Methods
AML patient and healthy donor materials
Peripheral blood mononuclear cells (PBMC) or bone marrow mononuclear cells
(BMMNC) were collected from healthy donors or AML patients enrolled in the UK MRC/NCRI
AML 14, 15, 16 and 17 clinical trials at point of diagnosis, before treatment and following
informed consent from patients in accordance with the 1964 Declaration of Helsinki.
Mononuclear cells were separated on Ficoll-Hypaque (Sigma-Aldrich, Poole Dorset U.K) as
previously described.1
Cell surface immunophenotyping of AML patient blasts and T-cell subpopulations
PBMC/BMMNC from AML patients or healthy donors were thawed and washed in prewarmed T-cell medium consisting of Iscove’s modified Dulbecco’s Medium supplemented with
10% heat inactivated human AB-serum (Invitrogen, Paisley, U.K.). Cells (106) were washed in
chilled staining buffer (PBS, 2% FCS, 1mM EDTA, 0.02% NaN3) before incubation with the
following anti-human antibodies purchased from BD Bioscience U.K. unless otherwise stated;
CD45-FITC, CD200-PE, PD-L1-APC, CD34-PerCP-Cy5.5 for AML blast cell phenotyping and
CD3-APC-H7, CD4 or CD8-pacific blue, (PD-1)-FITC, CD57-PE and CD28-PerCP-Cy5.5
(Biolegend, Cambridge, U.K.) for T-cell phenotyping. All incubations were carried out for 30
min and 4ºC after which cells were assessed by multi-parameter flow cytometry. For flow
cytometric gating, AML blast cells were analyzed as previously described using CD34 and CD45
bivariate analysis.2 CD200 and PD-L1 expression was assessed on the AML blast cell population
by means of an isotype match IgG control to set the negative population. For AML patient T cell
subpopulation analysis, T cells were identified using CD45++ low SSC, CD3+, CD4+ or CD8+,
followed by CD28/CD57 bivariate analysis. PD1 expression was assessed within CD28/CD57
regions by means of an isotype match IgG control to set the negative population (for full gating
strategy see Supplemental Material Fig S1). AquaLive (Invitrogen) was used to exclude dead
cells from the analysis. Cells were acquired using a FACSCantoII and analyzed post-acquisition
in FCS Express V4 (De Novo Software, Canada).
Generation of K562 cells expressing C200 and PD-L1
CD200 cDNA was kindly provided by IMAGE consortium; clone ID 5299899,1,3 and
was sub-cloned into the PINCO retroviral expression vector, which co-expresses GFP from a
CMV promoter as previously described.4 PD-L1 cDNA (Eurofins, Ebersberg, Germany) was
sub-cloned into the PINCO retroviral expression vector, which co-expresses DSred from a CMV
promoter using the same method. Phoenix packaging cells (gift from Garry Nolan, Stanford
University School of Medicine, Stanford, CA, USA) were used to generate replication-defective
retrovirus, before transducing K562 cells as previously described.1. PINCO co-expressing PD-L1
and DSred was use to co-transduce K562 cells stably expressing CD200 from a previous
transduction. In this way, five cultures were generated: K562-mock, K562-CD200-PD-L1+
(PINCO-expressing GFP alone with PINCO-expressing DSred and PD-L1), K562-CD200+PDL1+ (PINCO-expressing GFP and CD200 with PINCO-expressing DSred and PD-L1), K562CD200+PD-L1- (PINCO-expressing GFP and CD200 with PINCO-expressing DSred alone) and
K562-CD200-PD-L1- (PINCO-expressing GFP alone with PINCO-expressing DSred alone).
Transduced K562 cells were maintained in RPMI-1640 (Sigma-Aldrich), 10% (v/v) FCS
(Biosera, East Sussex, UK), 2 mM L-glutamine (Sigma-Aldrich) and cell sorted for DSred
positivity using a FACSAria (BD). Cell purity, CD200 and PD-L1 protein expression were
confirmed by flow cytometry.
Functional assessment of CD200 and PD-L1 in co-culture assays
In a 96-well flat bottomed plate, 5 x105 K562 cells were co-cultured with 105 7E7 CD8+
T cell clone5 at 37°C and 5% CO2 in the presence of Golgi-stop (1:1500) and 10 µg/ml brefeldinA. 7E7 cells are HPV16 E629-38 specific and produce IFNγ and TNFα following activation.6 In
the co-culture assay, 7E7 T cells were activated through CD3 (OKT3, eBioscience) and CD28
(28.2, R&D) co-stimulation (5µ/106 cells) for 6 h, after which cells were surface stained for
CD8, CD200R and PD1 (20ng/ml PMA and 1µ/ml ionomycin (Sigma-Aldrich) was used for
stimulation as a positive control). Intracellular TNFα staining was performed on the CD8+ cells
as previously described.2 For CD200 blocking experiments, incubations were carried out as
above in the presence of 5µg/106 cells un-conjugated anti-human CD200 (clone MRC OX-104,
BD) or IgG1, κ isotype matched control (BD).
References
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Coles SJ, Hills RK, Wang ECY, Burnett AK, Man S, Darley RL et al. Expression of
CD200 on AML blasts directly suppresses memory T-cell function. Leukemia 2012; 26:
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