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
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).
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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This work was funded by Leukaemia and Lymphoma Research UK and NISCHR, UK. RR is a CU/MRC UK funded student.
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.
The authors declare no conflict of interest.
Supplementary Information accompanies this paper on the Leukemia website
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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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