4 (21.1) 3 (13)
Serum creatinine level, median (range) mg/dL 2.3 (0.7-5.4) 1.1 (0.6-2.3)
Glomerular filtration rate by sex-adjusted MDRD, median (range) mL/min/1.72m2
44.8 (9.7-115.8) 74.1 (34.4-117.7)
Histologic characteristics
ISN/RPS lupus nephritis class
Class II 1 (5.3) 2 (8.7)
Class III (± V) 8 (42.1) 5 (21.7)
Class IV (± V) 7 (36.8) 11 (47.8)
Class V 1 (5.3) 3 (13.0)
Class VI 1 (5.3) 0 (0)
NIH activity index, mean±SD 6.0±4.3 6.3±3.7
NIH chronicity index, mean±SD 3.7±2.9 2.5±3.2
Values are the number (percentage) unless otherwise indicated. Glomerular filtration
rate calculation excludes patients under 18 years of age. MDRD = Modification of Diet in
Renal Disease; ISN/RPS = International Society of Nephrology/Royal Pathology
Society; NIH = National Institutes of Health.
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Table S2 – Digital image database used for CDM anal ysis.
LuN GC TCMR MR
TFH ICOS+
TFH PD-1+ TFH ICOS+
TFH PD-1+ TFH ICOS+
TFH ICOS+
Numbers examined
dHPFs 45 58 13 7 59 29
Biopsies 9 10 2 2 8 7
Minimal T:B distances 910 754 616 377 725 407
Minimal B:T distances 1579 1041 949 787 891 1305
All potential T:B interactions 1.4*106 7.8*105 5.8*105 2.9*105 6.4*105 5.3*105
Numbers of digital high-power fields (dHPFs), individual biopsies, and interactions
between identified T follicular helper (TFH) cells and B cells that comprised our analyzed
database. Numbers of analysis points are indicated for human lupus nephritis (LuN),
tonsil germinal center (GC), T cell-mediated (TCMR), and mixed cellular (MR) renal
allograft rejection cases.
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Table S3 – Direct statistical comparison of CDM dis tributions between clinical biopsy groups. LuN GC TCMR MR
TH ICOS+ TH PD-1+ TH ICOS+ TH PD-1+ TH ICOS+ TH ICOS+
LuN TH ICOS+ NA 0.5798 0.0023 0.0155 <0.0001 <0.0001
TH PD-1+ 0.5798 NA 0.0042 0.0382 <0.0001 <0.0001
GC TH ICOS+ 0.0023 0.0042 NA 0.3842 <0.0001 0.1892
TH PD-1+ 0.0155 0.0382 0.3842 NA <0.0001 0.0556
TCMR TH ICOS+ <0.0001 <0.0001 <0.0001 <0.0001 NA <0.0001
MR TH ICOS+ <0.0001 <0.0001 0.1892 0.0556 <0.0001 NA
P-values based on two-tailed unpaired t-test with a reference cutoff of 0.05 comparing
the average cumulative ICOS+ and PD-1+ T follicular helper (TFH) cell to B cell distance
distributions. Distance distributions of human lupus nephritis (LuN), tonsil germinal
center (GC), T cell-mediated (TCMR), and mixed cellular (MR) renal allograft rejection
cases were compared as indicated.
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Table S4 – Curve fit analysis for assessing kinetic s of CDM distributions between cases. LuN GC TCMR MR
TH ICOS+ TH PD-1+ TH ICOS+ TH PD-1+ TH ICOS+ TH ICOS+
Curve Fit Log Log Log Log Linear Log
Probability of Fit 99.93% >99.99% >99.99% >99.99% >99.99% >99.99%
R2 0.938 0.983 0.973 0.988 0.986 0.951
Y-intercept 49.16 54.72 73.17 67.09 15.62 81.73
95% CI 45.81-52.51 53.26-56.18 71.60-74.75 65.99-68.29 13.98-17.26 80.12-83.34
Curve fit analysis for average cumulative ICOS+ and PD-1+ T follicular helper (TFH) cell
to B cell distance distributions for human lupus nephritis (LuN), tonsil germinal center
(GC), T cell-mediated (TCMR), and mixed cellular (MR) renal allograft rejection cases.
A logarithmic curve fit was performed assuming 15 degrees of freedom for all cases and
was compared with straight line fit. Best fit was then selected based on probability of fit
versus a reference distribution and r2 values (indicated). Y-intercept reflects the
predicted zero conjugate rate for the best fit for each distribution with 95% confidence
intervals being listed for same.