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SUPPLEMENTAL METHODS AND FIGURES
Mononuclear cell isolation from blood: Venous blood (40 ml) was drawn into heparincontaining tubes and mononuclear cells isolated by density gradient centrifugation on 65%
Percoll gradient as previously described (1). Monocytes were depleted from the MNC fraction by
incubation in plastic flasks for 2 h at 37°C as the outgrowth of these cells interferes with the
colony formation in the methylcellulose assay. Non-adherent mononuclear cells (NAMNC) were
immunostained for flow cytometry and cultured for 14-day semi-solid methylcellulose colony
forming assay.
Sputum induction and processing: Sputum was induced and processed using the method
described by Pizzichini and coworkers (2). Briefly, following inhalation of hypotonic aerosolized
saline, cell plugs were collected from the expectorated sputum sample and processed using 0.1%
dithiothreitol (Sputolysin, Calbiochem) and Dulbecco’s phosphate buffered saline (Gibco) (1).
The dispersed sputum was filtered through Accufilter filter units (Cellometrics Inc.). Following
centrifugation (1500 rpm, 10 mins), cytospins were prepared from the pelleted cells on glass
slides and stained with Diff Quik (American Scientific Products, McGaw Park, IL) for
differential counts, which were enumerated by an observer blinded to the treatment period.
Means of duplicate slides were obtained (500 cells counted per slide) and expressed as absolute
counts (104cells /mL). The remaining sputum cell suspension was fixed in 1% paraformaldehyde
until immunostained for flow cytometry.
Eosinophil Colony Forming Assay: In methylcellulose culture assays, the outgrowth of
progenitors into clonally-derived aggregates of daughter cells defined as colony forming units
(CFUs) were enumerated based on distinct morphological features under light microscopy as
eosinophils and basophils (Eo/B)-CFU (tight, compact, round refractile cell aggregates) as
previously described (3, 4). Blood-derived NAMNC (1x106 cells/ plate) were cultured in
duplicate in Iscove's modified Dulbecco's medium (with 1% penicillin-streptomycin and 0.5 μm
of 2-ME), 20% FCS, 0.9% methylcellulose and rhIL-5 or diluent, for 14 days at 37o C and 5%
CO2. Day 14 granulocyte colonies of >40 cells were enumerated and classified as Eo/B-CFU(5).
Fluorescence Immunocytochemistry for EoP: Blood derived non-adherent mononuclear cells
(NAMNC; 1x106) and sputum cells (1-0.6x106) were stained with antibodies (CD45-fluorescein
isothiocyanate (FITC), CD34-allophycocyanin (APC) and CD125 (IL-5Rα)-phycoerythrin (PE);
BD Biosciences) or isotype control antibody in PBS plus 0.1% NaN3 and Fc block and then fixed
in PBS plus 1% paraformaldehyde and analyzed by a FACS Calibur flow cytometer (BDIS).
Using flow cytometric analysis 200 000 events in the lympho-mononuclear region (low side
scatter/low forward scatter) were counted and all events stored for subsequent analyses as
previously described (1, 6). Data plot analyses of EoP sputum samples from severe asthatmics
are described in supplemental Figure S2a and 2b).
In addition, further phenotyping of EoPs was performed showing Lin- expression and
co-expression of CD45RA, CD38 and CD123 (BD Biosciences) to confirm agreement of
identification of eosinophil precursors as described by Mori et al. previously (Supplemental
Figure S2c) (7).
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Flow cytometry gating: Sequential multi-gating flow cytometric assessments of progenitor cells
were performed as described previously (1, 6). Off-line analysis was performed using the Cell
Quest software as supplied by BDIS. True CD34+ blast cells were identified as cells with
lymphomononuclear cells with CD34high/CD45dull staining and eosinophil progenitors as
CD34high/CD45dull/IL5Rαhigh as previously described (1, 6).
Prednisone reduction: The prednisone reduction was performed as described in detail by Bel et
al., (8)
Sequential Time Course
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Prednisone/Prednisolone Reduction Phase
Oral Corticosteroid Dose (mg/day)
Optimized OCS dose
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30
25
20
15
12.5
10.
7.5
1st dose reduction
25.0 20.0
15.0
10.0
10.0
10.0
5.0
5.0
+ 4 Weeks
15.0 10.0
10.0
5.0
5.0
5.0
2.5
2.5
+ 4 Weeks
10.0
5.0
5.0
2.5
2.5
2.5
1.25* 1.25*
+ 4 Weeks
5.0
2.5
2.5
1.25* 1.25* 1.25*
0
0
+ 4 Weeks
2.5
2.5
2.5
0
0
0
0
0
*Subject taking 1.25mg/day should take this as 2.5mg administered every other day
5.0
2.5
1.25*
0
0
0
Asthma control questionnaire score: The ACQ-5 is a five-item self-completed questionnaire,
which has been developed as a measure of patients’ asthma control that can be quickly and easily
completed in clinical practice (9). The questionnaire was incorporated into the subject’s e-Diary
and subjects were prompted on a weekly basis to complete the questionnaire as described by Bel
et al.(8) The 5 questions enquire about the frequency and/or severity of symptoms (nocturnal
awakening on waking in the morning, activity limitation, shortness of breath, wheeze) over the
previous week. The response options for all these questions consisted of a zero (no
impairment/limitation) to six (total impairment/ limitation) scale. The questionnaire is scored
from 0 to 6, with higher numbers indicating poorer control and a minimally important difference
of 0.5.
References
1.
