CD200 expression suppresses natural killer cell function and directly inhibits patient anti-tumor response in acute myeloid leukemia

Abstract

Upregulation of the immunosuppressive cell surface glycoprotein, CD200, is a common feature of acute myeloid leukemia (AML) and is associated with poor patient outcome. We investigated whether CD200 overexpression on AML cells could specifically compromise patient natural killer (NK) cell anti-tumor responses. We found that CD200(hi) patients showed a 50% reduction in the frequency of activated NK cells (CD56(dim)CD16(+)) compared with CD200(lo) patients. Additionally, NK receptor expression (NKp44 and NKp46) on these cells was also significantly downregulated in CD200(hi) patients. To assess whether NK cell activity was directly influenced by CD200 expression, we examined the effect of ectopic expression of CD200. These assays revealed that both NK cell cytolytic activity and interferon-γ response were significantly reduced toward CD200(+) leukemic targets and that these targets showed increased survival compared with CD200(-) cells. Similarly, NK cells isolated from AML patients were less functionally active toward CD200(hi) autologous blasts from both cytolytic and immunoregulatory perspectives. Finally, blocking CD200 alone was sufficient to recover a significant proportion of NK cell cytolytic activity. Together, these findings provide the first evidence that CD200 has a direct and significant suppressive influence on NK cell activity in AML patients and may contribute to the increased relapse rate in CD200(+) patients.

Figures

Figure 1

Expression of CD200 on AML blasts relates to a change in the frequency of NK cell subsets. (a) Representative flow cytometric plots of NK cells in CD200lo and CD200hi AML patients (CD200 expression level is defined in Data acquisition and analysis). Five NK cell sub-populations are shown based on the bivariate expression of CD56 and CD16. Plots are gated on CD45+ + CD19− CD3− lymphocytes. (b) Summary box and whisker plot summarizing the frequency of the five NK cell sub-populations (as percentage of total lymphocytes) between CD200lo (filled boxes) and CD200hi (n=12 per group). ***P<0.001 analyzed by one-way analysis of variance (ANOVA) with Turkey’s multiple comparison test.

Figure 2

Comparisons of NK cell activation marker repertoire between CD200lo and CD200hi AML patients. NK cells from CD200lo and CD200hi AML patients were examined by eight color flow cytometry (see Materials and methods). Sub-populations 3, 4 and 5 in CD200lo (filled bars) and CD200hi (open bars) patients were assessed for expression of (a) NKp44, (b) NKp46, (c) NKp30 and (d) NKG2D. Data represent mean±1 s.d. (n=12, per group). *P<0.05 analyzed by one-way analysis of variance (ANOVA) with Turkey’s multiple comparison test.

Figure 3

Functional assessment of NK cell activity toward K562-CD200− and K562-CD200+ targets. (a) Summarized data illustrating percentage of degranulating NK cells in sub-populations 3, 4 and 5 (defined as in Figure 1a) in response to K562-CD200− or K562-CD200+ targets (Supplementary Figure S3). Background CD107a expression (PBMC control cultures with no targets) is also indicated. Flow cytometric gating strategy for this assay is shown in Supplementary Figure S4). (b) Representative flow cytometric plots illustrating expression of CD107a (open histograms) in sub-population 4 NK cells in response to K562-CD200− and K562-CD200+ targets, or PBMC control. Filled histograms represent isotype-matched controls. (c) IFNγ release in response to K562-CD200− targets compared with K562-CD200+ targets as measured by ELISPOT at different effector to target (E:T) ratios (see Materials and methods; representative data shown in Supplementary Figure S4). Data represent mean±1 s.d. (n=6, per group). *P<0.05 analyzed by one-way analysis of variance (ANOVA) with Turkey’s multiple comparison test.

Figure 4

Reduced NK cell degranulation corresponds to increased viability of CD200+ targets. K562-CD200− or K562-CD200+ targets were co-cultured with PBMC from healthy donors, for 18 h at 37 °C and 5% CO2 at an effector:target ratio of 2:1 (or in the absence of PBMC; K562 controls). Following this, cells were stained with annexin-V/7AAD and analyzed by flow cytometry, excluding lymphocytes, (deselected based on low FSC/side scatter (SSC)). (a) Summarized data (mean+1 s.d.; n=8). (b) Representative annexin-V/7AAD flow cytometric plots from PBMC co-cultured with K562-CD200− and PBMC co-cultured with K562-CD200+ targets. (c) Representative flow cytometric plots confirming that NK cells degranulated significantly more toward K562-CD200− targets compared with K562-CD200+ targets under the same assay conditions. Figures represent mean percent degranulation for each target type (n=8, P<0.05). *P<0.05 analyzed by one-way analysis of variance (ANOVA) with Turkey’s multiple comparison test.

Figure 5

CD200R is expressed on AML patient NK cell sub-populations. (a) Representative flow cytometric plots reveal that CD200R expression is expressed on NK cells on sub-populations 3, 4 and 5 in AML patients. (b) Summary data illustrating the CD200R relative expression level within the indicated NK cell sub-populations. Data represent mean+1 s.d. (n=20).

Figure 6

CD200 blocking antibody promotes NK cell activity in CD200hi patients. (a) BMMC from CD200lo and CD200hi AML patients were incubated in the presence of CD107a with 5 μg/ml of unconjugated anti-human CD200 or respective IgG isotype-matched control for 6 h at 37 °C and 5% CO2. The percentage of CD107a-positive NK cells in response to autologous AML blasts was determined by flow cytometric analysis. (b) NK cell IFNγ release from BMMC of AML patients in response to CD200lo or CD200hi autologous blasts, as measured by ELSIPOT. Incubations were carried out in the presence of unconjugated anti-human CD200 or respective IgG isotype-matched control for 18–20 h at 37 °C and 5% CO2. Data represent mean+1 s.d. (n=6). *P<0.05 one-way analysis of variance (ANOVA) with Turkey’s multiple comparison test.