Supplementary Material and Methods
Cell lines
Ramos, Raji, Daudi and CEM cells (DSMZ, The German Resource Centre for Biological
Material, Braunschweig, Germany) were cultured in RPMI 1640 Glutamax-I medium
(Invitrogen, Karlsruhe, Germany) containing 10% fetal calf serum (FCS; Invitrogen), 100
units/ml penicillin and 100 µg/ml streptomycin (Invitrogen). Lenti-X 293T cells (Clontech,
Saint-Germain-en-Laye, France) were maintained in Dulbecco's Modified Eagle Medium
(DMEM; Invitrogen) supplemented with 10% FCS, 100 units/ml penicillin and 100 µg/ml
streptomycin. CHO-K1 cells (DSMZ) were cultured in chemically defined CHO medium
(Invitrogen) containing 50 units/ml penicillin, 50 µg/ml streptomycin and HTT supplement
(Invitrogen).
Generation of recombinant proteins
The cDNA sequences encoding the natural secretion leaders and the ECDs of MICA
(spanning amino acids 1 – 275; NM_000247.1) and ULBP2 (amino acids 1 – 161;
NM_025217.2) were de novo synthesized (Entelechon, Regensburg, Germany) and ligated as
NheI/NotI cassettes into vector pSectag2HC/3G8 Fd-7D8-myc-his (M. Peipp, unpublished)
generating vectors pSecTag2/MICA:7D8-myc-his and pSecTag2/ULBP2:7D8-myc-his,
respectively. Vectors for control proteins were constructed by replacing the sequences for
scFv 7D8
1
by those encoding scFv TH69 2. For generation of a fusion protein between
NKG2D and human IgG1 Fc (NKG2D-Fc), the coding sequence for the ECD of NKG2D was
amplified from cDNA of mononuclear cells (MNC) by standard procedures using primers
5’-atgcatggcccagccggccttcaaccaagaagttcaaattcccttgacc-3’ and
5’-acgtacgtcgaccgcggccgcccacagtcctttgcatgcagatgtatgtatttgg-3’
and cloned as SfiI/NotI fragment into vector pSectag2-Fc 3. Correct sequences were confirmed
by Sanger sequencing.
Expression and purification
MICA:7D8, ULBP2:7D8, the control proteins MICA:TH69 and ULBP2:TH69 as well as
NKG2D-Fc were transiently expressed in Lenti-X 293T cells by calcium-phosphate
transfection as described
3
or in stably transfected CHO-K1 cells obtained after transfection
using Lipofectamine 2000 (Invitrogen). Positive cell clones were selected with 500 µg/ml
Hygromycin B (Roche, Grenzach-Wyhlen, Germany) and single clones were isolated by
limiting dilution. The His-tagged proteins were purified by affinity chromatography with
nickel-nitrilotriacetic acid (Ni-NTA) agarose beads (Qiagen, Hilden, Germany) as described 4.
NKG2D-Fc was purified using protein A columns (GE Healthcare, Munich, Germany)
according to manufacturer’s instructions. Concentrations of the purified proteins were
estimated against a standard curve of bovine serum albumin (BSA) or determined by
quantitative capillary electrophoresis using Experion™ Pro260 technology (BioRad,
Hercules, USA) in accordance with the manufacturer’s protocol.
Gelfiltration chromatography
Gelfiltration chromatography was performed on an ÄKTA purifier (GE Healthcare) using
phosphate buffered saline (PBS) as running buffer at a constant flow rate of 1 ml/min. 200 µg
protein were loaded in a volume of 1 ml on a Superdex 200 10/300 GL column (GE
Healthcare). Ferritin (440 kDa), human IgG1 (150 kDa), Conalbumin (75 kDa) and
Ribonuclease A (13.7 kDa) were used for calibration. Data were analyzed with Unicorn 5.1
software (GE Healthcare).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western
blot analysis
SDS-PAGE was performed by standard procedures. Proteins were visualized using colloidal
Roti®Blue Coomassie staining solution (Carl Roth, Karlsruhe, Germany). In Western transfer
experiments the recombinant proteins were transferred to PVDF membrane (BioRad) and
detected by mouse anti-penta-His (Qiagen) and secondary horse radish peroxidase-conjugated
goat anti-mouse IgG antibodies (Dianova, Hamburg, Germany). Blots were analyzed using
ECL reagents (Pierce, Rockford, USA) and a digital imaging system (Biorad).
Isolation of MNC and NK cells
Preparation of MNC from peripheral blood was performed as described previously 5. The
content of tumor cells in patient samples was determined by flow cytometry. NK cells were
enriched by negative selection using NK cell isolation kit (Miltenyi, Bergisch Gladbach,
Germany) and MACS technology following manufacturer’s protocols. Purities were analyzed
by 2-colour flow cytometry using CD16, CD3 and CD56 antibodies. Blood was drawn after
receiving the donors´ written informed consents and experiments reported here were approved
by the Ethics Committee of the Christian-Albrechts-University (Kiel, Germany), in
accordance with the Declaration of Helsinki.
