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The immunosuppressive ligands PD-L1 and CD200 are linked in AML T-cell immunosuppression: identification of a new immunotherapeutic synapse

Long-term remission in acute myeloid leukemia (AML) is generally not durable only being achieved in <50% of patients.1 Consequently there is a need to establish new treatments to prevent relapse. A promising approach is to augment the anti-tumor immune response in these patients; however, it is well established that overexpression of immunosuppressive molecules such as CD200 on the surface of AML cells directly suppresses the antitumor response.2, 3, 4 Nevertheless, blocking CD200:CD200R, only partially restores T-cell activity, suggesting that alternative immunosuppressive mechanisms need to be explored if the antitumor response in AML is to be optimally exploited.5

Recently, promising clinical outcomes using humanized antibodies targeting PD-1 have been reported for melanoma and even for non-small cell lung cancer.6 PD-1 suppresses immunological function via interaction with its cognate ligand PD-L1 (aka B7-H1, CD274) and previous work has indicated that PD-L1 may also suppress immunological function in AML.7, 8 Here we investigate whether the PD-1:PD-L1 axis cooperates with CD200 in mediating immunosuppression in AML patients.

Initially, we investigated whether CD200 and PD-L1 were co-expressed in AML blasts. Gene expression data from 158 AML diagnostic samples were analyzed and stratified into CD200hi and CD200lo based on upper and lower quartiles of expression.3 As shown in Figure 1a, CD200hi AML patients had 10-fold higher levels of PD-L1 mRNA compared to CD200lo patients. Flow cytometric analysis of CD200 and PD-L1 protein expression on AML blast cells confirmed this association at the protein level (Figure 1b). Taken together, these data establish that the immunosuppressive ligands CD200 and PD-L1 are coexpressed on patient AML blast cells, indicating that CD200 and PD-L1 could cooperate in AML cell mediated immunosuppression. A possible explanation for this observation is that CD200 and PD-L1 may be upregulated by AML as an adaptive mechanism following a T cell-meditated immune response.9

Figure 1
figure1

Assessment of CD200 and PD-L1 coexpression on AML patient blasts. The coexpression of CD200 and PD-L1 in AML patients was compared at the transcript level by microarray and at the protein level by flow cytometry. (a) Affymetrix gene expression data (U133 plus_2.0) from 158 AML patients was analyzed using GeneSpring v12.6 (Agilent Technologies). Gene expression data were normalized to median gene expression and expressed as Log2 as previously described.16 AML patients (Supplementary Table 1) were stratified to CD200hi and CD200lo (probe set; 209582_s_at) based on normalized expression level (n=39 for each) as previously described.4 Data were consistent for the alternative probe for CD200, 209583_s_at (not shown). Data illustrate a significant increase in PD-L1 normalized expression (probe sets; 223834_s_at and 227458_s_at) for CD200hi AML patients (mean±s.e). (b) The association between CD200 and PD-L1 expression level on AML patient blast cells isolated from peripheral blood was analyzed by flow cytometry. AML blast cells were identified through CD45/CD34 bivariate analysis as previously described.2 The data illustrate a positive correlation between CD200 and PD-L1 protein expression (normalized mean fluorescence intensity; MFI)2 on AML patient blast cells; Pearson’s correlation coefficient, r2=0.4901, P<0.01 (n=14). *P<0.05 analyzed by one-tailed unpaired t-test. See Supplementary Methods for detailed methods and supplementary Figure 1.

In common with other malignancies, robust CD8+ T-cell responses are thought to be important in AML antitumor immunity.10 We previously demonstrated that CD200 overexpression in AML suppresses memory CD8+ T-cell effector function.4 To investigate whether CD200 together with PD-L1 had the potential to inhibit CD8+ T-cell effector function, we first determined whether these cells expressed the respective negative coreceptors CD200R and PD-1 in AML patients. This analysis showed expression of both CD200R and PD-1 on CD8+ T cells from AML patients, interestingly, higher expression levels of PD-1 were observed for CD200hi AML patients (Supplementary Figures 2 and 3). To further characterize PD-1+ T cells, we analyzed several AML patient CD8+ T-cell subpopulations, including; CD57+ CD28 (late differentiated, poor antitumor function in AML)11 and CD57 CD28+ (early differentiated, important for robust antitumor function).12 Figure 2a shows that the mean frequency of CD57+ CD28 PD-1+ and CD57 CD28+ PD-1+ CD8+ T cells was almost twice that for CD200hi patients compared with CD200lo. These findings show for the first time a link between CD200 expression level on AML blast cells and the frequency of PD-1+ late differentiated and PD-1+ early differentiated CD8+ T cells, illustrating that CD200 and PD-1 are linked at multiple levels of CD8+ T-cell differentiation. A similar though less-marked effect was observed in CD4+ T cells (see Supplementary Figure 4).