Smith SG, Gugilla A, Mukherjee M, Merim K, Irshad A, Tang W, et al. Thymic stromal
lymphopoietin and IL-33 modulate migration of hematopoietic progenitor cells in patients with
allergic asthma. J Allergy Clin Immunol. 2015;135:1594-602
2.
Pizzichini E, Pizzichini MMM, Efthimiadis A, Evans S, Morris MM, Squillace D, et al.
Indices of airway inflammation in induced sputum: reproducibility and validity of cell and fluidphase measurements. AmJRespirCritCare Med. 1996;154:308-17.
3.
Smith SG, Hill M, Oliveria JP, Watson BM, Baatjes AJ, Dua B, Howie K, Campbell H,
Watson RM, Sehmi R, Gauvreau GM. Evaluation of peroxisome proliferator-activated receptor
agonists on interleukin-5-induced eosinophil differentiation.Immunology. 2014;142:484-91.
4.
Denburg JA, Messner H, Lim B, Jamal N, Telizyn S, Bienenstock J. Clonal origin of
human basophil/mast cells from circulating multipotent hemopoietic progenitors. ExpHematol.
1985;13:185-8.
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5.
Hui CC, Rusta-Sallehy S, Asher I, Heroux D, Denburg JA. The effects of thymic stromal
lymphopoietin and IL-3 on human eosinophil-basophil lineage commitment: Relevance to atopic
sensitization. Immun Inflamm Dis. 2014 ;2(1):44-55.
6.
Imaoka H, Campbell H, Babirad I, Watson RM, Mistry M, Sehmi R, et al. TPI ASM8
reduces eosinophil progenitors in the sputum after allergen challenge in mild asthmatics. Clinical
and Experimental Allergy. 2011;14:1740-6.
7.
Mori Y, Iwasaki H, Kohno K, Yoshimoto G, Kikushige Y, Okeda A, et al. Identification
of the human eosinophil lineage-committed progenitor: revision of phenotypic definition of the
human common myeloid progenitor. The Journal of Experimental Medicine. 2009;206(1):18393.
8.
Bel EH, Wenzel SE, Thompson PJ, Prazma CM, Keene ON, Yancey SW, et al. Oral
glucocorticoid-sparing effect of mepolizumab in eosinophilic asthma. N Engl J Med. 2014
371:1189-97.
9.
Juniper E, O'Byrne P, Guyatt G, Ferrie P, King D. Development and validation of a
questionnaire to measure asthma control. European Respiratory Journal. 1999;14(4):902-7.
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SUPPLEMENTAL FIGURES and LEGENDS
Primary Efficacy
Endpoint assessed
at week 24
Investigation Product given during this time
Visit 1
Week 0
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8
12
16
Visit
4
5
6
7
3
20
24
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8
9
Follow-up
Visit 2
OCS Optimisation
Phase
Induction
Phase
OCS Reduction Phase
-8 to -3 weeks
Phase 1
Phase 2
Lowest OCS
determined by
ACQ score
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Maintenance
Phase
Open label Phase
Drug
Placebo
Blood and Sputum Samples Collected
V1 – Pre-treatment, Screening
V4 – Start of Steroid Reduction
V9 – End of Treatment Study
Supplemental Figure S1 Study design: at baseline (Visit 1, screening pre-treatment time point)
venous blood and sputum sampling were performed for progenitor and eosinophil enumeration.
Following a corticosteroid optimization period, of 3 - 8 weeks in duration, subjects were
randomized to receive either mepolizumab (100mg sc) or placebo by subcutaneous injection.
Subsequent injections were administered monthly. Blood and sputum sampling were repeated at
Visit 4, 4 weeks after the first injection (reduction time-point), and at Visit 9, 24 weeks after the
first injection (post-treatment time point).
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Supplemental Figure S2: Sequential Multi-gating strategy of sputum cells: (A) identification of HPC
(CD34+45+ cells) using FlowJo software. Scatter plots show SSC and CD45 – region R1 identifies
nucleated white blood cells (WBC). Region R2 identifies CD34+ marker. True CD34+ are further
isolated based upon low granularity and low CD45+ marker expression-region R3. HPCs are
characterized by medium size (medium FSC) and low granularity (low SSC) –region R4. (B) EoP are
identified as HPC expressing CD125 compared to isotype control with a 98% confidence limit. (C) .
Further phenotyping of EoPs showing Lin- expression and co-expression of CD45RA, CD38 and
CD123 was performed to confirm identification of eosinophil precursors as described by Mori et al.
(7). The dotted vertical lines denote the isotype control marked 98% confidence limits.
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Drug
14 day Eo-CFU
(per 106 WBC)
40
Placebo
30
20
10
0
V1
V4
V9
V1
V4
V9
IL-5 (1 ng/mL)
Drug
14 day Eo-CFU
(per 106 WBC)
50
Placebo
40
30
20
10
0
V1
V4
V9
V1
V4
V9
IL-5 (10 ng/mL)
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Supplemental Figure S3: EoP clonogenic assay. Blood eosinophil progenitor cells (EoP) were
enumerated by clonogenic expansion in methylcellulose culture. Compared to pre-treatment levels
there was a trend for an increase in Eo/B-CFU grown with IL-5 (at 1 and 10 ng/ml) in the drug but not
the placebo treated group. These data suggest that inhibition of IL-5-driven terminal differentiation of
eosinophils results in the increased circulating levels of EoP. Data with IL-5 at 0.1ng/ml is shown in
Figure 4.