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: mclark@medicine.bsd.uchicago.edu

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.