Flow cytometric analysis
Flow cytometry was performed on flow cytometers FC 500 or Coulter EPICS XL (Brea,
USA). 3 x 105 cells were washed in PBS containing 1% BSA (Sigma-Aldrich Chemie GmbH,
Munich, Germany) and 0.1% sodium-azide (PBA buffer). To analyze binding of MICA:7D8
and ULBP2:7D8, Ramos cells were incubated with either protein on ice for 45 min, then
washed with 2 ml PBA buffer and subsequently stained with a secondary Alexa Fluor 488coupled anti-penta His antibody (Qiagen) on ice for 30 min. To demonstrate simultaneous
binding, Ramos cells were pre-incubated with MICA:7D8 or ULBP2:7D8 at 15 µg/ml
followed by a fusion protein NKG2D-Fc at 100 µg/ml. Finally, binding was visualized by
staining with a polyclonal FITC-coupled anti-human IgG F(ab´)2 fragment (Beckman Coulter,
Fullerton, USA). Cetuximab (Merck, Darmstadt, Germany) was used as an IgG1 Fccontaining non-relevant control protein. Expression of CD antigens with fluorescence-coupled
antibodies was analyzed according to manufacturer's protocols. Antibodies against CD38,
CD16 (both FITC-coupled), NKG2D and CD69 (PE-conjugated) were from Beckman
Coulter; antibodies specific for CD56 (PC7-conjugated), CD20 and CD3 (both FITC-coupled)
were from BD biosciences (Heidelberg, Germany) and PE-labelled antibodies against
MICA/B and ULBP2 from R&D systems (Wiesbaden-Nordenstadt, Germany). In indirect
staining experiments, the human CD20 and CD38 antibodies 7D8 and daratumumab,
respectively, were detected with a FITC- conjugated goat anti-human IgG F(ab’)2 fragment
(Beckmann Coulter).
NK cell activation assay
Un-stimulated NK cells or MNCs were mixed with Raji target cells at effector-to-target cell
(E:T) ratios of 1:1 or 2:1, respectively, and incubated with sensitizing proteins in a volume of
1 ml. After 20 h the expression levels of CD69 on CD56-positive / CD3-negative NK and
CD3-positive / CD56-negative T cells were analyzed by 3-colour flow cytometry. Specific
effects were determined by subtraction of basal activation of effector cells mediated by Raji
cells alone from all data points.
Detection of apoptosis
Twenty-five thousand Ramos cells labeled with the membrane labelling dye CellVue
Lavender (Polysciences, Inc., Eppelheim, Germany) were incubated with 2.5  105 NK cells
in presence of MICA:7D8, ULBP2:7D8 or the corresponding control proteins (each at 100
nM) in a total volume of 1 ml. After 3 h cells were stained using Vybrant FAM Poly Caspases
Kit and 7-Aminoactinomycin D (7-AAD) according to the manufacture’s protocols. Cells
within CellVue Lavender-positive population were analyzed for caspase cleavage and
membrane integrity on Navios flow cytometer using appropriate gates (Beckman Coulter).
Cytotoxicity assays
Cytotoxicity was analyzed in standard 4 h 51Cr release assays performed in 96-well microtiter
plates in a total volume of 200 µL as described 5. 25 µl of supernatant was mixed with
scintillation solution Supermix (Applied Biosystems, Darmstadt, Germany) and incubated for
15 min with agitation. 51Cr release from triplicates was measured in counts per minute (cpm).
Percentage of cellular cytotoxicity was calculated using the formula: % specific lysis =
(experimental cpm - basal cpm) / (maximal cpm - basal cpm) x 100. Maximal 51Cr release was
determined by adding Triton X-100 (1% final concentration) to target cells and basal release
was measured in the absence of sensitizing proteins and effector cells. In blocking
experiments, either the Fab 7D8 (M. Peipp, unpublished) or the murine anti-NKG2D IgG1
antibody (R&D systems) were added to the reactions at 20 and 50 µg/ml, respectively. Fab
4D5 (M. Peipp, unpublished) and the CD7-specifc IgG1 antibody TH69 2 were used as nonrelevant control proteins. To analyze synergistic effects of MICA:7D8 and ULBP2:7D8 on
ADCC, the molecules were added together with the CD38 antibody daratumumab to the
reactions at the indicated concentrations 6. Antibody 7D8 1 was included in some experiments
for comparison.
Data processing and statistical analyses
Graphical and statistical analyses were performed using GraphPad Prism 4.0 software. Pvalues were calculated using the student's t-test or repeated measures Anova and Bonferroni
post test when appropriate. The null hypothesis was rejected for p < 0.05. Calculation of
combination index (CI) was performed with CalcuSyn software (Biosoft Ferguson, USA)
according to Chou and Talalay 7.
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