Microarray
analysis
Median age
(range)
Sex
Male
Female
Functional
analysis
Median age
(range)
Sex
Male
Female
Total
CD200hi
CD200lo
53.5
52
(28-64)
54.5
(19-78)
34
44
15
24
19
20
53.5
54
(35-64)
53
(17-70)
21
19
8
9
13
10
Supplemental Table 1. Parameters of AML patients. Table shows patients stratified
according to level of CD200 expression (probe set; 209582_s_at) for PD-L1
expression analysis and functional analyses respectively. Patients were stratified into
CD200hi and CD200lo based on upper and lower quartiles of expression respectively.
All analyses were performed on diagnostic material.
A
B
C
40
CD3
30
CD200
CD8
CD3
20
10
CD8
CD200 hi
0
CD200 lo
Patient frequency (%)
PD-L1 (up-regulated)
PD-L1 (down-regulated)
PD-L1
Supplemental Figure 1. PD-L1 and CD200 co-expression on blast cells from AML
patients and CD8 T cell gating strategy. (A) Summary gene expression data
illustrating % patient PD-L1 change (up-regulated or down-regulated) between
CD200lo and CD200hi AML patients. (B) Representative flow cytometric data showing
the co-expression of PD-L1 and CD200 on blast cells AML patients.
(C)
+
Representative flow-cytometric bivariate plot identifying the CD8 T cell population
from CD200lo (upper) and CD200hi (lower) AML patients.
Supplemental Figure 2. Representative flow cytometric plots illustrating
CD200R expression on CD8+ T cells from AML patients. Isotype matched control
in shaded black.
Supplemental Figure 3. Representative flow cytometric plots illustrating PD1
expressio n on CD8+ T cells from AML patients. Isotype matched control in
shaded black
Supplemental Figure 4. Comparison of PD-1+ CD4+ T cells between CD200hi and
CD200lo AML patients. Using flow cytometry, the percentage of PD-1+ CD4+ T cell
subsets was evaluated between CD200hi and CD200lo AML age matched patients,
median age; 53 (range, 35-64) and 54 (range, 17-70).
A
B
Supplemental Figure 5. CD200R expression on 7E7 CD8+ T cells from co-culture
assays with K562 cells. The expression of CD200R on 7E7 CD8+ T cells from K652
co-culture assays was evaluated by flow cytometry. (A) 7E7 T cells were distinguished
as CD8+, low SSC. (B) Histogram illustrates CD200R expression on 7E CD8+ T cells.
Shaded plot represents IgG isotype matched control.
3
*
*
*
2
CD200- PD-L1+
CD200+ PD-L1+
0
CD200+ PD-L1-
1
CD200- PD-L1-
(normalized)
MFI
TNFα
TNF
MFI
(normailsed)
4
Supplemental Figure 6. The effect of K562 co-expression of CD200 and PD-L1
on TNFα release in 7E7 CD8+ T cells. CD200-PD-L1- (double negative), CD200+PDL1- (single positive), CD200+PD-L1+ (double positive) or CD200-PD-L1+ (single
positive) K562 cells were co-cultured with 7E7 CD8+ T cell and TNF-α production was
analysed by flow cytometry. The magnitude of the TNFα response (normalized mean
fluorescence intensity; MFI) was significantly lower in single positive and double
positive co-cultures compared with double negative. *p<0.05 analyzed by one-way
ANOVA with Tukey’s multiple comparison test.
B
6
4
2
CD200(hi)
0
CD200(lo)
Lymphocyte CD200 MFI (normalized)
A
Supplemental Figure 7. CD200 expression on lymphocytes from AML patients.
The expression of CD200 was evaluated on AML patient lymphocytes at diagnosis by
flow cytometry. (A) CD45 was used to distinguish lymphocytes (P1) from AML blast
cells. Lymphocyte frequency varied from 0.1% - 4.2% for the patients analysed. (B)
Summary of total CD4+ and CD8+ lymphocyte CD200 expression level (MFI) between
CD200lo and CD200hi AML patients.