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Short peptide vaccine induces CD4+ T helper cells in patients with different solid cancers –
desription of methods according to MIATA guidelines (MIATA checklist)
Information
on….
….the sample
patients
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techniques
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….the assay
detailed MFTC
assay
description
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HLA types: HLA-A1, HLA-A2, HLA-A3, HLA-A24, HLA-B7
Disease: patients had a variety of different solid survivin
expressing cancers
Treatment: vaccination with EMD640744 in Montanide ISA 51 VG
Co-medication: none
see detailed description of the trial in:
“Lennerz V, Gross S, Gallerani E, Sessa C, Mach N, Boehm S, et al.
Immunologic response to the survivin-derived multi-epitope
vaccine EMD640744 in patients with advanced solid tumors.
Cancer Immunol Immunother. 2014.”
Whole blood collection and PBMC processing was done in the
five Swiss study centers by trained personnel;
95 ml whole blood was obtained by venipuncture using LiHeparin blood collection tubes at different time points pre and
post vaccination;
Maximum time between collection and processing was two
hours.
PBMC were isolated using Ficoll densitiy gradient centrifugation
with Leukosep separation tubes (Greiner Bio-one, #227290);
whole blood was not diluted; leukaphereses were diluted 1:2
with PBS/EDTA;
PBMC were frozen in FCS/10% DMSO at 1x107/vial using 2propanol-filled cryobox freezing containers (Nalgene, #51000001) at
-80°C overnight and afterwards stored in liquid nitrogen until
transport to the experimental laboratories in Mainz and Erlangen
PBMC were transported from study centers to experimental lab
on dry ice within 24 hours
Upon arrival, all shipped vials were controlled for completeness
and proper condition, and the vials were rapidly transferred to
liquid nitrogen for storage until use; (every step of the shipment
was monitored, respective data loggers were signed and sent to
the study sponsor and a monitoring agency)
Samples for direct comparison (i.e. samples from different
timepoints of the same patient) were always thawed, stimulated
and assayed on the same day to ensure same assay conditions.
Vials with 10x106 cells each were thawed, washed and counted
(median recovery: 7.4x106 per vial) using a CasyCellCounter
(Schärfe Systems).
No dead cell determination was performed at this time point.
Cells were then seeded in a 12-Well-Plate (2–4 million cells per
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controls
reagents
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ml) in MLPC-Medium.
Cells were then prestimulated with the corresponding peptide or
the EMD640744 peptide mix (5-10 g/ ml). The next day, IL-2 and
IL-7 were added. Half of the medium was replaced every 3–4
days with fresh MLPC-medium containing IL-2. No IL-2 was given
to the in vitro stimulated PBMCs in the last two days before the
assay.
On day 12 – 15 cells were washed with MLPC medium and
stimulated overnight (37°C, 5% CO2) in sterile 5ml FACS tubes
(BD) in MLPC medium with or without the corresponding peptide
and in some cases blocking antibodies (as indicated in Fig.2) in
the presence of BrefeldinA and Monensin and CD107a- and
CD154- antibodies. In some experiments peptides were loaded
onto
HLA-matched
or
mismatched
EBV-transformed
lymphoblastic cell lines (EBV-LCLs). Loading was for one hour at
room temperature with 5 μg/ml peptide. Cells were then washed
and used at a ration of 1:1 in the assay.
The next day, cells were washed with PBS and stained with deadcell stain Live/Dead aqua (Invitrogen) according to the
manufacturer’s instructions. The cells were then spun down, the
supernatant was discarded and the cells were resuspended in
PBS containing surface-staining antibodies (CD8, CD4, CD14).
After 20 minutes at 4˚C cells were washed with PBS and fixed and
permeabilized with fix/perm solution and perm/wash (both
eBioscience) according to the manufacturer’s instructions.
Intracellular staining was performed in perm/wash with IL2-,
TNFalpha- and IFNgamma- antibodies, for 30 minutes. Cells were
then washed and resuspended in PBS.
For each sample all cells within a tube were acquired. For MFTC
assays total events ranged from 1.0 x105 to 4.1 x106 (mean: 1.8
x106).
For each sample a negative buffer control (prepared under the
same assay conditions but without peptide) was assessed.
MLPC-Medium (RPMI1640 with 10% pretested human pooled
serum (Lonza), gentamycin, pyruvate and nonessential amino
acids)
Survivin-peptides (Bachem) had a purity of >95%
HLA-A1-binding FTELTLGEF, Sur93-101/T2
HLA-A2-binding LMLGEFLKL, Sur96-104/M2
HLA-A3-binding RISTFKNWPK, Sur18-27/K10
HLA-A24-binding STFKNWPFL, Sur20-28
HLA-B7-binding LPPAWQPFL, Sur6-14
IL-2 (5 U/ml, Roche) and IL-7 (10 ng/ml, TEBU)
BrefeldinA (5ng/ml) and Monensin (750 pg/ ml), both from Sigma
Antibodies (vendor and concentration or dilution): anti-HLA-ABC
(BD, DX17, 10 μg/ ml), anti-HLA-DR/DP/DQ (BD, Tu39, 10 μg/ ml),
anti-HLA-DR (Biolegend, L243, 10 μg/ ml), anti-HLA-DQ (Beckman
Coulter, SPVL3, 10 μg/ ml), anti-HLA-DP (abcam, B7/21, 10 μg/
….data
acquisition
Materials/
Equipment
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Machines/
Software
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Calibration
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….results
Gating
Response
criteria
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Auditing
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Correlation
between data
sets
….the lab
environment
SOPs
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Qualification/
Training of
personnel
ml), anti-CD107a-FITC (BD, 1:50), anti-CD154-PE (BD, 1:50), antiCD8-PerCP (BD, 1: 10) and anti-CD4-V450 (BD, 1:20), anti-CD14PacificOrange (Invitrogen, 1:30), anti-IL2-APC (BD, 1:25), antiTNFalpha-PE-Cy7 (BD, 1:20) and anti-IFNgamma-AlexaFluor700
(BD, 1:30)
cell incubators, centrifuges and laminar flow cabinets were
state-of-the-art devices for the sterile handling of human cell
material
The Flow Cytometry assays were aquired on a FACS Canto II Flow
Cytometer (BD Biosciences) equipped with FACS Diva (6.1)
software; for Flow Cytometry analyses, FlowJo 9.7.5 was used;
the FACS Canto II Flow Cytometer was calibrated according to the
manufacturers recommendations on a regular basis (CS&T
Performance Check on every day of usage);
The gating strategy is shown in Supplemental Figure 1
The values obtained from negative controls were subtracted
from the corresponding test samples.
Criteria for a positive response were: results of individual
cytokine-producing subsets at least two times higher than the
corresponding subsets in the negative control (background), and
the sum of all cytokine-producing subsets greater than 0.03%.
Representative example data for an MFTC assay, including gating
strategy, can be seen in Supplemental Figure 1.
All plots were audited and cells within the cytokine gates were
regularly checked by backgating
A correlation of the immune response data with disease stage of
the patients and treatment was not analyzed since objective
clinical response rates are generally low after cancer
vaccination, clinical efficacy can only be reasonably
assessed in an appropriately designed trial with a time-toevent endpoint.
Standardized and validated working practices were in place for
cell thawing, counting, stimulation, the MFTC assay (staining and
data aquistion including quality-control for Canto II) and analysis
Only well trained personnel with more than five years’
experience with all methods and technologies performed the
experiments and data analyses as well as interpreted and
reported results;