Figure 2
figure2

Functional assessment of the relationship between CD200:CD200R and PD-L1:PD-1 co-expression. (a) Using flow cytometry, the percentage of PD-1+ CD8+ T-cell subsets from peripheral blood mononuclear cells was evaluated between CD200hi and CD200lo AML age matched patients, median age; 53 years (range, 35–64 years) and 54 years (range, 17–70 years), respectively (Supplementary Table 1). (b) Assessment of PD-1 expression induction upon coculture of the CD8+ T-cell clone 7E7 with either CD200+ or CD200- K562 cells.3 Assays were performed with or without prior CD3/CD28 receptor costimulation of 7E7 using 5 μg/106 cells anti-CD3 (OKT3) and anti-CD28 (28.2) as previously described.11 Data show an increase in PD-1+ 7E7 T cells when activated through CD3/CD28 receptor costimulation in CD200 coculture assays. (c) Effect of 5 μg/106 cells unconjugated anti-human CD200 (MRC OX-104), on the frequency of PD-1+ 7E7 CD8+ T cells following coculture with CD200+ or CD200 K562 cells. Isotype matched IgG antibody was used as a control. (d) Flow cytometric analysis of K562 cells (CD200PD-L1) following retroviral transduction with CD200 and/or PD-L1 to create, CD200+PD-L1 (CD200 single positive), CD200+PD-L1+ (double positive) or CD200-PD-L1+ (PD-L1 single positive) K562 cells. (e) The effect on 7E7 CD8+ T-cell activation of coculture with K562 cells expressing CD200 and/or PD-L1. This was assessed by secretion of TNFα (intracellular cytokine staining)4 in CD3/CD28 activated 7E7 CD8+ T cells following coculture with K562 cells (n=9). Data are mean±1 s.e. *P<0.05 analyzed by one-way ANOVA with Tukey’s multiple comparison test. P<0.05 and ††P<0.01 analyzed by one-tailed paired t-test.

The above data suggested that stimulation of the CD200:CD200R immune-axis may have the capacity to induce PD-1 expression on target CD8+ T cells. To investigate this, we assessed whether CD200:CD200R stimulation was directly capable of mediating PD-1 up-regulation on target CD8+ T cells. We carried out a refined coculture assay in which a CD8+ T cell clone (7E7)13 was incubated with CD200+ or CD200- K562 cells.3 CD200R expression on 7E7 T cells was confirmed by flow cytometry (Supplementary Figure 5). In these assays, 7E7 T-cell PD-1 expression was monitored by flow cytometry. Figure 2b illustrates that in the presence of CD200+ K562 cells, the frequency of PD-1+ 7E7 T cells was significantly increased 1.5-fold compared with 7E7 T cells cocultured with CD200- K562 cells and exceeded the level of PD-1 up-regulation achieved through CD3/CD28 receptor costimulation of 7E7. To verify these data, a CD200 blocking antibody3 was added to the CD200+ K562 7E7 T cell assay. Figure 2c illustrates a significant reduction in the frequency of PD-1+ 7E7 T cells in the presence of the CD200 blocking antibody, demonstrating that PD-1 expression on target CD8+ T cells can be reduced through CD200:CD200R blockade in a CD200hi setting. Taken together, our findings illustrate, for the first time, that CD200:CD200R interaction has the capacity to increase the frequency of PD-1+ CD8+ T cells.

To investigate the consequences of CD200 and PD-L1 coexpression on T-cell activation, we created a series of K562 lines expressing CD200 or PD-L1 or both molecules in combination (Figure 2d). The 7E7 CD8+ T-cell clone produces tumor necrosis factor alpha (TNFα) upon stimulation,13 and was used as an endpoint for 7E7 T-cell activation in our assays. Figure 2e shows that both CD200 and PD-L1 induced a similar (>50%) reduction in the frequency of activated 7E7 T cells (compared with cocultivation with K562 control cells expressing neither molecule); however, when both CD200 and PD-L1 were co-expressed 7E7 T-cell activation was almost ablated (~90% reduction). Moreover, the strength of the TNFα response was significantly reduced in coculture assays where either CD200 or PD-L1 were present, indicating a direct effect at the level of CD8+ T-cell function (Supplementary Figure 6). These data demonstrate that CD200:CD200R and PD-L1:PD-1 engagement on T cells can act in tandem to produce a greater immunosuppressive effect on CD8+ T cells when expressed on leukemia cells. This is of particular importance in AML, where both CD200 and PD-L1 are frequently co-expressed on AML cells. The expression level of CD200 on T cells was identical for CD200hi and CD200lo AML and is unlikely to contribute to the immunosuppressive effects observed, at least for newly diagnosed patients (Supplementary Figure 7).

Previous studies in AML indicate that multiple immunosuppressive mechanisms may work in conjunction; for example coexpression of PD-1 and the negative regulatory receptor, Tim-3, identify a dysfunctional CD8+ T-cell population;14 whereas in other contexts it has been shown that dual blockade of PD-L1:PD-1 and CTLA-4 is required to restore CD8+ effector T-cell antitumor responses.15 Here we propose that stimulation of the CD200:CD200R immune-axis augments the frequency of PD-1+ CD8+ T cells and that these in turn engage with PD-L1 on AML blasts, exacerbating immunosuppressive effects. Interestingly in AML, overexpression of both CD200 and PD-L1 in have been linked to a worse patient prognosis,.16, 17 Given the recent progress in PD-1 targeted immunotherapy (for example Nivolumab and Pembrolizumab) and also Samalizumab for CD200:CD200R blockade,6, 18 we propose a novel CD200/PD-L1 immunotherapeutic synapse in AML which should be targeted by combining CD200:CD200R and PD-L1:PD-1 blockade for future immunotherapy of AML.

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Acknowledgements

This work was funded by Leukaemia and Lymphoma Research UK and NISCHR, UK. RR is a CU/MRC UK funded student.

Author Contributions

SJC designed and performed the experiments, analyzed all data and cowrote the manuscript. MNG assisted with PD-L1 cloning and retroviral transduction. RR assisted with 7E7 T-cell expansion. SK and AKB provided resources and clinical insight. SM, AT and RLD contributed to experimental design and co-wrote the manuscript.

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Correspondence to R L Darley.

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Coles, S., Gilmour, M., Reid, R. et al. The immunosuppressive ligands PD-L1 and CD200 are linked in AML T-cell immunosuppression: identification of a new immunotherapeutic synapse. Leukemia 29, 1952–1954 (2015). https://doi.org/10.1038/leu.2015.62

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